Pyridine quaternary ammonium salt FK866 prodrug as well as preparation method and application thereof

Abstract

The invention provides a pyridine quaternary ammonium salt FK866 prodrug as well as a preparation method and application thereof. A large steric hindrance response group is introduced into a pyridine part of FK866 to form a quaternary ammonium salt prodrug, so that the combination of the drug and target protein is weakened, and the biological activity and toxicity are reduced. A self-cleavage connecting arm is used in the design of the prodrug, so that the drug activity can be quickly activated and recovered through a specific enzyme. The prodrug can be used for in-vitro drug activity regulation and control, clinical drug research and development, drug screening and optimization, drug safety evaluation, drug quality control, medical research and the like. The method has the advantages of simple preparation process, few side reactions and high yield, and can be further expanded and applied to the prodrug design of other drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical synthesis, and in particular to a pyridine quaternary ammonium salt FK866 prodrug, a preparation method and application thereof. Background Art

[0002] FK866 is a potent NAD (nicotinamide adenine dinucleotide) biosynthesis inhibitor. It blocks the production of NAD by inhibiting nicotinamide phosphoribosyltransferase (NAMPT), ultimately leading to a decrease in intracellular NAD levels, thereby causing cell apoptosis. However, since this effect is not specific and can damage normal cells, leading to side effects such as thrombocytopenia, its clinical progress remains in Phase II. Therefore, it is necessary to develop a prodrug of FK866 to enhance its therapeutic targeting.

[0003] At present, the prodrugization of highly toxic drugs is mainly achieved by modifying the hydroxyl, amino or carboxyl groups in the drug that have a significant impact on biological activity through ester bonds, amide bonds or ether bonds. However, the FK866 structure does not contain such modifiable functional groups. Pyridine, as its active functional group, plays a decisive role in the activity of the drug. In fact, all structural optimizations to date have been achieved by retaining the pyridine module. However, there is currently no prodrugization method for modifying the pyridine group in drugs. In addition, pyridine is easily metabolized and oxidized to nitrogen oxides, resulting in drug inactivation, which requires multiple doses of FK866 to ensure a stable blood drug concentration, which is not conducive to its application.

[0004] In summary, due to its special chemical properties, the pyridine group faces the following challenges during the modification process:

[0005] The nitrogen atom in the pyridine ring has a strong electron-withdrawing ability, making the entire ring relatively electron-poor and not easy to undergo electrophilic substitution reactions.

[0006] In some cases, substituents around the pyridine ring may create steric hindrance, further hindering the modification reaction.

[0007] Even if the pyridine ring is modified, it is extremely challenging to selectively modify a specific position without affecting other positions.

[0008] Due to the above reasons, the following strategies are usually adopted in the prior art to avoid direct modification on the pyridine ring:

[0009] Modifying the side chain or other functional groups on the pyridine ring instead of directly modifying the pyridine ring itself. Although this method can improve some physical and chemical properties, it has limited help in solving the metabolic stability problem of the pyridine ring itself.

[0010] The drug is converted into an inactive or low-activity form and then converted into an active form through specific in vivo conditions (such as pH changes, enzymatic cleavage, etc.). However, these prodrug designs usually rely on easily hydrolyzed connection methods such as ester bonds and amide bonds, and these methods are not applicable to the modification of pyridine rings.

[0011] Finding alternative compounds with similar pharmacological activities to avoid the modification of the pyridine ring often means re-evaluating the pharmacokinetic and pharmacodynamic properties of new compounds, increasing R&D costs and time.

[0012] In summary, although some studies have attempted to improve the metabolic stability of FK866 and reduce its toxic side effects through prodrugation, these methods have not effectively solved the metabolic stability and nonspecific cytotoxicity of the pyridine ring itself. Existing technical means have obvious limitations in solving the core problems of FK866 and need to be solved urgently. Summary of the invention

[0013] The purpose of the present invention is to provide a pyridinium quaternary ammonium salt prodrug for FK866, a preparation method and application thereof.

[0014] For FK866, pyridine as a skeleton structure has a significant effect on the binding of the drug to the target protein. The present invention modifies the pyridine in the drug by a large steric responsive group to form a pyridine quaternary ammonium salt, weakens the interaction between the drug and the target protein, and thus prepares a prodrug.

[0015] The design scheme of the present invention is to prepare an enzyme-responsive quaternary ammonium salt prodrug by condensing the response group derived from p-aminobenzyl bromide with pyridine in FK866. The response group and the parent drug are connected by a self-cleaving linker, which facilitates the insertion of the response group into the enzyme pocket and achieves rapid activation. At the same time, a prodrug without a self-cleaving linker is prepared as a control to explore the role of the self-cleaving linker in the design of FK866 prodrugs.

[0016] First, the present invention discloses a quaternary pyridine ammonium salt FK866 prodrug having the following molecular structure:

[0017]

[0018] The pyridine in FK866 and the self-cleavable linker arm p-aminobenzyl bromide or p-hydroxybenzyl bromide form a pyridinium quaternary ammonium salt, and the amino group or hydroxyl group in the self-cleavable linker is connected to the enzyme response group.

[0019] Another aspect of the present invention provides a method for preparing a prodrug, the method comprising the following steps: under the protection of an inert gas, dissolving 1-2 equivalents of a response group intermediate 4-1 and 0.8-1.2 equivalents of a pyridine drug FK866 in anhydrous acetonitrile, heating and refluxing until the reaction is complete, cooling, and purifying to obtain a target compound;

[0020] The response group has a molecular structure having the following formula 4-1:

[0021]

[0022] The FK866 has the molecular structure shown below:

[0023]

[0024] For the technical solution described above, it is further preferred that the ratio of the intermediate 4-1 to FK866 is: 1.5 equivalents of the intermediate 4-1 to 1.0 equivalent of FK866.

[0025] For the technical solution described above, it is further preferred that the reaction progress of the response group is monitored by thin layer chromatography.

[0026] For the technical solution described above, it is further preferred that the purification is performed by column chromatography, and the chromatography liquid consists of dichloromethane and methanol in a volume ratio of 100:8.

[0027] For the technical solution described above, it is further preferred that the step of removing the solvent is further included after the cooling.

[0028] The prodrug molecules provided by the present invention have a wide range of potential application scenarios, not only limited to the regulation of drug activity in in vitro experiments, but also in clinical drug development, including clinical application, drug screening and optimization, drug safety assessment, drug quality control and medical research.

[0029] 1 Regulation of drug activity in in vitro experiments

[0030] Cytotoxicity evaluation: The cytotoxicity of the prodrug molecules was evaluated in in vitro cell lines, and its cytotoxicity to different cell lines was determined by methods such as MTT assay and clone formation assay.

[0031] Metabolic stability test: An in vitro microsomal metabolism system was used to evaluate the metabolic stability of the prodrug molecules under different conditions.

[0032] 2Clinical drug development

[0033] Pharmacokinetic studies: Animal model experiments are used to evaluate the absorption, distribution, metabolism and excretion (ADME) characteristics of prodrug molecules in the body.

[0034] Pharmacodynamic studies: Animal model experiments are used to evaluate the pharmacodynamic properties of prodrug molecules in vivo, such as efficacy and safety.

[0035] Preclinical research: Conduct a series of preclinical studies, including safety assessment, toxicity testing, pharmacokinetic studies, etc., to lay the foundation for clinical trials.

[0036] 3 Clinical Application

[0037] Combination therapy: Develop combination products with other drugs to improve therapeutic effects and reduce side effects.

[0038] 4Drug screening and optimization

[0039] High-throughput screening: In the drug screening process, prodrug molecules are used as templates to screen new drug candidates.

[0040] Drug optimization: Search for better drug molecules by optimizing the structure of prodrug molecules. For example, based on the modification of the amino group in the p-aminobenzyl bromide linker, other enzyme or non-enzyme responsive fragments are constructed to design and prepare FK866 prodrugs, and based on the modification of the hydroxyl group in the p-hydroxybenzyl bromide linker, other enzyme or non-enzyme responsive fragments are constructed to design and prepare FK866 prodrugs.

[0041] 5. Drug Safety Assessment

[0042] Toxicity testing: A series of toxicity tests are performed to evaluate the safety of the prodrug molecules at different doses.

[0043] Long-term toxicity studies: Long-term toxicity studies are conducted to evaluate the safety of the prodrug molecules in long-term usage.

[0044] 6. Drug Quality Control

[0045] Production process: Optimize the production process of prodrug molecules to ensure their quality and stability.

[0046] Quality Control: Establish a strict quality control system to ensure the quality of prodrug molecules during production and storage.

[0047] 7. Medical Research

[0048] Mechanism research: Through the study of prodrug molecules, we can gain a deeper understanding of their mechanism of action and provide a theoretical basis for the development of new drugs.

[0049] Disease model research: Study the mechanism of action of prodrug molecules in disease models and explore their potential applications in disease treatment.

[0050] The present invention has the following advantages:

[0051] (1) The preparation reaction of pyridinium quaternary ammonium salt prodrug is simple, with fewer side reactions, high reaction yield, and can fully utilize the parent drug.

[0052] (2) By modifying the pyridine fragment of FK866 with a large sterically hindered response group, the contact between the drug and the target protein is spatially hindered. The constructed prodrug (PFK) can significantly reduce the biological activity of FK866 and weaken its toxicity by at least 742 times.

[0053] (3) Compared with the prodrug NFK without a self-cleaving linker, the enzyme-activated prodrug PFK designed with a self-cleaving linker is easy to insert into the PGA enzyme pocket, and can quickly release FK866 under its action, restoring cytotoxicity.

[0054] (4) The amino group in the self-cleavable linker has good modifiability and can be extended to the preparation of FK866 prodrugs activated by other enzymes, such as GGT. The self-cleavable linker itself is replaceable, such as by p-hydroxybenzyl bromide.

[0055] (5) Pyridine quaternary ammonium salt prodrug has better metabolic stability than FK866.

[0056] (6) Exogenous enzyme PGA can regulate the NAMPT inhibitory ability of PFK, affecting this pathway and can be used for cell biology research. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The present invention is attached Figure 8 width.

[0058] Figure 1 These are the results of the cytotoxicity evaluation experiments of the prodrugs PFK, NFK and the parent drug FK866 on SH-SY5Y cells.

[0059] Figure 2 These are the experimental results for evaluating the cytotoxicity of PFK in the presence of PGA.

[0060] Figure 3 These are the experimental results for evaluating the cytotoxicity of NFK in the presence of NTR / NADPH.

[0061] Figure 4 This is the HPLC spectrum before and after the reaction of PFK and PGA.

[0062] Figure 5 This is the HPLC spectrum before and after the reaction of NFK with NTR / NADPH.

[0063] Figure 6 These are the results of microsomal metabolic stability analysis of PFK, NFK and FK866.

[0064] Figure 7 These are the results of the cytotoxicity evaluation experiment when NMN was added during treatment with a lethal dose of PFK / PGA.

[0065] Figure 8It is the intracellular NAD+ content after PGA / PFK or single treatment. DETAILED DESCRIPTION

[0066] In the following specific implementation, the examples are only used to illustrate the technical solution of the present invention, and do not limit the protection scope of the present invention. The specific protection scope of this patent shall be subject to the claims in the claims. Unless otherwise specified, the reagents used are commercially available products; the solvents used in the experiment are of analytical grade; the experiments are carried out under standard laboratory conditions, and the temperature is controlled within the range of room temperature (about 20-25°C) unless otherwise specified.

[0067] Example 1 Synthesis of FK866 Pyridine Quaternary Ammonium Salt Prodrug PFK

[0068]

[0069] Under nitrogen protection, intermediate 4-1 (45 mg, 0.15 mmol) and FK866 (39 mg, 0.1 mmol) were dissolved in anhydrous acetonitrile and heated to reflux in an oil bath. The reaction progress was monitored by TLC. The reaction was stopped after 24 h, the oil bath was removed, and the mixture was naturally cooled to room temperature. The organic solvent was removed by rotary evaporation. The crude product was purified by column chromatography, and the elution system was V 二氯甲烷 :V 甲醇 =100:8, to give a white solid (63.35 mg, 80.72%).

[0070] ESI-HRMS (m / z): [M]+(C39H43N4O3+), calculated value 615.3330, found value 615.3339.

[0071] 1H NMR (400MHz, MeOD) δ9.31(s,1H),8.98(d,J=6.1Hz,1H),8.79(d,J=8.2Hz,1H),8.12(dd,J=8.2,6.1Hz,1H),7.76–7.70(m,2H),7.63(d,J=15.8H z,1H),7.53(dd,J=8.5,2.1Hz,2H),7.48(dd,J=5.1,2.0Hz,3H),7.41(d,J=2.1Hz,1H),7.40(d,J=3.8Hz,1H),7.36(s,2H),7.34(s,1H),7.27(t, J=6.9Hz,1H),6.99(dt,J=15.8,4.3Hz,1H),5.84(s,2H),4.69–4.55(m,1H),3.72(s,3H),3.37(d,J=6.9Hz,1H),3.11(t,J=13.0Hz,1H),2.86(t ,J=13.1Hz,1H),1.88(d,J=13.2Hz,1H),1.71(d,J=13.2Hz,1H),1.61(p ,J=7.0Hz,3H),1.50–1.40(m,2H),1.40–1.31(m,3H),1.30–1.08(m,3H).

[0072] 13 C NMR (126MHz, MeOD) δ171.11,170.92,164.94,143.74,143.58,142.86,140.27,136.48,135.99,135.24,131.86,129.71,129.50,128. 80,128.76,128.30,128.22,128.18,126.62,126.34,120.48,64.16,43.31,42.32,39.28,35.70,35.62,32.53,31.69,29.07,23.61.

[0073] Example 2 Synthesis of FK866 Pyridine Quaternary Ammonium Salt Prodrug NFK

[0074]

[0075] Under nitrogen protection, 4-2 (32 mg, 0.15 mmol) and FK866 (39 mg, 0.1 mmol) were dissolved in anhydrous acetonitrile in turn, and the reaction was carried out by heating the oil bath to reflux. The reaction progress was monitored by TLC. The reaction was stopped after 24 hours, the oil bath was removed, and the mixture was naturally cooled to room temperature. The organic solvent was removed by rotary evaporation. The crude product was purified by column chromatography, and the elution system was dichloromethane / methanol = 100 / 8 to obtain a white solid (48 mg, 72%).

[0076] HRMS(ESI):[M] + Calculated value C 31 H 35 N4O4 + :527.2653; Test value 527.2659.

[0077] 1 H NMR (400MHz, Methanol-d4) δ9.43(t,J=1.9Hz,1H),9.09(dt,J=6.1,1.3Hz,1H),8.89(dt,J=8.3,1.6Hz,1H),8.42(d,J=6.3Hz,1H),8.40–8.34(m,2H),8. 34–8.28(m,1H),8.22(dd,J=8.2,6.1Hz,1H),7.86–7.77(m,2H),7.67(d,J=1 5.8Hz,1H),7.50(dt,J=5.7,3.0Hz,3H),7.43(td,J=4.8,2.8Hz,2H),7.06(d, J=15.8Hz,1H),6.09(s,2H),4.65(d,J=13.2Hz,1H),3.75(d,J=13.6Hz,1H), 3.54(q,J=7.0Hz,2H),3.32(s,1H),3.15(d,J=14.0Hz,1H),2.88(t,J=12.5Hz ,1H),2.07(d,J=8.9Hz,1H),1.90(d,J=12.9Hz,1H),1.73(d,J=13.3Hz,1H), 1.64(p,J=6.7Hz,3H),1.47(td,J=14.5,14.0,6.3Hz,2H),1.43–1.35(m,2H).

[0078] Example 2 Cytotoxicity Evaluation of FK866 and PFK

[0079] The DMSO solutions of PFK and FK866 were diluted to different concentrations with DMEM / F12 medium and incubated with SH-SY5Y cells attached to the 96-well plate for 48 hours. The old medium was discarded and incubated with fresh medium containing MTT for 2 hours. The MTT solution was then discarded, 150 μL DMSO was added to dissolve formazan, and the absorbance at 490 nm was read by a microplate reader and the cell survival rate was calculated according to the following formula.

[0080]

[0081] In the formula, OD x Represents the absorbance of the test group, OD bc Represents the absorbance of the blank control group, OD c Represents the absorbance of the negative control group (pure cell group).

[0082] like Figure 1 As shown in the MTT results, compared with the high toxicity of FK866 (IC 50 <2.56E-5μM), the toxicity of PFK was significantly reduced, IC 50 The IC value of NFK is 19nM, which is 742 times higher than that of FK866. 50 The activity of FK866 was 370 nM, which was also much higher than that of FK866. This indicates that modification of the pyridine skeleton will significantly reduce the biological activity of FK866 and is an effective strategy for prodrugization of FK866.

[0083] Example 3 Evaluation of cytotoxicity of prodrug PFK in the presence of PGA

[0084] SH-SY5Y cells in the logarithmic growth phase were digested into single cell suspensions and seeded in 96-well plates at an appropriate density, with three replicates per group. After 24 hours of cell culture in a cell culture incubator, medium containing a fixed concentration of PGA and different concentrations of prodrugs was added and incubated for another 48 hours. The medium was discarded, and fresh MTT DMEM solution was added and incubated for 3 hours. The MTT solution was discarded, 150uL DMSO was added to dissolve formazan, and the plate was read at 490nm. The cell survival rate was calculated according to the following formula.

[0085]

[0086] In the formula, OD x Represents the absorbance of the test group, OD bc Represents the absorbance of the blank control group, OD c Represents the absorbance of the negative control group (pure cell group).

[0087] like Figure 2 As shown in Figure 2, in the presence of non-toxic doses of PGA, the cytotoxicity of PFK was significantly increased, with an IC 50<2.56E-5μM, which is consistent with free FK866. This shows that the exogenous enzyme PGA can effectively activate the prodrug PFK and restore its cytotoxicity. This is mainly due to the huge difference in the cytotoxicity of PFK and FK866. Only a small amount of drug needs to be released to kill tumor cells.

[0088] Example 4 Evaluation of cytotoxicity of prodrug NFK under hypoxia

[0089] SH-SY5Y cells in the logarithmic growth phase were digested with trypsin to form a single cell suspension, seeded in a 96-well plate at a density of 5000 cells per well, and incubated in a cell culture incubator under normal conditions for 24 hours to adhere to the wall. The cells were transferred to an anoxic culture tank, a real-time oxygen concentration measuring instrument was placed in the tank, and the gas in the tank was replaced with hypoxic balance gas to an oxygen content of 0.5%. The culture tank was sealed, surface sterilized, and transferred to a cell culture incubator for continued incubation for 12 hours to achieve the expression of endogenous NTR. Subsequently, the anoxic tank was opened, and fresh culture medium containing different concentrations of NFK or FK (final concentration of 10 μM) was quickly added, and the gas in the tank and the hypoxic balance gas were replaced again until the oxygen content in the tank was 0.5%. After the anoxic tank was sealed and surface sterilized, it was placed in a cell culture incubator for continued incubation for 48 hours. After that, discard the culture medium, add fresh MTT DMEM solution and incubate for 3 hours, carefully aspirate and discard the MTT solution, add 150uL DMSO to dissolve formazan and read the plate at 490nm, and calculate the cell survival rate according to formula 4.1

[0090] like Figure 3 As shown, NFK increased its cytotoxicity only to a limited extent under hypoxic conditions (107 nM), and the cytotoxicity of FK866 (IC 50 Further prolonging the hypoxic incubation time to 72 h did not significantly increase its cytotoxicity, indicating that NFK without a self-cleaving linker arm could not restore its cell killing ability in response to hypoxia, further illustrating the important role of the self-cleaving linker arm in designing the enzyme-activated prodrug of FK866.

[0091] Example 5 Analysis of the conversion of prodrug PFK in the presence of PGA

[0092] 0.1 mM PFK and 0.5 mg / mL PGA were added to PBS, and the mixed solution was reacted in a constant temperature shaker at 37°C for 2 hours, and then the sample was analyzed by HPLC with the detection wavelength set to 254 nm. The analysis results were compared with the retention time and UV absorption spectrum of the standard sample to confirm the transformation process of the substance.

[0093] from Figure 4It can be seen that after 30 minutes of PGA treatment, FK866 has been completely degraded, and the self-cleavage linker in the generated intermediate then undergoes a relatively slow cyclization process, gradually releasing FK866. After 24 hours, the intermediate has been completely converted to FK866. This shows that PGA can achieve the conversion of PFK to FK866, which further explains why PFK's cytotoxicity increases in the presence of PGA.

[0094] Example 6 Analysis of conversion of prodrug NFK in the presence of NTR / NADPH

[0095] In PBS, add 0.1mM NFK, 0.1units / mL NTR, and 1mM NADPH solution, mix well, react in a constant temperature shaker at 37°C for 24h, analyze by HPLC (detection wavelength is 254nm), and identify the substance by comparing the retention time and ultraviolet absorption spectrum with those of standard samples.

[0096] from Figure 5 It can be seen that after NTR / NADPH co-treatment, NFK still maintains good stability, and there is still no obvious release of FK866 even after 24 hours. Since the aromatic nitro group is a good substrate for NTR and is easily converted into an amino group, this shows that NFK and NTR do not interact, which may be due to excessive steric hindrance. Therefore, the cytotoxicity of NFK is not enhanced under hypoxic conditions, which further illustrates the importance of the self-cleaving linker in designing enzyme-activated FK866 prodrugs.

[0097] Example 7 Metabolic Stability Analysis of PFK, NFK and FK866

[0098] 5mM prodrug stock solution (DMSO) was diluted and dissolved in 300μL PBS to a concentration of 0.05mM, ensuring that the content of organic solvent in the system was less than 1%, and 3μL microsomes (20mg / mL stock solution) and 10μL NADPH (25mM stock solution) were added. After thorough mixing, the mixture was reacted in a 37°C constant temperature shaker for 20min, and then the precipitate was removed by centrifugation. The supernatant was taken for HPLC analysis with an injection volume of 10μL.

[0099] like Figure 6 As shown, after 20 minutes of incubation in the in vitro microsomal metabolic system, HPLC analysis showed that FK866 was almost completely degraded, while PFK still retained 70% and NFK still retained 79%. This indicates that the antioxidant capacity of pyridine in the pyridinium quaternary ammonium salt prodrug prepared by forming pyridinium quaternary ammonium salt is increased, avoiding a series of subsequent metabolic degradation processes. Compared with FK866, it has better metabolic stability and is conducive to maintaining blood drug concentration.

[0100] Example 8 Analysis of the effect of NAMPT product NMN on PGA / PFK cell toxicity

[0101] SH-SY5Y cells in the logarithmic growth phase were digested with trypsin to form a single cell suspension, and then seeded in a 96-well plate at a density of 5000 cells per well. The cells were incubated in the incubator for 24 hours before attachment, and then treated with a lethal dose of enzyme / prodrug (0.125 mg / mL PGA+1 nM PFK), and different concentrations (20, 15, 10, 5 μM) of NMN were added for incubation. Cells without PGA / PFK and only with the same concentration of NMN were used as controls. After continuing to incubate in the incubator for 48 hours, the culture medium was discarded, and a fresh MTT DMEM solution was added for incubation for 3 hours. The MTT solution was discarded, 150 μL DMSO was added to dissolve the formazan and the plate was read at 490 nm, and the cell survival rate was calculated according to the following formula.

[0102]

[0103] In the formula, OD x Represents the absorbance of the test group, OD bc Represents the absorbance of the blank control group, OD c Represents the absorbance of the negative control group (pure cell group).

[0104] like Figure 7 As shown, after the cells were treated with different concentrations of NMN, the survival rate was consistent with that of the control group, indicating that NMN itself had little effect on cell proliferation. When the cells were treated with a lethal dose of PGA / PFK (0.125mg / mL PGA+1nM PFK) and different concentrations of NMN (a downstream product of NAMPT), cell death was reversed in an NMN dose-dependent manner. With only 0.01mM NMN, the cell survival rate was increased to about 50%, and when the NMN concentration reached 1mM, the cell killing effect of PGA / PFK was almost completely inhibited (survival rate reached 90%). This shows that PGA / PFK, like FK866, kills tumor cells through the NAMPT pathway, further indicating that PGA can regulate the NAMPT inhibitory ability of the prodrug.

[0105] Example 9 Analysis of the effect of PGA / PFK on intracellular NAD+ content

[0106] The intracellular NAD+ content was determined using the NAD+ / NADH detection kit, which is based on the colorimetric reaction of WST-8 and detects the respective amounts, ratios, and total amounts of NAD+ and NDAH in cells or tissues by colorimetry. SH-SY5Y cells in the logarithmic growth phase were digested with trypsin to form a single cell suspension, seeded in a 6-well plate at a density of 20,000 cells per well, and placed in a cell culture incubator for 24 hours to allow the cells to adhere to the wall. Subsequently, the following treatments were performed: (1) untreated control group; (2) 0.125 mg / mL PGA treatment group; (3) 1 nM PFK treatment group; (4) 0.125 mg / mL PGA + 1 nM PFK combined treatment group. All treated cells were continued to be incubated in the incubator for 40 hours. After treatment, the culture medium was discarded and the cells were gently washed 3 times with PBS. Next, 300 μL of extraction solution was added to each well to lyse the cells on ice to extract NADH and NAD+, and the supernatant was collected by centrifugation. 150 μL of each sample was incubated at 60°C for 40 minutes to destroy NAD+, and the rest was temporarily stored at 4°C. Subsequently, the enzymatic reaction solution was prepared in a 96-well plate, 20 μL of sample was added to each well, and after incubation at room temperature in the dark for 10 minutes, 10 μL of color development solution was added to each well, and after incubation at room temperature in the dark for 30 minutes, the absorbance at 450 nm was measured by an enzyme reader, and the relative content of NAD+ in each group of cells was calculated by the absorbance value ratio.

[0107] like Figure 8 As shown, after 30 hours of treatment, there was basically no difference in the intracellular NAD+ levels among the control group, PGA treatment group, and PFK treatment group, while the intracellular NAD+ level in the PGA / PFK treatment group was only 4% of that in the control group, indicating that the exogenous enzyme PGA can regulate the NAD+ depletion ability of PFK and induce cell death by reducing NAD+, which can be used for cell biology research.

[0108] The above embodiments are only used to illustrate the technical solutions of the present invention and do not limit the protection scope of the present invention. Although the above embodiments provide detailed process flows and specific operating steps, they are only used to help understand the technical features of the present invention and do not limit the entire content of the present invention. The specific protection scope of the present invention shall be based on the claims in the claims. In addition, the specification and its drawings can be used to explain the contents of the claims in order to better understand the various aspects and implementation details of the present invention. Any unfinished matters or wider applicability should be reflected in the claims.

Claims

1. A pyridinium quaternary ammonium salt FK866 prodrug, characterized in that: It has the following molecular structure:

2. The method for preparing the quaternary pyridinium ammonium salt FK866 prodrug according to claim 1, characterized in that: The method comprises the following steps: under the protection of inert gas, dissolving 1-2 equivalents of intermediate 4-1 and 0.8-1.2 equivalents of FK866 in anhydrous acetonitrile, heating and refluxing until the reaction is complete, cooling, and purifying to obtain the target compound; The response group has a molecular structure having the following formula 4-1: The FK866 has the molecular structure shown below:

3. The method according to claim 2, characterized in that: The ratio of intermediate 4-1 to FK866 is: 1.5 equivalents of intermediate 4-1 and 1.0 equivalent of FK866.

4. The method according to claim 2, characterized in that: The progress of the responsive group reaction was monitored by thin layer chromatography.

5. The method according to claim 2, characterized in that: The purification is performed by column chromatography, and the chromatographic solution is composed of dichloromethane and methanol in a volume ratio of 100:

8.

6. The method according to claim 2, characterized in that: The method further comprises the step of removing the solvent after the cooling.

7. The use of the quaternary pyridinium ammonium salt FK866 prodrug as claimed in claim 1, characterized in that: The applications include: regulation of drug activity in in vitro experiments, and clinical drug development also includes clinical application, drug screening and optimization, drug safety assessment, drug quality control and medical research fields.

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