HK1237344A1 - Pyrrolopyrimidines for use in influenza virus infection - Google Patents

Description

Pyrrolopyrimidines for use in influenza virus infection

Influenza is a serious public health problem, with high morbidity among human populations, leading to regular, large-scale morbidity and mortality. Influenza is a highly contagious airborne disease that causes acute febrile illness. In terms of severity, systemic symptoms vary from mild fatigue to respiratory failure and death. According to the WHO, the average global burden of an epidemic per year can reach about 10 million cases, 300 to 500 million cases of severe disease, and 300,000-500,000 cases of death per year. Every year, influenza viruses circulate in humans, often affecting 5% -20% of the population in all age groups, with this figure rising to 30% during severe pandemic illness. Severe disease rates and mortality are highest among the following populations: people >65 years of age, children <2 years of age, and people of any age who have medical conditions (e.g., chronic heart disease, lung disease, kidney disease, hepatic hematological or metabolic disease, or weakened immune system) that increase their risk for influenza complications. Although childhood deaths are not frequent, for children <5 years of age, hospitalization rates range from about 100 to 500 out of every 10 million (depending on the presence or absence of co-morbidities). The hospitalization rates for children <24 months of age were comparable to those reported in people >65 years of age.

In the united states, influenza epidemics result in approximately 3000 million outpatient visits per year, thereby resulting in a medical cost of $ 100 million per year. Lost revenue due to disease and loss of life is expressed as costs in excess of $ 150 billion per year, and the total economic burden of the annual influenza epidemics in the united states amounts to $ 850 billion.

The causative agent of influenza is an antisense, single-stranded RNA virus, which belongs to the orthomyxoviridae family. There are three types of influenza viruses: type a, type b and type c. Influenza a viruses are the most common form, which can be transmitted in mammals and birds. Subtypes of influenza a are named by the types of surface proteins hemagglutinin (H) and neuraminidase (N). There are 18 different hemagglutinins and 11 known neuraminidases. Current seasonal influenza viruses found in humans are predominantly of subtypes H1N1 and H3N 2. Influenza b viruses are generally found only in humans. Influenza b viruses are not subtype but can be further subdivided into different strains. The prevalence of influenza virus is highly variable every year, and both influenza a and b cause seasonal epidemics worldwide. Influenza c virus is much less symptomatic and does not cause epidemics.

All three types of viruses have similar genomic structures. The genome comprises 8 segments that encode 9-11 proteins, depending on their type. Influenza a encodes 11 proteins, including surface proteins (hemagglutinin (HA) and Neuraminidase (NA)), polymerase complex (PA, PB1 and PB2), Nucleoprotein (NP), membrane proteins (M1 and M2), and other proteins (NS1, NS2, NEP). Of these three influenza virus types, influenza a has the highest mutation rate. Influenza b progresses slower than type a but faster than type c. The segmented genome allows gene exchange between different virus strains, which results in new variants of influenza virus.

Influenza viruses can be transmitted between humans by direct contact with infected individuals or virus-contaminated materials. Humans can also be infected by inhalation of airborne virus droplets. These droplets are produced by coughing, sneezing or speaking of infected individuals. Seasonal influenza is characterized by: sudden onset of high fever, cough (usually dry), headache, muscle and joint pain, severe discomfort (bad feeling), sore throat and runny nose. Coughing can be severe and can last for two or more weeks. Most people recover from fever and other symptoms within a week without medical assistance. However, influenza can cause serious illness or death, particularly in high risk groups as mentioned above. The time from infection to disease, called the incubation period, is approximately two days.

The most effective way to prevent the disease and/or serious consequences from the disease is vaccination. Safe and effective vaccines are available and have been used for more than 60 years. In healthy adults, influenza vaccines can provide reasonable protection. However, vaccination comes with several limitations. First, influenza vaccines may be less effective in preventing disease in the elderly, and may only reduce the severity of the disease and the incidence of complications and death. Furthermore, influenza vaccination is most effective when the epidemic virus is well matched to the vaccine virus, and the success of vaccination depends largely on good predictions of the most prevalent virus type for that season. The rapid and sustained evolution of influenza virus strains through antigenic drift, coupled with the short-term nature of the vaccine-induced immune response to current influenza vaccines, means that seasonal vaccination with appropriate strains is required for prophylaxis each year.

Current treatments for influenza use either direct antiviral drugs or drugs that alleviate influenza-induced symptoms. There are two classes of influenza antiviral drugs available on the market: neuraminidase inhibitors and M2 channel inhibitors. Neuraminidase inhibitors (oseltamivir or zanamivir) are the major antiviral agents recommended for the prevention and treatment of influenza. These are effective against both influenza a and b viruses. The development of resistance to these antiviral drugs has been identified during the treatment of seasonal influenza and sporadic oseltamivir-resistant 2009H1N1 virus, but public health impact has been limited to date. M2 channel inhibitors (e.g., amantadine and rimantadine) are active against influenza a strains and inactive against influenza b strains. Adamantane resistance in pandemic influenza a viruses began to increase rapidly worldwide during the year 2003-2004. Thus, amantadine and rimantadine are not recommended for antiviral treatment or chemoprevention of currently prevalent influenza a virus strains.

In 2009, the novel porcine H1N1 strain caused an unexpected influenza pandemic due to gene reassortment of H1N1 virus from humans, pigs and birds. This past pandemic, as well as the continuing prevalence of the highly pathogenic avian H5N1 strain and the recent emergence of H7N9 virus (a novel reassortant of avian origin isolated in china and associated with severe respiratory disease with 40% mortality that can potentially be adapted to interpersonal transmission) highlights the vulnerability of the world population to novel influenza strains. Although vaccination remains the primary prophylactic strategy for controlling influenza infection, a wider choice of anti-influenza drugs is required in order to cover a period of time before new vaccines become available and in order to treat severe influenza cases and in order to cope with problems with virus tolerance. Thus, the development of new influenza antiviral drugs is again a high priority and unmet medical need.

The present invention relates to compounds of formula (I) useful for treating or combating viral influenza infection:

a stereoisomeric form, a pharmaceutically acceptable salt, solvate or polymorph thereof, wherein

X is optionally substituted by-CN, -CF₃、-C_1-3alkyl-N-C (O) -C_1-3Alkyl, -C (O) -NH₂、-C(O)-NH-C_1-3Alkyl, -C (O) N- (dialkyl) or-CH₂-NC(O)-CH₃Substituted N or C;

R₁is H or CH₃；

R₂Is H or NH₂；

R₃Is C substituted by formic acid_1-8An alkyl group;

or by formic acid, -N-C_1-3Alkyl sulfone substituted C_3-8Cycloalkyl radicals, or

Optionally is covered with C_1-6Alkyl substituted-N-C (O) -C_3-6A heterocycle;

or is substituted by-N-C (O) -C_3-6Heterocycle-substituted C_3-6A heterocyclic ring.

Preferably, the compounds according to the invention are compounds according to formula (I), wherein R is₁And R₂Are all H.

Preferred compounds according to the invention have the formula

Also part of the invention is a pharmaceutical composition comprising a compound of formula (I) or a stereoisomeric form, a pharmaceutically acceptable salt, solvate or polymorph thereof together with one or more pharmaceutically acceptable excipients, diluents or carriers.

The pharmaceutical composition may also include an additional therapeutic agent, like another antiviral agent or an influenza vaccine, or both.

Also within the invention is a compound of formula (I) or a stereoisomeric form, a pharmaceutically acceptable salt, solvate or polymorph thereof or a pharmaceutical composition for use as a medicament.

In addition, the present invention relates to a compound of formula (I) or a stereoisomeric form, a pharmaceutically acceptable salt, solvate or polymorph thereof or a pharmaceutical composition for use in the treatment of influenza.

Accordingly, part of the present invention are compounds represented by the following structural formula (I)

A stereoisomeric form, a pharmaceutically acceptable salt, solvate or polymorph thereof, wherein

X isOptionally substituted by-CN, -CF₃、-C_1-3alkyl-N-C (O) -C_1-3Alkyl, -C (O) -NH₂、-C(O)-NH-C_1-3Alkyl, -C (O) N- (dialkyl) or-CH₂-NC(O)-CH₃Substituted N or C;

R₁is H or CH₃；

R₂Is H or NH₂；

R₃Is C substituted by formic acid_1-8An alkyl group;

or by formic acid, -N-C_1-3Alkyl sulfone substituted C_3-8Cycloalkyl radicals, or

Optionally is covered with C_1-6Alkyl substituted-N-C (O) -C_3-6A heterocycle;

or is substituted by-N-C (O) -C_3-6Heterocycle-substituted C_3-6Heterocyclic rings

Use for inhibiting replication of one or more influenza viruses in a biological sample or patient.

The use may further comprise co-administration of an additional therapeutic agent, wherein the additional therapeutic agent is selected from an antiviral agent or an influenza vaccine, or both.

The term "alkyl" refers to a straight or branched chain saturated aliphatic hydrocarbon containing the specified number of carbon atoms.

The term "cycloalkyl" refers to a carbocyclic ring containing the specified number of carbon atoms.

The term "heterocycle" refers to a saturated or partially saturated molecule comprising one or more heteroatoms selected from N, O or S, in particular from N and O. The heterocyclic ring may have 4,5, 6 or 7 ring atoms.

Pharmaceutically acceptable salts of the compounds of formula (I) include acid addition salts as well as base salts thereof. Suitable acid addition salts are formed from acids which form non-toxic salts. Suitable base salts are formed from bases which form non-toxic salts.

The compounds of the present invention may also exist in unsolvated forms as well as solvated forms. The term "solvate" is used herein to describe a molecular complex comprising a compound of the invention and one or more pharmaceutically acceptable solvent molecules (e.g., ethanol).

The term "polymorph" means that a compound of the invention can exist in more than one form or crystal structure.

The compounds of the present invention may be administered as crystalline or amorphous products. They can be obtained, for example, in the form of solid fillers, powders or films by processes such as precipitation, crystallization, freeze drying, spray drying or evaporation drying. They may be administered alone or in combination with one or more other compounds of the invention or in combination with one or more other drugs. Typically, they will be administered as a formulation in combination with one or more pharmaceutically acceptable excipients. The term "excipient" is used herein to describe any ingredient other than one or more compounds of the present invention. The choice of excipient will generally depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

The compounds of the invention or any subgroup thereof may be formulated in different pharmaceutical forms for administration purposes. As suitable compositions, all compositions usually used for systemic administration of drugs can be cited. To prepare the pharmaceutical compositions of this invention, an effective amount of the particular compound, optionally in addition salt form, as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of preparation desired for administration. Desirably, these pharmaceutical compositions are in unit dosage forms suitable for, e.g., oral, rectal, or transdermal administration. For example, in preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, and the like, in the case of oral liquid preparations (e.g., suspensions, syrups, elixirs, emulsions, and solutions); or solid carriers such as starches, sugars, kaolin, diluents, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules and tablets. Because of their ease of administration, tablets and capsules represent the most advantageous oral unit dosage form in which case solid pharmaceutical carriers are obviously employed. Also included are solid form preparations which can be converted to liquid form shortly before use. In compositions suitable for transdermal administration, the carrier may optionally include penetration enhancers and/or suitable wetting agents, optionally in combination with small proportions of suitable additives of any nature, which do not introduce significant deleterious effects on the skin. The additives may facilitate administration to the skin and/or may aid in the preparation of the desired composition. These compositions can be administered in different ways, e.g. as a transdermal patch, as drops, as an ointment. The compounds of the invention may also be administered via inhalation or insufflation by means of the methods and formulations employed in the present field for administration by this means. Thus, in general, the compounds of the invention can be administered to the lungs in the form of a solution, suspension or dry powder.

It is particularly advantageous to formulate the above pharmaceutical compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form, as used herein, refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of such unit dosage forms are tablets (including scored or coated tablets), capsules, pills, powder packets (powderpackets), wafers, suppositories, injectable solutions or suspensions and the like, and segregated multiples thereof.

One of ordinary skill in the art of treatment of infectious diseases will be able to determine the effective amount from the test results presented below. Generally, it is contemplated that the daily effective amount will be from 0.01mg/kg to 50mg/kg body weight, more preferably from 0.1mg/kg to 10mg/kg body weight. The desired dose may suitably be administered as two, three, four or more sub-doses at appropriate time intervals throughout the day. The sub-doses may be formulated in unit dosage forms, for example containing from 1mg to 1000mg, and in particular from 5mg to 200mg, of the active ingredient per unit dosage form.

As is well known to those of ordinary skill in the art, the precise dose and frequency of administration depends on the particular compound of formula (I) used, the particular condition being treated, the severity of the condition being treated, the age, weight and general physical condition of the particular patient, and other drugs that the individual may take. Furthermore, it will be apparent that the effective amount may be reduced or increased according to the response of the subject being treated and/or according to the evaluation of the physician prescribing the compounds of the instant invention. Accordingly, the effective amount ranges described above are merely guidance and are not intended to limit the scope or use of the invention in any way.

Examples of the invention

Preparation of Compounds of formula (I)

Scheme 1. preparation of Compound 7

Scheme 1.i) Br₂DMF, RT, 8h ii) NaH, TsCl, THF iii) Biboronic acid pinacol ester (bis (pinacolato) diboron), Pd (dppf) Cl₂KOAc, 1, 4-dioxane, 80 ℃, 16h iv) EtOH/THF, DIPEav) Na₂CO₃Tetrakis, Xantphos, 1, 4-dioxane, microwave 150 ℃ for 15min vi) NaOCH₃、CH₃OH

Preparation of intermediate 1

To 7H-pyrrolo [2,3-d ] at 0 DEG C]To a stirred solution of pyrimidine (11.5g, 73.92mmol) in DMF (350mL) was added bromine (11.8g, 73.84mmol) in DMF (50 mL). The cooling bath was removed and the reaction was stirred at 20 ℃ for 8 hours, then the reaction mixture was poured into ice water and Na was added₂CO₃Alkalization is carried out. The mixture was extracted with ethyl acetate. The combined organic matterLayer with 10% aqueous Na₂S₂O₃Washed with brine and over MgSO₄Drying, removing the solid by filtration, and concentrating the filtrate under reduced pressure to give 1, 5-bromo-7H-pyrrolo [2,3-d as a yellow solid]Pyrimidine, which was used in the next step without further purification.

¹H NMR(400MHz,DMSO-d₆)ppm 7.84(s,1H),8.84(s,1H),8.92(s,1H),12.57(br,1H)。

Preparation of intermediate 2

To 5-bromo-7H-pyrrolo [2,3-d ] at 0 ℃ under nitrogen]A stirred solution of pyrimidine (12.8g, 55.11mmol) in THF was added portionwise with NaH (4.48g, 112.01 mmol). The resulting mixture was stirred at 5 ℃ for 1 hour, then p-toluenesulfonyl chloride (11.6g, 60.85mmol) was added in portions. The reaction mixture was allowed to warm to 20 ℃ and stirred for 3 hours. The reaction mixture was poured into a mixture of ice and 1M aqueous HCl while stirring. The mixture was extracted with ethyl acetate. The combined organic layers were washed with brine, over MgSO₄Dry, remove the solids by filtration and concentrate the filtrate under reduced pressure. The residue was purified by crystallization from ethyl acetate to provide 2, 5-bromo-7-tosyl-7H-pyrrolo [2,3-d as a white solid]A pyrimidine.

¹H NMR(400MHz,DMSO-d₆)ppm 2.36(s,3H),7.47(d,J＝8.0Hz,2H),8.06(d,J＝8.0Hz,2H),8.31(s,1H),9.03(s,1H),9.06(s,1H)。LC-MS ES⁺351.8; rt: 1.16min, method D.

Preparation of intermediate 3

5-bromo-7-tosyl-7H-pyrrolo [2,3-d ] in 1, 4-dioxane (170mL, degassed with nitrogen) at 80 ℃ under nitrogen]Pyrimidine (10g, 28.39mmol), pinacol diboron (14.42g, 56.79mmol), potassium acetate (8.36g, 85.18mmol), Pd (dppf) Cl₂(1g, 1.37mmol) of the mixture was heated in a 500mL round-bottom flask equipped with a reflux condenser for 16 hours. The reaction mixture was cooled to room temperature, filtered through celite, and the solid was taken up with ethyl acetateAnd (4) ester rinsing. The filtrate was concentrated under reduced pressure and the residue was purified by silica column chromatography using a gradient of heptane to ethyl acetate. The desired fractions were collected and concentrated under reduced pressure to give 3, 5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -7-toluenesulfonyl-7H-pyrrolo [2,3-d ] -c]A pyrimidine.

¹H NMR(400MHz,DMSO-d₆)ppm 1.33(s,12H)2.37(s,3H)7.47(d,J＝8.36Hz,2H)8.11(d,J＝8.58Hz,2H)8.14(s,1H)9.00(s,1H)9.10(s,1H)。LC-MS ES⁺318.1; rt: 0.74min, method A.

Preparation of intermediate 5

A solution of 2, 4-dichloro-5-fluoro-pyrimidine (2.76g, 16.55mmol) in ethanol (70mL) and THF (70mL) was stirred at room temperature. To the reaction mixture was added dropwise (+/-) -cis-N- (3-aminocyclohexyl) pyrrolidine-1-carboxamide (4.1g, 16.55mmol) and DIPEA (8.56mL, 49.64mmol) and stirred at 70 ℃ for 1h and then at ambient temperature overnight. The solvent of the reaction mixture was removed under reduced pressure, the residue was reconstituted in water and extracted twice with DCM. The combined organic layers were washed with brine, over MgSO₄Drying, removing the solid by filtration, and removing the solvent in the filtrate under reduced pressure. By flash column chromatography on silica (gradient: CH)₂Cl₂To CH₂Cl₂/CH₃OH: 90/10) purifying the residue. The desired fractions were pooled and evaporated to dryness to afford 5 as a white solid. LC-MS ES⁺m/z is 342.3; rt: 0.75min, method A.

Preparation of intermediate 4

(+/-) -cis-3- (boc-amino) cyclohexanecarboxylic acid (9.51g, 39.09mmol), diphenylphosphoryl azide (12.61mL, 58.63mmol) and Et₃A mixture of N (7.61mL, 54.72mmol) in THF (250mL) was refluxed for 2 hours. The solution was allowed to reach room temperature, then pyrrolidine (9.81mL, 117.26mmol) was added and the mixture was refluxed for 1 hour. The mixture was cooled to 0 ℃, the precipitate was isolated by filtration and washed with THF, dried in vacuo to provide tert-butyl 4a, (+/-) - (cis-3- (pyrrolidine-1-carboxamido) cyclohexyl) carbamate as a white powder.

A solution of tert-butyl (+/-) - (cis-3- (pyrrolidine-1-carboxamido) cyclohexyl) carbamate (23.77g, 76.33mmol) in HCl (4M in 1, 4-dioxane, 344mL) was stirred at room temperature for 4 hours. The solution was concentrated under reduced pressure and then dried in vacuo to afford 4, (+/-) -N- ((cis) -3-aminocyclohexyl) pyrrolidine-1-carboxamide as a white solid. LC-MS ES⁺m/z is 212.2; rt: 1.06min, method C.

7 preparation of

At room temperature, under nitrogen, 3(799mg, 2mmol), 5(684mg, 2mmol) and Na₂CO₃(3mL, 2M, 6mmol) of the mixture was stirred in 1, 4-dioxane (10 mL). Tetrakis (triphenylphosphine) palladium (0) (116mg, 0.1mmol) and xanthphos (58mg, 0.1mmol) were then added and the mixture degassed for 10 min. The reaction was heated in a microwave at 150 ℃ for 15 minutes. The solvent was removed under reduced pressure and the residue was taken up in NaOCH₃(100mL, 0.5M in CH₃In OH) for 1 hour. The solvent was removed under reduced pressure and the residue was stirred in water and neutralized with acetic acid. The solution is treated with CH₂Cl₂Extracting for 3 times with MgSO₄Drying, removing the solid by filtration, and removing the solvent in the filtrate under reduced pressure. Using CH₂Cl₂/CH₃OH: a gradient of 98/2 to 90/10 purified the residue on silica.The best fractions were pooled and the solvent was removed under reduced pressure and the product recrystallized from acetonitrile. The off-white precipitate was collected by filtration and dried in vacuo to afford 7.

¹H NMR(400MHz,DMSO-d₆)ppm 1.06-1.52(m,4H),1.67-1.89(m,6H),1.97(d,J＝11.2Hz,1H),2.06-2.18(m,1H),3.11-3.23(m,5H),3.55-3.75(m,1H),4.02-4.27(m,1H),5.82(d,J＝7.9Hz,1H),7.54(d,J＝7.7Hz,1H),8.10-8.22(m,2H),8.80(s,1H),9.59(s,1H)。LC-MS ES⁺m/z is 425.4; rt: 1.42min, method B.

Preparation of intermediate 8

A solution of 2, 6-dichloro-5-fluoro-3-pyridinecarbonitrile (4.77g, 25mmol) in THF (40mL) was stirred at room temperature while a mixture of 4(6.19g, 25mmol) and DIPEA (8.62mL, 50mmol) in ACN (20mL) was added dropwise. The reaction was allowed to stir at ambient temperature for 2 days. The solvent was removed under reduced pressure. The crude product was dissolved in diisopropyl ether/ethyl acetate (1/1) and washed with water. The organic layer was dried (MgSO₄) The solid was removed by filtration, and the solvent in the filtrate was removed under reduced pressure. The residue was triturated in diisopropyl ether to afford 8, (+/-) -N- ((cis) -3- ((6-chloro-5-cyano-3-fluoropyridin-2-yl) amino) cyclohexyl) pyrrolidine-1-carboxamide as a white solid, which was dried in vacuo. LC-MS ES⁺366.1 m/z; rt: 0.88min, method A.

9 preparation of

To a thick-walled glass tube were added 3(500mg, 1.25mmol) and Pd (PPh)₃)₄(145mg，0.125mmol)、K₂CO₃(346mg, 2.51mmol) and 8(481mg, 1.32mmol) in DME (15mL) andthe mixture in water (5mL) was heated to 100 ℃ and stirred for 16 h. The solvent was removed under reduced pressure. The crude residue was stirred in DCM, filtered and purified by flash column chromatography on silica (first gradient: heptane-ethyl acetate; second gradient: DCM-DCM/CH)₃OH 100-90/10). The desired fractions were collected and evaporated to dryness to afford 9, (+/-) -N- ((cis) -3- ((5-cyano-3-fluoro-6- (7H-pyrrolo [2, 3-d)]Pyrimidin-5-yl) pyridin-2-yl) amino) cyclohexyl) pyrrolidine-1-carboxamide.

¹H NMR(600MHz,DMSO-d₆)ppm 1.15-1.31(m,2H)1.34-1.44(m,1H)1.48(q,J＝11.93Hz,1H)1.75-1.78(m,4H)1.78-1.84(m,2H)2.00(d,J＝11.30Hz,1H)2.03-2.06(m,1H)3.16-3.19(m,4H)3.53-3.56(m,1H)4.12-4.15(m,1H)5.84(d,J＝7.92Hz,1H)7.74(d,J＝7.04Hz,1H)7.87(d,J＝11.30Hz,1H)8.33(s,1H)8.85(s,1H)9.56(s,1H)12.66(br.s.,1H)。LC-MS ES⁺449.2; rt: 1.55min, method B.

Preparation of intermediate 10

(+/-) - ((cis) -3-aminocyclohexyl) carbamic acid tert-butyl ester (5g, 23.3mmol) and DMAP (7.1g, 58.3mmol) in CH₂Cl₂(100mL) the mixture was stirred at ambient temperature, then 1-methyl-1H-imidazole-4-carboxylic acid (2.9g, 23.3mmol) was added. After stirring at room temperature for 10 min, EDC (6.7g, 35mmol) was added. The mixture was stirred at room temperature for 18 hours. The reaction mixture was washed with citric acid (5% aqueous), the organic layer was removed and dried (MgSO)₄) The solid was removed by filtration, and the solvent in the filtrate was removed under reduced pressure to give 10a, (+/-) - ((cis) -3- (1-methyl-1H-imidazole-4-carboxamido) cyclohexyl) carbamic acid tert-butyl ester. LC-MS ES⁺323.5; rt: 0.75min, method A. The boc-group was removed by HCl in 1, 4-dioxane, as in the process for preparation of intermediate 4, to afford 10, (+/-) -N- ((cis) -3-aminocyclohexyl) -1-methyl-1H-imidazole-4-carboxamide.

Preparation of intermediate 11

Reacting (+/-) -N- [ (cis) -3-aminocyclohexyl]A solution of (E) -1-methyl-imidazole-4-carboxamide (4.64g, 15.7mmol) and 2, 6-dichloro-5-fluoro-3-pyridinecarbonitrile (3g, 15.7mmol) was stirred in ACN (50mL) at room temperature. DIPEA (10mL, 54mmol) was added and the reaction mixture was stirred at 50 ℃ for 24 h. The solvent was removed under reduced pressure. Addition of CH₂Cl₂And 11, (+/-) -N- ((cis) -3- ((6-chloro-5-cyano-3-fluoropyridin-2-yl) amino) cyclohexyl) -1-methyl-1H-imidazole-4-carboxamide, which precipitated as a white precipitate was isolated by filtration and used in the next step without further purification. LC-MS ES⁺m/z is 377.1; rt: 1.58min, method B.

12 preparation of

To a 20mL thick-walled glass vial was added 3(0.25g, 0.63mmol), Pd (PPh)₃)₄(72mg，0.0626mmol)、K₂CO₃(173mg, 1.25mmol), DME (5mL), water (1.5mL), and 11(0.25g, 0.63 mmol). The vial was sealed and heated in an oil bath at 100 ℃ for 18 hours. The reaction mixture was adjusted to pH 6 via addition of concentrated HCl. DMSO was added and the solution was filtered. By preparative HPLC (RP SunAire Prep C18 OBD-10 μm, 30X150mm, mobile phase 0.25% ammonium carbonate in water to CH₃OH) purifying the crude product. The best fractions were pooled and the solvent was removed under reduced pressure to afford 12 as a white solid.¹H NMR(400MHz,DMSO-d₆)ppm 1.18-1.54(m,3H),1.62(q,J＝11.7Hz,1H),1.84(d,J＝10.8Hz,2H),2.06(t,J＝14.2Hz,2H),3.67(s,3H),3.85(d,J＝8.6Hz,1H),4.20(dd,J＝7.8,3.6Hz,1H),7.59(d,J＝1.3Hz,1H),7.63(d,J＝0.9Hz,1H),7.68(d,J＝8.4Hz,1H),7.77(d,J＝7.0Hz,1H),7.87(d,J＝11.2Hz,1H),8.33(d,J＝2.4Hz,1H),8.86(s,1H),9.59(s,1H),12.65(br.s.,1H)。LC-MS ES⁺460.1 m/z; rt: 0.71min, method A.

Preparation of intermediate 13

A solution of 10(5.3g, 18mmol) and 2, 4-dichloro-5-fluoro-pyrimidine (3g, 18mmol) was stirred in ACN (50mL) at room temperature. DIPEA (10mL, 54mmol) was added and the reaction mixture was stirred at room temperature for 2 days. The solvent was removed under reduced pressure and the crude product was purified via silica gel chromatography using a gradient of heptane to ethyl acetate. The best fractions were pooled and the solvent was removed to afford 13 as an off-white solid. LC-MS ES⁺353.1; rt: 1.34min, method B.

14 preparation of

Intermediate 3(0.3g, 0.75mmol) was reacted with 13(0.265g, 0.75mmol) under the same Suzuki (Suzuki) reaction conditions as described for compound 12. By preparative HPLC (stationary phase: RP Vydac Denali C18-10 μm, 200g, 5cm), mobile phase: 0.25% NH₄HCO₃Aqueous solution, CH₃OH), the best fractions were pooled, and the solvent was removed under reduced pressure to afford 14.¹H NMR(400MHz,DMSO-d₆)ppm 1.27-1.44(m,2H)1.54(m,J＝11.70,11.70,11.70Hz,2H)1.84(m,J＝11.00Hz,2H)2.00(m,J＝12.30Hz,1H)2.14(m,J＝11.90Hz,1H)3.67(s,3H)3.89-4.04(m,1H)4.21(m,J＝7.80,3.40Hz,1H)7.53-7.71(m,4H)8.10-8.26(m,2H)8.81(s,1H)9.62(s,1H)12.47(br.s.,1H)。LC-MS ES⁺436.2; rt: 1.27min, method B.

Preparation of intermediate 15

2-bromo-3, 5, 6-trifluoropyridine (3g, 14.153mmol), 10(3.25g, 18.87mmol) and DIPEA (3.94mL, 28.31mmol) in THF/CH₃A solution in a mixture of OH (1/1) (50mL) was heated to 95 ℃ in a pressure vessel for 16 hours. The reaction mixture was dissolved in ethyl acetate with heating and washed with brine. The organic layer was dried (MgSO₄) The solid was removed by filtration, and the solvent in the filtrate was concentrated under reduced pressure. The crude product was purified via silica flash column chromatography using a heptane to ethyl acetate gradient. The desired fractions were collected and evaporated to dryness to afford compound 15 as a solid. LC-MS ES⁺414.1; rt: 0.91min, method A.

16 preparation of

Intermediate 3(0.20g, 0.50mmol) was reacted with 15(0.207g, 0.50mmol) under the same suzuki reaction conditions as described for the formation of compound 7. The crude product is further reacted with NaOCH in an ultrasonic bath₃(2.8mL, 0.5M in CH₃OH) for 1 hour, and then the solvent was removed under reduced pressure. The crude product was dissolved in ethyl acetate, neutralized with 1M HCl, and washed with brine. The organic layer was dried (MgSO₄) The solid was removed under reduced pressure to give a solid. The crude product was purified by silica flash column chromatography using DCM to DCM/CH₃Purification was performed with a gradient of OH 100-90/10. The desired fractions were collected and evaporated to dryness to afford 16.¹H NMR(400MHz,DMSO-d₆)ppm 1.17-1.42(m,2H)1.44-1.61(m,2H)1.76-1.93(m,2H)2.00-2.21(m,2H)3.65(s,3H)3.78-3.95(m,1H)3.95-4.17(m,1H)6.46-6.65(m,1H)7.50-7.79(m,4H)7.99(d,J＝2.42Hz,1H)8.71-8.89(m,1H)9.72(s,1H)12.46(br.s.,1H)。LC-MS ES⁺m/z＝453.0；Rt: 0.72min, method A

Preparation of (+/-) -3-amino-4, 4-dimethylpentanoate

To a solution of methyl 4, 4-dimethyl-3-oxopentanoate (2mL, 12.5mmol) in methanol (20mL) was added ammonium acetate (6.75g, 87.6mmol) and NaCNBH₃(944mg, 15.0 mmol). The reaction mixture was stirred at room temperature for 18 hours. The mixture was quenched by addition of water and the solvent was removed under reduced pressure. The residue was reconstituted in ethyl acetate and the organic layer was washed with NaOH (aq, 1M) and then MgSO₄Drying, removal of the solid by filtration and removal of the solvent from the filtrate under reduced pressure afforded (+/-) -3-amino-4, 4-dimethylpentanoate as a colorless liquid, which was used without further purification or characterization.

Preparation of intermediate 17

To a solution of 2, 6-dichloro-3-fluoro-5- (trifluoromethyl) pyridine (1g, 4.15mmol) and (+/-) -3-amino-4, 4-dimethylpentanoate (974mg, 4.98mmol) in DMA (5mL) was added DIPEA (2.86mL, 16.58 mmol). The mixture was heated in a sealed tube in a microwave at 140 ℃ for 45 minutes. The reaction mixture was quenched in ice water, and the product was extracted with ethyl acetate. The organic layer was separated and dried (MgSO)₄) The solid was removed by filtration, and the solvent in the filtrate was removed under reduced pressure. The crude product was purified by silica column chromatography using an isocratic dichloromethane. The desired fraction was collected and the solvent was removed to provide 17. LC-MS ES⁺357.2 m/z; rt: 0.92min, method A.

18 preparation of

Intermediate 3(0.35g, 0.88mmol) was reacted with 17(0.36g, 1.02mmol) under the same suzuki reaction conditions as described for the formation of compound 7. Adding the crude product to NaOCH in an ultrasonic bath₃(1.35mL, 0.5M in CH₃In OH) for 1 hour. The solution is treated with CH₃OH (10mL) and water (10 mL). LiOH (16mg, 0.67mmol) was added and the mixture was stirred at ambient temperature for 2 h. The reaction was treated with concentrated HCl until pH 4 and the reaction mixture was concentrated under reduced pressure. By preparative HPLC (stationary phase: RP Vydac Denali C18-10 μm, 200g, 5cm, mobile phase: 0.25% NH in water₄HCO₃Solution of CH₃OH) purifying the crude product. The best fractions were pooled and the solvent was removed under reduced pressure to afford 18.¹H NMR(400MHz,DMSO-d₆)ppm 0.76-0.93(m,9H)2.54-2.61(m,2H)4.60-4.77(m,1H)7.35(d,J＝8.58Hz,1H)7.64-7.81(m,1H)7.64-7.81(m,1H)8.82(s,1H)9.51(s,1H)。LC-MS ES⁺m/z is 426.2; rt: 1.42min, method B.

Preparation of 19

2-methyl-7H-pyrrolo [2,3-d]Pyrimidine (426mg, 3.2mmol) was dissolved in DMF (54mL), cooled in an ice bath and treated with N-bromosuccinimide (569mg, 3.2mmol) in portions under nitrogen. The resulting mixture was stirred for 20 minutes, allowed to warm to room temperature and stirred for 10 minutes. By adding CH₃The reaction was quenched with OH (5mL) and the solvent was removed under reduced pressure. The residue was purified by silica flash column chromatography using a heptane to ethyl acetate gradient. The desired fractions were collected and the solvent was removed under reduced pressure to give 5-bromo-2-methyl-7H-pyrrolo [2,3-d]Pyrimidine, 19.

Preparation of intermediate 20

A solution of (+/-) -3-amino-4, 4-dimethylvalerate (3.09g, 13.32mmol) and 2, 6-dichloro-5-fluoronicotinonitrile (3g, 15.71mmol) was stirred in a mixture of acetonitrile/THF/EtOH (50/25/25 mL). DIPEA (5.414mL, 31.42mmol) was added and the reaction mixture was stirred at 60 ℃ for 3 hours. The solvent was removed under reduced pressure, and the crude product was purified via silica gel chromatography using a gradient of heptane to ethyl acetate to afford 20 as a solid. LC-MS ES⁺m/z is 314.1; rt: 1.13min, method A.

21 preparation of

Intermediate 3(0.35g, 0.88mmol) was reacted with 17(0.36g, 1.16mmol) under the same suzuki reaction conditions as described for the formation of compound 7. Adding the crude product to NaOCH in an ultrasonic bath₃(4mL, 0.5M in CH₃In OH) for 1 hour. The solution is treated with CH₃OH (10mL) and water (10 mL). LiOH (16mg, 0.67mmol) was added and the mixture was stirred at ambient temperature for 2 h. By preparative HPLC (stationary phase: RP Vydac Denali C18-10 μm, 200g, 5cm, mobile phase: 0.25% NH in water₄HCO₃Solution of CH₃OH) purifying the crude product. The best fractions were pooled and the solvent was removed under reduced pressure to afford 21.¹H NMR(400MHz,DMSO-d₆)ppm 0.87-0.95(m,9H)2.54-2.62(m,2H)4.81(d,J＝5.28Hz,1H)7.83(d,J＝11.22Hz,1H)7.87(d,J＝9.02Hz,1H)8.29(s,1H)8.85(s,1H)9.82(s,1H)。LC-MS ES⁺383.2; rt: 0.66min, method A.

22 preparation of

2-bromo-3, 5, 6-trifluoropyridine (1.5g, 7.08mmol), 4(1645mg, 7.78mmol) and Et₃N (1.967mL, 14.15mmol) in THF/CH₃A solution in a mixture of OH (1/1, 50mL) was heated in a pressure vessel at 85 ℃ for 2 days. The reaction mixture was concentrated under reduced pressure. The crude product was purified by silica flash column chromatography using a heptane/ethyl acetate gradient. The desired fractions were collected and evaporated to dryness to afford intermediate 22. LC-MS ES⁺m/z is 403.1; rt: 1.90min, method B

Preparation of 23

Intermediate 3(0.40g, 1.0mmol) was reacted with 22(0.33g, 0.82mmol) under the same suzuki reaction conditions as described for the formation of compound 7. Adding the crude product to NaOCH in an ultrasonic bath₃(1.5mL, 0.5M in CH₃In OH) for 1 hour. The solvent was removed under reduced pressure. The crude product was dissolved in ethyl acetate, neutralized with HCl (1M aq), and washed with brine. The organic layer was dried (MgSO₄) The solid was removed by filtration, and the solvent in the filtrate was removed under reduced pressure. The solid was purified by flash column chromatography on silica: DCM-DCM/CH₃OH (100-90/10) is purified. The desired fractions were collected and evaporated to dryness to afford (cis) -3- ((2-chloro-5-fluoropyrimidin-4-yl) amino) bicyclo [2.2.2]Octane-2-carboxylic acid methyl ester, 23.¹H NMR(400MHz,DMSO-d₆)ppm 1.11-1.33(m,2H)1.35-1.51(m,2H)1.71-1.92(m,2H)1.71-1.92(m,4H)2.10(d,J＝11.22Hz,2H)3.18(t,J＝6.60Hz,4H)3.59(m,J＝7.80,3.80,3.80Hz,1H)3.93-4.11(m,1H)5.81(d,J＝7.92Hz,1H)6.51(d,J＝7.26Hz,1H)7.68(t,J＝10.45Hz,1H)7.99(d,J＝2.20Hz,1H)8.82(s,1H)9.70(s,1H)12.48(br.s.,1H)。LC-MS ES⁺441.0; rt: 1.64min, method B.

Preparation of 24

24a (283mg, 0.90mmol) (for preparation see journal of pharmaceutical chemistry (J.Med.chem.)2004, DOI: 10.1021/jm5007275) was reacted with intermediate 3(400mg, 1.00mmol) under the same conditions as described in the formation of 7. To the crude mixture was added the in CH in a vial₃KOAc (559mg, 5.69mmol) in CN (5mL), the vial was sealed and heated in a microwave at 120 ℃ for 10 minutes. The solvent was removed under reduced pressure. The compound was dissolved in ethyl acetate and treated with concentrated HCl until pH 5. The compound was extracted with ethyl acetate. The organic layer was dried (MgSO₄) The solid was removed by filtration, and the solvent in the filtrate was removed under reduced pressure. By preparative HPLC (stationary phase: RP Vydac DenaliC18-10 μm, 200g, 5cm, mobile phase: 0.25% NH in water₄HCO₃Solution of CH₃OH) purifying the crude product and collecting the desired fraction; the solvent was removed under reduced pressure to obtain 24 as a solid.¹H NMR(600MHz,DMSO-d₆)ppm 1.22-1.88(m,13H)1.92(br.s.,1H)1.94(br.s.,1H)1.99(br.s.,1H)2.01(br.s.,1H)2.81(dd,J＝10.27,2.20Hz,1H)2.84(d,J＝7.04Hz,1H)4.34-4.44(m,1H)4.71(t,J＝6.82Hz,1H)7.61(d,J＝6.75Hz,1H)8.11(d,J＝3.81Hz,1H)8.12(s,1H)8.14(s,1H)8.18(d,J＝3.81Hz,1H)8.34(s,1H)8.80(s,1H)8.81(s,1H)9.64(s,1H)9.68(s,1H)10.20(br.s.,1H)。LC-MS ES⁺383.2; rt: 0.55min, method A.

Preparation of intermediate 26

To a suspension of (+/-) -cis-3-aminocyclohexanecarboxylic acid (25g, 174.6mmol) in 1, 4-dioxane (200mL) in a 1L round bottom flask was added 1N NaOH (262mL, 1M aq, 262 mmol). Stirring the mixtureAfter stirring for 15min, the mixture became a clear solution and boc-anhydride (49.54g, 226.98mmol) was added. The reaction was stirred at room temperature for 16 hours. The volume of the reaction mixture was reduced under reduced pressure and the reaction mixture was made acidic (pH 5) with 1M HCl. The precipitate formed was isolated by filtration, washed with water and dried under vacuum to afford 25 as a white solid.¹H NMR(400MHz,DMSO-d₆)ppm 0.93-1.31(m,4H),1.37(s,9H),1.64-1.75(m,2H),1.79(d,J＝12.3Hz,1H),1.95(d,J＝12.3Hz,1H),2.15-2.30(m,1H),3.12-3.21(m,2H),6.75(d,J＝7.9Hz,1H)

Triethylamine (35mL, 251.6mmol) and diphenylphosphoryl azide (39.056mL, 181.09mmol) were added to (+/-) -cis-3- [ (tert-butoxycarbonyl) amino group]Cyclohexanecarboxylic acid (38.99g, 160.25mmol) in toluene (600mL) was added to a stirred solution, and the resulting mixture was stirred at room temperature for 3 h. Benzyl alcohol (33.17mL, 320.51mmol) was added and the mixture was heated to 100 ℃. After 12 hours, the reaction mixture was cooled to room temperature, diluted with ethyl acetate and the resulting mixture was washed with brine and dried (Na)₂SO₄) And concentrated in vacuo to provide a crude solid 26. LC-MS ES^-347.1 m/z; rt: 0.66min, method B.

Preparation of intermediate 27

Compound 27 was prepared according to the procedure of preparation 14.¹H NMR (400MHz, chloroform-d) ppm 1.06-1.33(m,4H),1.61(d, J ═ 13.4Hz,1H),1.78(s,1H),1.87-1.98(m,1H),2.08(d, J ═ 12.3Hz,1H),2.21(d, J ═ 10.3Hz,1H),2.38(s,3H),2.55(d, J ═ 11.4Hz,1H),3.65-3.82(m,1H),4.16-4.31(m,1H),4.74(br.s.,1H),5.00(dd, J ═ 7.7,1.8Hz,1H),5.05-5.16(m,2H),7.31(d, J ═ 8.1, 6H),8 (J ═ 8H), 8H, 1H, 8H, 1H), 1.05-5.16 (m,2H),7.31(d, J ═ 8.1H, 6, J ═ 8H), 8H, 1H, 8H), 1H, 8H, 1H

28 preparation of

Into a 20mL glass vial equipped with a magnetic stir bar was placed intermediate 27(500mg, 0.81mmol), CH₃OH (10mL) and KOAc (250mg, 2.55 mmol). The vial was sealed and heated to 150 ℃ in a microwave for 15 minutes. The solvent was removed under reduced pressure and purified by preparative HPLC (stationary phase: Uptisphere C18ODB-10 μm, 200g, 5cm, mobile phase: 0.5% NH)₄Ac solution in Water/10% CH₃CN，CH₃CN) purifying the crude product. The collected fractions were basified with ammonia, the volume was reduced under reduced pressure, and the precipitate was filtered and washed with water to remove the salts, yielding 28.¹H NMR(400MHz,DMSO-d₆)ppm 1.05-1.56(m,4H),1.75-1.91(m,2H),1.96(d,J＝10.3Hz,1H),2.14(d,J＝10.6Hz,1H),3.45-3.62(m,1H),4.05-4.26(m,1H),5.00(s,2H),7.15-7.44(m,6H),7.56(d,J＝7.7Hz,1H),8.10-8.26(m,2H),8.81(s,1H),9.61(s,1H),12.48(br.s.,1H)。LC-MS ES⁺m/z is 462.2; rt: 1.70min, method B.

General procedure A. Compound 27 was added to TFA at room temperature and allowed to stir for 2 days. The solvent was removed under reduced pressure and aqueous NaHCO was added to the residue₃And CH₂Cl₂. The organic layer was dried (MgSO₄) The solid was removed by filtration, and the solvent in the filtrate was removed under reduced pressure, followed by filtration through silica gel. The crude product is then reacted with an organic base (Et)₃N or DIPEA) and an electrophilic reagent (acid chloride, chloroformate or isocyanate) at 0 ℃ to room temperature in THF or acetonitrile. If the reactant is a carboxylic acid, an amide bond may also be formed using a coupling agent (e.g., HATU, EDC) in a polar solvent (e.g., DMF). The tosyl group was then removed by addition of excess potassium tert-butoxide (stirring at room temperature for 1 day). The product was chromatographed on a silica column on CH₂Cl₂CH in (1)₂Cl₂To 10% CH₃Gradient purification of OH.

Preparation of intermediate 29a

To a reaction flask containing intermediate 27(3g, 4.873mmol) was added TFA (30mL) at room temperature. The resulting solution was allowed to stir at ambient temperature for 2 days. The solvent was removed under reduced pressure and NaHCO was added₃And CH₂Cl₂. The organic layer was concentrated and dried. The organic layer was dried (MgSO₄) The solid was removed by filtration, and the solvent in the filtrate was removed under reduced pressure. The compound was purified by flash column chromatography using dichloromethane and methanol as eluent. The desired fractions were collected and the solvent was removed under reduced pressure, yielding 29 a. LC-MS ES⁺482 m/z; rt: 0.77min, method C.

Preparation of intermediate 29b

To a reaction flask containing intermediate 29a (100mg, 0.208mmol) in THF (4mL) were added HBTU (285mg, 0.75mmol) and N, N-diisopropylethylamine (0.131mL, 0.75 mmol). The resulting mixture was stirred at room temperature under an inert atmosphere for 5 minutes. Addition of pyrazolo [1,5-a]Pyrimidine-5-carboxylic acid (41mg, 0.25mmol) and the reaction mixture was stirred at room temperature for 16 h. THF was removed under reduced pressure and the resulting residue was extracted with DCM and water. The organic layer was concentrated under reduced pressure to give 29 b. LC-MS ES⁺m/z is 627; rt: 1.26min, method C.

Preparation of 29

To a reaction flask containing 29b (130mg, 0.207mmol) dissolved in THF (4mL) was added potassium tert-butoxide (1.04mL, 1.04 mmol). Will be provided withThe resulting mixture was stirred at room temperature for 24 hours. NaHCO is used for reaction₃And CH₂Cl₂And (4) quenching. The organic layer was dried (MgSO₄) The solid was removed by filtration, and the solvent in the filtrate was removed under reduced pressure. The compound was purified by flash column chromatography using dichloromethane and methanol as solvents. The desired fractions were collected and the solvent was removed under reduced pressure, yielding 29.

¹H NMR(300MHz,DMSO-d₆)ppm 1.18-2.09(m,8H)3.99-4.09(m,1H)4.25(br s,1H)6.90(d,J＝1.51Hz,1H)7.55(d,J＝7.29Hz,1H)8.17(d,J＝3.85Hz,1H)8.22(s,1H)8.37(d,J＝2.20Hz,1H)8.80(s,1H)8.83-8.93(m,1H)9.25(d,J＝7.15Hz,1H)9.61(s,1H)。LC-MS ES⁺m/z 473; rt: 2.16min, method C.

42 preparation of

To a 100mL round bottom flask was added 2, 6-dichloro-3-fluoro-5- (trifluoromethyl) pyridine (2g, 8.291mmol), 42a (for preparation see journal of medicinal chemistry (J.Med.chem.)2004, DOI: 10.1021/jm5007275, 1.822g, 7.794mmol), ACN (40mL), and DIPEA (3.215g, 24.874 mmol). The resulting mixture was allowed to stir at room temperature for 2 days. The solvent was removed under reduced pressure and the crude product was purified via silica column chromatography using a gradient of heptane to ethyl acetate. The best fractions were collected and the solvent was removed under reduced pressure to afford 42. LC-MS ES⁺394.1; rt: 1.37min, method A.

43 preparation of

Mixing 3(400mg, 0.98mmol), 42(400mg, 0.983mmol), Pd (PPh)₃)₄(116mg，0.1mmol)、Xantphos(58mg，0.1mmol)、Na₂CO₃A mixture of (2.8mL, 2M aqueous, 5.77mmol) and 1, 4-dioxane (10mL) was stirred under microwave irradiation at 150 ℃ for 10 minutes. The solvent was removed under reduced pressure. DCM was added and the mixture was filtered through a plug of silica and washed with 5 volumes of DCM. The solvent was removed under reduced pressure. KOAc (200mg) and ethanol (7mL) were added and heated to 120 ℃ in a microwave for 10 minutes. The residue was purified via silica column chromatography using a heptane to ethyl acetate gradient. The best fractions were collected and the solvent was removed under reduced pressure, yielding 43.

44 preparation of

In a 100mL flask, 43(1.6g, 2.533mmol) was stirred in 1, 4-dioxane (90mL) at 60 ℃ while a solution of LiOH (606.6mg, 25.33mmol) in water (10mL) was added. The mixture was refluxed for 1 hour and allowed to stir at ambient temperature overnight. The 1, 4-dioxane was evaporated and the residue was taken up in 20mL of ethyl acetate, stirred and neutralized with concentrated HCl. The residue was concentrated under reduced pressure. By preparative HPLC (stationary phase: RP Xbridge Prep C18ODB-5 μm, 30X250mm, mobile phase: 0.25% NH)₄HCO₃Solution in water, methanol). The desired fractions were collected and evaporated to dryness. After addition of methanol, the solution was concentrated a second time to provide 44 as a white solid.¹H NMR(400MHz,DMSO-d₆)ppm 1.27-1.45(m,4H)1.75(m,4H)1.78-1.86(m,1H)1.91-2.01(m,1H)2.74(m,1H)4.64(m,1H)7.42(m,1H)7.70-7.76(m,2H)8.82(s,1H)9.37(s,1H)。LC-MS ES⁺m/z 449; rt: 1.58min, method B.

46 preparation of

According toPreparation 7 preparation 46. LC-MS ES⁺m/z is 424.2; rt: 1.41min, method B.

48 preparation of

By preparative SFC (stationary phase: Chiralcel Diacel OD 20X250mm, mobile phase: CO)₂Isopropanol with 0.2% isopropylamine) purified compound 14. The desired fractions were collected and the solvent was removed under reduced pressure, yielding 48.

50 preparation of

A mixture of 3(4.39g, 11mmol), 2-chloro-5-fluoro-4-methylsulfanyl-pyrimidine (1.96g, 11mmol) and potassium phosphate (7g, 33mmol) was stirred in water (44mL) and Me-THF (132mL) at room temperature under a nitrogen atmosphere. Then, Xantphos (629mg, 1.32mmol) and Pd were added₂(dba)₃(302.19mg, 0.33mmol) and degassed by bubbling nitrogen through the mixture for 10 minutes. The reaction was heated to 80 ℃ and stirred in a closed vessel at this temperature for 2 hours. The mixture was allowed to cool for 1 hour, then reconstituted in 200mL ethyl acetate and washed twice with brine. The organic layer was purified over MgSO₄Dried, filtered and evaporated. The residue was purified on silica with dichloromethane/methanol (99/1) as eluent. The desired fractions were evaporated and the residue was crystallized from acetonitrile. The crystals were collected by filtration and dried in vacuo, 50. LC-MS ES⁺m/z 415; rt: 2.00min, method B.

51 preparation

A solution of 50(415.47mg, 1mmol) in methanol (2mL) and DCM (1.15mL) was stirred at room temperature, m-CPBA (739.58mg, 3mmol) was added portionwise over 10 min and the mixture was stirredStirring was continued overnight at ambient temperature. The mixture was diluted with 30mL of DCM and washed twice with saturated aqueous sodium bicarbonate and once with water. The organic layer was purified over MgSO₄Dried, filtered and evaporated. The residue was triturated in diisopropyl ether/acetonitrile (20/1). The yellow precipitate was collected by filtration and dried, yielding 51. LC-MS ES⁺m/z 447; rt: 1.59min, method B.

General procedure B. Compound 51 was added to 1, 4-dioxane at room temperature. The crude product is then reacted with an organic base (Et)₃N or DIPEA) and an amine. The resulting mixture was refluxed and stirred for 1 day. The solvent was removed under reduced pressure. The resulting crude product was purified by silica column chromatography. The tosyl group was then removed by addition of excess potassium tert-butoxide (stirring at room temperature for 1 day). The product was purified by silica gel column chromatography.

52 preparation of

To a solution of 51(1.683g, 3.76mmol) and DIPEA (2mL) in 1, 4-dioxane (30mL) was added ethyl 3-amino-3- (1-methyl-cyclopentyl) -propionate (1.500g, 7.527 mmol). The resulting reaction mixture was stirred at 70 ℃ overnight. Then, the reaction mixture was evaporated to dryness and purified by silica column chromatography (heptane: ethyl acetate 60:40) to provide 52.

Preparation of 3-amino-3- (1-methyl-cyclopentyl) -propionic acid ethyl ester

Step 1 LiHMDS (1M in THF) (189mL, 189mmol) was added dropwise to a solution of cyclopentanecarbonitrile (15g, 158mmol) in THF (64mL) under nitrogen at-78 ℃. Then, the mixture was stirred for 30 minutes, and CH was added at once₃I (14.7mL, 240mmol) and the mixture was slowly warmed to room temperature overnight. EtOAc (250mL) was added and NH was added slowly at 0 deg.C₄Cl 10% (200 mL). Then, water (100mL) was added to form a solution and the organic layer was evaporatedSeparated and washed with brine, dried and concentrated to give 1-methylcyclopentane carbonitrile (16.8g, yellow oil) which was used in the next step without purification.

Step 2 DIBAL (37mL, 37mmol) was added dropwise to 1-methylcyclopentanecarbonitrile (2.0g, 18mmol) in CH at-78 deg.C under nitrogen₂Cl₂(117mL), and the mixture was stirred at-78 ℃ for 15 minutes after the addition was terminated. Slowly add CH at-78 deg.C₃OH (37mL), and the reaction was warmed to room temperature. NaOH (1M)200mL was added and the aqueous solution was taken up with CH₂Cl₂Extracting twice with MgSO 2₄Dried and concentrated. The mixture was stirred in aqueous HCl (3M) for 1 hour with CH₂Cl₂Extracting over MgSO₄Dried and concentrated to give 1-methylcyclopentane carboxaldehyde (1.4g, yellow oil).

Step 3. 1-methylcyclopentanemaldehyde (1.4g, 12mmol), malonic acid (1.0g, 9.6mmol), NH in EtOH (5.6mL) in a sealed tube at 80 deg.C₄OAc (1.5g, 19mmol) was stirred overnight. The mixture was cooled to room temperature, filtered and washed with EtOH. H is to be₂SO₄(0.51mL, 9.6mmol) was added to the filtrate and the mixture was stirred at 80 ℃ for 2 hours. The mixture was concentrated, taken up in water and washed with DCM (3 ×). The organic mixture was discarded and the aqueous layer was basified with NaOH (3N) and extracted with DCM. The organic layer was purified over MgSO₄Dried, filtered and concentrated to give 3-amino-3- (1-methyl-cyclopentyl) -propionic acid (0.75g, colorless oil).

Step 4. mixing 3-amino-3- (1-methyl-cyclopentyl) -propionic acid with ethanol and adding dropwise SOCl thereto at 0 deg.C₂. After addition, the reaction mixture was heated to reflux and stirred for 6 hours. The solvent was removed under reduced pressure. The crude product was dissolved in DCM and taken up with saturated aqueous NaHCO₃And (6) washing. The organic layer was purified over MgSO₄Dried, filtered and evaporated to dryness to afford 3-amino-3- (1-methyl-cyclopentyl) -propionic acid ethyl ester, which was further purified for use in the next step.

Preparation of 53

To a closed vessel containing 52(400mg, 0.706mmol) were added potassium tert-butoxide (3.63mL, 3.63mmol) and THF (10mL) under an inert atmosphere. The mixture was stirred at room temperature for 1 hour. Then, a small amount of water (10 μ L) was added and the reaction mixture was heated to 60 ℃. The solvent of the reaction mixture was evaporated to dryness and purified by preparative HPLC (method: from 90% [ aqueous phase ]]-10% [ organic phase ]]To 54% [ AP%]-46％[OP].AP：25mM NH₄HCO₃OP: MeCN: methanol 1:1) for purification. The desired fractions were collected and the solvent was removed under reduced pressure, yielding 53.

¹H NMR(400MHz,DMSO-d₆)ppm 1.04(s,3H)1.17-1.43(m,2H)1.54-1.69(m,5H)1.73-1.83(m,1H)2.55-2.61(m,2H)4.87-4.94(m,1H)7.01-7.10(m,1H)8.04(s,1H)8.10(d,J＝3.96Hz,1H)8.77(s,1H)9.69(s,1H)。LC-MS ES⁺m/z is 384.5; rt: 1.24min, method B.

Preparation of intermediate 59

A solution of isopropylamine (2.66mL, 29.95mmol) and DIPEA (5.16mL, 29.95mmol) in THF (50mL) was stirred at-10 deg.C while 2, 4-dichloro-5-fluoropyrimidine (5g, 29.95mmol) was added portionwise. The resulting mixture was stirred at ambient temperature overnight. The mixture was diluted with 100mL of ethyl acetate and 50mL of diisopropyl ether. The solution was washed twice with water. The organic phase is passed over MgSO₄Dried, filtered and evaporated, yielding 67. The crude product was used as such. LC-MS ES⁺189 m/z; rt: 1.65min, method B.

60 preparation of

Under a nitrogen atmosphere, 3(798.6mg, 2mmol), 59(379.24mg, 2mmol) and Na were added at room temperature₂CO₃(3mL, 2M, 6mmol) of the mixture was stirred in 1, 4-dioxane (10 mL). Then, Pd (PPh) was added₃)₄(115.56mg, 0.1mmol) and Xantphos (57.86mg, 0.1mmol) and degassing was performed by bubbling nitrogen through the mixture for 10 minutes. The reaction was heated to 150 ℃ for 15 minutes under microwave irradiation. The mixture was diluted with water and extracted twice with ethyl acetate. The organic layer was washed once with brine, over MgSO₄Dried, filtered and evaporated. The residue was crystallized from diisopropyl ether containing about 10% acetonitrile. The off-white precipitate was collected by filtration and dried in vacuo to yield 60. LC-MS ES⁺426 m/z; rt: 2.22min, method B.

61 preparation of

A mixture of 60(300mg, 0.7mmol) and NaOMe (15mL, 0.5M, 7.5mmol) was sonicated for 10 min and stirred at room temperature for 1 h. The mixture was evaporated and reconstituted in ice water, stirred and neutralized with 7.5mL 1N aqueous HCl. The aqueous layer was extracted three times with dichloromethane. The combined organic layers were washed with MgSO₄Dried, filtered and evaporated. The residue was purified via silica column chromatography using a gradient of dichloromethane/methanol (98/2-95/5). The corresponding fractions were evaporated and the residue was crystallized from diisopropyl ether/acetonitrile (2/1). The crystals were collected by filtration and dried in vacuo to give 61¹H NMR(360MHz,DMSO-d₆)ppm 1.29(d,J＝6.59Hz,6H)4.40-4.51(m,1H)7.49(br d,J＝7.68Hz,1H)8.15-8.18(m,2H)8.82(s,1H)9.61(s,1H)12.51(br s,1H)。LC-MS ES⁺m/z is 272.1; rt: 1.45min, method B.

Preparation of methyl 3-amino-4, 4-dimethylpentanoate

Mixing trimethylacetaldehyde (33.25g, 386.027mmol), malonic acid (30.204g, 290.246mmol) and NH₄A mixture of OAc (44.746g, 580.492mmol) in 100mL EtOH was refluxed for 6 hours. The precipitate was isolated by filtration and washed with ethanol. The solution was used as such and H was added₂SO₄(15.5 mL). The resulting mixture was heated to reflux for 5 hours. The solvent was removed under reduced pressure and the crude product was added to 300mL of water and 150mL of Et₂And (4) in O. The aqueous layer was neutralized with NaOH (6N aqueous). The product was extracted with EtOAc. Passing the organic phase over Na₂SO₄Dried, filtered and concentrated under reduced pressure.

A solution of the residue (10g, 68.87mmol) in methanol (50mL) was cooled on an ice bath and SOCl was added dropwise₂(5mL, 68.87 mmol). After addition, the reaction mixture was heated to reflux and stirred for 6 hours. The reaction was completed, and the solvent was removed under reduced pressure. The crude product was dissolved in DCM and washed with saturated aqueous NaHCO₃And brine. The organic layer was purified over MgSO₄Dried, filtered and evaporated to dryness to afford methyl 3-amino-4, 4-dimethylpentanoate.

67 preparation of

A solution of 2, 4-dichloro-5-fluoro-pyrimidine (3g, 17.967mmol) was stirred at room temperature in EtOH (72mL) and THF (72 mL). Methyl 3-amino-4, 4-dimethylpentanoate (3.622g, 22.749mmol) and DIPEA (9.289mL, 53.90mmol) were added dropwise to the reaction mixture and stirred at 70 ℃ for 1 hour then overnight at ambient temperature. The reaction mixture was evaporated. Dissolving the residue in waterReconstituted and extracted twice with DCM. The combined organic layers were washed once with water and over MgSO₄Dried, filtered and evaporated. The residue was purified by silica flash column chromatography (eluent: DCM-DCM/methanol (100-90/10)). The desired fractions were collected and evaporated to dryness to afford 67. LC-MS ES⁺289.1; rt: 0.99min, method A.

68 preparation

In a pressure tube, 3(3.5g, 8.766mmol), Pd (PPh) in DME (50mL) and water (15mL)₃)₄(1013mg，0.877mmol)、K₂CO₃A mixture of (2423mg, 17.53mmol) and 67(2.667g, 9.204mmol) was heated to 100 ℃ and stirred for 16 h. The reaction was completed, and the solvent was removed under reduced pressure. The crude residue was taken up in DCM and filtered. The filtrate was purified by silica flash column chromatography (gradient: heptane-EtOAc (100-. The desired fractions were collected and evaporated to dryness. The residue was dissolved in a mixture of DCM/methanol (1/1) and purified by silica gel column chromatography (gradient: heptane-EtOAc (100-. The desired fractions were collected and the solvent was removed under reduced pressure, yielding 68.¹H NMR(400MHz,DMSO-d₆)ppm 0.97(s,9H)2.65(m,1H)2.78(m,1H)3.44(s,3H)4.76-4.94(m,1H)7.41(m,1H)8.07-8.27(m,2H)8.82(s,1H)9.73(s,1H)12.47(br.s.,1H)。LC-MS ES⁺m/z is 372.2; rt: 0.84min, method A.

69 preparation of

A solution of 68(150mg, 0.39mmol) and LiOH (37.379mg, 1.561mmol) in water (4mL) and 1, 4-dioxane (8mL) was stirred at room temperature for 16 hours. The organic solvent was removed under reduced pressure and the aqueous layer was acidified with 1N HCl. Will be describedThe precipitate formed was filtered off, washed with water and dried under vacuum at 50 ℃ to afford 69.¹H NMR(400MHz,DMSO-d₆)ppm 0.96(s,9H)2.54-2.62(m,2H)4.68-4.84(m,1H)7.69(m,1H)8.11-8.15(m,2H)8.81(s,1H)9.73(s,1H)。LC-MS ES⁺358.1 m/z; rt: 1.15min, method B.

70 preparation of

Intermediate 70 was prepared according to the procedure for preparation 67. LC-MS ES⁺315 for m/z; rt: 1.11min, method A.

71 preparation of

Preparation 71 was carried out according to the procedure of preparation 68.

87 preparation

Intermediate 72 was prepared according to the procedure for preparation 69. LC-MS ES⁺358.1 m/z; rt: 1.15min, method B.

Preparation of intermediate 74

Intermediate 74 was prepared according to the procedure for preparation of intermediate 2.

75 preparation of

74(580mg, 1.5mmol), 2, 4-dimethoxybenzylamine (276mg, 1.65mmol) and K₂CO₃A solution of (414mg, 3mmol) in 1, 4-dioxane (6mL) was stirred at 80 ℃ for 2 hours. The reaction mixture was allowed to reach room temperature and the solvent was removed under reduced pressure. The crude reaction mixture was purified by silica flash column chromatography (gradient: petroleum ether-EtOAc (100-50/50)). The desired fractions were collected and evaporated to dryness to afford 75.

76 preparation of

Preparation 76 was prepared according to the procedure for preparation of intermediate 3.

77 preparation of

Mixing 76(2.73g, 1.47mmol), 5(0.5g, 1.47mmol) and K₃PO₄(0.62g, 2.93mmol) and [1,1' -bis (di-tert-butylphosphino) ferrocene]A mixture of palladium (II) dichloride (96mg, 0.147mmol) in 1, 4-dioxane (80mL) and water (20mL) was heated to 70 ℃ for 10 hours. The reaction mixture was filtered through celite and concentrated. The reaction mixture was then diluted with DCM and washed with water. The organic layer was purified over MgSO₄Drying and purification by reverse phase HPLC (column: SYNERGI250X50X10 μm, flow rate: 80mL/min, mobile phase A: water (containing 0.1% TFA), mobile phase B: acetonitrile, gradient: 45% -75% (% B)). The desired fractions were collected and the solvent was removed under reduced pressure to afford 77.

Preparation of intermediate 78

To a solution of 77(120mg, 0.161mmol) in DCM (2mL) was added TFA (2mL) and the whole solution was stirred at room temperature for 16 h. The solvent was removed under reduced pressure and the residue containing 78 was used directly in the next step. LC-MSES⁺594.2; rt: 0.78min, method E.

79 preparation of

To a flask containing 78(90mg, 0.152mmol) in methanol (5mL) was added sodium methoxide (33mg, 0.608mmol) at room temperature. Then, the mixture was stirred at room temperature for 2 hours. The mixture was poured into water and extracted three times with EtOAc. The combined organic layers were washed with brine and dried (MgSO)₄) And concentrated. By reverse phase column chromatography: (Agela DuraShell C18150X 25X5 μm, flow rate: 35ml/min, mobile phase A: water (containing 0.05% NH)₃.H₂O), mobile phase B: acetonitrile, gradient: 26% -56% (% B)) of the crude mixture. The desired fractions were collected and the solvent was removed under reduced pressure, yielding 79.¹H NMR (400MHz, chloroform-d) ppm 0.97-1.35(m,3H)1.42-1.68(m,2H)1.86-1.95(m,4H)1.96-2.13(m,2H)2.64-2.76(m,1H)3.28-3.35(m,3H)3.83-3.97(m,1H)4.04-4.12(m,1H)4.94(br d, J ═ 7.03Hz,1H)7.94(d, J ═ 3.01Hz,1H)7.98(s,1H)8.26(s, 1H). LC-MS ES⁺m/z is 440.2; rt: 3.72min, method E.

80 preparation of

To 5-bromo-6-methyl-7H-pyrrolo [2,3-d]To a solution of pyrimidine (300mg, 1.415mmol) in toluene (15mL) was added tetrabutylammonium hydrogen sulfate (28.4mg, 0.113mmol), followed by NaOH (50% in water)(5mL) and the mixture was stirred vigorously. A solution of p-toluenesulfonyl chloride (378mg, 1.98mmol) in toluene (15mL) was added and the entire mixture was stirred at room temperature for 18 h. The organic layer was separated, washed with water and dried (MgSO)₄) And concentrated. The crude product containing 80 was used in the next step without further purification. LC-MS ES⁺367; rt: 1.031min, method C.

81 preparation of

Compound 81 was prepared according to the procedure of preparation 3. LC-MS ES⁺m/z 414; rt: 1.257min, method C.

82 preparation of

98(100mg, 0.242mmol), 5(107.7mg, 0.315mmol), Pd (PPh)₃)₄(140mg，0.121mmol)、Xantphos(70mg，0.121mmol)、2M Na₂CO₃A mixture of (0.363mL, 2M, 0.726mmol), dioxane (10mL) was stirred at 80 ℃ for 48 hours. The reaction mixture was filtered through a pad of celite and washed with ethyl acetate. The solvent was removed under reduced pressure. The residue containing 82 was used directly in the next step. LC-MS ES⁺m/z 593; rt: 1.094min, method C.

83 preparation of

Compound 83 was prepared according to the procedure for preparation 29.¹H NMR(300MHz,DMSO-d₆)ppm 1.20-1.44(m,5H)1.72-1.86(m,6H)1.87-2.06(m,1H)2.08-2.21(m,1H)2.86(s,2H)3.16-3.22(m,4H)3.50-3.68(m,1H)3.97-4.13(m,1H)5.82(br d,J＝7.97Hz,1H)7.54(br d,J＝7.56Hz,1H)8.21(d,J＝3.85Hz,1H)8.73(s,1H)9.57(s,1H)12.33(s,1H)。LC-MS ES⁺439 (m/z); rt: 2.160min, method C.

84 preparation of

At 0 deg.C, to 5-bromo-4-methyl-7H-pyrrolo [2,3-d]To a solution of pyrimidine (2g, 9.43mmol) in acetone (20mL) was added a 2N NaOH solution (9.43mL, 18.87mmol) followed by p-toluenesulfonyl chloride (1.98g, 10.38mmol) and the mixture was stirred for 18 h. The reaction mixture was concentrated and the aqueous layer was extracted with EtOAc. The organic layer was separated, washed with water and dried (MgSO)₄) And concentrated. The residue containing 84 was used directly in the next step.

85 preparation of

Preparation 85 was prepared according to the procedure for preparation of compound 3.

86 preparation of

84(180mg, 1.47mmol), 5(223.5mg, 0.65mmol) and K₃PO₄(278mg, 1.31mmol) and [1,1' -bis (di-tert-butylphosphino) ferrocene]A mixture of palladium (II) dichloride (28.5mg, 0.04mmol) in 1, 4-dioxane (15mL) and water (3mL) was heated to 100 ℃ under microwave irradiation for 20 minutes. The reaction mixture was filtered through celite and concentrated. The mixture containing 85 was used directly in the next step.

86 preparation of

To a mixture of 86(180mg, 0.304mmol) in methanol (10mL) was added NaOMe (66mg, 1.22mmol) and stirred at room temperature for 2 h. The reaction mixture was poured into water and extracted three times with EtOAc. The organic layer was separated and washed with water and dried (Na)₂SO₄) And concentrated. Through reverse phase column chromatography (column: Waters Xbridge C18150X20mmX5 μm, flow rate: 25mL/min, mobile phase A: water (containing 0.05% NH)₃.H₂O), mobile phase B: acetonitrile, gradient: 18% -48% (% B)) of the crude mixture. The desired fraction was concentrated to provide 87.¹H NMR (400MHz, chloroform-d) ppm 1.09(br d, J ═ 11.54Hz,4H)1.51-1.59(m,1H)1.85-1.94(m,6H)2.03-2.16(m,2H)2.60-2.69(m,1H)3.09(s,3H)3.27-3.36(m,4H)3.80-3.92(m,1H)4.04(d, J ═ 7.28Hz,1H)4.12-4.29(m,1H)4.90(br d, J ═ 5.52Hz,1H)8.00(s,1H)8.09(d, J ═ 3.01Hz,1H)8.79(s,1H)9.87(s, 1H). LC-MS ES⁺439.2; rt: 3.19min, method E.

88 preparation of

To a mixture of 2-methyl-7H-pyrrolo [2,3-d ] pyrimidine (400mg, 2.253mmol) in THF (4mL) was added NBS (480mg, 2.70mmol) and stirred at room temperature for 3 hours. The precipitate was isolated by filtration, washed with DCM and dried under vacuum to afford the desired compound 88.

89 preparation of

To a stirred solution of 88(230mg, 1.085mmol) in THF (25mL) at room temperature under nitrogen was added NaH (80mg, 2mmol) in portions. The resulting mixture was stirred at room temperature for 1 hour, then p-toluenesulfonyl chloride (264mg, 1.385mmol) was added. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure. The crude reaction mixture was purified by silica flash column chromatography (gradient: petroleum ether/EtOAc (65/35)). The desired fractions were collected and evaporated to dryness to afford 89.

107 preparation of

Intermediate 90 was prepared according to the procedure for preparation of intermediate 3.

91 preparation of

Mixing 90(165.4mg, 0.484mmol), 5(200mg, 0.484mmol) and K₃PO₄(308mg, 1.45mmol) and [1,1' -bis (di-tert-butylphosphino) ferrocene]A mixture of palladium (II) dichloride (31.5mg,0.049mmol) in 1, 4-dioxane (20mL) and water (5mL) was heated to 80 ℃ for 18 hours. The reaction mixture was filtered through celite and concentrated. The mixture containing 91 was used directly in the next step.

92 preparation of

Compound 92 was prepared according to the procedure for preparation 104.¹H NMR (400MHz, chloroform-d) ppm 1.08-1.29(m,4H)1.88-1.92(m,4H)2.08(m, J ═ 14.10Hz,1H)2.17-2.25(m,1H)2.62-2.69(m,1H)2.77-2.83(m,1H)2.81(s,2H)3.25-3.29(m,1H)3.29-3.34(m,3H)3.84-3.93(m,1H)4.04-4.09(m,1H)4.09-4.18(m,1H)4.88(m,1H)8.05-8.08(m,2H)9.58(s, 2H). LC-MS ES⁺439.2; rt: 3.44min, method E.

93 preparation of

By preparative SFC (stationary phase: Chiralcel Diacel OD 20X250mm, mobile phase: CO)₂Isopropyl alcohol containing 0.2% isopropylamine) purified compound 9. The desired fractions were collected and the solvent was removed under reduced pressure, yielding 93¹H NMR (400MHz, methanol-d)₄)ppm 1.22-1.53(m,9H)1.55-1.67(m,1H)1.85-1.91(m,4H)2.13-2.22(m,1H)2.24-2.32(m,1H)3.36-3.44(m,1H)3.70-3.80(m,1H)4.17-4.27(m,1H)5.81(m,1H)7.52(m,1H)8.39(s,1H)8.80(s,1H)9.62(s,1H)。LC-MS ES⁺448.2; rt: 1.55min, method A.

94 preparation of

A solution of benzyl tert-butyl (+/-) -cyclohexane-1, 3-diyl dicarbamate (52.27g, 150mmol) in methanol (1.5L) was stirred under a nitrogen atmosphere, Pd/C (10%) (1.6g, 1.5mmol) was added, and stirring was carried out under hydrogen (3.75L, 0.04M, 150mmol) at room temperature overnight. The catalyst was filtered off over celite and under nitrogen, rinsed twice with methanol, and the filtrate evaporated to dryness, yielding 94.

95 preparation of

A solution of 2, 6-dichloro-5-fluoro-pyridine-3-carbonitrile (19.1g, 100mmol) in THF (200mL) was stirred at room temperature while a mixture of 94(12.54g, 50mmol) and DIPEA (26mL, 150mmol) in ACN (100mL) was added dropwise. The reaction was allowed to stir at ambient temperature over the weekend. The solvent in the mixture was evaporated and the residue was triturated in water and stirred overnight. The precipitate was filtered off and dried in vacuo to yield 95.

96 preparation of

To a solution of HCl (6M in iPrOH) (16.7mL, 6M, 100mmol) in methanol (50mL) was added 95(4.61g, 10mmol) portionwise and the reaction was stirred at ambient temperature for 2 hours. The mixture was evaporated to dryness and the residue was triturated in diisopropyl ether/acetone. The crystals were collected by filtration and dried in vacuo to yield 96.

97 preparation of

To a flask containing picolinic acid (240.521mg, 41.954mmol) in THF (18mL) was added HBTU (1411mg, 3.721mmol) at room temperature. The resulting mixture was stirred under an inert atmosphere for 5 minutes. Then a solution of 96(500mg, 1.86mmol) and N, N-diisopropylethylamine (0.81mL, 4.652mmol) in DMSO (1mL) was added. The mixture was stirred at room temperature for 1 hour. Then, the reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was dried (Mg)₂SO₄) Filtered, and concentrated under reduced pressure. Purification was performed using a silica gel column (heptane: AcOEt 50: 50). The desired fractions were collected and the solvent was removed under reduced pressure, yielding 97. LC-MS ES⁺m/z 373.9; rt: 1.38min, method C.

155 preparation

Mixing 3(200mg, 0.501mmol), 97(187mg, 0.501mmol), K₃PO₄(33mg, 0.0501mmol) and [1,1' -bis (di-tert-butylphosphino) dicyclopentadieneIron]A mixture of palladium (II) dichloride (318mg, 1.503mmol) in 1, 4-dioxane (5mL) and water (0.5mL) was heated to 90 ℃ for 2 hours. The reaction mixture was filtered through celite and concentrated. The reaction mixture was then diluted with DCM and washed with water. The organic layer was purified over MgSO₄Dried and purified by reverse phase HPLC (method: MG3BIC, from 70% [ aqueous phase ]]-30% [ organic phase ]]To 27% [ AP%]-73％[OP]25mM aqueous NH₄HCO₃OP ═ acetonitrile: methanol 1:1) was purified. The desired fractions were collected and the solvent was removed under reduced pressure to afford 98. LC-MS ES⁺m/z is 457.2; rt: 2.39min, method C.

99 preparation of

Containing 2, 6-dichloro-5-fluoro-3-pyridinecarbonitrile (1g, 5.26mmol), triethylamine (1.61mL, 11.54mmol) and (+/-) -3-aminobicyclo [2.2.2 ] in ACN (30mL)]A flask of octane-2-carboxylic acid methyl ester (1.04g, 4.73mmol) was refluxed for 12 hours. The reaction mixture was diluted into EtOAc and brine. The organic phase is passed over MgSO₄Dry, filter and concentrate the solvent in vacuo. 99 was used in the next step without further purification.

100 preparation of

A solution containing 3(2.814g, 7.049mmol) and 99(2g, 54.92mmol) in THF (40mL) and water (10mL) was stirred at room temperature under an inert atmosphere for 10 minutes. Then, Pd was added₂(dba)₃(0.103g, 0.141mmol), tripotassium phosphate (3.753g, 17.69mmol) and XantPhos (0.336g, 0.705mmol), and the mixture was stirred at 120 ℃ for 12 hours. The crude product was concentrated under reduced pressure and 100 was used in the next step without further purification.

101 preparation of

To a flask containing 100(5.103g, 8.88mmol) in THF (10mL) was added sodium methoxide (11.5mL, 53.28 mmol). The resulting mixture was stirred at 40 ℃ for 3 hours. The reaction was then concentrated under vacuum. By reverse phase chromatography (start: 19% organic phase; finish: 55% organic phase; organic phase: 0.1% HCOOH: acetonitrile, aqueous phase: 1:1:25mM NH)₄HCO₃) The mixture is purified. The desired fractions were collected and the solvent was removed under reduced pressure, yielding 101. LC-MSES⁺m/z 407; rt: 2.69min, method C.

Table 1. compounds of formula (I) and corresponding analytical data. The compounds were prepared according to the methods described in the experimental section. Rt ═ retention time.

High Performance Liquid Chromatography (HPLC) measurements were performed using LC pumps, Diode Arrays (DADs) or UV detectors and columns as specified in the corresponding methods. Other detectors were included if necessary (see method tables below).

The flow from the column is directed to a Mass Spectrometer (MS) equipped with an atmospheric pressure ion source. It is within the knowledge of the skilled person to set tuning parameters (e.g. scan range, residence time, etc.) in order to obtain ions of nominal monoisotopic Molecular Weight (MW) that allow identification of compounds. Data acquisition is performed using suitable software.

By which the retention time (R) is determined_t) And an ion describing compound. If not specified differently in the data sheet, the reported molecular ion corresponds to [ M + H [ ]]⁺(protonated molecules) and/or [ M-H]^-(deprotonated molecules). In the case where the compound is not directly ionizable, the type of adduct is specified (i.e., [ M + NH ]₄]⁺、[M+HCOO]^-Etc.). For molecules with multiple isotopic patterns (Br, Cl, etc.), the reported values are the values obtained for the lowest isotopic mass. All results obtained have the experimental exception normally associated with the method usedCertainty.

"SQD" single quadrupole detector, "RT" refers to room temperature, "BEH" refers to a bridged ethylsiloxane/silica hybrid, "HSS" refers to high intensity silica, and "DAD" refers to a diode array detector. Flow is expressed in mL/min; the column temperature (T) is expressed in degrees Celsius; run time is expressed in minutes.

Biological activity of the compound of formula (I)

Cell-based antiviral assays were used to determine the in vitro antiviral activity of these compounds. In this assay, the cytopathic effect (CPE) in Madin-Darby canine kidney (MDCK) cells infected with influenza a virus/taiwan/1/86 (H1N1) was monitored in the presence or absence of these compounds. White 384-well microtiter plates (Greiner) were filled via sonic drop ejection using an echo liquid processor (Labcyte, sunnyvale, california). Two hundred nanoliters of compound stock (100% DMSO) were transferred to assay plates. MDCK cells were dispensed onto plates at final densities of 25,000 or 6,000 cells/well. Influenza a/taiwan/1/86 (H1N1) virus was then added at a multiplicity of infection of 0.001 or 0.01, respectively. These wells contained 0.5% DMSO per volume. Viral-infected and mock-infected controls were included in each test. At 37 ℃ in 5% CO₂And incubating the plates. Three days after virus exposure, cytopathic effects were quantified by measuring the decrease in ATP levels using the ATPliteTM kit (platinummer, zaftem, belgium) according to the manufacturer's instructions. Will IC₅₀Defined as 50% inhibitory concentration. In parallel, compounds were incubated in white 384-well microtiter plates for three days and passed through according to the manufacturerThe in vitro cytotoxicity of compounds in MDCK cells was determined using the ATPliteTM kit (platinum elmer, zaftem, belgium) to measure the ATP content of cells. Cytotoxicity is reported as CC₅₀This is the concentration that causes a 50% reduction in cell viability.

Table 2 biological activity of compounds of formula (I).

Claims (9)

1. A compound of formula (I)

(I)

A stereoisomeric form, a pharmaceutically acceptable salt, solvate or polymorph thereof, wherein

X is optionally substituted by-CN, -CF₃、-C_1-3alkyl-N-C (O) -C_1-3Alkyl, -C (O) -NH₂、-C(O)-NH-C_1-3Alkyl, -C (O) N- (dialkyl) or-CH₂-NC(O)-CH₃Substituted N or C;

R₁is H or CH₃；

R₂Is H or NH₂；

R₃Is C substituted by formic acid_1-8An alkyl group;

or by formic acid, -N-C_1-3Alkyl sulfone substituted C_3-8Cycloalkyl radicals, or

Optionally is covered with C_1-6Alkyl substituted-N-C (O) -C_3-6A heterocycle;

or is substituted by-N-C (O) -C_3-6Heterocycle-substituted C_3-6A heterocyclic ring.

2. The compound of claim 1, wherein R₁And R₂Are all H.

3. A compound according to claim 1 or 2, of formula

Or。

4. A pharmaceutical composition comprising a compound of formula (I) or a stereoisomeric form, a pharmaceutically acceptable salt, solvate or polymorph thereof according to claim 1 together with one or more pharmaceutically acceptable excipients, diluents or carriers.

5. A compound of formula (I) according to claim 1 or a stereoisomeric form, a pharmaceutically acceptable salt, solvate or polymorph thereof or a pharmaceutical composition according to claim 4 for use as a medicament.

6. A compound of formula (I) or a stereoisomeric form, a pharmaceutically acceptable salt, solvate or polymorph thereof according to claim 1 or a pharmaceutical composition according to claim 4 for use in the treatment of influenza.

7. A compound represented by the following structural formula (I)

A stereoisomeric form, a pharmaceutically acceptable salt, solvate or polymorph thereof, wherein

X is optionally substituted by-CN, -CF₃、-C_1-3alkyl-N-C (O) -C_1-3Alkyl, -C (O) -NH₂、-C(O)-NH-C_1-3Alkyl, -C (O) N- (dialkyl) or-CH₂-NC(O)-CH₃Substituted N or C;

R₁is H or CH₃；

R₂Is H or NH₂；

R₃Is C substituted by formic acid_1-8An alkyl group;

or by formic acid, -N-C_1-3Alkyl sulfone substituted C_3-8Cycloalkyl radicals, or

Optionally is covered with C_1-6Alkyl substituted-N-C (O) -C_3-6A heterocycle;

or is substituted by-N-C (O) -C_3-6Heterocycle-substituted C_3-6Heterocyclic rings

Use for inhibiting replication of one or more influenza viruses in a biological sample or patient.

8. The use of claim 7, further comprising co-administering an additional therapeutic agent.

9. The use of claim 8, wherein the additional therapeutic agent is selected from an antiviral agent or an influenza vaccine or both.