Patent Application
20230348493
The invention belongs to the field of pharmaceutical and chemical industry, which relates to a compound for adjusting the activity of an NMDA receptor, and a pharmaceutical composition and the use thereof. Specifically, the present invention relates to the compound capable of enhancing the activity of the NMDA receptor and use of the compound in preparation of medicaments for preventing/treating depression, anxiety, tension, learning and cognitive deficits, neuropathic pain and other diseases.
N-methyl-D-aspartic acid (NMDA) receptor is an ionotropic receptor for an excitatory neurotransmitter glutamate and is involved in many complex physiological and pathological mechanisms. The NMDA receptor can mediate Ca2+ influx, enhance synaptic plasticity, and participate in learning-memory and nervous system development; when excitatory amino acid of the body is increased sharply, a large influx of Ca2+ can be induced by stimulating the NMDA receptors, resulting in cell death. The imbalance of NMDA receptor activity regulation has been considered to be an important reason of central nervous system diseases such as neurodegenerative diseases, depression, epilepsy and ischemic brain injury.
The NMDA receptor has an important role during excitatory neurotoxic process. The excitatory neurotoxic process is characterized in that the excessive release of extracellular glutamate results in excessive activation of NMDA receptor, causes influx of Ca2+ and Na+ and K+ efflux, causes activation of intracellular signaling pathway, intracellular Ca2+ overload, and cell dysfunction, induces apoptosis or initiates cell death signal transduction pathway, and ultimately leads to neuronal death, resulting in neurodegenerative diseases, depression and other diseases, while an NMDA receptor antagonist can reduce the excitatory neurotoxic effect of cells by inhibiting the activity of NMDA receptor. In addition, by mediating Ca2+ influx, NMDA receptor can also enhance synaptic plasticity and neuronal excitability, induce long term promotion (LTP), and promote neural cell development, and NMDA receptor plays an important role in the treatment of cognitive dysfunction caused by the diseases such as neurodegenerative diseases, depression and schizophrenia. Therefore, reasonable regulation of the activity of NMDA receptor plays an important role in the treatment of neurological diseases.
Studies show that the NMDA receptor antagonist has rapid antidepressant effects, and an NMDA receptor agonist can also regulate the sensitivity of postsynaptic AMPA by increasing the release of the neurotransmitter mediated by NMDA receptor to generate the antidepressant effects. An NMDA receptor partial agonist can exert dual (antagonist/agonist) effects on NMDA receptor through allosteric sites.
In the field of antidepressant research, an NMDA receptor modulator which is designed and synthesized, such as the NMDA partial agonist/antagonist, is of great significance for the treatment of depression by exerting partial agonistic and/or antagonistic effects on NMDA receptors.
After intensive research and creative labor, inventors of the invention obtain a compound represented by formula I. The inventors are surprised to find that the compound of formula I or a pharmaceutically acceptable salt, a solvate thereof, or a mixture thereof can be the agonist of NMDA receptor, especially the partial agonist, and has a high affinity with NMDA receptor; while reflected in animal models, the compound can rapidly, effectively and persistently prevent and treat depression and anxiety states of the model animals.
One aspect of the present invention relates to a compound represented by formula I, a pharmaceutically acceptable salt or ester, stereoisomer or solvate thereof,
wherein,
Ring A is 3- to 8-membered aliphatic heterocycle;
R1 is selected from H, C1-C6 alkyl, aryl-C1-C4 alkyl, C2-C6 acyl, —CONRR′ and natural amino acid fragments;
R2 is selected from H, C1-C6 alkyl, C1-C6 alkoxycarbonyl, C2-C6 acyl, —CONRR′ and natural amino acid fragments;
R3 is selected from H, cyano, 3- to 8-membered nitrogen-containing aliphatic heterocycle-C1-C4 acyl, C1-C6 alkoxycarbonyl and —CONHR4;
R and R′ are each independently selected from H and C1-C6 alkyl;
R4 is selected from H, C1-C6 alkyl, natural amino acid fragments and carboxylic acid derivatives of the said natural amino acid fragments;
optionally, the C1-C6 alkyl, aryl-C1-C4 alkyl, C2-C6 acyl, 3- to 8-membered nitrogen-containing aliphatic heterocycle-C1-C4 acyl, C1-C6 alkoxycarbonyl, —CONRR′, —CONHR4 and natural amino acid fragments are each independently substituted by one or more substituenis selected from: halogen, amino, hydroxyl, cyano, carboxyl, nitro, C1-C6 alkyl, and C1-C6 alkoxy;
and, R1, R2 and R3 satisfy:
and R3 is H; R2 is not H,
and R3 is H; R2 is not C2-C6 acyl or —CONRR′;
R1 and R3 are H; R2 is not C1-C4 alkyl, acetyl,
In some embodiments, Ring A is selected from 4- to 7-membered aliphatic heterocycle, and preferably 4- to 6-membered aliphatic heterocycle.
In some embodiments, Ring A is selected from 4- to 7-membered nitrogen-containing aliphatic heterocycle, and preferably 4- to 6-membered nitrogen-containing aliphatic heterocycle.
In some embodiments, Ring A is selected from
In some embodiments, Ring A is selected from
In some embodiments, R1 is selected from H, C1-C6 alkyl, phenyl-C1-C4 alkyl, C2-C6 acyl and natural amino acid fragments.
In some embodiments, R1 is selected from H, C1-C6 alkyl and C2-C6 acyl.
In some embodiments, R1 is H.
In some embodiments, R1 is selected from carboxyl residues of natural amino acids.
In some embodiments, R1 is selected from H, acetyl, methyl,
In some embodiments, R1 is selected from H, acetyl, methyl,
In some embodiments, R1 is selected from H, acetyl, methyl,
In some embodiments, R2 is selected from H, C1-C6 alkyl, C1-C6 alkoxycarbonyl, C2-C6 acyl, and natural amino acid fragments; optionally, the C1-C6 alkyl, phenyl-C1-C4 alkyl, C2-C6 acyl and natural amino acid fragments are each independently substituted by one or more substituents selected from: halogen, amino, hydroxyl, cyano, carboxyl, nitro, C1-C6 alkyl and C1-C6 alkoxy.
In some embodiments, R2 is selected from H, C1-C4 alkoxycarbonyl, C2-C6 acyl and carboxyl residues of the natural amino acids.
In some embodiments, R2 is selected from H, C1-C6 alkyl, C2-C6 acyl and carboxyl residues of natural amino acids.
In some embodiments, R2 is selected from H, C1-C6 alkyl and C2-C6 acyl.
In some embodiments, R2 is selected from carboxyl residues of natural amino acids.
In some embodiments, R2 is selected from H, methyl, ethyl, propyl, butyl, tert-butoxycarbonyl, acetyl,
In some embodiments, R2 is selected from H, methyl, ethyl, tert-butoxycarbonyl, acetyl,
In some embodiments, R2 is selected from H methyl, tert-butoxycarbonyl, acetyl,
In some embodiments, R3 is selected from H, cyano, 5- to 7-membered (e.g. 6-membered) nitrogen-containing aliphatic heterocycle-C1-C3 acyl (e.g. formyl), C1-C4 alkoxycarbonyl and —CONHR4; R4 is selected from H, natural amino acid fragments, amide derivatives of the said natural amino acid fragments and cyano derivatives of the said natural amino acid fragments.
In some embodiments, R3 is selected from H, cyano, 5- to 7-membered nitrogen-containing aliphatic heterocycle-C1-C3 acyl, C1-C4 alkoxycarbonyl and —CONHR4; R4 is selected from H, natural amino acid fragments and amide derivatives of the said natural amino acid fragments.
In some embodiments, R3 is selected from H, cyano, 5- to 7-membered nitrogen-containing aliphatic heterocycle-C1-C3 acyl, C1-C4 alkoxycarbonyl and —CONHR4; R4 is selected from H and natural amino acid fragments.
In some embodiments, R3 is selected from H, cyano, 5- to 7-membered nitrogen-containing aliphatic heterocycle-C1-C3 acyl, C1-C4 alkoxycarbonyl and —CONHR4; R4 is selected from H and amide derivatives of natural amino acid fragments.
In some embodiments, R3 is H.
In some embodiments, R3 is —CONHR4; R4 is selected from amino residues of natural amino acids, and amide derivatives of the said amino residue of natural amino acids.
In some embodiments, RA s selected from H, cyano,
In some embodiments, R3 is selected from H, cyano,
In some embodiments, R3 is selected from H, cyano, C1-C4 alkoxycarbonyl and —CONHR4; R4 is selected from H, natural amino acid fragments, amide derivatives of the said natural amino acid fragments, and cyano derivatives of the said natural amino acid fragments.
In some embodiments, R3 is selected from H, cyano, C1-C4 alkoxycarbonyl and —CONHR4; R4 is selected from H, natural amino acid fragments and amide derivatives of the said natural amino acid fragments.
In some embodiments, R3 is selected from H, cyano, C1-C4 alkoxycarbonyl and —CONHR4; R4 is selected from H and natural amino acid fragments.
In some embodiments, R3 is selected from H, cyano, C1-C4 alkoxycarbonyl and —CONHR4; R4 is selected from H and amide derivatives of natural amino acid fragments.
In some embodiments, Ring A is selected from
R1 is independently selected from —H, C1-C6 alkyl and C2-C6 acyl;
R2 is selected from H, Boc, C2-C6 acyl and natural amino acid fragments;
R3 is H.
In some embodiments, Ring A is selected from
R1 is selected from natural amino acid fragments;
R2 is selected from H, C1-C6 alkyl, C2-C6 acyl and natural amino acid fragments;
R3 is H.
In some embodiments, Ring A is selected from
R1 is H;
R2 is selected from natural amino acid fragments;
R3 is selected from methyl ester, ethyl ester, propyl ester, isopropyl ester, formamide, cyano and CONHR4;
R4 is selected from natural amino acid fragments and carboxylic acid derivatives of the said natural amino acid fragments.
In some embodiments, Ring A is selected from
R1 is H;
R2 is selected from H, C1-C6 alkyl, C2-C6 acyl;
R3 is selected from CONHR4;
R4 is selected from natural amino acid fragments and carboxylic acid derivatives of the said natural amino acid fragments.
In some embodiments, the said natural amino acid fragments are selected from the carboxyl residues of the following amino acids: Thr, Ser, Val, Gly, Ala, Ile, Phe, Gln and Tyr.
In some embodiments, the said natural amino acid fragments are selected from the amino residues of the following amino acids: Thr, Ser, Val, Gly, Ala, Ile, Phe, Gln and Tyr.
In some embodiments, natural amino acid fragments are selected from
In some embodiments, natural amino acid fragments are selected form
In some embodiments, natural amino acid fragments are selected from
In some embodiments, natural amino acid fragments are selected from
In the embodiments of the present invention, the carboxylic acid derivative of the natural amino acid fragments is formed by replacing a part of carboxyl group in the natural amino acid fragment with other atomic group and hydrolyzed to obtain the carboxylic acid. Amide derivatives, cyano derivatives and carboxylate derivatives of the said natural amino acid fragments are included. In some embodiments, the carboxylic acid derivatives of the natural amino acid fragments include, but are not limited to, amide derivatives, cyano derivatives and carboxylate derivatives of the following amino acid fragments: Thr, Ser, Val, Gly, Ala, Ile, Phe, Gln and Tyr. In some embodiments, the carboxylic acid derivative of the natural amino acid fragments is, for example:
In some embodiments, Ring A is 3- to 8-membered aliphatic heterocycle;
R1 is selected from H, C1-C6 alkyl, aryl-C1-C4 alkyl, C2-C6 acyl, —CONRR′ and natural amino acid fragments;
R2 is selected from H, C1-C6 alkyl, C1-C6 alkoxycarbonyl, C2-C6 acyl, —CONRR′ and natural amino acid fragments;
R3 is selected from H, cyano, C1-C6 alkoxycarbonyl and —CONHR4;
R and R′ are each independently selected from H and C1-C6 alkyl;
R4 is selected from H, C1-C6 alkyl, natural amino acid fragments and carboxylic acid derivatives of the said natural amino acid fragments;
optionally, the C1-C6 alkyl, aryl-C1-C4 alkyl, C2-C6 acyl, C1-C6 alkoxycarbonyl, —CONRR′, —CONHR4 and natural amino acid fragments are each independently substituted by one or more substituents selected from: halogen, amino, hydroxyl, cyano, carboxyl, nitro, C1-C6 alkyl and C1-C6 alkoxy.
In some embodiments, R1, R2 and R3 satisfy:
and R3 is H; R2 is not H,
and R3 is H; R2 is not C2-C6 acyl or —CONRR′;
R1 and R3 are H; R2 is not C1-C4 alkyl, acetyl,
In some embodiments, R3 is selected from H, cyano, C1-C4 alkoxycarbonyl and —CONHR4; R4 is selected from H, natural amino acid fragments, amide derivatives of the said natural amino acid fragments, and cyano derivatives of the said natural amino acid fragments.
In some embodiments, the present invention provides the following compounds or salts thereof.
The compound of the invention can be prepared by various methods known in the field.
In some embodiments, when R1, R2 and R3 are H in the formula I, the compound of the formula I can be prepared by the following steps:
In some embodiments, the following series of the compounds of the formula I can be further obtained from the compound of the formula I-1:
In some embodiments, the following series of the compounds of the formula I can be obtained from L-Cysteine Methyl Ester Hydrochloride as a starting material:
In some embodiments, the compounds of the formulas I-7, I-8, and I-9 can be respectively further deprotected, and reacted with R2-LG or R3H to obtain the corresponding compound of the formula I.
In the above scheme, LG represents a leaving group of a nucleophilic substitution reaction, e.g., halogen, etc.; Pg represents an amino protecting group, e.g., Boc, Fmoc, etc.
In some embodiments, the sequence of the above experimental steps can be adjusted according to a target product structure.
Treatment and Pharmaceutical Composition
In another respect, the invention provides a pharmaceutical composition, comprising a compound of the invention, a pharmaceutically acceptable salt or ester, stereoisomer or solvate thereof; optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.
In some embodiments, the pharmaceutical composition of the invention comprises the compound of the invention, and its pharmaceutically acceptable excipients.
In some embodiments, the pharmaceutical composition of the present invention contains 0.1 to 90 weight % of the compound of the formula I and/or its physiologically acceptable salt.
The compound in the pharmaceutical composition of the present invention, a pharmaceutically acceptable salt or ester, stereoisomer or solvate thereof can be used for a subject for:
In another respect, the present invention provides use of the compound, a pharmaceutically acceptable salt or ester, stereoisomer or solvate thereof in preparation of medicaments for:
In another respect, the present invention provides a method for preventing and/or treating depression, anxiety, stroke, Huntington's disease, Alzheimer's disease, neuralgia or schizophrenia in subjects, the method includes administering an effective amount of the compound of the invention, pharmaceutically acceptable salt or ester, stereoisomer or solvate thereof or the pharmaceutical composition of the invention to the subjects in need.
In another respect, the present invention provides a method for regulating (e.g., up- or down-regulating) activity of NMDA receptor (e.g., a human NMDA receptor) in vivo or in vitro, including providing a subject, a mammalian cell or an NMDA receptor with an effective amount of the compound of the formula I of the invention, pharmaceutically acceptable salt or ester, stereoisomer or solvate thereof.
In another respect, the present invention provides the compound of the formula I, pharmaceutically acceptable salt or ester, stereoisomer or solvate thereof for use in treatment and/or prevention of depression, anxiety, stroke, Huntington's disease, Alzheimer's disease, neuralgia or schizophrenia.
In another respect, the present invention provides the compound of the formula I, pharmaceutically acceptable salt or ester, stereoisomer or solvate thereof for use in in-vivo or in-vitro regulation (e.g., up- or down-regulation) of the activity of an NMDA receptor (e.g., human NMDA receptor).
The compound of the present invention, a pharmaceutically acceptable salt or ester, stereoisomer or solvate thereof, or the pharmaceutical composition of the present invention can be prepared to any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixir, lozenges, suppositories, injections (including injections, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, etc. A preferred dosage form depends on an intended administration mode and therapeutic use. The pharmaceutical composition of the invention should be sterile and stable under production and storage conditions. One preferred dosage form is an injection. The injection can be a sterile injectable solution. For example, the sterile injectable solution can be prepared by the following steps: doping a required dose of the compound of the present invention, a pharmaceutically acceptable salt or ester, stereoisomer or solvate thereof, in a suitable solvent, and optionally, doping other desired constituents (including, but not limited to, a pH modifying agent, a surfactant, an ionic strength enhancing agent, an isotonic agent, a preservative, a diluent, or any combination thereof) at the same time, and then the solution is subjected to filtration and sterilization. In addition, the sterile injectable solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze-drying) to facilitate storage and use. The sterile lyophilized powder can be dispersed in a suitable carrier before use, such as sterile pyrogen-free water.
In addition, the compound of the present invention, a pharmaceutically acceptable salt or ester, stereoisomer or solvate thereof may exist in the pharmaceutical composition in a unit dose form for application.
The compound of the present invention, a pharmaceutically acceptable salt or ester, stereoisomer or solvate thereof, and the pharmaceutical composition of the present invention can be applied by any suitable method known in the field including, but not limited to, oral administration, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., powder, ointments, or drops), or nasal routes. However, for many therapeutic uses, the preferred administration route/mode is parenteral administration (e.g., intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The technician should understand that an administration route and/or mode can be changed according to an intended purpose. In a preferred embodiment, the compound of the present invention, a pharmaceutically acceptable salt or ester, stereoisomers or solvate thereof, and a pharmaceutical composition are administered by intravenous infusion or injection.
The pharmaceutical composition of the present invention may include the compound of the invention, a pharmaceutically acceptable salt or ester, stereoisomers, or solvate thereof with a “therapeutically effective amount” or a “prophylactically effective amount”. The term “prophylactically effective amount” refers to the amount sufficient to prevent, stop or delay the occurrence of diseases. The term “therapeutically effective amount” refers to the amount sufficient to treat or at least partially prevent a disease and its complications of a patient who already has the disease. The therapeutically effective amount of the compound, a pharmaceutically acceptable salt or ester, stereoisomer or solvate thereof may vary according to the following factors: severity of disease to be treated, an overall state of patient's own immune system, patient's general conditions such as age, weight and sex, an administration mode of medicine, and other concurrent treatment measures.
In the present invention, a dosage regimen can be adjusted to obtain the best target response (e.g., therapeutic or preventive response). For example, single administration can be applied, multiple administration over a period of time also can be applied, or a dose can be reduced or increased proportionally with the urgency of the treatment.
A typical non-limit range for prophylactically or therapeutically effective amount of the compound of the present invention, a pharmaceutically acceptable salt or ester, stereoisomer, or solvate thereof is 0.02 to 100 mg/kg, e.g., 0.1 to 100 mg/kg, 0.1 to 50 mg/kg, or 1 to 50 mg/kg. It should be noted that the dose can be changed according to the type and severity of symptoms of the disease to be treated. In addition, it is understood by technicians in the field that, for any particular patient, a particular dosage regimen should be adjusted over time in accordance with the patient's needs and the professional evaluation of a physician; and the dosage ranges given herein are for illustrative purposes only and do not limit the use or scope of the pharmaceutical composition of the present invention.
In the present invention, the subject can be a mammal such as a human.
In the present invention, unless otherwise stated, scientific and technical terms used herein have the meanings generally understood by the technicians in the field. Moreover, cell culture, biochemistry, nucleic acid chemistry, immunology laboratory and other operation steps used in this invention are all routine steps widely used in the corresponding fields. In the meantime, for a better understanding of the present invention, definitions and explanations of the relevant terms are provided below.
As used herein, the term “natural amino acids” mainly includes the following 20 kinds of common amino acids: alanine (Ala), arginine (Arg), aspartic acid (Asn), asparagine (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), leucine (Leu), isoleucine (Ile), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val). The amino acid molecules are known to contain two functional groups, an amino group and a carboxyl group. The “amino acid fragment” described in this invention refers to a remaining part of amino acid molecules after removing a hydrogen from the amino group or deleting a hydroxyl group from the carboxyl group. Therefore, the “carboxyl residue” refers to the structure remaining after one hydroxyl group is deleted from the carboxyl group in the amino acid molecule.
As used herein, the term “alkyl” is defined as a linear or branched saturated aliphatic hydrocarbon group. In some embodiments, the alkyl contains 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6) carbon atoms. For example, as used herein, the term “C1-C6 alkyl” refers to straight or branched alkyl groups of 1 to 6 carbon atoms (e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-amyl, isoamyl, neo-amyl or n-hexyl, etc.).
As used herein, the term “C1-C6 alkoxy” refers to the C1-C6 alkyl-O-group, wherein the C1-C6 alkyl is described previously. Unrestricted examples of suitable C1-C6 alkoxy include methoxy, ethoxy, and isopropoxy groups, and the like.
As used herein, the term “C2-C6 acyl” refers to a univalent group containing 2 to 6 (e.g. 2, 3, 4, 5, or 6) carbon atoms with a general formula R—C(O)—, wherein R can be an alkyl, enyl, etc. Unrestricted examples of suitable C2-C6 acyl include acetyl, propionyl, isopropionyl, n-butyryl, sec-butyryl, t-butyryl, n-pentanoyl, isovaleryl, and tervaloyl.
As used herein, the term “C1-C6 alkoxycarbonyl” refers to the C1-C6 alkyl-OC(O)-group, wherein the C1-C6 alkyl is described previously. Unrestricted examples of the suitable C1-C6 alkoxycarbonyl include methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, tert-butoxycarbonyl, etc.
As used herein, the term “aryl” refers to a monocyclic or condensed ring group with aromaticity. For example, phenyl, naphthyl, etc.
As used herein, the term “aliphatic heterocycle” refers to a cyclic aliphatic hydrocarbon that contains at least one ring heteroatom selected from N, O, and S. In some embodiments, the term “3- to 8-membered aliphatic heterocycle” refers to the above aliphatic heterocycle containing 3 to 8 ring members (e.g., 3, 4, 5, 6, 7, or 8), e.g., 3- to 8-membered N-containing aliphatic heterocycle, 4- to 6-membered N-containing aliphatic heterocycle. Specific examples include, but are not limited to:
As used herein, the term “nitrogen-containing aliphatic heterocycle” refers to the aliphatic heterocycle containing at least one N atom, wherein the aliphatic heterocycle is defined as previously; the term of 3- to 8-membered nitrogen-containing aliphatic heterocycle refers to the nitrogen-containing aliphatic heterocycle described above that contains 3 to 8 ring members (e.g., 3, 4, 5, 6, 7, or 8).
As used herein, the term “3- to 8-membered nitrogen-containing aliphatic heterocycle-C1-C4 acyl” refers to a monovalent group of the 3- to 8-membered nitrogen-containing aliphatic heterocycle-R—C(O)-structure, wherein, the 3- to 8-membered nitrogen-containing aliphatic heterocycle is described previously, and R does not exist or is alkyl, enyl, etc. Examples include, but are not limited to:
As used herein, the terms “substitution” and “substituted” refer to selective substitution of one or more (e.g., one, two, three, or four) hydrogens connected to a specified atom by an indicated group, provided that the normal valence of the specified atom in the current situation is not exceeded and that the substitution results in a stable compound. The combination of substituents and/or variables are only allowed if such combination forms a stable compound.
If substituents are described as “independently selected from” a set of groups, each substituent is selected independently of the other. Therefore, each substituent may be the same or different from another (other) substituent.
As used herein, the term “one or more” means 1 or more under reasonable conditions, e.g. 2, 3, 4, 5 or 10.
Unless otherwise indicated, as used herein, a connection point of the substituent may come from any suitable position of the substituent.
As used herein, the term “stereoisomer” refers to an isomer that has a same atom connection order but has different spatial arrangement modes of atoms, e.g., enantiomers, geometric isomers, etc. The term “enantiomer” indicates an isomer formed due to at least one asymmetric center. In a compound with one or more (e.g., one, two, three, or four) asymmetric centers, enantiomers and diastereomers can be generated. When the molecule contains double bonds, such as C═C, C═N or N═N double bonds, the two atoms connected by the double bonds cannot rotate freely and can also exist in the form of geometric isomers (cis/trans).
As used herein, a solid line (), a solid wedge line (
) or a dashed wedge line (
) can be used to delineate the chemical bonds of the compound of the present invention. The solid line is used to describe bonding to asymmetric carbon atoms, which indicates that all possible stereoisomers formed by the side carbon atoms are included. The solid or dashed wedge line is used to describe bonding to the asymmetric carbon atoms, which indicates the existence of the indicated stereoisomers.
The compound of the present invention may exist in the form of a solvate (preferably a hydrate), wherein the compound of the present invention contains a polar solvent as a structural element of crystal lattice of the compound, in particular water, methanol or ethanol. The amount of the polar solvent, especially water, can be existed in stoichiometric or non-stoichiometric ratios. It should be understood that, although any solvate of the compound of the formula I used in the treatment of the disease or condition of this application may provide different properties (including pharmacokinetic properties), once the solvate is absorbed into a subject, the compound of the formula I is obtained, such that the use of the compound of the formula I covers the use of any solvate of the compound of the formula I respectively.
It should also be understood that some of the compounds of the present invention may exist in free-form for therapeutic use or, where applicable, in the form of their pharmaceutically acceptable derivatives. In the present invention, the pharmaceutically acceptable derivative includes, but is not limited to, the pharmaceutically acceptable salt or ester that are capable of providing, directly or indirectly, the compound of the present invention or metabolites or residues thereof after the pharmaceutically acceptable derivative is administered to a patient in need of it.
The pharmaceutically acceptable salts of the compound include an acid addition salt and an alkali addition salt.
An overview of suitable salts can be seen in Stahl and Wermuth's “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” (Wiley-VCH, 2002), or S. M. Berge et al., “Pharmaceutical Salts”, J. Pharm. Sci. 1977, 66, 1-19. A method for preparing the pharmaceutically acceptable salts of the compound of the present invention is known to technicians in the field.
The suitable acid addition salt is formed from acids that form the pharmaceutically acceptable salts. The suitable base addition salt is formed from bases that form pharmaceutically acceptable salts. In some embodiments, suitable inorganic acids are hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, or nitric acid, etc.; organic acids are formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, enanthic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)-benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, glucaric acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, ecanoic acid, aminosulfonic acid, trifluoromethanesulphonic acid, dodecyl sulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalic acid, naphthalene disulfonic acid, camphoric acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, propandioic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-glucosylic acid, mandelic acid, ascorbic acid, glucoheptonic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, etc. For example, HCl (or hydrochloric acid), HBr (or a hydrobromic acid solution), methanesulfonic acid, sulfuric acid, tartaric acid, or fumaric acid can be used to form pharmaceutically acceptable salts with the compound represented by the formula I.
As used herein, the term “pharmaceutically acceptable ester” means an ester derived from the compound of the present invention, which includes a physiologically hydrolyzable ester (the ester can be hydrolyzed under physiological conditions to release the compound of the present invention in form of free acid or alcohol). The compound of the invention can also be an ester.
As used herein, the term “metabolic stability” refers to the ability of a compound to enter and stably exist in the body as a prototype drug and not be metabolized to other structural forms.
As used herein, the term “subject” can refer to a patient or other animals that receive a composition of the present invention to treat, prevent, mitigate, and/or alleviate a disease or condition described in the present invention, and the animals particularly refers to mammals such as humans, dogs, monkeys, cattle, horses, etc.
As used herein, the term “excipients” refers to an excipient or a medium used to administer the compound, including, but not limited to, diluents, disintegrants, precipitation inhibitors, surfactants, flow aids, adhesives, lubricants, coating materials, etc. The examples of excipients include but are not limited to aluminum monostearate, aluminum stearate, carboxymethylcellulose, sodium carboxymethylcellulose, crospovidone, glyceryl isostearate, glyceryl monostearate, hydroxyethyl cellulose, hydroxymethylcellulose, octacosyl hydroxystearate, hydroxypropyl cellulose, lactose, a lactose monohydrate, magnesium stearate, mannitol, microcrystalline cellulose, etc.
Specific Models for Carrying Out the Present Invention
The embodiments of the present invention will be completely described below in conjunction with examples, but it is understood by the technicians in the field that the following examples are intended only to illustrate the invention and are not considered to limit the scope of the invention. If no specific condition is indicated in the examples, the general condition or the condition recommended by a manufacturer shall be followed. The reagents or apparatuses used without indication of the manufacturer are conventional products that can be obtained by market purchase.
At −15° C., 1.14 g (10 mmol, 1.0eq) of a raw material mercaptoethylamine hydrochloride was dissolved in 100 ml of methanol, 1.75 ml (15 mmol, 1.5eq) of triethylamine was added and sufficiently dissociated, 1.8 g (10 mmol, 1.0eq) of 1-Boc-3-azetidinone was added, the materials were continuously reacted at a low temperature for 15 min and then transferred to room temperature; after the completion of the materials reaction was monitored by TLC, vacuum concentration was performed, a concentrate was dissolved with 100 ml of ethyl acetate, washed with a small amount of water for many times (15 ml×4), dried with anhydrous sodium sulfate, filtered, and then concentrated. A crude product was purified with a silica gel column (PE:EtOAc=2:1) to obtain a total of 2.25 g of a white solid A-002 with a yield of 92.6%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.37 (s, 9H, —CH3), 3.12 (t, 2H, J=8.2 Hz), 3.34 (t, 2H, J=6.1 Hz), 4.1-4.20 (d, 2H, J=9.2 Hz), 4.59-4.62 (d, 2H, J=9.5 Hz), 9.3 (s, 1H). M+1:231.11.
1 g (6 mmol) of an intermediate A-002 was dissolved in 10 mL of anhydrous ethyl acetate, and then 10 mL of 4 mol/L HCl/EtOAc was added and stirred overnight at room temperature, and a white solid was generated in the system; after the completion of the materials reaction was monitored by TLC, the reaction was terminated. Filtration and washing with anhydrous diethyl ether were performed, and then a total of 0.54 g of a white solid A-001 was obtained with a yield of 76%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 3.10 (t, 2H, J=6.8 Hz), 3.37 (t, 2H, J=6.4 Hz), 4.1-4.15 (d, 2H, J=11.2 Hz), 4.55-4.6 (d, 2H, J=10.5 Hz), 9.5 (s, 1H), 9.8 (s, 1H). M+1:131.06.
According to the preparation method of A-002, the corresponding products A-016, A-030 and A-044 of five-membered ring or six-membered ring can be prepared. The methods were the same as that of A-002, except that the corresponding cycloketone was replaced; similarly, the corresponding products A-015, A-029 and A-043 of five-membered ring or six-membered ring were prepared according to the preparation method of A-001.
A total of 0.4 g (2.4 mmol) of the compound A-001 was dissolved in 20 ml of anhydrous DCM, and 665 μL (4.8 mmol) of TEA was added for dissociation; 155 μL (2.2 mmol) of acetyl chloride was added to 5 ml of DCM, and it was slowly dripped to the above reaction system with a dropper funnel under ice bath conditions, and the materials were continuously reacted at low temperature for half an hour and then moved to room temperature; the reaction was performed for 4 h at room temperature; when the completion of the materials reaction was monitored by TLC, the reaction was terminated. A small amount of water was used for washing (5 ml×3), an organic phase was taken, dried, filtered, concentrated, and separated through a silica gel column to obtain a brown oily substance, after stirring with hydrochloric acid to generate salt, a solid was generated, and a total of 0.31 g of a product A-003 was obtained with a yield of 61.9%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 2.03 (s, 3H), 3.15 (t, 2H, J=6.8 Hz), 3.29 (t, 2H, J=7.4 Hz), 4.11-4.21 (d, 2H, J=7.1 Hz), 4.51-4.63 (d, 2H, J=8.1 Hz), 9.53 (s, 1H). M+1:173.07.
Examples of preparation of A-017 to A-019: A-017: A-001 in the synthesis step of A-003 was replaced with A-015, and the rest operations were the same as those in the synthesis of A-003. M+1: 187.08.
Examples of preparation of A-031 to A-033: A-031: A-001 in the synthesis step of A-003 was replaced with A-029, and the rest operations were the same as those in the synthesis of A-003. M+1: 201.10.
Examples of preparation of A-045 to A-047: A-045: A-001 in the synthesis step of A-003 was replaced with A-043, and the rest operations were the same as those in the synthesis of A-003. M+1: 201.10.
The example illustrates a synthesis method of compound A-006.
A total of 0.4 g (2.4 mmol) of the intermediate A-001 was dissolved in 20 ml of anhydrous DCM, 665 μL (4.8 mmol) of TEA was added for dissociation, and after stirring for 5 min, 0.63 g (2.3 mmol) of Boc-tBu-Thr, 0.49 g (3.6 mmol) of HOBt, and 0.62 g (3.6 mmol) of EDCI were added and the raw materials were reacted overnight at room temperature. When the completion of the raw materials reaction was monitored by TLC, the reaction was terminated. A saturated citric acid aqueous solution was used for washing (10 mL×2), and an organic phase was taken, dried with anhydrous sodium sulfate, filtered, concentrated, and separated through a silica gel column to obtain a total of 0.61 g of a colorless oily substance of intermediate 1a with a yield of 68.8%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.14-1.20 (m, 12H), 1.37 (s, 9H), 3.24 (t, 2H, J=7.2 Hz), 3.38-3.65 (m, 2H), 3.72-3.95 (m, 3H), 4.10-4.30 (m, 1H), 4.65-4.94 (m, 3H), 7.34 (s, 1H), 8.23 (s, 1H). M+1: 388.22.
A total of 0.5 g (1.3 mmol) of the intermediate 1a was dissolved in 5 ml of anhydrous ethyl acetate, and 10 mL of 4 mol/L HCl/EtOAc was added and the raw materials were reacted overnight at room temperature, and a white solid was generated in the system; when the completion of the materials reaction was monitored by TLC, the reaction was terminated. The product was filtered under reduced pressure and washed with anhydrous diethyl ether to obtain a total of 0.25 g of a white solid A-006 with a yield of 73%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.21 (m, 3H), 3.21 (t, 2H, J=6.4 Hz), 3.36-3.65 (m, 2H), 3.7-3.97 (m, 3H), 4.10-4.30 (m, 1H), 4.60-4.71 (m, 2H), 4.86-5.01 (m, 1H), 5.65 (s, 1H), 8.23-8.28 (d, 2H, J=21 Hz). M+1: 232.10.
in the synthesis step of A-006 was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 218.09.
in the synthesis step of A-006 was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 218.09.
Boc in the synthesis step of A-006 was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 230.12.
in the synthesis step of A-006 was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 230.12.
in the synthesis step of A-006 was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 188.08.
in the synthesis step of A-006 was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 202.09.
in the synthesis step of A-006 was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 244.14.
in the synthesis step of A-006 was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 278.12.
in the synthesis step of A-006 was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 245.10.
in the synthesis step of A-006 was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 294.12.
was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 232.10.
was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 244.14.
was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 202.09.
was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 216.11.
was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 258.16.
was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 292.14.
was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 259.12.
was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 308.14.
was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 246.12.
was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 258.16.
was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 216.11.
was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 230.12.
was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 272.17.
was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 306.16.
was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 273.13.
was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 322.15.
was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 246.12.
was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 258.16.
was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 216.11.
was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 230.12.
was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 272.17.
was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 306.16.
was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 273.13.
was replaced with
and the rest operations were the same as those in A-006 synthesis. M+1: 322.15.
The example illustrates a synthesis method of compound A-059.
A total of 0.6 g (2.6 mmol) of the compound A-002 was dissolved in 20 ml of anhydrous DCM, and 1.4 mL (10 mmol) of TEA was added; 350 μL (5 mmol) of acetyl chloride was added to 5 mL of DCM, and it was slowly dripped to the above reaction system with a dropper funnel under ice bath conditions, and the materials were reacted at low temperature for half an hour and then moved to room temperature, the reaction was continually performed for 4 h at room temperature; when the completion of the materials reaction was monitored by TLC, the reaction was terminated. After washing with water (10 mL×3), an organic phase was taken, dried, filtered, concentrated, and separated through a silica gel column to obtain a total of 0.57 g of a brown oily substance A-058 with a yield of 81.4%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.38 (s, 9H), 2.05 (s, 3H), 3.14 (t, 2H, J=7.8 Hz), 3.35 (t, 2H, J=5.8 Hz), 4.13-4.22 (d, 2H, J=8.7 Hz), 4.60-4.63 (d, 2H, J=10.2 Hz). M+1: 273.12.
A synthesis method of the end product A-059 was the same as that of the A-058. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.73 (s, 3H), 2.06 (s, 3H), 3.01 (t, 2H, J=6 Hz), 3.74-3.82 (m, 3H), 4.05-4.07 (d, 1H, J=10 Hz), 4.64-4.66 (d, 1H, J=9.6 Hz), 4.90-4.92 (d, 1H, J=8.8 Hz). M+1: 215.08.
According to the synthesis method of A-058, the following products were prepared:
According to the synthesis method of A-057, the following products were prepared:
According to the synthesis method of A-059, the following products were prepared:
TEA was replaced with potassium carbonate, and the rest operations were the same as those in A-062 synthesis. M+1: 230.09.
and the rest operations were the same as those in A-059 synthesis. M+1: 258.12.
The example illustrates a synthesis method of compound A-066.
Under ice bath conditions, a total of 0.3 g (1.74 mmol) of the compound A-057 was dissolved in 20 ml of anhydrous DCM, and 490 μL (3.5 mmol) of TEA was added and stirred for dissociation for 5 min, 0.49 g (1.74 mmol) of Boc-tBu-Thr, 0.35 g (2.6 mmol) of HOBt and 0.5 g (2.6 mmol) of EDCI were added, the raw materials were gradually returned to room temperature, the reaction was performed for 6 h at room temperature; when the completion of the raw materials reaction was monitored by TLC, the reaction was terminated. A saturated citric acid aqueous solution was used for washing (10 mL×3), and an organic phase was taken, dried with anhydrous sodium sulfate, filtered, concentrated, and separated through a silica gel column to obtain a total of 0.59 g of a colorless oily substance of an intermediate 2a with a yield of 78%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.13-1.22 (m, 12H), 1.37 (s, 9H), 2.34 (s, 3H), 3.25 (t, 2H, J=5.9 Hz), 3.37-3.98 (m, 4H), 4.09-4.28 (m, 1H), 4.58-4.89 (m, 3H), 7.89 (s, 1H). M+1: 430.23.
A total of 0.5 g (1.17 mmol) of the intermediate 2a was dissolved in 5 mL of anhydrous ethyl acetate, 10 mL of 4 mol/L HCl/EtOAc was added, and the materials were reacted overnight at room temperature, and a white solid was generated in the system; when the completion of the materials reaction was monitored by TLC, the reaction was terminated.
The product was filtered under reduced pressure and washed with anhydrous diethyl ether to obtain a total of 0.26 g of a white solid A-066 with a yield of 72.6%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.17-1.22 (m, 3H), 2.36 (s, 3H), 3.23 (t, 2H, J=6.7 Hz), 3.35-3.95 (m, 4H), 4.12-4.32 (m, 1H), 4.58-4.92 (m, 3H), 5.65 (s, 1H), 8.21-8.29 (d, 2H, J=19 Hz). M+1: 274.11.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 230.09.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 260.10.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 272.14.
was replaced with
and the rest operations were the same as those in A-066. M+1: 274.11.
was replaced with
and the rest operations were the same as those in A-066. M+1: 286.15.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 288.13.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 300.17.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 288.13.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 300.17.
was replaced with
and the rest operations were the same as those in A-066. M+1: 288.13.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 300.17.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 302.15.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 314.18.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 316.16.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 328.20.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 316.16.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 328.20.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 302.15.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 314.18.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 316.16.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 328.20.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 330.18.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 342.21.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 330.18.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 342.21.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 274.15.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 286.19.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 288.17.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 300.20.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 302.18.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 314.22.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 302.18.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 314.22.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 308.14.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 320.17.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 322.15.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 334.19.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 336.17.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 348.20.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 336.17.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 348.20.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 232.10.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 244.14.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 246.12.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 258.16.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 260.14.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 272.17.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 260.14.
was replaced with
and the rest operations were the same as those in A-066 synthesis. M+1: 272.17.
The example illustrates a synthesis method of compound A-253.
Under −15° C., 1.8 g (10 mmol) of a raw material L-cysteine methyl ester hydrochloride was dissolved in 100 mL of methanol, fully stirred to be dissolved, 1.75 mL (15 mmol) of TEA was added, after the raw material was fully dissociated, 1.8 g (10 mmol, 1.0 eq) of a raw material 1-Boc-3-azetidinone was added, then continued to be subjected to a low-temperature reaction for 15 min, and transferred to room temperature. After reaction at room temperature for 4 h, when the completion of the materials reaction was monitored by TLC, a reaction solution was subjected to rotary evaporation, a concentrate was dissolved with 100 mL of ethyl acetate, washed with a small amount of water for multiple times (15 ml×4), dried with anhydrous sodium sulfate, filtered, and then subjected to rotary evaporation. A crude product was purified with a silica column (PE:EtOAc=2:1) to obtain a total of 2.4 g of a yellow transparent oily intermediate 3a with a yield of 83%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.37 (s, 9H), 3.06-3.55 (m, 6H), 3.91-4.19 (m, 4H), 8.69 (s, 1H). M+1: 289.11.
A total of 0.8 g (2.8 mmol) of the intermediate 3a was dissolved in 5 mL of anhydrous methanol, 5 mL of 7 mol/L NH3/CH3OH solution was added, stirred overnight at room temperature; when the completion of the materials reaction was monitored by TLC, the reaction was terminated. After vacuum concentration, a white solid was obtained, the traits were improved by stirring with petroleum ether, and the material was filtered to obtain 0.68 g of a white solid, namely A-254, with a yield of 90.7%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.37 (s, 9H), 3.02-3.27 (m, 2H), 3.36-3.45 (m, 1H), 3.94-4.23 (m, 4H), 8.14 (s, 2H), 8.78 (s, 1H). M+1: 274.11.
0.4 g of A-254 was dissolved in 3 mL of anhydrous ethyl acetate, a total of 5 mL of 4 mol/L HCl/EtOAc was added, and stirred at room temperature for 4 h; when the completion of the materials reaction was monitored by TLC, a white solid was obtained in the system, then filtered and washed with the anhydrous ether to obtain 0.21 g of the end product A-253 with a yield of 70%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 3.04-3.28 (m, 2H), 3.37-3.42 (m, 1H), 3.91-4.22 (m, 4H), 8.15 (s, 2H), 8.54 (s, 1H). M+1: 174.06.
Similarly, in the synthesis of the intermediate 3a, the raw material 1-Boc-3-azetidinone was replaced with N-Boc-3-pyrrolidone, N-Boc-4-piperidone, and N-Boc-3-piperidone, and the other operations were the same as those of the synthesis of the intermediate 3a, and an intermediate 3b, an intermediate 3c, and an intermediate 3d were prepared respectively.
The example illustrates a synthesis method of compound A-255.
A total of 2.4 g (8.3 mmol) of the intermediate 3a was dissolved in 10 mL of anhydrous ethyl acetate, and 20 mL of 4 mol/L hydrochloric acid/ethyl acetate solution was added under ice bath, the raw materials were reacted overnight, an off-white solid was precipitated; when the completion of the raw materials reaction was monitored by TLC, 1.8 g of an intermediate 4a was obtained after filtration, washed with anhydrous ether, and dried. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 3.05-3.55 (m, 6H), 3.91-4.19 (m, 4H), 8.26 (s, 1H), 8.69 (s, 1H). M+1: 189.06.
1.6 g (7.15 mmol) of the intermediate 4a was dissolved in 20 mL DCM under ice bath, and after being added 4 mL (28 mmol) of TEA for dissociation, 1 mL (14 mmol) of acetyl chloride was slowly added dropwise, the raw materials were gradually returned to room temperature and reacted overnight; when the completion of the raw materials reaction was monitored by TLC, the reaction was terminated; a saturated citric acid aqueous solution was used for washing (10 mL×3), an organic phase was taken, dried with anhydrous sodium sulfate, filtered, concentrated, and separated through a silica gel column to obtain 1.53 g of a yellowish oily substance, namely an intermediate 5a, with a yield of 76.5%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 2.05 (s, 3H), 3.07-3.53 (m, 6H), 3.95-4.21 (m, 4H), 8.48 (s, 1H). M+1: 231.07.
The intermediate 5a was ammonolyzed, and the synthesis method of this step was the same as that of A-254. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 2.04 (s, 3H), 3.02-3.25 (m, 2H), 3.38-3.47 (m, 1H), 3.97-4.22 (m, 4H), 8.23 (s, 2H), 9.01 (s, 1H). M+1: 216.07.
In the synthesis of the intermediate 4a, the raw material intermediate 3a was replaced with the intermediate 3b, the intermediate 3c and the intermediate 3d respectively, and the other operations were the same as those in the synthesis of the intermediate 4a, and an intermediate 4b, an intermediate 4c and an intermediate 4d were prepared respectively;
similarly, in the synthesis process of the intermediate 5a, the raw material intermediate 4a was replaced with the intermediate 4b, the intermediate 4c and the intermediate 4d respectively, and the other operations were the same as those in the synthesis of the intermediate 5a, and an intermediate 5b, an intermediate 5c and an intermediate 5d were prepared respectively;
similarly, in the synthesis of the intermediate 5a, acetyl chloride was replaced with isobutyryl chloride and pivaloyl chloride respectively, and an intermediate 6a, an intermediate 6b, an intermediate 6c, and an intermediate 6d, and an intermediate 7a, an intermediate 7b, an intermediate 7c, and an intermediate 7d were prepared respectively.
The example illustrates a synthesis method of compound A-258.
A total of 1.8 g (8 mmol) of the intermediate 4a was dissolved in 50 mL of dichloromethane, 0.89 g (8.8 mmol) of triethylamine was added under ice bath, and the materials were fully dissociated, then 2.2 g (8 mmol) of Boc-tBu-Thr, 1.18 g (8.8 mmol) of HOBt and 1.68 g (8.8 mmol) of EDCI were added, and the raw materials were naturally returned to room temperature for a reaction; after the completion of the raw materials reaction was monitored by TLC, a citric acid aqueous solution, a saturated sodium bicarbonate solution and a saturated sodium chloride solution were used for washing in order, and the material was dried with anhydrous sodium sulfate, filtered and concentrated. A concentrate was purified with a silica gel column, wherein the ratio of petroleum ether to ethyl acetate was 1:1, and a total of 1.9 g of an intermediate 8a was obtained with a yield of 53%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.12-1.20 (m, 12H), 1.37 (s, 9H), 3.14-3.41 (m, 2H), 3.59-3.87 (m, 5H), 4.04-4.84 (m, 5H), 7.84 (s, 1H), 8.52 (s, 1H). M+1: 446.22.
A total of 1.5 g (3.37 mmol) of the intermediate 8a was dissolved in a small amount of methanol, and 10 mL of 7 mol/L NH3/CH3OH solution was added under ice bath, and the raw materials were reacted overnight; after the completion of the raw materials reaction was monitored by TLC, a reaction solution was subjected to rotary evaporation to obtain a total of 1.45 g of an off-white solid intermediate 9a with a yield of 99.4%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.12-1.22 (m, 12H), 1.37 (s, 9H), 3.15-3.43 (m, 2H), 3.59-3.89 (m, 2H), 4.07-4.81 (m, 5H), 7.84 (s, 1H), 8.24 (s, 2H), 8.57 (s, 1H). M+1: 431.22
A total of 1.3 g (3 mmol) of the intermediate 9a was dissolved in 10 mL of anhydrous ethyl acetate, and 10 mL of 4 mol/L HCl/EtOAc solution was added under ice bath, and the materials were gradually returned to room temperature and reacted for 6 h; when the completion of the raw materials reaction was monitored by TLC, a white solid was precipitated in the system, filtered, and washed with anhydrous diethyl ether to obtain a total of 0.81 g of the end product A-258 with a yield of 86.1%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.12-1.17 (m, 3H), 3.05-3.17 (m, 1H), 3.39-3.43 (m, 1H), 3.72-3.92 (m, 2H), 4.06-4.81 (m, 5H), 5.65 (s, 1H), 7.55 (s, 1H), 7.93 (s, 2H), 8.24 (s, 2H). M+1: 275.11.
The intermediate 4a was respectively replaced with the intermediate 4b, the intermediate 4c and the intermediate 4d, and an intermediate 8b, an intermediate 8c and an intermediate 8d were respectively prepared; and an intermediate 9b, an intermediate 9c and an intermediate 9d were prepared accordingly.
The intermediate 4a was respectively replaced with the intermediate 4b, the intermediate 4c and the intermediate 4d, the raw material
was replaced with
and an intermediate 10a, an intermediate 10b, an intermediate 10c and an intermediate 10d were prepared respectively; and an intermediate 11a, an intermediate 11b, an intermediate 11c and an intermediate 11d were prepared accordingly.
The intermediate 4a was respectively replaced with the intermediate 4b, the intermediate 4c and the intermediate 4d, the raw material
was replaced with
and an intermediate 12a, an intermediate 12b, an intermediate 12c and an intermediate 12d were prepared respectively; and an intermediate 13a, an intermediate 13b, an intermediate 13c and an intermediate 13d were prepared accordingly.
was replaced with
and the rest operations were the same as those in A-258 synthesis. M+1: 261.09.
was replaced with
and the rest operations were the same as those in A-258 synthesis. M+1: 273.13.
was replaced with
and the rest operations were the same as those in A-258 synthesis. M+1: 275.11.
was replaced with
and the rest operations were the same as those in A-258 synthesis. M+1: 287.15.
was replaced with
and the rest operations were the same as those in A-258 synthesis. M+1: 289.13.
was replaced with
and the rest operations were the same as those in A-258 synthesis. M+1: 301.16.
was replaced with
and the rest operations were the same as those in A-258 synthesis. M+1: 289.13.
was replaced with
and the the rest operations were the same as those in A-258 synthesis. M+1: 301.16.
The example illustrates a synthesis method of compound A-285.
Under ice bath conditions, 0.8 g (1.74 mmol) of the intermediate 8a was dissolved in 10 mL of anhydrous ethyl acetate, and a total of 5 mL of 4 mol/L HCl/EtOAc was added, and the raw materials were gradually returned to room temperature and reacted for 4 hours; when the completion of the raw materials reaction was monitored by TLC, a white solid was generated in the system. The reaction was terminated, after vacuum filtration a filter cake was taken, continuously stirred for 10 min in 15 mL of anhydrous ethyl acetate, and then filtered to obtain a total of 0.51 g of white solid A-285 with a yield of 90.1%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.12-1.17 (m, 3H), 3.07-3.19 (m, 1H), 3.38-3.42 (m, 1H), 3.68-3.91 (m, 5H), 4.07-4.83 (m, 5H), 5.63 (s, 1H), 7.53 (s, 1H), 7.88 (s, 2H). M+1: 290.11.
The example illustrates a synthesis method of compound A-302.
0.7 g (1.6 mmol) of the intermediate 9a was dissolved in 10 mL of anhydrous DCM, 832 μL (6 mmol) of TEA was added, 0.7 g of trifluoroacetic anhydride (TFAA, 3.2 mmol) was added under ice bath conditions, and the materials were gradually returned to room temperature, and stirred at the room temperature for 12 h; when the completion of the raw materials reaction was monitored by TLC, the reaction was terminated; an organic phase was washed with 10 mL of water and 10 mL of saturated aqueous sodium chloride respectively, dried with anhydrous sodium sulfate, filtrated, concentrated, and separated through a silica gel column (PE:EtOAc=2:1) to obtain a total of 0.62 g of an end-product intermediate 14a with a yield of 92%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.13-1.21 (m, 12H), 1.37 (s, 9H), 3.03-3.14 (m, 1H), 3.39-3.42 (m, 1H), 3.69-3.86 (m, 2H), 4.03-4.75 (m, 5H), 7.81 (s, 1H), 8.34 (s, 1H). M+1: 413.21.
0.5 g of the intermediate 14a was dissolved in 10 mL of anhydrous ethyl acetate under ice bath conditions, fully stirred and dissolved, a total of 5 mL of 4 mol/L HCl/EtOAc was added, the raw material was gradually returned to room temperature and reacted at room temperature for 5 hours, and a yellow solid was generated in the system; when the completion of the raw materials reaction was monitored by TLC, the reaction was terminated. After filtration, a filter cake was taken and stirred with anhydrous ethyl acetate for 15 min and then filtered to obtain a total of 0.26 g of A-302 with a yield of 83%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.13-1.18 (m, 3H), 3.06-3.18 (m, 1H), 3.39-3.42 (m, 1H), 3.74-3.91 (m, 2H), 4.05-4.79 (m, 5H), 5.68 (s, 1H), 8.29 (s, 2H), 8.52 (s, 1H). M+1: 257.10.
Under ice bath, 0.5 g (1.5 mmol) of Fmoc-Val was dissolved in 10 mL of anhydrous DCM, 2 drops of DMF were added for catalysis, then 230 μL (1.65 mmol) of TEA was added, 133 μL (1.58 mmol) of oxalyl chloride was added drop by drop, and the raw materials were returned to room temperature, and reacted overnight; when the completion of the raw materials reaction was monitored by TLC, 0.35 g (1.5 mmol) of compound A-002 was added to the reaction system, and the reaction system was continuously stirred at the room temperature for 4 h; when the completion of the raw materials reaction was monitored by TLC, the reaction was terminated; saturated citric acid was used for washing twice, saturated sodium chloride solution was used for washing once, and an organic phase was taken, dried with anhydrous sodium sulfate, filtered, concentrated, and separated through a silica gel column (PE:EtOAC=3:1) to obtain a total of 0.69 g of colorless oily substance with a yield of 83.3%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.12-1.23 (m, 6H), 1.37 (9H), 2.15-2.25 (m, 1H), 3.16-3.73 (m, 5H), 4.16-4.79 (m, 8H), 7.21-8.29 (m, 9H). M+1: 552.25.
0.48 g of the intermediate 15a was dissolved in 5 mL of anhydrous acetonitrile, 5 mL of diethylamine was added, and the raw materials were reacted at 50° C. for 4 h; when the completion of the raw materials reaction was monitored by TLC, the reaction was terminated; the reaction product was subjected to rotary evaporation under reduced pressure, 10 ml of DCM was added, and the product was continuously subjected to rotary evaporation, then the above steps were repeated twice; and then 0.25 g of an oily substance with a yield of 87.2% was obtained by separating through a silica gel column. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.12-1.20 (m, 6H), 1.37 (s, 9H), 2.14-2.27 (m, 1H), 3.13-3.77 (m, 5H), 4.13-4.57 (m, 4H), 8.23 (s, 2H). M+1: 330.18.
0.25 g of the intermediate 16 was dissolved in 4 mL of ethyl acetate, 3 mL of 4 mol/L HCl/EtOAc was added, and the raw materials were stirred at room temperature for 3 h; when the completion of the raw materials reaction was monitored by TLC, a white solid was generated in the system, filtered, and dried to obtain 0.16 g of the white solid with a yield of 79%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.12-1.19 (m, 6H), 2.14-2.25 (m, 1H), 3.13-3.75 (m, 5H), 4.16-4.61 (m, 4H), 8.29 (s, 2H), 9.01 (s, 1H). M+1: 230.12.
The synthesis method was similar to that of A-329, and a total of 0.65 g of a yellowish solid was obtained with a yield of 89.3%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.14-1.27 (m, 6H), 2.15-2.25 (m, 1H), 3.16-3.73 (m, 5H), 4.16-4.79 (m, 8H), 7.21-8.29 (m, 9H). M+1: 452.19.
Under ice bath conditions, a total of 0.62 g of the intermediate 17 was dissolved in 10 mL of anhydrous DCM, 300 μL of TEA was added for dissociation, 185 μL of acetic anhydride was slowly added dropwise, and the raw materials were gradually returned to room temperature and continuously reacted for 6 h; when the completion of the raw materials reaction was monitored by TLC, the reaction was terminated; the mixture was washed with a saturated citric acid aqueous solution and then with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, concentrated, and separated through a silica gel column to obtain 0.52 g of an oily substance with a yield of 83%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.13-1.27 (m, 6H), 2.05-2.27 (m, 4H), 3.15-3.70 (m, 5H), 4.12-4.75 (m, 8H), 7.21-8.28 (m, 9H). M+1: 494.2.
The synthesis method was similar to that of the intermediate 16a, and a total of 0.38 g of an oily substance was obtained, HCl/EtOAc was added and the mixture was stirred, then a white solid was generated in the system, filtered, and dried to obtain 0.34 g of a product A-330. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.12-1.18 (m, 6H), 2.05-2.24 (m, 4H), 3.11-3.78 (m, 5H), 4.12-4.65 (m, 4H), 8.60 (s, 2H). M+1: 272.14.
The synthesis method was similar to that of the intermediate 8a, and a total of 1.52 g of a colorless oily substance was obtained with a yield of 81.3%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.03-1.37 (m, 27H), 2.32-2.38 (m, 1H), 3.02-3.73 (m, 4H), 4.14-4.68 (m, 10H), 7.12-8.32 (m, 10H). M+1: 709.36.
The synthesis method was similar to that of the intermediate 16a, and 0.84 g of an oily substance was obtained with a yield of 86.9%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.01-1.38 (m, 27H), 2.32-2.38 (m, 1H), 3.05-3.77 (m, 4H), 4.12-4.71 (m, 7H), 7.76 (s, 1H), 8.35 (s, 2H). M+1: 487.29.
The synthesis method was similar to that of A-329, and 0.49 g of a white solid was obtained with a yield of 87.5%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.07-1.22 (m, 9H), 2.18-2.27 (m, 1H), 3.07-3.78 (m, 7H), 4.14-4.59 (m, 4H), 5.72 (s, 1H), 8.21-8.32 (m, 4H). M+1: 331.17.
in the synthesis steps of A-331 was replaced with
and the rest operation was the same as that in the A-331 synthesis. M+1: 317.16.
in the synthesis steps of A-331 was replaced with
and the rest operations were the same as those in A-331 synthesis. M+1: 329.20.
5.2 g (18 mmol) of the intermediate 3a was dissolved in 20 mL of methanol, 3.6 g (90 mmol) of NaOH was taken and dissolved in 10 mL of water to obtain an aqueous NaOH solution, the aqueous NaOH solution was slowly added to the system of the intermediate 3a under ice bath conditions and the color of the reaction solution became darker, and the raw materials were gradually returned to room temperature and reacted for 3 hours; when the completion of the raw materials reaction was monitored by TLC, the reaction was terminated; the mixture was concentrated under reduced pressure to remove methanol in the system, pH value of the concentrate was adjusted to slightly acidic with dilute hydrochloric acid, a white solid was precipitated from the system, filtered, and dried by infrared lamp, and 4.3 g of the white solid was obtained with a yield of 86.9%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.37 (s, 9H), 3.06-3.56 (m, 3H), 3.94-4.23 (m, 4H), 8.54 (s, 1H). M−1: 273.1.
Under ice bath conditions, a total of 0.8 g (2.9 mmol) of the intermediate 21a was dissolved in 20 mL of acetonitrile, 0.8 mL (5.8 mmol) of TEA and 1.1 g (3.5 mmol) of TBTU were added, then 0.53 g (3.1 mmol) of O-tert-butyl-L-serine methyl ester hydrochloride was added, and the raw materials were gradually returned to the room temperature and reacted for 4 hours; when the completion of the raw materials reaction was monitored by TLC, the reaction was terminated. The mixture was washed with saturated citric acid and then washed with saturated sodium chloride, dried with anhydrous sodium sulfate, filtered, concentrated, and separated through a silica gel column to obtain 0.86 g of an oily substance with a yield of 76.1%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.13 (s, 9H), 1.42 (s, 9H), 2.67-4.38 (m, 13H), 7.43 (s, 1H), 8.21 (s, 1H). M+1: 432.21.
The synthesis method was similar to that of the intermediate 9a, and a total of 0.76 g of an oily substance was obtained with a yield of 93.5%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.13 (s, 9H), 1.42 (s, 9H), 2.67-4.38 (m, 10H), 7.06-7.21 (m, 3H), 8.21 (s, 1H). M+1: 417.21.
The synthesis method was similar to that of A-329, and a total of 0.53 g of a white solid was obtained with a yield of 81.3%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 2.67-4.54 (m, 10H), 4.94 (s, 1H), 7.21-8.11 (m, 5H). M+1: 261.10.
in A-332 synthesis was replaced with
and the rest operations were the same as those of A-332. M+1: 275.11.
was replaced with
and the rest operations were the same as those in the A-332 synthesis. M+1: 289.13.
was replaced with
and the rest operations were the same as those in the A-332 synthesis. M+1: 303.14.
was replaced with
and the rest operations were the same as those in the A-332 synthesis. M+1: 303.14.
The synthesis method was similar to that of the intermediate 21a, and a total of 0.68 g of an oily substance was obtained with a yield of 76.7%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 2.10 (s, 3H), 2.67-2.92 (m, 2H), 3.67 (m, 1H), 4.12-4.38 (m, 4H), 7.06 (s, 1H), 12.39 (s, 1H). M+1: 217.06.
The synthesis method was similar to that of the intermediate 22a, and a total of 0.93 g of an oily substance was obtained with a yield of 79.1%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.13 (s, 12H), 2.10 (s, 3H), 2.67-2.92 (m, 2H), 3.67 (s, 3H), 3.69-4.38 (m, 7H), 7.06 (s, 1H), 8.32 (s, 1H). M+1: 374.17.
The synthesis method was similar to that of the intermediate 23a, and a total of 0.67 g of a substance was obtained with a yield of 75.2%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.13 (s, 12H), 2.10 (s, 3H), 2.67-2.92 (m, 2H), 3.33-4.38 (m, 7H), 7.06 (s, 1H), 7.21 (s, 2H), 8.32 (s, 1H). M+1: 359.17.
The synthesis method was similar to that of A-332, and a total of 0.48 g of a colorless oily substance was obtained, and then evacuated by an oil pump to obtain a white solid with a yield of 85.7%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 2.10 (s, 6H), 2.33-2.85 (m, 2H), 4.12-4.54 (m, 7H), 4.94 (s, 1H), 7.21 (s, 2H), 8.26 (s, 2H). M+1: 303.11.
in the synthesis steps of A-371 was replaced with
and the rest operations were the same as those of A-371. M+1: 317.12.
The synthesis method was similar to that of the intermediate 19a, and a total of 0.56 g of a colorless oily substance was obtained with a yield of 75.4%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.19 (s, 9H), 1.42 (s, 9H), 2.32-2.67 (m, 2H), 3.33-4.54 (m, 11H), 4.94 (s, 1H), 7.06 (s, 1H), 7.21 (s, 2H), 7.31 (s, 1H), 8.06 (s, 1H). M+1: 504.24.
The synthesis method was similar to that of A-371, and a total of 0.31 g of a white solid was obtained with a yield of 87.9%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 2.67-2.92 (m, 2H), 3.54-4.52 (m, 11H), 5.08 (s, 2H), 7.06-7.21 (m, 3H), 8.32 (s, 1H), 8.92 (s, 2H). M+1: 348.13.
Under ice bath conditions, a total of 0.7 g of the intermediate 8a was dissolved in 20 mL of DCM, 0.4 g of K2CO3 was added, 170 μL of acetyl chloride was dissolved in 5 mL of DCM to obtain a solution, and the solution was dripped to the reaction system and the raw materials were gradually returned to room temperature and reacted for 4 hours; when the completion of the raw materials reaction was monitored by TLC, the reaction was terminated; the mixture was washed with a saturated citric acid solution and then washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, concentrated, and separated by a silica gel column to obtain a total of 0.53 g of a colorless oily substance intermediate 27a with a yield of 69%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.13-1.23 (m, 12H), 1.37 (s, 9H), 2.05 (s, 3H), 3.15-3.43 (m, 2H), 3.61-3.87 (m, 5H), 4.05-4.87 (m, 5H), 7.84 (s, 1H). M+1: 488.24.
The synthesis method was similar to that of the intermediate 23a, and a total of 0.46 g of an oily substance was obtained with a yield of 92.6%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.13-1.25 (m, 12H), 1.37 (s, 9H), 2.04 (s, 3H), 3.16-3.44 (m, 2H), 3.62-3.87 (m, 2H), 4.06-4.85 (m, 5H), 7.84 (s, 1H), 8.35 (s, 2H). M+1: 473.24.
The synthesis method was similar to that of A-329, and a total of 0.25 g of a white solid was obtained with a yield of 87.5%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.13-1.17 (m, 3H), 2.03 (s, 3H), 3.07-3.18 (m, 1H), 3.39-3.43 (m, 1H), 3.73-3.92 (m, 2H), 4.07-4.83 (m, 5H), 5.63 (s, 1H), 7.57 (s, 1H), 7.95 (s, 1H), 8.25 (s, 2H). M+1: 317.12.
The synthesis method was similar to that of the intermediate 15a, and a total of 0.89 g of a yellow powdered solid was obtained with a yield of 78.6%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.13-1.42 (m, 21H), 2.7-3.57 (m, 4H), 4.26-4.51 (m, 9H), 7.28-7.91 (m, 9H). M+1: 610.29.
The synthesis method was similar to that of the intermediate 16a, and a total of 0.45 g of a yellow solid was obtained with a yield of 80.3%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.13-1.42 (m, 21H), 2.7-3.57 (m, 4H), 4.26-4.51 (m, 9H), 8.96 (s, 1H). M+1: 388.22.
The synthesis method was similar to that of A-329, and a total of 0.22 g of white powder was obtained with a yield of 70.9%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.16 (d, 3H), 2.70 (t, 2H), 3.47-4.21 (m, 8H), 5.37 (s, 1H), 8.96 (s, 3H). M+1: 232.11.
The synthesis method was similar to that of the intermediate 17a, and a total of 0.42 g of white powder was obtained with a yield of 102.4%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.43 (s, 3H), 2.7-3.57 (m, 4H), 4.26-4.51 (m, 9H), 5.44 (s, 1H), 7.28-7.91 (m, 10H). M+1: 454.18.
The synthesis method was similar to that of 18a, and a total of 0.33 g of a solid was obtained with a yield of 77.6%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.43 (s, 3H), 2.10 (s, 3H), 2.7-3.57 (m, 4H), 4.26-4.51 (m, 9H), 5.44 (s, 1H), 7.28-7.91 (m, 9H). M+1: 496.19.
The synthesis method was similar to that of A-330, and a total of 0.14 g was obtained with a yield of 70%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.43 (s, 3H), 2.10 (s, 3H), 2.7-3.57 (m, 4H), 4.26-4.51 (m, 9H), 5.44 (s, 1H), 7.8 (s, 2H). M+1: 274.12.
In the synthesis process of the intermediate 30a, the intermediate A-002 was replaced with the intermediate A-016, the intermediate A-030 and the intermediate A-044 respectively, and the other operations were the same as those of the synthesis of the intermediate 30a, and an intermediate 30b, an intermediate 30c and an intermediate 30d were prepared respectively;
similarly, in the synthesis of the intermediate 32a, the intermediate 30a was replaced with the intermediate 30b, the intermediate 30c and the intermediate 30d respectively, and the other operations were the same as those of the synthesis of the intermediate 32a, and the intermediate 32b, intermediate 32c and intermediate 32d were prepared respectively.
similarly, in the synthesis process of the intermediate 33a, the intermediate 30a was replaced with the intermediate 30b, the intermediate 30c and the intermediate 30d respectively, and the other operations were the same as those in the synthesis of the intermediate 33a, and an intermediate 33b, an intermediate 33c and an intermediate 33d were prepared respectively;
similarly, in the synthesis of the intermediate 33a, acetyl chloride was replaced with isobutyryl chloride and pivaloyl chloride respectively, and an intermediate 34a, an intermediate 34b, an intermediate 34c, and an intermediate 34d, and an intermediate 35a, an intermediate 35b, an intermediate 35c, and an intermediate 35d were prepared respectively.
in A-337 synthesis was replaced with
and the rest operations were the same as those in A-337 synthesis. M+1: 230.09.
in A-337 synthesis was replaced with
and the rest operations were the same as those in A-337 synthesis. M+1: 244.11.
in A-337 synthesis was replaced with
and the rest operations were the same as those in A-337 synthesis. M+1: 286.15.
in A-337 synthesis was replaced with
and the rest operations were the same as those in A-337 synthesis. M+1: 320.14.
in A-337 synthesis was replaced with
and the rest operations were the same as those of A-337. M+1: 287.11.
in A-337 synthesis was replaced with
and the rest operations were the same as those in A-337 synthesis. M+1: 336.13.
The synthesis method was similar to that of 1a, and a total of 0.22 g of yellow powder was obtained with a yield of 37%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.13-1.42 (m, 24H), 2.7-3.57 (m, 4H), 4.26-4.70 (m, 11H), 5.37 (s, 1H), 7.28-7.90 (m, 10H). M+1: 711.34.
The synthesis method was similar to that of the intermediate 16a, and 0.13 g of a solid was obtained with a yield of 41%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.13-1.42 (m, 24H), 2.7 (t, 2H), 3.47-4.64 (m, 10H), 5.37 (s, 1H), 7.28-7.90 (m, 3H). M+1: 489.27.
The synthesis method was similar to that of A-331, 59 mg of a substance was obtained with a yield of 22.3%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.16 (s, 6H), 2.7 (t, 2H), 3.47-4.64 (m, 10H), 5.37 (s, 2H), 8.95 (s, 4H). M+1: 333.16.
In the synthesis of an intermediate 36a, the intermediate 32a was replaced with 32b, 32c and 32d respectively, and the other operations were the same as those of the synthesis of the intermediate 36a, and an intermediate 36b-1, an intermediate 36c-1 and an intermediate 36d-1 were prepared respectively;
similarly, in the synthesis of the intermediate 36a, the raw material intermediate was respectively replaced with 32b, 32c and 32d,
was respectively replaced with
so that 36b-2, 36b-3, 36c-2, 36c-3, 36d-2 and 36d-3 were obtained respectively.
8.0 g (18 mmol) of the intermediate 8a was dissolved in 30 mL of methanol, 3.6 g (90 mmol) of NaOH was taken and dissolved in 10 mL of water to obtain an aqueous NaOH solution, the aqueous NaOH solution was slowly added to the reaction system of the intermediate 8a under ice bath conditions, the color of the reaction solution became darker, and the raw materials were gradually returned to room temperature and reacted for 3 hours; when the completion of the raw materials reaction was monitored by TLC, the reaction was terminated; the mixture was concentrated under reduced pressure to remove methanol in the system, pH value of the system was adjusted to slightly acidic by dilute hydrochloric acid, a white solid was precipitated from the system, filtered, and dried by infrared lamp, and 4.3 g of the white solid was obtained with a yield of 58.1%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.13 (s, 12H), 1.37 (s, 9H), 2.67-3.77 (m, 3H), 4.12-4.64 (m, 6H), 7.39-7.06 (m, 2H), 12.01 (s, 1H). M−1: 432.1.
Under ice bath conditions, a total of 4.3 g (10 mmol) of the intermediate 38a was dissolved in 20 mL of acetonitrile, 2.7 mL (20 mmol) of TEA and 3.78 g (12 mmol) of TBTU were added, then 1.83 g (10.7 mmol) of O-tert-butyl-L-serine methyl ester hydrochloride was added, and the raw materials were gradually returned to room temperature and reacted for 4 hours; when the completion of the raw materials reaction was monitored by TLC, the reaction was terminated. The mixture was washed with saturated citric acid and then washed with saturated sodium chloride, dried with anhydrous sodium sulfate, filtered, concentrated, and separated by a silica gel column to obtain 4.58 g of an oily substance with a yield of 76.1%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.13 (s, 24H), 1.37 (s, 9H), 2.67-3.84 (m, 3H), 3.67 (s, 3H), 4.12-4.64 (m, 8H), 7.39-8.06 (m, 3H). M+1: 603.34.
A total of 4.58 g (7.60 mmol) of the intermediate 39a was dissolved in a small amount of methanol, and 10 mL of 7 mol/L NH3/CH3OH solution was added under ice bath, and the raw materials were reacted overnight; after the completion of the raw materials reaction was monitored by TLC, a reaction solution was subjected to rotary evaporation to obtain a total of 4.04 g of an off-white solid intermediate 40a with a yield of 90.4%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.13 (s, 24H), 1.37 (s, 9H), 2.67-3.84 (m, 3H), 4.12-4.64 (m, 8H), 7.39-8.06 (m, 5H). M+1: 588.34.
1 g of the intermediate 40a was dissolved in 4 mL of ethyl acetate, 3 mL of 4 mol/L HCl/EtOAc was added, and the raw materials were stirred at room temperature for 3 h; when the completion of the raw materials reaction was monitored by TLC, a white solid was generated in the system, then filtered, and dried to obtain 0.51 g of a white solid with a yield of 79%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.16 (m, 6H), 2.67-4.62 (m, 11H), 5.32 (s, 2H), 7.3 (s, 2H), 8.96 (s, 4H). M+1: 376.16.
In the synthesis of the intermediate 38a, the raw material
was replaced with
respectively, and the other operations were the same as those of the synthesis of the intermediate 38a, so that an intermediate 38b and an intermediate 38c were prepared respectively;
similarly, in the synthesis of the intermediate 39a, the raw material
was respectively replaced with
and 39b, 39c and 39d were obtained respectively;
in the synthesis step of the intermediate 39a was replaced with
and the rest operations were the same as those of A-374. M+1: 422.18.
in the synthesis step of the intermediate 39a was replaced with
and the rest operations were the same as those of A-374. M+1: 389.16.
in the synthesis step of the intermediate 39a was replaced with
and the rest operations were the same as those of A-374. M+1: 438.18.
in the synthesis step of the intermediate 38a was replaced with
and the rest operations were the same as those of A-374. M+1: 376.16.
in the synthesis step of the intermediate 38a was replaced with
Boc, and the rest operations were the same as those of A-374. M+1: 388.2.
the synthesis step of the intermediate 38a was replaced with
and the rest operations were the same as those of A-374. M+1: 390.18.
in the synthesis step of the intermediate 38a was replaced with
and the rest operations were the same as those of A-374. M+1: 402.21.
in the synthesis step of the intermediate 38a was replaced with
and the rest operations were the same as those of A-374. M+1: 390.18.
in the synthesis step of the intermediate 38a was replaced with
and the rest operations were the same as those of A-374. M+1: 402.21.
21a (4.32 g, 15.7 mmol) was added to 100 ml of DCM (insoluble) under ice bath conditions, a solid in the solution was gradually dissolved after adding 2.5 eq of TEA, 1.1 eq of piperidine, 1.2 eq of HOBt and 1.2 eq of EDCI were added, and the raw materials were subjected to a thermal reaction for 10 min and then reacted overnight at room temperature; after the completion of the raw materials reaction was monitored by TLC (PE:EA=1:1), the mixture was washed sequentially with water, saturated citric acid solution and saturated saline, dried with anhydrous sodium sulfate and filtered, and a filtrate was concentrated and separated by column chromatography to obtain 4.9 g of a substance with a yield of 90%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.42 (s, 9H), 1.55-2.92 (m, 8H), 3.54-4.38 (m, 9H), 7.28 (s, 1H). M+1: 342.18.
The intermediate 41a (4.9 g, 14.4 mmol) was added to ethyl acetate for complete dissolution, then 28 ml of HCl-EtOAc solution was added, the raw materials were stirred at room temperature for 6 h; when the completion of the raw materials reaction was monitored by TLC (PE:EA=1:2), the mixture was filtered, and a filter cake was washed with ethyl acetate, and dried to obtain 4.69 g of a white solid with a yield of 93%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.55-2.92 (m, 8H), 3.54-4.38 (m, 9H), 7.28 (s, 2H). M+1: 242.13.
Under ice bath conditions, A-404 (0.5 g, 1.8 mmol) was added to 20 ml of DCM, a solid was insoluble, and the solid was gradually dissolved after adding 2.5 eq of TEA, 1.1 eq of Boc-tBu-Thr, 1.2 eq of HOBt and 1.2 eq of EDCI were added, and the raw materials were subjected to a thermal reaction for 10 min and reacted at room temperature for 5 h; after the completion of the raw materials reaction was monitored by TLC (DCM:MeOH=10:1), the mixture was sequentially washed with water, saturated citric acid solution and saturated saline, then dried with anhydrous sodium sulfate and filtered, and a filtrate was concentrated and separated by column chromatography to obtain 0.85 g substance with a yield of 94%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.16 (s, 21H), 1.55-2.92 (m, 8H), 3.54-4.64 (m, 11H), 7.28 (s, 2H). M+1: 499.29.
The intermediate 42a was added to 8 ml ethyl acetate for complete dissolution, 8 ml of HCl-EtOAc solution was added, the raw materials were stirred at room temperature for 6 h; after the completion of the raw materials reaction was monitored by TLC (PE:EA=1:2), the mixture was filtered, and a filter cake was washed with ethyl acetate, and then dried to obtain 0.45 g of a white solid with a yield of 69.6%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.16 (s, 3H), 1.55-2.92 (m, 8H), 3.54-4.38 (m, 11H), 5.37 (s, 1H), 7.28 (s, 1H), 8.93 (s, 2H). M+1: 343.18.
in A-404 synthesis was replaced with
and the rest operations were the same as those in A-404 synthesis. M+1: 244.11.
in A-404 synthesis was replaced with
and the rest operations were the same as those in A-404 synthesis. M+1: 257.14.
in A-404 synthesis was replaced with
and the rest operations were the same as those in A-404 synthesis. M+1: 260.08.
in A-405 synthesis was replaced with
and the rest operations were the same as those in A-405 synthesis. M+1: 341.20.
in A-405 synthesis was replaced with
and the rest operations were the same as those in A-405 synthesis. M+1: 329.16.
was replaced with
and the rest operations were the same as those of A-405. M+1: 343.18.
was replaced with
and the rest operations were the same as those of A-405. M+1: 331.14.
was replaced with
and the rest operations were the same as those in A-405 synthesis. M+1: 356.21.
was replaced with
and the rest operations were the same as those of A-405. M+1: 344.17.
was replaced with
and the rest operations were the same as those of A-405. M+1: 359.15.
was replaced with
and the rest operations were the same as those of A-405. M+1: 347.12.
was replaced with
and the rest operations were the same as those in A-405 synthesis. M+1: 355.21.
was replaced with
and the rest operations were the same as those in A-405 synthesis. M+1: 343.18.
was replaced with
and the rest operations were the same as those of A-404. M+1: 258.12.
was replaced with
and the rest operations were the same as those of A-404. M+1: 271.15.
was replaced with
and the rest operations were the same as those of A-404. M+1: 274.10.
was replaced with
and the rest operations were the same as those of A-405. M+1: 357.19.
was replaced with
and the rest operations were the same as those in A-405 synthesis. M+1: 345.16.
was replaced with
and the rest operations were the same as those of A-405. M+1: 370.22.
was replaced with
and the rest operations were the same as those in A-405 synthesis. M+1: 358.19.
was replaced with
and the rest operations were the same as those in A-405 synthesis. M+1: 373.17.
was replaced with
and the rest operations were the same as those in A-405 synthesis. M+1: 361.13.
replaced with
and the rest operations were the same as those in A-405 synthesis. M+1: 369.23.
was replaced with
and the rest operations were the same as those in A-405 synthesis. M+1: 357.19.
was replaced with
and the rest operations were the same as those of A-404, M+1: 272.14.
was replaced with
and the rest operations were the same as those of A-404. M+1: 285.17.
was replaced with
and the rest operations were the same as those of A-404. M+1: 288.12.
was replaced with
and the rest operations were the same as those in A-405 synthesis. M+1: 371.21.
was replaced with
and the rest operations were the same as those in A-405 synthesis. M+1: 359.17.
was replaced with
and the rest operations were the same as those in A-405 synthesis. M+1: 384.24.
was replaced with
and the rest operations were the same as those in A-405 synthesis. M+1: 372.20.
was replaced with
and the rest operations were the same as those in A-405 synthesis. M+1: 387.18.
was replaced with
and the rest operations were the same as those in A-405 synthesis. M+1: 375.15.
was replaced with
and the rest operations were the same as those of A-404. M+1: 272.14.
was replaced with
and the rest operations were the same as those of A-404. M+1: 285.17.
was replaced with
and the rest operations were the same as those of A-404. M+1: 288.12.
was replaced with
and the rest operations were the same as those in A-405 synthesis. M+1: 369.23.
A-455: A-404 in A-405 synthesis was replaced with A-452,
was replaced with
and the rest operations were the same as those in A-405 synthesis. M+1: 357.19.
was replaced with
and the rest operations were the same as those in A-405 synthesis. M+1: 371.21.
replaced with
and the rest operations were the same as those in A-405 synthesis. M+1: 359.17.
was replaced with
and the rest operations were the same as those in A-405 synthesis. M+1: 384.24.
was replaced with
and the rest operations were the same as those in A-405 synthesis. M+1: 372.20.
was replaced with
and the rest operations were the same as those in A-405 synthesis. M+1: 387.18.
replaced with
and the rest operations were the same as those in A-405 synthesis. M+1: 375.15.
Under ice bath conditions, the intermediate 38a (1.1 g, 2.55 mmol) was added into 50 ml of DCM, a solid was insoluble, and the solid was gradually dissolved after 2.5eq of TEA was added, then 1.1eq of N-Boc piperazine, 1.2eq of HOBt and 1.2eq of EDCI were added, and the raw materials were subjected to thermal reaction for 10 min and then reacted at room temperature overnight; after the completion of the raw materials reaction was monitored by TLC (DCM:MeOH=5:1), a mixture was washed sequentially with water, saturated citric acid solution and saturated saline, dried with anhydrous sodium sulfate, and filtered, and then a filtrate was concentrated and separated by column chromatography to obtain 0.87 g of substance with a yield of 57.2%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.16 (s, 12H), 1.42 (s, 18H), 2.67-4.64 (m, 17H), 7.38 (s, 2H). M+1: 600.34.
The intermediate 43a (0.87 g, 1.45 mmol) was added to 10 ml of ethyl acetate, 10 ml of HCl-EtOAc solution was added, and the raw materials were stirred at room temperature for 6 h; when the completion of the raw materials reaction was monitored by TLC (PE:EA=1:2), a mixture was filtered, and a filter cake was washed with ethyl acetate, and dried to obtain 0.74 mg of a white solid with a yield of 95%. 1H-NMR (400 MHz, DMSO-d6), δ ppm: 1.16 (s, 3H), 2.81-4.38 (m, 17H), 5.37 (s, 1H), 7.28 (s, 4H). M+1: 344.17.
in A-468 synthesis was replaced with
and the rest operations were the same as those of A-468. M+1: 342.19.
in A-468 synthesis was replaced with
and the rest operations were the same as those in A-468 synthesis. M+1: 330.16.
was replaced with
and the rest operations were the same as those of A-468. M+1: 356.21.
was replaced with
and the rest operations were the same as those of A-468. M+1: 344.17.
was replaced with
and the rest operations were the same as those of A-468. M+1: 370.22.
was replaced with
and the rest operations were the same as those of A-468. M+1: 358.19.
was replaced with
and the rest operations were the same as those of A-468. M+1: 370.2.
was replaced with
and the rest operations were the same as those of A-468. M+1: 358.19.
The affinity of the tested compounds on NMDA receptor was researched by receptor ligand binding assays.
Ten SD male rats of SPF grade were ordered and acclimated for 7 days for future use.
(1) Preparation of Crude Synaptosomes in Prefrontal Cortex and Hippocampus
After the rats were decapitated, the prefrontal cortex and hippocampus were rapidly separated on ice and weighed, 10 volumes of 50 mM Tris-HCl buffer (50 mM Tris-HCl, 5 mM MgCl2·6H2O, 1 mM EDTA, 0.5% (W/V) BSA, 1 mM PMSF, 0.32 M sucrose, pH 7.4) was added, and the mixture was homogenized for five times at 1500 revolutions/min with 30 s each time. A homogenate was centrifuged for 10 min at 1000×g, and a supernatant was taken and centrifuged for 10 min at 40,000×g, the precipitates were collected, re-suspended with volumes of Tris-HCl buffer, incubated at 37° C. for 10 min, and centrifuged for 10 min at 40,000×g, finally the precipitates were re-suspended with the above buffer, aliquoted and stored at −80° C. for later use.
(2) Detection of Inhibitory Function of Tested Medicines on Binding of Crude Synaptosomes in Rats to [3H]-MK-801
50 μg of rat crude synaptosomal proteins was added to all tubes respectively. A volume of 50 μl of MK-801 (dizocilpine) was added to a non-specific binding tube to achieve a final concentration of 100 μM, and the materials were reacted for 15 min in advance. 20 μL of control drug at corresponding concentration was added to the test tube for reaction for 15 min. A volume of 30 μl of labeled ligand [3H]-MK-801 was successively added to all tubes to achieve a final concentration of 10 nM. All reaction tubes were supplemented to a volume of 200 μl with 50 mM Tris-HCl buffer (50 mM Tris-HCl, 5 mM MgCl2·6H2O, 1 mM EDTA, 0.5% (WN) BSA, 0.1% NaN3, pH 7.4). A reaction was carried out at 37° C. for 10 min. A type-49 glass fiber filter membrane was prepared and a sample was applied at the same time. The filter membrane was placed in a multi-head cell collector, and a reaction system was subjected to suction filtration under negative pressure and washed with an ice-cold 50 mM Tris-HCl buffer, 10 ml each time, for a total of 5 times. After suction drying, 1 ml of scintillation liquid was added to the filter membrane, the filter membrane was placed on a shaker for shaking for 1.5 h and then placed in a liquid scintillation counter on the next day to determine the radioactive intensity.
(3) Detection of Influence of Tested Medicines on NMDA Receptor Agonistic Properties in Rat Crude Synaptosomal Proteins
100 μg of rat crude synaptosomal proteins was added to all tubes respectively. 50 μl of 5,7-dichlorokynurenic acid was added to a non-specific binding tube to achieve a final concentration of 10 μM. All tubes were preloaded with 50 μM of glutamic acid, and the materials were reacted for 15 min in advance. 20 μL of control drug at corresponding concentration was added to the test tube, and then reacted for 15 min. 1 mM glycine was added to the maximum reaction tube. A volume of 30 μl of labeled ligand [3H]-MK-801 was successively added to all tubes to achieve a final concentration of 10 nM, and reacted for 15 min. All reaction tubes were supplemented to a volume of 500 μl with 50 mM Tris-HCl buffer (50 mM Tris-HCl, 5 mM MgCl2·6H2O, 1 mM EDTA, 0.5% (WN) BSA, 0.1% NaN3, pH 7.4). A reaction was carried out at 37° C. for 15 min. A type-49 glass fiber filter membrane was prepared and a sample was applied at the same time. The filter membrane was placed in a multi-head cell collector, and the reaction system was subjected to suction filtration under negative pressure and washed with an ice-cold 50 mM Tris-HCl buffer, 10 ml each time, for a total of 5 times. After suction drying, 1 ml of scintillation liquid was added to the filter membrane, the filter membrane was placed on the shaker for shaking for 1.5 h and then placed in the liquid scintillation counter on the next day to determine the radioactive intensity.
(4) Data Processing and Statistical Analysis
GraphPad5.0 software was used to analyze the data, and the percentage of competitive inhibition was calculated through nonlinear fitting, wherein:
Percentage of competitive inhibition %=((total binding tube cpm−compound cpm)/(total binding tube cpm−non-specific binding tube cpm))×100%;
Specific binding amount=total binding amount−non-specific binding amount, wherein each binding site was determined by double compound tubes.
In an agonistic experiment, maximum [3H]-MK-801 binding %=((tested compounds cpm−5,7 dichlorokynurenic acid cpm)/(1 mM glycine cpm−5,7 dichlorokynurenic acid cpm))×100%.
The experimental results show that all the compounds of examples have NMDA receptor agonistic activity, and the maximum agonistic efficacy is between 12% and 78%, and the compounds belong to NMDA receptor partial agonists.
The pharmacokinetic parameters such as exposure of plasma concentrations of the compounds of examples were evaluated by oral/tail intravenous injection model tests in rats in order to evaluate the in-vivo metabolic stability in rats.
The male SD rats weighing 200±20 g and being SPF grade were used in this experiment and purchased from SPF.
Preparation of intravenous solution: 1 mg of each tested medicine was weighed accurately, and a volume was metered to 5 mL with 0.9% sodium chloride solution to prepare 0.2 mg/mL. A dose volume was 1 mL for per 200 g of weight, and the dose was 1 mg for per 1 kg of weight based on the concentration conversion.
Preparation of intragastric solution: 10 mg of each tested medicine was accurately weighed, and a volume was metered to 10 mL with 0.9% sodium chloride solution to prepare 1 mg/mL of the solution. A dose volume was 2 mL for per 200 g of weight, and the dose was 10 mg for per 1 kg of weight based on the concentration conversion.
Rats were randomly divided into 8 groups, 6 rats were in each group, oral gavage administration and tail intravenous administration were performed according to the above doses. About 0.2 mL of orbital blood was collected before administration and at 2 min, 5 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h and 6 h after administration, respectively, and centrifuged at 3000 rpm for 10 min at 4° C., and a supernatant was taken for LC/MS/MS determination. A DAS pharmacokinetics program was used to analyze the measured data and calculate the main pharmacokinetic parameters. The experimental results are shown in the following table.
The results shows that the compounds of examples have good pharmacokinetic properties after oral/injection in rats.
Pharmacodynamic evaluation was performed by the forced swimming tests in rats. The experimental animals were male SD rats weighing 150 g-180 g and being SPF grade, and acclimated for one week after purchase, and then subjected to forced swimming tests, the rats need be fasted for 12 hours before the tests. The compounds of examples were dissolved with normal saline, and blank control group (normal saline) and example compound administration group were established. 8-10 rats were provided for each group.
On the day before the tests, the rats were placed in a glass cylinder with a height of 40 cm, an inner diameter of 18 cm, and a water depth of 23 cm for pre-swimming for 15 min, and a water temperature was 28° C. After the pre-swimming the rats were removed, wiped with a dry cloth, and dried with an electrical heater, then returned to a rearing cage, and administered. A formal forced swimming test was performed on the next day for a total of 5 min. The accumulated immobility time within 5 min was recorded, and a second forced swimming test was performed on the seventh day; the criteria for determining immobility were that the rats stopped struggling in the water, were in a floating state, and had only slight limb movements to keep the head floating on the water surface. The results are shown in Table 4, and the experimental data were statistically analyzed by GraphPad Prism 5.0 software.
68%**
The experimental results show that intragastric administration with the compounds of examples is effective and the efficacy can be maintained after 2 to 7 days.
Although the embodiments of the invention have been described in detail, the technicians in the field will understand that various modifications and replacements may be made to those details according to all the instructions already disclosed, and these changes are within the scope of protection of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereto.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202010397265.5 | May 2020 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/093264 | 5/12/2021 | WO |
