Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/04—Ortho-condensed systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
  • A61P3/14—Drugs for disorders of the metabolism for electrolyte homeostasis for calcium homeostasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

• the present invention relates to the field of medicinal chemistry, and in particular to a class of 5-amino-1,4-dihydro-1,8-naphthyridines or pharmaceutically acceptable salts thereof and the same or a pharmaceutically acceptable salt thereof
• a pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof and a pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof as an L-type calcium channel blocker or/and an acetylcholinesterase inhibitor
• the application in particular, is for the preparation of a medicament for the treatment of cardiovascular diseases, cerebrovascular diseases or dementia. Background technique
• Alzheimer's disease is a central nervous system degenerative disease characterized by chronic, progressive cognitive impairment and memory impairment.
• the main pathological features are senile plaques, neurofibrillary tangles and neuronal loss, which seriously affect patients. Cognition, memory, language function and personal life ability and emotional personality.
• the more accepted pathology of Alzheimer's disease in the world is "the theory of cholinergic loss.” The theory is that the loss of choline acetate, a neurotransmitter in the brain of patients, is a key cause of Alzheimer's disease.
• Cholinesterase is a key enzyme in biological nerve conduction. In the cholinergic synapse, the enzyme can degrade acetylcholine, stop the excitatory effects of neurotransmitters on the postsynaptic membrane, and ensure that nerve signals are in vivo. The normal delivery. However, acetylcholinesterase can catalyze the cleavage reaction of acetylcholine, which leads to the loss of acetylcholine and the failure of neural signal transmission, thus affecting the cognitive and memory functions of the body.
• Vascular dementia is an acquired intelligent damage syndrome caused by various cerebrovascular diseases.
• the clinical manifestations are intelligent decline in memory, computational power, attention and executive function, which is second only to The second most common cause of dementia in Alzheimer's disease.
• the researchers believe that a mechanism of injury is: cerebral infarction, hypoxic hypoperfusion and hemorrhagic lesions, resulting in decreased brain tissue volume, delayed neuronal necrosis, and thus acetylcholine nerve damage in the brain, acetylcholine translation Reduced discharge, gradual emergence of memory impairment, cognitive impairment, social and daily life, decreased mobility. Taking an acetylcholinesterase inhibitor can effectively improve a patient's cognitive function, executive function, and daily living ability.
• the object of the present invention is to provide a compound and a pharmaceutical composition capable of inhibiting acetylcholinesterase activity and blocking extracellular calcium ions from flowing into cells through a calcium channel and as an L-type calcium channel blocker or/and acetylcholine Application of esterase inhibitors.
• the technical solution provided by the present invention is:
• R is selected from a C 6 -C 12 aryl group or a C 3 -C 12 heteroaryl group, the aryl or heteroaryl group optionally having a halogen atom, an amino group, a nitro group, a cyano group, a C 6 -C 12 aryl, C 3 -C 12 heteroaryl, CC 8 alkyl, C 3 -C 8 cycloalkyl, C 2 -C 8 chain or C 2 -C 8 alkynyl, optionally two
• the substituents at the position may be taken together to form an aliphatic ring, a heterocyclic ring, an aromatic ring or a heteroaryl ring, the C r C 8 alkyl group, a C 3 -C 8 cycloalkyl group, a C 2 -C 8 chain group or a C 2 group.
• the optional -CH 2 - in the -C 8 alkynyl group may be replaced by one or more of -0 -, -S -, -S0 2 -, -C(O)- or / and -NR 6 -, said dC Hydrogen at any position in the 8- alkyl, C 3 -C 8 cycloalkyl, C 2 -C 8 alkenyl or C 2 -C 8 alkynyl group may be substituted by one or more prime atoms, cyano, hydroxy, -NR 8 R 9 substitution;
• X is selected from -0-, -NR 7 -;
• R 3 is selected from hydrogen, C r C 8 alkyl or C 3 -C 8 cycloalkyl, and the optional -CH 2 - in the dC 8 alkyl or C 3 -C 8 cycloalkyl may be one or more a -0-, -S -, -C (O ) - and / or -NR 6 - substitution, the C r C 8 alkyl or C 3 -C 8 cycloalkyl group at any position in the hydrogen may be substituted with one or Substituted by a plurality of prime atoms;
• R 4 and R 5 are independently selected from -C 4 alkyl, C 3 -C 4 cycloalkyl, C 2 -C 4 chain or C 2 -C 4 alkynyl, said dC 4 Any of an alkyl group, a c 3 -c 4 cycloalkyl group, a c 2 -c 4 alkenyl group or a c 2 -
• R is selected from a C 6 -C 12 aryl group or a C 3 -C 12 heteroaryl group, and the ring of the aryl or heteroaryl group is optionally bonded to a halogen atom, a nitro group, an aryl group, a —C 8 alkyl group, C 3 -C 8 cycloalkyl, C 2 -C 8 alkenyl or C 2 -C 8 alkynyl substituted, wherein the substituents at two optional positions may be taken together to form an aliphatic ring, a heterocyclic ring, an aromatic ring or a hetero an aromatic ring, a C r C 8 alkyl, optionally substituted C 3 -C 8 cycloalkyl, C 2 -C 8 alkenyl or C 2 -C 8 alkynyl group of -CH 2 - may be substituted with one or more -0-, -S-, -so 2 -, -c(o)-
• R 1 is selected from dC 8 alkyl, C 3 -C 8 cycloalkyl, C 2 -C 8 alkenyl or C 2 -C 8 alkynyl, said C Cs alkyl, C 3 -C 8 cycloalkyl
• the optional -CH 2 - in the C 2 -C 8 alkenyl or C 2 -C 8 alkynyl group may be one or more of -0-, -S-, -C(O)- or/and-NR 6 -
• the hydrogen at any position in the -C 8 alkyl group, the C 3 -C 8 cycloalkyl group, the C 2 -C 8 alkenyl group or the 0 2 -0 8 alkynyl group may be substituted by one or more halogen atoms , an aryl group, a C 6 -C 12 aryl group, (3 ⁇ 4-0 12 heteroaryl, -NR 8 R 9 substituted;
• R 4 and R 5 are independently selected from -C 4 alkyl, C 2 -C 4 alkenyl or C 2 -C 4 alkynyl, said C C4 alkyl, C 2 -C 4 chain or C 2 -
• the optional -CH 2 - in the C 4 alkynyl group may be substituted by one or more of -0-, -S-, -S0 2 - or / and -C(O)-, the d-C4 alkyl group, c Of 2 -c 4 alkenyl or c 2 -c 4 alkynyl Hydrogen can be replaced by one or more prime atoms at any position.
• X is -0-
• R 8 and R 9 are independently selected from hydrogen, C r C 8 alkyl, C 3 -C 8 cycloalkyl, or R 8 and R 9 taken together to form a heteroalicyclic group of 5 to 9 ring atoms;
• R 1 is selected from dC 8 alkyl, C 3 -C 8 cycloalkyl, C 2 -C 8 alkenyl or C 2 -C 8 alkynyl, said C Cs alkyl, C 3 -C 8 cycloalkyl , optionally substituted C 2 -C 8 alkenyl or C 2 -C 8 alkynyl group of -CH 2 - may be substituted by one or more -0-, -S- and / or -NR 6 - replacing, the dC Hydrogen at any position in the 8- alkyl, C 3 -C 8 cycloalkyl, C 2 -C 8 alkenyl or c 2 -c 8 alkynyl group may be substituted by one or more atomic atoms, an aryl group, -NR 8 R 9 substitution;
• R 2 is selected from the group consisting of a trihaloalkyl group and a dC 8 alkyl group;
• R 4 and R 5 are independently selected from -C 4 alkyl, and optionally -CH 2 - in said C r C 4 alkyl may be one or more -0-, -S-, -S0 2 - or And -C(O)-substituted, the hydrogen at any position in the -C 4 alkyl group may be substituted by one or more ! 3 ⁇ 4 atom atoms.
• X is -0-
• R 6 is selected from the group consisting of hydrogen, C r C 8 alkyl, C 3 -C 6 cycloalkyl;
• R 2 is selected from the group consisting of trihalofluorenyl, dC 6 alkyl
• R 3 is selected from the group consisting of hydrogen, decyl, and ethyl
• R 4 and R 5 are independently selected from C r C 4 alkyl.
• alkyl refers to a saturated aliphatic hydrocarbon group, including straight-chain and branched hydrocarbon groups, including, but not limited to, mercapto, ethyl, propyl, isopropyl, n-butyl, isobutyl, t-butyl, An n-pentyl group, an n-hexyl group, etc.; an alkylene group means a divalent alkyl group;
• Alkenyl refers to an unsaturated straight or branched chain hydrocarbon group composed of at least one carbon-carbon double bond, including, but not limited to, ethylene, propylene, isopropylene, butylene, isobutylene, t-butene, n-pentene , isopentenyl, n-hexene, etc.;
• Alkenylene refers to a divalent alkenyl group
• alkynyl group refers to an unsaturated straight or branched chain hydrocarbon group consisting of one or two carbon-carbon porphins, including but not limited to ethynyl, propyne, isopropyne, butyne, isobutyne, Tert-butyne, pentyne, hexyne;
• Alkynylene refers to a divalent alkynyl group
• a cycloalkyl group refers to a monocyclic or fused ring that is all carbon (the fused ring means that each ring in the system shares an adjacent pair of carbon atoms with other rings in the system), one or more of which Without a fully linked pi-electron system, examples of cycloalkyl include, but are not limited to, cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, adamantane, cyclohexene, cyclohexadiene, Cycloheptane, cyclooctane, Cycloheptadiene and cycloheptatriene;
• Aryloxy represents -O-aryl and -0-heteroaryl, including but not limited to phenoxy, pyridyloxy, furanyloxy, thienyloxy, pyrimidinyloxy, pyrazinyloxy, etc. and derivatives thereof ;
• Substituted phenyl refers to a phenyl group substituted by one or more substituents, wherein the substituent includes, without limitation, C r C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkyne , dC 6 alkoxy, C 2 -C 6 alkenyloxy, phenoxy, benzyloxy, hydroxy, silk, peroxyhydroxy, ureido, aminodecanoyl, aminodecanoyl, carbonyl, amino, hydroxyamino , decanoylamino, decanoyl, fluorenyl, cyano, arylamino, isomeric, isocyanato, diazo, azide, decyl, triazide, nitrile, nitro, sub Nitro, isonitroso, nitrosylamino, imino, nitrosomino, oxo, dC 6 alkylthio, sulfo
• Heteroaryl means a monocyclic or fused ring radical of 5 to 12 ring atoms containing one, two, three or four ring heteroatoms selected from N, 0 or S, the remaining ring atoms being C, In addition, it has a fully conjugated ⁇ -electron system; the unsubstituted heteroaryl group includes, without limitation, pyrrole, furan, and ⁇ -plug.
• the heteroaryl group may be substituted or unsubstituted, and the substituent is preferably one or more, more preferably one, two or three, independently selected from (not limited to) a halogen atom, a nitrate Base, aryl, trihaloalkyl, acyl, heteroalicyclic, alkyl, cycloalkyl, alkenyl, alkynyl, amino, mono or dialkylamino, hydroxy, thiol, Alkoxy group, alkylthio group, etc.;
• a heteroalicyclic group means a monocyclic or fused ring group having 5 to 9 ring atoms in the ring, wherein one, two or three ring atoms are selected from N, 0 or S(0) m hetero atoms, wherein m is an integer from 0 to 2, and the remaining ring atoms are C.
• heteroalicyclic can be substituted or unsubstituted, when When substituted, the substituent is preferably one or two or more, more preferably one or two or three, still more preferably one or two, and includes, without limitation, a lower alkyl group, a trihaloalkyl group, a halogen, Hydroxy, alkoxy, decyl, alkylthio, cyano, acyl, thioacyl, 0-aminodecanoyl, fluorenyl-aminodecanoyl, 0-thioaminodecanoyl, fluorenyl-thioaminodecanoyl,
• Atom refers to fluorine, chlorine, bromine or a breaking group
• the trihaloalkyl group represents a -CX 3 group, wherein X is a halogen atom as defined above;
• An aliphatic ring represents a cyclic group of 3 to 9 carbon atoms, and the ring may have zero or one or more double bonds, but these rings do not have a fully conjugated ⁇ -electron system; the aliphatic ring may be substituted or not Substituted, the substituent is preferably one or two or more, more preferably one or two or three, and still more preferably one or two, independently selected from (not limited to) a halogen atom, a nitro group, a cyano group, Trihaloalkyl, acyl, heteroalicyclic, alkyl, cycloalkyl, alkenyl, alkynyl, amino, mono or dialkylamino, hydroxy, decyl, alkoxy, alkylthio, and the like.
• a heterocyclic ring means a hetero atom having 5 to 9 ring atoms in the ring, wherein one, two or three ring atoms are selected from ⁇ , 0 or S(0)m, wherein m is an integer from 0 to 2, and the remaining rings
• the atom is C, and the rings may have zero, one or more double bonds, but these rings do not have a fully conjugated ⁇ -electron system
• the unsubstituted heterocyclic ring includes, without limitation, pyrrolidine, piperidine, piperazine, Morpholine, thiomorpholine, homopiperazine, etc.
• the heterocyclic ring may be substituted or unsubstituted, and when substituted, the substituent is preferably one or two or more, more preferably one or two or three, More preferably, it is one or two, and includes, without limitation, a lower alkyl group, a trihaloalkyl group, a halogen group, a hydroxyl group, an alkoxy
• the aromatic ring means a cyclic aromatic hydrocarbon moiety having one or more ring closures, including, but not limited to, benzene, naphthalene, anthracene, phenanthrene, etc.; the aromatic ring may be substituted or unsubstituted; preferably one or more when substituted More preferably, one, two or three, the substituents are independently selected from (not limited to) halogen atoms, nitro groups, cyano groups, trihaloalkyl groups, acyl groups, heteroalicyclic groups, alkyl groups, naphthenes Alkyl, alkenyl, alkynyl, mono or dialkylamino, hydroxy, decyl, alkoxy, alkylthio, etc.;
• a heteroaryl ring means a monocyclic or fused ring group representing 5 to 12 ring atoms, containing one, two, three or four ring heteroatoms selected from N, 0 or S, the remaining ring atoms being C, in addition A fully conjugated ⁇ -electron system; the unsubstituted heteroaryl ring includes, without limitation, pyrrole, furan, ⁇ plug.
• the heteroaromatic ring may be substituted or unsubstituted, and the substituent is preferably one or more, more preferably one, two or three, independently selected from (not limited to) a halogen atom, a nitro group, Alkyl, trihaloalkyl, acyl, heteroalicyclic, alkyl, cycloalkyl, alkenyl, alkynyl, amino, mono or dialkylamino, hydroxy, decyl, alkoxy, alkylthio, etc. .
• the compounds provided herein also comprise a pharmaceutically acceptable equivalent of the compound or a mixture of two or more.
• the compounds provided herein are in a pharmaceutically acceptable equivalent, and the pharmaceutically acceptable salts comprise the acid or base salts of the compounds provided herein.
• the pharmaceutically acceptable salts have the pharmaceutical activity of the compound and are desirable in both biological and practical applications.
• a pharmaceutically acceptable acid Salts include, without limitation, acetates, sulfates, phosphates, citrates, propionates, adipates, succinates, tartrates, alginates, aspartates, benzoic acid Salt, terpene phthalate, sulfonate, benzoate, hydrogen sulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentane propionate, digluconate Salt, lauryl sulphate, ethyl sulphate, fumarate, glucoheptanoate, glycerol phosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide , hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, sulfonate, 2-naphthyl, nicot
• the compound provided by the present invention is in a pharmaceutically acceptable equivalent
• the pharmaceutically acceptable basic salt may comprise an ammonium salt, an alkali metal salt such as a sodium and potassium salt, an alkaline earth metal salt such as a calcium and magnesium salt, and an organic compound.
• a salt formed by a base such as a dicyclohexylamine salt, an N-mercapto-D glucosamine salt, and a salt with an amino acid such as arginine and lysine.
• the compounds provided herein are in a pharmaceutically acceptable equivalent, the prodrugs being derivatives of the compounds of the invention which require biological transformation, such as metabolism, prior to their pharmacological utility.
• Prodrugs are formulated from substances that improve chemical stability, improve patient acceptance and compliance, improve bioavailability, prolonged duration of action, improve organ selectivity, improve formulations such as enhanced water solubility, or reduce side effects such as toxicity.
• Prodrugs can be prepared from the compounds of the invention by conventional methods, see BURGER'S MEDICINAL CHEMISTRY AND DRUG CHEMISTRY, 5th Edition, Vol. 1, pp. 172-178, 949-982 (1995).
• isosteres refer to elements, functional groups, substituents, molecules or ions having different molecular formulas but exhibiting similar or identical physical properties.
• tetrazole is an isostere of a carboxylic acid because it has properties similar to those of a carboxylic acid, even though they have different molecular formulas.
• two equal rows of molecules have similar or identical sizes and shapes.
• the isosteric molecules will be isomorphic and capable of crystallizing together.
• Other physical properties of isostere molecules generally include boiling point, density, viscosity, and thermal conductivity. However, because the outer orbits can be hybridized differently, some properties may differ: dipole moment, polarity, polarization, size and shape.
• Isosteres including biological isles Things.
• Biological isosteres share certain biological properties in addition to physical similarities. Typically, bioisosteres interact with the same recognition sites or broadly produce similar biological effects.
• the present invention also provides the use of a 1,4-dihydro-1,8-naphthyridine derivative for the preparation of a medicament for regulating calcium homeostasis, treating cardiovascular diseases, cerebrovascular diseases or treating dementia.
• the dementia is preferably Alzheimer's disease or vascular dementia.
• the inventors examined the L-type Ca 2+ channel blocking activity of the 1,4-dihydro-1,8-naphthyridine derivative by using a patch clamp technique and a high content screening analyzer.
• the experimental results show that the 1,4-dihydro-1,8-naphthyridine derivative has a strong blocking activity on the L-type Ca 2+ channel, and its activity is better than the positive control drugs Nifedipine and Nimodipine.
• the inventors examined the inhibitory activity of the 1,4-dihydro-1,8-naphthyridine derivative on acetylcholinesterase.
• the experimental results show that the 1,4-dihydro-1,8-naphthyridine derivative can inhibit the activity of acetylcholinesterase.
• the compound of the present invention is a compound which inhibits the activity of acetylcholinesterase and blocks the extracellular calcium from flowing into the cell through the calcium channel, and has a dual activity.
• the substituent R in the formula of the present invention is an aryl group or a heteroaryl group
• the aryl group or the heteroaryl group is bonded to a halogen atom at any position.
• Substituents such as a hydroxyl group, a cyano group, an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group are substituted or ring-formed, and the obtained compounds are identical or similar in effect, and the preparation method is simple and easy.
• any position on the alkyl group, the cycloalkyl group, the alkenyl group or the alkynyl group may be substituted by a substituent or any -CH 2 - may be substituted.
• the substituent R 1 in the formula of the present invention is hydrogen, hydroxy, aryl, heteroaryl, -C 8 alkyl, C 3 -C 8 a cycloalkyl group, a C 2 -C 8 alkenyl group or a C 2 -C 8 alkynyl group, a -C 8 alkyl group, a C 3 -C 8 cycloalkyl group, a C 2 -C 8 alkenyl group or a C 2 -C Any position on the 8 alkynyl group is substituted by a substituent or any -CH 2 - thereof is substituted, and the obtained compounds are identical or similar in effect, and the preparation method is simple and easy.
• the substituent R 2 in the formula of the present invention is hydrogen, a halogen atom, a nitro group, an amino group, a hydroxyl group, a trihaloalkyl group, a -C 8 alkyl group.
• the substituent R 3 in the formula of the present invention is hydrogen, dC 8 alkyl or C 3 -C 8 cycloalkyl, dC 8 alkyl or Any position on the C 3 -C 8 cycloalkyl group is substituted by a substituent or any -CH 2 - thereof is substituted, and the obtained compounds are identical or similar in effect, and the preparation method is simple and easy.
• R 7 is hydrogen, aryl, heteroaryl, -C 8 alkyl, C 3 -C 8 cycloalkyl, C 2 -C 8 alkenyl or C 2 -C 8 alkynyl.
• Compound 16 5-amino-4-(2-decyloxyphenyl)-7-ethyl-2,6-dimercapto-1,4-2H-1,8-naphthyridine-3-carboxylic acid- Ethyl ester, such as S 1
• Step 2 Synthesis of intermediate D2 Add A (2.2 g, 0.015 mol), ammonium acetate (3.3 g, 0.043 mol) and 10 mL of sterol to the reaction flask, stir, reflux for 30 min, add C2 ( 3.47 g, 0.014 mol), and continue to reflux for 30 min. The residue was purified by EtOAc (EtOAc:EtOAc:EtOAc:EtOAc:ESI- MS: 312.4 [MH]"
• Step 2 Synthesis of intermediate D5 Add A (2.9 g, 0.019 mol), ammonium acetate (4.5 g, 0.058 mol), C5 (4.9 g, 0.019 mol) and 150 mL of sterol to the reaction flask, stir, reflux for 45 min, TLC detection, complete reaction, vacuum The residue was concentrated to give purified crystals eluted elut elut elut elut elut elut elut elut elut elut elut elut elut ESI-MS: 316.2 [MH] -.
• Example 24 Detection of L-type Ca 2+ channel block activity by high content screening analyzer High-content screening analyzer (HCS) platform, real-time fluorescence method for determination of compounds (@10 ⁇ ⁇ , @50 ⁇ ⁇ )
• HCS High-content screening analyzer
• the KC1 induced voltage-gated calcium ion influx inhibition activity of SH-SY5Y cells, thereby evaluating the calcium ion channel inhibitory activity of some of the compounds prepared in the examples.
• Fluo-4-AM calcium ion probe was used to load SH-SY5Y cells under physiological conditions, and then KC1 was used to induce voltage-gated calcium channel development. Calcium ions were flowed through the cell membrane calcium channel and combined with fluorescent probes. Fluorescence signal; at the same time, the high content records the intracellular real-time fluorescence signal, reflecting the calcium flow from the inflow intensity. Fluo-4-AM itself cannot be excited to produce fluorescence. When it enters the cell, it is cleaved by cytosolic esterase into Fluo-4, and then combined with calcium ions, it is excited by 488nm light to produce strong green fluorescence.
• KC1 acts as an agonist of the voltage-gated calcium channel: When K+ reaches a certain concentration, the voltage-gated calcium channel opens, and the calcium ion inflow enters the cell, which combines with the dye to produce fluorescence. If the calcium channel is inhibited by the compound, it enters the calcium ion inside the cell. Decreased, the fluorescence intensity decreases, and the degree of reduction is related to the degree of inhibition of the calcium channel by the compound.
• the L-type calcium channel is a voltage-gated calcium channel. In the SH-SY5Y cells used in this experiment, the voltage-gated calcium channel on the cell membrane surface is mainly L-type, so the calcium flux signal induced by KC1 is mostly L-type calcium channel signal.
• SH-SY5Y cells are from the Cell Biology Laboratory of Nanjing Medical University.
• Fluo-4 DirectTM Calcium Assay Kits purchased from Invitrogen, item number F 10471.
• KCL inorganic reagent purchased from sigma, formulated into 1M stock solution when used, diluted to 250 mM application solution when used.
• This experiment measures the inhibition rate of calcium channels in all compounds at a high concentration of 50 ⁇ and a low concentration of 10 ⁇ .
• the specific formulation procedure is as follows: All compounds were first prepared in DMSO with a stock solution of 0.01 , then each compound was diluted with DMSO. Two concentrations of 5000 ⁇ and 1000 ⁇ were used, and each concentration was further diluted 50 times with 1640 complete medium (using DMSO as a pure solvent control) to obtain a 2x compound solution for use.
• SH-SY5Y cells with a confluency of about 90% were trypsinized and incubated at 20,000 cells/well.
• the culture solution was removed, and 45 different concentrations of 2x compound solution (using DMSO as a pure solvent control) were added to each well, and 4 replicate wells were set for each concentration.
• the specific 96 wells were set as shown in Table 4. Show:
• Inhibition rate 100%* ( ⁇ control group - ⁇ administration group) / ⁇ control group
• Table 5 Compounds provided by the present invention for L-type Ca 2+ channel blocking activity
• the compound provided by the present invention is 10 ⁇ 50 ⁇
• Compound configuration According to the sample quality and molecular weight, the compound was configured into a 0.01 mol/L stock solution in DMSO. The concentration of 100 ⁇ compound was prepared: First, the compound stock solution was firstly set to 1000 mol/L, 200 ⁇ /L, 40 mol with DMSO. Concentration gradient of /L, 8 ⁇ /L, 1.6 ⁇ /L, 0.32 ⁇ /L, 0.064 ⁇ /L.
• the AchE stock solution was diluted with 1 X Reaction Buffer.
• each stock solution is mixed according to the ratio of 200 ⁇ A ⁇ ex Red reagent : 100 Horseradish peroxidas: 100 Choline Oxidase: 10 Ach : 9590 1 Reaction Buffer to obtain 2 x working fluid.
• Fluorescence detection The fluorescence value of each well at excitation wavelength 540 nm : emission wavelength 590 nm was measured under Infinite M200 enzyme labeling instrument, and the gain value was set to optimize.
• the positive vs. average 0D value-negative control average 0D value is used to find the logarithm of the dosing concentration based on the base 10, the logarithmic value is the abscissa, the inhibition rate is the ordinate, and the original 6.0 is drawn, and a pharmacological fit is fitted.
• the dose-effect relationship was determined by the sigmoid curve, and the drug concentration corresponding to the 50% inhibition rate was determined, that is, the IC50 value of the compound for inhibiting acetylcholinesterase activity.
• the test results of the compounds for acetylcholinesterase inhibitory activity are shown in Table 6.
• the compounds provided by the present invention inhibit the activity of IC 5 Q ( nmol / L ) ⁇
• the results of the tacrine 180 experiment showed that the compounds prepared in the examples were able to inhibit the activity of acetylcholinesterase.
• the compound of the present invention is a compound which can inhibit the activity of acetylcholinesterase and block the extracellular calcium from flowing into the cell through the calcium channel, has dual activity and has important potential therapeutic value.
• the above description is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can also make several improvements and retouchings without departing from the principles of the present invention. It should be considered as the scope of protection of the present invention.

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