CN103748081B - Infect and the quinazoline derivant of other disease for treating virus - Google Patents

Abstract

The present invention relates to quinazoline derivant, for preparing their method, pharmaceutical composition and their purposes in the treatment.

Description

Infect and the quinazoline derivant of other disease for treating virus

The present invention relates to quinazoline derivant, for prepare their method, pharmaceutical composition and they in the treatment Purposes.

The present invention relates to quinazoline derivant purposes in treatment virus infection, immunity or inflammatory imbalance, thus relate to The regulation of toll sample receptor (TLRs) or agonism.Toll-like receptor is main transmembrane protein, it is characterised in that a kind of Extracellular rich in leucic domain and comprise a conservative region Cytoplasm extend.Innate immune system can be via These TLR expressed on the cell surface of certain form of immunocyte identify pathogen associated molecular pattern.Exotic disease The identification of substance has activated the generation of cytokine and the rise of the costimulatory molecules in phagocyte.This causes T cell The regulation of behavior.

Estimate the Toll-like receptor type that major part mammal species has between ten kinds and 15 kinds.People Class is together with identifying 13 kinds of TLR (simple named TLR1 to TLR13) in mice, and in other mammalian species Middle discovery their equivalent form of many.But, the equivalent form of some TLR found in the mankind is not present in all sucklings and moves In thing.Such as, a kind of coding is similar to the gene of the protein of TLR10 in the mankind and is present in mice, but this expression Go out over and damaged by a kind of retrovirus retrovirus in certain site.On the other hand, mice expresses TLR11, TLR12 and TLR13, they The most it is not presented in the mankind.Other mammal can express the TLR not yet found in the mankind.Other nonmammalian Species can have the TLR different from mammal, and such as TLR14, (it is sent out in (Takifugu pufferfish) in the Orient Existing) confirmed.This is so that use laboratory animal to complicate as the method for mankind's innate immunity model.

Detailed overview for toll sample receptor sees following journal article.Hoffmann, J.A (Huffman, J.A) Nature (" naturally ") 426, p33-38,2003；Akira, S. (A Jila, S), Takeda, K. (bamboo field, K) and Kaisho, T. (shellfish is few, T) Annual Rev.Immunology (" immunology yearbook "), 21, p335-376,2003；Ulevitch, R.J. are (outstanding Le Weiqi, R.J.) Nature Reviews:Immunology (" summary naturally: immunology ") 4, p512-520,2004.

Compound activated to Toll-like receptor is indicated to be described, such as in WO 2,006 117670 Phonetic in adenine derivative in purine derivative, WO 98/01448 and WO 99/28321 and WO 2009/067081 Pyridine.

But, there is a kind of strong needs for novel Toll-like receptor instrumentality, these instrumentalities and existing skill The compound of art is compared has preferred selectivity, higher effect, higher metabolic stability and the safety of improvement Property.

In treatment that some virus infects, interferon (IFN α) regular injections can be given, such as hepatitis C virus Poison (HCV), (Fried etc., Peginterferon-alfa plus ribavirin for chronic hepatitis C Virus infection (Peg-IFN alpha-2b-α combines Ribavirin chronic hcv infection), N Engl J Med (New England Journal of Medicine) 2002；347:975-82).Orally available little molecule IFN inducer provides the immunity of reduction Originality and give the potential advantages of convenience.Therefore, novel IFN inducer be for treat virus infection potential effectively Novel drugs classification.About having an example of the document of the little molecule IFN inducer of antiviral effect, see De Clercq, E. (moral Clarke, E.)；Descamps, J. (enlightening SIKA Paasche, J.)；De Somer, P. (Durso Mel, P.) Science (" science ") 1978,200,563-565.

In the treatment of certain form of cancer, give the IFN α with other drug combination (see for example Eur.J.Cancer (Europe Journal of Cancer) 46,2849-57, and Cancer Res. (cancer research) 1992,52,1056). Due to the ability of TLR 7/8 agonist induction significant Th1 response, they are also vaccine adjuvants interested.

According to the invention provides a kind of compound with chemical formula (I)

Or its a kind of pharmaceutically acceptable salt, solvate or polymorph, wherein

R₁It is C_3-8Alkyl, C_3-8Alkoxyl, C_2-6Thiazolinyl or C_2-6Alkynyl, each of which is optionally taken by one or more Replacing for base, these substituent groups are independently selected from halogen, hydroxyl, amino, nitrile, ester, amide, C_1-3Alkyl, C_1-3Alkoxyl or C_3-6 Cycloalkyl,

R₂It is hydrogen, halogen, hydroxyl, amine, C_1-7Alkyl, C_1-7Alkylamino, C_1-6Alkoxyl, (C_1-4) alkoxyl-(C_1-4) alkane Base, C_3-6Cycloalkyl, C_4-7Heterocycle, aromatic series bicyclic heterocycles, aralkyl, heteroaryl, heteroarylalkyl, carboxylic acid amide, carboxylate, it Be each optionally substituted with one or more substituents, these substituent groups are independently selected from halogen, hydroxyl, amino, C_1-6Alkane Base, two-(C_1-6) alkylamino, C_1-6Alkylamino, C_1-6Alkyl, C_1-6Alkoxyl, C_3-6Cycloalkyl, carboxylic acid, carboxylate, carboxylic acid amide, Heterocycle, aryl, thiazolinyl, alkynyl, aralkyl, heteroaryl, heteroarylalkyl or nitrile,

R₃It is hydrogen, halogen, hydroxyl, amine, C_1-7Alkyl, C_1-7Thiazolinyl, C_1-7Alkynyl, C_1-7Alkylamino, C_1-6Alkoxyl, (C_1-4) Alkoxyl-(C_1-4) alkyl, C_3-6Cycloalkyl, C_4-7Heterocycle, aromatic series bicyclic heterocycles, aralkyl, heteroaryl, heteroarylalkyl, virtue oxygen Base, heteroaryl epoxide, ketone, nitrile, each of which is optionally substituted with one or more substituents, and these substituent groups are selected independently From halogen, hydroxyl, amino, C_1-6Alkyl, two-(C_1-6) alkylamino, C_1-6Alkylamino, C_1-6Alkyl, C_1-6Alkoxyl, C_3-6Cycloalkanes Base, carboxylic acid, carboxylate, carboxylic acid amide, heterocycle, aryl, thiazolinyl, alkynyl, aralkyl, heteroaryl, heteroarylalkyl or nitrile.

R₄It is hydrogen, halogen, hydroxyl, amine, C_1-7Alkyl, C_1-7Alkylamino, C_1-6Alkoxyl, (C_1-4) alkoxyl-(C_1-4) alkane Base, C_3-6Cycloalkyl, C_4-7Heterocycle, aromatic series bicyclic heterocycles, aralkyl, heteroaryl, heteroarylalkyl, aryloxy group, heteroaryl epoxide, Each of which is optionally substituted with one or more substituents, and these substituent groups are independently selected from halogen, hydroxyl, amino, C_1-6 Alkyl, two-(C_1-6) alkylamino, C_1-6Alkylamino, C_1-6Alkyl, C_1-6Alkoxyl, C_3-6Cycloalkyl, carboxylic acid, carboxylate, carboxylic acyloxy Amine, heterocycle, aryl, thiazolinyl, alkynyl, aralkyl, heteroaryl, heteroarylalkyl or nitrile, and

R₅Being hydrogen, fluorine, chlorine or methyl, its condition is:

R₂、R₃、R₄, and R₅Can not be H entirely.

In a first embodiment, the invention provides the compound with chemical formula (I), wherein R₁Be butyl, penta Base or 2-amyl group, and wherein R₂、R₃、R₄And R₅It is as specified above.

In a further embodiment, the present invention relates to the compound with chemical formula (I), wherein R₁By a hydroxyl The substituted C of base_4-8Alkyl, and wherein R₂、R₃、R₄, and R₅It is as specified above.

Another embodiment relates to the compound with chemical formula (I), wherein works as R₁It is the C being optionally substituted by a hydroxyl group_4-8During alkyl It is one below:

In another embodiment, the invention provides the compound with chemical formula (I), wherein R₅Preferably hydrogen or Fluorine, and R₁、R₂、R₃And R₄It is as described above.

There is compound and their pharmaceutically acceptable salt, solvate or the polymorph tool of chemical formula (I) There is the activity as medicine, especially as the activity of Toll-like receptor (especially TLR7 and/or TLR8) instrumentality.

Therefore, at an other aspect, the invention provides a kind of pharmaceutical composition, this pharmaceutical composition includes having The compound of chemistry formula (I) or its a kind of pharmaceutically acceptable salt, solvate or polymorph are together with one or more medicines Acceptable excipient, diluent or carrier on.

Additionally, according to the compound with chemical formula (I) of the present invention or its a kind of pharmaceutically acceptable salt, solvation Thing or polymorph, or include the described compound with chemical formula (I) or its a kind of pharmaceutically acceptable salt, solvent The pharmaceutical composition of compound or polymorph can serve as medicament.

Another aspect of the present invention is: have the compound of chemical formula (I) or its a kind of pharmaceutically acceptable salt, molten Agent compound or polymorph, or include the described compound with chemical formula (I) or its a kind of pharmaceutically acceptable salt, The described pharmaceutical composition of solvate or polymorph can be respectively used for treating the regulation relating to TLR7 and/or TLR8 Imbalance.

Term " alkyl " refers to comprise the straight or branched saturated aliphatic hydrocarbon specifying number carbon atom.

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

Term " thiazolinyl " refers to alkyl as defined above, and this alkyl is by least two carbon atom and at least one carbon-to-carbon Double bond forms.

Term " alkynyl " refers to alkyl as defined above, this alkyl by least two carbon former give and at least one carbon- Carbon three key forms.

Term " cycloalkyl " refers to comprise the carbocyclic ring specifying number carbon atom.

Term " alkoxyl " refers to that singly-bound is connected to alkyl (carbon and the hydrogen chain) group of oxygen, as such as methoxy group or second Epoxide group.

Term " aryl " refers to optionally include that one or two is selected from the miscellaneous former of N, O and S (being especially selected from N and O) The aromatic ring structure of son.Described aromatic ring structure can have 5,6 or 7 annular atomses.Especially, described aromatic ring knot Structure can have 5 or 6 annular atomses.

Term " aryloxy group " refers to a kind of aromatic ring structure.Described aromatic group is individually bonded on oxygen, as such as Phenol.

Term " heteroaryl epoxide " refers to optionally include that one or two is selected from the heteroatomic aromatic series of N, O and S Ring structure.Described aromatic group comprises 5 to 7 annular atomses, and one of them is individually bonded on oxygen, as such as hydroxyl pyrrole Pyridine.

Term " bicyclic heterocycles " refers to a kind of aromatic ring structure, as the term being made up of two fused aromatic rings " aryl " is defined.Each ring optionally includes the hetero atom selected from N, O and S (being especially selected from N and O).

Term " aralkyl " refers to as defined in the term " aryl " optionally replaced by a kind of alkyl group A kind of aromatic ring structure.

Term " heteroarylalkyl " refers to as determined for the term " heteroaryl " optionally replaced by a kind of alkyl group The aromatic ring structure of justice.

Heterocycle refers to following such molecule, and they are saturated or fractional saturation, and includes ether, tetrahydrochysene furan Mutter, dioxane or other cyclic ethers.The heterocycle comprising nitrogen include such as azetidine, morpholine, piperidines, piperazine, pyrrolidine and Analog.Other heterocycle includes such as thiomorpholine, Dloxole alkyl (dioxolinyl) and ring sulfone.

Heteroaryl groups is to be essentially aromatic heterocyclic group.These comprise one or more selected from N, O or S Heteroatomic monocycle, bicyclo-or multi-ring.Heteroaryl groups can be such as imidazole radicals, isoxazolyl, furyl, oxazolyl, pyrrole Cough up base, pyriconyl, pyridine radicals, pyridazinyl, pyrazinyl,

The pharmaceutically acceptable salt of the compound with chemical formula (I) includes its acid-addition salts and alkali salt.Suitably Acid-addition salts is formed from the acid formation of nontoxic salts.Suitably alkali salt is formed from the alkali formation of nontoxic salts.

The compound of the present invention can also be by presented in non-solvated and solvation.Term " solvent is used at this Compound " to describe compound and one or more the pharmaceutically acceptable solvent molecules (such as, ethanol) including the present invention Molecular complex.

Term " polymorph " refers to the ability that the compound of the present invention exists with form or the crystal structure of more than one.

The compound of the present invention can be given with crystalline state or amorphous product.Can by such as precipitating, crystallize, The method of lyophilization, spray drying or evaporation drying obtains in solid plug, powder or these compounds of film.They can be single Solely give or combine with other compounds of one or more present invention to give or give with the combination of one or more other drugs. Generally, they will give as the preparation combined with one or more pharmaceutically acceptable excipient.Use at this Term " excipient " is to describe any composition in addition to one or more compounds of the present invention.The selection of excipient is generally Depend on the most specifically giving pattern, excipient to factors such as the character of dissolubility and the impact of stability and dosage form.

The compound of the present invention or its any subgroup can be formulated as the different pharmaceutical form for giving purpose.Permissible Quote all of compositions of Formulations for systemic administration that is generally used for as suitable compositions.In order to prepare the drug regimen of the present invention Thing, using as active component, effective dose, the specific compound that is optionally in addition salt form is with pharmaceutically acceptable Carrier combinations be immixture, this carrier can depend on the desired object form for giving and take not similar shape Formula.It is desirable that these pharmaceutical compositions be in be suitable for such as being administered orally, unit dosage forms that per rectum or percutaneous give.Example As, in preparation is in the compositions of peroral dosage form, any common medicinal medium can be used, such as, as at liquid oral Water in the case of goods (such as suspending agent, syrup, elixir, Emulsion and solution), ethylene glycol, oils, alcohols and similar Thing；Or the solid carrier in the case of powder, pill, capsule and tablet, such as starch, sugar, Kaolin, diluent, Lubricant, binding agent, disintegrating agent and the like.Tablet and capsule represent best oral because they are prone to give Unit dosage forms, the most obviously uses solid pharmaceutical carriers.Be additionally included in use not long ago can be converted into liquid The solid form goods of bodily form formula.Be applicable to the compositions of percutaneous dosing, carrier optionally include penetration enhancers and/ Or the wetting agent being suitable for, optionally combining with the additive being suitable for any character accounting for small percentage, these add Skin is not introduced by agent significantly toxic action.Described additive can promote give skin and/or can aid in system Standby desired compositions.These compositionss can be given by different way, such as, as transdermal patch, as putting agent, as soft Unguentum.Or can also be blown into via oral cavity suction by means of the method for using in the administration field of thus mode and formula Method gives the compound of the present invention.Therefore, generally, the compound of the present invention can be with solution, suspending agent or dry powder Form and be given to pulmonary.

It is particularly advantageous to prepare the aforesaid pharmaceutical composition being in unit dosage forms, in order to give consistent with dosage Property.Unit dosage forms refers to be suitable as the discrete unit physically of unit dose as used herein, and each unit comprises pre- Quantitative active component, the active component of this scheduled volume is computed combining with required pharmaceutical carrier and producing desired treatment Effect.The example of this type of unit dosage forms is tablet (including the tablet of indentation or coating), capsule, pill, powder packets (powder Packet), wafer (wafer), suppository, Injectable solution or suspending agent and similar dosage form, and separate multiple.

Those of ordinary skill in infectious disease treatment field will be able to come really from test result presented below Determine effective dose.Generally, should be taken into account that day effective dose will be from 0.01mg/kg to 50mg/kg body weight, more preferably from 0.1mg/kg To 10mg/kg body weight.Can suitably by required dosage in whole day with appropriate intervals be two, three, four Individual or more sub-doses.Can be formulated as unit dosage forms by described sub-doses, the most each unit dosage forms comprises 1mg extremely The active component of 1000mg and particularly 5mg to 200mg.

As known to those of ordinary skill in the art, the exact dose of administration and frequency depend on the having used The particular compound of formula (I), treated concrete disease, the treated seriousness of disease, concrete patient Age, body weight and overall physical health situation, the other drug can taken together with individuality.Furthermore, it is to be understood that this effective dose is permissible Reducing or increase, this depends on the reaction of treated experimenter and/or depends on prescribing these compounds of the present invention The assessment of doctor.The scope of effective dose the most mentioned above only instruct rather than be intended to the scope of the present invention or Purposes limits to any degree.

The preparation of compound

There is the compound of chemical formula (I) according to scheme 1 preparation.2,4-dichloro-quinazoline can divide a few step react to carry 2,4-diaminourea quinazoline for acceptable yield.In the first step, will in the case of with or without transition-metal catalyst 2,4-dichloro-quinazolines mix with amine or heat, to provide 2-chloro-4-amido quinazoline.At processing thick 2-chloro-4-amino After quinazoline, this intermediate is had a kind of ammonia source (such as, the ammonia in methanol) and optionally having the pressure of CuO Force container heats.

Scheme 1

The compound of chemical formula (I) also can be had according to scheme 2 preparation.According at the document (JOC such as O ' Hara (Ou Hala) (1991) 56, p776) method described in uses a kind of alcoholic solvent (such as ethanol) or diethylene glycol dimethyl ether, in acid condition In the presence of excess cyanamide, substituted o-amino benzoyl ester (IV) is heated.Via some different approach, by 2-amino-4-hydroxyl Base quinazoline (V) carries out amine subsequently and replaces.In an example, can be by intermediate V at phosphorus oxychloride (POCl₃) existence Under in the case of with or without solvent heat.After removal of solvents, can add pure or in polar solvent (such as second Nitrile) in the presence of this amine, with room temperature or by heating provide VI.The second approach be in the presence of DBU and this amine will in Mesosome V reacts with a kind of coupling agent such as BOP or PyBOP.This reaction occurs in a kind of polar solvent (such as DMP).The third Method is the 2-amino group protected in intermediate V with a carboxyl groups.Intermediate V is anti-with anhydride (such as acetic anhydride) Should, the most some hours.This solvent is under reduced pressure removed and crude product can be stood as above with POCl₃Reaction subsequently.Protection will be had by the reaction in a kind of basic solvent (the such as Feldalat NM in methanol) The carboxyl groups of effect is leniently removed.

Scheme 2

Experimental section.

The preparation of intermediate A

Under agitation to 2,4-bis-chloro-6,7-dimethoxyquinazoline (500mg, 1.9mmol), diisopropylethylamine The mixture of (0.73mL, 4.2mmol) and acetonitrile (0.1mL) drips n-butylamine in acetonitrile (5mL) (0.19mL, 1.9mmol) solution.Allow this mixture is stirred one day at ambient temperature.Add ethyl acetate, with saturated aqueous ammonium chloride Wash this organic layer.This organic layer is removed, is dried with magnesium sulfate.By solid via filtering removal, to provide crude product A, just So in next step.

The preparation of compound 1

Intermediate A (0.5g, 1.7mmol) is placed on the 20mL pressure vessel of the 7N ammonia having in methanol (15mL) In, and it is added to CuO (242mg, 1.7mmol).Heat the mixture to 130 DEG C under this container is sealed and stirred hold Continuous 18 hours.Allow this reaction is cooled to room temperature.By solid via filtering removal, and the solvent of filtrate is under reduced pressure gone Remove.Thick material is purified via reversed-phase column chromatography method (Vydac Denali C18 tubing string 10 μm, 250g, 5cm).Flowing phase (0.25% NH in water₄HCO₃Solution, CH₃CN)。

The preparation of 9

Step 1.To equipped with the 500mL round-bottomed flask of magnetic stirring bar is placed 2-amino-6-methoxybenzoic acid first Ester (25g, 149.6mmol), ethanol (200mL), cyanamide (9.43g, 224mmol) and dense HCl (6mL).Allow this mixing Thing stirs 6 hours under reflux.Little at present at one, interval, add dense HCl (0.5mL).This reactant mixture is allowed to be cooled to Room temperature, and by this solid V-1 via filtering separation and using washing with alcohol.

LC-MS m/z=192 (M+H).

¹H NMR (400MHz, DMSO-d₆) δ ppm3.88 (s, 3H), 6.96 (dd, J=8.2,3.1Hz, 2H), 7.69 (t, J =8.3Hz, 1H), 8.28 (br.s., 2H), 12.67 (br.s., 1H)

Step 2.

In a 50mL bottle place V-1 (250mg, 1.24mmol), dry DMF (5mL), DBU (0.6g, 3.73mmol) and BOP (659mg, 1.49mmol).This mixture is stirred at room temperature 2 hours, adds n-butylamine (287mg, 3.37mmol), and allow to be stirred at room temperature this reaction 15 hours.This solvent is reduced volume, and by residual Excess uses dichloromethane to be purified to the gradient of the methanol of 10% in dichloromethane via silica gel column chromatography.By best Part carries out poly-pond, is under reduced pressure gone by solvent divided by providing 9.

Following intermediate is prepared according to the method preparing V-1.

LC-MS m/z=240/242

¹H NMR (400MHz, DMSO-d₆) δ ppm3.09-3.55 (m, 2H), 7.09 (br.s., 1H), 7.26 (dd, J= 7.9,1.3Hz, 1H), 7.37-7.48 (m, 2H)

LC-MS m/z=196 (M+H)

¹H NMR (400MHz, DMSO-d₆) δ ppm7.00 (br.s., 2H) 7.13 (d, J=7.78Hz, 1H) 7.18 (d, J= 8.28Hz, 1H) 7.50 (t, J=8.03Hz, 1H), do not observe phenol proton.

LC-MS m/z=176 (M+H)

LC-MS m/z=180 (M+H)

¹H NMR (400MHz, DMSO-d₆) δ ppm6.98 (dd, J=11.0,8.3Hz, 1H), 7.13 (d, J=8.3Hz, 1H), 7.51 (br.s., 2H), 7.64 (td, J=8.3,5.8Hz, 1H), 12.30 (br.s, 1H)

LC-MS m/z=180 (M+H)

LC-MS m/z=239/241 (M+H)

¹H NMR (400MHz, DMSO-d₆) δ ppm7.32 (d, J=8.8Hz, 1H), 7.49 (s, 2H), 7.71 (br.s., 1H), 7.81 (dd, J=8.6,24Hz, 1H), 8.00 (d, J=2.4Hz, 1H)

LC-MS m/z=192 (M+H)

LC-MS m/z=176 (M+H)

LC-MS m/z=180 (M+H)

¹H NMR (400MHz, DMSO-d₆) δ ppm7.01-7.16 (m, 2H), 7.56 (br.s., 2H), 7.99 (t, J= 7.7Hz, 1H), 10.38-13.48 (m, 1H)

LC-MS m/z=196 (M+H)

¹H NMR (400MHz, DMSO-d₆) δ ppm7.41 (dd, J=8.5,2.0Hz, 1H), 7.55 (d, J=2.0Hz, 1H), 7.98 (d, J=8.5Hz, 1H), 8.49 (br.s., 2H), 10.79-13.69 (m, 1H)

LC-MS m/z=176 (M+H)

¹H NMR (400MHz, DMSO-d₆) δ ppm2.43 (s, 3H), 7.22 (d, J=1.0Hz, 1H), 7.24 (s, 1H), 7.89 (d, J=8.0Hz, 1H), 8.29 (br.s., 2H), 12.65 (br.s, 1H)

LC-MS m/z=192 (M+H)

LC-MS m/z=220 (M+H)

¹H NMR (400MHz, DMSO-d₆) δ ppm3.87-3.95 (m, 3H), 7.12-7.47 (m, 1H), 7.83 (dd, J= 8.3,1.4Hz, 1H), 7.99 (d, J=1.3Hz, 1H), 8.07-8.13 (m, 1H), 8.43 (br.s., 2H)

LC-MS m/z=198 (M+H)

LC-MS m/z=298 (M+H)

¹H NMR (400MHz, DMSO-d₆) δ ppm3.85 (s, 3H), 5.10 (s, 2H), 6.17 (br.s., 2H), 6.70 (s, 1H), 7.30-7.36 (m, 2H), 7.40 (t, J=7.4Hz, 2H), 7.44-7.48 (m, 2H), 10.82 (br.s., 1H)

LC-MS m/z=180 (M+H)

¹H NMR (400MHz, DMSO-d₆) δ ppm6.51-6.67 (m, 2H), 7.00-7.08 (m, 1H), 7.42 (ddd, J= 11.2,7.91.3Hz, 1H), 7.69 (dd, J=7.9,0.6Hz, 1H), 11.08 (br.s., 1H)

LC-MS m/z=196 (M+H)

LC-MS m/z=176 (M+H)

¹H NMR (400MHz, DMSO-d₆) δ ppm2.41 (s, 3H), 7.15 (t, J=7.5Hz, 1H), 7.43 (br.s., 2H), 7.55 (d, J=7.0Hz, 1H), 7.80 (d, J=7.8Hz, 1H), 11.17-12.49 (m, 1H)

The preparation of 10

Step 1.The preparation of V-6.To equipped with in the 50mL bottle of magnetic stirring bar place V-3 (500mg, 2.16mmol), phenylboric acid (342mg, 2.8mmol), potassium carbonate (1.19g, 8.62mmol), dioxane (5.5mL), water (1.8mL) and tetrakis triphenylphosphine palladium (249mg, 0.215mmol).Blast nitrogen to this reactant mixture and continue 10 minutes. This bottle is sealed and is heated to 130 DEG C.This reaction is cooled to room temperature, and solvent is under reduced pressure removed.By thick material Via reversed-phase column chromatography method (RP Vydac Denali C18-10 μm, 200g, 5cm.Flowing is the 0.25%NH in water mutually₄HCO₃ Solution, CH₃CN) it is purified, to provide V-6.

LC-MS m/z=238 (M+H)

Step 2.To equipped with the 50mL bottle of magnetic stirring bar is placed V-6 (148mg, 0.624mmol), dry DMF (3.5mL), DBU (0.373mL, 2.5mmol), BOP (345mg, 0.78mmol), then placement (S)-2-aminopentanol (322mg, 3.12mmol).Allow this reactant mixture is stirred at room temperature 3 days.Volatile matter is under reduced pressure removed, and Crude product is made to be allocated between water and ethyl acetate.These organic layers are merged, is dried (magnesium sulfate), will be solid Body is via filtering removal, and is under reduced pressure removed by the solvent of filtrate.By crude product via reversed-phase column chromatography method (RP SunFire Prep C18 OBD-10 μm, 30 × 150mm) it is purified.Flowing phase (the 0.25%NH in water₄HCO₃Solution, CH₃CN) to provide 10.

The preparation of 11

Step 1.To equipped with the 1L round-bottomed flask of magnetic stirring bar is placed V-1 (8.8g, 46.03mmol) and acetic anhydride (150mL).Under this flask being equipped with a reflux condenser and stirring, this mixture is heated to backflow and continues 15 hours.Logical Filter and separate this precipitate and with diisopropyl ether, be then dried in a vacuum, to provide a kind of white solid V-9.

LC-MS m/z=234 (M+H)

Step 2.To equipped with the 250mL round-bottomed flask of magnetic stirring bar is placed V-9 (4.5g, 19.3mmol) and acetonitrile (100mL).POCl is dripped through 30 minutes₃(5.56mL, 59.8mmol), adds DIPEA (10.3mL, 59.8mmol) subsequently.Should Reactant mixture becomes a kind of brown solution and is stirred at room temperature 2 hours.This reactant mixture is poured into 1M NaOH (100mL) extract in and by ethyl acetate (2 × 100mL).By the organic layer MgSO of merging₄It is dried, by this solid Remove via filtering, and this filtrate is used in next step like this.

Step 3.By the filtering solution in ethyl acetate from step 2 with DIPEA (9.2mL, 53.6mmol) and just Butylamine (3.5mL, 35.8mmol) processes.This reactant mixture is stirred 16 hours at ambient temperature.By this solvent in decompression Lower removal, and in dichloromethane, regenerate crude product and wash with water.Organic layer is dried (MgSO₄), solid is led to Filter and remove, and be evaporated to the solvent of filtrate be dried to obtain orange solids V-11.

LC-MS m/z=289 (M+H)

Step 4.In a 30mL manometer tube place V-11 (2.8g, 9.71mmol), pyridine hydrochloride (6.73g, 58.26mmol) and pyridine (50mL) and this mixture is heated to 120 DEG C continue 16 hours.Pyridine is under reduced pressure removed. Crude product is dissolved in methylene chloride/methanol: in the mixture of 95/5, and washs with 1N HCl solution and water.To have Machine layer is dried (MgSO₄), by solid via filtering removal, and the solvent of filtrate is under reduced pressure gone divided by providing VI- 1。

LC-MS m/z=231 (M-H)

¹H NMR (400MHz, DMSO-d₆) δ ppm0.92 (t, J=7.37Hz, 3H) 1.33-1.43 (m, 2H) 1.50-1.59 (m, 2H) 3.41-3.49 (m, 2H) 5.79-5.88 (m, 1H) 6.02 (d, J=8.14Hz, 1H) 6.91 (br.s., 2H) 6.99- 7.04 (m, 1H) 10.78 (br.s., 1H) 13.35 (br.s., 1H)

Step 5.In a 100mL flask place VI-1 (175mg, 0.753mmol), cesium carbonate (0.74g, 2.26mmol) and DMF (15mL).Mixture is stirred at ambient temperature 30 minutes.Add 2-Bromoethyl methyl ether (0.089mL, 0.94mmol), and this mixture is stirred at room temperature 16 hours.This solvent is under reduced pressure removed, and By thick residue, by HPLC, (RP Vydac Denali C18-10 μm, 250g, 5cm, flow phase (0.25% in water NH₄HCO₃Solution, methanol) it is purified, collect The cream of the crop, and this solvent is under reduced pressure gone divided by obtaining 11, for one Solid.

The preparation of 12

Step 1.V-2 is dissolved in DMF (15mL) and in the oil bath of 80 DEG C and uses N₂Purge 10 minutes.Then add double (triphenylphosphine) Palladous chloride. (II) (69mg, 0.098mmol), triphenylphosphine (57.6mg, 0.22mmol) and Hydro-Giene (Water Science). (42.5mg, 0.22mmol).With N₂After purging 5 minutes, add diethylamine (3.15mL, 30.31mmol), add 2-subsequently Ethynyl pyridine (168mg, 1.63mmol).Stir 16 hours at 80 DEG C by this container closure and by this reaction.By this reaction Mixture pours in frozen water, and is isolated by filtration by precipitate, washes with water and is dried under vacuum.By product at dichloro Methane stirs 30 minutes.It is isolated by filtration precipitate, with dichloromethane and diisopropyl ether, and under vacuo at 50 DEG C Lower dry to obtain V-12.

LC-MS m/z=263 (M-H)

Step 2.At N₂Place in the V-12 (300mg, 1.15mmol) solution in THF (50mL) under (g) atmosphere 10%Pd/C (100mg).This reactant mixture is stirred at room temperature 16 hours, and filters with the kieselguhr filled subsequently.? Under decompression, the solvent of filtrate is removed, to provide crude product V-13, be used as such in next step.

LC-MS m/z=267 (M-H)

Step 3.Method according to preparation 9 prepares example 12.

The preparation of 14

Step 1.Intermediate VI-2 and VI-3 is prepared according to the method preparing VI-1.At room temperature VI-3 is used after agitation Diisopropyl ether separates.

VI-2:LC-MS m/z=337 (M+H)

VI-3:LC-MS m/z=379 (M+H)

Step 2.Compound 14 is prepared according to the method preparing intermediate V-12.

The preparation of 15

Step 1.To equipped with the 500mL round-bottomed flask of magnetic stirring bar is placed 3-aminophthalic acid hydrochlorate (25g, 115mmol), ethanol (250mL), cyanamide (7.25g, 172mmol) and dense HCl (6mL).This flask is equipped with one Reflux condenser and by this mixture return stirring 6 hours.Little at present at one, interval, added dense by glass pipet HCl(0.5mL).Allow this reaction is cooled to room temperature, solvent is under reduced pressure gone divided by providing a kind of yellow oil.By crude product With silica dehydrator, then dichloromethane is used to carry out portion to the gradient of the methanol of 10% in dichloromethane via silica gel column chromatography Separating and purifying.Crude product (yellow solid) V-14 is used as such in next step.

LC-MS m/z=234 (M+H).

Step 2.To equipped with the 100mL round-bottomed flask of magnetic stirring bar is placed V-14 (1.7g, 7.29mmol), anhydrous DMF (25mL), DBU (3.3g, 21.87mmol) and PyBOP (4.55g, 8.75mmol).Allow reactant mixture in room The lower stirring of temperature 1 hour.Then add n-butylamine (2.1g, 29.2mmol), and allow this mixture is stirred at room temperature 15 Hour.Solvent is under reduced pressure removed, and uses 20% methanol in dichloromethane to filter by silica gel this crude product. The solvent of filtrate is under reduced pressure removed and by crude oil (15,4g) via reversed-phase column chromatography method (RP Vydac Denali C18- 10 μm, 200g, 5cm) it is purified.Flowing phase (the 0.25%NH in water₄HCO₃Solution, CH₃CN)。

The preparation of 16

At N₂Add in 10% Pd/C suspension in methanol (25mL) under atmosphere compound 14 (111mg, 0.39mmol).Remove blanket of nitrogen and substituted by hydrogen.Allow to be stirred at room temperature until consuming the hydrogen of 2 equivalents this mixture Gas.The kieselguhr filled by this reactant mixture filters.The solvent of filtrate is under reduced pressure removed.By crude product via silica gel Column chromatography uses dichloromethane to be purified to provide 16 to the gradient of the methanol of 10% in dichloromethane.

The preparation of 18

17 (625mg, 2.28mmol) are dissolved in anhydrous THF (10mL).Dropping LAH (in THF 1M, 3.42mL, 3.42mmol), and by reactant mixture it is stirred at room temperature 3 hours.LC-MS illustrates and is fully converted to desired product. By this reactant mixture by saturated aqueous NH₄Cl carries out cancellation, solid by filtration is removed, and is being subtracted by the solvent of filtrate Subdue and remove.Through pre-HPLC, residue is purified, produces product, for a kind of white solid.

The preparation of 19

By the VI-2 (500mg, 1.48mmol) in DMF (5mL) in 10mL pipe, tetrakis triphenylphosphine palladium (86mg, 0.074mmol) and the mixture of zinc cyanide (106mg, 0.89mmol) is placed on microwave radiation at 160 DEG C lower 10 minutes.Will This mixture is cooled to room temperature and is concentrated under vacuum.Residue is made to be allocated between water and dichloromethane.This is had Machine layer separates, is dried (MgSO₄), solvent is removed by filtration, and the solvent of filtrate is concentrated under vacuum.By this product At CH₃CN grinds, solid by filtration is separated.The Feldalat NM being used in methanol provides after processing one hour at 60 DEG C Acyl group deprotection.By the cooling of this mixture precipitated product.White solid 19 is isolated by filtration and is dried under vacuum.

The preparation of 20

To equipped with in the 50mL bottle of magnetic stirring bar place VI-3 (300mg, 0.79mmol), borate (198mg, 0.95mmol), water (3mL, degassing) and DME (6mL, degassing), interpolation sodium bicarbonate (199mg, 2.37mmol) and PdCl₂ (PPh₃)₂(55mg, 0.079mmol), and this mixture is heated to 90 DEG C lasting 1 hour.This mixture is cooled down and adds second Acetoacetic ester.This organic layer is separated, is dried (MgSO₄), solid by filtration is removed, and by the solvent of filtrate under vacuo Remove.Dichloromethane is used to enter to the gradient of the methanol of 10% in dichloromethane (containing ammonia) via silica gel column chromatography residue Row purification.Collect this product section, and concentrate under vacuo.It is little that the Feldalat NM being used in methanol processes one at 60 DEG C Acyl group deprotection was provided time after.This solvent is under reduced pressure removed, and makes residue carry out between water and dichloromethane Distribution.This organic layer is separated, is dried (MgSO₄), by this solvent via filtering removal, and by the solvent of filtrate under vacuo Remove.From CH₃CN crystallized product, is isolated by filtration and is dried under vacuum, to provide a kind of white solid 20.

The preparation of 21

By Pd in the first bottle equipped with magnetic stirring bar and screw lid dividing plate₂(dba)₃(6mg, 0.007mmol) and 2-di-t-butyl phosphorus-3,4,5,6-tetramethyls-2 ', 4 ', 6 '-triisopropyl 1-1,1 '-xenyl (6mg, 0.013mmol) exists Solution N in toluene (0.5mL)₂Gas flushing, then stirs 3 minutes at 120 DEG C.Will be configured with magnetic stirring bar and spiral Second bottle of lid dividing plate 2-methylimidazole (104mg, 1.26mmol) and K₃PO₄(224mg, 1.05mmoL) fills, and then uses VI-3 (200mg, 0.53mmol) fills, and also uses N₂G () rinses.Via syringe by the catalyst solution of premixing, subsequently Dry toluene (0.5mL) and n-butyl alcohol (1.0mL) add to this second bottle (toluene of 2mL: t-BuOH 1: 1 solution altogether) In.This reaction is heated to 120 DEG C and continues 12 hours.This mixture is cooled down and adds Feldalat NM (30% in methanol).Will This mixture heats 1 hour at 60 DEG C.This mixture is cooled to room temperature and is concentrated under vacuum.Make residue at water and two It is allocated between chloromethanes.This organic layer is separated, is dried (MgSO₄), solid by filtration is removed, and by filtrate Solvent is concentrated under vacuum.Crude product is entered by pre-HPLC (RP SunFire Prep C18 OBD-10 μm, 30 × 150mm) Row purification.Flowing phase (the 0.25%NH in water₄HCO₃Solution, CH₃CN).Collect this product section, and carry out under vacuo Concentrate to provide compound 21.

The preparation of 22

By the VI-2 (500mg, 1.48mmol) in DMF (10mL), tributyl (1-ethoxy ethylene) stannum (0.626mL, 1.85mmol)、PdCl₂(PPh₃)₂The mixture of (220mg, 0.31mmol) is heated to 80 DEG C and continues 16 hours.This reaction is mixed Compound cools down and adds HCl (1N, 2mL).This mixture is stirred at room temperature 2 hours, then pours into saturated aqueous NaHCO₃(100mL) in, and by filtering, precipitate is separated, regenerate in dichloromethane, be dried (MgSO₄), by mistake Filter and solid is removed, and the solvent of filtrate is concentrated under vacuum.This product is used dichloromethane extremely via silica gel column chromatography In dichloromethane, the gradient of the methanol of 5% is purified, and collects product section and is concentrated under vacuum.By this product at DIPE Middle grinding, filters and is dried under vacuum to becoming a kind of faint yellow solid.

In this mixture, add methanol (6mL) and Feldalat NM (0.716mL) and stir 1 hour at 60 DEG C.This is mixed Compound cools down, and is then concentrated under vacuum.Residue is made to be allocated between water and dichloromethane.This organic layer is separated, It is dried (MgSO₄), solid by filtration is removed, and the solvent of filtrate is concentrated under vacuum.By this product in DIPE Grind, be isolated by filtration and be dried under vacuum to becoming a kind of yellow solid 22.

The preparation of 23

22 (59mg, 0.23mmol) are suspended in methanol (2mL) and add sodium borohydride (9mg, 0.23mmol).Should Mixture is at room temperature at N₂Stir two hours under (g).This mixture dichloromethane (5mL) is diluted, then adds saturated Aqueous NH₄Cl (0.5mL) adds NaHCO subsequently₃.This organic layer is dried (MgSO₄), by solid via filtering removal, and will The solvent of filtrate is concentrated under vacuum.This product is ground in DIPE, is isolated by filtration and is dried under vacuum to becoming A kind of faint yellow solid 23.

The preparation of 24

Step 1.Under nitrogen atmosphere by 75mL stainless steel autoclave VI-2 (626mg, 1.87mmol), Pd (OAc)₂ (8mg, 0.037mmol), 1, double (diphenylphosphine) propane (31mg, 0.074mmol) of 3-, potassium acetate (364mg, 3.71mmol), THF (20mL) and methanol (20mL) are filled.This autoclave cuts out and is forced into 30bar CO (g).By this reactant mixture Stir 16 hours at 120 DEG C.Allow that this reactant mixture is cooled to room temperature to be then concentrated under vacuum.Residue is dissolved In water, and extract with dichloromethane.This organic layer is dried (MgSO₄), solid by filtration is removed, and by filtrate Solvent be concentrated under vacuum.This product use on a silica gel column dichloromethane to the ladder of the methanol of 5% in dichloromethane Degree is purified.Collect this product section and concentrate in a vacuum, to obtain a kind of pale solid VI-4.

Step 2.Under nitrogen atmosphere at-75 DEG C to the VI-4 (190mg, 0.69mmol) solution in anhydrous THF (20mL) Middle interpolation LAH (1M in THF, 1.04mL, 1.04mmol).Allow it heats this reaction stirring for two hours simultaneously lentamente To 0 DEG C.Then this mixture cooled down in ice ethanol bath and add Na subsequently by adding 15mL ethyl acetate₂SO₄ 10H₂O (2g) careful cancellation.This mixture is stirred one hour and then uses MgSO₄It is dried, solid by filtration is removed, and will The solvent of filtrate is under reduced pressure removed.By residue by pre-HPLC (RP Vydac Denali C18-10 μm, 200g, 5cm) It is purified.Flowing phase (0.25% NH in water₄HCO₃Solution, CH₃CN), subsequently by SFC purification (Chiralpak Diacel AD 30×250mm)。

Flowing phase (CO₂, have the methanol of 0.2% 2-aminopropane .), desired part is collected, and solvent is being subtracted Subdue divided by providing 24.

The preparation of 25

Step 1.V-14 is reacted by the method according to preparing compound 14 with trimethyl acetylene, to provide V-15.

LC-MS m/z=258 (M+H)

Step 2.Example VI-5 is prepared according to the method preparing compound 9.NaHCO3, water, methane mixture perform The deprotection of TMS group.

LC-MS m/z=357 (M+H)

Step 3.Method according to preparation 16 performs hydrogenation.

The preparation of 26

Step 1.In addition to this reaction being run at 110 DEG C, perform 2-bromo-4-according to the program preparing VI-4 different The palladium chtalyst carbonylation of propyl benzene amine, to provide 2-amino-5-isopropyl acid.

LC-MS m/z=180 (M+H)

Step 2.Method according to preparing compound V-1 prepares V-16.

LC-MS m/z=204 (M+H)

Step 3.Method according to preparation 15 prepares example 26.

The preparation of 27

Step 1.Cyanamide is dissolved in ether, and this mixture is stirred under a nitrogen.At ambient temperature to this reaction Mixture drips HCl (2M is in ether), and at room temperature continues stirring 2 hours.Precipitate A-2 is isolated by filtration also It is dried at 50 DEG C under vacuo.

Step 2.By SO₂(CH₃)₂(20.4g, 217mmol) is heated to melting.Add A-2 (3.3g, 29mmol) and stir Gained mixture, and it is heated to 120 DEG C to being completely dissolved.5-(2-chloro-4-fluoroform is disposably added to this reactant mixture Phenoxyl) methyl 2-aminobenzoate (5g, 14.5mmol).Continue stirring 30 minutes.By this reactant mixture water (10mL) process and stir 10 minutes.Precipitate V-17 (a kind of white solid) is isolated by filtration and dry in vacuum drying oven Dry.

LC-MS m/z=356 (M+H)

Step 3.Method according to preparation 15 forms compound 27.

The preparation of 28

Step 1.A-3 (101g, 0.44mol) is dissolved in sulphuric acid (850mL).This solution is cooled to 0 DEG C.Little through 2 Time dropping HNO in sulphuric acid (200mL)₃(18.3mL, 0.44mol).This reactant mixture is stirred 45 points at-10 DEG C Clock, then pours in frozen water (6L).Solvent is fallen off and residue is dissolved in dichloromethane (1.5L).By water layer Extract with dichloromethane (1L).The organic layer of merging is dried (MgSO₄), solid by filtration is removed, and by molten Agent is under reduced pressure gone divided by providing A-4, and uses heptane to acetic acid second via silica gel column chromatography by-product isomers A-5 The gradient of ester separates.

Step 2.Methanol is placed in the 500mL conical flask sprayed equipped with magnetic stirring bar carrying out with nitrogen (100mL comprises 2% thiophene), 5%Pt/C (2g, 0.513mmol), be then placed under nitrogen atmosphere.This reactant mixture is existed Stir 16 hours under room temperature.Catalyst is removed by filtration, and the volatile matter of filtrate is under reduced pressure removed.By residue Silicon stone uses dichloromethane to dichloromethane: the gradient of methanol 9: 1 is purified, produce a kind of yellow oil IV-2.

LC-MS m/z=244 (M+H)

Step 3.Intermediate VI-18 is prepared according to the method preparing compound V-17.

LC-MS m/z=254 (M+H)

Step 4.The program preparing compound 9 is applied from V-18 synthesis 28.

The preparation of compound 29

Step 1.After the catalytic hydrogenation of 27, example 29 is provided according to the method described in the preparation of 25.

The preparation of 90

Step 1.17 (12.515g, 45.62mmol) are dissolved in THF (100mL).Add and be dissolved in water (20mL) LiOH (3.83g, 91.2mmol), add methanol (50mL) subsequently.This reactant mixture is stirred at room temperature overnight.To wave Stimulating food is under reduced pressure removed, and by solids washed with water and with DIPE grinding, to provide VI-6, for pale solid.

¹H NMR (400MHz, DMSO-d₆) δ ppm0.95 (t, J=7.4Hz, 3H), 1.40 (dq, J=14.9,7.3Hz, 2H), 1.68 (quin, J=7.3Hz, 2H), 3.54-3.65 (m, 2H), 7.89-8.05 (m, 2H), 8.14-8.31 (m, 2H), 9.11 (br.s., 1H), 11.10 (br.s., 1H), 16.37 (br.s., 1H)

Step 2.VI-6 (200mg, 0.768mmol), DMF (10mL), triethylamine is placed in a 50mL bottle (0.641mL, 4.61mmol), 3-aminopyridine (181mg, 1.92mmol) and diethyl phosphorocyanidate (0.233mL, 1.54mmol).Allow this reaction is stirred at room temperature 2 hours.Solvent is under reduced pressure removed and by crude product via anti-phase (Sunfire Prep C18, OBD 10 μm, 30 × 150mm, flow column chromatography the phase (0.25%NH in water₄HCO₃Solution, Methanol) it is purified, to provide 90.

Synthetic schemes for the preparation of AA-9

The synthesis of intermediate A A-3

AA-2 (200g, 532mmol) is added to the valeral (43g, 500mmol) solution in THF (1L), and will reaction Mixture is stirred at room temperature 16 hours.Solvent is evaporated, and residue is diluted in petroleum ether and carried out Filter.The solvent of filtrate is under reduced pressure removed, and uses petroleum ether in petroleum ether 3% by silica gel column chromatography residue The gradient of ethyl acetate be purified, to provide AA-3 (90g), for water white oil.

¹H NMR (400MHz, CDCl₃): δ ppm6.81-6.77 (m, 1H), 5.68-5.64 (td, J=1.2Hz, 15.6Hz, 1H), 2.11-2.09 (m, 2H), 1.406 (s, 9H), 1.38-1.26 (m, 4H), 0.85-0.81 (t, J=7.2Hz, 3H).

The synthesis of compound AA-5

At-78 DEG C, n-BuLi (290mL, 725mmol, 1.5 equivalent) is added to the AA-4 in THF (800mL) In the solution of the stirring of (165g, 781mmol).Then the stirring of this reactant mixture is added in THF (400mL) for 30 minutes AA-3 (90g, 488.4mmol), and reaction is stirred 2 hours at-78 DEG C.By this mixture by saturated aqueous NH₄Cl is carried out Cancellation is also heated up to room temperature.Product is allocated between ethyl acetate and water.By organic phases washed with brine, be dried and It is evaporated.Residue is purified by column chromatography (being used in the ethyl acetate eluting of in petroleum ether 5%), to provide colourless Oil, AA-5 (132g).

¹H NMR (400MHz, CDCl₃): δ ppm7.36-7.16 (m, 10H), 3.75-3.70 (m, 2H), 3.43-3.39 (d, J=15.2Hz, 1H), 3.33-3.15 (m, 1H), 1.86-1.80 (m, 2H), 1.47-1.37 (m, 2H), 1.32 (s, 9H), 1.26-1.17 (m, 7H), 0.83-0.79 (t, J=7.2Hz, 3H).

The synthesis of AA-6

AA-5 (130g, 328mmol) is dissolved in THF (1.5L) and 0 DEG C with several aliquots add LAH (20g, 526mmol).Gained mixture is stirred at that same temperature 2 hours, and then allow to be heated up to room temperature.By mixture Use saturated NH₄Cl aqueous solution carries out cancellation.Product is allocated between ethyl acetate and water.Organic facies salt is washed Wash, be dried and be evaporated.The organic layer sodium sulfate of merging is dried, by solid via filtering removal and carrying out Concentrate to provide rough AA-6 (100g), it is not further purified and is used in next step.

¹H NMR (400MHz, CDCl₃): δ ppm7.33-7.14 (m, 10H), 3.91-3.86 (m, 1H), 3.80-3.77 (d, J=13.6Hz, 1H), 3.63-3.60 (d, J=13.6Hz, 1H), 3.43-3.42 (m, 1H), 3.15-3.10 (m, 1H), 2.70- 2.63 (m, 2H), 1.65-1.28 (m, 10H), 0.89-0.81 (m, 3H).

The synthesis of AA-9

At 50 DEG C, by the AA-6 (38g, 116.75mmol) and 10%Pd/C solution in methanol (200mL) at 50PSI hydrogen Hydrogenate 24 hours under gas.Reactant mixture is filtered, and is evaporated solvent providing crude product AA-7 (17g).

Crude product is dissolved in dichloromethane (200mL), and adds triethylamine (26.17g, 259.1mmol) at 0 DEG C With Bis(tert-butoxycarbonyl)oxide (84.7g, 194.4mmol).Gained mixture is stirred at room temperature 16 hours.This mixture is made to exist It is allocated between dichloromethane and water.By organic phases washed with brine, it is dried and is evaporated.Residue is passed through silica gel Chromatography (being used in the ethyl acetate eluting of in petroleum ether 20%) is purified, to provide AA-8 (13g), for water white oil.

¹H NMR (400MHz, CDCl₃): δ ppm4.08-4.03 (br, 1H), 3.68 (m, 1H), 3.58-3.55 (m, 2H), 3.20-2.90 (br, 1H), 1.80-1.73 (m, 1H), 1.42-1.17 (m, 15H), 0.85-0.82 (t, J=6.8Hz, 3H).

AA-8 (42g, 0.182mol) is dissolved in dioxane (200mL), and 0 DEG C add dioxane/HCl (4M, 200mL).Gained mixture is stirred at room temperature 2h.It is evaporated providing crude product by solvent.By dichloromethane/oil Ether mixture (50mL, 1: 1, v/v) adds to this crude product, and is fallen off by supernatant.It is repeated twice to obtain by the method Obtain a kind of oil, AA-9 (26.6g).

¹H NMR (400MHz, DMSO-d₆): δ ppm8.04 (s, 3H), 3.60-3.49 (m, 2H), 3.16-3.15 (m, 1H), 1.71-1.67 (m, 2H), 1.60-1.55 (m, 2H), 1.33-1.26 (m, 4H), 0.90-0.87 (t, J=6.8Hz, 3H).

The preparation of AA-10

AA-10 is prepared in preparation according to AA-9, uses butyraldehyde to replace valeral.

¹H NMR (400MHz, DMSO-d₆): δ ppm8.07 (s, 3H), 4.85 (br, 1H), 3.57-3.45 (m, 2H), 3.14-3.12 (m, 1H), 1.70-1.64 (m, 2H), 1.56-1.49 (m, 2H), 1.38-1.30 (m, 2H), 0.90-0.80 (t, J =6.8Hz, 3H).

Table 1 has the compound of chemical formula (I).

Analysis method

All of compound is all characterized by LC-MS.Employ following LC-MS method:

Method A. is at the ethylsiloxane/Silicon stone mixture (BEH) C18 post (1.7 μm, 2.1 × 50mm of bridging；Water this (Waters) Acquity) on carry out anti-phase UPLC (ultra high efficiency liquid chromatography (LC)), flow velocity is 0.8ml/min.Use two flowing phases (10mM ammonium acetate is in H₂In O/ acetonitrile 95/5；Mobile phase B: acetonitrile) run a gradient condition: from 95% after 1.3 minutes A and 5%B to 5%A and 95%B, and keep 0.7 minute.Use the volume injected of 0.75 μ l.Taper hole voltage, for cation Pattern is 30V, and is 30V for negative ion mode.

Method B., on Xterra MS C18 post (3.5 μm, 4.6 × 100mm), carries out reversed-phase HPLC, adjoint The flow velocity of 1.6ml/min.Use three flowing phase (mobile phase A: 95%25mM ammonium acetate+5% acetonitrile；Mobile phase B: acetonitrile；Stream Dynamic phase C: methanol) run gradient condition: from 100%A to 50%B and 50%C after 6.5 minutes, to 100% after 0.5 minute B, keeps 100%B1 minute and with 100%A rebalancing 1.5 minutes.Use the volume injected of 10 μ l.

Method C. is at the ethylsiloxane/Silicon stone mixture (BEH) C18 post (1.7 μm, 2.1 × 50mm of bridging；Water this (Waters) Acquity) on carry out anti-phase UPLC (ultra high efficiency liquid chromatography (LC)), flow velocity is 0.8ml/min.Use two flowing phases (10mM ammonium acetate is in H₂In O/ acetonitrile 95/5；Mobile phase B: acetonitrile) run a gradient condition: from 95% after 1.3 minutes A and 5%B to 5%A and 95%B, and keep 0.2 minute.Use the volume injected of 0.5 μ l.

There is the biological activity of the compound of chemical formula (I)

Bioassay explanation

The assessment of TLR7 and TLR8 activity

Transient transfection TLR7 or TLR8 expression vector and NF κ B-luc report is used in cell reporter-gene assays The ability of these compound activatings people TLR7 and/or TLR8 is estimated by the HEK293 cell of gene construct.In a kind of feelings In condition, TLR expression construct expresses wild-type sequence or mutant sequences respectively, this mutant sequences TLR second rich in Leucine repetitive sequence includes a disappearance.Previously had shown that this type of saltant type TLR protein was more susceptible to agonist and lives The impact (US 7498409) changed.

In short, HEK293 cell is trained in culture medium (being supplemented with the DMEM of 10%FCS and 2mM glutamine) Support.For the transfection of the cell in 10cm culture dish, with trypsin-EDTA, cell is disperseed, with CMV-TLR7 or TLR8 Cell is transfected by the mixture of plasmid (750ng), NF κ B-luc plasmid (375ng) and transfection reagent, and 37 DEG C Moistening 5%CO₂24 hours are hatched under atmosphere.Then with trypsin-EDTA by the cell dispersion of transfection, wash in PBS And it is the most resuspended to 1.67 × 10⁵The density of cell/mL.Then the cell of 30 microlitres is distributed to 384-orifice plate In each hole in, hole has existed the compound (in 4% DMSO) of 10 μ L.At 37 DEG C, 5% CO₂Hatch 6 little Shi Hou, by adding Steady Lite Plus substrate (Perkinelmer Inc. (Perkin of 15 μ l in each hole Elmer)) and at ViewLux ultraHTS microwell plate imager (microplate imager) (Perkinelmer Inc.) On carry out reading out determining uciferase activity.Dose-effect curve is generated from by the measured value carried out in quadruplicate.To each Minimal effective concentration (LEC) value of compound is determined, and this minimal effective concentration value is defined as causing the standard beyond measuring The concentration of the effect of deviation at least twice.

In 384-orifice plate, use the similar dilution series of compound and the independent by CMV-TLR7 construct of every hole 30 μ L The cell (1.67 × 10 of transfection⁵Cells/mL) carry out toxicity of compound parallel determining.At 37 DEG C, 5%CO₂Lower through 6 hours After hatching, by every hole being added the ATP lite (Perkinelmer Inc.) of 15 μ l and at ViewLux ultraHTS micropore Cell viability is measured in the upper reading of plate imager (Perkinelmer Inc.).Data are with CC₅₀Report.

The suppression that HCV replicon replicates

The activation of people TLR7 causes being produced interferon strong by the plasmacytoid dendritic cells being present in human blood. By observing the antiviral in HCV Replicate Sub-system with the conditioned medium from peripheral blood lymphocytes (PBMC) when hatching Activity and the potentiality of these compound inducing interferons are estimated.It is based on a kind of bicistronic mRNA that this HCV replicon measures Expression construct, as by (Science (" science ") (1999) 285:110-113 described in Lohmann (Leman) et al.； Journal of Virology (" Journal of Virology ") (2003) 77:3007-153019), wherein have by Krieger (in gram Lattice) et al. described modification (Journal of Virology (" Journal of Virology ") (2001) 75:4614-4624)).Should Measure and utilize cell line Huh-7luc/neo of stable transfection, this cell line to contain a kind of bicistronic mRNA expression construct is carried out The RNA of coding, this bicistronic mRNA expression construct comprises from the internal ribosomal entry site from encephalomyocarditis virus (EMCV) The wild type NS3-NS5B district of the HCV 1b type that point (IRES) is translated, is a reporter gene (Lampyridea-fluorescein before this district Enzyme) and selected marker's (neoR, neomycin phosphotransferase).The flank of this construct is from HCV 1b type 5 ' and 3 ' NTR (untranslated region).In the presence of G418 (neoR), HCV is depended in the continuation cultivation to these replicon cells The duplication of RNA.By independently replicate HCV RNA and reach high levels of replication stable transfection type replicon cell (coding especially Luciferase) for analysis condition cell culture medium.

In short, use the Ficoll centrifugation protocol of standard to prepare PBMC from the buffy coat of at least two donor. The PBMC of separation is resuspended in the RPMI culture medium being supplemented with 10% people's AB serum, and by 2 × 10⁵Cells/well is distributed extremely In the 384-orifice plate of inclusion compound (70 μ L cumulative volume).After overnight incubation, the supernatant of 10 μ L is transferred to comprise 2.2 × 10³The 384-orifice plate of replicon cells/well (30 μ L) (bed board the previous day).After 24 hours hatch, by measuring luciferase Activity (use 40 μ L/ holes Steady Lite Plus substrate (Perkinelmer Inc.), and micro-with ViewLux ultraHTS Orifice plate imager (Perkinelmer Inc.) is measured) and duplication is measured.Each compound is to Huh7-luc/neo cell Inhibitory activity be reported as EC₅₀Value, this EC₅₀Value is defined as being applied to PBMC and causes uciferase activity to reduce by the change of 50% Compound concentration, this and then the instruction duplication degree of replicon rna when shifting the PBMC culture medium of defined amount.By recombinant interferon α-2a (Recomvinated Interferon α-2a-A) is used as standard control compound.

During HEK 293 TOX that the biological activity of the compound with chemical formula (I) is described above measures, all chemical combination Thing display CC50 ＞ 24 μMs.

The activation of ISRE promoter element

Also by the activation measuring the IFN-stimulated response element (ISRE) caused by the conditioned medium from PBMC And the potentiality of these compounds induction IFN-I are assessed.The ISRE element heights with sequence GAAACTGAAACT is rung Their receptor IFNAR (Clontech, PT3372-5W) should be bound at IFN-I in STAT1-STAT2-IRF9 transcription factor Time be activated.Plasmid pISRE-Luc from Clontech (reference number 631913) comprises 5 this ISRE element copies, with After be LUC Photinus pyralis LUC Photinus pyralis FL ORF.The HEK293 cell line setting up a kind of stable transfection pISRE-Luc (HEK-ISREluc) is come Analyze PBMC conditioned cell culture medium.

In short, use the Ficoll centrifugation protocol of standard to prepare PBMC from the buffy coat of at least two donor. The PBMC of separation is resuspended in the RPMI culture medium being supplemented with 10% people's AB serum, and by 2 × 10⁵Cells/well is distributed extremely In the 384-orifice plate of inclusion compound (70 μ L cumulative volume).After overnight incubation, the supernatant of 10 μ L is transferred to comprise 5 × 10³The 384-orifice plate of HEK-ISREluc cells/well (30 μ L) (bed board the previous day).After 24 hours hatch, by measuring fluorescence Element enzymatic activity (uses 40 μ L/ holes Steady Lite Plus substrate (Perkinelmer Inc.), and uses ViewLux UltraHTS microwell plate imager (Perkinelmer Inc.) measures) and activation to ISRE element measures.Each chemical combination Thing is reported as LEC value to the stimulating activity of HEK-ISREluc cell, and this LEC value is defined as being applied to PBMC and causes fluorescein Enzymatic activity is at least beyond the compound concentration of measurement standard deviation twice.LEC and then instruction are cultivated at the PBMC of transfer defined amount The degree of ISRE activation during base.Interferon Alfa-2a (Recomvinated Interferon α-2a-A) is used as standard control compound.

For a given compound, measure, from this, the LEC value obtained and be in and obtain with from " HCV replicates mensuration to be suppressed " The EC obtained₅₀It is worth in identical scope.Therefore, it is possible to by 2 kinds measure in any one measured compound via The potentiality of PBMC induction IFN-I compare.

^*In the 48 little mensuration run at present

Claims (4)

1. a compound with chemical formula (I)

(I)

Or its pharmaceutically acceptable salt, wherein

R₁It is one of following radicals:

、、Or,

R₂It is hydrogen, halogen, hydroxyl, amine, C_1-7Alkyl, C_1-7Alkylamino, C_1-6Alkoxyl or (C_1-4) alkoxyl-(C_1-4) alkyl,

R₃It is hydrogen, halogen, hydroxyl, amine, C_1-7Alkyl, C_2-7Thiazolinyl, C_2-7Alkynyl, C_1-7Alkylamino, C_1-6Alkoxyl or (C_1-4) alcoxyl Base-(C_1-4) alkyl,

R₄It is hydrogen, halogen, hydroxyl, amine, C_1-7Alkyl, C_1-7Alkylamino, C_1-6Alkoxyl or (C_1-4) alkoxyl-(C_1-4) alkyl, and And

R₅Being hydrogen, fluorine, chlorine or methyl, its condition is:

R₂、R₃、R₄, and R₅Can not be H entirely.

The compound with chemical formula (I) the most according to claim 1, wherein R₅It is hydrogen or fluorine, and R₁、R₂、R₃, and R₄It is as described in claim 1.

3. a pharmaceutical composition, including the compound with chemical formula (I) according in claim 1-2 Or its pharmaceutically acceptable salt is together with one or more pharmaceutically acceptable excipient, diluent or carrier.

4. according to the described compound with chemical formula (I) of in claim 1-2 or it is pharmaceutically acceptable Salt or pharmaceutical composition according to claim 3 are directed to the regulation of TLR7 and/or TLR8 in preparation for treatment Imbalance medicament in purposes.