A pf-00835231 conjugate and pharmaceutical compositions thereof

Abstract

The present application relates to a polyethylene glycol-amino acid-PF-00835231 conjugate or a pharmaceutically acceptable salt thereof, a preparation method thereof and application thereof in preparing an anti-novel coronavirus pneumonia drug. The conjugate can improve drug loading rate and solubility, prolong half-life, slow down metabolic decomposition of the drug, can effectively play a therapeutic effect, and has a good application prospect in treating novel coronavirus pneumonia. Meanwhile, the preparation method of the polyethylene glycol-amino acid-PF-00835231 conjugate has the advantages of short synthesis line, mild reaction condition, high yield, high purity, simple post-treatment, strong substrate adaptability and environmental friendliness.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to a polyethylene glycol-amino acid-PF-00835231 composition, a pharmaceutical composition, its preparation method, and its application. Background Technology

[0002] Researchers are developing a vaccine against SARS-CoV-1M. pro In the process of inhibitory action, PF-00835231 (Formula I-1) was identified as recombinant SARS-CoV-1M in substrate cleavage analysis based on fluorescence resonance energy transfer (FRET). pro Effective inhibitors.

[0003]

[0004] PF-00835231 has also been shown to be effective against recombinant SARS-CoV-2M. pro It exhibits effective inhibitory activity [inhibition constant (K i) = 0.271 nM], with excellent activity and metabolic stability. Because SARS-CoV-1 and SARS-CoV-2 M... pro It has 100% sequence homology at the binding site, so the inhibitory effect is in line with expectations. However, experiments also confirmed that PF-00835231 has poor permeability (Papp < ​​0.207 ± 0.048 × 10-6 cm / sec) and poor oral absorption (1.4 ± 0.8%) [Owen et al., Science 374, 1586–1593 (2021)].

[0005] Currently approved and developing formulations for COVID-19 (SARS-CoV-2) focus on oral formulations. These formulations generally exhibit a first-pass effect. Although PF-00835231 shows activity against SARS-CoV-2 M... pro The virus has excellent inhibitory effects, but its poor water solubility leads to slow absorption, short drug half-life, low permeability of passive absorption in animals, and poor oral absorption. Therefore, PF-00835231 is not suitable for oral formulation. Thus, it is urgent and necessary to develop fast-acting dosage forms such as injections. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a polyethylene glycol-amino acid-PF-00835231 conjugate. This conjugate can improve drug loading rate and solubility, slow down enzyme-mediated drug metabolism, prolong half-life, and slow down drug metabolism and decomposition. Furthermore, by preparing it into an injectable dosage form, it can overcome the defects of low permeability of passive absorption in animals and poor oral absorption of PF-00835231.

[0007] To achieve the above objectives, a first aspect of the present invention provides a polyethylene glycol-amino acid-PF-00835231 conjugate represented by the following formula I-2:

[0008]

[0009] in:

[0010] m represents the number of CH2CH2O molecules in the polyethylene glycol fragment; n represents the number of PEG chains; G represents amino acid residues; m is an integer from 2 to 200, n is an integer from 1 to 4, and k is an integer from 0 to 3.

[0011] In some embodiments, the number-average molecular weight of the polyethylene glycol in formula (I-2) is 100-20000 Daltons.

[0012] In some embodiments, G represents an amino acid residue selected from at least one of glycine, alanine, valine, leucine, isoleucine, methionine, tryptophan, serine, tyrosine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

[0013] In some embodiments, the carbon atom to which the G group is attached is a chiral carbon with an R or S configuration.

[0014] Preferably, the polyethylene glycol-amino acid-PF-00835231 is a single-arm, double-arm, triple-arm, or quadruple-arm type, selected from any one of Formulas II-V:

[0015]

[0016]

[0017] It is understandable that the PF-00835231 structure contains a hydroxymethyl carbonyl group, which can directly combine with the carboxyl group of the end-modified polyethylene glycol-modified amino acid to form an ester bond, thereby achieving effective protection and rational utilization of the PF-00835231 molecule. In this conjugate, each polyethylene glycol end group can be linked to 1-4 PF-00835231 residues, greatly increasing the drug loading rate and solubility. This conjugate does not need to be used in combination with ritonavir to slow down the enzyme-mediated drug metabolism, prolong the half-life, slow down the metabolic breakdown of the drug, and allow high drug concentrations to remain in the patient's body for a longer period of time, thus better combating the virus. Because it uses an injectable formulation, it avoids the shortcomings of low permeability in passive absorption in animals and poor oral absorption of PF-00835231, further enhancing the clinical efficacy and application value of PF-00835231 in antiviral therapy.

[0018] A second aspect of the present invention provides a method for preparing a polyethylene glycol-amino acid-PF-00835231 conjugate, comprising the following steps:

[0019] (1) Modify the hydrophilic polymer polyethylene glycol to introduce the active functional group carboxyl group;

[0020] (2) PEGylated carboxylic acids are bonded to amino groups on amino acids to obtain PEGylated amino acids with exposed carboxyl groups.

[0021] (3) The hydroxyethyl carbonyl group on PF-00835231 reacts with the carboxyl group on the PEGylated amino acid to form an ester.

[0022] Generally, the compounds described herein can be prepared by methods known in the field of chemistry, especially in light of the descriptions contained herein. Certain methods for preparing the compounds described herein are provided as additional features of the embodiments and are illustrated in the reaction schemes and experimental sections provided below.

[0023] Unless otherwise stated, the variables in Scheme A have the same meaning as defined herein.

[0024] Option A:

[0025]

[0026] As illustrated in Scheme A, polyethylene glycol carboxylic acid and amino acid tert-butyl ester undergo a condensation reaction under the action of a condensing agent, followed by hydrolysis under acid to obtain a polyethylene glycol amino acid conjugate. The polyethylene glycol amino acid conjugate is then condensed with PF-00835231 under the action of a condensing agent to obtain a polyethylene glycol-amino acid-PF-00835231 compound. Specific examples can be found in this document.

[0027] Compared with existing technologies, the preparation method of the compound has the following advantages: 1) The synthetic route is shorter, consisting of two steps; 2) The reaction conditions are mild; 3) Post-processing does not require column chromatography purification, and high-yield and high-purity products can be obtained by conventional filtration and drying, which is convenient and fast; 4) The substrate adaptability is strong; 5) Since no column purification is used, only conventional filtration, less wastewater is generated, which is environmentally friendly.

[0028] For some steps of the methods described above for preparing the compounds of the present invention, it is necessary to protect potentially reactive functional groups that are not desired to react, and thus cleave the protecting groups. In such cases, any compatible protecting group can be used.

[0029] All the above-described reactions and preparations of the novel starting materials used in the aforementioned methods are conventional and appropriate reagents and reaction conditions for their performance or preparation, and the procedures for separating the desired products will be well known to those skilled in the art through reference to precedents and examples and preparations thereof.

[0030] A third aspect of the present invention provides a pharmaceutical composition comprising the aforementioned polyethylene glycol-amino acid-PF-00835231 conjugate and a pharmaceutically acceptable carrier, excipient, adjuvant, mediator, or any combination thereof.

[0031] In some embodiments, the combinations of the present invention can be administered in the form of pure compounds or suitable pharmaceutical compositions, using any acceptable route of administration or reagents for similar purposes.

[0032] In some embodiments, the administration method may be selected through oral, intranasal, local, or transdermal delivery, and the form of the drug may be solid, semi-solid, lyophilized powder, or liquid, such as powders, suppositories, injections, solutions, suspensions, ointments, patches, nebulizers, etc.

[0033] In some implementations, a unit dose form suitable for precise dosage administration by injection is used.

[0034] Another aspect of the present invention provides the use of the aforementioned polyethylene glycol-amino acid-PF-00835231 conjugate in the preparation of drugs against COVID-19 (SARS-CoV-2).

[0035] The present invention provides a novel polyethylene glycol-amino acid-PF-00835231 conjugate, which improves drug loading rate and solubility, prolongs half-life, and slows down drug metabolism and decomposition, thus effectively achieving a therapeutic effect. Therefore, the polyethylene glycol-amino acid-PF-00835231 conjugate of the present invention has good application prospects in the treatment of COVID-19. Furthermore, the preparation method of the above-mentioned polyethylene glycol-amino acid-PF-00835231 conjugate of the present invention has the advantages of short synthetic route, mild reaction conditions, high yield, high purity, simple post-processing, strong substrate adaptability, and environmental friendliness.

[0036] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0037] In this invention, the terms "compound A" and "compound represented by formula A" refer to the same compound. Detailed Implementation

[0038] To enable those skilled in the art to better understand the technical solutions of the present invention, some non-limiting embodiments are further disclosed below to provide a more detailed description of the present invention.

[0039] All reagents used in this invention are commercially available or can be prepared by the methods described herein. Specifically, PF-00835231 used in the examples was purchased from Suzhou Opto New Materials Co., Ltd., amino acids and their derivatives were purchased from Jier Biochemical (Shanghai) Co., Ltd., polyethylene glycol and its derivatives were synthesized, and the remaining reagents were commercially available.

[0040] In this invention, TLC represents thin-layer liquid chromatography.

[0041] In this invention, DMF represents N,N-dimethylformamide; DCC represents dicyclohexylcarbodiimide; DMAP represents 4-dimethylaminopyridine; and HOBt represents 1-hydroxybenzotriazole.

[0042] Example 1: Preparation of a single-arm polyethylene glycol (number average molecular weight 2000)-glycine-PF-00835231 conjugate (PF-1)

[0043] 20.0 g (10 mmol) of single-arm polyethylene glycol acetic acid (EPEG2000-CH2COOH) was dissolved in 200 mL of DMF solvent, followed by the addition of 2.45 g (20 mmol) of DMAP and 2.7 g (20 mmol) of HOBt. After complete dissolution, 4.12 g (20 mmol) of DCC was added. After stirring at room temperature for 30 min, 3.9 g (30 mmol) of glycine tert-butyl ester was added, and the reaction was stirred at room temperature overnight. The reaction progress was monitored by TLC and ninhydrin was used for colorimetric tracking. After the reaction was completed, the insoluble solid was filtered off, and 2500 mL of isopropanol was slowly added to the residue, followed by freezing at low temperature for half an hour. After filtration, a white solid was obtained. The white solid was dissolved in a small amount of dichloromethane and then slowly added to ice-cold methyl tert-butyl ether to precipitate a white solid. The white solid product was dried under vacuum. The intermediate product was dissolved in 200 mL of acetonitrile and 5 mL of water was added. 1 mL of trifluoroacetic acid was added at 0 °C, and the reaction was carried out at room temperature for 24 h. The reaction endpoint was monitored by NMR. After the reaction was complete, the reaction solution was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. 2500 mL of ice-cold isopropanol was added to the residue, precipitating a large amount of white solid. This solid was filtered, and the product was dried under vacuum to obtain 14.5 g of single-arm polyethylene glycol (2000)-glycine. The yield was 72.5%. 1 H NMR (400MHz, DMSO-d6)δ:5.43(s,1H),4.09(s,2H),3.69(s,2H),3.65-3.60(m,184H), 2.52(s,2H),1.13(s,3H).

[0044] The single-arm polyethylene glycol (2000)-glycine (2.12 g, 1 mmol), HOBt (0.27 g, 2 mmol), and DMAP (0.244 g, 2 mmol) were added sequentially to a reaction flask and dissolved in a mixed solvent of dichloromethane and DMF. The mixture was cooled in an ice bath under nitrogen protection for 30 min. Then, 0.412 g (2 mmol) of DCC in DMF solution and 0.94 g (2 mmol) of PF-00835231 compound were added dropwise. After the addition was complete, the mixture was allowed to rise to room temperature and reacted for 16 hours. The reaction solution was concentrated, and the insoluble solids were removed by filtration. The residue was dissolved in 1000 ml of dichloromethane and frozen at low temperature for 30 min. The insoluble solids were filtered off again, and the solvent was concentrated and slowly poured into ice-cold isopropanol for precipitation and crystallization, yielding 1.62 g of a white solid single-arm polyethylene glycol (number average molecular weight 2000)-glycine-PF-00835231 conjugate (PF-1). The yield was 75.3%. 1H NMR(400MHz, DMSO-d6)δ:10.92-10.58(m,1H),8.46 (d,J=7.4Hz,1H),8.34(d,J=7.2Hz,1H),7.64(s,1H),7.32(d,J=2.4Hz,1H),7.12(t, J=8.2Hz,1H),7.02(d,J=7.4Hz,1H),6.52(d,J=8.0Hz,1H),5.40(s,1H),4.52(dd,J= 9.2,5.4Hz,1H),4.32(dt,J=10.4,7.2Hz,1H),4.11(s,2H),3.82(s,3H),3.68(s,2H), 3.64-3.60(m,187H),3.21-3.02(m,2H),2.52(s,2H),2.42-2.26(m,1H),2.18-2.02( m,2H),1.76-1.56(m,5H),1.14(s,3H),0.94(d,J=6.4Hz,3H),0.88(d,J=6.2Hz,3H).

[0045] Example 2: Preparation of a single-arm polyethylene glycol (number average molecular weight 5000)-tryptophan-PF-00835231 conjugate (PF-2)

[0046] 50.0 g (10 mmol) of single-arm polyethylene glycol propionic acid (EPEG5000-CH2CH2COOH) was dissolved in 500 mL of DMF solvent, followed by the addition of 2.45 g (20 mmol) of DMAP and 2.7 g (20 mmol) of HOBt. After complete dissolution, 4.12 g (20 mmol) of DCC was added. After stirring at room temperature for 60 min, 8.88 g (30 mmol) of tryptophan tert-butyl ester was added, and the reaction was stirred overnight at room temperature. The reaction progress was monitored by TLC and ninhydrin was used for colorimetric tracking. After the reaction was completed, the insoluble solid was filtered off, and 5000 mL of isopropanol was slowly added to the residue, followed by freezing at low temperature for half an hour. After filtration, a white solid was obtained. The white solid was dissolved in a small amount of dichloromethane and then slowly added to ice-cold methyl tert-butyl ether to precipitate a white solid. The white solid product was dried under vacuum. The intermediate product was dissolved in 400 mL of acetonitrile and 5 mL of water was added. 1 mL of trifluoroacetic acid was added at 0 °C, and the reaction was carried out at room temperature for 24 h. The reaction endpoint was monitored by NMR. After the reaction was complete, the reaction solution was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. 5000 mL of ice-cold isopropanol was added to the residue, precipitating a large amount of white solid. This solid was filtered, and the product was dried under vacuum to obtain 44.5 g of single-arm polyethylene glycol (5000)-tryptophan. The yield was 87.2%. 1H NMR (400MHz, DMSO-d6)δ:7.72(s,1H),7.63-7.47(m,1H),7.41-7.24(m,1H),7.22-7.07 (m,2H),7.04-6.82(m,1H),6.31(s,1H),4.71(s,1H),3.61(q,J=15.0Hz,2H),3.56– 3.52(m,462H),3.38–3.25(m,1H),3.13-2.91(m,1H),2.54(q,J=15.0Hz,2H),1.13 (q,J=11.8Hz,3H).

[0047] Single-arm polyethylene glycol (5000)-tryptophan (5.22 g, 1 mmol), HOBt (0.27 g, 2 mmol), and DMAP (0.244 g, 2 mmol) were added sequentially to a reaction flask and dissolved in a mixture of dichloromethane and DMF. The mixture was cooled in an ice bath under nitrogen protection for 30 min. Then, 0.412 g (2 mmol) of DCC in DMF solution and 0.94 g (2 mmol) of PF-00835231 compound were added dropwise. After the addition was complete, the mixture was allowed to rise to room temperature and reacted for 16 hours. The reaction solution was concentrated, and insoluble solids were removed by filtration. The residue was dissolved in 1000 ml of dichloromethane and frozen at low temperature for 30 min. The insoluble solids were filtered off again. The solvent was concentrated and slowly poured into ice-cold isopropanol for precipitation and crystallization, yielding 4.12 g of a white solid single-arm polyethylene glycol (number average molecular weight 5000)-tryptophan-PF-00835231 conjugate (PF-2). The yield was 66.5%. 1H NMR(400MHz, DMSO-d6)δ:10.63–10.50(m,1H), 8.52(d,J=8.0Hz,1H),8.34(d,J=7.0Hz,1H),7.72(s,1H),7.65(s,1H),7.62-7.47(m, 1H),7.41-7.24(m,1H),7.35(d,J=1.6Hz,1H),7.22-7.08(m,2H),7.08(t,J=8.0 Hz,1H),7.04-6.82(m,1H),7.02(d,J=7.2Hz,1H),6.52(d,J=7.8Hz,1H),6.32(s, 1H),4.72(s,1H),4.52(ddd,J=10.1,8.2,5.0Hz,1H),4.36(ddt,J=11.4,7.9,3.3Hz, 1H),3.88(s,3H),3.62-3.61(m,6H),3.58–3.52(m,462H),3.35-3.22(m,1H),3.18 –2.92(m,3H),2.56(q,J=12.0Hz,2H),2.44–2.29(m,1H),2.20–2.02(m,2H), 1.74–1.53(m,5H),1.12(q,J=10.8Hz,3H),0.93(d,J=6.4Hz,3H),0.89(d,J=6.4Hz,3H).

[0048] Example 3: Preparation of a two-arm polyethylene glycol (number average molecular weight 5000)-leucine-PF-00835231 conjugate (PF-3)

[0049] 50.0 g (10 mmol) of bi-arm polyethylene glycol propionic acid (HOOCCH2CH2PEG5000-CH2CH2COOH) was dissolved in 500 mL of DMF solvent, followed by the addition of 4.9 g (40 mmol) of DMAP and 5.4 g (40 mmol) of HOBt. After complete dissolution, 8.24 g (40 mmol) of DCC was added. After stirring at room temperature for 30 min, 6.69 g (30 mmol) of leucine tert-butyl ester was added, and the reaction was stirred overnight at room temperature. The reaction progress was monitored by TLC and ninhydrin was used for colorimetric tracking. After the reaction was completed, the insoluble solid was filtered off, and 5000 mL of isopropanol was slowly added to the residue, followed by freezing at low temperature for half an hour. After filtration, a white solid was obtained. The white solid was dissolved in a small amount of dichloromethane and then slowly added to ice-cold methyl tert-butyl ether to precipitate a white solid. The white solid product was dried under vacuum. The intermediate product was dissolved in 200 mL of acetonitrile and 5 mL of water was added. 1 mL of trifluoroacetic acid was added at 0 °C, and the reaction was carried out at room temperature for 24 h. The reaction endpoint was monitored by NMR. After the reaction was complete, the reaction solution was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. 5000 mL of ice-cold isopropanol was added to the residue, precipitating a large amount of white solid. This solid was filtered, and the product was dried under vacuum to obtain 44.5 g of two-arm polyethylene glycol (5000)-leucine. The yield was 82.4%. 1 H NMR (400MHz, DMSO-d6)δ:6.33(s,1H),4.55(s,1H),3.62(s,2H),3.58-3.54(m,462 H),3.35(s,2H),2.44(s,2H),1.75(d,J=5.0Hz,3H),1.49(s,1H),0.90(s,6H).

[0050] Two-arm polyethylene glycol (5000)-leucine (5.12 g, 1 mmol), HOBt (0.27 g, 2 mmol), and DMAP (0.244 g, 2 mmol) were added sequentially to a reaction flask and dissolved in a mixture of dichloromethane and DMF. The mixture was cooled in an ice bath under nitrogen protection for 30 min. Then, 0.412 g (2 mmol) of DCC in DMF solution and 1.88 g (4 mmol) of PF-00835231 compound were added dropwise. After the addition was complete, the mixture was allowed to warm to room temperature and reacted for 16 hours. The reaction solution was concentrated, and insoluble solids were removed by filtration. The residue was dissolved in 1000 ml of dichloromethane and frozen at low temperature for 30 min. The insoluble solids were filtered off again, and the solvent was concentrated and slowly poured into ice-cold isopropanol for precipitation and crystallization, yielding 4.32 g of a white solid, a two-arm polyethylene glycol (number average molecular weight 5000)-leucine-PF-00835231 conjugate (PF-3). The yield was 61.7%. 1H NMR(400MHz, DMSO-d6)δ:11.03–10.85(m,1H), 8.44(d,J=8.2Hz,1H),8.36(d,J=7.0Hz,1H),7.64(s,1H),7.36(d,J=2.3Hz,1H), 7.12(t,J=7.9Hz,1H),7.02(d,J=8.2Hz,1H),6.52(d,J=6.6Hz,1H),6.33(s, 1H),4.55(s,1H),4.52(ddd,J=10.1,8.2,5.0Hz,1H),4.38(d,J=8.8Hz,1H),3.86(s,3H ),3.64(s,2H),3.60(s,3H),3.58-3.55(m,462H),3.33(s,2H),3.21-3.03(m,2H),2.46 –2.29(m,3H),2.22–2.02(m,2H),1.78(d,J=5.2Hz,3H),1.75–1.58(m,5H),1.48(s, 1H),0.96(d,J=5.4Hz,3H),0.92(s,6H),0.88(d,J=5.4Hz,3H).

[0051] Example 4: Preparation of a two-arm polyethylene glycol (number average molecular weight 10000)-tryptophan-PF-00835231 conjugate (PF-4)

[0052] 100.0 g (10 mmol) of bi-arm polyethylene glycol acetic acid (HOOCCH2-PEG10000-CH2COOH) was dissolved in 500 mL of DMF solvent, followed by the addition of 4.9 g (40 mmol) of DMAP and 5.4 g (40 mmol) of HOBt. After complete dissolution, 8.24 g (40 mmol) of DCC was added. After stirring at room temperature for 60 min, 8.88 g (30 mmol) of tryptophan tert-butyl ester was added, and the reaction was stirred overnight at room temperature. The reaction progress was monitored by TLC and ninhydrin was used for colorimetric tracking. After the reaction was completed, the insoluble solid was filtered off, and 2500 mL of isopropanol was slowly added to the residue, followed by freezing at low temperature for half an hour. After filtration, a white solid was obtained. The white solid was dissolved in a small amount of dichloromethane and then slowly added to ice-cold methyl tert-butyl ether to precipitate a white solid. The white solid product was dried under vacuum. The intermediate product was dissolved in 200 mL of acetonitrile and 5 mL of water was added. 1 mL of trifluoroacetic acid was added at 0 °C, and the reaction was carried out at room temperature for 24 h. The reaction endpoint was monitored by NMR. After the reaction was complete, the reaction solution was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. 2500 mL of ice-cold isopropanol was added to the residue, precipitating a large amount of white solid. This solid was filtered, and the product was dried under vacuum to obtain 94.8 g of two-arm polyethylene glycol (10000)-tryptophan. The yield was 87.8%. 1 H NMR(400MHz, DMSO-d6)δ:7.60(d,J=22.5Hz,4H),7.33(s,2H),7.19(d,J=5.0Hz, 4H), 6.98 (s, 2H), 6.15 (s, 2H), 4.72 (s, 2H), 3.62 (m, 912H), 3.33 (d, J = 17.0Hz, 6H), 3.06 (s, 2H), 1.76 (s, 2H).

[0053] Bis-arm polyethylene glycol (10000)-tryptophan (10.54 g, 1 mmol), HOBt (0.54 g, 4 mmol), and DMAP (0.488 g, 4 mmol) were added sequentially to a reaction flask and dissolved in a mixture of dichloromethane and DMF. The mixture was cooled in an ice bath under nitrogen protection for 30 min. Then, 0.824 g (4 mmol) of DCC in DMF solution and 1.88 g (4 mmol) of PF-00835231 compound were added dropwise. After the addition was complete, the mixture was allowed to warm to room temperature and reacted for 16 hours. The reaction solution was concentrated, and insoluble solids were removed by filtration. The residue was dissolved in 1000 ml of dichloromethane and frozen at low temperature for 30 min. The insoluble solids were filtered off again. The solvent was concentrated and slowly poured into ice-cold isopropanol for precipitation and crystallization, yielding 8.72 g of a white solid, a two-arm polyethylene glycol (number average molecular weight 10000)-tryptophan-PF-00835231 conjugate (PF-4). Yield: 79.3%. 1H NMR(400MHz, DMSO-d6)δ:11.03–10.84(m, 1H),8.45(d,J=6.8Hz,1H),8.32(d,J=8.4Hz,1H),7.63(s,1H),7.56(d,J=12.5Hz, 4H),7.32(d,J=2.6Hz,1H),7.31(s,2H),7.17(d,J=5.2Hz,4H),7.08(t,J=7.6Hz,1H), 7.02(d,J=7.2Hz,1H),6.96(s,2H),6.53(d,J=7.6Hz,1H),6.18(s,2H),4.70(s,2H), 4.50(ddd,J=10.1,8.2,5.0Hz,1H),4.34(ddt,J=11.4,7.9,3.3Hz,1H),3.86(s,3H), 3.65-3.62(m,912H),3.31(d,J=17.0Hz,6H),3.21–3.03(m,4H),2.44–2.30(m,1H), 2.20–2.04(m,2H),1.78(s,2H),1.72–1.53(m,5H),0.94(d,J=6.4Hz,3H),0.88(d,J=7.4Hz,3H).

[0054] Example 5: Preparation of a three-arm polyethylene glycol (number average molecular weight 10000)-threonine-PF-00835231 conjugate (PF-5)

[0055] 50.0 g (5 mmol) of three-arm polyethylene glycol propionate (3Arm-PEG10000-CH2CH2COOH) was dissolved in 300 mL of DMF solvent, followed by the addition of 2.45 g (20 mmol) of DMAP and 2.7 g (20 mmol) of HOBt. After complete dissolution, 4.12 g (20 mmol) of DCC was added. After stirring at room temperature for 30 min, 8.44 g (40 mmol) of threonine tert-butyl ester was added, and the reaction was stirred overnight at room temperature. The reaction progress was monitored by TLC and ninhydrin was used for colorimetric tracking. After the reaction was completed, the insoluble solid was filtered off, and 3500 mL of isopropanol was slowly added to the residue, followed by freezing at low temperature for half an hour. After filtration, a white solid was obtained. The white solid was dissolved in a small amount of dichloromethane and then slowly added to ice-cold methyl tert-butyl ether to precipitate a white solid. The white solid product was dried under vacuum. The intermediate product was dissolved in 300 mL of acetonitrile and 5 mL of water was added. 1 mL of trifluoroacetic acid was added at 0 °C, and the reaction was carried out at room temperature for 24 h. The reaction endpoint was monitored by NMR. After the reaction was complete, the reaction solution was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. 3500 mL of ice-cold isopropanol was added to the residue, precipitating a large amount of white solid. This solid was filtered, and the product was dried under vacuum to obtain 44.8 g of three-armed polyethylene glycol (10000)-threonine. The yield was 80%. 1 H NMR (400MHz, DMSO-d6)δ:6.32(s,1H),6.04(s,1H),4.85(d,J=16.5Hz,2H),3.65 (s,2H),3.58(m,304H),3.35(s,2H),2.72(s,1H),2.52(s,2H),1.16(s,3H).

[0056] Three-arm polyethylene glycol (10000)-threonine (10.42 g, 1 mmol), HOBt (0.54 g, 4 mmol), and DMAP (0.488 g, 4 mmol) were added sequentially to a reaction flask and dissolved in a mixture of dichloromethane and DMF. The mixture was cooled in an ice bath under nitrogen protection for 60 min. Then, 0.824 g (4 mmol) of DCC in DMF solution and 2.82 g (6 mmol) of PF-00835231 compound were added dropwise. After the addition was complete, the mixture was allowed to warm to room temperature and reacted for 16 hours. The reaction solution was concentrated, and insoluble solids were removed by filtration. The residue was dissolved in 1000 ml of dichloromethane and frozen at low temperature for 30 min. The insoluble solids were filtered off again. The solvent was concentrated and slowly poured into ice-cold isopropanol for precipitation and crystallization, yielding 9.65 g of a white solid, a three-arm polyethylene glycol (number average molecular weight 10000)-threonine-PF-00835231 conjugate (PF-5). Yield: 74.2%. 1H NMR (400MHz, DMSO-d6) δ: 11.23–11.09 (m, 1H), 8.50 (d, J = 8.0Hz, 1H), 8.32 (d, J = 6.0Hz, 1H), 7.63 (s, 1H), 7.32 (d, J = 2.6Hz, 1H),7.12(t,J=7.2Hz,1H),7.10(d,J=5.2Hz,1H),6.48(d,J=7.3Hz,1H),6.33(s,1H), 6.11(s,1H),4.82(d,J=16.2Hz,2H),4.51(d,J=12.0Hz,1H),4.32(dt,J=7.9,3.3Hz, 1H),3.86(s,3H),3.63-3.61(m,5H),3.59(m,304H),3.37(s,2H),3.22–3.05(m,2H), 2.75(s,1H),2.54(s,2H),2.44–2.29(m,1H),2.20–2.02(m,2H),1.76–1.57(m, 5H), 1.18 (s, 3H), 0.97 (d, J = 6.4Hz, 3H), 0.87 (d, J = 6.5Hz, 3H).

[0057] Example 6 Preparation of three-arm polyethylene glycol (number average molecular weight 20,000)-tryptophan-PF-00835231 conjugate (PF-6)

[0058] 50 g (2.5 mmol) of three-arm polyethylene glycol acetic acid (3Arm-PEG20000-CH2COOH) was dissolved in 400 mL of DMF solvent, followed by the addition of 1.28 g (10 mmol) of DMAP and 1.35 g (10 mmol) of HOBt. After complete dissolution, 2.06 g (10 mmol) of DCC was added. After stirring at room temperature for 30 min, 3.17 g (15 mmol) of tryptophan tert-butyl ester was added, and the reaction was stirred overnight at room temperature. The reaction progress was monitored by TLC and ninhydrin was used for colorimetric tracking. After the reaction was completed, the insoluble solid was filtered off, and 2500 mL of isopropanol was slowly added to the residue, followed by freezing at low temperature for half an hour. After filtration, a white solid was obtained. The white solid was dissolved in a small amount of dichloromethane and then slowly added to ice-cold methyl tert-butyl ether to precipitate a white solid. The white solid product was then dried under vacuum. The intermediate product was dissolved in 200 mL of acetonitrile and 5 mL of water was added. 1 mL of trifluoroacetic acid was added at 0 °C, and the reaction was carried out at room temperature for 24 h. The reaction endpoint was monitored by NMR. After the reaction was complete, the reaction solution was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. 2500 mL of ice-cold isopropanol was added to the residue, precipitating a large amount of white solid. This solid was filtered, and the product was dried under vacuum to obtain 48.5 g of three-arm polyethylene glycol (20000)-tryptophan. The yield was 91.5%.1 H NMR(400MHz,DMSO-d6)δ:7.66(s,1H),7.51(s,1H),7.34(s,1H),7.18(d,J=5.2Hz,2H ),6.94(s,1H),6.24(s,1H),4.72(s,1H),3.58(m,636H),3.41-3.24(m,3H),3.06(s, 1H).

[0059] Three-arm polyethylene glycol (20000)-tryptophan (20.24 g, 1 mmol), HOBt (0.54 g, 4 mmol), and DMAP (0.488 g, 4 mmol) were added sequentially to a reaction flask and dissolved in a mixture of dichloromethane and DMF. The mixture was cooled in an ice bath under nitrogen protection for 30 min. Then, 0.824 g (4 mmol) of DCC in DMF solution and 1.88 g (4 mmol) of PF-00835231 compound were added dropwise. After the addition was complete, the mixture was allowed to warm to room temperature and reacted for 16 hours. The reaction solution was concentrated, and insoluble solids were removed by filtration. The residue was dissolved in 1000 ml of dichloromethane and frozen at low temperature for 30 min. The insoluble solids were filtered off again. The solvent was concentrated and then slowly poured into ice-cold isopropanol for precipitation and crystallization, yielding 17.2 g of a white solid three-arm polyethylene glycol (number average molecular weight 10000)-tryptophan-PF-00835231 conjugate (PF-6). Yield: 78.2%. 1 H NMR(400MHz, DMSO-d6)δ:11.53–11.44(m, 1H),8.54(d,J=8.0Hz,1H),8.25(d,J=8.6Hz,1H),7.65(s,1H),7.58(s,1H),7.50(s, 1H),7.44(d,J=1.6Hz,1H),7.36(s,1H),7.14(d,J=5.2Hz,2H),7.06(t,J=5.9Hz,1H), 7.11(d,J=8.2Hz,1H),6.93(s,1H),6.50(d,J=7.6Hz,1H),6.23(s,1H),4.74(s,1H), 4.52(ddd,J=10.1,8.2,5.0Hz,1H),3.41-3.27(m,3H),4.36(ddt,J=11.4,7.9,3.3Hz, 1H),3.88(s,3H),3.62(s,3H),3.58(m,636H),3.23–3.02(m,3H),2.45–2.26 (m,1H),2.22–2.05(m,2H),1.75–1.53(m,5H),0.93(d,J=6.4Hz,3H),0.89(d,J=6.4Hz,3H).

[0060] Example 7 Preparation of a four-arm polyethylene glycol (number average molecular weight 10000)-lysine-PF-00835231 conjugate (PF-7)

[0061] 20.0 g (10 mmol) of four-arm polyethylene glycol propionate (4Arm-PEG10000-CH2CH2COOH) was dissolved in 200 mL of DMF solvent, followed by the addition of 2.45 g (20 mmol) of DMAP and 2.7 g (20 mmol) of HOBt. After complete dissolution, 4.12 g (20 mmol) of DCC was added. After stirring at room temperature for 30 min, 13.5 g (30 mmol) of N(e)-Fmoc-lysine tert-butyl hydrochloride was added, and the reaction was stirred at room temperature overnight. The reaction progress was monitored by TLC and ninhydrin was used for colorimetric tracking. After the reaction was completed, the insoluble solid was filtered off, and 2500 mL of isopropanol was slowly added to the residue, followed by freezing at low temperature for half an hour. After filtration, a white solid was obtained. The white solid was dissolved in a small amount of dichloromethane and then slowly added to ice-cold methyl tert-butyl ether to precipitate a white solid. The white solid product was dried under vacuum. The intermediate product was dissolved in 200 mL of acetonitrile and 5 mL of water was added. 1 mL of trifluoroacetic acid was added at 0 °C, and the reaction was carried out at room temperature for 24 h. The reaction endpoint was monitored by NMR. After the reaction was complete, the reaction solution was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. 2500 mL of ice-cold isopropanol was added to the residue, precipitating a large amount of white solid. This solid was filtered, and the product was dried under vacuum to obtain 19.1 g of tetra-armed polyethylene glycol (10000)-N(e)-Fmoc-lysine. The yield was 63.7%. 1 H NMR (400MHz, DMSO-d6) δ: 7.90 (s, 2H), 7.44-7.10 (m, 6H), 5.52 (s, 1H), 5.31 (s, 2H), 4.54 (d, J = 13.3Hz, 2H), 4.25 (s, 1H), 3.70 (s, 2H), 3.62 (s,2H),3.58(m,228H),3.45(s,2H),3.18(s,2H),2.59(s,2H),1.83(d,J=14.2Hz,2H), 1.65(s,2H),1.50(s,1H),1.25(s,2H).

[0062] Four-arm polyethylene glycol (10000)-N(e)-Fmoc-lysine (11.24 g, 1 mmol), HOBt (0.81 g, 6 mmol), and DMAP (0.732 g, 6 mmol) were added sequentially to a reaction flask and dissolved in a mixture of dichloromethane and DMF. The mixture was cooled in an ice bath under nitrogen protection for 30 min. Then, 1.236 g (6 mmol) of DCC in DMF solution and 2.82 g (6 mmol) of PF-00835231 compound were added dropwise. After the addition was complete, the mixture was allowed to warm to room temperature and reacted for 16 hours. The reaction solution was concentrated, and insoluble solids were removed by filtration. The residue was added to 300 ml of acetonitrile and 10 ml of 0.5 mol / L sodium hydroxide and stirred continuously for 12 hours. The system was then dried with anhydrous sodium sulfate. The residue after concentration in the organic solvent was dissolved in 1000 ml of dichloromethane and frozen at low temperature for 30 min. The insoluble solids were filtered off again. After solvent concentration, the residue was slowly poured into ice-cold isopropanol for precipitation and crystallization, yielding 8.92 g of a white solid, a four-arm polyethylene glycol (number average molecular weight 10000)-lysine-PF-00835231 conjugate (PF-7). Yield: 68.6%. 1 H NMR (400MHz, DMSO-d6) δ: 11.22-11.10 (m, 1H), 8.49 (d, J = 7.6Hz, 1H), 8.34(d,J=7.0Hz,1H),7.92(s,2H),7.66(s,1H),7.44-7.10(m,7H),7.07(t,J=6.8 Hz,1H),7.02(d,J=7.2Hz,1H),6.52(d,J=6.6Hz,1H),5.54(s,1H),5.32(s,2H),4.56 (d,J=10.3Hz,2H),4.50(d,J=8.6Hz,1H),4.32(dt,J=12.0,4.2Hz,1H),4.26(s,1H),3 .82(s,3H),3.72(s,2H),3.52(s,3H),3.54(m,228H),3.48(s,2H),3.21-3.03(m,4H), 2.56(s,2H),2.40-2.28(m,1H),2.21-2.04(m,2H),1.86(d,J=14.2Hz,2H),1.76-1.6 5(m,5H),1.63(s,2H),1.48(s,1H),1.26(s,2H),0.93(d,J=5.4Hz,3H),0.88(d,J=5.4 Hz,3H).

[0063] Example 8: Preparation of a four-arm polyethylene glycol (number average molecular weight 20,000)-tyrosine-PF-00835231 conjugate (PF-8)

[0064] 80.0 g (4 mmol) of four-arm polyethylene glycol acetic acid (4Arm-PEG20000-CH2COOH) was dissolved in 500 mL of DMF solvent, followed by the addition of 2.45 g (20 mmol) of DMAP and 2.7 g (20 mmol) of HOBt. After complete dissolution, 4.12 g (20 mmol) of DCC was added. After stirring at room temperature for 30 min, 7.11 g (30 mmol) of tert-butyl tyrosine was added, and the reaction was stirred overnight at room temperature. The reaction progress was monitored by TLC and ninhydrin was used for colorimetric tracking. After the reaction was completed, the insoluble solid was filtered off, and 2500 mL of isopropanol was slowly added to the residue, followed by freezing at low temperature for half an hour. After filtration, a white solid was obtained. The white solid was dissolved in a small amount of dichloromethane and then slowly added to ice-cold methyl tert-butyl ether to precipitate a white solid. The white solid product was dried under vacuum. The intermediate product was dissolved in 200 mL of acetonitrile and 5 mL of water was added. 1 mL of trifluoroacetic acid was added at 0 °C, and the reaction was carried out at room temperature for 24 h. The reaction endpoint was monitored by NMR. After the reaction was complete, the reaction solution was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. 2500 mL of ice-cold isopropanol was added to the residue, precipitating a large amount of white solid. This solid was filtered, and the product was dried under vacuum to obtain 74.5 g of tetra-armed polyethylene glycol (20000)-tyrosine. The yield was 85.6%. 1 H NMR (400MHz, DMSO-d6)δ:7.00(s,2H),6.74(s,2H),5.27(s,1H),4.71(s,1H),4.56(s,1H), 3.69(s,2H),3.59(m,464H),3.46(s,2H),3.11(s,1H),2.86(s,1H),1.28(s,1H).

[0065] Four-arm polyethylene glycol (20000)-tyrosine (21.34 g, 1 mmol), HOBt (0.81 g, 6 mmol), and DMAP (0.732 g, 6 mmol) were added sequentially to a reaction flask and dissolved in a mixture of dichloromethane and DMF. The mixture was cooled in an ice bath under nitrogen protection for 30 min. Then, 1.224 g (6 mmol) of DCC in DMF solution and 2.82 g (6 mmol) of PF-00835231 compound were added dropwise. After the addition was complete, the mixture was allowed to warm to room temperature and reacted for 16 hours. The reaction solution was concentrated, and insoluble solids were removed by filtration. The residue was dissolved in 1000 ml of dichloromethane and frozen at low temperature for 30 min. The insoluble solids were filtered off again. The solvent was concentrated and slowly poured into ice-cold isopropanol for precipitation and crystallization, yielding 15.82 g of a white solid, a four-arm polyethylene glycol (number average molecular weight 20000)-tyrosine-PF-00835231 conjugate (PF-8). Yield: 65.9%. 1HNMR(400MHz, DMSO-d6)δ:11.53-11.42(m, 1H),8.46(d,J=7.6Hz,1H),8.33(d,J=7.4Hz,1H),7.60(s,1H),7.32(d,J=2.8Hz, 1H),7.12(t,J=6.8Hz,1H),7.00(m,3H),6.72(s,2H),6.52(d,J=6.6Hz,1H),5.25(s, 1H),4.70(s,1H),4.56(s,1H),4.50(ddd,J=9.0,7.2,5.2Hz,1H),4.32(ddt,J=9.4, 7.2,3.2Hz,1H),3.82(s,3H),3.68(s,2H),3.61(s,3H),3.58(m,464H),3.44(s,2H), 3.24-3.06(m,3H),2.88(s,1H),2.42-2.30(m,1H),2.22-2.04(m,2H),1.7 6-1.56(m,5H),1.27(s,1H),0.94(d,J=6.4Hz,3H),0.88(d,J=6.6Hz,3H).

[0066] Example 9: Solubility of different polyethylene glycol PF-00835231 conjugates in water and various solvents

[0067] Weigh approximately 1g of sample into a 100ml volumetric flask. First, add 0.8ml of water (within the very soluble range: 1g dissolves in less than 1ml of solvent). Observe the dissolution within 30 minutes according to the pharmacopoeia test method. If it is not completely dissolved, add approximately 8ml of water (within the easily soluble range: 1g dissolves in 1 to less than 10ml of solvent). Continue the test for another 30 minutes and observe the dissolution. If it is still not completely dissolved, add approximately 20ml of water (within the soluble range). Because a 100ml volumetric flask is used, if it is still not completely dissolved when 90ml of water is added (within the slightly soluble range), the sample needs to be prepared again and the test continued. In this case, the amount of drug weighed can be appropriately reduced, which can also save solvent. For example, weigh approximately 10mg of sample into a 100ml volumetric flask, add 9ml of water (within the slightly soluble range), and operate in the same way. If it is still not completely dissolved, add approximately 80ml of water (within the very slightly soluble range). If it is not completely dissolved, add water to the mark (within the range of near insolubility). Following this method, we tested the solubility of PF-00835231 and various PEGylated PF-00835231 from the above examples as follows:

[0068] Table 1 Solubility of PF-00835231 and various PEGylated PF-00835231 in the above examples

[0069]

[0070] It can be seen that PEG-modified PF-00835231 has a significant improvement in solubility, especially the PEG-modified PF-00835231 with excellent solubility.

[0071] Example 10: Different polyethylene glycol PF-00835231 conjugates paired with 3CL pro Study on the inhibitory effect of proteases

[0072] 3C-like proteases are crucial proteases in the replication process of other coronaviruses, such as Middle East Respiratory Syndrome Coronavirus (MERS-CoV). This internally encoded 3C-like protease... pro The function of proteases is indispensable for the processing of these proteins and is crucial for viral replication. SARS-CoV-1's 3CL... pro It shares high structural homology and similar substrate specificity with coronavirus 3C-like cysteine ​​proteases such as hCoV229E and TGEV8, but with SARSCoV-2 3C-like cysteine ​​proteases. pro Most similar. Specifically, SARS-CoV-1 and SARS-CoV-2 each have 3 CLs. pro The sequence homology was 96%, and the homology of the active site was 100%. The SARS-CoV-2 genome encodes two polyproteins and four structural proteins. The short non-structural proteins produced after the polyproteins are cleaved by proteases are crucial for SARS-CoV-2 replication. PF-07321332 targets this main protease M. pro / 3CL pro Because there is no suitable substance in the human body to be absorbed by M. pro / 3CL pro The substrate was cut, so PF-07321332 and PEGylated PF-07321332 theoretically have specific selectivity for the SARS-CoV-2 virus. We only need to test the selectivity of PF-07321332 and PEGylated PF-07321332 for 3CL. pro Inhibition of proteases can be used to evaluate the antiviral activity of drugs. Therefore, we designed a FRET assay and analysis for the inhibition of SARS-CoV-2 protease by PF-00835231 and PEGylated PF-00835231.

[0073] Monitoring of the major SARS-CoV-2 protease 3CL using continuous fluorescence resonance neural transfer (FRET) methodpro The proteolytic activity of SARS-CoV-2 3CL. pro Inhibitor detection was used to assess the activity of the full-sequence SARS-CoV-2 3CL protease. The protease cleaved the protein to synthesize a fluorescent substrate peptide with the shared sequence DABCYL-KTSAVLQ-SGFRKME-EDANS. The fluorescence of the cleaved EDANS peptide was measured using a fluorescence intensity detector (excitation 340 nm / emission 490 nm). The activity was further assessed in the presence of PF-00835231 and PEGylated PF-00835231 (as a SARS-CoV-2 3CL inhibitor). pro (The inhibitor) will inhibit the 3CL pro Hydrolysis and cleavage resulted in a decrease in fluorescence signal. The detection reaction buffer contained 20 mM Tris-HCl (pH 7.3), 100 nM sodium chloride, 1 mM EDTA, 5 mM TCEP, and 25 μM peptide substrate. 15 nM of SARSCoV-2 3CL protease was added to initiate the enzyme reaction, which was then incubated at 25 °C for 60 min. Inhibition rate or activity was calculated from control wells containing no compound (0% inhibition / 100% activity) and control compound (100% inhibition / 0% activity). The half-inhibition concentration (HIC) was generated using a four-parameter fitting model of the half-inhibition concentration software (IDBS). K i The values ​​conform to the Morrison equation, and the enzyme concentration parameter is fixed at 15 nM, K m The parameters were fixed at 14 μM, and the substrate concentration parameter was fixed at 25 μM.

[0074] Table 2. PEGylated PF-00835231 for 3CL derived from SARS-CoV-2 pro Inhibition of proteases

[0075]

[0076] 3CL was treated with PF-00835231 and PEGylated PF-00835231 as described above. pro Comparing the half-maximal inhibitory concentrations (WMCs), it is easy to see that PF-00835231, after modification with 3Arm-PEG and 4Arm-PEG (PF-5, PF-6, PF-7, and PF-8), still exhibits generally high selectivity for 3CL. pro The protease showed a low half-inhibitory concentration (HIC) value, and both single-arm and two-arm PEG-modified PF-00835231 showed resistance to 3CL. pro The proteases (PF-1 half-inhibitory concentration = 0.13 μM; PF-2 half-inhibitory concentration = 0.17 μM) exhibited moderate inhibitory levels. 3CL proThe high similarity between protease catalytic sites suggests the potential of PEGylated PF-00835231 as a SARS-CoV-2 coronavirus inhibitor. Detection showed that PF-6 is effective against related 3CLs. pro The protease (half-inhibitory concentration = 0.00032 μM) has an effective inhibitory effect.

[0077] Compared with the positive control PF-00835231 (10 mg / kg), the relative 3CL of the test drugs PF-5, PF-6, PF-7, and PF-8 (10 mg / kg) pro The protease inhibition rates (μM) were 94%, 93%, 94%, and 94%, respectively, indicating that all PEG compounds had statistically significant anti-3CL activity. pro Compared with PF-00835231 (10mg / kg), the antiviral effects of the active pharmaceutical ingredients contained in the same doses of PF-5, PF-6, PF-7, and PF-8 (10mg / kg) are more significant.

[0078] Example 11: Determination of Drug Plasma Half-Life

[0079] Materials and Methods

[0080] Laboratory animals: One healthy domestic rabbit, male or female, weighing 2-4 kg. Items include: a No. 7 scalp vein needle, a three-way stopcock, a syringe, test tubes; 500 U / ml heparin sodium saline solution; a spectrophotometer; and a benchtop centrifuge.

[0081] Experimental Objective

[0082] Based on the principle that most drugs are eliminated in vivo according to first-order kinetics, the plasma half-life of the drug was determined using the drug concentration data of rabbits measured at various time points after administration.

[0083] Experimental methods

[0084] Take seven test tubes and number them 0-6. Add 3 ml of physiological saline to tube 0, and 4 ml of physiological saline to each of the remaining tubes. Take one rabbit, weigh it, and fix it in a rabbit box. Insert a No. 7 scalp vein needle filled with heparin into the small artery in the center of the rabbit's ear and secure it with an infusion clamp. Inject 1 ml / kg of 500 U / ml heparin sodium saline solution into the small artery through the three-way stopcock to induce systemic heparinization in the rabbit for anticoagulation. After repeatedly aspirating 3-5 times with a syringe connected to the three-way stopcock, accurately draw 0.2 ml of pre-drug blood from the three-way stopcock (artery direction) into tube 1 as a pre-drug control. Inject 2 ml / kg of 0.5 μM pre-drug solution into the marginal ear vein of the rabbit using a syringe. At 5, 10, 20, 40, and 60 minutes after drug administration, accurately aspirate 0.2 ml of blood from the three-way stopcock (arterial direction) into test tubes numbered 2, 3, 4, 5, and 6 respectively. Shake well, let stand, and record the time of blood collection. Because the three-way stopcock contains residual blood from the previous administration, aspirate repeatedly 3-5 times with a syringe before each blood collection. Centrifuge each test tube at 3000 rpm for 5 minutes. Accurately aspirate 3 ml of the supernatant after centrifugation into another set of numbered test tubes, using tube number 0 as a control. Measure the optical density of the supernatant in each tube after drug administration using a spectrophotometer. On semi-logarithmic coordinate paper, plot time on the x-axis (equal squares) and drug concentration on the y-axis (logarithmic value). Connect the points of the six concentration measurements to form the drug-time curve. The time corresponding to a 50% decrease in drug concentration on this line is the plasma half-life of the drug.

[0085] Table 3. Plasma half-life of PEGylated PF-00835231

[0086]

[0087]

[0088] PF-00835231 showed poor plasma clearance (CL) in rabbits. p Elimination half-life (t) after intravenous administration 1 / 2 The half-life of PEGylated PF-00835231 prodrug after intravenous administration was 107 min, while the half-life of PEGylated PF-00835231 prodrug after intravenous administration generally exceeded 5 h. This shows that PEGylated PF-00835231 can significantly prolong the drug half-life.

Claims

1. A polyethylene glycol-amino acid-PF-00835231 conjugate or a pharmaceutically acceptable salt thereof, represented by general formula I-2: in: m is an integer between 2 and 200; n is an integer from 1 to 4; k is an integer between 0 and 3; G stands for tryptophan residue; The number average molecular weight of the polyethylene glycol is 10,000-20,000 Daltons.

2. The conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The carbon atom attached to the G group is a chiral carbon with an R or S configuration.

3. The conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The polyethylene glycol-amino acid-PF-00835231 is a single-arm, double-arm, triple-arm, or quadruple-arm type.

4. The conjugate according to claim 3 or a pharmaceutically acceptable salt thereof, characterized in that, The polyethylene glycol-amino acid-PF-00835231 conjugate is selected from any one of II-V:

5. A method for preparing the complex as described in claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, Includes the following steps: Where m is an integer between 2 and 200; n is an integer from 1 to 4; k is an integer between 0 and 3; G represents an amino acid residue.

6. A pharmaceutical composition comprising the combination of any one of claims 1-4 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, excipient, adjuvant, mediator, or any combination thereof.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition is at least one of the following: powder, suppository, injection, solution, suspension, ointment, patch, and nebulizer.

8. The use of the combination of any one of claims 1-4 or a pharmaceutically acceptable salt thereof, or the composition of claim 6, in the preparation of an anti-COVID-19 drug.

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