A methylisoindole derivative as an Mpro inhibitor and its application in antiviral drugs

Abstract

This invention discloses an isoindigo derivative as an Mpro inhibitor and its application in antiviral drugs, having a structure as shown in Formula I or a pharmaceutically acceptable salt thereof. The isoindigo derivative of this invention can effectively inhibit coronavirus Mpro, and can serve as a novel and potent coronavirus Mpro inhibitor with high activity, good stability, and a simple preparation method, showing great promise for application in the preparation of antiviral drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a methylisoindole derivative as an Mpro inhibitor and its application in antiviral drugs. Background Technology

[0002] Developing effective and broad-spectrum anti-coronavirus drugs is key to defeating the coronavirus. However, coronaviruses are highly diverse and mutate rapidly, making drug development extremely challenging.

[0003] Currently, the approved small molecule drugs are Pfizer's Paxlovid and Merck's Molnupinavir. Paxlovid can only be used for mild and moderate cases within 5 days of onset, and for high-risk adult patients with a risk of progressing to severe illness; moreover, the drug is expensive. Oral anti-COVID-19 drugs under development are also mainly targeted at mild to moderate cases, with primary targets including coronavirus Mpro, PLpro, RNA polymerase (RdRp), and androgen receptor (AR) antagonists. With the continuous emergence of SARS-CoV-2 mutant strains, drug resistance will inevitably become a major problem in future SARS-CoV-2 treatment, necessitating the development of broad-spectrum, highly effective novel anti-coronavirus drugs.

[0004] Currently, the Mpro protein in coronaviruses is receiving considerable attention. The Mpro protein is a cysteine ​​protease encoded by the Nsp5 gene. In the early stages of coronavirus invasion of host cells, Mpro is responsible for cleaving the viral polyproteins pp1a and pp1ab at 11 different sites, allowing the virus to complete normal transcription and replication. Therefore, Mpro plays a crucial role in viral proliferation. Inhibiting Mpro activity can effectively suppress SARS-CoV-2 replication and infection in humans, and is a major strategy in the discovery of anti-SARS-CoV-2 drugs.

[0005] Several Mpro inhibitors have been reported, mainly small molecules or short peptides. Important advances include: Ebselen, a known drug obtained through high-throughput screening, and N3, a computer-aided design, which effectively inhibit Mpro activity and SARS-CoV-2 virus replication; 13b, designed based on α-ketoamide inhibitors, exhibiting good Mpro enzyme activity inhibition, viral replication inhibition, and pharmacokinetic properties; and 11a and 11b, designed based on the Mpro active pocket, which can covalently bind to Mpro and have better IC50 and EC50 values. FRET and in vitro experiments revealed that Boceprevir, GC-376, calpain inhibitor II, and XII also exhibited Mpro inhibitory effects and anti-SARS-CoV-2 activity. Cinnamyl thiamine, obtained through computer-generated virtual screening, also showed some inhibitory activity against SARS-CoV-2. In a recent report, researchers designed and synthesized 32 new Mpro inhibitors based on the structures of Mpro and its complexes with Boceprevir or Telaprevir. All compounds exhibited good SARS-CoV-2 Mpro enzyme inhibitory activity, with IC50 values ​​ranging from 7.6 to 748.5 nM. Among them, MI-09 and MI-30 showed excellent antiviral activity in cell and mouse models and demonstrated good pharmacokinetic properties and safety in rats.

[0006] Indigofera methylbenzene is a derivative of indirubin, the active ingredient in the traditional Chinese medicine Indigofera tinctoria. It exhibits good in vitro and in vivo inhibitory activity against various leukemia cell lines. Indigofera methylbenzene has been used clinically to treat CML (Chronic Myelocytic Leukemia) and also shows some efficacy against APL (Acute Promyelocytic Leukemia) and AML. Currently, there are no reports on the use of indigofera methylbenzene derivatives for antiviral therapy. Summary of the Invention

[0007] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a methylisoindigo derivative.

[0008] The present invention also proposes a method for preparing the above-mentioned isoindigo derivative.

[0009] The present invention also proposes an application of the above-mentioned isoindigo derivative.

[0010] The present invention also proposes a composition.

[0011] The present invention also proposes a medicine box for treating coronavirus.

[0012] In a first aspect of the invention, a methylisoindigo derivative or a pharmaceutically acceptable salt thereof is provided, said methylisoindigo derivative having a structure as shown in Formula I or a pharmaceutically acceptable salt thereof:

[0013]

[0014] Among them, R 1 R 4 Selected independently from H and C1-C 20 Straight-chain or branched alkyl, C3-C6 alkynyl; wherein, the C1-C 20 Straight-chain or branched alkyl groups are optionally surrounded by 0, 1, 2 or 3 R groups. a replace;

[0015] R a The groups are independently selected from Br, Cl, F, hydroxyl, amino, carboxyl, C1-C3 alkyl-substituted piperazine ring, and C1-C3 alkyl-substituted morpholine ring;

[0016] R 2 R 3 Each group is independently selected from H, halogen, nitro, hydroxyl, carboxyl, C1-C5 straight-chain or branched alkyl, C1-C5 straight-chain or branched alkoxy, and C1-C5 straight-chain or branched heterocyclic groups; wherein the heterocyclic group refers to phenyl, pyridinyl, furanyl, thiophene, imidazolyl, thiazolyl, oxazolyl, pyrazinyl, and piperazinyl.

[0017] X is selected from CH2, O, NH, S, NOR, NOH, NNHR, and R is a C1-C5 straight-chain or branched alkyl group;

[0018] X1 and X2 form carbon-carbon double bonds or saturated six-membered rings.

[0019] In some embodiments of the present invention, the isoindigo derivative has a structure as shown in either Formula I or Formula II, or a pharmaceutically acceptable salt thereof:

[0020]

[0021] Among them, R 1 R 4 Selected independently from H and C1-C 20 Straight-chain or branched alkyl, C3-C6 alkynyl; wherein, the C1-C 20 Straight-chain or branched alkyl groups are optionally surrounded by 0, 1, 2 or 3 R groups. a replace;

[0022] R aThe groups are independently selected from Br, Cl, F, hydroxyl, amino, carboxyl, C1-C3 alkyl-substituted piperazine ring, and C1-C3 alkyl-substituted morpholine ring;

[0023] R 2 R 3 Each group is independently selected from H, halogen, nitro, hydroxyl, carboxyl, C1-C5 straight-chain or branched alkyl, C1-C5 straight-chain or branched alkoxy, and C1-C5 straight-chain or branched heterocyclic groups; wherein the heterocyclic group refers to phenyl, pyridinyl, furanyl, thiophene, imidazolyl, thiazolyl, oxazolyl, pyrazinyl, and piperazinyl.

[0024] R 2 R 3 The positions on the benzene ring are independently selected from the C4, C5, C6, and C7 positions, respectively;

[0025] X is selected from CH2, O, NH, S, NOR, NOH, NNHR, and R is a C1-C5 straight-chain or branched alkyl group.

[0026] In some embodiments of the present invention, the halogen includes at least one of Br, Cl and F.

[0027] In some embodiments of the present invention, the isoindigo derivative has a structure as shown in formula (III):

[0028]

[0029] Among them, R 1 R 4 Selected independently from H and C1-C 20 Straight-chain or branched alkyl, C3-C6 alkynyl; wherein, the C1-C 20 Straight-chain or branched alkyl groups are optionally surrounded by 0, 1, 2 or 3 R groups. a replace;

[0030] R a The groups are independently selected from Br, Cl, F, hydroxyl, amino, carboxyl, C1-C3 alkyl-substituted piperazine ring, and C1-C3 alkyl-substituted morpholine ring;

[0031] Preferably, the methylisoindigo derivative is one of the following compounds: 2b-2e, 2h, 2i, 2k, 2p, 2q, 2s, 2u, and 2v.

[0032]

[0033]

[0034] In some embodiments of the present invention, the isoindigo derivative has a structure as shown in formula (Ⅳ):

[0035]

[0036] Among them, R 1 Selected from H, C1-C 20 Straight-chain or branched alkyl, C3-C6 alkynyl; wherein, the C1-C 20 Straight-chain or branched alkyl groups are optionally surrounded by 0, 1, 2 or 3 R groups. a replace;

[0037] R a The groups are independently selected from Br, Cl, F, hydroxyl, amino, carboxyl, C1-C3 alkyl-substituted piperazine ring, and C1-C3 alkyl-substituted morpholine ring;

[0038] R 3 It is selected from H, halogen, nitro, hydroxyl, carboxyl, C1-C5 straight-chain or branched alkyl, C1-C5 straight-chain or branched alkoxy, and C1-C5 straight-chain or branched heterocyclic groups; wherein the heterocyclic group refers to phenyl, pyridinyl, furanyl, thiophene, imidazolyl, thiazolyl, oxazolyl, pyrazinyl, and piperazinyl.

[0039] In some embodiments of the present invention, the isoindigo derivative is one of the following compounds: 2g, 2j, 2l, 2m, 2n, and 2o.

[0040]

[0041]

[0042] In some embodiments of the present invention, the isoindigo derivative has a structure as shown in formula (V):

[0043]

[0044] Among them, R 2 It is selected from H, halogen, nitro, hydroxyl, carboxyl, C1-C5 straight-chain or branched alkyl, C1-C5 straight-chain or branched alkoxy, and C1-C5 straight-chain or branched heterocyclic groups; wherein the heterocyclic group refers to phenyl, pyridinyl, furanyl, thiophene, imidazolyl, thiazolyl, oxazolyl, pyrazinyl, and piperazinyl.

[0045] In some embodiments of the present invention, the isoindigo derivative is one of the following compounds: 2f, 2s, and 2t:

[0046]

[0047] In some embodiments of the present invention, the isoindigo derivative is one of compounds 3a to 3c of the following formulas:

[0048]

[0049] In some embodiments of the present invention, the pharmaceutically acceptable salt is a salt formed from inorganic acids, organic acids, alkali metals, alkaline earth metals, and basic amino acids.

[0050] In some embodiments of the present invention, the inorganic acid includes at least one of hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, and hydrobromic acid.

[0051] In some embodiments of the present invention, the organic acid includes at least one selected from maleic acid, fumaric acid, tartaric acid, lactic acid, citric acid, acetic acid, methanesulfonic acid, p-toluenesulfonic acid, adipic acid, palmitic acid, and tannic acid.

[0052] In some embodiments of the present invention, the alkali metal includes at least one of lithium, sodium, and potassium.

[0053] In some embodiments of the present invention, the alkaline earth metal includes at least one of calcium and magnesium.

[0054] In some embodiments of the present invention, the basic amino acid includes arginine, histidine, or lysine.

[0055] A second aspect of the present invention provides a method for preparing the above-mentioned isoindigo derivative, the method comprising the following steps:

[0056] 1) When X1 and X2 form a double bond, the preparation method of the iso-indigo derivative includes the following steps: adding an indole compound to the compound shown in Formula 1 and reacting to obtain the iso-indigo derivative shown in Formula (I).

[0057]

[0058] R2 and R4 are defined as described above; the indole compounds include 2-indoleone and indole;

[0059] 2) When X1 and X2 form a six-membered ring, the preparation method of the methylisoindigo derivative includes the following steps:

[0060] Reacting the compound shown in Formula 2 with cyclo-butene sulfone yields the isocyanate derivative shown in Formula (II).

[0061]

[0062] The definitions of R1, R2, R3, R4 and X are as described above.

[0063] A third aspect of the present invention proposes the application of the above-mentioned isoindigo derivative, namely, the application of the isoindigo derivative in the preparation of Mpro inhibitors.

[0064] In some embodiments of the present invention, the application is the use of methylisoindole derivatives in the preparation of antiviral drugs.

[0065] In some embodiments of the present invention, the virus is a coronavirus.

[0066] In some embodiments of the present invention, the coronavirus is HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, SARS-CoV, SARS-CoV-2, and MERS-CoV and their mutant strains.

[0067] In some embodiments of the present invention, SARS-CoV-2 includes variants such as alpha, beta, gamma, delta, ..., ormicron, 1.1, 1.2, 4, 5, etc.

[0068] In some embodiments of the present invention, the raw materials for preparing the drug also include a pharmaceutical carrier.

[0069] In some embodiments of the present invention, the pharmaceutical carrier is a conventional drug carrier in the pharmaceutical field.

[0070] In some embodiments of the present invention, the pharmaceutical carrier includes at least one of a diluent, excipient, filler, binder, disintegrant, absorption enhancer, surfactant, adsorbent, lubricant, sweetener, and flavoring agent.

[0071] In some embodiments of the invention, the excipient comprises water.

[0072] In some embodiments of the present invention, the filler includes at least one of starch and sucrose.

[0073] In some embodiments of the present invention, the adhesive includes at least one of cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone.

[0074] In some embodiments of the present invention, the wetting agent includes glycerin.

[0075] In some embodiments of the present invention, the disintegrant includes at least one of agar, calcium carbonate, and sodium bicarbonate.

[0076] In some embodiments of the present invention, the absorption enhancer includes a quaternary ammonium compound.

[0077] In some embodiments of the present invention, the surfactant includes hexadecyl alcohol.

[0078] In some embodiments of the present invention, the adsorbent carrier includes at least one of kaolin and soap clay.

[0079] In some embodiments of the present invention, the lubricant includes at least one of talc, calcium stearate, magnesium stearate, and polyethylene glycol.

[0080] In some embodiments of the present invention, the mass fraction of the isoindigo derivative in the drug is 0.1% to 99%.

[0081] In some embodiments of the present invention, the mass fraction of the isoindigo derivative in the drug is 0.5% to 90%.

[0082] In some embodiments of the present invention, the standard dosage of the novel coronavirus drug is: 0.01-10 mg / kg body weight of methyl isoindigo derivative.

[0083] In some embodiments of the present invention, the standard dosage of the novel coronavirus drug is: 0.1-5 mg / kg body weight of methyl isoindigo derivative.

[0084] In some embodiments of the present invention, the dosage form of the drug is a variety of dosage forms conventional in the art, preferably in the form of a solid, semi-solid or liquid, and may be an aqueous solution, a non-aqueous solution or a suspension, more preferably a tablet, capsule, soft capsule, granule, pill, oral liquid, dry suspension, drop pill, dry extract, injection or infusion.

[0085] In some embodiments of the present invention, the administration method of the drug can be a conventional administration method in the art, including but not limited to injection or oral administration. The injection administration can be via intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection.

[0086] In a fourth aspect of the invention, a composition comprising the above-described isocyanate derivative is provided.

[0087] In a fifth aspect of the invention, a kit for treating coronavirus is provided, wherein the active ingredients include the aforementioned coronavirus Mpro inhibitor and / or the aforementioned anticoronavirus drug.

[0088] In some embodiments of the present invention, the coronavirus is SARS-CoV, SARS-CoV-2, or MERS-CoV. Mutants are also mentioned.

[0089] According to the application described in the embodiments of the present invention, it has at least the following beneficial effects: the isoindigo derivative of the present invention can effectively inhibit coronavirus Mpro, and can be used as a novel and potent coronavirus Mpro inhibitor. It has high activity, good stability, and a simple preparation method, and has good application prospects in the preparation of various antiviral drugs. Attached Figure Description

[0090] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0091] Figure 1 This is a graph showing the detection results of the inhibition rate of the methylisoindole derivative against Mpro protein in the test examples of this invention. Detailed Implementation

[0092] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0093] Example 1

[0094] This embodiment prepared a methylisoindigo derivative. The reaction process for preparation is shown below, and the specific synthesis process is as follows:

[0095]

[0096] 1. Synthesis of compound 2a-2v

[0097] (1) Synthesis of methylisoindigo 2a, the synthesis steps are as follows:

[0098] Step 1: Weigh indigo 1 (1.47 g, 10 mmol) and Cs₂CO₃ (4.89 g, 15 mmol) into a 100 mL round-bottom flask, add DMF (30 mL), and add chloropropyne (0.87 mL, 12 mmol) dropwise at 0 °C. Then, move the flask to room temperature and stir. After about 6 hours of reaction, monitor the reaction with TLC until the starting material has completely reacted. Quench the reaction with 80 mL of water, then extract three times with CH₂Cl₂ (40 mL × 3), wash with saturated NaCl (40 mL × 3), and combine the organic phases. Dry the organic layer with anhydrous Na₂SO₄, concentrate under reduced pressure to remove CH₂Cl₂, and obtain the crude product. The crude product does not require purification and can be used directly in the next step.

[0099] Step 2: Add 1.31 g of 2-indolone (1.31 g, 10 mmol) and glacial acetic acid (30 mL) to the crude product (1.61 g, 10 mmol) obtained in Step 1, followed by two drops of concentrated hydrochloric acid. Heat to 120 °C and reflux with stirring for 12 hours. A red solid precipitates out. Filter to remove the reaction solution. Wash the red solid with EtOH to obtain the target compound 2a.

[0100]

[0101] (E)-1-methyl-[3,3'-biindolinylidene]-2,2'-dione(2a):

[0102] Red solid, 2.0 g, 73%. 1 H NMR (500MHz, DMSO-d6) δ10.97 (s, 1H), 9.07 (dt, J = 7.5, 3.2 Hz,2H),7.44–7.38(m,1H),7.33(t,J=7.9Hz,1H),6.98(dq,J=21.7,8.0Hz,3H),6.84(d,J=7.9Hz,1H),3.20(d,J=3.0Hz,3H).

[0103] (2) The synthesis of the isoindigo derivative 2b is performed according to the steps in 2a.

[0104]

[0105] (E)-1-(prop-2-yn-1-yl)-[3,3'-biindolinylidene]-2,2'-dione(2b):

[0106] Red solid, 990 mg, 33%. 1 H NMR (500MHz, DMSO-d6) δ10.95(s,1H),9.11(d,J=8.0Hz, 1H),9.02(d,J=8.1Hz,1H),7.47(t,J=7.7Hz,1H),7.35(t,J=7.7Hz,1H),7.15–7.05 (m, 2H), 6.97 (t, J = 7.8Hz, 1H), 6.84 (d, J = 7.7Hz, 1H), 4.64 (s, 2H), 3.37 (s, 1H).

[0107] (3) The synthesis of the isoindigo derivative 2c is performed according to the steps in 2a.

[0108]

[0109] (E)-1-(but-3-yn-1-yl)-[3,3'-biindolinylidene]-2,2'-dione(2c):

[0110] Red solid, 660 mg, 10%. 1H NMR (500MHz, DMSO-d6) δ10.90 (s, 1H), 9.06 (dd, J = 23.7, 8.0Hz,2H),7.36(dt,J=29.4,7.6Hz,2H),7.11(d,J=7.8Hz,1H),7.02(t,J=7.8Hz,1H) ,6.95(t,J=7.8Hz,1H),6.82(d,J=7.7Hz,1H),3.91(t,J=6.9Hz,2H),2.84(t,J=2.5Hz, 1H),2.61–2.53(m,2H).

[0111] (4) Synthesis of methylisoindole derivative 2d, the synthesis steps are the same as those in 2a.

[0112]

[0113] (E)-1-(pent-4-yn-1-yl)-[3,3'-biindolinylidene]-2,2'-dione(2d):

[0114] Red solid, 1.25 g, 38%. 1 H NMR (500MHz, DMSO-d6) δ10.89 (s, 1H), 9.07 (dd, J = 17.5, 8.0Hz,2H),7.40(s,1H),7.33(s,1H),7.07–6.98(m,2H),6.95(s,1H),6.82(d,J=7.7H z,1H),3.81(d,J=7.3Hz,2H),2.84(d,J=2.4Hz,1H),2.26(d,J=7.1Hz,2H),1.79(t,J= 7.2Hz,2H).

[0115] (5) The synthesis of the isoindigo derivative 2e is performed according to the steps in 2a.

[0116]

[0117] (E)-1-methyl-1'-(prop-2-yn-1-yl)-[3,3'-biindolinylidene]-2,2'-dione(2e):

[0118] Red solid, 910 mg, 29%. 1H NMR(400MHz,DMSO-d6)δ9.11(d,J=8.0Hz,1H),9.02(d,J=8.0Hz,1H),7.50–7.38(m,2H ),7.13–6.94(m,4H),4.61(d,J=2.5Hz,2H),3.33–3.30(m,1H),3.17(d,J=2.1Hz,3H).

[0119] (5) Synthesis of methylisoindole derivative 2f, the synthesis steps are the same as those in 2a.

[0120]

[0121] (E)-5'-bromo-1-methyl-[3,3'-biindolinylidene]-2,2'-dione(2f)

[0122] Red solid 1 H NMR (600MHz, DMSO-d6) δ 11.05 (s, 1H), 9.31 (d, J = 21.5Hz, 1H), 9.09 (s, 1H), 7.48 (d, J = 42.2Hz, 2H), 7.02 (s, 2H), 6.80 (s, 1H), 3.22 (s, 3H).

[0123] (6) The synthesis of 2g of methylisoindole derivative is as follows: the synthesis steps are the same as those in 2a.

[0124]

[0125] (E)-5-bromo-1-methyl-[3,3'-biindolinylidene]-2,2'-dione(2g)

[0126] Red solid, 0.43 g, 52%; 1 H NMR (600MHz, DMSO-d6) δ10.88 (d, J=2.7Hz, 1H), 9.25(d,J=2.1Hz,1H),9.10–8.86(m,1H),7.52(dd,J=8.4,2.7Hz,1H),7.32(td,J=7. 8, 2.3Hz, 1H), 6.99–6.85 (m, 2H), 6.79 (dd, J = 7.8, 2.4Hz, 1H), 3.14 (d, J = 2.5Hz, 3H).

[0127] (7) Synthesis of methylisoindole derivative 2h, the synthesis steps are the same as those in 2a.

[0128]

[0129] (E)-1-(2-bromoethyl)-[3,3'-biindolinylidene]-2,2'-dione(2h)

[0130] Red solid, 0.61 g, 42%; 1 H NMR (400MHz, DMSO-d6) δ10.90(s,1H),9.09(dd,J=8.1,1.2Hz,1H),9.02(d,J=8.0Hz,1H),7.41(td,J=7.7,1.2Hz,1H),7.34(td,J=7.6,1.3 Hz,1H),7.14(d,J=7.8Hz,1H),7.07–7.00(m,1H),6.99–6.92(m,1H),6.83(d,J=7.4Hz,1H),4.20(t,J=6.5Hz,2H),3.76(t,J=6.5Hz,2H).

[0131] (8) Synthesis of methylisoindole derivative 2i, the synthesis steps are the same as those in 2a.

[0132]

[0133] (E)-1-(2-(4-methylpiperazin-1-yl)ethyl)-[3,3'-biindolinylidene]-2,2'-dione(2i)

[0134] Dark red solid, 240 mg, 50%; 1 H NMR (600MHz, CDCl3) δ9.13(dd,J=8.1,1.2Hz,1H),9.07(dd,J=8.1,1.2Hz,1H),8.56(s,1H),7.34(td,J=7.7,1.2Hz,1H),7.28–7.19(m,1H),7.01(td d,J=7.5,5.8,1.1Hz,2H),6.82(d,J=7.8Hz,1H),6.75(dd,J=7.7,1.1Hz,1 H), 3.92 (t, J = 7.2Hz, 2H), 2.68 (t, J = 7.2Hz, 2H), 2.48 (s, 4H), 2.28 (s, 3H).

[0135] (9) Synthesis of methylisoindole derivative 2j, the synthesis steps are the same as those in 2a.

[0136]

[0137] (E)-5-methoxy-1-methyl-[3,3'-biindolinylidene]-2,2'-dione(2j)

[0138] Dark red solid, 912 mg, 75%; 1 H NMR(400MHz, DMSO-d6)δ10.86(s,1H),9.09(d,J=8.1Hz,1H),8.90–8.80(m,1H),7.33(t,J=7.8 Hz, 1H), 6.98 (dd, J = 16.1, 8.1 Hz, 2H), 6.85 (dd, J = 16.9, 8.1 Hz, 2H), 3.74 (s, 3H), 3.16 (s, 3H).

[0139] (10) Synthesis of the isoindigo derivative 2k, the synthesis steps are the same as in 2a.

[0140]

[0141] (E)-6-(2,2'-dioxo-[3,3'-biindolinylidene]-1-yl)hexanoic acid(2k)

[0142] Red solid, 82 mg, 73%; 1 H NMR (400MHz, DMSO-d6) δ12.01(s,1H),10.91(s,1H),9.12–9.02(m,2H),7.40(td,J=7.7,1.2Hz,1H),7.34(td,J=7.6, 1.2Hz, 1H), 7.01 (ddd, J=8.8, 7.8, 1.9Hz, 2H), 6.96 (td, J=7.8, 1.2Hz, 1H), 6.83 (dd, J=7.8, 1.0Hz, 1H), 3.73 (t, J=7.1 Hz, 2H), 2.20 (t, J = 7.3 Hz, 2H), 1.66–1.49 (m, 2H), 1.33 (ddd, J = 12.3, 7.6, 4.6 Hz, 2H).

[0143] (11) Synthesis of methylisoindole derivative 2l, the synthesis steps are the same as those in 2a.

[0144]

[0145] (E)-5-chloro-1-methyl-[3,3'-biindolinylidene]-2,2'-dione(2l)

[0146] Red solid, 74 mg, 50%;1 H NMR (500MHz, DMSO-d6) δ10.95(s,1H),9.19(d,J=2.3Hz,1H),9.08(d,J=8.0Hz,1H),7.47(dd,J=8.4,2.2Hz,1 H),7.36(td,J=7.6,1.2Hz,1H),7.03(d,J=8.4Hz,1H),6.99–6.91(m,1H),6.84(d,J=8.1Hz,1H),3.20(s,3H).

[0147] (12) The synthesis of the methylisoindole derivative 2m is performed according to the steps in 2a.

[0148]

[0149] (E)-8-(5-bromo-2,2'-dioxo-[3,3'-biindolinylidene]-1-yl)octanoic acid(2m)

[0150] Red solid, 68 mg, 87%; 1 H NMR (600MHz, DMSO-d6) δ12.24–11.69 (m, 1H), 10.93 (s, 1H), 9.31 (d, J = 2.2Hz, 1H),9.05(d,J=8.1Hz,1H),7.56(dd,J=8.4,2.1Hz,1H),7.35(t,J=7.6Hz,1H) ,7.00(d,J=8.4Hz,1H),6.95(t,J=7.8Hz,1H),6.83(d,J=7.7Hz,1H),3.71(t, J=7.3Hz,2H),2.16(t,J=7.4Hz,2H),1.57(t,J=7.2Hz,2H),1.46(p,J=7.4Hz, 2H), 1.31–1.22(m,6H).

[0151] (13) The synthesis of the methylisoindole derivative 2n is performed according to the steps in 2a.

[0152]

[0153] (E)-1-methyl-5-nitro-[3,3'-biindolinylidene]-2,2'-dione(2n)

[0154] Red solid, 320 mg, 67%; 1H NMR (400MHz, DMSO-d6) δ10.99(s,1H),10.08(d,J=2.4Hz,1H),9.05(d,J=8.1Hz,1H),8.31(dd,J=8.7,2.4Hz,1 H),7.38(td,J=7.6,1.2Hz,1H),7.20(d,J=8.8Hz,1H),7.04–6.92(m,1H),6.84(d,J=7.7Hz,1H),3.27(s,3H).

[0155] (14) The synthesis of the iso-indigo derivative 2o is performed according to the steps in 2a.

[0156]

[0157] (E)-8-(5-methoxy-2,2'-dioxo-[3,3'-biindolinylidene]-1-yl)octanoicacid(2o)

[0158] Red solid, 0.35g, 76%; 1 H NMR(400MHz,DMSO-d6)δ11.95(s,1H),10.85(s,1H),9.07 (d,J=8.1Hz,1H),8.84(d,J=2.7Hz,1H),7.31(td,J=7.6,1.2Hz,1H),7.01–6.89(m,2H ),6.84(dd,J=16.7,8.1Hz,2H),3.73(s,3H),3.65(t,J=7.2Hz,2H),2.16(t,J=7.4Hz, 2H), 1.55 (t, J = 7.1Hz, 2H), 1.45 (p, J = 7.3Hz, 2H), 1.31–1.18 (m, 6H).

[0159] (15) The synthesis of the methylisoindole derivative 2p is performed according to the steps in 2a.

[0160]

[0161] (E)-10-(2,2'-dioxo-[3,3'-biindolinylidene]-1-yl)decanoic acid(2p)

[0162] Red solid, 134 mg, 81%; 1H NMR(400MHz,DMSO-d6)δ11.97(s,1H),10.91(s,1H),9.14 –9.02(m,2H),7.39(td,J=7.7,1.2Hz,1H),7.33(td,J=7.6,1.2Hz,1H),7.01(ddd,J=7.3 ,4.1,2.8Hz,2H),6.95(td,J=7.8,1.2Hz,1H),6.83(dd,J=7.8,1.1Hz,1H),3.72(t,J=7.1 Hz, 2H), 2.15 (t, J = 7.3Hz, 2H), 1.58 (t, J = 7.0Hz, 2H), 1.44 (t, J = 7.2Hz, 2H), 1.27 (q, J = 6.2Hz, 5H), 1.21 (s, 7H).

[0163] (16) The synthesis of the methylisoindole derivative 2q is performed according to the steps in 2a.

[0164]

[0165] (E)-8-(2,2'-dioxo-[3,3'-biindolinylidene]-1-yl)octanoic acid(2q)

[0166] Red solid, 65 mg, 79%; 1 H NMR(400MHz,DMSO-d6)δ11.98(s,1H),10.91(s,1H),9.13–9.04(m,2H),7.41(t d,J=7.7,1.2Hz,1H),7.35(td,J=7.6,1.2Hz,1H),7.06–7.00(m,2H),6.97(td, J=7.8,1.2Hz,1H),6.84(dd,J=7.8,1.1Hz,1H),3.74(t,J=7.2Hz,2H),2.17(t, J=7.3Hz,2H),1.60(t,J=7.1Hz,2H),1.47(p,J=7.3Hz,2H),1.37–1.18(m,7H).

[0167] (17) The synthesis of the methylisoindole derivative 2r is performed according to the steps in 2a.

[0168]

[0169] (E)-12-(2,2'-dioxo-[3,3'-biindolinylidene]-1-yl)dodecanoic acid(2r)

[0170] Red solid, 96 mg, 83%; 1 H NMR(500MHz,DMSO-d6)δ11.95(s,1H),10.91(s,1H),9.09(dd,J=12.8,8.0Hz,2H) ,7.42(t,J=7.6Hz,1H),7.35(t,J=7.6Hz,1H),7.07–7.01(m,2H),6.97(t,J=7.7H z,1H),6.85(d,J=7.7Hz,1H),3.76(t,J=7.2Hz,2H),2.17(t,J=7.5Hz,3H),1.62( t,J=7.0Hz,2H),1.50–1.42(m,5H),1.29(d,J=5.0Hz,4H),1.22(d,J=5.6Hz,14H).

[0171] (18) Synthesis of the methylisoindole derivative 2s, the synthesis steps are the same as those in 2a.

[0172]

[0173] (E)-5'-hydroxy-1-methyl-[3,3'-biindolinylidene]-2,2'-dione(2s)

[0174] 1 H NMR (400MHz, DMSO-d6) δ10.57(s,1H),9.07(dd,J=8.1,1.2Hz,2H),8.66(d,J=2.6H z,1H),7.36(td,J=7.7,1.3Hz,1H),7.07–6.87(m,2H),6.79(dd,J=8.3,2.5Hz,1H), 6.63(d,J=8.3Hz,1H),3.19(s,3H).

[0175] (19) The synthesis of the methylisoindole derivative 2t is performed according to the steps in 2a.

[0176]

[0177] (E)-5'-methoxy-1-methyl-[3,3'-biindolinylidene]-2,2'-dione(2t)

[0178] 1H NMR (500MHz, DMSO-d6) δ10.67 (s, 1H), 9.09 (dd, J=8.1, 1.2Hz, 1H), 8.84 (d, J= 2.7Hz,1H),7.39(td,J=7.6,1.2Hz,1H),7.03–6.90(m,3H),6.71(d,J=8.4Hz,1H),3.73(s,3H),3.19(s,3H).

[0179] (20) Synthesis of the methylisoindole derivative 2u, the synthesis steps are the same as in 2a.

[0180]

[0181] (E)-1-(2-morpholinoethyl)-[3,3'-biindolinylidene]-2,2'-dione(2u)

[0182] 1 H NMR (400MHz, DMSO-d6) δ10.91(s,1H),9.16–8.98(m,2H),7.41(td,J=7.7,1.3Hz,1H),7.35(td,J=7.6,1.2Hz,1H),7.11–6. 91 (m, 3H), 6.84 (d, J = 7.3Hz, 1H), 3.89 (t, J = 6.6Hz, 2H), 3.51 (t, J = 4.5Hz, 4H), 2.55 (t, J = 6.6Hz, 2H), 2.46 (t, J = 4.6Hz, 4H).

[0183] (21) The synthesis of the methylisoindole derivative 2v is performed according to the steps in 2a.

[0184]

[0185] (E)-4-(2-(2,2'-dioxo-[3,3'-biindolinylidene]-1-yl)ethyl)-4-iodomorpholin-4-ium-3-ide(2v)

[0186] 1H NMR (400MHz, DMSO-d6) δ10.93 (s, 1H), 9.12 (dd, J=8.1, 1.2Hz, 1H), 9.03 (d, J= 8.0Hz,1H),7.48(td,J=7.7,1.2Hz,1H),7.36(td,J=7.6,1.3Hz,1H),7.24(d,J=7.8H z,1H),7.15–7.05(m,1H),7.03–6.93(m,1H),6.85(d,J=7.7Hz,1H),4.29(t,J=7.3Hz, 2H), 3.98(dt,J=8.0,4.1Hz,4H), 3.81(t,J=7.3Hz,2H), 3.67–3.54(m,4H).

[0187] 2. Synthesis of methylisoindigo derivatives 3a-3d

[0188] (1) Synthesis of methylisoindole derivative 3a, the synthetic steps are as follows:

[0189]

[0190] Under a nitrogen atmosphere, a 1,4-dioxane (10 mL) solution of compound 2a (290 mg, 1.05 mmol) was added to a sealed tube, followed by the sequential addition of cyclo-3-butene sulfone (1.57 g, 12.0 mmol) and hydroquinone (130 mg, 1.05 mmol). The reaction system was heated to 140 °C and reacted for 12 hours. After the reaction was completed by TLC monitoring, the system was cooled to room temperature, and the solvent in the reaction system was concentrated under pressure. The crude product was directly separated by silica gel column chromatography (petroleum ether: ethyl acetate = 2:1) to obtain the target compound 3a (310 mg, 89% yield).

[0191]

[0192] 1-methyldisspiro[indoline-3,1'-cyclohexane-2',3”-indolin]-4’-ene-2,2”-dione(3a)

[0193] Compound 3a: white solid, 310 mg, 89%; 1H NMR(500MHz, CDCl3)δ8.41(s,1H),7.29(d, J=7.5Hz,1H),7.11(t,J=7.8Hz,2H),7.03(t,J=7.7Hz,1H),6.87(dt,J=10.0,7.6Hz,2H),6.63(d ,J=7.7Hz,1H),6.57(d,J=7.8Hz,1H),6.02(d,J=2.3Hz,2H),3.13(s,5H),2.35(t,J=21.7Hz,2H).

[0194] (2) The synthesis of the methylisoindole derivative 3b is performed by referring to the steps in 3a.

[0195]

[0196] Compound 3b: white solid, 100 mg, 21%; 1 H NMR (600MHz, CDCl3) δ8.35 (s, 1H), 7.10 (s,1H),7.03(t,J=7.6Hz,1H),6.94(s,1H),6.85(t,J=7.6Hz,1H),6.63(d,J=8.2H z, 2H), 6.47 (d, J = 8.4Hz, 1H), 6.00 (s, 2H), 3.66 (s, 3H), 3.11 (s, 5H), 2.34 (s, 2H).

[0197] (3) The synthesis of the isoindigo derivative 3c is performed according to the steps in 3a.

[0198]

[0199] Compound 3c: white solid, 90 mg, 65%; 1 H NMR (600MHz, CDCl3) δ8.52(s,1H),7.24(s,1H),7.15(q,J=7.7Hz,2H),6.90(t,J=7.6Hz,1H),6 .62(d,J=7.8Hz,1H),6.51(d,J=8.2Hz,1H),6.01(s,2H),3.16(s,5H),2.38(d,J=28.5Hz,2H).

[0200] Example 2

[0201] This embodiment prepares an injection solution, and the preparation steps are as follows:

[0202] The salt prepared by combining the methylisoindole derivative (one of 2a-2v, 3a-3c) obtained in Example 1 with an organic acid (tartaric acid, citric acid, formic acid, oxalic acid, etc.) or an inorganic acid (hydrochloric acid, sulfuric acid, phosphoric acid, etc.) with water for injection according to conventional methods, followed by fine filtration, filling, and sterilization to prepare an injection solution.

[0203] Example 3

[0204] This embodiment prepares a powder injection, and the preparation steps are as follows:

[0205] The salt prepared by the methylisoindole derivative (one of 2a-2v, 3a-3c) obtained in Example 1 and an organic acid (tartaric acid, citric acid, formic acid, oxalic acid, etc.) or an inorganic acid (hydrochloric acid, sulfuric acid, phosphoric acid, etc.) was dissolved in sterile water for injection, stirred until dissolved, filtered through a sterile suction funnel, then sterilely filtered again, dispensed into 2 ampoules, freeze-dried at low temperature, and then sterilely sealed to obtain a powder for injection.

[0206] Example 4

[0207] This embodiment prepares a powder, and the preparation steps are as follows:

[0208] The salt prepared by combining the isoindigo derivative (one of 2a-2v, 3a-3c) obtained in Example 1 with an organic acid (tartaric acid, citric acid, formic acid, oxalic acid, etc.) or an inorganic acid (hydrochloric acid, sulfuric acid, phosphoric acid, etc.) is added to the excipient in a weight ratio of 9:1 to prepare a powder.

[0209] Example 5

[0210] This embodiment prepares a tablet, and the preparation steps are as follows:

[0211] The salt prepared by Example 1 and organic acids (tartaric acid, citric acid, formic acid, oxalic acid, etc.) or inorganic acids (hydrochloric acid, sulfuric acid, phosphoric acid, etc.) is added to the excipient at a weight ratio of 1:5 to 1:10, and then granulated and compressed into tablets.

[0212] Example 6

[0213] This embodiment prepares an oral liquid, and the preparation steps are as follows:

[0214] The isoindigo derivatives (one of 2a-2v, 3a-3c) prepared in Example 1 and salts made from organic acids (tartaric acid, citric acid, formic acid, oxalic acid, etc.) or inorganic acids (hydrochloric acid, sulfuric acid, phosphoric acid, etc.) are prepared according to conventional oral liquid preparation methods.

[0215] Example 7

[0216] This embodiment prepares a capsule, granule, or powder, and the preparation steps are as follows:

[0217] The salt prepared by Example 1, consisting of an isoindigo derivative (one of 2a-2v, 3a-3c) and an organic acid (tartaric acid, citric acid, formic acid, oxalic acid, etc.) or an inorganic acid (hydrochloric acid, sulfuric acid, phosphoric acid, etc.), is added to the excipient at a weight ratio of 5:1 to make capsules, granules, or powders.

[0218] Example 8

[0219] This embodiment prepares a capsule, granule, or powder, and the preparation steps are as follows:

[0220] The salt prepared by Example 1, consisting of a methylisoindigo derivative (one of 2a-2v, 3a-3c) and an organic acid (tartaric acid, citric acid, formic acid, oxalic acid, etc.) or an inorganic acid (hydrochloric acid, sulfuric acid, phosphoric acid, etc.), is added to the excipient at a weight ratio of 3:1 to make capsules, granules, or powders.

[0221] Test case

[0222] 1. FRET method for determining the inhibition rate of methylisoindole derivatives against Mpro protease

[0223] FRET fluorescence resonance energy transfer was performed using light-protected 96-well plates. The assay was performed with 100 μL of each well containing 10 μM of the compound (2a, 2b, 2f, 2g, 2h, 2i, 2j, 2k, 2m, 2o, 2p, 2q, 2r, 2s, 2t, 2u, 2v, 3a, 3b) at a concentration of 10 μM. Each system contained 10 μM of the compound, 1 μg of Mpro protein, 20 μM of Mpro substrate, and Assay Buffer.

[0224] First, weigh 1.5 mg of compound 2a and dissolve it in μL of DMSO to obtain 10 mM. Then, take 2 μL of 10 mM compound 2a and dissolve it in 98 μL of DMSO to obtain 200 μM.

[0225] The second step involves mixing 837 μL of Assay Buffer and 9 μg of Mpro protein thoroughly, then distributing 93 μL of the mixture evenly into six wells. Three wells are designated for enzyme activity assay, three for compound assay, and three for positive control (Ebselen). Then, distributing another 93 μL of Assay Buffer into three wells as blanks.

[0226] In the third step, add 5 μL of DMSO to the Blank and enzyme activity assay wells, add 5 μL of 200 μM compound 2a to the compound test wells, and add 5 μL of 200 μM Ebselen to the positive control wells. Incubate at 37°C in the dark for 5-10 min.

[0227] In the fourth step, add 2 μL of 1 mM Mpro substrate to each well and incubate at 37°C with shaking at 40-60 rpm in the dark for 15 min.

[0228] The fifth step involves using a microplate reader to detect the fluorescence value. The program is set to excitation wavelength 320 nm and emission wavelength 405 nm. Record the fluorescence value and calculate the inhibition rate: Inhibition rate (%) = (RFU) 100％酶活性对照 -RFU 样品 ) / (RFU 100％酶活性对照 -RFU 空白对照 )×100%.

[0229] Following the above method, the inhibition rate of the synthesized isoindigo and its derivatives against Mpro enzyme activity at a concentration of 10 μM was determined, and the results are as follows:

[0230] Table 1

[0231]

[0232] The results are shown in Table 1 and Figure 1 As shown in the figure, methylisoindole and its derivatives generally have an inhibitory rate on Mpro protein. Among them, compound 2m has an inhibitory rate of 85.06% on Mpro enzyme, and compound 2p has an inhibitory rate of 69.52% on Mpro protein.

[0233] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A methylisoindigo derivative or a pharmaceutically acceptable salt thereof, characterized in that, The methylisoindigo derivative is one of the compounds 2m and 2p: 。 2. The isoindigo derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The pharmaceutically acceptable salt is a salt formed by the methylisoindole derivative with inorganic acids, organic acids, alkali metals, alkaline earth metals, and basic amino acids.

3. The isoindigo derivative or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that, The inorganic acid includes at least one of hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, and hydrobromic acid.

4. The isoindigo derivative or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that, The organic acid includes at least one of maleic acid, fumaric acid, tartaric acid, lactic acid, citric acid, acetic acid, methanesulfonic acid, p-toluenesulfonic acid, adipic acid, palmitic acid, and tannic acid.

5. The isocyanate derivative or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that, The alkali metal includes at least one of lithium, sodium, and potassium.

6. The isocyanate derivative or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that, The alkaline earth metal includes at least one of calcium and magnesium.

7. The isoindigo derivative or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that, The basic amino acids include arginine, histidine, or lysine.

8. A composition, characterized in that, Includes the isoindigo derivatives or pharmaceutically acceptable salts thereof as described in any one of claims 1-7.

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