Compound containing nitrogen heterocyclic skeleton, and preparation method and application thereof

Abstract

The invention relates to a compound containing a nitrogen heterocyclic skeleton as well as a preparation method and application of the compound. Specifically, the compound disclosed by the invention has a structure as shown in a formula (I), and the definition of each group or substituent group is described in the specification. The invention provides a preparation method of the compound and application of the compound in pharmacy, and further discloses application of the compound in prevention and / or treatment of respiratory tract related diseases or symptoms caused by viruses (such as SARS-CoV-2, SARS-CoV and MERS-CoV), and application of the compound in inhibition of RNA polymerase or a compound thereof dependent on non-structural proteins of the related viruses such as RNA. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of drug research of nitrogen heterocyclic skeletons and their derivatives, and specifically relates to a class of compounds containing nitrogen heterocyclic skeletons, and their preparation methods and uses. The present invention further relates to the use mainly for coronaviruses containing RNA-dependent RNA polymerase or containing Nsp7 / 8 / 12 protein complexes. Background Art

[0002] SARS-CoV-2 is a single-stranded positive-sense RNA virus whose genome includes dozens of genes encoding structural and non-structural proteins. Among them, non-structural proteins (Nsp) play a key role in viral replication and spread.

[0003] Nsp7, Nsp8, and Nsp12 are considered to be important nonstructural proteins in SARS-CoV-2. Nsp12 encodes RNA-dependent RNA polymerase (RdRp), a key enzyme for viral replication and transcription. Nsp7 and Nsp8 form an auxiliary complex that works with Nsp12 to promote the activity and stability of RdRp. Due to the key role of the Nsp7 / 8 / 12 complex in the viral replication process, they have become important targets for the development of antiviral drugs. By interfering with the function of the Nsp7 / 8 / 12 complex, the replication and spread of SARS-CoV-2 can be effectively inhibited, thus providing new possibilities for the treatment of COVID-19.

[0004] At present, the research on Nsp7 / 8 / 12 complex mainly focuses on the relationship between its structure and function, potential antiviral drug screening and drug design. By deeply understanding the structure and function of Nsp7 / 8 / 12 complex, it can provide important theoretical basis and technical support for the development of therapeutic drugs for COVID-19.

[0005] Therefore, there is an urgent need in the art to develop a COVID-19 drug with a high inhibition rate against the SARS-CoV-2Nsp7 / 8 / 12 complex. Summary of the invention

[0006] The purpose of the present invention is to provide a compound with a nitrogen-containing heterocyclic skeleton having a high inhibition rate against the SARS-CoV-2 Nsp7 / 8 / 12 complex, a preparation method thereof and its use. This research has important scientific significance and clinical application prospects, and is expected to provide new targets and strategies for the development of antiviral drugs.

[0007] In a first aspect of the present invention, there is provided a compound represented by formula (I), or a pharmaceutically acceptable salt or ester, tautomer, enantiomer, diastereomer, racemate, hydrate, solvate, prodrug or deuterated compound thereof:

[0008]

[0009] in,

[0010] X1, X2 and X3 are each independently selected from the group consisting of CH, CR1, N, and at least one of X1, X2 and X3 is N;

[0011] R1 is one or more groups selected from the group consisting of H, halogen, -NO2, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, -N(R 5 )2.-OR 5 、-SR 5 、-C(=O)N(R 5 )2, -C(=O)OR 5 、-NR 5 -C(=O)R 5 、-NR 5 -C(=O)OR 5 、-NR 5 -S(=O)2-R 5 、-C1-C6 alkylene-N(R 5 )2, -C1-C6 alkylene-OR 5 、-C2-C6 alkenylene-OR 5 、-O-C1-C6 alkylene-N(R 5 )2.-NR 5 -C1-C6 alkylene-OR 5 ;

[0012] Among them, R 5 Each is independently selected from the following group: H, guanidinyl, C1-C6 alkyl, saturated or partially unsaturated C3-C8 cycloalkyl, saturated or partially unsaturated C3-C10 heterocyclyl containing 1-3 heteroatoms selected from N, O or S, C6-C10 aryl, C5-C10 heteroaryl;

[0013] Y is selected from the following group: CH, CR 10 , N, O, S;

[0014] When Y is O or S, R2 does not exist;

[0015] When Y is CH or CR 10When R2 is selected from the group consisting of H, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted saturated or partially unsaturated C3-C10 cycloalkyl, substituted or unsubstituted saturated or partially unsaturated C3-C10 heterocyclic group containing 1 to 3 heteroatoms selected from N, O or S, -C(=O)R 6 、-OC(=O)R 6 、-C(=O)OR 6 、-OR 6 、-SR 6 、-S(=O)R 6 、-S(=O)2R 6 、-S(=O)2N(R 6 )2、-N(R 6 )2, -C(=O)N(R 6 )2.-NR 6 -C(=O)R 6 、-NR 6 -C(=O)OR 6 、-NR 6 -S(=O)2-R 6 、C(=O)-N(R 6 )2, -C1-C6 alkylene-N(R 6 )2, -C1-C6 alkylene-C(=O)N(R 6 )2, -C1-C6 alkylene-C(=O)OR 6 、-C1-C6 alkylene-OR 6 、-C2-C6 alkenylene-OR 6 and -O-C1-C6 alkylene-N(R 6 )2;

[0016] Among them, R 6 Each is independently selected from the following group: H, guanidino, C1-C6 alkyl, saturated or partially unsaturated C3-C8 cycloalkyl, saturated or partially unsaturated C3-C10 heterocyclyl containing 1-3 heteroatoms selected from N, O or S;

[0017] R 10 Selected from the group consisting of halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl;

[0018] When Y is N, R2 is selected from the following group: H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted saturated or partially unsaturated C3-C10 cycloalkyl, substituted or unsubstituted saturated or partially unsaturated C3-C10 heterocyclic group containing 1-3 heteroatoms selected from N, O or S, -C(=O)R 8 、-C(=O)OR 8 、-S(=O)R 8 、-S(=O)2R 8 、-S(=O)2N(R 8 )2, -C(=O)N(R 8 )2, -C1-C6 alkylene-N(R 6 )2, -C1-C6 alkylene-C(=O)N(R 8 )2, -C1-C6 alkylene-C(=O)OR 8 、-C1-C6 alkylene-OR 8 、-C2-C6 alkenylene-OR 8 and -O-C1-C6 alkylene-N(R 8 )2;

[0019] Among them, R 8 Each is independently selected from the following group: H, guanidino, C1-C6 alkyl, saturated or partially unsaturated C3-C8 cycloalkyl, saturated or partially unsaturated C3-C10 heterocyclic group containing 1-3 N, O, S heteroatoms;

[0020] Or, R1 and R2 together with the carbon atom to which they are attached and the Y atom form a C3-C6 cycloalkyl group or a C3-C6 heterocyclic group containing 1-3 heteroatoms selected from N, O or S, which may be R 10 optionally substituted;

[0021] R3 and R4 are each independently selected from the following group: H, halogen, -OH, -CN, -NO2, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted saturated or partially unsaturated C3-C10 cycloalkyl, substituted or unsubstituted saturated or partially unsaturated C3-C10 heterocyclic group containing 1-3 heteroatoms selected from N, O or S, -N(R 7 )2.-OR 7 、-C(=O)R 7 、-OC(=O)R 7 、-C(=O)OR 7 、-SR 7 、-S(=O)R 7 、-S(=O)2R7 、-S(=O)2N(R 7 )2、-N(R 7 )2, -C(=O)N(R 7 )2.-NR 7 -C(=O)R 7 、-NR 7 -C(=O)OR 7 、-NR 7 -S(=O)2-R 7 、C(=O)-N(R 7 )2;

[0022] R 7 Selected from the following group: H, guanidino, C1-C6 alkyl, saturated or partially unsaturated C3-C8 cycloalkyl, saturated or partially unsaturated C3-C10 heterocyclyl containing 1-3 heteroatoms selected from N, O or S;

[0023] A is selected from the following group:

[0024] In another preferred embodiment, when R5, R6, R7 and R8 are connected to N or O, said R5, R6, R7 and R8 are not guanidine groups.

[0025] In another preferred embodiment, R1 is selected from one or more groups of the following groups: H, halogen, -NO2, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, -N(R 5 )2.-OR 5 、-SR 5 、-C(=O)N(R 5 )2, -C(=O)OR 5 、-NR 5 -C(=O)R 5 、-NR 5 -C(=O)OR 5 ; R 5 Selected from the following group: H, C1-C6 alkyl, saturated or partially unsaturated C3-C8 cycloalkyl, saturated or partially unsaturated C3-C10 heterocyclic group containing 1-3 N, O, S heteroatoms, and C6-C10 aryl.

[0026] In another preferred embodiment, Y is selected from the following group: N, O.

[0027] In another preferred embodiment, when Y is N, R2 is selected from the following group: H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted saturated or partially unsaturated C3-C10 cycloalkyl, substituted or unsubstituted saturated or partially unsaturated C3-C10 heterocyclic group containing 1-3 heteroatoms selected from N, O or S, -C(=O)R 8 、-S(=O)R 8 、-S(=O)2R 8 、-C1-C6 alkylene-N(R 8 )2, -C1-C6 alkylene-C(=O)N(R 8 )2, -C1-C6 alkylene-C(=O)OR 8 、-C1-C6 alkylene-OR 8 and -O-C1-C6 alkylene-N(R 8 )2; where R 8 Selected from the following group: H, C1-C6 alkyl, saturated or partially unsaturated C3-C8 cycloalkyl, saturated or partially unsaturated C3-C10 heterocyclic group containing 1-3 heteroatoms selected from N, O or S.

[0028] In another preferred embodiment, R 8 Each is independently selected from the following group: H, guanidino, C1-C6 alkyl, saturated or partially unsaturated C3-C8 cycloalkyl, saturated or partially unsaturated C3-C10 heterocyclic group containing at least one N heteroatom.

[0029] In another preferred embodiment, R3 and R4 are each independently selected from the following group: H, halogen, -OH, -CN, -NO2, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted saturated or partially unsaturated C3-C10 cycloalkyl, substituted or unsubstituted saturated or partially unsaturated C3-C10 heterocyclic group containing 1-3 heteroatoms selected from N, O or S, -N(R 7 )2.-OR 7 、-C(=O)R 7 、-OC(=O)R 7 、-C(=O)OR 7 、-C(=O)N(R 7 )2.-NR 7 -C(=O)R 7 、-NR 7 -S(=O)2-R 7 ; Among them, R 7Selected from the following group: H, C1-C6 alkyl, saturated or partially unsaturated C3-C8 cycloalkyl, saturated or partially unsaturated C3-C10 heterocyclic group containing 1-3 heteroatoms selected from N, O or S.

[0030] In another preferred embodiment, A is selected from the following group:

[0031] In another preferred embodiment, R1 is selected from the following group: H, halogen, -NO2, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl.

[0032] In another preferred embodiment, Y is N.

[0033] In another preferred embodiment, R2 is selected from the following group: H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, -C1-C6 alkylene-N(R 8 )2, -C1-C6 alkylene-C(=O)N(R 8 )2, -C1-C6 alkylene-C(=O)OR 8 、-C1-C6 alkylene-OR 8 and -O-C1-C6 alkylene-N(R 8 )2; where R 8 Each is independently selected from the following group: H, C1-C6 alkyl.

[0034] In another preferred embodiment, R3 and R4 are each independently selected from the following group: H, halogen, -CN, -NO2, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted saturated or partially unsaturated C3-C10 heterocyclic group containing 1-3 heteroatoms selected from N, O or S.

[0035] In another preferred embodiment, A is selected from the following group:

[0036] In another preferred embodiment, R1 is selected from the following group: H, Cl, -NO2, -CH3, methylamino.

[0037] In another preferred embodiment, Y is N.

[0038] In another preferred embodiment, R2 is selected from one or two groups of the following groups: H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, -C1-C6 alkylene-C(=O)N(R 8 )2, -C1-C6 alkylene-C(=O)OR 8 .

[0039] In another preferred embodiment, R3 and R4 are each independently selected from the following group: H, halogen, -CF3, substituted or unsubstituted saturated or partially unsaturated C3-C10 heterocyclic group containing 1-3 heteroatoms selected from N, O or S.

[0040] In another preferred embodiment, A is

[0041] In another preferred embodiment, the compound is selected from the following group:

[0042]

[0043]

[0044] The second aspect of the present invention provides a method for preparing the compound, comprising the following steps:

[0045]

[0046] in,

[0047] X1, X2, X3, R1, R2, R3, R4, Y and A are as defined in the first aspect of the present invention.

[0048] The third aspect of the present invention provides a pharmaceutical composition comprising

[0049] (1) The compound according to claim 1, or a pharmaceutically acceptable salt or ester, tautomer, enantiomer, diastereomer, racemate, hydrate, solvate, prodrug or deuterated compound thereof;

[0050] (2) A pharmaceutically acceptable carrier.

[0051] The fourth aspect of the present invention provides a use of the compound described in the first aspect of the present invention, or its pharmaceutically acceptable salt or ester, tautomer, enantiomer, diastereomer, racemate, hydrate, solvate, prodrug or deuterated compound, or the pharmaceutical composition described in the third aspect of the present invention, for preparing a drug or pharmaceutical composition for treating diseases caused by viral infection.

[0052] In another preferred embodiment, the disease is selected from the group consisting of coronavirus, calicivirus, and picornavirus infection.

[0053] In another preferred embodiment, the disease is an infection caused by a virus selected from the group consisting of SARS-CoV-2 virus, SARS-CoV virus, and MERS-CoV virus.

[0054] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Compound W2 showed an inhibitory effect on RNA extension. DETAILED DESCRIPTION

[0056] After extensive and in-depth research, the inventors have provided a class of compounds with nitrogen-containing heterocyclic skeletons as shown in formula (I), specifically, the compounds have a high inhibition rate against the ARS-CoV-2Nsp7 / 8 / 12 complex, and the present invention also discloses a method for preparing the compounds and their use in treating related diseases caused by coronavirus infection. On this basis, the inventors have completed the present invention.

[0057] the term

[0058] In the present invention, unless otherwise specified, the terms used have the general meanings well known to those skilled in the art.

[0059] In the present invention, the term "halogen" refers to F, Cl, Br or I.

[0060] In the present invention, “C 1-6 The term "alkyl" refers to a straight or branched chain alkyl group containing 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, neopentyl, tert-pentyl, or the like.

[0061] In the present invention, the term "C 2-6 The term "alkenyl" refers to a straight or branched alkenyl group having 2 to 6 carbon atoms and containing one double bond, including but not limited to ethenyl, propenyl, butenyl, isobutenyl, pentenyl and hexenyl.

[0062] In the present invention, the term "C 2-6 The term "alkynyl" refers to a straight or branched chain alkynyl group having 2 to 6 carbon atoms and containing one triple bond, including but not limited to ethynyl, propynyl, butynyl, isobutynyl, pentynyl and hexynyl.

[0063] In the present invention, the term "C 3-6 The "cycloalkyl" group includes a group selected from the group consisting of: C 3-6 Cycloalkyl, C 3-6 Cycloalkenyl, C 3-6 The term "C 3-10 "Cycloalkyl" has a similar meaning.

[0064] In the present invention, the term "C3-6 "Cycloalkyl" refers to a cyclic alkyl group having 3 to 6 carbon atoms in the ring, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. The term "C 3-10 "Cycloalkyl" has a similar meaning.

[0065] In the present invention, the term "C 1-6 "Alkoxy" refers to a straight or branched alkoxy group having 1 to 6 carbon atoms, including but not limited to methoxy, ethoxy, propoxy, isopropoxy and butoxy. Preferably, C 1-4 Alkoxy.

[0066] In the present invention, the term "heterocyclic group" refers to a 4-8 membered heterocyclic group containing 1, 2 or 3 heteroatoms selected from N, O and S, including (but not limited to) the following groups:

[0067] In the present invention, the term "aromatic ring" or "aryl group" has the same meaning, preferably "C 6-10 The term "C 6-10 The term "aryl" refers to an aromatic ring group having 6 to 10 carbon atoms and containing no heteroatoms in the ring, such as phenyl, naphthyl, etc.

[0068] In the present invention, the term "aromatic heterocycle" or "heteroaryl" has the same meaning and refers to a heteroaromatic group containing one to multiple heteroatoms. For example, "5-14 membered heteroaryl" refers to an aromatic heterocycle containing 1 to 3 heteroatoms selected from oxygen, sulfur and nitrogen and 2 to 13 carbon atoms. Non-limiting examples include: furanyl, thienyl, pyridyl, pyrazolyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring can be fused to an aryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring. The heteroaryl group can be optionally substituted or unsubstituted.

[0069] In the present invention, the term "C 6-12 "Arylalkyl" refers to a group having the structure C 6-12 Aryl-C 1-6 Alkylene-group.

[0070] In the present invention, the term "deuterated" means substituted with deuterium.

[0071] In the present invention, the term "substituted" refers to one or more hydrogen atoms on a specific group being replaced by a specific substituent. The specific substituent is the substituent described above, or the substituent appearing in each embodiment. Unless otherwise specified, a substituted group may have a substituent selected from a specific group at any substitutable position of the group, and the substituent may be the same or different at each position. It should be understood by those skilled in the art that the combination of substituents contemplated by the present invention is those stable or chemically feasible combinations. The substituents include, for example (but not limited to): halogen, hydroxyl, carboxyl (-COOH), C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, 3- to 12-membered heterocyclic radical, aryl, heteroaryl, C1-C8 aldehyde, C2-C10 acyl, C2-C10 ester, amino, C1-C6 alkoxy, C1-C10 sulfonyl, etc.

[0072] In the present invention, the term 1-6 refers to 1, 2, 3, 4, 5 or 6. Other similar terms independently have similar meanings. The term "plurality" refers to 2-6, such as 2, 3, 4, 5 or 6.

[0073] It should be understood that when a group is present in multiple different positions of a compound at the same time, its definition at each position is independent of each other and may be the same or different. That is, the term "selected from the following group:" has the same meaning as the term "each independently selected from the following group:".

[0074] Compound

[0075] The present invention provides a compound represented by formula (I), or a pharmaceutically acceptable salt, tautomer, enantiomer, diastereomer, racemate, hydrate, ester, solvate, metabolic precursor, prodrug or deuterated compound thereof:

[0076]

[0077] wherein each group is as defined above.

[0078] In another preferred embodiment, in the compound, any one of X1, X2, X3, Y, R1, R2, R3, R4, and A is independently a corresponding group in the specific compound described in the present invention.

[0079] As used herein, the term "pharmaceutically acceptable salt" refers to a salt formed by a compound of the present invention and an acid or base that is suitable for use as a drug. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts is a salt formed by a compound of the present invention and an acid. Suitable acids for forming salts include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, and naphthalenesulfonic acid; and amino acids such as proline, phenylalanine, aspartic acid, and glutamic acid.

[0080] Another preferred salt is a salt of the compound of the present invention and a base, such as an alkali metal salt (e.g., sodium salt or potassium salt), an alkaline earth metal salt (e.g., magnesium salt or calcium salt), an ammonium salt (e.g., lower alkanolammonium salt and other pharmaceutically acceptable amine salts), for example, methylamine salt, ethylamine salt, propylamine salt, dimethylamine salt, trimethylamine salt, diethylamine salt, triethylamine salt, tert-butylamine salt, ethylenediamine salt, hydroxyethylamine salt, dihydroxyethylamine salt, trihydroxyethylamine salt, and amine salts formed from morpholine, piperazine, and lysine, respectively.

[0081] Replacing hydrogen atoms with deuterium atoms to change the physical and chemical properties of compounds has become a well-known structural modification method for those skilled in the art. Unless otherwise specified, the present invention intends to include the deuterated form of the compound represented by general formula (I) in the invention content.

[0082] Preparation method

[0083] The preparation method of the compound of formula I described below does not constitute any limitation to the present invention. The compound of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known in the art, and such combination can be easily performed by a technician in the field to which the present invention belongs.

[0084] It should be understood that the raw materials and reagents used in the preparation process of the compounds of the present invention can be purchased through commercial channels unless otherwise specified.

[0085] The compounds of the present invention can be prepared by a variety of synthetic methods. The methods shown in the following schemes 1 and 2 are representative general schemes, which, combined with the synthetic methods of the specific compounds in the examples, constitute the preparation methods of the compounds of the present invention. It should be understood that the preparation methods of the compounds of the present invention are not limited to those shown in schemes 1 and 2, which are only used to explain and not to limit the present invention in any way.

[0086] Option 1:

[0087]

[0088] In the first step, the compound of general formula (I-1) reacts with 1-Boc-piperazine under the action of a base at room temperature to obtain a compound of general formula (I-2);

[0089] In the second step, the compound of general formula (I-2) reacts with the substrate shown in the figure under the action of a base at high temperature to obtain a compound of general formula (I-3);

[0090] In the third step, the compound of general formula (I-3) is firstly subjected to room temperature and acidic conditions to remove the protective group tert-butyloxycarbonyl, and then is reacted with a covalent linker under alkaline conditions without purification and then dried to obtain a compound of general formula (I-4).

[0091] Option 2:

[0092]

[0093] In the first step, the compound of general formula (II-1) reacts with the substrate shown in the figure under the action of a base at room temperature to obtain a compound of general formula (II-2);

[0094] In the second step, the compound of the general formula (II-2) is oxidized by an oxidizing agent at room temperature to obtain a compound of the general formula (II-3);

[0095] The third step is to obtain a compound of the general formula (II-4) under the action of a nitrosating agent at room temperature with the compound of the general formula (II-3);

[0096] The fourth step is to react the compound of general formula (II-4) with 1-Boc-piperazine under the action of a base at room temperature to obtain a compound of general formula (II-5);

[0097] The fifth step is to obtain a compound of the general formula (II-6) by treating the compound of the general formula (II-5) with dilute sulfuric acid at high temperature;

[0098] In the sixth step, the compound of the general formula (II-6) reacts with a covalent linker under low temperature and alkaline conditions to obtain a compound of the general formula (II-7).

[0099] In another preferred embodiment, the reagent providing alkaline conditions is an organic base or an inorganic base. The organic base is selected from the following group: triethylamine, N,N-diisopropylethylamine, lithium diisopropylamide, sodium tert-butoxide, sodium methoxide and potassium tert-butoxide, or a combination thereof; the inorganic base is selected from the following group: sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, potassium acetate, cesium carbonate, sodium hydroxide, potassium hydroxide, sodium bicarbonate, lithium hydroxide, butyl lithium, or a combination thereof.

[0100] In another preferred embodiment, the reagent providing acidic conditions is selected from the following group: hydrogen chloride, 1,4-dioxane solution of hydrogen chloride, methanol solution of hydrogen chloride, trifluoroacetic acid, formic acid, acetic acid, hydrochloric acid, sulfuric acid, methanesulfonic acid, nitric acid, phosphoric acid, or a combination thereof.

[0101] In another preferred embodiment, the oxidant is selected from the following group: hydrogen peroxide, ammonium persulfate, potassium permanganate, m-chloroperbenzoic acid, potassium permanganate, or a combination thereof.

[0102] Pharmaceutical compositions and methods of administration

[0103] The pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention or a pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Usually, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, and more preferably, contains 5-1000 mg of the compound of the present invention per dose. Preferably, the "one dose" is a capsule or tablet.

[0104] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0105] The pharmaceutical composition is in the form of injection, capsule, tablet, pill, powder or granule.

[0106] There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administrations include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0107] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; I solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0108] Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microencapsulated form with one or more of the above-mentioned excipients.

[0109] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0110] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0111] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances, and the like.

[0112] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0113] Dosage forms for topical administration of the compounds of the invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0114] The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

[0115] The treatment method of the present invention can be used alone or in combination with other treatment methods or therapeutic drugs.

[0116] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the daily dosage is usually 1 to 2000 mg, preferably 5 to 1000 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill range of a skilled physician.

[0117] Compared with the prior art, the present invention has the following main advantages:

[0118] 1. The compounds of the present invention have good inhibitory activity against the novel coronavirus nsp7 / 8 / 12 protein complex, thereby effectively inhibiting the replication and spread of SARS-CoV-2;

[0119] 2. The compounds of the present invention have an inhibitory effect on RNA-dependent RNA polymerase (RdRp), thereby effectively terminating viral replication and transcription.

[0120] 3. The compounds of the present invention have a direct covalent inhibitory effect on Nsp12.

[0121] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0122] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0123] Unless otherwise specified, the experimental materials and reagents used in the following examples can be obtained from commercial channels.

[0124] In the present invention, 1 H NMR spectra were measured using a Bruker instrument (400 MHz), and chemical shifts were expressed in ppm using tetramethylsilane as an internal standard (0.00 ppm). 1 H NMR notation: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad, dd = doublet of a doublet, dt = doublet of a triplet. Coupling constants, when given, are given in Hz.

[0125] In the present invention, the mass spectrum is measured by LC / MS instrument, and the ionization method is ESI.

[0126] In the present invention, the high performance liquid chromatograph model is: Agilent 1260, Thermo Fisher U3000; the chromatographic column model is: Waters xbrige C18 (4.6*150mm, 3.5μm); the mobile phase is: A:ACN, B:Water (0.1%H3PO4); the flow rate is: 1.0mL / min; the gradient is: 5%A for 1min, increase to 20%Awithin 4min, increase to 80%Awithin 8min, 80%A for 2min, back to 5%A ​​within0.1min; the wavelength is: 220nm; the column oven is: 35°C.

[0127] In the present invention, the thin layer chromatography silica gel plate (HSGF254) used in the reaction detection comes from Sinopharm Chemical Reagent Co., Ltd.; the 200-300 mesh silica gel of Sinopharm Chemical Reagent Co., Ltd. is used for compound separation.

[0128] Preparation of intermediates

[0129] Example 1 Preparation of Intermediate S2

[0130]

[0131] Step 1: 4,6-dichloro-N-methyl-1,3,5-triazine-2-amine (S1)

[0132] At 0°C, methylamine hydrochloride (1.83 g, 27.11 mmol) was added to a solution of cyanuric chloride 1a (5.0 g, 27.11 mmol) and DIPEA (7.01 g, 54.22 mmol) in tetrahydrofuran (100 mL). The mixture was reacted for 1 hour. TLC showed that the reaction was complete. The reaction solution was quenched with water (100 mL) and extracted with ethyl acetate (50 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography (PE / EA=10 / 1) to obtain the title compound S1 (2.1 g, yield 43.3%) as a white solid.

[0133] LC-MS: m / z=179.0 [M+H] +

[0134] 1 H NMR (400MHz, DMSO) δ9.02–8.95 (m, 1H), 2.82 (d, J = 4.7Hz, 3H). 13 C NMR (101MHz, DMSO) δ169.92,168.72,166.02,165.98,28.07,28.04.

[0135] Step 2: 4-[4-chloro-6-(methylamino)-1,3,5-triazacyclohexane-2-yl]piperazine-1-carboxylic acid-2-methylpropane-2-yl ester (S2)

[0136] Compound S1 (1.5 g, 8.4 mmol) was dissolved in acetone / water (8 mL + 2 mL), and 1-Boc-piperazine (1.7 g, 8.8 mmol) and potassium carbonate (1.7 g, 12.5 mmol) were added. After reacting at room temperature overnight, water was added and the aqueous phase was extracted with ethyl acetate. After extraction three times, the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography to obtain the title compound S2 (2.5 g, yield 83%) as a white solid.

[0137] LC-MS: m / z = 273.1 [M-55] +

[0138] 1 H NMR (400MHz, CDCl3) δ6.83(s,1H),3.88–3.70(m,4H),3.46(ddt,J=11.9,8.1,4.9Hz,4H),2.97(d,J=4.9Hz,3H),1.48(d,J=2.1Hz,9H). 13C NMR (101MHz, CDCl3) δ168.54,165.98,164.52,154.66,80.23,77.40,77.08,76.76,43.29,28.39,27.71.

[0139] Example 2 Preparation of Intermediate 4-(2,6-dichloropyrimidin-4-yl)piperazine-1-carboxylic acid 2-methylpropan-2-yl ester (S3)

[0140]

[0141] 1-Boc-piperazine (2.1 g, 11.5 mmol) was added to a tetrahydrofuran solution of trichloropyrimidine (2.0 g, 10.9 mmol) and DIPEA (1.4 g, 32.7 mmol), and the reaction was carried out at room temperature for 3 h. The reaction was completed by TLC detection. The reaction solution was directly added to a silica gel sand column for chromatography (PE / EA=10 / 1) and purified to obtain a pure white solid title compound S3 (3.1 g, yield 86.2%).

[0142] LC-MS: m / z=333.1[M+H] +

[0143] 1 H NMR (400MHz, DMSO) δ7.02 (s, 1H), 3.78–3.55 (m, 4H), 3.42 (dd, J = 6.8, 3.8Hz, 4H), 1.42 (s, 9H). 13 C NMR (101MHz, DMSO) δ163.36,159.61,158.94,154.21,101.36,79.76,28.49.

[0144] Preparation of compounds

[0145] Example 1 Preparation of W1

[0146]

[0147] Step 1: 4-[6-(ethyl{[4-(trifluoromethyl)phenyl]methyl}amino)-4-(methylamino)-1,3,5-triazacyclohexyl-2-yl]piperazine-1-carboxylic acid-2-methylpropan-2-yl ester (1a)

[0148] The intermediate S2 (200 mg, 0.61 mmol) was dissolved in 5 mL of DMF, and ethyl {[4-(trifluoromethyl)phenyl]methyl}amine (186 mg, 0.91 mmol) and potassium carbonate (252 mg, 1.83 mmol) were added. The reaction solution was heated at 120 degrees Celsius for overnight reaction, and then 20 mL of ice water was added to cool and stand until the product was completely precipitated. The precipitated product was washed with cold water and flash purified (PE / EA=1:1) to finally obtain a white product 1a.

[0149] LC-MS: m / z = 496.2 [M+H] +

[0150] Step 2: 2-Chloro-1-{4-[6-(ethyl{[4-(trifluoromethyl)phenyl]methyl}amino)-4-(methylamino)-1,3,5-triazacyclohexane-2-yl]piperazin-1-yl}ethan-1-one (W1)

[0151] The reactant 1a (200 mg, 0.4 mmol) was dissolved in 4 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added dropwise to the reaction solution under stirring, and the reaction was allowed to react at room temperature for 4 h. After the reaction was complete as detected by LCMS, the solvent was dried under reduced pressure. 4 mL of dichloromethane was added to the reaction bottle after drying, and chloroacetyl chloride (48 mg, 0.42 mmol) was added dropwise while stirring under an ice bath, and the reaction was allowed to react at 0°C for 2 h. The reaction was complete as monitored by LCMS. The crude product was purified by Pre-HPLC to obtain the final product W1 (73 mg, yield 38%).

[0152] LC-MS: m / z = 472.2 [M+H] +

[0153] 1 H NMR(500MHz,DMSO)δ7.70(d,J=8.0Hz,2H),7.51(d,J=8.0Hz,2H),4.87(s,2H),4.46–4.39( m,2H),3.62–3.39(m,10H),3.18(d,J=1.6Hz,1H),2.88–2.71(m,3H),1.11(t,J=6.8Hz,3H).

[0154] Preparation of Example 2W2

[0155]

[0156] The experimental operation was the same as in Example 1. 2a was synthesized from intermediate S2. The subsequent de-Boc and amidation reactions were the same. The product was purified by HPLC to obtain compound W2.

[0157] LC-MS: m / z=472.1[M+H]+

[0158] 1 H NMR(500MHz,DMSO)δ7.64(s,1H),7.40(d,J=8.4Hz,1H),7.25–7.18(m,1H),4.81(s,2 H),4.48–4.39(m,2H),3.65–3.40(m,10H),2.89–2.68(m,3H),1.12(q,J=5.9Hz,3H).

[0159] Preparation of Example 3W3

[0160]

[0161] Step 1: 4-Bromo-2-(1,4-oxazacyclohexane-4-yl)benzene-1-carboxaldehyde (3a)

[0162] 2-Fluoro-4-bromo-benzaldehyde (1.0 g, 4.92 mmol), morpholine (493 mg, 5.67 mmol), and potassium carbonate (783 mg, 5.67 mmol) were dissolved in DMF and stirred at 160°C for 4 h. After the reaction was completed as detected by LCMS, the product 3a (1.1 g, yield 83%) was obtained by flash purification (PE / EA=10:1).

[0163] LC-MS: m / z = 270.0 [M+H] +

[0164] 1 H NMR (400MHz, CDCl3) δ10.23(s,1H),7.66(d,J=8.2Hz,1H),7.27(dd,J=8.4,1.5Hz,1H),7.24(d,J=1.6Hz,1H),3.98-3.76(m,4H),3.20-2.93(m,4H). 13 C NMR (101MHz, CDCl3) δ189.94,155.82,131.90,130.05,127.21,126.08,122.33,66.75,53.98.

[0165] Step 2: 2-({[4-bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]methyl}amino)-N-methylacetamide (3b)

[0166] The reactant 3a (120 mg, 0.45 mmol), 2-amino-N-methylacetamide (79 mg, 0.89 mmol), and DIPEA (173 mg, 1.34 mmol) were dissolved in 4 mL of dichloromethane and stirred at room temperature for 8 h. Sodium triacetoxyborohydride (284 mg, 1.34 mmol) was slowly added to the above solution and the reaction was continued at room temperature for 4 h. After the reaction was completed by LCMS detection, the product 3b (138 mg, yield 91%) was obtained by flash purification (PE / EA=5:1).

[0167] LC-MS: m / z=342.1[M+H] +

[0168] Step 3: 4-(2-{1-[4-bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]-4-oxyde-2,5-diazepine-2-yl}-6-chloropyrimidin-4-yl)piperazine-1-carboxylic acid-2-methylpropane-2-yl ester (3c)

[0169] The reactant 3b (138 mg, 0.40 mmol), intermediate S3 (122 mg, 0.37 mmol), and potassium carbonate (168 mg, 1.21 mmol) were dissolved in 4 mL of DMF and heated and stirred at 95°C for overnight reaction. The reaction was complete as detected by LCMS. The product 3c (144 mg, yield 56%) was obtained by flash purification (PE / EA=3:1).

[0170] LC-MS: m / z=638.2[M+H] +

[0171] 1 H NMR (400MHz, CDCl3) δ7.23(d,J=2.0Hz,1H),7.19(dd,J=8.2,2.0Hz,1H),7.08(d,J=8.3Hz,1H),5.98(s,1H),4.93(s,2H), 4.19–3.95(m,2H),3.87–3.78(m,4H),3.66–3.36(m,8H),2.91(dd,J=5.5,3.4Hz,4H),2.75(d,J=4.8Hz,3H),1.49(s,9H). 13 C NMR (101MHz, CDCl3) δ170.41,163.08,161.02,154.59,152.67,131.37,127.42, 123.47,92.17,80.37,77.24,67.15,53.07,51.79,46.64,43.88,28.39,26.09.

[0172] Step 4: 2-({[4-bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]methyl}{6-chloro-4-[4-(2-chloroacetyl)piperazin-1-yl]pyrimidin-2-yl}amino)-N-methylacetamide (W3)

[0173] The reactant 3c (144 mg, 0.23 mmol) was dissolved in 4 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added dropwise to the reaction solution under stirring. The reaction was allowed to react at room temperature for 4 h. After the reaction was complete as detected by LCMS, the solvent was dried under reduced pressure. 4 mL of dichloromethane was added to the reaction bottle after drying, and chloroacetyl chloride (25 mg, 0.25 mmol) was added dropwise while stirring under ice bath. The reaction was allowed to react at 0°C for 2 h. The reaction was monitored by TLC spot plate. The crude product was purified by Pre-HPLC to obtain the final product W3 (80 mg, yield 58%).

[0174] LC-MS: m / z=614.1[M+H] +

[0175] 1 H NMR (500MHz, CDCl3) δ7.23(d,J=2.0Hz,1H),7.18(d,J=8.1Hz,1H),7.07(s,1H),6.00(s,1H),4.93(s,2 H),4.09(s,2H),4.04(s,2H),3.85–3.80(m,4H),3.71–3.51(m,8H),2.91(t,J=4.5Hz,4H),2.74(s,3H). 13 C NMR (126MHz, CDCl3) δ170.34,165.42,163.03,161.00,160.84,152.64,131.26,127.43,123.5 0,92.23,67.12,60.41,53.06,51.78,46.73,45.57,43.76,41.54,40.72,26.13,21.07,14.21.

[0176] Preparation of Example 4W4

[0177]

[0178] Step 1: 2-({[4-bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]methyl}(2,6-dichloropyrimidin-4-yl)amino)-N-methylacetamide (4a)

[0179] The reactant 3b (120 mg, 0.35 mmol), trichloropyrimidine (64 mg, 0.35 mmol), and DIPEA (136 mg, 1.06 mmol) were dissolved in 4 mL of acetonitrile and stirred at 0°C for 1 h. The reaction was completed after LCMS detection. The product 4a (122 mg, yield 71%) was obtained after flash purification (PE / EA=1:1).

[0180] LC-MS: m / z=488.0[M+H] +

[0181] 1 H NMR (400MHz, CDCl3) δ7.31(d,J=2.0Hz,1H),7.25(dd,J=8.3,1.9Hz,1H),7.07(d,J=7.5Hz,1H),6.46(s,1 H),6.17(s,1H),4.69(s,2H),4.52–4.07(m,2H),3.95–3.78(m,4H),3.01–2.91(m,4H),2.90–2.80(m,3H).

[0182] Step 2: 4-(6-{1-[4-bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]-4-oxyde-2,5-diazepine-2-yl}-4-chloropyrimidin-2-yl)piperazine-1-carboxylic acid-2-methylpropane-2-yl ester (4b)

[0183] The reactant 4a (122 mg, 0.25 mmol), 1-Boc-piperazine (49 mg, 0.26 mmol) and potassium carbonate (104 mg, 0.75 mmol) were dissolved in DMF solution and reacted at 90°C for 8 h. The reaction was completed after LCMS detection. The product 4b (119 mg, yield 75%) was obtained after flash purification (PE / EA=3:1).

[0184] LC-MS: m / z=638.2[M+H] +

[0185] 1 H NMR (400MHz, CDCl3) δ7.30–7.28(m,1H),7.27–7.19(m,1H),7.03(dd,J=15.2,8.2Hz,1H),6.07(s,1H),5.89(s,1H),4.83–4.53 (m,2H),4.24–4.07(m,2H),3.93–3.70(m,8H),3.49–3.42(m,4H),2.95–2.87(m,4H),2.81(d,J=4.9Hz,3H),1.52–1.48(m,9H).13 CNMR(101MHz,CDCl3)δ163.33,160.95,160.50,154.77,152.33,127.83,123.97,12 2.30,91.37,80.12,77.24,67.12,53.18,43.70,29.06,28.42,28.38,26.21,22.63.

[0186] Step 3: 2-({[4-bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]methyl}{6-chloro-2-[4-(2-chloroacetyl)piperazin-1-yl]pyrimidin-4-yl}amino)-N-methylacetamide (W4)

[0187] The reactant 4b (119 mg, 0.19 mmol) was dissolved in 4 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added dropwise to the reaction solution under stirring. The reaction was allowed to react at room temperature for 4 h. After the reaction was complete as detected by LCMS, the solvent was dried under reduced pressure. 4 mL of dichloromethane was added to the reaction bottle after drying, and chloroacetyl chloride (22 mg, 0.20 mmol) was added dropwise while stirring under ice bath. The reaction was allowed to react at 0°C for 2 h. The reaction was completed after monitoring by TLC spot plate. The crude product was purified by Pre-HPLC to obtain the final product W4 (66 mg, yield 57%).

[0188] LC-MS: m / z=614.1[M+H] +

[0189] 1 H NMR(500MHz, CDCl3)δ7.29(d,J=2.4Hz,1H),7.22(dd,J=8.2,2.0Hz,1H),7.08–6.98(m,1H),6.13–5.81(m,2H),4.88–4.51(m ,2H),4.16(s,2H),4.10(d,J=8.1Hz,2H),4.00–3.73(m,8H),3.69–3.53(m,4H),2.92(q,J=5.2Hz,4H),2.81(d,J=4.4Hz,3H). 13 C NMR (126MHz, CDCl3) δ165.39,163.37,160.36,152.31,127.84,123.99,122.35,67.11,53.20,46.01,43.84,43.39,41.95,40.86,26.27.

[0190] Preparation of Example 5W5

[0191]

[0192] Step 1: 4-(2-chloro-5-nitropyrimidin-4-yl)piperazine-1-carboxylic acid-2-methylpropan-2-yl ester (5a)

[0193] 2,4-Dichloro-5-nitropyrimidine (150 mg, 0.78 mmol), 1-Boc-piperazine (153 mg, 0.82 mmol), and DIPEA (301 mg, 2.33 mmol) were dissolved in 4 mL of acetonitrile and stirred at room temperature for 2 h. The reaction was completed after LCMS detection. The product 5a (174 mg, yield 65%) was obtained after flash purification (PE / EA=10:1).

[0194] LC-MS: m / z=344.1[M+H] +

[0195] 1 H NMR (400MHz, DMSO) δ8.89 (s, 1H), 3.56–3.42 (m, 8H), 1.42 (d, J = 4.1Hz, 9H). 13 CNMR(101MHz,DMSO)δ160.13,158.18,155.15,154.22,130.90,79.79,47.31,28.49.

[0196] Step 2: 4-(2-{1-[4-bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]-4-oxyde-2,5-diazepine-2-yl}-5-nitropyrimidin-4-yl)piperazine-1-carboxylic acid-2-methylpropane-2-yl ester (5b)

[0197] The reactant 5a (150 mg, 0.44 mmol), intermediate 3b (164 mg, 0.48 mmol), and potassium carbonate (181 mg, 1.31 mmol) were dissolved in DMF solution and reacted at 90°C overnight. LCMS detected that the reaction was complete. The product 6b (133 mg, yield 47%) was obtained by flash purification (PE / EA=3:1).

[0198] LC-MS: m / z=649.2[M+H] +

[0199] 1H NMR (500MHz, CDCl3) δ8.88–8.83(m,1H),7.24(d,J=2.4Hz,1H),7.22–7.14(m,1H),7.12–6.97(m,1H),5.09–4.91(m,2H),4.2 2–4.05(m,2H),3.82(dd,J=6.3,3.1Hz,4H),3.55–3.38(m,8H),2.96–2.85(m,7H),2.81–2.74(m,3H),1.46(d,J=3.0Hz,9H).

[0200] Step 3: 2-({[4-bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]methyl}{4-[4-(2-chloroacetyl)piperazine-1-yl]-5-nitropyrimidin-2-yl}amino)-N-methylacetamide (W5)

[0201] The reactant 5b (133 mg, 0.20 mmol) was dissolved in 4 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added dropwise to the reaction solution under stirring, and the reaction was allowed to react at room temperature for 4 h. After the reaction was complete as detected by LCMS, the solvent was dried under reduced pressure. 4 mL of dichloromethane was added to the reaction bottle after drying, and chloroacetyl chloride (25 mg, 0.23 mmol) was added dropwise while stirring under ice bath, and the reaction was allowed to react at 0°C for 2 h. The reaction was monitored by TLC spot plate. The crude product was purified by Pre-HPLC to obtain the final product W5 (78 mg, yield 61%).

[0202] LC-MS: m / z=624.1[M+H] +

[0203] 1 H NMR (400MHz, DMSO) δ8.83 (d, J=12.5Hz, 1H), 7.98–7.85 (m, 1H), 7.30 (dd, J=5 .8,2.0Hz,1H),7.23(ddd,J=7.9,5.7,1.9Hz,1H),7.18–7.07(m,1H),5.66(s, 1H),5.02–4.83(m,2H),4.41(d,J=14.6Hz,2H),4.31–4.00(m,2H),3.71(q,J =4.7Hz,4H),3.60–3.37(m,8H),2.86(t,J=4.4Hz,4H),2.62(t,J=4.8Hz,3H). 13C NMR(101MHz,DMSO)δ168.85,165.45,165.38,160.51,160.37,159.02,158.88,15 8.64,156.25,156.20,153.12,152.83,132.08,132.01,130.20,129.87,127.21,1 27.03,124.29,124.18,123.61,123.10,121.16,120.90,66.92,52.86,51.43,51.23,48.31,47.84,47.22,46.88,44.81,44.61,42.44,41.72,41.54,26.00,25.96.

[0204] Preparation of Example 6W6

[0205]

[0206] Step 1: 4-(2,6-dichloro-5-methylpyrimidin-4-yl)piperazine-1-carboxylic acid 2-methylpropan-2-yl ester (6a)

[0207] 2,4,6-Trichloro-5-methylpyrimidine (150 mg, 0.77 mmol), 1-Boc-piperazine (157 mg, 0.84 mmol), and DIPEA (298 mg, 2.31 mmol) were dissolved in 4 mL of acetonitrile and stirred at room temperature for 2 h. The reaction was completed after LCMS detection. The product 6a (182 mg, yield 69%) was obtained after flash purification (PE / EA=10:1).

[0208] LC-MS: m / z=347.1[M+H] +

[0209] 1 H NMR (400MHz, DMSO) δ3.45 (d, J = 1.7Hz, 8H), 2.20 (s, 3H), 1.42 (s, 9H). 13 C NMR (101MHz, DMSO) δ166.87,160.63,155.07,154.35,114.33,79.66,48.03,28.50,16.46.

[0210] Step 2: 4-(2-{1-[4-bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]-4-oxyde-2,5-diazepine-2-yl}-6-chloro-5-methylpyrimidin-4-yl)piperazine-1-carboxylic acid-2-methylpropane-2-yl ester (6b)

[0211] The reactant 6a (151 mg, 0.44 mmol), intermediate 3b (164 mg, 0.48 mmol), and potassium carbonate (181 mg, 1.31 mmol) were dissolved in DMF solution and reacted at 90°C overnight. LCMS detected that the reaction was complete. The product 6b (113 mg, yield 40%) was obtained by flash purification (PE / EA=3:1).

[0212] LC-MS: m / z=652.2[M+H] +

[0213] 1 H NMR (400MHz, CDCl3) δ7.22(d,J=1.9Hz,1H),7.16(dd,J=8.2,2.0Hz,1H),7.04(d,J=8.3Hz,1H),4.90(s,2H),3.88–3.79(m,4H), 3.47(dd,J=6.7,3.6Hz,4H),3.24(t,J=5.1Hz,4H),2.90(dd,J=5.4,3.5Hz,4H),2.74(d,J=4.9Hz,3H),2.14(s,3H),1.47(s,9H). 13 C NMR (101MHz, CDCl3) δ170.52,167.27,161.48,158.57,154.71,152.68,131.47,127.37,123 .49,105.30,80.14,77.28,67.14,53.03,51.62,48.28,46.65,28.40,26.09,15.44,14.20.

[0214] Step 3: 2-({[4-bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]methyl}{4-chloro-6-[4-(2-chloroacetyl)piperazine-1-yl]-5-methylpyrimidin-2-yl}amino)-N-methylacetamide (W6)

[0215] The reactant 6b (113 mg, 0.17 mmol) was dissolved in 4 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added dropwise to the reaction solution under stirring, and the reaction was allowed to react at room temperature for 4 h. After the reaction was complete as detected by LCMS, the solvent was dried under reduced pressure. 4 mL of dichloromethane was added to the reaction bottle after drying, and chloroacetyl chloride (21 mg, 0.19 mmol) was added dropwise while stirring under ice bath, and the reaction was allowed to react at 0°C for 2 h. The reaction was monitored by TLC spot plate. The crude product was purified by Pre-HPLC to obtain the final product W6 (79 mg, yield 73%).

[0216] LC-MS: m / z=628.1[M+H] +

[0217] 1 H NMR (500MHz, DMSO) δ7.81(d,J=5.5Hz,1H),7.28–7.17(m,2H),7.09(d,J=7.9Hz,1H),5.72(s,1H),4.78(s,2H),4.38(d,J=14.2 Hz,2H),4.11–3.92(m,2H),3.70(t,J=4.3Hz,4H),3.58–3.43(m,4H),3.36–3.17(m,4H),2.85(s,4H),2.60(s,3H),2.07(s,3H). 13 C NMR (126MHz, DMSO) δ169.74,166.60,165.28,160.78,159.02,158.71,132.8 7,114.48,103.94,66.91,52.87,47.99,47.19,42.29,41.73,25.95,15.42.

[0218] Preparation of Example 7W7

[0219]

[0220] Step 1: Methyl ({[4-bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]methyl}amino)acetate (7a)

[0221] The reactant 3a (120 mg, 0.45 mmol), glycine methyl ester hydrochloride (112 mg, 0.89 mmol), and DIPEA (173 mg, 1.34 mmol) were dissolved in 4 mL of dichloromethane and stirred at room temperature for 8 h. Sodium triacetoxyborohydride (284 mg, 1.34 mmol) was slowly added to the above solution and the reaction was continued at room temperature for 4 h. After the reaction was completed by LCMS detection, the product 7a (129 mg, yield 84%) was obtained by flash purification (PE / EA=5:1).

[0222] LC-MS: m / z=343.1[M+H] +

[0223] 1 H NMR (500MHz, CDCl3) δ7.19(d,J=8.1Hz,1H),7.14(d,J=6.9Hz,2H),3.81–3.75(m,4H),3.73(s,2H),3.66(s,3H),3.34(s,2H),2.91–2.80(m,4H). 13C NMR (126MHz, CDCl3) δ172.71,152.75,133.34,131.51,127.03,123.38,121.42,67.19,52.98,51.72,50.05,48.66.

[0224] Step 2: Methyl ({[4-bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]methyl}[6-chloro-4-(4-{[(2-methylpropan-2-yl)oxy]carbonyl}piperazin-1-yl)pyrimidin-2-yl]amino)acetate (7b)

[0225] The reactant 7b (129 mg, 0.38 mmol), intermediate S3 (114 mg, 0.34 mmol), and potassium carbonate (156 mg, 1.13 mmol) were dissolved in 4 mL of DMF and heated and stirred at 95° C. for overnight reaction. The reaction was complete as detected by LCMS. The product 7b (136 mg, yield 57%) was obtained by flash purification (PE / EA=3:1).

[0226] LC-MS: m / z=639.2[M+H] +

[0227] 1 H NMR (400MHz, CDCl3) δ7.19(s,1H),7.15(d,J=7.0Hz,2H),5.92(d,J=4.8Hz,1H),4.99–4.83(m,2H),4.25–3. 96(m,2H),3.81–3.77(m,4H),3.69(d,J=16.4Hz,3H),3.53–3.41(m,8H),2.88(t,J=4.5Hz,4H),1.46(s,9H). 13 CNMR(101MHz, CDCl3)δ170.90,163.14,162.91,160.99,160.68,154.60,152.69,152.42,131.75,131.56,130 .71,130.15,127.17,123.10,121.45,91.85,80.23,80.12,77.36,67.12,53.00,51.87,46.31,43.80,28.39.

[0228] Step 3: ({[4-bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]methyl}{6-chloro-4-[4-(2-chloroacetyl)piperazin-1-yl]pyrimidin-2-yl}amino)methyl acetate (W7)

[0229] The reactant 7b (136 mg, 0.21 mmol) was dissolved in 4 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added dropwise to the reaction solution under stirring. The reaction was allowed to react at room temperature for 4 h. After the reaction was complete as detected by LCMS, the solvent was dried under reduced pressure. 4 mL of dichloromethane was added to the reaction bottle after drying, and chloroacetyl chloride (26 mg, 0.23 mmol) was added dropwise while stirring under ice bath. The reaction was allowed to react at 0°C for 2 h. The reaction was monitored by TLC spot plate. The crude product was purified by Pre-HPLC to obtain the final product W7 (77 mg, yield 59%).

[0230] LC-MS: m / z=615.1[M+H] +

[0231] 1 H NMR (500MHz, DMSO) δ7.34–7.25(m,1H),7.25–7.12(m,2H),6.35–6.23(m,1H),5.68(s,1H),4.89–4.75(m,2H),4.42(d,J=16.7H z,2H),4.37–4.09(m,2H),3.72(d,J=6.1Hz,4H),3.65(d,J=12.4Hz,4H),3.53(s,4H),3.50–3.35(m,3H),2.85(p,J=4.1Hz,4H). 13 C NMR (126MHz, DMSO) δ170.96,165.36,162.97,162.89,160.83,160.62,160.03 ,159.69,159.00,158.70,153.12,152.62,132.94,132.72,130.48,130.00,1 27.20,126.81,123.59,122.71,121.03,120.57,92.19,66.94,52.92,52.85,52.19,52.09,50.04,47.62,47.21,45.04,43.70,42.43,41.60,41.46,40.45.

[0232] Preparation of Example 8W8

[0233]

[0234] Step 1: ({[4-bromo-2-(1,4-oxazepine-4-yl)phenyl]methyl}amino)acetic acid-2-methylpropan-2-yl ester (8a)

[0235] The reactant 3a (120 mg, 0.45 mmol), glycine tert-butyl ester (117 mg, 0.89 mmol), and DIPEA (173 mg, 1.34 mmol) were dissolved in 4 mL of dichloromethane and stirred at room temperature for 8 h. Sodium triacetoxyborohydride (284 mg, 1.34 mmol) was slowly added to the above solution and the reaction was continued at room temperature for 4 h. After the reaction was completed by LCMS, the product 8a (133 mg, yield 78%) was obtained by flash purification (PE / EA=5:1).

[0236] LC-MS: m / z=385.1[M+H] +

[0237] 1 H NMR (500MHz, CDCl3) δ7.10(d,J=4.4Hz,2H),6.53(d,J=7.4Hz,1H),3.73(d,J=4.1Hz ,4H),3.21(s,2H),2.79(t,J=4.6Hz,4H),1.91–1.82(m,2H),1.33(d,J=1.7Hz,9H). 13 C NMR (126MHz, CDCl3) δ174.99,170.51,152.76,132.14,131.74,127.29,123.75,121.82,81.77,81.42,67.03,52.92,49.94,48.33,27.95,21.63.

[0238] Step 2: ({[4-bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]methyl}[6-chloro-4-(4-{[(2-methylpropan-2-yl)oxy]carbonyl}piperazin-1-yl)pyrimidin-2-yl]amino)acetic acid-2-methylpropan-2-yl ester (8b)

[0239] The reactant 8a (133 mg, 0.35 mmol), intermediate S3 (96 mg, 0.29 mmol), and potassium carbonate (143 mg, 1.04 mmol) were dissolved in 4 mL of DMF and heated with stirring at 95° C. to react overnight. LCMS detected that the reaction was complete. The product 8b (168 mg, yield 71%) was obtained by flash purification (PE / EA=3:1).

[0240] LC-MS: m / z=681.2[M+H] +

[0241] 1H NMR (400MHz, CDCl3) δ7.16(d,J=1.7Hz,1H),7.13(d,J=5.5Hz,2H),5.88(d,J=8.7Hz,1H),4.97–4.77(m,2H),3.77(dd,J=5.8,3.1Hz, 4H),3.55–3.48(m,4H),3.47–3.42(m,4H),3.41–3.34(m,2H),2.85(dt,J=8.0,4.5Hz,4H),1.44(d,J=2.5Hz,9H),1.43–1.37(m,9H). 13 C NMR (101MHz, CDCl3) δ169.49,163.04,162.93,161.18,160.56,154.56,154.39,152.66,152.33,132.00,131.85,130.79, 130.21,127.10,123.07,122.93,121.30,121.17,99.92,91.60,81.08,80.13,67.13,52.97,49.80,43.75,28.35,28.09.

[0242] Step 3: N-{[4-bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]methyl}-N-{6-chloro-4-[4-(2-chloroacetyl)piperazin-1-yl]pyrimidin-2-yl}glycine (W8)

[0243] The reactant 8b (168 mg, 0.25 mmol) was dissolved in 4 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added dropwise to the reaction solution under stirring. The reaction was allowed to react at room temperature for 4 h. After the reaction was complete as detected by LCMS, the solvent was dried under reduced pressure. 4 mL of dichloromethane was added to the reaction bottle after drying, and chloroacetyl chloride (30 mg, 0.27 mmol) was added dropwise while stirring under ice bath. The reaction was allowed to react at 0°C for 2 h. The reaction was monitored by TLC spot plate. The crude product was purified by Pre-HPLC to obtain the final product W8 (64 mg, yield 43%).

[0244] LC-MS: m / z=601.0 [M+H] +

[0245] 1H NMR (500MHz, DMSO) δ7.30–7.25(m,1H),7.24–7.19(m,1H),7.15(t,J=9.0Hz,1H),6.30–6.21(m,1H),4.86–4.73(m,2H),4.42(d,J =13.2Hz,2H),4.27–4.02(m,2H),3.72(dt,J=8.7,4.0Hz,4H),3.68–3.57(m,2H),3.55–3.39(m,6H),2.86(dq,J=8.8,4.9Hz,4H). 13 C NMR (126MHz, DMSO) δ171.83,171.77,165.37,165.33,162.99,160.99,160.77,1 59.98,159.72,159.00,158.69,153.20,152.69,133.10,132.86,130.60,130.1 3,127.23,126.83,123.58,122.72,121.01,120.55,92.05,91.92,66.92,52.96,52.88,49.89,49.65,47.46,46.82,45.07,43.79,42.45,42.43,41.63,41.49.

[0246] Preparation of Example 9W9

[0247]

[0248] Step 1: 4-{5-bromo-2-[(ethylamino)methyl]phenyl}-1,4-oxazacyclohexane (9a)

[0249] The reactant 3a (120 mg, 0.45 mmol), glycine methyl ester hydrochloride (40 mg, 0.89 mmol), and DIPEA (173 mg, 1.34 mmol) were dissolved in 4 mL of dichloromethane and stirred at room temperature for 8 h. Sodium triacetoxyborohydride (284 mg, 1.34 mmol) was slowly added to the above solution and the reaction was continued at room temperature for 4 h. After the reaction was completed by LCMS detection, the product 9a (111 mg, yield 84%) was obtained by flash purification (PE / EA=1:1).

[0250] LC-MS: m / z=299.1[M+H] +

[0251] 1H NMR (500MHz, CDCl3) δ10.25(s,1H),7.30(d,J=8.2Hz,1H),7.15(d,J=2.0Hz,1H),7.12(dd,J=8.2,1. 9Hz,1H),3.98(s,2H),3.69(dd,J=5.8,3.3Hz,4H),2.77–2.69(m,6H),1.22(dd,J=10.3,7.0Hz,3H). 13 C NMR (126MHz, CDCl3) δ176.53,152.94,132.10,128.12,127.13,124.42,123.30,66.92,53.15,44.88,41.41,22.86.

[0252] Step 2: 4-[2-({[4-bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]methyl}(ethyl)amino)-6-chloropyrimidin-4-yl]piperazine-1-carboxylic acid-2-methylpropan-2-yl ester (9b)

[0253] The reactant 9a (111 mg, 0.37 mmol), intermediate S3 (112 mg, 0.34 mmol), and potassium carbonate (154 mg, 1.12 mmol) were dissolved in 4 mL of DMF and heated with stirring at 95° C. to react overnight. LCMS detected that the reaction was complete. The product 9b (101 mg, yield 46%) was obtained by flash purification (PE / EA=1:1).

[0254] LC-MS: m / z = 595.2 [M+H] +

[0255] 1 H NMR (400MHz, CDCl3) δ7.16(d,J=2.0Hz,1H),7.13(s,1H),7.01(d,J=8.3Hz,1H),5.83(s,1H),4.77(s,1H), 3.86–3.77(m,4H),3.64–3.38(m,12H),2.87(t,J=4.4Hz,4H),1.45(d,J=5.2Hz,9H),1.10(t,J=7.0Hz,3H). 13C NMR (101MHz, CDCl3) δ163.20,163.05,160.86,160.56,160.53,159.81,154.59,154.41,132. 58,126.97,122.80,120.82,99.93,90.68,80.50,80.12,67.19,52.90,43.78,28.38,28.35.

[0256] Step 3: 1-{4-[2-({[4-bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]methyl}(ethyl)amino)-6-chloropyrimidin-4-yl]piperazine-1-yl}-2-chloroethan-1-one (W9)

[0257] The reactant 9b (101 mg, 0.17 mmol) was dissolved in 4 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added dropwise to the reaction solution under stirring. The reaction was allowed to react at room temperature for 4 h. After the reaction was complete as detected by LCMS, the solvent was dried under reduced pressure. 4 mL of dichloromethane was added to the reaction bottle after drying, and chloroacetyl chloride (21 mg, 0.19 mmol) was added dropwise while stirring under ice bath. The reaction was allowed to react at 0°C for 2 h. The reaction was monitored by TLC spot plate. The crude product was purified by Pre-HPLC to obtain the final product W9 (43 mg, yield 44%).

[0258] LC-MS: m / z=571.1[M+H] +

[0259] 1 H NMR(500MHz,DMSO)δ7.31–7.16(m,2H),7.00(d,J=8.3Hz,1H),6.17(s,1H),4.72(s, 2H),4.40(s,2H),3.74(s,4H),3.70–3.15(m,10H),3.03–2.73(m,4H),1.09(s,3H). 13 C NMR (126MHz, DMSO) δ165.31,163.08,160.49,160.02,159.02,158.71,152.68,133.31,129.38,126.73, 122.56,116.76,114.47,91.17,66.96,52.85,45.60,45.03,43.56,42.40,41.54,40.36,13.27,12.66.

[0260] Example 10 Preparation of W10

[0261]

[0262] Step 1: 4-{5-bromo-2-[(methylamino)methyl]phenyl}-1,4-oxazacyclohexane (10b)

[0263] The reactant 3a (120 mg, 0.45 mmol), methylamine hydrochloride (60 mg, 0.89 mmol), and DIPEA (173 mg, 1.34 mmol) were dissolved in 4 mL of dichloromethane and stirred at room temperature for 8 h. Sodium triacetoxyborohydride (284 mg, 1.34 mmol) was slowly added to the above solution and the reaction was continued at room temperature for 4 h. After the reaction was completed by LCMS, the product 10a (105 mg, yield 83%) was obtained by flash purification (PE / EA=1:1).

[0264] LC-MS: m / z=285.1[M+H] +

[0265] 1 H NMR (500MHz, CDCl3) δ7.77(s,1H),7.31(d,J=8.1Hz,1H),7.20–7.16(m,2H),3.98(s,2H),3.76–3.70(m,4H),2.79–2.74(m,4H),2.41(s,3H). 13 C NMR (126MHz, CDCl3) δ177.01,153.04,132.10,128.19,127.20,124.53,123.43,66.96,53.15,52.96,47.37.

[0266] Step 2: 4-[2-({[4-bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]methyl}(methyl)amino)-6-chloropyrimidin-4-yl]piperazine-1-carboxylic acid-2-methylpropan-2-yl ester (10b)

[0267] The reactant 10a (105 mg, 0.37 mmol), intermediate S3 (112 mg, 0.34 mmol), and potassium carbonate (153 mg, 1.11 mmol) were dissolved in 4 mL of DMF and heated and stirred at 95° C. for overnight reaction. The reaction was completed as detected by LCMS. The product 10b (76 mg, yield 35%) was obtained after flash purification (PE / EA=1:1).

[0268] LC-MS: m / z=581.2[M+H] +

[0269] 1H NMR (400MHz, CDCl3) δ7.16(d,J=1.9Hz,1H),7.10(dd,J=8.2,1.9Hz,1H),6.95(d,J=8.2Hz,1H),5.84(s,1H),4 .79(s,1H),3.84–3.75(m,4H),3.56–3.38(m,11H),2.98(s,2H),2.85(t,J=4.5Hz,4H),1.44(d,J=5.2Hz,9H). 13 C NMR (101MHz, CDCl3) δ163.13,163.03,161.40,160.52,160.46,159.78,154.60,154.43,152.35,131.89,129.31 ,126.97,122.89,120.91,99.93,90.70,80.52,80.16,79.91,77.43,67.12,52.81,50.47,43.76,28.37,28.34.

[0270] Step 3: 1-{4-[2-({[4-bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]methyl}(methyl)amino)-6-chloropyrimidin-4-yl]piperazine-1-yl}-2-chloroethan-1-one (W10)

[0271] The reactant 10b (76 mg, 0.13 mmol) was dissolved in 4 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added dropwise to the reaction solution under stirring, and the reaction was allowed to react at room temperature for 4 h. After the reaction was complete as detected by LCMS, the solvent was dried under reduced pressure. 4 mL of dichloromethane was added to the reaction bottle after drying, and chloroacetyl chloride (16 mg, 0.14 mmol) was added dropwise while stirring under ice bath, and the reaction was allowed to react at 0°C for 2 h. The reaction was monitored by TLC spot plate. The crude product was purified by Pre-HPLC to obtain the final product W10 (35 mg, yield 48%).

[0272] LC-MS: m / z=557.1[M+H] +

[0273] 1 H NMR(500MHz,DMSO)δ7.25(s,1H),7.19(d,J=8.3Hz,1H),6.94(d,J=8.4Hz,1H),6.60(s,1H),6.19(s,1H), 4.77(s,2H),4.41(s,2H),3.73(t,J=4.5Hz,4H),3.66–3.37(m,8H),3.12–2.96(m,3H),2.87–2.83(m,4H). 13C NMR (126MHz, DMSO) δ165.31,163.14,159.95,159.32,159.01,158.71,158.40,152.90,129.31,1 26.85,120.60,116.72,114.43,91.17,66.89,52.78,48.17,45.06,43.87,43.56,42.39,41.58.

[0274] Example 11 Preparation of W11

[0275]

[0276] Step 1: [4-Bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]methanol (11a)

[0277] The reactant 3a (300 mg, 1.12 mmol) was dissolved in 4 mL of methanol, and sodium borohydride (127 mg, 3.35 mmol) was added in batches at 0° C. After the reaction solution returned to room temperature, the reaction was continued for 4 h. The reaction was complete when LCMS detected that the reaction was complete, and the product 11a (299 mg, yield 99%) was obtained by flash purification (PE / EA=3:1).

[0278] LC-MS: m / z=272.0 [M+H] +

[0279] 1 H NMR(500MHz,DMSO)δ7.39(d,J=8.2Hz,1H),7.26(dd,J=8.2,2.0Hz,1H),7.15(d,J=2.0Hz ,1H),5.18(t,J=5.6Hz,1H),4.49(d,J=5.6Hz,2H),3.75–3.68(m,4H),2.87–2.80(m,4H). 13 C NMR (126MHz, DMSO) δ152.01,136.20,130.66,126.18,121.98,120.56,66.92,58.64,52.73.

[0280] Step 2: 4-[2-(Azidomethyl)-5-bromophenyl]-1,4-oxazacyclohexane (11b)

[0281] The reactant 11a (299 mg, 1.10 mmol) and diphenylphosphoryl azide (364 mg, 1.32 mmol) were dissolved in 5 mL of tetrahydrofuran, and DBU (185 mg, 1.21 mmol) was added dropwise to the above system under nitrogen protection, and the reaction was allowed to proceed overnight at room temperature. The reaction was complete by LCMS detection, and the product 11b (302 mg, yield 92%) was obtained by flash purification (pure PE).

[0282] LC-MS: m / z=297.1[M+H] +

[0283] 1 H NMR (400MHz, CDCl3) δ7.27 (dd, J=6.1, 2.1Hz, 2H), 7.22 (d, J=8.7Hz, 1H), 4.41 (s, 2H), 3.85–3.81 (m, 4H), 2.91–2.87 (m, 4H). 13 C NMR (101MHz, CDCl3) δ152.89,131.59,130.33,129.89,129.47,127.57,12 4.63,124.27,122.93,120.83,67.31,67.11,53.42,53.22,50.56,50.09.

[0284] Step 3: [4-Bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]methanamine (11c)

[0285] The reactant 11b (302 mg, 1.02 mmol) and triphenylphosphine (401 mg, 1.53 mmol) were dissolved in 6 mL of THF / H2O (v / v=9:1), and heated under reflux for 3 h under nitrogen protection. The reaction was complete when detected by LCMS. 10 mL of 10% dilute hydrochloric acid was added to the above system, stirred for 30 min, extracted three times with petroleum ether, and the aqueous phase was retained. After adjusting the pH of the aqueous phase to alkaline with saturated sodium hydroxide aqueous solution, the aqueous phase was extracted with ethyl acetate, the organic phases were combined and flash purified (DCM / MeOH=10:1) to obtain the product 11c (258 mg, yield 94%).

[0286] LC-MS: m / z=271.1[M+H] +

[0287] 1 H NMR (400MHz, MeOD) δ7.36–7.25(m,3H),3.91(s,2H),3.88–3.81(m,4H),2.94–2.87(m,4H). 13C NMR (101MHz, MeOD) δ152.51,135.04,130.33,127.29,123.55,121.23,66.96,52.82,40.57.

[0288] Step 4: 4-[2-({[4-bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]methyl}amino)-6-chloropyrimidin-4-yl]piperazine-1-carboxylic acid-2-methylpropan-2-yl ester (11d)

[0289] The reactant 11c (220 mg, 0.81 mmol), intermediate S3 (271 mg, 0.81 mmol), and potassium carbonate (335 mg, 2.43 mmol) were dissolved in 4 mL of DMF and heated and stirred at 95°C for overnight reaction. The reaction was complete as detected by LCMS. The product 11d (217 mg, yield 47%) was obtained after flash purification (PE / EA=1:1).

[0290] LC-MS: m / z = 566.9 [M+H] +

[0291] 1 H NMR (400MHz, CDCl3) δ7.22–7.07(m,3H),5.82(d,J=4.0Hz,1H),4.56(dd,J=6.0,2.5Hz,2H),3.80(dd ,J=6.0,3.2Hz,4H),3.51–3.44(m,4H),3.42–3.37(m,4H),2.86(q,J=3.8Hz,4H),1.46–1.43(m,9H). 13 C NMR (101MHz, CDCl3) δ163.20,161.51,154.55,152.03,133.17,130.15,126. 94,122.90,121.10,80.18,67.16,60.32,52.83,43.70,28.36,22.61,14.19.

[0292] Step 5: 1-{4-[2-({[4-bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]methyl}amino)-6-chloropyrimidin-4-yl]piperazine-1-yl}-2-chloroethan-1-one (W11)

[0293] The reactant 11d (150 mg, 0.27 mmol) was dissolved in 4 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added dropwise to the reaction solution under stirring. The reaction was allowed to react at room temperature for 4 h. After the reaction was complete as detected by LCMS, the solvent was dried under reduced pressure. 4 mL of dichloromethane was added to the reaction bottle after drying, and chloroacetyl chloride (33 mg, 0.29 mmol) was added dropwise while stirring under ice bath. The reaction was allowed to react at 0°C for 2 h. The reaction was monitored by TLC spot plate. The crude product was purified by Pre-HPLC to obtain the final product W11 (98 mg, yield 68%).

[0294] LC-MS: m / z=543.1[M+H] +

[0295] 1 H NMR(500MHz,DMSO)δ7.22(dd,J=13.1,8.1Hz,3H),6.16(s,1H),6.05(s,1H),4.46(s ,2H),4.41(s,2H),3.75(t,J=4.3Hz,4H),3.63–3.38(m,8H),2.86(t,J=4.4Hz,4H). 13 C NMR (126MHz, DMSO) δ165.34,163.13,159.42,159.12,158.82,158.51,152.47,134.23,130.05,1 26.52,122.20,120.36,116.81,114.51,91.39,66.94,52.78,45.01,43.83,43.51,42.39,41.53.

[0296] Example 12 Preparation of W12

[0297]

[0298] Step 1: 4-(2-{[(4-bromo-2-chlorophenyl)methyl]amino}-6-chloropyrimidin-4-yl)piperazine-1-carboxylic acid-2-methylpropan-2-yl ester (12a)

[0299] The reactant 4-bromo-2-chlorobenzylamine (120 mg, 0.55 mmol), intermediate S3 (166 mg, 0.50 mmol), and potassium carbonate (227 mg, 1.64 mmol) were dissolved in 4 mL of DMF and heated with stirring at 95 °C to react overnight. The reaction was complete when detected by LCMS. The product 12a (100 mg, yield 35%) was obtained after flash purification (PE / EA=3:1).

[0300] LC-MS: m / z=516.1[M+H] +

[0301] 1 H NMR(400MHz, CDCl3) δ7.49(d,J=1.8Hz,1H),7.32–7.24(m,2H),5.86(d,J=1.6Hz,1H),4.6 0(d,J=6.3Hz,2H),3.53–3.48(m,4H),3.42(dd,J=6.7,3.5Hz,4H),1.47(d,J=2.1Hz,9H). 13 C NMR (101MHz, CDCl3) δ163.18,161.33,160.58,160.17,154.62,136.30,133.88,131.75,129.92,120.72,80.25,79.95,43.82,42.49,28.40.

[0302] Step 2: 1-[4-(2-{[(4-bromo-2-chlorophenyl)methyl]amino}-6-chloropyrimidin-4-yl)piperazin-1-yl]-2-chloroethan-1-one (W12)

[0303] The reactant 12a (100 mg, 0.19 mmol) was dissolved in 4 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added dropwise to the reaction solution under stirring. The reaction was allowed to react at room temperature for 4 h. After the reaction was complete as detected by LCMS, the solvent was dried under reduced pressure. 4 mL of dichloromethane was added to the reaction bottle after drying, and chloroacetyl chloride (24 mg, 0.21 mmol) was added dropwise while stirring under ice bath. The reaction was allowed to react at 0°C for 2 h. The reaction was monitored by TLC spot plate. The crude product was purified by Pre-HPLC to obtain the final product W12 (52 mg, yield 55%).

[0304] LC-MS: m / z=492.0 [M+H] +

[0305] 1 H NMR(500MHz,DMSO)δ7.79(s,1H),7.69(d,J=2.0Hz,1H),7.52(dd,J=8.2,2.0Hz,1H),7 .29(d,J=8.6Hz,1H),6.16(s,1H),4.52–4.36(m,4H),3.68(s,2H),3.58–3.43(m,6H). 13 C NMR (126MHz, DMSO) δ165.33,163.31,159.97,137.42,133.45,131.56,130.86,130.61,120.35,45.04,43.90,43.60,42.46,42.05,41.59.

[0306] Example 13 Preparation of W13

[0307]

[0308] Step 1: 4-(6-chloro-2-{[(2,4-dichlorophenyl)methyl]amino}pyrimidin-4-yl)piperazine-1-carboxylic acid-2-methylpropan-2-yl ester (13a)

[0309] The reactant 2,4-dichloro-benzylamine (120 mg, 0.69 mmol), intermediate S3 (208 mg, 0.62 mmol), and potassium carbonate (284 mg, 2.06 mmol) were dissolved in 4 mL of DMF and heated and stirred at 95 °C for overnight reaction. LCMS detected that the reaction was complete. The product 13a (193 mg, yield 60%) was obtained by flash purification (PE / EA=3:1).

[0310] LC-MS: m / z=472.1[M+H] +

[0311] 1 H NMR(500MHz, CDCl3)δ7.35(d,J=2.1Hz,1H),7.34–7.29(m,1H),7.16(dd,J=8.3,2.2Hz,1H),5.8 7(d,J=2.3Hz,1H),4.62(d,J=6.3Hz,2H),3.52(t,J=5.1Hz,4H),3.45–3.41(m,4H),1.47(s,9H). 13 C NMR (126MHz, CDCl3) δ163.19,161.28,160.73,160.58,154.82,154.62,154.46,135.67,133 .16,129.05,127.02,99.90,91.20,80.26,80.24,43.83,43.68,42.47,28.42,28.39,28.36.

[0312] Step 2: 2-Chloro-1-[4-(6-chloro-2-{[(2,4-dichlorophenyl)methyl]amino}pyrimidin-4-yl)piperazin-1-yl]ethan-1-one (W13)

[0313] The reactant 13a (193 mg, 0.41 mmol) was dissolved in 4 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added dropwise to the reaction solution under stirring. The reaction was allowed to react at room temperature for 4 h. After the reaction was complete as detected by LCMS, the solvent was dried under reduced pressure. 4 mL of dichloromethane was added to the reaction bottle after drying, and chloroacetyl chloride (50 mg, 0.45 mmol) was added dropwise while stirring under ice bath. The reaction was allowed to react at 0°C for 2 h. The reaction was monitored by TLC spot plate. The crude product was purified by Pre-HPLC to obtain the final product W13 (95 mg, yield 52%).

[0314] LC-MS: m / z=448.0 [M+H] +

[0315] 1 H NMR(400MHz,DMSO)δ7.79(s,1H),7.59–7.56(m,1H),7.46–7.29(m,2H),6.18 (s,1H),4.88(s,2H),4.47(d,J=4.2Hz,2H),4.42(s,2H),3.60–3.44(m,6H). 13 C NMR (101MHz, DMSO) δ165.34,163.28,159.77,159.00,158.63,136.95,133.23,132.32,128.89,127.70,45.04,43.91,43.62,42.43,41.99,41.58.

[0316] Example 14 Preparation of W14

[0317]

[0318] Step 1: 4-(6-chloro-2-{[(2-chloro-4-fluorophenyl)methyl]amino}pyrimidin-4-yl)piperazine-1-carboxylic acid-2-methylpropan-2-yl ester (14a)

[0319] The reactant 4-fluoro-2-chlorobenzylamine (100 mg, 0.63 mmol), intermediate S3 (190 mg, 0.57 mmol), and potassium carbonate (260 mg, 1.89 mmol) were dissolved in 4 mL of DMF and heated with stirring at 95 °C for overnight reaction. The reaction was complete when detected by LCMS. The product 14a (137 mg, yield 48%) was obtained after flash purification (PE / EA=3:1).

[0320] LC-MS: m / z=456.1[M+H] +

[0321] 1H NMR (400MHz, CDCl3) δ7.37(td,J=7.2,4.1Hz,1H),7.09(dd,J=8.5,2.6Hz,1H),6.91(td,J=8.3,2.6Hz,1H),5.86(d,J=2.9Hz, 1H), 4.61 (d, J = 6.3Hz, 2H), 3.76–3.69 (m, 1H), 3.52 (dd, J = 6.6, 3.7Hz, 4H), 3.44 (dd, J = 6.7, 3.8Hz, 4H), 1.47 (d, J = 1.9Hz, 9H). 13 C NMR (101MHz, CDCl3) δ163.23,161.30,160.72,160.28,154.63,132.85,116.77,116. 52,113.98,113.78,99.91,91.20,80.27,43.82,43.67,42.42,28.41,28.38,28.35.

[0322] Step 2: 2-Chloro-1-[4-(6-chloro-2-{[(2-chloro-4-fluorophenyl)methyl]amino}pyrimidin-4-yl)piperazin-1-yl]ethan-1-one (W14)

[0323] The reactant 14a (137 mg, 0.30 mmol) was dissolved in 4 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added dropwise to the reaction solution under stirring, and the reaction was allowed to react at room temperature for 4 h. After the reaction was complete as detected by LCMS, the solvent was dried under reduced pressure. 4 mL of dichloromethane was added to the reaction bottle after drying, and chloroacetyl chloride (37 mg, 0.33 mmol) was added dropwise while stirring under ice bath, and the reaction was allowed to react at 0°C for 2 h. The reaction was monitored by TLC spot plate. The crude product was purified by Pre-HPLC to obtain the final product W14 (74 mg, yield 57%).

[0324] LC-MS: m / z=432.1[M+H] +

[0325] 1 H NMR (500MHz, DMSO) δ7.85–7.52(m,1H),7.39(dt,J=11.8,5.9Hz,2H),7.18(td,J=8 .5,2.6Hz,1H),6.15(s,1H),4.46(d,J=6.1Hz,2H),4.42(s,2H),3.62–3.43(m,8H). 13C NMR (126MHz, DMSO) δ165.34,163.33,162.19,161.50,160.23,159.99,134.08,132.98,132.9 0,130.61,116.78,116.58,114.71,114.54,91.37,45.06,43.91,43.60,42.44,41.86,41.60.

[0326] Example 15 Preparation of W15

[0327]

[0328] Step 1: [4-Bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]methanol (15a)

[0329] The reactant 4-trifluoromethyl-2-chloro-benzaldehyde (300 mg, 1.44 mmol) was dissolved in 4 mL of methanol, and sodium borohydride (164 mg, 4.33 mmol) was added in batches at 0° C. After the reaction solution returned to room temperature, the reaction was continued for 4 h. LCMS detected that the reaction was complete, and the product 15a (291 mg, yield 96%) was obtained by flash purification (PE / EA=3:1).

[0330] LC-MS: m / z = 211.0 [M+H] +

[0331] 1 H NMR (400MHz, DMSO) δ7.78(d,J=8.0Hz,1H),7.75–7.66(m,2H),5.65(t,J=5.6Hz,1H),4.63(d,J=5.4Hz,2H). 13 C NMR(101MHz,DMSO)δ145.01,131.86,129.45,129.13,128.89,125.86,125.8 2,125.78,125.74,125.28,124.33,124.29,124.25,124.22,122.58,60.48.

[0332] Step 2: 1-(Azidomethyl)-2-chloro-4-(trifluoromethyl)benzene (15b)

[0333] The reactant 15a (280 mg, 1.33 mmol) and diphenylphosphoryl azide (440 mg, 1.60 mmol) were dissolved in 5 mL of tetrahydrofuran, and DBU (223 mg, 1.47 mmol) was added dropwise to the above system under nitrogen protection, and the reaction was allowed to proceed overnight at room temperature. The reaction was complete by LCMS detection, and the product 15b (273 mg, yield 87%) was obtained by flash purification (pure PE).

[0334] LC-MS: No ion response from ESI ionization

[0335] 1 H NMR (500MHz, CDCl3) δ7.71 (s, 1H), 7.59 (d, J = 1.1Hz, 2H), 4.59 (s, 2H). 13 C NMR (126MHz, CDCl3) δ137.46,133.99,131.98,131.71,129.91,126.76,126.72,124.08,124.04,51.84.

[0336] Step 3: 2-Chloro-4-(trifluoromethyl)-benzylamine (15c)

[0337] The reactant 15b (273 mg, 1.16 mmol) and triethylamine (50 μL) were dissolved in 5 mL of methanol, and Raney-Ni (300 μL) and hydrazine hydrate (300 μL) were added to the above reaction solution at 0°C. After the addition was completed, the mixture was returned to room temperature and stirred for 6 hours. The reaction was complete when detected by LCMS, and the product 15c (165 mg, yield 68%) was obtained by flash purification (PE / EA=1:1).

[0338] LC-MS: m / z = 210.1 [M+H] +

[0339] 1 H NMR (400MHz, CDCl3) δ7.67–7.62(m,1H),7.61–7.50(m,2H),4.01(s,2H). 13 CNMR (101MHz, CDCl3) δ144.43,133.74,130.71,130.39,129.07,126.50,126.46,123.95,123.91,44.10.

[0340] Step 4: 4-[6-chloro-2-({[2-chloro-4-(trifluoromethyl)phenyl]methyl}amino)pyrimidin-4-yl]piperazine-1-carboxylic acid-2-methylpropan-2-yl ester (15d)

[0341] The reactant 15c (165 mg, 0.79 mmol), intermediate S3 (238 mg, 0.71 mmol), and potassium carbonate (325 mg, 2.36 mmol) were dissolved in 4 mL of DMF and heated with stirring at 95° C. to react overnight. LCMS detected that the reaction was complete. The product 15d (125 mg, yield 32%) was obtained by flash purification (PE / EA=3:1).

[0342] LC-MS: m / z=506.2[M+H] +

[0343] 1 H NMR (400MHz, CDCl3) δ7.63–7.58(m,1H),7.53(d,J=8.1Hz,1H),7.48–7.40(m,1H ),5.86(s,1H),4.72(d,J=6.2Hz,2H),3.51–3.39(m,8H),1.46(d,J=5.2Hz,9H). 13 C NMR (101MHz, CDCl3) δ163.19,163.16,161.38,160.73,160.58,154.59,141.51,133.31,126.21,126.17, 124.72,123.55,123.51,123.47,122.02,80.23,77.30,60.34,43.81,43.67,42.69,28.38,28.35,28.33.

[0344] Step 5: 2-Chloro-1-{4-[6-chloro-2-({[2-chloro-4-(trifluoromethyl)phenyl]methyl}amino)pyrimidin-4-yl]piperazin-1-yl}ethan-1-one (W15)

[0345] The reactant 15d (125 mg, 0.25 mmol) was dissolved in 4 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added dropwise to the reaction solution under stirring. The reaction was allowed to react at room temperature for 4 h. After the reaction was complete as detected by LCMS, the solvent was dried under reduced pressure. 4 mL of dichloromethane was added to the reaction bottle after drying, and chloroacetyl chloride (30 mg, 0.27 mmol) was added dropwise while stirring under ice bath. The reaction was allowed to react at 0°C for 2 h. The reaction was monitored by TLC spot plate. The crude product was purified by Pre-HPLC to obtain the final product W15 (68 mg, yield 57%).

[0346] LC-MS: m / z=482.0 [M+H] +

[0347] 1H NMR (500MHz, DMSO) δ7.81 (d, J=1.8Hz, 1H), 7.68 (dd, J=8.2, 1.8Hz, 1H), 7.59–7.48 (m, 1H), 6. 30–6.11(m,1H),4.69(s,1H),4.56(d,J=5.6Hz,2H),4.41(d,J=9.2Hz,2H),3.71–3.37(m,8H). 13 C NMR (126MHz, DMSO) δ165.33,163.27,161.39,159.94,142.81,133.03,129.85,129.46,129.20,12 6.25,126.22,124.98,124.42,124.39,122.81,120.65,91.58,45.01,43.91,43.58,42.40,41.54.

[0348] Example 16 Preparation of W16

[0349]

[0350] Step 1: 2-({[4-bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]methyl}amino)-4-(methylthio)pyrimidine (16a)

[0351] The reactants [4-bromo-2-(1,4-oxazacyclohexan-4-yl)phenyl]methanamine (500 mg, 1.85 mmol), 2-chloro-4-(methylthio)pyrimidine (294 mg, 1.85 mmol), and potassium carbonate (767 mg, 5.56 mmol) were dissolved in 6 mL of DMF and heated with stirring at 120° C. to react overnight. LCMS detected that the reaction was complete. The reaction solution was added with water and extracted with EA three times, and then flash purified (PE / EA=3:1) to obtain the product 16a (555 mg, yield 76%).

[0352] LC-MS: m / z=394.9[M+H] +

[0353] 1 H NMR (400MHz, CDCl3) δ7.65 (s, 1H), 7.18–7.10 (m, 2H), 7.07 (dd, J = 8.2, 2.0Hz, 1H), 6.31 (d ,J=5.4Hz,1H),4.57(d,J=5.8Hz,2H),3.82–3.61(m,4H),2.88–2.72(m,4H),2.31(s,3H). 13C NMR (101MHz, CDCl3) δ170.73,161.47,155.24,152.18,133.03,130.26,126.95,123.10,121.23,107.75,67.08,60.25,52.76,40.87.

[0354] Step 2: 2-({[4-bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]methyl}amino)-4-(methyldioxy-λ 6 -thio)pyrimidine (16b)

[0355] The reactant 16a (555 mg, 1.41 mmol) and m-chloroperbenzoic acid (608 mg, 3.52 mmol) were dissolved in 6 mL of DMF and stirred at room temperature overnight. The reaction was complete when LCMS detected that the reaction was complete. After adding water to the reaction solution, a solid was precipitated. The crude product was flash purified (PE / EA=1:1) to obtain the product 16b (497 mg, yield 83%).

[0356] LC-MS: m / z=426.8[M+H] +

[0357] 1 H NMR(500MHz, CDCl3)δ8.31(s,1H),7.15–7.10(m,2H),7.07(dt,J=8.3,1.7Hz,1H),7.01(d,J=4.8 Hz,1H),4.58(s,2H),3.72(dd,J=5.4,3.5Hz,4H),3.11–2.89(m,3H),2.80(dd,J=5.6,3.6Hz,4H). 13 C NMR (126MHz, CDCl3) δ166.06,162.06,161.31,152.35,132.12,130.16,127.14,123.56,121.56,105.00,67.04,52.79,41.16.

[0358] Step 3: 2-({[4-bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]methyl}(nitroso)amino)-4-(methyldioxy-λ 6 -thio)pyrimidine (16c)

[0359] The reactant 16b (250 mg, 0.59 mmol) was dissolved in 5 mL of acetic acid, and sodium nitrite (121 mg, 1.76 mmol) was added in batches at room temperature. The reaction was stirred for 4 h. The reaction was completed by LCMS. After adding water to the reaction solution, a solid was precipitated. The crude product was washed three times with water to obtain the product 16c (202 mg, yield 76%) without further purification.

[0360] LC-MS: No target TIC response under ESI ionization

[0361] 1 H NMR (500MHz, CDCl3) δ9.11(d,J=4.9Hz,1H),7.91(d,J=4.8Hz,1H),7.27(d,J=1.9Hz,1H),7.09(dd,J=8. 3,2.0Hz,1H),6.56(d,J=8.2Hz,1H),5.42(s,2H),3.88–3.82(m,4H),3.15(s,3H),2.95(t,J=4.6Hz,4H). 13 CNMR (126MHz, CDCl3) δ167.02,162.41,159.34,152.19,128.37,127.98,127.55,124.30,121.91,113.82,67.17,52.89,40.71,38.95.

[0362] Step 4: 2-({[4-bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]methyl}amino)-4-(piperazin-1-yl)pyrimidine (16d)

[0363] The reactant 16c (202 mg, 0.44 mmol), 1-Boc-piperazine (124 mg, 0.67 mmol), potassium carbonate (184 mg, 1.33 mmol) were dissolved in 5 mL DMF and stirred at 90 ° C for 4 h. The reaction was complete when LCMS detected. After the reaction solution was cooled to room temperature, 10 mL 10% H2SO4 was added, and the temperature was continued to rise to 120 ° C and stirred for 1 h. After the reaction was completed, it was cooled to room temperature, and the pH of the reaction solution was adjusted to alkaline with saturated NaOH, and extracted three times with ethyl acetate. The organic phase was flash purified (DCM / MeOH=10:1) to obtain the product 16d (97 mg, yield 51%)

[0364] LC-MS: m / z=433.2[M+H] +

[0365] 1H NMR (500MHz, CDCl3) δ7.78(d,J=6.3Hz,1H),7.16(d,J=8.1Hz,1H),7.12–7.09(m,2H),5.93(d,J=6.5Hz ,1H),4.51(d,J=5.3Hz,2H),3.78(dt,J=12.6,4.7Hz,8H),3.06(d,J=7.3Hz,4H),2.82(t,J=4.6Hz,4H). 13 C NMR (126MHz, CDCl3) δ161.80,152.09,132.69,130.30,127.07,123.16,121.29,93.67,67.15,52.81,43.63,40.75.

[0366] Step 5: 1-{4-[2-({[4-bromo-2-(1,4-oxazacyclohexane-4-yl)phenyl]methyl}amino)pyrimidin-4-yl]piperazine-1-yl}-2-chloroethan-1-one (W16)

[0367] The reactant 16d (97 mg, 0.22 mmol) and triethylamine (68 mg, 0.67 mmol) were dissolved in 4 mL of dichloromethane, stirred under an ice bath and chloroacetyl chloride (28 mg, 0.25 mmol) was added dropwise, and the mixture was reacted at 0°C for 3 h. The reaction was monitored by TLC spot plate until completion. The crude product was purified by Pre-HPLC to obtain the final product W16 (86 mg, yield 75%).

[0368] LC-MS: m / z=509.1[M+H] +

[0369] 1 H NMR (500MHz, DMSO) δ9.03(s,1H),7.93(d,J=7.4Hz,1H),7.26(d,J=4.0Hz,3H),6.51(s,1H),4.58( d,J=5.8Hz,2H),4.45(s,2H),3.81(s,4H),3.75(s,4H),3.61–3.46(m,4H),2.85(t,J=4.4Hz,4H). 13 C NMR (126MHz, DMSO) δ165.48,161.29,153.32,152.71,143.58,132.52,130.26,126.84,122.87,121.05,95.02,66.89,52.85,42.43,41.39.

[0370] Example 17 Preparation of W17

[0371]

[0372] Step 1: 2-{[(4-bromo-2-chlorophenyl)methyl]amino}-4-(methylthio)pyrimidine (17a)

[0373] The reactants (4-bromo-2-chlorophenyl)methanamine (500 mg, 2.28 mmol), 2-chloro-4-(methylthio)pyrimidine (365 mg, 2.28 mmol), and potassium carbonate (947 mg, 6.85 mmol) were dissolved in 8 mL of DMF and heated with stirring at 120° C. to react overnight. LCMS detected that the reaction was complete. The reaction solution was added with water and extracted with EA three times, and then flash purified (PE / EA=5:1) to obtain the product 17a (600 mg, yield 77%).

[0374] LC-MS: m / z=344.0 [M+H] +

[0375] 1 H NMR (400MHz, DMSO) δ7.98 (d, J=5.3Hz, 1H), 7.78 (s, 1H), 7.71 (dd, J=11.9, 2.0Hz, 1H), 7.51 (dd, J= 8.3, 2.0Hz, 1H), 7.26 (d, J = 8.3Hz, 1H), 6.55 (d, J = 5.3Hz, 1H), 4.50 (d, J = 6.1Hz, 2H), 2.40 (s, 3H). 13 C NMR(101MHz,DMSO)δ161.81,161.64,137.40,135.96,133.74,133.41,131.76,131.57, 131.22,130.74,130.59,130.41,120.92,120.29,107.84,42.13,40.47,38.88,12.02.

[0376] Step 2: 2-{[(4-bromo-2-chlorophenyl)methyl]amino}-4-(methyldioxy-λ 6 -thio)pyrimidine (17b)

[0377] The reactant 17a (600 mg, 1.75 mmol) and m-chloroperbenzoic acid (755 mg, 4.37 mmol) were dissolved in 8 mL of DMF and stirred at room temperature overnight. The reaction was completed by LCMS. After adding water to the reaction solution, a solid was precipitated. The crude product was flash purified (PE / EA=5:1) to obtain the product 17b (575 mg, yield 88%).

[0378] LC-MS: m / z=376.0 [M+H] +

[0379] 1 H NMR (500MHz, DMSO) δ8.69(d,J=11.2Hz,1H),8.54(d,J=11.1Hz,1H),7.72(d,J=1.9Hz,1H),7.52( dd,J=8.3,2.0Hz,1H),7.37–7.27(m,1H),7.12(s,1H),4.56(d,J=6.0Hz,2H),3.29–3.13(m,3H). 13 C NMR (126MHz, DMSO) δ162.64,162.31,136.36,131.75,130.68,120.71,105.00,42.29,38.84.

[0380] Step 3: 2-{[(4-bromo-2-chlorophenyl)methyl](nitroso)amino}-4-(methyldioxy-λ 6 -thio)pyrimidine (17c)

[0381] The reactant 17b (250 mg, 0.67 mmol) was dissolved in 5 mL of acetic acid, and sodium nitrite (137 mg, 2.0 mmol) was added in batches at room temperature. The reaction was stirred for 4 h. The reaction was completed by LCMS. After adding water to the reaction solution, a solid was precipitated. The crude product was washed three times with water to obtain the product 17c (266 mg, yield 99%) without further purification.

[0382] LC-MS: No target TIC response under ESI ionization

[0383] 1 H NMR (400MHz, DMSO) δ9.32(d,J=4.9Hz,1H),8.10(d,J=4.9Hz,1H),7.78(d,J=2.0Hz ,1H),7.42(dd,J=8.3,2.1Hz,1H),6.89(d,J=8.4Hz,1H),5.36(s,2H),3.35(s,2H). 13 C NMR (101MHz, DMSO) δ166.88,164.03,158.84,133.40,132.12,131.75,130.89,130.06,121.37,115.27,43.38.

[0384] Step 4: 2-{[(4-bromo-2-chlorophenyl)methyl]amino}-4-(piperazin-1-yl)pyrimidine (17d)

[0385] The reactant 17c (240 mg, 0.59 mmol), 1-Boc-piperazine (166 mg, 0.89 mmol), and potassium carbonate (246 mg, 1.78 mmol) were dissolved in 5 mL DMF and stirred at 90 ° C for 4 h. The reaction was complete when LCMS detected. After the reaction solution was cooled to room temperature, 10 mL 10% H2SO4 was added, and the temperature was continued to rise to 120 ° C and stirred for 1 h. After the reaction was completed, it was cooled to room temperature, and the pH of the reaction solution was adjusted to alkaline with saturated NaOH, and extracted three times with ethyl acetate. The organic phase was flash purified (DCM / MeOH=15:1) to obtain the product 17d (115 mg, yield 51%)

[0386] LC-MS: m / z=382.0 [M+H] +

[0387] 1 H NMR(500MHz, CDCl3)δ7.80(d,J=6.2Hz,1H),7.48(d,J=1.7Hz,1H),7.31–7.27(m,2H),5.86(d, J=6.2Hz,1H),4.71(s,1H),4.58(d,J=4.1Hz,2H),3.57(t,J=5.2Hz,4H),2.91(t,J=5.2Hz,4H). 13 C NMR (126MHz, CDCl3) δ162.28,161.16,155.64,136.49,133.93,131.74,130.25,129.92,120.67,93.61,45.14,44.17,42.51.

[0388] Step 5: 1-[4-(2-{[(4-bromo-2-chlorophenyl)methyl]amino}pyrimidin-4-yl)piperazin-1-yl]-2-chloroethan-1-one (W17)

[0389] The reactant 17d (115 mg, 0.30 mmol) and triethylamine (101 mg, 0.91 mmol) were dissolved in 4 mL of dichloromethane, stirred under an ice bath and chloroacetyl chloride (33 mg, 0.33 mmol) was added dropwise, and the mixture was reacted at 0°C for 3 h. The reaction was monitored by TLC spot plate until completion. The crude product was purified by Pre-HPLC to obtain the final product W17 (82 mg, yield 59%).

[0390] LC-MS: m / z=458.0[M+H] +

[0391] 1H NMR (500MHz, DMSO) δ9.19(s,1H),7.96(d,J=7.4Hz,1H),7.73(s,1H),7.55(d,J=8.3Hz,1H),7.39(d, J=8.3Hz,1H),6.55(s,1H),4.61(d,J=5.6Hz,2H),4.45(s,2H),3.93–3.65(m,4H),3.63–3.44(m,4H). 13 C NMR (126MHz, DMSO) δ165.44,161.33,159.85,159.58,153.43,143.74,135.51,133.83,131.89,131.64,1 31.41,130.79,121.19,117.90,115.57,95.33,45.60,45.28,44.72,43.60,43.26,42.44,41.95,41.46.

[0392] Example 18 Preparation of W18

[0393]

[0394] Step 1: 2-{[(2,4-dichlorophenyl)methyl]amino}-4-(methylthio)pyrimidine (18a)

[0395] The reactants (2,4-dichlorophenyl)methaneamine (500 mg, 2.86 mmol), 2-chloro-4-(methylthio)pyrimidine (457 mg, 2.86 mmol), and potassium carbonate (1183 mg, 8.57 mmol) were dissolved in 8 mL of DMF and heated and stirred at 120°C for overnight reaction. LCMS detected that the reaction was complete. The reaction solution was added with water and extracted with EA three times, and then flash purified (PE / EA=10:1) to obtain the product 18a (627 mg, yield 73%).

[0396] LC-MS: m / z=300.0[M+H] +

[0397] Step 2: 2-{[(2,4-dichlorophenyl)methyl]amino}-4-(methyldioxy-λ 6 -thio)pyrimidine (18b)

[0398] The reactant 18a (600 mg, 2.00 mmol) and m-chloroperbenzoic acid (866 mg, 5.02 mmol) were dissolved in 8 mL of DMF and stirred at room temperature overnight. The reaction was completed by LCMS. After adding water to the reaction solution, a solid was precipitated. The crude product was flash purified (PE / EA=5:1) to obtain the product 18b (529 mg, yield 80%).

[0399] LC-MS: m / z=332.0 [M+H] +

[0400] 1 H NMR (400MHz, DMSO) δ8.71–8.66(m,1H),8.55(s,1H),7.65–7.59(m,1H),7.39( dt,J=9.6,4.8Hz,2H),7.12(s,1H),4.58(d,J=6.0Hz,2H),3.30–3.07(m,3H). 13 C NMR (101MHz, DMSO) δ162.63,162.32,135.95,133.67,132.66,130.97,130.16,129.08,127.78,104.98,42.22.

[0401] Step 3: 2-{[(2,4-dichlorophenyl)methyl](nitroso)amino}-4-(methyldioxy-λ 6 -thio)pyrimidine (18c)

[0402] The reactant 18b (250 mg, 0.76 mmol) was dissolved in 5 mL of acetic acid, and sodium nitrite (156 mg, 2.27 mmol) was added in batches at room temperature. The reaction was stirred for 4 h and the reaction was complete as detected by LCMS. After adding water to the reaction solution, a solid was precipitated. The crude product was washed three times with water to obtain the product 18c (271 mg, yield 99%) without further purification.

[0403] LC-MS: m / z=361.0 [M+H] +

[0404] 1 H NMR (400MHz, DMSO) δ9.32(d,J=4.9Hz,1H),8.10(d,J=4.9Hz,1H),7.67(d,J=2.2Hz ,1H),7.29(dd,J=8.4,2.2Hz,1H),6.97(d,J=8.4Hz,1H),5.38(s,2H),3.38(s,3H). 13C NMR (101MHz, DMSO) δ166.88,164.03,158.85,133.28,133.22,131.35,129.79,129.45,127.99,115.27,43.31.

[0405] Step 4: 2-{[(4-bromo-2-chlorophenyl)methyl]amino}-4-(piperazin-1-yl)pyrimidine (18d)

[0406] The reactant 18c (260 mg, 0.72 mmol), 1-Boc-piperazine (202 mg, 1.08 mmol), and potassium carbonate (299 mg, 2.17 mmol) were dissolved in 5 mL DMF and stirred at 90 ° C for 4 h. The reaction was complete when LCMS detected. After the reaction solution was cooled to room temperature, 10 mL 10% H2SO4 was added, and the temperature was continued to rise to 120 ° C and stirred for 1 h. After the reaction was completed, it was cooled to room temperature, and the pH of the reaction solution was adjusted to alkaline with saturated NaOH, and extracted three times with ethyl acetate. The organic phase was flash purified (DCM / MeOH=15:1) to obtain the product 18d (128 mg, yield 53%)

[0407] LC-MS: No target TIC response under ESI ionization

[0408] 1 H NMR(400MHz, CDCl3)δ7.82(d,J=6.2Hz,1H),7.38–7.30(m,2H),7.16(dd,J=8.3,2.1Hz,1H), 5.88(d,J=6.2Hz,1H),4.62(d,J=5.7Hz,2H),3.53(dd,J=6.4,3.8Hz,4H),2.90–2.86(m,4H). 13 C NMR (101MHz, CDCl3) δ162.42,161.15,135.95,133.77,133.11,129.97,129.06,127.02,93.72,77.25,45.66,44.85,42.50.

[0409] Step 5: 2-Chloro-1-[4-(2-{[(2,4-dichlorophenyl)methyl]amino}pyrimidin-4-yl)piperazin-1-yl]ethan-1-one (W18)

[0410] The reactant 18d (128 mg, 0.38 mmol) and triethylamine (115 mg, 1.14 mmol) were dissolved in 4 mL of dichloromethane, stirred under an ice bath and chloroacetyl chloride (47 mg, 0.42 mmol) was added dropwise, and the mixture was reacted at 0°C for 3 h. The reaction was monitored by TLC spot plate until completion. The crude product was purified by Pre-HPLC to obtain the final product W18 (103 mg, yield 66%).

[0411] LC-MS: m / z=414.1[M+H] +

[0412] 1 H NMR(500MHz,DMSO)δ9.20(d,J=6.0Hz,1H),7.97(d,J=7.5Hz,1H),7.62(d,J=1.9Hz,1H),7.49–7.41 (m,2H),6.56(d,J=7.2Hz,1H),4.63(d,J=5.7Hz,2H),4.45(s,2H),3.89–3.68(m,4H),3.57(s,4H). 13 C NMR (126MHz, DMSO) δ165.45,161.34,153.45,143.77,135.11,133.63,133.1 1,131.24,129.21,127.90,95.33,45.28,44.72,43.62,42.45,41.89,41.46.

[0413] Example 19 Preparation of W19

[0414]

[0415] Step 1: 2-{[(2-chloro-4-fluorophenyl)methyl]amino}-4-(methylthio)pyrimidine (19a)

[0416] The reactants (2-chloro-4-fluorophenyl)methanamine (500 mg, 3.13 mmol), 2-chloro-4-(methylthio)pyrimidine (500 mg, 3.13 mmol), and potassium carbonate (1294 mg, 9.38 mmol) were dissolved in 8 mL of DMF and heated with stirring at 120° C. to react overnight. LCMS detected that the reaction was complete. The reaction solution was added with water and extracted with EA three times, and then flash purified (PE / EA=10:1) to obtain the product 19a (607 mg, yield 69%).

[0417] LC-MS: m / z=284.1[M+H] +

[0418] 1H NMR (400MHz, DMSO) δ7.98 (d, J=5.3Hz, 1H), 7.76 (s, 1H), 7.45–7.32 (m, 2H), 7.17 ( td,J=8.6,2.7Hz,1H),6.54(d,J=5.3Hz,1H),4.52(d,J=6.1Hz,2H),2.39(s,3H). 13 C NMR(101MHz,DMSO)δ162.43,161.69,159.99,156.53,134.02,132.94,132.83, 130.21,130.12,116.86,116.61,114.70,114.49,107.76,41.93,21.52,12.01.

[0419] Step 2: 2-{[(2-chloro-4-fluorophenyl)methyl]amino}-4-(methyldioxy-λ 6 -thio)pyrimidine (19b)

[0420] The reactant 19a (607 mg, 2.14 mmol) and m-chloroperbenzoic acid (925 mg, 5.36 mmol) were dissolved in 8 mL of DMF and stirred at room temperature overnight. The reaction was complete when LCMS detected that the reaction was complete. After adding water to the reaction solution, a solid was precipitated. The crude product was flash purified (PE / EA=5:1) to obtain the product 19b (533 mg, yield 79%).

[0421] LC-MS: m / z=316.0 [M+H] +

[0422] 1 H NMR(500MHz,DMSO)δ8.68(d,J=4.8Hz,1H),8.53(s,1H),7.51–7.37(m,2H),7.1 9(td,J=8.5,2.7Hz,1H),7.11(s,1H),4.58(d,J=6.0Hz,2H),3.30–3.13(m,3H). 13 CNMR (126MHz, DMSO) δ162.62,162.38,162.33,160.43,133.07,130.36,117.01,116.82,114.78,114.62,104.90,42.13,38.87.

[0423] Step 3: 2-{[(2-chloro-4-fluorophenyl)methyl](nitroso)amino}-4-(methyldioxy-λ 6 -thio)pyrimidine (19c)

[0424] The reactant 19b (250 mg, 0.79 mmol) was dissolved in 5 mL of acetic acid, and sodium nitrite (164 mg, 2.38 mmol) was added in batches at room temperature. The reaction was stirred for 4 h and the reaction was completed by LCMS. After adding water to the reaction solution, a solid was precipitated. The crude product was washed three times with water to obtain the product 19c (272 mg, yield 99%) without further purification.

[0425] LC-MS: No target TIC response under ESI ionization

[0426] 1 H NMR (400MHz, DMSO) δ9.32(d,J=4.9Hz,1H),8.10(d,J=4.9Hz,1H),7.49(dd,J=8.7,2.6Hz,1H),7.14–6.98(m,2H),5.38(s,2H). 13 C NMR(101MHz,DMSO)δ166.87,164.02,162.82,160.35,158.89,133.09,132.99,1 30.08,129.99,128.56,128.52,117.43,117.17,115.25,115.12,114.91,43.18.

[0427] Step 4: 2-{[(2-chloro-4-fluorophenyl)methyl]amino}-4-(piperazin-1-yl)pyrimidine (19d)

[0428] The reactant 19c (252 mg, 0.73 mmol), 1-Boc-piperazine (204 mg, 1.10 mmol), and potassium carbonate (303 mg, 2.20 mmol) were dissolved in 5 mL DMF and stirred at 90 ° C for 4 h. The reaction was complete when LCMS detected. After the reaction solution was cooled to room temperature, 10 mL 10% H2SO4 was added, and the temperature was continued to rise to 120 ° C and stirred for 1 h. After the reaction was completed, it was cooled to room temperature, and the pH of the reaction solution was adjusted to alkaline with saturated NaOH, and extracted three times with ethyl acetate. The organic phase was flash purified (DCM / MeOH=15:1) to obtain the product 19d (131 mg, yield 56%)

[0429] LC-MS: m / z=322.1[M+H] +

[0430] 1H NMR (500MHz, CDCl3) δ7.68(d,J=6.2Hz,1H),7.30(dd,J=8.6,6.2Hz,1H),6.99(dd,J=8.5,2.6Hz,1H),6.79(td,J=8.4,2 .7Hz,1H),5.74(d,J=6.2Hz,1H),4.52(d,J=5.5Hz,2H),3.59–3.50(m,1H),3.43(t,J=5.2Hz,4H),2.76(t,J=5.2Hz,4H). 13 C NMR (126MHz, CDCl3) δ162.37,162.28,161.58,160.31,155.99,133.56,133.47,133.42 ,133.39,130.15,130.08,116.51,116.31,113.79,113.63,93.29,45.45,44.60,42.25.

[0431] Step 5: 2-Chloro-1-[4-(2-{[(2-chloro-4-fluorophenyl)methyl]amino}pyrimidin-4-yl)piperazin-1-yl]ethan-1-one (W19)

[0432] The reactant 19d (131 mg, 0.41 mmol) and triethylamine (124 mg, 1.22 mmol) were dissolved in 4 mL of dichloromethane, stirred under ice bath and chloroacetyl chloride (50 mg, 0.45 mmol) was added dropwise, and reacted at 0°C for 3 h. The reaction was monitored by TLC spot plate. The crude product was purified by Pre-HPLC to obtain the final product W19 (95 mg, yield 59%).

[0433] LC-MS: m / z=398.1[M+H] +

[0434] 1 H NMR(500MHz,DMSO)δ9.06(d,J=6.0Hz,1H),7.95(d,J=7.4Hz,1H),7.53–7.44(m,2H),7.23(td,J=8.5,2.7Hz,1 H), 6.56 (s, 1H), 4.63 (d, J = 5.7Hz, 2H), 4.46 (s, 2H), 3.96–3.82 (m, 2H), 3.80–3.67 (m, 2H), 3.63–3.47 (m, 4H). 13C NMR (126MHz, DMSO) δ165.45,162.61,161.34,160.65,153.36,143.74,133.49,132 .26,131.46,117.17,116.97,114.98,114.81,95.32,44.74,42.45,41.81,41.47.

[0435] Test Example 1 Pharmacological Activity Test Method

[0436] (1) Test method

[0437] In this experiment, the single-stranded RNA synthesized by GenScript that spontaneously forms a partial hairpin structure was used as the substrate, with the sequence of 5'-bioUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUAACAGGUUCUAGAA CCUGUU-3'. SARS-CoV-2RdRp catalyzes the substrate in the presence of ATP to form a completely complementary hairpin RNA product, which is then used The product was detected by fluorescent dye of dsDNA system (Promega). The dye can bind to hairpin RNA with double-stranded characteristics, but not to single-stranded RNA. Finally, the enzymatic activity of SARS-CoV-2RdRp was reflected by fluorescent signal, and the inhibitory activity test of the inhibitor was performed.

[0438] (2) Experimental procedures

[0439] The total reaction system of the experiment was 20 μL, and the specific steps were as follows: First, SARS-CoV-2RdRp was diluted to 15 μg / mL with a standard reaction solution (20 mM Tris-HCl pH 8.0, 10 mM KCl, 6 mM MgCl2, 0.01% Triton-X100), and incubated with inhibitors or an equal amount of DMSO for 5 minutes; then 20 μM ATP and 0.2 μM RNA substrate were added, and the incubation continued for 30 minutes; finally, 15 μL of the reaction system was added to 200 μL of 1× fluorescent dye, and after shaking at room temperature for 5 minutes, the fluorescence signal was immediately recorded using BioTek Synergy H1, with excitation and emission wavelengths of 504 nm and 531 nm, respectively. In the experiment, the reaction with different inhibitors was set as the experimental group, the reaction with DMSO was set as the control group, and the reaction containing only RNA substrate and ATP was set as the blank group. The inhibition rate was calculated by the formula (control group-experimental group) / (control group-blank).

[0440] (3) Experimental results

[0441] The inhibition rate (IC 50) as shown in Table 1.

[0442] Table 1

[0443]

[0444]

[0445]

[0446] Test Example 2 Gel imaging experimental method (RNA extension inhibition experiment)

[0447] (1) Test method

[0448] According to the method described in the literature (Structural basis for inhibition of the RNA-dependent RNApolymerase from SARS-CoV-2 by remdesivir. Science. 2020, 368 (6498): 1499-1504.), 5'-FAM-labeled RNA double-stranded (template sequence is 5'-UUUUUUUUUUAUA CUUAAUCUCACAUAGC-3', primer sequence is 5'-FAM-GCUAUGUGAGAUUAAGUU AU-3') and recombinant protein SARS-CoV-2RdRp were prepared.

[0449] (2) Experimental procedures

[0450] Prepare 4X reaction buffer (4XRB) containing 80mM Tris (pH 8.0), 40mM KCl, 24mM MgCl2 and 0.04% Triton-X100 with DEPC water. During the test, SARS-CoV-2RdRp and RNA duplex were diluted to 4μM and 12μM respectively using 4XRB. 4.5μL RdRp (4μM), 4.5μL RNA duplex (12μM) and 9μL of different concentrations of the test compound were added to each reaction. The reaction system was then incubated at room temperature in the dark for 1 hour. Then 2μL ATP (100mM) was added to each reaction, and the reactants were placed in a 37°C water bath for 1 hour. Finally, 40μL of stop solution (94% formamide, 30mM EDTA) was added to terminate the reaction. Take 10μL of the reaction product and mix it with 2μL 6X DNA loading buffer (Thermo Scientific). Prepare a polyacrylamide gel containing 15% urea. Load 10 μL of sample. Run electrophoresis at 150 V for 90 minutes, and image using a Bio-Rad gel imager.

[0451] The above reagents were purchased from Sigma-Aldrich, RNA was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the gel imager model was Bio-Rad ChemiDoc XRS+.

[0452] (3) Experimental results

[0453] Compound W2 inhibits RNA elongation Figure 1 shown.

[0454] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A compound represented by formula (I), or a pharmaceutically acceptable salt or ester, tautomer, enantiomer, diastereomer, racemate, hydrate, solvate, prodrug or deuterated compound thereof: in, X1, X2 and X3 are each independently selected from the group consisting of CH, CR1, N, and at least one of X1, X2 and X3 is N; R1 is one or more groups selected from the group consisting of H, halogen, -NO2, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, -N(R 5 )2.-OR 5 、-SR 5 、-C(=O)N(R 5 )2, -C(=O)OR 5 、-NR 5 -C(=O)R 5 、-NR 5 -C(=O)OR 5 、-NR 5 -S(=O)2-R 5 、-C1-C6 alkylene-N(R 5 )2, -C1-C6 alkylene-OR 5 、-C2-C6 alkenylene-OR 5 、-O-C1-C6 alkylene-N(R 5 )2.-NR 5 -C1-C6 alkylene-OR 5 ; Among them, R 5 Each is independently selected from the following group: H, guanidinyl, C1-C6 alkyl, saturated or partially unsaturated C3-C8 cycloalkyl, saturated or partially unsaturated C3-C10 heterocyclyl containing 1-3 heteroatoms selected from N, O or S, C6-C10 aryl, C5-C10 heteroaryl; Y is selected from the following group: CH, CR 10 , N, O, S; When Y is O or S, R2 does not exist; When Y is CH or CR 10 When R2 is selected from the group consisting of H, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted saturated or partially unsaturated C3-C10 cycloalkyl, substituted or unsubstituted saturated or partially unsaturated C3-C10 heterocyclic group containing 1 to 3 heteroatoms selected from N, O or S, -C(=O)R 6 、-OC(=O)R 6 、-C(=O)OR 6 、-OR 6 、-SR 6 、-S(=O)R 6 、-S(=O)2R 6 、-S(=O)2N(R 6 )2、-N(R 6 )2, -C(=O)N(R 6 )2.-NR 6 -C(=O)R 6 、-NR 6 -C(=O)OR 6 、-NR 6 -S(=O)2-R 6 、C(=O)-N(R 6 )2, -C1-C6 alkylene-N(R 6 )2, -C1-C6 alkylene-C(=O)N(R 6 )2, -C1-C6 alkylene-C(=O)OR 6 、-C1-C6 alkylene-OR 6 、-C2-C6 alkenylene-OR 6 and -O-C1-C6 alkylene-N(R 6 )2; Among them, R 6 Each is independently selected from the following group: H, guanidino, C1-C6 alkyl, saturated or partially unsaturated C3-C8 cycloalkyl, saturated or partially unsaturated C3-C10 heterocyclyl containing 1-3 heteroatoms selected from N, O or S; R 10 Selected from the group consisting of halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl; When Y is N, R2 is selected from the following group: H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted saturated or partially unsaturated C3-C10 cycloalkyl, substituted or unsubstituted saturated or partially unsaturated C3-C10 heterocyclic group containing 1-3 heteroatoms selected from N, O or S, -C(=O)R 8 、-C(=O)OR 8 、-S(=O)R 8 、-S(=O)2R 8 、-S(=O)2N(R 8 )2, -C(=O)N(R 8 )2, -C1-C6 alkylene-N(R 6 )2, -C1-C6 alkylene-C(=O)N(R 8 )2, -C1-C6 alkylene-C(=O)OR 8 、-C1-C6 alkylene-OR 8 、-C2-C6 alkenylene-OR 8 and -O-C1-C6 alkylene-N(R 8 )2; Among them, R 8 Each is independently selected from the following group: H, guanidino, C1-C6 alkyl, saturated or partially unsaturated C3-C8 cycloalkyl, saturated or partially unsaturated C3-C10 heterocyclic group containing 1-3 heteroatoms of N, O or S; Or, R1 and R2 together with the carbon atom to which they are attached and the Y atom form a C3-C6 cycloalkyl group or a C3-C6 heterocyclic group containing 1-3 heteroatoms selected from N, O or S, which may be R 10 optionally substituted; R3 and R4 are each independently selected from the following group: H, halogen, -OH, -CN, -NO2, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted saturated or partially unsaturated C3-C10 cycloalkyl, substituted or unsubstituted saturated or partially unsaturated C3-C10 heterocyclic group containing 1-3 heteroatoms selected from N, O or S, -N(R 7 )2.-OR 7 、-C(=O)R 7 、-OC(=O)R 7 、-C(=O)OR 7 、-SR 7 、-S(=O)R 7 、-S(=O)2R 7 、-S(=O)2N(R 7 )2、-N(R 7 )2, -C(=O)N(R 7 )2.-NR 7 -C(=O)R 7 、-NR 7 -C(=O)OR 7 、-NR 7 -S(=O)2-R 7 、C(=O)-N(R 7 )2; R 7 Each is independently selected from the following group: H, guanidino, C1-C6 alkyl, saturated or partially unsaturated C3-C8 cycloalkyl, saturated or partially unsaturated C3-C10 heterocyclyl containing 1-3 heteroatoms selected from N, O or S; A is selected from the following group:

2. The compound according to claim 1, or a pharmaceutically acceptable salt or ester, tautomer, enantiomer, diastereomer, racemate, hydrate, solvate, prodrug or deuterated compound thereof, characterized in that: X1, X2 and X3 are each independently selected from the group consisting of CH, CR1, N, and at least one of X1, X2 and X3 is N; R1 is one or more groups selected from the group consisting of H, halogen, -NO2, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, -N(R 5 )2.-OR 5 、-SR 5 、-C(=O)N(R 5 )2, -C(=O)OR 5 、-NR 5 -C(=O)R 5 、-NR 5 -C(=O)OR 5 ; Among them, R 5 Selected from the following group: H, C1-C6 alkyl, saturated or partially unsaturated C3-C8 cycloalkyl, saturated or partially unsaturated C3-C10 heterocyclic group containing 1-3 N, O, S heteroatoms, C6-C10 aryl; Y is selected from the group consisting of N, O; When Y is O, R2 does not exist; When Y is N, R2 is selected from the following group: H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted saturated or partially unsaturated C3-C10 cycloalkyl, substituted or unsubstituted saturated or partially unsaturated C3-C10 heterocyclic group containing 1-3 heteroatoms selected from N, O or S, -C(=O)R 8 、-S(=O)R 8 、-S(=O)2R 8 、-C1-C6 alkylene-N(R 8 )2, -C1-C6 alkylene-C(=O)N(R 8 )2, -C1-C6 alkylene-C(=O)OR 8 、-C1-C6 alkylene-OR 8 and -O-C1-C6 alkylene-N(R 8 )2; Among them, R 8 Each is independently selected from the following group: H, C1-C6 alkyl, saturated or partially unsaturated C3-C8 cycloalkyl, saturated or partially unsaturated C3-C10 heterocyclyl containing 1-3 heteroatoms selected from N, O or S; R3 and R4 are each independently selected from the following group: H, halogen, -OH, -CN, -NO2, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted saturated or partially unsaturated C3-C10 cycloalkyl, substituted or unsubstituted saturated or partially unsaturated C3-C10 heterocyclic group containing 1-3 heteroatoms selected from N, O or S, -N(R 7 )2.-OR 7 、-C(=O)R 7 、-OC(=O)R 7 、-C(=O)OR 7 、-C(=O)N(R 7 )2.-NR 7 -C(=O)R 7 、-NR 7 -S(=O)2-R 7 ; Among them, R 7 Each is independently selected from the following group: H, C1-C6 alkyl, saturated or partially unsaturated C3-C8 cycloalkyl, saturated or partially unsaturated C3-C10 heterocyclyl containing 1-3 heteroatoms selected from N, O or S; A is selected from the following group:

3. The compound according to claim 1, or a pharmaceutically acceptable salt or ester, tautomer, enantiomer, diastereomer, racemate, hydrate, solvate, prodrug or deuterated compound thereof, characterized in that: X1, X2 and X3 are each independently selected from the group consisting of CH, CR1, N, and at least one of X1, X2 and X3 is N; R1 is selected from the group consisting of H, halogen, -NO2, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl; Y is N; R2 is selected from the group consisting of H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, -C1-C6 alkylene-N(R 8 )2, -C1-C6 alkylene-C(=O)N(R 8 )2, -C1-C6 alkylene-C(=O)OR 8 、-C1-C6 alkylene-OR 8 and -O-C1-C6 alkylene-N(R 8 )2; Among them, R 8 Each independently selected from the group consisting of: H, C1-C6 alkyl; R3 and R4 are each independently selected from the following group: H, halogen, -CN, -NO2, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted saturated or partially unsaturated C3-C10 heterocyclic group containing 1-3 heteroatoms selected from N, O or S; A is selected from the following group:

4. The compound of claim 1, or a pharmaceutically acceptable salt or ester, tautomer, enantiomer, diastereomer, racemate, hydrate, solvate, prodrug or deuterated compound thereof, characterized in that: X1, X2 and X3 are each independently selected from the group consisting of CH, CR1, N, and at least one of X1, X2 and X3 is N; R1 is selected from the group consisting of H, Cl, -NO2, -CH3, methylamino; Y is N; R2 is one or two groups selected from the following groups: H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, -C1-C6 alkylene-C(=O)N(R 8 )2, -C1-C6 alkylene-C(=O)OR 8 ; Among them, R 8 Each independently selected from the group consisting of: H, C1-C6 alkyl; R3 and R4 are each independently selected from the following group: H, halogen, -CF3, substituted or unsubstituted saturated or partially unsaturated C3-C10 heterocyclic group containing 1-3 heteroatoms selected from N, O or S; A is 5. The compound according to claim 1, or a pharmaceutically acceptable salt or ester, tautomer, enantiomer, diastereomer, racemate, hydrate, solvate, prodrug or deuterated compound thereof, characterized in that: The compound is selected from the group consisting of: 。 6. The compound according to claim 1, or a pharmaceutically acceptable salt or ester, tautomer, enantiomer, diastereomer, racemate, hydrate, solvate, prodrug or deuterated compound thereof, characterized in that: The preparation method of the compound comprises the following steps: in, X1, X2, X3, R1, R2, R3, R4, Y and A are as defined in claim 1.

7. A pharmaceutical composition, characterized in that include (1) The compound according to claim 1, or a pharmaceutically acceptable salt or ester, tautomer, enantiomer, diastereomer, racemate, hydrate, solvate, prodrug or deuterated compound thereof; (2) A pharmaceutically acceptable carrier.

8. The use of the compound according to claim 1, or a pharmaceutically acceptable salt or ester, tautomer, enantiomer, diastereomer, racemate, hydrate, solvate, prodrug or deuterated compound thereof, characterized in that: Used for preparing medicines or pharmaceutical compositions for treating diseases caused by viral infections.

9. The use according to claim 8, characterized in that The disease is selected from the group consisting of coronavirus, calicivirus, and picornavirus infection.

10. The use according to claim 8, characterized in that The disease is an infection caused by a virus selected from the group consisting of SARS-CoV-2 virus, SARS-CoV virus, and MERS-CoV virus.

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