A pyridine quaternary ammonium salt FK866 prodrug, a preparation method and application thereof

Abstract

The application provides a pyridine quaternary ammonium salt FK866 prodrug, a preparation method and application thereof. By introducing a large steric response group into the pyridine part of FK866, a quaternary ammonium salt prodrug is formed, the binding of the drug to the target protein is weakened, and the biological activity and toxicity are reduced. A self-cleavage linker is used in the prodrug design to facilitate rapid activation by a specific enzyme and restore the drug activity. The prodrug can be used in vitro drug activity regulation, clinical drug research and development, drug screening and optimization, drug safety evaluation, drug quality control and medical research and the like. The prodrug has the advantages of simple preparation process, few side reactions, high yield, and can be further applied to the prodrug design of other drugs.

Description

Technical Field

[0001] This invention relates to the field of chemical synthesis technology; specifically, it relates to a pyridine quaternary ammonium salt prodrug FK866, its preparation method, and its application. Background Technology

[0002] FK866 is a potent inhibitor of NAD (nicotinamide adenine dinucleotide) biosynthesis. It works by inhibiting nicotinamide phosphoribosyltransferase (NAMPT), blocking NAD production and ultimately leading to a decrease in intracellular NAD levels, thereby inducing apoptosis. However, because this effect lacks specificity and can damage normal cells, causing 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] Currently, the prodrugation of highly toxic drugs is mainly achieved by modifying the hydroxyl, amino, or carboxyl groups in the drug that have a significant impact on its biological activity, such as ester, amide, or ether bonds. However, FK866 does not contain such modifiable functional groups. Pyridine, as its active functional group, plays a decisive role in drug activity. In fact, to date, all structural optimizations have been achieved by retaining the pyridine module. However, there is currently no prodrugation method specifically targeting the modification of the pyridine group in the drug. Furthermore, pyridine is highly susceptible to metabolic oxidation to nitrogen oxides, leading to drug inactivation. This necessitates multiple dosings of FK866 to maintain a stable blood drug concentration, which is also detrimental to its application.

[0004] In summary, due to the unique chemical properties of the pyridine group, the following challenges are faced 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 less susceptible to electrophilic substitution reactions.

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

[0007] Even when the pyridine ring is modified, selectively modifying specific sites without affecting other sites is extremely challenging.

[0008] For the reasons mentioned above, the following strategies are commonly used in the prior art to avoid direct modification on the pyridine ring:

[0009] Modifying the side chains or other functional groups on the pyridine ring, rather than directly modifying the pyridine ring itself, can improve certain physicochemical properties, but it offers limited help in addressing the metabolic stability issues of the pyridine ring itself.

[0010] This involves converting a drug into an inactive or less active form, and then converting it into its active form under specific in vivo conditions (such as pH changes, enzymatic cleavage, etc.). However, these prodrug designs typically rely on easily hydrolyzed linkages such as ester bonds and amide bonds, which are not suitable for modifications of the pyridine ring.

[0011] The search for alternative compounds with similar pharmacological activities aims to avoid the modification issues associated with the pyridine ring. However, this often necessitates a reassessment of the pharmacokinetic and pharmacodynamic properties of the new compound, increasing research and development costs and time.

[0012] In summary, although existing studies have attempted to improve the metabolic stability and reduce the toxic side effects of FK866 through prodrugation methods, these methods have not effectively addressed the metabolic stability and non-specific cytotoxicity issues of the pyridine ring itself. Current technological approaches have significant limitations in solving the core problems of FK866 and urgently need to be addressed. Summary of the Invention

[0013] The purpose of this invention is to provide a pyridine quaternary ammonium salt prodrug for FK866, its preparation method, and its application.

[0014] For FK866, pyridine, as a backbone structure, significantly affects the binding of the drug and the target protein. This invention modifies the pyridine in the drug with sterically hindered responsive groups to form a pyridine quaternary ammonium salt, thereby weakening the interaction between the drug and the target protein and preparing a prodrug.

[0015] The design of this invention involves condensing a responsive group derived from aminobenzyl bromide with pyridine in FK866 to prepare an enzyme-responsive quaternary ammonium salt prodrug. The responsive group and the parent drug are connected by a self-cleaving linker arm, facilitating the embedding of the responsive group into the enzyme pocket for rapid activation. Simultaneously, a prodrug without the self-cleaving linker arm was prepared as a control to investigate the role of the self-cleaving linker arm in the design of the FK866 prodrug.

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

[0017]

[0018] In FK866, pyridine and the self-cleaving linker form a pyridine quaternary ammonium salt with either aminobenzyl bromide or hydroxybenzyl bromide. The amino or hydroxyl group in the self-cleaving linker is linked to an enzyme-responsive group.

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

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

[0021]

[0022] The FK866 has the following molecular structure:

[0023]

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

[0025] In a further preferred embodiment of the above-described technical solution, the reaction process of the responsive group is monitored by thin-layer chromatography.

[0026] In a further preferred embodiment of the above-described technical solution, the purification is performed by column chromatography, and the chromatography solvent is composed of dichloromethane and methanol in a volume ratio of 100:8.

[0027] A further preferred embodiment of the above-described technical solution is that the cooling process further includes a solvent removal step.

[0028] The prodrug molecule provided by this invention has a wide range of potential applications, not limited to drug activity regulation in in vitro experiments, but also including clinical application, drug screening and optimization, drug safety assessment, drug quality control and medical research in clinical drug development.

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

[0030] Cytotoxicity assessment: The cytotoxicity of prodrug molecules is evaluated in in vitro cell lines. The cytotoxicity to different cell lines is determined by methods such as MTT assay and colony formation assay.

[0031] Metabolic stability testing: The metabolic stability of prodrug molecules under different conditions was evaluated using an in vitro microsomal metabolism system.

[0032] 2. Clinical drug development

[0033] Pharmacokinetic studies: using animal model experiments to evaluate the absorption, distribution, metabolism, and excretion (ADME) characteristics of prodrug molecules in vivo.

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

[0035] Preclinical studies: Conducting a series of preclinical studies, including safety assessments, toxicity tests, and pharmacokinetic studies, to lay the foundation for clinical trials.

[0036] 3. Clinical Application

[0037] Combination therapy: Developing combination products with other drugs to improve treatment efficacy and reduce side effects.

[0038] 4. Drug screening and optimization

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

[0040] Drug optimization: By optimizing the structure of prodrug molecules, better drug molecules can be found. For example, based on the modification of the amino group in the linker arm of p-aminobenzyl bromide, other enzyme- or non-enzymatic response fragments can be constructed to design and prepare FK866 prodrugs. Similarly, based on the modification of the hydroxyl group in the linker arm of p-hydroxybenzyl bromide, other enzyme- or non-enzymatic response fragments can be constructed to design and prepare FK866 prodrugs.

[0041] 5. Drug safety assessment

[0042] Toxicity testing: A series of toxicity tests are conducted to assess the safety of the prodrug molecule at different doses.

[0043] Long-term toxicity studies: Conduct long-term toxicity studies to assess the safety of prodrug molecules during long-term use.

[0044] 6. Drug Quality Control

[0045] Manufacturing process: Optimize the manufacturing 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: By studying prodrug molecules, we can gain a deeper understanding of their mechanisms of action and provide a theoretical basis for new drug development.

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

[0050] The present invention has the following advantages:

[0051] (1) The preparation of pyridine quaternary ammonium salt prodrugs is simple, with few side reactions, high reaction yield, and can make full use of the parent drug.

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

[0053] (3) Compared with NFK, a prodrug that does not have a self-cleaving linker arm, PFK, an enzyme activation prodrug designed based on a self-cleaving linker arm, is easy to insert into the PGA enzyme pocket and can rapidly release FK866 under its action, thus restoring cytotoxicity.

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

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

[0056] (6) The exogenous enzyme PGA can regulate the NAMPT inhibition ability of PFK, thus affecting the pathway and can be used for cell biology research. Attached Figure Description

[0057] This invention appendix Figure 8 width.

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

[0059] Figure 2 This is the result of an experimental assessment of the cytotoxicity of PFK in the presence of PGA.

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

[0061] Figure 4 These are HPLC spectra of PFK and PGA before and after the reaction.

[0062] Figure 5 These are HPLC spectra of NFK before and after the reaction with NTR / NADPH.

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

[0064] Figure 7 This is the result of an experimental assessment of cytotoxicity when NMN was added to treatment with a lethal dose of PFK / PGA.

[0065] Figure 8It refers to the intracellular NAD+ content after PGA / PFK or single treatment. Detailed Implementation

[0066] The following specific embodiments are only used to illustrate the technical solution of the present invention and do not limit the scope of protection of the present invention. The specific scope of protection of this patent shall be determined by the claims in the claims. Unless otherwise stated, all reagents used are commercially available products; the solvents used in the experiments are of analytical grade; the experiments are conducted under standard laboratory conditions, and unless otherwise stated, the temperature is controlled within the range of room temperature (approximately 20-25°C).

[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 sequentially dissolved in anhydrous acetonitrile, and the mixture was heated to reflux in an oil bath. The reaction progress was monitored by TLC. After 24 h, the reaction was stopped, the oil bath was removed, and the mixture was allowed to cool naturally to room temperature. The organic solvent was removed by rotary evaporation. The crude product was purified by column chromatography, with an elution system of V... 二氯甲烷 :V 甲醇 =100:8, yielding a white solid (63.35 mg, 80.72%).

[0070] ESI-HRMS (m / z): [M]+(C39H43N4O3+), calculated value 615.3330, tested 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 sequentially dissolved in anhydrous acetonitrile. The reaction was carried out by heating to reflux in an oil bath, and the reaction progress was monitored by TLC. After 24 h, the reaction was stopped, the oil bath was removed, and the mixture was allowed to cool naturally to room temperature. The organic solvent was removed by rotary evaporation. The crude product was purified by column chromatography with an elution system of dichloromethane / methanol = 100 / 8 to give 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 assessment of FK866 and PFK

[0079] PFK and FK866 solutions in DMSO were diluted to different concentrations with DMEM / F12 medium and incubated with adherent SH-SY5Y cells in 96-well plates for 48 h. The old medium was discarded, and the cells were incubated with fresh medium containing MTT for 2 h. Then, the MTT solution was discarded, and 150 μL LDMSO was added to dissolve formazan. The absorbance at 490 nm was read using a microplate reader, and the cell viability was calculated according to the following formula.

[0080]

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

[0082] like Figure 1 As shown, the MTT results indicate that, compared to the high toxicity (IC50) of FK866, 50 <2.56E-5μM), PFK toxicity is significantly reduced, IC50 50 It has a capacity of 19nm, which is more than 742 times that of the FK866. NFK's IC... 50 The value was 370 nM, which is also much higher than that of FK866. This indicates that modifying the pyridine skeleton leads to a significant decrease in the biological activity of FK866, making it an effective strategy for the prodrugation of FK866.

[0083] Example 3 Cytotoxicity assessment of the prodrug PFK in the presence of PGA

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

[0085]

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

[0087] like Figure 2 As shown, in the presence of a non-toxic dose of PGA, PFK exhibits significantly increased cytotoxicity, IC50... 50<2.56E-5μM, consistent with free FK866, indicating that the exogenous enzyme PGA can effectively activate the prodrug PFK and restore its cytotoxicity. This is mainly due to the huge difference in cytotoxicity between PFK and FK866, meaning that only a small amount of drug needs to be released to kill tumor cells.

[0088] Example 4: Cytotoxicity assessment of the prodrug NFK under hypoxic conditions

[0089] SH-SY5Y cells in logarithmic growth phase were digested with trypsin into a single-cell suspension and seeded at a density of 5000 cells per well in 96-well plates. After incubation for 24 hours under standard conditions in a cell culture incubator, the cells adhered. The cells were then transferred to a hypoxic culture vessel, where a real-time oxygen concentration meter was placed. The gas inside the vessel was replaced with a hypoxic balanced gas until the oxygen content reached 0.5%. The vessel was then sealed, surface sterilized, and transferred to a cell culture incubator for further incubation for 12 hours to achieve endogenous NTR expression. Subsequently, the hypoxic vessel was opened, and fresh medium containing different concentrations of NFK or FK (final concentration 10 μM) was quickly added. The gas inside the vessel and the hypoxic balanced gas were replaced again until the oxygen content reached 0.5%. The vessel was then sealed, surface sterilized, and placed in a cell culture incubator for further incubation for 48 hours. Afterwards, the culture medium was discarded, and fresh MTT in DMEM solution was added and incubated for 3 hours. The MTT solution was carefully aspirated, and 150 μL of DMSO was added to dissolve formazan. The cell viability was read at 490 nm, and the cell viability was calculated according to Formula 4.1.

[0090] like Figure 3 As shown, NFK showed only a limited increase in cytotoxicity under hypoxic conditions (107 nM), and the cytotoxicity of FK866 (IC50) was similar. 50 The difference between <2.56E-5μM) was huge, and further extending the hypoxic incubation time to 72h did not significantly improve its cytotoxicity. This indicates that NFK without the self-cleavage linker arm cannot restore its cell-killing ability in response to hypoxia, further illustrating the important role of the self-cleavage linker arm in the design of enzyme activation prodrugs for FK866.

[0091] Example 5: Transformation analysis 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 mixture was reacted in a shaker at 37 °C for 2 h. The sample was then analyzed by HPLC at a detection wavelength of 254 nm. The analytical results were compared with the retention time and UV absorption spectrum of standard samples to confirm the transformation process.

[0093] from Figure 4As can be seen, after 30 minutes of PGA treatment, FK866 was completely degraded. The self-cleaving linker in the generated intermediate then underwent a relatively slow cyclization process, gradually releasing FK866. After 24 hours, the intermediate was completely converted to FK866. This indicates that PGA can facilitate the conversion of PFK to FK866, further explaining the increased cytotoxicity of PFK in the presence of PGA.

[0094] Example 6: Transformation analysis of prodrug NFK in the presence of NTR / NADPH

[0095] Add 0.1 mM NFK, 0.1 units / mL NTR, and 1 mM NADPH solution to PBS, mix thoroughly, and react in a shaker at 37°C for 24 h. Analyze by HPLC (detection wavelength 254 nm), and identify the substances by comparing the retention time and UV absorption spectrum with those of standard samples.

[0096] from Figure 5 As can be seen, NFK maintained good stability after NTR / NADPH co-treatment, with no significant release of FK866 even after 24 hours. Since aryl nitro groups are good substrates for NTRs and readily convert to amino groups, this indicates that NFK and NTR did not interact, possibly due to excessive steric hindrance. Therefore, the cytotoxicity of NFK was not enhanced under hypoxic conditions, further highlighting the importance of the self-cleaving linker in designing prodrugs for enzyme activation of FK866.

[0097] Example 7: Metabolic stability analysis of PFK, NFK, and FK866

[0098] Dilute 5 mM prodrug stock solution (DMSO) in 300 μL PBS to a concentration of 0.05 mM, ensuring that the organic solvent content in the system is less than 1%. Add 3 μL of microparticles (20 mg / mL stock solution) and 10 μL of NADPH (25 mM stock solution), mix thoroughly, and react in a shaker at 37 °C for 20 min. Centrifuge to remove the precipitate, and take the supernatant for HPLC analysis. The injection volume is 10 μL.

[0099] like Figure 6 As shown, after incubation in the in vitro microsomal metabolic system for 20 min, HPLC analysis showed that FK866 was almost completely degraded, while PFK retained 70% and NFK retained 79%. This indicates that the antioxidant capacity of pyridine in the pyridine quaternary ammonium salt prodrug prepared by forming pyridine 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 cytotoxicity

[0101] SH-SY5Y cells in logarithmic growth phase were trypsinized into single-cell suspensions and seeded at a density of 5000 cells per well in 96-well plates. After incubation for 24 h, the cells adhered and were then treated with a lethal dose of enzyme / prodrug (0.125 mg / mL PGA + 1 nM PFK). Simultaneously, different concentrations (20, 15, 10, 5 μM) of NMN were added for incubation. Cells without PGA / PFK but with only the same concentration of NMN served as a control. After further incubation for 48 h, the culture medium was discarded, and fresh MTT in DMEM solution was added for 3 h of incubation. The MTT solution was then discarded, and 150 μL of DMSO was added to dissolve formazan. The cell viability was read at 490 nm, and the cell viability was calculated using the following formula.

[0102]

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

[0104] like Figure 7 As shown, the cell survival rate after treatment with different concentrations of NMN was consistent with that of the control group, indicating that NMN itself has virtually no effect on cell proliferation. When cells were treated with a lethal dose of PGA / PFK (0.125 mg / mL PGA + 1 nM PFK) simultaneously with different concentrations of NMN (a downstream product of NAMPT), cell death was reversed in an NMN dose-dependent manner. Only 0.01 mM of NMN was required to increase cell survival to approximately 50%, and when the NMN concentration reached 1 mM, the cell-killing effect of PGA / PFK was almost completely inhibited (survival rate reached 90%). This indicates that PGA / PFK, like FK866, kills tumor cells through the NAMPT pathway, further demonstrating that PGA can regulate the NAMPT inhibitory ability of prodrugs.

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

[0106] Intracellular NAD+ content was determined using an NAD+ / NADH assay kit, which is based on the WST-8 colorimetric reaction and uses a colorimetric method to detect the amount, ratio, and total amount of NAD+ and NADH in cells or tissues. SH-SY5Y cells in logarithmic growth phase were digested with trypsin into a single-cell suspension and seeded at a density of 20,000 cells per well in 6-well plates. The plates were then incubated for 24 hours to allow cell adhesion. 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 incubated for 40 hours. After treatment, the culture medium was discarded and the cells were gently washed three times with PBS. Next, 300 μL of extraction buffer was added to each well and cells were lysed on ice to extract NADH and NAD+. The supernatant was collected by centrifugation, and 150 μL of each sample was aliquoted and incubated at 60 °C for 40 min to destroy NAD+. The remainder was temporarily stored at 4 °C. Subsequently, enzyme reaction solution was prepared in 96-well plates. 20 μL of sample was added to each well and incubated at room temperature in the dark for 10 min. Then, 10 μL of chromogenic solution was added to each well and incubated at room temperature in the dark for 30 min. The absorbance at 450 nm was measured using a microplate reader, and the relative NAD+ content in each group was calculated by the ratio of absorbance values.

[0107] like Figure 8 As shown, after 30 hours of treatment, there was essentially no difference in intracellular NAD+ levels among the control group, PGA-treated group, and PFK-treated group. However, the intracellular NAD+ level in the PGA / PFK-treated group was only 4% of that in the control group. This indicates that the exogenous enzyme PGA can regulate the NAD+ depletion capacity of PFK and reduce NAD+-induced cell death, 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 scope of protection 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 are not intended to limit all contents of the present invention. The specific scope of protection of the present invention should be determined by the claims in the claims. In addition, the specification and its drawings can be used to interpret the content of the claims in order to better understand the various aspects and implementation details of the present invention. Any matters not covered or broader applicability should be reflected in the claims.

Claims

1. A pyridine quaternary ammonium salt prodrug FK866, characterized in that: It has a molecular structure of the following formula I:

2. The method for preparing the pyridine quaternary ammonium salt prodrug FK866 as described in claim 1, characterized in that: The method includes the following steps: under inert gas protection, 1-2 equivalents of intermediate 4-1 and 0.8-1.2 equivalents of FK866 are dissolved in anhydrous acetonitrile, heated to reflux until complete reaction, cooled, and purified to obtain the target compound; The responsive group has a molecular structure with the following formula 4-1: The FK866 has the following molecular structure:

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

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

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

8.

6. The method according to claim 2, characterized in that: The cooling process also includes a step of removing the solvent.

7. The application of the pyridine quaternary ammonium salt prodrug FK866 as described in claim 1 in non-disease diagnosis and treatment, characterized in that: The application is for regulating drug activity in in vitro experiments, or for drug screening and optimization.

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