Quinone compounds and their pharmaceutical uses

Abstract

The present invention discloses a quinone compound having the chemical structure shown in general formula (I) and its pharmaceutical use. The present invention has medicinal value, has unique analgesic effect, suppresses inflammation, and inhibits the invasion and growth of tumor cells. It is useful for the manufacture of medicines for analgesia, antitumor, anti-inflammation, autoimmune disease treatment, etc. [Formula 1] TIFF2024511529000027.tif83155

Description

[Technical Field]

[0001] The present invention relates to the field of medicinal chemistry, and specifically to novel structural compounds having the chemical structure set forth in general formula (I). [Background technology]

[0002] Pyrroloquinoline quinone (PQQ) is the third prosthetic group discovered in bacterial dehydrogenases, following flavin nucleotide (FAD) and nicotinamide nucleotide (NAD). Its chemical name is 4,5-dihydro-4,5-dioxy-1-hydropyrrole(2,3f)quinone-2,7,9-tricarboxylic acid. Its structural formula is: [ka]

[0003] Although only a few bacteria can synthesize PQQ under natural conditions, trace amounts of PQQ have been observed in various plants, animals, and humans, with a particularly high relative concentration in breast milk. PQQ is a prosthetic group for oxidoreductases that is widely present in animal and plant tissues, and although its concentration is very low, it plays an important role. As a newly discovered component of the mitochondrial respiratory chain, it not only participates in catalysis of redox reactions in the body, but also has a wide variety of biological activities, and its deficiency can cause various disorders in mammals, making it a recognized new vitamin.

[0004] Research on PQQ dates back to 1959, when a glucose dehydrogenase (GDH) independent of NAD(P) and FAD was observed in Acinetobacter calcoaceticus during studies of glucose metabolism in non-phosphorylating bacteria (bacteria generally metabolize glucose via the glucose-6-phosphate pathway). Hauge subsequently isolated a separable prosthetic group from this enzyme and speculated that this prosthetic group might be a naphthoquinone derivative (Hauge, JG, Glucose dehydrogenase of bacterium anitratum: an enzyme with a novel prosthetic group. J Biol Chem. 1964 Nov;239:3630-9).

[0005] In 1979, Duine et al. declared that the prosthetic group in question was a quinone-type substance containing two nitrogen atoms (Duine JA, Frank J, van Zeeland JK. Glucose dehydrogenase from Acinetobacter calcoaceticus: a 'quinoprotein'. FEBS Lett. 1979 Dec 15;108(2): 443-6). At the same time, Salisbury et al. analyzed the crystalline acetone adduct of the prosthetic group using X-ray crystal diffraction techniques and determined that the prosthetic group had a tricarboxypyrroloquinoline quinone structure with the structural formula 4,5-dihydro-4,5-1-dioxo-hydropyrrolo(2,3-f)quinoline-2,7,9-tricarboxylic acid (Salisbury SA, Forrest HS, Cruse WB, Kennard O. Nature. 1979 Aug 30; 280 (5725): 843-4).

[0006] The discovery of PQQ was a significant event in the history of quinone enzyme research, introducing a new prosthetic group to the general public and marking the emergence of a new branch of enzymology: quinone enzymes, i.e., oxidoreductases that use PQQ and other quinone compounds as prosthetic groups. Prior to the discovery of PQQ, oxidoreductases were thought to have only two prosthetic groups: NAD / NADP and FAD / FMN.

[0007] PQQ has a wide range of functions, which are briefly described below.

[0008] 1. PQQ has a wide range of nutritional functions. PQQ promotes the growth of microorganisms and plants and can affect the growth and reproduction of animals. PQQ is an essential nutritional factor that animals cannot produce endogenously. PQQ deficiency can cause poor growth and development in mice and reduced reproductive ability. Female mice have half the number of offspring of mice whose diet contains PQQ, and half of the offspring of PQQ-deficient female mice do not survive the weaning period (4 weeks).

[0009] When the PQQ content in mouse diets falls below 200 ng / g, cytokines necessary for cell proliferation, especially IL-2, which promotes lymphocyte division and proliferation, decrease, inhibiting the maturation and processing of T cells, ultimately resulting in defects in mature T cells and a weakened immune response in the mouse body (Steinberg, FM et al. Dietary pyrroloquinoline quinone: growth and immune response in BALB / c mice. Journal of Nutrition, 1994. 124(5): p 744).

[0010] PQQ deficiency in pregnant or newborn mice significantly reduces lysyl oxidase content in the skin (only 10%-30% of normal skin levels), doubles collagen solubility, and causes dry, flaking skin. Therefore, PQQ is important for collagen cross-linking and skin health (Nishigori et al., Preventive effect of pyrroloquinoline quinone (PQQ) on biliverdin accumulation in the liver of chick embryos after glucocorticoid administration. Life Sciences, 1993. 52 (3): p 305-12).

[0011] 2. Antioxidant function The production and removal of various radicals in the body should be in a state of dynamic equilibrium, meaning that radicals in the body must always be maintained at a certain level. An excess or deficiency of radicals is detrimental to the body. An excess can accelerate aging and lead to the development of various diseases, including cancer and heart disease. An excess or deficiency can affect health, impede normal metabolism, or even induce other diseases. PQQ prevents oxidative damage to the body through several mechanisms:

[0012] Free PQQ in animal tissues and body fluids exists in the quinone, hydroquinone, and semiquinone forms, and can catalyze the interconversion of oxygen and O2-, helping to maintain the radical equilibrium in the body. [ka]

[0013] PQQ and superoxide dismutase (SOD) combine to form a broad-spectrum oxidase system that generates hydrogen oxide and suppresses the damage caused by superoxide anion radicals in cells. SOD functions as an apoenzyme protein, and PQQ functions as a non-covalently bound redox prosthetic group.

[0014] PQQ accelerates the reaction "NAD+ → NADH" and converts oxidized glutathione (GSSG) into reduced glutathione (GSH) more quickly after use. [ka]

[0015] PQQ itself has a strong ability to remove these radicals, 50 to 100 times stronger than ascorbic acid (Akaike T, Sato K, Maeda H, et al. PQQ as a Generator and a Scavenger of Oxygen Radicals: Determination with ESR Spectroscopy using a Spin Trap Agent(M) / / Enzymes Dependent on Pyridoxal Phosphate and Other Carbonyl Compounds As Cofactors. 1991).

[0016] 3. PQQ and Disease Due to its nutritional and antioxidant properties, PQQ has shown some preventive and therapeutic effects in many laboratory disease models.

[0017] (1) Protecting the heart from oxidative damage The cardiac protective effect of PQQ is related to its radical scavenging ability. PQQ can scavenge reactive oxygen species (ROS) generated by hypoxia-reperfusion and significantly reduce the release of lactate dehydrogenase in the heart. Under the catalysis of flavin reductase, its catalytic product can reduce hemoglobin hyperoxidation and eliminate the damage caused by hypoxia-reperfusion to the myocardium. Research has shown that when PQQ is used to protect the hearts of ischemia-reperfusion mice, the area of ​​myocardial infarction is significantly reduced, the rate of rise and fall of left ventricular pressure and left ventricular diastolic pressure is increased, ventricular fibrillation is reduced, and the level of malondialdehyde in myocardial tissue is reduced. PQQ can also reduce oxidative stress, inhibit mitochondrial inactivation, and protect rat cardiomyocytes by inhibiting hydrogen peroxide-induced ROS generation and a decrease in mitochondrial membrane potential (Zhu BQ, Zhou HZ, Teerlink JR, Karliner JS. Cardiovasc Drugs Ther. 2004 Nov;18(6):421-31, Xu X, Chen C, Lu WJ, Su YL, Shi JY, Liu YC, Wang L, Xiao CX, Wu X, Lu Q. Cardiovasc Diagn Ther. 2020 Jun;10(3):453-469).

[0018] (2) Prevent and treat liver damage Experimental liver injury in rats induced by toxins such as carbon tetrachloride (CCl), galactosamine, and acetamide sulfide can be prevented by intraperitoneal injection of a certain dose of PQQ or its derivatives. PQQ reduces ROS production caused by hepatotoxic substances, significantly reduces serum bilirubin glutamic pyruvic transamine (GPT) and lactate dehydrogenase levels, and prevents hepatocellular necrosis without affecting normal biochemical indices (blood glucose, blood nitrogen, urinary nitrogen, etc.) in rats (Tsuchida, T., et al., "The protective effect of pyrroloquinoline quinone and its derivatives against carbon tetrachloride-induced liver injury of rats." J Gastroenterol Hepatol, 2010. 8(4): pp. 342-347).

[0019] (3) Promotes nerve growth and protects the nervous system Nerve growth factor (NGF), the first neurotrophic factor discovered and the most thoroughly studied, has the dual biological functions of neurotrophy and neuroprotection, and plays an important regulatory role in the growth, development, differentiation, regeneration, and function-specific expression of central and peripheral neurons. Experiments have shown that PQQ can stimulate NGF production by LM cells and Schwann cells in vitro (Urakami T, Tanaka A, Yamaguchi K, et al. Synthesis of esters of coenzymes PQQ and IPQ, and stimulation of nerve growth factor production. (J. Biofactors, 1995, 5(3):139).

[0020] (4) Prevent acetaldehyde poisoning Acetaldehyde is an intermediate metabolite of alcohol in the animal body and is toxic. Many people have a mutation in the acetaldehyde dehydrogenase gene, which causes it to malfunction, resulting in the accumulation of acetaldehyde after drinking alcohol, causing mild symptoms of acetaldehyde poisoning such as blushing and dizziness.

[0021] Experiments using rodents have shown that PQQ aids in the metabolism of acetaldehyde. Rats were given an intraperitoneal injection of PQQ (11.5 mg / kg body weight) before receiving ethanol via the stomach. Blood and liver ethanol concentrations in the treated group were not significantly different from those in the control group, but acetaldehyde concentrations in the former were significantly lower than those in the latter. Similar effects were observed when other quinone derivatives, such as coenzyme Q10, were used instead of PQQ (Hobara N, Watanab A, Kobayashi M, et al. Quinone derivatives lower blood and liver acetaldehyde but not ethanol concentrations following ethanol loading to rats. (J. Pharmacology, 1988, 37(4):264-267).

[0022] (5) Analgesic effect Neuropathic pain is chronic pain caused by damage to the somatosensory nervous system. Gong et al. found that PQQ exerted an analgesic effect in the rat CCI model, which may be related to PQQ's NMDA receptor inhibitory effect (Gong D, Geng C, Jiang L, Aoki Y, Nakano M, Zhong L. Effect of pyrroloquinoline quinone on europathic pain following chronic constriction injury of the sciatic nerve in rats. Eur J Pharmacol. 2012 Dec 15;697(1-3):53-8).

[0023] The first PQQ supplement was approved in the United States in 2009. Because PQQ is slightly soluble in water, the supplement's ingredient is PQQ sodium salt (PQQ-2Na+), which has higher solubility. In 2018, the European Union also approved PQQ-2Na+ as a health food for adults, excluding pregnant and lactating women. The commercially available PQQ sodium salt dosage is ≤20 mg / day. When taken orally, PQQ is primarily absorbed in the small intestine (~60%), and 80% is excreted via the kidneys (urine) 24 hours after administration. The safe dosage in rats is shown in the table below.

[0024] [Table 1]

[0025] In a human controlled double-blind clinical trial, volunteers took 60 mg of PQQ per day for one month, and no side effects occurred, nor did kidney damage markers change. Regarding the FDA's safe dose, a healthy adult weighing approximately 60 kg can take 240 mg of PQQ daily with almost no adverse events (FDA, Generally Recognized as Safe (GRAS) notice of pyrroloquinoline quinone disodium salt as a food ingredient).

[0026] More than 40 years have passed since the discovery of PQQ, but while it has been marketed in the United States as a health food for 10 years, it has never been developed or used as a pharmaceutical. After investigating the reasons for this, the inventors concluded that the following three factors may be at play: 1. Low bioavailability. PQQ prototypes have low water solubility, and according to the inventors' research, the bioavailability of PQQ in beagle dogs is approximately 12%. 2. The target and molecular mechanism are unknown. 3. The PQQ structure has been publicly available for more than 40 years, and is not protected, limiting its commercial value for pharmaceutical development. Therefore, reconstructing PQQ to obtain a novel structure with higher bioavailability, a clear target, a clear molecular mechanism, significant efficacy, and patent protection is of great scientific and commercial significance.

[0027] AMP-dependent protein kinase (AMPK) is a key molecule in the regulation of bioenergetic metabolism and is a key research focus for diabetes and other metabolic disorders. It is expressed in various metabolically relevant organs and can be activated by various biological stimuli, including cellular stress, migration, and various hormones and substances that can affect cellular metabolism. Genetic and pharmacological studies have shown that AMPK is essential for maintaining glucose balance in the body. In addition to metabolic diseases, AMPK is also involved in neurodegenerative diseases (Curry DW, Stutz B, Andrews ZB, Ellsworth JD. Targeting AMPK signaling as a neuroprotective strategy in Parkinson's disease. J Parkinsons Dis. 2018;8(2):161-181), cardiovascular disease (Feng Y, Zhang Y, Xiao H. AMPK and cardiacre modeling. Sci China Life Sci. 2018 Jan;61(1):14-23), tumors (Wang Z, Wang N, Liu P, Xie X. AMPK and cancer. Exp Suppl. 2016;107:203-226), and pathological pain (Asiedu MN, Dussor G, Price TJ. Targeting AMPK for the alleviation of pathological pain. Exp Suppl. 2016;107:257-285) and pathogenic microbial (including viral) infections (Silwal P, Kim JK, Yuk JM, Jo EK. AMP-Activated Protein Kinase and Host Defense against Infection. Int J Mol Sci. 2018 Nov 6;19(11):3495). AMPK is also an important target in anti-aging and lifespan extension research (Burkewitz K, Zhang Y, Mair WB. AMPK at the nexus of energetics and aging. Cell Metab. 2014 Jul 1;20(1):10-25).Cheng et al. reported that PQQ can promote mitochondrial biogenesis by activating AMPK in a rotenone-induced Parkinson's disease model, thereby having therapeutic effects (Cheng Q, Chen J, Guo H, Lu JL, Zhou J, Guo XY, Shi Y, Zhang Y, Yu S, Zhang Q, Ding F. Pyrroloquinoline quinone promotes mitochondrial biogenesis in rotenone-induced Parkinson's disease model via AMPK activation. Acta Pharmacol Sin. 2020 Aug 28). The inventors discovered that 100 nM PQQ and its reconstituted compounds SP3101 and SP3102 significantly increased the phosphorylation level of AMPK (AMPK activation state) in various cell types.

[0028] It is generally believed that PQQ exerts its range of biological benefits by stimulating antioxidant signaling pathways in the body. However, when used as an analgesic, PQQ has a slow onset of action, making it unsuitable for use as an acute analgesic. Summary of the Invention

[0029] The object of the present invention is to obtain a compound represented by structural formula (I) by reconstructing PQQ, which has the characteristics of rapid analgesia and long analgesia duration.

[0030] In one aspect, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt, ester, stereoisomer, solvate or prodrug thereof: [ka] During the ceremony, R 1 is selected from -OH, -OR, -OC(O)R, -NH2, -NHR, -SH or -SR; R 2is selected from H, C1-C3 alkyl, NH2, NHR or —COOH; R 3 is selected from H, C1-C3 alkyl, OH, NH2, NHR, —COOH, or SH; R 4 is selected from H, C1-C3 alkyl, or C6-C10 aryl; R 5 is selected from H, C1-C3 alkyl, OH, NH2, NHR, —COOH, or SH; R 6 is selected from H, C1-C3 alkyl, OH, NH2, NHR, —COOH, or SH; wherein R is C1-C6 alkyl, and in some specific embodiments, R is C1-C3 alkyl.

[0031] In some embodiments, the present invention further provides a compound of Formula (II) or a pharmaceutically acceptable salt, ester, stereoisomer, solvate, or prodrug thereof: [ka] In the formula, R 1 is selected from -OH, -OR, -OC(O)R, -NH2, -NHR, -SH, or -SR, and R is C1-C6 alkyl, and in some specific embodiments, R is C1-C3 alkyl.

[0032] The present invention further provides several compounds shown in the results below or pharmaceutically acceptable salts, esters, stereoisomers, solvates or prodrugs thereof. [ka] [ka] [ka]

[0033] In another aspect of the present invention, there is further provided a method for preparing a compound of formula (II). [ka]

[0034] Salts that can be formed by the compounds of the present invention also fall within the scope of the present invention. Unless otherwise specified, the compounds of the present invention are understood to include their salts. For example, a compound of formula (I) is reacted with a certain amount, e.g., an equivalent amount, of an acid or base, and a salt is precipitated from the medium or obtained by lyophilization of an aqueous solution.

[0035] The compounds of the present invention are obtained by sequential production, separation, and purification, and have a weight content of 90% or more, for example, 95% or more, or 99% or more ("highly pure" compounds), as described herein. Such "highly pure" compounds of the present invention also constitute part of the present invention.

[0036] The present specification further provides a pharmaceutical composition comprising a compound according to the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, solvate, or prodrug thereof, and a pharmaceutically acceptable carrier.

[0037] The present invention further provides the use of a compound according to the present invention, or a pharmaceutically acceptable salt, ester, stereoisomer, solvate or prodrug thereof, as an AMPK agonist.

[0038] The present invention further provides the use of a compound according to the present invention, or a pharmaceutically acceptable salt, ester, stereoisomer, solvate or prodrug thereof, as an inhibitor of the Cox-2 enzyme.

[0039] The present invention further provides the use of a compound according to the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, solvate or prodrug thereof for the manufacture of an analgesic medicament.

[0040] The analgesic agent according to the present invention can treat various types of pain caused by various factors, such as discomfort due to any physical injury, disease, or adverse external stimuli, including surgery, inflammation, tumor, myocardial infarction injury, bacterial infection, viral infection, and neurological disease. Pain caused by neurological disease includes, but is not limited to, pain caused by neurodegeneration, nerve alteration, epilepsy, migraine, and the like, and pain caused by various liver injuries and myocardial infarction injury.

[0041] The present invention further provides use of a compound according to the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, solvate or prodrug thereof for the manufacture of a medicament for preventing and treating an inflammatory disease, wherein the inflammatory disease according to the present invention may be an inflammatory response caused by bacterial or viral infection, including, but not limited to, respiratory inflammation such as lung inflammation caused by bacterial or viral infection, viral myocarditis, hepatitis or decreased liver function caused by bacteria or viruses, or an excessive inflammatory response caused by tumor immunotherapy, or an inflammatory response caused by autoimmune disease, or rejection of allogeneic organ transplants and tissue transplants.

[0042] The present invention further provides the use of a compound according to the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, solvate or prodrug thereof for the manufacture of an antitumor medicament.

[0043] The present invention further provides use of a compound according to the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, solvate or prodrug thereof for the manufacture of a medicament for preventing and treating various liver damages and liver function declines caused by cytotoxic tumor chemotherapy, chemical substances or medicines, and chronic hepatitis, and for preventing and treating myocardial infarction damage and viral myocarditis.

[0044] The present invention further provides the use of a compound according to the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, solvate or prodrug thereof for the manufacture of a medicament for the prevention and treatment of neurodegenerative and neurotransmitter diseases, epilepsy and migraine.

[0045] The present invention further provides the use of a compound according to the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, solvate or prodrug thereof for the manufacture of a medicament for the prevention and treatment of viral infections and inflammatory conditions resulting from viral infections.

[0046] The present invention further provides the use of a compound according to the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, solvate or prodrug thereof for the manufacture of a medicament for the prevention and treatment of chemotherapy-induced peripheral neuropathy, such as chemotherapy-induced pain.

[0047] The present invention further provides the use of a compound according to the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, solvate or prodrug thereof for the manufacture of a medicament for the prevention and treatment of depression, and for the manufacture of a sedative or hypnotic agent.

[0048] The terms of the present invention are defined as follows unless otherwise specified. The terms "C1-C3 alkyl," "C1-C6 alkyl," "C2-C6 alkyl," and "C3-C6 alkyl" refer to saturated straight- or branched-chain monovalent hydrocarbon radicals having 1 to 3, 1 to 6, 2 to 6, or 3 to 6 carbon atoms, respectively. Examples include, but are not limited to, methyl, ethyl, 1-propyl, isopropyl, 1-butyl, isobutyl, sec-butyl, tert-butyl, 2-methyl-2-propyl, pentyl, neopentyl, and hexyl. Alkyl groups are substituted or unsubstituted. If substituted, the substituents are preferably one or more, more preferably one, two, or three, and even more preferably one or two, and are independently selected from lower alkyl, trihaloalkyl, halogen, hydroxyl, lower alkoxy, thiol, (lower alkyl)sulfanyl, cyano, acyl, thioacyl, O-carbamoyl, N-carbamoyl, and the like.

[0049] The term "aryl" refers to an all-carbon monocyclic or fused polycyclic group of 1 to 12 carbon atoms having a completely conjugated π-electron system. Non-limiting examples of aryl include phenyl, naphthyl, and anthracenyl. Aryl can be substituted or unsubstituted. If substituted, the substituents are preferably one or more, more preferably one, two, or three, and even more preferably one or two, and are independently selected from lower alkyl, trihaloalkyl, halogen, hydroxyl, lower alkoxy, thiol, (lower alkyl)sulfanyl, cyano, acyl, thioacyl, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-acylamino, N-acylamino, nitro, N-sulfonylamino, S-sulfonylamino, and the like. Preferably, the aryl is optionally substituted by one or two substituents, which are independently selected from halogen, lower alkyl, trihaloalkyl, hydroxyl, thiol, cyano, N-acylamino, mono- or di-alkylamino, carboxyl, or N-sulfonylamino.

[0050] Compared with the prior art, the beneficial effects of the present invention are as follows: The compounds described in this invention are highly water-soluble and bioavailable after salt formation. Biologically, these novel structures not only activate AMPK, but also unexpectedly inhibit Cox-2 enzyme, resulting in unique anti-inflammatory and analgesic effects. In an inflammatory pain model established with complete Freund's adjuvant, a single dose of SP3101, SP3102, and SP3103 rapidly exerted analgesic effects, with the analgesic effect becoming apparent 1.5 hours after oral administration and lasting for 72 hours. Interestingly, injection of an AMPK inhibitor via the tail vein of rats 0.5 hours before administration had no effect on the short-term analgesic effects of these analgesics, but the analgesic effect completely disappeared after 24 hours. This suggests that their long-term analgesic effects depend on AMPK activation. Furthermore, these drugs exert their analgesic effects through at least two targets: short-term (1.5 hours) analgesia and long-term (24-72 hours) analgesia target different targets. [Brief explanation of the drawings]

[0051] [Figure 1] FIG. 1 shows the analgesic effect of a single oral administration of 20 mg / kg of SP3101 to rats. [Figure 2] FIG. 1 is a graph showing the analgesic effect of repeated oral administration of 25 mg / kg of ibuprofen to rats. [Figure 3] FIG. 1 shows the analgesic effect of SP330 (PQQ) administered orally at 20 mg / kg / day to rats for three consecutive days. [Figure 4] Inhibition of the long-term analgesic effect of SP3101 by the AMPK inhibitor compound c. [Figure 5] This is the molecular mechanism of the series of molecular actions of SP3101. [Figure 6] Inhibition of cox-2 activity by SP3101. [Figure 7] SP3101 inhibits mouse hepatitis virus (MHV)-induced pneumonia in mice. [Figure 8] FIG. 1 shows the analgesic effect of a single oral administration of 40 mg / kg of SP3101 in a chronic sciatic nerve compression (CCI) model in mice. [Figure 9] These are the results of a sedative experiment using SP3101. [Figure 10] This shows the effect of SP3101 on an experimental pain model induced by oxaliplatin chemotherapy. [Figure 11] This figure shows statistics obtained by using a 1.0 g von Frey filament for a specific pain threshold, performing 10 detections per mouse, recording the number of positive reactions per mouse, and calculating the percentage of the total number of detections per mouse. [Figure 12] This figure shows statistics obtained by using a 0.4 g von Frey filament for a specific pain threshold, performing 10 detections per mouse, recording the number of positive reactions per mouse, and calculating the percentage of the total number of detections per mouse. [Figure 13]A 0.07 g von Frey filament was selected, and each mouse was subjected to 10 detections. The number of positive reactions of the mouse was recorded, and the percentage of the total number of detections was calculated. This figure shows statistics. [Figure 14] A 0.4 g von Frey filament was selected, and each mouse was subjected to 10 detections. The number of positive reactions of each mouse was recorded, and the percentage of the total number of detections was calculated. This figure shows statistics. DETAILED DESCRIPTION OF THE INVENTION

[0052] The following examples are provided to facilitate understanding of the present invention, but are not intended to limit the present invention. Unless otherwise specified, all experimental methods in the following examples are conventional methods. Unless otherwise specified, all test materials used in the following examples were purchased from conventional biochemical reagent stores.

[0053] 1. Equipment and reagents: ALMEMO2490 data acquisition device: AHLBORN, Germany. 2. PQQ disodium salt was purchased from Hisheng Pharmaceutical. 3. MHV-A59 virus strain was purchased from Suzhou Xishan Biotechnology Co., Ltd., with a PFU / milliliter of 1.4×107. 4. RAW264.7 was purchased from Beijing Union Cell Center.

[0054] Example 1 Compound SP3101 (Chinese name: [ka] Preparation and Identification of (4,5-Dioxo-4,5-dihydro-1-hydro-pyrrole[2,3-f]quinoline-7-hydroxy-9-carboxylic acid)

[0055] PQQ samples were dissolved in 1M NaOH aqueous solution to a concentration of 100 mg / ml, poured into multiple reaction flasks, and reacted in a microwave oven at 250°C for 1 hour. The samples were then removed. LCMS analysis revealed that the major decomposition product had a molecular weight of 258. The samples were filtered and purified several times by high-pressure prep-LC using high-pressure C18-acetonitrile / 2 mM ammonium acetate with a gradient of 2% to 30%. The resulting samples were centrifuged to remove the organic phase, then freeze-dried. The freeze-dried samples were then oven-dried at 105°C for 2 hours and weighed.

[0056] Mass spectrometry was performed in the positive ion mode using electrospray ionization, and the compound was detected as m / z=259.2 (M+1)+, which indicates that the free molecular weight of the compound is 258.

[0057] The nuclear magnetic resonance spectrum is shown in Table 1.

[0058] [Table 2]

[0059] After comparing and analyzing the nuclear magnetic resonance data, we further added maleic acid to the sample to create a hydrogen spectrum, which revealed two NH4 + The signal of is clearly shown, which indicates that the compound is a diamine salt.

[0060] The TGA weight loss analysis showed that the sample lost 5.97% weight at 150°C, which, combined with the fact that the sample was previously dried at 105°C and the nuclear magnetic resonance, suggests that the sample contains one molecule of bound water.

[0061] After purification, the impurities were analyzed by UPLC-MS, nuclear magnetic resonance spectroscopy, and TGA weight loss analysis to confirm the following structure, which was named the diammonium salt of SP3101, and could be converted to the SP3101 form by conventional treatment. [ka]

[0062] Example 2 Compound SP3102 (Chinese name: [ka] Preparation and Identification of (7-Acetoxy-4,5-dioxo-4,5-dihydro-1-hydro-pyrrole[2,3-f]quinoline-9-carboxylic acid)

[0063] The raw material SP3101 (3.1 g, 0.01 mol) was dissolved in dry pyridine (6 mL), followed by the addition of acetic anhydride (6 mL) and the mixture was stirred at room temperature for 20 hours. After the reaction was complete, the solvent was centrifuged and dried, and the resulting solid was separated by column chromatography to obtain compound SP3102. [ka]

[0064] Mass spectrometry was performed in the positive ion mode using electrospray ionization, and the compound was detected as m / z=300.1 (M+1)+, indicating that the free molecular weight of compound SP3102 is 300. Its nuclear magnetic resonance spectrum is shown in Table 2.

[0065] [Table 3]

[0066] Example 3 Compound SP3103 (Chinese name: [ka] Preparation and Identification of (7-Methoxy-4,5-dioxo-4,5-dihydro-1-hydro-pyrrole[2,3-f]quinoline-9-carboxylic acid)

[0067] Dimethyl sulfate (1.26 grams, 0.01 moles) was first added to a reaction flask, and the temperature was raised to 80°C. The raw material SP3101 (3.1 grams, 0.01 moles) was then added in batches. After the raw material had reacted as monitored by TLC, a certain amount of water was added to the reaction solution at 0°C, and then ammonia solution was slowly added dropwise, and sodium bicarbonate solution was added dropwise to adjust the pH of the reaction solution to about 4. The reaction solution was then filtered under suction to obtain the product SP3103. [ka]

[0068] Mass spectrometry was performed in the positive ion mode using electrospray ionization, and the compound was detected as m / z=273.1 (M+1)+, indicating that the free molecular weight of the compound was 272. Its nuclear magnetic resonance spectrum is shown in Table 3.

[0069] [Table 4]

[0070] Activity Experiment 1: Inflammatory pain model and pharmaceutical analgesic research Four SD rats weighing 200-250 grams were injected with saline into the plantar of their left paws to serve as normal controls (NC). Eighteen rats were used to construct an inflammatory pain model. 100 microliters of complete Freund's adjuvant was injected into the plantar of their left paws. 24 hours later, the mechanical pain threshold of the left paw was measured using an ALMEMO 2490 data acquisition device. Two rats with a pain threshold above 15 grams were discarded. The remaining 16 rats were randomly divided into four groups, each containing four rats. Each group received water (PC), SP3101 (20 mg / kg), PQQ (20 mg / kg), or ibuprofen (25 mg / kg / day) intragastrically.

[0071] The results are shown in FIGS. Figure 1 shows the analgesic effect of a single oral dose of 20 mg / kg SP3101 in rats. A clear analgesic effect was observed 1.5 hours after a single oral dose, and persisted for 72 hours (1-4 days) and 5-7 days. The analgesic effect then decreased, but still showed a significant difference compared to the model group (PC) (* indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and so forth).

[0072] Figure 2 shows the analgesic effect of repeated oral administration of 25 mg / kg ibuprofen to rats. The ibuprofen dose was 25 mg per kilogram of body weight, administered orally once daily for three consecutive days, with pain thresholds measured 1.5 hours after each administration. Similar to SP3101, ibuprofen was effective immediately, producing significant analgesia 1.5 hours after oral administration. Significant analgesia was observed after administration for one, two, or three consecutive days. However, there was no analgesic effect prior to administration (data not shown), and no administration on the fourth day resulted in no analgesic effect.

[0073] Figure 3 shows the analgesic effect of oral administration of 20 mg / kg / day SP330 (i.e., PQQ) to rats for three consecutive days. On the first day, no analgesic effect was observed 1.5 hours after oral administration, but on the second, third, fourth, fifth, sixth, and seventh days, a clear analgesic effect was observed.

[0074] The results show that the compound SP3101 described in the present invention has rapid and long-lasting analgesic effects, and the inventors have discovered that the compounds SP3102 and SP3103 also have similar effects.

[0075] Activity Experiment 2: Research into the mechanism of long-term analgesic action Similar to the modeling method in Experiment 1, 200-250 g SD rats were injected with 100 microliters of complete Freund's adjuvant into the plantar area of ​​the left paw. 24 hours later, mechanical pain thresholds of the left paw were measured using an ALMEMO 2490 data acquisition device. Rats with a pain threshold above 15 g were discarded. On the second day of modeling, each rat was injected with 100 microliters of the AMPK inhibitor compound c (Dorsomorphin, CAS No. 866405-64-3, CC, 0.4 mg / ml) via the tail vein. Half an hour later, SP3101 (20 mg / kg) was administered orally. Pain thresholds were measured 1.5 and 24 hours later, respectively.

[0076] The results are shown in Figure 4, which shows that inhibition of AMPK does not affect the short-term (1.5 hours) analgesic effect of SP3101, but abolishes its long-term (24 hours) analgesic effect.

[0077] Activity experiment 3: AMPK agonist RAW264.7 cells were seeded in two 6-well plates and grown to approximately 90% confluency. Different concentrations of SP3101 and SP3102 were added. After 24 hours, cells were disrupted with RIPA and protein expression was detected by protein hybridization.

[0078] Phosphorylation of serine 127 in AMPK is a sign of activation, and as shown in Figure 5, SP3101 and SP3102 at a concentration of 0.1 μM significantly activated AMPK (increased p-AMPK expression), and the expression of the antioxidant transcription factor Nrf2 increased in a dose-dependent manner, as did the expression of the autophagy-related protein LC3B. Therefore, the compounds described in this invention, represented by SP3101 and SP3102, are AMPK agonists.

[0079] Activity experiment 4 Cox-2 inhibitor 1. Sample production (1) Thaw RAW264.7 cells to a cell density of approximately 100% and wash twice with PBS. (2) After culturing for 3 hours in serum-free DMEM medium containing 1 μg / ml LPS, the cells were cultured for 21 hours in 2% serum-containing DMEM medium containing 1 μg / ml LPS. (3) Prepare 1110 μl of cell lysate, pipette it over the cells, shake it on an ice bath shaker for 15 minutes, and start the centrifuge to cool it down. (4) Scrape the large culture dish with a tip, aspirate the treatment solution, dispense it into two EP tubes, treat it at 12,000 r, 15 min, and 4°C, and then store it on ice.

[0080] 2. COX activity detection Cyclooxygenase COX activity was detected using an Abcam kit (ab204699 COX activity assay kit, fluorometric), which was handled according to the instructions, and finally fluorescent detection was performed at Ex / Em = 535 / 587 nm.

[0081] The results are shown in FIG. 6, which demonstrate that SP3101 has the same inhibitory effect on COX-2 activity as the positive control celecoxib.

[0082] Activity test 5: Inhibition of inflammation by SP3101 Viral pneumonia can be induced in C57BL / B6 mice by nasal instillation of MHV-A59 virus, and this model will be used to study the inhibitory ability of SP3101 on virus-induced pneumonia in mice.

[0083] The MHV-A59 virus strain was purchased from Suzhou Xishan Biotechnology Co., Ltd., with a PFU / milliliter of 1.4×107.

[0084] Six-week-old mice were divided into three groups of eight mice each. After anesthetizing the mice, saline was administered intranasally to the normal control group, and the remaining two groups were treated with a viral pneumonia model as follows: MHV was administered intranasally at a PFU of 1.4 x 10 5The mice were administered 10 μl of the solution into each nostril, 5 μl each, and then awoke approximately 3 to 6 hours later. The model treatment group received intragastric administration of SP3101 solution, with the dose for the SP3101 group being 20 milligrams per kilogram of body weight per day (mg / kg / d).

[0085] The mice were administered for five consecutive days, and four hours after the final administration, they were anesthetized and dissected. The lungs were fixed with paraformaldehyde and preserved for HE staining. Pathological sections are shown in Figure 7, demonstrating that SP3101 has a significant inhibitory effect on virus-induced pneumonia.

[0086] Activity Test 6: Study of neuropathic pain and medicinal analgesic effects Neuropathic pain was evaluated using the chronic sciatic nerve compression (CCI) model. Six- to eight-week-old B6 mice were anesthetized. A small horizontal incision was made from the base of the left leg approximately 1-2 mm above the horizontal line of the coccyx. The muscles were bluntly separated using hemostats. The sciatic nerve was located and removed by prodding with elbow tweezers. Three 4-0 chromic catgut ligatures, presoaked in sterile saline, were inserted into the sciatic nerve and ligated at 1 mm intervals above, below, and beneath the nerve. The ligation was tight enough to induce mild, brief spasms in the surrounding muscles. The sciatic nerve was then positioned under the muscle and the wound was sutured. The wound was disinfected with iodine tincture and observed.

[0087] Seven days after surgery, mechanical pain was detected in mice using Von Frey filaments. Mice were first allowed to adapt to a 20-minute session in a transparent organic glass plantar test apparatus. A series of Von Frey filaments were used to stimulate the mice longitudinally, resulting in modeling of the forepaw on one side (i.e., the right hind paw). Stimulation was initiated at a lower intensity. If a positive response was not evoked at this intensity, stimulation was initiated at a higher intensity. A sudden withdrawal of the paw was considered a positive response. In this case, stimulation was repeated at a lower intensity close to the intensity of the initial stimulation. The intensity interval between the initial positive and negative responses was recorded. A positive response at a force of less than 0.4 grams was considered successful modeling.

[0088] The mice were divided into three groups: a non-modeling group, a modeling vehicle group, and a modeling administration group. The vehicle was 0.5% methylcellulose, and the drug was SP3101. A single dose of 40 mg / kg was administered according to body weight, and pain thresholds were measured 1.5, 24, 48, and 72 hours after administration. The results are shown in Figure 8. SP3101 exerted significant analgesic effects 1.5, 24, and 48 hours after intragastric administration to the mice (**p<0.01).

[0089] Activity Test 7 SP3101 Sedation Experiment A total of 15 12-week-old B6 male mice were selected and allowed to adapt to the environment for three days. They were randomly divided into three groups: (1) normal control group (n=5), (2) low-dose SP3101 group (20 mg / kg) (n=5), and (3) high-dose SP3101 group (50 mg / kg) (n=5). Each group received a single dose of SP3101 via intraperitoneal injection. The normal control group received an equal volume of saline intraperitoneally. Two hours after administration, mice were tracked in a mouse open field test (OFT) using a video tracking and detection system for behavioral evaluation.

[0090] The results are shown in Figure 9. Intraperitoneally injected SP3101 significantly reduced open field activity in mice, demonstrating a clear sedative effect.

[0091] Activity Test 8: Experimental effect of SP3101 on pain model using oxaliplatin chemotherapy A total of 30 10-week-old C57LB6 wild-type mice, all male, were selected.

[0092] DAY 0: The mice were first allowed to adapt to the environment for 7 days. The basic mechanical pain threshold was measured, which was approximately 1-1.4 g. There were 30 mice in total in this experiment, and no abnormal mice were found.

[0093] Days 1–5: Mice were randomly divided into three groups: a normal control group (n=10), a model control group (n=10), and an SP3101 group (40 mg / kg, n=10). The SP3101 group received an intragastric administration of SP3101 solution, while the model control group received intragastric administration of double-distilled water. 0.5 h later, the model control group and the SP3101 group received an intraperitoneal injection of oxaliplatin (3 mg / kg). The same procedure was repeated for five consecutive days. On day 5, pain thresholds were measured using a Von Frey test.

[0094] Days 6 to 10: SP3101 and water alone were administered intragastrically.

[0095] Days 11-15: The procedures of days 1-5 were repeated, and on day 11, the pain threshold was measured using a Von Frey test.

[0096] Days 16-20: Observation only will be performed to detect mechanical pain on days 16 and 20.

[0097] The results are shown in FIG. 10. During the administration period of SP3101, hyperalgesia caused by oxaliplatin was significantly prevented, and the pain threshold of the model mice was clearly improved.

[0098] Activity Test 9: Effect of SP3101 on pain model of cisplatin chemotherapy Twenty 12-week-old male B6 mice were selected and allowed to adapt to the environment for 3 days.

[0099] Day 1: The basal mechanical pain threshold was determined using a Von Frey filament. Mice with abnormal pain thresholds (not present in this batch) were screened out. The pain threshold is generally between 1.4g and 2.0g. Based on the basal pain threshold, subsequent pain thresholds of 0.4g and 1.0g were determined in this experiment. Behaviors such as paw lifting and paw licking were considered positive responses. The proportion of mice with positive responses to the Von Frey filaments at the two corresponding thresholds was calculated to determine whether intragastrically administered SP3101 had a preventive effect on cisplatin-induced hyperalgesia.

[0100] DAY 1-DAY 5: After detecting the basal mechanical pain threshold, mice were randomly divided into (1) a model control group (n=10) and (2) a group receiving intragastric administration of SP3101 at 40 mg / kg / day (n=10). The model control group received intragastric administration of double-distilled water, and 0.5 h later, both groups received intraperitoneal injections of cisplatin (2.3 mg / kg / day) prepared in saline.

[0101] Days 6 to 10: SP3101 and water alone were administered intragastrically, and the mechanical pain threshold was detected on day 7.

[0102] Days 11-15: The procedures from days 1 to 5 were repeated, and on day 15 the mechanical pain threshold was detected.

[0103] Days 16-21: Observation only was performed, and on day 21 the mechanical pain threshold was detected.

[0104] The results are shown in Figures 11 and 12, and demonstrate that SP3101 has a significant alleviating effect on cisplatin-induced hyperalgesia.

[0105] Activity Study 10: SP3101 Paclitaxel Chemotherapy Pain Model Data A total of 30 B6 male mice aged 8 to 10 weeks were selected and allowed to adapt to the environment for 3 days.

[0106] On Day 0, mechanical pain thresholds were measured using a Von Frey filament. Mice with abnormal pain thresholds were screened out. The baseline pain threshold is generally between 0.6g and 1.0g. Based on the baseline pain threshold, 0.07g and 0.4g were selected as the subsequent pain thresholds for this experiment. Behaviors such as paw lifting and paw licking were considered positive responses. The proportion of mice with positive responses to the Von Frey filament at the two corresponding thresholds was calculated to determine whether intragastrically administered SP3101 had a preventive effect against paclitaxel-induced hyperalgesia. Mice were randomly divided into (1) a model control group (PC, n = 10), (2) a group receiving 40mg / kg of SP3101 intragastrically (n = 10), and (3) a normal control group (NC). The model control group received intragastric administration of double-distilled water. 0.5 hours later, the model control group and the SP3101 group received intraperitoneal injections of paclitaxel (4 mg / kg), while the normal control group received intraperitoneal injections of saline. Paclitaxel chemotherapy pain modeling was performed by intraperitoneal injection of paclitaxel (4 mg / kg) every other day, four times over seven days, for a total dose of 16 mg / kg. Modeling day 1 was designated day 0. SP3101 was administered intragastrically from day 0 onward, once daily, for a total of 15 days. From day 14 onward, animals were observed only. Mechanical pain was measured on days 0, 3, 7, 9, 14, 18, and 21.

[0107] The results are shown in Figures 13 and 14. Comparing the SP3101 group with the model control group, the pain threshold response rate was significantly reduced in both groups, indicating that SP3101 has a significant alleviating effect on paclitaxel-induced hyperalgesia.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof. 【Chemistry 1】 (In the formula, R １ is -OH, -OR, -OC(O)R, -NH ２ , —NHR, —SH, and —SR; R ２ But H, C １ ~C ３ Alkyl, NH ２ , NHR and —COOH; R ３ But H, C １ ~C ３ Alkyl, OH, NH ２ , NHR, —COOH and SH; R ４ But H, C １ ~C ３ Alkyl and C ６ ~C １０ aryl; R ５ But H, C １ ~C ３ Alkyl, OH, NH ２ , NHR, —COOH and SH; R ６ But H, C １ ~C ３ Alkyl, OH, NH ２ , NHR, —COOH and SH; where R is C １ ~C ６ It is alkyl.)

2. A compound of formula (II) or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof: 【Chemistry 2】 (In the formula, R １ is -OH, -OR, -OC(O)R, -NH ２ , —NHR, —SH and —SR, and R is selected from C １ ~C ６ It is alkyl.)

3. A compound shown below or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof. 【Transformation 3】

4. 4. A pharmaceutical composition comprising a compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, and a pharmaceutically acceptable carrier.

5. A pharmaceutical comprising the compound of any one of claims 1 to 3 or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof as an AMPK agonist.

6. A medicament comprising a compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof as an inhibitor of the Cox-2 enzyme.

7. 10. Use of a compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof for the manufacture of an analgesic medicament.

8. 10. Use of a compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof for the manufacture of a medicament for the prevention and treatment of inflammatory diseases.

9. Use of a compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof for the manufacture of an antitumor drug, or for the manufacture of a medicament for preventing and treating various liver damages and liver function declines caused by cytotoxic tumor chemotherapy, chemicals or medicines, and chronic hepatitis, as well as for preventing and treating myocardial infarction damage and viral myocarditis, or for the manufacture of a medicament for preventing and treating neurodegeneration, epilepsy, and migraine.

10. 10. Use of a compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof for the manufacture of a medicament for the prevention and treatment of chemotherapy-induced peripheral neuropathy.

11. 10. Use of a compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof for the manufacture of a medicament for the prevention and treatment of depression, and for the manufacture of a sedative or hypnotic drug.

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