substituted indoline derivatives as inhibitors of dengue viral replication

专利摘要:
the present invention relates to substituted indoline derivatives, methods to prevent or treat dengue viral infections when using said compounds and also refers 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:BR112019019928A2
申请号:R112019019928
申请日:2018-03-29
公开日:2020-04-22
发明作者:Didier M Marchand Arnaud；Rudolf Romanie Kesteleyn Bart；Alice Marie-Eve Bardiot Dorothée；Bonfanti Jean-François；Jean-Marie Bernard Raboisson Pierre
申请人:Janssen Pharmaceuticals Inc；Univ Leuven Kath；
IPC主号:

专利说明:

Descriptive Report of the Invention Patent for INDOLINAN DERIVATIVES SUBSTITUTED COIVIO INHIBITORS OF DENGUE'S VIRAL REPLICATION.
[0001] The present invention relates to substituted indoline derivatives, methods to prevent or treat viral dengue infections when using said compounds and also refers 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 [0002] 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 infection by DENV (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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2/77 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.
[0003] Although progress is being made in the development of dengue vaccines 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). The recovery from an infection by a serotype provides immunity to the yeast of life against that serotype but provides only partial and transient protection against subsequent infection by one of the other three serotypes.
[0004] 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 most elevated 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.
[0005] In locations with two or more serotypes circulating simultaneously, also referred to as hyper-endemic regions, the risk of severe dengue disease is significantly higher due to an increased risk of experiencing a secondary infection
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3/77 daily, more serious. Furthermore, in a situation of hyperendemicity, the likelihood of the emergence of more virulent strains is increased, which in turn increases the likelihood of dengue hemorrhagic fever (DHF) or dengue shock syndrome. [0006] Dengue-carrying mosquitoes, 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 carrying dengue is not confined to the USA, but has also been observed in Europe.
[0007] Dengvaxia®, the dengue vaccine produced by Sanofi Pasteur, was first approved in Mexico and has since been approved 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.
[0008] 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.
[0009] Furthermore, specific antiviral drugs are currently not available for the treatment or prevention of infection by the dengue fever virus. What's more, current are not available
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4/77 specific antiviral drugs for the treatment or prevention of infection by the dengue fever virus. There is clearly still a great 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 fiavivirus, more particularly Dengue virus. 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 derivatives and indoiine as antagonists of cold menthol receptors for the treatment of central and inflammatory diseases. WO2013 / 045516 discloses indole and indoiine derivatives for use in the treatment of dengue viral infections.
[0011] The present invention now provides compounds, substituted indoindin 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 virus infections.
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5/77 of Dengue.
[0014] The present invention also relates to the use of such compounds as medicines and their use for 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:
where [0017] R ¹ is fluorine, R ² is -CH2CH2OH, R ³ is trifluoromethyl and R ⁴ is hydrogen or [0018] R ¹ is fluorine, R ² is -CH2CH2OH, R ³ is trifluoromethyl and R ⁴ is methoxy or [0019] R ¹ is fluorine, R ² is -CH2CH2OH, R ³ is trifluoromethoxy and R ⁴ is hiPetition 870190095446, from 24/09/2019, p. 10/25
6/77 ου hydrogen [0020] R ¹ is chlorine, R ² is -CH ₂ CH ₂ OH, R ³ is trifluoromethyl and R ⁴ is hydrogen or [0021] R ¹ is chlorine, R ² is -CH2CH2OH, R ³ is trifluoromethyl and R ⁴ is methoxy or [0022] R ¹ is chlorine, R ² is -CH2CH2OH, R ³ is trifluoromethoxy and R ⁴ is hydrogen or [0023] R ¹ is chlorine, R ² is - (CH ₂ ) ₃ COOH, R ³ is trifluoromethyl and R ⁴ is hydrogen or [0024] R ¹ is chlorine, R ² is - (CH ₂ ) 3COOH, R ³ is trifluoromethyl and R ⁴ is methoxy or [0025] R ¹ is chlorine, R ² is - (CH ₂ ) ₃ COOH, R ³ is trifluoromethoxy and R ⁴ is hydrogen;
[0026] or a pharmaceutically acceptable salt, solvate or polymorph thereof.
[0027] The compounds specifically mentioned above are
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[0028] 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 in conjunction with one or more pharmaceutically acceptable excipients, diluents or vehicles.
[0029] The 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.
[0030] 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, p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic acid and similar acids.
[0031] The compounds of the invention can also exist in unsolvated and solvated forms. 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.
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[0032] The term polymorph refers to the ability of the compound of the invention to exist in more than one crystal shape or structure. [0033] 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. They will generally be administered as a formulation in combination with one or more pharmaceutically acceptable excipients. The term excipient is used herein to describe any ingredient other than 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 addition salt, as the active ingredient is combined in admixture with a pharmaceutically acceptable carrier, which vehicle can take a wide variety of forms depending on the form of preparation desired for administration. These pharmaceutical compositions are desirably in unitary dosage form suitable, for example, for oral or rectal administration. For example, in the preparation of compositions in the oral dosage form, any of the usual pharmaceutical means such as, for example, water, glycols, oils, alcohols can be employed
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9/77 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.
[0035] 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.
[0036] 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, more preferably 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. These underdoses can be formulated as dosage forms
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10/77 unit gem, for example, containing 1 to 1000 mg, and in particular 5 to 200 mg of active ingredient per unit dosage form.
[0037] 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.
[0038] 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.
[0039] 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 said compounds may have. [0040] 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 as a mixture
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11/77 with each other, are intended to fall within the scope of the present invention, including any racemic mixtures or racemates.
[0041] 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 a stereoisomeric excess of 100% (that is, 100% of an isomer and none of the others), more particularly, to compounds or intermediates with a stereoisomeric excess of 90% to 100%, even more particularly 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.
[0042] 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. 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 corrections
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12/77 in 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.
[0043] 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 *:
[0044] Due to the presence of 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 absolute (R) or (S) 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.
[0045] In one aspect, the present invention relates to a first group of compounds of formula (I) in which the compounds of formula (I) have specific rotation (+).
[0046] 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 (-).
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Examples
LC / EM METHODS [0047] 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).
[0048] 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.
[0049] Compounds are described by their retention times (Rt) 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 + ΝΗ4Γ, [M + HCOO] ·, etc.). In the case of molecules with multiple isotopic patterns (Br, Cl), the reported value is 0 obtained for the lowest isotopic mass. All results were obtained with experimental uncertainties that are commonly associated with the method used. [0050] Hereinafter, “SQD” stands for Single Quadropole Detector, “MSD” Selective Mass Detector, “TA” at room temperature, “Ethyl siloxane / silica bridge hybrid”, “DAD” Diode Array Detector, “HSS ”High Resistance silica.
[0051] LC / EM method codes (Flow expressed in mL / min; column temperature (T) in ° C; Operating time in minutes).
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Method code instrument Column Mobile phase Gradient FlowCol T Run time (MIN) LC-A Waters: Acquity® UPLC® - DADQuattro Micro ™ Waters: BEH®C18 (1.7 pm, 2.1 x 100 mm) A: 95% CH3COONH4 7mM / 5% CH ₃ CN, B: CH3CN A at 84.2% for 0.49 min, up to A at 10.5% in 2.18 min, hold for 1.94 min, from voita to A at 84.2% in 0.73 min, hold for 0 , 73 min. 0.343 mL / min40 ° C 6.2 LC-B Waters: Acquity® Class H - DAD and SQD2TM Waters: BEH®C18 (1.7 pm, 2.1 x100 mm) A: 95% CH3COONH4 7mM / 5% CH ₃ CN, B: CH3CN 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 at 15.8% in 0.73 min, hold for 0.49 min. 0.343 mL / min40 ° C 6.1 LC-C Waters: Acquity® UPLC® DAD- Acquity® TQ detector Waters: UPLCHSS C18 (1.8 pm, 2.1 x 50 mm) A: 0.1% HCOOHB: CH3CN 50% A to 10% A in3.5 min, keep for1.5 min. 0.5 mL / min40 ° C 5 LC-D Waters: Acquity®UPLC® - DAD-SQD Waters: BEH C18(1.7 pm, 2.1 x 50mm) A: 10mM CH3COONH4 in 95% H2O + 5% CH3CNB: CH3CN From A to 95% to A to 5% in 1.3 min, keep for 0.7 min. 0.8 mL / min55 ° C 2
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SFC / EM methods [0052] 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 , a diode array detector 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.
[0053] Analytical methods of SFC / MS (Flow expressed in mL / min; column temperature (T) in ° C; Operating time in minutes, Backpressure (BPR) in bars).
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Method code column mobile phase gradient Flow Runtime ColT BPR SFC-A Daicel Chíralpak® AD-H column (5 pm,150 x 4.6 mm) A: CO ₂ B: EtOH (+ 0.3% iPrNH ₂ ) 20% B keep 7 min, 335 7100 SFC-B Regis Whelk O1® (S, S) column (3 pm, 100 X 4.6 mm) A: CO ₂ B: EtOH (+ 0.3% iPrNH ₂ ) 50% B keep 3 min, 3.535 3103 SFC-C Daicel Chíralpak® AD-H column (5 pm, 150 X 4.6 mm) A: CO ₂ B: EtOH (+ 0.3% iPrNH ₂ ) 30% B keep 7 min, 335 7100 SFC-D Daicel Chíralpak® IC-3 column (3 pm, 100 x 4.6 mm) A: CO ₂ B: EtOH (+ 0.3% iPrNH ₂ ) B at 40% keep 3 min, 3.535 3103 SFC-E Daicel Chíralpak® IA column (5 pm, 150x4.6 mm) A: CO ₂ B: iPrOH (+ 0.3% iPrNH ₂ ) 30% B keep 7 min, 3.535 7103
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Method code column mobile phase gradient Flow Runtime ColT BPR SFC-F Daicel Chiraipak® IC column (5 pm, 150 x 4.6 mm) A: CO ₂ B: MeOH 30% B keep 7 min, 335 7100 SFC-G Daicel Chiraipak® IC-3 column (3 pm,100 X 4.6 mm) A: CO ₂ B: IPrOH (+ 0.3% IPrNH ₂ ) 40% B retention for 5 minutes, 3.535 ⁵ 103 SFC-H Daicel Chiraipak® iC speaker (5 pm,150 X 4.6 mm) A: CO ₂ B: EtOH / IPrOH 50/50 (+ 0.3% IPrNH ₂ ) 25% B keep 7 min, 335 7100 SFC-I Daicel Chiraipak® 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
LLIL1
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FUSION POINTS [0054] The values are peak values or melting ranges, and are obtained with experimental uncertainties that are commonly associated with this analytical method.
DSC823e (indicated as DSC) [0055] 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 ° C.
Optical Rotations:
[0056] Optical rotations were measured in a PerkinElmer 341 polarimeter with a sodium lamp and reported as follows: [α] ° (λ, c g / 100 mL, solvent, T ° C).
[0057] [α] χ ^τ = (100α) / (/ xc): where / is the path length in dm and c is the concentration in g / 100 mL for a sample at a temperature T (° C) 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. The 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) ⁺ protonated molecular ion hci hydrochloric acid aq. aqueous / aqueous HPLC high performance liquid chromatography : Boc ferc-butyloxycarbonyl iPrNH ₂ isopropylamine : BOC2O di-tert-butyl dlcarbonate IPrOH 2-propanol br extended K ₂ CO ₃ Potassium carbonate CH3CN acetonitrile LÍAIH4 aluminum hydride and lithium ί CHCb chloroform m / z mass-to-charge ratio CH2CH2 dichloromethane Me methyl CH3OH methanol MeOH methanol
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co ₂ ; carbon dioxide MgSCU i magnesium sulfate d i voicemail 1 min i minute (s) DCM i dichloromethane | N ₂ i nitrogen Dl EA ; diisopropylethylamine Na ₂ COs and sodium carbonate DIPE diisopropyl ether i; Na ₂ SO4; sodium sulfate DMA i dimethylacetamide i; NaBHk i sodium borohydride DMAP ; 4-dimethylaminopyridine NaHCOs i sodium bicarbonate DME ; 1,2-dimethoxyethane ii NaOH; sodium hydroxide DMF i dimethylformamide i; NH4CI i ammonium chloride DMSO i dimethylsulfoxide | q i quartet eq.EW ; equivalent ii ta or TA; room temperaturediethyl ether i; s i singlet Et ₃ N i triethylamine 11 i triplet EtOAc ; tBuOK ethyl acetate; potassium ferc-butanolate EtOH ethanol i; TEA i triethylamine h ₂ o i water | TFA i trifluoroacetic acid H2SO4 ; sulfuric acid THF; tetrahydrofuranO- (7- | i hexafluorophosphate HATU aza-1H-benzotriazol-1-yl) - ii i,,,; , _k „... _Λ . ii TMSCI; trimethylsihla chloride i Ν, Ν, Ν, N -tetramethyl-uromo - [i CAS [148893-10-1] í
Example 1: synthesis of 2- (4-fluoro-2- (2-hydroxyphenyl) 2 ((3-methoxy ”5” (methyl · sulfonyl) phenH) amino) “1“ (6- (trifluoromethyl) indole! N-1-yl) etan “1“ Ona (Compound 1) and chiral separation in Enantiomers 1A and 1B.
Ό— —-QBn
TSf ·. < 8'C
ISn.fn
3) Νβδ. -7 «to -4ίΓ '£
ÍS :: ·
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^ Fí3Í '
Synthesis of intermediate 1a:
[0058] A solution of 4-fluoro-2-methoxyphenylacetic acid [CAS 886498-61-9] (10 g, 54.30 mmol) in EtOH (200 mL) and H2SO4 (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 filtered MgSCX 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 in the next step without further purification.
Synthesis of intermediate 1b:
[0059] Boron tribromide (109.3 mL, 109.3 mmol) was added dropwise to a chilled solution (-30 ° C) of ethyl 2- (4-fluor-2-methoxyphenyljacetate) (11.6 g , 54.7 mmol) in CH2Cl2 (300 mL) The reaction was stirred at -20 ° C for 1 h and quenched with CH3OH. The pH was adjusted to 8 by the addition of a saturated solution of NaHCOs. The solution was extracted with CH2Cl2 and the combined organic layers were dried over MgSO *, 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 in the step without further purification.
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Synthesis of intermediate 1c:
[0060] To a mixture of 2- (4-fluoro-2-hydroxyphenyl) ethyl acetate 1b (1.24 g, 6.26 mmol) and cesium carbonate (4.08 g, 12.5 mmol) in DMF (20 ml) Benzyl ether and 2-bromoethyl [CAS 146237-9] (1.61 g, 7.51 mmol) were added. The reaction mixture was stirred at room temperature overnight. Water 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 CH2 Cl2 (15% to 100%) in heptane to give 2- (2- (2- (benzyloxy) ethoxy) -4-fluorophenyl) ethyl acetate 1c ( 1.55 g).
Synthesis of intermediate 1d:
[0061] To a 1 M solution of lithium bis (trimethylsilyl) amide in THF (4.51 mL, 4.51 mmol) cooled (-78 ° C), a solution of 2- (2- (2 - ethyl (benzyloxy) ethoxy) -4-fluorophenyl) acetate 1c (0.750 g, 2.26 mmol) in THF (4 mL). After 1 h at -78 ° C, chlorotrimethylsilane (0.458 ml, 3.61 mmol) was added. The reaction mixture was stirred at 78 ° C for 15 min. / -Bromosuccinimide (0.482 g, 2.71 mmol) was added and stirring was continued at -40 ° C for 2 h. The reaction mixture was poured into H2O and extracted twice with EtOAc. The organic phases were combined, dried over Na2SO4, filtered and concentrated under reduced pressure to provide 2- (2- (2- (benzyloxy) ethoxy) 4-fluorophenyl) -2-bromoacetate 1d (0.920 g), which was used in the step without further purification.
Synthesis of intermediate 1e:
[0062] A mixture of 2- (2- (2- (benzyloxy) ethoxy) -4-fluorophenii) -2-bromoacetate 1d (0.920 g, 2.24 mmol) and 3-methoxy-5- (methylsulfonll) aniline [ CAS 62606-02-4] (1.35 g, 6.71 mmol) in CH3CN (5 ml) and THF (5 ml) was stirred at 60 ° C overnight. The reaction mixture was diluted with EtOAc and washed with 1 N HCI. The organic phase was washed with 1 HCI
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N, a saturated aqueous solution of NaHCCh, 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 (5% to 50%) in heptane to give 2- (2- (2- (benzyloxy) ethoxy) -4-fluorophenyl) -2 - (( 3-methoxy-5 (methylsulfonyl) phenyl) amino) ethyl acetate 1e (0.870 g).
Synthesis of intermediate 1f:
[0063] A mixture of 2- (2- (2- (benzyloxy) ethoxy) -4-fluorophenyl) -2 - ((3-methoxy-5- (methylsulfonyl) phenyl) amino) ethyl acetate 1e (0.868 g, 1, 63 mmol) and 10% palladium on carbon (0.180 g) in EtOAc (30 mL) was stirred at room temperature under an atmosphere of H2. The readonal mixture was filtered through celite®. The filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel using a gradient of EtOAc (30% to 100%) in heptane to give 2- (4-fluorine-2- (2-hydroxyethoxy) phenyl) -2 - ((3- methoxy-5- (methylsulfonyl) phenyl) amino) ethyl acetate if, quantitatively.
Synthesis of intermediate 1q:
[0064] To a solution of 2- (4-fluorine-2- (2-hydroxyethoxy) phenyl) -2 - ((3 methoxy · 5- (methylsulfonyl) phenyl) amino) ethyl acetate 1f (0.910 g, 2.06 mmol) in THF (6 mL), MeOH (6 mL) and HzO (6 mL) were added lithium hydroxide monohydrate (0.432 g, 10.3 mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was partially concentrated under reduced pressure to remove THF and MeOH. The residual aqueous solution was acidified with 1N HCI and extracted with CH2Cl2. The organic phase was dried over Na ₂ SO4, filtered and concentrated under reduced pressure to provide 2- (4-fluoro-2- (2hydroxyethoxy) phenyl) -2 - ((3-methoxy-5- (methylsulfonyl) phenyl) amino acid ) acetic 1g (0.736 g), which was used in the next step without further purification. Synthesis of Compound 1 and chiral separation in Enantiomers 1A and 1B: [0065] Method 1: To a solution of 2- (4-fluorine-2- (2-hydroxy) acidPetition 870190095446, of 09/24/2019, page 42 / 103
23/77 toxic) phenyl) -2 - ((3-methoxy-5- (methylsulfonyl) phenyl) amino) acetic 1g (0.200 g, 0.484 mmol) in DMF (4 mL) HATU (0.184 g, 0.484 mmol) was added , triethylamine (0.267 ml, 1.94 mmol) and 6- (trifluorornethyl) indoline [CAS 181513-29-1] (0.091 g, 0.484 mmol). The reaction mixture was stirred at room temperature for 4 h. The reaction mixture was diluted with EtOAc and washed with 1N HCI. The organic phase was washed with a saturated aqueous solution of NaHCO3, Η ₂ Ο and a saturated aqueous solution of sodium chloride, dried over NasSCU filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel using a gradient of EtOAc (2% to 40%) in CH2 Cl2. Fractions containing the desired product were combined and concentrated under reduced pressure. The residue was purified by preparative TLC using a mixture of EtOAc (50%) in CH2 Cl2 as eluent. Subsequent purification by preparative HPLC (column: XBridge® C18 - 5 pm 100 x 19 mm, mobile phase: 10% NH4OAc solution in H2O, CH3CN) provided 2- (4-fluorine-2- (2-hydroxyethoxy) phenyl) - 2 - ((3-methoxy ~ 5 ~ (methylsulfonyl) phenyl) amino) -1- (6 ~ (trifluoromethyl) indolin ~ 1-yl) ethanone (Compound 1, 0.043 g) as a racemic mixture.
[0066] Method 2: To a solution of 2- (4-fluorine-2- (2-hydroxyethoxy) phenyl) -2 - ((3-methoxy-5- (methylsulfonyl) phenyl) amino) acetic acid 1g (0.300 g , 0.726 mmol) in Me-THF (5.4 mL) under N ₂ flow, 6 (trifluoromethyl) indoline [CAS 181513-29-1] (0.136 g, 0.726 mmol), / V-diisopropylethylamine (264 pL) was added , 1.556 mmol) and propylphosphonic anhydride (653 pL, 1.09 mmol). The reaction was stirred at room temperature for 16 h. The mixture was poured into water and extracted with EtOAc. The combined organic layers were washed with a 10% solution of K2CO3 in water and then with water. The organic solution was dried over MgSO4, filtered, and the solvent was concentrated under reduced pressure. This fraction (0.47 g) was combined with a second batch (total amount: 0.585 g) and purified by flash chromatography on silica gel (15
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24/77 pm, 24 g, CHzCh / MeOH 99.5 / 0.5). The pure fractions were combined and concentrated under reduced pressure to provide 2- (4-fluoro-2 (2-hydroxyethoxy) phenyl) ~ 2 ~ ((3-methoxy-5- (methylsulfonyl) phenyl) amino) -1- (6 (trifluoromethyl) indolin-1-yl) ethanone (Compound 1, 0.160 g) as a racemic mixture.
[0067] The Enantiomers of Compound 1 (160 mg) were separated by means of Preparative Chiral SFC (stationary phase: Chiralpak® AD-H 5 pm 250 x 20 mm, mobile phase: 75% CO ₂ , 25% EtOH (+0 , 3% iPrNHs)). The first eluted product (72 mg) was solidified from heptane / diisopropyl ether to give Enantiomer 1Â (50 mg). The second eluted product (80 mg) was solidified from heptane / diisopropyl ether to give Enantiomer 1B (43 mg).
Compound 1:
[0068] ¹ H NMR (300 MHz, DMSO-cfe) δ ppm 3.10 (s, 3 H) 3.22 (m, 2 H) 3.62 - 3.92 (m, 5 H) 3.97 - 4.22 (m, 3 H) 4.46 (m, 1 H) 4.98 (s I, 1 H) 5.82 (d, J-7.9 Hz, 1 H) 6.56 (s , 1 H) 6.62 (s, 1 H) 6.80 (t, J-7.7 Hz, 1 H) 6.92 (s, 1 H) 6.95 - 7.11 (m, 2 H ) 7.29 - 7.53 (m, 3 H) 8.39 (s, 1 H) [0069] LC-MS (LC-C method): Rt 1.37 min, MH ⁺ 583
Enantiomer 1A:
[0070] ¹ H NMR (500 MHz, DMSO-de) 6 ppm 3.10 (s, 3 H) 3.14 3.29 (m, 2 H) 3.73 (s, 3 H) 3.75 - 3.78 (m, 1 H) 3.78 - 3.89 (m, 1 H) 3.98 - 4.23 (m, 3 H) 4.37 - 4.55 (m, 1 H) 4, 97 (t, J = 5.4 Hz, 1 H) 5.82 (d, J = 8.2 Hz, 1 H) 6.56 (s, 1 H) 6.62 (s, 1 H) 6, 79 (dt, J = 2.2, 8.5 Hz, 1 H) 6.91 (s, 1 H) 6.98 - 7.04 (m, 2 H) 7.32 - 7.43 (m, 2 H) 7.46 (d, J = 7.9 Hz, 1 H) 8.39 (s, 1 H).
[0071] LC / MS (LC-A method): Rt 3.04 min, MH * 583 [0072] [a] _D ²⁰ : -49.6 ° (c 0.25, DMF) [0073] Chiral SFC ( SFC-A method): Rt 2.76 min, MH ⁺ 583, chiral purity 100%.
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Enantiomer 1B:
[0074] ¹ H NMR (500 MHz, DMSO-c / e) δ ppm 3.10 (s, 3 H) 3.14 3.29 (m, 2 H) 3.73 (s, 3 H) 3, 75 - 3.78 (m, 1 H) 3.78 - 3.89 (m, 1 H) 3.98 - 4.23 (m, 3 H) 4.37 - 4.55 (m, 1 H) 4.97 (t, J = 5.4 Hz, 1 H) 5.82 (d, J = 8.2 Hz, 1 H) 6.56 (s, 1 H) 6.62 (s, 1 H) 6.79 (dt, J = 2.2, 8.5 Hz, 1 H) 6.91 (s, 1H) 6.98 - 7.04 (m, 2 H) 7.32 - 7.43 (m , 2 H) 7.46 (d, J = 7.9 Hz, 1 H) 8.39 (s, 1 H).
[0075] LC / MS (LC-A method): Rt 3.04 min, MH * 583 [0076] [a] _D ²⁰ : +51.7 ^o (c 0.23, DMF) [0077] Chiral SFC ( method SFC-A): Rt 4.16 min, MH ⁺ 583, chiral purity 100%.
Example 2: synthesis of 2 (4 "fluorine-2- (2hydroxyethoxy) phenyl) ~ 2 ~ ((3methoxy" 5 (methylsulfonyl) phenyl) amino) 1 (5 "methoxy6 (trifluoromethyl) indolin-1-yl) ethanone (Compound 2) and chiral separation in Enantiomers 2A and 2B.

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Synthesis of intermediate 2a:
[0078] 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 mm) in DMF (200 ml) at 10 ° C (2-bromoethoxy) (phen> butyl) dimethyl · silane [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 MgSÜ4, 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 2- (2- (2 ~ ((ferc ~ butyldimethylsilyl) oxy) ethoxy) -4-fluorophenyl) ethyl acetate 2a (17.7 g).
Synthesis of intermediate 2b:
[0079] To a 1 M solution of lithium bis (trimethylsilyl) amide in THF (28.05 ml, 28.05 mmol), cooled to -78 ° C, a solution of 2- (2- (2 - ((ferc-butyldimethylsilyl) oxy) ethoxy) -4-fluorophenyl) ethyl acetate 2a (5 g, 14.03 mmol) in THF (30 ml). After stirring for 1 h at -78 ° C, chlorotrimethylsilane (2.85 ml, 22.44 mmol) was added. The reaction mixture was stirred at -78 ° C for 15 min. / V-Bromosuccinimide (3 g, 16.83 mmol) in THF (30 mL) was added and stirring was continued at -55 ° C 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 - ((ferc-butyldimethylsilyl) oxy) ethoxy) -4-fluorophenyl ) ethyl acetate 2b (6.57 g) which was used in the next step without further purification.
Synthesis of intermediate 2c:
[0080] A mixture of 2-bromo-2- (2- (2 - ((tert-butyldimethylsilyl) oxy) ethoxy) -4-fluorophenyl) ethyl acetate 2b (3 g, 6.89 mmol), 3-methoxy ~ 5 ~ (methylsulfonyl) aniline [CAS 62606-02-4] (2.08 g, 10.3 mmol) and diisopropyl
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27/77 piletylamine (2.37 ml, 13.8 mmol) in CH3CN (60 ml) was stirred at 50 ° C overnight. The reaction mixture was concentrated under reduced pressure. The residue was absorbed with EtOAc and washed with 0.5 N HCI and water. The organic phase was dried over MgSCU, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 μΜ, 120 g, heptane / EtOAc 90/10 to 80/20) to give 2- (2- (2 - ((ferc-butyldimethylsilyl) oxy) ethoxy ) -4-fluorophenyl) -2 - ((3-methoxy-5 (methylsulfonyl) phenyl) amino) ethyl acetate 2c (2.6 g).
Synthesis of intermediate 2d:
[0081] Lithium hydroxide monohydrate (205 mg, 4.8 mmol) was added in portions to a solution of 2- (2 ~ (2 - ((tert-butyldimethylsilyl) oxy) ethoxy) -4-fluorophenyl) -2 - ((3-methoxy-5- (methylsulfonyl) phenyl) amino) ethyl acetate 2c (2,227 g, 4.09 mmol) in THF / CH3OH / H2O (1/1/1) (100 mL) at 10 ° C. The reaction was stirred at room temperature for 4 h and diluted with water. After cooling to 0 ° C, the aqueous solution was slowly acidified to pH 6 with 0.5 N HCI 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 - ((tert-butyldimethylsilyl) oxy) ethoxy) -4-fluorophenyl) -2 - ((3- methoxy-5- (methylsulfonyl) phenyl) amino) acetic 2d (2 g). The compound was used in the next step without further purification.
Synthesis of intermediate 2e:
[0082] A mixture of 1 ~ methoxy ~ 4 ~ nitro-2- (trifluorometH) benzene [CAS 654-76-2] (24.5 g, 110.8 mmol) and 4-chlorophenoxyacetonitrile [CAS 3598-13-8 ] (20.4 g, 121.9 mmol) in DMF (100 mL) was added dropwise for 30 min to a stirred solution of tBuOK (27.35 g, 243.7 mmol) in DMF (100 mL) at -10 ° C. After addition, the purple solution was kept at -10 ° C 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
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28/77 nitro-4- (trifluoromethyl) phenyl) acetonitrile 2e (used as such in the next step).
Synthesis of intermediate 2f:
[0083] A solution of 2- (5-methoxy-2-nitro-4- (trifluoromethyl) phenyl) acetonitrile 2e (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 CH 3 Cl and CH 3 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) -1H-indole 2f (15.6 g).
Synthesis of intermediate 2g:
[0084] At 0 ° C, BFh-Pyridine (23.5 mL, 232.4 mmol) was added dropwise to a solution of 5-methoxy-6- (trifluoromethyl) -1H-indole 2f (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 ° C). The precipitate was filtered off, washed with water (twice) and coevaporated under reduced pressure with toluene to give 5-methoxy-6 (trifluoromethyl) indoline 2g (9 g).
Synthesis of intermediate 2h:
[0085] To a solution of 2- (2- (2 - ((ferc-butyldimethylsilyl) oxy) ethoxy) -4-fluorophenyl) -2 - ((3-methoxy-5 ”(methylsulfonyl) phenll) amino) acetic acid 2d (1 g, 1.90 mmol) in DMF (10 mL) were added HATU (1.08 g, 2.84 mmol), diisopropylethylamine (940 pL, 5.69 mmol) and 5-methoxy-6
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29/77 (trifluoromethyl) indoline 2g (412 mg, 1.90 mmol). The reaction mixture was stirred at room temperature for 4 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 1N HCL, water, dried over MgSCA, filtered and the solvent was concentrated under reduced pressure to give 2- (2- (2 ~ ((tert ~ butyldimethylsilyl) oxy) ethoxy) -4fluorophenyl) “2“ ((3 ~ methoxy · 5 ~ (methylsulfonyl) phenyl) amino) “1“ (5 ~ methoxy · 6- (trl · fluoromethyl) indolin1yl) ethanone 2h (1.36 g, LC purity: 70%). The impure compound was used directly in the next reaction step. Synthesis of Compound 2 and chiral separation in Enantiomers 2A and 2B: [0086] A solution of 2- (2 ~ (2 ~ ((tert ~ butyldimethylsilyl) oxy) ethoxy) -4-fluorophenyl) -2 - ((3 -methoxy-5- (methylsulfonyl) phenyl) amino) -1- (5-methoxy-6- (trifluoromethyl) indolin-1-yl) ethanone 2h (1.29 g, 1.77 mmol) in 4M HCI in dioxane ( 30 ml) and dioxane (100 ml) was stirred at room temperature for 1 h. The solvent was removed under reduced pressure. EtOAc and 10% K2CO3 aqueous solution were added. 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, 80 g, CHsCb / MeOH / NhUOH 99/1 / 0.1) to give, after crystallization from CH3CN, 2- (4-fluorine ~ 2 ~ (2-hydroxyethyl ~ xi) phenyl) -2 - ((3-methoxy-5- (methylsulfonyl) phenyl) amino) -1- (5-methoxy-6- (trifluoromethyl) indolin-1-yl) ethanone (Compound 2.595 mg) as a racemate. [0087] The Compound 2 Enantiomers (560 mg) were separated by means of Chiral Preparative SFC (stationary phase: Whelk 01 (S, S) ® 5 pm 250 x 21.1 mm, Mobile phase: 50% CO ₂₎ 50 % EtOH (+ 0.3% iPrNHb)). The first eluted enantiomer (288 mg) was crystallized from CHsCN / diisopropyl ether to give the 2A Enantiomer (240 mg). The second eluted enantiomer (293 mg) was crystallized from CHsCN / diisopropyl ether to give the 2B Enantiomer (232 mg).
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Compound 2:
[0088] ¹ H NMR (500 MHz, DMSO-cfe) δ ppm 3.09 (s, 3 H) 3.12 -
3.29 (m, 2 H) 3.73 (s, 3 H) 3.74 - 3.80 (m, 1 H) 3.80 - 3.90 (m, 4 H) 4.02 (td, > 10.4, 7.2 Hz, 1 H) 4.05 - 4.17 (m, 2 H) 4.42 (td,> 10.4, 6.2 Hz, 1 H) 4.97 (t ,> 5.7 Hz, 1 H) 5.79 (d,> 8.20 Hz, 1 H) 6.56 (s, 1 H) 6.61 (s, 1 H) 6.78 (td,> 8.51, 2.52 Hz, 1 H) 6.90 (s, 1 H) 6.95 - 7.04 (m, 2 H) 7.24 (s, 1 H) 7.37 (dd,> 8.67, 6.78 Hz, 1 H) 8.35 (s, 1 H) [0089] LC-MS (LC-A method): Rt 3.02 min, MH ⁺ 613 [0090] Melting point: 215 ° C
2A Enantiomer:
[0091] ¹ H NMR (500 MHz, DMSO-de) δ ppm 3.09 (s, 3 H) 3.12 -
3.29 (m, 2 H) 3.73 (s, 3 H) 3.74 - 3.80 (m, 1 H) 3.80 - 3.90 (m, 4 H) 4.02 (td, > 10.4, 7.2 Hz, 1 H) 4.05 - 4.17 (m, 2 H) 4.42 (td,> 10.4, 6.2 Hz, 1 H) 4.97 (t ,> 5.7 Hz, 1 H) 5.79 (d,> 8.20 Hz, 1 H) 6.56 (s, 1 H) 6.61 (s, 1 H) 6.78 (td,> 8.51, 2.52 Hz, 1 H) 6.90 (s, 1 H) 6.95 - 7.04 (m, 2 H) 7.24 (s, 1 H) 7.37 (dd,> 8.67, 6.78 Hz, 1 H) 8.35 (s, 1 H) [0092] LC / MS (LC-A method): Rt 3.00 min, MH ⁺ 613 [0093] [a] _D ²⁰ : + 53.5 ° (c 0.2392, DMF) [0094] SFC quirai (SFC-B method): Rt 1.43 min, ΜΗ ^1- 613, 100% quirai purity.
[0095] Melting point: 204 ° C
2B Enantiomer:
[0096] ¹ H NMR (500 MHz, DMSO-dg) δ ppm 3.09 (s, 3 H) 3.12 -
3.29 (m, 2 H) 3.73 (s, 3 H) 3.74 - 3.80 (m, 1 H) 3.80 - 3.90 (m, 4 H) 4.02 (td, > 10.4, 7.2 Hz, 1 H) 4.05 - 4.17 (m, 2 H) 4.42 (td,> 10.4, 6.2 Hz, 1 H) 4.97 (t ,> 5.7 Hz, 1 H) 5.79 (d,> 8.20 Hz, 1 H) 6.56 (s, 1 H) 6.61 (t,> 1.73 Hz, 1 H) 6 .78 (td,> 8.51, 2.52 Hz, 1 H) 6.90 (s, 1 H) 6.95 - 7.04 (m, 2 H) 7.24 (s, 1 H) 7 , 37 (dd,> 8.67, 6.78 Hz, 1 H) 8.35 (s, 1 H) [0097] LC / MS (LC-A method): Rt 3.00 min, MH ⁺ 613 [ 0098] [a] _D ²⁰ : -56.5 ° (c 0.255, DMF)
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31/77 [0099] Chiral SFC (SFC-B method): Rs 1.72 min, MH ⁺ 613, chiral purity 99.8%.
[00100] Melting point: 206 ° C
Example 3: synthesis of 2- (4-fluoro-2- (2-hydroxyethoxy) phen! 1) -2 - ((3-methoxy-5 (methylsulfonyl) phenyl) amino) -1- (6- (trifluoromethoxy) indolin -1-yl) ethanone (Compound 3) and chiral separation in Enantiomers 3A and 3B.

Synthesis of intermediate 3a:
[00101] To a solution of 2- (2 ~ (2 - ((tert-butyldimethylsilyl) oxy) ethoxy) -4-fluorophenyl) -2 - ((3-methoxy-5- (methylsulfonyl) phenyl) amino) acetic acid 2d (1 g, 1.90 mmol) in DMF (10 mL) were added HATU (1.08 g, 2.84 mmol), diisopropylethylamine (940 pL, 5.69 mmol) and 6 ~ (trifluoro ~ methoxy ) indoline [CAS 959235-95-1] (385 mg, 1.90 mmol). The reaction mixture was stirred at room temperature for 4 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% K2CO3 solution in water, saturated NaCl solution in water, water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure to give 2- (2- (2 - (( ferc-butyldimethylsHI) oxy) ethoxy) -4-fluorophenyl) -2 - ((3-methoxy-5- (methylsu Ifon H) phen H) amine) -1- (6- (trifluoromethoxy) indolin ~ 1 ~ il) ethanone 3a (1.32 g). The crude compound was used without purification in the next reaction step.
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Synthesis of Compound 3 and chiral separation in Enantiomers 3A and 3B: [00102] A solution of 2- (2- (2 - ((ferc-butyldimethylsilyl) oxy) ethoxy) -4fluorophenyl) -2 - ((3-methoxy-5 - (methylsulfonyl) phenyl) amino) -1- (6- (trifluoromethoxy) indolin-1-yl) ethanone 3a (1.17 g, 1.64 mmol) in 4M HCI in dioxane (3.3 mL) and dioxane (50 ml) was stirred at room temperature for 1 h. The solvent was removed by evaporation under reduced pressure. EtOAc and 10% K2CO3 solution in water were added. 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, CHhCb / MeOH 99.5 / 0.5) to give 2 ~ (4-fluorine-2- (2 ~ hydroxyethoxy) phenyl ) -2 - ((3 ~ methoxy ~ 5 ~ (methyl ~ sulfonyl) phenyl) amino) -1- (6- (trifluoromethoxy) indolin-1-yl) ethanone (Compound 3, 508 mg) as a racemate. An analytical sample of Compound 3 was solidified from CHaCN / diisopropyl ether (35 mg). The remaining amount was used to separate Compound 3 enantiomers via Preparative Chiral SFC (stationary phase: Chiraipak® AD-H 5 pm 250 x 20 mm, mobile phase: 70% CO ₂ , 30% EtOH (+ 0.3% iPrNHs )). The first eluted enantiomer (166 mg) was solidified from heptane / diiisopropyl ether to give the 3Â Enantiomer (130 mg). The second eluted enantiomer (165 mg) was solidified from heptane / diisopropyl ether to give the 3B Enantiomer (110 mg).
Compound 3:
[00103] ¹ H NMR (500 MHz, DMSO-cfe) δ ppm 3.10 (s, 3 H) 3.11 -
3.25 (m, 2 H) 3.73 (s, 3 H) 3.74 - 3.79 (m, 1 H) 3.79 - 3.88 (m, 1 H)
3.96 - 4.19 (m, 3 H) 4.45 (dt, J = 6.3, 10.4 Hz, 1 H) 4.95 (t, J-5.52 Hz, 1 H) 5 , 81 (d, J ~ 8.51 Hz, 1 H) 6.57 (s, 1 H) 6.62 (t, J = 1.89 Hz, 1 H) 6.80 (td, J-8.43, 2.36 Hz, 1 H) 6.91 (s, 1 H) 6.96 - 7.05 (m, 3 H) 7.28 - 7.46 (m, 2 H) 8.05 (s, 1 H) [00104] LC-EM (LC-A method): Rt 3.15 min, MH ⁺ 599
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3A Enantiomer:
[00105] ¹ H NMR (500 MHz, DMSO-c / e) δ ppm 3.10 (s, 3 H) 3.11 -
3.25 (m, 2 H) 3.73 (s, 3 H) 3.74 - 3.79 (m, 1 H) 3.79 - 3.88 (m, 1 H)
3.96 - 4.19 (m, 3 H) 4.45 (dt,> 6.3, 10.4 Hz, 1 H) 4.97 (t,> 5.52 Hz, 1 H) 5.81 (d,> 8.51 Hz, 1 H) 6.57 (s, 1 H) 6.62 (t,> 1.89 Hz, 1 H) 6.80 (td,> 8.43, 2.36 Hz, 1 H) 6.91 (s, 1 H) 6.96 - 7.11 (m, 3 H) 7.28 - 7.46 (m, 2 H) 8.05 (s, 1 H) [ 00106] LC / MS (LC-A method): Rt 3.13 min, MH * 599 [00107] [a] _D ²⁰ : -59.0 ° (c 0.2542, DMF) [00108] Chiral SFC (method SFC-C): Rt 1.87 min, MH ⁺ 599, chiral purity 100%.
3B Enantiomer:
[00109] ¹ H NMR (500 MHz, DMSO-de) δ ppm 3.10 (s, 3 H) 3.11 -
3.25 (m, 2 H) 3.73 (s, 3 H) 3.74 - 3.79 (m, 1 H) 3.79 - 3.88 (m, 1 H)
3.96 - 4.19 (m, 3 H) 4.45 (dt,> 6.3, 10.4 Hz, 1 H) 4.97 (t,> 5.52 Hz, 1 H) 5.81 (d,> 8.51 Hz, 1 H) 6.57 (s, 1 H) 6.62 (t,> 1.89 Hz, 1 H) 6.80 (td,> 8.43, 2.36 Hz, 1 H) 6.91 (s, 1 H) 6.96 - 7.11 (m, 3 H) 7.28 - 7.46 (m, 2 H) 8.05 (s, 1 H) [ 00110] LC / MS (LC-A method): Rt 3.13 min, MH ⁺ 599 [00111] [a] _D ²⁰ : + 56.8 ° (c 0.2467, DMF) [00112] Chiral SFC (method SFC-C): Rt 2.34 min, MH ⁺ 599, chiral purity 100%.
Example 4 (method 1): synthesis of 2- (4-chloro-2- (2 ~ hydroxyethoxy) phenyl) -2 ((3-methoxy-5- (methylsulfonyl) phenyl) amino) -1- (6- (trifluoromethyl) ) indolin-1-yl) ethanone (Compound 4).
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Synthesis of intermediate 4a:
[00113] To a mixture of 2- (4-chloro-2-hydroxyphenyl) ethyl acetate [CAS 1261826-30-5] (2.82 g, 3.28 mmol) and cesium carbonate (8.56 g, 26.3 mmol) in DMF (50 mL) was added benzyl ether and 2bromoethyl [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 4a (4 , 17 g).
Synthesis of intermediate 4b:
[00114] To a 1 M solution of lithium bis (trimethylsilyl) amide in THF (11.0 mL, 11.0 mmol) cooled (-78 ° C), a solution of 2- (2 ~ (2 - (benzyloxy) ethoxy) ~ 4 ~ chlorophenyl) ethyl acetate 4a (1.82 g, 5.22 mmol) in THF (9 mL). After stirring for 1 h at -78 ° C, chlorotrimethylsilane (1.1 ml, 8.67 mmol) was added. The reaction mixture was stirred at -78 ° C for 15 min. / -Bromosuccinimide (1.11 g, 8.67 mmol) was added and stirring was continued at -78 ° C for 2 h. The reaction mixture was poured into H2O and extracted with EtOAc. Organic phase was dried over MgSO4, filtered and concentrated under reduced pressure to provide 2- (2- (2- (benzyloxy) ethoxy) -4-chlorophenyl) -2-bromoacetate 4b (2.23 g), which was used in the step next without purification
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Synthesis of intermediate 4c:
[00115] To a solution of 2- (2 ~ (2 ~ (benzyloxy) ethoxy) ~ 4 ~ chlorophenyl) -2bromoacetate 4b (2.23 g, 5.22 mmol) in CH3CN (22.5 mL) and THF (22.5 ml) was added 3-methoxy-5- (methylsulfonyl) aniline [CAS 62606-02-4] (3.12 ml, 15.5 mmol). The reaction mixture was stirred at 60 ° C overnight. The reaction mixture was concentrated under reduced pressure. The residue was partitioned between EtOAc and 1 N HCI. The aqueous phase was extracted with EtOAc. The organic phases were combined, dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by fiash chromatography on silica gel using a gradient of EtOAc (0% to 40%) in heptane to give 2- (2- (2 (benzyloxy) ethoxy) -4-chlorophenyl) -2 - ((3 -methoxy-5- (methylsulfonyl) phenyl) amino) ethyl acetate 4c (1.57 g).
Synthesis of intermediate 4d:
[00116] A mixture of 2- (2- (2- (benzyloxy) ethoxy) -4-chlorophenyl) -2 - ((3-methoxy-5- (methylsulfonyl) phenyl) amino) ethyl acetate 4c (1.57 g, 2.86 mmol) and 10% palladium on carbon (0.320 g) in EtOAc (40 mL) was stirred at room temperature under an atmosphere of H2. The reaction mixture was filtered through celite®. The filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel using a gradient of EtOAc (30% to 100%) in heptane to give 2- (4-chloro-2- (2 ~ hydroxyethoxy) phenyl) -2 - ((3 ~ methoxy ~ 5 ~ (methylsulfonyl) phenyl) amino) ethyl acetate 4d (1.13 g).
Synthesis of intermediate 4e:
[00117] To a solution of 2- (4-chloro-2- (2-hydroxyethoxy) phenyl) -2 - ((3-methoxy-5- (methylsulfonyl) phenyl) amine) ethyl acetate 4d (1.14 g, 2 , 49 mmol) in THF (8 mL), MeOH (8 mL) and H2O (8 mL) were added lithium hydroxide monohydrate (0.522 g, 12.5 mmol). The reaction mixture was stirred at room temperature for 3 h. HCI 1N and
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EtOAc and the phases were separated. The aqueous phase was extracted with EtOAc. The organic phases were combined, washed with saturated aqueous sodium chloride solution, dried over MgSO4, filtered and concentrated under reduced pressure to give 2- (4-chloro-2- (2-hydroxyethoxy) phenyl) -2 - (( 3-methoxy-5- (methylsulfonyl) phenyl) amino) acetic 4e _s quantitatively, which was used in the next step without further purification. Synthesis of Compound 4:
[00118] To a solution of 6-trifluroromethylindoline [CAS 181513-291] (0.200 g, 1.07 mmol), 2- (4-chloro-2- (2-hydroxyethoxy) phenyl) -2 - ((3-methoxy- 5- (methylsulfonyl) phenyl) amino) acetic 4e (0.478 g, 1.11 mmol) and triethylamine (0.593 mL, 4.28 mmol) in DMF (10 mL) was added HATU (0.406 g, 1.07 mmol). The reaction mixture was stirred overnight. The reaction mixture was diluted with H2O and was extracted with ethyl acetate. The organic phase was washed with HCl 1N, a saturated aqueous solution of NaHCCh and saturated sodium chloride solution, dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel using a gradient of EtOAc (60% to 70%) in heptane. Fractions containing the desired product were combined and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel using a gradient of EtOAc (0% to 25%) in CH2 Cl2 to give 2- (4 chloro-2- (2-hydroxyethoxy) phenyl) -2 - ((3-methoxy- 5- (methylsulfonyl) phenyl) amino) -1 (6- (trifluoromethyl) indolin-1-yl) ethan ~ 1 ~ one (Compound 4, 0.162 g) as a racemic mixture.
Compound 4:
[00119] ¹ H NMR (300 MHz, DMSO-cfe) δ ppm 3.11 (s, 3 H) 3.22 (m, 2 H) 3.67 - 3.88 (m, 5 H) 4.00 - 4.22 (m, 3 H) 4.44 (m, 1 H) 4.98 (t,> 5.5 Hz, 1 H) 5.83 (d,> 8.3 Hz, 1 H) 6 , 56 (s, 1 H) 6.63 (s, 1 H) 6.92 (s, 1 H) 7.04 (m, 2 H) 7.17 (m, 1 H) 7.31-7, 50 (m, 3 H) 8.38 (s, 1 H) [00120] LC-MS (LC-C method): Rt 1.89 min, MH ⁺ 599
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Example 4 (method 2): synthesis of 2- (4-chloro-2- (2-hydroxyethoxy) phenyl) -2 ((3-ηβΐόχί-5- (ηΊβίίΐ5υΙΤοηϋ) ίβπΗ) 3ηΊΐηο) -1- (6- (ίη ^ υοΓθΠ6ίΗ) ίηάοΙίη-1-ΙΙ) ethanone (Compound 4) and chiral separation in Enantiomers 4A and 4B.
st Ç , 4 g .... O ^: Χ. · Ϊ '· / '«ÈSÈfe Wi W _s tãW THE41
S,
Bí: í _.
· $
x'Cs, ^ <5G1 Vtj & <Q ·
Synthesis of intermediate 4f:
[00121] To a solution of 2- (2- (2 "(benzyloxy) ethoxy) -4-chlorophenyl) ethyl acetate 4a (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 MgSCX, filtered and concentrated under reduced pressure to give 2 ”(2 (2- (benzyloxy) ethoxy) 4 Chlorophenyl) acetic acid 4f (3.83 g).
Synthesis of intermediate 4q:
[00122] A solution of 2- (2 ~ (2- (benzyloxy) ethoxy) ~ 4 ~ chlorophenyl) acetic acid 4f (7.12 g, 22.2 mmol) in thionyl chloride (50 mL, 689 mmol)
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38/77 was stirred at room temperature for 18h. The reaction mixture was concentrated under reduced pressure and coevaporated with toluene to provide 2- (2 ~ (2- (benzyloxy) ethoxy) ~ 4 ~ chlorophenyl) acet 4 to 4g (7.53 g) chloride which was used in the next step without further purification.
Synthesis of intermediate 4h:
[00123] A solution of 2- (2- (2 ~ (benzyloxy) ethoxy) -4-chlorophenyl) acetia 4g (5.29 g, 15.6 mmol) in ChhCN (50 mL) was added dropwise under N2 atmosphere 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 EtzO. The combined organic layers were washed with saturated aqueous sodium chloride solution, dried over MgSCX, filtered and evaporated under reduced pressure. The residue solidified after standing. The product was stirred in diisopropyl ether (25 ml), filtered, washed (3x) with diisopropyl ether, and dried under vacuum at 45 ° C to obtain 2- (2- (2 (benzyloxy ) ethoxy) -4-chlorophenyl) -1 - (6- (trifluoromethyl) indoHn-1-yl) ethanone 4h (6.97 g).
Synthesis of intermediate 4i:
[00124] A solution of 2- (2- (2- (benzyloxy) ethoxy) -4-chloro-phenyl) -1- (6 (trifluoromethyl) indolin-1-yl) ethanone 4h (1.0 g, 2, 04 mmol) in 2-Me-THF (100 mL) was stirred under N ₂ flow and cooled to -78 ° C. A solution of 1 M lithium bis (trimethylsilyl) amide in THF (4.08 mL, 4.08 mmol) was added dropwise and the resulting mixture was stirred at -78 ° C for 15 minutes. Chlorotrimethylsilane (417 µL; 3.27 mmol) was added dropwise and the mixture was stirred at -78 ° C for 15 minutes. A solution of AZ-bromosuccinimide (400 mg, 2.25 mmol) in 2-Me-THF (25 mL) was added dropwise and the reaction mixture was stirred at -78 ° C for 50 min. A saturated aqueous solution was added
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39/77 of NH4CI (40 ml) in one go, and the resulting mixture was stirred without cooling until the temperature reached 0 ° C. Water (10 ml) was added and the layers were separated. The organic layer was dried over MgSCh, filtered, evaporated under reduced pressure and coevaporated with CH3CN to provide 2- (2- (2- (benzyloxy) ethoxy) -4-chlorophenyl) -2bromo-1 ~ (6 ~ (trifluoromethyl) indoiin -1-yl) ethanone 4i (1.16 g). The product was used without further purification in the next step.
Synthesis of intermediate 4j:
[00125] To a stirred solution of 2- (2- (2- (benzyloxy) ethoxy) -4-chlorophenii) -2-bromo-1- (6- (trifluoromethyl) indoiin-1-yl) ethanone 4i (1, 16 g, 2.04 mmol) in CH3CN (50 mL) under N2 atmosphere 3 methoxy-5- (methylsulfonyl) aniline [CAS 62606-02-4] (0.82 g, 4.08 mmol) and dHSopropylethylamine ( 703 pL, 4.08 mmol) and the reaction mixture was stirred at 60 ° C for 65 h. The mixture was cooled to room temperature and poured into H2O with stirring (250 ml). The product was extracted (2x) with Et ₂ O. The combined organic layers were dried over MgSCu, filtered and evaporated under reduced pressure. The residue was purified by flash chromatography on silica (40 g) using a gradient of heptane / EtOAc / EtOH from 100/0/0 to 40/45/15. The desired fractions were combined and the solvent was evaporated under reduced pressure in a rotavapor® to a residual volume of 35 ml. The product crystallized upon standing. The precipitate was removed by filtration, washed (3x) with 1/1 EtOAc / heptane and dried under vacuum at 45 ° C to provide 2 (2- (2- (benzyloxy) ethoxy) -4-chlorophenyl) -2 - (( 3-methoxy-5- (methylsulfonyl) phenii) amino) -1- (6- (trifluoromethyl) indolin-1-yl) ethanone 4j (870 mg).
Synthesis of Compound 4 and chiral separation in Enantiomers 4A and 4B: [00126] A solution of 2- (2- (2- (benzyloxy) ethoxy) -4-cyiorophenyl) -2 - ((3 methoxy-5- (methylsififonyl) phenyl ) amino) -1- (6- (trifluoromethyl) indolin-1-yl) ethanone 4j (210 mg, 0.28 mmol) in THF (30 mL) was added to a stirred mixture of Pd / C (0.5 g ) in EtOAc (10 ml). The mixture was hydrogen
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40/77 swim for 10 min at room temperature under atmospheric pressure. The catalyst was removed by filtration over dicalite® and the solvents were evaporated under reduced pressure. The residue was combined with another iote (total amount: 1.0 g) and purified by reverse phase HPLC (Stationary phase: Kromasil C18 100A 5 um (Eka Nobel), Mobile phase: 50% ammonia-bicarbonate gradient (0 , 25% in water), 50% acetonitrile to 20% ammonia-bicarbonate (0.25% in water), 80% acetonitrile) yielding Compound 4 (700 mg). The enantiomers of Compound 4 (700 g) were separated through Quirai separation in Normal Phase (stationary phase: Whelk-01 (SS) 5 pm with peak cutting recycling technique, Mobile phase: 100% ethanol). Fractions containing the first eluted enantiomer were combined and evaporated under reduced pressure. The residue was further purified by flash chromatography on silica gel (4 g) using a gradient of heptane / EtOAc / EtOH from 100/0/0 to 40/45/15. The desired fractions were combined and evaporated, and coevaporated with MeOH. The residue was triturated at 45 ° C in HzO (4 ml) and MeOH (1 ml), the precipitate was filtered, washed (3x) with 4/1 HbO / MeOH and dried under vacuum at 45 ° C to provide the 4A Enantiomer (197 mg). Fractions containing the second eluted enantiomer were combined and evaporated under reduced pressure. The residue was further purified by flash chromatography on silica gel (4 g) using a gradient of heptane / EtOAc / EtOH from 100/0/0 to 40/45/15. The desired fractions were combined and evaporated, and coevaporated with MeOH / water. The residue was stirred in H2O (4 ml) and MeOH (1 ml), the precipitate was filtered, washed (3x) with H2O / MeOH 4/1 and dried under vacuum at 45 ° C to provide the 4B Enantiomer (209 mg) .
4A Enantiomer:
[00127] ¹ H NMR (400 MHz, DMSO-cfe) δ ppm 3.10 (s, 3 H) 3.16 3.27 (m, 2 H) 3.68 - 3.85 (m, 5 H) 4.04 - 4.20 (m, 3 H) 4.44 (td,> 10.2,
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6.6 Hz, 1 Η) 4.94 (t, J ^ 5, Q Hz, 1 H) 5.83 (d,> 8.4 Hz, 1 H) 6.56 (t,> 2.1 Hz , 1 H) 6.63 (t,> 1.8 Hz, 1 H) 6.91 (t,> 1.4 Hz, 1 H) 6.97 7.08 (m, 2 H) 7.17 ( d,> 2.0 Hz, 1 H) 7.36 (d,> 8.1 Hz, 1 H) 7.39 (dd,> 7.9, 0.9 Hz, 1 H) 7.43 - 7 , 49 (m, 1 H) 8.38 (s I, 1 H) [00128] LC / MS (LC-D method): Rt 1.17 min, MH ⁺ 599 [00129] [a] _D ²⁰ : + 59.8 ° (c 0.435, DMF) [00130] Chiral SFC (SFC-I method): Rt 2.84 min, MH ⁺ 599, 100% chiral purity.
4B Enantiomer:
[00131] Ή NMR (400 MHz, DMSO-cfe) δ ppm 3.10 (s, 3 H) 3.16 -
3.26 (m, 2 H) 3.70 - 3.85 (m, 5 H) 4.02 - 4.19 (m, 3 H) 4.44 (td,> 10.2, 6.4 Hz , 1 H) 4.94 (t,> 5.6 Hz, 1 H) 5.83 (d,> 8.4 Hz, 1 H) 6.56 (t,> 2.0 Hz, 1 H) 6 , 63 (t,> 1.8 Hz, 1 H) 6.91 (t,> 1.7 Hz, 1 H) 6.99 7.07 (m, 2 H) 7.16 (d,> 2, 0 Hz, 1 H) 7.36 (d,> 8.1 Hz, 1 H) 7.37 7.41 (m, 1 H) 7.44 - 7.48 (m, 1 H) 8.38 ( s, 1 H) [00132] LC / MS (LC-D method): Rt 1.17 min, MH ⁺ 599 [00133] [a] _D ²⁰ : -56.4 ° (c 0.47, DMF) [ 00134] Chiral SFC (SFC-I method): Rt 3.14 min, MH ⁺ 599, chiral purity 97.0%.
Example 5: synthesis of 2- (4 ~ chloro ~ 2 ~ (2-hydroxyethoxy) phenyl) -2 - ((3-methoxy-5 (methylsulfonyl) phenyl) amnno) -1- (5-methoxy-6- (trifluoromethyl) ) indolin-1-yl) ethanone (Compound 5) and chiral separation in Enantiomers 5A and SB.

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Synthesis of intermediate 5a:
[00135] To a mixture of 2- (4-chloro-2-hydroxyphenyl) ethyl acetate [CAS 1261826-30-5] (5.2 g, 24.2 mmol) and cesium carbonate (15.8 g, 48.5 mmol) in DMF (90 mL) at 10 ° C (2-bromoethoxy) (ferc-butyl) dirnetylsilane [CAS 86864-60-0] (6.26 mL, 29.1 mmol) was added. The reaction mixture was stirred at room temperature overnight. Water was added and the reaction mixture was extracted with EtOAc. The organic phase was dried with filtered MgSCU and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 80 g, heptane / EtOAc 80/20). The pure fractions were combined and the solvent was removed under reduced pressure to give 2- (2- (2 - ((fercbutyldimethylsilyl) oxy) ethoxy) -4-chlorophenyl) ethyl acetate 5a (7.8 g).
Synthesis of intermediate 5b:
[00136] To a 1 M solution of lithium bis (trimethylsilyl) amide in THF (41.8 mL, 41.8 mmol) cooled (-78 ° C), a solution of 2- (2- (2 - ((tert-butyldimethylsilyl) oxy) ethoxy) -4-chlorophenyl) ethyl acetate 5a (7.8 g, 20.9 mmol) in THF (45 mL). After 1 h at -70 ° C, chlorotrimethylsilane (4.24 ml, 33.5 mmol) was added. The reaction mixture was stirred at -70 ° C for 15 min. / V-Bromosuccinimide (4.46 g, 25.1 mmol) in THF (45 mL) was added and stirring was continued at ~ 55 ° C for 2 h. The reaction mixture was poured into HzO 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 - ((ferc-butyldimethylsilyl) oxy) ethoxy) -4-chlorophenyl ) ethyl acetate 5b (10.1 g) which was used in the next step without further purification.
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Synthesis of intermediate 5c:
[00137] A mixture of 2-bromo-2- (2- (2 - ((tert-butyldimethylsilyl) oxy) ethoxy) ~ 4 ~ chlorophenyl) ethyl acetate 5b (4.75 g, 10.5 mmoi), 3 -methoxy-5 (methylsulfonyl) aniline [CAS 62606-02-4] (3.17 g, 15.8 ml) and diisopropylethylamine (3.62 ml, 21.0 ml) in CHsCN (90 ml) was stirred at 50 ° C overnight. The reaction mixture was concentrated under reduced pressure. The residue was absorbed with EtOAc and washed with 0.5 N HCI and water. The organic phase was dried over MgSCU, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 120 g, heptane / EtOAc 90/10 at 80/20) to give 2- (2 ~ (2 - ((tert-butyldimethylsilyl) oxy) ethoxy) ) -4chlorophenyl) ~ 2 ~ ((3-methoxy -5 (methylsulfonyl) phenyl) amino) ethyl acetate 5c (3.5 g).
Synthesis of intermediate 5d:
[00138] Lithium hydroxide monohydrate (513 mg, 12.2 mmoi) was added in portions to a solution of 2- (2- (2 - ((tert-butyldimethylsilyl) oxy) ethoxy) -4chlorophenyl) -2 - ethyl (5-methoxy-5- (methylsulfonyl) phenyl) amino) acetate 5c (3.5 g, 6.12 mmol) in THF / CH3OH / H2O (1/1/1) (75 mL) at 10 ° C. The reaction was stirred at room temperature for 2 h, diluted with water and cooled to 0 ° C. The solution was slowly acidified to pH 6 with 0.5 N HCI 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 - ((ferc-butyldimethylsilyl) oxy) ethoxy acid) -4οΙθΓθΤοηίΙ) -2 - ((3 ^ β1όχί ~ 5- ^ θ1ίΐ3υΙΤοηίΙ) Τ8ηίΙ) 3ΠΊ! Ηο) 3θέ1ίοο 5d (2.85 g). The compound was used without further purification in the next step. Synthesis of intermediate 5e:
[00139] To a solution of 2- (2- (2 - ((tert-butyldimethylsilyl) oxy) ethoxy) -4-chlorophenyl) -2 - ((3-methoxy-5- (methylsulfonyl) phenyl) amino) acetic acid 5d (1 g, 1.84 mmol) in DMF (10 mL) were added HATU (1.05 g, 2.76 ml), diisopropylethylamine (913 pL, 5.53 ml) and 5-methoxy-6 ( trifluoromethyl) indoline 2g (412 mg, 1.90 mmol). The reaction mixture was
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44/77 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 and water, dried over MgSCu, filtered and the solvent was concentrated under reduced pressure to give 2- (2- (2 - ((tert-butyldimethylsilyl) oxy) ethoxy) - 4-chlorophenyl) -2 - ((3-methoxy ~ 5 ~ (methylsulfonyl) phenyl) amino) -1 (S-methoxy-and-Orifluoromethyljindolin-l-yljetanone 5e (1.4 g). The compound was used without purification additional in the next reaction step.
Synthesis of Compound 5 and chiral separation in Enantiomers 5A and 5B: [00140] Under N ₂ flow, at 5 ° C, 4 M HCI in dioxane (4.71 mL, 18.8 mmol) was added dropwise to a 2- (2- (2 ~ ((fer ~ butyldimethylsilyl) oxy) ethoxy) -4-chlorophenyl) -2 - ((3-methoxy-5- (methylsulfonyl) phenyl) amino) -1- (5-methoxy -6- (tr! Fluoromethyl) indolin-1-yl) ethanone 5e (1.4 g, 1.88 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 over MgSOd, filtered, and the solvent was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 40 g, CHzCL / MeOH 98.5 / 1.5). The pure fractions were combined and the solvent was removed under reduced pressure to provide 2- (4-chloro-2- (2-hydroxyethoxy) phenyl) -2 - ((3-methoxy-5- (methylsulfonyl) phenyl) amino) -1- (5-methoxy-6- (trifluoromethyl) indolinyl) ethanone (Compound 5, 1.0 g) as a racemate. An analytical sample of Compound 5 was crystallized from MeOH (60 mg). The remaining amount was used to separate the Enantiomers via Preparative Chiral SFC (Stationary phase: Chiralpak® IC 5 pm 250 x 30 mm, Mobile phase: 70% CO2, 30% EtOH (+ 0.3% IPrNH ₂ )). The first eluted enantiomer (400 mg) was solidified from diiisopropyl ether to give the 5A Enantiomer (351 mg). The second eluted enantiomer (430 mg) was solidified from diiisopropyl ether to give 0B Enantiomer (336 mg).
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Compound 5:
[00141] ¹ H NMR (500 MHz, DMSO-cfe) δ ppm 3.10 (s, 3 H) 3.13 -
3.27 (m, 2 H) 3.73 (s, 3 H) 3.73 - 3.78 (m, 1 H) 3.78 - 3.84 (m, 1 H)
3.84 (s, 3 H) 3.98 - 4.22 (m, 3 H) 4.41 (dt,> 6.1, 10.1 Hz, 1 H) 4.95 (t,> 5, 6 Hz, 1 H) 5.80 (d,> 8.08 Hz, 1 H) 6.55 (s, 1 H) 6.61 (s, 1 H) 6.90 (s, 1 H) 6, 96 - 7.05 (m, 2 H) 7.16 (d,> 1.52 Hz, 1 H) 7.24 (s, 1 H) 7.35 (d,> 8.08 Hz, 1 H) 8.34 (s, 1 H) [00142] LC-MS (LC-A method): Rt 3.15 min, MH ⁺ 629 [00143] Melting point: 220 ° C
5A Enantiomer:
[00144] ¹ H NMR (400 MHz, DMSO-de) δ ppm 3.10 (s, 3 Η) 3.13 -
3.27 (m, 2 H) 3.73 (s, 3 H) 3.73 - 3.78 (m, 1 H) 3.78 - 3.84 (m, 1 H)
3.84 (s, 3 H) 3.98 - 4.22 (m, 3 H) 4.41 (dt,> 6.1, 10.1 Hz, 1 H) 4.95 (t,> 5, 6 Hz, 1 H) 5.80 (d,> 8.08 Hz, 1 H) 6.55 (s, 1 H) 6.61 (s, 1 H) 6.90 (s, 1 H) 6, 96 - 7.05 (m, 2 H) 7.16 (d,> 1.52 Hz, 1 H) 7.24 (s, 1 H) 7.35 (d,> 8.08 Hz, 1 H) 8.34 (s, 1 H) [00145] LC / MS (LC-A method): Rt 3.13 min, MH ⁺ 629 [00146] [a] _D ²⁰ : -60.4 ° (c 0.28 , DMF) [00147] Chiral SFC (SFC-D method): Rt 1.02 min, ΜΗ ^1- 629, 100% chiral purity.
Enantiomer 5B:
[00148] ¹ H NMR (400 MHz, DMSO-de) δ ppm 3.10 (s, 3 H) 3.13 -
3.27 (m, 2 H) 3.73 (s, 3 H) 3.73 - 3.78 (m, 1 H) 3.78 - 3.84 (m, 1 H)
3.84 (s, 3 H) 3.98 - 4.22 (m, 3 H) 4.41 (dt,> 6.1, 10.1 Hz, 1 H) 4.95 (t I,> 5 , 6 Hz, 1 H) 5.80 (d,> 8.08 Hz, 1 H) 6.55 (s, 1 H) 6.61 (s, 1 H) 6.90 (s, 1 H) 6 , 96 - 7.05 (m, 2 H) 7.16 (d,> 1.52 Hz, 1 H) 7.24 (s, 1 H) 7.35 (d,> 8.08 Hz, 1 H ) 8.34 (s, 1 H) [00149] LC / MS (LC-A method): Rt 3.15 min, MH ⁺ 629 [00150] [a] _D ²⁰ : + 56.7 ° (c 0, 3, DMF) [00151] Chiral SFC (SFC-D method): Rt 1.22 min, MH ⁺ 629, purity
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46/77 chiral 99.7%.
Example 6 (method 1): synthesis of 2- (4-chloro-2- (2-hydroxyethoxy) phenyl) -2 ((3 ~ meioxy ~ 5 ~ (methylsulfonH) phenyr) amino) -1- (6 ~ (trifluorQmethoxy ) indQHn-1yl) ethanone (Compound 6).
F
Synthesis of intermediate 6a:
[00152] To a solution of 2 ~ (2 ~ (2 ~ ((ferc ~ butyldimetHsilil) oxy) ethoxy) -4-chlorophenyl) -2 - ((3-methoxy-5- (methylsulfonyl) phenyl) amino) acetic acid Sd (1.07 g, 1.97 mmol) in DMF (10 mL) were added HATU (1.12 g, 2.95 mmol), diisopropylethylamine (976 pL, 5.91 mmol) and 6 (trifluoromethoxy) indoline [CAS 959235-95-1] (400 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% K2CO3 solution, water, dried over MgSÜ4, filtered and the solvent was concentrated under reduced pressure to give 2- (2- (2 ((fe / r-butyldimethylsiloxyethoxyH-chlorophenyl ^ - ^ S-methoxy-S ^ methylsulfonylphenyl) amino) -1- (6- (trifluoromethoxy) indolin-1-yl) ethanone 6a (1.36 g) The crude compound was used without purification in the next reaction step.
Synthesis of Compound 6:
[00153] Under a flow of N ₂ at 5 ° C, 4 M HCI in dioxane (4.66 mL, 18.6 mmol) was added dropwise to a solution of 2- (2- (2 - ((fercbutildimet ! lsilyl) oxy) ethoxy) -4-chlorophenyl) ~ 2 ~ (((3-methoxy-5- (methylsulfonyl) phenyl)
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47/77 amino) -1- (6- (trifluoromethoxy) indolin-1-yl) ethanone 6a (1.36 g, 1.87 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 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, CHaCb / MeOH 99.5 / 0.5). The pure fractions were combined and the solvent was removed under reduced pressure to provide 2- (4-chloro-2- (2-hydroxyethoxy) phenyl) -2 - ((3-methoxy-5 (methylsulfonyl) phenyl) amino) -1 - (6- (tnfluoromethoxy) indolin-1-yl) ethanone (Compound 6, 540 mg) as a racemate. An analytical sample of Compound 6 was obtained by crystallization from MeOH (34 mg). Compound 6:
[00154] ¹ H NMR (400 MHz, DMSO-cfe) δ ppm 3.07 - 3.23 (m, 5 H)
3.70 - 3.83 (m, 5 H) 4.06 - 4.19 (m, 3 H) 4.42 (td,> 10.23, 6.32 Hz, 1 H) 4.92 (t ,> 5.31 Hz, 1 H) 5.81 (d,> 8.59 Hz, 1 H) 6.56 (s, 1 H) 6.61 (s, 1 H) 6.90 (s. 1 H) 6.99 - 7.05 (m, 3 H) 7.16 (d,> 2.02 Hz, 1 H) 7.30 7.40 (m, 2 H) 8.03 (s, 1 H ) [00155] LC-EM (LC-A method): Rt 3.28 min, MH ⁺ 615 [00156] Melting point: 191 ° C
Example 6 (method 2): synthesis of 2- (4-chloro-2- (2-hydroxyethoxy) phenyl) -2 ((3-methoxy-5- (methylsulfonyl) phenyl) amino) -1- (6- (trifluoromethoxy ) indolin-1-yl) ethanone (Compound 6) and chiral separation in Enantiomers 6A and 6B.

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Η ·>. Ρ'Λ'ί ·. ·
Synthesis of intermediate 6b:
[00157] To a 1.5 M solution of lithium bis (trimethylsilyl) amide in THF (23 mL, 34.4 mmol) cooled (-70 ° C) under N ₂ flow was added a 2- ( 2- (2- (benzyloxy) ethoxy) -4-chlorophenyl) ethyl acetate 4a (6 g, 17.2 mmol) in THF (35 mL). After 1 h at -70 ° C, chlorotrimethylsilane (3.5 ml, 27.5 mmol) was added. The reaction mixture was stirred at -70 ° C for 15 min. / V-Bromosuccinimide (3.7 g, 20.6 mmol) in THF (35 mL) was added and stirring was continued at -70 ° C for 2 h. The reaction mixture was poured into H ₂ O and extracted with EtOAc. The organic phases were combined, dried over MgSCu, 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:
[00158] A mixture of 2- (2- (2- (benzyloxy) ethoxy) -4-chlorophenyl) -2-bromoacetate 6b (7.36 g, 17.2 mmol), 3-methoxy-5- (methylsulfonyl) aniline [CAS 62606-02-4] (5.2 g, 25.8 mmol) and diisopropylethylamine (5.9 mL, 25.8 mmol) in CH3CN (150 mL) was stirred at 50 ° C overnight . The solvent was concentrated under reduced pressure. The residue was diluted with EtOAc and washed with 0.5N HCl and water. The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 220 g, CH ₂ CI ₂ / IVIeOH 99/1). The pure fractions were combined and the solvent was concentrated under reduced pressure to give 2- (2- (2 (όβηζΠόχΙ) βίόχί) -4-οΙθΓθΤβηίΙ) -2 - ((3 ^ θίόχί-5- (ΓηβίΙΐ3υΙΤοηίΙ) ίβηο) 8Γηίηο) ethyl acetate 6c (5.52 g).
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Synthesis of intermediate 6d:
[00159] At 10 ° C, lithium hydroxide monohydrate (845 mg, 20.1 mmol) was added to a solution of 2- (2- (2- (benzyloxy) ethoxy) -4chlorophenyl) -2- ( (3-methoxy-5- (methylsulfonyl) phenyl) amino) ethyl acetate 6c (5.52 g, 10.1 mmol) in MeOH / THF / water (1/1/1) (90 mL). The mixture was stirred at room temperature for 2 h. The mixture was diluted with ice water and cooled to 0 ° C. 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) acid -5- (methylsulfonyl) phenyl) amine) acetic 6d (5.26 g). The compound was used in the next reaction step without further purification.
Synthesis of intermediate 6e:
[00160] A mixture of 6- (trifluoromethoxy) indoline [CAS 959235-951] (1.85 g, 9.12 mmol), 2- (2- (2- (benzyloxy) ethoxy) -4-chlorophenyl) - 2 ((3-methoxy-5- (methylsulfonyl) phenyl) amino) acetic 6d (5.69 g, 10.9 mmol), HATU (5.2 g, 13.7 mmol) and dl-isopropylethylamyl (4.52 ml, 27.4 mmol) in DMF (40 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% K2CO3 solution in water, water, dried over MgSO4, filtered and the solvent was evaporated under reduced pressure. Purification by flash chromatography on silica gel (15-40 pm, 220 g, heptane / EtOAc 70/30) was performed. The pure fractions were combined and concentrated to dryness to provide 2- (2- (2- (benzyloxy) ethoxy) -4-chlorophenyl) -2 - ((3 methoxy-5- (methylsulfonyl) phenyl) amino) -1- ( 6- (trifluoromethoxy) indolin-1yl) ethanone 6e (5.6 g).
Synthesis of Compound 6 and chiral separation in Enantiomers 6A and 6B: [00161] A mixture of 2- (2- (2- (benzyloxy) ethoxy) -4-chlorophenyl) -2 - ((3
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50/77 methoxy-5- (methylsulfonyl) phenyl) amino) -1- (6- (trifluoromethoxy) indolin-1-yl) ethanone 6e (5.6 g, 7.94 mmol) in EtOAc (100 mL) was hydrogenated at atmospheric pressure of H ₂ in the presence of Pd / C (10%) (1.7%, 1.59 mmol) as a catalyst for 6 min (until the end of H ₂ consumption). The reaction was diluted with EtOAc and filtered through a pad of celite®. The filtrate was concentrated under reduced pressure to provide 2 "(4ClorO2 (2hydroxyethoxy) phenyl)" 2 "(((3methoxy5 (methylsulfonyl) phenyl) amine) ~ 1 ~ (6 (trifluoromethoxy) indolin ~ 1-yl) ethanone (Compound 6) as a racemate (4.6 g), crude compound). The Compound 6 Enantiomers were separated via chiral SFC (Stationary phase: Chiralcel® OJ-H 5 pm 250 x 20 mm, mobile phase: 80% CO ₂ , 20% MeOH (+ 0.3% iPrNH ₂ )). The first eluted enantiomer (1.96 g) was further purified by chiral SFC (Stationary phase: Chiralpak® IA 5 pm 250 x 20 mm, mobile phase: 74% CO ₂ , 26% IPrOH (+ 0.3% iPrNH ₂ )), to obtain after precipitation from heptane / diisopropyl ether, Enantiomer 6A (1.527 g). The second eluted enantiomer (2.10 g) was solidified from heptane / diisopropyl ether to give Enantiomer 6B (1.708 g).
6A Enantiomer:
[00162] Ή NMR (500 MHz, DIVISE »δ ppm 3.08 - 3.18 (m, 5 H)
3.70 - 3.83 (m, 5 H) 4.05 - 4.19 (m, 3 H) 4.43 (td,> 10.32, 6.46 Hz, 1 H) 4.97 (t ,> 5.52 Hz, 1 H) 5.82 (d,> 8.20 Hz, 1 H) 6.56 (s, 1 H) 6.62 (s, 1 H) 6.91 (s, 1 H) 7.00 - 7.08 (m, 3 H) 7.16 (d,> 1.58 Hz, 1 H) 7.34 (d,> 8.20 Hz, 2 H) 8.04 (s , 1H) [00163] LC / MS (LC-A method): Rt 3.32 min, MH ⁺ 615 [00164] [a] _D ²⁰ : + 64.3 ° (c 0.305, DMF) [00165] SFC chiral (SFC-E method): Rt 2.82 min, MH ⁺ 615, chiral purity 100%.
6B Enantiomer:
[00166] ¹ H NMR (500 MHz, DIVISION »δ ppm 3.08 - 3.18 (m, 5 H)
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3.70 - 3.83 (m, 5 Η) 4.05 - 4.19 (m, 3 Η) 4.43 (td,> 10.32, 6.46 Hz, 1 H) 4.97 (t ,> 5.52 Hz, 1 H) 5.82 (d,> 8.20 Hz, 1 H) 6.56 (s, 1 H) 6.62 (s, 1 H) 6.91 (s, 1 H) 7.00 - 7.08 (m, 3 H) 7.16 (d,> 1.58 Hz, 1 H) 7.34 (d,> 8.20 Hz, 2 H) 8.04 (s , 1H) [00167] LC / MS (LC-A method): Rt 3.31 min, MH * 615 [00168] [a] _D ²⁰ : -53.7 ° (c 0.3, DMF) [00169 ] Chiral SFC (SFC-E method): Rt 3.34 min, MH ⁺ 615, chiral purity 95.7%.
Example 7: 4- (5-chloro-2- (1 - ((3-methoxy-5- (methylsulfonyl) phenyl) -amino) -2-oxo ~ 2 ~ (6- (tnfluoromethyl) indolin-1 acid synthesis) -yl) ethyl) phenoxy) butanoic (Compound 7) and chiral separation in Enantiomers 7A and 7B.

..........
3) Wc W

Synthesis of intermediate 7a:
[00170] To a suspension of 2- (4-chloro-2-hydroxyphenyl) acetate [CAS 1261826-30-5] (8.5 g, 39.6 mmol), Cs ₂ CO ₃ (25.8 g, 79 , 2 mmol) in DMF (130 mL) at 10 ° C, tert-butyl 4-bromobutanoate [CAS 110611-91-1] (7 mL, 39.6 mmol) was added dropwise. The mixture was stirred
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52/77 at room temperature overnight. The mixture was diluted with EtOAc and water. The layers were separated. The organic layer was washed with water, dried over MgSCh, filtered, and the solvent was concentrated under reduced pressure. Purification by flash chromatography on silica gel (15-40 pm, 120 g, heptane / EtOAc 90/10) was performed. The pure fractions were combined and concentrated to dryness to give ferc-butyl 4- (5chloro-2- (2-ethoxy-2-oxoethyl) phenoxy) butanoate 7a (12.7 g). Synthesis of intermediate 7b:
[00171] A flask was charged with 1.5 M LiHMDS in THF (23.5 mL, 35.3 mmol) under N2 flow and was cooled to -78 ° C. A solution of ferc-butyl 4- (5-chloro-2- (2-ethoxy-2-oxoethyl) phenoxy) butanoate 7a (6.3 g, 17.6 mmol) in THF (60 mL) was added dropwise drop and the mixture was stirred at -78 ° C for 15 min. Chlorotrimethylsilane (3.6 ml, 28.3 mmol) was added. After 15 min at ~ 78 ° C, A / -bromos ~ succinimide (3.77 g, 21.2 mmol) in THF (40 mL) was added and the mixture was stirred at -70 ° C for 1 h. The reaction was quenched with water and extracted with EtOAc. The organic layer was washed with water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure to produce 4 (2- (1-bromo-2-ethoxy-2-oxoethyl) -5-chlorophenoxy!) butyl 7b (7.6 g). The compound was used in the next reaction step without further purification.
Synthesis of intermediate 7c:
[00172] To a solution of fer-butyl 4- (2- (1 ~ bromo-2-ethoxy ~ 2 ~ oxoethyl) ~ 5 ~ chloro-phenoxy) butanoate 7b (7.6 g, 17.4 mmol) in CH3CN (140 mL) at room temperature, diisopropylethylamine (4.8 mL, 27.9 mmol) was added and then 3-methoxy-5- (methylsulfonyl) aniline [CAS 62606-02-4] (4.2 g, 20.9 mmol). The mixture was stirred at 65 ° C for 24 h. The mixture was diluted with EtOAc, then 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. It was made
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53/77 purification by flash chromatography on silica gel (15-40 pm, 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-methoxy5- (methylsulfonyl) phenyl) amino) -2-oxoethyl) phenoxy) butanoate tert-butyl 7c (7.3 g).
Synthesis of intermediate 7d:
[00173] Tc-butyl 4- (5-Chloro-2- (2-ethoxy-1 - ((3-methoxy-5- (methylsulfonyl) phenyl) amino) -2 ”oxoethyl) phenoxy) butanoate 7c (7, 3 g, 13.1 mmol) and lithium hydroxide monohydrate (1.65 g, 39.4 mmol) in THF / water (1/1) (180 mL) was stirred at room temperature for 3 h. The mixture was diluted with water. The aqueous layer was slowly acidified with 3N HCI and extracted with EtOAc. The organic layers were dried over MgSCA, filtered and the solvent was concentrated under reduced pressure to give 2- (2- (4 ~ (tert-butoxy) -4-oxobutoxy) -4-chlorophenyl) -2 - ((3 ~ methoxy-5- (methylsulfonyl) phenyl) amino) acetic 7d (6.9 g). The product was used in the next reaction step without further purification.
Synthesis of intermediate 7e:
[00174] A mixture of 6- (trifluoromethoxy) indoline [CAS 181513-291] (390 g, 2.08 mmol), 2- (2- (4- (tert-butoxy) -4-oxobutoxy) -4- acid chlorophenyl) -2 - ((3-methoxy-5- (methylsulfonyl) phenyl) amino) acetic 7d (1.1 g, 2.08 mmol), HATU (1.2 g, 3.12 mmol) and diisopropylethylamine (1 ml, 6.25 mmol) in DMF (40 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 10% aqueous K2CO3 solution, water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. Purification was performed by flash chromatography on silica gel (15-40 pm, 80 g, ChhCb / MeOH 99.5 / 0.5) to give, after crystallization of CH3CN, 4- (5chloro-2- (1- ( Tert-butyl (3-methoxy-5- (methylsulfonyl) phenyl) amino) -2-oxo-2- (6- (trifluoromethyl) indolin-1-yl) ethyl) phenoxy) butanoate 7e (700 mg).
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Synthesis of Compound 7 and chiral separation in Enantiomers 7A and 7B: [00175] A solution of 4- (5 ~ οΙθΓθ-2 ~ (1 - (((3-ηΊ61όχΙ ~ 5 ~ (ΠΊβ * Η3υίίοηΙΙ) phenyl) amino) ~ 2 ~ oxo ~ 2 ~ (6- (trifluoromethyl) indolin-1-yl) ethyl) phenoxy) tert-butyl 7e butanoate (0.6 mg, 0.143 mmol) in 4M HCI in dioxane (6 mL) was stirred at 5 ° C for 3 h and at room temperature for 8 h. The solvent was removed under reduced pressure and the product was crystallized from diisopropyl ether to produce 4- (5-chloro-2- (1 - ((3-methoxy-5- (methylsulfonyl) phenyl) amino) -2- acid oxo-2- (6- (trifluoromethyl) indolin-1yl) et! 1) phenoxy) butanoic (Compound 7, 530 mg) as a racemate. The Enantiomers were separated by SFC Quirai Preparativa (Stationary phase: Chiralpak® IC 5 pm 250 x 30 mm, mobile phase: 65% COz, 35% MeOH). The first eluted enantiomer (264 mg) was crystallized from CHsCN / diisopropyl ether to give Enantiomer 7Â (207 mg). The second eluted enantiomer (269 mg) was crystallized from CHaCN / diisopropyl ether to give Enantiomer 7B (212 mg). Compound 7:
[00176] ¹ H NMR (500 MHz, DMSO-cfe) δ ppm 1.90 - 2.09 (m, 2 H)
2.31 - 2.43 (m, 2 H) 3.12 (s, 3 H) 3.17 - 3.28 (m, 2 H) 3.74 (s, 3 H) 3.88
- 4.07 (m, 1 H) 4.07 - 4.15 (m, 2 H) 4.35 - 4.45 (m, 1 H) 5.73 (di,> 7.88 Hz, 1 H ) 6.55 (s I, 1 H) 6.64 (s I, 1 H) 6.90 (s I, 1 H) 7.04 (s I, 2 H) 7.16 (s I, 1 H ) 7.31 (d I,> 7.88 Hz, 1 H) 7.39 (d I,> 7.25 Hz, 1 H) 7.46 (d I,> 7.25 Hz, 1 H) 8 , 39 (s I, 1 H) 12.12 (s I, 1 H) [00177] LC-MS (LC-A method): Rt 2.73 min, MH ⁺ 641 [00178] Melting point: 210 ° Ç
Enantiomer 7A:
[00179] ¹ H NMR (400 MHz, DMSO-cfe) δ ppm 1.99 (dq,> 13.26, 6.86 Hz, 2 H) 2.30 - 2.46 (m, 2 H) 3, 10 (s, 3 H) 3.15 - 3.37 (m, 2 H) 3.74 (s, 3 H) 3.95 - 4.06 (m, 1 H) 4.07 - 4.17 ( m, 2 H) 4.34 - 4.43 (m, 1 H) 5.72 (d,> 8.08 Hz, 1 H) 6.54 (s, 1 H) 6.63 (s, 1 H ) 6.89 (s, 1 H) 6.99
- 7.05 (m, 2 H) 7.14 (d,> 1.52 Hz, 1 H) 7.31 (d,> 8.08 Hz, 1 H) 7.38 (d,
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55/77> 7.58 Hz, 1 Η) 7.45 (d,> 8.08 Hz, 1 Η) 8.38 (s, 1 Η) 12.09 (s I, 1 Η) [00180] LC / MS (LC-A method): Rt 2.73 min, MH * 641 [00181] [a] _D ²⁰ : -49.8 ° (c 0.225, DMF) [00182] Chiral SFC (SFC-F method): Rt 3.13 min, without EM response, 100% chiral purity.
[00183] Melting point: 182 ° C
Enantiomer 7B:
[00184] ¹ H NMR (400 MHz, DMSO-de) δ ppm 1.99 (dq,> 13.26, 6.86 Hz, 2 H) 2.30 - 2.46 (m, 2 H) 3, 10 (s, 3 H) 3.15 - 3.37 (m, 2 H) 3.74 (s, 3 H) 3.95 - 4.06 (m, 1 H) 4.07 - 4.17 ( m, 2 H) 4.34 - 4.43 (m, 1 H) 5.72 (d,> 8.08 Hz, 1 H) 6.54 (s, 1 H) 6.63 (s, 1 H ) 6.89 (s, 1 H) 6.99 - 7.05 (m, 2 H) 7.14 (d,> 1.52 Hz, 1 H) 7.31 (d,> 8.08 Hz , 1 H) 7.38 (d,> 7.58 Hz, 1 H) 7.45 (d,> 8.08 Hz, 1 H) 8.38 (s, 1 H) 12.09 (s I, 1 H) [00185] LC / MS (LC-A method): Rt 2.73 min, MH ⁺ 641 [00186] [a] _D ²⁰ : + 49.3 ° (c 0.2333, DMF) [00187] Chiral SFC (SFC-F method): Rt 4.34 min, without EM response, 100% chiral purity.
[00188] Melting point: 180 ° C
Example 8: synthesis of 4- (5-chloro-2- (1 ~ ((3-methoxy-5- (methylsulfonyl) pheniO-amino ^ - ^ - methoxy-e ^ trifluoromethiQindolin-1-iQ ^ -oxoetiQphenoxy) butanoic acid (Compound 8) and chiral separation in Enantiomers §A and 8B.
$
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Synthesis of intermediate 8a:
[00189] A mixture of 5-methoxy-6- (trifluoromethoxy) indoHna 2g (617 g, 2.84 mmol), 2- (2- (4 ~ (ferc-butoxy) -4-oxobutoxy) ~ 4 ~ chlorophenyl acid ) -2 ((3-methoxy-5- (methylsulfonyl) phenyl) amino) acetic 7d (1.5 g, 2.84 mmol), HATU (1.62 g, 4.26 mmol) and diisopropylethylamine (1 , 4 mL, 8.5 mmol) in DMF (60 mL) was stirred at room temperature for 12 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% aqueous K2CO3 solution, water, dried over filtered MgSCX and the solvent was concentrated under reduced pressure. Purification was carried out by flash chromatography on silica gel (15-40 pm, 120 g, heptane / EtOAc 60/40) to give, after crystallization from petroleum ether / diisopropyl ether, 4- (5-chlorine -2- (1 - ((3-methoxy-5- (methylsulfonyl) phenyl) am! No) ~ 2 ~ (5 ~ methoxy-6- (trifluoromethyl) indolin-1-yl) ~ 2 ~ oxoethyl) phenoxy) bu fer-butyl tanoate 8a (1.36 g).
Synthesis of Compound 8 and chiral separation in Enantiomers 8A and 8B: [00190] A solution of 4- (5 ~ chloro ~ 2 ~ (1 - ((3 ~ methoxy! ~ 5 ~ (methylsulfonyl) phenyl) amino) -2- Ferric-butyl 8a (1.36 mg, 1.87 mmol) (5-methoxy-6- (trifluoromethyl) indolin-1-yl) -2-oxoethyl) phenanoxy (1.36 mg, 1.87 mmol) in 4M HCI in dioxane (12 mL) was stirred at 5 ° C for 3 h and at room temperature for 14 h. The precipitate was removed by filtration and washed with dioxane / diisopropyl ether to produce 4- (5-chloro-2- (1 - ((3 methoxy! ~ 5 ~ (methylsulfonyl) fenn) amino) -2- (5 ~ methoxy! ~ 6 ~ (trifluoromethyl) indolin1-ii) -2-oxoethyl) phenoxy) butanoic (Compound 8, 1.2 g) as a racemate (contaminated with 2.2% of intermediate 8a). The smaller fraction (40 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 produce, after crystallization from ChhCN / diisopropyl ether, 28 mg of compound 8. The remaining amount of Compound 8 was used to separate enantiomers via Preparative Chiral
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SFC (Stationary phase: Chiralpak® IC 5 pm 250 x 30 mm, mobile phase: 60% CO ₂ , 40% MeOH). The first eluted enantiomer (340 mg) was solidified in petroleum ether / diisopropyl ether to give the 8A Enantiomer (285 mg). The second eluted enantiomer (334 mg) was solidified in petroleum ether / diisopropyl ether to give 0B Enantiomer (210 mg).
Compound 8:
[00191] ¹ H NMR (400 MHz, DMSO-cfe) δ ppm 1.95 - 2.08 (m, 2 H)
2.32 - 2.44 (m, 2 H) 3.08 - 3.27 (m, 5 H) 3.73 (s, 3 H) 3.84 (s, 3 H) 3.92 - 4, 00 (m, 1 H) 4.12 (d I,> 3.54 Hz, 2 H) 4.32 - 4.40 (m, 1 H) 5.69 (d I,> 8.08 Hz, 1 H) 6.54 (s I, 1 H) 6.62 (s, 1 H) 6.87 (s, 1 H) 6.98 - 7.04 (m, 2 H) 7.14 (s, 1 H) 7.22 (s, 1 H) 7.31 (d,> 8.08 Hz, 1 H) 8.34 (s, 1 H) 12.07 (s I, 1 H) [00192] LC- MS (LC-A method): Rt 2.74 min, MH ⁺ 671 [00193] Melting point: 232 ° C
Enantiomer 8A:
[00194] ¹ H NMR (500 MHz, DMSO-de) δ ppm 1.95 - 2.07 (m, 2 H) 2.35 - 2.47 (m, 2 H) 3.11 (s, 3 H ) 3.15 - 3.31 (m, 2 H) 3.74 (s, 3H) 3.85 (s, 3 H) 3.91 - 4.02 (m, 1 H) 4.06 - 4, 19 (m, 2 H) 4.37 (td,> 10.25, 6.31 Hz, 1 H) 5.70 (d,> 8.20 Hz, 1 H) 6.54 (s, 1 H) 6.63 (s, 1 H) 6.88 (s, 1 H) 7.02 (d,> 8.20 Hz, 2 H) 7.12 - 7.17 (m, 1 H) 7.23 ( s, 1 H) 7.31 (d,> 8.20 Hz, 1 H) 8.34 (s, 1 H) 12.13 (s I, 1 H) [00195] LC / MS (LC-A method ): Rt 2.75 min, MH ⁺ 671 [00196] [a] _D ²⁰ : -52.9 ° (c 0.28, DMF) [00197] chiral SFC (SFC-G method): Rt 2.50 min , MH * 671, chiral purity 100%.
Enantiomer 8B:
[00198] Ή NMR (500 MHz, DMSO-de) δ ppm 1.95 - 2.07 (m, 2 H) 2.35 - 2.47 (m, 2 H) 3.11 (s, 3 H) 3.15 - 3.31 (m, 2 H) 3.74 (s, 3H) 3.85 (s, 3 H) 3.91 - 4.02 (m, 1 H) 4.06 - 4.19 (m, 2 H) 4.37 (td,> 10.25, 6.31
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Hz, 1 Η) 5.70 (d,> 8.20 Hz, 1 Η) 6.54 (s, 1 Η) 6.63 (s, 1 Η) 6.88 (s, 1 Η) 7.02 (d,> 8.20 Hz, 2 Η) 7.12 - 7.17 (m, 1 Η) 7.23 (s, 1 Η) 7.31 (d,> 8.20 Hz, 1 Η) 8 , 34 (s, 1 Η) 11.44 (s I, 1 Η) [00199] LC / MS (LC-A method): Rt 2.73 min, MH ⁺ 671 [00200] [a] _D ²⁰ : + 46.4 ° (c 0.28, DMF) [00201] Chiral SFC (SFC-G method): Rt 3.31 min, MH ⁺ 671, 100% chiral purity.
Example 9: Synthesis of 4 (5 "Chloro-2" (1 ((3-methoxy-5- (methylsulfonyl) phenyl) -amino) -2-oxo-2- (6- (trifluoromethoxy) indolin-1-yl) ethyl ) phenoxy) butanol · co (Compound 9) and chiral separation in Enantiomers 9A and 9B.
O . fô · O Ç: v  6 ' ® ^Λ ·· 4 .... <45Q. 7. · ''> ϊ4 · ·· '<· .. >> R. · '···· -.-.! <C- H EWF.fctzn ^ 4
HO:
Q
Ç,
Synthesis of intermediate 9a:
[00202] A mixture of 6- (trifluoromethoxy) indoline [CAS 959235-951] (577 g, 2.84 mmol), 2- (2- (4- (tert-butoxy) -4-oxobutoxy) -4-chlorophenyl acid) -2 - ((3-methoxy · 5- (methylsulfonyl) phenyl) amino) acetic 7d (1.5 g, 2.84 mmol), HATU (1.62 g, 4.26 mmol) and diisopropylethylamine (1 , 4 mL, 8.5 mmol) in DMF (60 mL) was stirred at room temperature for 12 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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59/77 with a 10% aqueous K2CO3 solution, water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. Purification was carried out by flash chromatography on silica gel (15-40 pm, 120 g, heptane / EtOAc 60/40) to give, after crystallization from petroleum ether / diisopropyl ether, 4- (5-chlorine -2- (1 - ((3-methoxy-5- (methylsulfonyl) phenyl) amino) -2-oxo-2- (6- (trifluorornethoxy) indolin-1-yl) ethyl) phenoxy) ferc-butyl butanoate 9a (1.02 g).
Synthesis of Compound 9 and chiral separation in Enantiomers 9A and 9B: [00203] A solution of 4- (5-chloro-2- (1 - ((3-methoxy-5- (methylsulfonyl) phenyl) amino) -2-oxo -2- (6- (trifluoromethoxy) indolin-1-yl) ethyl) phenoxy) ferc-butyl butanoate 9a (1.02 g, 1.43 mmol) in 4M HCI in dioxane (10 mL) was stirred at 5 ° C for 3 h and at room temperature for 12 h. The precipitate was removed by filtration and washed with dioxane / diisopropyl ether to give 4- (5-chloro-2- (1 - ((3-methoxy-5- (methylsulfonyl) phenii) -amino) -2-oxo acid -2- (6- (trifluoromethoxy) indolin-1-yl) ethyl) phenoxy) butanoic (Compound 9, 930 mg, 0.78 equiv. HCI, 0.08 equiv. H2O, 0.162 equiv. Dioxane (determined by titration) ) as a racemate. The Enantiomers were separated by Preparative Chiral SFC (Stationary phase: Chiralpak® IC 5 pm 250 x 30 mm, Mobile phase: 70% COz, 30% EtOH / iPrOH (50/50)). The first eluted enantiomer 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 compound was crystallized from CHsCN / diisopropyl ether to give the 9A Enantiomer (145 mg). The second eluted enantiomer 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 compound was crystallized from CHaCN / diisopropyl ether to give the 9B Enantiomer (156 mg). Compound 9:
[00204] ¹ H NMR (400 MHz, DMSO-cfe) δ ppm 1.99 (dq, J = 13.71,
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7.05 Hz, 2 Η) 2.32 - 2.46 (m, 2 Η) 3.08 - 3.20 (m, 5 Η) 3.74 (s, 3 Η) 4.00 (td,> 10.23, 7.33 Hz, 1 H) 4.07 - 4.15 (m, 2 H) 4.38 (td,> 10.23, 6.82 Hz, 1 H) 5.70 (s, 1 H) 6.54 (s, 1 H) 6.63 (s, 1 H) 6.88 (s, 1 H) 6.95 - 7.09 (m, 2 H) 7.14 (d,> 1.52 Hz, 1 H) 7.30 (d I,> 8.08 Hz, 1 H) 7.33 (d I,> 8.59 Hz, 1 H) 8.03 (s, 1 H) [ 00205] LC-EM (LC-A method): Rt 2.87 min, MH ⁺ 657 [00206] Melting point: 173 ° C
9A Enantiomer:
[00207] ¹ H NMR (500 MHz, DMSO-d ₆ ) δ ppm 1.91 - 2.06 (m, 2 H)
2.32 - 2.44 (m, 2 H) 3.09 - 3.23 (m, 5 H) 3.74 (s, 3 H) 3.98 - 4.16 (m, 3 H) 4, 39 (td,> 10.17, 6.78 Hz, 1 H) 5.71 (d,> 8.20 Hz, 1 H) 6.55 (s, 1 H) 6.63 (s, 1 H) 6.89 (s, 1 H) 7.00 - 7.08 (m, 2 H) 7.15 (s, 1 H) 7.30 (d,> 8.20 Hz, 1 H) 7.34 ( d,> 8.20 Hz, 1 H) 8.04 (s, 1 H) 12.11 (s I, 1 H) [00208] LC / MS (LC-A method): Rt 2.86 min, MH ⁺ 657 [00209] [a] _D ²⁰ : -56.5 ° (c 0.255, DMF) [00210] Chiral SFC (SFC-H method): Rt 4.85 min, MH ⁺ 657, 100% chiral purity.
[00211] Melting point: 154 ° C
9B Enantiomer:
[00212] ¹ H NMR (500 MHz, DMSO-de) δ ppm 1.91 - 2.06 (m, 2 H)
2.32 - 2.44 (m, 2 H) 3.09 - 3.23 (m, 5 H) 3.74 (s, 3 H) 3.98 - 4.16 (m, 3 H) 4, 39 (td,> 10.17, 6.78 Hz, 1 H) 5.71 (d,> 8.20 Hz, 1 H) 6.55 (s, 1 H) 6.63 (s, 1 H) 6.89 (s, 1 H) 7.00 - 7.08 (m, 2 H) 7.15 (s, 1 H) 7.30 (d,> 8.20 Hz, 1 H) 7.34 ( d,> 8.20 Hz, 1 H) 8.04 (s, 1 H) 12.11 (s I, 1 H) [00213] LC / MS (LC-A method): Rt 2.86 min, MH ⁺ 657 [00214] [a] _D ²⁰ : + 55.3 ° (c 0.302, DMF) [00215] Chiral SFC (SFC-H method): Rt 6.34 min, MH ⁺ 657, 100% chiral purity.
[00216] Melting point: 155 ° C
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Table: compounds prepared as described above
Compound Structure Optical rotation 1 F och ₃ Q, Á- / “ H V_ / ίΑΥ « A II »cA u _CH3 racemic 1A F p _CH ., .... Ύ ί ΥΆ Ά ^ζ ch ₃ [cc] _D ²⁰ - -49.6 ° 1B F OCH-. UV λ _Ηι [a] _D ²⁰ - + 51.7 ° 2 F ç> ch ₃ FC YY h “O rlv / X ^ · Ν _n 11) oA CH» · '- ^ ⁷ CH ₃ racemic 2A F 11 _OCH ^ | (+) ^ _z FC ../Ylíj ^ · Ν _n Tí s X Y-ss · '*''11) oA CH ^' ^ ' ⁷ CH ₃ [box] d ²⁰ + 53.5 ° 2B F _n JLl pCH ₃ 1 (-) o JL [cx] d ²⁰ = -56.5 °
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Compound Structure Optical rotation 3 F OCH ₃ .ΟΟγ / υΚ) ₀ ^v CH ₃ racemic 3A F OCH ₃ ^< ^ Y ^Xn A / ^{, #} Υγ ^{H v} V W _{oA cHi} [a] o ²⁰ - -59.0 ° 3B och _{3 F} , O0 <t (JO X; [a] _D ²⁰ = + 56.8 ° 4 Ç! ^Ho x / x. _o jC ^ OCH _{3 rc} ΎΟ ·· 0 Όν JA racemic 4A Çl ^H Xx ^ _rY jC ^ och ₃ 'come 7kJ-ζ ^! ~! ₃ [a] o ²⁰ ··· * 59.8 ^o 4B Çl J p _{CH; s} c. ! (-) / == ( _F ~ Ί KO ^υ CHj [a] _D ²⁰ -56.4 ° 5 Ci ^HC V ^ _O 0C | - | ₃ η m „jí 0 ^ CH ₃ O '^ ^{XíS #} ^ ^{/ ch} 3 racemic
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Compound Structure Optical rotation 5A Cl och ₃ T (-) / == / _o JLAy A. CH ₃ O ^ch 3 [a] o ²⁰ -60.4 ° 5B Cl OCH ₃ | (+) / == ( ^Γ 'ΎΥ CH.O' · ^ '' CH ₃ [a] o ²⁰ * 56.7 ° 6 Cl HO _x _x _q .JI _s ^ _í J och ₃ CK Jx, Zx o Y ti # ^ ^z - CH ₃ racemic 6A Cl och ₃ | (+) ch ₃ [a] o ²⁰ “+ 64.3 ° 6B Cl och ₃ ^ χο'Υ [α] ο ²⁰ -53.7 ° 7 __och ₃ ό cv Â. Λχ t rw ' ³ ' ^ V ^% r ^ ^r CH ₃ racemic 7A IA / <n _H3 [ _a ] _D 2Q -49.8 °
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Compound Structure Optical rotation 7B _Ύ [ _a ] _D 2nd 4-49.3 ° 8 __OCHjvw χχ> Ία racemic 8A OCH ₃ ο ./x -vÇv to Ji J / 'CH ₃ [a] _D ²⁰ - -52.9 ° 8B och ₃ Lw AJV [α] ϋ ^ζ0 ~ + 46.4 ° 9 P ^CH = racemic 9A pCH ₃ Uv [α] ο ²⁰ -56.5 ° 9B Ci ^ Vx ^ / U OCH ₃ & Olh _Fca I Τλ / IL) ^J CH ₃ [a] _D ²⁰ - + 55.3 °
ANTIVIRAL ACTIVITY OF THE COMPOUNDS OF THE INVENTION
DENV-2 antiviral assay [00217] The antiviral activity of all compounds of the invention was
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65/77 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 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: 25pM - 0.000064 μΜ or 2.5 pM - 0.0000064 pM 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 are 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 ° C, 5% CO ₂ ) until the next day. Then, the 16681 DENV-2 strain, 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 parallel, 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 ° C, 5% CO2). On the day of reading, the
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66/77 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).
[00218] 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 (CCso) 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 calculated as follows: SI = CC50 / EC50.
Table 1: EC50, CC50 and SI for the compounds of the invention in the DENV-2 antiviral assay
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# of the compound EC50 (μΜ) N CC50 (μΜ) N SI N 1 0.0032 14 4397 1A 2.4 12 4.8 1B 0.0014 3.4 > 1980 2 0.0010 12 13700 2A 0.00093 9.6 16400 2B 0.17 18 105 3 0.00074 7.9 10700 3A 0.57 11 19 3B 0.00061 8.7 16300 4 0.00066 7 7 11600 4A 0.00041 6.0 15000 4B 0.016 11 693 5 0.00070 11 15700 5A 0.076 16 210 5B 0.00023 8.5 > 16800 6 0.00043 3.6 7070 6A 0.00023 7.3 > 12000 6B 0.020 10 492 7 0.00058 13 21800 7A 0.069 11 165 7B 0.00025 11 90600 8 0.0019 14 7460 8A 0.095 12 126 8B 0.0012 14 6780 9 0.00031 12 40200 9A 0.12 12 93 9B 0.00015 13 830004 4 43 3 33 3 34 4 43 4 33 3 33 3 33 3 3
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# of the compound ECso (mM) N CC50 (μΜ) N SI N3 3 33 3 37 7 76 7 63 3 33 3 34 4 44 4 47 8 75 6 53 3 33 4 35 7 53 3 34 5 43 3 33 3 33 3 34 4 4
N = the number of independent experiments in which the compounds were tested.
Quantitative PCR assay with tetravalent reverse transcriptase (RT-qPCR) [00219] The antiviral activity of the compounds of the invention was tested against the DENV-1 strain TC974 # 666 (NCPV), DENV-2 strain 16681, DENV- strain 3 H87 (NCPV) and DENV-4 H241 (NCPV) strain 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. At day 3 post-infection, the cells were used and the used cells were used to prepare cDNA from both a viral target (at 3'UTR of DENV; Table 2) and a cell reference gene (βactin, Table 2). Subsequently, a real-time duplex PCR
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69/77 was performed on a Lightcycler480 instrument. The generated Cp value 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 3'LJTR 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 ÁÁCp method is used to calculate the EC50, which is based on the relative gene expression of the target gene (3 ^! RTU) 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 '- / Afí / ÍFQ Factin743 β-actin 5 ‘-GGCCAGGTCATCACCATT-3‘ Ractin876 β-actin 5'-ATGTCCACGTCACACTTCATG-3 ' Pactin773 β-actin HEX-5'-TTCCGCTGC-ZEW-CCTGAGGCTCTC-3 '- / ABkFQ
^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 3TJTR 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).
[00220] The culture medium consisted of a minimum essential medium supplemented with 2% fetal calf serum inactivated by heat,
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70/77 0.04% gentamicins (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 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% DIVISO (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 ° C, 5% CO2) 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 pL of virus suspension was added to all wells containing test compound and to the wells assigned as virus control. In parallel, 25 pL of culture medium was added to the cell controls. Then, the plates were incubated for 3 days in a fully humidified incubator (37 ° C, 5% CO2). After 3 days, the supernatant was removed from the wells and the cells washed twice with ice-cold PBS (~ 100 pL). 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.
[00221] 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,
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71/77 a five minute denaturation step was carried out at 75 ° C (table 3B). Then 7.43 pL of mixture B was added (table 3C) and the reverse transcription step was started (table 3D) to generate cDNA. [00222] 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 pL of cDNA was added and the qPCR was performed according to the conditions in table 4B on a LightCycler 480.
[00223] Using LightCycler software and an internal LIMS system, dose response curves were calculated for each compound and determined half of the maximum effective concentration (ECso) and half of the maximum cytotoxic concentration (CCso) (Tables 5-8).
Table 3: Synthesis of cDNA using Mixture A, denaturation, Mixture B and reverse transcription.
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Mixture A
Signs 8 Samples 828Reaction Vol. (PL) 20 Mixing Item Concentration Volume to (pL)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 0.15 124.20Mixing volume / well (pL) 7.57Cell lysates 5.00
zz / zz
B Denaturation step:
Step Temp Time Denaturation 75 ° C 5 ' Maintenance 4 ° C maintenance
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C Mix Β
Samples 864Mixing Item Concentration Volume to (pL) unity Stock Final 1 sample x samples Expand HIFI 2 buffer 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 Rnase Inhibitor U / pL 40.00 1.00 0.50 432.0 Expand RT U / pL 50.00 0.33 0.13 112.3Total Mix Volume (pL) 7.43
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D cDNA Synthesis Protocol
Step Temp Time Transc rev 42 ° C 30 ' Creamed feed 99 ° C 5 ’ Maintenance 4 ° C maintenance
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Table 4: Mixture and qPCR protocol.
The Mixture C
Samples 833Reaction Vol. (PL) 25 Mixing Item Concentration Volume to (pL)unity Stock Final 1 sample x samples H2Õ grade PCR Roche 7.74 6447.42 Mixture 2xMM Roche X 2 1 12.50 10412.50 F3utr258 pM 20 0.3 0.38 316.54 R3utr425 pM 20 0.3 0.38 316.54 P3utr343 pM 20 0.1 0.13 108.29 Factin743 pM 20 0.3 0.38 316.54 Ractin876 pM 20 0.3 0.38 316.54 Pactina773 pM 20 0.1 0.13 108.29Volume Mixture / Tube (pL) 22.02cDNA 3.00
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QPCR3 protocol
Step Temp Time Ramp ratepre-incub / skim 95 ° C 10 mL / min 4.4 Denaturation 95 ° C 10 sec 4.4 40 cycles pairing 58 ° C 1 min 2.2 Stretching 72 ° C 1 sec 4.4 Cooling 40 ° C 10 sec 1.5
Table 5: EC50, CC50 and SI for compounds against 0 serotype 1 in RT-qPCR assays
RT-qPCR serotype 1 TC974 # 666 # of the compound EC50 (μΜ) N CC50 (μΜ) N Si N 1B 0.0015 3 > 2.5 3 > 2860 3 2A 0.0046 5 > 2.5 4 > 981 4 3B 0.0011 3 > 2.5 3 > 3640 3 4A 0.00075 3 > 2.5 3 > 5470 3 5B 0.00094 4 5.1 4 8640 4 6A 0.00016 3 > 2.5 3 > 41300 3 7B 0.00013 3 > 2.5 3 > 19200 3 8B 0.0012 3 14 3 10500 3 9B 0.00010 3 > 2.5 3 > 45500 3
N = The number of independent experiments in which the compounds were tested.
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Table 6: ECso, CC50 and SI for compounds against 0 serotype 2 in RT-qPCR assays
RT-qPCR serotype 2 16681 # of the compound EC50 (μΜ) N CC50 (μΜ) N SI N 1B 0.0011 3 4.1 3 4670 3 2A 0.0013 4 12 4 4910 4 3B 0.00090 3 3.6 3 4760 3 4A 0.00045 3 2.7 3 11700 3 5B 0.00024 5 4.2 5 > 17100 5 6A 0.00016 3 4.2 3 > 12600 3 7B 0.00019 3 > 2.5 2 > 13500 2 8B 0.00030 3 16 2 54300 2 9B 0.000068 3 > 2.5 3 > 56500 3
N The number of independent experiments in which the compounds were tested.
Table 7: EC50, CC50 and SI for compounds against 0 serotype 3 in RT-qPCR assays
RT-qPCR serotype 3 H87 # of the compound EC50 (μΜ) N CC50 (μΜ) N SI N 1B 0.020 3 > 2.5 3 > 127 3 2A 0.020 3 > 2.5 3 > 157 3 3B 0.013 3 > 2.5 3 > 444 3 4A 0.013 3 > 2.5 3 > 234 3 5B 0.0067 4 > 2.5 4 > 752 4 6A 0.0026 3 > 2.5 3 > 1480 3 7B 0.0052 3 > 2.5 3 > 473 3 8B 0.019 3 12 3 796 3 9B 0.0017 3 > 2.5 3 > 1840 3
N The number of independent experiments in which the compounds were tested.
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Table 8: ECso, CC50 and SI for compounds against 0 serotype 4 in RT-qPCR assays
RT-qPCR serotype 4 H241 # of the compound EC50 (μΜ) N CC50 (μΜ) N SI N 1B 0.13 3 > 2.4 3 23 3 2A 0.10 3 2.8 3 35 3 3B 0.072 3 > 2.3 3 > 32 3 4A 0.044 4 2.2 4 69 4 5B 0.026 4 2.5 2 86 2 6A 0.026 4 2.3 4 119 4 7B 0.024 3 > 2.5 3 > 186 3 8B 0.084 3 7.4 3 88 3 9B 0.0072 3 5.0 2 1390 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), including any stereochemically isomeric form of it,

[2]
2/4 table.
2. Compound according to claim 1, characterized by the fact that said compound is selected from

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

[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 vehicles.
[6]
6. Pharmaceutical composition according to claim 5, characterized by the fact that it comprises a second or more active ingredients.
[7]
7. Pharmaceutical composition according to claim 6, characterized by the fact that the second or more active ingredients are an antiviral agent.
[8]
Compound of formula (I) according to any one of claims 1 to 4, characterized in that it is for use as a medicament.
[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.
Petition 870190095446, of 24/09/2019, p. 100/103
4/4
[10]
10. Compound of formula (I) for use, according to claim 9, characterized by the fact that the 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:
2021-04-20| B08F| Application dismissed because of non-payment of annual fees [chapter 8.6 patent gazette]|Free format text: REFERENTE A 3A ANUIDADE. |

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2021-08-10| B08K| Patent lapsed as no evidence of payment of the annual fee has been furnished to inpi [chapter 8.11 patent gazette]|Free format text: REFERENTE AO DESPACHO 8.6 PUBLICADO NA RPI 2624 DE 20/04/2021. |

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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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EP17164045|2017-03-31|

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PCT/EP2018/058077|WO2018178238A1|2017-03-31|2018-03-29|Substituted indoline derivatives as dengue viral replication inhibitors|

---