CN100363346C - Small molecular inhibiting agent for coronavirus main proteinase, its preparation method and uses - Google Patents
Small molecular inhibiting agent for coronavirus main proteinase, its preparation method and uses Download PDFInfo
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- CN100363346C CN100363346C CNB2004100877217A CN200410087721A CN100363346C CN 100363346 C CN100363346 C CN 100363346C CN B2004100877217 A CNB2004100877217 A CN B2004100877217A CN 200410087721 A CN200410087721 A CN 200410087721A CN 100363346 C CN100363346 C CN 100363346C
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- 238000002360 preparation method Methods 0.000 title claims abstract description 17
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
- C07K5/0205—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)3-C(=0)-, e.g. statine or derivatives thereof
-
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D207/18—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member
- C07D207/22—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D207/24—Oxygen or sulfur atoms
- C07D207/26—2-Pyrrolidones
- C07D207/263—2-Pyrrolidones with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms
- C07D207/27—2-Pyrrolidones with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms with substituted hydrocarbon radicals directly attached to the ring nitrogen atom
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
- C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Virology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pulmonology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention provides a series of small molecular inhibitors designed on the basis of crystal texture of SARS coronavirus main proteinase, and a texture general formula is disclosed as a formula (I). The present invention also provides a preparation method for the small molecular inhibitors and an application for preparing medicaments for treating or preventing the infection of various kinds of coronavirus.
Description
Technical Field
The invention provides a series of small molecule inhibitors designed based on the crystal structure of SARS coronavirus main protease, a preparation method and application thereof in preparing medicaments for treating or preventing various coronavirus infections.
Background
A novel coronavirus has been identified as a murder of Severe Acute Respiratory Syndrome (SARS) and is named SARS coronavirus (SARS-CoV). SARS Coronavirus belongs to the order of Nidovirales, the family of Coronaviridae, the genus of Coronaviridae, in the genus of genus species. It is a new variant of the coronavirus family, with a length of 29,736bp (Urbani Strain). SARS is a virulent infectious disease with a great threat to human beings, and no specific medicine or vaccine is available on the market at present.
According to the serological classification, the coronavirus family (coronavirus genus) includes four types (Group) in total. Type I includes porcine transmissible gastroenteritis virus (TGEV), human coronavirus (HCoV) strain 229E, Feline Infectious Peritonitis Virus (FIPV), and the like; type II includes bovine coronavirus (BCoV for short), Murine hepatitis virus (MHV for short), and the like; type III currently includes only three viruses, one of which is known as Avian Infectious Bronchitis Virus (AIBV); SARS-CoV is type IV. Various coronaviruses have great threat to the life health of people and livestock.
The genome of the SARS coronavirus encodes two large replicase polyproteins pp1a (486kDa) and pp1ab (790kDa), which are encoded by the genome comprising coronaviruses 2/3 to 3/4. These two proteins are hydrolyzed to produce many functional subunits of the viral replication complex. In this hydrolysis process, SARThe major proteolytic enzyme of S-coronavirus (main protease, abbreviated as M)pro33.8kDa, sometimes also referred to as 3C-Like Protein) plays a very critical role. The main protease, also a stretch of pp1a and pp1ab, is released by autocatalytic hydrolysis, which occurs at the Gln (Ser, Ala) site flanking the protease, by trans-splicing (bimolecular reaction). Under the action of the main protease, the replicase polyproteins pp1a and pp1ab are hydrolyzed into more than ten functional peptides, thereby further playing a role. If the hydrolysis of SARS coronavirus main protease can be inhibited, then the infection of SARS coronavirus to human body can be effectively resisted. Therefore, SARS coronavirus main protease is an ideal target for designing anti-SARS drugs.
Disclosure of Invention
It is an object of the present invention to provide a small molecule inhibitor that is effective in inhibiting the activity of the coronavirus main protease.
The invention also aims to provide a preparation method of the small molecule inhibitor.
It is a further object of the invention to provide the use of said small molecule inhibitor for the manufacture of a medicament for the treatment or prevention of a coronavirus infection.
Accordingly, the present invention provides a compound of the general formula (I):
wherein,
u is
The invention also provides a preparation method of the compound of the general formula (I), which comprises the following steps:
the protecting group R of amino in the compound of formula (II)6Removing R therein6Selected from the group consisting of: t-butoxycarbonyl, trifluoroacetyl, benzyloxycarbonyl;
condensing the product of the last step with a compound of formula (III) in the presence of a condensing agent to obtain a compound of formula (I),
wherein R in the formula (II) and the formula (III)1、R2、R4And U is as defined for formula (I).
The invention also provides the use of a compound of formula (I) in the manufacture of a medicament for the treatment or prophylaxis of coronavirus infection.
Experiments prove that the compound can obviously inhibit the activity of main protease of coronavirus such as TGEV, HcoV, FIPV, AIBV, SARS-CoV and the like, and has good application prospect in the aspect of preparing medicaments for treating or preventing coronavirus infection.
Drawings
The drawings are not necessarily to scale, emphasis instead being placed upon better illustrating the present invention for the convenience of the reader. The invention may be better understood by considering the drawings in conjunction with the detailed description.
FIG. 1 shows small molecule N1 and SARS-CoV MproSurface map of monomer a binding;
FIG. 2 is a graph of electron density of N1 binding to the active pocket of monomer A;
FIG. 3 shows the small molecules N1, N2, N3 and N4 against SARS-CoV MproWherein SARS _3CL represents SARS coronavirus main protease, namely SARS-CoVMpro；
FIG. 4 shows the inhibitory activity curve of small molecule N1 against the transmissible gastroenteritis virus (TGEV) main protease (abbreviated as TGEV-3 CL in the figure);
FIG. 5 shows the inhibitory activity curves of small molecule N1 against human coronavirus 229E (HCoV) main protease (abbreviated as HCoV-3 CL in the figure);
FIG. 6 shows the inhibitory activity curve of small molecule N1 against Feline Infectious Peritonitis Virus (FIPV) major protease (abbreviated as FIPV _3CL in the figure);
FIG. 7 shows the inhibitory activity profile of small molecule N1 against Avian Infectious Bronchitis Virus (AIBV) main protease (abbreviated as AIBV _3CL in the figure).
Detailed Description
Definition of terms
For descriptive convenience, specific terminology is used herein and is explained below on a case-by-case basis.
"N1", "N2", "N3" and "N4" are particularly preferred small molecule inhibitors of the present invention having the structural formulas:
structural formula of N1
Structural formula of N2
Structural formula of N3
Structural formula of N4
As used herein, the terms "major proteolytic enzyme of SARS coronavirus", "SARS-CoV Mpro”、“SARS-CoV 3CLpro"," SARS coronavirus main protease "and the like, refer to the major proteolytic enzyme of SARS coronavirus.
“C3～C6Alkyl of (a) "means a straight or branched chain alkyl group having between 3 and 6 carbon atoms, including but not limited to: n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl and the like.
“C1～C4The "alkyl group" of (a) means a linear or branched alkyl group having between 1 and 4 carbon atoms, including: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, and the like.
"Fluorobenzyl" includes p-fluorobenzyl, m-fluorobenzyl, o-fluorobenzyl and the like.
"fluorophenyl" includes p-fluorophenyl, m-fluorophenyl, o-fluorophenyl, and the like.
In a partial structural formula, the following compounds,ipr represents isopropyl, Et represents ethyl, "Bn" represents benzyl, and "Boc" represents t-butyloxycarbonyl.
"TFA" represents trifluoroacetic acid, "THF" represents tetrahydrofuran, "DMF" represents N, N-dimethylformamide, "DMSO" represents dimethyl sulfoxide, "PhH" represents benzene, "iPr represents2NEt "stands for diisopropylethylamine," NEt3"represents triethylamine.
"DCC" represents dicyclohexylcarbodiimide (dicyclohexylcarbodiimide);
"DIEA" represents diisopropylethylamine (diisopropylethylamine);
"DMAP" represents 4-N, N-dimethylaminopyridine (4-N, N-dimethylammonylidine);
"EDCI" represents 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (1-ethyl-3- (3-dimethylamino propyl) carbodiimide hydrochloride);
"HATU" represents N- [ (dimethylamino) (3H-1, 2, 3-triazole (4, 5-b) pyridin-3-yloxy) methylene ] -N-methylbetaine hexafluorophosphate (N- [ (dimethyllamino) (3H-1, 2, 3-triazo (4, 5-b) pyridine-3-yloxy) methyl ] -N-methylethaneaminonium hexafluorophosphate;
"HBTU" represents O- (benzotriazol-1-yl) -1, 1, 3, 3-tetramethyluronium hexafluorophosphate (O- (benzotriazol-1-yl) -1, 1, 3, 3-tetramethyluronium hexafluorophosphate);
"HOBt" represents 1-hydroxybenzotriazole (1-hydroxybenzotriazole).
Structure of the Compound of the present invention
As mentioned above, the structure of the compound of the invention is shown in the general formula (I).
Certain preferred compounds of the invention correspond to the following formula,
further preferred compounds of the invention correspond to the following general formula,
further preferred compounds of the invention correspond to the following general formula,
some particularly preferred specific compounds of the invention are listed below,
process for the preparation of the compounds of the invention
The preparation method of the compound of formula (I) provided by the invention comprises the following steps:
the protecting group R of amino in the compound of formula (II)6Removing R therein6Selected from the group consisting of: t-butoxycarbonyl, trifluoroacetyl, benzyloxycarbonyl;
condensing the product of the last step with a compound of formula (III) in the presence of a condensing agent to obtain a compound of formula (I),
wherein R in the formula (II) and the formula (III)1、R2、R4And U is as defined for formula (I).
The synthesis of compounds of formula (II) can be referred to: qingping Tian, Naresh K.Nayyar, Srinivasan Babu, Lijian Chen, Junhua Tao, Steven Lee, Anthony Tibbetts, Terence Moran, Jason Liou, Ming Guo and Timothy P.Kennedy tetrahedron Lett.2001, 42, 6808-. The synthesis of compounds of formula (III) can be referred to: dawei Ma, Weiqing Xie, Bin Zou, Qiong Lei and Duanqing Pei Tetrahedron Lett.2004, 45, 8103-.
In some preferred embodiments of the present invention, the compound of formula (II) is reacted with an acid (e.g., a mixture of dichloromethane and trifluoroacetic acid at a volume ratio of 1-4: 1) in an organic solvent at room temperature for 1-4 hours; extracting the solvent, dissolving the compound of formula (III) in an aprotic solvent (e.g. CH)2Cl2，THF，CHCl3) Adding a condensing agent such as HATU, HBTU, EDCI, and adding an organic base such asiPr2NEt，NEt3And reacting at room temperature for 8-24 hours to obtain the compound shown in the formula (I).
R in the compounds of formula (I) may also be derivatized1Group exchange to the desired group R1', to give a series of derivatives of the compound of formula (I) wherein R1' is selected from the group consisting of: c3～C6Alkylcarbonyl, t-butoxycarbonyl, benzyloxycarbonyl, isoxazolylcarbonyl, furylcarbonyl, trifluoromethylcarbonyl,
The derivatization comprises the following steps: r in the obtained compound of formula (I)1Removing; reacting the product of the previous step with a carboxylic acid R in the presence of a condensing agent1' -OH to give a derivatized compound of formula (I) wherein R1Radical is replaced by R1' group; wherein R is1' is selected from the group consisting of: c3～C6Alkylcarbonyl, t-butoxycarbonyl, benzyloxycarbonyl, isoxazolylcarbonyl, furylcarbonyl, trifluoromethylcarbonyl,
In some preferred embodiments of the present invention, the compound of formula (I) to be derivatized is reacted with an acid (e.g., a mixture of dichloromethane and trifluoroacetic acid at a volume ratio of 1-4: 1) in an organic solvent at room temperature for 1-4 hours; extracting the solvent with a carboxylic acid R1' -OH is dissolved in an aprotic solvent (e.g., CH)2Cl2，THF，CHCl3) Adding a condensing agent such as HATU, HBTU, EDCI, and adding an organic base such asiPr2NEt，NEt3And reacting at room temperature for 8-24 hours to obtain a series of derivatives of the compound shown in the formula (I).
Wherein the acid is preferably trifluoroacetic acid or hydrochloric acid; preferred organic solvents are selected from the group consisting of: CH (CH)2Cl2Tetrahydrofuran, tetrahydrofuran、CHCl3N, N-dimethylformamide, dioxane; preferred aprotic solvents are selected from the group consisting of: CH (CH)2Cl2Tetrahydrofuran, CHCl3N, N-dimethylformamide, dimethyl sulfoxide, benzene; preferred condensation reagents are selected from the group consisting of: HATU, HBTU, EDCI; preferred organic bases are selected from the group consisting of: diisopropylethylamine and triethylamine.
In order to explain the present invention in more detail, specific embodiments thereof will be given below with reference to the accompanying drawings. In describing these embodiments, well-known experimental methods, instruments, reagents, materials and the like have not been described in detail so as not to obscure the present invention.
Example 1
42mg ofDissolved in 2ml of CH2Cl20.5ml of TFA was added thereto, and the mixture was reacted at room temperature for 1 hour, and the solvent was then drained. The resulting de-Boc substrate was dissolved in 2ml of CH2Cl2In (1), 40mg of
Then 71 mul ofiPr2NEt, then 63mg of HATU. The reaction was carried out at room temperature for 12 hours, followed by 1M HCl and saturated NaHCO3Aqueous solution, saturated brine and Na2SO4And (5) drying. Filtering, evaporating solvent under reduced pressure, and performing flash column chromatography to obtain 52mg of product
The yield was 80%.
The spectrum data are as follows:
H NMR：δ(500MHz，CDCl3)0.76(d，3H，J＝6.9Hz)，0.98(d，3H，J＝5.7Hz)，1.30(t，3H，J＝7.3Hz)，1.42(s，9H)，1.74-1.94(m，4H)，2.10-2.40(m，2H)，2.49-2.52(m，1H)，2.67-2.71(m，1H)，2.85-3.00(m，1H)，3.05-3.17(m，1H)，3.23-3.39(m，3H)，4.18(q，2H，J＝7.1Hz)，4.41-4.51(m，1H)，4.58-4.65(m，1H)，5.03-5.10(m，1H)，5.49(t，1H，J＝14.7Hz)，5.98(dd，1H，J＝15.8Hz，4.4Hz)，6.70-7.00(m，3H)，7.12-7.16(m，3H)；
ESI-MS：[M+H+]590.2，HRMS found m/z 612.3062，C31H44N3O7FNarequires 612.3065；
[α]D 24.0(c 0.69，CHCl3)。
example 2
Mixing 45mg
Dissolved in 2ml of CH2Cl20.5ml of TFA was added thereto, and the mixture was reacted at room temperature for 1 hour, and the solvent was then drained. The resulting de-Boc substrate was dissolved in 2ml of CH2Cl2In (1), 12mg of
Then 48. mu.l ofiPr2NEt, then 44mg of HATU. The reaction was carried out at room temperature for 12 hours, followed by 1M HCl and saturated NaHCO3Aqueous solution, saturated brine and Na2SO4And (5) drying. Filtering, evaporating the solvent under reduced pressure, and performing flash column chromatography to obtain 34mg of product
The yield was 73%.
The spectrum data are as follows:
H NMR：δ(300MHz，CDCl3)0.85(d，3H，J＝6.6Hz)，1.03(d，3H，J＝6.9Hz)，1.30(t，3H，J＝7.2Hz)，1.52-1.61(m，1H)，1.71-1.90(m，2H)，2.26-2.42(m，2H)，2.48(s，3H)，2.53-2.73(m，3H)，2.84-2.98(m，2H)，3.13-3.23(m，1H)，3.30-3.42(m，2H)，4.18(q，2H，J＝7.2Hz)，4.41-4.56(m，1H)，4.66-4.76(m，1H)，5.50(d，1H，J＝15.6Hz)，5.91(s，1H)，6.39(s，1H)，6.63(dd，1H，J＝15.3Hz，4.8Hz)，6.94-7.03(m，2H)，7.11-7.35(m，4H)；
ESI-MS：[M+H+]599.3，[M+Na+]621.3，HRMS found m/z 621.2717，C31H44N3O7FNa requires 621.2695；
[α]D 32.8(c 0.51，CHCl3)。
examples 1 and 2 can be briefly summarized by the following reaction schemes:
example 3
In describing this example, reference is made to the following reaction scheme (where the one or two digit arabic number below certain compounds is their numbering):
preparation of Compound 9
321mg (1mmol) of Compound 8 was dissolved in 2ml of dichloromethane, and 1ml of trifluoroacetic acid was added thereto, followed by stirring at room temperature for 1 hour. The solvent was evaporated under reduced pressure and pumped to dryness. The resulting product was dissolved in 4ml of dichloromethane and 260mg of the compound were added in order0.4ml ofDiisopropylethylamine, 162mg of HOBt, and finally 247mg of DCC were added, and the mixture was stirred at room temperature overnight. The reaction system was filtered and 10ml of dichloromethane was added, washed with 1M HCl, saturated aqueous sodium bicarbonate solution, and saturated aqueous sodium chloride solution in this order, dried over anhydrous sodium sulfate, filtered, the solvent was evaporated, and flash column chromatography was performed to obtain 387mg of compound 9 with a yield of 92%.
The spectrum data are as follows:
H NMR：δ(300MHz，CDCl3)0.90-0.95(m，12H)，1.44(s，9H)，1.45-1.68(m，3H)，2.03-2.1 5(m，1H)，3.88(dd，1H，J＝6.6Hz，9.3Hz)，4.64-4.72(m，1H)，5.06(d，1H，J＝9.9Hz)，5.16(d，2H，J＝3.3Hz)，6.24(d，1H，J＝10.5Hz)，7.29-7.30(m，5H)。
preparation of Compound 10
331mg of Compound 9 was dissolved in 4ml of dichloromethane, and 1ml of trifluoroacetic acid was added thereto, followed by stirring at room temperature for 1 hour. The solvent was evaporated under reduced pressure and pumped to dryness. The resulting product was dissolved in 4ml of dichloromethane and 19mg of the compound were added in succession
0.3ml of diisopropylethylamine, 128mg of HOBt, and finally 195mg of DCC were added and stirred at room temperature overnight. The reaction system was filtered, 10ml of dichloromethane was added, washed with 1M HCl, saturated aqueous sodium bicarbonate solution, and saturated aqueous sodium chloride solution in this order, dried over anhydrous sodium sulfate, filtered, the solvent was evaporated, and flash column chromatography was performed to obtain 357mg of compound 10 with a yield of 92%.
The spectrum data are as follows:
H NMR：δ(300MHz，CDCl3)0.89-0.94(m，12H)，1.35(d，3H，J＝7.2Hz)，1.44(s，9H)，1.56-1.69(m，3H)，2.1 3-2.24(m，1H)，4.09-4.19(m，1H)，4.24(dd，1H，J＝6.6Hz，8.7Hz)，4.92-5.03(m，1H)，4.95(d，2H，J＝5.4Hz)，6.35-6.44(m，1H)，6.69-6.78(m，2H)，7.31-7.42(m，5H)。
preparation of Compound 11
Dissolving 310mg of compound 10 in 5ml of methanol, adding 62mg of 20% palladium on carbon, hydrogenating at normal pressure for 3 hours, filtering off the palladium on carbon, evaporating the solvent, and carrying out flash column chromatography to obtain 258mg of compound 11 with a yield of 100%.
The spectrum data are as follows:
H NMR：δ(300MHz，CDCl3)0.9 1-0.96(m，12H)，1.34(d，3H，J＝6.9Hz)，1.44(s，9H)，1.60-1.75(m，3H)，2.08-2.20(m，1H)，4.1 8-4.28(m，1H)，4.33(t，1H，J＝9.0Hz)，4.51-4.61(m，1H)，5.27-5.35(m，1H)，7.08-7.17(m，1H)，7.27-7.33(m，1H)。
preparation of Compound N2
50mg of
Dissolved in 2ml of CH2Cl20.5ml of TFA was added thereto, and the mixture was reacted at room temperature for 1 hour, and the solvent was then drained. The resulting de-Boc substrate was dissolved in 2ml of CH2Cl2To this, 62mg of Compound 11 was added, and 97. mu.l of the mixture was addediPr2NEt, then 75mg of HATU. The reaction was carried out at room temperature for 12 hours, followed by 1M HCl and saturated NaHCO3Aqueous solution, saturated brine and Na2SO4And (5) drying. Filtration, evaporation of the solvent under reduced pressure and flash column chromatography gave 76mg of product N2 in 81% yield.
The spectrum data are as follows:
ESI-MS：[M+H+]610.4。
preparation of Compound N1
41mg of Compound N2 were dissolved in 2ml of CH2Cl20.5ml of TFA was added thereto, and the mixture was reacted at room temperature for 1 hour, and the solvent was then drained. The resulting Boc-removed substrate was solubilizedTo 2ml of THF were added 10mg of
Then 42. mu.l ofiPr2NEt, then 33mg of HATU. The reaction was carried out at room temperature for 12 hours, followed by 1M HCl and saturated NaHCO3Washing the solution with saturated saline water, Na2SO4And (5) drying. Filtration, evaporation of the solvent under reduced pressure and flash column chromatography gave 34mg of product N1 in 82% yield.
The spectrum data are as follows:
H NMR：δ(300MHz，CDCl3)0.89(s，12H)，1.25-1.29(m，3H)，1.44(d，3H，J＝7.4Hz)，1.50-1.92(m，5H)，2.04-2.17(m，2H)，2.20-2.41(m，3H)，2.47(s，3H)，3.20-3.41(m，2H)，4.18(q，2H，J＝7.2Hz)，4.32-4.43(m，1H)，4.57-4.79(m，2H)，4.85-4.97(m，1H)，5.93-6.00(m，1H)，6.46(s，1H)，6.82-6.92(m，1H)，7.47-7.93(m，3H)；
ESI-MS：[M+H+]619.3。
EXAMPLE 4 SARS-CoV MproCrystal preparation, data collection and structure resolution of complexes with small molecule inhibitors
Materials and methods
1.SARS-CoV MproCrystal preparation of complexes with Small molecule Compound N1
SARS-CoV MproFurther isolation, purification and crystallization were carried out after expression in the E.coli strain BL21(DE3) (see: Yang H et al 2003.the Crystal Structures of SARSVirus Main Protease Mpro and Its Complex with an inhibitor. PNAS, 100 (23): 13190-. After N1 was dissolved in 10mM solution prepared from 7.5% PEG6000, 6% DMSO and 0.1M MES (pH6.0), the solution was added in an equal volume to the suspension of the grown crystals, and left to stand at 291K for 2 days.
2.SARS-CoV MproData collection and Structure of Complex with Small molecule Compound N1Parse
(1) Data collection and data processing
The data collection is completed by a Rigaku CuK alpha rotating target X-ray diffractometer, and the system parameters during data collection are voltage 40KV, current 20mA, wavelength 1.5418 Å and temperature 100K. The crystals were immersed in 30% PEG400, 0.1M MES (pH6.0) antifreeze solution and rapidly cooled to 100K with a stream of liquid nitrogen. The diffraction resolution of the crystal was 1.88 Å. All final diffraction data were treated with HKL2000 to 2.0 Å.
(2) Structural analysis
The structure of the complex is analyzed by using SARS-CoV MproThe parent structure (PDBcode: 1UJ1) of (2) was used as an initial search model, and the structure was analyzed by a molecular replacement method. A clear solution of symmetric disomy can be obtained by performing a rotation function and translation function search using CNS procedures. Then, a difference graph of 2Fo-Fc and Fo-Fc is used in the program O to build a small molecule model. After correction, Rwork20.0，Rfree23.8。
SARS-CoV MproThe methods for crystal preparation, data collection and structure analysis of the compound of N2, N3 and N4 are basically the same as those of N1, and are not described in detail herein.
Second, structural analysis
1、SARS-CoV MproStructural analysis of Complex with Small molecule Compound N1
SARS-CoV MproIs a homodimeric structure containing two monomers a and B in an asymmetric unit, since the binding of small molecule compound N1 to A, B two monomers is substantially similar, only the binding pattern of N1 and a is now analyzed.
In the 2Fo-Fc difference plot (counted at 1. sigma.) for monomer A, it was clearly observed that N1 was bound into the substrate binding pocket of the enzyme. Wherein C beta in N1 and S gamma of A145-Cys form a covalent bond with the bond length of 1.8 Å. The carbonyl oxygen in the N1 ester group and the amino group on the main carbon chain of A145-Cys form a hydrogen bond of 3.1 Å; the ethyl group in the ester forms a hydrophobic interaction with the side chains of A27-Leu, His-A41 and Thr-A25. At the P1 site, the oxygen in the five-membered ring of the lactam forms a 2.6 Å hydrogen bond with NE2 of His-163. One water molecule near the lactam ring can form hydrogen bonds with the carbonyl oxygen of N, His-172, NE2 and 140-Phe and Ser-B1 on the lactam ring of N1, respectively, to form 2.6 Å, 3.2 Å, 2.6 Å and 2.7 Å, so that the lactam ring is firmly bound in the S1 pocket. Since the P1 site of the main protease of the entire coronavirus family is well conserved and always appears as Q, occupying the corresponding substrate binding pocket S1 is a key to inhibiting protease activity. For the P2 site of N1, the Leu side chain of the small molecule was easily inserted into a hydrophobic pocket consisting of the His-41, Met-49 and Phe-181 side chains, and the alkyl portions of the Gln-189 and Asp-187 side chains. The carbonyl oxygen in His-164 forms a hydrogen bond of 2.9 Å with N in a peptide bond on the side close to the ester group in N1, the carbonyl oxygen of Val in N1 forms a hydrogen bond of 2.9 Å with N in Glu-A166, the N in a Val peptide bond forms a hydrogen bond of 3.0 Å with the Glu-A166 carbonyl oxygen, and the N in an Ala peptide bond in N1 forms a hydrogen bond of 3.2 Å with the carbonyl oxygen of Thr-190. The side chain of Ala is inserted into a hydrophobic pocket consisting of the side chains of Phe-185, Glu-192, Leu-167, Met-165. The terminal heterocycle in N1 has a hydrophobic interaction with the five-membered ring of Pro-168. All of these covalent, hydrogen bonding and hydrophobic interactions result in the inhibitor compound N1 being firmly bound within the active pocket of the enzyme substrate, thereby causing inactivation of the enzyme.
2. The combination of N2 and N3 is substantially similar to N1 and will not be described in detail.
Example 5 pairs of small molecule compounds N1, N2, N3 and N4 were derived from various sources
Inhibitory Activity of the Main protease of different coronaviruses
Materials and methods
(1) N1, N2, N3 and N4 for SARS-CoV MproMeasurement of inhibitory Activity of
To the buffer (20mM Tris-HCl pH7.0, 1mM DTT) was added 1. mu.M (final concentration) of SARS-CoV M pro100 μ M (final concentration)After compound N1, 298K was allowed to stand for 10 minutes, 5. mu.M of fluorescently labeled substrate (MCA-AVLQ ↓ SGFRL (DNP) L-NH2) was added rapidly. The excitation and emission wavelengths were 330nm and 395nm, respectively, and the temperature was maintained at 298K, and fluorescence readings were recorded every 2 seconds.
The inhibitory activities of N2, N3 and N4 were determined in substantially the same manner as N1.
Comparison: no inhibitor was added and the remaining conditions were the same. The results are shown in FIG. 3.
(2) Measurement of inhibitory Activity of N1 against Transmissible Gastroenteritis Virus (TGEV) Main protease.
mu.M of the protease, 100. mu.M of Compound N1, 298K, were added to a buffer (20mM Tris-HCl pH7.0, 1mM DTT) and after 10 minutes of standing, 5. mu.M of a fluorescently labeled substrate (MCA-AVLQSGFRL(DNP) L-NH) was rapidly added2). The excitation and emission wavelengths were 330nm and 395nm, respectively, and the temperature was maintained at 298K, and fluorescence readings were recorded every 2 seconds. The inhibitor concentration was then varied and the inhibitory activity was determined at 10nM, 1. mu.M, respectively. Comparison: no inhibitor was added and the remaining conditions were the same. The results are shown in FIG. 4.
(3) Determination of inhibitory Activity of N1 against Human Coronavir (HCoV) 229E Primary protease
mu.M of the protease, 100. mu.M of Compound N1, 298K, were added to a buffer (20mM Tris-HCl pH7.0, 1mM DTT) and after 10 minutes of standing, 5. mu.M of a fluorescently labeled substrate (MCA-AVLQSGFRL(DNP) L-NH2) was added rapidly. The excitation and emission wavelengths were 330nm and 395nm, respectively, and the temperature was maintained at 298K, and fluorescence readings were recorded every 2 seconds. Comparison: no inhibitor was added and the remaining conditions were the same. The results are shown in FIG. 5.
(4) Measurement of inhibitory Activity of N1 against major protease of Feline Infectious Peritonitis Virus (FIPV)
The procedure is as in (3), and the results are shown in FIG. 6.
(5) Determination of inhibitory Activity of N1 on Main protease of Avian Infectious Bronchitis Virus (AIBV)
mu.M of the protease, 100. mu.M of Compound N1, 298K, were added to a buffer (20mM Tris-HCl pH7.0, 1mM DTT) and after 10 minutes of standing, 5. mu.M of a fluorescently labeled substrate (MCA-AVLQSGFRL(DNP) L-NH2) was added rapidly. The excitation and emission wavelengths were 330nm and 395nm, respectively, and the temperature was maintained at 298K, and fluorescence readings were recorded every 2 seconds. Comparison: no inhibitor was added and the remaining conditions were the same. The results are shown in FIG. 7.
Expression purification references for FIPV, HCoV, TGEV: constellation of substripes specificities, ammonium coronavirus main proteins, J Gen Virol, 2002; 83(Pt 3): 595-9.
Second, results and analysis
1. N1, N2, N3 and N4 for SARS-CoV MproInhibiting activity of
As can be seen in FIG. 3, N1, N2, N3 and N4 are directed against SARS-CoV MproAll had inhibitory activity, and N1 had the strongest inhibitory activity.
2. Inhibitory Activity of N1 against other coronavirus Main proteases
From the measurement of the inhibitory activity of N1 on other coronavirus main proteases (i.e., FIG. 4-FIG. 7), it can be seen that N1 has inhibitory activity on TGEV, HcoV, FIPV and AIBV main proteases, wherein the inhibitory activity on TGEV main protease is strongest, and N1 still has inhibitory activity on TGEV main protease under the condition of 10 nM.
Since TGEV, HcoV and FIPV belong to the type I (serotype) of the family Coronaviridae, AIBV belongs to the type III and SARS-CoV belongs to the type IV, it is concluded that N1 has inhibitory activity against the major proteases of the entire family Coronaviridae.
References referred to herein, including patent documents, academic papers, publications, and the like, are hereby incorporated by reference in their entirety.
It should be noted that the various experimental procedures involved in the present invention are all conventional in the art, and if not specifically mentioned herein, those skilled in the art can refer to various common tool books, scientific literatures or related specifications, manuals, etc. before the filing date of the present invention.
Manufacturers, references, or detailed manufacturing methods for those particular or otherwise unavailable from the various experimental articles (including, but not limited to, chemical reagents, biological products, cells, organisms, instruments, etc.) referred to herein are noted; unless otherwise specified, are all conventional laboratory supplies and may be readily obtained by various means (e.g., purchased, self-prepared, etc.) prior to the filing date of the present application.
It will be understood by those skilled in the art that various changes and modifications in form and detail may be made therein without departing from the spirit and scope of the invention, and these are to be considered as falling within the scope of the invention.
Claims (12)
1. A compound of the following general formula (I):
wherein,
u is
Wherein X is NH or CH2；
R1Selected from the group consisting of: c3～C6Alkylcarbonyl, t-butoxycarbonyl, benzyloxycarbonyl, isoxazolylcarbonyl, furylcarbonyl, trifluoromethylcarbonyl,
R2Selected from the group consisting of: c1～C4Alkyl, phenyl, benzyl, fluorobenzyl;
R3selected from the group consisting of: c1～C4Alkyl, phenyl, benzyl, p-methylphenyl, fluorobenzyl;
R4selected from the group consisting of: c1～C4Alkyl, phenyl, benzyl, p-methylphenyl, fluorobenzyl;
R5selected from the group consisting of: c1～C4Alkyl, phenyl, benzyl, p-methylphenyl, fluorobenzyl, fluorophenyl.
2. The compound of claim 1, wherein:
R1selected from the group consisting of: t-butyloxycarbonyl group, isoxazolylcarbonyl group, trifluoromethylcarbonyl group,
R2Is methyl or isopropyl;
R3is methyl or isopropyl;
R4is ethyl or benzyl;
R5selected from the group consisting of: isobutyl, p-fluorobenzyl, phenyl, fluorophenyl.
3.The compound of claim 1, wherein the compound is selected from the group consisting of:
wherein Boc is tert-butyloxycarbonyl, Ph is phenyl, Et is ethyl, and Bn is benzyl.
4. The compound of claim 1, wherein the compound has the formula:
5. the compound of claim 1, wherein the compound has a formula according to the following formula:
wherein R is1、R2、R3、R4、R5As defined in claim 1.
6. The compound of claim 2, wherein the compound has a formula according to the following formula:
wherein R is1、R2、R3、R4、R5As defined in claim 2.
7. A process for the preparation of a compound according to claim 1, comprising the steps of:
the protecting group R of amino in the compound of formula (II)6Removing R therein6Selected from the group consisting of: t-butoxycarbonyl, trifluoroacetyl, benzyloxycarbonyl;
condensing the product of the last step with a compound of formula (III) in the presence of a condensing agent to obtain a compound of formula (I),
wherein R in the formula (II) and the formula (III)1、R2、R4And U is as defined for formula (I).
8. The method of claim 7, comprising the steps of:
in an organic solvent, reacting the compound of the formula (II) with an acid at room temperature for 1-4 hours, and removing the protecting group R of amino6Extracting the solvent, said R6Selected from the group consisting of: t-butoxycarbonyl, trifluoroacetyl, benzyloxycarbonyl;
and (3) dissolving the product and the compound of the formula (III) in an aprotic solvent, adding a condensation reagent and organic base, and reacting at room temperature for 8-24 hours to obtain the compound of the formula (I).
9. The process of claim 7, further comprising further derivatizing the resulting compound of formula (I) to convert R therein1Group exchange to the desired group R1', wherein R1' is selected from the group consisting of: c3～C6Alkylcarbonyl, t-butoxycarbonyl, benzyloxycarbonyl, isoxazolylcarbonyl, furylcarbonyl, trifluoromethylcarbonyl,
The derivatization comprises the following steps:
a process according to claim 7R in the compound of formula (I) obtained in (1)1Removing;
reacting the product of the previous step with a carboxylic acid R in the presence of a condensing agent1' -OH to give a derivatized compound of formula (I) wherein R1Radical is replaced by R1' group (a).
10. The method of any one of claims 7-9, wherein,
the acid is trifluoroacetic acid or hydrochloric acid;
the organic solvent is selected from the group consisting of: CH (CH)2Cl2Tetrahydrofuran, CHCl3N, N-dimethylformamide, dioxane;
the aprotic solvent is selected from the group consisting of: CH (CH)2Cl2Tetrahydrofuran, CHCl3N, N-dimethylformamide, dimethyl sulfoxide, benzene;
the condensing agent is selected from the group consisting of: HATU, HBTU, EDCI;
the organic base is selected from the group consisting of: diisopropylethylamine and triethylamine.
11. Use of a compound according to claims 1-6 in the manufacture of a medicament for the treatment or prevention of a coronavirus infection.
12. Use according to claim 11, wherein the coronavirus is a SARS coronavirus, a transmissible gastroenteritis virus of swine, a human coronavirus, a feline infectious peritonitis virus or an avian infectious bronchitis virus.
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| CNB2004100877217A CN100363346C (en) | 2004-10-22 | 2004-10-22 | Small molecular inhibiting agent for coronavirus main proteinase, its preparation method and uses |
| PCT/CN2005/001748 WO2006042478A1 (en) | 2004-10-22 | 2005-10-24 | Small-molecule inhibitors of coronaviral main protease, their preparation and use |
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| CNB2004100877217A CN100363346C (en) | 2004-10-22 | 2004-10-22 | Small molecular inhibiting agent for coronavirus main proteinase, its preparation method and uses |
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| CN103044338B (en) * | 2012-12-12 | 2016-08-03 | 天津医科大学总医院 | MiR-21 micromolecular inhibitor and application |
| CN104592349A (en) * | 2015-02-15 | 2015-05-06 | 天津国际生物医药联合研究院 | Small molecule inhibitor of coronavirus main proteases as well as preparation method and application of small molecule inhibitor |
| CN105412151A (en) * | 2015-12-11 | 2016-03-23 | 天津国际生物医药联合研究院 | Inhibitor aiming at coronavirus main protease, and application of Zn<2+> |
| CN105837487A (en) * | 2016-03-17 | 2016-08-10 | 天津国际生物医药联合研究院 | Small-molecule inhibitor against MERS-CoV main protease, and preparation method and application thereof |
| CA3024120A1 (en) * | 2016-05-13 | 2017-11-16 | Emory University | Peptidomimetics for the treatment of norovirus infection |
| CN115645396A (en) * | 2020-02-26 | 2023-01-31 | 上海科技大学 | Application of propyl-2-imidazole disulfide in resisting coronavirus |
| EP3912628A1 (en) * | 2020-05-20 | 2021-11-24 | Institut de Recherche en Semiochimie et Ethologie Appliquée | Nucleoside analogues to inhibit the main protease of a coronavirus |
| US11351149B2 (en) * | 2020-09-03 | 2022-06-07 | Pfizer Inc. | Nitrile-containing antiviral compounds |
| CN114149415A (en) * | 2021-07-26 | 2022-03-08 | 中国药科大学 | Peptide-like compound and derivative, preparation method, pharmaceutical composition and application thereof |
| CN117209558B (en) * | 2023-08-14 | 2024-09-17 | 北京大学深圳研究生院 | Use of Mpro protein inhibitors in medicaments for the treatment of novel coronavirus infections |
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| WO1998043950A1 (en) * | 1997-03-28 | 1998-10-08 | Agouron Pharmaceuticals, Inc. | Antipicornaviral compouds, compositions containing them, and methods for their use |
| EE200000621A (en) * | 1998-04-30 | 2002-06-17 | Agouron Pharmaceuticals, Inc. | Anti-picornaviral compounds, their preparation and use |
| US6355807B1 (en) * | 1999-08-24 | 2002-03-12 | Agouron Pharmaceuticals, Inc. | Efficient synthetic routes for the preparation of rhinovirus protease inhibitors and key intermediates |
| CA2550515A1 (en) * | 2003-12-31 | 2005-07-21 | Taigen Biotechnology | Protease inhibitors |
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Non-Patent Citations (1)
| Title |
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| an efficinet and runabler oute to ag 70888 a rhinovirusproteaseinhi bitor. dawet ma et al.Tetrahedron letters,Vol.45 . 2004 * |
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| WO2006042478A1 (en) | 2006-04-27 |
| WO2006042478A8 (en) | 2006-07-13 |
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