CN115636831A - Fused pyrimidinediones, their use and pharmaceutical compositions - Google Patents
Fused pyrimidinediones, their use and pharmaceutical compositions Download PDFInfo
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Abstract
The invention provides a condensed pyrimidinedione compound and pharmaceutically acceptable salts thereof, the structural formula of the compound is shown as formula (I),
the compound and the pharmaceutically acceptable salt thereof can be used as a TRPC4/5 inhibitor and for preparing a medicament for treating skin diseases related to keratinocyte pathological changes.
Description
Technical Field
The invention relates to the field of pharmaceutical chemistry, in particular to a fused pyrimidinedione compound, and application and a pharmaceutical composition thereof.
Background
Transient Receptor Potential (TRP) ion channels are six-time transmembrane channel proteins widely distributed in the peripheral and central nervous systems, and are molecular receptors for sensing environmental changes of cells, transmitting signals and maintaining cell homeostasis. Mammalian TRP ion channels can be divided into six subfamilies of TRPA, TRPC, TRPM, TRPML, TRPP, TRPV, etc. according to sequence similarity. The classical Transient Receptor Potential (TRPC) channel is a non-selective cation channel, allowing Ca 2+ 、Na + And (4) permeating. The mammal TRPC consists of six members of TRPC1, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7, wherein the amino acid sequence of the TRPC3, the TRPC6 and the TRPC7 has higher consistency, and the sequence of the TRPC1, the TRPC4 and the TRPC5 has higher consistency. TRP family proteins exist in a homozygous or heterotetrameric form and exert an ion channel effect, and TRPC5 is easy to form a heterotetramer with TRPC1 and TRPC4 (J Med chem.2019,62, 7589.).
TRPC4/5 is expressed in a plurality of parts in brain, including hippocampus, amygdala, frontal cortex and the like, and is related to the occurrence of central nervous system diseases such as anxiety, depression, epilepsy, addiction and the like. The TRPC4 or TRPC5 gene knockout, or the application of TPRC4/5 inhibitor can relieve anxiety and depression symptoms in mice (PLoS ONE.2018,13, e0191225.). Recent studies have found that TRPC5 is also expressed in glomerular podocytes, and its dysfunction is linked to the development of chronic kidney disease. TRPC5 gene knock-out or application of TPRC5 inhibitor can reduce proteinuria, protect podocyte, and inhibit renal disease progression (science.2017, 358, 1332.). The prior art is studied for inhibitors of TRPC4/5, for example, patent applications WO2014143799, WO2019011802 disclose xanthine TRPC4/5 inhibitors, and patent applications WO2019055966, WO2020061162 disclose pyridazinone TRPC4/5 inhibitors.
TRPC4 and TRPC5 are expressed in skin keratinocytes and peripheral sensory neurons, and play an important role in regulating keratinocyte differentiation and pain perception (Pharmaceuticals (Basel). Keratinocytes are the major component of the epidermal layer of the skin, which not only constitute the natural barrier of the skin against physical, chemical and biological invasion, but also participate in the initiation of the skin's immune inflammatory response. Abnormal differentiation and proliferation of keratinocytes lead to the formation of keratoderma manifested as skin keratinosis, dry and cracked skin, scaling, itching, etc. Keratoderma is divided into hereditary and acquired types, and mainly comprises ichthyosis, palmoplantar keratosis, olmsted disease, toad skin disease, menopausal keratoderma and the like. Psoriasis is a common chronic inflammatory skin disease characterized by excessive immune cell infiltration and abnormal keratinocyte differentiation, proliferation in the dermis and epidermis. There is increasing evidence that keratinocyte dysfunction is an important factor in the development, progression and recurrence of psoriasis, and thus targeted modulation of keratinocytes is a new option for treating psoriasis (J Leukoc biol.2020,108, 485). No published reports have been made on the use of TRPC4/5 inhibitors and drugs containing the inhibitor components and capable of targeted regulation of keratinocytes.
Disclosure of Invention
The invention discloses an 8-site ethynyl or vinyl substituted condensed pyrimidinedione compound which can be used as a TRPC4/5 inhibitor and used for preparing a compound for treating skin diseases related to keratinocyte pathological changes. Compared with the prior TRPC4/5 inhibitor, the compound disclosed by the invention has higher in vitro activity and faster liver microsome metabolism rate. When the compound is used for preparing a skin external medicament, the compound can be rapidly cleared by liver metabolism after being absorbed through skin. Since TRPC4 and TRPC5 are widely distributed in human physiological tissue structures and also play an important role in maintaining normal physiological functions, rapidly metabolized TRPC4/5 inhibitors can avoid systemic side effects to a greater extent.
The technical scheme adopted by the invention is as follows:
condensed pyrimidinedione compounds and pharmaceutically acceptable salts thereof, wherein the structural formula of the compounds is shown as formula (I),
wherein R is 1 Is C 1 -C 6 Alkyl groups of (a);
each R is 2 Independently a halogen atom;
R 3 selected from substituted or unsubstituted C 1 -C 6 Alkyl, substituted or unsubstituted C 3 -C 10 Substituted or unsubstituted 6-to 10-membered aryl and substituted or unsubstituted 5-to 8-membered heteroaryl, wherein R 3 The substituent(s) is 1 to 3 groups selected from the group consisting of: c 1 -C 6 Alkyl of (C) 3 -C 8 Cycloalkyl of (ii), -OR 4 、-N(R 4 ) 2 And a halogen atom;
each R 4 Each independently selected from H, substituted or unsubstituted C 1 -C 6 Alkyl, substituted or unsubstituted C 3 -C 8 Cycloalkyl, substituted or unsubstituted phenyl, wherein R 4 The substituents of (a) are 1 to 3 groups of the following group: c 3 -C 8 Cycloalkyl, halogen atom, hydroxyl and = O; alternatively, the first and second liquid crystal display panels may be,
two R 4 Taken together to form a 4-to 8-membered substituted or unsubstituted heterocyclic ring, the heteroatoms of which are selected from N, O, and S and contain at least 1N, the number of heteroatoms being 1 or 2; the substituents of the heterocycle are 1-3 groups independently selected from the group consisting of: c 1 -C 6 Alkyl groups and halogen atoms of (a);
n is an integer of 1 to 3;
x is N or CH;
y is selected from one of ethynyl and substituted or unsubstituted vinyl, of which vinylThe substituent is 1-2C 1 -C 6 An alkyl group.
As a further improvement of the technical scheme, R 1 Is methyl, ethyl or isopropyl.
As a further improvement of the technical scheme, R 3 Is C 3 -C 10 Cycloalkyl OR-OR of 4 、-N(R 4 ) 2 Substituted C 1 -C 6 Alkyl group of (1).
As a further improvement of the technical solution, said compound is selected from the structural formulae (II a) to (IIc):
wherein the content of the first and second substances,
R 1 is methyl, ethyl or isopropyl;
each R 2 Each independently a halogen atom;
ring C is substituted or unsubstituted C 3 -C 10 Substituted or unsubstituted phenyl or substituted or unsubstituted 5-to 8-membered heteroaryl, wherein the substituents of ring C are 1 to 3 groups selected from: c 1 -C 6 Alkyl groups and halogen atoms of (a);
R 4 each independently selected from substituted or unsubstituted C 1 -C 6 Alkyl, substituted or unsubstituted C 3 -C 8 A cycloalkyl group, a substituted or unsubstituted phenyl group of (a), wherein R 4 The substituent(s) is 1 to 3 groups selected from the group consisting of: c 3 -C 8 Cycloalkyl groups and halogen atoms of (a); alternatively, the first and second liquid crystal display panels may be,
two R 4 Together with N to form a 4-to 8-membered substituted or unsubstituted heterocycle having 1 to 3C substituents 1 -C 6 Alkyl groups of (a);
R 5 、R 6 each independently is H or C 1 -C 3 Alkyl groups of (a);
n is an integer of 1 to 3.
As a further improvement in the technical solution, said compound is selected from the group consisting of:
unless otherwise indicated, the terms used in the present invention have the following definitions:
in the present invention, unless otherwise specified, the terms used have the ordinary meanings well known to those skilled in the art.
In the present invention, the term "C1-C3" means having 1, 2 or 3 carbon atoms, the term "C1-C6" means having 1, 2,3, 4, 5 or 6 carbon atoms, and so on. "4-8 membered" means having 4, 5, 6,7 or 8 ring atoms, and so on.
In the present invention, the "alkyl group" is a branched and straight chain hydrocarbon group having a specific number of carbon atoms, and representative examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, tert-butyl, and the like.
In the present invention, the "haloalkyl group" means a group in which a hydrogen atom in an "alkyl group" having a specific number of carbon atoms is partially or completely substituted with a "halogen atom".
In the present invention, the "cycloalkyl" represents a non-aromatic cyclic aliphatic hydrocarbon group having a specific number of ring-forming carbon atoms, and includes monocyclic, fused, bridged, and spiro ring systems. Representative examples are, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, decahydronaphthyl, adamantyl, and the like.
In the present invention, "aryl" is defined as monocyclic and bicyclic ring systems consisting of a specified number of carbon atoms and complying with Huckel's rule, including but not limited to benzene, naphthalene rings.
In the present invention, "heteroaryl" is defined as monocyclic and bicyclic ring systems having a specified number of ring members and containing 1, 2,3 or 4 heteroatoms (selected from N, O, S) while complying with the huckel rule, including but not limited to pyridine, pyrrole, imidazole, thiophene.
In the present invention, "heterocycle" is defined as a saturated or partially unsaturated non-aromatic cyclic group consisting of a specified number of ring-forming atoms, and contains 1, 2,3 or 4 hetero atoms (selected from N, O, S) as ring-forming atoms, and the others are carbon atoms. Examples of heterocycles include, but are not limited to, morpholine, piperazine, piperidine, pyrrolidine, and the like.
In the present invention, the "halogen atom" includes 4 atoms of fluorine, chlorine, bromine and iodine.
The term "substituted" as used herein means substituted with one or more groups. If it is not specified that it occurs at a particular atom, it is meant that it may occur at any atom for which the number of substituents has not yet reached saturation.
The term "independently" as used herein means that when a plurality of substituents are selected from the same group, they may be the same or different.
The "pharmaceutically acceptable salts" of the present invention may be salts of anions with positively charged groups on the compounds of formula (I). Suitable anions are chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methylsulfonate, trifluoroacetate, acetate, malate, tosylate, tartrate, fumarate, glutamate, glucuronate, lactate, glutarate or maleate. Similarly, salts may be formed from cations with negatively charged groups on the compounds of formula (I). Suitable cations include sodium, potassium, magnesium, calcium, and ammonium ions, such as tetramethylammonium. Preferably, "pharmaceutically acceptable salt" refers to a salt of a compound of formula (I) with an acid selected from the group consisting of: hydrofluoric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, acetic acid, oxalic acid, sulfuric acid, nitric acid, methanesulfonic acid, sulfamic acid, salicylic acid, trifluoromethanesulfonic acid, naphthalenesulfonic acid, maleic acid, citric acid, acetic acid, lactic acid, tartaric acid, succinic acid, oxalic acid, pyruvic acid, malic acid, glutamic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, malonic acid, fumaric acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, isethionic acid and the like.
The compound of the general formula (I) or the pharmaceutically acceptable salt thereof is distilled out, crystallized or recrystallized from water or an organic solvent, and the compound may contain solvent molecules used. In addition, different crystallization conditions may result in different crystalline forms of the compound. Therefore, the compound shown in the general formula (I) or the pharmaceutically acceptable salt thereof containing different chemical doses of crystallization solvents and all crystal forms are within the scope of the invention.
In the present invention, some compounds are asymmetric, e.g., having one or more stereogenic centers, with an olefinic bond. Unless otherwise indicated, the present invention is intended to include all stereo, optical and geometric isomers, such as, but not limited to, enantiomers, diastereomers, racemates, E/Z isomers, as well as mixtures of the individual isomers in varying proportions, and pharmaceutically acceptable salts thereof. Compounds of the present invention containing asymmetrically substituted carbon atoms may be isolated in optically active, racemic or cis-trans isomeric forms.
In the present invention, the "effective therapeutic dose" means that a subject treated with the dose is cured, improved, effectively prevented from suffering from a disease, a side effect, or the like, or the incidence thereof is significantly reduced, as compared with a subject not treated with the dose; in addition, it includes an effective amount to enhance normal physiological function.
It is another object of the present invention to provide a process for the preparation of a compound of formula (I), said process being selected from scheme 1 or 2;
the reaction procedure of scheme 1 is shown in formula A:
wherein PG represents a hydroxyl protecting group such as, but not limited to, tetrahydropyran (THP), tert-butyldimethylsilyl (TBDMS), 2-trimethylsilylethoxymethyl (SEM); halo is a halogen atom; the other substituents are as defined above for formula (I);
the reaction steps of scheme 1 include:
(1.1) intermediate INT-A1 (see WO2014143799 Synthesis) with acetylenic starting Material
(purchased commercially or synthesized according to literature procedures) to undergo a Sonogashira coupling reaction to produce the intermediate INT-A2;
the Sonogashira coupling reaction is usually carried out under the action of a palladium catalyst, a copper salt and an organic base; such as but not limited to PdCl 2 (Ph 3 P) 2 、PdCl 2 (dppf)、Pd(Ph 3 P) 4 (ii) a Such as, but not limited to, cuI, cuBr; such as, but not limited to triethylamine, N-Diisopropylethylamine (DIPEA); the reaction is carried out in an excess of organic base or with the addition of a suitable amount of organic solvent, such as N, N-Dimethylformamide (DMF); the reaction temperature is generally between room temperature and 80 ℃; the reaction conditions can be suitably adjusted by those skilled in the art based on literature or actual conditions encountered during the synthetic process;
(1.2) removing a protecting group of the intermediate INT-A2 to generate a compound shown in a general formula (I); the deprotection reaction of the hydroxyl group is usually carried out under the action of an acidic reagent or a fluorine ion reagent; such as, but not limited to, trifluoroacetic acid, hydrogen chloride, p-toluenesulfonic acid; such as, but not limited to, tetrabutylammonium fluoride, KF, HF;
(1.3) in another alternative, the intermediate INT-A1 may be prepared by first removing the hydroxy protecting group to form intermediate INT-A3, and then reacting with an acetylenic starting material
Carrying out Sonogashira coupling reaction to generate a compound shown in a general formula (I); the reaction conditions are the same as those of the steps (1) and (2);
scheme 2 applies to compounds of general formulae (II B) and (II c) according to the reaction scheme shown in formula B:
wherein L is a leaving group including sulfonate and halogen atoms such as, but not limited to, mesylate, triflate, p-tosylate, chlorine atom, bromine atom, iodine atom; PG, halo are as defined above for formula A; the other substituents are as defined above for the general formulae (II b) and (II c);
the reaction steps of scheme 2 include:
(2.1) intermediate INT-A1 (synthesized with reference to WO 2014143799) with acetylenic starting material
(purchased commercially or synthesized according to literature procedures) to undergo a Sonogashira coupling reaction to produce an intermediate INT-B1; the reaction conditions are the same as in the reaction step (1.1);
(2.2) carrying out sulfonylation reaction or halogenation reaction on the intermediate INT-B1 to generate an intermediate INT-B2; the sulfonylation reaction is carried out by a sulfonylation reagent under the action of a base; such sulfonylating agents as, but not limited to, methanesulfonyl chloride, trifluoromethanesulfonic anhydride, p-toluenesulfonyl chloride; the base includes organic bases such as but not limited to triethylamine, DIPEA, p-Dimethylaminopyridine (DMAP) and inorganic bases such as but not limited to K 2 CO 3 、Cs 2 CO 3 (ii) a The halogenation reaction is carried out under the action of a halogenating agent; such as, but not limited to, thionyl chloride, phosphorus tribromide, I 2 /Ph 3 P, etc.; the reaction solvent is typically an aprotic solvent such as, but not limited to, dichloromethane, tetrahydrofuran; the reaction temperature is generally from-20 ℃ to room temperature;
(2.3) intermediate INT-B2 with HOR 4 Substitution reaction is carried out to generate an intermediate INT-B3 or an intermediate INT-B2 and HN (R) 4 ) 2 Carrying out substitution reaction to generate an intermediate INT-B4; the substitution reaction is carried out under the action of alkali; the base comprisesOrganic bases such as but not limited to triethylamine, DIPEA, DMAP and inorganic bases such as but not limited to K 2 CO 3 、Cs 2 CO 3 NaH; the reaction solvent is typically a polar solvent such as, but not limited to, tetrahydrofuran, acetonitrile, DMF; the reaction temperature is generally 0 ℃ to 80 ℃;
(2.4) deprotecting the intermediate INT-B3 or INT-B4 to produce a compound of formula (II B) or formula (II c); the reaction conditions were the same as in the aforementioned reaction step (1.2).
The invention also discloses an application of the compound or the pharmaceutically acceptable salt thereof, and the compound or the pharmaceutically acceptable salt thereof is used as a TRPC4 and TRPC5 inhibitor or is used for preparing a medicament for treating diseases related to abnormal function and expression of TRPC4 or/and TRPC 5.
In the invention, the compound shown in the general formula (I), the salt thereof and the pharmaceutical composition thereof can be used in combination with other drugs with action mechanisms when being used for treating skin diseases; such as, but not limited to, glucocorticoid based drugs, such as mometasone, fluocinolone acetonide, triamcinolone acetonide, hydrocortisone, clobetasol, and the like; janus kinase inhibitors such as lucentitinib, tofacitinib, uppatinib, and the like; calcineurin inhibitors such as tacrolimus, pimecrolimus; biological immunomodulators, such as adalimumab, infliximab, etanercept, etc.
In the present invention, the compound of formula (I) is usually used in the form of a pharmaceutical composition for the preparation of a medicament, which comprises one or more therapeutically effective amounts of the compound of formula (I) and one or more pharmaceutical excipients; the pharmaceutic adjuvant is a pharmaceutically acceptable carrier, an excipient, a sustained release agent, a flavoring agent and the like.
In the pharmaceutical composition, the compound shown in the general formula (I) is used as an active component, the weight of the compound accounts for 0.1-99.9% of the total weight of the pharmaceutical composition, and the balance is pharmaceutically acceptable auxiliary materials; the compound shown in the general formula (I) and pharmaceutically acceptable auxiliary materials are preferably mixed in a proportion that the compound shown in the general formula (I) as an active ingredient accounts for more than 60 percent of the total weight, the rest accounts for 0 to 40 percent of the total weight, and the amount of the rest is preferably 1 to 20 percent, and most preferably 1 to 10 percent.
The compounds and pharmaceutical compositions provided herein can be formulated into a variety of dosage forms based on procedures customary in the pharmaceutical formulation art, such as tablets, capsules, powders, syrups, solutions, suspensions, sprays, creams, ointments, gels, transdermal patches, and the like, and can be in a suitable solid or liquid carrier or diluent. The pharmaceutical compositions of the present invention may also be stored in a suitable injection or drip sterilization device.
As a further improvement of the technical solution, the pharmaceutical composition is used for treating skin diseases associated with keratinocyte abnormalities.
As a further improvement of the technical solution, the disease is psoriasis, ichthyosis, palmoplantar keratosis, olmsted's disease, toad's skin disease or menopausal keratoderma.
The compound shown in the general formula (I) or the pharmaceutical composition containing the compound shown in the general formula (I) can be clinically used for mammals including human and animals, and the administration route can comprise oral administration, nasal cavity inhalation, skin local administration, lung administration, gastrointestinal tract administration and the like. The preferred route of administration is topical application to the skin. In the present invention, topical application means applying the drug to a body surface, such as skin or mucous membranes.
When the compound shown in the general formula (I) or the pharmaceutical composition thereof is used for preparing skin topical application, the compound can be prepared into a suitable pharmaceutical dosage form, such as but not limited to cream, ointment, gel, transdermal patch, spray and the like. The drug carrier can be selected from starch, oil, cream, foam, ointment, skin lotion, gel, etc., and can also be drug delivery system, such as liposome, niosome, microsponge, microemulsion, microsphere, solid lipid nanoparticle, etc.
When used as a pharmaceutical formulation, the compounds of formula (I) of the present invention may be effectively dissolved, suspended or dispersed in a liquid medium to form a solution, suspension or dispersion for topical application to the skin by application or spraying, and may be delivered as an aid in a sponge, patch, pad or other cosmetic device. In some cases, a slow release delivery system for a compound may be important to exert drug efficacy.
When used as a pharmaceutical preparation, the compound represented by the general formula (I) of the present invention is preferably used in a unit dose, each dose containing 0.01mg to 200mg, preferably 0.5mg to 50mg of the active ingredient, once or in divided portions. Regardless of the method of administration, the optimal dosage for an individual will depend on the particular treatment. Usually, the dosage is increased gradually starting from a small dosage until the most suitable dosage is found.
Compared with the prior art, the fused pyrimidinedione compound and the pharmaceutically acceptable salt thereof have outstanding substantive characteristics and remarkable progress, and particularly can be used as a TRPC4/5 inhibitor or for preparing a medicine for treating skin diseases related to keratinocyte pathological changes. Further, the compounds disclosed herein have higher in vitro activity and faster rate of liver microsome metabolism compared to existing TRPC4/5 inhibitors. Furthermore, when the compound is used for preparing a skin external medicament, the compound can be rapidly metabolized and cleared by the liver after being absorbed by the skin, and systemic side effects are avoided.
Description of the drawings:
FIG. 1 is a graph showing the effect of compound E37 on the inhibition of IL-17-induced proliferation of human immortalized keratinocytes HaCaT and the quantitative results.
Figure 2 is a graph of the improvement in the appearance of psoriasis induced by imiquimod by the cream with compound E37 as active ingredient.
FIG. 3 is a graph showing the effect of the cream containing the compound E37 as an active ingredient on the improvement of the dorsal skin disease of mice induced by imiquimod and the quantitative results.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The invention will be further illustrated by the following examples. It is specifically noted that these examples are only intended to illustrate the invention and do not limit it in any way. All parameters in the examples and the remaining description are based on mass unless otherwise stated. If not, all the fillers used for column chromatography are silica gel. Experimental procedures without specific conditions noted in the following examples, generally according to conventional conditions, or according to conditions recommended by the manufacturer.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are exemplary only.
Abbreviations: DCM: dichloromethane; DIPEA: n, N-diisopropylethylamine; DMAP: 4-dimethylaminopyridine; DMF: n, N-dimethylformamide; DMSO, DMSO: dimethyl sulfoxide; TBAF: tetrabutylammonium fluoride; TFA: trifluoroacetic acid; THF: and (4) tetrahydrofuran.
Example E1- (4-chlorobenzyl) -1- (3-hydroxypropyl) -8- (3-methoxyprop-1-yn-1-yl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
The starting material INT 1-1 was synthesized according to the method described in patent application WO 2014143799. INT 1-1 (43mg, 0.1mmol), pdCl 2 (Ph 3 P) 2 (10.5mg, 0.015mmol), cuI (5.7mg, 0.03mmol), 1.5mL of triethylamine, and 0.5mL of DMF were placed in a pressure-resistant tube, degassed with nitrogen for 3min, then 3-methoxy-1-propyne (42. Mu.L, 0.5 mmol) was added, and further degassed for 0.5min. The pressure tube was sealed and heated at 60 ℃ for 6h. After cooling, diluting with ethyl acetate, filtering to remove insoluble substances, and evaporating the filtrate under reduced pressure to obtain a crude product. Then, the mixture was purified by preparative liquid chromatography (C18 packing), eluted with acetonitrile/water system as mobile phase, and the target product fraction was collected and evaporated to dryness to obtain 20mg of white solid. 1 H-NMR(500MHz,Chloroform-d)δ7.41(d,J＝8.5Hz,2H),7.34(d,J＝8.5Hz,2H),5.59(s,2H),4.45(s,2H),4.20(t,J＝4.0Hz,2H),3.60(s,3H),3.54(m,2H),3.49(s,3H),3.37(brs,1H),1.91(m,2H)；MS(ESI):m/z417.1[M+H] + .
Example E2- (4-chlorobenzyl) -8- (3-cyclobutoxyprop-1-yn-1-yl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
Sodium hydride (60% in oil, 1.44g, 36mmol) was suspended in 20mL dry THF, cyclobutanol (2g, 27.78mmol) was added dropwise to the reaction mixture, the gas was evolved and dropped over 5min, and stirring was continued for 0.5h. Bromopropyne (2.63mL, 30.5 mmol) was then added dropwise over 5min and heated at 50 ℃ for 4h. TLC showed disappearance of the starting material, after cooling, the reaction was quenched by dropping 2mL of saturated ammonium chloride solution and drying with anhydrous magnesium sulfate. After 1h, the inorganic salts were removed by filtration, washed with THF, and THF was added to about 28mL to give a 1mol/L THF solution of intermediate INT 2-1.
Intermediate INT 2-2 was synthesized according to the method described in patent application WO 2014143799.
INT 2-2(102mg，0.2mmol)、PdCl 2 (Ph 3 P) 2 (21mg, 0.03mmol), cuI (11.4mg, 0.06mmol), triethylamine 3mL, and DMF 1mL were placed in a pressure tube, degassed with nitrogen for 3min, then intermediate INT 2-1 (1mL, 1mmol) was added, and further degassed for 1min. The pressure tube was sealed and heated at 60 ℃ for 4h. After cooling, diluting with ethyl acetate, filtering to remove insoluble substances, and evaporating the filtrate under reduced pressure to obtain a crude product. And separating by using a flash silica gel chromatographic column, eluting by using MeOH/DCM as a mobile phase, collecting a target product part, and evaporating to dryness to obtain 86mg of white solid, namely the intermediate INT 2-3.
Intermediate INT 2-3 (86mg, 0.16mmol) was dissolved in 2mL DCM, then 0.4mL TFA was added, stirred at RT for 1h, TLC indicated complete reaction. The solvent was evaporated under reduced pressure and the residue was purified by preparative liquid chromatography (C18 packing) eluting with acetonitrile/water system as the mobile phase, the target product fraction was collected and evaporated to dryness to give 42mg of a white solid, example E2. 1 H-NMR(500MHz,Chloroform-d)δ7.40(d,J＝8.5Hz,2H),7.33(d,J＝8.5Hz,2H),5.59(s,2H),4.39(s,2H),4.20(t,J＝4.0Hz,2H),4.15(m,1H),3.59(s,3H),3.54(m,2H),3.33(brs,1H),2.26(m,2H),2.01(m,2H),1.76(m,1H),1.57(m,1H)；MS(ESI):m/z 457.2[M+H] + .
Example E3- (4-chlorobenzyl) -8- (3-cyclobutoxyprop-1-yn-1-yl) -1- (2-hydroxyethyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
8-bromo-3-methyl-3, 7-dihydro-1H-purine-2, 6-dione (244mg, 1mmol) was dissolved in 10mL of DMF, and KHCO was added 3 (150mg, 1.5 mmol) and 4-chlorobenzyl bromide (204mg, 1mmol) at 60 ℃ for 3 hours. After TLC detection, insoluble matter was removed by filtration, and the mixture was partitioned with ethyl acetate/saturated aqueous NaCl solution to separate an organic phase, dried over anhydrous sodium sulfate and then separated by flash silica gel chromatography, eluted with MeOH/DCM as a mobile phase, and the target product fraction, intermediate INT3-1, was collected as about 330mg.
INT3-1 (330mg, 0.89mmol) described above was dissolved in DMF and K was added 2 CO 3 (249mg, 1.8mmol), and then 2- (2-bromoethoxy) tetrahydro-2H-pyran (225mg, 1.08mmol) was added thereto, followed by reaction at 60 ℃ for 6 hours. After cooling, the mixture was partitioned with ethyl acetate/saturated aqueous NaCl, the organic phase was separated, dried over anhydrous sodium sulfate and separated by flash chromatography on silica gel to give the intermediate INT3-2 of about 400mg.
The synthesis method of the intermediate INT 3-3 is the same as INT 2-3, except that the intermediate INT3-2 is used for replacing INT 2-2 as a raw material.
Example E3 was synthesized according to the same method as E2, except that INT 2-3 was replaced by INT 3-3 as the starting intermediate.
H-NMR(500MHz,DMSO-d6)δ7.50–7.41(m,2H),7.36–7.30(m,2H),5.57(s,2H),4.76(t,J＝6.0Hz,1H),4.40(s,2H),4.04–3.98(m,1H),3.96(t,J＝6.5Hz,2H),3.52(q,J＝6.5Hz,2H),3.41(s,3H),2.16–2.07(m,2H),1.84(ttd,J＝10.0,7.9,2.8Hz,2H),1.68–1.58(m,1H),1.45(qt,J＝10.5,8.0Hz,1H).MS(ESI):m/z 443.1[M+H] + .
Example E4- (4-chlorobenzyl) -8- (3-cyclobutoxyprop-1-yn-1-yl) -1- (4-hydroxybutyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
The synthesis method of the intermediate INT 4-1 is the same as INT3-2 except that (4-bromobutoxy) (tert-butyl) dimethylsilane is used as a raw material instead of 2- (2-bromoethoxy) tetrahydro-2H-pyran.
The synthesis method of the intermediate INT 4-2 is the same as INT 2-3, except that the intermediate INT 4-1 is used for replacing INT 2-2 as a raw material.
Intermediate INT 4-2 (117mg, 0.2mmol) was dissolved in 8mL THF, then 1mol/L TBAF tetrahydrofuran solution was added dropwise at room temperature, stirred at room temperature for 1h, TLC indicated completion of the reaction. Saturated NH 4 The solution was quenched with aqueous Cl, extracted 2 times with ethyl acetate, washed 1 time with saturated aqueous NaCl and dried over anhydrous magnesium sulfate. Filtering, evaporating to obtain crude product, purifying with preparative liquid chromatography column (C18 filler), eluting with acetonitrile/water system as mobile phase, collecting target product part, and evaporating to obtain 55mg white solid as example E4. 1 H-NMR(600MHz,DMSO-d6)δ7.43(d,J＝8.4Hz,2H),7.31(d,J＝8.4Hz,2H),5.56(s,2H),4.39(s,2H),4.38(t,J＝5.7Hz,1H),3.99(p,J＝7.3Hz,1H),3.86(t,J＝7.4Hz,2H),3.42–3.36(m,5H),2.10(dtt,J＝9.4,7.2,2.6Hz,2H),1.84(dtd,J＝12.5,10.0,7.9Hz,2H),1.66–1.59(m,1H),1.55(td,J＝8.1,5.7Hz,2H),1.45–1.37(m,3H).MS(ESI):m/z 471.2[M+H] + .
Example E5- (3-Cyclobutoxyprop-1-yn-1-yl) -7- (4-fluorobenzyl) -1- (2-hydroxyethyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
The intermediate INT 5-1 was synthesized in the same manner as INT3-1 except that 4-fluorobenzyl bromide was used instead of 4-chlorobenzyl bromide.
The intermediate INT 5-2 is synthesized by the same method as INT3-2 except that INT3-1 is replaced by the intermediate INT 5-1.
The synthesis method of the intermediate INT 5-3 is the same as INT 2-3, except that the intermediate INT 5-2 is used for replacing INT 2-2 as a raw material.
Example E5 was synthesized according to the same method as E2, except that INT 2-3 was replaced by INT 5-3 as the starting intermediate. 1 H-NMR(500MHz,DMSO-d6)δ7.42–7.35(m,2H),7.24–7.17(m,2H),5.56(s,2H),4.76(t,J＝6.0Hz,1H),4.42(s,2H),4.08–4.00(m,1H),3.97(t,J＝6.6Hz,2H),3.52(q,J＝6.5Hz,2H),3.41(s,3H),2.13(dddd,J＝10.6,6.8,5.2,2.5Hz,2H),1.86(dddd,J＝19.3,10.0,7.9,2.8Hz,2H),1.69–1.58(m,1H),1.45(qt,J＝10.5,8.0Hz,1H).MS(ESI):m/z 427.2[M+H] + .
EXAMPLE E6- (3-Cyclobutoxyprop-1-yn-1-yl) -7- (4-fluorobenzyl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
The synthesis method of the intermediate INT 6-1 is the same as INT3-2, except that INT 5-1 is used for replacing INT3-1, and 2- (3-bromopropoxy) tetrahydro-2H-pyran is used for replacing 2- (2-bromoethoxy) tetrahydro-2H-pyran as a raw material.
The synthesis method of the intermediate INT 6-2 is the same as INT 2-3, except that the intermediate INT 6-1 is used for replacing INT 2-2 as a raw material.
Example E6 was synthesized according to the same method as E2, except that INT 2-3 was replaced by INT 6-2, an intermediate, as a starting material. 1 H-NMR(500MHz,DMSO-d6)δ7.360～7.387(m,2H),7.178～7.213(m,2H),5.546(s,2H),4.447～4.467(t,J＝5Hz,1H),4.408(s,2H),3.997～4.055(m,1H),3.902～3.932(t,J＝7Hz,2H),3.413～3.449(q,J＝6Hz,2H),3.397(s,3H),2.093～2.150(m,2H),1.810～1.869(m,2H),1.671～1.712(m,2H),1.414～1.468(m,2H).MS(ESI):m/z 441.2[M+H] + .
Example E7- (3-Cyclobutoxyprop-1-yn-1-yl) -7- (4-fluorobenzyl) -1- (4-hydroxybutyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
The synthesis method of the intermediate INT 7-1 is the same as INT3-2, except that INT3-1 is replaced by the intermediate INT 5-1, and 2- (2-bromoethoxy) tetrahydro-2H-pyran is replaced by (4-bromobutoxy) (tert-butyl) dimethylsilane as a raw material.
The synthesis method of the intermediate INT 7-2 is the same as INT 2-3, except that the intermediate INT 7-1 is used for replacing INT 2-2 as a raw material.
The synthesis of example E7 was performed as in E4, except that INT 4-2 was replaced by INT 7-2 as the starting material. 1 H-NMR(600MHz,DMSO-d6)δ7.40–7.32(m,2H),7.19(t,J＝8.8Hz,2H),5.55(s,2H),4.41(s,2H),4.38(t,J＝5.2Hz,1H),4.03(p,J＝7.3Hz,1H),3.86(t,J＝7.4Hz,2H),3.39(m,5H),2.12(dddd,J＝11.1,9.4,6.9,2.5Hz,2H),1.85(dtd,J＝12.5,10.2,8.0Hz,2H),1.66–1.59(m,1H),1.59–1.52(m,2H),1.47–1.37(m,3H).MS(ESI):m/z 455.2[M+H] + .
Example E8- (3-Cyclobutoxyprop-1-yn-1-yl) -7- (3, 4-difluorobenzyl) -1- (2-hydroxyethyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
The intermediate INT 8-1 was synthesized in the same manner as INT3-1 except that 3, 4-difluorobenzyl bromide was used as the starting material instead of 4-chlorobenzyl bromide.
The synthesis method of the intermediate INT 8-2 is the same as INT3-2, except that the intermediate INT 8-1 is used as a raw material to replace INT 3-1.
The synthesis method of the intermediate INT 8-3 is the same as INT 2-3, except that the intermediate INT 8-2 is used for replacing INT 2-2 as a raw material.
Example E8 was synthesized according to the same method as E2, except that INT 2-3 was replaced by INT 8-3 as the starting intermediate. 1 H-NMR(600MHz,DMSO-d6)δ7.48–7.37(m,2H),7.18–7.11(m,1H),5.56(s,2H),4.74(t,J＝6.0Hz,1H),4.40(s,2H),4.05–3.99(m,1H),3.96(t,J＝6.6Hz,2H),3.52(q,J＝6.5Hz,2H),3.40(s,3H),2.12(dtt,J＝11.5,6.9,2.5Hz,2H),1.84(ttd,J＝10.0,7.8,2.8Hz,2H),1.67–1.58(m,1H),1.48–1.41(m,1H).MS(ESI):m/z 445.2[M+H] + .
Example E9- (3-Cyclobutoxyprop-1-yn-1-yl) -7- (3, 4-difluorobenzyl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
The synthesis method of the intermediate INT 9-1 is the same as INT3-2, except that the intermediate INT 8-1 is used for replacing INT3-1, and 2- (3-bromopropoxy) tetrahydro-2H-pyran is used for replacing 2- (2-bromoethoxy) tetrahydro-2H-pyran as a raw material.
The synthesis method of the intermediate INT 9-2 is the same as INT 2-3, except that the intermediate INT 9-1 is used as a raw material to replace INT 2-2.
Example E9 was synthesized according to the same method as E2, except that INT 2-3 was replaced by INT 9-2, an intermediate, as a starting material. 1 H-NMR(600MHz,DMSO-d6)δ7.48–7.37(m,2H),7.14(m,1H),5.55(s,2H),4.45(t,J＝5.3Hz,1H),4.40(s,2H),4.01(p,J＝7.2Hz,1H),3.92(t,J＝6.0,H),3.43(q,J＝6.1Hz,2H),3.40(s,3H),2.15–2.08(m,2H),1.84(m,2H),1.72–1.66(m,2H),1.66–1.58(m,1H),1.44(m,J 1H)；MS(ESI):m/z 459.2[M+H] + .
Example E10- (3-Cyclobutoxyprop-1-yn-1-yl) -7- (3, 4-difluorobenzyl) -1- (4-hydroxybutyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
The synthesis method of the intermediate INT 10-1 is the same as INT3-2, except that the intermediate INT 8-1 is used for replacing INT3-1, and (4-bromobutoxy) (tert-butyl) dimethyl silane is used for replacing 2- (2-bromoethoxy) tetrahydro-2H-pyran as a raw material.
The synthesis method of the intermediate INT 10-2 is the same as INT 2-3, except that the intermediate INT 10-1 is used for replacing INT 2-2 as a raw material.
The synthesis of example E10 is the same as E4, except that INT 4-2 is replaced by the intermediate INT 10-2. 1 H-NMR(600MHz,DMSO-d6)δ7.47–7.37(m,2H),7.14(ddd,J＝9.8,4.6,2.2Hz,1H),5.55(s,2H),4.40(s,2H),4.38(t,J＝5.2Hz,1H),4.01(p,J＝7.3Hz,1H),3.87(t,J＝7.4Hz,2H),3.42–3.36(m,5H),2.11(ddt,J＝14.7,6.9,2.5Hz,2H),1.89–1.80(m,2H),1.66–1.60(m,1H),1.59–1.53(m,2H),1.47–1.38(m,3H).MS(ESI):m/z 473.2[M+H] + .
Example E11- (4-chloro-2-fluorobenzyl) -8- (3-cyclobutoxyprop-1-yn-1-yl) -1- (2-hydroxyethyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
The synthesis method of the intermediate INT 11-1 is the same as INT3-1, except that 4-chloro-2-fluorobenzyl bromide is used as a raw material instead of 4-chlorobenzyl bromide.
The synthesis method of the intermediate INT 11-2 is the same as INT3-2, except that the intermediate INT 11-1 is used as a raw material to replace INT 3-1.
The synthesis method of the intermediate INT 11-3 is the same as INT 2-3, except that the intermediate INT 11-2 is used for replacing INT 2-2 as a raw material.
Example E11 was synthesized according to the same method as E2, except that INT 2-3 was replaced by INT 11-3 as the starting intermediate. 1 H-NMR(600MHz,DMSO-d6)δ7.52(dd,J＝10.1,2.0Hz,1H),7.26(dd,J＝8.3,2.0Hz,1H),7.09(t,J＝8.2Hz,1H),5.61(s,2H),4.73(t,J＝5.9Hz,1H),4.34(s,2H),3.95(m,3H),3.49(q,J＝6.4Hz,2H),3.41(s,3H),2.08(dtd,J＝11.7,7.5,6.2,2.6Hz,2H),1.82(dq,J＝13.2,10.1Hz,2H),1.61(q,J＝10.1Hz,1H),1.43(dtd,J＝18.5,10.5,8.1Hz,1H).MS(ESI):m/z 461.1[M+H] + .
Example E12- (4-chloro-2-fluorobenzyl) -8- (3-cyclobutoxyprop-1-yn-1-yl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
The synthesis method of the intermediate INT 12-1 is the same as INT3-2, except that the intermediate INT 11-1 is used for replacing INT3-1, and 2- (3-bromopropoxy) tetrahydro-2H-pyran is used for replacing 2- (2-bromoethoxy) tetrahydro-2H-pyran as a raw material.
INT 12-1(100mg，0.1894mmol)、PdCl 2 (Ph 3 P) 2 (19.9mg, 0.0284mmol), cuI (10.8mg, 0.0568mmol), triethylamine 3mL and DMF 1mL were placed in a pressure-resistant tube, degassed with nitrogen for 3min, then intermediate INT 2-1 (0.95mL, 0.95mmol) was added and degassed for 1min. The pressure tube was sealed and heated at 60 ℃ for 4h. Cooling, diluting with ethyl acetate, filtering to remove insoluble substances, and evaporating the filtrate under reduced pressure to obtain crude extractAnd (5) preparing the product. And separating by using a flash silica gel chromatographic column, eluting by using MeOH/DCM as a mobile phase, collecting a target product part, and evaporating to dryness to obtain 86mg of a white solid, namely the intermediate INT12-2.
Intermediate INT12-2 (84mg, 0.15mmol) was dissolved in 2mL DCM, then 0.4mL TFA was added, stirred at room temperature for 1h, TLC indicated complete reaction. The solvent was evaporated under reduced pressure and the residue was purified by preparative liquid chromatography using acetonitrile/water system as mobile phase, the target product fraction was collected and evaporated to dryness to obtain 41mg of white solid, example E12. 1 H-NMR(600MHz,DMSO-d6)δ7.52(dd,J＝10.1,2.1Hz,1H),7.26(dd,J＝8.2,2.1Hz,1H),7.09(t,J＝8.3Hz,1H),5.61(s,2H),4.44(t,J＝5.2Hz,1H),4.34(s,2H),3.96(p,J＝7.3Hz,1H),3.89(m,t,J＝6.5Hz,2H),3.41(m,5H),2.12–2.04(m,2H),1.82(dtd,J＝12.7,10.2,7.9Hz,2H),1.67(p,J＝6.5Hz,2H),1.63–1.57(m,1H),1.43(qt,J＝10.5,8.0Hz,1H)；(ESI):m/z 475.2[M+H] + .
Example E13- (4-chloro-2-fluorobenzyl) -8- (3-cyclobutoxyprop-1-yn-1-yl) -1- (4-hydroxybutyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
The synthesis method of the intermediate INT 13-1 is the same as INT3-2, except that the intermediate INT 11-1 is used for replacing INT3-1, and (4-bromobutoxy) (tert-butyl) dimethyl silane is used for replacing 2- (2-bromoethoxy) tetrahydro-2H-pyran as a raw material.
INT 13-1(100mg，0.175mmol)、PdCl 2 (Ph 3 P) 2 (18.4mg, 0.0263mmol), cuI (10mg, 0.0526mmol), 3mL of triethylamine and 1mL of DMF were placed in a pressure-resistant tube, degassed with nitrogen for 3min, then intermediate INT 2-1 (0.9mL, 0.9mmol) was added, and further degassed for 1min. The pressure tube was sealed and heated at 60 ℃ for 4h. After cooling, diluting with ethyl acetate, filtering to remove insoluble substances, and evaporating the filtrate under reduced pressure to obtain a crude product. And separating by using a flash silica gel chromatographic column, eluting by using MeOH/DCM as a mobile phase, collecting a target product part, and evaporating to dryness to obtain 90mg of a white solid, namely the intermediate INT13-2.
The synthesis of example E13 was performed as in E4, except that INT13-2 was used as the starting material instead of INT 4-2. 53mg of example E13, white solid, were obtained from 90mg of intermediate INT13-2. 1 H-NMR(600MHz,DMSO-d6)δ7.51(dd,J＝10.1,2.1Hz,1H),7.26(dd,J＝8.4,2.1Hz,1H),7.08(t,J＝8.3Hz,1H),5.61(s,2H),4.38(t,J＝5.2Hz,1H),4.34(s,2H),4.00–3.92(m,1H),3.84(t,J＝7.4Hz,2H),3.42(s,3H),3.38(td,J＝6.4,5.0Hz,2H),2.08(ddt,J＝14.6,6.8,2.5Hz,2H),1.81(qdd,J＝10.2,5.0,2.2Hz,2H),1.65–1.58(m,1H),1.57–1.50(m,2H),1.41(dddd,J＝21.9,15.8,11.8,7.3Hz,3H).MS(ESI):m/z 489.2[M+H] + .
Example E14- (4-chloro-3-fluorobenzyl) -8- (3-cyclobutoxyprop-1-yn-1-yl) -1- (2-hydroxyethyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
The intermediate INT 14-1 was synthesized in the same manner as INT3-1 except that 4-chloro-3-fluorobenzyl bromide was used instead of 4-chlorobenzyl bromide.
The synthesis method of the intermediate INT 14-2 is the same as INT3-2, except that the intermediate INT 14-1 is used for replacing INT3-1 as a raw material.
The synthesis method of the intermediate INT 14-3 is the same as INT 2-3, except that the intermediate INT 14-2 is used for replacing INT 2-2 as a raw material.
Example E14 was synthesized according to the same method as E2, except that INT 2-3 was replaced by INT 14-3 as the starting intermediate. 1 H-NMR(600MHz,DMSO-d6)δ7.59(t,J＝8.0Hz,1H),7.36(dd,J＝10.0,2.0Hz,1H),7.12(d,J＝8.4Hz,1H),5.58(s,2H),4.73(t,J＝6.0Hz,1H),4.38(s,2H),3.96(m,3H),3.51(q,J＝6.5Hz,2H),3.41(s,3H),2.13–2.05(m,2H),1.87–1.78(m,2H),1.61(q,J＝10.3Hz,1H),1.43(dtd,J＝18.4,10.4,7.9Hz,1H).MS(ESI):m/z 461.1[M+H] + .
Example E15- (4-chloro-3-fluorobenzyl) -8- (3-cyclobutoxyprop-1-yn-1-yl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
The synthesis method of the intermediate INT 15-1 is the same as INT3-2, except that the intermediate INT 14-1 is used for replacing INT3-1, and 2- (3-bromopropoxy) tetrahydro-2H-pyran is used for replacing 2- (2-bromoethoxy) tetrahydro-2H-pyran as a raw material.
INT 15-1(100mg，0.1893mmol)、PdCl 2 (Ph 3 P) 2 (19.9mg, 0.0284mmol), cuI (10.8mg, 0.0568mmol), triethylamine 3mL and DMF 1mL were placed in a pressure-resistant tube, degassed with nitrogen for 3min, then intermediate INT 2-1 (0.95mL, 0.95mmol) was added and degassed for 1min. The pressure tube was sealed and heated at 60 ℃ for 4h. After cooling, diluting with ethyl acetate, filtering to remove insoluble substances, and evaporating the filtrate under reduced pressure to obtain a crude product. And separating by using a flash silica gel chromatographic column, eluting by using MeOH/DCM as a mobile phase, collecting a target product part, and evaporating to dryness to obtain 81mg of a white solid, namely the intermediate INT15-2.
Intermediate INT15-2 (81mg, 0.145mmol) was dissolved in 2mL DCM, then 0.4mL TFA was added, stirred at RT for 1h and TLC indicated completion of the reaction. The solvent was evaporated under reduced pressure and the residue was purified by preparative liquid chromatography using acetonitrile/water system as mobile phase, the target product fraction was collected and evaporated to dryness to give 39mg of white solid as example E15. 1 H-NMR(500MHz,Chloroform-d)δ7.40(t,J＝8.0Hz,1H),7.26(d,J＝9.4Hz,1H),7.22(d,J＝8.4Hz,1H),5.58(s,2H),4.39(s,2H),4.21(t,J＝6.0Hz,2H),4.15(t,J＝7.3Hz,1H),3.60(s,3H),3.56(t,J＝5.5Hz,2H),2.26(t,J＝7.8Hz,3H),2.05–2.00(m,2H),1.93(m,2H),1.67(m,1H),1.61–1.52(m,1H).MS(ESI):m/z 475.2[M+H] + .
Example E16- (4-chlorobenzyl) -8- (3-cyclobutoxyprop-1-yn-1-yl) -1- (3-hydroxypropyl) -3-isopropyl-3, 7-dihydro-1H-purine-2, 6-dione
2,6-dihydroxypurine (15g, 0.0987mol) was suspended in 240mL of water, and 7.5mL of liquid bromine (0.1464 mol) was added dropwise at room temperature, and after completion of the dropwise addition, the reaction was allowed to proceed overnight at 100 ℃. After cooling to room temperature, the solid was filtered, washed several times with distilled water and dried to give the intermediate INT 16-1.
Intermediate INT 16-1 (2.3 g, 10mmol) was dissolved in DMSO, and KHCO was added 3 (1.2g, 12mmol), 4-chlorobenzyl bromide (2.04g, 10mmol) was further added, and the mixture was reacted at 35 ℃ overnight. The reaction solution was poured into a saturated aqueous NaCl solution, extracted 3 times with ethyl acetate, the organic phases were combined, and washed 3 times with a saturated aqueous NaCl solution. Anhydrous MgSO (MgSO) 4 And (3) drying, separating by using a flash silica gel chromatographic column, eluting by using MeOH/DCM as a mobile phase, and collecting a target product part to obtain an intermediate INT 16-2.
Intermediate INT 16-2 (2.48g, 7mmol) was dissolved in DMF and K was added 2 CO 3 (1.16g, 8.4 mmol) and iodoisopropane (1.19g, 7 mmol) were reacted at 40 ℃ overnight. The reaction solution was poured into a saturated aqueous NaCl solution, extracted 3 times with ethyl acetate, and the organic phases were combined and washed 3 times with a saturated aqueous NaCl solution. Anhydrous MgSO (MgSO) 4 And (3) drying, separating by using a flash silica gel chromatographic column, eluting by using MeOH/DCM as a mobile phase, and collecting a target product part to obtain an intermediate INT 16-3.
Intermediate INT 16-3 (1.386g, 3.5mmol) was dissolved in DMF and K was added 2 CO 3 (0.967 g,7 mmol) and 2- (3-bromopropoxy) tetrahydro-2H-pyran (0.932g, 4.2mmol) were reacted at 60 ℃ overnight. The reaction solution was then poured into a saturated aqueous NaCl solution, extracted 3 times with ethyl acetate, the organic phases were combined and washed 3 times with a saturated aqueous NaCl solution. Anhydrous MgSO (MgSO) 4 And (4) drying, separating by using a flash silica gel chromatographic column, eluting by using MeOH/DCM as a mobile phase, and collecting a target product part to obtain an intermediate INT 16-4.
INT 16-4(108mg，0.2mmol)、PdCl 2 (Ph 3 P) 2 (21mg, 0.03mmol), cuI (11.4mg, 0.06mmol), triethylamine 3mL and DMF 1mL were placed in a pressure resistant tube, degassed with nitrogen for 3min, then intermediate INT 2-1 (1mL, 1mmol) was added and degassed for 1min. The pressure tube was sealed and heated at 60 ℃ for 4h. After cooling, diluting with ethyl acetate, filtering to remove insoluble substances, and evaporating the filtrate under reduced pressure to obtain a crude product. Separating with flash silica gel chromatography column, eluting with MeOH/DCM as mobile phase, collecting target product, and evaporating to dryness to obtain 80mg of white solid, i.e. mediumIntermediate INT16-5.
Intermediate INT16-5 (80mg, 0.14mmol) was dissolved in 2mL DCM, then 0.4mL TFA was added, stirred at RT for 1h, TLC indicated complete reaction. The solvent was evaporated under reduced pressure and the residue was purified by preparative liquid chromatography using acetonitrile/water system as mobile phase, the target product fraction was collected and evaporated to dryness to obtain 40mg of white solid, example E16. 1 H-NMR(500MHz,DMSO-d6)δ7.48–7.42(m,2H),7.37–7.32(m,2H),5.57(s,2H),5.05(m,1H),4.47(t,J＝5.2Hz,1H),4.41(s,2H),4.00(m,1H),3.92(t,J＝7.5Hz,2H),3.44(q,J＝6.1Hz,2H),2.17–2.07(m,2H),1.85(m,2H),1.69(m,2H),1.65–1.59(m,1H),1.49(d,J＝7.0Hz,6H),1.47–1.39(m,1H)；MS(ESI):m/z 485.2[M+H] + .
Example E17- (4-chlorobenzyl) -8- (3-cyclobutoxyprop-1-yn-1-yl) -3-ethyl-1- (3-hydroxypropyl) -3, 7-dihydro-1H-purine-2, 6-dione
The synthesis method of the intermediate INT 17-1 is the same as INT 16-3, except that iodoethane is used as a raw material to replace iodoisopropane.
The synthesis method of the intermediate INT 17-2 is the same as INT 16-4, except that the intermediate INT 17-1 is used for replacing INT 16-3 as a raw material.
The synthesis method of the intermediate INT 17-3 is the same as INT16-5, except that the intermediate INT 17-2 is used for replacing INT 16-4 as a raw material.
The synthesis of example E17 was performed as in E16, except that INT16-5 was replaced by INT 17-3 as the starting material. 1 H-NMR(500MHz,DMSO-d6)δ7.44(d,J＝8.4Hz,2H),7.34(d,J＝8.2Hz,2H),5.57(s,2H),4.48(t,J＝5.2Hz,1H),4.40(s,2H),4.12(q,J＝5.2Hz,1H),4.01(m,2H),3.93(t,J＝7.0Hz,2H),3.44(q,J＝6.1Hz,2H),2.12(m,2H),1.84(m,2H),1.69(m,2H),1.62(m,1H),1.45(m,1H),1.22(t,J＝7.6Hz,3H)；MS(ESI):m/z 471.2[M+H] +.
Example E18- (4-chlorobenzyl) -8- (3- (cyclopentyloxy) prop-1-yn-1-yl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
Sodium hydride (60% in oil, 1.44g, 36mmol) was suspended in 20mL of dry THF, cyclopentanol (2.41g, 28mmol) was added dropwise to the reaction mixture, the gas evolved, and the reaction was stirred for an additional 0.5h over 5min. Bromopropyne (2.63mL, 30.5 mmol) was then added dropwise over 5min and heated at 50 ℃ for 4h. TLC showed disappearance of the starting material, and after cooling, the reaction was quenched by addition of 2mL of saturated ammonium chloride solution dropwise, and dried by addition of anhydrous magnesium sulfate. After 1h, the inorganic salts were removed by filtration, washed with THF, and THF was added to about 28mL to give a 1mol/L THF solution of intermediate INT 18-1.
INT 2-2(100mg，0.196mmol)、PdCl 2 (Ph 3 P) 2 (20.6mg, 0.0294mmol), cuI (11.17mg, 0.0588mmol), 3mL of triethylamine, and 1mL of DMF were placed in a pressure-resistant tube, degassed with nitrogen for 3min, then intermediate INT 18-1 (1mL, 1mmol) was added, and further degassed for 1min. The pressure pipe is sealed and heated for 4-6 h at 60 ℃. After cooling, the mixture was diluted with ethyl acetate, the insoluble matter was removed by filtration, and the filtrate was evaporated to dryness under reduced pressure to give a crude product. And separating by using a flash silica gel chromatographic column, eluting by using MeOH/DCM as a mobile phase, collecting a target product part, and evaporating to dryness to obtain 78mg of a white solid, namely the intermediate INT 18-2.
Intermediate INT 18-2 (78mg, 0.14mmol) was dissolved in 2mL DCM, then 0.4mL TFA was added, stirred at RT for 1h, TLC indicated complete reaction. The solvent was evaporated under reduced pressure and the residue was purified by preparative liquid chromatography using acetonitrile/water system as mobile phase, the target product fraction was collected and evaporated to dryness to obtain 32mg of white solid, example E18. 1 H-NMR(500MHz,DMSO-d6)δ7.43(d,J＝8.5Hz,2H),7.32(d,J＝8.2Hz,2H),5.57(s,2H),4.48(t,J＝5.2Hz,1H),4.45(s,2H),4.02(m,1H),3.92(t,J＝7.5Hz,2H),3.48–3.42(m,2H),3.41(s,3H),1.74–1.51(m,8H),1.47(m,2H)；MS(ESI):m/z 471.2[M+H] + .
Example E19- (3- (cyclopentyloxy) prop-1-yn-1-yl) -7- (4-fluorobenzyl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
Example E19 was synthesized according to the same method as E18, except that INT 2-2 was replaced with INT 6-1 as the starting intermediate. 1 H-NMR(500MHz,DMSO-d6)δ7.42–7.36(m,2H),7.23–7.16(m,2H),5.56(s,2H),4.47(m,3H),4.05(tt,J＝5.8,2.9Hz,1H),3.96–3.90(m,2H),3.44(q,J＝6.2Hz,2H),3.41(s,3H),1.74–1.54(m,8H),1.49(m,2H)；MS(ESI):m/z 455.2[M+H] + .
Example E20- (4-chlorobenzyl) -8- (3- (cyclohexyloxy) prop-1-yn-1-yl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
Sodium hydride (60% in oil, 1.44g, 36mmol) was suspended in 20mL of dry THF, cyclohexanol (2.8g, 27.78mmol) was added dropwise to the atmosphere, the gas evolved over 5min, and stirring was continued for an additional 0.5h. Bromopropyne (2.63mL, 30.5 mmol) was then added dropwise over 5min and heated at 50 ℃ for 4h. TLC showed disappearance of the starting material, after cooling, the reaction was quenched by dropping 2mL of saturated ammonium chloride solution and drying with anhydrous magnesium sulfate. After 1h, the inorganic salts were removed by filtration, washed with THF, and THF was added to about 28mL to give a 1mol/L THF solution of intermediate INT 20-1.
INT 2-2(100mg，0.196mmol)、PdCl 2 (Ph 3 P) 2 (20.6mg, 0.0294mmol), cuI (11.2mg, 0.0588mmol), 3mL of triethylamine, and 1mL of DMF were placed in a pressure-resistant tube, degassed with nitrogen for 3min, then intermediate INT 20-1 (1mL, 1mmol) was added, and further degassed for 1min. The pressure pipe is sealed and heated for 4-6 h at 60 ℃. After cooling, diluting with ethyl acetate, filtering to remove insoluble substances, and evaporating the filtrate under reduced pressure to obtain a crude product. And then separating by using a flash silica gel chromatographic column, eluting by using MeOH/DCM as a mobile phase, collecting a target product part, and evaporating to dryness to obtain 66mg of a white solid, namely an intermediate INT 20-2.
Intermediate INT 20-2 (66mg, 0.12mmol) was dissolved in 2mL DCM, then 0.4mL TFA was added, stirring at room temperature for 1h, TLC indicated complete reaction. The solvent was evaporated under reduced pressure and the residue was purified by preparative liquid chromatography using acetonitrile/water system as mobile phase, the target product fraction was collected and evaporated to dryness to give 30mg of white solid as example E20. 1 H-NMR(500MHz,DMSO-d6)δ7.43(d,J＝8.3Hz,2H),7.32(d,J＝8.2Hz,2H),5.56(s,2H),4.52(s,2H),4.47(t,J＝5.2Hz,1H),3.92(t,J＝7.4Hz,2H),3.48–3.36(m,6H),1.82(m,2H),1.72–1.65(m,2H),1.63(m,2H),1.46(m,1H),1.20(m,5H).MS(ESI):m/z 485.2[M+H] + .
Example E21- (3- (cyclohexyloxy) prop-1-yn-1-yl) -7- (4-fluorobenzyl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
Example E21 was synthesized as in E20, except that INT 2-2 was replaced by INT 6-1, the intermediate. 1 H-NMR(500MHz,DMSO-d6)δ7.42–7.35(m,2H),7.23–7.16(m,2H),5.55(s,2H),4.54(s,2H),4.48(t,J＝5.2Hz,1H),3.96–3.89(m,2H),3.47–3.42(m,2H),3.41(s,3H),1.86–1.80(m,2H),1.73–1.58(m,4H),1.49–1.43(m,1H),1.26–1.15(m,5H).MS(ESI):m/z 469.2[M+H] + .
Example E22- (3- (cyclohexyloxy) prop-1-yn-1-yl) -7- (3, 4-difluorobenzyl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
The synthesis of example E22 was performed as in E20, except that INT 2-2 was replaced by INT 9-1 as the starting intermediate. 1 H-NMR(500MHz,DMSO-d6)δ7.48–7.37(m,2H),7.14(d,J＝8.0Hz,1H),5.56(s,2H),4.52(s,2H),4.48(t,J＝5.2Hz,1H),3.96–3.89(m,2H),3.43(m,5H),1.84–1.79(m,2H),1.69(m,2H),1.66–1.58(m,2H),1.46(m,1H),1.28–1.14(m,5H).MS(ESI):m/z 487.2[M+H] + .
Example E23- (4-chlorobenzyl) -8- (3- (cyclohexyloxy) prop-1-yn-1-yl) -1- (3-hydroxypropyl) -3-isopropyl-3, 7-dihydro-1H-purine-2, 6-dione
The synthesis of example E23 was performed as in E20, except that INT 2-2 was replaced by INT 16-4, an intermediate. 1 H-NMR(500MHz,DMSO-d6)δ7.46–7.41(m,2H),7.34(d,J＝8.4Hz,2H),5.56(s,2H),5.04(m,1H),4.52(s,2H),4.48(t,J＝5.2Hz,1H),3.94–3.88(m,2H),3.44(q,J＝6.1Hz,2H),1.84–1.79(m,2H),1.72–1.65(m,2H),1.62(m,2H),1.49(d,J＝6.0Hz,6H),1.46(m,1H),1.23–1.12(m,5H)；MS(ESI):m/z 513.2[M+H] + .
Example E24- (4-chlorobenzyl) -8- (3-hydroxybut-1-yn-1-yl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
INT 2-2(100mg，0.196mmol)、PdCl 2 (Ph 3 P) 2 (20.6mg, 0.0294mmol), cuI (11.17mg, 0.0588mmol), 3mL of triethylamine, and 1mL of DMF were placed in a pressure tube, degassed with nitrogen for 3min, then 3-butyn-2-ol (28mg, 0.4mmol) was added, and further degassed for 1min. The pressure pipe is sealed and heated for 4-6 h at 60 ℃. After cooling, diluting with ethyl acetate, filtering to remove insoluble substances, and evaporating the filtrate under reduced pressure to obtain a crude product. And separating by using a flash silica gel chromatographic column, eluting by using MeOH/DCM as a mobile phase, collecting a target product part, and evaporating to dryness to obtain 65mg of jelly, namely an intermediate INT 24-1.
Intermediate INT 24-1 (65mg, 0.13mmol) was dissolved in 2mL DCM, then 0.4mL TFA was added, stirred at RT for 1h, TLC indicated complete reaction. The solvent was evaporated under reduced pressure and the residue was purified by preparative liquid chromatography using acetonitrile/water system as mobile phase, the target product fraction was collected and evaporated to dryness to obtain 41mg of white solid, example E24. 1 H-NMR(500MHz,DMSO-d6)δ7.47–7.41(m,2H),7.38(d,J＝8.6Hz,2H),5.82(d,J＝5.6Hz,1H),5.54(s,2H),4.75–4.66(m,1H),4.48(t,J＝5.2Hz,1H),3.96–3.89(m,2H),3.44(q,J＝6.1Hz,2H),3.40(s,3H),1.74–1.65(m,2H),1.41(d,J＝6.7Hz,3H).MS(ESI):m/z 417.1[M+H] + .
Example E25- (4-chlorobenzyl) -1- (3-hydroxypropyl) -3-methyl-8- (3-phenoxypropyl-1-yn-1-yl) -3, 7-dihydro-1H-purine-2, 6-dione
Phenol (2.64g, 30mmol) was dissolved in 20mL dry DMF and K was added 2 CO 3 (4.97g, 36mmol), stirring for 0.5h. Then, bromopropyne (2.63mL, 30.5 mmol) was added dropwise thereto over 5min, and the reaction was carried out at room temperature. TLC showed disappearance of the starting material, saturated brine was added, extraction was performed three times with ethyl acetate, and the organic layers were combined, washed three times with saturated brine, and dried over anhydrous sodium sulfate. Filtering, evaporating the filtrate under reduced pressure, and then diluting to 30mL with THF to obtain a THF solution with the concentration of the intermediate INT 25-1 being about 1 mol/L.
INT 2-2(100mg，0.196mmol)、PdCl 2 (Ph 3 P) 2 (20.6mg, 0.0294mmol), cuI (11.2mg, 0.0588mmol), 3mL of triethylamine, and 1mL of DMF were placed in a pressure-resistant tube, degassed with nitrogen for 3min, then intermediate INT 25-1 (1mL, 1mmol) was added, and further degassed for 1min. The pressure pipe is sealed and heated for 4-6 h at 60 ℃. After cooling, the mixture was diluted with ethyl acetate, the insoluble matter was removed by filtration, and the filtrate was evaporated to dryness under reduced pressure to give a crude product. And then separating by using a flash silica gel chromatographic column, eluting by using MeOH/DCM as a mobile phase, collecting a target product part, and evaporating to dryness to obtain 87mg of white solid, namely an intermediate INT 25-2.
Intermediate INT 25-2 (87mg, 0.159mmol) was dissolved in 2mL DCM, then 0.4mL TFA was added, stirred at room temperature for 1h, TLC indicated complete reaction. The solvent was evaporated under reduced pressure and the residue was purified by preparative liquid chromatography using acetonitrile/water system as the mobile phase, the target product fraction was collected and evaporated to dryness to obtain 40mg of white solid, example E25. 1 H-NMR(500MHz,DMSO-d6)δ7.38–7.30(m,4H),7.26(d,J＝8.4Hz,2H),7.08–7.03(m,2H),7.01(t,J＝7.3Hz,1H),5.48(s,2H),5.23(s,2H),4.48(t,J＝5.2Hz,1H),3.94–3.86(m,2H),3.43(q,J＝6.2Hz,2H),3.39(s,3H),1.73–1.64(m,2H).MS(ESI):m/z 479.1[M+H] + .
Example E26- (4-chlorobenzyl) -8- (3- ((1-hydroxypropyl-2-oxy) prop-1-yn-1-yl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
Example E26 was synthesized as in E24, except that 2- (2-propyn-1-yloxy) propan-1-ol was used as starting material instead of 3-butyn-2-ol. 1 H-NMR(500MHz,DMSO-d6)δ7.43(d,J＝8.2Hz,2H),7.35(d,J＝8.2Hz,2H),5.56(s,2H),4.73(d,J＝4.7Hz,1H),4.54(s,2H),4.48(t,J＝5.2Hz,1H),3.97–3.87(m,2H),3.75(p,J＝5.7Hz,1H),3.47–3.39(m,5H),3.30(m,2H),1.69(p,J＝6.7Hz,2H),1.02(d,J＝6.3Hz,3H).MS(ESI):m/z 461.2[M+H] + .
Example E27- (4-chlorobenzyl) -1- (3-hydroxypropyl) -3-methyl-8- (4-methylpent-1-yn-1-yl) -3, 7-dihydro-1H-purine-2, 6-dione
The synthesis of example E27 is identical to E24, except that starting from 4-methyl-1-pentyne instead of 3-butyn-2-ol. 1H-NMR (500MHz, DMSO-d 6) delta 7.47-7.39 (m, 2H), 7.30 (d, J =8.3Hz, 2H), 5.54 (s, 2H), 4.47 (t, J =5.2Hz, 1H), 3.95-3.88 (m, 2H), 3.47-3.39 (m, 5H), 2.48 (d, J =6.4Hz, 2H), 1.88 (hept, J =6.6Hz, 1H), 1.69 (p, J =6.5Hz, 2H), 0.95 (d, J =6.7Hz, 6H). MS (ESI): m/z.2 [ M ]. M + H ], (M + H)] + .
Example E28- (4-chlorobenzyl) -1- (3-hydroxypropyl) -3-methyl-8- (3- ((4-oxocyclohexyl) oxy) prop-1-yn-1-yl) -3, 7-dihydro-1H-purine-2, 6-dione
The synthesis of example E28 is as for E20, except that 4-hydroxycyclohexanone acetal is used instead of cyclohexanol as starting material. 1 H-NMR(500MHz,DMSO-d6)δ7.46–7.41(m,2H),7.36–7.30(m,2H),5.58(s,2H),4.63(s,2H),4.47(t,J＝5.2Hz,1H),3.95–3.90(m,2H),3.87(tt,J＝6.6,3.4Hz,1H),3.47–3.39(m,5H),2.35(ddd,J＝14.7,8.9,5.9Hz,2H),2.24–2.15(m,2H),2.05–1.84(m,4H),1.69(p,J＝6.6Hz,2H).MS(ESI):m/z 499.2[M+H] + .
Example E29- (4-chlorobenzyl) -1- (3-hydroxypropyl) -3-methyl-8- (3- (pyrrolidin-1-yl) but-1-yn-1-yl) -3, 7-dihydro-1H-purine-2, 6-dione
Intermediate INT 24-1 (300mg, 0.6 mmol) and triethylamine (0.34mL, 2.4 mmol) were dissolved in 20mL of ultra-dry dichloromethane and treated with N 2 Protecting, and cooling in ice bath. Methanesulfonyl chloride (89mg, 0.78mmol) was dissolved in 2mL of dichloromethane and added dropwise over 2min to the flask and stirred for about half an hour to complete the reaction as detected by TLC. Quenching with saturated aqueous ammonium chloride solution, separating dichloromethane phase, washing with brine for 2 times, anhydrous MgSO 4 Dried and then chromatographed on flash silica gel to give 312mg of gum, intermediate INT 29-1.
Intermediate INT 29-1 (312mg, 0.54mmol) was dissolved in 10mL acetonitrile and K was added 2 CO 3 (149mg, 1.08mmol) followed by the addition of pyrrolidine (58mg, 0.81mmol), N 2 The reaction was heated at 75 ℃ for 4 hours under protection and TLC indicated completion. The inorganic material was removed by filtration and the filtrate was chromatographed on flash silica gel to give 89mg of gum, intermediate INT 29-2.
Intermediate INT 29-2 (89mg, 0.162mmol) was dissolved in 2mL DCM, then 0.4mL TFA was added, stirred at room temperature for 1h, TLC indicated complete reaction. The solvent was evaporated under reduced pressure and the residue was purified by preparative liquid chromatography using acetonitrile/water system as mobile phase, the target product fraction was collected and evaporated to dryness to give 42mg of white solid as example E29. 1 H-NMR(500MHz,DMSO-d6)δ7.46–7.41(m,2H),7.28–7.22(m,2H),5.58(s,2H),4.45(t,J＝5.2Hz,1H),4.00(d,J＝6.8Hz,1H),3.95–3.88(m,2H),3.43(m,5H),2.48(m,4H),1.69(dq,J＝9.0,6.6Hz,2H),1.61(s,4H),1.36(d,J＝7.1Hz,3H).MS(ESI):m/z 470.2[M+H] + .
Example E30- (4-chlorobenzyl) -8- (3- ((4-hydroxycyclohexyl) oxy) prop-1-yn-1-yl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
EXAMPLE Compound E28 (50mg, 0.1mmol) was dissolved in 2mL of methanol and NaBH was added 4 (7.6mg, 0.2mmol), stirred for 5min and TLC indicated completion of the reaction. Pouring into saturated NaCl aqueous solution, extracting with ethyl acetate for 3 times, combining organic phases, anhydrous MgSO 4 Drying, separating with flash silica gel chromatography column, eluting with dichloromethane/methanol system as mobile phase, collecting target product, and evaporating to dryness to obtain 38mg white solid, i.e. example E30. 1 H-NMR(500MHz,DMSO-d6)δ7.46–7.39(m,2H),7.33(dq,J＝9.1,2.7Hz,2H),4.51(s,2H),4.47(t,J＝5.2Hz,1H),3.95–3.89(m,2H),3.59–3.37(m,7H),1.90(dt,J＝13.2,4.8Hz,1H),1.80–1.64(m,4H),1.55–1.39(m,4H),1.21–1.12(m,1H).MS(ESI):m/z 501.2[M+H] + .
Example E31- (4-chlorobenzyl) -8- (3, 3-dimethylbut-1-yn-1-yl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
INT 2-2(100mg，0.196mmol)、PdCl 2 (Ph 3 P) 2 (20.6mg, 0.0294mmol), cuI (11.17mg, 0.0588mmol), 3mL of triethylamine, and 1mL of DMF were placed in a pressure-resistant tube, degassed with nitrogen for 3min, then 3, 3-dimethyl-1-butyne (82mg, 1mmol) was added, and further degassed for 1min. The pressure tube was sealed and heated at 60 ℃ for 6h. Cooling, diluting with ethyl acetate, filtering to remove insoluble substances, and filteringEvaporating to dryness under reduced pressure to obtain crude product. And then separating by using a flash silica gel chromatographic column, eluting by using MeOH/DCM as a mobile phase, collecting a target product part, and evaporating to dryness to obtain 91mg of a white solid, namely the intermediate INT 31-1.
Intermediate INT 31-1 (91mg, 0.178mmol) was dissolved in 2mL DCM, followed by the addition of 0.4mL TFA, stirring at RT for 1h, and TLC indicated completion of the reaction. The solvent was evaporated under reduced pressure and the residue was purified by preparative liquid chromatography using acetonitrile/water system as the mobile phase, the target product fraction was collected and evaporated to dryness to give 52mg of white solid, example E31. 1 H-NMR(500MHz,DMSO-d6)δ7.45(d,J＝8.4Hz,2H),7.35(d,J＝8.2Hz,2H),5.53(s,2H),4.48(t,J＝5.2Hz,1H),3.93(t,J＝7.5Hz,2H),3.44(q,J＝6.0Hz,2H),3.40(s,3H),1.70(p,J＝6.6Hz,2H),1.30(s,9H)；MS(ESI):m/z 429.2[M+H] + .
Example E32- (4-chlorobenzyl) -8- (cyclopentylacetylene) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
The synthesis of example E32 is the same as E31 except that starting from cyclopentylacetylene instead of 3, 3-dimethyl-1-butyne. 1 H NMR(500MHz,DMSO-d6)δ7.44(d,J＝8.0Hz,2H),7.32(d,J＝8.5Hz,2H),5.52(s,2H),4.48(t,J＝5.2Hz,1H),3.92(t,J＝7.5Hz,2H),3.44(q,J＝6.1Hz,2H),3.40(s,3H),3.01(p,J＝7.4Hz,1H),1.99(m,2H),1.73–1.54(m,8H)；MS(ESI):m/z 441.2[M+H] + .
Example E33- (4-chlorobenzyl) -8- (cyclohexylacetylene) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
The synthesis of example E33 is identical to E31, except that cyclohexylacetylene is used instead of 3, 3-dimethyl-1-butyne as starting material. 1 H-NMR(500MHz,DMSO-d6)δ7.44(d,J＝8.4Hz,2H),7.32(d,J＝8.5Hz,2H),5.53(s,2H),4.48(t,J＝5.2Hz,1H),3.92(t,J＝7.5Hz,2H),3.45(m,2H),3.40(s,3H),2.80(m,1H),1.81(m,2H),1.69(m,2H),1.59(m,2H),1.49(m,3H),1.34(m,3H)；MS(ESI):m/z 455.2[M+H] + .
Example E34- (4-chlorobenzyl) -8- (3-cyclohexylpropyl-1-yn-1-yl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
The synthesis of example E34 is as for E31, except that 3-cyclohexylpropyne is used instead of 3, 3-dimethyl-1-butyne as starting material. 1 H-NMR(500MHz,DMSO-d6)δ7.45–7.40(m,2H),7.27(d,J＝8.2Hz,2H),5.54(s,2H),4.47(t,J＝5.2Hz,1H),3.95–3.88(m,2H),3.44(q,J＝6.2Hz,2H),3.41(s,3H),2.46(d,J＝6.5Hz,2H),1.73–1.55(m,7H),1.49(m,1H),1.18(m2H),1.06(m,1H),1.00–0.89(m,2H)；MS(ESI):m/z 469.2[M+H] + .
Example E35- ((1R, 3S,5r, 7r) -adamantan-2-yl) ethynyl) -7- (4-chlorobenzyl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
The synthesis of example E35 is as for E31, except that 1-ethynyladamantane is used as starting material instead of 3, 3-dimethyl-1-butyne. 1 H-NMR(500MHz,Chloroform-d)δ7.46–7.40(m,2H),7.34(d,J＝6.6Hz,1H),4.19(t,J＝6.0Hz,2H),3.58(s,3H),3.53(m,2H),3.41(m,1H),2.06(m,3H),2.03(m,6H),1.91(p,J＝5.7Hz,2H),1.81–1.73(m,6H)；MS(ESI):m/z 507.2[M+H] + .
Example E36- (4-chlorobenzyl) -1- (3-hydroxypropyl) -3-methyl-8- (3- (4-methylpiperidin-1-yl) but-1-yn-1-yl) -3, 7-dihydro-1H-purine-2, 6-dione
Example E36 SynthesisThe preparation method is the same as E29 except that 4-methylpiperidine is used as a raw material instead of pyrrolidine. 1 H-NMR(500MHz,Chloroform-d)δ7.33(s,4H),5.59(s,2H),4.19(t,J＝6.0Hz,2H),3.83(q,J＝7.1Hz,1H),3.60(s,3H),3.53(t,J＝5.5Hz,2H),2.93(d,J＝10.9Hz,1H),2.78(d,J＝11.0Hz,1H),2.38(d,J＝2.8Hz,1H),2.09(d,J＝2.6Hz,1H),1.91(p,J＝5.7Hz,2H),1.67(m,6H),1.50(d,J＝7.1Hz,3H),0.94(d,J＝5.4Hz,3H)；MS(ESI):m/z 498.2[M+H] + .
Example E37- (4-chlorobenzyl) -8- (3-ethoxybut-1-yn-1-yl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
Potassium hydroxide (364mg, 6.5 mmol) was placed in 8mL DMSO, 3-butyn-2-ol (0.37mL, 5 mmol) was added, and stirring was carried out at room temperature for 20min, followed by iodoethane (440. Mu.L, 5.5 mmol), and stirring was carried out overnight at room temperature. Ice water was added to the flask and neutralized with acetic acid. Then extracted with 8mL of methyl tert-butyl ether, washed twice with brine and dried over anhydrous magnesium sulfate. Magnesium sulfate was removed by filtration, and the filtrate was made to volume of 10mL to obtain a methyl tert-butyl ether solution of intermediate INT 37-1 at a concentration of 0.5 mol/L.
INT 2-2(100mg，0.196mmol)、PdCl 2 (Ph 3 P) 2 (20.6mg, 0.0294mmol), cuI (11.17mg, 0.0588mmol), 3mL of triethylamine, and 1mL of DMF were placed in a pressure-resistant tube, degassed with nitrogen for 3min, then intermediate INT 37-1 (1.6mL, 0.8mmol) was added, and further degassed for 1min. The pressure tube was sealed and heated at 60 ℃ for 4h. After cooling, the mixture was diluted with ethyl acetate, the insoluble matter was removed by filtration, and the filtrate was evaporated to dryness under reduced pressure to give a crude product. Separation was then carried out by preparative plate TLC, meOH/DCM was developed and the desired product fractions were collected as 30mg of gum, intermediate INT 37-2.
Intermediate INT 37-2 (30mg, 0.057mmol) was dissolved in 2mL DCM and 0.4mL TFA was added and stirred at RT for 1h, TLC indicated completion of the reaction. Evaporating under reduced pressure to remove solvent, purifying the residue with preparative liquid chromatography column, eluting with acetonitrile/water system as mobile phase, collecting target product,evaporation to dryness afforded 12mg of a white solid, example E37. 1 H-NMR(500MHz,CD 3 OD)δ7.38(s,4H),5.65(m,2H),4.52(q,J＝6.5Hz,1H),4.11(t,J＝7.0Hz,2H),3.73(m,1H),3.62(t,J＝6.5Hz,2H),3.54(s,3H),3.51(m,1H),1.87(m,2H),1.53(d,J＝7.0Hz,3H),1.21(t,J＝7.0Hz,3H)；MS(ESI):m/z 445.2[M+H] + .
Example E38- (4-chlorobenzyl) -8- (3- (cyclopropylmethoxy) but-1-yn-1-yl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
The synthesis of example E38 is as for E37, except that bromomethylcyclopropane is used instead of iodoethane as starting material. 1 H-NMR(500MHz,Chloroform-d)δ7.38(d,J＝8.5Hz,2H),7.33(d,J＝8.5Hz,2H),5.57(m,2H),4.52(q,J＝6.5Hz,1H),4.20(t,J＝6.0Hz,2H),3.59(s,3H),3.54(m,3H),3.33(m,2H),1.91(m,2H),1.61(d,J＝7.0Hz,3H),1.06(m,1H),0.59(m,2H),0.23(m,2H)；MS(ESI):m/z 471.2[M+H] + .
Example E39- (4-chlorobenzyl) -1- (3-hydroxypropyl) -3-methyl-8- (3-methyl-3- (4-methylpiperidin-1-yl) but-1-yn-1-yl) -3, 7-dihydro-1H-purine-2, 6-dione
3-chloro-3-methyl-1-butyne (1.02g, 10mmol), 4-methylpiperidine (0.991g, 10mmol) were dissolved in 10mL of ultra-dry tetrahydrofuran, triethylamine (1.67mL, 12mmol) was added, and the mixture was stirred with N 2 Protecting, cooling to 0 deg.C in ice bath, suspending CuI (0.38g, 2mmol) in 5mL tetrahydrofuran, adding dropwise into the reaction system with constant pressure dropping funnel, reacting overnight at room temperature, detecting with thin layer chromatography, developing with potassium permanganate developer, pouring saturated NaCl aqueous solution, extracting with ethyl acetate for 2 times, anhydrous MgSO, and allowing to stand for a while 4 Drying, filtering and evaporating to dryness, and then making the volume to 10mL to obtain a tetrahydrofuran solution of which the concentration of the intermediate INT 39-1 is 1 mol/L.
INT 2-2(100mg，0.196mmol)、PdCl 2 (Ph 3 P) 2 (20.6mg, 0.0294mmol), cuI (11.17mg, 0.0588mmol), 3mL of triethylamine, and 1mL of DMF were placed in a pressure-resistant tube, degassed with nitrogen for 3min, then intermediate INT 39-1 (1mL, 1mmol) was added, and further degassed for 1min. The pressure tube was sealed and heated at 60 ℃ for 4h. After cooling, diluting with ethyl acetate, filtering to remove insoluble substances, and evaporating the filtrate under reduced pressure to obtain a crude product. Separating with flash silica gel chromatography column, eluting with MeOH/DCM as mobile phase, collecting target product, and evaporating to dryness to obtain 91mg of jelly, namely intermediate INT 39-2.
Intermediate INT 39-2 (91mg, 0.153mmol) was dissolved in 2mL DCM, then 0.4mL TFA was added, stirred at RT for 1h, TLC indicated complete reaction. The solvent was evaporated under reduced pressure and the residue was purified by preparative liquid chromatography using acetonitrile/water system as the mobile phase, the target product fraction was collected and evaporated to dryness to give 46mg of a white solid, example E39. 1 H-NMR(500MHz,Chloroform-d)δ7.33(s,4H),5.59(s,2H),3.61(s,3H),3.53(q,J＝5.5Hz,3H),3.10(d,J＝10.8Hz,2H),2.16–2.08(m,2H),2.04(m,1H),1.91(p,J＝5.7Hz,2H),1.68(m,4H),1.54(s,6H),1.36–1.32(m,2H),0.94(d,J＝5.8Hz,3H).MS(ESI):m/z 512.2[M+H] + .
Example E40- (4-chlorobenzyl) -8- (cyclopropylethynyl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
The synthesis of example E40 is as for E31, except that cyclopropylacetylene is used instead of 3, 3-dimethyl-1-butyne as starting material. 1 H-NMR(500MHz,DMSO-d6)δ7.45(m,2H d,J＝8.5Hz,2H),7.32(d,J＝8.5Hz,2H),5.51(s,2H),4.46(t,J＝5.3Hz,1H),3.92(t,J＝7.5Hz,2H),3.44(m,2H),3.39(s,3H),1.74–1.65(m,3H),1.01(m,2H),0.89–0.82(m,2H)；MS(ESI):m/z 413.1[M+H] + .
Example E41- (4-chlorobenzyl) -8- (3- (cyclopentylamino) -3-methylbut-1-yn-1-yl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
The synthesis of example E41 is as for E39, except that cyclopentylamine is used as starting material instead of 4-methylpiperidine. 1 H-NMR(500MHz,DMSO-d6)δ7.46–7.41(m,2H),7.29(d,J＝8.4Hz,2H),5.55(s,2H),4.46(t,J＝5.2Hz,1H),3.91(dd,J＝8.3,6.5Hz,2H),3.43(d,J＝12.8Hz,5H),3.18(d,J＝5.2Hz,1H),3.06(p,J＝7.2Hz,1H),1.73–1.65(m,4H),1.57–1.48(m,2H),1.36(s,6H),1.21(m,2H)；MS(ESI):m/z 498.2[M+H] + .
Example E42- (4-chlorobenzyl) -8- (3- ((3, 3-difluorocyclobutyl) amino) -3-methylbut-1-yn-1-yl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
The synthesis of example E42 is as for E39, except that 3, 3-difluorocyclobutylamine is used as starting material instead of 4-methylpiperidine. 1 H-NMR(500MHz,DMSO-d6)δ7.47–7.41(m,2H),7.31(d,J＝8.4Hz,2H),5.55(s,2H),4.47(t,J＝5.2Hz,1H),4.12(q,J＝5.3Hz,1H),3.96–3.89(m,2H),3.44(q,J＝6.1Hz,2H),3.41(s,3H),3.18(d,J＝4.6Hz,2H),2.71(m,2H),2.37(m,2H),1.69(p,J＝6.6Hz,2H),1.35(s,6H).MS(ESI):m/z 520.2[M+H] + .
Example E43- (4-chlorobenzyl) -8- (3- (cyclopropyl (methyl) amino) -3-methylbut-1-yn-1-yl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
The synthesis of example E43 is as for E39, except that N-methylcyclopropane is used instead of 4-methylpiperidine as starting material. 1 H-NMR(500MHz,DMSO-d6)δ7.47–7.40(m,2H),7.30–7.24(m,2H),5.59(s,2H),4.48(t,J＝5.2Hz,1H),3.97–3.88(m,2H),3.44(d,J＝9.5Hz,5H),2.25(s,3H),1.74–1.66(m,2H),1.61(tt,J＝6.8,3.8Hz,1H),1.43(s,6H),0.52–0.45(m,2H),0.38–0.31(m,2H).MS(ESI):m/z 484.2[M+H] + .
Example E44- (4-chlorobenzyl) -8- (3- (cyclobutylmethoxy) but-1-yn-1-yl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
The synthesis of example E44 is the same as E37 except bromomethylcyclobutane is used instead of iodoethane. 1 H-NMR(500MHz,Chloroform-d)δ7.39(d,J＝8.5Hz,2H),7.33(d,J＝8.5Hz,2H),5.58(m,2H),4.45(q,J＝7.0Hz,1H),4.20(t,J＝6.0Hz,2H),3.69(dd,J＝7.0,2.0Hz,1H),3.60(s,3H),3.55(t,J＝6.0Hz,2H),3.45(dd,J＝7.0,2.0Hz,1H),3.32(brs,1H,-OH),2.60(m,1H),2.08(m,2H),1.92(m,4H),1.74(m,2H),1.59(d,J＝6.5Hz,3H)；MS(ESI):m/z 485.2[M+H] + .
EXAMPLE E45- (4-chlorobenzyl) -8- (3- (cyclopropylmethoxy) -3-methylbut-1-yn-1-yl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
The synthesis of example E45 is carried out as in E37, except that bromomethylcyclopropane is used instead of iodoethane and 2-methyl-3-butyn-2-ol is used instead of 3-butyn-2-ol as starting material. 1 H-NMR(500MHz,Chloroform-d)δ7.37(d,J＝8.5Hz,2H),7.34(d,J＝8.5Hz,2H),5.58(s,2H),4.20(t,J＝6.0Hz,2H),3.60(s,3H),3.54(m,2H),3.41(d,J＝7.0Hz,2H),3.31(t,1H,-OH),1.91(m,2H),1.62(s,6H),1.08(m,1H),0.57(m,2H),0.21(m,2H)；MS(ESI):m/z 485.2[M+H] + .
Example E46- (4-chlorobenzyl) -8- (3- (cyclopentyloxy) but-1-yn-1-yl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
The synthesis of example E46 is as for E37 except that bromocyclopentane is used as the starting material instead of iodoethane. 1 H-NMR(500MHz,Chloroform-d)δ7.41(d,J＝8.5Hz,2H),7.33(d,J＝8.5Hz,2H),5.58(m,2H),4.48(q,J＝6.5Hz,1H),4.20(m,3H),3.60(s,3H),3.55(t,J＝5.5Hz,2H),2.04(m,1H),1.92(m,2H),1.81(m,1H),1.74(m,4H),1.67(m,1H),1.57(d,J＝6.5Hz,3H),1.54(m,1H)；MS(ESI):m/z 485.2[M+H] + .
Example E47:7- (4-chlorobenzyl) -8- (4-fluorophenyl) ethynyl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
The synthesis of example E47 is as for E31, except that 1-ethynyl-4-fluorobenzene is used as starting material instead of 3, 3-dimethyl-1-butyne. 1 H-NMR(500MHz,Chloroform-d)δ7.63(dd,J＝8.5,5.5Hz,2H),7.44(d,J＝8.5Hz,2H),7.35(d,J＝8.5Hz,2H),7.16(t,J＝9.0Hz,2H),5.64(s,2H),4.22(t,J＝6.0Hz,2H),3.63(s,3H),3.56(m,2H),3.33(brs,1H,-OH),1.93(m,2H)；MS(ESI):m/z 467.1[M+H] + .
Example E48:7- (4-chlorobenzyl) -1- (3-hydroxypropyl) -3-methyl-8- (pyridin-2-ylethynyl) -3, 7-dihydro-1H-purine-2, 6-dione
The synthesis of example E48 is as for E31, except that 2-ethynylpyridine is used as starting material instead of 3, 3-dimethyl-1-butyne. 1 H-NMR(500MHz,Chloroform-d)δ8.74(d,J＝6.0Hz,1H),7.81(t,J＝7.5Hz,1H),7.68(d,J＝7.0Hz,1H),7.55(d,J＝8.5Hz,2H),7.42(m,1H),7.33(d,J＝8.5Hz,2H),5.70(s,2H),4.23(t,J＝6.0Hz,2H),3.63(s,3H),3.56(m,2H),3.32(brs,1H,-OH),1.94(m,2H)；MS(ESI):m/z 450.1[M+H] + .
Example E49:7- (4-chlorobenzyl) -1- (3-hydroxypropyl) -3-methyl-8- (thien-2-ylethynyl) -3, 7-dihydro-1H-purine-2, 6-dione
The synthesis of example E49 is as for E31, except that 2-ethynylthiophene is used as starting material instead of 3, 3-dimethyl-1-butyne. 1 H-NMR(500MHz,Chloroform-d)δ7.52(d,J＝5.0Hz,1H),7.50(d,J＝3.5Hz,1H),7.45(d,J＝8.5Hz,2H),7.35(d,J＝8.0Hz,2H),7.14(t,J＝4.5Hz,1H),5.63(s,2H),4.22(t,J＝6.0Hz,2H),3.62(s,3H),3.57(m,2H),3.37(brs,1H,-OH),1.94(m,2H)；MS(ESI):m/z 455.1[M+H] + .
Example E50:7- (4-chlorobenzyl) -1- (3-hydroxypropyl) -3-methyl-8- (3-oxobut-1-yn-1-yl) -3, 7-dihydro-1H-purine-2, 6-dione
INT 2-2(255mg，0.5mmol)、PdCl 2 (Ph 3 P) 2 (52.5mg, 0.074mmol), cuI (28.5mg, 0.15mmol), 3mL of triethylamine and 1mL of DMF were placed in a pressure-resistant tube, degassed with nitrogen for 3min, followed by addition of 3-butyn-2-ol (175mg, 2.5mmol) and further degassing for 1min. The pressure tube was sealed and heated at 60 ℃ for 4h. After cooling, diluting with ethyl acetate, filtering to remove insoluble substances, and evaporating the filtrate under reduced pressure to obtain a crude product. Separating with flash silica gel chromatography column, eluting with MeOH/DCM as mobile phase, collecting target product, and evaporating to dryness to obtain 91mg of jelly, namely intermediate INT 50-1.
Intermediate INT 50-1 (100mg, 0.2mmol) was dissolved in 2mL DCM, dess-Martin oxidant (170mg, 0.4mmol) was added, stirring was carried out at RT for 3h, TLC indicated completion of the reaction. DCM/saturated NaHCO 3 And (3) distributing the aqueous solution, separating an organic phase, drying by using anhydrous sodium sulfate, separating by using a flash silica gel chromatographic column, eluting by using MeOH/DCM as a mobile phase, collecting a target product part, and evaporating to dryness to obtain 73mg of jelly, namely an intermediate INT 50-2.
Intermediate INT 50-2 (73mg, 0.151mmol) was dissolved in 2mL DCM, then 0.4mL TFA was added,the reaction was stirred at room temperature for 1h and TLC showed completion. The solvent was evaporated under reduced pressure and the residue was purified by preparative liquid chromatography using acetonitrile/water system as mobile phase, the target product fraction was collected and evaporated to dryness to obtain 32mg of white solid, example E50. 1 H-NMR(500MHz,Chloroform-d)δ7.45–7.39(m,2H),7.37–7.31(m,2H),5.62(s,2H),4.22(t,J＝6.1Hz,2H),3.60(s,3H),3.56(t,J＝5.6Hz,2H),2.54(s,3H),1.93(p,J＝5.8Hz,2H)；MS(ESI):m/z 415.1[M+H] + .
Example E51: (E) -7- (4-chlorobenzyl) -8- (2-cyclopropylvinyl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
INT 2-2 (100mg, 0.196mmol), (E) -2-cyclopropylvinylboronic acid pinacol ester (95mg, 0.49mmol), pd (dppf) Cl 2 (21.5mg，0.029mmol)、Cs 2 CO 3 (191mg, 0.588 mmol), 1, 4-dioxane 10mL, and H 2 O3 mL was placed in a pressure tube, degassed with nitrogen for 3min, sealed, and heated at 100 ℃ for 2h. After cooling, diluting with ethyl acetate, filtering to remove insoluble substances, and evaporating the filtrate under reduced pressure to obtain a crude product. And separating by using a flash silica gel chromatographic column, eluting by using MeOH/DCM as a mobile phase, collecting a target product part, and evaporating to dryness to obtain 85mg of jelly, namely an intermediate INT 51-1.
Intermediate INT 51-1 (85mg, 0.176mmol) was dissolved in 2mL DCM, then 0.4mL TFA was added, stirred at RT for 1h, TLC indicated complete reaction. The solvent was evaporated under reduced pressure and the residue was purified by preparative liquid chromatography using acetonitrile/water system as the mobile phase, the target product fraction was collected and evaporated to dryness to afford 40mg of a white solid, example E51. 1 H-NMR(500MHz,Chloroform-d)δ7.38–7.32(m,2H),7.22–7.13(m,2H),6.56(dd,J＝15.1,10.0Hz,1H),6.37(d,J＝15.1Hz,1H),5.56(s,2H),4.23–4.13(m,2H),3.61(s,3H),3.56–3.49(m,2H),1.91(p,J＝5.8Hz,2H),1.68–1.62(m,1H),1.07–0.95(m,2H),0.76–0.71(m,2H)；MS(ESI):m/z 413.1[M+H] + .
Example E52- (4-chlorophenyl) -1- (3-hydroxypropyl) -3-methyl-8- (3-methyl-3- (methylsulfonyl) but-1-yn-1-yl) -3, 7-dihydro-1 h-purine-2, 6-dione
Sodium methanesulfonate (830mg, 7.033mmol) and CuCl (77mg, 0.778mmol) were dissolved in 10mL of DMF, and 3-chloro-3-methyl-1-butyne (1g, 9.8mmol) was slowly added dropwise thereto, and the mixture was heated at 40 ℃ for 12 hours. TLC showed the reaction was complete. And (3) filtering, concentrating the filtrate, separating by using a flash silica gel chromatographic column, eluting by using MeOH/DCM as a mobile phase, collecting a target product part, and evaporating to dryness to obtain 184mg of jelly, namely an intermediate INT 52-1.
INT 2-2(100mg，0.196mmol)、PdCl 2 (Ph 3 P) 2 (20.6mg, 0.0294mmol), cuI (11.17mg, 0.0588mmol), 3mL of triethylamine, and 1mL of DMF were placed in a pressure-resistant tube, degassed with nitrogen for 3min, then intermediate INT 52-1 (85mg, 0.582mmol) was added, and degassed for 1min. The pressure tube was sealed and heated at 60 ℃ for 4h. After cooling, diluting with ethyl acetate, filtering off insoluble substances, and evaporating the filtrate under reduced pressure to obtain a crude product. Separating with flash silica gel chromatography column, eluting with MeOH/DCM as mobile phase, collecting target product, and evaporating to dryness to obtain 50mg of jelly, namely intermediate INT 52-2.
Intermediate INT 52-2 (50mg, 0.089mmol) was dissolved in 2mL DCM, then 0.4mL TFA was added, stirred at RT for 1h, TLC indicated complete reaction. The solvent was evaporated under reduced pressure and the residue was purified by preparative liquid chromatography using acetonitrile/water system as the mobile phase, the target product fraction was collected and evaporated to dryness to obtain 25mg of white solid, example E52. 1 H-NMR(500MHz,Chloroform-d)δ7.44–7.37(m,2H),7.37–7.32(m,2H),5.60(s,2H),4.22(t,J＝6.1Hz,2H),3.60(s,3H),3.58–3.53(m,2H),3.00(s,3H),1.93(m,2H),1.82(s,6H)；MS(ESI):m/z 493.1[M+H] + .
Example E53 (E) -7- (4-chlorobenzyl) -1- (3-hydroxypropyl) -3-methyl-8- (pent-1-en-1-yl) -3, 7-dihydro-1H-purine-2, 6-dione
INT 2-2 (100mg, 0.196mmol), trans-1-penten-1-ylboronic acid pinacol ester (96.1mg, 0.49mmol), pd (dppf) Cl 2 (21.5mg，0.029mmol)、Cs 2 CO 3 (191mg, 0.588 mmol), 1, 4-dioxane 10mL, and H 2 O3 mL was placed in a pressure tube, degassed with nitrogen for 3min, sealed, and heated at 100 ℃ for 2h. After cooling, diluting with ethyl acetate, filtering to remove insoluble substances, and evaporating the filtrate under reduced pressure to obtain a crude product. Separating with flash silica gel chromatography column, eluting with MeOH/DCM as mobile phase, collecting target product, and evaporating to dryness to obtain 100mg of jelly, i.e. intermediate INT 51-1.
Intermediate INT 51-1 (100mg, 0.2mmol) was dissolved in 2mL DCM, then 0.4mL TFA was added, stirred at room temperature for 1h, and TLC indicated complete reaction. The solvent was evaporated under reduced pressure and the residue was purified by preparative liquid chromatography using acetonitrile/water system as the mobile phase, the target product fraction was collected and evaporated to dryness to give 50mg of a white solid, example E51. 1 H-NMR(500MHz,Chloroform-d)δ7.37–7.31(m,2H),7.20–7.14(m,2H),7.10(dt,J＝15.4,7.1Hz,1H),6.31(dt,J＝15.4,1.6Hz,1H),5.57(s,2H),4.26–4.09(m,2H),3.63(s,3H),3.57–3.49(m,2H),2.30(qd,J＝7.2,1.6Hz,2H),1.91(p,J＝5.8Hz,2H),1.56(p,J＝7.4Hz,2H),0.98(t,J＝7.3Hz,3H)；MS(ESI):m/z 417.2[M+H] + .
EXAMPLE E54 Ethyl propionate 3- (7- (4-chlorobenzyl) -1- (3-hydroxypropyl) -3-methyl-2, 6-dioxo-2, 3,6, 7-tetrahydro-1H-purin-8-yl)
Trimethylsilyne (980mg, 10mmol) was dissolved in 20mL of diethyl ether, isopropylmagnesium bromide (1.617g, 11mmol) was added thereto, the mixture was stirred at room temperature for 1 hour, heated at 150 ℃ and refluxed for 15 minutes, cooled to room temperature, tetraethyl orthocarbonate (2.208g, 11.5 mmol) was added thereto, and the mixture was stirred at 45 ℃ for 12 hours. TLC showed the reaction was complete. And (3) distributing EA/saturated NaCl aqueous solution, separating an organic phase, drying by anhydrous sodium sulfate, separating by using a flash silica gel chromatographic column, eluting by using PE/EA as a mobile phase, collecting a target product part, and evaporating to dryness to obtain 1.05g of liquid, namely an intermediate INT 54-1.
INT 54-1 (1g, 4.098mmol) was dissolved in 5mL of THF, TBAF (5.3mL, 6.147mmol) was added, and the mixture was stirred at room temperature for 15min. TLC shows that the reaction is finished, the solvent is removed by reduced pressure distillation, the separation is carried out by a flash silica gel chromatographic column, PE/EA is used as a mobile phase for elution, the target product part is collected, and 700mg of liquid, namely the intermediate INT 54-2, is obtained by evaporation.
INT 2-2(100mg，0.196mmol)、PdCl 2 (Ph 3 P) 2 (20.6 mg, 0.0294mmol), cuI (11.17mg, 0.0588mmol), triethylamine 3mL, and 1mL of DMF were placed in a pressure tube, degassed with nitrogen for 3min, then intermediate INT 54-2 (100mg, 0.582mmol) was added, and degassed for 1min. The pressure tube was sealed and heated at 60 ℃ for 4h. After cooling, the mixture was diluted with ethyl acetate, insoluble matter was filtered off, and the filtrate was evaporated to dryness under reduced pressure to give a crude product. Separating with flash silica gel chromatography column, eluting with MeOH/DCM as mobile phase, collecting target product, and evaporating to dryness to obtain 80mg of jelly, namely intermediate INT 54-3.
Intermediate INT 52-2 (80mg, 0.133mmol) was dissolved in 2mL DCM, then 0.4mL TFA was added, stirred at room temperature for 1h, TLC indicated complete reaction. The solvent was evaporated under reduced pressure and the residue was purified by preparative liquid chromatography using acetonitrile/water system as the mobile phase, the target product fraction was collected and evaporated to dryness to obtain 45mg of white solid, example E54. 1 H-NMR(500MHz,Chloroform-d)δ7.48–7.43(m,2H),7.38–7.32(m,2H),5.63(s,2H),4.40(q,J＝7.1Hz,2H),4.22(t,J＝6.1Hz,2H),3.60(s,3H),3.58–3.54(m,2H),1.93(p,J＝5.8Hz,2H),1.42(t,J＝7.1Hz,3H)；MS(ESI):m/z 445.1[M+H] + .
Example E55- (4-chlorobenzyl) -6- (cyclopentylethynyl) -3- (3-hydroxypropyl) -1-methyl-1, 5-dihydro-2H-pyrrolo [3,2-d ] pyrimidine-2, 4 (3H) -dione
6-Chlorouracil (10g, 68.493mmol)Dissolved in 40mL DMSO, K was added sequentially 2 CO 3 (4.8g，34.783mmol)、CH 3 I (12.7mL, 205.48mmol) was stirred at room temperature for 3h. TLC showed the reaction was complete, 50mL H was added 2 And O, performing suction filtration, and washing a filter cake to obtain 7.9g of white solid, namely the intermediate INT 55-1.
Intermediate INT 55-1 (1g, 6.25mmol) was dissolved in 10mL DMF and K was added 2 CO 3 (1.73g, 12.5 mmol) and 3-bromopropyl methyl ether (1.07mL, 9.375 mmol), and heated at 60 ℃ for 3 hours. TLC shows that the reaction is finished, EA/saturated NaCl aqueous solution is distributed, an organic phase is separated, anhydrous sodium sulfate is dried and then is separated by a flash silica gel chromatographic column, DCM/MeOH is used as a mobile phase for elution, a target product part is collected and is evaporated to dryness to obtain 800mg of jelly, namely an intermediate INT 55-2.
Intermediate INT 55-2 (1g, 4.31mmol) was dissolved in 3mL of concentrated sulfuric acid, stirred at 0 ℃ for 5min, 3mL of concentrated nitric acid was slowly added dropwise, and stirred at 0 ℃ for 1h. TLC shows that the reaction was complete, EA/ice water was partitioned, the organic layer was washed with saturated brine four times, dried over anhydrous sodium sulfate and separated by flash column chromatography on silica gel, eluted with DCM/MeOH as mobile phase, the target product fractions were collected and evaporated to dryness to obtain 800mg of gum, intermediate INT55-3.
Diethyl malonate (3.24mL, 21.66mmol) was dissolved in 10mL of dried 1, 4-dioxane, potassium tert-butoxide (2.1lg, 19.494mmol) was added and stirred at room temperature for 10min, then intermediate INT55-3 (3g, 10.83mmol) was added and stirred at room temperature for 1h. TLC shows that the reaction is finished, 2M HCl is adjusted to be acidic, EA/saturated NaCl aqueous solution is distributed, an organic phase is separated, anhydrous sodium sulfate is dried and then separated by a flash silica gel chromatographic column, DCM/MeOH is used as a mobile phase for elution, a target product part is collected, and 3.2g of jelly, namely the intermediate INT 55-4, is obtained by evaporation.
The intermediate INT 55-4 (3 g, 7.463mmol) is dissolved in 15mL of acetic acid, heated to 60 ℃, added with zinc powder (7.3g, 111.94mmol) and reacted for 12h in a pressure resistant tube at 120 ℃. TLC shows that the reaction is finished, and the crude product is obtained by reduced pressure evaporation. Then separating with flash silica gel chromatographic column, eluting with MeOH/DCM as mobile phase, collecting target product, and evaporating to dryness to obtain 1.8g of jelly, namely intermediate INT 55-5.
Intermediate INT55-5 (100mg, 7.463mmol) was dissolved in 3mL of dry 1, 4-dioxane, tribromooxyphosphorus (340mg, 22.389mmol) was added under nitrogen, and stirring was carried out at 100 ℃ for 1h. TLC showed completion of the reaction, EA/saturated NaHCO 3 Distributing the aqueous solution, separating an organic phase, drying by anhydrous sodium sulfate, separating by using a flash silica gel chromatographic column, eluting by using DCM/MeOH as a mobile phase, collecting a target product part, and evaporating to dryness to obtain 90mg of jelly, namely an intermediate INT 55-6.
Intermediate INT 55-6 (90mg, 0.286mmol) was dissolved in 5mL DMF and K was added 2 CO 3 (1.73g, 12.5 mmol) and 4-chlorobenzyl bromide (116mg, 0.572mmol), and heating at 60 ℃ for 2 hours. TLC shows that the reaction is finished, EA/saturated NaCl aqueous solution is distributed, an organic phase is separated, anhydrous sodium sulfate is dried and then is separated by a flash silica gel chromatographic column, DCM/MeOH is used as a mobile phase for elution, a target product part is collected and is evaporated to dryness to obtain 80mg of white solid, namely an intermediate INT 55-7.
Intermediate INT 55-7 (40mg, 0.091mmol) was dissolved in 3mL dry DCM and 182. Mu.L BBr 1M was added at 0 deg.C 3 The solution was stirred at this temperature for 1h. TLC shows that the reaction is finished, and the crude product is obtained by reduced pressure evaporation. Separating with flash silica gel chromatography column, eluting with MeOH/DCM as mobile phase, collecting target product, and evaporating to dryness to obtain 30mg white solid, namely intermediate INT 55-8.
INT 55-8(50mg，0.118mmol)、PdCl 2 (Ph 3 P) 2 (12mg, 0.0177mmol), cuI (7mg, 0.0354mmol), triethylamine 3mL and DMF 1mL were placed in a pressure tube, degassed with nitrogen for 3min, then cyclopentylacetylene (56mg, 0.588mmol) was added and degassed for 1min. The pressure tube was sealed and heated at 60 ℃ for 4h. After cooling, diluting with ethyl acetate, filtering to remove insoluble substances, and evaporating the filtrate under reduced pressure to obtain a crude product. Then separating with flash silica gel chromatography column, eluting with MeOH/DCM as mobile phase, collecting the target product, and evaporating to dryness to obtain 40mg of gum. The crude product was purified by preparative liquid chromatography, eluted with acetonitrile/water system as mobile phase, the target product fraction was collected and evaporated to dryness to obtain 25mg of white solid, example E55. 1 H-NMR(500MHz,Chloroform-d)δ7.30(s,4H),6.11(s,1H),5.61(s,2H),4.26–4.07(m,2H),3.51(t,J＝5.5Hz,2H),3.45(s,3H),2.91(p,J＝7.4Hz,1H),2.05(d,J＝10.7Hz,3H),1.90(p,J＝5.7Hz,2H),1.84–1.54(m,3H)；MS(ESI):m/z 440.2[M+H] + .
Example E56 (E) -5- (4-chlorobenzyl) -6- (4-chlorophenylvinyl) -3- (3-hydroxypropyl) -1-methyl-1, 5-dihydro-2H-pyrrolo [3,2-d ] pyrimidine-2, 4 (3H) -dione
INT 55-8 (25mg, 0.049 mmol), (E) - (4-chlorostyryl) boronic acid (22.3mg, 0.122mmol), pd (dppf) Cl 2 (5.38mg，0.007mmol)、Cs 2 CO 3 (48mg, 0.147mmol), 1, 4-dioxane 10mL, and H 2 O3 mL was placed in a pressure tube, degassed with nitrogen for 3min, sealed, and heated at 100 ℃ for 2h. After cooling, diluting with ethyl acetate, filtering to remove insoluble substances, and evaporating the filtrate under reduced pressure to obtain a crude product. Then separating with flash silica gel chromatography column, eluting with MeOH/DCM as mobile phase, collecting the target product, and evaporating to dryness to obtain 25mg of gum. The crude product was purified by preparative liquid chromatography, eluted with acetonitrile/water system as mobile phase, the target product fraction was collected and evaporated to dryness to give 15mg of a white solid, example E56. 1 H-NMR(500MHz,Chloroform-d)δ7.37(s,4H),7.34–7.30(m,2H),7.16–7.03(m,3H),6.91(d,J＝16.1Hz,1H),6.31(s,1H),5.78(s,2H),4.22(t,J＝5.9Hz,2H),3.55(s,3H),3.53(s,2H),1.96–1.87(m,2H)；MS(ESI):m/z 484.1[M+H] + .
Example E57 (E) -5- (4-chlorobenzyl) -6- (2-cyclopentylprop-1-en-1-yl) -3- (3-hydroxypropyl) -1-methyl-1, 5-dihydro-2H-pyrrolo [3,2-d ] pyrimidine-2, 4 (3H) -dione
Pinacol diboron (1.05g, 4.134mmol), potassium tert-butoxide (536mg, 4.785mmol) and IMesCuCl (5mg, 0.012mmol) were dissolved in THF in this order, cooled to 0 ℃ under nitrogen protection and stirred for 10min. Cyclopentylacetylene (300mg, 3.191mmol) and CH were added 3 I (1.359g, 9.574mmol) and stirred at room temperature for 12h. TLC showed completion of the reaction, EA/saturated NH 4 And (3) distributing the Cl aqueous solution, separating an organic phase, drying by using anhydrous sodium sulfate, separating by using a flash silica gel chromatographic column, eluting by using PE/EA as a mobile phase, collecting a target product part, and evaporating to dryness to obtain 350mg of jelly, namely an intermediate INT 57-1.
INT 55-8(25mg，0.049mmol)、INT 57-1(34.7mg，0.147mmol)、Pd(dppf)Cl 2 (5.38mg，0.007mmol)、Cs 2 CO 3 (48mg, 0.147mmol), 1, 4-dioxane 10mL, and H 2 O3 mL was placed in a pressure tube, degassed with nitrogen for 3min, sealed, and heated at 100 ℃ for 2h. After cooling, diluting with ethyl acetate, filtering to remove insoluble substances, and evaporating the filtrate under reduced pressure to obtain a crude product. Then, the mixture was separated by flash silica gel chromatography, eluted with MeOH/DCM as a mobile phase, and the desired product fractions were collected and evaporated to dryness to obtain 25mg of a gum. The crude product was purified by preparative liquid chromatography, eluted with acetonitrile/water system as mobile phase, the target product fraction was collected and evaporated to dryness to give 18mg of a white solid, example E57. 1 H-NMR(500MHz,Chloroform-d)δ7.29–7.25(m,2H),7.13–6.99(m,2H),6.05(d,J＝1.6Hz,1H),5.94(s,1H),5.59(s,2H),4.20(q,J＝6.1Hz,2H),3.53(d,J＝5.3Hz,2H),3.51(s,3H),2.65–2.50(m,1H),1.92(d,J＝5.8Hz,2H),1.89(d,J＝1.3Hz,3H),1.87–1.78(m,2H),1.77–1.68(m,2H),1.67(s,2H),1.50–1.36(m,2H)；MS(ESI):m/z 456.2[M+H] + .
Example E58 (E) -7- (4-chlorobenzyl) -8- (2-cyclopentylprop-1-en-1-yl) -1- (3-hydroxypropyl) -3-methyl-3, 7-dihydro-1H-purine-2, 6-dione
INT 2-2(100mg，0.196mmol)、INT 57-1(115.6mg，0.49mmol)、Pd(dppf)Cl2(21.5mg，0.029mmol)、Cs 2 CO 3 (191mg, 0.588 mmol), 1, 4-dioxane 10mL, and H 2 O3 mL was placed in a pressure tube, degassed with nitrogen for 3min, sealed, and heated at 100 ℃ for 2h. After cooling, it was diluted with ethyl acetate and filteredRemoving insoluble substances, and evaporating the filtrate under reduced pressure to obtain crude product. Separating with flash silica gel chromatography column, eluting with MeOH/DCM as mobile phase, collecting target product, and evaporating to dryness to obtain 80mg of jelly, i.e. intermediate INT 58-2.
Intermediate INT 58-2 (80mg, 0.191mmol) was dissolved in 2mL DCM, then 0.4mL TFA was added, stirred at RT for 1h, TLC indicated complete reaction. The solvent was evaporated under reduced pressure and the residue was purified by preparative liquid chromatography using acetonitrile/water system as mobile phase, the target product fraction was collected and evaporated to dryness to obtain 50mg of white solid, example E58. 1 H-NMR(500MHz,Chloroform-d)δ7.31(d,J＝8.2Hz,2H),7.17(d,J＝8.3Hz,2H),6.10(s,1H),5.52(s,2H),4.20(h,J＝7.1,6.6Hz,2H),3.63(s,3H),3.56(t,J＝5.5Hz,2H),2.63(ddd,J＝17.0,9.6,7.4Hz,1H),2.30–2.12(m,3H),1.99–1.79(m,4H),1.78–1.56(m,4H),1.54–1.40(m,2H)；MS(ESI):m/z 457.2[M+H] + .
EXAMPLE E59 (E) -5- (4-chlorobenzyl) -6- (3, 3-dimethylbut-1-en-1-yl) -3- (3-hydroxypropyl) -1-methyl-1, 5-dihydro-2H-pyrrolo [3,2-d ] pyrimidine-2, 4 (3H) -dione
The synthesis of example E59 was performed as in E56, except that (E) - (4-chlorostyryl) boronic acid was replaced with pinacol trans- (3, 3-dimethylbuten-1-yl) boronic acid ester. 1 H NMR(500MHz,Chloroform-d)δ7.30(d,J＝8.0Hz,2H),7.12–7.06(m,2H),6.37(d,J＝16.0Hz,1H),6.19(d,J＝16.0Hz,1H),6.10(s,1H),5.67(s,2H),4.26–4.12(m,2H),3.53(d,J＝5.4Hz,2H),3.51(d,J＝2.2Hz,3H),1.91(p,J＝5.8Hz,2H),1.10(s,9H)；MS(ESI):m/z 430.2[M+H] + .
Example E60- (4-chlorobenzyl) -6- (3, 3-dimethylbut-1-yn-1-yl) -3- (3-hydroxypropyl) -1-methyl-1, 5-dihydro-2H-pyrrolo [3,2-d ] pyrimidine-2, 4 (3H) -dione
INT 55-8(40mg，0.094mmol)、PdCl 2 (Ph 3 P) 2 (9.9mg, 0.014mmol), cuI (5.36mg, 0.028mmol), triethylamine 3mL, and DMF 1mL were placed in a pressure resistant tube and degassed with nitrogen for 3min, then 3, 3-dimethyl-1-butyne (38.8mg, 0.471mmol) was added and degassed for 1min. The pressure tube was sealed and heated at 60 ℃ for 4h. After cooling, the mixture was diluted with ethyl acetate, the insoluble matter was removed by filtration, and the filtrate was evaporated to dryness under reduced pressure to give a crude product. Then, the mixture was separated by flash silica gel chromatography, eluted with MeOH/DCM as a mobile phase, and the desired product fractions were collected and evaporated to dryness to give 35mg of a gum. The crude product was purified by preparative liquid chromatography, eluted with acetonitrile/water system as mobile phase, the target product fraction was collected and evaporated to dryness to give 20mg of a white solid, example E60. 1 H-NMR(500MHz,Chloroform-d)δ7.30(t,J＝2.3Hz,4H),6.12(s,1H),5.61(s,2H),4.19(q,J＝6.1Hz,2H),3.52(t,J＝5.5Hz,2H),3.45(s,3H),1.90(p,J＝5.6Hz,2H),1.35(s,9H)；MS(ESI):m/z 428.2[M+H] + .
Example E61- (4-chlorobenzyl) -3- (3-hydroxypropyl) -1-methyl-6- (phenylethynyl) -1, 5-dihydro-2H-pyrrolo [3,2-d ] pyrimidine-2, 4 (3H) -dione
The synthesis of example E61 is as for E60 except that phenylacetylene is used as starting material instead of 3, 3-dimethyl-1-butyne. 1 H-NMR(500MHz,Chloroform-d)δ7.56–7.51(m,2H),7.47–7.40(m,3H),7.39–7.30(m,4H),6.29(s,1H),5.72(s,2H),4.30–4.14(m,2H),3.57–3.52(m,2H),3.50(s,3H),1.92(p,J＝5.6Hz,2H)；MS(ESI):m/z 448.1[M+H] + .
2. Pharmacological examples
Determination of TRPC4 and TRPC5 inhibitory Activity
HEK-293 cells expressing hTRPC4 or hTRPC5 were inoculated into PDL-coated black-walled-bottom-permeable 96-well plates, 2X 10 cells per well 4 Culturing the cells for 8 hr, discarding the original culture medium, adding 60 μ L of Fluo-4/AM dye with final concentration of 4 μ M, incubating at 37 deg.C for 60 min, rinsing with calcium flow detection buffer 5 times, and mixingThe cell plate is placed in a preheating chamber at 30 DEG C
(Molecular Devices, sunnyvale, calif., USA), excited at a wavelength of 488nm and fluorescent signals were recorded continuously at a sampling frequency of 1s over the range of 515-535 nm. After 60 seconds of recording, the signal acquisition is continued for 300s by adding the solvent control, test compound and positive inhibitor HC608 (final concentration of 100 nM), followed by the addition of the agonist Englerin A (EA) (final concentration of 0.3 nM) and continued for 600 seconds fluorescence signal. Trace plot fluorescence signal F/F 0 Wherein F is the fluorescence signal at different time points, F 0 The base fluorescence signal, i.e. the average of the fluorescence signals at the first 10 time points, is used. Dose-effect diagram is firstly expressed as F/F 0 With =1 as baseline, the area under the curve of the change in fluorescence intensity after addition of EA was calculated, and log [ Inhibitor ] was used in combination with the area under the curve and the log of the compound concentration]response-Variable slope computing IC 50 The value is obtained.
2. Determination of stability of liver microsomes
First, 0.1M Tris buffer, pH 7.4, was prepared, and then MgCl was prepared at a concentration of 100mM using this buffer 2 A solution and a coenzyme factor NADPH solution with a concentration of 10 mM. Test compounds were first formulated from DMSO into stock solutions, which were then diluted with water and 0.1% bsa to working concentrations as applicable. For the measurement, TRIS buffer (final concentration of microsomal protein 0.33 mg/mL) of liver microsomes and MgCl were added 2 The solution (final concentration: 5 mM), the test compound solution (final concentration: 1. Mu.M) and the NADPH solution (final concentration: 1 mM) were incubated at 37 ℃ and the reaction was stopped by adding methanol for 0, 7, 17, 30 and 60 minutes, respectively. The remaining concentration of the test compound was determined by LC/MS/MS method. Calculation of half-life: t is a unit of 1/2 ＝0.693/k e 。k e Is the slope (absolute value) of the linear regression line when plotted semilogarithmically. The remaining percentage of substrate was taken as a semilog and a semilog plot was made of reaction time.
3. Evaluation results of Activity and microsomal stability
TRPC4/5 inhibitory activity and liver microsome stability are shown in the following table:
note: -, not tested.
Compound C31 in the above table is compound 31 at pages 169, 256 and C297 is compound 297 at pages 210, 301 in patent application WO2014143799, which are both potent TRPC5 inhibitors. C31 is also known in the literature as HC-608 and Pico145 (PLoS ONE.2018,13, e0191225.). As can be seen from the assay results, many compounds represented by the general formula (I) of the present invention, including compound E37 structurally close to C297, unexpectedly exhibited stronger TRPC4 and TRPC5 inhibitory activities and faster liver microparticle metabolic rates than C31 and C297. Patent application WO2014143799 discloses the use of compound C31 in the treatment of neuropsychiatric diseases such as depression, anxiety and the like, and patent application US20220031706 discloses the use of compound C31 in the treatment of metabolic diseases such as diabetes, obesity and the like. The invention discloses application of a compound represented by a general formula (I) and represented by a compound E37 to treatment of skin diseases such as psoriasis. When used as a skin topical drug, the compound is absorbed transdermally to exert its pharmacological effect and then taken into the blood circulation. If the compound is cleared more rapidly by liver metabolism, it is predicted that systemic side effects can be avoided more effectively with better safety.
4. Evaluation of hepatorenal cytotoxicity of Compounds
4.1 isolation of mouse Primary hepatocytes and evaluation of toxicity
Male C57/BL-6 mice 2 months old were anesthetized with tribromoethanol, and were fixed on the bench with the abdomen facing up. First 100mL of Ca-free solution 2+ HBSS buffer (containing 5.5mM glucose and 2mM HEPES, pH = 7.4) was fed at a rate of 4-5mL/minPerfusing the liver with a solution containing 5mM Ca 2+ And 0.05% collagenase IV were perfused in the liver for 10min. The resulting cell suspension was filtered through two nylon meshes with a mesh size of 70 μm, and then centrifuged at 50 Xg for 2 minutes at 4 ℃ to collect cell masses. The cell pellet was resuspended in 50mL of solution, 12.5mL of which was taken and 10.8mL of Percoll solution and 1.2mL of 10-fold concentrated DMEM solution (containing 4.5g/l glucose) were added. The mixture was centrifuged at 500g for 5 minutes and the cell pellet was washed twice with DMEM solution. By morphological analysis, 95% of the isolated cells were judged as hepatocytes. Cell viability was between 85% and 95% as determined by trypan blue exclusion.
Isolated mouse primary hepatocytes were seeded in 96-well culture plates (1.5X 10) 4 Individual cells/well) were cultured in low sugar DMEM containing 10% fetal bovine serum and 100 units/ml penicillin and streptomycin. After 4 hours of culture, serum-free DMEM was replaced. Test compounds were pre-dissolved in DMSO to make 100mM stock solutions, which were then diluted to the indicated concentrations with serum-free DMEM. mu.L of the compound was added to the wells to give a final concentration of 1 or 10. Mu.M, and the incubation was continued for 24 hours. The medium was then removed and the cells were incubated with 0.5mg/mL MTT (in medium) for an additional 2 hours at 37 ℃. After removing the supernatant, 150. Mu.L of dimethyl sulfoxide was added to each well, and the absorbance at 570nm was measured.
4.2 HK-2 Kidney cell culture and toxicity evaluation
After recovery of commercially available HK-2 kidney cells, 96-well plate cultures were performed using DMEM/F12 complete medium containing penicillin/streptomycin, HEPES (4-hydroxyethylpiperazineethanesulfonic acid), and 10% fetal bovine serum, seeded at approximately 2.5X 10 per well 3 And (4) cells. After culturing for about 24 hours, the culture medium was removed, 100. Mu.L of a medium diluent of the test compound prepared in advance was added to the medium diluent so that the final concentration of the test compound was 1 or 10. Mu.M, and the culture was continued for 24 hours. The medium was then removed and the cells were incubated with 0.5mg/mL MTT (in medium) for an additional 2 hours at 37 ℃. After removing the supernatant, 150. Mu.L of dimethyl sulfoxide was added to each well, and the absorbance at 570nm was measured.
4.3 toxicity assay results
The results of the hepatorenal cytotoxicity assays for the compounds are shown in the following table:
note: -, cell viability is not reduced; +, cell viability decreased by 10-30%; + + +, cell viability decreased 30-60%; (ii) a + + + +, cell viability decreased 60-90%.
From the measurement results, compared with the compounds C31 and C297 disclosed in patent application WO2014143799, the compound represented by the general formula (I) in the invention has smaller influence on the viability of liver and kidney cells and shows lower cytotoxicity.
5. Effect of Compounds on keratinocyte proliferation
5.1 culture of the human immortalized keratinocyte cell line HaCaT
HaCaT cells cultured in DMEM medium containing 10% FBS, 10mM HEPES and 100U/mL penicillin/streptomycin at 37 ℃ 5% CO 2 And cultured in 95% humidity. When the cells grow to 70-80% confluence, discarding the old culture medium, rinsing with PBS for 2 times, adding pancreatin-EDTA, digesting at 37 ℃ for 5-7 minutes, when the cells become round and the cell gaps become larger, adding DMEM containing FBS to stop digestion, blowing the cells with a pipette, collecting the cell suspension, centrifuging at 800rpm for 5 minutes, discarding the supernatant, resuspending the cells with 10% FBS of DMEM, taking a part of the cell suspension, transferring into a new cell bottle, and continuing culturing. Or after adjusting the cell concentration with DMEM containing 2% FBS, the cells were plated on a cell plate and used for the next experiment.
5.2 BrdU incorporation experiments
Adding 10 μ MBrdU (5-bromodeoxyuridine) into cell culture environment, culturing at 37 deg.C for 3 hr, removing the solution in the wells, and rinsing with PBS for 3 times; fixing the cells with 4% paraformaldehyde for 30 minutes, and rinsing with PBS for 3 times; 0.1% TritonX-100 cells were treated for 15 minutes and rinsed 3 times with PBS; then, the mixture was treated with 2M HCl for 30 minutes, neutralized with 0.1M boric acid for 10 minutes, rinsed with PBS for 3 times, and then blocked with 5% BSA dropwise at room temperature for 30 minutes; after discarding the liquid, 1. The next day, primary antibody is discarded, and PBS is used for rinsing for 5 times; dripping 1; under the condition of keeping out of the light, 1. Under an inverted fluorescence microscope, 5-6 fields were randomly selected for observation and photographing.
5.3 results of the experiment
As shown in fig. 1, the proliferating HaCaT cells contained BrdU, so the positive nuclei exhibited red fluorescence (as indicated by white triangles); while Hoechst dye stains all nuclei blue. The number of positive cells in HaCaT cells in the Veh group accounts for 1.7 +/-1.2% of the total number of cells, and the ratio of the proliferated HaCaT cells is increased to 5.8 +/-1.0% after being stimulated by 100ng/mL IL-17 for 12 hours; haCaT cells were treated alone at 100nM E37 for 12 hours, with a cell proliferation rate of 2.0 + -1.4%, with insignificant differences (P > 0.05) relative to the Veh group; haCaT cells treated by 100nM E37 and 100ng/mL IL-17 for 12 hours have a cell proliferation rate of 2.7 + -0.6%, which is significantly lower than that of IL-17 treated group (P < 0.01).
6. Evaluation of efficacy of compound in treating psoriasis
6.1 pharmaceutical cream formulation Process
The pharmaceutical cream formulations are shown in the following table:
preparation of an aqueous phase: weighing ethanol according to the prescription amount, adding E37, stirring for dissolving, sequentially adding Tween 80, sodium dodecyl sulfate, glycerol, isopropyl myristate, methyl hydroxybenzoate, azone and purified water into a beaker, and stirring until the mixture is uniformly dispersed.
Preparing an oil phase: weighing the glyceryl monostearate, the palmitic acid and the white vaseline with the prescription amount in a beaker, and heating and dissolving the mixture in a water bath at the temperature of 80 ℃ until the mixture is liquid.
Slowly adding the water phase into the oil phase under stirring, taking out from the water bath, and stirring at room temperature until cooling to obtain cream.
6.2 Imiquemode-induced psoriasis model in mice
24C 57BL/6 mice, each half male and female, purchased from the laboratory animal center of Nanjing university of medical science, weighing 22-23g. The mice were shaved of their back hair and were divided into 4 groups by sex: control group (Vas), modeling group (IMQ), modeling group + mometasone furoate group (IMQ + Mome), and modeling group + E37 cream group (IMQ + E37), each group containing 6 animals. Before the experiment, the mice are placed in a quiet environment and a light-proof environment for feeding for 3 days to adapt to the environment, the time ratio of illumination to darkness is 1, the room temperature is controlled to be 25 ℃, the humidity is 55%, and the mice can freely eat and drink water.
62.5mg of 5% (0.1g. After 6 hours of application of IMQ or Vas each day, 100mg of positive drug Mometasone furoate cream (Mometasone, mome, shanghai zhengda general pharmaceutical products gmbh) or E37 cream was applied, and the Vas group was administered with the same dose of Vas. The skin of the back of the mice was recorded by photographing on the day 1,3,5 of molding, and the appearance of the skin on the day 5 of molding is shown in fig. 2.
6.3 evaluation of clinical symptoms
The clinical symptoms of the Psoriasis model of the mice are evaluated from 3 indexes of erythema, scale and infiltration thickening by adopting a Psoriasis Area and Severity Index (PASI) scoring standard, scoring is carried out according to 0-4 minutes, and the integrals of the 3 indexes are added to obtain a total integral. PASI scoring criteria are as follows: 0 point, no symptom; 1 minute, light; 2 points, medium; 3 points, severe; 4 points, extremely severe. Single-blind evaluations and photographic recordings were performed daily by trained experimenters prior to application of the IMQ cream. The scoring results are shown in the following table: the measured data are expressed as mean ± standard deviation (mean ± SEM), and measured by two-way anova, in which analysis results, ap<0.01, relative to the Vas group; # P<0.05， ## P<0.01, relative to the IMQ group.
6.4 histopathological examination
Skin tissues of the drug administration area on the back of the mouse were collected, fixed in 4% (mass fraction) paraformaldehyde solution for 48 hours, embedded in paraffin and sectioned at 3 μm for Hematoxylin-Eosin (H)&E) And (6) dyeing. The thickness of the epidermis layer of the skin was measured using NIS-element BR software by observing and photographing using an Eclipse Ti type inverted microscope at a magnification of 100, and 3 fields of view were randomly selected for each section. Note: the measured data are expressed as mean + -standard deviation (mean + -SEM) and measured using one-way anova, where P is the measured result<0.01, relative to the Vas group; ## P<0.01, relative to the IMQ group.
6.5 results of the experiment
6.5.1E37 cream Effect on Back skin appearance and PASI score in psoriasis mice
Throughout the experiment, the back skin of Vas mice was consistently smooth and flat without erythema, scaling, thickening, etc. (fig. 2). The back skin of IMQ group mice has obvious psoriasis-like symptoms such as erythema, scale, thickening and the like on the 3 rd day of model building, the symptoms are obviously increased along with the model building (figure 2), and the erythema, scale (shown as black triangles) and thickening are the most serious on the 5 th day; their PASI scores showed a gradual increase in PASI scores in IMQ group mice, and the IMQ group was significantly different from the Vas group starting on day 3 (P < 0.01) (table 1). The smearing of the positive medicament Mome can improve the appearance of psoriasis-like symptoms such as erythema, scale, thickening and the like caused by IMQ (figure 2); PASI scores showed a slow increase in PASI scores for IMQ + Mome group mice, with statistically significant (P < 0.01) scores for IMQ + Mome group PASI significantly lower than IMQ group starting on day 4 (table 1). The application of the E37 cream can slightly improve erythema, scales and thickened psoriasis-like symptoms of the back skin of the mice (figure 2), and the erythema, scales (shown by black triangles) and thickened symptoms of the IMQ + E37 group are reduced to some extent compared with the IMQ group at the 5 th day (figure 2), but the improvement effect is weaker than that of the positive medicine Mome; PASI scores showed a slow increase in PASI scores for IMQ + E37 group mice, with PASI scores for IMQ + E37 group being lower than IMQ group by day 4, statistically significant (P < 0.05) (table 1).
TABLE 1 dorsal skin PASI score in groups of mice
6.5.2 Effect of E37 cream on Back skin pathology in psoriasis mice
As shown in fig. 3, the tissue structure of the back skin of Vas mice was not significantly abnormal, the horny layer was completely keratinized, the boundary between the epidermis layer and the dermis layer was clear, and the infiltration of inflammatory cells was less; the thickness of the epidermis layer was measured to be 25.72. + -. 1.19. Mu.m. IMQ group mice had excessive thickening of the epidermal layer of the dorsal skin tissue (as indicated by white lines), keratosis of the stratum corneum with visible accumulation of inflammatory cells in the stratum corneum (as indicated by black arrows), edema of the dermis with massive inflammatory infiltration (as indicated by black triangles); the thickness of the epidermal layer is 108.48 +/-3.51 mu m, and is obviously increased compared with the Vas group (P < 0.01). The thickness of the epidermal layer of the dorsal skin tissue of mice in the IMQ + Mome group is 57.71 +/-2.66 mu m, the thickness is obviously reduced compared with that of the IMQ group (P is less than 0.01), no edema is seen in the dermal layer, and the infiltration degree of inflammatory cells is reduced compared with that of the IMQ group. The stratum corneum structure of the dorsal skin tissue of mice in the IMQ + E37 group was slightly restored, the thickness of the epidermal layer was 85.38 +/-3.61 μm, which was reduced compared with the IMQ group, and was statistically significant (P < 0.01), and the edema of the dermal layer was reduced compared with the IMQ group, but inflammatory cell infiltration was still observed (as shown by black triangles).
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention and not to limit it; although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; without departing from the spirit of the present invention, it is intended to cover all aspects of the invention as defined by the appended claims.
Claims (10)
1. Condensed pyrimidinedione compounds and pharmaceutically acceptable salts thereof are characterized in that the structural formula of the compounds is shown as formula (I),
wherein, the first and the second end of the pipe are connected with each other,
R 1 is C 1 -C 6 Alkyl groups of (a);
each R is 2 Independently a halogen atom;
R 3 selected from substituted or unsubstituted C 1 -C 6 Alkyl, substituted or unsubstituted C 3 -C 10 A substituted or unsubstituted 6-to 10-membered aryl group and a substituted or unsubstituted 5-to 8-membered heteroaryl group, wherein R 3 The substituent(s) is 1 to 3 groups selected from the group consisting of: c 1 -C 6 Alkyl of (C) 3 -C 8 Cycloalkyl of (ii), -OR 4 、-N(R 4 ) 2 And a halogen atom;
each R 4 Each independently selected from H, substituted or unsubstituted C 1 -C 6 Alkyl, substituted or unsubstituted C 3 -C 8 A cycloalkyl group, a substituted or unsubstituted phenyl group of (a), wherein R 4 The substituents of (a) are 1 to 3 groups of the following group: c 3 -C 8 Cycloalkyl, halogen atom, hydroxyl and = O; alternatively, the first and second liquid crystal display panels may be,
two R 4 Taken together to form a 4-to 8-membered substituted or unsubstituted heterocyclic ring, the heteroatoms of which are selected from N, O, and S and contain at least 1N, the number of heteroatoms being 1 or 2; the substituents of the heterocycle are 1-3 groups independently selected from the group consisting of: c 1 -C 6 Alkyl groups and halogen atoms of (a);
n is an integer of 1 to 3;
x is N or CH;
y is selected from one of ethynyl and substituted or unsubstituted ethenyl, the stituent of the ethenyl is 1-2C 1 -C 6 An alkyl group.
2. The compound and pharmaceutically acceptable salts thereof according to claim 1, wherein R is 1 Is methyl, ethyl or isopropyl.
3. The compound and pharmaceutically acceptable salts thereof according to claim 1, wherein R is 3 Is C 3 -C 10 Cycloalkyl OR-OR of 4 、-N(R 4 ) 2 Substituted C 1 -C 6 Alkyl group of (1).
4. The compound of claim 1, and pharmaceutically acceptable salts thereof, wherein the compound is selected from the group consisting of structural formulas (IIa) - (IIc):
wherein the content of the first and second substances,
R 1 is methyl, ethyl or isopropyl;
each R 2 Each independently is a halogen atom;
ring C is substituted or unsubstituted C 3 -C 10 Substituted or unsubstituted phenyl or substituted or unsubstituted 5-to 8-membered heteroaryl, wherein the substituents of ring C are 1 to 3 groups selected from: c 1 -C 6 Alkyl groups and halogen atoms of (b);
R 4 each independently selected from substituted or unsubstituted C 1 -C 6 Alkyl, substituted or unsubstituted C 3 -C 8 A cycloalkyl group, a substituted or unsubstituted phenyl group of (a), wherein R 4 The substituent(s) is 1 to 3 groups selected from the group consisting of: c 3 -C 8 Cycloalkyl group and halogen atom of (b); alternatively, the first and second electrodes may be,
two R 4 Together with N to form a 4-to 8-membered substituted or unsubstituted heterocycle having 1 to 3C substituents 1 -C 6 Alkyl groups of (a);
R 5 、R 6 each independently is H or C 1 -C 3 Alkyl groups of (a);
n is an integer of 1 to 3.
5. The compound and pharmaceutically acceptable salts thereof according to claim 1, wherein the compound is selected from the group consisting of:
6. use of a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, for use as a TRPC4 and TRPC5 inhibitor or for the manufacture of a medicament for the treatment of a disease associated with abnormal TRPC4 or/and TRPC5 function, expression.
7. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
8. The pharmaceutical composition according to claim 7, wherein the pharmaceutical composition is for the treatment of a skin disorder associated with keratinocyte cell abnormalities.
9. The pharmaceutical composition of claim 8, wherein the disease is psoriasis, ichthyosis, keratopalmoplantar, olmsted, cutis Bufonis, or menopausal keratoderma.
10. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition is for topical application to the skin.
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| CN101631788A (en) * | 2006-12-22 | 2010-01-20 | 施瓦茨制药有限公司 | 8-alkynyl xanthine and derivative |
| WO2014152287A2 (en) * | 2013-03-14 | 2014-09-25 | Hydra Biosciences, Inc. | Substituted xanthines and methods of use thereof |
| CN105143229A (en) * | 2013-03-15 | 2015-12-09 | 海德拉生物科学有限公司 | Substituted xanthines and methods of use thereof |
| CN112771032A (en) * | 2018-10-29 | 2021-05-07 | 江苏先声药业有限公司 | Pyrimidine pyrazole compounds as fourth generation EGFR inhibitors |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101631788A (en) * | 2006-12-22 | 2010-01-20 | 施瓦茨制药有限公司 | 8-alkynyl xanthine and derivative |
| WO2014152287A2 (en) * | 2013-03-14 | 2014-09-25 | Hydra Biosciences, Inc. | Substituted xanthines and methods of use thereof |
| CN105143229A (en) * | 2013-03-15 | 2015-12-09 | 海德拉生物科学有限公司 | Substituted xanthines and methods of use thereof |
| CN112771032A (en) * | 2018-10-29 | 2021-05-07 | 江苏先声药业有限公司 | Pyrimidine pyrazole compounds as fourth generation EGFR inhibitors |
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