CN112972648A - Application of protease inhibitor in inhibiting novel coronavirus - Google Patents

Application of protease inhibitor in inhibiting novel coronavirus Download PDF

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CN112972648A
CN112972648A CN202110200746.7A CN202110200746A CN112972648A CN 112972648 A CN112972648 A CN 112972648A CN 202110200746 A CN202110200746 A CN 202110200746A CN 112972648 A CN112972648 A CN 112972648A
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substituted
aryl
alkyl
heteroaryl
independently selected
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黄子为
阿龙·切哈诺沃
安静
徐岩
梁柏强
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Nobel Institute Of Biomedicine Co ltd
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Nobel Institute Of Biomedicine Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

Abstract

The invention provides an application of a proteasome inhibitor in inhibiting novel coronavirus or preparing the novel coronavirus, wherein the proteasome inhibitor has a structure shown in a formula (I) or an isomer, a pharmaceutically acceptable salt and a prodrug thereof. The proteasome inhibitor is used for inhibiting the novel coronavirus, obtains good inhibitory activity, and provides a new treatment idea for diseases such as pneumonia and the like caused by the novel coronavirus.

Description

Application of protease inhibitor in inhibiting novel coronavirus
Technical Field
The invention relates to the technical field of medicinal chemistry, in particular to application of a protease inhibitor in inhibiting novel coronavirus.
Background
The novel coronavirus spreads globally, and according to statistics of data of Weijian Commission of China, over 4300 million people are infected with the novel coronavirus in the world in 26 days 10 and 26 months 2020, wherein over 115 million people die of the novel coronavirus.
The coronaviruses belong to the order of the nested viruses (Nidovirales) and the family of coronaviridae in the phylogenetic classification
(Coronaviridae) and Coronaviridae (Coronaviridus), the virus of the genus Coronaviridae is an RNA virus with an envelope (enceope) and a linear single-strand positive strand genome, the 5 'end of the genome has a methylated cap structure, the 3' end of the genome has a poly (A) tail, the genome has the total length of about 27-32kb, and the virus has the largest genome among the RNA viruses known at present. The surface of the fat membrane that surrounds the virion has three glycoproteins: spinous process glycoprotein (S, SpikeProtein, which is a receptor binding site, cytolytic and major antigenic site); small envelope glycoproteins (E, enveloppeprotein, smaller, envelope-bound proteins); membrane glycoproteins (M, membrane proteins, responsible for transmembrane transport of nutrients, budding release of nascent viruses and formation of viral envelope). A few species also have hemagglutinin glycoproteins (HE proteins). Viruses mediate viral invasion primarily through the binding of Spike proteins (S proteins) to host cell receptors and determine the tissue or host tropism of the virus. Both the N-terminal domain (S1-NTD) and the C-terminal domain (S1-CTD) of the S1 subunit of coronavirus protein serve as Receptor Binding Domains (RBDs). It is believed that S1-NTD binds to carbohydrate receptors and S1-CTD binds to protein receptors.
Potential anti-coronavirus therapies can be divided into two categories: one directly targets the new coronavirus, and the other is human-oriented. Because viruses must enter host cells to be replicated to function, when designing antiviral drugs, research and development can be respectively carried out on a series of targets in the processes of virus and cell receptor interaction, gene replication, transcription, translation and the like when viruses invade the host cells and complete the life cycle. In addition to the above drug development against viruses, therapeutic drugs can be sought against the immune response of the body caused by viruses, using the human body as a guide. In the early stage of viral infection, the virus can be prevented by appropriately enhancing/activating the human body's own immunity through the activation of interferon or innate immune receptors. For critically advanced/immune-hyperactive patients, the use of immunosuppressive drugs or mechanisms may be considered for modulation due to the patient being over-challenged by the autoimmune system. In addition, treatment may be directed against mechanisms/targets where the host and pathogen act against each other. Currently, the main treatment modes of diseases include small molecule targeted drugs, biomacromolecule drugs, gene therapy, cell therapy, traditional Chinese medicine therapy, medical instruments and the like. At present, small molecule targeted drugs are one of the currently expected drug types for treating novel coronavirus pneumonia. The invention provides application of a protease inhibitor to novel coronavirus, and finds that the structure has good activity of inhibiting the novel coronavirus, and is expected to be applied to development of anti-novel coronavirus medicines.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a proteasome inhibitor for inhibiting a novel coronavirus, wherein the proteasome inhibitor has good inhibitory activity against the novel coronavirus.
The invention provides an application of a proteasome inhibitor in inhibiting novel coronavirus or preparing the novel coronavirus, wherein the proteasome inhibitor has a structure shown in a formula (I) or an isomer, a pharmaceutically acceptable salt and a prodrug thereof:
Figure BDA0002948706340000021
the invention provides an application of a composition comprising a proteasome inhibitor in inhibiting novel coronavirus or preparing the novel coronavirus, wherein the proteasome inhibitor has a structure shown in a formula (I) or an isomer, a pharmaceutically acceptable salt and a prodrug thereof:
Figure BDA0002948706340000022
wherein R is1Is substituted or unsubstituted arylA group or heteroaryl; preferably substituted or unsubstituted aryl of C6-12 or heteroaryl of C5-C12.
Preferably, R1Selected from the following groups:
Figure BDA0002948706340000031
wherein, X1、X2、X3、X4And X5Any one or two of them are N, and the rest are C; or X1、X2、X3、X4And X5All are C.
In certain embodiments of the invention, X1Is N, and the rest is C; in other embodiments of the invention, X2Is N, and the rest is C; in other embodiments of the invention, X3Is N, and the rest is C.
R11And R12Independently selected from:
H. halogen, -OCF3Cyano, nitro, alkenyl, alkynyl, alkyl, haloalkenyl, haloalkynyl, haloalkyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, cycloalkylalkyl, arylalkyl, heteroarylalkyl, cycloalkylheteroalkyl, heterocycloalkylheteroalkyl, arylheteroalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkoxyaryl, alkenyloxy, alkynyloxy, cycloalkoxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, aralkoxy, carboxy, acyl, sulfonyl, amino, -C (O) OR13、-OC(O)R13、-COR13、-NHS(O)mR13、-NHC(O)R13、-NHC(O)OR13、-NR14R15、-OC(O)NR14R15、-OC(O)R14R15、-SH、-SR14；
Wherein the content of the first and second substances,
m is 1 or 2;
R13selected from aryl, heteroaryl, alkenyl, alkynyl, alkyl, haloalkyl,The cycloalkyl or heteroalkyl, or heterocycloalkyl, is further substituted with one or more substituents selected from the group consisting of halogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, hydroxy, hydroxyalkyl, alkoxy, acyl.
R14Or R15Each independently selected from a hydrogen atom, a substituted or unsubstituted aryl, heteroaryl, alkenyl, alkynyl, alkyl, haloalkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl.
Or R11、R12Any one of (1) and X1、X2、X3、X4And X5Any two adjacent rings in the (b) are fused to form a 3-to 6-membered substituted or unsubstituted aliphatic or aromatic ring.
Preferably, R1Selected from any one of the following groups:
Figure BDA0002948706340000041
wherein the content of the first and second substances,
R16、R17independently selected from:
H. f, Cl, Br, methyl, methoxy, trifluoromethyl, trifluoromethoxy,
Or
Figure BDA0002948706340000042
Or R16、R17And the attached carbon atoms are linked to form a substituted or unsubstituted aromatic ring.
More preferably, R1Selected from any one of the following groups:
Figure BDA0002948706340000043
wherein the content of the first and second substances,
R16and R17Independently preferably:
H. f, Cl, Br, methyl, methoxy, trifluoromethyl, trifluoromethoxy,
Or
Figure BDA0002948706340000044
Or when R is16And R17When in ortho position, R16And R17And the attached carbon atoms are joined together to form an aliphatic or aromatic ring.
R16And R17More preferably ethyl, methoxy or
Figure BDA0002948706340000045
R2、R3Independently selected from hydrogen atom, C1-10 alkyl, aryl substituted C1-12 alkyl, C6-12 aryl or substituted aryl, C6-12 heteroaryl or substituted heteroaryl.
Preferably, said R is2、R3Independently selected from:
H、CH3、
Figure BDA0002948706340000051
in the present invention, when R is2，R3When the groups are different, such as independently selected from hydrogen atoms and are not H at the same time, the structure shown in formula (I) comprises a mixture of two conformations (formula Ia or Ib) of the chiral center in different proportions or one of the monomers:
Figure BDA0002948706340000052
R5and R7Independently selected from hydrogen atom, C1-10 alkyl, aryl substituted C1-12 alkyl, C6-12 aryl or substituted aryl, C6-12 heteroaryl or substituted heteroaryl.
Preferably, said R is5H, C1-5 alkyl, C1-5 haloalkyl, benzyl, substituted benzyl, indolylmethyl; the substituent of the substituted benzyl is preferably halogen, nitro, C1-5 alkyl, C1-5 alkoxy orA hydroxyl group.
In certain embodiments of the invention, R is5Or R7Each independently selected from:
H、CH3、
Figure BDA0002948706340000053
Figure BDA0002948706340000054
in certain embodiments of the invention, R is7Is methyl or isobutyl, cyclohexane methyl.
R4And R6Independently selected from hydrogen atoms.
In certain embodiments of the invention, R4And R6And is also H.
R8Is N (R)9)LQR10。
Wherein R is9Is a hydrogen atom.
And L is C ═ O.
Q is O, NH, N-alkyl or CH2(ii) a The N-alkyl group is preferably an N-C1-12 alkyl group.
R10Is aryl substituted C1-12 alkyl, heteroaryl substituted C1-12 alkyl, heterocyclyl substituted C1-12 alkyl, substituted or unsubstituted heterocyclyl or aryl. Preferably, said R is10The aryl group is substituted or unsubstituted benzyl, substituted or unsubstituted morpholinyl methylene, substituted or unsubstituted aryl or heteroaryl of C5-12.
In certain embodiments of the invention, R9Is hydrogen atom, L is C ═ O, Q is NH, R10Is a substituted aromatic ring or aromatic heterocycle.
More preferably, R is10Is any one of the following groups:
Figure BDA0002948706340000061
in the invention, the halogen is fluorine, chlorine, bromine or iodine;
the alkyl group is a straight or branched aliphatic hydrocarbon group;
the heteroalkyl group is a straight-chain or branched-chain alkyl-containing group and at least contains one or more heteroatoms, and the heteroatoms are S, O or N atoms;
the cycloalkyl is a saturated or partially saturated monocyclic, fused or spiro carbocyclic ring;
the heterocycloalkyl group is a cycloalkyl group containing at least one heteroatom;
the aryl group is an optionally substituted monocyclic, fused polycyclic or aromatic carbocyclic ring containing 5 to 12 carbon atoms;
the heteroaryl group is an optionally substituted aromatic ring-containing group having one or more heteroatoms in a ring atom of the aromatic ring, the heteroatom being S, O or an N atom.
The aralkyl group is an alkyl group having an aryl substituent.
In certain embodiments of the invention, the proteasome inhibitor has any one of the following structures:
Figure BDA0002948706340000071
the invention provides a preparation method of the proteasome inhibitor, which comprises the following steps:
oxidizing the compound shown in the formula (I-a) to obtain a compound shown in the formula (I);
Figure BDA0002948706340000081
in the present invention, the conditions for the oxidation reaction are not particularly limited, and may be reaction conditions for oxidizing a hydroxyl group to a carbonyl group, which are well known to those skilled in the art.
In certain embodiments of the invention, R4And R6Is a hydrogen atom, R8Is composed of
N(R9)LQR10，R9Is hydrogen atom, L is C ═ O, Q is single bond, the synthetic route is as follows:
Scheme1
Figure BDA0002948706340000082
the compound IV can be obtained by carrying out deprotection, condensation, oxidation and other steps on the compound I.
Or according to the following route:
Scheme2
Figure BDA0002948706340000083
in the present invention, the composition comprising the proteasome inhibitor may further comprise a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, vehicle or combination thereof.
In the present invention, the proteasome inhibitors are suitable for use in non-therapeutic applications when used to inhibit novel coronaviruses.
Preferably, the novel coronavirus inhibitor is used for preventing, treating or alleviating any one or more of diseases related to the novel coronavirus.
Compared with the prior art, the invention provides the application of a proteasome inhibitor in inhibiting novel coronavirus or preparing the novel coronavirus, wherein the proteasome inhibitor has a structure shown in a formula (I) or an isomer, a pharmaceutically acceptable salt and a prodrug thereof. The proteasome inhibitor is used for inhibiting the novel coronavirus, obtains good inhibitory activity, and provides a new treatment idea for diseases such as pneumonia and the like caused by the novel coronavirus.
Drawings
FIG. 1 shows the toxicity test results of Compound 1 against VeroE6 cells;
FIG. 2 is an image of a plaque reduction experiment;
FIG. 3 shows the in vitro inhibitory activity of Compound 1 against SARS-CoV-2.
Detailed Description
To further illustrate the present invention, the use of the proteasome inhibitors provided by the present invention for inhibiting novel coronaviruses is described in detail below with reference to the examples.
In the following examples, all temperatures are in degrees celsius unless otherwise indicated. The various starting materials and reagents used are commercially available and, unless otherwise indicated, are used without further purification; the glassware is oven dried and/or heat dried.
The specification of the silica gel plate for thin layer chromatography is 0.15-0.2mm, and the specification of the silica gel for column chromatography is 200-300 meshes; the mass spectrum is measured by an MS instrument, and the ionization mode can be ESI or APCI.
The following examples are merely illustrative of the synthesis of specific compounds of the invention. But there is no limitation on the synthesis method. The compounds not shown in the examples can be prepared by selecting appropriate starting materials and adjusting reaction conditions slightly appropriately and universally as necessary by the same synthetic route and synthetic method as described below. In general, the compounds of the present invention can be prepared by the methods described herein, or by other conventional techniques well known to those skilled in the art, wherein the substituents are defined as shown in formula (I), unless otherwise specified.
EXAMPLE 1 Synthesis of Compound 1
Figure BDA0002948706340000101
1) Synthesis of methyl (S) -2-amino-3- (4-methoxyphenyl) propionate hydrochloride
A250 mL round bottom flask was charged with (S) -2- ((tert-butoxycarbonyl) amino) -3- (4-methoxyphenyl) propionic acid (10g, 33.8604mmol, 1eq) and 13mol/L methanolic hydrochloric acid solution (100mL), and the reaction was stirred at 70 ℃ for 3 hours. The reaction was monitored by TLC and after completion of the reaction solution was concentrated in vacuo to give 8.1g of a pale yellow solid which was used directly in the next reaction.
Synthesis of methyl (S) -2- ((S) -2- ((tert-butoxycarbonyl) amino) propionylamino) -3- (4-methoxyphenyl) propanoate
Figure BDA0002948706340000102
A100 mL round bottom flask was charged with (S) -methyl 2-amino-3- (4-methoxyphenyl) propionate hydrochloride (3.12g, 12.6843mmol, 1.2eq), N-tert-butoxycarbonyl-L-alanine (2.0g, 10.5703mmol, 1.0eq), tetrahydrofuran (20mL), 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (4.82g, 412.6843mmol, 1.2eq), 1-hydroxybenzotriazole (1.71g, 12.6843mmol, 1.2eq), N, N-diisopropylethylamine (5.2mL, 31.7109mmol, 3.0 eq). The reaction was carried out at room temperature for 2 hours, monitored by TLC, and after completion of the reaction, ethyl acetate (30mL) and water (30mL) were added for extraction, the aqueous layer was extracted twice with ethyl acetate (2 × 20mL), and the organic layers were combined and washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The crude product was purified on a silica gel column using a eluent (petroleum ether: ethyl acetate ═ 2:1) to give 3.87g of a pale yellow oil.
Synthesis of methyl (S) -2- ((S) -2- (aminopropionamide) -3- (4-methoxyphenyl) propionate hydrochloride
Figure BDA0002948706340000103
A100 mL round bottom flask was charged with methyl (S) -2- ((S) -2- ((tert-butoxycarbonyl) amino) propionylamino) -3- (4-methoxyphenyl) propanoate (3.87g, 10.1724mmol, 1.0eq), and dichloromethane solution (30mL), and the reaction was stirred at room temperature for 2 hours with excess hydrochloric acid gas. The reaction was monitored by TLC and after completion of the reaction solution was concentrated in vacuo to give 3.3g of a white solid, which was used directly in the next reaction.
4) Synthesis of methyl (S) -3- (4-methoxyphenyl) -2- (S) -2- (2-morpholineacetamide) propionamide) propionate
Figure BDA0002948706340000111
To a 100mL round bottom flask was added (S) -methyl 2- ((S) -2- (aminopropionamide) -3- (4-methoxyphenyl) propionate hydrochloride (2.0g, 6.3103mmol, 1.0eq), morpholineacetic acid (1.1g, 7.5724mmol, 1.2eq), tetrahydrofuran (20mL), water (4mL), 2- (7-azabenzotriazole) -N, N' -tetramethylurea hexafluorophosphate (3.6g, 9.4655mmol, 1.5eq), 1-hydroxybenzotriazole (1.28g, 9.4655mmol, 1.5eq), N-diisopropylethylamine (3.1mL, 18.9309mmol, 3.0eq), reacted at room temperature for 2 hours, monitored by TLC, after completion of the reaction was extracted with dichloromethane: methanol ═ 1:50(30mL) and water (30mL), the aqueous layer was extracted twice with dichloromethane: methanol ═ 1:50(2 × 20mL), the organic layers were combined and washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The crude product was purified on a silica gel column with eluent (dichloromethane: methanol ═ 40:1) to give 5.27g of a yellow liquid.
5) Synthesis of (S) -3- (4-methoxyphenyl) -2- (S) -2- (2-morpholineacetamido) propanamide) propanoic acid
Figure BDA0002948706340000112
A100 mL round bottom flask was charged with methyl (S) -3- (4-methoxyphenyl) -2- (S) -2- (2-morpholineacetamido) propionamide) propionate (5.27g, 12.9338mmol, 1.0eq), tetrahydrofuran (30mL), water (10mL), lithium hydroxide monohydrate (1086mg, 25.8676mmol, 2.0 eq). Stirred at room temperature for 2 hours. TLC monitoring, after the reaction was completed, vacuum concentration was carried out to remove most of tetrahydrofuran, 15mL of dichloromethane was added to extract impurities, then 1M hydrochloric acid was added to the aqueous layer under ice bath to adjust to acidity, and the aqueous layer was concentrated to obtain 6.45g of a white solid which was directly used in the next reaction.
6) Synthesis of N-benzamide
Figure BDA0002948706340000113
A100 mL round-bottomed flask was charged with aniline (10g,10.74mmol, 1.0eq) and formic acid (11.85g,25.77mmol, 2.4eq), and the reaction was stirred at 60 ℃ for 4 hours. TLC monitored the progress of the reaction, and when the reaction was complete, dichloromethane (150mL) and water (50mL) were added for extraction. The organic layer was washed successively with a saturated aqueous sodium hydrogencarbonate solution (50mL) and saturated brine (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to give 13.4g N-benzamide. The crude product was used directly in the next reaction.
7) Synthesis of Isobenzonitrile
Figure BDA0002948706340000121
In a 250mL three-necked round bottom flask was added N-benzamide (13.4g,11.1mmol, 1.0eq), dried dichloromethane (80 mL). After the temperature of the reaction mixture had cooled to 0 deg.C, DIEA (42.91g,33.3mmol, 3eq) was added and POCl3(18.72g,12.21mmol, 1.1eq) was slowly added dropwise at 0 deg.C. The obtained reaction solution reacts for 1 hour at 0 ℃, and then is placed at room temperature until the reaction is complete. Dichloromethane (150mL) and ice water (50mL) were added to conduct extraction. The organic layer was separated, washed with saturated brine (50mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting crude product was purified by a silica gel column using a eluent (dichloromethane) to give 6.9g of a pale yellow oil.
8) (S) -tert-butyl (1-oxo-3-phenylpropyl-2-yl) carbamate
Figure BDA0002948706340000122
Adding dimethyl sulfoxide (5.6g, 71.52mmol, 6.0eq) into a 250mL three-neck round-bottom flask, dissolving in 60mL dichloromethane, then cooling to-78 ℃, dropwise adding a solution consisting of oxalyl chloride (4.63g, 35.85mmol, 3.0eq) and 20mL dichloromethane, controlling the temperature to be completely dropped at-70 ℃, stirring at the same temperature for 20 minutes, dropwise adding a solution consisting of (S) -tert-butyl (1-hydroxy-3-phenylpropyl-2-yl) carbamate (3.0g, 11.95mmol, 1.0eq) and dichloromethane (10mL), controlling the temperature to be completely dropped at-70 ℃, completing the reaction at the same temperature for 30 minutes, dropwise adding triethylamine (14.48g, 143.40mmol, 12.0eq) at-70 ℃, naturally returning to the temperature, monitoring TLC after the reaction for 30 minutes, dropwise adding saturated ammonium chloride solution after the reaction, and quenching the reaction at low temperature, the layers were separated by standing, the aqueous layer was extracted with dichloromethane (2 × 30ml), the organic layers were combined and washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. Concentration gave 4.3g of a yellow oil which was used directly in the next reaction.
9) Synthesis of t-butyl ((2S) -3-hydroxy-4-oxo-1-phenyl-4- (phenylamino) butyl-2-yl) carbamate
Figure BDA0002948706340000131
Adding (S) -tert-butyl (1-oxo-3-phenylpropyl-2-yl) carbamate (2.4g, 9.70mmol, 1.0eq), dichloromethane (30mL), isobenzonitrile (1.5g, 14.55mmol, 1.5eq) and pyridine (3.1g, 38.8mmol, 4.0eq) into a 100mL round bottom flask, cooling to-15 ℃, then adding trifluoroacetic acid (2.2g, 19.4mmol, 2.0eq) dropwise, after completion of the reaction at 0 ℃ for 2 hours, removing the ice bath and continuing to stir the reaction at room temperature for 4 hours, monitoring by TLC, cooling to 0 ℃ after completion of the reaction, adding dilute hydrochloric acid to adjust the pH to acidity, removing the excess pyridine, standing for layering, extracting the aqueous layer with dichloromethane (2 x 30mL), combining the organic layers and washing with saturated common salt water, drying over anhydrous sodium sulfate, filtering, and concentrating. The crude product was purified on a silica gel column with eluent (petroleum ether: ethyl acetate ═ 5:1) to give 1.4g of a yellow solid.
10) (S) -tert-butyl (3, 4-dioxy-1-phenyl-4- (phenylamino) butan-2-yl) carbamate
Figure BDA0002948706340000132
Tert-butyl ((2S) -3-hydroxy-4-oxo-1-phenyl-4- (phenylamino) butyl-2-yl) carbamate (500mg, 1.35mmol, 1.0eq), dimethyl sulfoxide (4mL) were added to a 25mL round-bottomed flask, after ultrasonic dissolution, 2-iodoxybenzoic acid (568mg, 2.03mmol, 1.5eq) was added, the reaction was stirred at room temperature for 3 hours, monitored by TLC, ethyl acetate (30mL) and water (30mL) were added after completion of the reaction, shaking was conducted, the mixture was allowed to stand for separation, the aqueous layer was extracted with ethyl acetate (2X 20mL), the organic layers were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified on a silica gel column with eluent (petroleum ether: ethyl acetate ═ 5:1) to give 218mg of a white solid.
11) (S) -3-amino-2-oxy-N, 4-diphenylbutanamide hydrochloride
Figure BDA0002948706340000133
A50 mL round bottom flask was charged with (S) -tert-butyl (3, 4-dioxy-1-phenyl-4- (phenylamino) butan-2-yl) carbamate (52mg, 0.1411mmol, 1.0eq), dichloromethane (2mL), dissolved and clear, and excess hydrochloric acid gas was bubbled through, after 30 minutes of reaction, a large amount of solid precipitated, monitored by TLC, and after completion of the reaction, the solvent and hydrochloric acid gas were concentrated to remove 45.7mg of white solid.
12) (S) -3- ((S) -3- (4-methoxyphenyl) -2- ((S) -2- (2-morpholinoacetylamino) propionamido) propionamide) -2-oxo-N, 4-diphenylbutylamine
Figure BDA0002948706340000141
Adding (S) -3- (4-methoxyphenyl) -2- (S) -2- (2-morpholineacetamido) propionamide) propionic acid (660mg, 1.6763mmol, 3.0eq) and tetrahydrofuran (10mL) into a 50mL round-bottom flask, dissolving and clarifying, adding 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (637mg, 1.6763mmol, 3.0eq), 1-hydroxybenzotriazole (226mg, 1.6763mmol, 3.0eq) and (S) -3-amino-2-oxy-N, 4-diphenylbutanamide hydrochloride (150mg, 0.5588mmol, 1.0eq), dropping N, N-diisopropylethylamine (216mg, 1.6763mmol, 3.0eq) when the temperature is reduced to 0 ℃, returning to the room temperature, stirring and reacting for 2 hours after the temperature is reduced to 0 ℃, TLC, after completion of the reaction was extracted by adding ethyl acetate (30mL) and water (30mL), the aqueous layer was extracted twice more with ethyl acetate (2 × 20mL), the organic layers were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by silica gel column using eluent (ethyl acetate) to give 210mg of a yellow solid, and recrystallized (dichloromethane: ether ═ 1:5) to give 100mg of a pale yellow solid, which was purified by preparative liquid phase to give 20mg of a white solid.
The mass spectrum detection results are as follows: MS ([ M + H)+])＝644.3080
The nuclear magnetic resonance detection results are as follows:1H NMR(400MHz,CDCl3)δ8.64(s,1H),7.64(d,J＝7.4Hz,2H),7.38(t,J＝7.7Hz,2H),7.15-7.24(m,4H),7.03(d,J＝7.4Hz,5H),6.77(d,J＝7.8Hz,4H),5.60-5.61(m,1H),4.48-4.65(m,1H),5.22-4.43(m,1H),3.68-3.80(m,7H),3.19-3.31(m,2H),2.81-3.17(m,5H),2.29-2.71(m,3H),1.28-1.33(m,3H).
EXAMPLE 2 Synthesis of Compound 2
1) Synthesis of (S) -2-amino-4-methyl pentanoic acid methyl ester hydrochloride
Figure BDA0002948706340000151
A250 mL round bottom flask was charged with (S) -2- ((tert-butoxycarbonyl) amino) -4-methylpentanoic acid (20g, 86.4715mol, 1eq) and 10mol/L methanol hydrochloride solution (170mL), and the reaction was stirred at 70 ℃ for 3 hours. The reaction was monitored by TLC and after completion the reaction was concentrated in vacuo to give 18.54g of crude product which was used directly in the next reaction.
2) Synthesis of methyl (S) -2- ((S) -2- (((benzyloxy) carbonyl) amino) -4-methylpentanamido) -4-methylpentanoate
Figure BDA0002948706340000152
In a 250mL round bottom flask was added (S) -methyl 2-amino-4-methylpentanoate hydrochloride (710g, 4.8970mmol, 1.0eq) and tetrahydrofuran (20mL), after dissolution and clarification 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (2.23g, 5.8760mmol, 1.2eq), 1-hydroxybenzotriazole (0.79g, 5.8760mmol, 1.2eq), (S) -2- ((benzyloxy) carbonyl) amino) -4-methylpentanoic acid (1.60g, 5.8760mmol, 1.2eq), N, N-diisopropylethylamine (972. mu.l, 5.8760mmol, 1.2eq) was added at 0 ℃ and after completion of the reaction at room temperature for 2 hours, TLC was monitored, ethyl acetate (30mL) and water (30mL) were added after completion of the reaction and extracted twice with ethyl acetate (2mL), the organic layers were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified on a silica gel column with eluent (petroleum ether: ethyl acetate ═ 3:1) to give 1.45g of a white solid.
3) Synthesis of (S) -2- ((S) -2- ((benzyloxy) carbonyl) amino) -4-methylpentanoic acid
Figure BDA0002948706340000153
A50 mL round bottom flask was charged with methyl (S) -2- ((S) -2- (((benzyloxy) carbonyl) amino) -4-methylpentamamido) -4-methylpentanoate (1.45g, 3.6940mmol, 1.0eq), tetrahydrofuran (10mL), water (2.5mL), lithium hydroxide monohydrate (155mg, 3.6940mmol, 1.0 eq). Stir at room temperature for 1 hour. TLC, after completion of the reaction, concentrated in vacuo to remove most of the tetrahydrofuran, extracted with ethyl acetate (20mL) and water (20mL), the aqueous layer was extracted twice with ethyl acetate (2 x 10mL), the organic layers were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give 1.15g of a white solid.
4) Synthesis of (S) -2-amino-4-methylpentane-1-ol
Figure BDA0002948706340000161
Dimethyl sulfoxide (4.3g, 55.20mmol, 6.0eq) was added to a 250mL three-necked round-bottomed flask, dissolved in 50mL dichloromethane, and then cooled to-78 deg.C under N2Dropping solution of oxalyl chloride (3.6g, 27.61mmol, 3.0eq) and 20mL of dichloromethane at-70 deg.C, stirring at the same temperature for 20 min, dropping solution of (S) -tert-butyl (1-hydroxy-4-methylpentane-2-yl) carbamate (2.0g, 9.2mmol, 1.0eq) and dichloromethane (10mL), reacting at the same temperature for 30 min, and adding triethylamine(11.15g, 110.40mmol, 12.0eq) is controlled to be dripped at-70 ℃, natural temperature return is finished, TLC monitoring is carried out after reaction for 30 minutes, saturated ammonium chloride aqueous solution is dripped at low temperature after the reaction is finished to quench the reaction, standing and layering are carried out, an aqueous layer is extracted by dichloromethane (2 x 30mL), organic layers are combined and washed by saturated saline solution, dried by anhydrous sodium sulfate, filtered and concentrated to obtain 2.66g of yellow oily matter which is directly used for the next reaction.
5) Synthesis of benzyl ((S) -1- (((S) -1- ((S) -1-hydroxy-4-methylpentane-2-yl) amino) -4-methyl-1-oxopentan-2-yl) carbamate
Figure BDA0002948706340000162
A25 mL round bottom flask was charged with (S) -2- ((S) -2- ((benzyloxy) carbonyl) amino) -4-methylpentanoic acid (560mg, 1.4919mmol, 1.0eq), tetrahydrofuran (4mL), after dissolution and clarification 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (624mg, 1.6411mmol, 1.1eq), 1-hydroxybenzotriazole (222mg, 1.6411mmol, 1.1eq), (S) -2-amino-4-methylpentane-1-ol (184mg, 1.5665mmol, 1.05eq), N, N-diisopropylethylamine (212mg, 1.6411mmol, 1.1eq) was added at 0 ℃ for 2 hours at room temperature, TLC was monitored, after completion of the reaction, ethyl acetate (20mL) and water (20mL) were added for extraction, the aqueous layer was extracted twice more with ethyl acetate (2 × 10mL), and the organic layers were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting crude product was purified by column on silica gel using an eluent (ethyl acetate) to give 782mg of a white solid.
6) Synthesis of ((S) -4-methyl-1- (((S) -4-methyl-1-oxopentan-2-yl) amino) -1-oxopentan-2-yl) -amino) -1-oxopentan-2-yl) carbamate
Figure BDA0002948706340000171
To a 25mL round bottom flask was added benzyl ((S) -1- (((S) -1-hydroxy-4-methylpentan-2-yl) amino) -4-methyl-1-oxopentan-2-yl) carbamate (782mg, 1.6394mmol, 1.0eq), dimethylsulfoxide (4mL), after sonication was added 2-iodoxybenzoic acid (689mg, 2.4591mmol, 1.5eq), the reaction was stirred at room temperature overnight, monitored by TLC, after completion of the reaction was added ethyl acetate (20mL) and water (20mL), shaking was done, the layers were allowed to stand, the aqueous layer was extracted with ethyl acetate (2 x 10mL), the organic layers were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column using eluent (petroleum ether: ethyl acetate: 3:1) to give 560mg of a yellow solid, and recrystallized (dichloromethane: diethyl ether: cyclohexane: 1:3:40) to give 237mg of a pale yellow solid.
7) Synthesis of N-benzamide
Figure BDA0002948706340000172
A100 mL round-bottomed flask was charged with aniline (10g,10.74mmol, 1.0eq) and formic acid (11.85g,25.77mmol, 2.4eq), and the reaction was stirred at 60 ℃ for 4 hours. TLC monitored the progress of the reaction, and when the reaction was complete, dichloromethane (150mL) and water (50mL) were added for extraction. The organic layer was washed successively with a saturated aqueous sodium hydrogencarbonate solution (50mL) and saturated brine (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to give 13.4g N-benzamide. The crude product was used directly in the next reaction.
8) Synthesis of Isobenzonitrile
Figure BDA0002948706340000173
In a 250mL three-necked round bottom flask was added N-benzamide (13.4g,11.1mmol, 1.0eq), dried dichloromethane (80 mL). After the temperature of the reaction mixture had cooled to 0 deg.C, DIEA (42.91g,33.3mmol, 3eq) was added and POCl3(18.72g,12.21mmol, 1.1eq) was slowly added dropwise at 0 deg.C. The obtained reaction solution reacts for 1 hour at 0 ℃, and then is placed at room temperature until the reaction is complete. Dichloromethane (150mL) and ice water (50mL) were added to conduct extraction. The organic layer was separated, washed with saturated brine (50mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting crude product was purified by a silica gel column using a eluent (dichloromethane) to give 6.9g of a pale yellow oil.
9) Synthesis of benzyl ((2S) -1- (((((3S) -2-hydroxy-5-methyl-1-oxo-1- (phenylamino) hex-3-yl) amino) -4-methyl-1-oxopentan-2-yl) carbamate
Figure BDA0002948706340000181
To a 50mL round bottom flask was added benzyl ((S) -4-methyl-1- (((S) -4-methyl-1-oxopentan-2-yl) amino) -1-oxopentan-2-yl) -amino) -1-oxopentan-2-yl) carbamate (237mg, 0.4990mmol, 1.0eq), dichloromethane (5mL), isothiocyanatobenzene (158mg, 1.9960mmol, 2.0eq) and pyridine (158mg, 1.9960mmol, 4.0eq), cooled to-15 deg.C and then trifluoroacetic acid (114mg, 0.9979mmol, 2.0eq) was added dropwise, after completion, the reaction was continued at 0 deg.C for 2 hours and stirring was continued at room temperature overnight, monitored by TLC, cooled to 0 deg.C after completion of the reaction, adding dilute hydrochloric acid dropwise to adjust pH to acidity, removing excessive pyridine, standing for layering, extracting the water layer with dichloromethane (2 × 10mL), combining the organic layers, washing with saturated saline, drying over anhydrous sodium sulfate, filtering, and concentrating. The crude product was purified on a silica gel column with eluent (petroleum ether: ethyl acetate ═ 2:1) to give 221g of a yellow solid.
10) Synthesis of benzyl ((S) -4-methyl-1- (((S) -5-methyl-1, 2-dioxy-1- (phenylamino) hex-3-yl) amino) -1-oxopentan-2-yl) carbamate
Figure BDA0002948706340000182
To a 25mL round bottom flask was added benzyl ((2S) -1- ((((2S) -1- ((((3S) -2-hydroxy-5-methyl-1-oxo-1- (phenylamino) hex-3-yl) amino) -4-methyl-1-oxopentan-2-yl) carbamate (221mg, 0.3716mmol, 1.0eq) and dimethyl sulfoxide (6mL), after sonication was added 2-iodoxybenzoic acid (156mg, 0.5574mmol, 1.5eq), the reaction was stirred overnight at room temperature, monitored by TLC, after completion of the reaction was added ethyl acetate (20mL) and water (20mL), shaken, allowed to stand for layering, the aqueous layer was extracted with ethyl acetate (2 × 10mL), and the organic layers were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified on a silica gel column with eluent (petroleum ether: ethyl acetate ═ 5:1) to give 88mg of a white solid.
The mass spectrum detection results are as follows: MS ([ M + H)+])＝595.3497
The nuclear magnetic resonance detection results are as follows:1H NMR(400MHz,CDCl3)δ8.65(s,1H),7.66(s,2H),7.46–7.33(m,7H),7.20(t,J＝7.4Hz,1H),6.73(s,1H),6.34(s,1H),5.41(d,J＝3.4Hz,1H),5.14(s,2H),4.48(d,J＝6.2Hz,1H),4.18(s,1H),1.87–1.66(m,6H),1.57–1.48(m,3H),0.96(ddt,J＝23.6,17.6,8.7Hz,18H).
EXAMPLE 3 Synthesis of Compound 3
1) Synthesis of (S) -2-amino-3-phenylpropionic acid methyl ester hydrochloride
Figure BDA0002948706340000191
A100 mL round-bottom flask was charged with (S) -2- ((tert-butoxycarbonyl) amino) -3-phenylpropionic acid (1g, 3.7692mmol, 1.0eq) and 10mol/L methanol hydrochloride solution (10mL), and the reaction was stirred at 70 ℃ for 3 hours. The reaction was monitored by TLC, and after completion of the reaction, the reaction solution was concentrated in vacuo and the resulting crude was used directly in the next reaction.
2) Synthesis of methyl (S) -methyl 2- ((S) -2- ((benzyloxy) carbonyl) amino) -4-methylpentanyl) -3-phenylpropionate
Figure BDA0002948706340000192
In a 100mL round bottom flask was added (S) -methyl 2-amino-3-phenylpropionate hydrochloride (1g, 3.7692mmol, 1.0eq) and tetrahydrofuran (15mL), after dissolution and clarification 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (1.72g, 4.5230mmol, 1.2eq), 1-hydroxybenzotriazole (0.61g, 4.5230mmol, 1.2eq), (S) -2- ((benzyloxy) carbonyl) amino) -4-methylpentanoic acid (1.0g, 3.7690mmol, 1.0eq), N, N-diisopropylethylamine (1.46g, 11.3100mmol, 3.0eq) was added at 0 ℃ and after completion of the reaction at room temperature for 2 hours, TLC was monitored, ethyl acetate (30mL) and water (30mL) were added after completion of the reaction and extracted twice with ethyl acetate (2mL), the organic layers were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified on a silica gel column with eluent (petroleum ether: ethyl acetate ═ 3:1) to give 1.31g of a white solid.
3) Synthesis of (S) -2- ((S) -2- (((benzyloxy) carbonyl) amino) -4-methylpentanamido) -3-phenylpropionic acid
Figure BDA0002948706340000201
A25 mL round bottom flask was charged with methyl (S) -methyl 2- ((S) -2- ((benzyloxy) carbonyl) amino) -4-methylpentyl) -3-phenylpropionate (300mg, 0.7034mmol, 1.0eq), tetrahydrofuran (6mL), water (2mL), lithium hydroxide monohydrate (44mg, 1.0551mmol, 1.5 eq). Stir at room temperature for 1 hour. TLC monitoring, after completion of the reaction, vacuum concentration to remove most of the tetrahydrofuran was added, ethyl acetate (20mL) and water (20mL) were added, the pH was adjusted to acidity with dilute hydrochloric acid, the mixture was allowed to stand for separation, the aqueous layer was extracted twice with ethyl acetate (2X 10mL), the organic layers were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give 323g of crude product.
4) Synthesis of (S) -2-amino-4-methylpentane-1-ol
Figure BDA0002948706340000202
Dimethyl sulfoxide (4.3g, 55.20mmol, 6.0eq) was added to a 250mL three-necked round-bottomed flask, dissolved in 50mL dichloromethane, and then cooled to-78 deg.C under N2A solution of oxalyl chloride (3.6g, 27.61mmol, 3.0eq) and 20mL of dichloromethane was added dropwise to the environment, with temperature controlAfter finishing dropping at-70 ℃, stirring for 20 minutes at the same temperature, dropwise adding a solution consisting of (S) -tert-butyl (1-hydroxy-4-methylpentane-2-yl) carbamate (2.0g, 9.2mmol, 1.0eq) and dichloromethane (10mL), controlling the temperature to be completely dropped at-70 ℃, finishing the reaction, reacting for 30 minutes at the same temperature, dropwise adding triethylamine (11.15g, 110.40mmol, 12.0eq) and controlling the temperature to be completely dropped at-70 ℃, naturally returning to the temperature, monitoring by TLC after reacting for 30 minutes, dropwise adding a saturated ammonium chloride aqueous solution after the reaction is finished, quenching the reaction, standing for layering, extracting an aqueous layer by dichloromethane (2 x 30mL), combining the organic layers, washing by saturated salt water, drying by anhydrous sodium sulfate, filtering, concentrating to obtain 2.66g of yellow oily matter, and directly using the yellow oily matter for the next reaction.
5) Synthesis of benzyl ((S) -1- (((S) -1- (((S) -1-hydroxy-4-methylpentane-2-yl) amino) -1-oxo-3-phenylpropan-2-yl) amino) -4-methyl-1-oxopentan-2-yl) carbamate
Figure BDA0002948706340000211
(S) -2- ((S) -2- (((benzyloxy) carbonyl) amino) -4-methylpentanoylamino) -3-phenylpropionic acid (500mg, 1.2122mmol, 1.0eq) and tetrahydrofuran (15mL) were charged into a 50mL round-bottomed flask, and after dissolution and clarification, 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (553mg, 1.4546mmol, 1.2eq) and 1-hydroxybenzotriazole (196mg, 1.4546mmol, 1.2eq) were added and (S) -2-amino-4-methylpentane-1-ol (186mg, 1.2122mmol, 1.0eq) were added and N, N-diisopropylethylamine (469mg, 3.6366mmol, 3.0eq) was added at 0 ℃ and the reaction was completed at room temperature for 2 hours, followed by TLC monitoring, and after completion of the reaction, ethyl acetate (30mL) and water (30mL) were added and extracted, the aqueous layer was extracted twice more with ethyl acetate (2 × 20mL), and the organic layers were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified on a silica gel column with eluent (petroleum ether: ethyl acetate 1:1) to yield 540mg of a white solid.
6) Synthesis of benzyl ((S) -4-methyl-1- (((S) -1- (((S) -4-methyl-1-oxopentan-2-yl) amino) -1-oxo-3-phenylpropan-2-yl) amino) -1-oxopentan-2-yl) carbamate
Figure BDA0002948706340000212
To a 25mL round bottom flask was added benzyl ((S) -1- (((S) -1-hydroxy-4-methylpentan-2-yl) amino) -1-oxo-3-phenylpropan-2-yl) amino) -4-methyl-1-oxopentan-2-yl) carbamate (540mg, 1.0372mmol, 1.0eq), dimethylsulfoxide (5mL), after sonication was added 2-iodoxybenzoic acid (463mg, 1.5558mmol, 1.5eq), the reaction was stirred at room temperature overnight, monitored by TLC, after completion of the reaction was added ethyl acetate (20mL) and water (20mL), shaken well, allowed to stand for separation, the aqueous layer was extracted with ethyl acetate (2 x 10mL), the organic layers were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified on a silica gel column with eluent (petroleum ether: ethyl acetate ═ 2:1) to give 240mg of a white solid.
7) Synthesis of N- (pyridin-3-yl) carboxamides
Figure BDA0002948706340000221
Pyridine-3-amine (1g,10.6259mmol, 1.0eq) and formic acid (1.53g,25.5021mmol, 2.4eq) were added to a 100mL round bottom flask and the reaction was stirred at 60 ℃ for 4 hours. TLC monitored the progress of the reaction, and when the reaction was complete, dichloromethane (10mL) and water (10mL) were added for extraction, the aqueous layer was extracted with dichloromethane (2 x 10mL), and the combined organic layers were washed with saturated aqueous sodium bicarbonate (10mL) and saturated brine (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated and the crude product was used directly in the next reaction.
8) Synthesis of 3-isocyano pyridine
Figure BDA0002948706340000222
A50 mL three-necked round bottom flask was charged with N- (pyridin-3-yl) formamide (1.0g,6.4276mmol, 1.0eq), dried dichloromethane (10 mL). After the temperature of the reaction mixture was cooled to 0 ℃, N-diisopropylethylamine (5.3mL,32.1380mmol, 3.0eq) was added and phosphorus oxychloride (1.48g,9.6414mmol, 1.5eq) was slowly added dropwise at 0 ℃. The obtained reaction solution reacts for 1 hour at 0 ℃, and then is placed at room temperature until the reaction is complete. Dichloromethane (150mL) and ice water (50mL) were added to conduct extraction. The organic layer was separated, washed with saturated brine (50mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting crude product was purified by column on silica gel using eluent (dichloromethane) to give 752mg of pale yellow oil.
9) Synthesis of benzyl ((2S) -1- (((2S) -1- (((3S) -2-hydroxy-5-methyl-1-oxo-1- (pyridin-3-ylamino) hex-3-yl) amino) -1-oxo-3-phenylpropan-2-yl) amino) -4-methyl-1-oxopentan-2-yl) carbamate
Figure BDA0002948706340000223
To a 50mL round bottom flask was added benzyl ((S) -4-methyl-1- (((S) -1- (((S) -4-methyl-1-oxopentan-2-yl) amino) -1-oxo-3-phenylpropan-2-yl) amino) -1-oxopentan-2-yl) carbamate (240mg, 0.4628mmol, 1.0eq), dichloromethane (10mL), 3-isocyanopyridine (106mg, 0.9256mmol, 2.0eq) and pyridine (146mg, 1.8512mmol, 4.0eq), cooled to-15 deg.C and trifluoroacetic acid (106mg, 0.9256mmol, 2.0eq) was added dropwise, after completion, the reaction was continued at 0 deg.C for 2 hours and the reaction was allowed to continue overnight at room temperature, monitored by TLC, cooled to 0 deg.C after completion of the reaction, adding dilute hydrochloric acid dropwise to adjust pH to acidity, removing excessive pyridine, standing for layering, extracting the water layer with dichloromethane (2 × 10mL), combining the organic layers, washing with saturated saline, drying over anhydrous sodium sulfate, filtering, and concentrating. The crude product was purified on a silica gel column with eluent (petroleum ether: ethyl acetate ═ 2:1) to give 232g of a yellow solid.
10) Synthesis of benzyl ((S) -4-methyl-1- (((S) -1- (((S) -5-methyl-1, 2-dioxy-1- (pyridin-3-ylamino) hex-3-yl) amino) -1-oxy-3-phenylpropan-2-yl) amino) -1-oxopentan-2-yl) carbamate
Figure BDA0002948706340000231
Benzyl ((2S) -1- (((2S) -1- (((3S) -2-hydroxy-5-methyl-1-oxo-1- (pyridin-3-ylamino) hex-3-yl) amino) -1-oxo-3-phenylpropan-2-yl) amino) -4-methyl-1-oxopentan-2-yl) carbamate (232mg, 0.3441mmol, 1.0eq) and dimethyl sulfoxide (5mL) were added to a 25mL round bottom flask, after sonication 2-iodoxybenzoic acid (145mg, 0.5162mmol, 1.5eq) was added, the reaction was stirred overnight at room temperature, monitored by TLC, after completion of the reaction ethyl acetate (20mL) and water (20mL) were added, shaken well, the mixture was allowed to stand for separation, and the aqueous layer was extracted with ethyl acetate (2 × 10mL), and the organic layers were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column using eluent (petroleum ether: ethyl acetate ═ 3:1) to give 260mg of a white solid, and recrystallized (dichloromethane: cyclohexane ═ 1:5) to give 113mg of a white solid.
The mass spectrum detection results are as follows: MS ([ M + H)+])＝630.3293
The nuclear magnetic resonance detection results are as follows:1HNMR(400MHz,DMSO)δ10.90(d,J＝9.9Hz,1H),8.98(dd,J＝10.3,2.2Hz,1H),8.61–8.10(m,3H),7.77–7.10(m,7H),5.19–5.00(m,1H),4.60(d,J＝29.6Hz,1H),4.01(s,1H),3.09(d,J＝7.2Hz,2H),1.38(dt,J＝53.1,37.5Hz,4H),1.02–0.78(m,11H).
example 4 biological Activity
4.1 inhibition of proteasome by Compounds 1,2, 3
NCI-H226 (1250/well, 45. mu.L/well) cells were plated in 96-well plates (bottom-penetrating white plates) overnight, after which 5. mu.L of test compound at different concentrations were added, each set of 2 replicate wells. At 37 ℃ with 5% CO2Incubated at concentration for 100 minutes, removed and tested by adding the Chymotrypsin-Like kit (Promega G8661 kit). Addition of Proteasome-GloTMCell-Based Reagent, 50. mu.L/well, protected from light, incubated at room temperature for 10 minutes, and then assayed for Luminescences using a Promega microplate reader. IC for enzyme activity data50Or inhibition rate.
TABLE 1 enzymatic Activity data for Compounds
Compound numbering Chymotrypsin-Like IC50(nM)
Compound 1 365.10
Compound 2 34.01
Compound 3 55.84a
Inhibition of ChTL (. beta.5) by Compounds at 50nM concentration
4.2 assay of the anti-New coronavirus Activity of Compounds
The compounds were first subjected to a cytotoxicity assay to rule out non-specific inhibition of virus-infected cells by the compounds. As shown in FIG. 1, it can be seen from FIG. 1 that Compound 1 has the lowest toxicity to VeroE6 cells and CC50The value was 71.09. mu.M, therefore, Compound 1 was selected for further testing against neocoronavirus activity.
VeroE6 cells were cultured at 4X 10 in anti-neocoronavirus experiments5Cell/well density was incubated in 12-well plates 1 day in advance. After 24 hours of incubation, 50 units of the novel coronavirus were added to the cells, and different concentrations of the compound were added.
The results are shown in FIGS. 2 to 3, in which Compound 1 exhibited a better anti-neocoronavirus activity, and its EC was50The value was 1.28. mu.M.
4.3 Compounds 1,2, 3 against novel coronavirus (SARS-CoV-2) Mpro/3CLproProtease activity test of
Using a 96-well blackboard, 100. mu.l of the system per well, 92. mu.L of buffer and 1. mu.L of Mpro/3CLpro were added to the experimental, 100% enzyme activity control and positive inhibitor (Ebselen) control, 93. mu.L of buffer was added to the blank control, 5. mu.L of test compound (DMSO solution), 5. mu.L of positive inhibitor (DMSO solution) or 5. mu.L of DMSO was added to each well, and incubation was carried out at 37 ℃ for 10 minutes. And quickly adding 2 mu L of substrate into each well, uniformly mixing, incubating for 10 minutes at 37 ℃ in a dark place, performing fluorescence measurement by using a multifunctional microplate reader (PerkinElmer), wherein the excitation wavelength is 340nm, the emission wavelength is 490nm, and recording the fluorescence intensity value. The inhibition rate of each compound on the enzymatic activity of the novel coronavirus Mpro/3CLpro at a certain concentration was calculated according to the inhibition rate (%) (RFU 100% enzymatic activity control-RFU sample)/(RFU 100% enzymatic activity control-RFU blank) × 100%.
The results are shown in Table 2. Wherein Compound 1 is administered to a novel coronavirus (SARS-CoV-2) M at a concentration of 100. mu.Mpro/3CLproThe protease had an inhibition rate of 48.26%.
TABLE 2 different concentrations of Compound 1,2, 3 against novel coronavirus Mpro/3CLproInhibition rate of protease
Figure BDA0002948706340000251
As can be seen from the above examples, the above compounds have high inhibitory activity against the novel coronavirus.
The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.

Claims (10)

1. Use of a proteasome inhibitor for inhibiting a novel coronavirus, or for the preparation of a novel coronavirus inhibitor, wherein said proteasome inhibitor has the structure of formula (i) or an isomer, a pharmaceutically acceptable salt, or a prodrug thereof:
Figure FDA0002948706330000011
wherein R is1Is substituted or unsubstituted aryl or heteroaryl;
R2、R3independently selected from hydrogen atom, C1-10 alkyl, aryl substituted C1-12 alkyl, C6-12 aryl or substituted aryl, C6-12 heteroaryl or substituted heteroaryl;
R5and R7Independently selected from hydrogen atom, C1-10 alkyl, aryl substituted C1-12 alkyl, C6-12 aryl or substituted aryl, C6-12 heteroaryl or substituted heteroaryl;
R4and R6Independently selected from hydrogen atoms;
R8is N (R)9)LQR10；
R9Is a hydrogen atom;
l is C ═ O;
q is O, NH, N-alkyl or CH2；
R10Is aryl substituted C1-12 alkyl, heteroaryl substituted C1-12 alkyl, heterocyclyl substituted C1-12 alkyl, substituted or unsubstituted heterocyclyl or aryl.
2. Use of a composition comprising a proteasome inhibitor for inhibiting a novel coronavirus, or for preparing a novel coronavirus inhibitor, wherein the proteasome inhibitor has the structure of formula (i) or an isomer, a pharmaceutically acceptable salt, or a prodrug thereof:
Figure FDA0002948706330000012
wherein R is1Is substituted or unsubstituted aryl or heteroaryl;
R2、R3independent of each otherIs selected from hydrogen atom, C1-10 alkyl, aryl substituted C1-12 alkyl, C6-12 aryl or substituted aryl, C6-12 heteroaryl or substituted heteroaryl;
R5and R7Independently selected from hydrogen atom, C1-10 alkyl, aryl substituted C1-12 alkyl, C6-12 aryl or substituted aryl, C6-12 heteroaryl or substituted heteroaryl;
R4and R6Independently selected from hydrogen atoms;
R8is N (R)9)LQR10；
R9Is a hydrogen atom;
l is C ═ O;
q is O, NH, N-alkyl or CH2；
R10Is aryl substituted C1-12 alkyl, heteroaryl substituted C1-12 alkyl, heterocyclyl substituted C1-12 alkyl, substituted or unsubstituted heterocyclyl or aryl.
3. Use according to claim 1 or 2, wherein R is1Selected from the following groups:
Figure FDA0002948706330000021
wherein, X1、X2、X3、X4And X5Any one or two of them are N, and the rest are C; or X1、X2、X3、X4And X5All are C;
R11and R12Independently selected from:
H. halogen, -OCF3Cyano, nitro, alkenyl, alkynyl, alkyl, haloalkenyl, haloalkynyl, haloalkyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, cycloalkylalkyl, arylalkyl, heteroarylalkyl, cycloalkylheteroalkyl, heterocycloalkylheteroalkyl, arylheteroalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkoxyaryl, alkenyloxy, alkynyloxy, cycloalkyl, heterocycloalkyl, heterocycloalkenyl, cycloalkylalkyl, heteroarylalkyl, alkoxyaryl, alkenyloxy, alkynyloxy, and the likeAryl, cycloalkoxy, heterocycloalkoxy, aryloxy, heteroaryloxy, aralkoxy, carboxyl, acyl, sulfonyl, amino, -C (O) OR13、-OC(O)R13、-COR13、-NHS(O)mR13、-NHC(O)R13、-NHC(O)OR13、-NR14R15、-OC(O)NR14R15、-OC(O)R14R15、-SH、-SR14；
Wherein the content of the first and second substances,
m is 1 or 2;
R13selected from aryl, heteroaryl, alkenyl, alkynyl, alkyl, haloalkyl, cycloalkyl or heteroalkyl, or heterocycloalkyl further substituted with one or more substituents selected from halo, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, hydroxy, hydroxyalkyl, alkoxy, acyl;
R14or R15Each independently selected from a hydrogen atom, a substituted or unsubstituted aryl, heteroaryl, alkenyl, alkynyl, alkyl, haloalkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl;
or R11、R12Any one of (1) and X1、X2、X3、X4And X5Any two adjacent rings in the (b) are fused to form a 3-to 6-membered substituted or unsubstituted aliphatic or aromatic ring.
4. Use according to claim 3, wherein R is1Selected from any one of the following groups:
Figure FDA0002948706330000031
wherein the content of the first and second substances,
R16、R17independently selected from:
H. f, Cl, Br, methyl, methoxy, trifluoromethyl, trifluoromethoxy,
Figure FDA0002948706330000032
5. Use according to claim 4, wherein R is1Selected from any one of the following groups:
Figure FDA0002948706330000033
wherein the content of the first and second substances,
R16and R17Independently selected from:
H. f, Cl, Br, methyl, methoxy, trifluoromethyl, trifluoromethoxy,
Or
Figure FDA0002948706330000034
Or when R is16And R17When in ortho position, R16And R17And the attached carbon atoms are joined together to form an aliphatic or aromatic ring.
6. Use according to claim 1 or 2, wherein R is2、R3Independently selected from:
H、CH3、
Figure FDA0002948706330000041
7. use according to claim 1 or 2, wherein R is5Or R7Each independently selected from:
H、CH3、
Figure FDA0002948706330000042
Figure FDA0002948706330000043
8. use according to claim 1 or 2, wherein R is10Selected from any one of the following groups:
Figure FDA0002948706330000044
9. the use according to claim 1 or 2, characterized in that the proteasome inhibitor has any one of the following structures:
Figure FDA0002948706330000045
Figure FDA0002948706330000051
10. use according to claim 1 or 2, wherein the novel coronavirus inhibitor is for use in the prevention, treatment or alleviation of any one or more of the diseases associated with the novel coronavirus.
CN202110200746.7A 2021-02-23 2021-02-23 Application of protease inhibitor in inhibiting novel coronavirus Pending CN112972648A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113912670A (en) * 2021-12-10 2022-01-11 深圳市小分子新药创新中心有限公司 Angiotensin converting enzyme 2 inhibitor, application thereof and anti-coronavirus infection drug

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JUAN WANG等: "A new class of a-ketoamide derivatives with potent anticancer and anti-SARS-CoV-2 activities", EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY, vol. 215, pages 1 - 2 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113912670A (en) * 2021-12-10 2022-01-11 深圳市小分子新药创新中心有限公司 Angiotensin converting enzyme 2 inhibitor, application thereof and anti-coronavirus infection drug
CN113912670B (en) * 2021-12-10 2022-04-08 深圳市小分子新药创新中心有限公司 Angiotensin converting enzyme 2 inhibitor, application thereof and anti-coronavirus infection drug

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