An N-(tert-butyl)-2-(N-acetamido)formamide compound, its preparation method and application

Abstract

The present invention provides an N-(tert-butyl)-2-(N-acetylamino)formamide compound and a 3CL derivative of the N-(tert-butyl)-2-(N-acetylamino)formamide compound. pro The experimental results show that the compound synthesized by the present invention has an inhibitory effect on 3CL pro The compounds 1, 8, 13, 16 and 20 showed the strongest inhibitory activity against 3CL protease, with IC 50 The concentrations of compound 1 and its derivatives were all below 200 nM, and the in vitro toxicity test showed that compound 1 was safer than the positive controls PF-07321332 and S-217622, indicating that it can be developed and applied as an anti-coronavirus drug.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal chemistry, and specifically relates to the synthesis of an N-(tert-butyl)-2-(N-acetamido)formamide compound that inhibits the activity of coronavirus 3CL protease by using the Ugi reaction, and its preparation method and application. Background Art

[0002] Coronaviruses are a class of RNA viruses with envelopes and linear single-stranded positive-sense genomes, with irregular shapes and particles being round or oval.

[0003] 3CL protease, also known as 3CL pro or M pro , belongs to cysteine protease, can process polyprotein precursors pp1a and pp1ab, is responsible for releasing most non-structural functional proteins (NSPs), and participates in the viral replication and transcription processes. Due to its key role in the coronavirus life cycle and the absence of homologous proteins in human cells, 3CL protease has become one of the most attractive targets for inhibiting novel coronaviruses.

[0004] Currently, some drugs that inhibit the activity of 3CL protease have been disclosed. For example, Pfizer's Nirmatrelvir Paxlovid, but it is easily metabolized and degraded rapidly by liver enzyme CYP3A, resulting in a rapid loss of drug efficacy activity in the body. Therefore, it needs to be combined with ritonavir to inhibit the activity of liver enzyme CYP3A, protect Nirmatrelvir from being rapidly metabolized in the body, and make Nirmatrelvir have a longer and stronger drug effect time. Ensitrelvir is a non-covalent small molecule 3CLpro inhibitor, but there is a possibility of teratogenesis. Summary of the Invention

[0005] The purpose of the present invention is to provide an N-(tert-butyl)-2-(N-acetamido)formamide compound, its preparation method and application. The above compound can inhibit the activity of coronavirus 3CL protease and can be used to treat diseases caused by coronaviruses.

[0006] The present invention is achieved through the following technical solutions:

[0007] An N-(tert-butyl)-2-(N-acetamido)formamide compound, which is a compound of formula I or a pharmaceutically acceptable salt thereof, or a solvate, enantiomer, diastereomer, tautomer of the compound of formula I or a pharmaceutically acceptable salt thereof, or a mixture thereof in any proportion;

[0008] The structural formula of the compound of formula I is:

[0009]

[0010] Among them, R1 is one of pyridyl, pyrazolo[1,5 - a]pyrimidinyl, 1H - tetrazolyl, pyrazolyl, pyrazinyl, 1H - imidazolyl, thienyl, oxazolyl, furyl, imidazolyl, thiazolyl, and 2 - hydroxypyridyl;

[0011] R2 is one of 2 - thienyl, 2 - pyrrolyl, 1 - methyl - 1H - pyrazolyl, 4 - (1H - imidazol - 1 - yl)phenyl, 3 - thienyl, 1H - pyrazolyl, 3 - furyl, 4 - imidazolyl, and 3 - pyridyl;

[0012] R3 is one of 4 - bromophenyl, 4 - methyldiphenyl ether, diphenylmethane, 4 - (imidazol - 1 - yl)phenyl, 3 - fluoro - 4 - (4 - morpholino)phenyl, 4 - acetophenonyl, 4 - (tert - butoxycarbonylamino)phenyl, and p - aminobenzonitrile;

[0013] Preferably, the compound of formula I is one of the following compounds:

[0014]

[0015] Preferably, the pharmaceutically acceptable salt is a salt formed by the compound of formula I and hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, oxalic acid, malonic acid, succinic acid, citric acid, malic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, glutamic acid, or aspartic acid.

[0016] For the preparation method of the N - (tert - butyl)-2-(N - acetamido)formamide compound, a Ugi reaction is carried out on a substituted aldehyde, a substituted amine, a substituted acid, and tert - butyl isocyanate in a solvent to obtain the N - (tert - butyl)-2-(N - acetamido)formamide compound.

[0017] Preferably, the molar ratio of the substituted aldehyde, the substituted amine, the substituted acid, and tert - butyl isocyanate is 1:1:1:1.

[0018] Preferably, it specifically includes the following steps:

[0019] Add the substituted aldehyde and the substituted amine into a methanol solvent, react at room temperature for 30 min, then add the substituted acid and tert - butyl isocyanate, and continue to react at room temperature for 8 - 14 h. After separation and purification, the N - (tert - butyl)-2-(N - acetamido)formamide compound shown in formula I is obtained.

[0020] Application of the N - (tert - butyl)-2-(N - acetamido)formamide compound in the preparation of a coronavirus 3CL protease inhibitor.

[0021] Application of the N - (tert - butyl)-2-(N - acetamido)formamide compound in the preparation of an anti - coronavirus drug.

[0022] Preferably, the coronavirus is the novel coronavirus SARS-CoV-2.

[0023] Preferably, the inhibitor or drug is in the form of tablets, capsules, granules, syrups, premixes, pellets, liniments or injections.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention provides an N-(tert-butyl)-2-(N-acetamido)formamide compound, and also provides the 3CL pro inhibitory effect of the N-(tert-butyl)-2-(N-acetamido)formamide compound. The experimental results show that the compounds synthesized in the present invention have a strong inhibitory effect on 3CL pro Among them, compounds 1, 8, 13, 16 and 20 have the strongest inhibitory activity against 3CL protease, and the IC 50 are all below 200 nM. Moreover, the in vitro toxicity experiment of compound 1 shows that its safety is better than that of the positive controls PF-07321332 and S-217622, and it can be developed and applied as an anti-coronavirus drug.

[0026] The N-(tert-butyl)-2-(N-acetamido)formamide compound of the present invention is synthesized by using the Ugi reaction, with simple operation and mild reaction conditions. Description of the Drawings

[0027] Figure 1 is the 1H NMR spectrum of compound 1 in deuterated DMSO in the present invention;

[0028] Figure 2 is the 13C NMR spectrum of compound 1 in deuterated DMSO in the present invention;

[0029] Figure 3 is the 1H NMR spectrum of compound 6 in deuterated DMSO in the present invention;

[0030] Figure 4 is the 13C NMR spectrum of compound 6 in deuterated DMSO in the present invention;

[0031] Figure 5 is the 1H NMR spectrum of compound 7 in deuterated DMSO in the present invention;

[0032] Figure 6 is the 13C NMR spectrum of compound 7 in deuterated DMSO in the present invention;

[0033] Figure 7 is the 1H NMR spectrum of compound 9 in deuterated DMSO in the present invention;

[0034] Figure 8 It is the carbon-13 NMR spectrum of Compound 9 in deuterated DMSO in the present invention;

[0035] Figure 9 It is the proton NMR spectrum of Compound 10 in deuterated DMSO in the present invention;

[0036] Figure 10 It is the carbon-13 NMR spectrum of Compound 10 in deuterated DMSO in the present invention;

[0037] Figure 11 It is the proton NMR spectrum of Compound 14 in deuterated DMSO in the present invention;

[0038] Figure 12 It is the carbon-13 NMR spectrum of Compound 14 in deuterated DMSO in the present invention;

[0039] Figure 13 It is the inhibitory effect of Compound 1 on 3CL in the present invention pro in the present invention;

[0040] Figure 14 It is the inhibitory effect of Compound 8 on 3CL in the present invention pro in the present invention;

[0041] Figure 15 It is the inhibitory effect of Compound 16 on 3CL in the present invention pro in the present invention;

[0042] Figure 16 It is the survival rate of A549 cells treated with PF-07321332, S-217622 and Compound 1 at different concentrations;

[0043] Figure 17 It is the survival rate of HEK2932 cells treated with PF-07321332, S-217622 and Compound 1 at different concentrations;

[0044] Figure 18 It is the survival rate of HepG2 cells treated with PF-07321332, S-217622 and Compound 1 at different concentrations. Detailed implementation manners

[0045] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0046] It should be noted that in the description and claims of the present invention, the terms "comprising" and "having" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product or device comprising a series of steps or units need not be limited to those steps or units clearly listed, but may include other steps or units not clearly listed or inherent to these processes, methods, products or devices.

[0047] The present invention will be further described in detail below with reference to the accompanying drawings:

[0048] The N-(tert-butyl)-2-(N-acetamido)formamide compound of the present invention for inhibiting the activity of coronavirus 3CL protease is a compound of formula I or a pharmaceutically acceptable salt thereof, as well as a solvate, enantiomer, diastereoisomer, tautomer or any mixture thereof in any ratio of the compound of formula I or a pharmaceutically acceptable salt thereof, including a racemic mixture;

[0049] The structural formula of the compound of formula I is:

[0050]

[0051] Wherein, R1 is one of pyridyl, pyrazolo[1,5-a]pyrimidinyl, 1H-tetrazolyl, pyrazolyl, pyrazinyl, 1H-imidazolyl, thienyl, oxazolyl, furyl, imidazolyl, thiazolyl and 2-hydroxypyridyl;

[0052] R2 is one of 2-thienyl, 2-pyrrolyl, 1-methyl-1H-pyrazolyl, 4-(1H-imidazol-1-yl)phenyl, 3-thienyl, 1H-pyrazolyl, 3-furyl, 4-imidazolyl and 3-pyridyl;

[0053] R3 is one of 4-bromophenyl, 4-methyldiphenyl ether, diphenylmethane, 4-(imidazol-1-yl)phenyl, 3-fluoro-4-(4-morpholinyl)phenyl, 4-acetophenone, 4-(tert-butoxycarbonylamino)phenyl and p-aminobenzonitrile.

[0054] The pharmaceutically acceptable salt of the present invention is a salt formed by the diamide compound for inhibiting the activity of coronavirus 3CL protease and hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, oxalic acid, malonic acid, succinic acid, citric acid, malic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, glutamic acid or aspartic acid.

[0055] The preparation method of the N-(tert-butyl)-2-(N-acetamido)formamide compound for inhibiting the activity of coronavirus 3CL protease provided by the present invention includes the following operating steps:

[0056] Methanol was added to the reactor, and then the four components of the Ugi reaction were added in sequence, 1 equivalent of a substituted aldehyde and 1 equivalent of a substituted amine. After reacting at room temperature, 1 equivalent of a substituted acid and 1 equivalent of tert-butyl isocyanate were added, and the reaction was continued at room temperature for 8 - 14 h. After separation and purification, an N-(tert-butyl)-2-(N-acetamido)formamide compound with inhibitory activity against coronavirus 3CL protease having the structure of Formula I was obtained.

[0057] The described N-(tert-butyl)-2-(N-acetamido)formamide compound can be used to prepare a coronavirus 3CL protease inhibitor or an anti-coronavirus drug.

[0058] The described inhibitor or drug is used alone or in combination with other anti-coronavirus drugs, or mixed with a pharmaceutically acceptable excipient or diluent to form tablets, capsules, granules, syrups, premixes or pellets for oral administration, or prepared into liniments or injections for non-oral administration.

[0059] It can also form a pharmaceutical composition, and the pharmaceutical composition contains the N-(tert-butyl)-2-(N-acetamido)formamide compound with inhibitory activity against coronavirus 3CL protease as an active ingredient.

[0060] 1. Specific examples for synthesizing Compounds 1 - 20

[0061] The structural formula of the representative compound of the present invention is shown as follows

[0062]

[0063] The following are the synthetic examples of the above compounds

[0064] Example 1

[0065] Compound 1: Preparation of N-(4-benzylphenyl)-N-(2-(tert-butylamino)-2-oxo-1-(thiophen-3-yl)ethyl)pyrazine-2-carboxamide

[0066]

[0067] At room temperature, 3-thiophenecarboxaldehyde (112.1 mg, 1 mmol) and 4-aminodiphenylmethane (183.3 mg, 1 mmol) were placed in a reactor containing methanol, and the mixture was stirred for 30 min. Subsequently, 2-pyrazinecarboxylic acid (124.1 mg, 1 mmol) and tert-butyl isocyanate (99.7 mg, 1 mmol) were successively added to the reactor, and the mixture was stirred for 8 h. After the reaction was completed, the solvent methanol was removed under reduced pressure, and the residue was separated and purified by column chromatography (ethyl acetate: cyclohexane: methanol (V:V:V) as the mobile phase = 6:1:1). After drying, 287.37 mg of compound 1 was obtained, and the reaction yield was 59.3%. The 1H NMR spectrum of compound 1 in deuterated DMSO is as shown in Figure 1 , and the 13C NMR spectrum of compound 1 in deuterated DMSO is as shown in Figure 2 .

[0068] 1 H NMR(600MHz,DMSO)δ8.55(s,1H),8.43(d,J=30.5Hz,2H),7.85(s,1H),7.30–7.18(m,4H),7.13(t,J=7.4Hz,1H),7.07–6.97(m,1H),6.93–6.69(m,5H),6.26(s,1H),3.73(s,2H),1.26(s,9H).

[0069] 13 C NMR(151MHz,DMSO)δ168.80,166.48,150.88,144.71,144.04,143.88,141.44,140.06,137.65,136.07,131.37,129.27,128.83,128.79,128.68,126.46,126.30,125.94,60.29,50.90,40.53,28.87.

[0070] Example 2

[0071] Compound 2: Preparation of N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)-N-(4-cyanophenyl)pyrazine-2-carboxamide

[0072]

[0073] The preparation method was referred to Example 1, and only the corresponding raw materials needed to be replaced. The yield of the obtained compound 2 was 41.39%.

[0074] 11H NMR (600 MHz, DMSO) δ 8.98 (s, 1H), 8.40 (d, J = 30.3 Hz, 2H), 8.07 (s, 1H), 7.63 (t, J = 7.1 Hz, 1H), 7.24–7.03 (m, 1H), 6.78–6.54 (m, 4H), 6.13 (s, 1H), 5.86 (s, 2H), 1.37 (s, 9H).

[0075] 13 13C NMR (151 MHz, DMSO) δ 168.63, 165.75, 150.88, 145.73, 143.14, 142.86, 141.42, 140.85, 138.24, 135.24, 132.24, 128.28, 126.24, 124.27, 123.97, 122.87, 65.57, 57.85, 34.68, 28.87.

[0076] Example 3

[0077] Compound 3: Preparation of N-(1-(4-(1-(1H-imidazol-1-yl)phenyl)-2-(tert-butylamino)-2-oxoethyl)-N-(4-benzylphenyl)oxazole-5-carboxamide

[0078]

[0079] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained Compound 3: 40.71%.

[0080] 1 1H NMR (600 MHz, DMSO) δ 8.67 (s, 1H), 8.35 (d, J = 30.2 Hz, 2H), 7.71 (s, 1H), 7.50–7.38 (m, 4H), 7.22–7.16 (m, 4H), 7.12 (t, J = 7.2 Hz, 1H), 7.02–6.87 (m, 1H), 6.56–6.43 (m, 5H), 6.14 (s, 1H), 3.12 (s, 2H), 1.22 (s, 9H).

[0081] 1313C NMR (151 MHz, DMSO) δ 169.83, 167.82, 152.42, 145.75, 144.67, 143.24, 141.67, 140.17, 138.59, 137.57, 136.38, 133.83, 132.78, 131.18, 129.37, 128.68, 127.58, 127.01, 126.58, 125.37, 119.27, 65.37, 54.67, 40.37, 26.57.

[0082] Example 4

[0083] Compound 4: Preparation of N-(2-(tert-butylamino)-1-(furan-3-yl)-2-oxoethyl)-N-(4-(p-tolyloxy)phenyl)oxazole-5-carboxamide

[0084]

[0085] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained Compound 4: 42.55%.

[0086] 1 1H NMR (600 MHz, DMSO) δ 8.72 (s, 1H), 8.27 (d, J = 30.1 Hz, 2H), 7.38 (s, 1H), 7.25–7.11 (m, 4H), 7.03 (t, J = 7.5 Hz, 1H), 6.98–6.91 (m, 1H), 6.87–6.54 (m, 5H), 6.38 (s, 1H), 3.77 (s, 1H), 1.23 (s, 9H).

[0087] 13 13C NMR (151 MHz, DMSO) δ 167.82, 164.38, 153.83, 148.39, 145.38, 143.34, 141.95, 138.43, 136.93, 131.39, 129.47, 128.13, 127.38, 126.68, 126.01, 125.67, 124.68, 64.68, 55.39, 36.97, 21.34.

[0088] Example 5

[0089] Compound 5: Preparation of N-(2-(tert-butylamino)-2-oxo-1-(1H-pyrrol-2-yl)ethyl)-N-(4-(p-tolyloxy)phenyl)nicotinamide

[0090]

[0091] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained compound 5: 48.63%.

[0092] 1 H NMR(600MHz,DMSO)δ8.97(s,1H),8.43(d,J=30.1Hz,2H),7.38(s,1H),7.21–7.03(m,3H),6.96(t,J=7.3Hz,1H),6.93–6.82(m,1H),6.76–6.31(m,4H),6.12(s,1H),3.64(s,2H),2.25(s,3H),1.33(s,9H).

[0093] 13 C NMR(151MHz,DMSO)δ169.28,167.37,155.34,148.37,145.34,143.88,141.31,138.34,137.13,135.13,131.93,129.45,128.87,128.12,127.34,126.39,126.15,125.86,68.74,57.37,35.78,22.65.

[0094] Example 6

[0095] Compound 6: Preparation of N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)-N-(4-(p-tolyloxy)phenyl)nicotinamide

[0096]

[0097] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained compound 6: 47.31%. The 1H NMR spectrum of compound 6 in deuterated DMSO is as shown in Figure 3 , and the 13C NMR spectrum in deuterated DMSO is as shown in Figure 4 .

[0098] 1 H NMR(600MHz,DMSO)δ8.72(s,1H),8.45(d,J=30.2Hz,2H),7.69(s,1H),7.40–7.34(m,4H),7.12(t,J=7.3Hz,1H),7.05–6.92(m,1H),6.84–6.63(m,4H),6.37(s,1H),3.38(s,2H),2.21(s,3H),1.37(s,9H).

[0099] 1313C NMR (151 MHz, DMSO) δ 169.68, 168.21, 155.34, 153.31, 148.37, 147.36, 146.89, 145.83, 143.34, 141.78, 138.61, 137.39, 135.36, 134.41, 131.14, 129.14, 128.86, 127.45, 126.78, 126.46, 125.67, 67.85, 58.58, 31.47, 23.85.

[0100] Example 7

[0101] Compound 7: Preparation of N-(1-(4-(1-(1H-imidazol-1-yl)phenyl)-2-(tert-butylamino)-2-oxoethyl)-N-(4-benzylphenyl)nicotinamide

[0102]

[0103] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained Compound 7: 46.39%. The 1H NMR spectrum of Compound 7 in deuterated DMSO is as shown in Figure 5 , and the 13C NMR spectrum in deuterated DMSO is as shown in Figure 6 .

[0104] 1 1H NMR (600 MHz, DMSO) δ 8.56 (s, 1H), 8.33 (d, J = 30.3 Hz, 2H), 7.54 (s, 2H), 7.48–7.37 (m, 4H), 7.21–7.08 (m, 4H), 7.01 (t, J = 6.9 Hz, 1H), 6.95–6.72 (m, 1H), 6.38–6.24 (m, 5H), 6.12 (s, 1H), 3.87 (s, 2H), 1.32 (s, 9H).

[0105] 13 13C NMR (151 MHz, DMSO) δ 169.38, 168.37, 151.37, 148.43, 144.43, 141.38, 139.89, 138.39, 138.01, 137.68, 136.93, 135.38, 133.39, 132.94, 131.34, 129.69, 128.14, 127.65, 127.34, 126.67, 125.48, 119.64, 118.94, 75.64, 56.43, 42.34, 26.34.

[0106] Example 8

[0107] Preparation of Compound 8: Tributyl(4-(N-(2-(tert-butylamino)-2-oxo-1-(thiophen-3-yl)ethyl)thiophene-2-carboxamido)phenyl)carbamate

[0108]

[0109] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained Compound 8: 42.75%.

[0110] 1 H NMR(600MHz,DMSO)δ8.74(s,1H),8.64(d,J=30.4Hz,2H),7.57(s,1H),7.32–7.22(m,2H),7.27(t,J=7.4Hz,1H),7.11–7.01(m,3H),6.89(s,1H),6.56(s,1H),1.49(s,9H),1.28(s,9H).

[0111] 13 C NMR(151MHz,DMSO)δ168.24,164.37,152.34,139.38,138.37,137.65,136.44,135.31,133.25,131.37,129.34,128.21,127.17,127.01,126.37,126.12,125.24,72.36,69.24,57.24,29.24,27.38.

[0112] Example 9

[0113] Preparation of Compound 9: N-(4-(1H-imidazol-1-yl)phenyl)-N-(2-(tert-butylamino)-1-(furan-3-yl)-2-oxoethyl)thiophene-2-carboxamide

[0114]

[0115] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained Compound 9: 45.21%. The 1H NMR spectrum of Compound 9 in deuterated DMSO is as shown in Figure 7 , and the 13C NMR spectrum in deuterated DMSO is as shown in Figure 8 .

[0116] 11H NMR (600 MHz, DMSO) δ 8.35 (s, 1H), 7.85–7.38 (m, 8H), 7.12 (s, 1H), 6.92–6.86 (m, 1H), 6.64 (dd, J = 3.8, 1.2 Hz, 1H), 6.12 (s, 2H), 1.27 (s, 9H).

[0117] 13 13C NMR (151 MHz, DMSO) δ 169.14, 161.66, 143.50, 143.24, 138.46, 138.32, 136.88, 135.93, 133.67, 132.58, 132.40, 130.58, 127.50, 120.39, 119.90, 118.11, 112.01, 57.44, 50.87, 28.90.

[0118] Example 10

[0119] Compound 10: Preparation of N-(2-(tert-butylamino)-1-(furan-3-yl)-2-oxoethyl)-N-(4-(p-tolyloxy)phenyl)thiophene-2-carboxamide

[0120]

[0121] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained Compound 10: 45.33%. The 1H NMR spectrum of Compound 10 in deuterated DMSO is as shown in Figure 9 , and the 13C NMR spectrum in deuterated DMSO is as shown in Figure 10 .

[0122] 1 1H NMR (600 MHz, DMSO) δ 8.35 (s, 1H), 8.57 (d, J = 30.3 Hz, 2H), 7.39 (s, 1H), 7.30–7.15 (m, 2H), 7.08 (t, J = 7.4 Hz, 2H), 6.87–6.61 (m, 1H), 6.45–6.37 (m, 4H), 6.21 (s, 1H), 5.82 (s, 1H), 2.11 (s, 3H), 1.34 (s, 9H).

[0123] 1313C NMR (151 MHz, DMSO) δ 168.27, 166.37, 155.37, 145.68, 141.28, 140.23, 139.28, 138.27, 137.38, 136.73, 132.34, 129.37, 128.34, 128.01, 127.37, 126.63, 126.74, 125.16, 124.37, 67.12, 59.37, 26.34, 18.34.

[0124] Example 11

[0125] Compound 11: Preparation of tributyl(4-(N-(2-(tert-butylamino)-1-(1H-imidazol-4-yl)-2-oxoethyl)thiophene-2-carboxamido)phenyl)carbamate

[0126]

[0127] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained Compound 11: 42.69%.

[0128] 1 1H NMR (600 MHz, DMSO) δ 8.72 (s, 1H), 8.54 (d, J = 30.2 Hz, 2H), 7.71 (s, 1H), 7.45–7.33 (m, 2H), 7.27 (t, J = 7.2 Hz, 1H), 7.12–7.02 (m, 1H), 6.75 (s, 2H), 5.36 (s, 2H), 1.50 (s, 9H), 1.33 (s, 9H).

[0129] 13 13C NMR (151 MHz, DMSO) δ 169.28, 165.38, 153.38, 141.35, 140.38, 139.54, 138.38, 137.43, 136.46, 135.54, 132.28, 131.58, 129.58, 127.45, 126.82, 125.14, 78.36, 72.14, 59.28, 29.28, 21.28.

[0130] Example 12

[0131] Compound 12: Preparation of N-(4-benzylphenyl)-N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)thiophene-2-carboxamide

[0132]

[0133] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained compound 12: 45.33%.

[0134] 1 H NMR(600MHz,DMSO)δ8.31(s,1H),8.02(d,J=30.1Hz,2H),7.25(s,1H),7.12–7.04(m,4H),6.72(t,J=7.3Hz,1H),6.65–6.52(m,1H),6.45–6.36(m,3H),6.27(s,1H),5.38(s,2H),3.21(s,3H),1.24(s,9H).

[0135] 13 C NMR(151MHz,DMSO)δ169.86,165.75,145.28,141.58,140.66,138.34,137.64,136.28,135.58,134.58,133.58,132.58,131.38,130.58,130.14,129.02,128.58,127.38,126.21,125.34,73.82,58.58,41.04,25.74.

[0136] Example 13

[0137] Preparation of Compound 13: N-(2-(tert-Butylamino)-1-(furan-3-yl)-2-oxoethyl)-N-(3-fluoro-4-morpholinophenyl)thiophene-2-carboxamide

[0138]

[0139] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained compound 13: 42.96%.

[0140] 1 H NMR(600MHz,DMSO)δ8.31(s,1H),7.39–7.21(m,8H),7.11(s,1H),6.95–6.75(m,1H),6.57(dd,J=3.5,1.2Hz,1H),6.14(s,2H),3.77(s,2H),3.27(s,2H),1.27(s,9H).

[0141] 1313C NMR (151 MHz, DMSO) δ 168.28, 163.85, 153.85, 143.58, 138.17, 137.64, 136.68, 135.53, 134.55, 132.69, 127.67, 120.38, 119.17, 118.23, 113.17, 67.58, 65.47, 57.44, 50.87, 28.90.

[0142] Example 14

[0143] Compound 14: Preparation of N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)-N-(4-(p-tolyloxy)phenyl)thiazole-5-carboxamide

[0144]

[0145] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained Compound 14: 40.66%. The 1H NMR spectrum of Compound 14 in deuterated DMSO is as shown in Figure 11 , and the 13C NMR spectrum in deuterated DMSO is as shown in Figure 12 .

[0146] 1 1H NMR (600 MHz, DMSO) δ 9.08 (s, 1H), 8.42–8.32 (m, 2H), 7.96 (s, 2H), 7.58 (s, 1H), 7.42 (dt, J = 7.9, 2.0 Hz, 1H), 7.25–7.15 (m, 3H), 7.04–6.61 (m, 5H), 6.20 (s, 1H), 2.28 (s, 3H), 1.25 (s, 9H).

[0147] 13 13C NMR (151 MHz, DMSO) δ 168.49, 160.78, 159.42, 157.55, 154.43, 151.84, 149.29, 147.92, 137.93, 134.16, 134.04, 133.40, 133.37, 131.47, 130.92, 123.42, 118.98, 63.16, 50.98, 28.84, 20.71.

[0148] Example 15

[0149] Compound 15: Preparation of N-(4-(1H-imidazol-1-yl)phenyl)-N-(2-(tert-butylamino)-1-(furan-3-yl)-2-oxoethyl)thiazole-5-carboxamide

[0150]

[0151] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained compound 15: 53.63%.

[0152] 1 H NMR(600MHz,DMSO)δ8.47(s,1H),7.98–7.56(m,7H),7.17(s,1H),6.91–6.72(m,1H),6.17(dd,J=3.1,1.2Hz,1H),6.47(s,2H),1.37(s,9H).

[0153] 13 C NMR(151MHz,DMSO)δ169.98,162.17,143.27,143.27,138.83,138.47,136.14,135.43,134.47,132.14,132.01,128.24,127.24,120.04,119.48,116.47,111.56,62.47,55.24,27.43.

[0154] Example 16

[0155] Preparation of Compound 16: N-(4-Benzylphenyl)-N-(2-(tert-butylamino)-1-(1-methyl-1H-pyrazol-4-yl)-2-oxoethyl)thiazole-5-carboxamide

[0156]

[0157] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained compound 16: 50.12%.

[0158] 1 H NMR(600MHz,DMSO)δ8.83(s,1H),8.24(d,J=30.2Hz,2H),7.86(s,1H),7.41–7.11(m,4H),6.86(t,J=7.2Hz,1H),6.47–6.32(m,1H),6.25–6.16(m,3H),6.04(s,1H),5.47(s,2H),3.36(s,3H),1.35(s,9H).

[0159] 1313C NMR (151 MHz, DMSO) δ 168.14, 166.23, 143.58, 141.87, 138.24, 137.47, 136.36, 135.25, 134.58, 133.45, 132.12, 131.72, 130.38, 129.14, 127.78, 126.26, 115.27, 72.25, 59.28, 43.42, 41.36, 27.27.

[0160] Example 17

[0161] Compound 17: Preparation of N-(2-(tert-butylamino)-2-oxo-1-(1H-pyrazol-3-yl)ethyl)-N-(3-fluoro-4-morpholinophenyl)furan-3-carboxamide

[0162]

[0163] The preparation method refers to Example 1, only the corresponding raw materials need to be replaced. The yield of the obtained compound 17: 48.69%.

[0164] 1 1H NMR (600 MHz, DMSO) δ 8.12 (s, 1H), 7.28–7.01 (m, 7H), 6.98 (s, 1H), 6.87–6.77 (m, 1H), 6.56 (dd, J = 3.4, 1.2 Hz, 1H), 6.24 (s, 2H), 3.74 (s, 2H), 3.19 (s, 2H), 1.32 (s, 9H).

[0165] 13 13C NMR (151 MHz, DMSO) δ 170.51, 165.28, 155.59, 143.98, 138.47, 137.39, 135.72, 134.27, 132.15, 127.35, 120.57, 119.17, 118.48, 113.77, 77.15, 65.14, 54.45, 46.13, 27.43.

[0166] Example 18

[0167] Compound 18: Preparation of N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)-N-(4-(p-tolyloxy)phenyl)furan-3-carboxamide

[0168]

[0169] The preparation method refers to Example 1, only the corresponding raw materials need to be replaced. The yield of the obtained compound 18: 47.93%.

[0170] 1 1H NMR (600 MHz, DMSO) δ 8.78 (s, 1H), 8.45–8.31 (m, 2H), 7.38 (s, 2H), 7.12 (s, 1H), 7.02 (dt, J = 7.9, 2.0 Hz, 2H), 6.94–6.85 (m, 3H), 6.74–6.60 (m, 5H), 6.36 (s, 1H), 2.25 (s, 3H), 1.37 (s, 9H).

[0171] 13 13C NMR (151 MHz, DMSO) δ 169.24, 167.24, 155.47, 152.28, 151.27, 150.35, 149.99, 147.17, 137.46, 135.37, 134.57, 133.47, 132.10, 131.47, 130.24, 123.58, 116.85, 107.96, 73.57, 59.25, 28.28, 22.45.

[0172] Example 19

[0173] Compound 19: Preparation of N-(1-(4-(1-(1H-imidazol-1-yl)phenyl)-2-(tert-butylamino)-2-oxoethyl)-N-(4-(p-tolyloxy)phenyl)thiazole-5-carboxamide

[0174]

[0175] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained Compound 19: 50.13%.

[0176] 1 1H NMR (600 MHz, DMSO) δ 8.64 (s, 1H), 8.32 (d, J = 30.1 Hz, 2H), 7.86 (s, 1H), 7.45–7.39 (m, 4H), 7.24–7.15 (m, 4H), 7.07 (t, J = 7.3 Hz, 1H), 6.98–6.84 (m, 1H), 6.61–6.47 (m, 4H), 6.36 (s, 1H), 2.22 (s, 3H), 1.22 (s, 9H).

[0177] 1313C NMR (151 MHz, DMSO) δ 168.27, 167.24, 153.85, 145.45, 144.27, 142.38, 141.83, 140.14, 138.96, 137.58, 136.17, 133.37, 132.27, 131.14, 129.64, 128.38, 127.36, 126.14, 124.58, 119.24, 117.23, 75.24, 58.14, 29.75, 23.13.

[0178] Example 20

[0179] Compound 20: Preparation of N-(1-(4-(1-(1H-imidazol-1-yl)phenyl)-2-(tert-butylamino)-2-oxoethyl)-N-(4-cyanophenyl)thiophene-2-carboxamide

[0180]

[0181] The preparation method refers to Example 1, and only the corresponding raw materials need to be replaced. The yield of the obtained compound 20: 50.36%.

[0182] 1 1H NMR (600 MHz, DMSO) δ 8.41 (s, 1H), 8.03 (d, J = 30.3 Hz, 2H), 7.83 (s, 1H), 7.66–7.54 (m, 4H), 7.40–7.24 (m, 2H), 7.15 (t, J = 7.1 Hz, 1H), 7.02–6.94 (m, 1H), 6.64–6.49 (m, 2H), 6.23 (s, 1H), 1.32 (s, 9H).

[0183] 13 13C NMR (151 MHz, DMSO) δ 168.38, 166.89, 153.55, 146.38, 139.33, 137.54, 136.15, 135.69, 134.12, 133.58, 132.24, 131.55, 130.38, 129.93, 128.17, 127.26, 126.54, 111.52, 107.36, 73.85, 56.25, 26.57.

[0184] 2. Biological activity assay

[0185] (1) 3CL pro Inhibitory activity test

[0186] The inhibitory activity of the compound against SARS-CoV-2 3CL was determined using fluorescence resonance energy transfer technology pro .

[0187] Weigh appropriate amounts of the test compound and positive control drugs (S-216722 and PF-07321332), and prepare solutions with appropriate concentration gradients using DMSO. Take 5 μL of each of the above-prepared solutions and 91 μL of Assay Reagent (Assay Buffer: SARS-CoV-2 3CLpro = 90:1, purchased from Shanghai Beyotime Biotechnology Co., Ltd.) and add them successively to a black 96-well plate, and mix well. After incubating in the dark at 37 °C for 10 minutes, quickly add 4 μL of Substrate (100 μM Dabcyl-KTSAVLQSGFRKME-Edans, purchased from Shanghai Beyotime Biotechnology Co., Ltd.) to each well on ice, and mix well. After incubating in the dark at 37 °C for 5 min, the signal becomes stable. Use a multifunctional microplate reader (Thermo Fisher Scientific, Varioskan Flash) to perform fluorescence measurement, and calculate the inhibition percentage of the sample (excitation wavelength: 340 nm, emission wavelength: 490 nm). Use the Assay Reagent without the compound as the 100% enzyme activity control, the Assay Buffer without SARS-CoV-2 3CLpro as the blank control, S-216722 (Shandong Xuanshuo Pharmaceutical Technology Co., Ltd.) and PF-07321332 (Jinan Jianfeng Chemical Co., Ltd.) as the positive controls, and the remaining treatment methods are the same. Use GraphPad Prism software to perform nonlinear regression analysis to calculate the IC50 values of the samples (compounds 1-20 synthesized in the present invention).

[0188]

[0189] The experimental results are shown in Table 1 and Table 2 (in Table 1, in the column where IC 50 is located, A: IC 50 < 200 nM, B: IC 50 = 200 - 500 nM, C: IC 50 = 500 - 1000 nM, D: IC 50 > 1000 nM). The compounds in the examples all have inhibitory activity against 3CL pro Among them, compounds 1, 8, 13, 16, and 20 have stronger inhibitory effects on 3CL pro , and the IC 50 values are all below 200 nM.

[0190] Table 1 Inhibitory activity of compounds 1-20 against 3CL pro

[0191]

[0192] ​

[0193] Table 2 Inhibitory activities of Compound 1, 8, 16, S-217622 and PF-07321332 against 3CL pro

[0194]

[0195] The data in Table 1-2 show that Compounds 1-20 have inhibitory effects on 3CL to varying degrees. Compounds 1, 8, 13, 16, and 20 have IC pro values against 3CL pro all less than 200 nM. All 20 N-(tert-butyl)-2-(N-acetamido)formamide compounds tested have 3CL 50 inhibitory activity, and at high concentrations, the inhibitory effect of Compound 1 is better than that of the control group PF-07321332. pro Figure 13 - 15 Shown are the inhibitory effects of Compounds 1, 8, and 16 against 3CL pro respectively, among which Compound 1 has the best inhibitory activity (IC 50 = 103.01 nM) and can be developed and applied as an anti-coronavirus drug.

[0196] (2) 3CL pro Cytotoxicity test

[0197] The MTT method was used to evaluate the in vitro cytotoxicity of Compounds 1, 8, and 16. HepG2, HEK293, and A549 cells in the logarithmic growth phase were taken, digested with trypsin to make cell suspensions, and the cell density was adjusted to 5×10 4 cells / mL. 180 μL per well was inoculated into a sterile 96-well cell culture plate and cultured in a 37°C, 5% CO2 constant temperature incubator for 24 h. After the cells adhered to the bottom of the well plate, 20 μL of the drug solution with a concentration gradient was added to each well, and 6 parallel replicates were set. At the same time, a zero-adjustment group (without cells and drugs) and a control group (without drugs) were set. After incubation in a 37°C, 5% CO2 constant temperature incubator for 24 h, 20 μL of 5 g / L MTT solution was added to each well and cultured for another 4 h. After the culture was completed, the culture medium in the wells was gently aspirated, 100 μL of DMSO was added to each well, and the plate was placed on a shaker and shaken at a low speed for 10 min to fully dissolve the crystals. Then, the absorbance of each well was measured at OD 490 nm of the enzyme-linked immunosorbent detector, and the cell survival rate was calculated. The results are shown in Tables 3-5, and Figure 16 - Figure 18 .

[0198]

[0199] Table 3 Tested drugs on A549 cells​​

[0200]

[0201] Table 4 Test drug on HEK293 cells

[0202]

[0203] Table 5 Test drug on HepG2 cells

[0204]

[0205] Tables 3 - 5 respectively show the inhibition rates of PF - 07321332, S - 217622, representative compounds 1, 8, and 16 on A549 cells, HEK293, and HepG2 cells at different concentrations. From the above data and Figure 16 - Figure 18 , it can be known that the inhibition rates of compound 1 on A549 cells, HepG2 cells, and HEK293 cells are lower than those of PF - 07321332 and S - 217622 at any concentration. The cytotoxicities of compound 8 and compound 16 on the three tested cells are slightly higher than those of PF - 07321332 and S - 217622 at the tested concentrations. Based on the above data, the tested compound 1 has good inhibitory activity against 3CL protease and low toxicity, and can be further studied.

[0206] The above content is only to illustrate the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any modification made on the basis of the technical solution according to the technical idea proposed by the present invention falls within the protection scope of the claims of the present invention.

Claims

1. A compound of N-(tert-butyl)-2-(N-acetamido)formamide, characterized in that, It is a compound of Formula 1: 。 2. The preparation method of the N-(tert-butyl)-2-(N-acetamido) formamide compound according to claim 1, characterized in that, Performing a Ugi reaction on 3-thiophenecarboxaldehyde, 4-aminodiphenylmethane, pyrazine-2-carboxylic acid, and tert-butyl isocyanate in a solvent to obtain an N-(tert-butyl)-2-(N-acetamido)formamide compound.

3. The preparation method of the N-(tert-butyl)-2-(N-acetamido) formamide compound according to claim 2, characterized in that, The molar ratio of 3-thiophenecarboxaldehyde, 4-aminodiphenylmethane, pyrazine-2-carboxylic acid, and tert-butyl isocyanate is 1:1:1:

1.

4. The preparation method of the N-(tert-butyl)-2-(N-acetamido)formamide compound according to claim 2, characterized in that, Specifically, it includes the following steps: Adding 3-thiophenecarboxaldehyde and 4-aminodiphenylmethane into a methanol solvent, reacting at room temperature for 30 min, then adding pyrazine-2-carboxylic acid and tert-butyl isocyanate, and continuing to react at room temperature for 8 - 14 h. After separation and purification, the N-(tert-butyl)-2-(N-acetamido)formamide compound shown in Formula 1 is obtained.

5. Use of the N-(tert-butyl)-2-(N-acetamido)formamide compound according to claim 1 in the preparation of a coronavirus 3CL protease inhibitor.

6. Use of the N-(tert-butyl)-2-(N-acetamido)formamide compound according to claim 1 in the preparation of an anti-coronavirus drug.

7. The application according to claim 5 or 6, characterized in that, The coronavirus is the novel coronavirus SARS-CoV-2.

8. The application according to claim 5 or 6, characterized in that, The inhibitor or drug is in the form of tablets, capsules, granules, syrups, pellets, liniments, or injections.

9. The application according to claim 5 or 6, characterized in that, The inhibitor or drug is a premix.

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