WO2023185282A1 - Fatty acid prodrug of nucleoside broad-spectrum antiviral drug and preparation method therefor and use thereof - Google Patents

Abstract

Disclosed in the present invention is a long-chain fatty acid prodrug of N4-hydroxycytidine isobutyl ester, the prodrug being freely selected from at least one of a lauroyl prodrug, a myristoyl prodrug, and a palmitoyl prodrug represented by the following structural formulae. According to the three prodrugs disclosed by the present invention, long-chain alkyl is introduced to hydroxylamine in an N4-hydroxycytidine isobutyl ester molecule, such that the biomembrane permeability is greatly improved. Moreover, it is found in the present invention that cytotoxicity is easily caused if a carbon chain is too long, and the effect of improving a biopermeable membrane cannot be achieved if the carbon chain is too short. In addition, a hydroxylamine structure of N4-hydroxycytidine isobutyl ester is easy to oxidize and inactivate in vivo, and is designed into a hydroxylamine ester prodrug, such that the metabolic stability is improved, the affinity of the prodrug to a cell membrane is improved, and good lung tissue targeted distribution for inhalation administration is provided. Therefore, the compound of the present invention has a better antiviral effect and a wide application prospect.

Description

Fatty acid prodrugs of nucleoside-like broad-spectrum antiviral drugs and preparation methods and uses thereof

References to related applications

This application requires the priority rights and interests of the Chinese patent application with the application number 202210342433. , the entire contents of which are hereby incorporated herein by reference.

Technical field

The invention belongs to the field of pharmaceuticals, and specifically relates to fatty acid prodrugs of nucleoside broad-spectrum antiviral drug N4-hydroxycytidine isobutyl ester.

Background technique

Molnupiravir, generic N4-hydroxycytidine isobutyl ester (EIDD-2801), chemical name is 1((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxy Methyl)tetrahydrofuran-2-yl)-4-(hydroxyamino)-pyrimidin-2(1H)one, CAS number: 2349386-89-4, is the isopropyl derivative of the nucleoside analog N4-hydroxycytidine (NHC) Ester prodrug, against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome virus (SARS-CoV) and influenza A virus etc. have broad-spectrum antiviral activity. In in vitro models, NHC can effectively inhibit the replication of MERS-CoV, SARS-CoV and SARS-CoV-2 (including remdesivir-resistant strains), and also has a significant inhibitory effect on Venezuelan equine encephalitis virus and murine hepatitis virus. , has no obvious cytotoxicity and has poor oral bioavailability in crab-eating macaques. N4-Hydroxycytidine isobutyl ester is designed to improve the in vivo pharmacokinetics and oral bioavailability of NHC in humans and non-human primates. N4-Hydroxycytidine isobutyl ester is hydrolyzed to NHC in the body and metabolized into the pharmacologically active NHC 5'-triphosphate form after entering host cells. Currently, N4-hydroxycytidine isobutyl ester has entered clinical research and is a promising broad-spectrum antiviral drug including SARS-CoV-2.

N4-Hydroxycytidine isobutyl ester is an isobutyrate prodrug of NHC, its active ingredient, and is a carrier prodrug. Its purpose is to enhance oral absorption and availability by increasing fat solubility. However, N4-hydroxycytidine isobutyl ester still has high polarity and poor membrane permeability. The present invention utilizes endogenous fatty acids to further improve its lipid-water distribution coefficient on the basis of N4-hydroxycytidine isobutyl ester, thereby increasing Its biofilm permeability enhances the affinity of drugs with lung tissue cells and improves the drug's lung-targeted distribution capability.

Contents of the invention

According to one aspect of the present invention, an object of the present invention is to provide long-chain fatty acid prodrugs of N4-hydroxycytidine isobutyl ester. After calculation of the lipid-water partition coefficient and compound toxicity evaluation, lauroyl prodrugs and myristoyl prodrugs are respectively preferred. Prodrugs and palmitoyl prodrugs, the structural formula is as follows:

According to one aspect of the present invention, one object of the present invention is to provide a preparation method for the lauroyl prodrug (compound 19), myristoyl prodrug (compound 20) and palmitoyl prodrug (compound 21), the method Proceed according to the following reaction equation 1:

Reaction 1

Dissolve N4-hydroxycytidine isobutyl ester in a solvent, add an organic base such as triethylamine or diisopropylethylamine to catalyze the reaction, and slowly add acid chloride dropwise at low temperature (0-10°C). After the addition is completed, stir at room temperature for 2-24 hours, remove the solvent, extract, wash with ammonium chloride solution, hydrolyze the impurities produced by the secondary acylation, and then perform column chromatography to obtain the target product.

Among them, R-C(=O)-Cl is selected from lauroyl chloride, myristoyl chloride, and palmitoyl chloride;

The solvent is selected from dichloromethane, tetrahydrofuran, acetonitrile or ethyl acetate.

According to one aspect of the present invention, an object of the present invention is to provide the lauroyl prodrug (compound 19), myristoyl prodrug (compound 20) and palmitoyl prodrug (compound 21) as N4-hydroxycytidine isopropyl Uses of prodrugs of butyl ester.

According to one aspect of the invention, an object of the invention is to provide the lauroyl prodrug (compound 19), myristoyl prodrug (compound 20) and palmitoyl prodrug (compound 21) in the preparation of N4-hydroxycytidine Uses in isobutyl ester.

According to one aspect of the present invention, an object of the present invention is to provide the prodrug compounds, mainly lauroyl prodrug (compound 19), myristoyl prodrug (compound 20) and palmitoyl prodrug (compound 21). The use of medicinal ingredients in the preparation of broad-spectrum antiviral pharmaceutical preparations, including capsules, inhalants, tablets, sustained-release preparations, etc.

beneficial effects

Severe respiratory viral infections are treated through systemic administration. Due to the influence of drug distribution and metabolism, effective drug concentrations are often not reached in the target organs. Inhaled drug delivery has become a potentially safe and efficient way to combat severe viral infections of the respiratory tract. However, inhalation administration puts forward new requirements for the physical and chemical properties of drugs, requiring better membrane permeability. Lauroyl prodrug (compound 19) according to the present invention, Myristoyl prodrug (Compound 20) and Palmitoyl prodrug (Compound 21) significantly improve biomembrane permeability by introducing long-chain alkyl groups to the hydroxylamine in the N4-hydroxycytidine isobutyl ester molecule. Moreover, the present invention found that a carbon chain that is too long can easily cause cytotoxicity, while a carbon chain that is too short cannot achieve the effect of improving bioisopermeable membranes. In addition, the N4-hydroxycytidine isobutyl ester hydroxylamine structure is easily oxidized and inactivated in the body, producing toxic groups. Designed as a prodrug, it is beneficial to improve metabolic stability. Therefore, the compound of the present invention has better antiviral effect and has broad application prospects.

Description of drawings

In order to more clearly explain the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description The drawings illustrate some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without exerting any creative effort.

Figure 1 is a graph showing the cytotoxicity test of N4-hydroxycytidine isobutyl ester and compounds 19, 20, and 21;

Figure 2 is a photograph showing viral plaques at different dilutions in the viral titer determination of influenza virus Pr8 stock solution;

Figure 3 is a schematic diagram showing the cytopathic effects of N4-hydroxycytidine isobutyl ester and compounds 19, 20, and 21 against influenza virus Pr8;

Figure 4 is a schematic diagram showing the results of N4-hydroxycytidine isobutyl ester and compound 19 inhibiting influenza virus pr8 in vivo;

Figure 5-1 is a graph showing the results of the effect of N4-hydroxycytidine isobutyl ester on the survival rate of virus-infected mice;

Figure 5-2 is a graph showing the results of the effect of compound 19 on the survival rate of virus-infected mice;

Figure 6 is a schematic diagram showing the effects of N4-hydroxycytidine isobutyl ester and compound 19 on the expression of influenza virus nucleoprotein NP mRNA in mouse lung tissue;

Figure 7 is the infrared spectrum of the lauroyl prodrug (compound 19) prepared in Example 1;

Figure 8 is the ¹ H NMR spectrum of the lauroyl prodrug (compound 19) prepared in Example 1;

Figure 9 is a ¹ H NMR spectrum of myristoyl prodrug (compound 20) prepared in Example 2;

Figure 10 is the ¹ H NMR spectrum of the palmitoyl prodrug (compound 21) prepared in Example 3;

Figure 11 is the experimental principle diagram of the phospholipid bimolecular membrane adsorption test;

Figure 12 is a schematic diagram of the results of the metabolism experiment of compound 19 after inhalation administration.

Detailed ways

Hereinafter, the present invention will be described in detail. Before proceeding to the description, it is to be understood that the terms used in this specification and the appended claims should not be construed to be limited to ordinary and dictionary meanings, but rather to allow the inventor to appropriately define the terms for best interpretation. On the basis of the principles, explanations are made according to the meanings and concepts corresponding to the technical aspects of the present invention. Accordingly, the descriptions set forth herein are preferred examples for illustrative purposes only and are not intended to limit the scope of the invention, so that it will be understood that others may be derived therefrom without departing from the spirit and scope of the invention. price or improvement methods.

N4-Hydroxycytidine isobutyl ester is still highly polar and has poor membrane permeability. In vivo metabolism studies show that it is easily metabolized into uridine triphosphate (UTP) and cytidine triphosphate (CTP). Although the literature (Antimicrobial agents and chemotherapy, 2004, 4636–4642) believe that the latter two can also be incorporated into viral RNA (metabolic reaction 2), however, the inventors believe that uridine triphosphate (UTP) and cytidine triphosphate (CTP) are endogenous substances , which is detrimental to the antiviral efficacy because N4-hydroxycytidine isobutyl ester (EIDD-2801) is metabolically inactivated, which increases its antiviral efficacy. Therefore, it is necessary Optimize its metabolic defects.

The following reaction equation 2 is the metabolic change reaction of N4-hydroxycytidine isobutyl ester.

Reaction 2

In particular, the inventor used ADME/T Predictor 9.5 software to conduct calculation studies on the metabolism site of N4-hydroxycytidine isobutyl ester. The results showed that the hydroxylamine group on the pyrimidine ring of N4-hydroxycytidine isobutyl ester may be in the CYP3A4 enzyme. Oxidation reaction occurs under the action of reaction formula 3, producing toxic metabolic groups, affecting the safety of drug use.

The following reaction equation 3 is the oxidative metabolism process of N4-hydroxycytidine isobutyl hydroxylamine group.

Reaction 3

In summary, regarding the metabolism of the hydroxylamine group of N4-hydroxycytidine isobutyl ester, the inventor conducted research on blocking the metabolic site in order to extend the drug's action time and reduce potential toxicity risks. The present invention introduces protective side chains on the hydroxylamine group. There are two possible sites for introducing side chains, one is the N atom of the hydroxylamine group, and the other is the O atom of the hydroxylamine group. The present invention found through research that when introducing side chains At this time, chemical reactions can occur at both positions, but the side chain on the N atom can be removed using silica gel or saturated ammonium chloride to obtain a pure O atom side chain prodrug. For prodrugs, the choice of protective side chains is very important. It must be easy to fall off in the body to release the active ingredients, and must have low toxicity and high safety. At the same time, it must increase the lipid-water partition coefficient as much as possible. Natural long-chain fatty acids are usually non-toxic in the body and can greatly increase the logP value of the compound. Therefore, the present invention naturally selects long-chain fatty acids and designs three long-chain fatty acid ester prodrugs through toxicological calculations and metabolic property calculations.

ADME/T Predictor 9.5 software was used to predict the lipid-water partition coefficient coefficients of N4-hydroxycytidine isobutyl ester and three prodrugs according to the present invention, and the results are shown in Table 1.

Table 1 Lipophilicity prediction results

It can be seen from the table that when a long-chain aliphatic chain is connected to the pyrimidine ring of N4-hydroxycytidine isobutyl ester, the MlogP, S+logP, and S+logD values are greatly improved, which indicates that the modification of this compound can improve the membrane permeability.

The following examples are merely examples of embodiments of the present invention and do not constitute any limitation to the present invention. Those skilled in the art can understand that modifications within the scope that do not deviate from the essence and concept of the present invention fall within the protection of the present invention. scope. Unless otherwise stated, the reagents used in the following examples and instruments are commercially available products.

¹ H NMR and ¹³ C NMR were measured using LSI-IST/JNM-ECA400 nuclear magnetic resonance instrument, and electrospray mass spectrometry (ESI-MS) was measured on LSI-IST-003/G6230A ion trap mass spectrometer. The high-performance liquid chromatograph uses Agilent, model 1260InfinityⅡ, and the C18 reversed-phase chromatographic column specification is 250×4.6mm. Infrared testing uses a Fourier transform infrared spectrometer FTIR-650. The thin layer chromatography plate uses GF254 fluorescent plate. In the experiments of preparing compounds, the chemical reagents used were of chemical or analytical grade and were used without further purification. The canine kidney epithelial MDCK cells used in this study were purchased from ATCC, and the mice used in the experiment were purchased from Beijing Vitong Lihua Technology Co., Ltd., high-glucose DMEM (CM10017, MACGENE), 4% paraformaldehyde (P1110, MACGENE), Fetal calf serum (10091148, Gibco), crystal violet dye (C0121, Beyotime), TPCK (T1426, Sigma), agar (V2111, Promega), methylcellulose (M0512, Sigma), Trizol (T9424, sigma) , PrimeScript RT Master Mix (RR036A, TaKaLa), PowerUpTM Green (A25778, Applied BiosystemsTM), MTS (G3580, Promega). Microplate reader (Thermo Scientific), real-time fluorescence quantitative PCR instrument (Applied Biosystem), NanoDrop (Thermo Scientific), centrifuge (Eppendorf).

Example

Example 1: Preparation of lauroyl prodrug (compound 19)

Add N4-hydroxycytidine isobutyl ester (2.64g, 8mmol) into a 100ml reaction bottle, add dichloromethane (30ml), triethylamine (3.36ml, 24mmol), and dissolve lauroyl chloride at low temperature (0-10°C). (1.67ml, 7.2mmol) was slowly added dropwise into the reaction system, and then the temperature was naturally raised for 2-24h. After the reaction is complete, add Quench with water, wash the organic phase with saturated aqueous ammonium chloride solution, dry with anhydrous sodium sulfate, filter and concentrate. The resulting residue is purified by column chromatography (DCM:MeOH (v/v) = 50:1) to obtain a solid product. Yield 57%.

¹ H NMR (600MHz, chloroform-d) δ9.02 (s, 1H), 7.08 (d, J = 8.3Hz, 1H), 5.87–5.70 (m, 2H), 4.36–4.17 (m, 5H), 2.58 (p,J＝7.0Hz,1H),2.48(t,J＝7.6Hz,2H),1.69(q,J＝7.5Hz,2H),1.24-1.29(m,16H),1.18(d,J＝ 7.0Hz, 6H), 0.88 (t, J = 7.0Hz, 3H). MS (ESI): m/z = 512.28 (M+H) ⁺ . ¹ H NMR spectrum is shown in Figure 8.

At the same time, the infrared spectrum further verified that the generated ester bond was not an amide bond. The analysis results are as follows:

Figure 7 shows the FT-IR spectrum of the product in the range of 4000-400cm ^-1 . Among them, the peaks at 3392cm ^-1 and 3302cm ^-1 are attributed to OH stretching vibration and come from the hydroxyl structure. The signals at 3114cm ^-1 and 3053cm ^-1 are assigned to the CH stretching vibration on the benzene ring. The peaks at 2922cm ^-1 and 2852cm ^-1 are attributed to CH stretching vibration and come from the alkyl carbon skeleton structure. The strong absorption at 1743cm ^-1 and 1728cm ^-1 comes from the C=O stretching vibration in the ester group. The strong peak at 1657cm ^-1 is attributed to the C=O stretching vibration of the carbonyl group and comes from the diamide structure. Multiple signals in the range of 1288cm ^-1 -1032cm ^-1 are respectively attributed to CO asymmetric and symmetric stretching vibrations, which are one of the signs of the existence of esters, hydroxyl and ether bonds. also. The array signals near 901cm ^-1 and 769cm ^-1 belong to CH out-of-plane bending vibration.

Example 2: Preparation of myristoyl prodrug (compound 20)

Compound 20 was prepared in the same manner as in Example 1, except that lauroyl chloride was replaced by myristoyl chloride. Finally, a solid product was obtained. Yield 52%. ¹ H NMR (600MHz, chloroform-d) δ9.02 (s, 1H), 7.08 (d, J = 8.3Hz, 1H), 5.87–5.70 (m, 2H), 4.36–4.17 (m, 5H), 2.58 (p,J ＝7.0Hz,1H),2.48(t,J＝7.6Hz,2H),1.69(q,J＝7.5Hz,2H),1.24-1.29(m,20H),1.18(d,J＝7.0Hz,6H ),0.88(t,J＝7.0Hz,3H).MS(ESI):m/z＝540.30(M+H) ⁺ . The ¹ H NMR spectrum is shown in Figure 9.

Example 3: Preparation of palmitoyl prodrug (compound 21)

Compound 21 was prepared in the same manner as in Example 1, except that lauroyl chloride was replaced by palmitoyl chloride. Finally, a solid product was obtained. Yield 55%.

¹ H NMR (600MHz, chloroform-d) δ9.02 (s, 1H), 7.08 (d, J = 8.3Hz, 1H), 5.87–5.70 (m, 2H), 4.36–4.17 (m, 5H), 2.58 (p,J＝7.0Hz,1H),2.48(t,J＝7.6Hz,2H),1.69(q,J＝7.5Hz,2H),1.24-1.29(m,24H),1.18(d,J＝ 7.0Hz, 6H), 0.88 (t, J = 7.0Hz, 3H). MS (ESI): m/z = 568.34 (M+H) ⁺ . The ¹ H NMR spectrum is shown in Figure 10.

Example 4: Preparation of lauroyl prodrug (compound 19)

Add N4-hydroxycytidine isobutyl ester (2.64g, 8mmol) into a 100ml reaction bottle, add tetrahydrofuran (30ml), N,N-diisopropylethylamine (4ml, 24mmol) at low temperature (0-10°C ) Lauroyl chloride (1.67ml, 7.2mmol) was slowly added dropwise to the reaction system, and then the temperature was naturally raised for 2-24h. After the reaction is completed, water is added to quench, the organic phase is washed with saturated aqueous ammonium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue is purified by column chromatography (DCM:MeOH (v/v) = 50:1) to obtain a solid product. Yield 56%. MS (ESI): m/z=512.28(M+H) ⁺ .

Example 5: Preparation of myristoyl prodrug (compound 20)

Compound 20 was prepared in the same manner as in Example 4, except that lauroyl chloride was replaced by myristoyl chloride. Finally, a solid product was obtained. Yield 60%. MS (ESI): m/z=540.30(M+H) ⁺ .

Example 6: Preparation of palmitoyl prodrug (compound 21)

Compound 21 was prepared in the same manner as in Example 4, except that lauroyl chloride was replaced by palmitoyl chloride. Finally, a solid product was obtained. Yield 56%. MS (ESI): m/z=568.34(M+H) ⁺ .

Example 7: Preparation of lauroyl prodrug (compound 19)

Add N4-hydroxycytidine isobutyl ester (2.64g, 8mmol) into a 100ml reaction bottle, add acetonitrile (30ml), N,N-diisopropylethylamine (4ml, 24mmol), at low temperature (0-10°C ) Lauroyl chloride (1.67ml, 7.2mmol) was slowly added dropwise to the reaction system, and then the temperature was naturally raised for 2-24h. After the reaction is completed, water is added to quench, the organic phase is washed with saturated aqueous ammonium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated, and the resulting residue is purified by column chromatography (DCM: MeOH (v/v) = 50:1) to obtain solid product. The yield is 58%. MS (ESI): m/z=512.28(M+H) ⁺ .

Example 8: Preparation of myristoyl prodrug (compound 20)

Compound 20 was prepared in the same manner as in Example 7, except that lauroyl chloride was replaced by myristoyl chloride. Finally, a solid product was obtained. Yield 54%. MS (ESI): m/z=540.30(M+H) ⁺ .

Example 9: Preparation of palmitoyl prodrug (compound 21)

Compound 21 was prepared in the same manner as in Example 7, except that lauroyl chloride was replaced by palmitoyl chloride. Finally, a solid product was obtained. Yield 52%. MS (ESI): m/z=568.34(M+H) ⁺ .

Test Example 1: Cytotoxicity Experiment

MDCK cells (purchased from ATCC) were cultured in high-glucose DMEM medium (CM10017, MACGENE) containing 10% fetal calf serum (10091148, Gibco). When the cell density reached about 90%, they were spread on a 96-well plate with 2 × 10 ⁴ cells/well, culture for 12 hours at 37°C and 5% _CO2 . Discard the old culture medium after the cells adhere to the wall. Add 200 μl of compound of corresponding concentration to each well, set up 3 duplicate wells, and set up a solvent control group. After culturing for 72 hours at 37°C, discard the supernatant, add 100 μl of newly prepared MTS (G3580, Promega) detection solution to each well, and set a background control group. Place the 96-well plate in the incubator and incubate for 1 hour. The OD value at 490 nm was measured with a microplate reader (Thermo Scientific), and the value measured by the microplate reader was the value obtained after automatically subtracting the background value. Cell viability (%) = OD value of drug experimental group/OD value of solvent control group × 100%.

The results are shown in Figure 1. When the concentration of N4-hydroxycytidine isobutyl ester and compound 19 is 160 μM, the cell viability still maintains a high level, indicating that compound 19 has low toxicity to MDCK cells and is highly safe. As the carbon chain lengthens, the cytotoxicity increases significantly, indicating that as the side chain of the compound lengthens, the chemical The lipid solubility of the compound is increased and the membrane permeability is enhanced, thereby causing cytotoxicity.

Test Example 2: Anti-influenza virus Pr8 experiment

Determination of virus titer of influenza virus Pr8 stock solution

After the influenza virus is amplified in chicken embryos, its virus titer is unknown, and its titer needs to be determined experimentally. This article uses the plaque method for determination. The principle of the plaque assay is that when cells are infected by a virus, due to the limitations of the solid medium, the released virus can only spread from the initially infected cells to the surroundings. After several proliferation cycles, a localized diseased cell area is formed. That's viral plaque. A plaque is formed by a virus particle that initially infects a cell. Therefore, the inoculated virus liquid must be fully dispersed and diluted. Calculate the number of plaques and multiply them by the dilution factor to obtain the original virus infection unit concentration.

Plaque forming unit (PFus/mL) = (X ₁ +X ₂ +...+X _n )*d/nV

_{X 1 +}

MDCK cells (purchased from ATCC) were cultured in high-glucose DMEM medium (CM10017, MACGENE) containing 10% fetal calf serum (10091148, Gibco). When the cell density reached about 90%, they were spread on a 12-well plate, 2.3 × 10 ⁵ cells/well, cultured at 37°C and 5% CO ₂ for 24 hours, and then the plaque experiment was performed after the cells grew into a monolayer. Influenza virus Pr8 (gifted by Mr. Zhao Zhongpeng from the Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences) was subjected to a series of gradients at 10 times in serum-free high-glucose DMEM medium (CM10017, MACGENE) containing 1 μg/ml TPCK (T1426, Sigma). Dilute, add 1 ml of diluted virus solution to each well of a 12-well plate, infect MDCK cells, and adsorb for 1 hour at 37°C and 5% _CO2 , shaking every 15 minutes. After adsorption, discard the virus liquid, wash it with PBS (10010049, Thermo), and then add 2× DMEM medium (CM10013.2, MACGENE) and 2% agar (V2111, Promega) containing 2 μg/ml TPCK, serum-free ) mixed at a ratio of 1:1 and added to the cells. After the agar has completely solidified, place it in a cell culture incubator for 60 hours. The cells were fixed with 4% paraformaldehyde (P1110, MACGENE), left at room temperature for 1 hour, the agar was removed, and stained with 1% crystal violet (C0121, Beyotime) for 30 minutes. The cells were slowly rinsed with tap water, and then recorded on the plate after drying. Number of plaques. Calculate the virus titer according to the formula.

The results in Figure 2 show that when the virus was at dilution 1 (expressed as 10 ^-1 in Figure 2) and 2 (expressed as 10 ^-2 in Figure 2, other dilutions are similar), almost all cells were damaged and died, and at dilution 3 , the plaques are flaky and cannot be calculated. At dilutions 5, 6, and 7 the cells were undisturbed. At dilution 4 (shown as 10 ^-4 in Figure 2), the number and size of plaques were just right. The calculated virus titer at this dilution is: 54×10 ⁴ /(3×1)=1.8×10 ⁵ pfu/ml

Test Example 3: Cytopathy experiment against influenza virus Pr8

MDCK cells (purchased from ATCC) were cultured in high-glucose DMEM medium (CM10017, MACGENE) containing 10% fetal calf serum (10091148, Gibco). When the cell density reached about 90%, they were spread on a 96-well plate at 3.5× 10 ⁴ cells/well, cultured at 37°C, 5% CO ₂ for 12 hours, set up solvent control group, pr8 control group, and drug administration group. Experiments were performed after cells had grown into a monolayer. In the pr8 control group and the treatment group, MDCK cells were infected with pr8 with a virus titer of 50 PFU, and adsorbed in a 37°C, 5% CO ₂ incubator for 1 hour, shaking every 15 minutes. After adsorption, discard the virus liquid, wash it with PBS (10010049, Thermo), and then add the compound, 1 μg/ml TPCK (T1426, Sigma), and serum-free high-glucose DMEM medium (CM10017, MACGENE) to 96 In the well plate, place it in a cell culture incubator for 48 hours. When the virus control group is completely diseased, add 100 μl MTS (G3580, Promega) detection solution to each well to detect cell viability to characterize the inhibitory effect of the compound on the virus. Virus inhibition rate (%) = (OD value of the administration group - OD value of the pr8 control group) / (OD value of the solvent control group - OD value of the pr8 control group) × 100%

It can be seen from the results in Figure 3 that when the dosage concentration of N4-hydroxycytidine isobutyl ester is 20 μM, The virus was not effectively inhibited, and the cells were completely damaged and died under the action of the virus. However, the modified prodrug showed antiviral efficacy at a dosage concentration of 20 μM. Among them, compound 20 and compound 21 are more toxic than N4-hydroxycytidine isobutyl ester and compound 19, and their protective effect on cells is not very good at high concentrations. The results are shown in Figure 3.

Test Example 4: Experiment on the inhibitory effect of compounds N4-hydroxycytidine isobutyl ester and 19 on influenza virus pr8 in vivo

The least toxic prodrug compound 19 was selected to continue antiviral experiments in mice. BALB/C mice (purchased from Beijing Weitong Lihua Technology Co., Ltd.) were randomly divided into 3 groups according to body weight, namely solvent group, 240 mg/kg N4-hydroxycytidine isobutyl ester, and 240 mg/kg compound 19. Each group 10. After the mice were anesthetized with isoflurane (r510-22-10, Shenzhen Reward Life Technology Co., Ltd.), each mouse was intranasally inoculated with 40 μl of 2×10 ³ PFU influenza virus pr8. 2 hours after infection with the virus, the compound was administered orally, once in the morning and once in the evening. Among them, N4-hydroxycytidine isobutyl ester and compound 19 were dissolved in 1% methylcellulose (M0512, Sigma). Weigh and record the mice's food intake, body weight and death status every day. Observe the mouse's hair, excrement and mental state until the 10th day. Plot the food intake, body weight and survival curves of mice. The results are shown in Figure 4.

As can be seen from Figure 4, the mice in the solvent control group gained weight in the first 2 days, were in good mental state, and moved quickly without significant changes. However, 72 hours after being infected with the virus, the weight of the mice began to drop sharply and continued until death. The mice's food and water intake were significantly reduced, and the mice's hair stood on end, rough and dull. In the later stages of infection there is shortness of breath and increased discharge from the eyes. The body weight of the mice in the two treatment groups first decreased and then increased. At the same dose, compared with the N4-hydroxycytidine isobutyl ester group, the weight increase of the mice in the compound 19 group was slightly greater, suggesting the safety of the compound. Even better, considering that the molecular weight of compound 19 is nearly twice that of N4-hydroxycytidine isobutyl ester, this suggests that the compound is produced in the body after intragastric administration The active ingredient nucleoside analog N4-hydroxycytidine (NHC) is nearly twice as efficient as N4-hydroxycytidine isobutyl ester.

As shown in Figure 5-1 and Figure 5-2, the mice in the virus control group developed severe morbidity after being challenged by the virus, and all died on the 7th day. The mice in the compound 19 (240 mg/kg) and N4-hydroxycytidine isobutyl ester (240 mg/kg) treatment groups showed a 100% survival rate throughout the entire experiment.

In order to compare the differences before and after compound modification and reduce the dosage, BALB/C mice were randomly divided into 3 groups according to body weight, namely solvent group, 50 mg/kg N4-hydroxycytidine isobutyl ester group, and 50 mg/kg compound 19 Groups of 3 animals each. After the mice were anesthetized with isoflurane, each mouse was intranasally inoculated with 40 μl of 2×10 ⁴ PFU influenza virus pr8. 2 hours after infection with the virus, the compound was administered orally for 3 consecutive days, once in the morning and once in the evening. After 72 hours, the mice were euthanized by cervical dislocation. The left lobe lungs were removed and rinsed in pre-cooled PBS, and then placed in RNase-free EP tubes (Eppendorf). 0.5 ml Trizol (T9424, Sigma), use a high-throughput homogenizer (JXFSTPRP-192L) to lyse the lung tissue, centrifuge at 4°C, 15000 rpm for 15 min, and transfer the supernatant to a new RNase-free EP tube. Add 100 μl chloroform (Sinopharm Group) to each tube, let stand at room temperature for 6 minutes, centrifuge at 4°C, 15000rpm, 15min, collect the aqueous phase, precipitate with an equal volume of isopropyl alcohol (Sinopharm Group), let stand at room temperature for 12min, centrifuge at 4°C, 15000rpm for 15min , discard the supernatant, wash the pellet twice with 75% anhydrous ethanol, 4°C, 8000rpm, 5min each time, blow dry and dissolve with DEPC water (A220, Kangrun Biotechnology), measure the RNA concentration and perform reverse transcription. The reverse transcription process is: transfer 500ng total RNA, the total volume is 10μl, according to the measured RNA concentration, add the required RNA volume to a 0.2ml EP tube (Eppendorf), then add 2μl RT Master Mix (RR036A, TaKaLa), and the remaining The volume was made up with DEPC water. Reverse transcription program: 37°C for 15min, 85°C for 5sec, and incubation at 4°C. The obtained cDNA was used to detect the expression changes of influenza virus nucleoprotein NP mRNA using real-time fluorescence quantitative PCR method. The specific process is as follows: the reaction system of each well is 10 μl, and the Mix 0.3 μl cDNA and 5 μl SYBR Green mix (A25778, Applied Biosystems ^TM ) to form system A, mix 0.5 μl primer (Shanghai Sangon Bioengineering Technology Service Co., Ltd.) and 4.2 μl distilled water to form system B, and mix systems A and B respectively. Add to 96-well QPCR plate (HC96112-05, Genestar), two-step PCR amplification program: Holding Stage Step1: 95℃ 30sec; Cycling Stage Step1: 95℃ 5sec, Step2: 60℃ 30sec, Cycle numbers: 40; Melt Curve Stage. Determine whether the results meet the standards based on the amplification curve and melting curve. In order to quantify the expression changes of influenza virus NP, the mouse internal reference gene β-actin is used to normalize, and then the relative expression of the target gene is analyzed according to the 2 ^-ΔΔCT method. The results are shown in Figure 6.

As shown in Figure 6, compared with the virus control group, the expression of influenza virus and nucleoprotein NP mRNA in the lung tissue of mice in the drug administration group was significantly down-regulated (P=0.0001). Moreover, at the same dose, the expression of influenza virus and nucleoprotein NP mRNA was significantly lower than that of N4-hydroxycytidine. Compared with butyl ester, compound 19 slightly inhibited influenza virus NP mRNA expression in mice treated with compound 19 (P=0.0431).

By introducing a long-chain aliphatic chain into the side chain of the N4-hydroxycytidine isobutyl pyrimidine ring, the lipid-water partition coefficient can be significantly increased, the fat solubility of the compound can be improved, and the membrane permeability of the compound can be increased, thus increasing the concentration of the compound in cells. Through the action of a series of enzymes in the cells, the active metabolite NHC is released, which shows an increase in antiviral activity. However, excessive membrane permeability will cause increased cytotoxicity, so compound 19 with a moderate chain length was selected for in vivo experiments in mice. In the in vivo experiments, at high doses, both compound 19 and N4-hydroxycytidine isobutyl ester were able to completely protect the cells. mice. At low doses, compound 19 is slightly better than N4-hydroxycytidine isobutyl ester.

Test Example 5 Cell Membrane (Phospholipid Bilayer) Adsorption Experiment

Compared with other tissues, lung tissue has large blood flow and good permeability, and drugs are easily diffused. Influenza viruses, coronaviruses, etc. are respiratory viruses, which cause lung inflammation and are a serious threat to human health. Threat. By enhancing the interaction between drugs and cell membranes and preparing antiviral drug inhalants, the drug can be retained in the lungs. Therefore, a cell membrane (phospholipid bimolecular membrane) adsorption experiment was used to compare the difference in cell membrane adsorption capacity between the compound of the present invention and N4-hydroxycytidine isobutyl ester.

Add the drug to physiological saline (PBS, pH7.4) to prepare a 10uM solution. Add 0.1mL of the solution to the outer pool, and add 0.1mL of blank physiological saline to the inner pool. As shown in Figure 7, the drug will pass through the phospholipids. The molecular membrane diffuses into the inner pool. After incubation and equilibrium at 25°C for 16 hours, the non-adsorbed drugs will be distributed in the inner pool and the outer pool, and can be extracted and recovered from the liquid using liquid chromatography mass spectrometry (Shimadzu liquid chromatography separation system equipped with degasser DGU-20A5R, solvent delivery The device LC-30AD, system controller CBM-20A, column furnace CTO-30A and CTC Analytics HTC PAL-XT system) are analyzed to determine the recovery rate; according to the adsorbed drugs, they stay on the membrane and are not recovered. Therefore, the higher the recovery rate, the stronger the interaction between the molecules and the phospholipid bimolecular membrane. As shown in the table below, the recovery rate of Example Compound 19 was 2.83%, showing strong adsorption to the phospholipid bimolecular membrane. Therefore, artificial phospholipid bimolecular membrane experiments indicate that the cell membrane adsorption capacity of Compound 19 is significantly better than that of N4-hydroxycytidine isobutyl ester, which is beneficial to the drug's strong cell membrane adsorption capacity in the lungs after inhalation administration. stay. Figure 11 is the experimental principle diagram of the phospholipid bimolecular membrane adsorption test.

Table 2 Phospholipid bimolecular membrane adsorption test results

Test Example 6 Rat Inhalation Administration Lung Targeted Distribution Test

Compounds with strong adsorption capacity to cell membranes (phospholipid bilayers) can enhance drug delivery after inhalation and administration. lung residence time of the substance. In this test example, Compound 19 was selected as an example to detect its drug concentration in the lungs and blood of rats after inhalation administration, as well as its ability to improve the distribution of the antiviral active ingredient N4-hydroxycytidine in the lungs.

Experimental materials: 1.5mL EP tube, 5mL EP tube, 0.5mm capillary glass tube, EDTAK2 anticoagulation tube, centrifuge, balance, pipette, anesthesia machine, tracheal administration nebulizer, small animal laryngoscope, and surgical instruments.

Experimental reagents: Compound 19, isoflurane, Tween-80, physiological saline, 1% sodium pentobarbital.

Experimental animals: rats, male, 27.

Drug preparation: 1% Tween-80, 99% normal saline. For example: weigh 20mg NHA-1 and place it in a mortar, add 40μL Tween-80, grind thoroughly and evenly, slowly add 3960μL physiological saline while grinding to obtain a 5mg/mL suspension, shake well before use .

Experimental steps:

1) Dosing regimen

As shown in Table 3 below, there were 3 rats at each time point. After inhalation administration, the animals were sacrificed according to the time points, and blood and lung tissue were taken to test the blood drug concentration and lung tissue drug concentration.

table 3

2) Administration method: The rats were induced anesthetized with isoflurane and placed on the support board, and the anesthetized state was maintained with isoflurane (concentration: 2.5%); use a quantitative limiter to quantitatively absorb 0.1mL of the medicinal solution, and Insert the laryngoscope into the pharynx of the rat. When the airway is observed to be open, insert the nebulizer needle into the airway; observe the chest expansion process, and at the end of exhalation, quickly inject the nebulizer to complete the administration and time it. .

3) Sample collection: Blood is collected from the orbit of the animals at time points, plasma is separated after anticoagulation, and frozen at -80°C for testing. The animals were then euthanized with pentobarbital, the lungs were dissected, and the lungs were cryopreserved at -80°C until testing.

4) Liquid chromatography mass spectrometry analysis

5) Test result analysis:

Figure 12 is a schematic diagram of the results of the inhalation administration metabolism experiment of Compound 19. As shown in Figure 12, the rat inhalation drug metabolism experiment proved that Compound 19 can reside in the lungs and not diffuse into the blood (proving the conclusion of Test Example 5). After inhalation administration of Compound 19, the drug concentration in the lung tissue was higher than its concentration in blood (compare Figure 12A and B). Compound 19 can quickly and efficiently release the antiviral active ingredient NHC in the lung tissue (Figure 12C); the active ingredient NHC released by compound 19 has a targeted distribution in the lung tissue (Figure 12C and D), and the concentration of NHC in the blood is significantly lower than the concentration in the lung tissue. . Compound 19 has strong lung targeting distribution properties, which can significantly improve the antiviral respiratory virus efficacy of its inhalants.

The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person familiar with the technical field can easily think of changes or replacements within the technical scope disclosed by the present invention. are covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the protection scope of the claims.

Claims (4)

A long-chain fatty acid prodrug of N4-hydroxycytidine isobutyl ester, the prodrug is selected from one of the following compounds: lauroyl prodrug (compound 19), myristoyl prodrug (compound 20) and palmitoyl prodrug Drug (compound 21), the structural formula is as follows:
The preparation method of prodrug according to claim 1, said method is carried out according to reaction formula 1:
Dissolve N4-hydroxycytidine isobutyl ester in the solvent, add organic base triethylamine or diisopropylethylamine as catalyst, slowly add acid chloride dropwise at low temperature (0-10°C), complete the addition, and stir at room temperature for 2- 24 hours, remove the solvent, extract, wash with ammonium chloride solution, hydrolyze the impurities produced by the secondary acylation, and then column chromatography to obtain the target product; Among them, R-C(=O)-Cl is selected from lauroyl chloride, myristoyl chloride, and palmitoyl chloride; The solvent is selected from dichloromethane, tetrahydrofuran, acetonitrile or ethyl acetate, etc. The prodrug compound according to claim 1, lauroyl prodrug, myristoyl prodrug and brown Use of palmitoyl prodrugs in the preparation of broad-spectrum antiviral drugs. The prodrug compound according to claim 1, the use of lauroyl prodrug, myristoyl prodrug and palmitoyl prodrug as the main active ingredients in the preparation of broad-spectrum antiviral drug preparations, said preparations including capsules, Inhalants, tablets, extended release formulations.