INDOLINE DERIVATIVES REPLACED AS INHIBITORS OF DENGUE VIRAL REPLICATION

专利摘要:
the present invention relates to substituted indoline derivatives, methods to prevent or treat viral dengue infections using said compounds, and also relates to said compounds for use as a medicament, more preferably for use as a medicament to treat or prevent dengue viral infections. the present invention also relates to pharmaceutical compositions or preparations of combinations of the compounds, compositions or preparations for use as a medicament, more preferably for the prevention or treatment of dengue viral infections. the invention also relates to processes for the preparation of the compounds.
公开号:BR112019020175A2
申请号:R112019020175-0
申请日:2018-03-29
公开日:2020-06-02
发明作者:Rudolf Romanie Kesteleyn Bart；Bonfanti Jean-François；Coesemans Erwin；Jean-Marie Bernard Raboisson Pierre；Didier M. Marchand Arnaud；Alice Marie-Eve Bardiot Dorothée
申请人:Janssen Pharmaceuticals, Inc.；Katholieke Universiteit Leuven；
IPC主号:

专利说明:

Descriptive Report of the Invention Patent for INDOLINE DERIVATIVES REPLACED AS INHIBITORS OF DENGUE'S VIRAL REPLICATION.
[001] The present invention relates to substituted indoline derivatives, methods to prevent or treat viral dengue infections using said compounds, and it also relates to said compounds for use as a medicine, more preferably for use as a medicine to treat or prevent dengue viral infections. The present invention also relates to pharmaceutical compositions or preparations of combinations of the compounds, to compositions or preparations for use as a medicament, more preferably for the prevention or treatment of dengue viral infections. The invention also relates to processes for the preparation of the compounds.
BACKGROUND OF THE INVENTION
[002] Flaviviruses, which are transmitted by mosquitoes or ticks, cause fatal infections in humans, such as encephalitis and hemorrhagic fever. Four distinct but closely related serotypes of dengue flavivirus are known, the so-called DENV-1, -2, -3 and -4. Dengue is endemic in most tropical and subtropical regions around the world, predominantly in urban and semi-urban areas. According to the World Health Organization (WHO), 2.5 billion people, including 1 billion children, are at risk of DENV infection (WHO, 2002). It is estimated that 50 to 100 million cases of dengue fever [DF] occur worldwide each year, half a million cases of severe dengue disease (ie dengue hemorrhagic fever [DHF] and shock syndrome). dengue [DSS]), and more than 20,000 deaths. DHF has become a leading cause of hospitalization and death among children in endemic regions. Altogether, dengue represents the most common cause of
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2/109 arboviral disease. Due to large recent outbreaks in countries located in Latin America, Southeast Asia and the Western Pacific (including Brazil, Puerto Rico, Venezuela, Cambodia, Indonesia, Vietnam, Thailand), the numbers of dengue cases have increased dramatically over the past few years . Not only is the number of dengue cases increasing as the disease is spreading to new areas, but outbreaks tend to be more serious.
[003] Although progress is being made in the development of vaccines against dengue with the availability of the Dengvaxia® vaccine, many difficulties are encountered. These include the existence of a phenomenon referred to as antibody-dependent enhancement (ADE). Recovery from infection by a serotype provides lifelong immunity against that serotype, but provides only partial and transient protection against subsequent infection by one of the other three serotypes.
[004] After infection with another serotype, the pre-existing heterologous antibodies form complexes with the newly infected dengue virus serotype, but do not neutralize the pathogen. Instead, it is believed that virus entry into cells is facilitated, resulting in uncontrolled viral replication and spikes in higher viral titers. In both primary and secondary infections, the highest viral titers are associated with the most severe dengue disease. Since maternal antibodies can pass easily to children through breastfeeding, this may be one of the reasons why children are more affected by severe dengue disease than adults.
[005] In locations with two or more serotypes circulating simultaneously, also referred to as hyperendemic regions, the risk of severe dengue disease is significantly higher due to an increased risk of experiencing a secondary infection
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3/109 daily, more serious. Additionally, in a situation of hyperendemicity, the probability of the emergence of more virulent strains is increased, which in turn increases the probability of dengue hemorrhagic fever (DHF) or dengue shock syndrome. [006] Mosquitoes that carry dengue, including Aedes aegypti and Aedes albopictus (tiger mosquito), are moving north across the globe. According to the Centers for Disease Control and Prevention (CDC) in the United States (USA), both mosquitoes are currently ubiquitous in southern Texas. The spread to the north of mosquitoes that carry dengue is not confined to the USA, but has also been observed in Europe.
[007] Dengvaxia®, the dengue vaccine produced by Sanofi Pasteur, was first approved in Mexico and has, however, received approval in more countries. Nevertheless, the vaccine leaves a lot of room for improvement due to its limited efficacy, especially against DENV-1 and -2, low efficacy in naive patients for flavivirus and the prolonged dosing schedule.
[008] Despite these deficiencies, the vaccine is decisive in endemic areas because it will offer protection to a large part of the population, but probably not to very young children, on whom the greatest incidence of dengue falls. In addition, the dosing schedule and the very limited efficacy in subjects naive to flavivirus make it inadequate and probably not very rewarding / profitable for travelers from non-endemic areas who travel to dengue-endemic areas. The aforementioned deficiencies in dengue vaccines are the reason why there is a need for a prophylactic antiviral against dengue pre-exposure.
[009] In addition, specific antiviral drugs are currently not available for the treatment or prevention of infection by the dengue fever virus. Clearly, there is still a large
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4/109 unmet medical need for therapies for the prevention or treatment of viral infections in animals, more particularly in humans and especially for viral infections caused by flaviviruses, more particularly Dengue viruses. Compounds with good antiviral potency, no or low levels of side effects, broad spectrum activity against multiple Dengue virus serotypes, low toxicity and / or good pharmacokinetic or dynamic properties are highly needed.
[0010] WO-2010/021878 discloses 2-phenylpyrrolidine and indoline derivatives as cold menthol receptor antagonists for the treatment of inflammatory and central diseases. WO-2013/045516 discloses indole and indoline derivatives for use in the treatment of dengue viral infections.
[0011] The present invention now provides compounds, substituted indoline derivatives, which have high potent activity against the four (4) serotypes of the Dengue virus.
SUMMARY OF THE INVENTION
[0012] The present invention is based on the unexpected discovery that at least one of the problems mentioned above can be solved by the current compounds of the invention.
[0013] The present invention provides compounds that have been shown to have potent antiviral activity against the four (4) serotypes currently known. The present invention further demonstrates that these compounds effectively inhibit the proliferation of the Dengue virus (DENV). Therefore, these compounds constitute a useful class of potent compounds that can be used in the treatment and / or prevention of viral infections in animals, mammals and humans, more specifically in the treatment and / or prevention of infections with Dengue virus.
[0014] The present invention also relates to the use of such compositions
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5/109 as medicines and their use in the manufacture of medicines for the treatment and / or prevention of viral infections, in particular with viruses belonging to the family of Dengue viruses in animals or mammals, more particularly in humans. The invention also relates to methods for the preparation of all of these compounds and to pharmaceutical compositions comprising them in an effective amount.
[0015] The present invention also relates to a method of treating or preventing viral dengue infections in humans by administering an effective amount of one or more such compounds, or a pharmaceutically acceptable salt thereof, in combination with one or more others medications, as another antiviral agent, to a patient in need.
[0016] One aspect of the invention is the supply of compounds of formula (I), including any stereochemically isomeric form thereof:
[0018] R ¹ is chlorine or fluorine,
[0019] R ² is -CH2CH2OH or C ₃ - ₅ alkylCOOH;
[0020] R ³ is trifluoromethyl or trifluoromethoxy;
[0021] R ⁴ is hydrogen, fluorine or methoxy;
[0022] R ⁵ is hydrogen or methyl;
[0023] or a pharmaceutically acceptable salt, solvate or polymorph thereof.
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[0024] The term Cs-salkyl as used herein, defines straight and branched chain saturated hydrocarbon radicals with 3 to 5 carbon atoms, such as, for example, propyl, butyl, pentyl,
1,1-dimethylpropyl, 2-methylpropyl, 2-methylbutyl and the like.
[0025] The compounds specifically mentioned above are selected from the group comprising:
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[0026] Part of the present invention is also a pharmaceutical composition comprising a compound mentioned above or a stereoisomeric form thereof, salt, solvate or pharmaceutically acceptable polymorph together with one or more pharmaceutically acceptable excipients, diluents or vehicles.
[0027] Pharmaceutically acceptable salts of said compounds include their acid and base addition salts. Suitable acid addition salts are formed from acids that form non-toxic salts. Suitable base salts are formed from bases that form non-toxic salts.
[0028] The pharmaceutically acceptable acid salts as mentioned above are intended to comprise the therapeutically active non-toxic acid addition salt forms that the compounds of formula (I) are capable of forming. These pharmaceutically acceptable acid addition salts can be conveniently obtained by treating the base form with such an appropriate acid. Suitable acids comprise, for example, inorganic acids such as hydrochloric acids, for example, hydrochloric or hydrobromic acid, sulfuric, nitric, phosphoric acid and the like; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e., ethanedioic), malonic, succinic (i.e., butanedioic acid), maleic, fumaric, malic, tartaric, citric, methanesulfonic acid , ethanesulfonic, benzenesulfonic, ptoluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic acid and similar acids.
[0029] The compounds of the invention may also exist in strength
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8/109 but not solvated and solvated. The term solvate is used here to describe a molecular complex comprising the compound of the invention and one or more pharmaceutically acceptable solvent molecules, for example, ethanol.
[0030] The term polymorph refers to the ability of the compound of the invention to exist in more than one crystal shape or structure.
[0031] The compounds of the present invention can be administered as crystalline or amorphous products. They can be obtained, for example, as solid buffers, powders, or films, by methods such as precipitation, crystallization, lyophilization, spray drying or evaporative drying. They can be administered alone or in combination with one or more other compounds of the invention or in combination with one or more other drugs. Generally, they will be administered as a formulation in association with one or more pharmaceutically acceptable excipients. The term excipient is used herein to describe any ingredient other than the compound (s) of the invention. The choice of excipient depends largely on factors such as the particular mode of administration, the effect of the excipient on solubility and the stability and nature of the dosage form. The compounds of the present invention or any subgroup thereof can be formulated in various dosage forms for administration purposes. As appropriate compositions, all compositions usually employed for systemic drug administration can be cited. To prepare the pharmaceutical compositions of this invention, an effective amount of the particular compound, optionally in the form of the addition salt, as the active ingredient is combined in admixture with a pharmaceutically acceptable carrier, which carrier can take a wide variety of forms depending on the form of preparation desired for administration. These pharmaceutical compositions are want
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9/109 in unitary dosage form suitable, for example, for oral or rectal administration. For example, in the preparation of compositions in oral dosage form, any of the usual pharmaceutical means such as, for example, water, glycols, oils, alcohols and the like in the case of liquid oral preparations such as suspensions, syrups, elixirs, emulsions and solutions; or solid vehicles such as starches, sugars, kaolin, thinners, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules and tablets. Due to their ease of administration, tablets and capsules represent the most advantageous oral dosage unit forms, in which case solid pharmaceutical carriers are obviously employed. Also included are preparations in solid form that can be converted, shortly before use, into liquid forms.
[0033] It is especially advantageous to formulate the pharmaceutical compositions mentioned above 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 amount 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 grooved or coated tablets), capsules, pills, powder packs, cachets, suppositories, injectable solutions and suspensions and the like and their multiple secretions.
[0034] Experts in the treatment of infectious diseases will be able to determine the effective amount from the test results presented hereinafter. In general, it is contemplated that an effective daily amount would be 0.01 mg / kg to 50 mg / kg of body weight,
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10/109 more preferably from 0.1 mg / kg to 10 mg / kg of body weight. It may be appropriate to administer the required dose as two, three, four or more underdoses at appropriate intervals throughout the day. Said underdoses can be formulated as unit dosage forms, for example, containing 1 to 1000 mg, and in particular 5 to 200 mg of active ingredient per unit dosage form.
[0035] The exact dosage and frequency of administration depend on the particular compound of the invention used, the particular pathological condition being treated, the severity of the pathological condition being treated, the age, weight and general physical condition of the particular patient, as well as other medication that the individual may be taking, as is well known to those skilled in the art. Furthermore, it is evident that the effective amount can be decreased or increased depending on the response of the treated subject and / or depending on the judgment of the physician who prescribes the compounds of the present invention. The ranges of effective amounts mentioned above are therefore guidelines only, and are not intended to limit the scope or use of the invention to any extent.
[0036] The present disclosure is also intended to include any isotopes of atoms present in the compounds of the invention. For example, hydrogen isotopes include tritium and deuterium, and carbon isotopes include C-13 and C-14.
[0037] The present compounds used in the present invention can also exist in their stereochemically isomeric form, defining all possible compounds consisting of the same atoms linked by the same sequence of bonds but having different three-dimensional structures, which are not interchangeable. Unless otherwise mentioned or indicated, the chemical designation of compounds encompasses the mixture of all possible stereochemically isomeric forms, which the said compounds may possess. 870190096418, of 26/09/2019, p. 27/134
11/109 go.
[0038] Said mixture may contain all diastereoisomers and / or enantiomers of the basic molecular structure of said compound. All stereochemically isomeric forms of the compounds used in the present invention, both in pure form and in admixture with each other, are intended to fall within the scope of the present invention, including any racemic or racemate mixtures.
[0039] The pure stereoisomeric forms of the compounds and intermediates as mentioned herein are defined as isomers substantially free of other enantiomeric or diastereoisomeric forms of the same basic molecular structure of said compounds or intermediates. In particular, the term "stereoisomerically pure" refers to compounds or intermediates with a stereoisomeric excess of at least 80% (that is, a minimum of 90% of an isomer and a maximum of 10% of the other possible isomers) to an excess stereoisomeric of 100% (that is, 100% of an isomer and none of the others), more in particular, to compounds or intermediates with a stereoisomeric excess of 90% to 100%, even more in particular with a stereoisomeric excess of 94% to 100%, and more in particular with a stereoisomeric excess of 97% to 100%. The terms "enantiomerically pure" and "diastereoisomerically pure" should be understood in a similar way, but then taking into account the enantiomeric excess, respectively the diastereoisomeric excess, of the mixture in question.
[0040] Pure stereoisomeric forms of the compounds and intermediates used in this invention can be obtained by applying procedures known in the art. For example, enantiomers can be separated from each other by selective crystallization of their diastereoisomeric salts with optically active acids or bases
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12/109 you. Examples of these are tartaric acid, dibenzoyltartaric acid, ditoluoyltartaric acid and camphorsulfonic acid. Alternatively, enantiomers can be separated by chromatographic techniques using chiral stationary phases. Said pure stereochemically isomeric forms can also be derived from the corresponding pure stereochemically isomeric forms of the appropriate starting materials, provided that the reaction occurs stereospecifically. Preferably, if a specific stereoisomer is desired, said compound will be synthesized by stereospecific methods of preparation. These methods will advantageously employ enantiomerically pure starting materials.
[0041] The compounds of formula (I) of the present invention all have at least one chiral carbon atom as indicated in the figure below by the carbon atom marked with *:
[0042] Due to the presence of the said chiral carbon atom, a "compound of formula (I)" can be the enantiomer (R), the enantiomer (S), the racemic form or any possible combination of the two individual enantiomers for any reason . When the (R) or (S) absolute configuration of an enantiomer is not known, this enantiomer can also be identified by indicating whether the enantiomer is dextrorotatory (+) - or levorotatory (-) - after measuring the specific optical rotation of the referred particular enantiomer.
[0043] In one aspect, the present invention relates to a first group of compounds of formula (I) in which the compounds of formula
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13/109 (I) have specific rotation (+).
[0044] In a further aspect, the present invention relates to a second group of compounds of formula (I) in which the compounds of formula (I) have specific rotation (-).
Examples
LC / EM METHODS
[0045] Measurement by High Performance Liquid Chromatography (HPLC) was performed using an LC pump, a diode array (DAD) or a UV detector and a column as specified in the respective methods. If necessary, additional detectors have been included (see method table below).
[0046] The flow from the column was conducted to the Mass Spectrometer (EM) that was configured with an ion source at atmospheric pressure. It is within the knowledge of the expert to define the adjustment parameters (for example, scanning range, retention time ...) in order to obtain ions that allow the identification of the nominal monoisotopic molecular weight (PM) of the compound. Data acquisition was performed with appropriate software.
[0047] Compounds are described by their retention times (R _t ) and ions. If not specified differently in the data table, the reported molecular ion corresponds to [M + H] ⁺ (protonated molecule) and / or [MH] '(deprotonated molecule). In case the compound is not directly ionizable, the type of adduct is specified (ie, [Μ + ΝΗψ, [M + HCOO] ', etc ...). In the case of molecules with multiple isotopic patterns (Br, Cl), the reported value is that obtained for the lowest isotopic mass. All results were obtained with experimental uncertainties that are commonly associated with the method used.
[0048] Hereinafter, “SQD” means Single Quadropole Detector, “MSD” Selective Mass Detector, “TA” at room temperature, “BEH”
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14/109 Ethylsiloxane / Silica Bridged Hybrid, “DAD” Arrangement Detector
Diodes, “HSS” High Resistance silica.
[0049] LC / EM method codes (Flow expressed in mL / min; column temperature (T) in Ό; Operating time in minutes).
Method code Instrument Column Mobile phase Gradient FlowColT Run time (MIN) LC-A Waters: Acquity® UPLC®DAD-Quattro Micro ™ Waters: BEH® C18(1.7pm,2.1 x 100mm) A: 95% CH3COONH 4 7mM / 5% CH ₃ CN, B: CH ₃ CN A at 84.2% for 0.49 min, until A at 10.5% at 2.18 min, keep for1.94 min, back to A at 84.2% in 0.73 min, hold for 0.73 min. 0.343 mL / min40Ό 6.2 LC-B Waters: Acquity® Class H DAD and SQD2TM Waters:BEH®C18 (1.7 gm, 2.1 x 100 mm) A: 95% CH3COONH 4 7mM / 5% CH ₃ CN, B: CH ₃ CN A at 84.2% / B at 15.8% to A at 10.5% in 2.18 min, hold for 1.96 min, back to A at 84.2% / B at15.8% in 0.73 min, hold for 0.49 min. 0.343 mL / min40Ό 6.1 LC-C Waters: Acquity® UPLC®DAD-SQD Waters: BEH C18 (1.7pm, 2.1 x 50 mm) A: 10mM CH3COONH 4 in 95% H ₂ O + 5% CH ₃ CN B: CH ₃ CN From A to 95% to A to 5% in 1.3 min, keep for 0.7 min. 0.8 mL / min55Ό 2
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Method code Instrument Column Mobile phase Gradient FlowColT Run time (MIN) LC-D Waters: Acquity® UPLC®DAD-SQD Waters:HSST3 (1.8 gm, 2.1x100 mm) A: 10mM CH3COONH 4 in 95% H ₂ O + 5% CH ₃ CN B: CH ₃ CN From A to 100% up5% A in 2.10 min, up to A at 0% in 0.90 min, up to Aat 5% in 0.5 min 0.7 mL / min55Ό 3.5
CFS / MS methods
[0050] The SFC measurement was performed using an Analytical Chromatography system with supercritical fluid (SFC) composed of a binary pump for administration of carbon dioxide (CO2) and modifier, a self-sampler, a column oven, an array detector diode equipped with a high pressure flow cell resisting up to 400 bars. If configured with a Mass Spectrometer (EM), the flow from the column was conducted to 0 (EM). It is within the knowledge of the expert to define the adjustment parameters (for example, scanning range, retention time ...) in order to obtain ions that allow the identification of the nominal monoisotopic molecular weight (PM) of the compound. Data acquisition was performed with appropriate software.
[0051] Analytical methods of SFC / EM (Flow expressed in mL / min; column temperature (T) in Ό; Time of operation in minutes, Backpressure (BPR) in bars).
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Method code column mobile phase gradient FlowColT RuntimeBPR SFC-A Column DaicelChiralpak® IA (5 pm, 150 x4.6 mm) A: CO ₂ B: EtOH (+ 0.3% iPrNH ₂ ) retention of 30% B for 7 min, 335 7100 SFC-B Daicel Chiralpak® AD-H column (5 pm, 150 x 4.6 mm) A: CO ₂ B: EtOH (+ 0.3% iPrNH ₂ ) retention of 30% B for 7 min, 335 7100 SFC-C Daicel Chiralpak® AD-H column (5 pm, 150 x 4.6 mm) A: CO ₂ B: EtOH (+ 0.3% iPrNH ₂ ) 40% B retention for 7 min, 335 7100 SFC-D Daicel Chiralpak® IC column (5 pm, 150 x4.6 mm) A: CO ₂ B: iPrOH (+ 0.3% iPrNH ₂ ) 25% B retention for 7 min, 335 7100 SFC-E Daicel Chiralpak® IC column (5 pm, 150 x4.6 mm) A: CO ₂ B: iPrOH (+ 0.3% iPrNH ₂ ) retention of 30% B for 7 min, 335 7100 SFC-F Daicel Chiralpak® AD-3 column (3 pm, 100 x 4.6 mm) A: CO ₂ B: iPrOH (+ 0.3% iPrNH ₂ ) retention of 30% B for 3 min, 3.535 3103 SFC-G Daicel Chiralpak® AD-H column (5 pm, 150 x 4.6 mm) A: CO ₂ B: EtOH retention of 30% B for 7 min, 335 7100
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Method code column mobile phase gradient FlowColT RuntimeBPR SFC-H Daicel Chiralpak® AS-3 column (3 pm, 100 x 4.6 mm) A: CO ₂ B: iPrOH (+ 0.3% iPrNH ₂ ) retention of 20% B for 10 min, 3.535 10103 SFC-I Daicel Chiralpak® AS-3 column (3 pm, 100 x 4.6 mm) A: CO ₂ B: EtOH (+ 0.3% iPrNH ₂ ) retention of 10% B for 10 min, 3.535 10103 SFC-J Daicel Chiralcel® OD-3 column (3 pm, 100 x 4.6 mm) A: CO ₂ B: MeOH (+ 0.3% iPrNH ₂ ) retention of 20% B for 6 min, 3.535 6103 SFC-K Daicel Chiralpak® AS3 column (3.0 pm, 150 x 4.6 mm) A: CO ₂ B: EtOH (+ 0.2% iPrNH ₂ + 3% H ₂ O) B at 10% -50% in 6 min, keep 3.5 min 2.540 9.5110
FUSION POINTS
[0052] The values are peak values or melting intervals and are obtained with uncertainties that are commonly associated with this analytical method.
DSC823e (indicated as DSC)
[0053] For some compounds, melting points were determined with a DSC823e (Mettler-Toledo). Melting points were measured with a temperature gradient of 10 ° C / minute. The maximum temperature was 300 Ό.
Optical Rotations:
[0054] Optical rotations were measured on a PerkinElmer 341 polarimeter with a sodium lamp and reported as if
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18/109 that: [α] ² (λ, cg / 100 ml_, solvent, T Ό).
[0055] [α] χ ^τ = (100α) / (/ χ c): where I is the path length in dm and c is the concentration in g / 100 ml_ for a sample at a temperature T (O) and a length wave λ (in nm). If the wavelength of the light used is 589 nm (line D of sodium), then the symbol D can be used instead. The rotation sign (+ or -) must always be provided. When using this equation, the concentration and solvent are always provided in parentheses after rotation. Rotation is reported using degrees and no concentration units are given (assumed to be g / 100 ml_).
Abbreviations used in the experimental part
(M + H) ⁺ aq. aqueous / aqueous protonated molecular ion HCIhplc hydrochloric acid liquid chromatographyhigh performance Boc tert-butyloxycarbonyl iPrNH ₂ isopropylamine BOC2O di-carbonatetert-butyl iPrOH 2-propanol br extended K2CO3 Potassium carbonate CH ₃ CN acetonitrile L1AIH4 aluminum hydride andlithium CHCI3 chloroform m / z mass-to-ratiocharge CH2CH2 dichloromethane Me methyl CH3OH methanol MeOH methanol CO ₂ carbon dioxide MgSÜ4 magnesium sulfate d doublet min minute (s) DCM dichloromethane n ₂ nitrogen DIEA diisopropylethylamine Na2COs sodium carbonate DIPE diisopropyl ether Na2SO4 sodium sulfate
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| DMA dimethylacetamide NaBH ₄ sodium borohydride | DMAP | ⁴ dimethylaminopyridine NaHCO ₃ baking soda | DME 1,2-dimethoxyethane NaOH sodium hydroxide | DMF dimethylformamide NH4CI ammonium chloride | DMSO dimethylsulfoxide q quartet | eq. equivalent ta or TA room temperature Et ₂ O diethyl ether s singlet | Et ₃ N triethylamine t triplet | EtOAc ethyl acetate tBuOK potassium tert-butanolate [EtOH ethanol TEA triethylamine | h ₂ o Water TFA trifluoroacetic acid | H2SO4 sulfuric acid THF tetrahydrofuran 1 HATU hexafluorophosphate | O- (7-aza-1H-benzotriazol-1-yl) -Ν, Ν, Ν ', Ν'-tetramethyluronium - CAS[148893-10-1] TMSCI trimethylsilyl chloride
Example 1: synthesis of 2- (4-fluoro-2- (2-hydroxyethoxy) phenyl) -2 - ((3-methoxy-5 (1H-1,2,4-triazol-1-i I) pheni I) ami no) -1 - (6- (trifluoromethyl) i ndol i n-1-yl) ethanone (Compound 1) and chiral separation in enantiomers 1A and 1B.
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HCí (4M in dsD-xanoí
MéQH ts, 1h
separation:
Ensntsomers IA and 18
Synthesis of intermediate 1a:
[0056] A solution of 4-fluoro-2-methoxyphenylacetic acid [CAS 886498-61-9] (10 g, 54.3 mmol) in EtOH (200 ml_) and H ₂ SO ₄ (2 ml_) was heated under reflux for 12 h. Water was added and the mixture was concentrated under reduced pressure to half the original volume. Ice was added. The solution was basified with K2CO3 and extracted with EtOAc. The organic layer was washed with saturated aqueous sodium chloride solution, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure to give ethyl 2- (4-fluor-2-methoxyphenyl) acetate 1a (11.6 g). The compound was used directly in the next step.
Synthesis of intermediate 1b:
[0057] A 1 M solution of boron tribromide in CH2 Cl2 (109.3 ml_, 109.3 mmol) was added dropwise to a solution of
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21/109 2- (4-fluoro-2-methoxyphenyl) ethyl acetate 1a (11.6 g, 54.7 mmol) in CH2 Cl2 (300 ml) at -30 Ό. The reaction was stirred at -20 Ό for 1 h, and then quenched with CH3OH. The pH was adjusted to 8 by adding a saturated solution of NaHCOs. The solution was extracted with CH2 Cl2 and the combined organic layers were dried over MgSO4, filtered and the solvent was concentrated under reduced pressure to give ethyl 2- (4-fluoro-2-hydroxyphenyl) acetate 1b (10.8 g). The compound was used directly in the next step without further purification.
Synthesis of intermediate 1c:
[0058] To a mixture of 2- (4-fluor-2-hydroxyphenyl) ethyl acetate 1b (10.6 g, 53.5 mmol) and cesium carbonate (34.8 g, 106.9 mmol) in DMF (200 ml_) at 10 Ό (2-bromoethoxy) (tert-butyl) dimethylsilane [CAS 86864-60-0] (13.8 ml_, 64.2 mmol) was added. The reaction mixture was stirred at room temperature overnight. H2O was added and the reaction mixture was extracted with EtOAc. The organic phase was dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 μΜ, 40 g, heptane / EtOAc 80/20). The pure fractions were combined and the solvent was removed under reduced pressure to give ethyl 2- (2- (2 - ((tert-butyldimethylsilyl) oxy) ethoxy) -4-fluorophenyl) acetate (17.7 g).
Synthesis of intermediate 1d:
[0059] To a 1 M solution of lithium bis (trimethylsilyl) amide in THF (28.05 ml_, 28.05 mmol), cooled to -78 Ό, a solution of 2- (2- (2- ((ether-butyldimethylsilyl) oxy) ethoxy) -4-fluorophenyl) ethyl acetate 1c (5 g, 14.03 mmol) in THF (30 ml). After stirring for 1 h at -78 Ό, chlorotrimethylsilane (2.85 ml, 22.4 mmol) was added. The reaction mixture was stirred at -78 Ό for 15 min. NBromosuccinimide (3 g, 16.8 mmol) in THF (30 ml) was added and stirring was continued at -55 Ό for 2 h. The reaction mixture was
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22/109 poured into H2O and extracted twice with EtOAc. The organic phases were combined, dried over MgSO4, filtered and concentrated under reduced pressure to give 2-bromo-2- (2- (2 - ((tert-butyldimethylsilyl) oxy) ethoxy) -4-fluorophenyl) acetate ethyl acetate (6.57 g) which was used in the next step without further purification.
Synthesis of intermediate 1e:
[0060] A mixture of 2-bromo-2- (2- (2 - ((tert-butyldimethylsilyl) oxy) ethoxy) -4-fluorophenyl) ethyl acetate 1d (3.1 g, 7.12 mmol), 3-methoxy -5- (1 H-1,2,4-triazol-1-yl) aniline [CAS 1220630-56-7] (2.03 g, 10.7 mmol) and diisopropylethylamine (2.45 ml_, 14 , 2 mmol) in CH3CN (60 ml) was stirred at 50 ° for 18 h. The reaction mixture was concentrated under reduced pressure. The residue was absorbed with EtOAc and washed with 0.5 N HCI, water and saturated aqueous sodium chloride solution. The organic phase was dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 120 g, heptane / EtOAc gradient 80/20 to 60/40) to give 2- (2- (2 - ((ether-butyldimethylsilyl) oxy) ethoxy) -4fluorophenyl) -2 - ((3-methoxy-5- (1 H-1,2,4-triazol-1-yl) phenyl) amino) ethyl acetate 1e (2.5 g).
Synthesis of intermediate 1f:
[0061] A solution of lithium hydroxide monohydrate (226 mg, 5.397 mmol) in water (25 ml_) was added in portions to a solution of 2- (2- (2 - ((ether-butyldimethylsilyl) oxy) ethoxy) -4-fluorophenyl) -2 - ((3 methoxy-5- (1 H-1,2,4-triazol-1-yl) phenyl) amino) ethyl acetate 1e (2.45 g, 4.498 mmol) in a solvent mixture of THF / CH3OH (1/1) (50 ml) at 10 Ό. The reaction was stirred at room temperature for 6 h, diluted with water and cooled to 0 ° C. The solution was slowly acidified with 0.5 N HCI to pH 6 and extracted with EtOAc. The organic layer was dried over MgSO4, filtered and the solvent was concentrated under reduced pressure to give 2- (2- (2 - ((ether-butyldimethylsilyl) oxy) ethoxy) -4 acid
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23/109 fluorophenyl) -2 - ((3-methoxy-5- (1 H-1,2,4-triazol-1-yl) phenyl) amino) acetic 1f (2.05 g). The compound was used directly in the next step without further purification.
Synthesis of intermediate 1g:
[0062] To a solution of 2- (2- (2 - ((etherbutyldimethylsilyl) oxy) ethoxy) -4-fluorophenyl) -2 - ((3-methoxy-5- (1 H-1,2,4- triazole-
1-yl) phenyl) amino) acetic 1f (1.58 g, 3.06 mmol) in DMF (20 ml), HATU (1.74 g, 4.60 mmol), diisopropylethylamine (1.5 ml) were added , 9.17 mmol) and 6- (trifluoromethyl) indoline [CAS 181513-29-1] (572 mg, 3.06 mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with water. The precipitate was removed by filtration, washed with water and taken up with EtOAc. The organic layer was washed with a 10% solution of K2CO3 in water, water, dried over MgSCU, filtered and the solvent was concentrated under reduced pressure to give 2- (2- (2 - ((ether-butyldimethylsilyl) oxy) ethoxy) ) -4fluorophenyl) -2 - ((3-methoxy-5- (1 H-1,2,4-triazol-1-yl) phenyl) amino) -1 - (6 (trifluoromethyl) indolin-l-yl) ethanone 1g (2.1 g). The crude compost was used directly in the next step.
Synthesis of Compound 1 and chiral separation in Enantiomers 1A and 1B:
[0063] Under a flow of N2, at 5 Ό, 4 M HCI in dioxane (7.65 ml_,
30.6 mmol) was added dropwise to a solution of 2- (2- (2 - ((etherbutyldimethylsilyl) oxy) ethoxy) -4-fluorophenyl) -2 - ((3-methoxy-5- (1 H- 1,2,4-triazole-
1-yl) phenyl) amino) -1- (6- (trifluoromethyl) indolin-1-yl) ethanone 1g (2.1 g, 3.06 mmol) in MeOH (40 ml). The reaction was stirred at room temperature for 1 h. The mixture was cooled to 0 Ό, basified with 10% aqueous K2CO3 solution and extracted with EtOAc. The organic phase was separated, dried over MgSO4, filtered, and the solvent was concentrated under reduced pressure. The residue was crystallized from CHsCN / diisopropyl ether to give 2- (4-fluorine-2- (2-hydroxyethoxy) phenyl) -2
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24/109 ((3-methoxy-5- (1 H-1,2,4-triazol-1-yl) phenyl) amino) -1 - (6 (trifluoromethyl) indolin-1-yl) ethanone 1 (800 mg) as a racemate.
[0064] Compound 1 enantiomers (720 mg) were separated through Preparative Chiral SFC (Stationary phase: Chiralpak® IA 5 pm 250 x 20 mm, mobile phase: 70% CO2, 30% EtOH (+ 0.3% iPrNHz )). The first eluted enantiomer (303 mg) was crystallized from Et2Ü to give 0 Enantiomer 1A (270 mg). The second eluted enantiomer (320 mg) was crystallized from ΕϊςΟ to give Enantiomer 1B (274 mg).
Compound 1:
[0065] ¹ H NMR (500 MHz, DMSO-c / ₆ ) δ ppm 3.15 - 3.30 (m, 2 H)
3.73 (s, 3 H) 3.74 - 3.85 (m, 2 H) 4.06 - 4.17 (m, 3 H) 4.45 (td, J = 10.3,
6.1 Hz, 1 H) 4.98 (t, J = 5.4 Hz, 1 H) 5.83 (d, J = 8.5 Hz, 1 H) 6.35 (s, 1 H)
6.66 (t, J = 1.9 Hz, 1 H) 6.76 - 6.82 (m, 2 H) 6.84 (s, 1 H) 6.98 (dd, J = 11.2, 2.4 Hz, 1 H) 7.37 - 7.42 (m, 2 H) 7.44 - 7.49 (m, 1 H) 8.16 (s, 1 H) 8.39 (s, 1 H) 9.13 (s, 1 H)
[0066] LC / MS (LC-A method): Tr 3.06 min, MH ⁺ 572
[0067] Melting point: 151Ό
Enantiomer 1 A:
[0068] ¹ H NMR (500 MHz, DMSO-d ₆ ) δ ppm 3.15 - 3.29 (m, 2 H)
3.73 (s, 3 H) 3.74 - 3.86 (m, 2 H) 4.06 - 4.19 (m, 3 H) 4.45 (td, J = 10.2,
6.3 Hz, 1 H) 4.97 (t, J = 5.5 Hz, 1 H) 5.83 (d, J = 8.8 Hz, 1 H) 6.36 (s, 1 H)
6.66 (t, J = 1.9 Hz, 1 H) 6.76 - 6.82 (m, 2 H) 6.84 (s, 1 H) 6.98 (dd, J = 11.3, 2.5 Hz, 1 H) 7.37 - 7.42 (m, 2 H) 7.46 (d, J = 7.9 Hz, 1 H) 8.16 (s, 1 H) 8.39 ( s, 1 H) 9.13 (s, 1 H)
[0069] LC / MS (LC-A method): Tr 3.06 min, MH ⁺ 572
[0070] [a] _D ²⁰ : -44.8 ^o (c 0.2525, DMF)
[0071] Chiral SFC (SFC-A method): Tr 2.59 min, MH ⁺ 572, chiral purity 100%.
[0072] Melting point: 163Ό
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Enantiomer 1B:
[0073] ¹ H NMR (500 MHz, DMSO-c / ₆ ) δ ppm 3.15 - 3.30 (m, 2 H)
3.73 (s, 3 H) 3.74 - 3.85 (m, 2 H) 4.06 - 4.19 (m, 3 H) 4.45 (td, J = 10.3,
6.1 Hz, 1 H) 4.97 (t, J = 5.5 Hz, 1 H) 5.83 (d, J = 8.5 Hz, 1 H) 6.36 (s, 1 H)
6.66 (t, J = 1.9 Hz, 1 H) 6.76 - 6.82 (m, 2 H) 6.84 (s, 1 H) 6.98 (dd, J = 11.2, 2.4 Hz, 1 H) 7.36 - 7.43 (m, 2 H) 7.46 (d, J = 7.9 Hz, 1 H) 8.16 (s, 1 H) 8.39 ( s, 1 H) 9.13 (s, 1 H)
[0074] LC / MS (LC-A method): Tr 3.06 min, MH ⁺ 572
[0075] [a] _D ²⁰ : + 36.2 ° (c 0.2567, DMF)
[0076] Chiral SFC (SFC-A method): Tr 3.15 min, MH ⁺ 572, chiral purity 98.07%.
[0077] Melting point: 162Ό
Example 2: synthesis of 2- (4-fluoro-2- (2-hydroxyethoxy) phenyl) -2 - ((3-methoxy-5 (1H-1,2,4-triazol-1-i) pheni I) ami no) -1 - (5-methoxy-6- (trifluoromethyl) i ndol i n-1 yl) ethanone (Compound 2) and chiral separation in Enantiomers 2A and 2B.
.-140½
Mia
H ·,. · Ν
CJ tBcOK. LW
2s
AcDH
EtOH / water
1 am, Iftbar
2b
2c
2c
IT
HCl (4M in Doxane)
MeOH
F-.
tnantiomers 2A and 2S n-n
Synthesis of intermediate 2a:
[0078] A mixture of 1-methoxy-4-nitro-2- (trifluoromethyl) benzene
[CAS 654-76-2] (24.5 g, 110.8 mmol) and 4-chlorophenoxyacetonitrile [CAS
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3598-13-8] (20.4 g, 121.9 mmol) in DMF (100 ml) was added dropwise over 30 min to a stirred tBuOK solution (27.35 g,
243.7 mmol) in DMF (100 ml) at -10 O. After addition, the purple solution was kept at -10 Ό for 1 h. 500 ml of ice water and 500 ml of 6N HCI were added and the precipitate was filtered off, washed with water and dried under reduced pressure to give 40.4 g of 2 (5-methoxy-2-nitro-4 - (trifluoromethyl) phenyl) acetonitrile 2a (used as such in the next step).
Synthesis of intermediate 2b:
[0079] A solution of 2- (5-methoxy-2-nitro-4 (trifluoromethyl) phenyl) acetonitrile 2a (26 g, 99.9 mmol) in ethanol / water (9/1) (500 ml) and AcOH ( 5.2 ml) was hydrogenated for 1 h at a pressure of 3.5 Bar with 10% Pd / C (15.3 g) as a catalyst. The reaction mixture was filtered through a pad of celite® and the filter cake was washed with a solvent mixture of CH3 Cl and CH3 OH. The filtrate was concentrated under reduced pressure. The residue was filtered through a glass filter loaded with 60-200 pm silica and using 80/20 heptane / EtOAc as the eluent. Fractions containing the expected compound were combined and the solvent concentrated under reduced pressure to give 5-methoxy-6- (trifluoromethyl) -1 H-indole 2b (15.6 g) ·
Synthesis of intermediate 2c:
[0080] At 0 Ό, BHs-Pyridine (23.5 ml_,
232.4 mmol) to a solution of 5-methoxy-6- (trifluoromethyl) -1 H-indole 2b (10 g, 46.5 mmol) in EtOH (60 ml). 6N HCI (140 ml) was added slowly while maintaining the temperature below 10 ° C. The mixture was stirred at 0 ° C for 2 h. Water (200 ml) was added and the mixture was basified to pH 8-9 with a concentrated aqueous solution of NaOH (the reaction temperature was kept below 20 Ό). The precipitate was filtered off, washed with water (twice) and
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27/109 coevaporated under reduced pressure with toluene to give 5-methoxy-6 (trifluoromethyl) indoline 2c (9 g).
Synthesis of intermediate 2d:
[0081] To a solution of 2- (2- (2 - ((iercbutyldimethylsilyl) oxy) ethoxy) -4-fluorophenyl) -2 - ((3-methoxy-5- (1 H-1,2,4- triazole-
1-yl) phenyl) amino) acetic 1f (1.02 g, 1.97 mmol) in DMF (10 ml_) HATU (1.13 g, 2.96 mmol), diisopropylethylamine (979 μΙ_, 5 , 92 mmol) and 5-methoxy-6- (trifluoromethyl) indoline 2c (429 mg, 1.97 mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with water. The precipitate was removed by filtration, washed with water and taken up with EtOAc. The organic layer was washed with a 10% solution of K2CO3 in water, water, dried over MgSCU, filtered and the solvent was concentrated under reduced pressure to give 2- (2- (2 - ((ether-butyldimethylsilyl) oxy) ethoxy) ) -4-fluorophenyl) -
2- ((3-methoxy-5- (1 H-1,2,4-triazol-1-i) pheni I) amine) -1 - (5-methoxy-6 (trifluoromethyl) indolin-l- il) 2d ethanone (1.36 g). The compound was used as such in the next reaction step.
Synthesis of Compound 2 and chiral separation in Enantiomers 2A and 2B:
[0082] Under a flow of N2, at 5 O, 4 M HCI in dioxane (4.75 ml_,
18.99 mmol) was added dropwise to a solution of 2- (2- (2 ((tert-butyldimethylsilyl) oxy) ethoxy) -4-fluorophenyl) -2 - ((3-methoxy-5- (1 H -1,2,4 triazole-1 -i I) phenyl I) amine) -1 - (5-methoxy-6- (trifluoromethyl I) i ndol i n-1 -yl) ethanone 2d (1.36 g, 1.9 mmol) in MeOH (25 ml). The reaction was stirred at room temperature for 1 h. The mixture was cooled to 0 O, basified with a 10% aqueous solution of K2CO3 and extracted with EtOAc. The organic phase was separated, dried over MgSO4, filtered, and the solvent was concentrated under reduced pressure. The residue was crystallized from MeOH to give 2- (4-fluoro-2- (2-hydroxyethoxy) phenyl) -2 - ((3-methoxy-5- (1 H-1,2,4-triazole-1-yl ) phenyl) amino) -1 - (5-methoxy-6-
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28/109 (trifluoromethyl) indolin-1-yl) ethanone 2 (850 mg) as racemate.
[0083] Compound 2 enantiomers (800 mg) were separated by Preparative Chiral SFC (Stationary phase: Chiralpak® pm 5 pm 250 x 20 mm, mobile phase: 6% CH2CI2, 70% CO2, 24% EtOH (+0 , 3% iPrNFE)). The first eluted enantiomer (370 mg) was solidified by trituration with diisopropyl ether to give 0 Enantiomer 2A (329 mg). The second eluting enantiomer (400 mg) was further purified by flash chromatography on silica gel (15-40 pm, 24 g, CFkCb / MeOH 99/1). The pure fractions were combined and the solvent was concentrated under reduced pressure. The residue (320 mg) was solidified by trituration with diisopropyl ether to give the 2B Enantiomer (262 mg).
Compound 2:
[0084] ¹ H NMR (500 MHz, DMSO-c / ₆ ) δ ppm 3.15 - 3.30 (m, 2 H)
3.72 (s, 3 H) 3.74 - 3.83 (m, 2 H) 3.84 (s, 3 H) 4.04 - 4.18 (m, 3 H) 4.43 (td, J = 10.4, 6.3 Hz, 1 H) 4.99 (t, J = 5.7 Hz, 1 H) 5.81 (d, J = 8.5 Hz, 1 H)
6.35 (s, 1 H) 6.65 (t, J = 1.9 Hz, 1 H) 6.75 - 6.81 (m, 2 H) 6.83 (s, 1 H) 6.98 (dd, J = 11.2, 2.4 Hz, 1 H) 7.24 (s, 1 H) 7.39 (dd, J = 8.5, 6.9 Hz, 1 H) 8.16 ( s, 1 H) 8.35 (s, 1 H) 9.13 (s, 1 H)
[0085] LC / MS (LC-A method): Tr 2.99 min, MH ⁺ 602
[0086] Melting point: 192Ό
2A Enantiomer:
[0087] ¹ H NMR (400 MHz, DMSO-cfe) δ ppm 3.16 - 3.28 (m, 2 H)
3.72 (s, 3 H) 3.74 - 3.83 (m, 2 H) 3.84 (s, 3 H) 4.03 - 4.18 (m, 3 H) 4.37 - 4, 49 (m, 1 H) 4.97 (t, J = 5.6 Hz, 1 H) 5.81 (d, J = 8.1 Hz, 1 H) 6.35 (s, 1 H) 6, 65 (s, 1 H) 6.73 - 6.81 (m, 2 H) 6.83 (s, 1 H) 6.97 (dd, J = 11.1.2.0 Hz, 1 H) 7 , 23 (s, 1 H) 7.39 (t, J = 7.6 Hz, 1 H) 8.15 (s, 1 H) 8.35 (s, 1 H)
9.12 (s, 1 H)
[0088] LC / MS (LC-A method): Tr 2.97 min, MH ⁺ 602
[0089] [a] _D ²⁰ : -45.0 ^o (c 0.2425, DMF)
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[0090] Chiral SFC (SFC-A method): Tr 4.14 min, MH ⁺ 602, 100% chiral purity.
2B Enantiomer:
[0091] ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 3.16 - 3.28 (m, 2 H)
3.72 (s, 3 H) 3.74 - 3.83 (m, 2 H) 3.84 (s, 3 H) 4.02 - 4.20 (m, 3 H) 4.42 (td, J = 10.2, 6.3 Hz, 1 H) 4.97 (t, J = 5.6 Hz, 1 H) 5.81 (d, J = 8.6 Hz, 1 H)
6.35 (s, 1 H) 6.65 (s, 1 H) 6.73 - 6.81 (m, 2 H) 6.83 (s, 1 H) 6.97 (dd, J = 11.4, 2.3 Hz, 1 H) 7.23 (s, 1 H) 7.36 - 7.43 (m, 1 H) 8.15 (s, 1 H)
8.35 (s, 1 H) 9.12 (s, 1 H)
[0092] LC / MS (LC-A method): Tr 2.97 min, MH ⁺ 602
[0093] [a] _D ²⁰ : + 43.4 ° (c 0.2007, DMF)
[0094] Chiral SFC (SFC-A method): Tr 5.08 min, MH ⁺ 602, 100% chiral purity.
Example 3: synthesis of 2- (4-fluoro-2- (2-hydroxyethoxy) phenyl) -2 - ((3-methoxy-5 (1 H-1,2,4-triazol-1-i) pheni I ) ami no) -1 - (6- (trifluoromethoxy) indol i n-1 yl) ethanone (Compound 3) and chiral separation in Enantiomers 3A and 3B.

Synthesis of intermediate 3a:
[0095] To a solution of 2- (2- (2 - ((iercbutyldimethylsilyl) oxy) ethoxy) -4-fluorophenyl) -2 - ((3-methoxy-5- (1 H-1,2,4- triazol1-yl) phenyl) amino) acetic 1f (1.02 g, 1.954 mmol) in DMF (10 ml_) HATU (1.13 g, 2.96 mmol), diisopropylethylamine (979 μΙ_,
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5.92 mmol) and 6- (trifluoromethoxy) indoline [CAS 959235-95-1] (401 mg, 1.97 mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with water. The precipitate was removed by filtration, washed with water and taken up with EtOAc. The organic layer was washed with a 10% solution of K2CO3 in water, water, dried over MgSCU, filtered and the solvent was concentrated under reduced pressure to give 2- (2- (2 - ((ether-butyldimethylsilyl) oxy) ethoxy) ) -4fluorophenyl) -2 - ((3-methoxy-5- (1 H-1,2,4-triazol-1-yl) phenyl) amino) -1 - (6 (trifluoromethoxy) indolin-1-yl) ethanone 3a (1.34 g). The impure compound was used directly in the next reaction step.
Synthesis of Compound 3 and chiral separation in Enantiomers 3A and 3B:
[0096] Under a flow of N2, at 5 Ό, 4 M HCI in dioxane (4.27 ml_,
17.1 mmol) was added dropwise to a solution of 2- (2- (2 - ((methylbutyldimethylsilyl) oxy) ethoxy) -4-fluorophenyl) -2 - ((3-methoxy-5- (1 H- 1,2,4-triazol1-yl) phenyl) amino) -1- (6- (trifluoromethoxy) indolin-1-yl) ethanone 3a (1.2 g, 1.71 mmol) in MeOH (25 ml). The reaction was stirred at room temperature for 1 h. The mixture was cooled to 0 Ό, basified with 10% aqueous K2CO3 solution and extracted with EtOAc. The organic phase was separated, dried over MgSO4, filtered, and the solvent was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 40 g, CHbCb / MeOH gradient from 99.5 / 0.5 to 99/1). The pure fractions were combined and concentrated to dryness under reduced pressure to give 2- (4-fluoro-2- (2-hydroxyethoxy) phenyl) -2 - ((3-methoxy-5- (1 H-1,2,4- triazol-1 yl) phenyl) amino) -1- (6- (trifluoromethoxy) indolin-1-yl) ethanone 3 (550 mg) as a racemate. Part of this fraction was crystallized from MeOH to provide Compound 3 (36 mg).
[0097] The remaining material was used to separate the Compound 3 enantiomers via Preparative Chiral SFC (stationary phase: Chi
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31/109 ralpak® AD-H 5 pm 250 x 20 mm, mobile phase: 65% CO2, 35% EtOH (+ 0.3% iPrNHE)). The first eluted enantiomer (210 mg) was solidified by trituration with diisopropyl ether / heptane to give the 3A Enantiomer (182 mg). The second eluting enantiomer (230 mg) was further purified by flash chromatography on silica gel (15-40 pm, 24 g, CHEWMeOH 99/1). The pure fractions were combined and the solvent was concentrated under reduced pressure. The residue (180 mg) was solidified by trituration with diisopropyl ether / heptane to give 0 Enantiomer 3B (137 mg).
Compound 3:
[0098] ¹ H NMR (500 MHz, DMSO-d ₆ ) δ ppm 3.06 - 3.25 (m, 2 H)
3.73 (s, 3 H) 3.75 - 3.86 (m, 2 H) 4.06 - 4.17 (m, 3 H) 4.38 - 4.50 (m, 1 H) 4, 95 (s I, 1 H) 5.81 (d, J = 8.6 Hz, 1 H) 6.35 (s, 1 H) 6.65 (s, 1 H) 6.75 - 6.81 ( m, 2 H) 6.83 (s, 1 H) 6.94 - 7.04 (m, 2 H) 7.33 (d, J = 8.6 Hz, 1 H)
7.36 - 7.43 (m, 1 H) 8.04 (s, 1 H) 8.15 (s, 1 H) 9.11 (s, 1 H)
[0099] LC / MS (LC-A method): Tr 3.13 min, MH ⁺ 588
[00100] Melting point: 178Ό
3A Enantiomer:
[00101] ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 3.06 - 3.26 (m, 2 H)
3.72 (s, 3 H) 3.74 - 3.86 (m, 2 H) 4.05 - 4.18 (m, 3 H) 4.38 - 4.50 (m, 1 H) 4, 97 (t, J = 5.3 Hz, 1 H) 5.82 (d, J = 8.6 Hz, 1 H) 6.35 (s, 1 H) 6.65 (s, 1 H) 6, 74 - 6.86 (m, 3 H) 6.94 - 7.04 (m, 2 H) 7.34 (d, J = 8.1 Hz, 1 H)
7.36 - 7.42 (m, 1 H) 8.04 (s, 1 H) 8.15 (s, 1 H) 9.12 (s, 1 H)
[00102] LC / MS (LC-A method): Tr 3.11 min, MH ⁺ 588
[00103] [a] _D ²⁰ : -38.2 ° (c 0.28, DMF)
[00104] Chiral SFC (SFC-B method): Tr 3.38 min, MH ⁺ 588, 100% chiral purity.
3B Enantiomer:
[00105] 1H NMR (400 MHz, DMSO-d ₆ ) δ ppm 3.07 - 3.26 (m, 2H)
3.72 (s, 3 H) 3.74 - 3.86 (m, 2 H) 4.04 - 4.20 (m, 3 H) 4.38 - 4.50 (m, 1
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Η) 4.97 (t, J = 5.6 Hz, 1 H) 5.81 (d, J = 8.6 Hz, 1 H) 6.35 (s, 1 H) 6.65 (s, 1 H) 6.74 - 6.87 (m, 3 H) 6.94 - 7.03 (m, 2 H) 7.34 (d, J = 8.1 Hz, 1 H)
7.36 - 7.42 (m, 1 H) 8.04 (s, 1 H) 8.15 (s, 1 H) 9.12 (s, 1 H)
[00106] LC / MS (LC-A method): Tr 3.11 min, MH ⁺ 588
[00107] [a] D20: + 40.9 ° (c 0.23, DMF)
[00108] Chiral SFC (SFC-B method): Tr 5.31 min, MH ⁺ 588, chiral purity 100%.
Example 4: synthesis of 2- (4-chloro-2- (2-hydroxyethoxy) phenyl) -2 - ((3-methoxy5- (1 H-1,2,4-triazol-1-i I) pheni I) ami no) -1 - (6- (trifluoromethyl) i ndol i n-1 yl) ethanone (Compound 4) and chiral separation in Enantiomers 4A and 4B.
Synthesis of intermediate 4a:
[00109] A solution of 2- (4-chloro-2-methoxyphenyl) acetic acid [CAS 170737-95-8] (20 g, 101 mmol) in dry THF (300 ml) was cooled to 0 Ό. Oxalyl chloride (18 ml, 202 mmol) and two drops of DMF were added. The reaction mixture was stirred at room temperature
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33/109 for 30 min. The solvent was evaporated under reduced pressure. The residue was dissolved in methanol (300 ml) and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure to provide ethyl 2- (4-chloro-2-methoxyphenyl) acetate 4a (23 g), which was used in the next step without further purification.
Synthesis of intermediate 4b:
[00110] To a solution of 2- (4-chloro-2-methoxyphenyl) ethyl acetate 4a (10 g, 44 mmol) in CH2 Cl2 (350 ml), cooled to -30 Ό, was added dropwise a solution of 1 M BBra in CH2 Cl2 (87.5 ml_, 87.5 mmol) while maintaining the temperature below -20 Ό. The reaction mixture was stirred at -30 Ό for 1 h before quenching with methanol. The pH was adjusted to 8 by the addition of a saturated aqueous solution of NaHCOs. The phases were separated. The aqueous phase was extracted with CH2 Cl2. The organic phases were combined, dried over MgSO4, filtered and concentrated under reduced pressure to provide ethyl 2- (4-chloro-2-hydroxyphenyl) acetate 4b (9.5 g), which was used in the next step without further purification.
Synthesis of intermediate 4c:
[00111] To a mixture of 2- (4-chloro-2-hydroxyphenyl) ethyl acetate 4b [CAS 1261826-30-5] (2.82 g, 13.1 mmol) and cesium carbonate (8.56 g , 26.3 mmol) in DMF (50 ml) was added benzyl ether and
2-bromoethyl [CAS 1462-37-9] (2.29 g, 14.5 mmol). The reaction mixture was stirred at room temperature for 24 h. H2O was added and the reaction mixture was extracted with EtOAc. The organic phase was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel using a gradient of EtOAc (2% to 20%) in heptane to give 2- (2- (2- (benzyloxy) ethoxy) 4-chlorophenyl) ethyl acetate 4c (4 , 17 g).
Synthesis of intermediate 4d:
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[00112] To a solution of 2- (2- (2- (benzyloxy) ethoxy) -4chlorophenyl) ethyl acetate 4c (4.17 g, 12.0 mmol) in a mixture of EtOH (80 ml_) and THF ( 40 ml (0.5 N NaOH) (72 ml, 36.0 mmol) was added. The reaction mixture was stirred at room temperature for 3 h. The reaction mixture was partially concentrated under reduced pressure to remove organic solvents. The residue was acidified to pH 2-3 with 1N HCI and the mixture was extracted with EtOAc. The organic phase was dried over MgSO4, filtered and concentrated under reduced pressure to give 2- (2- (2- (benzyloxy) ethoxy) -4chlorophenyl) acetic acid 4d (3.83 g).
Synthesis of intermediate 4e:
[00113] A solution of 2- (2- (2- (benzyloxy) ethoxy) -4chlorophenyl) acetic acid 4d (7.12 g, 22.2 mmol) in thionyl chloride (50 ml, 689 mmol) was stirred at room temperature for 18 h. The reaction mixture was concentrated under reduced pressure and co-evaporated with toluene to provide 2- (2- (2- (benzyloxy) ethoxy) -4chlorophenyl) acetyl chloride 4e (7.53 g) which was used in the next step without purification additional.
Synthesis of intermediate 4f:
[00114] A solution of 2- (2- (2- (benzyloxy) ethoxy) -4chlorophenyl) acetyl 4e chloride (5.29 g, 15.6 mmol) in CH3CN (50 ml) was added dropwise under atmosphere of N2 to a stirred mixture of 6 (trifluoromethyl) indoline [CAS 181513-29-1] (2.92 g, 15.6 mmol) and sodium bicarbonate (1.44 g, 17.1 mmol) in CH3CN (50 ml_). The reaction mixture was stirred at room temperature for 65 h and poured into water (500 ml). The product was extracted (2x) with Et20. The combined organic layers were washed with saturated aqueous sodium chloride solution, dried over MgSO4, filtered and evaporated under reduced pressure. The residue solidified after standing. The product was stirred in diisopropyl ether (25 ml), filtered, washed (3x) with di ether
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35/109 isopropyl, and dried under vacuum at 45 O to obtain 2- (2- (2 (benzyloxy) ethoxy) -4-chlorophenyl) -1 - (6- (trifluoromethyl) i ndol i n- 1-yl) ethanone 4f (6.97 g).
Synthesis of intermediate 4g:
[00115] A solution of 2- (2- (2- (benzyloxy) ethoxy) -4-chloro-phenyl) -1- (6 (trifluoromethyl) indolin-1-yl) ethanone 4f (1.5 g, 3, 06 mmol) in 2-Me-THF (125 ml) was stirred under N ₂ flow and cooled to -78 Ό. A solution of 1 M lithium bis (trimethylsilyl) amide in THF (6.12 ml, 6.12 mmol) was added dropwise and the resulting mixture was stirred at -78 Ό for 20 minutes. Chlorotrimethylsilane (626, 4.90 mmol) was added dropwise and the mixture was stirred at -78 Ό for 25 min. A solution of A / -bromosuccinimide (599 mg, 3.37 mmol) in 2-Me-THF (50 ml) was added dropwise and the reaction mixture was stirred at -78 Ό for 1 h. A saturated aqueous solution of NH4 Cl (60 ml) was added in one go, and the resulting mixture was stirred without cooling until the temperature reached 0 Ό. Water (20 ml) was added and, after stirring for 30 min, the layers were separated. The organic layer was dried over MgSO4, filtered, evaporated under reduced pressure and co-evaporated with CH3CN to provide 2- (2- (2 (benzyloxy) ethoxy) -4-chlorophenyl) -2-bromo-1- (6- ( trifluoromethyl) indolin-1-yl) ethanone 4g (1.16 g). The product was used without further purification in the next step.
Synthesis of intermediate 4h:
[00116] To a stirred solution of 2- (2- (2- (benzyloxy) ethoxy) -4chlorophenyl) -2-bromo-1- (6- (trifluoromethyl) indolin-1-yl) ethanone 4g (1.74 g , 3.06 mmol) in CH3CN (100 ml_) under N ₂ atmosphere 3-methoxy-5- (1 H-1,2,4-triazol-1-yl) aniline [CAS 1220630-56-7] (874 mg, 4.59 mmol) and diisopropylethylamine (1.06 ml, 6.12 mmol) and the reaction mixture was stirred at room temperature for 20 h and then at 50 Ό for 7 h. The mixture was cooled to room temperature
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36/109 and poured into H2O with stirring (400 ml). The product was extracted (2x) with EtzO. The combined organic layers were dried over MgSO4, filtered and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel (40 g) using a heptane / EtOAc / EtOH gradient from 100/0/0 to 40/45/15. The desired fractions were combined and the solvent was evaporated under reduced pressure and coevaporated with toluene. The residue was dried under vacuum at 50 Ό to obtain 2- (2- (2- (benzyloxy) ethoxy) -4-chlorophenyl) -2 - ((3-methoxy5- (1 H-1,2,4- triazol-1-i I) phenyl I) amine) -1 - (6- (trifluoromethyl) i ndol i n-1 yl) ethanone 4h (1.43 g).
Synthesis of Compound 4 and chiral separation in Enantiomers 4A and 4B:
[00117] A solution of 2- (2- (2- (benzyloxy) ethoxy) -4-chlorophenyl) -2 - ((3 methoxy-5- (1 H-1,2,4-triazol-1-yl) phenyl ) amino) -1 - (6- (trifluoromethyl) i ndol i n-1 yl) ethanone 4h (1.43 g, 2.11 mmol) in a solvent mixture of THF (15 ml) in EtOAc (75 ml) it was hydrogenated for 5 hours at room temperature under atmospheric pressure of H2 using Pd / C (0.5 g) as a catalyst. The catalyst was removed by filtration over dicalite®. The filter cake was washed several times with THF and the combined filtrates were evaporated under reduced pressure. The obtained solid was stirred in a 1/2/1 C ^Cb / EtOAc / methanol solvent mixture. The precipitate was filtered off, washed (2x) with EtOAc and dried under vacuum at 45 O to provide 2- (4-chloro-2- (2-hydroxyethoxy) phenyl) 2 - ((3-methoxy-5) - ( 1 H-1,2,4-triazole-1-i I) pheni I) amine) -1 - (6 (trifluoromethyl) indolin-1-yl) racemic ethanone (Compound 4, 600 mg).
[00118] Compound 4 enantiomers (700 mg) were separated through Preparative Chiral SFC (Stationary phase: Chiralpak® Diacel AD 20 x 250 mm, Mobile phase: CO2, MeOH / iPrOH (50/50) + 0.4% iPrNH2). The product fractions were combined and evaporated under reduced pressure to provide 0 Enantiomer 4A as 0
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37/109 product eluted first and Enantiomer 4B as the product eluted second. Both enantiomers were further purified by flash chromatography on silica gel (4 g) using a heptane / EtOAc / EtOH gradient from 100/0/0 to 40/45/15. The desired fractions were combined and evaporated under reduced pressure. The residue was stirred in water (3 ml) and MeOH (0.75 ml). The solids were filtered off, washed (3x) with water and dried under vacuum at 50 Ό to provide Enantiomer 4A (81 mg) and Enantiomer 4B (132 mg).
4A Enantiomer:
[00119] ¹ H NMR (400 MHz, DMSO-c / ₆ ) δ ppm 3.18 - 3.28 (m, 2 H)
3.73 (s, 3 H) 3.74 - 3.84 (m, 2 H) 4.08 - 4.19 (m, 3 H) 4.44 (td, J = 10.2,
6.7 Hz, 1 H) 4.96 (t, J = 5.6 Hz, 1 H) 5.84 (d, J = 8.6 Hz, 1 H) 6.35 (t, J = 2, 1 Hz, 1 H) 6.66 (t, J = 1.7 Hz, 1 H) 6.79 - 6.87 (m, 2 H) 7.02 (dd, J = 8.3, 1.9 Hz, 1 H) 7.15 (d, J = 1.8 Hz, 1 H) 7.34 - 7.43 (m, 2 H) 7.43 7.51 (m, 1 H) 8.15 ( s, 1 H) 8.38 (s I, 1 H) 9.12 (s, 1 H)
[00120] LC / MS (LC-C method): Tr 1.16 min, MH ⁺ 588
[00121] [a] _D ²⁰ : -42.9 ° (c 0.515, DMF)
[00122] Chiral SFC (SFC-K method): Tr 2.91 min, MH ⁺ 588, chiral purity 100%.
4B Enantiomer:
[00123] ¹ H NMR (400 MHz, DMSO-c / ₆ ) δ ppm 3.15 - 3.28 (m, 2 H)
3.73 (s, 3 H) 3.74 - 3.84 (m, 2 H) 4.07 - 4.22 (m, 3 H) 4.44 (td, J = 10.1,
6.6 Hz, 1 H) 4.96 (t, J = 5.5 Hz, 1 H) 5.84 (d, J = 8.6 Hz, 1 H) 6.35 (t, J = 2, 0 Hz, 1 H) 6.67 (t, J = 1.9 Hz, 1 H) 6.79 - 6.87 (m, 2 H) 7.03 (dd, J = 8.1, 2.0 Hz, 1 H) 7.15 (d, J = 2.0 Hz, 1 H) 7.34 - 7.42 (m, 2 H) 7.43 7.49 (m, 1 H) 8.16 ( s, 1 H) 8.38 (s I, 1 H) 9.13 (s, 1 H)
[00124] LC / MS (LC-C method): Tr 1.15 min, MH ⁺ 588
[00125] [a] _D ²⁰ : + 39.5 ° (c 0.595, DMF)
[00126] Chiral SFC (SFC-K method): Tr 2.78 min, MH ⁺ 588, purity
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38/109 chiral 100%.
Example 5: synthesis of 2- (4-chloro-2- (2-hydroxyethoxy) phenyl) -2 - ((3-methoxy5- (1 / 7-1,2,4-triazol-1-i I) pheni I ) ami no) -1 - (5-methoxy-6- (trifluoromethyl) indolin1-yl) ethanone (Compound 5) and chiral separation in Enantiomers 5A and 5B.
Br
SiD IN L.WCJS
THF, 7 0 * C 1 h iTMF
OH
4b you at night
3) NBS.
Et.Q
Er
Cí
OFfe
HO
N-N te
HO
N'-N
5c
HO í »fe
Dictate
DMF.íaSb .s ,. Ήί
Ν '
H fsW
Hí sepa ration qt
5A and 5S Fetal Enasythioni
Synthesis of intermediate 5a:
[00127]
To a mixture of ethyl 2- (4-chloro-2-hydroxyphenyl) acetate
4b (5.2 g, 24.2 mmol) and cesium carbonate (15.8 g, 48.5 mmol) in
DMF (90 ml_) at 10 Ό (2-bromoethoxy) (tert-butyl) dimethylsilane [CAS 86864-60-0] (6.26 ml_, 29.1 mmol) was added. The reaction mixture was stirred at room temperature overnight. H2O was added and the reaction mixture was extracted with EtOAc. The organic phase
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39/109 was dried with MgSO 4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 μηπ, 80 g, heptane / EtOAc 80/20). The pure fractions were combined and the solvent was removed under reduced pressure to give 2- (2- (2 - ((tert-butyldimethylsilyl) oxy) ethoxy) -4-chlorophenyl) ethyl acetate 5a (7.8 g).
Synthesis of intermediate 5b:
[00128] To a 1 M solution of lithium bis (trimethylsilyl) amide in THF (41.8 ml_, 41.8 mmol), cooled to -70 Ό, a solution of 2- (2- (2- ((ether-butyldimethylsilyl) oxy) ethoxy) -4-chlorophenyl) ethyl acetate 5a (7.8 g, 20.9 mmol) in THF (45 ml). After 1 h at -70 Ό, chlorotrimethylsilane (4.24 ml, 33.5 mmol) was added. The reaction mixture was stirred at -70 Ό for 15 min. / -Bromosuccinimide (4.46 g, 25.1 mmol) in THF (45 ml) was added and stirring was continued at 55 Ό for 2 h. The reaction mixture was poured into H2O and extracted twice with EtOAc. The organic phases were combined, dried over MgSO4, filtered and concentrated under reduced pressure to give 2-bromo-2- (2- (2 - ((tert-butyldimethylsilyl) oxy) ethoxy) -4-chlorophenyl) acetate of ethyl 5b (10.1 g) which was used in the next step without further purification.
Synthesis of intermediate 5c:
[00129] A mixture of 2-bromo-2- (2- (2 - ((tert-butyldimethylsilyl) oxy) ethoxy) -4-chlorophenyl) ethyl acetate 5b (4.75 g, 10.5 mmol), 3-methoxy -5- (1 H-1,2,4-triazol-1-yl) aniline [CAS 1220630-56-7] (3 g, 15.8 mmol) and diisopropylethylamine (3.62 ml_, 21.0 mmol) in CH3CN (90 ml) was stirred at 50 ° for 24 h. The reaction mixture was concentrated under reduced pressure. The residue was absorbed with EtOAc and washed with 0.5 N HCI, water and saturated aqueous sodium chloride solution. The organic phase was dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 μηπ, 120 g, heptane / EtOAc gradient 80/20 a
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10/40
70/30) to give 2- (2- (2 - ((tert-butyldimethylsilyl) oxy) ethoxy) -4-chlorophenyl) -2 ((3-methoxy-5- (1 H-1,2,4-triazole -1-yl) phenyl) amino) ethyl acetate 5c (3.7 g) ·
Synthesis of intermediate 5d:
[00130] Lithium hydroxide monohydrate (523 mg, 12.5 mmol) in water (25 ml_) was added in portions to a solution of 2- (2- (2 ((tert-butyldimethylsilyl) oxy) ethoxy) -4-chlorophenyl) -2 - ((3-methoxy-5- (1 H-1,2,4triazol-1-yl) phenyl) amino) ethyl acetate 5c (3.5 g, 6.24 mmol) in THF / CH3OH (1/1) (50 ml) at 10 Ό. The reaction was stirred at room temperature for 2 h, diluted with water and cooled to 0 Ό. The solution was slowly acidified with 0.5 N HCI to pH 6 and extracted with EtOAc. The organic layer was dried over MgSO4, filtered and the solvent was concentrated under reduced pressure to give 2- (2- (2 - ((tercbuti Idi meti Isi I i) oxide) ethoxy) -4-chloropheni I) -2 - ((3-methoxy-5- (1H-1,2,4-triazol-1 yl) phenyl) amino) acetic 5d (3.1 g). The compound was used as such in the next step.
Synthesis of intermediate 5e:
[00131] A mixture of 5-methoxy-6- (trifluoromethyl) indoline 2c (400 mg, 1.84 mmol), 2- (2- (2 - ((ether-butyldimethylsilyl) oxy) ethoxy) -4chlorophenyl) - 2 - ((3-methoxy-5- (1 H-1,2,4-triazol-1-yl) phenyl) amino) acetic 5d (982 mg, 1.84 mmol), HATU (1.05 g, 2 , 76 mmol) and diisopropylethylamine (913 μΙ_, 5.53 mmol) in DMF (10 ml_) was stirred at room temperature for 2 h. The mixture was diluted with water. The precipitate was filtered off and washed with water. The precipitate was removed with EtOAc, washed with a 10% solution of K2CO3 in water, water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure to give 2- (2- (2 - ((force-butyldimethylsilyl)) oxy) ethoxy) -4-chlorophenyl) -2 ((3-methoxy-5- (1 H-1,2,4-triazol-1-i) phenyl I) amine) -1 - (5-methoxy- 6 (trifluoromethyl) indolin-1-yl) ethanone 5e (1.35 g). The compound was used as such in the next reaction step.
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Synthesis of Compound 5 and chiral separation in Enantiomers 5A and 5B:
[00132] Under a flow of N2, 5 O, 4 M HCI in dioxane (4.6 ml_,
18.4 mmol) was added dropwise to a solution of 2- (2- (2 - ((tercbuti Idi meti Isi I i I) oxy) ethoxy) -4-chloropheni I) -2 - ((3-methoxy -5- (1H-1,2,4-triazol-1 yl) phenyl) amino) -1 - (5-methoxy-6- (trifluoromethyl) indolin-1-yl) ethanone 5e (1.35 g, 1, 84 mmol) in MeOH (25 ml). The reaction was stirred at room temperature for 1 h. The mixture was cooled to 0 ° C, basified with 10% aqueous K2CO3 solution and extracted with EtOAc. The organic phase was separated, dried with MgSCU, filtered, and the solvent was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 40 g, CHbCb / MeOH 98.5 / 1.5). The pure fractions were combined and concentrated to dryness under reduced pressure. The residue (980 mg) was crystallized from MeOH to give 2- (4-chloro-2- (2-hydroxyethoxy) phenyl) -2 - ((3-methoxy-5- (1 H-1,2,4triazole -1 -i I) phenyl I) amine) -1 - (5-methoxy-6- (trifluoromethyl)) nidol-n-1-yl) ethanone 5 (805 mg) as a racemate.
[00133] Compound 5 enantiomers (771 mg) were separated by Preparative Chiral SFC (Stationary phase: Chiralpak® IA, 5 pm, 250x20 mm, Mobile phase: 6% CH2CI2, 70% CO2, 24% EtOH (+0 , 3% iPrNH2)). The first eluted enantiomer (375 mg) was solidified by trituration with diisopropyl ether to give the 5A Enantiomer (308 mg). The second eluting enantiomer (400 mg) was further purified by flash chromatography on silica gel (15-40 pm, 24 g, C CWMeOH 99/1). The pure fractions were combined and the solvent was concentrated under reduced pressure. The residue (340 mg) was solidified by trituration with diisopropyl ether to give the 5B Enantiomer (291 mg).
Compound 5:
[00134] ¹ H NMR (500 MHz, DMSO-c / ₆ ) δ ppm 3.15 - 3.30 (m, 2 H)
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3.72 (s, 3 Η) 3.74 - 3.83 (m, 2 Η) 3.85 (s, 3 Η) 4.06 - 4.20 (m, 3 Η) 4.41 (td, J = 10.2, 6.3 Hz, 1 H) 4.99 (t, J = 5.5 Hz, 1 H) 5.82 (d, J = 8.5 Hz, 1 H)
6.35 (s, 1 H) 6.65 (t, J = 1.9 Hz, 1 H) 6.80 - 6.85 (m, 2 H) 7.02 (dd, J = 8.2, 1.9 Hz, 1 H) 7.15 (d, J = 2.2 Hz, 1 H) 7.24 (s, 1 H) 7.37 (d, J = 8.5 Hz, 1 H) 8 , 16 (s, 1 H) 8.34 (s, 1 H) 9.13 (s, 1 H)
[00135] LC / MS (LC-A method): Tr 3.13 min, MH ⁺ 618
[00136] Melting point: 228Ό
5A Enantiomer:
[00137] ¹ H NMR (400 MHz, DMSO-cfe) δ ppm 3.16 - 3.28 (m, 2 H)
3.72 (s, 3 H) 3.74 - 3.83 (m, 2 H) 3.84 (s, 3 H) 4.06 - 4.21 (m, 3 H) 4.35
- 4.46 (m, 1 H) 4.97 (t, J = 5.6 Hz, 1 H) 5.81 (d, J = 8.6 Hz, 1 H) 6.35 (s, 1 H ) 6.65 (s, 1 H) 6.77 - 6.86 (m, 2 H) 7.02 (dd, J = 8.3, 1.8 Hz, 1 H) 7.14 (d, J = 1.5 Hz, 1 H) 7.24 (s, 1 H) 7.38 (d, J = 8.6 Hz, 1 H) 8.15 (s, 1 H)
8.34 (s, 1 H) 9.12 (s, 1 H)
[00138] LC / MS (LC-A method): Tr 3.11 min, MH ⁺ 618
[00139] [a] _D ²⁰ : -40.3 ° (c 0.2383, DMF)
[00140] Chiral SFC (SFC-C method): Tr 2.75 min, MH ⁺ 618, 100% chiral purity.
5B Enantiomer:
[00141] ¹ H NMR (400 MHz, DMSO-cfe) δ ppm 3.16 - 3.28 (m, 2 H)
3.72 (s, 3 H) 3.74 - 3.83 (m, 2 H) 3.84 (s, 3 H) 4.06 - 4.20 (m, 3 H) 4.36
- 4.46 (m, 1 H) 4.97 (t, J = 5.6 Hz, 1 H) 5.81 (d, J = 8.6 Hz, 1 H) 6.35 (s, 1 H ) 6.65 (s, 1 H) 6.78 - 6.85 (m, 2 H) 7.01 (dd, J = 8.3, 1.8 Hz, 1 H) 7.14 (d, J = 1.5 Hz, 1 H) 7.24 (s, 1 H) 7.38 (d, J = 8.1 Hz, 1 H) 8.15 (s, 1 H)
8.34 (s, 1 H) 9.12 (s, 1 H)
[00142] LC / MS (LC-A method): Tr 3.11 min, MH ⁺ 618
[00143] [a] _D ²⁰ : + 40.0 ° (c 0.22, DMF)
[00144] Chiral SFC (SFC-C method): Tr 3.60 min, MH ⁺ 618, chiral purity 98.47%.
Example 6 (Method 1): synthesis of 2- (4-chloro-2- (2-hydroxyethoxy) phenyl) -2
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43/109 ((3-methoxy-5- (1 H-1,2,4-triazol-1-i) phenyl I) amine) -1 - (6 (trifluoromethoxy) indolin-1-yl) ethanone ( Compound 6) and chiral separation in Enantiomers 6A and 6B.
Synthesis of intermediate 6a:
[00145] A mixture of 6- (trifluoromethoxy) indoline [CAS 959235-951] (837 mg, 4.12 mmol), 2- (2- (2 - (((tert-butyldimethylsilyl) oxy) ethoxy) -4chlorophenyl) -2 - ((3-methoxy-5- (1 H-1,2,4-triazol-1-yl) phenyl) amino) acetic 5d (2.196 g, 4.12 mmol), HATU (2.35 g, 6.18 mmol) and diisopropylethylamine (2 ml, 12.36 mmol) in DMF (20 ml) was stirred at room temperature for 2 h. The mixture was diluted with water. The resulting gummy material was absorbed with EtOAc, washed with a 10% K2CO3 solution in water, water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 80 g, 70/30 to 60/40 heptane / EtOAc gradient). The pure fractions were combined and the solvent was concentrated under reduced pressure to give 2- (2- (2 - ((tercbuti Idi meti Isi I i I) oxy) ethoxy) -4-chloropheni I) -2 - ((3- methoxy-5- (1 H-1,2,4-triazol-1 yl) phenyl) amino) -1 - (6- (trifluoromethoxy) indolin-1-yl) ethanone 6a (1.65 g).
Synthesis of Compound 6 and chiral separation in Enantiomers 6A and 6B:
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[00146] Under a flow of N2, at 5 Ό, 4 M HCI in dioxane (6.5 ml_,
21.6 mmol) was added dropwise to a solution of 2- (2- (2 - ((tercbuti Idi meti Isi II i) oxy) ethoxy) -4-chloropheni I) -2 - ((3-methoxy -5- (1H-1,2,4-triazol-1 yl) phenyl) amino) -1- (6- (trifluoromethoxy) indolin-1-yl) ethanone 6a (1.85 g, 2.58 mmol) in MeOH (40 ml). The reaction was stirred at room temperature for 1 h. The mixture was cooled to 0 Ό, basified with 10% aqueous K2CO3 solution and extracted with EtOAc. The organic phase was separated, dried over MgSO4, filtered, and the solvent was concentrated under reduced pressure. The compound was crystallized from CH2Cl2 to provide 2- (4-chloro-2- (2-hydroxyethoxy) phenyl) -2 - ((3-methoxy-5- (1 H-1,2,4-triazole-1-yl ) phenyl) amino) -1 - (6- (trifluoromethoxy) indolin1 -yl) ethanone 6 (1.47 g) as a racemate.
[00147] Compound 6 enantiomers were separated by Preparative Chiral SFC (stationary phase: Chiralpak® IC 5 pm 250 x 30 mm, mobile phase: 70% CO2, 30% iPrOH (+ 0.3% iPrNFE)). The first eluting enantiomer (585 mg) was further purified by flash chromatography on silica gel (15-40 pm, 24 g, CFkCb / MeOH 99/1) to provide, after solidification in MeOH / diisopropyl ether / heptane, 0 Enantiomer 6A (491 mg). The second eluting enantiomer (400 mg) was further purified by flash chromatography on silica gel (15-40 pm, 24 g, CFkCb / MeOH 99/1) to provide, after solidification in MeOH / diisopropyl ether / heptane, 0 6B enantiomer (467 mg).
Compound 6:
[00148] ¹ H NMR (400 MHz, DMSO-c / ₆ ) δ ppm 3.08 - 3.23 (m, 2 H)
3.73 (s, 3 H) 3.75 - 3.84 (m, 2 H) 4.07 - 4.22 (m, 3 H) 4.37 - 4.49 (m, 1 H) 4, 94 (s I, 1 H) 5.82 (d, J = 8.6 Hz, 1 H) 6.35 (s, 1 H) 6.64 - 6.68 (m, 1 H) 6.79 - 6.86 (m, 2 H) 6.98 - 7.05 (m, 2 H) 7.15 (d, J = 2.0 Hz, 1 H) 7.34 (d, J = 8.6 Hz , 1 H) 7.37 (d, J = 8.1 Hz, 1 H) 8.04 (s, 1 H) 8.15 (s, 1 H) 9.11 (s, 1 H)
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[00149] LC / MS (LC-A method): Tr 3.27 min, MH ⁺ 604
[00150] Melting point: 161Ό
6A Enantiomer:
[00151] ¹ H NMR (500 MHz, DMSO-d ₆ ) δ ppm 3.10 - 3.25 (m, 2 H)
3.73 (s, 3 H) 3.74 - 3.84 (m, 2 H) 4.06 - 4.23 (m, 3 H) 4.39 - 4.48 (m, 1 H) 4, 99 (t I, J = 5.4 Hz, 1 H) 5.83 (d I, J = 8.5 Hz, 1 H) 6.36 (s I, 1 H) 6.67 (s, 1 H ) 6.84 (s, 1 H) 6.88 (d I, J = 8.5 Hz, 1 H) 7.03 (t I, J = 7.6 Hz, 2 H)
7.16 (s, 1 H) 7.36 (dd, J = 11.8, 8.4 Hz, 2 H) 8.04 (s I, 1 H) 8.17 (s, 1 H)
9.14 (s, 1 H)
[00152] LC / MS (LC-A method): Tr 3.25 min, MH ⁺ 604
[00153] [a] _D ²⁰ : + 45.9 ° (c 0.29, DMF)
[00154] Chiral SFC (SFC-D method): Tr 4.20 min, MH ⁺ 604, chiral purity 100%.
6B Enantiomer:
[00155] ¹ H NMR (500 MHz, DMSO-d ₆ ) δ ppm 3.09 - 3.25 (m, 2 H)
3.73 (s, 3 H) 3.74 - 3.83 (m, 2 H) 4.06 - 4.21 (m, 3 H) 4.43 (td, J = 10.2,
6.6 Hz, 1 H) 4.98 (t, J = 5.4 Hz, 1 H) 5.83 (d, J = 8.5 Hz, 1 H) 6.35 (s, 1 H)
6.66 (s, 1 H) 6.83 (s, 1 H) 6.88 (d, J = 8.8 Hz, 1 H) 6.99 - 7.06 (m, 2 H)
7.15 (d, J = 1.6 Hz, 1 H) 7.36 (dd, J = 12.6, 8.2 Hz, 2 H) 8.04 (s, 1 H) 8.16 (s, 1 H) 9.14 (s, 1 H)
[00156] LC / MS (LC-A method): Tr 3.31 min, MH ⁺ 604
[00157] [a] _D ²⁰ : -46.3 ° (c 0.3, DMF)
[00158] Chiral SFC (SFC-D method): Tr 5.30 min, MH ⁺ 604, chiral purity 100%.
Example 6 (Method 2): synthesis of 2- (4-chloro-2- (2-hydroxyethoxy) phenyl) -2 ((3-methoxy-5- (1 H-1,2,4-triazol-1 -i I) phenyl I) amine) -1 - (6 (trifluoromethoxy) indolin-1-yl) ethanone (Compound 6).
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V »^ .5M UHMDSemTHF
THF _r -7O ^u 'C

2) Cfôiihtes, -70 ^and C 15 iw;
EtO
OSn

Synthesis of intermediate 6b:
[00159] To a solution of 1.5 M lithium bis (trimethylsilyl) amide in THF (23 ml_, 34.4 mmol), cooled to -70 Ό under a flow of N2, a solution of 2- (4 -chloro-2-hydroxyphenyl) ethyl acetate 4c (6 g, 17.2 mmol) in THF (35 ml). After 1 h at -70 Ό, chlorotrimethylsilane (3.5 ml, 27.5 mmol) was added. The reaction mixture was stirred at 70 Ό for 15 min. / -Bromosuccinimide (3.7 g, 20.6 mmol) in THF (35 ml) was added and stirring was continued at -70 Ό for 2 h. The reaction mixture was poured into H2O and extracted with EtOAc. The organic phases were combined, dried over MgSO4, filtered and concentrated under reduced pressure to provide 2- (2- (2 (benzyloxy) ethoxy) -4-chlorophenyl) -2-bromoacetate 6b (8.2 g), which was used in the next step without further purification.
Synthesis of intermediate 6c:
[00160] A mixture of 2- (2- (2- (benzyloxy) ethoxy) -4-chlorophenyl) -2bromoacetate 6b (6.5 g, 15.2 mmol), 3-methoxy-5- (1 H-1 , 2,4-triazol-1-yl) aniline [CAS 1220630-56-7] (4.6 g, 24.1 mmol) and diisopropylethylamine (5.3 ml_, 30.4 mmol) in CH3CN (130 ml_) was stirred at 50 Ό for
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H. The reaction mixture was concentrated under reduced pressure. The residue was diluted with EtOAc. The solution was filtered to remove solid particles (residual aniline). The organic layer was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 120 g, heptane / EtOAc gradient 80/20 to 70/30). The pure fractions were combined and the solvent was concentrated under reduced pressure to give 2- (2- (2- (benzyloxy) ethoxy) -4chlorophenyl) -2 - ((3-methoxy-5- (1 H-1,2, 4-triazol-1-yl) phenyl) amino) ethyl acetate 6c (4.8 g).
Synthesis of intermediate 6d:
[00161] At 10 Ό, lithium hydroxide monohydrate (500 mg, 11.9 mmol) was added to a solution of 2- (2- (2- (benzyloxy) ethoxy) -4chlorophenyl) -2 - (( 3-methoxy-5- (1 H-1,2,4-triazol-1-yl) phenyl) amino) ethyl acetate 6c (3.2 g, 5.96 mmol) in MeOH / THF / water (1 / 1/1) (50 ml). The mixture was stirred at room temperature for 2 h. The mixture was diluted with ice water and cooled to 0 Ό. The resulting mixture was acidified to pH 6-7 with 0.5N HCI and extracted with EtOAc. The organic layers were combined, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure to give 2- (2- (2 (benzyloxy) ethoxy) -4-chlorophenyl) -2 - ((3-methoxy-5- (1 H-1,2,4-triazol-1 yl) phenyl) amino) acetic 6d (2.75 g). The compound was used in the next reaction step without further purification.
Synthesis of intermediate 6e:
[00162] A mixture of 6- (trifluoromethoxy) indoline [CAS 959235-951] (1.2 g, 5.89 mmol), 2- (2- (2- (benzyloxy) ethoxy) -4-chlorophenyl) - 2 - ((3-methoxy-5- (1 H-1,2,4-triazol-1-yl) phenyl) amino) acetic 6d (2.5 g, 4.91 mmol), HATU (2.29 g, 6 , 01 mmol) and diisopropylethylamine (1.99 ml, 12.0 mmol) in DMF (18 ml) was stirred at room temperature for 2 h. The mixture was diluted with water. The precipitate was filtered off and washed with water. The precipitate was removed with EtOAc, washed
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48/109 with a 10% K2CO3 solution in water, water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. The compound was purified by flash chromatography on silica gel (15-40 μηπ, 220 g, 50/50 heptane / EtOAc). The pure fractions were combined and the solvent was concentrated under reduced pressure to give 2- (2- (2 (benzyloxy) ethoxy) -4-chlorophenyl) -2 - ((3-methoxy-5- (1 H-1,2 , 4-triazol-1-yl) phenyl) amino) -1- (6- (trifluoromethoxy) indolin-1-yl) ethanone 6e (2.5 g).
Synthesis of Compound 6:
[00163] A mixture of 2- (2- (2- (benzyloxy) ethoxy) -4-chlorophenyl) -2 - ((3 methoxy-5- (1 H-1,2,4-triazol-1-yl) phenyl ) amino) -1 - (6- (trifluoromethoxy) indolin1-yl) ethanone 6e (2 g, 2.88 mmol) in EtOAc / MeOH / THF (1/1/1) (100 ml) was hydrogenated for 50 min under atmospheric pressure of H2 with Pd / C (10%) (3.07 g, 2.88 mmol) as catalyst. The reaction was diluted with MeOH and filtered through a pad of celite®. The filtrate was concentrated under reduced pressure. The residue (1.42 g) was combined with another batch (total amount: 1.65 g) and purified by achiral SFC (stationary phase: NH2 5 μιτι 150x30 mm), mobile phase: 70% CO2, 30% iPrOH ( + 0.3% iPrNH2)). The pure fractions were combined and the solvent was concentrated under reduced pressure to give 2- (4-chloro-2- (2-hydroxyethoxy) phenyl) -2 - ((3-methoxy-5- (1 H-1,2,4- triazol-1 yl) phenyl) amino) -1- (6- (trifluoromethoxy) indolin-1-yl) ethanone 6 (1.36 g) as a racemate.
Example 7: synthesis of 2- (4-chloro-2- (2-hydroxyethoxy) phenyl) -1- (5-fluoro6 (trifluoromethyl) I ndol i n-1 -yl) -2 - ((3-methoxy -5- (1 H-1,2,4-triazol-1 yl) phenyl) amino) ethanone (Compound 7) and chiral separation in Enantiomers 7A and 7B.
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Synthesis of intermediate 7a:
[00164] At 0 Ό, BH 3-Pyridine (10.45 ml_, 103.4 mmol) was added dropwise to a solution of 5-fluor-6- (trifluoromethyl) -1 H-indole [CAS 1493800-10 -4] (7 g, 34.5 mmol) in EtOH (45 ml). 6N HCI (105 ml) was added slowly while maintaining the temperature below 10 ° C. The mixture was stirred at 0 ° C for 2 h. Water (210 ml) was added and the mixture was basified to pH 8-9 with a concentrated aqueous solution of NaOH (the reaction temperature was kept below 20 Ό). EtOAc was added. The organic layer was separated, washed with water, dried over MgSO4, filtered, and the solvent concentrated under reduced pressure. The residue was co-evaporated under reduced pressure with toluene. The crude product was purified by flash chromatography on silica gel (20-45 pm, 120 g, CHzCb / MeOH 98.5 / 1.5). The pure fractions were combined and the solvent was removed under reduced pressure to give 5-fluorine-6- (trifluoromethyl) indoline 7a (3.5 g).
Synthesis of intermediate 7b:
[00165] A mixture of 5-fluorine-6- (trifluoromethyl) indoline 7a (385 mg,
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1.88 mmol), 2- (2- (2 - ((tert-butyldimethylsilyl) oxy) ethoxy) -4-chlorophenyl) -2 ((3-methoxy-5- (1 H-1,2,4- triazol-1-yl) phenyl) amino) acetic 5d (1 g, 1.88 mmol), HATU (1.07 g, 2.814 mmol) and diisopropylethylamine (930 pL, 5.63 mmol) in DMF (10 mL ) was stirred at room temperature for 2 h. The mixture was diluted with water. The resulting gummy material was removed with EtOAc. The organic layer was washed with a 10% solution of K2CO3 in water, water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure to give 2- (2- (2 - ((etherbutyldimethylsilyl) oxy) ethoxy) - 4-chlorophenyl) -1- (5-fluor-6- (trifluoromethyl) indolin1 -yl) -2 - ((3-methoxy-5- (1 H-1,2,4-triazol-1-yl) phenyl) amino) ethanone 7b (1.4 g).
Synthesis of Compound 7 and chiral separation in Enantiomers 7A and 7B:
[00166] Under a flow of N2, 5 O, 4 M HCI in dioxane (4.9 mL,
19.4 mmol) was added dropwise to a solution of 2- (2- (2 - ((etherbutyldimethylsilyl) oxy) ethoxy) -4-chlorophenyl) -1 - ((5-fluor-6- (trifluoromethyl) indolin1 -yl) -2 - ((3-methoxy-5- (1 H-1,2,4-triazol-1-yl) phenyl) amino) ethanone 7b (1.4 g, 1.94 mmol) in MeOH ( The reaction was stirred at room temperature for 1 hr. The mixture was cooled to 0 ° C, basified with 10% K2CO3 aqueous solution and extracted with EtOAc. The organic layer was separated, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure.The residue was purified by flash chromatography on silica gel (15-40 pm, 40 g, CFhCb / MeOH 98/2). The pure fractions were combined and the solvent was concentrated under reduced pressure to give 2- (4-chloro-2- (2-hydroxyethoxy) phenyl) -1 (5-fluor-6- (trifluoromethyl) i ndol i n-1-yl) -2 - (((3-methoxy-5- (1 H-1,2,4-triazol-1 yl) phenyl) amino) ethanone 7 (737 g) as a racemate.
[00167] Compound 7 enantiomers were separated by Preparative Chiral SFC (stationary phase: Chiralpak® AD-H 5 pm 250 x 30 mm, mobile phase: 60% CO2, 40% EtOH (+ 0.3% iPrNFE)) . The first eluted enantiomer (325 mg) was crystallized from ether di
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51/109 isopropyl / petroleum ether to give Enantiomer 7A (244 mg). The second eluted enantiomer (310 mg) was crystallized from diisopropyl ether / petroleum ether to give Enantiomer 7B (220 mg).
Compound 7:
[00168] ¹ H NMR (500 MHz, DMSO-c / ₆ ) δ ppm 3.19 - 3.30 (m, 2 H) 3.63 - 3.87 (m, 5 H) 4.05 - 4, 24 (m, 3 H) 4.40 - 4.49 (m, 1 H) 4.97 (s I, 1 H) 5.83 (d I, J = 6.9 Hz, 1 H) 6.35 (s I, 1 H) 6.67 (s I, 1 H) 6.80 - 6.89 (m, 2 H) 7.03 (d I, J = 7.3 Hz, 1 H) 7.15 (s I, 1 H) 7.37 (d I, J = 7.6 Hz, 1 H) 7.47 (d I, J = 9.5 Hz, 1 H) 8.16 (s I, 1 H ) 8.39 (d I, J = 4.4 Hz, 1 H) 9.14 (s I, 1 H)
[00169] LC / MS (LC-B method): Tr 3.14 min, MH ⁺ 606
[00170] Melting point: 140Ό
Enantiomer 7A:
[00171] ¹ H NMR (500 MHz, DMSO-d ₆ ) δ ppm 3.20 - 3.30 (m, 2 H)
3.69 - 3.86 (m, 5 H) 4.06 - 4.23 (m, 3 H) 4.40 - 4.50 (m, 1 H) 4.98 (t I, J = 5, 2 Hz, 1 H) 5.83 (d I, J = 8.8 Hz, 1 H) 6.35 (s I, 1 H) 6.67 (s, 1 H) 6.82
- 6.89 (m, 2 H) 7.03 (d I, J = 8.2 Hz, 1 H) 7.16 (s, 1 H) 7.37 (d, J = 8.2 Hz, 1 H) 7.47 (d I, J = 10.1 Hz, 1 H) 8.17 (s, 1 H) 8.39 (d I, J = 6.3 Hz, 1 H) 9.14 (s , 1 H)
[00172] LC / MS (LC-A method): Tr 3.27 min, MH ⁺ 606
[00173] [a] _D ²⁰ : -44.3 ° (c 0.282, DMF)
[00174] Chiral SFC (SFC-B method): Tr 2.89 min, MH ⁺ 606, chiral purity 100%.
[00175] Melting point: 166Ό
7B Enantiomer:
[00176] ¹ H NMR (500 MHz, DMSO-d ₆ ) δ ppm 3.18 - 3.30 (m, 2 H)
3.69 - 3.86 (m, 5 H) 4.07 - 4.22 (m, 3 H) 4.40 - 4.50 (m, 1 H) 4.97 (t I, J = 5, 2 Hz, 1 H) 5.83 (d I, J = 8.8 Hz, 1 H) 6.35 (s I, 1 H) 6.67 (s, 1 H) 6.81
- 6.89 (m, 2 H) 7.03 (d I, J = 8.2 Hz, 1 H) 7.16 (s, 1 H) 7.37 (d I, J = 8.2 Hz, 1 H) 7.47 (d I, J = 10.1 Hz, 1 H) 8.17 (s, 1 H) 8.39 (d I, J = 6.3 Hz, 1
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,1) 9.14 (s, 1 Η)
[00177] LC / MS (LC-A method): Tr 3.27 min, MH ⁺ 606
[00178] [a] _D ²⁰ : + 35.6 ° (c 0.281, DMF)
[00179] Chiral SFC (SFC-B method): Tr 4.92 min, MH ⁺ 606, 100% chiral purity.
[00180] Melting point: 100Ό
Example 8: synthesis of 2- (4-chloro-2- (2-hydroxyethoxy) phenyl) -2 - ((3-methoxy5- (1 H-1,2,4-triazol-1-i I) pheni I) ami no) -1 - (5-methoxy-6 (trifluoromethoxy) indolin-1-yl) ethanone (Compound 8) and chiral separation in Enantiomers 8A and 8B.
F ,. .. OF ... O ..
..ΙΉ
CuL PtfC & PPh-ík '
NEts. EMF
THE
3d rüuOK NMP
F._ ... O.
F ,. ..THE.
CS
CM®
BOH. 0C
3d
HO
Ν 'H
HATU.
8e
HCI (4M eiii doxane)
THE
Q,
OMe separation. there inantS & neros SA and 8B
MeOH, ^F '. ·
NH
Synthesis of intermediate 8a:
[00181] A solution of 4-methoxy-3- (trifluoromethoxy) aniline [CAS 647855-21-8] (3.1 g, 15.0 mmol) in toluene (50 ml_) was treated with Nbromosuccinimide (2.8 g , 15.7 mmol) at 5 Ό and the resulting mixture was stirred at 5-10 Ό for 2 h. The mixture was diluted with water and extracted
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53/109 with EtOAc. The combined extracts were dried over MgSO4, filtered and evaporated under reduced pressure. Purification was carried out by flash chromatography on silica gel (15-40 pm, 24 g, heptane / EtOAc gradient from 95/5 to 90/10). The pure fractions were combined and evaporated to dryness to give 2-bromo-4-methoxy-5 (trifluoromethoxy) aniline 8a (2.5 g).
Synthesis of intermediate 8b:
[00182] A solution of 2-bromo-4-methoxy-5- (trifluoromethoxy) aniline 8a (2.72 g, 9.51 mmol) in DMF (30 ml) was degassed with N ₂ for 15 min. Dichlorobis (triphenylphosphine) palladium (ll) (667 mg, 0.95 mmol), copper (I) iodide (362 mg, 1.90 mmol), triethylamine (3.96 ml, 28.53 mmol) and trimethylsilylacetylene (3.95 ml, 28.5 mmol). The reaction mixture was heated to 70 O for 12 h under a flow of N ₂ . After cooling to room temperature, the reaction mixture was diluted with H ₂ O and extracted with EtOAc. The organic phases were combined, dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 80 g, heptane / EtOAc 85/15). The pure fractions were combined and evaporated to dryness to give 4-methoxy-5- (trifluoromethoxy) 2 - ((trimethylsilyl) ethynyl) aniline 8b (1.4 g).
Synthesis of intermediate 8c:
[00183] To a solution of 4-methoxy-5- (trifluoromethoxy) -2 ((trimethylsilyl) ethynyl) aniline 8b (1.2 g, 3.96 mmol) in NMP (11 ml_) under a flow of N ₂ tBuOK (1.33 g, 11.9 mmol) is added in one portion. The reaction mixture was heated to 80 O for 4 h, and poured into ice / water and acidified with 3 N HCI to pH 4-5. The reaction mixture was extracted with EtOAc. The organic phases were combined, washed with H ₂ O, dried with MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 40 g, heptane / EtOAc 85/15). Pure fractions were
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54/109 were combined and evaporated to dryness to give 5-methoxy-6 (trifluoromethoxy) -1 H-indole 8c (490 mg).
Synthesis of intermediate 8d:
[00184] At 0 O, BHs-Pyridine (10.5 ml_, 103.8 mmol) was added dropwise to a solution of 5-methoxy-6- (trifluoromethoxy) -1 H-indole 8c (8 g , 34.6 mmol) in EtOH (45 ml). 6 N HCI (6 ml) was added dropwise while maintaining the temperature below 10 Ό. The mixture was stirred at 0 Ό for 3 h. Water (210 ml) was added and the mixture was basified to pH 8-9 with a concentrated solution of NaOH in water (the reaction temperature was kept below 20 Ό). The mixture was extracted with EtOAc. The organic layer was washed with water, dried over MgSO4, filtered, and the solvent evaporated under reduced pressure. Toluene was added and the solution was concentrated under reduced pressure to give 5-methoxy-6- (trifluoromethoxy) indoline 8d (7.5 g) ·
Synthesis of intermediate 8e:
[00185] A mixture of 5-methoxy-6- (trifluoromethoxy) indoline 8d (437 mg, 1.88 mmol), acid 2- (2- (2 - ((ether-butyldimethylsilyl) oxy) ethoxy) -4chlorophenyl) - 2 - ((3-methoxy-5- (1 H-1,2,4-triazol-1-yl) phenyl) amino) acetic 5d (1 g, 1.88 mmol), HATU (1.07 g, 2 , 81 mmol) and diisopropylethylamine (930 μΙ_, 5.63 mmol) in DMF (10 ml_) was stirred at room temperature for 2 h. The mixture was diluted with water. The resulting gummy material was removed with EtOAc. The organic solution was washed with a 10% K2CO3 solution in water, water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure to give 2- (2- (2 - ((tercbuti Idi meti Isi I i I I ) oxy) ethoxy) -4-chloropheni I) -2 - ((3-methoxy-5- (1 H-1,2,4-triazol-1 yl) phenyl) amino) -1 - (5-methoxy-6 - (trifluoromethoxy) indolin-1-yl) ethanone 8e (1.5 g). The compound was used as such in the next reaction step.
Synthesis of Compound 8 and chiral separation in Enantiomers 8A and 8B:
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[00186] Under a flow of N2, at 5 Ό, 4 M HCI in dioxane (4.9 ml_,
19.4 mmol) was added dropwise to a solution of 2- (2- (2 - ((tercbuti Idi meti Isi II i) oxy) ethoxy) -4-chloropheni I) -2 - ((3-methoxy -5- (1H-1,2,4-triazol-1 yl) phenyl) amino) -1 - (5-methoxy-6- (trifluoromethoxy) indolin-1-yl) ethanone 8e (1.4 g, 1, 94 mmol) in MeOH (25 ml). The reaction was stirred at room temperature for 1 h. The mixture was cooled to 0 ° C, basified with 10% aqueous K2CO3 solution and extracted with EtOAc. The organic layer was separated, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 40 g, CH2Cl2 / MeOH / NH4OH at 98.4 / 1.5 / 0.1). The pure fractions were combined and the solvent was concentrated under reduced pressure to give, after crystallization from CH2 Cl2, 2- (4-chloro-2- (2-hydroxyethoxy) phenyl) 2 - ((3-methoxy-5- ( 1 H-1,2,4-triazol-1-i I) pheni I) amine) -1 - (5-methoxy-6 (trifluoromethoxy) indolin-1-yl) ethanone 8 (850 g) as a racemate .
[00187] Compound 8 enantiomers were separated by Preparative Chiral SFC (stationary phase: Chiralpak® IC 5 pm 250 x 30 mm, mobile phase: 60% CO2, 40% iPrOH). The first eluted enantiomer (410 mg) was solidified by trituration with diisopropyl ether to give the 8A Enantiomer (314 mg). The second eluted enantiomer (388 mg) was solidified by trituration with diisopropyl ether to give the 8B Enantiomer (300 mg).
Compound 8:
[00188] ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 3.07 - 3.27 (m, 2 H)
3.70 - 3.85 (m, 8 H) 4.07 - 4.19 (m, 3 H) 4.35 - 4.45 (m, 1 H) 4.97 (t, J = 5.6 Hz, 1 H) 5.80 (d, J = 8.6 Hz, 1 H) 6.34 (s, 1 H) 6.63 - 6.67 (m, 1 H)
6.80 - 6.87 (m, 2 H) 7.02 (dd, J = 8.1.2.0 Hz, 1 H) 7.14 (d, J = 2.0 Hz, 1 H) 7 , 20 (s, 1 H) 7.37 (d, J = 8.6 Hz, 1 H) 8.06 (s, 1 H) 8.15 (s, 1 H) 9.12 (s, 1 H )
[00189] LC / MS (LC-A method): Tr 3.20 min, MH ⁺ 634
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[00190] Melting point: 181Ό
8A Enantiomer:
[00191] ¹ H NMR (500 MHz, DMSO-cfe) δ ppm 3.09 - 3.26 (m, 2 H)
3.70 - 3.85 (m, 8 H) 4.08 - 4.20 (m, 3 H) 4.40 (td, J = 10.3, 6.5 Hz, 1 H)
4.99 (t I, J = 5.4 Hz, 1 H) 5.81 (d, J = 8.5 Hz, 1 H) 6.35 (s, 1 H) 6.66 (s, 1 H ) 6.83 (s, 1 H) 6.86 (d, J = 8.8 Hz, 1 H) 7.03 (dd, J = 8.2, 1.3 Hz, 1 H)
7.15 (d, J = 1.6 Hz, 1 H) 7.21 (s, 1 H) 7.38 (d, J = 8.2 Hz, 1 H) 8.07 (s, 1 H) 8.16 (s, 1 H) 9.13 (s, 1 H)
[00192] LC / MS (LC-B method): Tr 3.09 min, MH ⁺ 634
[00193] [a] _D ²⁰ : + 39.3 ° (c 0.3, DMF)
[00194] Chiral SFC (SFC-E method): Tr 3.39 min, MH ⁺ 634, 100% chiral purity.
8B Enantiomer:
[00195] ¹ H NMR (500 MHz, DMSO-cfe) δ ppm 3.09 - 3.27 (m, 2 H)
3.70 - 3.85 (m, 8 H) 4.06 - 4.19 (m, 3 H) 4.40 (td, J = 10.2, 6.6 Hz, 1 H)
4.99 (t I, J = 5.2 Hz, 1 H) 5.81 (d, J = 8.5 Hz, 1 H) 6.35 (s, 1 H) 6.66 (s, 1 H ) 6.83 (s, 1 H) 6.86 (d, J = 8.8 Hz, 1 H) 7.03 (dd, J = 8.2, 1.6 Hz, 1 H)
7.15 (d, J = 1.6 Hz, 1 H) 7.21 (s, 1 H) 7.38 (d, J = 8.2 Hz, 1 H) 8.07 (s, 1 H) 8.16 (s, 1 H) 9.13 (s, 1 H)
[00196] LC / MS (LC-A method): Tr 3.07 min, MH ⁺ 634
[00197] [a] _D ²⁰ : -44.4 ° (c 0.295, DMF)
[00198] Chiral SFC (SFC-E method): Tr 5.69 min, MH ⁺ 634, 100% chiral purity.
Example 9: synthesis of 2- (4-chloro-2- (2-hydroxyethoxy) phenyl) -1- (5-fluoro6 (trifluoromethoxy) indol i n-1 -yl) -2 - ((3-methoxy- 5- (1 H-1,2,4-triazol-1 yl) phenyl) amino) ethanone (Compound 9) and chiral separation in Enantiomers 9A and 9B.
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Synthesis of intermediate 9a:
[00199] A solution of 4-bromo-2-fluor-1 (trifluoromethoxy) benzene [CAS 105529-58-6] (98.7 g, 381.1 mmol) in concentrated H2SO4 (98%, 200 ml_) was cooled to 0 Ό with an ice bath. KNO3 (43.0 g; 425.3 mmol) was added portion by portion. After the addition, the ice bath was removed and the mixture was stirred at room temperature for 16 h. The reaction mixture was poured into ice water (2 L) while stirring. The mixture was extracted with CH2 Cl2 (3 x 500 ml). The combined organic layers were washed with a saturated aqueous solution of NaHCOs (2 x 500 ml), saturated aqueous sodium chloride solution (500 ml), dried over MgSO4, filtered and concentrated under reduced pressure to provide 1-bromo-5- fluorine-2-nitro-4- (trifluoromethoxy) benzene 9a (117.2 g), which was used in the next step without further purification.
Synthesis of intermediate 9b:
[00200] To a stirred suspension of 1-bromo-5-fluor-2-nitro-4 (trifluoromethoxy) benzene 9a (70.0 g, 230 mmol) and NH4CI (123.2 g, 2.30
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58/109 mol) in iPrOH (1 L) and water (330 ml), reducing iron powder (64.3 g, 1.15 mol) was added under N2 atmosphere. The reaction mixture was stirred at 60 Ό for 16 h. The reaction mixture was diluted with EtOAc (1L) and filtered through Celite®. The filtrate was concentrated under reduced pressure. The residue was partitioned between EtOAc (1 L) and water (800 ml). The layers were separated and the organic phase was washed with brine (1 L), dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by distillation under reduced pressure (oil pump, b.p. 60-64 Ό). 2-Bromo4-fluoro-5- (trifluoromethoxy) aniline 9b (47.3 g) was obtained as a yellow oil.
Synthesis of intermediate 9c:
[00201] To a mixture of 2-bromo-4-fluor-5- (trifluoromethoxy) aniline 9b (18.4 g, 67.2 mmol), ethinyl (trimethyl) silane (19.9 g, 202.4 mmol, 28.00 ml_) in EtaN (300 ml_) Cul (1.28 g, 6.72 mmol) and Pd (PPh3) 2Cl2 (2.40 g, 3.42 mmol) were added. The reaction mixture was heated under an atmosphere of N2 at 90 Ό for 16 h. After cooling to room temperature, the mixture was diluted with MTBE (300 ml) and filtered through Celite®. The filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (ISCO®, 220 g SepaFlash® Silica Flash Column, eluent: gradient from 0 to 5% EtOAc in petroleum ether @ 100 mL / min). 4-Fluor-5 (trifluoromethoxy) -2 - ((trimethylsilyl) ethynyl) aniline 9c (16.1 g, 90% purity) was obtained as a brown oil.
Synthesis of intermediate 9d:
[00202] A mixture of 4-fluor-5- (trifluoromethoxy) -2 ((trimethylsilyl) ethynyl) aniline 9c (16.1 g, 55.3 mmol) and tBuOK (18.6 g, 165.8 mmol) in NMP (220.00 mL) was heated to 90 O for 16 h under N2 atmosphere. After cooling to room temperature, the reaction mixture was poured into ice water (1 L) and extracted with MTBE (3 x
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300 ml). The combined organic phases were washed with water (2 x 200 ml), saturated aqueous sodium chloride solution (300 ml), dried over MgSCU, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (ISCO®, 120 g SepaFlash® Silica Flash Column, eluent: 0 to 5% EtOAc gradient in petroleum ether at 85 mL / min) to give 5-fluorine-6 (trifluoromethoxy)) - 1 H-indole 9d (11 g) as a dark green oil. The residue was combined with another fraction (total amount = 17.2 g) and then purified by distillation under reduced pressure (oil pump, bp 60 ~ 64 Ό) to provide 5-fluorine-6- (trifluoromethoxy) -1 H -indole 9d (14.7 g, 95% purity) as a colorless oil.
Synthesis of intermediate 9e:
[00203] At 0 O, BHs-Pindine (13.8 ml_, 136.9 mmol) was added dropwise to a solution of 5-fluorine-6- (trifluoromethoxy) -1 H-indole 9d (6 g , 27.4 mmol) in EtOH (40 ml). 6 N HCI (90 ml) was added dropwise while maintaining the temperature below 10 Ό. The mixture was stirred at 0 Ό for 2 h. Water (100 ml) was added and the mixture was basified to pH 8-9 with a concentrated solution of NaOH in water (the reaction temperature was kept below 20 Ό). The mixture was extracted with CH2 Cl2. The organic layer was washed with water, dried over MgSCU, filtered and the solvent evaporated under reduced pressure. Toluene was added and the solution was concentrated under reduced pressure to give 5.52 g of 5-fluorine-6- (trifluoromethoxy) indoline 9e. The compound was used in the next reaction step without further purification.
Synthesis of intermediate 9f:
[00204] A mixture of 5-fluor-6- (trifluoromethoxy) indoline 9e (169 mg, 0.76 mmol), acid 2- (2- (2 - ((ether-butyldimethylsilyl) oxy) ethoxy) -4chlorophenyl) 2 - ((3-methoxy-5- (1 H-1,2,4-triazol-1-yl) phenyl) amino) acetic 5d (407 mg, 0.76 mmol), HATU (435 mg, 1.15 mmol) and di
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60/109 isopropylethylamine (379 μΙ_, 2.29 mmol) in DMF (3.9 ml_) was stirred at room temperature for 2 h. The mixture was diluted with water. The resulting gummy material was removed with EtOAc. The organic solution was washed with a 10% solution of K2CO3 in water, water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 μηπ, 24 g, heptane / EtOAc 70/30). The pure fractions were combined and the solvent concentrated under reduced pressure to give 2- (2- (2 - ((etherbutyldimethylsilyl) oxy) ethoxy) -4-chlorophenyl) -1- (5-fluor-6 (trifl uoromethoxy) i ndol i n-1-yl) -2 - ((3-methoxy-5- (1 H-1,2,4-triazol-1 yl) phenyl) amino) ethanone 9f (257 mg).
Synthesis of Compound 9 and chiral separation in Enantiomers 9A and 9B:
[00205] Under a flow of N2, at 5 Ό, 4 M HCI in dioxane (873 μΙ_, 3.49 mmol) was added dropwise to a solution of 2- (2- (2 - ((tercbutyldimethylsilyl) oxy) ethoxy) -4-chlorophenyl) -1 - ((5-fluor-6 (trifluoromethoxy) i ndol i n-1 -yl) -2 - ((3-methoxy-5- (1 H-1,2,4 -triazol-1 yl) phenyl) amino) ethanone 9f (257 mg, 0.35 mmol) in MeOH (4 ml) The reaction was stirred at room temperature for 1 h. The mixture was cooled to 0 Ό, basified with a 10% aqueous solution of K2CO3 and extracted with EtOAc. The organic layer was separated, washed with water, dried over MgSO4, filtered, and the solvent concentrated under reduced pressure. The residue was purified by flash chromatography on silica (15-40 μηπ, 12 g, C ^ Cb / MeOH 98.5 / 1.5). The pure fractions were combined and the solvent was concentrated under reduced pressure to give 2- (4-chloro-2- (2 -hydroxyethoxy) phenyl) -1- (5-fluor-6 (trifluoromethoxy) indol i n-1 -yl) -2 - ((3-methoxy-5- (1 H-1,2,4-triazole 1 il) phenyl) amino) ethanone 9 (210 mg) as a racemate. o (17 mg) was further purified via reverse phase chromatography (Stationary phase: YMC-actus Triart-C18 10 pm 30 x 150mm, Phase
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61/109 mobile: 50% gradient of 0.2% NH4HCO3, 50% CH3CN to 0% 0.2% NH4HCO3, 100% CH3CN) to give 7 mg. The residue was solidified by lyophilization from a solvent mixture of CH3CN (1 ml) and water (4 ml).
[00206] Compound 9 enantiomers (190 mg) were separated by Preparative Chiral SFC (Stationary phase: Chiralpak® AD-H 5 pm 250 x 30 mm, mobile phase: 65% CO2, 35% EtOH (+0.3 % iPrNFE)). The first eluted enantiomer (58 mg) was dissolved in CH 3 CN (2 ml), water (8 ml) was added and the mixture was lyophilized to give the 9A enantiomer (58 mg) as a powder. The second eluted enantiomer (59 mg) was dissolved in CH 3 CN (2 ml), water (8 ml) was added and the mixture was lyophilized to give the 9B enantiomer (59 mg) as a powder.
Compound 9:
[00207] ¹ H NMR (500 MHz, DMSO-c / ₆ ) δ ppm 3.13 - 3.30 (m, 2 H)
3.72 (s, 3H) 3.70 - 3.80 (m, 2 H) 4.06 - 4.22 (m, 3 H) 4.42 (td, J = 10.4,
6.3 Hz, 1 H) 4.97 (s I, 1 H) 5.82 (d, J = 8.5 Hz, 1 H) 6.34 (s, 1 H) 6.66 (t, J = 1.9 Hz, 1 H) 6.82 (s, 1 H) 6.87 (d, J = 8.2 Hz, 1 H) 7.03 (dd, J = 8.2, 1.9 Hz , 1 H) 7.15 (d, J = 2.2 Hz, 1 H) 7.36 (d, J = 8.2 Hz, 1 H) 7.45 (d, J = 9.8 Hz, 1 H) 8.14-8.18 (m, 2 H)
[00208] LC / MS (LC-A method): Tr 3.32 min, MH ⁺ 622
9A Enantiomer:
[00209] ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 3.12 - 3.28 (m, 2 H)
3.72 (s, 3H) 3.69 - 3.82 (m, 2 H) 3.99 - 4.22 (m, 3 H) 4.42 (td, J = 10.0,
6.8 Hz, 1 H) 4.96 (t, J = 5.3 Hz, 1 H) 5.81 (d, J = 9.1 Hz, 1 H) 6.34 (s, 1 H)
6.66 (t, J = 1.8 Hz, 1 H) 6.80 - 6.88 (m, 2 H) 7.02 (dd, J = 8.1, 2.0 Hz, 1 H) 7 , 15 (d, J = 2.0 Hz, 1 H) 7.36 (d, J = 8.6 Hz, 1 H) 7.45 (d, J = 10.1 Hz, 1 H) 8.13 - 8.18 (m, 2 H) 9.13 (s, 1 H)
[00210] LC / MS (LC-B method): Tr 3.17 min, MH ⁺ 622
[00211] [a] _D ²⁰ : -35.1 ° (c 0.276, DMF)
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[00212] Chiral SFC (SFC-B method): Tr 2.75 min, MH ⁺ 622, 100% chiral purity.
9B Enantiomer:
[00213] ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 3.12 - 3.28 (m, 2 H)
3.72 (s, 3H) 3.69 - 3.82 (m, 2 H) 3.99 - 4.22 (m, 3 H) 4.42 (td, J = 10.0,
6.8 Hz, 1 H) 4.96 (t, J = 5.3 Hz, 1 H) 5.81 (d, J = 9.1 Hz, 1 H) 6.34 (s, 1 H)
6.66 (t, J = 1.8 Hz, 1 H) 6.80 - 6.88 (m, 2 H) 7.02 (dd, J = 8.1, 2.0 Hz, 1 H) 7 , 15 (d, J = 2.0 Hz, 1 H) 7.36 (d, J = 8.6 Hz, 1 H) 7.45 (d, J = 10.1 Hz, 1 H) 8.13 - 8.18 (m, 2 H) 9.13 (s, 1 H)
[00214] LC / MS (LC-B method): Tr 3.17 min, MH ⁺ 622
[00215] [a] _D ²⁰ : + 32.3 ° (c 0.254, DMF)
[00216] Chiral SFC (SFC-B method): Tr 3.75 min, MH ⁺ 622, chiral purity 100%.
Example 10: synthesis of 2- (4-chloro-2- (2-hydroxyethoxy) phenyl) -2 - ((3-methoxy5- (1 H-1,2,4-triazol-1-i I) pheni I) ami no) -1 - (4-methyl-6- (trifluoromethoxy) indolin1-yl) ethanone (Compound 10) and chiral separation in Enantiomers 10A and 10B.

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Synthesis of intermediate 10a:
[00217] To a solution of 2-methyl-4- (trifluoromethoxy) aniline [CAS 86256-59-9] (10.0 g, 52.3 mmol) in dioxane (20 ml) was added trifluoroacetic anhydride (8 ml, 57.2 mmol). The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure. The residue was partitioned between EtOAc and 1 N HCI. The phases were separated. The organic phase was washed with a saturated solution of NaHCOs in water, H2O and a saturated aqueous solution of sodium chloride, dried over Na2SO4, filtered and concentrated under reduced pressure to give 14.7 g of 2,2,2-trifluorine. -A / (2-methyl-4- (trifluoromethoxy) phenyl) acetamide 10a as a white powder. The compound was used in the next step without further purification.
Synthesis of intermediate 10c:
[00218] To acetic anhydride (11.4 ml_, 61.1 mmol), cooled to 0 Ό 70% nitric acid (3.9 ml_) was added dropwise. 2,2,2-TrifluorA / - (2-methyl-4- (trifluoromethoxy) phenyl) acetamide 10a (5 g, 17.4 mmol) was added in portions and the reaction mixture was heated at 55 ° for 12 h. After cooling to room temperature, the reaction mixture was diluted with EtOAc and washed with H2O. The organic phase was washed with saturated aqueous sodium chloride solution, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was dissolved in methanol (46 ml). 2M K2CO3 (23 ml, 46 mmol) was added and the reaction mixture was heated to 70 Ό for 4 h. More 2M K2CO3 (10 ml, 20 mmol) was added and the reaction mixture was heated at 70 ° for 12 h. The reaction mixture was partially concentrated under reduced pressure to remove methanol. The residue was extracted with EtOAc. The organic phase was washed with H2O and saturated aqueous sodium chloride solution, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel using a gradient of EtOAc (20% to 50%) in heptane to
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64/109 give 3.6 g of 2-methyl-6-nitro-4- (trifluoromethoxy) aniline 10c as a yellow solid.
Synthesis of intermediate 10d:
[00219] To a solution of 2-methyl-6-nitro-4- (trifluoromethoxy) aniline 10c (1.8 g, 7.69 mmol) in acetic acid (10.9 ml) was added dropwise a solution of sodium nitrite (0.806 g, 11.7 mmol) in H2SO4 / H2O (ml_ 2, 1/1). The reaction mixture was stirred at room temperature for 30 min. H2O (22 ml) and urea (0.802 g,
13.4 mmol). After 10 min at room temperature, a solution of potassium iodide (1.7 g, 10.2 mmol) in H2O (11 ml) was added dropwise. The reaction mixture was stirred at room temperature for 30 min. The yellow solid was removed by filtration, washed with H2O and dried to give 2.4 g of 2-iodo-1-methyl-3-nitro-5- (trifluoromethoxy) benzene 10d.
Synthesis of intermediate 10e:
[00220] To a solution of 2-iodo-1-methyl-3-nitro-5 (trifluoromethoxy) benzene 10d (3.5 g, 10.0 mmol) in EtOH (30 ml_) was added a solution of NH4CI (2 , 7 g, 49.9 mmol) in H2O (30 ml). The reaction mixture was heated to 50 Ό. Iron (2.6 g, 46.9 mmol) was added and the reaction mixture was heated to reflux for 40 min. After cooling to room temperature, the reaction mixture was filtered through celite®. The solids were washed with EtOH. The filtrate was partially concentrated under reduced pressure to remove EtOH. The residue was partitioned between EtOAc and a saturated solution of NaHCO without water. The phases were separated. The organic phase was washed with H2O and saturated aqueous sodium chloride solution, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel using a gradient of EtOAc (0% to 25%) in heptane to give 2.9 g of 2-iodo-3-methyl-5- (trifluoromethoxy) aniline 10e as an oil yellow.
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Synthesis of intermediate 10f:
[00221] A solution of 2-iodo-3-methyl-5- (trifluoromethoxy) aniline 10e (2.9 g, 9.1 mmol) in triethylamine (23 ml) was degassed with argon for 15 min. Dichlorobis (triphenylphosphine) palladium (ll) (0.327 g, 0.47 mmol), copper (l) iodide (0.164 g, 0.86 mmol) and trimethylsilylacetylene (1.8 ml_, 13.1 mmol) were added . The reaction mixture was heated to 65 ° C for 12 h. After cooling to room temperature, the reaction mixture was diluted with Η ₂ Ο and extracted with EtOAc (3x). The organic phases were combined, washed with H2O and saturated aqueous sodium chloride solution, dried with Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel using a gradient of EtOAc (0% to 20%) in heptane to give 2.6 g of 3-methyl-5- (trifluoromethoxy) -2 ((trimethylsily) ethynyl) aniline 10f as an orange oil.
Synthesis of intermediate 10g:
[00222] To a solution of 3-methyl-5- (trifluoromethoxy) -2 ((trimethylsilyl) ethynyl) aniline 10f (2.7 g, 9.3 mmol) in NMP (27 ml_) was added tBuOK (3.1 g, 27.8 mmol). The reaction mixture was heated to 80 O for 4 h. After cooling to room temperature, the reaction mixture was diluted with H2O and extracted with EtOAc (2x). The organic phases were combined, washed with H2O and saturated aqueous sodium chloride solution, dried with Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel using a gradient of EtOAc (0% to 20%) in heptane to give 1.7 g of 4-methyl-6- (trifluoromethoxy) -1 Hindol 10g as an orange oil.
Synthesis of the intermediate 10h:
[00223] At 0 O, BH 3-Pyridine (1.2 ml, 11.6 mmol) was added dropwise to a solution of 4-methyl-6- (trifluoromethoxy) -1 H-indole 10g (0.5 g, 2.32 mmol) in EtOH (3 ml). 6N HCI (6 ml_) was slowly added
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66/109 drop by drop while maintaining the reaction temperature below 10 Ό. The mixture was stirred at 0 Ό for 3 h. Water (12 ml) was added and the mixture was basified to pH 8-9 with a concentrated solution of NaOH in water (the reaction temperature was kept below 20 Ό). The mixture was extracted with EtOAc. The organic layer was washed with water, dried over MgSO4, filtered, and the solvent evaporated under reduced pressure. Toluene was added and the solution was concentrated under reduced pressure to give 450 mg of 4-methyl-6 (trifluoromethoxy) indoline 10h.
Synthesis of intermediate 10i:
[00224] A mixture of 4-methyl-6- (trifluoromethoxy) indoline 10h (163 mg, 0.75 mmol), 2- (2- (2 - ((ether-butyldimethylsilyl) oxy) ethoxy) -4chlorophenyl) - 2 - ((3-methoxy-5- (1 H-1,2,4-triazol-1-yl) phenyl) amino) acetic 5d (400 mg, 0.75 mmol), HATU (428 mg, 1.13 mmol) and diisopropylethylamine (372 μΙ_, 2.25 mmol) in DMF (3.8 ml_) was stirred at room temperature for 2 h. The mixture was diluted with water. The resulting gummy material was removed with EtOAc. The organic layer was washed with a 10% solution of K2CO3 in water, water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. The compound was purified by flash chromatography on silica gel (15-40 pm, 24 g, heptane / EtOAc gradient from 80/20 to 70/30). The pure fractions were combined and the solvent was concentrated under reduced pressure to give 2- (2- (2 - ((force-butyldimethylsilyl) oxy) ethoxy) -4-chlorophenyl) -2 ((3-methoxy-5- (1 H-1,2,4-triazol-1-i I) phenyl I) amine) -1 - (4-methyl-6 (trifluoromethoxy) indolin-1-yl) ethanone 10i (226 mg).
Synthesis of Compound 10 and chiral separation in the 10A and 10B Enantiomers:
[00225] Under a flow of N2, at 5 Ό, HCI 4 M in dioxane (772 μΙ_,
3.1 mmol) was added dropwise to a solution of 2- (2- (2 - ((iercbuti Idi meti Isi I i I) ox) ethoxy) -4-chloropheni I) -2 - ((3-methoxy -5- (1 H-1,2,4-triazole-1
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67/109 iI) pheniI) amino) -1 - (4-methyl-6- (trifluoromethoxy) indoliη-1-i) ethanone 10i (226 mg, 0.31 mmol) in MeOH (4 mL). The reaction was stirred at room temperature for 1 h. The mixture was cooled to 0 ° C, basified with 10% aqueous K2CO3 solution and extracted with EtOAc. The organic layer was separated, washed with water, dried over MgSO4, filtered, and the solvent concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 12 g, CHkCb / MeOH 98.5 / 1.5). A second purification was performed using reverse phase chromatography (Stationary phase: YMC-actus Triart-C18 10 pm 30 x 150mm, Mobile phase: 55% gradient of NH4HCO3 0.2%, 45% CH3CN to 0% NH4HCO3 0.2%, 100% CH3CN). The pure fractions were combined and the solvent was concentrated under reduced pressure to give 2- (4-chloro-2- (2-hydroxyethoxy) phenyl) 2 - ((3-methoxy-5- (1 H-1,2,4 -triazole-1 -i I) pheni I) amine) -1 - (4-methyl-6 (trifluoromethoxy) indolin-1-yl) ethanone 10 (78 mg) as a racemate. A small fraction was solidified by trituration with CHsCN / diisopropyl ether, to obtain compound 10 (9 mg). The remaining amount was used to separate the Compound 10 Enantiomers via Preparative Chiral SFC (stationary phase: Chiralpak® AD-H 5 pm 250 x 30 mm, mobile phase: 60% CO2, 40% iPrOH). The first eluted enantiomer (27 mg) was dissolved in CH3CN (2 ml), water (8 ml) was added and the mixture was lyophilized to give the 10A enantiomer (25 mg) as a powder. The second eluted enantiomer (28 mg) was dissolved in CH3CN (2 ml), water (8 ml) was added and the mixture was lyophilized to give the 10B enantiomer (22 mg) as a powder.
Compound 10:
[00226] ¹ H NMR (500 MHz, DMSO-c / ₆ ) δ ppm 2.21 (s, 3 H) 3.01 -
3.13 (m, 2 H) 3.72 (s, 3H) 3.70 - 3.82 (m, 2 H) 4.08 - 4.22 (m, 3 H) 4.40 - 4.48 (m, 1 H) 4.98 (t, J = 5.4 Hz, 1 H) 5.82 (d, J = 8.5 Hz, 1 H) 6.35 (s I, 1 H) 6, 66 (s, 1 H) 6.82 - 6.90 (m, 3 H) 7.02 (dd, J = 8.4, 1.7 Hz, 1 H)
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7.15 (d, J = 1.6 Hz, 1 Η) 7.36 (d, J = 8.2 Hz, 1 Η) 7.89 (s, 1 Η) 8.16 (s, 1 Η) 9.13 (s, 1 Η)
[00227] LC / MS (LC-B method): Tr 3.26 min, MH ⁺ 618
10 A enantiomer:
[00228] ¹ H NMR (500 MHz, DMSO-c / ₆ ) δ ppm 2.21 (s, 3 H) 3.01 -
3.14 (m, 2 H) 3.72 (s, 3H) 3.71 - 3.83 (m, 2 H) 4.07 - 4.22 (m, 3 H) 4.44 (td, J = 10.2, 6.5 Hz, 1 H) 4.98 (t, J = 5.5 Hz, 1 H) 5.82 (d, J = 8.5 Hz, 1 H)
6.35 (s, 1 H) 6.66 (t, J = 1.9 Hz, 1 H) 6.81 - 6.90 (m, 3 H) 7.02 (dd, J = 8.4, 2.1 Hz, 1 H) 7.15 (d, J = 1.9 Hz, 1 H) 7.36 (d, J = 8.2 Hz, 1 H) 7.89 (s, 1 H) 8 , 16 (s, 1 H) 9.13 (s, 1 H)
[00229] LC / MS (LC-B method): Tr 3.26 min, MH ⁺ 618
[00230] [a] _D ²⁰ : -38.4 ^o (c 0.279, DMF)
[00231] Chiral SFC (SFC-F method): Tr 1.41 min, MH ⁺ 618, 100% chiral purity.
10B Enantiomer:
[00232] ¹ H NMR (500 MHz, DMSO-c / ₆ ) δ ppm 2.21 (s, 3 H) 3.00 -
3.14 (m, 2 H) 3.72 (s, 3H) 3.71 - 3.82 (m, 2 H) 4.06 - 4.23 (m, 3 H) 4.44 (td, J = 10.1, 6.9 Hz, 1 H) 4.95 - 5.02 (m, 1 H) 5.82 (d, J = 8.5 Hz, 1 H)
6.35 (s, 1 H) 6.66 (t, J = 1.7 Hz, 1 H) 6.82 - 6.90 (m, 3 H) 7.02 (dd, J = 8.5, 1.9 Hz, 1 H) 7.15 (d, J = 1.9 Hz, 1 H) 7.36 (d, J = 8.2 Hz, 1 H) 7.89 (s, 1 H) 8 , 16 (s, 1 H) 9.13 (s, 1 H)
[00233] LC / MS (LC-B method): Tr 3.26 min, MH ⁺ 618
[00234] [a] _D ²⁰ : + 37.5 ° (c 0.299, DMF)
[00235] Chiral SFC (SFC-F method): Tr 1.82 min, MH ⁺ 618, 100% chiral purity.
Example 11: 4- (5-chloro-2- (1 - ((3-methoxy-5- (1 H-1,2,4triazol-1-yl) phenyl) amino) -2-oxo-2 acid synthesis - (6- (trifluoromethyl) indolin-1 yl) ethyl) phenoxy) butanoic (Compound 11) and chiral separation in Enantiomers 11A and 11B.
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·: $ Α S S.
Synthesis of intermediate 11a:
[00236] To a suspension of 2- (4-chloro-2-hydroxyphenyl) acetate 4b (8.5 g, 39.6 mmol) and CS2CO3 (25.8 g, 79.2 mmol) in DMF (130 ml_) at 10 °, tert-butyl 4-bromobutanoate [CAS 110611-91-1] (7 ml, 39.6 mmol) was added dropwise. The mixture was stirred at room temperature overnight. The mixture was diluted with EtOAc and water. The layers were decanted. The organic layer was washed with water, dried over MgSCU, filtered, and the solvent was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 120 g, heptane / EtOAc 90/10). The pure fractions were combined and concentrated to dryness to give tert-butyl 4- (5chloro-2- (2-ethoxy-2-oxoethyl) phenoxy) butanoate 11a (12.7 g).
Synthesis of intermediate 11b:
[00237] A flask was charged with 1.5 M LiHMDS in THF (23.5 ml_, 35.3 mmol) under a flow of N2 and the solution was cooled to -78 Ό. A solution of tert-butyl 4- (5-chloro-2- (2-ethoxy-2-oxoethyl) phenoxy) butanoate 11a (6.3 g, 17.6 mmol) in THF (60 ml) was added dropwise. drop and the mixture was stirred at -78 Ό for 15 min. Clo added
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70/109 rotrimethylsilane (3.6 ml, 28.3 mmol). After 15 min at -78 O, A / -bromosuccinimide (3.77 g, 21.2 mmol) in THF (40 ml) was added and the mixture was stirred at -70 O for 1 h. The reaction was quenched with water and extracted with EtOAc. The organic layer was separated, washed with water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure to produce tert- 4- (2- (1-bromo-2-ethoxy-2-oxoethyl) -5chlorophenoxy) butanoate buty 11b (7.6 g). The compound was used directly in the next reaction step without further purification.
Synthesis of intermediate 11c:
[00238] To a solution of tert-butyl 4- (2- (1-bromo-2-ethoxy-2-oxoethyl) -5chlorophenoxy) butanoate 11b (7.6 g, 17.4 mmol) in CH3CN (140 ml_ ) at room temperature, diisopropylethylamine (4.8 ml_, 27.9 mmol) and 3-methoxy-5- (1 H-1,2,4-triazol-1-yl) aniline [CAS 1220630-56 -7] (4 g, 20.9 mmol). The mixture was stirred at 65 ° for 24 h. The mixture was diluted with EtOAc, washed with 0.5 N HCI (twice) and water. The organic layer was dried over MgSO4, filtered, and the solvent was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 μηπ, 120 g, heptane / EtOAc 85/15 to 70/30). The pure fractions were combined and concentrated to dryness to give 4- (5-chloro-2- (2-ethoxy-1 - ((3-methoxy-5- (1 H-1,2,4triazol-1-yl) tert -butyl phenyl) amino) -2-oxoethyl) phenoxy) butanoate 11c (6.6 g).
Synthesis of intermediate 11 d:
[00239] A mixture of 4- (5-chloro-2- (2-ethoxy-1 - ((3-methoxy-5- (1 H1,2,4-triazol-1-yl) phenyl) amino) -2 -oxoethyl) phenoxy) tert-butyl 11ano butanoate (6.6 g, 12.1 mmol) and lithium hydroxide monohydrate (1.52 g,
36.3 mmol) in THF / water (1/1) (160 ml) was stirred at room temperature for 1 h. The mixture was diluted with water. The aqueous solution was slowly acidified with 3N HCI and extracted with EtOAc. The organic layers were dried over MgSO4, filtered and the solvent was
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71/109 concentrated under reduced pressure to give 2- (2- (4- (tert-butoxy) 4-oxobutoxy) -4-chlorophenyl) -2 - ((3-methoxy-5- (1 H-1,2 , 4-triazol-1 yl) phenyl) amino) acetic 11 d (6.2 g). The impure product was used without further purification in the next step.
Synthesis of intermediate 11e:
[00240] A mixture of 6- (trifluoromethyl) indoline [CAS 181513-29-1] (290 mg, 1.55 mmol), 2- (2- (4 - ((tert-butoxy) -4-oxobutoxy) acid -4chlorophenyl) -2 - ((3-methoxy-5- (1 H-1,2,4-tri-azol-1-yl) phenyl) amino) acetic 11 d (800 mg, 1.55 mmol), HATU ( 880 mg, 2.32 mmol) and diisopropylethylamine (770 μΙ_, 4.64 mmol) in DMF (30 ml_) was stirred at room temperature for 2 h. The mixture was diluted with water. The precipitate was removed by filtration, washed with water and taken up with EtOAc. The organic layer was washed with water, dried over MgSO4, filtered, and the solvent was concentrated under reduced pressure. The residue was crystallized from diisopropyl ether to give 4- (5-chloro-2 - (1 (((3-methoxy-5- (1 H-1,2,4-triazol-1-yl) phenyl) amino) -2-oxo-2- (6 (trifluoromethyl) indolin-1-yl) ethyl ) tert -butyl phenoxy) butanoate 11e (500 mg).
Synthesis of Compound 11 and chiral separation in 11A and 11B Enantiomers:
[00241] A solution of 4- (5-chloro-2- (1 - ((3-methoxy-5- (1 H-1,2,4triazol-1-yl) phenyl) amino) -2-oxo-2 - tert -butyl (6- (trifluoromethyl) indolin-1 yl) ethyl) phenoxy) butanoate 11e (500 mg, 0.729 mmol) in 4M HCI in dioxane (5 ml) was stirred at 5 ° for 3 h at room temperature 8 h. The precipitate was removed by filtration, washed with dioxane / diisopropyl ether and dried to give 4- (5-chloro-2- (1 - ((3-methoxy-5- (1 H-1,2,4-triazole- 1 -yl) phenyl) amino) -2-oxo-2- (6 (trifluoromethyl) indolin-1-yl) ethyl) phenoxy) butanoic 11 (430 mg, 0.4 H2O (determined by titration)) as a racemate.
[00242] The Compound 11 Enantiomers were separated by
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72/109 Preparative Chiral SFC (stationary phase: Chiralpak® AD-H 5 pm 250 x 30 mm, Mobile phase: 65% CO2, 35% EtOH). The first eluted enantiomer (80 mg) was solidified by titration with petroleum ether / diisopropyl ether to give the 11A Enantiomer (65 mg). The second eluted enantiomer (126 mg) was solidified by titration with petroleum ether / diisopropyl ether to give the 11B Enantiomer (110 mg).
Compound 11:
[00243] ¹ H NMR (400 MHz, DMSO-c / ₆ ) δ ppm 1.94 - 2.02 (m, 2 H)
2.31 - 2.42 (m, 2 H) 3.15 - 3.35 (m, 2 H) 3.73 (s, 3 H) 4.01 - 4.24 (m, 3 H) 4, 22 - 4.49 (m, 1 H) 5.73 (s, 1 H) 6.34 (s, 1 H) 6.67 (s, 1 H) 6.82 (s, 1 H) 7.02 (dd, J = 8.1,2.0 Hz, 1 H) 7.13 (d, J = 2.0 Hz, 1 H) 7.33 (d, J = 8.0 Hz, 1 H) 7 , 38 (d, J = 7.6 Hz, 1 H) 7.45 (d, J = 7.6 Hz, 1 H) 8.16 (s, 1 H) 8.38 (s, 1 H) 9 , 15 (s, 1 H)
[00244] LC / MS (LC-A method): Tr 2.70 min, MH ⁺ 630
11 A enantiomer:
[00245] ¹ H NMR (500 MHz, DMSO-cfe) δ ppm 1.98 (s I, 2 H) 2.28 -
2.45 (m, 2 H) 3.13 - 3.29 (m, 2 H) 3.74 (s, 3 H) 4.02 - 4.17 (m, 3 H)
4.36 - 4.44 (m, 1 H) 5.74 (d I, J = 8.5 Hz, 1 H) 6.35 (s I, 1 H) 6.68 (s, 1 H)
6.80 - 6.88 (m, 2 H) 7.03 (d I, J = 7.9 Hz, 1 H) 7.14 (s, 1 H) 7.33 (d, J = 7.8 Hz, 1 H) 7.37 - 7.40 (m, 1 H) 7.46 (d I, J = 7.6 Hz, 1 H) 8.16 (s, 1 H) 8.39 (s, 1 H) 9.16 (s, 1 H) 12.13 (s I, 1 H)
[00246] LC / MS (LC-A method): Tr 2.79 min, MH ⁺ 630
[00247] [a] _D ²⁰ : -28.9 ° (c 0.26, DMF)
[00248] Chiral SFC (SFC-G method): Tr 3.31 min, MH ⁺ 630, chiral purity 100%.
11B Enantiomer:
[00249] ¹ H NMR (500 MHz, DMSO-cfe) δ ppm 1.91 - 2.04 (m, 2 H) 2.28 - 2.46 (m, 2 H) 3.16 - 3.30 ( m, 2 H) 3.73 (s, 3 H) 4.02 - 4.18 (m, 3 H) 4.35 - 4.44 (m, 1 H) 5.73 (d I, J = 8 , 5 Hz, 1 H) 6.34 (s I, 1 H) 6.68 (s, 1 H) 6.80 - 6.87 (m, 2 H) 7.02 (d I, J = 7, 9 Hz, 1 H) 7.13 (s, 1 H) 7.31
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7.41 (m, 2 Η) 7.45 (d I, J = 7.9 Hz, 1 Η) 8.16 (s, 1 Η) 8.38 (s, 1 Η) 9.16 (s, 1 Η) 12.13 (s I, 1 Η)
[00250] LC / MS (LC-A method): Tr 2.79 min, MH ⁺ 630
[00251] [a] _D ²⁰ : + 23.8 ° (c 0.29, DMF)
[00252] Chiral SFC (SFC-G method): Tr 4.32 min, MH ⁺ 630, 100% chiral purity.
Example 12: 4- (5-chloro-2- (1 - ((3-methoxy-5- (1 H-1,2,4triazol-1-yl) phenyl) amino) -2- (5- methoxy-6- (trifluoromethyl) indolin-1-yl) -2oxoethyl) phenoxy) butanoic (Compound 12) and chiral separation in Enantiomers 12A and 12B.

Synthesis of intermediate 12a:
[00253] A mixture of 5-methoxy-6- (trifluoromethylindoline 2c (630 mg, 2.9 mmol), 2- (2- (4 - ((tert-butoxy) -4-oxobutoxy) -4-chlorophenyl) -2 ((3-methoxy-5- (1 H-1,2,4-triazol-1-yl) phenyl) amino) acetic 11d (1.5 g, 2.9 mmol), HATU (1.65 g , 4.35 mmol) and diisopropylethylamine (1.45 ml, 8.7 mmol) in DMF (30 ml) was stirred at room temperature for 2 h. The mixture was diluted with water. The precipitate was removed by filtration, washed with water and taken up with EtOAc. The organic solution was washed with water, dried over MgSCU, filtered, and the solvent was concentrated
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74/109 under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 120 g, heptane / EtOAc 60/40). The pure fractions were combined and concentrated to dryness to give, after crystallization from ether / diisopropyl ether, 4- (5-chloro-2- (1 - (((3-methoxy-5- (1 H-1,2 , 4-triazol-1-yl) phenyl) amino) -2- (5-methoxy-6 (trifluoromethyl) indolin-1-yl) -2-oxoethyl) phenoxy) tert-butyl butanoate 12a (1.45 g) .
Synthesis of Compound 12 and chiral separation in Enantiomers 12A and 12B:
[00254] A solution of 4- (5-chloro-2- (1 - ((3-methoxy-5- (1 H-1,2,4triazol-1-yl) phenyl) amino) -2- (5- methoxy-6- (trifluoromethyl) indolin-1-yl) -2oxoethyl) phenoxy) tert-butyl butanoate 12a (1.45 g, 2.03 mmol) in 4M HCI in dioxane (12 ml) was stirred at 5 Ό per 3 h at room temperature for 12 h. The precipitate was removed by filtration, washed with dioxane / diisopropyl ether and dried to give crude Compound 12 (1.02 g). The smaller part (90 mg) was further purified by achiral SFC (Stationary phase: 2-Ethylpyridine 6 pm 150 x 21.2 mm, Mobile phase: 60% CO2, 40% iPrOH) to give, after solidification by trituration with CHaCN / diisopropyl ether, 4- (5-chloro-2 (1 - ((3-methoxy-5- (1 H-1,2,4-triazol-1-yl) phenyl) amino) -2- ( 5-methoxy-6 (trifluoromethyl) indolin-1-yl) -2-oxoethyl) phenoxy) butanoic 12 (70 mg) as a racemate. The remaining amount was used to separate the enantiomers.
[00255] Compound 12 enantiomers were separated by Preparative Chiral SFC (stationary phase: Chiralpak® AS-H 5 pm 250 x 20 mm, Mobile phase: 63% CO2, 37% iPrOH). The first eluted enantiomer (458 mg) was stirred in a mixture of 1 N HCI and EtOAc. The organic layer was separated, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. The residue was solidified by trituration with diisopropyl ether / petroleum ether to give 0 Enan
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75/109 thiomer 12A (270 mg). The second eluted enantiomer (405 mg) was stirred in a mixture of 1 N HCI and EtOAc. The organic layer was separated, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. The residue was solidified by trituration with diisopropyl ether / petroleum ether to give Enantiomer 12B (272 mg).
Compound 12:
[00256] ¹ H NMR (400 MHz, DMSO-c / ₆ ) δ ppm 1.95 - 2.04 (m, 2 H)
2.31 - 2.45 (m, 2 H) 3.15 - 3.28 (m, 2 H) 3.73 (s, 3 H) 3.84 (s, 3 H) 3.98
- 4.17 (m, 3 H) 4.33 - 4.41 (m, 1 H) 5.70 (d I, J = 8.6 Hz, 1 H) 6.33 (s, 1 H) 6 , 66 (s, 1 H) 6.77 - 6.83 (m, 2 H) 7.01 (d I, J = 8.6 Hz, 1 H) 7.12 (s, 1 H) 7.23 (s, 1 H) 7.33 (d, J = 8.6 Hz, 1 H) 8.15 (s, 1 H) 8.34 (s, 1 H) 9.13 (s, 1 H) 12 , 07 (s I, 1 H)
[00257] LC / MS (LC-A method): Tr 2.72 min, MH ⁺ 660
12A Enantiomer:
[00258] ¹ H NMR (500 MHz, DMSO-d ₆ ) δ ppm 1.94 - 2.05 (m, 2 H)
2.31 - 2.46 (m, 2 H) 3.16 - 3.31 (m, 2 H) 3.73 (s, 3 H) 3.85 (s, 3 H) 3.99
- 4.18 (m, 3 H) 4.38 (td, J = 10.2, 6.5 Hz, 1 H) 5.71 (d, J = 8.5 Hz, 1 H)
6.34 (s, 1 H) 6.67 (s, 1 H) 6.82 (s, 1 H) 6.83 (d, J = 9.5 Hz, 1 H) 7.02 (dd, J = 8.2, 1.6 Hz, 1 H) 7.13 (s, 1 H) 7.23 (s, 1 H) 7.33 (d, J = 8.2 Hz, 1 H) 8.16 (s, 1 H) 8.34 (s, 1 H) 9.15 (s, 1 H) 12.11 (s I, 1 H)
[00259] LC / MS (LC-A method): Tr 2.70 min, MH ⁺ 660
[00260] [a] _D ²⁰ : + 30.4 ° (c 0.257, DMF)
[00261] Chiral SFC (SFC-H method): Tr 3.71 min, MH ⁺ 660, chiral purity 100%.
12B Enantiomer:
[00262] ¹ H NMR (500 MHz, DMSO-d ₆ ) δ ppm 1.91 - 2.08 (m, 2 H)
2.32 - 2.44 (m, 2 H) 3.16 - 3.31 (m, 2 H) 3.73 (s, 3 H) 3.85 (s, 3 H) 3.99
- 4.17 (m, 3 H) 4.38 (td, J = 10.3, 6.6 Hz, 1 H) 5.71 (d, J = 8.5 Hz, 1 H)
6.34 (s, 1 H) 6.67 (s, 1 H) 6.79 - 6.85 (m, 2 H) 7.02 (d, J = 8.1 Hz, 1 H)
7.13 (s, 1 H) 7.23 (s, 1 H) 7.33 (d, J = 8.2 Hz, 1 H) 8.16 (s, 1 H) 8.34 (s,
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Η) 9.15 (s, 1 Η) 12.12 (s I, 1 Η)
[00263] LC / MS (LC-A method): Tr 2.70 min, MH ⁺ 630
[00264] [a] _D ²⁰ : -36.9 ° (c 0.287, DMF)
[00265] Chiral SFC (SFC-H method): Tr 5.91 min, MH ⁺ 660, chiral purity 100%.
Example 13: 4- (5-chloro-2- (1 - ((3-methoxy-5- (1H-1,2,4triazol-1-yl) phenyl) amino) -2-oxo-2- acid synthesis (6- (trifluoromethoxy) indolin-1yl) ethyl) phenoxy) butanoic (Compound 13) and chiral separation in Enantiomers 13A and 13B.

ΗΑΤΙΛ iPr-.NEt
DMFjs 2h

Enantomers 13A and 138
Synthesis of intermediate 13a:
[00266] A mixture of 6- (trifluoromethoxy) indoline [CAS 959235-951] (590 mg, 2.9 mmol), 2- (2- (4 - (((tert-butoxy) -4-oxobutoxy) -4-chlorophenyl acid ) -2 - ((3-methoxy-5- (1 / 7-1,2,4-tri azol-1-yl) phenyl) amino) acetic 11 d (1.5 g, 2.9 mmol), HATU (1.65 g, 4.35 mmol) and diisopropylethylamine (1.45 ml_, 8.7 mmol) in DMF (60 ml_) was stirred at room temperature for 12 h. The mixture was diluted with water. was removed by filtration, washed with water and taken up with EtOAc. The organic layer was washed with water, dried over MgSO4, filtered, and the solvent was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15- 40 pm, 120 g, heptane / EtOAc
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60/40). The pure fractions were combined and concentrated to dryness to give, after crystallization from ether / diisopropyl ether, 4 (5-chloro-2- (1 - (((3-methoxy-5- (1 H-1, 2,4-triazol-1-yl) phenyl) amino) -2-oxo-2- (6 (trifluoromethoxy) indolin-1-yl) ethyl) phenoxy) -butanoate tert-butyl 13a (1.05 g).
Synthesis of Compound 13 and chiral separation in 13A and 13B Enantiomers:
[00267] A solution of 4- (5-chloro-2- (1 - ((3-methoxy-5- (1H-1,2,4triazol-1-yl) phenyl) amino) -2-oxo-2- Tera-butyl (6- (trifluoromethoxy) indolin-1yl) ethyl) phenoxy) butanoate 13a (1.05 g, 1.50 mmol) in 4M HCI in dioxane (9.5 ml) was stirred at 5 ° for 3 h at room temperature for 12 h. The precipitate was removed by filtration, washed with dioxane / diisopropyl ether and dried to give 4- (5-chloro-2- (1 ((3-methoxy-5- (1 H-1,2,4-triazole acid) -1 -yl) phenyl) amino) -2-oxo-2- (6 (trifluoromethoxy) indolin-1-yl) ethyl) phenoxy) butanoic 13 (965 mg, 0.23 H2O (determined by titration)) as a racemate .
[00268] Compound 13 enantiomers were separated by Preparative Chiral SFC (stationary phase: Chiralpak® AS-H 5 pm 250 x 20 mm, Mobile phase: 80% CO2, 20% EtOH). The first eluted enantiomer (390 mg) was solidified by trituration with petroleum ether / diisopropyl ether to give the 13A Enantiomer (260 mg). The second eluted enantiomer (350 mg) was solidified by trituration with petroleum ether / diisopropyl ether to give the 13B Enantiomer (188 mg).
Compound 13:
[00269] ¹ H NMR (500 MHz, DMSO-c / ₆ ) δ ppm 1.94 - 2.03 (m, 2 H)
2.33 - 2.41 (m, 2 H) 3.10 - 3.24 (m, 2 H) 3.73 (s, 3 H) 4.04 - 4.26 (m, 3 H) 4, 39 (td, J = 10.2, 6.5 Hz, 1 H) 5.71 (s, 1 H) 6.34 (s, 1 H) 6.67 (s, 1 H)
6.81 (s, 1 H) 6.99 - 7.07 (m, 2 H) 7.13 (d, J = 1.6 Hz, 1 H) 7.27-7.39 (m, 2 H ) 8.04 (s, 1 H) 8.16 (s, 1 H) 9.15 (s, 1 H) 12.08 (s I, 1 H)
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[00270] LC / MS (LC-B method): Tr 2.73 min, MH ⁺ 646
13A Enantiomer:
[00271] ¹ H NMR (500 MHz, DMSO-d ₆ ) δ ppm 1.93 - 2.03 (m, 2 H)
2.34 - 2.47 (m, 2 H) 3.13 - 3.21 (m, 2 H) 3.73 (s, 3 H) 4.03 - 4.17 (m, 3 H) 4, 39 (td, J = 10.1, 6.6 Hz, 1 H) 5.72 (d, J = 8.5 Hz, 1 H) 6.35 (s, 1 H) 6.68 (s, 1 H) 6.82 (s, 1 H) 6.87 (d, J = 8.2 Hz, 1 H) 7.02 (t, J = 8.4 Hz, 1 H) 7.13 (s, 1 H) 7.27 - 7.39 (m, 2 H) 8.04 (s, 1 H) 8.16 (s, 1 H) 9.15 (s, 1 H) 12.10 (s I, 1 H)
[00272] LC / MS (LC-A method): Tr 2.83 min, MH ⁺ 646
[00273] [a] _D ²⁰ : + 38.4 ° (c 0.276, DMF)
[00274] Chiral SFC (SFC-I method): Tr 4.96 min, MH ⁺ 646, 100% chiral purity.
13B Enantiomer:
[00275] ¹ H NMR (500 MHz, DMSO-d ₆ ) δ ppm 1.91 - 2.02 (m, 2 H)
2.33 - 2.41 (m, 2 H) 3.10 - 3.24 (m, 2 H) 3.73 (s, 3 H) 4.04 - 4.17 (m, 3 H) 4, 39 (td, J = 10.1, 6.6 Hz, 1 H) 5.72 (d, J = 8.5 Hz, 1 H) 6.35 (s, 1 H) 6.68 (s, 1 H) 6.81 - 6.89 (m, 2 H) 6.99 - 7.05 (m, 2 H) 7.12 - 7.15 (m, 1 H) 7.27-7.40 (m , 2 H) 8.04 (s, 1 H) 8.16 (s, 1 H) 9.16 (s, 1 H) 12.11 (s I, 1 H)
[00276] LC / MS (LC-A method): Tr 2.83 min, MH ⁺ 646
[00277] [a] _D ²⁰ : -43.2 ° (c 0.273, DMF)
[00278] Chiral SFC (SFC-I method): Tr 6.56 min, MH ⁺ 646, 100% chiral purity.
Example 14: 4- (5-chloro-2- (1 - ((3-methoxy-5- (1H-1,2,4triazol-1-yl) phenyl) amino) -2-oxo-2- acid synthesis (6- (trifluoromethoxy) indolin-1yl) ethyl) phenoxy) -2,2-dimethylbutanoic (Compound 14) and chiral separation in Enantiomers 14A and 14B.
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Synthesis of intermediate 14a:
[00279] To a mixture of methyl 2- (4-chloro-2-hydroxyphenyl) acetate [CAS 518979-09-4] (1 g, 4.99 mmol) and cesium carbonate (3.25 g, 9, 97 mmol) in DMF (30 ml) methyl 4-bromo-2,2-dimethylbutanoate [CAS 4833-99-2] (1.09 g, 5.23 mmol) was added. The reaction mixture was stirred at room temperature for 20 h. The reaction mixture was poured into water with stirring (150 ml) and the product was extracted (2x) with EtzO. The combined organic layers were washed with saturated aqueous sodium chloride solution, dried over MgSCU, filtered and evaporated under reduced pressure. The product crystallized upon standing at room temperature. The solid residue was stirred in 5 ml of diisopropyl ether. The precipitate was removed by filtration, washed (3x) with diisopropyl ether and dried under vacuum at 45 Ό to provide 4 (5-chloro-2- (2-methoxy-2-oxoethyl) phenoxy) -2,2-dimethylbutanoate 14a (0.897 g) ·
Synthesis of intermediate 14b:
[00280] A solution of 4- (5-chloro-2- (2-methoxy-2-oxoethyl) phenoxy) -
Methyl 2,2-dimethylbutanoate 14a (0.897 g, 2.73 mmol) in a solvent mixture of MeOH (10 ml) and dioxane (5 ml) was cooled in an ice bath. AOO, 1M NaOH (2.73 ml, 2.73 mmol) was added
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80/109 carefully. The reaction mixture was stirred at 0 Ό for 2 h, and at room temperature for 14 h. The reaction mixture was poured into water (50 ml), stirred for 15 minutes and left to stand for 30 minutes. The solid fraction (intermediate 14a that did not react) was filtered and washed (3x) with water. The combined filtrates were acidified by dropwise addition of 1N HCl (2.8 ml) while stirring. After 10 minutes, the precipitate was filtered, washed (3x) with water and dried under vacuum at 45 O to provide 2- (4-chloro-2- (4-m ethoxy-3,3-dimethyl-4oxobutoxy) phenyl) acid acetic 14b (0.576 g).
Synthesis of intermediate 14c:
[00281] To a stirring solution of 2- (4-chloro-2- (4-methoxy-
3,3-dimethyl-4-oxobutoxy) phenyl) acetic 14b (576 mg, 1.83 mmol), 6 (trifluoromethoxy) indoline [CAS 959235-95-1] (409 mg, 2.01 mmol) and diisopropylethylamine (907 μΙ_, 5.49 mmol) in DMF (7.5 ml_) under N2-atm HATU (1.07 g, 2.75 mmol) was added and the reaction mixture was stirred at room temperature for 5 h. Water (30 ml) was added and the product was extracted (2x) with Et20. The combined organic layers were washed with saturated aqueous sodium chloride solution, dried over MgSO4, filtered and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel (40 g) using a heptane / EtOAc gradient from 100/0 to 0/100. The desired fractions were combined and evaporated under reduced pressure, and coevaporated with toluene. The residue was dried under vacuum at 450 to provide 4- (5-chloro-2- (2-oxo-2- (6- (trifluoromethoxy) indolin-1 yl) ethyl) phenoxy) -2,2-dimethylbutanoate14c ( 790 mg) as a powder.
Synthesis of intermediate 14d:
[00282] A solution of methyl 4- (5-chloro-2- (2-oxo-2- (6 (trifluoromethoxy) i ndol i n-1 -yl) ethyl) phenoxy) -2,2-dimethylbutanoate ( 790 mg, 1.58 mmol) in 2-Me-THF (30 ml) was stirred under N2 flow and cooled to -78 O. A solution was added dropwise
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81/109 1M lithium bis (trimethylsilyl) amide in THF (3.16 mL, 3.16 mmol) and the resulting mixture was stirred at -78 Ό for 30 minutes. Chlorotrimethylsilane (323, 2.53 mmol) was added dropwise and the mixture was stirred at -78 Ό for 25 min. A solution of A / -bromosuccinimide (352 mg, 1.98 mmol) in 2-Me-THF (7.5 mL) and THF (2.5 mL) was added dropwise and the reaction mixture was stirred at - 78 Ό for 45 min. A saturated aqueous NH4 Cl solution (50 mL) was added slowly, and the resulting mixture was stirred without cooling until the temperature reached 0 Ό. Water (10 ml) was added and, after stirring for 30 min, the layers were separated. The organic layer was dried over MgSO4, filtered, evaporated under reduced pressure and coevaporated with CH3CN to provide 4- (2- (1-bromo-2-oxo-2- (6 (trifluoromethoxy) indolin-1-yl) ethyl) - Methyl 5-chlorophenoxy) -2,2-dimethylbutanoate 14d (915 mg). The product was used without further purification in the next step.
Synthesis of intermediate 14e:
[00283] To a stirred solution of methyl 4- (2- (1-bromo-2-oxo-2- (6 (trifluoromethoxy) indolin-1-yl) ethyl) -5-chlorophenoxy) -2,2-dimethylbutanoate 14d (915) mg, 1.58 mmol) in CH3CN (40 mL), under N2-atm, 3-methoxy-5- (1 H-1,2,4-triazol-1-yl) aniline [CAS 1220630-56-7] (301 mg, 1.58 mmol) and diisopropylethylamine (545 pL,
3.16 mmol) and the reaction mixture was stirred at 50 ° C for 18 h and 70 Ό for 6 h. The mixture was cooled to room temperature and stirred with H2O (200 ml). The product was extracted (2x) with Et20. The combined organic layers were washed with saturated aqueous sodium chloride solution, dried over MgSCU, filtered and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel (40 g) using a heptane / EtOAc / EtOH gradient from 100/0/0 to 40/45/15. The desired fractions were combined and the solvent was evaporated under reduced pressure and coeval
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82/109 porated with dioxane to provide 4- (5-chloro-2- (1 - ((3-methoxy-5- (1 H1,2,4) -triazol-1-yl) phenyl) amino) -2- methyl oxo-2- (6- (trifluoromethoxy) indolin-1) ethyl) phenoxy) -2,2-dimethylbutanoate 14e (1.09 g). The product was used in the next step without purification.
Synthesis of Compound 14 and chiral separation in 14A and 14B Enantiomers:
[00284] 1M NaOH (3.95 ml_, 3.95 mmol) was added to a stirred solution of 4- (5-chloro-2- (1 - ((3-methoxy-5- (1 / - / - 1 , 2,4) -triazole-1yl) phenyl) amino) -2-oxo-2- (6- (trifluoromethoxy) indolin-1-yl) ethyl) phenoxy) -2,2 methyl dimethylbutanoate 14e (1.09 g, 1.58 mmol) in dioxane (6.5 ml). The reaction mixture was stirred at room temperature for 40 h. Water (21 ml) and HCl 1 N (4.1 ml) were added and after stirring for 10 minutes, the precipitate was removed by filtration and washed (3x) with water. The solid residue (0.9 g) was stirred in CH2 Cl2 (7.5 ml) for 45 minutes, filtered, washed (3x) with CH2 Cl2 and dried under vacuum at 45 Ό to provide 4- (5-chloro-2- acid) (1 - ((3-methoxy-5- (1H-1,2,4-triazol-1yl) phenyl) amino) -2-oxo-2- (6- (trifluoromethoxy) indolin-1-yl) ethyl) phenoxy ) Racemic -2,2-dimethylbutanoic (Compound 14, 590 mg).
[00285] Compound 14 enantiomers (557 mg) were separated by Preparative Chiral SFC (Stationary phase: Chiralpak® Diacel AD 20 x 250 mm; Mobile phase: CO2, EtOH + 0.4% iPrNFE). Fractions containing the first eluted product were combined, evaporated under reduced pressure and coevaporated with CH3CN. The residue was crystallized from Et2O / 3/1 heptane, filtered off, washed (3x) with Et2Ü and dried under vacuum at 50 ° to provide 0 14A Enantiomer (96 mg). Fractions containing the second eluted product were combined, evaporated under reduced pressure and coevaporated with CH3CN. The residue was crystallized from Et20, filtered off, washed (3x) with Et2Ü and dried under vacuum at 50 Ό to provide the 14B Enantiomer (35 mg + 106 mg (second crop)).
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Compound 14:
[00286] ¹ H NMR (400 MHz, DMSO-c / ₆ ) δ ppm 1.11 (d, J = 5.1 Hz, 6 H) 1.86 - 2.03 (m, 2 H) 3.09 - 3.28 (m, 2 H) 3.73 (s, 3 H) 4.01 - 4.18 (m, 3 H) 4.38 (td, J = 10.2, 6.6 Hz, 1 H) 5.69 (d, J = 8.6 Hz, 1 H) 6.33 (t, J = 2.0 Hz, 1 H) 6.67 (t, J = 1.9 Hz, 1 H) 6.79 - 6.86 (m, 2 H) 6.97 - 7.05 (m, 2 H) 7.18 (d, J = 1.8 Hz, 1 H) 7.31 (d, J = 8.1 Hz, 1 H) 7.34 (d, J = 8.1 Hz, 1 H) 8.03 (s I, 1 H) 8.15 (s, 1 H) 9.14 (s, 1 H) 12.25 (s I, 1 H)
[00287] LC / MS (LC-C method): Tr 1.12 min, MH ⁺ 674
14 A Enantiomer:
[00288] ¹ H NMR (400 MHz, DMSO-c / ₆ ) δ ppm 1.11 (d, J = 5.1 Hz, 6 H) 1.87 - 2.02 (m, 2 H) 3.09 - 3.26 (m, 2 H) 3.73 (s, 3 H) 4.02 - 4.18 (m, 3 H) 4.37 (td, J = 10.2, 6.6 Hz, 1 H) 5.69 (d, J = 8.8 Hz, 1 H) 6.33 (t, J = 2.2 Hz, 1 H) 6.67 (t, J = 1.8 Hz, 1 H) 6.79 - 6.86 (m, 2 H) 6.99 - 7.04 (m, 2 H) 7.18 (d, J = 2.0 Hz, 1 H) 7.31 (d, J = 8.1 Hz, 1 H) 7.34 (d, J = 8.4 Hz, 1 H) 8.03 (s I, 1 H) 8.15 (s, 1 H) 9.14 (s, 1 H) 12.25 (s I, 1 H)
[00289] LC / MS (LC-D method): Tr 2.09 min, MH ⁺ 674
[00290] [a] _D ²⁰ : -32.8 ^o (c 0.528, DMF)
[00291] Chiral SFC (SFC-K method): Tr 3.63 min, MH ⁺ 674, chiral purity 100%.
[00292] Melting point: 111Ό
14B Enantiomer:
[00293] ¹ H NMR (400 MHz, DMSO-c / ₆ ) δ ppm 1.11 (d, J = 5.1 Hz, 6 H) 1.87 - 2.02 (m, 2 H) 3.10 - 3.27 (m, 2 H) 3.73 (s, 3 H) 4.03 - 4.18 (m, 3 H) 4.38 (td, J = 10.2, 6.5 Hz, 1 H) 5.69 (d, J = 8.6 Hz, 1 H) 6.33 (t, J = 1.9 Hz, 1 H) 6.67 (t, J = 1.9 Hz, 1 H) 6.79 - 6.86 (m, 2 H) 6.98 - 7.05 (m, 2 H) 7.18 (d, J = 1.8 Hz, 1 H) 7.31 (d, J = 8.4 Hz, 1 H) 7.34 (d, J = 8.1 Hz, 1 H) 8.03 (s I, 1 H) 8.15 (s, 1 H) 9.14 (s, 1 H) 12.25 (s I, 1 H)
[00294] LC / MS (LC-D method): Tr 2.08 min, MH ⁺ 674
[00295] [a] _D ²⁰ : + 32.8 ° (c 0.515, DMF)
[00296] Chiral SFC (SFC-K method): Tr 4.22 min, MH ⁺ 674, purity
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84/109 chiral 100%.
[00297] Melting point: 177Ό
Example 15: 4- (5-chloro-2- (2- (5-fluoro-6 (trifluoromethoxy) acid) n-1-yl) -1 - ((3-methoxy-5- (1 H-1,2,4-triazol-1 yl) phenyl) amino) -2-oxoethyl) phenoxy) butanoic (Compound 15) and chiral separation in Enantiomers 15A and 15B.

HCJ (4 M donsno}
5 ^Q C3h, ta12h
2) LiOH. TH r / water
15A and 15S Ensntiomers
Synthesis of intermediate 15a:
[00298] A mixture of 5-fluor-6- (trifluoromethoxy) indoline 9e (321 mg, 1.45 mmol), 2- (2- (4 - ((tert-butoxy) -4-oxobutoxy) -4- acid chlorophenyl) 2 - ((3-methoxy-5- (1 H-1,2,4-triazol-1-yl) phenyl) amino) acetic 11d (750 mg,
1.45 mmol), HATU (827 mg, 2.18 mmol) and diisopropylethylamine (719 μΙ_, 4.35 mmol) in DMF (30 ml_) was stirred at room temperature for 12 h. The mixture was diluted with water. The precipitate was removed by filtration, washed with water and taken up with EtOAc. The organic solution was washed with a 10% solution of K2CO3 in water, water, dried over MgSCU, filtered and the solvent was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 40 g, C2 WMeOH 98/2). The pure fractions were combined and concentrated to dryness to give 4- (5-chloro-2- (2- (5floro-6- (trifluoromethoxy) i ndol i n-1 -yl) -1 - ((3- methoxy-5- (1 H-1,2,4-triazole-1
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85/109 yl) phenyl) amino) -2-oxoethyl) phenoxy) tert-butyl butanoate 15a (1.05 g).
Synthesis of Compound 15 and chiral separation in Enantiomers 15A and 15B:
[00299] A solution of 4- (5-chloro-2- (2- (5-fluoro-6 (trifluoromethoxy) i ndol i n-1 -yl) -1 - ((3-methoxy-5- (1 Tert-Butyl H-1,2,4-triazol-1yl) phenyl) amino) -2-oxoethyl) phenoxy) butanoate 15a (1.05 g,
1.46 mmol) in 4M HCI in dioxane (8 ml) was stirred at 50 ° for 3 h and at room temperature for 12 h. The precipitate was removed by filtration, washed with dioxane / diisopropyl ether and dried. The residue (580 mg) was dissolved in THF (2.5 ml) and a solution of lithium hydroxide monohydrate (180 mg, 4.277 mmol) in water (2.5 ml) was added dropwise. The mixture was stirred at room temperature for 18h. The reaction was cooled to 0 ° C and water and ice were added. The pH was adjusted to 6 by the addition of 3N HCI. The product was extracted with EtOAc. The organic layer was separated, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. A small fraction of the residue was crystallized from CH2Cl2 to give 4- (5-chloro-2- (2- (5-fluoro-6- (trifluoromethoxy) indolin-1-yl) -1 - ((3-methoxy-5- ( 1 H-1,2,4-triazol-1-yl) phenyl) amino) -2-oxoethyl) phenoxy) butanoic 15 (24 mg) as a racemate. The remaining amount was further purified by flash chromatography on silica gel (20-45 pm, 24 g, CH2Cl2 / MeOH gradient from 99.5 / 0.5 to 90/10). The pure fractions were combined and concentrated to dryness to provide a second fraction of Compound 15 (382 mg).
[00300] The enantiomers of Compound 15 (382 mg) were separated by Preparative Chiral SFC (Stationary phase: Chiralcel® OD-H 5 pm 250 x 30 mm, mobile phase: 75% CO2, 25% MeOH (+0.3 % iPrNH2)). The first eluted enantiomer (178 mg) was solidified by titration with heptane / diisopropyl ether to give the 15A Enantiomer (140 mg). The second eluted enantiomer (187 mg) was solidified by
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86/109 titration with heptane / diisopropyl ether to give Enantiomer 15B (136 mg).
Compound 15:
[00301] ¹ H NMR (500 MHz, DMSO-c / ₆ ) δ ppm 1.90 - 2.04 (m, 2 H) 2.28 - 2.46 (m, 2 H) 3.11 - 3, 28 (m, 2 H) 3.73 (s, 3 H) 4.03 - 4.18 (m, 3
H) 4.33 - 4.42 (m, 1 H) 5.71 (d, J = 8.8 Hz, 1 H) 6.34 (s, 1 H) 6.67 (s, 1 H) 6 , 81 (s, 1 H) 6.87 (d I, J = 8.8 Hz, 1 H) 7.02 (dd, J = 8.2, 1.9 Hz, 1 H) 7.12 (s , 1 H) 7.31 (d, J = 8.2 Hz, 1 H) 7.43 (d, J = 9.8 Hz, 1 H) 8.12-8.20 (m, 2 H) 9 , 16 (s, 1 H) 12.12 (s I, 1 H)
[00302] LC / MS (LC-B method): Tr 2.72 min, MH ⁺ 664
[00303] Melting point: 188Ό
15A Enantiomer:
[00304] ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.88 - 2.12 (m, 2 H)
2.19 - 2.42 (m, 2 H) 2.96 - 3.24 (m, 2 H) 3.73 (s, 3 H) 3.89 - 4.16 (m, 3
H) 4.25 - 4.43 (m, 1 H) 5.72 (d, J = 8.6 Hz, 1 H) 6.34 (s, 1 H) 6.66 (s, 1 H) 6 , 79 - 6.86 (m, 2 H) 7.01 (dd, J = 8.3, 1.8 Hz, 1 H) 7.09 - 7.14 (m, 1
H) 7.32 (d, J = 8.1 Hz, 1 H) 7.41 (d, J = 9.6 Hz, 1 H) 8.12 - 8.17 (m, 2 H)
9.16 (s, 1 H)
[00305] LC / MS (LC-B method): Tr 2.75 min, MH ⁺ 664
[00306] [a] _D ²⁰ : -31.5 ^o (c 0.267, DMF)
[00307] Chiral SFC (SFC-J method): Tr 2.66 min, MH ⁺ 664, chiral purity 99.69%.
15B Enantiomer:
[00308] ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.88 - 2.04 (m, 2 H) 2.22 - 2.43 (m, 2 H) 3.12 - 3.40 (m, 2 H) 3.72 (s, 3H) 4.03 - 4.17 (m, 3
H) 4.33 - 4.43 (m, 1 H) 5.72 (d, J = 8.6 Hz, 1 H) 6.34 (s, 1 H) 6.67 (s, 1 H) 6 , 78 - 6.87 (m, 2 H) 7.01 (dd, J = 8.3, 1.8 Hz, 1 H) 7.09 - 7.13 (m, 1
H) 7.32 (d, J = 8.1 Hz, 1 H) 7.41 (d, J = 9.6 Hz, 1 H) 8.12 - 8.18 (m, 2 H)
9.16 (s, 1 H)
[00309] LC / MS (LC-B method): Tr 2.73 min, MH ⁺ 664
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[00310] [a] _D ²⁰ : + 28.2 ° (c 0.262, DMF)
[00311] Chiral SFC (SFC-J method): Tr 3.53 min, MH ⁺ 664, chiral purity 98.93%.
[00312] Example 16: 4- (5-chloro-2- (1 - ((3-methoxy-5 (1 H-1,2,4-triazol-1-i)) phenyl I) amino acid synthesis ) -2- (4-methyl-6- (trifluoromethoxy) indolin n-1yl) -2-oxoethyl) phenoxy) butanoic (Compound 16) and chiral separation in 16A and 16B Enantiomers.

Synthesis of intermediate 16a:
[00313] A mixture of 4-methyl-6- (trifluoromethoxy) indoline 10h (336 mg, 1.55 mmol), 2- (2- (4 - ((tert-butoxy) -4-oxobutoxy) -4- acid chlorophenyl) 2 - ((3-methoxy-5- (1 H-1,2,4-triazol-1-yl) phenyl) amino) acetic 11d (800 mg, 1.55 mmol), HATU (883 mg, 2 , 32 mmol) and diisopropylethylamine (767 μΙ_, 4.64 mmol) in DMF (30 ml_) was stirred at room temperature for 12 h. The mixture was diluted with water. The precipitate was removed by filtration, washed with water and The organic solution was washed with a 10% solution of K2CO3 in water, water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure.The residue was purified by flash chromatography on silica gel (15- 40 pm, 40 g, heptane / EtAOc gradient 90/10 to 70/30) The pure fractions were combined and concentrated to dryness to give 4- (5-chloro-2- (1 - ((3-methoxy- 5- (1 H-1,2,4-triazol-1-yl) phenyl) amino) -2- (4
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88/109 methyl-6- (trifluoromethoxy) indoliη-1-yl) -2-oxoethyl) phenoxy) tert-butyl butanoate 16a (816 mg).
Synthesis of Compound 16 and chiral separation in 16A and 16B Enantiomers:
[00314] A solution of 4- (5-chloro-2- (1 - ((3-methoxy-5- (1 H-1,2,4triazol-1-yl) phenyl) amino) -2- (4- methyl-6- (trifluoromethoxy) indolin-1-yl) -2oxoethyl) phenoxy) tert -butyl butanoate 16a (816 mg, 1.14 mmol) in 4M HCI in dioxane (7 ml) was stirred at 5 ° for 2 h at room temperature for 12 h. The precipitate was removed by filtration, washed with dioxane / diisopropyl ether and dried. The residue was purified by flash chromatography on silica gel (20-45 pm, 40 g, CHbCb / MeOH gradient from 100/0 to 95/5). The pure fractions were combined and the solvent was evaporated under reduced pressure to give, after crystallization from ChhCN / diisopropyl ether, 4- (5-chloro-2- (1 - ((3 methoxy-5- (1 H -1,2,4-triazol-1-yl) phenyl) amino) -2- (4-methyl-6 (trifluoromethoxy) indolin-1-yl) -2-oxoethyl) phenoxy) butanoic 16 (495 mg).
[00315] Compound 16 enantiomers were separated using Preparative Chiral SFC (stationary phase: Chiralpak® AD-H 5 pm 250 x 30 mm, mobile phase: 65% CO2, 35% iPrOH (+ 0.3% iPrNH2)) . The enantiomer that eluted first (145 mg) was further purified by flash chromatography on silica gel (10-40 pm, 24 g, CH2Cl2 / MeOH 98/2). The pure fractions were combined and the solvent was concentrated under reduced pressure to give, after solidification by trituration with diisopropyl ether / pentane, the 16A Enantiomer (99 mg). The second eluting enantiomer (149 mg) was further purified by flash chromatography on silica gel (10-40 pm, 24 g, CH2Cl2 / MeOH 98/2). The pure fractions were combined and the solvent was concentrated under reduced pressure to give, after solidification by trituration with diisopropyl ether / pentane, the 16B Enantiomer (95 mg).
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Compound 16:
[00316] ¹ H NMR (400 MHz, DMSO-c / ₆ ) δ ppm 1.91 - 2.05 (m, 2 H)
2.20 (s, 3 H) 2.29 - 2.43 (m, 2 H) 3.01 - 3.27 (m, 2 H) 3.72 (s, 3 H) 4.02
- 4.18 (m, 3 H) 4.35 - 4.44 (m, 1 H) 5.71 (d I, J = 8.6 Hz, 1 H) 6.34 (s I, 1 H) 6.67 (s, 1 H) 6.78 - 6.90 (m, 3 H) 7.01 (d I, J = 7.1 Hz, 1 H) 7.12 (s, 1 H) 7, 31 (d, J = 8.1 Hz, 1 H) 7.88 (s I, 1 H) 8.15 (s, 1 H) 9.14 (s, 1 H) 12.07 (s I, 1 H)
[00317] LC / MS (LC-A method): Tr 2.93 min, MH ⁺ 660
[00318] Melting point: 214Ό
16 A enantiomer:
[00319] ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.92 - 2.04 (m, 2 H)
2.20 (s, 3 H) 2.30 - 2.44 (m, 2 H) 2.99 - 3.16 (m, 2 H) 3.73 (s, 3 H) 4.04
- 4.17 (m, 3 H) 4.35 - 4.44 (m, 1 H) 5.71 (d I, J = 8.6 Hz, 1 H) 6.34 (s I, 1 H) 6.67 (s, 1 H) 6.79 - 6.89 (m, 3 H) 7.02 (d I, J = 8.6 Hz, 1 H) 7.12 (s, 1 H) 7, 32 (d, J = 8.1 Hz, 1 H) 7.89 (s, 1 H) 8.15 (s, 1 H) 9.14 (s, 1 H) 12.09 (s I, 1 H )
[00320] LC / MS (LC-A method): Tr 2.94 min, MH ⁺ 660
[00321] [a] _D ²⁰ : -35.4 ° (c 0.263, DMF)
[00322] Chiral SFC (SFC-F method): Tr 1.61 min, MH ⁺ 660, 100% chiral purity.
16B Enantiomer:
[00323] ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.91 - 2.04 (m, 2 H)
2.20 (s, 3 H) 2.30 - 2.44 (m, 2 H) 3.00 - 3.15 (m, 2 H) 3.73 (s, 3 H) 4.03
- 4.17 (m, 3 H) 4.35 - 4.44 (m, 1 H) 5.71 (d, J = 8.6 Hz, 1 H) 6.34 (s, 1 H)
6.67 (s, 1 H) 6.79 - 6.89 (m, 3 H) 7.02 (dd, J = 8.1, 1.5 Hz, 1 H) 7.10 -
7.14 (m, 1 H) 7.32 (d, J = 8.59 Hz, 1 H) 7.89 (s, 1 H) 8.15 (s, 1 H) 9.14 (s, 1 H) 12.10 (s I, 1 H)
[00324] LC / MS (LC-A method): Tr 2.94 min, MH ⁺ 660
[00325] [a] _D ²⁰ : + 34.3 ° (c 0.274, DMF)
[00326] Chiral SFC (SFC-F method): Tr 2.22 min, MH ⁺ 660, purity
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90/109 chiral 99.47%.
Table: compounds prepared as described above
Compound Structure Optical rotation 1 F OCH ₃ FC. π N racemic 1A F P ^ch 3 θΐω / = Ç FC. π N [a] _D ²⁰ = -44.8 ° 1B F ^HC V ^ (/ xZ P ^ch 3 qIw / = Ç fc. Π N [a] _D ²⁰ = + 36.2 ° 2 F OCH ₃ FC. ³ XX) π ch ₃ c ^ - V racemic 2A F P ^ch 3 qI »/ = Ç fc. ³ ΧϊΟ π ch ₃ <^^ V [a] _D ²⁰ = -45.0 ° 2B F P ^ch 3 qIw / = Ç fc. ³ ΧϊΟ π ch ₃ <^^ V [a] _D ²⁰ = + 43.4 °
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Compound Structure Optical rotation 3 F OCH ₃ ΚΛ) Π racemic 3A F pch ₃ hV_ ^ u. Π [a] _D ²⁰ = -38.2 ° 3B F pch ₃ lp, = / hV_ζ u. Π [a] _D ²⁰ = + 40.9 ° 4 ÇI PCH ₃ FC. Xx) π N racemic 4A Çi P ^ch 3 θΐω / = <FC. I'XW XX> π N [a] _D ²⁰ = -42.9 ° 4B Çi P ^ch 3 qIw / = Ç FC. I'XW XX> π N [a] _D ²⁰ = + 39.5 °
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Compound Structure Optical rotation 5 ÇI OCH ₃ fc. ³ XX) π CH3 <^^ V racemic 5A ÇI P ^ch 3 qI »/ = Ç FC. ΙΐΓνγ ³ XX π CH3 <^^ V [a] _D ²⁰ = -40.3 ° 5B ÇI P ^ch 3 qIw / = Ç FC. ³ XX π CH3 <^^ V [a] _D ²⁰ = + 40.0 ° 6 ÇI PCH ₃ hV_ζ kA) Π racemic 6A Çi SHPs θΐω, = / ^Γ · hV_ kA) Π [a] _D ²⁰ = + 45.9 ° 6B ÇiSHPs θΐω, = / hV_ ^ kA) Π [a] _D ²⁰ = -46.3 °
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Compound Structure Optical rotation 7 ÇI OCH ₃ fc. '^ 03 π N racemic 7A ÇI P ^ch 3 θΐω / = Ç fc. ΙΐΓνγ '^ 03 π N [a] _D ²⁰ = -44.3 ° 7B ÇI ^{Η (} ν ^ (/ Μ P ^ch 3 qIw / = Ç fc. '^ 03 π N [a] _D ²⁰ = + 35.6 ° 8 ÇI OCH ₃ 1Λ) Π ch ₃ <^^ V racemic 8A Ç ¹ P ^ch 3 θΐω / = <Ç ^F 3 ^C % ^ Z ί ^ -Λ ΑΛ / Π ch ₃ <^^ V [a] _D ²⁰ = + 39.3 ° 8B Ç ¹ P ^ch 3 qIh / = Ç ^f 3 ^c % ^ Z ί ^ -Λ ΑΛ / Π ch ₃ <^^ V [a] _D ²⁰ = -44.4 °
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Compound Structure Optical rotation 9 Çl OCH ₃ AA> π N racemic 9A Çl SHP ₃ AA> π N [a] _D ²⁰ = -35.1 ° 9B Çl PCH ₃ {vJW / = ( ^F 3 ^{C (} X ^ Zn ^ AA> π N [a] _D ²⁰ = + 32.3 ° 10 Çl och ₃ ^f 3C0 <^ T (II I> N— N W v ch ₃ racemic 10A Çl P ^ch 3 oil ·) / = ( ^f 3C0 <^ Z (II I> N ^- N W v ch ₃ [a] _D ²⁰ = -38.4 ° 10B Çi P ^ch 3 q] (+) / = ( ^f 3C0 <^ Z (II I> N— N W v ch ₃ [a] _D ²⁰ = + 37.5 °
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Compound Structure Optical rotation 11 ÇI, OCH ₃ oo _5is Js. / ⁼⁼ fc. I π N racemic 11A ÇI ^H0 Y ^ —PCh3 ⁰ t— / ⁼⁼ fc. I π N [a] _D ²⁰ = -28.9 ° 11B ÇI ^H0 Y ^ —PCh ₃ FC. I ^ V7 π N [a] _D ²⁰ = + 23.8 ° 12 Çi ^{H &} V ^x ^ ' ^cr i / _0CH3 ⁰ O _5is Js. / ⁼ FC. I ³ ^ O0 n CHaC ^^ V racemic 12A Çi P ^CH3 0 CkA ^+: / = FC. I ³ ^ O0 n CHaC ^^ V [a] _D ²⁰ = + 30.4 ° 12B Çi P ^CH3 FC. I ^ V7 ³ ^ O0 n CHaC ^^ V [a] _D ²⁰ = -36.9 °
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Compound Structure Optical rotation 13 LC / wn ^0CH3 N racemic 13A Çl Pch ₃ ο% A ⁺ ^ / = Ç ^F 3 ^{C (} x% J wn N [a] _D ²⁰ = + 38.4 ° 13B Çl P ^ch 3 I ^F 3 ^{C (} x% J wn N [a] _D ²⁰ = -43.2 ° 14 Çl, OCH ₃ ^F 3 ^{C (} x% J wn N racemic 14A Çl ^ho Y ^: ^^ ot ^ P ^ch 3 I ^F 3 ^{C (} x% J w Π N [a] _D ²⁰ = -32.8 ° 14B Çi ^ho Y ^: ^^ ot ^ P ^ch 3 ο ^f 3 ^{c (} x% J w π N [a] _D ²⁰ = + 32.8 °
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Compound Structure Optical rotation 15 CL / ^{0 0CH3 F} 3 ^{C (} x% J H ~ V ^ AA> Π N racemic 15A Çl Pch ₃ I ^F 3 ^{C (} x% J H ~ V ^ AA> Π N [a] _D ²⁰ = -31.5 ^o 15B Çl P ^ch 3 ο ^F 3 ^{C (} x% J H ~ V ^ AA> Π N [a] _D ²⁰ = + 28.2 ° 16 Çl __ OCH ₃ hCCl ^^ JT h ”V ^ II T> N ^- N W v ch ₃ racemic 16A Çl ^HC x ~ · - '^' cr ^ r, och ₃ I hCCl ^^ JT li I> NN W v ch ₃ [a] _D ²⁰ = -35.4 ° 16B Çi ^HC x ~ · - '^' cr ^ rz ^och 3 o% A ⁺ _| j / = <hCCl ^^ JT li T> NN W v ch ₃ [a] _D ²⁰ = + 34.3 °
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ANTIVIRAL ACTIVITY OF THE COMPOUNDS OF THE INVENTION
DENV-2 antiviral assay
[00327] The antiviral activity of all compounds of the invention was tested against the 16681 DENV-2 strain that was labeled with enhanced green fluorescent protein (eGFP). The culture medium consists of a minimum essential medium supplemented with 2% heat-inactivated fetal calf serum, 0.04% gentamicin (50 mg / mL) and 2 mM L-glutamine. Vero cells, obtained from ECACC, were suspended in culture medium and 25 pL were added to 384-well plates (2500 cells / well), which already contain the antiviral compounds. Typically, these plates contain a 5-fold serial dilution of 9 steps of diluting the test compound to 200 times the final concentration in 100% DMSO (200 nL). In addition, the concentration of each compound is tested in quadruplicate (range of final concentrations: 25μΜ - 0.000064 μΜ or 2.5 μΜ - 0.0000064 μΜ for the most active compounds). Finally, each plate contains wells that are assigned as virus controls (containing cells and viruses in the absence of compound), cell controls (containing cells in the absence of viruses and compound) and medium controls (containing medium in the absence of cells, viruses) and compounds). To the wells assigned as media control, 25 µl of culture medium was added instead of Vero cells. Once the cells were added to the plates, the plates were incubated for 30 minutes at room temperature to allow the cells to distribute evenly within the wells. Then, the plates were incubated in a fully humidified incubator (37 Ό, 5% CO 2) until the next day. Then, the DENV-2 strain 16681, marked with eGFP, was added to a multiplicity of infection (MOI) of 0.5. Therefore, 15 pL of virus suspension was added to all wells containing test compound and to the wells assigned as virus control. In pa
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99/109 thinning, 15 pL of culture medium was added to the media and cell controls. Then, the plates were incubated for 3 days in a fully humidified incubator (37 Ό, 5% CO 2). On the day of reading, eGFP fluorescence was measured using an automated fluorescence microscope at 488 nm (blue laser). Using an internal LIMS system, inhibition dose response curves were calculated for each compound and half of the maximum effective concentration (EC50) was determined. Therefore, the percentage of inhibition (I) for each test concentration is calculated using the following formula: I = 100 * (StScc) / (Svc-Scc); St, Scc and Svc are the amount of eGFP signal in the wells with test compounds, cell control and virus control, respectively. The EC50 represents the concentration of a compound to which virus replication is inhibited by 50%, as measured by a 50% reduction in the fluorescent intensity of eGFP compared to the virus control. EC50 is calculated using linear interpellation (Table 1).
[00328] In parallel, the toxicity of the compounds was evaluated on the same plates. Once the eGFP signal was read, 40 pL of ATPlite, a cell viability dye, was added to all wells of the 384-well plates. ATP is present in all metabolically active cells and the concentration decreases very quickly when the cells undergo necrosis or apoptosis. The ATPLite assay system is based on the production of light caused by the reaction of ATP with added luciferase and D-luciferin. The plates were incubated for 10 minutes at room temperature. Then, the plates were measured in a ViewLux. Half of the maximum cytotoxic concentration (CC50) was also determined, defined as the concentration required to reduce the luminescent signal by 50% compared to that of wells with cell control. Finally, the selectivity index (SI) was determined for the compounds, having been
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100/109 calculated as follows: SI = CC50 / EC50.
Table 1: EC50, CC50 and SI for the compounds of the invention in the DENV-2 antiviral assay
# of the compound EC50 (pM) N CC50 (pM) N SI N 1 0.0031 4 12 4 3350 4 1A 1.00 3 7.7 3 7.8 3 1B 0.0028 4 9.8 2 3530 2 2 0.0028 4 13 4 4000 4 2A 0.27 3 12 3 43 3 2B 0.0018 3 10 3 4180 3 3 0.0019 4 8.5 4 5210 4 3A 0.52 3 11 3 22 3 3B 0.00099 3 8.7 3 11400 3 4A 1.2 4 7.8 4 6.4 4 4B 0.0010 3 11 3 16100 3 5 0.00082 3 11 3 11200 3 5A 0.042 3 11 3 275 3 5B 0.00062 3 9.2 3 14400 3 6 0.00097 3 3.3 3 3370 3 6A 0.00059 9 8.5 9 14900 9 6B 0.092 7 7.3 7 79 7 7 0.0030 3 10 3 3410 3 7A 0.43 3 11 3 26 3 7B 0.0018 3 7.6 3 4180 3 8 0.0021 3 9.5 3 4410 3 8A 0.0015 3 12 3 7360 3 8B 0.25 3 12 3 47 3 9 0.0016 4 3.9 3 2850 3 9A 0.25 3 4.2 4 17 3 9B 0.00060 3 10 3 13800 3 10 0.00035 3 10 3 26600 3 10A 0.063 3 10 3 165 3 10B 0.00025 3 11 3 51400 3
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# of the compound EC50 (pM) N CC50 (pM) N SI N 11 0.00031 3 15 3 53500 3 11A 0.045 3 11 3 246 3 11B 0.00020 3 12 3 45200 3 12 0.0022 3 13 3 5660 3 12A 0.0012 3 13 3 11000 3 12B 0.32 3 12 3 37 3 13 0.00023 3 11 3 59500 3 13A 0.00012 4 12 4 103601 4 13B 0.012 3 12 3 972 3 14 0.00041 3 10 3 24900 3 14A 0.42 3 9.1 3 22 3 14B 0.00027 4 9.4 4 35500 4 15 0.00022 3 11 3 58700 3 15A 0.0053 3 10 3 1970 3 15B 0.00013 3 11 4 94800 3 16 0.00011 3 11 3 95800 3 16A 0.014 3 11 3 800 3 16B 0.000069 5 6.2 5 > 174673 5
N = the number of independent experiments in which the compounds were tested.
Quantitative PCR assay with tetravalent reverse transcriptase (RT-qPCR): Protocol A.
[00329] The antiviral activity of the compounds of the invention was tested against the DENV-1 TC974 # 666 (NCPV) strain, DENV-2 16681 strain, DENV-3 H87 (NCPV) strain and the DENV-4 H241 strain ( NCPV) in an RT-qPCR assay. Therefore, Vero cells were infected with DENV-1 or -2 or -3 or -4 in the presence or absence of test compounds. On day 3 post-infection, cells were lysed and cell lysates were used to prepare cDNA for both a viral target (at DENV 3'LJTR; Table 2) and a cell reference gene (β-actin, Table 2 ). Subsequently, a time PCR
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102/109 duplex was performed on a Lightcycler480 instrument. The C _p value generated is inversely proportional to the amount of RNA expression of these targets. Inhibition of DENV replication by a test compound results in a shift of Cps to the 3TJTR gene. On the other hand, if a test compound is toxic to cells, a similar effect will be observed on the expression of β-actin. The comparative AACp method is used to calculate the EC50, which is based on the relative gene expression of the target gene (3'UTR) normalized with the cell maintenance gene (β-actin). In addition, CC50 values are determined based on the C _p values acquired for the β-actin maintenance gene.
Table 2: Primers and probes used for quantitative RT-PCR, in real time ·
Initiator / probe Target Sequence ³ ' ^b F3utr258 DENV 3'-UTR 5'-CGGTTAGAGGAGACCCCTC-3 ' R3utr425 DENV 3'-UTR 5'-GAGACAGCAGGATCTCTGGTC-3 ' P3utr343 DENV 3'-UTR FAM-5'-AAGGACTAG-ZEN-AGGTTAGAGGAGACCCCCC-3'-lABkFQ Factin743 β-actin 5'-GGCCAGGTCATCACCATT-3 ' Ractin876 β-actin 5'-ATGTCCACGTCACACTTCATG-3 ' Pactin773 β-actin HEX-5'-TTCCGCTGC-ZE / V-CCTGAGGCTCTC-3'-lABkFQ
^a Reporter (FAM, HEX) and inactivating dyes (ZEN and lABkFQ) are indicated in bold and italics.
^b The nucleotide sequence of the primers and probes was selected from the region conserved in the 3'UTR region of the dengue virus genome, based on the alignment of 300 nucleotide sequences of the four dengue serotypes deposited at Genbank (Gong et al., 2013 , Methods Mol Biol, Chapter 16).
[00330] The culture medium consisted of a minimum essential medium supplemented with 2% fetal calf serum inactivated by heat, gen
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103/109 0.04% tamicin (50 mg / ml) and 2 mM L-glutamine. Vero cells, obtained from ECACC, were suspended in culture medium and 75pL / well was added to 96-well plates (10,000 cells / well) already containing the antiviral compounds. Typically, these plates contain a 5-fold serial dilution of 9 steps of dilution of the test compound to 200 times the final concentration in 100% DMSO (500 nl_; final concentration range: 25μΜ - 0.000064 μΜ or 2, 5 μΜ - 0.0000064 μΜ for the most active compounds). In addition, each plate contains wells that are assigned as virus controls (containing cells and viruses in the absence of compound) and cell controls (containing cells in the absence of virus and compound). Once the cells were added to the plates, the plates were incubated in a fully humidified incubator (37 Ό, 5% CO 2) until the next day. Serotype-1, 2, 3 and 4 dengue virus were diluted to obtain a Cp of -22-24 in the assay. Therefore, 25 μΙ_ of virus suspension was added to all wells containing test compound and to the wells assigned as virus control. In parallel, 25 μΙ_ of culture medium was added to the cell controls. Then, the plates were incubated for 3 days in a fully humidified incubator (37 Ό, 5% CO 2). After 3 days, the supernatant was removed from the wells and the cells washed twice with ice-cold PBS (-100 μΙ_). The cell pellets inside the 96-well plates were stored at -80 ° C for at least 1 day. Then, the RNA was extracted using the Cells-to-CT ™ lysis kit, according to the manufacturer's guidance (Life Technologies). Cell lysates can be stored at 80 ° C or used immediately in the reverse transcription step.
[00331] In the preparation of the reverse transcription step, mixture A (table 3A) was prepared and 7.57 pL / well was distributed in a 96-well plate. After adding 5 pL of the cell lysates, a five minute denaturation step was performed at 75 Ό (table 3B).
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Then, 7.43 μΙ_ of mixture B was added (table 3C) and the reverse transcription step was started (table 3D) to generate cDNA. [00332] Finally, a mixture of RT-qPCR was prepared, mixture C (table 4A), and 22.02 pL / well was distributed in 96-well LightCycler qPCR plates to which 3 μΙ_ of cDNA was added and the qPCR was performed according to the conditions in table 4B on a LightCycler 480.
[00333] Using LightCycler software and an internal LIMS system, dose response curves were calculated for each compound and determined half of the maximum effective concentration (EC50) and half of the maximum cytotoxic concentration (CC50) (Tables 5-8).
Table 3: Synthesis of cDNA using Mixture A, denaturation, Mixture B and reverse transcription.
Mixture A
Signs 8Samples 828 Reaction Vol. (PL) 20 Mis- Concentration Volume to (pL)ture unity Stock Final 1 sample x samples H ₂ O Milli-Q 7.27 6019.56 R3utr425 μΜ 20 0.27 0.15 124.20 Ractin876 μΜ 20 0.27 0.15 124.20 Mixing volume / well (pL) 7.57 Cell lysates 5.00
B Denaturation step:
Step Temp Time Denaturation 75Ό Maintenance 4Ό maintenance
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Mixture B
Samples 864 Mixing Item Concentration Volume to (μΙ) unity Stock Final 1 sample x samples Expand capHIFI 2 X 10.00 1.00 2.00 1728.0 MgCb mM 25.00 3.50 2.80 2419.2 dNTPs mM 10.00 1.00 2.00 1728.0 InhibitorRnase U / pL 40.00 1.00 0.50 432.0 Expand RT U / μΙ- 50.00 0.33 0.13 112.3Total Mix Volume (pL) 7.43
D cDNA Synthesis Protocol
Step Temp Time Transc rev 42Ό: 30 ' Denaturation 99Ό I j ' Maintenance 4Ό maintenance
Table 4: Mixture and qPCR protocol ·
The Mixture C
Samples 833Reaction Vol. (ΜΙ) 25 Mixing Item Concentration Volume to (μΙ)unity Stock Final 1 sample x samples H ₂ The Roche PCR grade 7.74 6447.42 Mixture 2xMM Roche X 2 1 12.50 10412.50 F3utr258 μΜ 20 0.3 0.38 316.54 R3utr425 μΜ 20 0.3 0.38 316.54 P3utr343 μΜ 20 0.1 0.13 108.29 Factin743 μΜ 20 0.3 0.38 316.54 Ractin876 μΜ 20 0.3 0.38 316.54 Pactina773 μΜ 20 0.1 0.13 108.29Mixing Volume / Tube (μ!) 22.02cDNA 3.00
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B qPCR3 Protocol
Step Temp Time Ramp ratepre-incub / skim 95Ό 10 min 1.4 Denaturation 95Ό 10 sec 1.4 40 cycles pairing 58Ό 1 min 2.2 Stretching 72Ό I sec 1.4 Cooling 40Ό I0 sec 1.5
Table 5: EC50, CC50 and SI for compounds against 0 serotype 1 in RT-gPCR assays
Protocol A RT-qPCR serotype 1 TC974 # 666 # of the compound EC50 (μΜ) N CC50 (μΜ) N SI N 1B 0.0015 4 > 2.5 4 > 2290 4 2B 0.0060 5 > 2.5 5 > 744 5 3B 0.0024 3 > 2.5 2 > 1550 2 4B 0.00057 4 > 2.5 4 > 8060 4 5B 0.0020 4 > 2.5 4 > 981 4 6A 0.00064 4 > 2.5 4 > 10900 4 7B 0.00088 3 > 2.5 3 > 6750 3 8A 0.0020 3 > 2.5 3 > 1570 3 9B 0.00099 3 > 2.5 3 > 2860 3 10B 0.00036 3 > 2.5 3 > 8670 3 11B 0.000095 3 > 2.5 3 > 41800 3 12A 0.0021 3 11 3 3850 3 13A 0.00012 3 > 2.5 3 > 32500 3 14B 0.00022 3 2.3 3 8720 3 15B 0.00013 3 4.9 3 46500 3 16B 0.000092 3 > 1.0 3 > 23100 3
N = The number of independent experiments in which the compounds were tested.
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Table 6: EC50, CC50 and SI for compounds against 0 serotype 2 in RT-gPCR assays
Protocol A RT-qPCR serotype 2 1 3681 # of the compound EC50 (μΜ) N CC50 (μΜ) N SI N 1B 0.0024 3 > 2.5 1 > 1480 1 2B 0.0021 3 4.3 3 2070 3 3B 0.0014 3 13 3 5680 3 4B 0.00045 3 2.4 3 6270 3 5B 0.00052 3 2.8 3 6730 3 6A 0.00049 5 11 5 18700 5 7B 0.0019 3 > 2.5 2 > 9150 2 8A 0.00038 3 > 0.99 3 3620 3 9B 0.00047 3 > 2.5 2 > 4250 2 10B 0.00015 3 > 1.0 3 > 10700 3 11B 0.000059 3 5.0 3 64900 3 12A 0.00042 3 14 3 19800 3 13A 0.000057 3 > 2.5 3 > 53100 3 14B 0.00016 4 2.6 3 > 9320 3 15B 0.000100 3 6.3 4 > 22600 3 16B 0.000055 3 > 1.0 3 > 44200 3
N = The number of independent experiments in which the compounds were tested.
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Table 7: EC50, CC50 and SI for compounds against 0 serotype 3 in RT-gPCR assays
Protocol A RT-qPCR serotype 3 h 187 # of the compound EC50 (μΜ) N CC50 (μΜ) N SI N 1B 0.025 3 > 2.5 3 > 123 3 2B 0.038 4 > 2.5 4 > 118 4 3B 0.023 3 > 2.5 3 > 136 3 4B 0.011 3 2.3 3 228 3 5B 0.015 4 > 2.2 4 > 116 4 6A 0.0081 4 > 2.5 4 > 227 4 7B 0.013 3 > 2.5 3 > 259 3 8A 0.015 3 > 2.5 3 > 203 3 9B 0.0064 4 > 1.0 1 > 103 1 10B 0.0059 3 > 2.5 3 > 414 3 11B 0.0065 3 > 2.5 3 > 991 3 12A 0.036 3 12 3 384 3 13A 0.0018 3 > 2.5 3 > 1580 3 14B 0.0055 3 > 2.5 3 > 644 3 15B 0.0028 3 5.8 3 3130 3 16B 0.00087 3 > 1.0 3 > 1780 3
N = The number of independent experiments in which the compounds were tested.
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Table 8: EC50, CC50 and SI for compounds against 0 serotype 4 in RT-gPCR assays
Protocol A RT-qPCR serotype 4 H 241 # of the compound EC50 (pM) N CC50 (pM) N SI N 1B 0.18 3 2.2 2 10 2 2B 0.15 3 > 2.5 3 > 11 3 3B 0.098 3 > 2.5 2 > 25 2 4B 0.076 3 1.8 3 23 3 5B 0.090 3 > 2.5 2 > 33 2 6A 0.060 6 > 2.5 4 > 53 4 7B 0.12 3 1.1 3 10 3 8A 0.056 3 1.1 2 21 2 9B 0.087 3 2.5 3 27 3 10B 0.032 3 1.1 2 27 2 11B 0.020 3 3.3 3 184 3 12A 0.10 3 5.5 3 96 3 13A 0.010 3 2.6 3 251 3 14B 0.023 3 1.4 3 61 3 15B 0.015 3 2.3 3 174 3 16B 0.0053 3 1.2 2 181 2
N = The number of independent experiments in which the compounds were tested.

权利要求:
Claims (10)
[1]
1. Compound, characterized by the fact that it has the formula (I)

[2]
2. Compound or its stereoisomeric form, its pharmaceutically acceptable salt, solvate or polymorph according to claim 1, characterized in that said compound is selected

[3]
3. Compound, according to claim 1, characterized by the fact that said compound has a specific rotation (+).
[4]
4. Compound, according to claim 1, characterized by the fact that said compound is selected from:

[5]
Pharmaceutical composition, characterized in that it comprises a compound, as defined in any one of claims 1 to 4, together with one or more pharmaceutically acceptable excipients, diluents or carriers.
[6]
6. Pharmaceutical composition according to claim 5, characterized by the fact that it comprises a second or more active ingredient (s).
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[7]
7. Pharmaceutical composition according to claim 6, characterized by the fact that the second or more active ingredient (s) is an antiviral agent.
[8]
A compound of formula (I) according to any one of claims 1 to 4, characterized by the fact that it is for use as a medicine.
[9]
Compound of formula (I) according to any one of claims 1 to 4, characterized by the fact that it is for use in the treatment of dengue infection and for the prevention or treatment of diseases associated with Dengue infection.
[10]
10. Compound of formula (I) for use, according to claim 9, characterized by the fact that dengue infection is an infection by viruses of the strains DENV-1, DENV-2, DENV-3 or DENV-4.

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法律状态:
2021-10-19| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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EP17164048.5|2017-03-31|

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EP17164048|2017-03-31|

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PCT/EP2018/058079|WO2018178240A1|2017-03-31|2018-03-29|Substituted indoline derivatives as dengue viral replication inhibitors|

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