Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
  • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
  • C07D209/04—Indoles; Hydrogenated indoles
  • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
  • C07D209/18—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
  • C07D209/26—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with an acyl radical attached to the ring nitrogen atom
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
  • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
  • A61K31/404—Indoles, e.g. pindolol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
  • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
  • A61K31/404—Indoles, e.g. pindolol
  • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/76—Viruses; Subviral particles; Bacteriophages
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to substituted indoline derivatives, methods to prevent or treat dengue viral infections by using said compounds and also relates to said compounds for use as a medicine, more preferably for use as a medicine to treat or prevent dengue viral infections.
• the present invention furthermore relates to pharmaceutical compositions or combination preparations of the compounds, to the compositions or preparations for use as a medicine, more preferably for the prevention or treatment of dengue viral infections.
• the invention also relates to processes for preparation of the compounds.
• Flaviviruses which are transmitted by mosquitoes or ticks, cause life-threatening infections in man, such as encephalitis and hemorrhagic fever.
• Four distinct, but closely related serotypes of the flavivirus dengue are known, so-called DENV-1 , -2, -3, and -4.
• Dengue is endemic in most tropical and sub-tropical regions around the world, predominantly in urban and semi-urban areas. According to the World Health Organization (WHO), 2.5 billion people of which 1 billion children are at risk of DENV infection (WHO, 2002).
• WHO World Health Organization
• DHF dengue hemorrhagic fever
• DSS dengue shock syndrome
• the mosquitoes that carry dengue including Aedes aegypti and Aedes albopictus (tiger mosquito), are moving north on the globe. According to the United States (US) Centers for Disease Control and Prevention (CDC), both mosquitoes are currently omnipresent in southern Texas. The spread north of dengue-carrying mosquitoes is not confined to the US, but has also been observed in Europe.
• US United States
• CDC Centers for Disease Control and Prevention
• Dengvaxia® the dengue vaccine produced by Sanofi Pasteur was first approved in Mexico and has received in the meantime approval in more countries.
• the vaccine leaves considerable room for improvement due to limited efficacy, especially against DENV-1 and -2, low efficacy in flavivirus-na ' fve subjects and the lengthy dosing schedule.
• the vaccine is a game changer in endemic settings as it will offer protection to a large part of the population, but likely not to very young infants, who bear the largest burden of dengue.
• the dosing schedule and very limited efficacy in flavivirus-na ' fve subjects make it unsuitable and likely not worthwhile/cost-effective for travelers from non-endemic areas to dengue-endemic areas.
• the above mentioned shortcomings of the dengue vaccines are the reason why there is a need for a pre-exposure prophylactic dengue antiviral.
• specific antiviral drugs for the treatment or prevention of dengue fever virus infection are not available.
• WO-2010/021878 discloses 2-phenylpyrrolidine and indoline derivatives as cold menthol receptor antagonists for treatment of inflammatory and central diseases.
• WO-2013/045516 discloses indole or indoline derivatives for use in the treatment of dengue viral infections.
• the present invention now provides compounds, substituted indoline derivatives, which show high potent activity against all four (4) serotypes of the Dengue virus.
• the present invention is based on the unexpected finding that at least one of the above-mentioned problems can be solved by the current compounds of the invention.
• the present invention provides compounds which have been shown to possess potent antiviral activity against all four (4) serotypes currently known.
• the present invention furthermore demonstrates that these compounds efficiently inhibit proliferation of 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 for the treatment and/or prevention of infections with Dengue viruses.
• DEV Dengue virus
• the present invention furthermore relates to the use of such compounds as medicines and to their use for the manufacture of medicaments for treating and/or preventing viral infections, in particular with viruses belonging to the family of the Dengue viruses in animals or mammals, more in particular in humans.
• the invention also relates to methods for the preparation of all such compounds and to pharmaceutical compositions comprising them in an effective amount.
• the present invention also relates to a method of treatment or prevention of dengue viral infections in humans by the administration an effective amount of one or more such compounds, or a pharmaceutically acceptable salt thereof optionally combination with one or more other medicines, like another antiviral agent, to patient in need thereof.
• One aspect of the invention is the provision of compounds having formula (I), a stereoisomeric form, a pharmaceutically acceptable salt, solvate or polymorph thereof :
• R is trifluoromethyl, R 2 is hydrogen, R 3 is hydrogen, and R is methoxy; or
• R is trifluoromethyl, R 2 is hydrogen, R 3 is hydrogen, and R is fluoro; or
• R is trifluoromethoxy, R 2 is hydrogen, R 3 is hydrogen, and R is hydrogen; or R is trifluoromethyl, R 2 is hydrogen, R 3 is hydrogen, and R is hydrogen; or R is trifluoromethoxy, R 2 is hydrogen, R is hydrogen, and R is methoxy; or R is trifluoromethoxy, R 2 is hydrogen, R is hydrogen, and R is fluoro; or
• R is trifluoromethyl, R 2 is methoxy, R 3 is hydrogen, and R is hydrogen;
• R is trifluoromethyl, R 2 is methoxy, R 3 is hydrogen, and R is fluoro; or
• R is trifluoromethoxy, R 2 is hydrogen, R is methyl, and R is hydrogen; or
• R is trifluoromethyl, R 2 is hydrogen, R is methyl, and R is hydrogen; or
• R is trifluoromethyl, R 2 is methoxy, R 3 is hydrogen, and R is methoxy; or
• R is trifluoromethyl, R 2 is fluoro, R 3 is hydrogen, and R is hydrogen;
• R is trifluoromethoxy, R 2 is methoxy, R 3 is hydrogen, and R is hydrogen.
• Part of the current invention is also a pharmaceutical composition
• a pharmaceutical composition comprising a compound mentioned above or a stereoisomeric form, a pharmaceutically acceptable salt, solvate or polymorph thereof together with one or more
• Pharmaceutically acceptable salts of said compounds include the acid addition and base salts thereof. Suitable acid addition salts are formed from acids which form non-toxic salts. Suitable base salts are formed from bases which form non- toxic salts.
• the compounds of the invention may also exist in un-solvated and solvated forms.
• solvate is used herein to describe a molecular complex comprising the compound of the invention and one or more pharmaceutically acceptable solvent molecules, for example, ethanol.
• polymorph refers to the ability of the compound of the invention to exist in more than one form or crystal structure.
• the compounds of the present invention may be administered as crystalline or amorphous products. They may be obtained for example as solid plugs, powders, or films by methods such as precipitation, crystallization, freeze drying, spray drying, or evaporative drying. They may be administered alone or in combination with one or more other compounds of the invention or in combination with one or more other drugs. Generally, they will be administered as a formulation in association with one or more pharmaceutically acceptable excipients.
• 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 stability, and the nature of the dosage form.
• compositions of the present invention may be formulated into various pharmaceutical forms for administration purposes.
• compositions there may be cited all compositions usually employed for systemically administering drugs.
• an effective amount of the particular compound, optionally in addition salt form, as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of preparation desired for administration.
• compositions are desirably in unitary dosage form suitable, for example, for oral or rectal administration.
• unitary dosage form suitable, for example, for oral or rectal administration.
• compositions in oral dosage form any of the usual pharmaceutical media may be employed such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid preparations such as suspensions, syrups, elixirs, emulsions, and solutions; or solid carriers such as starches, sugars, kaolin, diluents, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules, and tablets. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit forms, in which case solid pharmaceutical carriers are obviously employed. Also included are solid form preparations that can be converted, shortly before use, to liquid forms.
• Unit dosage form refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
• unit dosage forms are tablets (including scored or coated tablets), capsules, pills, powder packets, wafers, suppositories, injectable solutions or suspensions and the like, and segregated multiples thereof.
• an effective daily amount would be from 0.01 mg/kg to 50 mg/kg body weight, more preferably from 0.1 mg/kg to 10 mg/kg body weight. It may be appropriate to administer the required dose as two, three, four or more sub-doses at appropriate intervals throughout the day. Said sub-doses may be formulated as unit dosage forms, for example, containing 1 to 1000 mg, and in particular 5 to 200 mg of active ingredient per unit dosage form. The exact dosage and frequency of administration depends on the particular compound of the invention used, the particular condition being treated, the severity of the condition being treated, the age, weight and general physical condition of the particular patient as well as other medication the individual may be taking, as is well known to those skilled in the art.
• the effective amount may be lowered or increased depending on the response of the treated subject and/or depending on the evaluation of the physician prescribing the compounds of the instant invention.
• the effective amount ranges mentioned above are therefore only guidelines and are not intended to limit the scope or use of the invention to any extent.
• isotopes of hydrogen include tritium and deuterium and isotopes of carbon include C-13 and C-14.
• the present compounds used in the current invention may also exist in their stereochemically isomeric form, defining all possible compounds made up of the same atoms bonded 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 might possess.
• Said mixture may contain all diastereomers and/or enantiomers of the basic molecular structure of said compound.
• All stereochemically isomeric forms of the compounds used in the present invention either in pure form or in admixture with each other are intended to be embraced within the scope of the present invention including any racemic mixtures or racemates.
• Pure stereoisomeric forms of the compounds and intermediates as mentioned herein are defined as isomers substantially free of other enantiomeric or
• Pure stereoisomeric forms of compounds and intermediates used in this invention may be obtained by the application of art-known procedures.
• enantiomers may be separated from each other by the selective crystallization of their diastereomeric salts with optically active acids or bases. Examples thereof are tartaric acid, dibenzoyltartaric acid, ditoluoyltartaric acid and camphosulfonic acid.
• enantiomers may be separated by chromatographic techniques using chiral stationary phases.
• Said pure stereochemically isomeric forms may also be derived from the corresponding pure stereochemically isomeric forms of the appropriate starting materials, provided that the reaction occurs
• stereospecifically Preferably, if a specific stereoisomer is desired, said compound will be synthesized by stereospecific methods of preparation. These methods will advantageously employ enantiomerically pure starting materials.
• 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 labelled with * :
• a "compound of formula (I)” can be the (R)-enantiomer, the (S)-enantiomer, the racemic form, or any possible combination of the two individual enantiomers in any ratio.
• this enantiomer can also be identified by indicating whether the enantiomer is dextrorotatory (+)- or levorotatory (-)- after measuring the specific optical rotation of said particular enantiomer.
• the present invention relates to a first group of compound of formula (I) wherein the compounds of formula (I) have the (+) specific rotation.
• the present invention relates to a second ground of compounds of formula (I) wherein the compounds of formula (I) have the (-) specific rotation.
• HPLC High Performance Liquid Chromatography
• MS Mass Spectrometer
• SQL Single Quadrupole Detector
• MSD Mass Selective Detector
• RT room temperature
• BEH bridged ethylsiloxane/silica hybrid
• DAD Diode Array Detector
• HSS High Strength silica.
• the SFC measurement was performed using an Analytical Supercritical fluid chromatography (SFC) system composed by a binary pump for delivering carbon dioxide (CO2) and modifier, an autosampler, a column oven, a diode array detector equipped with a high-pressure flow cell standing up to 400 bars. If configured with a Mass Spectrometer (MS) the flow from the column was brought to the (MS). It is within the knowledge of the skilled person to set the tune parameters (e.g.
• DSC823e (indicated as DSC)
• [ ⁇ ] ⁇ ⁇ (100a) / (/ x c) : 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 wavelength ⁇ (in nm). If the wavelength of light used is 589 nm (the sodium D line), then the symbol D might be used instead.
• T temperature
• ⁇ in nm
• the wavelength of light used is 589 nm (the sodium D line)
• the symbol D might be used instead.
• the sign of the rotation (+ or -) should always be given. When using this equation the concentration and solvent are always provided in parentheses after the rotation. The rotation is reported using degrees and no units of concentration are given (it is assumed to be g/100 ml).
• Example 1 synthesis of 2-(4-chloro-2-methoxyphenyl)-2-((3-(2-hydroxyethoxy)- 5-methoxyphenyl)amino)-1 -(6-(trifluoromethyl)indolin-1 -yl)ethanone (Compound 1 ) and chiral separation into Enantiomers 1A and 1 B.
• the enantiomers were separated via Preparative Chiral SFC (Stationary phase: Chiralcel ® OD-H 5 ⁇ 250 x 30mm, Mobile phase: 55% CO 2 , 45% MeOH).
• the first eluted enantiomer was further purified by flash chromatography on silica gel (15-40 ⁇ , 40 g,
• Example 2.1 synthesis of 2-(4-chloro-2-fluorophenyl)-2-((3-(2-hydroxyethoxy)-5- methoxyphenyl)amino)-1 -(6-(trifluoromethyl)indolin-1 -yl)ethanone (Compound 2).
• Example 2.2 synthesis of 2-(4-chloro-2-fluorophenyl)-2-((3-(2-hydroxyethoxy)- 5-methoxyphenyl)amino)-1 -(6-(trifluoromethyl)indolin-1 -yl)ethanone (Compound 2) and chiral separation into Enantiomers 2A and 2B.
• LiHMDS 1 M in THF (38.5 ml_, 38.5 mmol) was added dropwise to a mixture of methyl 2-(4-chloro-2-fluorophenyl)acetate [CAS 917023-
• HATU (1 .85 g, 4.87 mmol) was added to a mixture of 6-(trifluoromethyl)indoline [CAS 181513-29-1 ] (607 mg, 3.24 mmol), 2-(4-chloro-2-fluorophenyl)-2-((3-(2- hydroxyethoxy)-5-methoxyphenyl)amino)acetic acid 2e (1 .2 g, 3.24 mmol) and diisopropylethylamine (1 .6 ml_, 9.74 mmol) in DMF (35 ml_). The resulting mixture was stirred at room temperature for 12 h. The mixture was diluted with water and the precipitate was filtered off.
• a second purification was performed via achiral SFC (Stationary phase: 2-Ethylpyridine 5 ⁇ 150 x 30mm, Mobile phase: 70% CO 2 , 30% MeOH) to afford 2-(4-chloro-2-fluorophenyl)-2-((3- (2-hydroxyethoxy)-5-methoxyphenyl)amino)-1 -(6-(trifluoromethyl)indolin-1 -yl)- ethanone (Compound 2, 550 mg) as a racemic mixture. This batch was combined with another batch (total amount: 950 mg).
• the enantiomers were separated via Preparative Chiral SFC (Stationary phase: Chiralpak ® IA 5 ⁇ 250 x 20 mm, Mobile phase: 70% CO 2 , 30% iPrOH (+ 0.3% iPrNH 2 )) to give, after solidification in petroleum ether/diisopropyl ether, the first eluted Enantiomer 2A (384 mg) and the second eluted Enantiomer 2B (375 mg).
• Benzoyl peroxide (5 mg) was added to a mixture of methyl 2-(4-chlorophenyl)- acetate [CAS 52449-43-1 ] (5.0 g, 29.7 mmol) and NBS (4.82 g, 27.1 mmol) in CH3CN (80 mL). The mixture was heated under reflux for 48 h and the solvent was evaporated under reduced pressure. The mixture was taken up in
• the compound was purified by flash chromatography on silica gel (15-40 ⁇ , 40 g, heptane/EtOac 90/10). The pure fractions were combined and evaporated to dryness to give methyl 2-((3-(2-(fe/t-butoxy)ethoxy)-5-methoxyphenyl)amino)-2-(4-chlorophenyl)- acetate 3b (4.8 g).
• HATU (2.10 g, 5.52 mmol) was added to a mixture of 6-(trifluoromethoxy)indoline [CAS 953906-76-8] (747 mg, 3.68 mmol), 2-((3-(2-(terf-butoxy)ethoxy)-5-methoxy- phenyl)amino)-2-(4-chlorophenyl)acetic acid 3c (1 .5 g, 3.68 mmol) and
• the enantiomers were separated via Preparative Chiral SFC (Stationary phase: Chiralpak ® IA 5 ⁇ 250 x 20 mm, Mobile phase: 55% CO 2 , 40% EtOH (+ 0.3% iPrNh ), 5% CH2CI2). The enantiomers were further separated via preparative chiral SFC (Stationary phase: Chiralcel ® OD-H 5 ⁇ 250 x 30 mm, Mobile phase: 50% CO2, 50% EtOH (+ 0.3% iPrNH 2 )) to give, after solidification in petroleum ether/diisopropyl ether, the first eluted Enantiomer 3A (294 mg) and the second eluted Enantiomer 3B (244 mg).
• Example 4 synthesis of 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxy- phenyl)amino)-1 -(6-(thfluoromethyl)indolin-1 -yl)ethanone (Compound 4) and chiral separation into Enantiomers 4A and 4B.
• HATU (2.24 g, 5.88 mmol) was added to a mixture of 6-(trifluoromethyl)indoline [CAS 181513-29-1 ] (734 mg, 3.92 mmol), 2-((3-(2-(terf-butoxy)ethoxy)-5-methoxy- phenyl)amino)-2-(4-chlorophenyl)acetic acid 3c (1 .6 g, 3.92 mmol) and
• the enantiomers were separated via Preparative Chiral SFC (Stationary phase: Chiralcel ® OJ-H 5 ⁇ 250 x 20 mm, Mobile phase: 60% CO 2 , 40% EtOH (+ 0.3% iPrNh )).
• the first eluted enantiomer (413 mg) was solidified in heptane/ to give Enantiomer 4A (327 mg).
• the second eluted enantiomer (410 mg) was solidified in heptane/diisopropyl ether to give Enantiomer 4B (330 mg).
• Example 5 synthesis of 2-(4-chloro-2-methoxyphenyl)-2-((3-(2-hydroxyethoxy)- 5-methoxyphenyl)amino)-1 -(6-(trifluoromethoxy)indolin-1 -yl)ethanone (Compound 5) and chiral separation into Enantiomers 5A and 5B.
• the first eluted enantiomer (453 mg) was solidified in petroleum ether/diisopropyl ether to give Enantiomer 5A (355 mg).
• the second eluted enantiomer (436 mg) was solidified in petroleum ether/diisopropyl ether to give Enantiomer 5B (342 mg).
• HATU (1 .54 g, 4.06 mmol) was added to a mixture of 6-(trifluoromethoxy)indoline [CAS 953906-76-8] (550 mg, 2.70 mmol), 2-(4-chloro-2-fluorophenyl)-2-((3- (2-hydroxyethoxy)-5-methoxyphenyl)amino)acetic acid 2e (1 g, 2.70 mmol) and diisopropylethylannine (1 .34 mL, 8.1 1 mmol) in DMF (30 mL). The resulting mixture was stirred at room temperature for 12 h. The mixture was diluted with water. The precipitate was filtered off, and washed with water.
• Example 6.2 synthesis of 2-(4-chloro-2-fluorophenyl)-2-((3-(2-hydroxyethoxy)- 5-methoxyphenyl)annino)-1 -(6-(thfluoromethoxy)indolin-1 -yl)ethanone (Compound 6) and chiral separation into Enantiomers 6A and 6B.
• HATU (7.02 g, 18.5 mmol) was added to a mixture of 6-(trifluoromethoxy)indoline [CAS 953906-76-8] (2.5 g, 12.31 mmol), 2-(4-chloro-2-fluorophenyl)acetic acid [CAS 194240-75-0] (2.32 g, 12.3 mmol) and diisopropylethylamine (6.1 ml_, 36.9 mmol) in DMF (100 ml_). The resulting mixture was stirred at room
• the first eluted enantiomer (1 .45 g) was solidified by trituration with MeOH/water to give Enantiomer 6A (1 .409 g).
• the second eluted enantiomer (1 .41 g) was solidified by trituration with MeOH/water to give Enantiomer 6B (1 .37 g).
• Example 7.1 synthesis of 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxy- phenyl)amino)-1 -(5-methoxy-6-(trifluoromethyl)indolin-1 -yl)ethanone (Compound 7)
• Bhb-Pyridine (23.5 mL, 232.4 mmol) was added dropwise to a solution of 5-methoxy-6-(trifluoromethyl)-1 /-/-indole 7b (10 g, 46.5 mmol) in EtOH (60 mL).
• 6N HCI 140 mL was slowly added while maintaining the reaction temperature below 10°C.
• the mixture was stirred at 0°C for 2 h.
• Water (200 mL) was added and the mixture was basified until pH 8-9 with a concentrated solution of NaOH in water, while keeping the reaction temperature below 20°C.
• the precipitate was filtered off, washed with water (twice) and co-evaporated under reduced pressure with toluene to give 5-methoxy-6-(trifluoromethyl)indoline 7c (9 g).
• HATU (0.84 g, 2.21 mmol) was added to a mixture of 5-methoxy-6-(trifluoro- methyl)indoline 7c (320 mg, 1 .47 mmol), 2-((3-(2-(terf-butoxy)ethoxy)-5-methoxy- phenyl)amino)-2-(4-chlorophenyl)acetic acid 3c (631 mg, 1 .55 mmol) and diisopropylethylamine (731 ⁇ , 4.42 mmol) in DMF (18 mL). The reaction mixture was stirred at room temperature for 12 h. The reaction was diluted with water and EtOAc.
• Example 7.2 synthesis of 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxy- phenyl)amino)-1 -(5-methoxy-6-(trifluoromethyl)indolin-1 -yl)ethanone (Compound 7) and chiral separation into Enantiomers 7A and 7B.
• the first eluted enantiomer (980 mg) was crystallized from MeOH to afford Enantiomer 7A (71 1 mg).
• the second eluted enantiomer (1 .08 g) was further purified by flash chromatography on silica gel (15-40 ⁇ , 40 g, ChbC /MeOH 99.5/0.5). The pure fractions were combined and evaporated to dryness (950 mg) to afford, after crystallization from MeOH, Enantiomer 7B (770 mg).
• Example 8.1 synthesis of 2-(4-chloro-2-fluorophenyl)-2-((3-(2-hydroxyethoxy)-5- methoxyphenyl)amino)-1 -(5-methoxy-6-(trifluoromethyl)indolin-1 -yl)ethanone (Compound 8).
• HATU (308 mg, 0.81 mmol) was added to a mixture of 5-methoxy-6-(trifluoro- methyl)indoline 7c (1 17 mg, 0.54 mmol), 2-(4-chloro-2-fluorophenyl)-2-((3- (2-hydroxyethoxy)-5-methoxyphenyl)amino)acetic acid 2e (200 mg, 0.54 mmol) and diisopropylethylamine (0.267 ml_, 1 .61 mmol) in DMF (10 ml_). The reaction mixture was stirred at room temperature for 12 h. The reaction was diluted with water, causing precipitation. The precipitate was filtered off and washed with water.
• Example 8.2 synthesis of 2-(4-chloro-2-fluorophenyl)-2-((3-(2-hydroxyethoxy)- 5-methoxyphenyl)amino)-1 -(5-methoxy-6-(trifluoromethyl)indolin-1 -yl)ethanone (Compound 8) and chiral separation into Enantiomers 8A and 8B.
• Example 9 synthesis of 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxy- phenyl)amino)-1 -(4-methyl-6-(trifluoromethoxy)indolin-1 -yl)ethanone (Compound 9) and chiral separation into Enantiomers 9A and 9B.
• 2M K2CO3 (23 ml_, 46 mmol) was added and the reaction mixture was heated at 70°C for 4 h. More 2M K2CO3 (10 ml_, 20 mmol) was added and the reaction mixture was heated at 70°C for 12 h. The reaction mixture was partially concentrated under reduced pressure to remove methanol. The residue was extracted with EtOAc. The organic phase was washed with H2O and brine, dried over Na2SO 4 , filtered and concen- trated under reduced pressure.
• BH3-Pyridine (1 .2 mL, 1 1 .6 mmol) was added dropwise to a solution of 4-methyl-6-(trifluoromethoxy)-1 /-/-indole 9g (0.5 g, 2.32 mmol) in EtOH (3 mL).
• 6N HCI (6 mL) was slowly added dropwise while maintaining the reaction temperature below 10°C.
• the mixture was stirred at 0°C for 3 h. Water (12 mL) was added and the mixture was basified until pH 8-9 with a concentrated solution of NaOH in water (the reaction temperature was kept below 20°C). The mixture was extracted with EtOAc.
• the enantiomers of Compound 9 were separated via Preparative Chiral SFC (Stationary phase: Chiralpak ® IA 5 ⁇ 250 x 20 mm, Mobile phase: 50% CO 2 , 50% iPrOH (+ 0.3% iPrNH 2 )).
• the first eluted enantiomer (180 mg) was solidified from heptane/diisopropyl ether to afford Enantiomer 9A (121 mg).
• the second eluted enantiomer (180 mg) was solidified from heptane/diisopropyl ether to afford Enantiomer 9B (132 mg).
• Example 10 synthesis of 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxy- phenyl)amino)-1 -(4-methyl-6-(trifluoromethyl)indolin-1 -yl)ethanone (Compound 10) and chiral separation into Enantiomers 10A and 10B.
• the first eluted enantiomer (180 mg) was solidified from heptane/diisopropyl ether to afford Enantiomer 10A (145 mg).
• the second eluted enantiomer (170 mg) was solidified from heptane/diisopropyl ether to afford Enantiomer 10B (1 13 mg).
• Example 11 synthesis of 2-(4-chloro-2-methoxyphenyl)-2-((3-(2-hydroxyethoxy)- 5-methoxyphenyl)amino)-1 -(5-methoxy-6-(thfluoromethyl)indolin-1 -yl)ethanone (Compound 11 ) and chiral separation into Enantiomers 11A and 11 B.
• HATU (0.692 g, 1 .82 mmol) was added to a mixture of 5-methoxy-6-(trifluoro- methyl)indoline 7c (264 mg, 1 .21 mmol), 2-(4-chloro-2-methoxyphenyl)-2-((3-(2- hydroxyethoxy)-5-methoxyphenyl)amino)acetic acid 11c (520 mg, 1 .36 mmol) and diisopropylethylamine (0.602 mL, 3.64 mmol) in DMF (14 mL). The resulting mixture was stirred at room temperature for 12 h. The mixture was diluted with water and EtOAc.
• Enantiomer 11A (194 mg).
• the second eluted enantiomer (240 mg) was crystallized from CHsCN/diisopropyl ether to give Enantiomer 11 B (189 mg).
• Example 12 synthesis of 2-(4-chlorophenyl)-1 -(5-fluoro-6-(thfluoronnethyl)indolin- 1 -yl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)annino)ethanone (Compound 12) and chiral separation into Enantiomers 12A and 12B.
• Bhb-Py dine (10.5 mL, 103.5 mmol) was added dropwise to a solution of 5-fluoro-6-(trifluoromethyl)-1 H-indole [CAS 1493800-10-4] (7 g, 34.5 mmol) in EtOH (45 mL).
• 6N HCI 105 mL was slowly added dropwise while maintaining the reaction temperature below 10°C.
• the mixture was stirred at 0°C for 3 h.
• Water 210 mL was added and the mixture was basified until pH 8-9 with a concentrated solution of NaOH in water (the reaction temperature was kept below 20°C). The mixture was extracted with EtOAc.
• the enantiomers of Compound 12 were separated via Preparative Chiral SFC (Stationary phase: Chiralcel ® OJ-H 5 ⁇ 250 x 20 mm, Mobile phase: 60% CO 2 , 40% EtOH (+ 0.3% iPrNH 2 )).
• the first eluted enantiomer (193 mg) was solidified from heptane/diisopropyl ether to give Enantiomer 12A (164 mg).
• the second eluted enantiomer (190 mg) was solidified from heptane/diisopropyl ether to give Enantiomer 12B (131 mg).
• Example 13 synthesis of 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxy- phenyl)amino)-1 -(5-methoxy-6-(trifluoromethoxy)indolin-1 -yl)ethanone (Compound 13) and chiral separation into Enantiomers 13A and 13B.
• the enantiomers of Compound 13 were separated via Preparative Chiral SFC (Stationary phase: Chiralpak® IC 5 ⁇ 250 x 30 mm, Mobile phase: 55% CO 2 , 45% EtOH (+ 0.3% iPrNH 2 )).
• the first eluted enantiomer 400 mg was solidified from heptane/diisopropyl ether to give Enantiomer 13A (332 mg).
• the second eluted enantiomer (397 mg) was solidified from
• the antiviral activity of all the compounds of the invention was tested the against DENV-2 16681 strain which was labeled with enhanced green fluorescent protein (eGPF).
• the culture medium consists of minimal essential medium supplemented with 2% of heat-inactivated fetal calf serum, 0.04% gentamycin (50 mg/mL) and 2 mM of L-glutamine.
• Vero cells, obtained from ECACC, were suspended in culture medium and 25 ⁇ _ was added to 384-well plates (2500 cells/well), which already contain the antiviral compounds. Typically, these plates contain a 5-fold serial dilution of 9 dilution steps of the test compound at 200 times the final concentration in 100% DMSO (200 nL).
• each compound concentration is tested in quadruplicate (final concentration range: 25 ⁇ - 0.000064 ⁇ or 2.5 ⁇ - 0.0000064 ⁇ for the most active compounds).
• each plate contains wells which are assigned as virus controls (containing cells and virus in the absence of compound), cell controls (containing cells in the absence of virus and compound) and medium controls (containing medium in the absence of cells, virus and compounds).
• virus controls containing cells and virus in the absence of compound
• cell controls containing cells in the absence of virus and compound
• medium controls containing medium in the absence of cells, virus and compounds.
• 25 ⁇ _ of culture medium was added instead of Vero cells. Once the cells were added to the plates, the plates were incubated for 30 minutes at room temperature to allow the cells to distribute evenly within the wells. Next, the plates were incubated in a fully humidified incubator (37°C, 5% CO 2 ) until the next day.
• DENV-2 strain 16681 labeled with eGFP, was added at a multiplicity of infection (MOI) of 0.5. Therefore, 15 ⁇ _ of virus suspension was added to all the wells containing test compound and to the wells assigned as virus control. In parallel, 15 ⁇ _ of culture medium was added to the medium and cell controls. Next, the plates were incubated for 3 days in a fully humidified incubator (37°C, 5% CO2). At the day of the read out, the eGFP fluorescence was measured using an automated fluorescence microscope at 488 nm (blue laser). Using an in-house LIMS system, inhibition dose response curves for each compound were calculated and the half maximal effective concentration (EC50) was determined.
• MOI multiplicity of infection
• the EC50 represents the concentration of a compound at which the virus replication is inhibited with 50%, as measured by a 50% reduction of the eGFP fluorescent intensity compared to the virus control.
• the EC50 is calculated using linear interpolation (Table 1 ).
• 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. Next, the plates were measured on a ViewLux. The half maximal cytotoxic concentration (CC50) was also determined, defined as the concentration required to reduce the luminescent signal by 50% compared to that of the cell control wells. Finally, the selectivity index (SI) was determined for the compounds, which was calculated as followed:
• Table 1 EC50, CC50, and SI for the compounds of the invention in the DENV-2 antiviral assay compound# ⁇ 0 5 ⁇ ( ⁇ ) N CCso ( ⁇ ) N SI N
• N the number of independent experiments in which the compounds were tested.
• the antiviral activity of the compounds of the invention was tested against DENV-1 strain TC974#666 (NCPV), DENV-2 strain 16681 , DENV-3 strain H87 (NCPV) and DENV-4 strain H241 (NCPV) in a RT-qPCR assay. Therefore, Vero cells were infected with either DENV-1 , or -2, or -3, or -4 in the presence or absence of test compounds. At day 3 post-infection, the cells were lysed and cell lysates were used to prepare cDNA of both a viral target (the 3'UTR of DENV; Table 2) and a cellular reference gene ( ⁇ -actin, Table 2).
• a duplex real time PCR 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 Cp's for the 3'UTR gene. On the other hand, if a test compound is toxic to the cells, a similar effect on ⁇ -actin expression will be observed.
• the comparative ⁇ method is used to calculate EC50, which is based on the relative gene expression of the target gene (3'UTR) normalized with the cellular housekeeping gene ( ⁇ -actin). In addition, CC50 values are determined based on the Cp values acquired for the
• Reporter dyes FAM, HEX
• quenchers ZEN and lABkFQ
• nucleotide sequence of the primers and probes were selected from the conserved region in the 3'UTR region of the dengue virus genome, based on the alignment of 300 nucleotide sequences of the four dengue serotypes deposited in Genbank (Gong et al., 2013, Methods Mol Biol, Chapter 16).
• the culture medium consisted of minimal essential medium supplemented with 2% of heat-inactivated fetal calf serum, 0.04% gentamycin (50mg/mL) and 2mM of L-glutamine.
• Vera cells obtained from ECACC, were suspended in culture medium and 75 L/well was added in 96-well plates (10000 cells/well), which already contain the antiviral compounds. Typically, these plates contain a 5-fold serial dilution of 9 dilution steps of the test compound at 200 times the final concentration in 100% DMSO (500nl_; final concentration range: 25 ⁇ - 0.000064 ⁇ or 2.5 ⁇ - 0.0000064 ⁇ for the most active compounds).
• each plate contains wells which are assigned as virus controls
• the cell lysates can be stored at -80°C or immediately used in the reverse transcription step.
• LightCycler 480 Using the LightCycler software and an in-house LIMS system, dose response curves for each compound were calculated and the half maximal effective concentration (EC50) and the half maximal cytotoxic concentration (CC50) were determined (Tables 5-8). Table 3: cDNA synthesis using Mix A, denaturation, Mix B and reverse transcription.
• N the number of independent experiments in which the compounds were tested.
• N the number of independent experiments in which the compounds were tested.
• Table 7 EC50, CC50, and SI for the compounds against serotype 3 in the RT-qPCR assays
• N the number of independent experiments in which the compounds were tested.
• Table 8 EC50, CC50, and SI for the compounds against serotype 4 in the RT-qPCR assays
• Table 9 EC50, CC50, and SI for compounds (56) and(170) disclosed in the

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• Animal Behavior & Ethology (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Medicinal Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Pharmacology & Pharmacy (AREA)
• Virology (AREA)
• Epidemiology (AREA)
• Oncology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Communicable Diseases (AREA)
• Molecular Biology (AREA)
• Microbiology (AREA)
• Mycology (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Indole Compounds (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Priority Applications (16)

Applications Claiming Priority (2)

Publications (1)

Family

ID=55646444

Family Applications (1)

Country Status (20)

Cited By (14)

Citations (2)

Family Cites Families (42)

• 2017
  • 2017-03-31 WO PCT/EP2017/057663 patent/WO2017167953A1/en active Application Filing
  • 2017-03-31 EA EA201892216A patent/EA201892216A1/ru unknown
  • 2017-03-31 SG SG11201808270PA patent/SG11201808270PA/en unknown
  • 2017-03-31 MA MA044498A patent/MA44498A/fr unknown
  • 2017-03-31 KR KR1020187026918A patent/KR102359735B1/ko active IP Right Grant
  • 2017-03-31 EP EP17714475.5A patent/EP3436436B1/en active Active
  • 2017-03-31 CR CR20180496A patent/CR20180496A/es unknown
  • 2017-03-31 CN CN201780021312.2A patent/CN109195949B/zh active Active
  • 2017-03-31 ES ES17714475T patent/ES2923771T3/es active Active
  • 2017-03-31 CA CA3013407A patent/CA3013407A1/en not_active Abandoned
  • 2017-03-31 MX MX2018011788A patent/MX2018011788A/es unknown
  • 2017-03-31 US US16/088,459 patent/US10913716B2/en active Active
  • 2017-03-31 JP JP2018550333A patent/JP6931357B2/ja active Active
  • 2017-03-31 AU AU2017240077A patent/AU2017240077A1/en not_active Abandoned
  • 2017-03-31 BR BR112018069601A patent/BR112018069601A2/pt not_active Application Discontinuation
• 2018
  • 2018-08-10 CO CONC2018/0008421A patent/CO2018008421A2/es unknown
  • 2018-09-20 PH PH12018502016A patent/PH12018502016A1/en unknown
  • 2018-09-25 CL CL2018002718A patent/CL2018002718A1/es unknown
  • 2018-09-26 IL IL261943A patent/IL261943A/en unknown
  • 2018-09-28 EC ECSENADI201873366A patent/ECSP18073366A/es unknown

Patent Citations (2)

Also Published As

Similar Documents

Legal Events