CN108948016A - Purine ketones derivant, preparation method and its application in medicine - Google Patents

Abstract

The present invention relates to purine ketones derivant, preparation method and its applications in medicine.Specifically, the present invention relates to purine ketones derivant new shown in a kind of logical formula (I), preparation method and pharmaceutical compositions as well as therapeutic agent containing the derivative, especially as the purposes of TLR7 agonist, each substituent group of formula of (I) is identical as the definition in specification.

Description

Purine ketones derivant, preparation method and its application in medicine

Technical field

The invention belongs to field of medicaments, it is related to purine ketones derivant new shown in a kind of logical formula (I), preparation method And pharmaceutical composition as well as therapeutic agent containing the derivative, especially as the purposes of TLR7 agonist.

Background technique

Toll-like receptor (toll-like receptors；It TLRs) is a kind of key protein point for participating in congenital immunity Son.TLRs is the non-catalytic receptor of monomer cross-film, is usually expressed in sentry post cell such as macrophage and Dendritic Cells, can To identify the molecule of the structural conservation generated by microorganism.Once these microorganisms break through the physics screen such as skin or intestinal mucosa Barrier, will be identified, then immune cell activated response (Mahla, R S. et al., Front Immunol.4:248 by TLRs (2013)).Why immune system has the ability of identification pathogenic microorganism extensively, is since Toll-like is immune in a way Receptor is widely present.

At least 10 kinds of different TLRs in mammals.The ligand of this some receptoroid and corresponding signal cascade are put It is accredited out greatly.TLR7 is the member of TLRs (TLRs 3,7,8 and 9) subgroup, is confined to the thin of the non-own nucleic acid of special detection The endosome compartment of born of the same parents.TLR7 by identification ssRNA antiviral defense in terms of play a crucial role (Diebold S.S. etc., Science,2004:303,1529-1531；With Lund J.M. etc., PNAS, 2004:101,5598-5603).TLR7 is in the person It is upper that there is limited expression and distribution, and mainly expressed by B cell and plasmacytoid dendritic cells (pDC), and lower degree Pass through monocytes.Plasmacytoid DCs is unique group (peripheral blood of 0.2-0.8% of dendritic cells derived from lymph Monocyte (PBMCs)), it is in response to virus infection and secreting high levels interferon-' alpha ' (IFN α) and interferon-beta (IFN β) Initial I type interferon cellulation (Liu Y-J, Annu.Rev.Immunol., 2005:23,275-306).

Many diseases, obstacle are related with the exception of TLRs, such as melanoma, non-small cell lung cancer, hepatocellular carcinoma, substrate Cell cancer (basalcellcarcinoma), clear-cell carcinoma, myeloma, allergic rhinitis, asthma, chronic obstructive pneumonia (COPD), ulcerative colitis, liver fibrosis, HBV, flaviviridae (Flaviviridae) virus, HCV, HPV, RSV, SARS, HIV or the virus infection of influenza etc..It therefore is very promising with the agonist treatment related disease of TLRs.

Due to TLR7 and TLR8 very high homology, TLR7 ligand, in most cases and TLR8 ligand.TLR8 thorn Swash main induction and generates cell factor such as tumor necrosis factor α (TNF-α) and chemotactic factor (CF).Interferon-' alpha ' is treatment chronic type b One of hepatitis or the key agents of hepatitis C, and TNF-α is a kind of proinflammatory cytokine, excessive secretion may cause serious Side effect.So to the selectivity of TLR7 and TLR8 for exploitation TLR7 agonist for treating disease of viral infection to closing weight It wants.

Have relevant TLR7 agonist patent application at present, as WO2005025583, WO2007093901, WO2008011406、WO2009091032、WO2010077613、WO2010133882、WO2011031965、WO2012080730 And WO2010018133.But it still needs to further research and develop safer and more effective TLR7 agonist.

In view of the above technical problems, it is low to provide a kind of action concentration by the present invention, good, the apparent purine of activation effect of selectivity Ketone medical compounds.

Summary of the invention

The purpose of the present invention is to provide a kind of logical formula (I) compounds represented:

Or its tautomer, mesomer, racemic modification, enantiomter, diastereoisomer, or mixtures thereof shape Formula or its pharmaceutical salt,

Wherein:

For cis or trans；

L¹Selected from-O- ,-C (O)-,-S (O)_t-、-NR⁶-、-N(R⁶)C(O)-、-C(O)N(R⁶)-、-N(R⁶)S(O)₂And-S (O)₂N(R⁶)-；

R¹Selected from hydrogen atom, alkyl, halogenated alkyl, alkenyl, alkynyl, naphthenic base, heterocycle, aryl and heteroaryl, wherein Alkyl, alkenyl, alkynyl, naphthenic base, heterocycle, aryl and the heteroaryl is optionally selected from alkyl, alcoxyl each independently One in base, halogen, halogenated alkyl, hydroxyl, hydroxyalkyl, cyano, amino, nitro, naphthenic base, heterocycle, aryl and heteroaryl Replaced a or multiple substituent groups；

R²Selected from hydrogen atom, halogen, alkyl, alkoxy, halogenated alkyl, hydroxyl, hydroxyalkyl, cyano, amino, nitro, ring Alkyl, heterocycle, aryl and heteroaryl, wherein alkyl, naphthenic base, heterocycle, aryl and the heteroaryl are each independently Optionally by selected from alkyl, alkoxy, halogen, halogenated alkyl, hydroxyl, hydroxyalkyl, cyano, amino, nitro, naphthenic base, heterocycle, Replaced one or more substituent groups in aryl and heteroaryl；

R³Selected from alkyl, halogenated alkyl, hydroxyalkyl, naphthenic base, heterocycle, aryl and heteroaryl, wherein the alkyl, Naphthenic base, heterocycle, aryl and heteroaryl each independently optionally by selected from alkyl, alkoxy, halogen, halogenated alkyl, hydroxyl, Hydroxyalkyl, cyano, amino, nitro, naphthenic base, heterocycle, aryl, heteroaryl ,-NR⁷R⁸、-C(O)NR⁷R⁸、-C(O)R⁹、-C(O) OR⁹With-S (O)_tR⁹In one or more substituent groups replaced；

R⁴And R⁵It is identical or different, and it is each independently selected from hydrogen atom, halogen, alkyl, alkoxy, halogenated alkyl, hydroxyl Base, hydroxyalkyl, cyano, amino, nitro, naphthenic base, heterocycle, aryl and heteroaryl；

R⁶Selected from hydrogen atom, alkyl, halogenated alkyl, naphthenic base, heterocycle, aryl and heteroaryl；

R⁷And R⁸It is identical or different, and it is each independently selected from hydrogen atom, alkyl, halogenated alkyl, naphthenic base, heterocycle, virtue Base and heteroaryl, wherein alkyl, naphthenic base, heterocycle, aryl and the heteroaryl each independently optionally by selected from alkyl, One or more in alkoxy, halogen, amino, cyano, nitro, hydroxyl, hydroxyalkyl, naphthenic base, heterocycle, aryl and heteroaryl Replaced a substituent group；

Alternatively, the R⁷And R⁸It is formed together heterocycle with the nitrogen-atoms being connected, wherein the heterocycle, which removes, contains 1 Except a nitrogen-atoms, also optionally containing 1~2 identical or different hetero atom selected from N, O and S, and the heterocycle Optionally by selected from alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxyl, hydroxyalkyl, naphthenic base, heterocycle, aryl and miscellaneous Replaced one or more substituent groups in aryl；

R⁹Selected from hydrogen atom, alkyl, halogenated alkyl, amino, hydroxyl, hydroxyalkyl, naphthenic base, heterocycle, aryl and heteroaryl Base；And

T is 0,1 or 2.

In a preferred embodiment of the present invention, the logical formula (I) compound represented is shown in logical formula (II) Compound:

Or its tautomer, mesomer, racemic modification, enantiomter, diastereoisomer, or mixtures thereof shape Formula or its pharmaceutical salt,

Wherein:

Alk is alkylidene, is optionally selected from alkyl, alkoxy, halogen, halogenated alkyl, hydroxyl, hydroxyalkyl, cyano, ammonia Replaced one or more substituent groups in base, nitro, naphthenic base, heterocycle, aryl and heteroaryl；

R⁷And R⁸It is identical or different, and it is each independently selected from hydrogen atom, alkyl, halogenated alkyl, naphthenic base, heterocycle, virtue Base and heteroaryl, wherein alkyl, naphthenic base, heterocycle, aryl and the heteroaryl each independently optionally by selected from alkyl, One or more in alkoxy, halogen, amino, cyano, nitro, hydroxyl, hydroxyalkyl, naphthenic base, heterocycle, aryl and heteroaryl Replaced a substituent group；

Alternatively, the R⁷And R⁸It is formed together heterocycle with the nitrogen-atoms being connected, wherein the heterocycle, which removes, contains 1 Except a nitrogen-atoms, also optionally containing 1~2 identical or different hetero atom selected from N, O and S, and the heterocycle Optionally by selected from alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxyl, hydroxyalkyl, naphthenic base, heterocycle, aryl and miscellaneous Replaced one or more substituent groups in aryl；

L¹、R¹、R²、R⁴And R⁵As defined in logical formula (I).

In a preferred embodiment of the present invention, the logical formula (I) compound represented is shown in logical formula (III) Compound:

Or its tautomer, mesomer, racemic modification, enantiomter, diastereoisomer, or mixtures thereof shape Formula or its pharmaceutical salt,

Wherein:

For cis or trans；

R⁷And R⁸Be formed together heterocycle with the nitrogen-atoms being connected, wherein the heterocycle remove containing 1 nitrogen-atoms it Outside, also optionally containing 1~2 identical or different hetero atom selected from N, O and S, and the heterocycle is optionally selected from One in alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxyl, hydroxyalkyl, naphthenic base, heterocycle, aryl and heteroaryl Replaced a or multiple substituent groups；

The integer that n is 1 to 6；

L¹、R¹、R²、R⁴And R⁵As defined in logical formula (I).

In a preferred embodiment of the present invention, the logical formula (I) compound represented, wherein the R⁴With R⁵It is hydrogen atom.

In a preferred embodiment of the present invention, the logical formula (I) compound represented, wherein the L¹ For-O-.

In a preferred embodiment of the present invention, the logical formula (I) compound represented is shown in logical formula (IV) Compound:

Or its tautomer, mesomer, racemic modification, enantiomter, diastereoisomer, or mixtures thereof shape Formula or its pharmaceutical salt,

Wherein:

R¹⁰Selected from alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxyl, hydroxyalkyl, naphthenic base, heterocycle, aryl And heteroaryl；

Q is 0,1,2 or 3, and preferably q is 0；

M is 0,1,2 or 3, preferably 1 or 2；

The integer that n is 1 to 6；

R¹And R²As defined in logical formula (I).

In a preferred embodiment of the present invention, the logical formula (I) compound represented, to lead to formula (V) institute The compound shown:

Or its tautomer, mesomer, racemic modification, enantiomter, diastereoisomer, or mixtures thereof shape Formula or its pharmaceutical salt,

Wherein:

R¹⁰Selected from alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxyl, hydroxyalkyl, naphthenic base, heterocycle, aryl And heteroaryl；

Q is 0,1,2 or 3, and preferably q is 0；

M is 0,1,2 or 3, preferably 1 or 2；

The integer that n is 1 to 6；

R¹And R²As defined in logical formula (I).

In a preferred embodiment of the present invention, the logical formula (II) compound represented, wherein the n is 2 or 3.

In a preferred embodiment of the present invention, the logical formula (I) compound represented, wherein the R¹For Alkyl.

In a preferred embodiment of the present invention, the logical formula (I) compound represented, wherein the R²For Alkyl, preferably methyl or ethyl.

Typical compound of the invention includes but is not limited to:

Or its tautomer, mesomer, racemic modification, enantiomter, diastereoisomer, or mixtures thereof shape Formula or its pharmaceutical salt.

In a preferred embodiment of the present invention, a kind of general formula (IA) compound represented, to prepare logical formula (I) The intermediate of compound:

Or its tautomer, mesomer, racemic modification, enantiomter, diastereoisomer, or mixtures thereof shape Formula or its pharmaceutical salt,

Wherein:

L¹、R¹、R²、R⁴And R⁵As defined in logical formula (I).

General formula (IA) compound represented includes, but are not limited to:

Another aspect of the present invention relates to a kind of methods for preparing logical formula (I) compound represented, this method comprises:

The compound and general formula (IB) compound of general formula (IA) react to obtain the compound of logical formula (I)；

Wherein:

R¹~R⁵And L¹As defined in logical formula (I).

Another aspect of the present invention relates to a kind of methods for preparing logical formula (II) compound represented, this method comprises:

The compound and general formula (IB) compound of general formula (IA) react to obtain the compound of logical formula (I)；

Wherein:

L¹、R¹、R²、R⁴、R⁵、R⁷、R⁸With n as defined in logical formula (II).

Another aspect of the present invention relates to a kind of pharmaceutical composition, described pharmaceutical composition contains the general formula of therapeutically effective amount (I) compound represented or its tautomer, mesomer, racemic modification, enantiomter, diastereoisomer or its Form of mixtures or its pharmaceutical salt and one or more pharmaceutically acceptable carriers, diluent or excipient.

The invention further relates to compound shown in logical formula (I) or its tautomers, mesomer, racemic modification, right Reflect isomers, diastereoisomer, or mixtures thereof form or its pharmaceutical salt or prepared comprising its pharmaceutical composition For the purposes in the drug of exciting TLR7.

The invention further relates to compound shown in logical formula (I) or its tautomers, mesomer, racemic modification, right Reflect isomers, diastereoisomer, or mixtures thereof form or its pharmaceutical salt or prepared comprising its pharmaceutical composition The purposes in drug for treating infection caused by virus.

The invention further relates to compound shown in logical formula (I) or its tautomers, mesomer, racemic modification, right Reflect isomers, diastereoisomer, or mixtures thereof form or its pharmaceutical salt or prepared comprising its pharmaceutical composition The purposes in drug for treating or preventing tumour.

The invention further relates to the methods of excitement TLR7 a kind of comprising by logical formula (I) compound represented or its mutually Tautomeric, mesomer, racemic modification, enantiomter, diastereoisomer, or mixtures thereof form or its is pharmaceutically acceptable The step of salt or the pharmaceutical composition for including it and TLR7 are contacted.

The invention further relates to a kind of methods for treating infection caused by virus, and the method includes giving required trouble The logical formula (I) compound represented of person's therapeutically effective amount or its tautomer, mesomer, racemic modification, enantiomter, Diastereoisomer, or mixtures thereof form or its officinal salt or the pharmaceutical composition comprising it.

The invention further relates to a kind of methods for treating or preventing tumour comprising gives required bacterium Logical formula (I) compound represented or its tautomer, mesomer, racemic modification, enantiomter, diastereo-isomerism Body, or mixtures thereof form or its officinal salt or the pharmaceutical composition comprising it.

The invention further relates to a kind of logical formula (I) compound represented or its tautomer, mesomer, racemics Body, enantiomter, diastereoisomer, or mixtures thereof form or its officinal salt or the drug comprising it, be used as medicine Object.

The invention further relates to a kind of logical formula (I) compound represented or its tautomer, mesomer, racemics Body, enantiomter, diastereoisomer, or mixtures thereof form or its officinal salt or the drug comprising it, be used as TLR7 agonist.

The invention further relates to a kind of logical formula (I) compound represented or its tautomer, mesomer, racemics Body, enantiomter, diastereoisomer, or mixtures thereof form or its officinal salt or the drug comprising it, be used to control Treat or prevent infection caused by virus.

The invention further relates to a kind of logical formula (I) compound represented or its tautomer, mesomer, racemics Body, enantiomter, diastereoisomer, or mixtures thereof form or its officinal salt or the drug comprising it, be used to control It treats or tumour is selected in prevention.

Heretofore described virus be preferably selected from dengue fever virus, yellow fever virus, west nile virus, japanese encephalitis virus, Tick-brone encephalitis virus, elder brother's saliva virus, Murray valley encephalitis virus, St. Louis encephalitis virus, msk haemorrhagia fever virus, cattle disease Viral diarrhea virus, zika virus, HIV, HBV, HCV, HPV, RSV, SARS and influenza virus, are more preferably selected from HBV and HCV.

Heretofore described tumour is preferably selected from melanoma, non-small cell lung cancer, hepatocellular carcinoma, basal-cell carcinoma, kidney Cell cancer and myeloma.

Pharmaceutical composition containing active constituent, which can be, is suitable for oral form, such as tablet, dragee, pastille, water Or oil suspension, dispersible powder or particle, lotion, hard or soft capsule or syrup or elixir.It can be any according to this field Know that the method for preparing Pharmaceutical composition prepares Orally administered composition, such composition can containing it is one or more it is selected from the following at Point: sweetener, corrigent, colorant and preservative, to provide pleasing and palatable pharmaceutical formulation.Tablet contain active constituent and The suitable nontoxic pharmaceutical excipient for preparing tablet for mixing.

Water slurry contains active material and the suitable excipient for preparing water slurry for mixing.Aqueous suspension can also To contain one or more preservatives, one or more colorants, one or more corrigents and one or more sweeteners.

Oil suspension can be formulated by being suspended in active constituent in vegetable oil.Oil suspension can contain thickener. Above-mentioned sweetener and corrigent can be added, to provide palatable preparation.

Pharmaceutical composition of the invention is also possible to the form of oil in water emulsion.

Pharmaceutical composition can be sterile injectable aqueous form.The acceptable solvent or solvent that can be used have water, Ringer's solution and isotonic sodium chlorrde solution.Aseptic injection preparation can be the aseptic injection water packet that wherein active constituent is dissolved in oily phase Oil microemulsion.Such as active constituent is dissolved in the mixture of soybean oil and lecithin.Then water and glycerol is added in oil solution Processing forms micro emulsion in mixture.Can be by a large amount of injections in part, it will be in injection or the blood flow of micro emulsion injection patient.Alternatively, Preferably solution and micro emulsion are given in the way of it can keep the compounds of this invention constant circulating concentration.To keep this constant density, Continuous intravenous delivery device can be used.The example of this device is the intravenous injection of Deltec CADD-PLUS.TM.5400 type Pump.

Pharmaceutical composition can be for intramuscular and the aseptic injection water of subcutaneous administration or the form of oil suspension.It can be by Know technology, the dispersing agent or wetting agent and suspending agent for being suitable for those described above prepare the suspension.Aseptic injection preparation can also be with It is the aseptic injectable solution or suspension prepared in the acceptable non-toxic diluent of parenteral or solvent.Furthermore, it is convenient to Use sterile fixed oil as solvent or suspension media.

The compounds of this invention can be given by the suppository form for rectally.It can be by by drug and at normal temperatures For solid but in the rectum it is liquid, thus can dissolves and discharge the suitable nonirritant excipient mixing of drug in the rectum To prepare these pharmaceutical compositions.Substance of this kind includes the poly- second of cocoa butter, glycerin gelatine, hydrogenated vegetable oil, various molecular weight The mixture of the aliphatic ester of two pure and mild polyethylene glycol.

As it is well known to the skilled in the art, the dosage of drug depends on many factors, including but and non-limiting In following factor: the activity of particular compound used, the age of patient, the weight of patient, the health status of patient, patient row For, the diet of patient, administration time, administration mode, the rate of excretion, the combination of drug etc.；In addition, optimal therapeutic modality is such as The type of the mode for the treatment of, the consumption per day of general formula compound (I) or pharmaceutical salt can be tested according to traditional therapeutic scheme Card.

Detailed description of the invention

Unless stated to the contrary, the term used in the specification and in the claims has following meanings.

Term " alkyl " refers to saturated aliphatic hydrocarbons group, is the linear chain or branched chain group comprising 1 to 20 carbon atom, excellent Select the alkyl containing 1 to 12 carbon atom, the alkyl of further preferably 1 to 6 carbon atom.Non-limiting example include methyl, Ethyl, n-propyl, isopropyl, normal-butyl, isobutyl group, tert-butyl, sec-butyl, n-pentyl, 1,1- dimethyl propyl, 1,2- diformazan Base propyl, 2,2- dimethyl propyl, 1- ethyl propyl, 2- methyl butyl, 3- methyl butyl, n-hexyl, 1- Ethyl-2-Methyl third Base, 1,1,2- thmethylpropyl, 1,1- dimethylbutyl, 1,2- dimethylbutyl, 2,2- dimethylbutyl, 1,3- dimethyl butyrate Base, 2- ethyl-butyl, 2- methyl amyl, 3- methyl amyl, 4- methyl amyl, 2,3- dimethylbutyl, n-heptyl, 2- methyl oneself Base, 3- methylhexyl, 4- methylhexyl, 5- methylhexyl, 2,3- dimethyl amyl group, 2,4- dimethyl amyl group, 2,2- dimethyl Amyl, 3,3- dimethyl amyl group, 2- ethylpentyl, 3- ethylpentyl, n-octyl, 2,3- dimethylhexanyl, 2,4- dimethyl oneself Base, 2,5- dimethylhexanyl, 2,2- dimethylhexanyl, 3,3- dimethylhexanyl, 4,4- dimethylhexanyl, 2- ethylhexyl, 3- Ethylhexyl, 4- ethylhexyl, 2- methyl -2- ethylpentyl, 2- methyl -3- ethylpentyl, n-nonyl, 2- methyl -2- ethyl Hexyl, 2- methyl -3- ethylhexyl, 2,2- diethyl amyl group, positive decyl, 3,3- diethylhexyl, 2,2- diethylhexyl, and Its various branched isomer etc..Low alkyl group containing 1 to 6 carbon atom, non-limiting embodiment include methyl, ethyl, just Propyl, isopropyl, normal-butyl, isobutyl group, tert-butyl, sec-butyl, n-pentyl, 1,1- dimethyl propyl, 1,2- dimethyl propyl, 2,2- dimethyl propyl, 1- ethyl propyl, 2- methyl butyl, 3- methyl butyl, n-hexyl, 1- Ethyl-2-Methyl propyl, 1,1, 2- thmethylpropyl, 1,1- dimethylbutyl, 1,2- dimethylbutyl, 2,2- dimethylbutyl, 1,3- dimethylbutyl, 2- second Base butyl, 2- methyl amyl, 3- methyl amyl, 4- methyl amyl, 2,3- dimethylbutyl etc..Alkyl can be substituted or non- Replace, when substituted, substituent group can be substituted on any workable tie point, and the substituent group is preferably independently It is optionally chosen from alkyl, alkenyl, alkynyl, alkoxy, alkylthio group, alkyl amino, halogen, mercaptan, hydroxyl, nitro, cyano, cycloalkanes Base, heterocycle, aryl, heteroaryl, cycloalkyloxy, heterocyclylalkoxy groups, cycloalkylthio, heterocycle alkylthio group, oxo base ,-NR⁷R⁸、-C (O)NR⁷R⁸、-C(O)R⁹、-C(O)OR⁹With-S (O)_tR⁹In one or more substituent groups replaced.

Term " alkylidene " refers to the linear chain or branched chain aliphatic alkyl of saturation, and the same carbon with 2 from parent alkane is former Residue derived from two hydrogen atoms is removed on son or two different carbon atoms, is the straight chain comprising 1 to 20 carbon atom Or branched group, preferably comprise 1 to 12 carbon atom, the alkylidene of further preferably 1 to 6 carbon atom.The non-limit of alkylidene Property example processed includes but is not limited to methylene (- CH₂), 1,1- ethylidene (- CH (CH₃) -), 1,2- ethylidene (- CH₂CH₂)-、 1,1- propylidene (- CH (CH₂CH₃) -), 1,2- propylidene (- CH₂CH(CH₃) -), 1,3- propylidene (- CH₂CH₂CH₂-)、1,4- Butylidene (- CH₂CH₂CH₂CH₂) etc..Alkylidene can be substituted or non-substituted, and when substituted, substituent group can be Be substituted on any workable tie point, the substituent group be preferably independently optionally chosen from alkyl, alkenyl, alkynyl, alkoxy, Alkylthio group, alkyl amino, halogen, mercaptan, hydroxyl, nitro, cyano, naphthenic base, heterocycle, aryl, heteroaryl, cycloalkyloxy, Heterocyclylalkoxy groups, cycloalkylthio, heterocycle alkylthio group, oxo base ,-NR⁷R⁸、-C(O)NR⁷R⁸、-C(O)R⁹、-C(O)OR⁹With-S (O)_tR⁹In one or more substituent groups replaced.

Term " alkenyl " refers to that the unsaturated alkyl compound in molecule containing carbon-carbon double bond, alkyl are as defined above. Alkenyl can be substituted or non-substituted, and when substituted, substituent group is preferably one or more following groups, independently Selected from hydrogen atom, alkyl, alkoxy, halogen, halogenated alkyl, hydroxyl, hydroxyalkyl, cyano, amino, nitro, naphthenic base, heterocycle Base, aryl, heteroaryl ,-NR⁷R⁸、-C(O)NR⁷R⁸、-C(O)R⁹、-C(O)OR⁹With-S (O)_tR⁹In one or more substituent groups It is replaced.

Term " alkynyl " refers to the unsaturated alkyl compound in molecule containing triple carbon-carbon bonds, alkyl be as defined above on institute It states.Alkynyl can be substituted or non-substituted, and when substituted, substituent group is preferably one or more following groups, solely On the spot selected from hydrogen atom, alkyl, alkoxy, halogen, halogenated alkyl, hydroxyl, hydroxyalkyl, cyano, amino, nitro, naphthenic base, miscellaneous Ring group, aryl, heteroaryl ,-NR⁷R⁸、-C(O)NR⁷R⁸、-C(O)R⁹、-C(O)OR⁹With-S (O)_tR⁹In one or more replace Replaced base.

Term " naphthenic base " refers to the unsaturated monocycle of saturation or part or polycyclic cyclic hydrocarbon substituent, cycloalkyl ring include 3 to 20 carbon atoms, preferably comprise 3 to 12 carbon atoms, include more preferably 3 to 6 carbon atoms, most preferably comprise 5 to 6 carbon originals Son.The non-limiting example of monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopenta, cyclopentenyl, cyclohexyl, cyclohexenyl group, Cyclohexadienyl, suberyl, cycloheptatriene base, cyclooctyl etc.；Polycyclic naphthene base includes the naphthenic base of loop coil, condensed ring and bridged ring.

Term " spiro cycloalkyl group " refers to the polycyclic moiety that a carbon atom (claiming spiro-atom) is shared between 5 to 20 yuan of monocycle, It can be containing one or more double bonds, but none ring has the pi-electron system of total conjugated.Preferably 6 to 14 yuan, more Preferably 7 to 10 yuan.Spiro cycloalkyl group is divided into single spiro cycloalkyl group, double spirocyclanes according to the number for sharing spiro-atom between ring and ring Base or more spiro cycloalkyl groups, preferably single spiro cycloalkyl group and double spiro cycloalkyl groups.More preferably 4 yuan/4 yuan, 4 yuan/5 yuan, 4 yuan/6 yuan, 5 Member/5 yuan or 5 yuan/6 yuan single spiro cycloalkyl groups.The non-limiting example of spiro cycloalkyl group includes:

Term " cycloalkyl " refers to 5 to 20 yuan, each ring in system and the shared a pair adjoined of other rings in system The full carbon polycyclic moiety of carbon atom, wherein one or more rings can be containing one or more double bonds, but none ring has The pi-electron system of total conjugated.Preferably 6 to 14 yuan, more preferably 7 to 10 yuan.It can be divided into according to a group cyclic number double Ring, tricyclic, Fourth Ring or polycyclic fused ring alkyl, preferably bicyclic or tricyclic, more preferably 5 yuan/5 yuan or 5 yuan/6 membered bicyclic alkyl. The non-limiting example of cycloalkyl includes:

Term " bridge ring alkyl " refers to 5 to 20 yuan, and the full carbon that any two ring shares two carbon atoms being not directly connected is more Cyclic group, can be containing one or more double bonds, but none ring has the pi-electron system of total conjugated.Preferably 6 to 14 yuan, more preferably 7 to 10 yuan.Bicyclic, tricyclic, Fourth Ring or polycyclic bridge ring alkyl can be divided into according to a group cyclic number, it is excellent It is selected as bicyclic, tricyclic or Fourth Ring, more preferably bicyclic or tricyclic.The non-limiting example of bridge ring alkyl includes:

The cycloalkyl ring can be condensed on aryl, heteroaryl or heterocycloalkyl ring, wherein being connected to precursor structure Ring together is naphthenic base, and non-limiting example includes indanyl, tetralyl, benzocyclohepta alkyl etc.；It is preferred that phenyl and ring Amyl, tetralyl.Naphthenic base can be it is optionally replacing or non-substituted, when substituted, substituent group be preferably one or Multiple following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio group, alkyl amino, halogen, sulfydryl, hydroxyl Base, nitro, cyano, naphthenic base, Heterocyclylalkyl, aryl, heteroaryl, cycloalkyloxy, heterocyclylalkoxy groups, cycloalkylthio, heterocycle alkane Sulfenyl, oxo base, carboxyl or carboxylate.

Term " heterocycle " refers to the unsaturated monocycle of saturation or part or polycyclic cyclic hydrocarbon substituent, and it includes 3 to 20 rings Atom, wherein one or more annular atoms are selected from nitrogen, oxygen or S (O)_tThe hetero atom of (wherein t is integer 0 to 2), but do not wrap The loop section of-O-O- ,-O-S- or-S-S- are included, remaining annular atom is carbon.3 to 12 annular atoms are preferably comprised, wherein 1~4 It is hetero atom；3 to 8 annular atoms are most preferably comprised, wherein 1~3 is hetero atom；5 to 6 annular atoms are most preferably comprised, In 1~2 or 1~3 be hetero atom.The non-limiting example of monocyclic heterocycles base includes pyrrolidinyl, imidazolidinyl, tetrahydrofuran Base, THP trtrahydropyranyl, tetrahydro-thienyl, glyoxalidine base, dihydrofuryl, pyrazoline base, pyrrolin base, piperidyl, Piperazinyl, morpholinyl, thio-morpholinyl, high piperazine base etc., preferably THP trtrahydropyranyl, piperidyl, pyrrolidinyl.Multiring heterocyclic Heterocycle including loop coil, condensed ring and bridged ring.

Term " spiro heterocyclic radical " refers to the multiring heterocyclic that an atom (claiming spiro-atom) is shared between 5 to 20 yuan of monocycle Group, wherein one or more annular atoms are selected from nitrogen, oxygen or S (O)_tThe hetero atom of (wherein t is integer 0 to 2), remaining annular atom For carbon.It can be containing one or more double bonds, but none ring has the pi-electron system of total conjugated.Preferably 6 to 14 Member, more preferably 7 to 10 yuan.Spiro heterocyclic radical is divided into single spiro heterocyclic radical, double according to the number for sharing spiro-atom between ring and ring Spiro heterocyclic radical or more spiro heterocyclic radicals, preferably single spiro heterocyclic radical and double spiro heterocyclic radicals.More preferably 4 yuan/4 yuan, 4 yuan/5 yuan, 4 Member/6 yuan, 5 yuan/5 yuan or 5 yuan/6 yuan single spiro heterocyclic radicals.The non-limiting example of spiro heterocyclic radical includes:

Term " condensed hetero ring base " refers to 5 to 20 yuan, each ring in system and the shared a pair adjoined of other rings in system The polycyclic heterocyclic group of atom, one or more rings can be containing one or more double bonds, but none ring is with completely total The pi-electron system of yoke, wherein one or more annular atoms are selected from nitrogen, oxygen or S (O)_tThe miscellaneous original of (wherein t is integer 0 to 2) Son, remaining annular atom are carbon.Preferably 6 to 14 yuan, more preferably 7 to 10 yuan.According to a group cyclic number can be divided into it is bicyclic, Tricyclic, Fourth Ring or polycyclic condensed hetero ring base, preferably bicyclic or tricyclic, more preferably 5 yuan/5 yuan or 5 yuan/6 membered bicyclic condensed hetero rings Base.The non-limiting example of condensed hetero ring base includes:

Term " bridge heterocycle " refers to 5 to 14 yuan, and any two ring shares the polycyclic heterocycle of two atoms being not directly connected Group, can be containing one or more double bonds, but none ring has the pi-electron system of total conjugated, one of them or Multiple annular atoms are selected from nitrogen, oxygen or S (O)_tThe hetero atom of (wherein t is integer 0 to 2), remaining annular atom are carbon.Preferably 6 To 14 yuan, more preferably 7 to 10 yuan.Bicyclic, tricyclic, Fourth Ring or polycyclic bridge heterocycle can be divided into according to a group cyclic number, Preferably bicyclic, tricyclic or Fourth Ring, more preferably bicyclic or tricyclic.The non-limiting example of bridge heterocycle includes:

The heterocyclic ring can be condensed on aryl, heteroaryl or cycloalkyl ring, wherein being connected to one with precursor structure The ring risen is heterocycle, and non-limiting example includes:

Deng.

Heterocycle can be it is optionally replacing or non-substituted, when substituted, substituent group be preferably it is one or more with Lower group, be independently optionally chosen from alkyl, alkenyl, alkynyl, alkoxy, alkylthio group, alkyl amino, halogen, mercaptan, hydroxyl, Nitro, cyano, naphthenic base, Heterocyclylalkyl, aryl, heteroaryl, cycloalkyloxy, heterocyclylalkoxy groups, cycloalkylthio, heterocycle alkane sulphur Base, oxo base ,-NR⁷R⁸、-C(O)NR⁷R⁸、-C(O)R⁹、-C(O)OR⁹With-S (O)_tR⁹In one or more substituent groups taken Generation.

Term " aryl " refers to that 6 to 14 yuan of full carbon monocycles of the pi-electron system with conjugation or fused polycycle are (namely shared The ring of adjacent carbon atoms pair) group, preferably 6 to 10 yuan, more preferable 5 to 6 yuan, such as phenyl and naphthalene.The aryl rings can To condense on heteroaryl, heterocycle or cycloalkyl ring, wherein being aryl rings, non-limit with the ring that precursor structure links together Property example processed includes:

Aryl can be substituted or non-substituted, and when substituted, substituent group is preferably one or more following groups, It is independently optionally chosen from alkyl, alkenyl, alkynyl, alkoxy, alkylthio group, alkyl amino, halogen, mercaptan, hydroxyl, nitro, cyanogen Base, naphthenic base, Heterocyclylalkyl, aryl, heteroaryl, cycloalkyloxy, heterocyclylalkoxy groups, cycloalkylthio, heterocycle alkylthio group ,- NR⁷R⁸、-C(O)NR⁷R⁸、-C(O)R⁹、-C(O)OR⁹With-S (O)_tR⁹In one or more substituent groups replaced.

Term " amino protecting group " is in order to which amino remains unchanged when reacting molecule other positions, with being easy to slough Group amino is protected.Non-limiting embodiment includes tertbutyloxycarbonyl, acetyl group, benzyl, allyl and to methoxy Benzyl etc..These groups are optionally replaced the 1-3 substituent group in halogen, alkoxy or nitro.The amino Protecting group is preferably to methoxybenzyl.

Term " heteroaryl " refers to the heteroaromatic system comprising 1 to 4 hetero atom, 5 to 14 annular atoms, and wherein hetero atom selects From oxygen, sulphur and nitrogen.Heteroaryl is preferably 5 to 10 yuan, contains 1 to 3 hetero atom；It is more preferably 5- or 6-membered, contain 1 to 2 miscellaneous original Son；It is preferred that such as imidazole radicals, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, pyrrole radicals, tetrazole radical, pyridyl group, phonetic Piperidinyl, thiadiazoles, pyrazinyl etc., preferably imidazole radicals, pyrazolyl or pyrimidine radicals, thiazolyl；More preferable pyrazolyl.The heteroaryl Basic ring can be condensed on aryl, heterocycle or cycloalkyl ring, wherein be heteroaryl ring with the ring that precursor structure links together, Its non-limiting example includes:

Heteroaryl can be it is optionally replacing or non-substituted, when substituted, substituent group be preferably it is one or more with Lower group, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio group, alkyl amino, halogen, mercaptan, hydroxyl, nitro, Cyano, naphthenic base, Heterocyclylalkyl, aryl, heteroaryl, cycloalkyloxy, heterocyclylalkoxy groups, cycloalkylthio, heterocycle alkylthio group ,- NR⁷R⁸、-C(O)NR⁷R⁸、-C(O)R⁹、-C(O)OR⁹With-S (O)_tR⁹In one or more substituent groups replaced.

Term " alkoxy " refers to-O- (alkyl) and-O- (non-substituted naphthenic base), and wherein the definition of alkyl and naphthenic base is such as It is upper described.The non-limiting example of alkoxy includes: methoxyl group, ethyoxyl, propoxyl group, butoxy, cyclopropyl oxygroup, ring fourth oxygen Base, cyclopentyloxy, cyclohexyloxy.Alkoxy can be optionally replacing or non-substituted, and when substituted, substituent group is preferably One or more following groups, independently selected from hydrogen atom, halogen, alkyl, alkoxy, halogenated alkyl, hydroxyl, hydroxyalkyl, Cyano, amino, nitro, naphthenic base, heterocycle, aryl, replaced one or more substituent groups in heteroaryl.

Term " halogenated alkyl " refers to that alkyl is replaced by one or more halogens, and wherein alkyl is as defined above.Term " hydroxyl Base " refers to-OH group.

Term " hydroxyalkyl " refers to the alkyl being optionally substituted by a hydroxyl group, and wherein alkyl is as defined above.

Term " halogen " refers to fluorine, chlorine, bromine or iodine.

Term " amino " refers to-NH₂。

Term " cyano " refers to-CN.

Term " nitro " refers to-NO₂。

Term " oxo base " refers to=O.

" optional " or " optionally " mean event or environment described later can with but need not occur, which includes The event or environment generation or not spot occasion.For example, meaning that alkyl can be with " optionally by alkyl-substituted heterocyclic group " But necessarily exist, the explanation include heterocyclic group by alkyl-substituted situation and heterocyclic group not by alkyl-substituted situation.

" substituted " refers to one or more hydrogen atoms in group, preferably at most 5, more preferably 1~3 hydrogen atom Replaced independently of one another by the substituent group of respective number.Self-evident, substituent group is only in their possible chemical position, this Field technical staff, which can determine in the case where not paying excessive make great efforts and (pass through experiment or theoretical), may or impossible take Generation.It may be unstable when for example, amino or hydroxyl with free hydrogen are in conjunction with the carbon atom with unsaturated (such as olefinic) key Fixed.

" pharmaceutical composition " indicate containing one or more compounds described herein or its physiologically/pharmaceutical salt or The mixture and other components such as physiology/pharmaceutical carrier and excipient of pro-drug and other chemical constituents.Medicine The purpose of compositions is the administration promoted to organism, plays bioactivity in turn conducive to the absorption of active constituent.

" officinal salt " refers to the salt of the compounds of this invention, has safety when this kind of salt is used in the mammalian body and has Effect property, and there is due bioactivity.

T and R⁷~R⁹As defined in logical formula (I) compound.

The synthetic method of the compounds of this invention

In order to complete the purpose of the present invention, the present invention adopts the following technical scheme:

Scheme one

The present invention lead to formula (I) compound represented or its tautomer, mesomer, racemic modification, enantiomter, Diastereoisomer, or mixtures thereof form or its pharmaceutical salt preparation method, comprising the following steps:

The first step, the compound or its salt (preferably trifluoroacetate) of general formula (I-1) and the compound of general formula (I-2) exist Under alkaline condition, nucleophilic substitution occurs and obtains the compound of general formula (I-3)；

The compound of second step, general formula (I-3) sloughs R in acid condition^aObtain the compound of general formula (IA)；

In acid condition, reaction obtains logical formula (I) to the compound of third step, the compound of general formula (IA) and general formula (IB) Compound；

Wherein:

The reagent for providing alkaline condition includes organic base and inorganic base, and the organic bases include but is not limited to three second Amine, pyridine, 4-dimethylaminopyridine, N, N- diisopropylethylamine, n-BuLi, lithium diisopropylamine, double trimethyl silicon substrates Amido lithium, potassium acetate, sodium tert-butoxide, potassium tert-butoxide and n-butanol sodium, the inorganic base include but is not limited to sodium hydride, phosphorus Sour potassium, sodium carbonate, potassium carbonate, potassium acetate, cesium carbonate, sodium hydroxide and lithium hydroxide；

The reagent for providing acid condition includes but is not limited to hydrochloric acid, the 1,4- dioxane solution of hydrogen chloride, trifluoro second Acid, formic acid, acetic acid, hydrochloric acid, sulfuric acid, methanesulfonic acid, nitric acid, phosphoric acid, to benzene methanesulfonic acid, Me₃SiCl and TMSOTf；

Above-mentioned reaction preferably carries out in a solvent, and solvent for use includes but is not limited to: acetic acid, trifluoroacetic acid, methanol, second Alcohol, n-butanol, toluene, tetrahydrofuran, methylene chloride, petroleum ether, ethyl acetate, n-hexane, dimethyl sulfoxide, 1,4- dioxy six Ring, water, N,N-dimethylformamide and its mixture；

R^aFor alkyl, preferably methyl；

X is halogen, preferably bromine；

L¹And R¹~R⁵As defined in logical formula (I).

Scheme two

The present invention leads to formula (II) compound represented or its tautomer, mesomer, racemic modification, enantiomerism Body, diastereoisomer, or mixtures thereof form or its pharmaceutical salt preparation method, comprising the following steps:

In acid condition, reaction obtains the change of logical formula (II) to the compound of the compound of general formula (IA) and general formula (IIB) Close object；

Wherein:

The reagent for providing acid condition includes but is not limited to hydrochloric acid, the 1,4- dioxane solution of hydrogen chloride, trifluoro second Acid, formic acid, acetic acid, hydrochloric acid, sulfuric acid, methanesulfonic acid, nitric acid, phosphoric acid, to benzene methanesulfonic acid, Me₃SiCl and TMSOTf；

Above-mentioned reaction preferably carries out in a solvent, and solvent for use includes but is not limited to: acetic acid, trifluoroacetic acid, methanol, second Alcohol, n-butanol, toluene, tetrahydrofuran, methylene chloride, petroleum ether, ethyl acetate, n-hexane, dimethyl sulfoxide, 1,4- dioxy six Ring, water, N,N-dimethylformamide and its mixture；

alk、L¹、R¹、R²、R⁴、R⁵、R⁷And R⁸As defined in logical formula (II).

Scheme three

The present invention leads to formula (III) compound represented or its tautomer, mesomer, racemic modification, enantiomerism Body, diastereoisomer, or mixtures thereof form or its pharmaceutical salt preparation method, comprising the following steps:

In acid condition, reaction obtains logical formula (III) to the compound of the compound of general formula (IA) and general formula (IIIB) Compound；

Wherein:

The reagent for providing acid condition includes but is not limited to hydrochloric acid, the 1,4- dioxane solution of hydrogen chloride, trifluoro second Acid, formic acid, acetic acid, hydrochloric acid, sulfuric acid, methanesulfonic acid, nitric acid, phosphoric acid, to benzene methanesulfonic acid, Me₃SiCl and TMSOTf；

Above-mentioned reaction preferably carries out in a solvent, and solvent for use includes but is not limited to: acetic acid, trifluoroacetic acid, methanol, second Alcohol, n-butanol, toluene, tetrahydrofuran, methylene chloride, petroleum ether, ethyl acetate, n-hexane, dimethyl sulfoxide, 1,4- dioxy six Ring, water, N,N-dimethylformamide and its mixture；

L¹、R¹、R²、R⁴、R⁵、R⁷、R⁸With n as defined in logical formula (III).

Scheme four

The present invention leads to formula (IV) compound represented or its tautomer, mesomer, racemic modification, enantiomerism Body, diastereoisomer, or mixtures thereof form or its pharmaceutical salt preparation method, comprising the following steps:

In acid condition, reaction obtains the change of logical formula (IV) to the compound of the compound of general formula (IVA) and general formula (IVB) Close object；

Wherein:

The reagent for providing acid condition includes but is not limited to hydrochloric acid, the 1,4- dioxane solution of hydrogen chloride, trifluoro second Acid, formic acid, acetic acid, hydrochloric acid, sulfuric acid, methanesulfonic acid, nitric acid, phosphoric acid, to benzene methanesulfonic acid, Me₃SiCl and TMSOTf；

Above-mentioned reaction preferably carries out in a solvent, and solvent for use includes but is not limited to: acetic acid, trifluoroacetic acid, methanol, second Alcohol, n-butanol, toluene, tetrahydrofuran, methylene chloride, petroleum ether, ethyl acetate, n-hexane, dimethyl sulfoxide, 1,4- dioxy six Ring, water, N,N-dimethylformamide and its mixture；

R¹⁰Selected from alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxyl, hydroxyalkyl, naphthenic base, heterocycle, aryl And heteroaryl；

Q is 0,1,2 or 3, and preferably q is 0；

R¹、R², m and n be as defined in logical formula (IV).

Specific embodiment

Embodiment

The structure of compound is by nuclear magnetic resonance (NMR) or/and mass spectrum (MS) come what is determined.NMR is displaced (δ) with 10^-6 (ppm) unit provides.The measurement of NMR is to use Bruker AVANCE-400 nuclear magnetic resonance spectrometer, and measurement solvent is deuterated dimethyl sulfoxide (DMSO-d₆), deuterated chloroform (CDCl₃), deuterated methanol (CD₃), OD inside it is designated as tetramethylsilane (TMS).

The measurement of MS is with FINNIGAN LCQAd (ESI) mass spectrograph (manufacturer: Thermo, model: Finnigan LCQ advantage MAX)。

The measurement of HPLC uses Agilent 1200DAD high pressure liquid chromatograph (Sunfire C18 150 × 4.6mm chromatography Column) and Waters 2695-2996 high pressure liquid chromatograph (Gimini 150 × 4.6mm of C18 chromatographic column).

Chiral HPLC measurement uses LC-10A vp (Shimadzu) or SFC-analytical (Berger Instruments Inc.)。

Tlc silica gel plate uses Yantai Huanghai Sea HSGF254 or Qingdao GF254 silica gel plate, and thin-layered chromatography (TLC) makes The specification that silica gel plate uses is 0.15mm~0.2mm, thin-layer chromatography isolate and purify product use specification be 0.4mm~ 0.5mm。

Column chromatography is generally carrier using 200~300 mesh silica gel of Yantai Huanghai Sea silica gel.

Chiral preparatory column chromatography uses Prep Star SD-1 (Varian Instruments Inc.) or SFC- multigram(Berger Instruments Inc.)。

Quick preparing instrument uses the quick preparing instrument of CombiFlash, model RF+UV.

Chiral preparatory column use CHIRALPAK AS-H 20*250mm, 5 μm.

Kinases average inhibition and IC₅₀The measurement of value is with NovoStar microplate reader (German BMG company).

Known starting material of the invention can be used or be synthesized according to methods known in the art, or can purchase certainly ABCR GmbH&Co.KG, Acros Organics, Aldrich Chemical Company, splendid remote chemical science and technology (Accela ChemBio Inc), up to the companies such as auspicious chemicals.

Without specified otherwise in embodiment, reaction can be carried out under argon atmospher or nitrogen atmosphere.

Argon atmospher or nitrogen atmosphere refer to that reaction flask connects the argon gas or nitrogen balloon of an about 1L volume.

Nitrogen atmosphere refers to that reaction flask connects the hydrogen balloon of an about 1L volume.

Pressure hydration reaction uses Parr 3916EKX type hydrogenation instrument and clear indigo plant QL-500 type hydrogen generator or HC2-SS Type hydrogenates instrument.

Hydrogenation usually vacuumizes, and is filled with hydrogen, operates 3 times repeatedly.

Microwave reaction uses 908860 type microwave reactor of CEM Discover-S.

Without specified otherwise in embodiment, solution refers to aqueous solution.

Without specified otherwise in embodiment, it is 20 DEG C~30 DEG C that the temperature of reaction, which is room temperature,.

The monitoring of reaction process in embodiment uses thin-layered chromatography (TLC), reacts used solvent, purifyingization The system of eluant, eluent and the solvent system of thin-layered chromatography for closing the column chromatography that object uses include: A: methylene chloride/methanol body The volume ratio of system, solvent is different according to the polarity of compound and is adjusted, and a small amount of triethylamine and acetic acid etc. can also be added Alkalinity or acid reagent are adjusted.

Embodiment 1,2

(E) -6- amino -2- butoxy -9- (2- ((2- (pyrrolidin-1-yl) ethyoxyl) ammonia subunit) propyl) -7,9- two Hydrogen -8H- purine -8- ketone 1

(Z) -6- amino -2- butoxy -9- (2- ((2- (pyrrolidin-1-yl) ethyoxyl) ammonia subunit) propyl) -7,9- two Hydrogen -8H- purine -8- ketone 2

The first step

1- (6- amino -2- butoxy -8- methoxyl group -9H- purine -9- base) propane -2- ketone 1b

Under the conditions of 0 DEG C, by 2- butoxy -8- methoxyl group -9H- purine -6- amine trifluoroacetate 1a (450mg, 1.89mmol is prepared using method disclosed in PCT Patent Application " WO2016075661 ") it is dissolved in n,N-Dimethylformamide In, it is added potassium carbonate (1.3g, 9.45mmol), adds 1- N-Propyl Bromide -2- ketone (519mg, 3.79mmol), reaction is stirred at room temperature 12 hours.50mL water is added in reaction solution, is extracted with ethyl acetate (50mL), organic phase washed with water (30mL × 3) and saturation Sodium chloride solution (30mL × 3) washing, anhydrous sodium sulfate dry, filter, and filtrate decompression concentration is quickly prepared with CombiFlash Instrument with eluant, eluent system A purify gained residue, then with thin-layered chromatography with solvent system A purify obtained by residue, obtain Title compound 1b (140mg), yield: 25.3%.

MS m/z(ESI):294.4[M+1]

Second step

6- amino -2- butoxy -9- (2- oxopropyl) -7,9- dihydro -8H- purine -8- ketone 1c

Compound 1b (140mg, 0.4mmol) is dissolved in 8mL hydrochloric acid, is stirred to react 4 hours.Reaction solution is concentrated under reduced pressure, 10mL water is added in gained residue, it is 7~8 that saturated sodium bicarbonate solution, which is added dropwise, to pH, it filters, collection filter cake, vacuum drying, It obtains crude title compound 1c (100mg), product directly carries out next step reaction without further purification.

MS m/z(ESI):280.4[M+1]

Third step

(E) -6- amino -2- butoxy -9- (2- ((2- (pyrrolidin-1-yl) ethyoxyl) ammonia subunit) propyl) -7,9- two Hydrogen -8H- purine -8- ketone 1

(Z) -6- amino -2- butoxy -9- (2- ((2- (pyrrolidin-1-yl) ethyoxyl) ammonia subunit) propyl) -7,9- two Hydrogen -8H- purine -8- ketone 2

Crude Compound 1c (110mg, 0.39mmol) is dissolved in 10mL toluene, O- (2- (pyrrolidin-1-yl) second is added Base) azanol 1d (51mg, 0.39mmol are prepared using method disclosed in U.S. Patent application " US20040116710 ") and P-methyl benzenesulfonic acid (3.7mg, 0.0195mmol) is warming up to 110 DEG C and is stirred to react 12 hours.Reaction solution is cooled to room temperature, with nothing Aqueous sodium persulfate dries, filters, filtrate decompression concentration, with thin-layered chromatography with solvent system A purify gained residue, progress hand Property prepare (separation condition: chiral preparatory column CHIRALPAK AS-H 20*250mm, 5 μm；Mobile phase: n-hexane: ethyl alcohol: diethyl Amine=85:15:0.01, flow velocity: 15mL/min), collect its respective components, be concentrated under reduced pressure, obtain title compound 1 (80mg) and 2(45mg)。

Compound 1:

MS m/z(ESI):392.5[M+1]；

¹H NMR(400MHz,DMSO-d₆)δ10.18(s,1H),6.58(s,2H),4.38(s,2H),4.13(t,2H), 4.08-4.11(m,2H),2.87-2.56(m,6H),1.77(s,3H),1.76-1.68(m,4H),1.68-1.57(m,2H), 1.44-1.32(m,2H),0.92(t,3H)。

Compound 2:

MS m/z(ESI):392.5[M+1]；

¹H NMR(400MHz,DMSO-d₆)δ10.08(s,1H),6.54(s,2H),4.54(s,2H),4.14(t,4H), 2.92-2.38(m,6H),1.71-1.68(m,4H),1.68-1.62(m,2H),1.54(s,3H),1.43-1.36(m,2H), 0.91(t,3H)。

Embodiment 3,4

(E) -6- amino -2- butoxy -9- (2- ((3- (pyrrolidin-1-yl) propoxyl group) ammonia subunit) propyl) -7,9- two Hydrogen -8H- purine -8- ketone 3

(Z) -6- amino -2- butoxy -9- (2- ((3- (pyrrolidin-1-yl) propoxyl group) ammonia subunit) propyl) -7,9- two Hydrogen -8H- purine -8- ketone 4

The first step

2- (3- (pyrrolidin-1-yl) propoxyl group) iso-indoles -1,3- diketone 2c

It is cooled to 0 DEG C, by 2- hydroxyl iso-indoles -1,3- diketone 2a (2.5g, 15.33mmol), 3- (pyrrolidin-1-yl) third Alcohol 2b (1.98g, 15.33mmol, using well known method " Catalysis Communications, 2007,8 (11), 1671- 1674 " are prepared) and triphenylphosphine (4.02g, 15.33mmol) be dissolved in 50mL tetrahydrofuran, be added dropwise to N, N- diisopropyl Reaction 12 hours is stirred at room temperature in base ethamine (3.1g, 15.33mmol).Reaction solution is concentrated under reduced pressure, and ether, filtering is added, and filtrate subtracts Pressure concentration gained residue, is beaten with ether, filters, repeat above operation three times, and filtrate decompression concentration is fast with CombiFlash Fast preparing instrument purifies gained residue with eluant, eluent system A, obtains title compound 2c (1.1g), yield: 26.2%.

MS m/z(ESI):275.2[M+1]

Second step

O- (3- (pyrrolidin-1-yl) propyl) azanol 2d

Compound 2c (1.1g, 4mmol) is dissolved in 15mL ethyl alcohol, hydrazine hydrate is added, and (342mg, 6mmol, 88% are molten Liquid), it is warming up to 80 DEG C and is stirred to react 12 hours.Reaction solution is cooled to room temperature, filtering, filtrate decompression concentration, in gained residue The mashing of 10mL ether, filtering is added, filtrate decompression is concentrated, 10mL ether and the mashing of 10mL n-hexane are added in gained residue, Filtering, filtrate decompression concentration, obtains crude title compound 2d (400mg), and product directly carries out next step reaction without further purification.

MS m/z(ESI):145.4[M+1]

Third step

(E) -6- amino -2- butoxy -9- (2- ((3- (pyrrolidin-1-yl) propoxyl group) ammonia subunit) propyl) -7,9- two Hydrogen -8H- purine -8- ketone 3

(Z) -6- amino -2- butoxy -9- (2- ((3- (pyrrolidin-1-yl) propoxyl group) ammonia subunit) propyl) -7,9- two Hydrogen -8H- purine -8- ketone 4

Crude Compound 1c (240mg, 0.86mmol) is dissolved in 25mL toluene, addition crude Compound 2d (124mg, 0.86mmol) with p-methyl benzenesulfonic acid (8mg, 0.043mmol), it is warming up to 100 DEG C and is stirred to react 96 hours.Reaction solution is cooled to room Temperature is dried, filtered with anhydrous sodium sulfate, and filtrate decompression concentration is purified with the quick preparing instrument of CombiFlash with eluant, eluent system A Gained residue, carry out chirality prepare (separation condition: chiral preparatory column CHIRALPAK AS-H 20*250mm, 5 μm；Flowing Phase: n-hexane: ethyl alcohol: diethylamine=85:15:0.01, flow velocity: 15mL/min), its respective components is collected, is concentrated under reduced pressure, obtains Title compound 3 (50mg) and 4 (35mg).

Compound 3:

MS m/z(ESI):406.5[M+1]；

¹H NMR(400MHz,DMSO-d₆)δ9.95(s,1H),6.45(s,2H),4.36(s,2H),4.13(t,2H), 3.93(t,2H),2.44-2.28(m,6H),1.74(s,3H),1.72-1.55(m,8H),1.48-1.32(m,2H),0.91(t, 3H)。

Compound 4:

MS m/z(ESI):406.5[M+1]；

¹H NMR(400MHz,DMSO-d₆)δ10.04(s,1H),6.51(s,2H),4.51(s,2H),4.14(t,2H), 4.04(t,2H),2.48-2.37(m,6H),1.80-1.73(m,2H),1.71-1.66(m,4H),1.66-1.58(m,5H), 1.44-1.35(m,2H),0.91(t,3H)。

Embodiment 5,6

(Z) -6- amino -2- butoxy -9- (2- ((2- (pyrrolidin-1-yl) ethyoxyl) ammonia subunit) butyl) -7,9- two Hydrogen -8H- purine -8- ketone 5

(E) -6- amino -2- butoxy -9- (2- ((2- (pyrrolidin-1-yl) ethyoxyl) ammonia subunit) butyl) -7,9- two Hydrogen -8H- purine -8- ketone 6

The first step

1- (6- amino -2- butoxy -8- methoxyl group -9H- purine -9- base) butane -2- ketone 3a

Under the conditions of 0 DEG C, compound 1a (2g, 5.7mmol) is dissolved in n,N-Dimethylformamide, potassium carbonate is added (3.93g, 28.5mmol) adds 1- bromobutane -2- ketone (1.72g, 11.4mmol), and reaction 1 hour is stirred at room temperature.Reaction solution Middle addition 50mL water is extracted with ethyl acetate (50mL), organic phase washed with water (30mL × 3) and saturated sodium chloride solution (30mL × 3) washing, anhydrous sodium sulfate dry, filter, filtrate decompression concentration, with the quick preparing instrument of CombiFlash with eluant, eluent System A purifying gained residue, then with high performance liquid chromatography purify obtained by residue, obtain title compound 3a (420mg), Yield: 24%.

MS m/z(ESI):308.4[M+1]

Second step

- 8 ketone 3b of 6- amino -2- butoxy -9- (2- oxo butyl) -7,9- dihydro -8H- purine

Compound 3a (420mg, 1.37mmol) is dissolved in 10mL hydrochloric acid, is stirred to react 12 hours.Reaction solution decompression is dense It contracts, 10mL water is added in gained residue, it is 7~8 that saturated sodium bicarbonate solution, which is added dropwise, to pH, is filtered, collection filter cake, and vacuum is dry It is dry, it obtains crude title compound 3b (340mg), product directly carries out next step reaction without further purification.

MS m/z(ESI):294.4[M+1]

Third step

(Z) -6- amino -2- butoxy -9- (2- ((2- (pyrrolidin-1-yl) ethyoxyl) ammonia subunit) butyl) -7,9- two Hydrogen -8H- purine -8- ketone 5

(E) -6- amino -2- butoxy -9- (2- ((2- (pyrrolidin-1-yl) ethyoxyl) ammonia subunit) butyl) -7,9- two Hydrogen -8H- purine -8- ketone 6

Crude Compound 3b (180mg, 0.61mmol) is dissolved in 20mL toluene, addition compound 1d (80mg, 0.61mmol) with p-methyl benzenesulfonic acid (6mg, 0.03mmol), it is warming up to 105 DEG C and is stirred to react 12 hours.Reaction solution is cooled to room Temperature is dried, filtered with anhydrous sodium sulfate, and filtrate decompression concentration is purified with the quick preparing instrument of CombiFlash with eluant, eluent system A Gained residue, carry out chirality prepare (separation condition: chiral preparatory column CHIRALPAK AS-H 20*250mm, 5 μm；Flowing Phase: n-hexane: ethyl alcohol: diethylamine=85:15:0.01, flow velocity: 15mL/min), its respective components is collected, is concentrated under reduced pressure, obtains Title compound 5 (170mg) and 6 (20mg).

Compound 5:

MS m/z(ESI):406.2[M+1]；

¹H NMR(400MHz,DMSO-d₆)δ9.92(s,1H),6.44(s,2H),4.39(s,2H),4.13(t,2H), 3.97(t,2H),2.57-2.52(m,2H),2.44-2.34(m,4H),2.20(q,2H),1.68-1.58(m,6H),1.42- 1.35(m,2H),0.96(t,3H),0.91(t,3H)。

Compound 6:

MS m/z(ESI):406.2[M+1]；

¹H NMR(400MHz,DMSO-d₆)δ10.02(s,1H),6.51(s,2H),4.54(s,2H),4.13(dd,4H), 2.78-2.66(m,2H),2.50-2.45(m,4H),2.08-1.91(m,4H),1.77-1.57(m,6H),0.88(m,6H)。

Embodiment 7,8

(E) -6- amino -2- butoxy -9- (2- ((3- (pyrrolidin-1-yl) propoxyl group) ammonia subunit) butyl) -7,9- two Hydrogen -8H- purine -8- ketone 7

(Z) -6- amino -2- butoxy -9- (2- ((3- (pyrrolidin-1-yl) propoxyl group) ammonia subunit) butyl) -7,9- two Hydrogen -8H- purine -8- ketone 8

Crude Compound 3b (150mg, 0.51mmol) is dissolved in 20mL toluene, addition compound 2d (74mg, 0.51mmol) with p-methyl benzenesulfonic acid (5mg, 0.026mmol), it is warming up to 115 DEG C and is stirred to react 12 hours.Reaction solution is cooled to room Temperature is dried, filtered with anhydrous sodium sulfate, and filtrate decompression concentration is purified with the quick preparing instrument of CombiFlash with eluant, eluent system A Gained residue, carry out chirality prepare (separation condition: chiral preparatory column CHIRALPAK AS-H 20*250mm, 5 μm；Flowing Phase: n-hexane: ethyl alcohol: diethylamine=85:15:0.01, flow velocity: 15mL/min), its respective components is collected, is concentrated under reduced pressure, obtains Title compound 7 (45mg) and 8 (50mg).

Compound 7:

MS m/z(ESI):420.3[M+1]；

¹H NMR(400MHz,DMSO-d₆)δ9.93(s,1H),6.44(s,2H),4.38(s,2H),4.13(t,2H), 3.91(t,2H),2.40-2.30(m,6H),2.20(q,2H),1.72-1.56(m,8H),1.44-1.36(m,2H),0.97(t, 3H),0.91(t,3H)。

Compound 8:

MS m/z(ESI):420.3[M+1]；

¹H NMR(400MHz,DMSO-d₆)δ10.03(s,1H),6.51(s,2H),4.53(s,2H),4.13(t,2H), 4.05(t,2H),2.48-2.35(m,6H),1.98(q,2H),1.76(p,2H),1.71-1.57(m,6H),1.44-1.35(m, 2H),0.90(td,6H)。

Embodiment 9,10

(E) -6- amino -2- butoxy -9- (2- ((2- (piperidin-1-yl) ethyoxyl) ammonia subunit) propyl) -7,9- dihydro - 8H- purine -8- ketone 9

(Z) -6- amino -2- butoxy -9- (2- ((2- (piperidin-1-yl) ethyoxyl) ammonia subunit) propyl) -7,9- dihydro - 8H- purine -8- ketone 10

Crude Compound 1c (200mg, 0.716mmol) is dissolved in 15mL toluene, O- (2- (piperidin-1-yl) second is added Base) azanol 4a (206.54mg, 1.43mmol, using the preparation of method disclosed in U.S. Patent application " US20040116710 " ) and p-methyl benzenesulfonic acid (6.17mg, 35.8mmol), it is warming up to 110 DEG C and is stirred to react 12 hours.Reaction solution is cooled to room temperature, It is dried, filtered with anhydrous sodium sulfate, filtrate decompression concentration, institute is purified with eluant, eluent system A with CombiFlash quick preparing instrument Residue, carry out chirality prepare (separation condition: chiral preparatory column CHIRALPAK AS-H 20*250mm, 5 μm；Mobile phase: N-hexane: ethyl alcohol: diethylamine=90:10:0.01, flow velocity: 15mL/min), its respective components is collected, is concentrated under reduced pressure, is marked Inscribe compound 9 (130mg) and 10 (50mg).

Compound 9:

MS m/z(ESI):406.2[M+1]；

¹H NMR(400MHz,DMSO-d₆)δ9.92(s,1H),6.44(s,2H),4.36(s,2H),4.13(t,2H), 3.98(t,2H),2.42(t,2H),2.35-2.25(m,4H),1.74(s,3H),1.70-1.56(m,2H),1.50-1.29(m, 8H),0.92(t,3H)。

Compound 10:

MS m/z(ESI):406.2[M+1]；

¹H NMR(400MHz,DMSO-d₆)δ10.01(s,1H),6.50(s,2H),4.51(s,2H),4.12(dt,4H), 2.57-2.51(m,2H),2.44-2.34(m,4H),1.68-1.57(m,5H),1.53-1.44(m,4H),1.43-1.31(m, 4H),0.91(t,3H)。

Test case:

Biological assessment

The measurement of test case 1, the compounds of this invention to source of people TLR agonist activity

The compounds of this invention is to HEK-Blue^TMHTLR7 surely turns the hTLR7 protein activation effect of plant cell expression and right HEK-Blue^TMThe hTLR8 protein activation effect that hTLR8 surely turns plant cell expression uses following determination of experimental method:

One, experimental material and instrument

1.DMEM (Gibco, 10564-029),

2. fetal calf serum (GIBCO, 10099),

3. trypan blue solution (Sigma, T8154-100ML),

3 multi-function microplate reader of 4.Flexstation (Molec μ lar Devices),

5.HEK-Blue^TMHTLR7 cell line (InvivoGen, hkb-hTLR7),

6.HEK-Blue^TMHTLR8 cell line (InvivoGen, hkb-hTLR8),

7.HEK-Blue detection reagent (InvivoGen, hb-det3),

Phosphate buffer 8. (PBS) pH7.4 (the Shanghai biotech inc Yuan Pei, B320).

Two, experimental procedure

It configures HEK-Blue and detects culture medium, HEK-Blue is taken to detect one bag of dry powder, 50ml is added, endotoxin water is gone to dissolve, 37 DEG C of incubators are placed into, are sterile filtered after ten minutes.Compound is first configured to the stoste of 20mM；It is diluted to most with pure DMSO again High concentration is 6x10⁶NM, through 3 times of gradient dilutions, totally 10 points.

With culture medium first above-mentioned 20 times of prepared diluted chemical compound, then the chemical combination after 20 μ l dilution is added in every hole Object.HEK-Blue is taken respectively^TMHTLR7 cell and HEK-Blue^TMHTLR8 cell, first removes supernatant, adds 2-5ml preheating PBS, be put into incubator 1-2 minutes, gently blow and beat cell, Trypan Blue counts.It is resuspended with HEK-Blue detection culture medium It is 2.2x 10 that cell, which adjusts concentration,⁵A cell/ml adds 180 μ l cells to the above-mentioned 96 hole cell culture that 20 μ l drugs have been added In plate, 37 DEG C, 6-16h is cultivated.

Microplate reader reading, wavelength 620nm.It can get corresponding OD value, drug be calculated through Graphpad Prism EC₅₀Value.The compounds of this invention can be measured source of people TLR7 and TLR8 activation by above test, measure EC₅₀Value is shown in Table 1.

EC of 1 the compounds of this invention of table to source of people TLR7 and TLR8₅₀Value.

Conclusion: the compounds of this invention shows have apparent activation to source of people TLR7, compared to source of people TLR8's Activation, the compounds of this invention have higher selectivity to the activation of TLR7.

The measurement of IFN-α ability is secreted in compound stimulation peripheral blood mononuclear cells (PBMC) in test case 2, the present invention

Compound stimulation PBMC secretes IFN-α ability using following determination of experimental method in the present invention:

One, experimental material and instrument

1.RPMI 1640(Invitrogen,11875),

2.FBS(Gibco,10099-141),

3.Ficoll-Paque PREMIUM(GE,17-5442-02),

4. trypan blue solution (Sigma, T8154-100ML),

5.SepMateTM-50(Stemcell,15460),

6.Bright-Line^TMBlood-counter system (Sigma, Z359629-1EA),

7.96 hole flat undersides (Corning, 3599),

8.96 hole v bottom plates (Corning, 3894),

9. source of people IFN-α kit (cisbio, 6FHIFPEB),

10.PHERAStar multi-function microplate reader (BMG, PHERAStar).

Two, experimental procedure

Compound is diluted with pure DMSO, maximum concentration 5mM, 4 times of gradient dilutions, totally 9 points.Then 4 μ l compounds are taken, It is added in 1640 culture medium of RMPI of the 196 μ l containing 10%FBS, mixes.Every hole takes 50 μ l to 96 new porocyte culture plates.

All reagents equilibrate to room temperature, take 250ml culture bottle, 60ml blood and PBS+2%FBS are added thereto, gently Piping and druming mixes dilution.50ml PBMC separating pipe SepMateTM-50 is taken, 15ml lymphocyte separation medium Ficoll-Paque is added Then blood after 30ml dilution is added in PREMIUM.1200g is centrifuged 10 minutes, room temperature.Supernatant is taken, then 300g, is centrifuged 8 points Clock.It is resuspended and is counted with 1640 culture medium of RMPI containing 10%FBS, adjustment PBMC quantity to 3.33 × 10⁶A cell/ml, takes 150 μ l are into the tissue culture plate that compound has been added, and 37 DEG C, 5.0%CO₂Incubator in cultivate for 24 hours.

Tissue culture plate is put into a centrifuge, 1200rpm, room temperature is centrifuged 10 minutes.Take out 150 μ l supernatants in every hole.First The reagent in source of people IFN-α kit is balanced to room temperature, anti-IFN-α-is prepared according to kit specification under the conditions of being protected from light Eu³⁺Cave-shaped conjugate (Cryptate conjugate) and anti-IFN-α-d2- conjugate, both with the ratio of 1:40 and knot Buffer (conjugate Buffer) is closed to mix.Then the supernatant that the centrifuging and taking of 16 μ l obtains is added in every hole.Every hole is added 2 again Anti- IFN-α-the Eu that μ l has just been prepared³⁺Cave-shaped conjugate and anti-IFN-α-d2- conjugate, concussion mix, and room temperature is protected from light incubation 3h。

It is read on PHERAStar with HTRF mode.We will stimulation generate minimum detection limit at least 3 times or more cells because Sub horizontal lowest concentration of drug, is defined as MEC (minimum effective concentration of the compound on the cell factor stimulation test Minimal Effective Concentration) value.

The ability that the compounds of this invention stimulation PBMC secretes IFN-α is measured by above test, the MEC value measured It is shown in Table 2.

2 the compounds of this invention of table stimulates the MEC of PBMC secretion IFN-α

Embodiment number	MEC(nM)
		1	1.4
3	<0.4

Conclusion: from the active data that stimulation PBMC secretes IFN-α, the compounds of this invention can preferably cause IFN-α release.

The inhibition of test case 3, the compounds of this invention to the enzymatic activity in people's hepatomicrosome CYP3A4 midazolam metabolism site Effect

The compounds of this invention is to the enzymatic activity in people's hepatomicrosome CYP3A4 midazolam metabolism site using following experiment side Method measurement:

One, experimental material and instrument

1. phosphate buffer (PBS),

2.NADPH(Sigma N-1630),

3. people's hepatomicrosome (Corning Gentest),

4000 liquid matter double-purpose instrument (AB Sciex) of 4.ABI QTrap,

5.Inertsil C8-3 column, 4.6 × 50mm, 5 μm (Di Ma company of the U.S.),

6.CYP probe substrate (15 μM of midazolam, SIGMA UC429) and positive control inhibitor (ketoconazole, SIGMA K1003)。

Two, experimental procedure

The PBS buffer solution of 100mM is configured, it is molten with the particle liquid solution of buffer 2.5mg/ml and the NADPH of 5mM Liquid, with the compound working solutions (150,50,15,5,1.5,0.15,0.015,0 μM) of PBS gradient dilution 5X concentration.With PBS ladder The ketoconazole working solution (150,50,15,5,1.5,0.15,0.015,0 μM) of degree dilution 5X concentration.With PBS be diluted to 15 μM it is dense The midazolam working solution of degree.

The particle liquid solution of 2.5mg/ml, 15 μM of midazolam working solution, MgCl are taken respectively₂Solution and compound work Liquid (150,50,15,5,1.5,0.15,0.015,0 μM, different reaction systems is arranged in each concentration) each 20 μ l is uniformly mixed. The ketoconazole of positive controls same concentrations replaces compound.Simultaneously by the NADPH solution of 5mM together in 37 DEG C of preincubates 5 Minute.20 μ l NADPH are taken to be added in a hole after 5 minutes, starting reaction is incubated for 30 minutes.All samples of incubation set double samples This.The acetonitrile of 250 μ l containing the internal standards is added within 30 minutes in backward all samples, mixes, 800rpm shakes 10 minutes, then 3700rpm Centrifugation 10 minutes.The supernatant for taking 80 μ l is transferred to LC-MS/MS analysis.

Drug is calculated to the IC in CYP3A4 midazolam metabolism site through Graphpad Prism in numerical value₅₀Value is shown in Table 3.

IC of 3 the compounds of this invention of table to CYP3A4 midazolam metabolism site₅₀Value

Embodiment number	IC50(μM)
		1	7.7
3	24

Conclusion: the compounds of this invention does not have inhibiting effect or suppression to the midazolam metabolism site of people's hepatomicrosome CYP3A4 It is weaker to make use, shows better safety, prompts the generation that will not occur to be metabolized midazolam metabolism site based on CYP3A4 The drug interaction of thanking property.

Test case 4, the compounds of this invention are to the inhibiting effect of people's hepatomicrosome CYP2D6 enzymatic activity

The compounds of this invention uses following determination of experimental method to people's hepatomicrosome CYP2D6 enzymatic activity:

One, experimental material and instrument

1. phosphate buffer (PBS),

2.NADPH(Sigma N-1630),

3. people's hepatomicrosome (Corning Gentest),

4000 liquid matter double-purpose instrument (AB Sciex) of 4.ABI QTrap,

5.Inertsil C8-3 column, 4.6 × 50mm, 5 μm (Di Ma company of the U.S.),

6.CYP probe substrate (20 μM of dextromethorphan, SIGMA Q0750) and positive control inhibitor (quinindium, SIGMA D9684)。

Two, experimental procedure

The PBS buffer solution of 100mM is configured, it is molten with the particle liquid solution of buffer 2.5mg/ml and the NADPH of 5mM Liquid, with the compound working solutions (150,50,15,5,1.5,0.15,0.015,0 μM) of PBS gradient dilution 5X concentration.With PBS ladder The quinindium working solution (150,50,15,5,1.5,0.15,0.015,0 μM) of degree dilution 5X concentration.With PBS be diluted to 20 μM it is dense The dextromethorphan working solution of degree.

The particle liquid solution of 2.5mg/ml, 20 μM of dextromethorphan working solution, MgCl are taken respectively₂Solution and compound work Liquid (150,50,15,5,1.5,0.15,0.015,0 μM, different reaction systems is arranged in each concentration) each 20 μ l is uniformly mixed. The quinindium of positive controls same concentrations replaces compound.Simultaneously by the NADPH solution of 5mM together in 37 DEG C of preincubates 5 Minute, take 20 μ l NADPH to be added in a hole after 5 minutes, starting reaction is incubated for 30 minutes.All samples of incubation set double samples This.The acetonitrile of 250 μ l containing the internal standards is added within 30 minutes in backward all samples, mixes, 800rpm shakes 10 minutes.3700rpm centrifugation 10 minutes.The supernatant for taking 80 μ l is transferred to LC-MS/MS analysis.

Drug is calculated to the IC in CYP2D6 metabolism site through Graphpad Prism in numerical value₅₀Value is shown in Table 4.

IC of 4 the compounds of this invention of table to CYP2D6 metabolism site₅₀Value

Embodiment number	IC50(μM)
		1	4.6
3	5.2

Conclusion: the compounds of this invention is weak to the inhibition of enzyme activity effect of people's hepatomicrosome CYP2D6, shows preferably to pacify Quan Xing, prompt will not occur that metabolic drug interaction occurs based on CYP2D6.

Test case 5, the compounds of this invention are to the blocking effect of hERG potassium current

1, experiment purpose

The compounds of this invention is tested on the stable cell line of transfection hERG potassium channel using system for automatic patch-clamp to hERG The blocking effect of potassium current.

2, experimental method

2.1 experimental materials and instrument

2.1.1 experimental material:

Reagent name	Supply of material company	Article No.
			FBS	GIBCO	10099
Sodium pyruvate solution	sigma	S8636-100ML
			MEM nonessential amino acid solution (100 ×)	sigma	M7145-100ML
G418 sulfate	Enzo	ALX-380-013-G005
			MEM	Hyclone	SH30024.01B
hERG cDNA	Origene	-

2.1.2 laboratory apparatus:

2.2 system for automatic patch-clamp experimental procedures

HEK293-hERG stable cell line is according to the density of 1:4 in MEM/EBSS culture medium (10%FBS, 400 μ g/ml G418,1%MEM nonessential amino acid solution (100 ×), 1% sodium pyruvate solution) in carry out secondary culture, culture 48-72 it is small When within carry out system for automatic patch-clamp experiment.After experimental day digests cell with 0.25% pancreatin, cell is collected by centrifugation, with thin Extracellular fluid (140mM NaCl, 4mM KCl, 1mM MgCl₂, 2mM CaCl₂, 5mMD Dextrose Monohydrate, 10mM Hepes, pH7.4, Cell 298mOsmol) is resuspended, cell suspension is made.Cell suspension is placed on the cell bank of Patchliner instrument, Cell is added on chip (NPC-16) by Patchliner instrument using negative pressure controller, and negative pressure attracts individual cells in chip Aperture on.After forming full cell pattern, instrument will obtain hERG electric current according to the hERG Current Voltage program of setting, then Instrument automatically by low concentration to high concentration, carries out compound perfusion.Pass through HEAK Patchmaster, HEAK EPC10 diaphragm It is soft to clamp the data analysis that amplifier (Nanion) and Pathlinersoftware and Pathcontrol HTsoftware are provided Part, under each concentration of compound electric current and blank control electric current analyze.

2.3 test result

The compounds of this invention is measured the blocking effect of hERG potassium current by above test, the IC measured₅₀Value It is shown in Table 5.

IC of 5 the compounds of this invention of table to the blocking effect of hERG potassium current₅₀

Embodiment number	IC50(μM)
		1	> 30
3	> 30

Conclusion: the compounds of this invention is weak to the inhibiting effect of hERG, and the side effect possibility as caused by hERG access is small.

Claims (20)

1. a kind of logical formula (I) compound represented:

Or its tautomer, mesomer, racemic modification, enantiomter, diastereoisomer, or mixtures thereof form or Its pharmaceutical salt,

Wherein:

For cis or trans；

L¹Selected from-O- ,-C (O)-,-S (O)_t-、-NR⁶-、-N(R⁶)C(O)-、-C(O)N(R⁶)-、-N(R⁶)S(O)₂And-S (O)₂N (R⁶)-；

R¹Selected from hydrogen atom, alkyl, halogenated alkyl, alkenyl, alkynyl, naphthenic base, heterocycle, aryl and heteroaryl, wherein described Alkyl, alkenyl, alkynyl, naphthenic base, heterocycle, aryl and heteroaryl are optionally selected from alkyl, alkoxy, halogen each independently One or more in element, halogenated alkyl, hydroxyl, hydroxyalkyl, cyano, amino, nitro, naphthenic base, heterocycle, aryl and heteroaryl Replaced a substituent group；

R²Selected from hydrogen atom, halogen, alkyl, alkoxy, halogenated alkyl, hydroxyl, hydroxyalkyl, cyano, amino, nitro, naphthenic base, Heterocycle, aryl and heteroaryl, wherein alkyl, naphthenic base, heterocycle, aryl and the heteroaryl optional quilt each independently Selected from alkyl, alkoxy, halogen, halogenated alkyl, hydroxyl, hydroxyalkyl, cyano, amino, nitro, naphthenic base, heterocycle, aryl and Replaced one or more substituent groups in heteroaryl；

R³Selected from alkyl, halogenated alkyl, hydroxyalkyl, naphthenic base, heterocycle, aryl and heteroaryl, wherein the alkyl, cycloalkanes Base, heterocycle, aryl and heteroaryl are optionally selected from alkyl, alkoxy, halogen, halogenated alkyl, hydroxyl, hydroxyl alkane each independently Base, cyano, amino, nitro, naphthenic base, heterocycle, aryl, heteroaryl ,-NR⁷R⁸、-C(O)NR⁷R⁸、-C(O)R⁹、-C(O)OR⁹ With-S (O)_tR⁹In one or more substituent groups replaced；

R⁴And R⁵It is identical or different, and it is each independently selected from hydrogen atom, halogen, alkyl, alkoxy, halogenated alkyl, hydroxyl, hydroxyl Alkyl, cyano, amino, nitro, naphthenic base, heterocycle, aryl and heteroaryl；

R⁶Selected from hydrogen atom, alkyl, halogenated alkyl, naphthenic base, heterocycle, aryl and heteroaryl；

R⁷And R⁸It is identical or different, and be each independently selected from hydrogen atom, alkyl, halogenated alkyl, naphthenic base, heterocycle, aryl and Heteroaryl, wherein alkyl, naphthenic base, heterocycle, aryl and the heteroaryl are optionally selected from alkyl, alcoxyl each independently One or more of base, halogen, amino, cyano, nitro, hydroxyl, hydroxyalkyl, naphthenic base, heterocycle, aryl and heteroaryl take Replaced Dai Ji；

Alternatively, the R⁷And R⁸It is formed together heterocycle with the nitrogen-atoms being connected, wherein the heterocycle, which removes, contains 1 nitrogen Except atom, also optionally containing 1~2 identical or different hetero atom selected from N, O and S, and the heterocycle is optional It is selected from alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxyl, hydroxyalkyl, naphthenic base, heterocycle, aryl and heteroaryl In one or more substituent groups replaced；

R⁹Selected from hydrogen atom, alkyl, halogenated alkyl, amino, hydroxyl, hydroxyalkyl, naphthenic base, heterocycle, aryl and heteroaryl；And And

T is 0,1 or 2.

2. logical formula (I) compound represented according to claim 1, to lead to formula (II) compound represented:

Or its tautomer, mesomer, racemic modification, enantiomter, diastereoisomer, or mixtures thereof form or Its pharmaceutical salt,

Wherein:

For cis or trans；

Alk is alkylidene, optionally by selected from alkyl, alkoxy, halogen, halogenated alkyl, hydroxyl, hydroxyalkyl, cyano, amino, Replaced one or more substituent groups in nitro, naphthenic base, heterocycle, aryl and heteroaryl；

R⁷And R⁸It is identical or different, and be each independently selected from hydrogen atom, alkyl, halogenated alkyl, naphthenic base, heterocycle, aryl and Heteroaryl, wherein alkyl, naphthenic base, heterocycle, aryl and the heteroaryl are optionally selected from alkyl, alcoxyl each independently One or more of base, halogen, amino, cyano, nitro, hydroxyl, hydroxyalkyl, naphthenic base, heterocycle, aryl and heteroaryl take Replaced Dai Ji；

Alternatively, the R⁷And R⁸It is formed together heterocycle with the nitrogen-atoms being connected, wherein the heterocycle, which removes, contains 1 nitrogen Except atom, also optionally containing 1~2 identical or different hetero atom selected from N, O and S, and the heterocycle is optional It is selected from alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxyl, hydroxyalkyl, naphthenic base, heterocycle, aryl and heteroaryl In one or more substituent groups replaced；

L¹、R¹、R²、R⁴And R⁵As defined in claim 1.

3. logical formula (I) compound represented according to claim 1 or 2, to lead to formula (III) compound represented:

Or its tautomer, mesomer, racemic modification, enantiomter, diastereoisomer, or mixtures thereof form or Its pharmaceutical salt,

Wherein:

For cis or trans；

R⁷And R⁸It is formed together heterocycle with the nitrogen-atoms being connected, wherein the heterocycle is in addition to containing 1 nitrogen-atoms, Also optionally containing 1~2 identical or different hetero atom selected from N, O and S, and the heterocycle is optionally selected from alkane One in base, alkoxy, halogen, amino, cyano, nitro, hydroxyl, hydroxyalkyl, naphthenic base, heterocycle, aryl and heteroaryl Or replaced multiple substituent groups；

The integer that n is 1 to 6；

L¹、R¹、R²、R⁴And R⁵As defined in claim 1.

4. logical formula (I) compound represented described in any one of claim 1 to 3, wherein the R⁴And R⁵It is hydrogen Atom.

5. logical formula (I) compound represented according to any one of claims 1 to 4, wherein the L¹For-O-.

6. logical formula (I) compound represented according to any one of claims 1 to 5, to change shown in logical formula (IV) Close object:

Or its tautomer, mesomer, racemic modification, enantiomter, diastereoisomer, or mixtures thereof form or Its pharmaceutical salt,

Wherein:

R¹⁰Selected from alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxyl, hydroxyalkyl, naphthenic base, heterocycle, aryl and miscellaneous Aryl；

Q is 0,1,2 or 3, and preferably q is 0；

M is 0,1,2 or 3, preferably 1 or 2；

The integer that n is 1 to 6；

R¹And R²As defined in claim 1.

7. logical formula (I) compound represented described according to claim 1~any one of 6, for chemical combination shown in logical formula (V) Object:

Or its tautomer, mesomer, racemic modification, enantiomter, diastereoisomer, or mixtures thereof form or Its pharmaceutical salt,

Wherein:

R¹⁰Selected from alkyl, alkoxy, halogen, amino, cyano, nitro, hydroxyl, hydroxyalkyl, naphthenic base, heterocycle, aryl and miscellaneous Aryl；

Q is 0,1,2 or 3, and preferably q is 0；

M is 0,1,2 or 3, preferably 1 or 2；

The integer that n is 1 to 6；

R¹And R²As defined in claim 1.

8. the logical formula (I) compound represented according to any one of claim 2~7, wherein the n is 2 or 3.

9. logical formula (I) compound represented described according to claim 1~any one of 8, wherein the R¹For alkyl.

10. logical formula (I) compound represented described according to claim 1~any one of 9, wherein the R²For alkyl.

11. logical formula (I) compound represented described according to claim 1~any one of 10, is selected from:

12. a kind of general formula (IA) compound represented:

Or its tautomer, mesomer, racemic modification, enantiomter, diastereoisomer, or mixtures thereof form or Its pharmaceutical salt,

Wherein:

L¹、R¹、R²、R⁴And R⁵As defined in claim 1.

13. general formula (IA) compound represented according to claim 12, is selected from:

14. a kind of method for preparing logical formula (I) compound represented according to claim 1, this method comprises:

The compound and general formula (IB) compound of general formula (IA) react to obtain the compound of logical formula (I)；

Wherein:

R¹~R⁵And L¹As defined in claim 1.

15. a kind of pharmaceutical composition, described pharmaceutical composition contain therapeutically effective amount according to claim 1~any one of 11 It is the logical formula (I) compound represented or its tautomer, mesomer, racemic modification, enantiomter, diastereomeric different Structure body, or mixtures thereof form or its pharmaceutical salt and one or more pharmaceutically acceptable carriers, diluent or tax Shape agent.

16. logical formula (I) compound represented or its tautomer described according to claim 1~any one of 11 interior disappear Revolve body, racemic modification, enantiomter, diastereoisomer, or mixtures thereof form or its pharmaceutical salt or according to right It is required that purposes of the pharmaceutical composition described in 15 in the drug that preparation is used for excitement TLR7.

17. logical formula (I) compound represented or its tautomer described according to claim 1~any one of 11 interior disappear Revolve body, racemic modification, enantiomter, diastereoisomer, or mixtures thereof form or its pharmaceutical salt or according to right It is required that pharmaceutical composition described in 15 is preparing the purposes in the drug for treating infection caused by virus.

18. purposes according to claim 17, wherein the virus is selected from: dengue fever virus, yellow fever virus, Xi Niluo Virus, japanese encephalitis virus, tick-brone encephalitis virus, elder brother's saliva virus, Murray valley encephalitis virus, St. Louis encephalitis virus, E Mu This gram of hemorrhagic fever viruse, bovine viral diarrhea virus, zika virus, HIV, HBV, HCV, HPV, RSV, SARS and influenza virus.

19. logical formula (I) compound represented or its tautomer described according to claim 1~any one of 11 interior disappear Revolve body, racemic modification, enantiomter, diastereoisomer, or mixtures thereof form or its pharmaceutical salt or according to right It is required that pharmaceutical composition described in 15 is preparing the purposes in the drug for treating or preventing tumour.

20. purposes according to claim 19, wherein the tumour is selected from melanoma, non-small cell lung cancer, liver cell Cancer, basal-cell carcinoma, clear-cell carcinoma and myeloma.