Patent Application
20210323977
The present invention relates to organic compounds useful for therapy and/or prophylaxis in a mammal, and in particular to antagonist of TLR7 and/or TLR8 and/or TLR9 useful for treating systemic lupus erythematosus or lupus nephritis.
Autoimmune connective tissue disease (CTD) include prototypical autoimmune syndromes such as Systemic Lupus Erythematosus (SLE), primary Sjögren's syndrome (pSjS), mixed connective tissue disease (MCTD), Dermatomyositis/Polymyositis (DM/PM), Rheumatoid Arthritis (RA), and systemic sclerosis (SSc). With the exception of RA, no really effective and safe therapies are available to patients. SLE represents the prototypical CTD with a prevalence of 20-150 per 100,000 and causes broad inflammation and tissue damage in distinct organs, from commonly observed symptoms in the skin and joints to renal, lung, or heart failure. Traditionally, SLE has been treated with nonspecific anti-inflammatory or immunosuppressive drugs. However, long term usage of immunosuppressive drug, e.g. corticosteroids is only partially effective, and is associated with undesirable toxicity and side effects. Belimumab is the only FDA-approved drug for lupus in the last 50 years, despite its modest and delayed efficacy in only a fraction of SLE patients (Navarra, S. V. et al Lancet 2011, 377, 721). Other biologics, such as anti-CD20 mAbs, mAbs against or soluble receptors of specific cytokines, have failed in most clinical studies. Thus, novel therapies are required that provide sustained improvement in a greater proportion of patient groups and are safer for chronic use in many autoimmune as well as auto-inflammation diseases.
Toll Like Receptors (TLR) are an important family of pattern recognition receptors (PRR) which can initiate broad immune responses in a wide variety of immune cells. As natural host defense sensors, endosomal TLRs 7, 8 and 9 recognize nucleic acids derived from viruses, bacteria; specifically, TLR7/8 and TLR9 recognize single-stranded RNA (ssRNA) and single-stranded CpG-DNA, respectively. However, aberrant nucleic acid sensing of TRL7/8/9 is considered as a key node in a broad of autoimmune and auto-inflammatory diseases (Krieg, A. M. et al. Immunol. Rev. 2007, 220, 251. Jiménez-Dalmaroni, M. J. et al Autoimmun Rev. 2016, 15, 1. Chen, J. Q., et al. Clinical Reviews in Allergy & Immunology 2016, 50, 1.) Therefore, TLR7/8/9 represents a new therapeutic target for autoimmune and auto-inflammatory diseases, for which no effective steroid-free and non-cytotoxic oral drugs exist, and inhibition of these pathways from the very upstream may deliver satisfying therapeutic effects. From a safety perspective, because there are multiple nucleic acid sensing pathways (e.g. other TLRs, cGAS/STING), such redundancy should still allow responses to infection in the presence of TLR7/8/9 inhibition. As such, we proposed and invented oral compounds that target and suppress TLR7/8/9 for the treatment of autoimmune and auto-inflammatory diseases.
The present invention relates to novel compounds of formula (I),
wherein
wherein
R5 is cyano, C1-6alkyl, halogen, haloC1-6alkyl or nitro;
X is N or CH;
Another object of the present invention is related to novel compounds of formula (I), their manufacture, medicaments based on a compound in accordance with the invention and their production as well as the use of compounds of formula (I) as TLR7 and/or TLR8 and/or TLR9 antagonist, and for the treatment or prophylaxis of systemic lupus erythematosus or lupus nephritis. The compounds of formula (I) show superior TLR7 and/or TLR8 and/or TLR9 antagonism activity. In addition, the compounds of formula (I) also show good cytotoxicity, solubility, human microsome stability and SDPK profiles, as well as low CYP inhibition.
The term “C1-6alkyl” denotes a saturated, linear or branched chain alkyl group containing 1 to 6, particularly 1 to 4 carbon atoms, for example methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl and the like. Particular “C1-4alkyl” groups are methyl, ethyl and isopropyl.
The term “halogen” and “halo” are used interchangeably herein and denote fluoro, chloro, bromo, or iodo.
The term “haloC1-4alkyl” denotes an alkyl group wherein at least one of the hydrogen atoms of the alkyl group has been replaced by same or different halogen atoms, particularly fluoro atoms. Examples of haloC1-6alkyl include monofluoro-, difluoro- or trifluoro-methyl, -ethyl or -propyl, for example 3,3,3-trifluoropropyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, fluoromethyl, difluoromethyl, trifluoromethyl and trifluoroethyl.
The term “C3-7cycloalkyl” denotes a saturated carbon ring containing from 3 to 7 carbon atoms, particularly from 3 to 6 carbon atoms, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like. Particular “C3-7cycloalkyl” groups are cyclopropyl and cyclohexyl.
The term “halopyrrolidinyl” denotes a pyrrolidinyl group wherein at least one of the hydrogen atoms of the pyrrolidinyl group has been replaced by same or different halogen atoms, particularly fluoro atoms. Examples of halopyrrolidinyl include fluoropyrrolidinyl and difluoropyrrolidinyl.
The term “heterocyclyl” denotes a monovalent saturated or partly unsaturated mono- or bicyclic ring system of 3 to 12 ring atoms, comprising 1, 2, or 3 ring heteroatoms selected from N, O and S, the remaining ring atoms being carbon. In particular embodiments, heterocyclyl is a monovalent saturated monocyclic ring system of 4 to 10 ring atoms, comprising 1, 2, or 3 ring heteroatoms selected from N, O and S, the remaining ring atoms being carbon. Examples for monocyclic saturated heterocyclyl are aziridinyl, oxiranyl, azetidinyl, oxetanyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydro-thienyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperazinyl, morpholinyl, thiomorpholinyl, 1,1-dioxo-thiomorpholin-4-yl, azepanyl, diazepanyl, homopiperazinyl, or oxazepanyl. Heterocyclyl can be fully or partially saturated. Examples for bicyclic saturated heterocyclyl are bicyclo[3.1.0]hexanyl, spiro[3.3]heptanyl, azabicyclo[2.2.1]heptanyl, azabicyclo[3.2.1]octanyl, azabicyclo[3.3.1]nonanyl, azaspiro[3.5]nonanyl, diazaspiro[4.4]nonanyl, diazaspiro[5.5]undecanyl, 1,2,3,3a,4,5,6,6a-octahydrocyclopenta[c]pyrrolyl, oxaazabicyclo[3.3.1]nonanyl. Examples for partially saturated heterocyclyl are dihydrofuryl, imidazolinyl, dihydro-oxazolyl, tetrahydropyridinyl, and dihydropyranyl. Monocyclic or bicyclic heterocyclyl can be further substituted by halogen, hydroxy, amino, C1-6alkyl, haloC1-6alkyl or heterocyclyl.
The term “heterocyclylamino” denotes heterocyclyl-NH—, wherein “heterocyclyl” is defined as above.
The term “enantiomer” denotes two stereoisomers of a compound which are non-superimposable mirror images of one another.
The term “diastereomer” denotes a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, e.g. melting points, boiling points, spectral properties, and reactivities.
The term “pharmaceutically acceptable salts” denotes salts which are not biologically or otherwise undesirable. Pharmaceutically acceptable salts include both acid and base addition salts.
The term “pharmaceutically acceptable acid addition salt” denotes those pharmaceutically acceptable salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, and organic acids selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes of organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid, maleic acid, maloneic acid, succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelic acid, embonic acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicyclic acid.
The term “pharmaceutically acceptable base addition salt” denotes those pharmaceutically acceptable salts formed with an organic or inorganic base. Examples of acceptable inorganic bases include sodium, potassium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Salts derived from pharmaceutically acceptable organic nontoxic bases includes salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperizine, piperidine, N-ethylpiperidine, and polyamine resins.
The term “A pharmaceutically active metabolite” denotes a pharmacologically active product produced through metabolism in the body of a specified compound or salt thereof. After entry into the body, most drugs are substrates for chemical reactions that may change their physical properties and biologic effects. These metabolic conversions, which usually affect the polarity of the compounds of the invention, alter the way in which drugs are distributed in and excreted from the body. However, in some cases, metabolism of a drug is required for therapeutic effect.
The term “therapeutically effective amount” denotes an amount of a compound or molecule of the present invention that, when administered to a subject, (i) treats or prevents the particular disease, condition or disorder, (ii) attenuates, ameliorates or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition or disorder described herein. The therapeutically effective amount will vary depending on the compound, the disease state being treated, the severity of the disease treated, the age and relative health of the subject, the route and form of administration, the judgement of the attending medical or veterinary practitioner, and other factors.
The term “pharmaceutical composition” denotes a mixture or solution comprising a therapeutically effective amount of an active pharmaceutical ingredient together with pharmaceutically acceptable excipients to be administered to a mammal, e.g., a human in need thereof.
Antagonist of TLR7 and/or TLR8 and/or TLR9
The present invention relates to a compound of formula (I),
wherein
wherein
R5 is cyano, C1-6alkyl, halogen, haloC1-6alkyl or nitro;
X is N or CH;
A further embodiment of present invention is (ii) a compound of formula (I), wherein
wherein
R5 is cyano, C1-6alkyl, halogen, haloC1-6alkyl or nitro;
X is N or CH;
or R2 and R3 together with the carbon they are attached to form C3-7cycloalkyl;
A further embodiment of present invention is (iii) a compound of formula (I) according to (ii), wherein R4 is (C1-6alkylhaminoC1-6alkylamino, (C1-6alkylmorpholinyl)C1-6alkylamino, (C1-6alkylpiperidyl)C1-6alkylamino, (morpholinylC1-6alkyl)amino, [(C1-6alkyl)2aminoC1-6alkyl]C3-7cycloalkylamino, aminoazetidinyl, aminobicyclo[3.1.0]hexanylamino, aminoC1-6alkylamino, aminoC3-7cycloalkylamino, aminoC3-7cycloalkylC1-6alkylamino, aminospiro[3.3]heptanylamino, azabicyclo[2.2.1]heptanylamino, azabicyclo[3.2.1]octanylamino, azabicyclo[3.3.1]nonanylamino, azaspiro[3.5]nonanylamino, azepanylamino, C1-6alkoxycarbonylpyrrolidinylamino, C1-6alkylazaspiro[2.4]heptanylamino, C1-6alkylpiperidylamino, diazaspiro[4.4]nonanyl, diazaspiro[5.5]undecanyl, halopyrrolidinylamino, morpholinylC1-6alkylamino, octahydrocyclopenta[c]pyrrolylamino, oxaazabicyclo[3.3.1]nonanylamino or piperidylpiperidylamino.
A further embodiment of present invention is (iv) a compound of formula (I) according to (iii), wherein R5 is cyano, methyl, chloro, trifluoromethyl or nitro.
A further embodiment of present invention is (v) a compound of formula (I) according to (iv), wherein R3 is H, methyl, ethyl, isopropyl, difluoromethyl, trifluoromethyl or cyclopropyl; or R2 and R3 together with the carbon they are attached to form cyclopropyl.
A further embodiment of present invention is (vi) a compound of formula (I) according to (v), wherein R3 is methyl or trifluoromethyl; or R2 and R3 together with the carbon they are attached to form cyclopropyl.
A further embodiment of present invention is (vii) a compound of formula (I) according to (v) or (vi), wherein R4 is (3-aminocyclobutyl)methylamino, (dimethylamino)ethylamino, (methylmorpholinyl)methylamino, (methylpiperidyl)methylamino, (morpholinylethyl)amino, (morpholinylmethyl)amino, [(dimethylamino)methyl]cyclobutylamino, 1,2,3,3a,4,5,6,6a-octahydrocyclopenta[c]pyrrol-5-ylamino, 2,7-diazaspiro[4.4]nonanyl, 2-azabicyclo[2.2.1]heptan-5-ylamino, 3,9-diazaspiro[5.5]undecanyl, 3-azabicyclo[3.2.1]octan-8-ylamino, 3-azabicyclo[3.3.1]nonan-7-ylamino, 3-azabicyclo[3.3.1]nonan-9-ylamino, 3-oxa-7-azabicyclo[3.3.1]nonan-9-ylamino, 3-oxa-9-azabicyclo[3.3.1]nonan-7-ylamino, 5-methyl-5-azaspiro[2.4]heptan-7-ylamino, 6-aminospiro[3.3]heptan-2-ylamino, 7-azaspiro[3.5]nonan-2-ylamino, 8-azabicyclo[3.2.1]octan-3-ylamino, 9-azabicyclo[3.3.1]nonan-3-ylamino, amino-2-bicyclo[3.1.0]hexanylamino, aminoazetidinyl, aminocyclobutylamino, aminocyclohexylamino, aminomethylpropylamino, azepanylamino, fluoropyrrolidinylamino, methoxycarbonylpyrrolidinylamino, methylpiperidylamino or piperidylpiperidylamino.
A further embodiment of present invention is (viii) a compound of formula (I) according to (vii), wherein R4 is methylpiperidylamino.
Another embodiment of present invention is that (ix) particular compounds of formula (I) are selected from:
The compounds of the present invention can be prepared by any conventional means. Suitable processes for synthesizing these compounds as well as their starting materials are provided in the schemes below and in the examples. All substituents, in particular, R1 to R5 are as defined above unless otherwise indicated. Furthermore, and unless explicitly otherwise stated, all reactions, reaction conditions, abbreviations and symbols have the meanings well known to a person of ordinary skill in organic chemistry.
General synthetic route for preparing the compound of formula (I) is shown below.
wherein R6 is C1-6alkyl; R7 and R8 are independently selected from H, heterocyclyl, heterocyclylC1-6alkyl, (C1-6alkyl)2aminoC1-6alkyl, aminoC3-7cycloalkyl, [(C1-6alkyl)2aminoC1-6alkyl]C3-7cycloalkyl, aminoC3-7cycloalkylC1-6alkyl or aminoC1-6alkyl; or R7 and R8 together with the nitrogen they are attached to form heterocyclyl.
The coupling of halide (IV) with compound of formula (III) can be achieved by direct coupling in the presence of a base, such as DIPEA or K2CO3, or under Buchwald-Hartwig amination conditions (ref: Acc. Chem. Res. 1998, 31, 805-818; Chem. Rev. 2016, 116, 12564-12649; Topics in Current Chemistry, 2002, 219, 131-209; and references cited therein) with a catalyst, such as Ruphos Pd-G2, and a base, such as Cs2CO3, to provide compound of formula (V). Hydrolysis of compound of formula (V) in basic condition, such as LiOH in THF/water, gives carboxylic acid (VI), which is condensed with amine (VII) in the presence of a coupling reagent, such as HATU, to give the compound of formula (II). In some embodiment, the coupling of compound of formula (VI) and amine (VII) may give a product containing a protecting group, e.g. Boc, originated from amine (VII), which will be removed before affording the final compound of formula (II).
This invention also relates to a process for the preparation of a compound of formula (I) comprising the following step:
a) condensation of carboxylic acid (VI),
In step a), the coupling reagent can be for example HATU.
A compound of formula (I) or (II) when manufactured according to the above process is also an object of the invention.
Compounds of this invention can be obtained as mixtures of diastereomers or enantiomers, which can be separated by methods well known in the art, e.g. (chiral) HPLC or SFC.
The present invention provides compounds that can be used as TLR7 and/or TLR8 and/or TLR9 antagonist, which inhibits pathway activation through TLR7 and/or TLR8 and/or TLR9 as well as respective downstream biological events including, but not limited to, innate and adaptive immune responses mediated through the production of all types of cytokines and all forms of auto-antibodies. Accordingly, the compounds of the invention are useful for blocking TLR7 and/or TLR8 and/or TLR9 in all types of cells that express such receptor(s) including, but not limited to, plasmacytoid dendritic cell, B cell, T cell, macrophage, monocyte, neutrophil, keratinocyte, epithelial cell. As such, the compounds can be used as a therapeutic or prophylactic agent for systemic lupus erythematosus and lupus nephritis.
The present invention provides methods for treatment or prophylaxis of systemic lupus erythematosus and lupus nephritis in a patient in need thereof.
Another embodiment includes a method of treating or preventing systemic lupus erythematosus and lupus nephritis in a mammal in need of such treatment, wherein the method comprises administering to said mammal a therapeutically effective amount of a compound of formula (I), a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt thereof.
The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention.
The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention.
Abbreviations used herein are as follows:
ACN: acetonitrile
Boc2O: di-tert-butyl dicarbonate
DCM: dichloromethane
DIPEA or DIEA: N,N-diisopropylethylamine
TEA triethylamine
EA or EtOAc: ethyl acetate
FA: formic acid
HATU: 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate
IC50: half inhibition concentration
LCMS liquid chromatography-mass spectrometry
MS: mass spectrometry
PE: petroleum ether
prep-HPLC: preparative high performance liquid chromatography
rt: room temperature
RT: retention time
RuPhos Pd G2: chloro(2-dicyclohexylphosphino-2′,6′-diisopropoxy-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) 2nd generation
SFC: supercritical fluid chromatography
TFA: trifluoroacetic acid
v/v volume ratio
DDI drug-drug-interaction
LYSA lyophilisation solubility assay
HLM human liver microsome
General Experimental Conditions
Intermediates and final compounds were purified by flash chromatography using one of the following instruments: i) Biotage SPI system and the Quad 12/25 Cartridge module. ii) ISCO combi-flash chromatography instrument. Silica gel brand and pore size: i) KP-SIL 60 Å, particle size: 40-60 μm; ii) CAS registry NO: Silica Gel: 63231-67-4, particle size: 47-60 micron silica gel; iii) ZCX from Qingdao Haiyang Chemical Co., Ltd, pore: 200-300 or 300-400.
Intermediates and final compounds were purified by preparative HPLC on reversed phase column using XBridgeJm Prep-C18 (5 μm, OBD™ 30×100 mm) column, SunFire™ Prep-C18 (5 μm, OBD™ 30×100 mm) column, Phenomenex Synergi-C18 (10 μm, 25×150 mm) or Phenomenex Gemini-C18 (10 μm, 25×150 mm). Waters AutoP purification System (Sample Manager 2767, Pump 2525, Detector: Micromass ZQ and UV 2487, solvent system: acetonitrile and 0.1% ammonium hydroxide in water, acetonitrile and 0.1% FA in water or acetonitrile and 0.1% TFA in water). Or Gilson-281 purification System (Pump 322, Detector: UV 156, solvent system: acetonitrile and 0.05% ammonium hydroxide in water; acetonitrile and 0.225% FA in water, acetonitrile and 0.05% HCl in water; acetonitrile and 0.075% TFA in water; or acetonitrile and water).
For SFC chiral separation, intermediates were separated by chiral column (Daicel chiralpak IC, 5 μm, 30×250 mm), AS (10 μm, 30×250 mm) or AD (10 μm, 30×250 mm) using Mettler Toledo Multigram III system SFC, Waters 80Q preparative SFC or Thar 80 preparative SFC, solvent system: CO2 and IPA (0.5% TEA in IPA) or CO2 and MeOH (0.1% NHrH2O in McOH), back pressure 100 bar, detection UV@ 254 or 220 nm.
LC/MS spectra of compounds were obtained using a LC/MS (Waters™ Alliance 2795-Micromass ZQ, Shimadzu Alliance 2020-Micromass ZQ or Agilent Alliance 6110-Micromass ZQ), LC/MS conditions were as follows (running time 3 or 1.5 mins):
Acidic condition I: A: 0.1% TFA in H2O; B: 0.1% TFA in acetonitrile;
Acidic condition II: A: 0.0375% TFA in H2O; B: 0.01875% TFA in acetonitrile;
Basic condition I: A: 0.1% NH3H2O in H2O; B: acetonitrile;
Basic condition II: A: 0.025% NH3.H2O in H2O; B: acetonitrile;
Neutral condition: A: H2O; B: acetonitrile.
Mass spectra (MS): generally only ions which indicate the parent mass are reported, and unless otherwise stated the mass ion quoted is the positive mass ion (MH)+.
NMR Spectra were obtained using Bruker Avance 400 MHz.
The microwave assisted reactions were carried out in a Biotage Initiator Sixty microwave synthesizer. All reactions involving air-sensitive reagents were performed under an argon or nitrogen atmosphere. Reagents were used as received from commercial suppliers without further purification unless otherwise noted.
The following examples are intended to illustrate the meaning of the present invention but should by no means represent a limitation within the meaning of the present invention:
A detailed synthetic route is provided in the following Scheme
To a solution of 2-bromo-5-(trifluoromethyl)aniline (7.4 g, 30.0 mmol, 1 eq) and TEA (6.1 g, 60.0 mmol, 2 eq) in DCM (160 mL) was added acetyl chloride (7.0 g, 90.0 mmol, 3 eq) dropwise at 0° C. The mixture was stirred at 20° C. for 2 hrs, then basified with NaHCO3 and washed with water (100 mL). The aqueous layer was extracted with DCM (100 mL) twice. The combined organic layer was dried and concentrated under vacuum. The crude product was triturated in PE to give compound A1 (6.2 g, 75.6%) as a white solid. MS: calc'd 282 (MH+), measured 282 (MH+).
H2SO4 (40 mL) was added slowly into a flask containing HNO3 (25 mL) at 0° C. followed by the addition of compound A1 (6.2 g, 22.1 mmol, 1 eq) in small portions over 40 mins. After the mixture was stirred at 0° C. for additional 20 mins, it was warmed to 25° C. and stirred for 1 hour, and extracted with EA (200 mL) twice. The combined organic layer was washed with sat. NaHCO3 and brine (100 mL) twice, dried and concentrated under vacuum to give compound A2 (8.1 g, crude) as a red solid. MS: calc'd 325 (MH+), measured 325 (MH+).
A solution of compound A2 (8.1 g, 24.8 mmol, 1 eq) and HCl (6 M, 40 mL) in MeOH (160 mL) was stirred at 90° C. for 18 hrs. The mixture was basified with aq. NaHCO3 and extracted with EA (200 mL) twice. The combined organic layer was washed with brine (100 mL) twice, dried and concentrated under vacuum to give the crude product, which was purified by column chromatography to give compound A3 (1.9 g, 26.7%) as a yellow solid. MS: calc'd 285 (MH+), measured 285 (MH+).
A solution of compound A3 (1.9 g, 6.7 mmol, 1 eq) and Fe (1.9 g, 33.5 mmol, 5 eq) in AcOH (60 mL) was stirred at 50° C. for 3 hours. The solid was removed by filtration and the filtrate was neutralized with sat. NaHCO3 and extracted with EA (200 mL) twice. The combined organic layer was washed with brine (100 mL) twice, dried over Na2SO4, and concentrated to give a crude product which was purified by column chromatography (EA/PE=1/10) to give compound A4 (1.3 g, 76.5%) as a yellow solid. MS: calc'd 255 (MH+), measured 255 (MH+).
A solution of compound A4 (1.9 g, 4.7 mmol, 1 eq) and oxaldehyde (6.8 g, 47.0 mmol, 10 eq) in EtOH (30 mL) was stirred at 80° C. for 2 hrs. The mixture was neutralized with aq. NaHCO3 (100 mL) and extracted with EA (200 mL) twice. The organic layers were combined and washed with brine (100 mL) twice. The organic layer was dried and concentrated under vacuum to give the crude product, which was purified by column chromatography (EA/PE=1/8) to give Intermediate A (0.95 g, 67.8%) as a yellow solid. MS: calc'd 277 (MH+), measured 277 (MH+).
The title compound was prepared according to the following scheme:
To a solution of methyl (3R,4R)-4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (compound 1b, 22 mg, 94 μmol, Pharmablock, PBXA3229-1) and 8-bromoquinoxaline-5-carbonitrile (compound 1a, 20 mg, 85.5 μmol) (Reference: WO2017/106607) in 1,4-dioxane (2 mL) was added K2CO3 (59 mg, 427 μmol). The mixture was degassed three times, then Ruphos Pd G2 (CAS: 1375325-68-0, 6.6 mg, 8.55 μmol) was added. After the reaction mixture was stirred at 90° C. for 5 hrs under N2, it was cooled to rt, diluted with EA (50 mL) and washed with water. The organic layer was evaporated to give a crude product (30 mg) which was used in the next step without purification. MS: calc'd 351 (MH+), measured 351 (MH+).
To a solution of (3R,4R)-methyl 1-(8-cyanoquinoxalin-5-yl)-4-(trifluoromethyl)pyrrolidine-3-carboxylate (compound 1c, 30 mg, 85.6 μmol) in acetonitrile (10 mL) was added lithium hydroxide (20.5 mg, 856 μmol) in water (2 mL). After the mixture was stirred at rt for 4 hrs, the solvent was removed under vacuum to give a crude product. The crude product was dissolved in 10 mL H2O and washed with EA. The aqueous layer was acidified to PH 4-5 by use of 1M HCl. The aqueous solution was extracted with 100 mL EA. The organic layer was separated, dried over Na2SO4 and evaporated under vacuum to give the crude product (28 mg) which was used in the next step without further purification. MS: calc'd 337 (MH+), measured 337 (MH+).
To a solution of (3R,4R)-1-(8-cyanoquinoxalin-5-yl)-4-(trifluoromethyl)pyrrolidine-3-carboxylic acid (compound 1d, 28 mg, 83.3 μmol) and 1-methylpiperidin-4-amine (compound 1e, 10 mg, 91.6 μmol, WuXi Apptec, CAS:41838-46-4) in DCM (10 mL) was added HATU (95 mg, 250 μmol) and triethylamine (84 mg, 833 μmol). The mixture was stirred at rt overnight, then diluted with DCM (50 mL) and washed with water (50 mL). The organic layer was evaporated under vacuum to give an oil, which was purified by prep-HPLC to give Example 1 (30 mg) as a yellow solid. MS: calc'd 433 (MH+), measured 433 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.88 (d, J=1.7 Hz, 1H), 8.81 (d, J=1.7 Hz, 1H), 8.02 (d, J=8.7 Hz, 1H), 6.85 (d, J=8.7 Hz, 1H), 4.45-4.21 (m, 3H), 4.10-3.99 (m, 2H), 3.69-3.53 (m, 3H), 3.24-3.08 (m, 2H), 2.91 (s, 3H), 2.29-2.02 (m, 3H), 1.85-1.66 (m, 2H).
The title compound was prepared in analogy to the preparation of Example 1 by using 5-bromo-8-(trifluoromethyl)quinoxaline (Intermediate A) instead of bromoquinoxaline-5-carbonitrile (compound 1a). Example 2 (40 mg) was obtained as a yellow solid. MS: calc'd 476 (MH+), measured 476 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.74 (d, J=1.7 Hz, 1H), 8.69 (d, J=1.7 Hz, 1H), 7.86 (d, J=8.7 Hz, 1H), 6.78-6.66 (m, 1H), 4.27-4.15 (m, 2H), 4.14 (d, J=8.4 Hz, 2H), 3.96-3.82 (m, 2H), 3.56-3.38 (m, 2H), 3.14-2.99 (m, 2H), 2.79 (s, 3H), 2.19-1.92 (m, 3H), 1.77-1.51 (m, 2H).
The title compound was prepared in analogy to the preparation of Example 1 by using methyl (3R,4R)-4-methylpyrrolidine-3-carboxylate HCl salt (Pharmablock, PBXA3221-1) instead of methyl (3R,4R)-4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (compound 1b). Example 3 (18 mg) was obtained as a yellow solid. MS: calc'd 379 (MH+), measured 379 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.82 (d, J=1.7 Hz, 1H), 8.75 (d, J=1.6 Hz, 1H), 7.95 (d, J=8.7 Hz, 1H), 6.74 (d, J=8.7 Hz, 1H), 4.28-4.12 (m, 3H), 4.07-3.93 (m, 1H), 3.66-3.45 (m, 3H), 3.25-3.11 (m, 2H), 2.91 (s, 3H), 2.77-2.53 (m, 2H), 2.31-2.04 (m, 2H), 1.86-1.70 (m, 2H), 1.24-1.15 (m, 3H).
The title compound was prepared in analogy to the preparation of Example 1 by using 5-bromoquinoline-8-carbonitrile instead of bromoquinoxaline-5-carbonitrile (compound 1a). Example 4 (62 mg) was obtained as a yellow solid. MS: calc'd 432 (MH+), measured 432 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.72 (br dd, J=1.5, 8.6 Hz, 1H), 8.66-8.53 (m, 1H), 8.09 (d, J=8.2 Hz, 1H), 7.60 (dd, J=4.2, 8.6 Hz, 1H), 7.12-6.99 (m, 1H), 4.08-3.81 (m, 5H), 4.05-3.45 (m, 3H), 3.23-3.06 (m, 2H), 2.90 (s, 3H), 2.33-2.02 (m, 3H), 1.87-1.62 (m, 2H).
The title compound was prepared in analogy to the preparation of Example 1 by using methyl (3R,4R)-4-methylpyrrolidine-3-carboxylate HCl salt (Pharmablock, PBXA3221-1) and 5-bromo-8-(trifluoromethyl)quinoxaline (Intermediate A) instead of methyl (3R,4R)-4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (compound 1b) and bromoquinoxaline-5-carbonitrile (compound 1a). Example 5 (35 mg) was obtained as a yellow solid. MS: calc'd 422 (MH+), measured 422 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.80 (d, J=1.7 Hz, 1H), 8.75 (d, J=1.7 Hz, 1H), 7.92 (d, J=8.7 Hz, 1H), 6.78-6.68 (m, 1H), 4.22-3.94 (m, 4H), 3.64-3.50 (m, 3H), 3.17 (dt, J=2.2, 13.0 Hz, 2H), 2.91 (s, 3H), 2.74-2.53 (m, 2H), 2.31-2.01 (m, 2H), 1.85-1.69 (m, 2H), 1.21 (d, J=6.4 Hz, 3H).
The title compound was prepared according to the following scheme:
To a solution of 5-(tert-butoxycarbonyl)-5-azaspiro[2.4]heptane-7-carboxylic acid (compound 6a, 0.5 g, 2.07 mmol, Pharmablock, PBLJ7032) in DCM (5 mL) was added 2,2,2-trifluoroacetic acid (1.65 g, 1.08 mL, 14.5 mmol). The reaction mixture was stirred at rt for 3 hrs, then concentrated in vacuo to give the crude product (282 mg) which was used in the next step without purification. MS: calc'd 142 (MH+), measured 142 (MH+).
A mixture of 5-bromo-8-(trifluoromethyl)quinoxaline ((Intermediate A, 554 mg, 2 mmol), 5-azaspiro[2.4]heptane-7-carboxylic acid (compound 6b, 282 mg, 2 mmol) and TEA (1.01 g, 1.39 mL, 10 mmol) in DMSO (10 mL) was charged with argon. The mixture was heated with microwave at 130° C. for 3 hrs, then partitioned between EA and water. The separated aqueous layer was extracted with EA twice. The combined organic layer was dried over Na2SO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with a gradient of PE:EA (100:10 to 100:50) to give 5-(8-(trifluoromethyl)quinoxalin-5-yl)-5-azaspiro[2.4]heptane-7-carboxylic acid (compound 6c, 200 mg) as a yellow solid. MS: calc'd 338 (MH+), measured 338 (MH+).
To a solution of 5-(8-(trifluoromethyl)quinoxalin-5-yl)-5-azaspiro[2.4]heptane-7-carboxylic acid (compound 6c, 50 mg, 148 μmol), tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d, 72 mg, 296 μmol) and DIEA (38 mg, 51.8 sL, 296 μmol) in CH2C12 (2 mL) was added 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate(V) (113 mg, 296 μmol). The reaction mixture was stirred at rt for 2 hrs. The mixture was partitioned between EA and Water. The separated aqueous layer was extracted twice with EA. The combined organic phase was dried over Na2SO4 and concentrated in vacuo. The residue was purified by silica gel chromatography on a 12 g column using a MPLC system (CombiFlash Companion, Isco Inc.) eluting with a gradient of PE:EA (100:10 to 100:50) to give tert-butyl 7-(5-(8-(trifluoromethyl)quinoxalin-5-yl)-5-azaspiro[2.4]heptane-7-carboxamido)-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6e, 80 mg) as a yellow solid. MS: calc'd 562 (MH+), measured 562 (MH+).
To a solution of tert-butyl 7-(5-(8-(trifluoromethyl)quinoxalin-5-yl)-5-azaspiro[2.4]heptane-7-carboxamido)-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6e, 80 mg, 142 μmol) in CH2Cl2 (3 mL) was added 2,2,2-trifluoroacetic acid (49 mg, 427 μmol). The reaction mixture was stirred at rt for 3 hrs. Then the reaction mixture was concentrated in vacuo. The residue was purified by prep-HPLC to give Example 6 (70 mg) as a yellow solid. MS: calc'd 462 (MH+), measured 462 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ 8.82 (d, J=1.59 Hz, 1H), 8.77 (d, J=1.71 Hz, 1H), 8.37 (d, J=9.54 Hz, 1H), 6.79 (d, J=8.68 Hz, 1H), 4.42-4.51 (m, 1H), 4.37 (dd, J=1.65, 11.31 Hz, 1H), 4.20-4.31 (m, 2H), 3.95-4.02 (m, 1H), 3.84-3.93 (m, 3H), 3.51-3.62 (m, 3H), 2.65-2.73 (m, 1H), 2.43-2.58 (m, 2H), 1.96 (t, J=15.53 Hz, 2H), 0.95-1.04 (m, 1H), 0.74-0.93 (m, 3H).
The title compound was prepared in analogy to the preparation of Example 6 by using 8-bromoquinoxaline-5-carbonitrile and 1-methylpiperidin-4-amine instead of 5-bromo-8-(trifluoromethyl)quinoxaline (Intermediate A) and tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 7 (200 mg) was obtained as a yellow solid. MS: calc'd 391 (MH+), measured 391 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.83 (d, J=1.5 Hz, 1H), 8.75 (d, J=1.5 Hz, 1H), 7.98 (d, J=8.7 Hz, 1H), 6.76 (d, J=8.8 Hz, 1H), 4.43-4.33 (m, 1H), 4.33-4.26 (m, 1H), 4.15 (d, J=11.2 Hz, 1H), 3.99-3.86 (m, 1H), 3.77 (d, J=11.3 Hz, 1H), 3.60-3.50 (m, 2H), 3.21-3.06 (m, 2H), 2.81 (s, 3H), 2.80-2.73 (m, 1H), 2.24-2.10 (m, 2H), 1.83-1.67 (m, 2H), 0.91-0.75 (m, 4H).
SFC-HPLC separation gave two isomers Example 7A (17 mg) and Example 7B (20 mg). The compound names of the two isomers are (7R)-5-(8-Cyanoquinoxalin-5-yl)-N-(1-methyl-4-piperidyl)-5-azaspiro[2.4]heptane-7-carboxamide and (7S)-5-(8-Cyanoquinoxalin-5-yl)-N-(1-methyl-4-piperidyl)-5-azaspiro[2.4]heptane-7-carboxamide.
Example 7A: MS: calc'd 391 (MH+), measured 391 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.81 (d, J=1.5 Hz, 1H), 8.73 (d, J=1.8 Hz, 1H), 7.96 (d, J=8.8 Hz, 1H), 6.74 (d, J=8.8 Hz, 1H), 4.39-4.31 (m, 1H), 4.31-4.24 (m, 1H), 4.17-4.08 (m, 1H), 3.94-3.84 (m, 1H), 3.79-3.70 (m, 1H), 3.53 (br d, J=12.5 Hz, 2H), 3.16-3.04 (m, 2H), 2.85 (s, 3H), 2.74 (dd, J=3.1, 6.9 Hz, 1H), 2.20-2.08 (m, 2H), 1.75 (q, J=12.8 Hz, 2H), 0.86-0.73 (m, 4H).
Example 7B: MS: calc'd 391 (MH+), measured 391 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.82 (d, J=1.8 Hz, 1H), 8.74 (d, J=1.8 Hz, 1H), 7.96 (d, J=8.5 Hz, 1H), 6.75 (d, J=8.8 Hz, 1H), 4.46-4.33 (m, 1H), 4.33-4.23 (m, 1H), 4.18-4.09 (m, 1H), 3.91 (tt,J=4.0, 11.9 Hz, 1H), 3.78-3.71 (m, 1H), 3.55 (br d, J=12.5 Hz, 2H), 3.16-3.05 (m, 2H), 2.86 (s, 3H), 2.77 (dd, J=3.5, 7.0 Hz, 1H), 2.24-2.09 (m, 2H), 1.85-1.72 (m, 2H), 0.88-0.75 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 6 by using tert-butyl 9-amino-3-azabicyclo[3.3.1]nonane-3-carboxylate instead of tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 8 (14 mg) was obtained as a yellow solid. MS: calc'd 460 (MH+), measured 460 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.69 (d, J=1.71 Hz, 1H), 8.64 (d, J=1.71 Hz, 1H), 7.82 (d, J=8.80 Hz, 1H), 6.64 (d, J=8.68 Hz, 1H), 4.20-4.29 (m, 1H), 4.12-4.19 (m, 1H), 3.94-4.05 (m, 2H), 3.57 (d, J=10.76 Hz, 1H), 3.35-3.42 (m, 2H), 3.26-3.33 (m, 2H), 2.82 (dd, J=3.30, 6.85 Hz, 1H), 1.87-2.11 (m, 4H), 1.59-1.72 (m, 4H), 0.67-0.81 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 6 by using tert-butyl 3-amino-9-azabicyclo[3.3.1]nonane-9-carboxylate instead of tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 10 (62 mg) was obtained as a yellow solid. MS: calc'd 460 (MH+), measured 460 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.67-8.71 (m, 1H), 8.63 (d, J=1.71 Hz, 1H), 7.81 (d, J=8.68 Hz, 1H), 6.62 (d, J=8.68 Hz, 1H), 4.58 (dt, J=6.24, 12.23 Hz, 1H), 4.21-4.29 (m, 1H), 4.10-4.16 (m, 1H), 3.94-4.02 (m, 1H), 3.60 (s, 3H), 2.60-2.68 (m, 1H), 2.05-2.23 (m, 2H), 1.94 (m, 8H), 0.62-0.78 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 6 by using 1-methylpiperidin-4-amine instead of and tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 11 (20 mg) was obtained as a yellow solid. MS: calc'd 434 (MH+), measured 434 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.92 (d, J=1.71 Hz, 1H), 8.81 (d, J=1.71 Hz, 1H), 7.84 (d, J=7.70 Hz, 1H), 6.70 (d, J=8.68 Hz, 1H), 4.13-4.22 (m, 1H), 4.06 (dd, J=4.40, 11.37 Hz, 1H), 3.92 (d, J=11.13 Hz, 1H), 3.71 (d, J=10.64 Hz, 1H), 3.48 (s, 1H), 2.64-2.79 (m, 3H), 2.13 (s, 3H), 1.91 (m, 2H), 1.67 (m, 2H), 1.30-1.45 (m, 2H), 0.59-0.80 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 6 by using tert-butyl 8-amino-3-azabicyclo[3.2.1]octane-3-carboxylate instead of and tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 12 (65 mg) was obtained as a yellow solid. MS: calc'd 446 (MH+), measured 446 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.80 (d, J=1.71 Hz, 1H), 8.74 (d, J=1.59 Hz, 1H), 7.93 (d, J=8.80 Hz, 1H), 6.74 (d, J=8.80 Hz, 1H), 4.31-4.44 (m, 1H), 4.21-4.31 (m, 1H), 4.06-4.15 (m, 1H), 3.75-3.97 (m, 1H), 3.70 (d, J=10.76 Hz, 1H), 3.36-3.42 (m, 2H), 3.21-3.28 (m, 1H), 3.01-3.13 (m, 1H), 2.79-2.96 (m, 1H), 2.37-2.60 (m, 2H), 2.00-2.19 (m, 2H), 1.76-1.89 (m, 2H), 0.74-0.94 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 6 by using bromoquinoxaline-5-carbonitrile and tert-butyl 2-(aminomethyl)morpholine-4-carboxylate (CAS: 140645-53-0) instead of 5-bromo-8-(trifluoromethyl)quinoxaline (Intermediate A) and tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 13 (44 mg) was obtained as a yellow solid. MS: calc'd 393 (MH+), measured 393 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.72 (d, J=1.83 Hz, 1H), 8.63 (d, J=1.71 Hz, 1H), 7.86 (d, J=8.68 Hz, 1H), 6.64 (d, J=8.80 Hz, 1H), 4.15-4.30 (m, 2H), 3.96-4.11 (m, 2H), 3.57-3.73 (m, 3H), 3.30-3.41 (m, 1H), 3.23-3.29 (m, 1H), 3.12-3.18 (m, 2H), 2.95-3.07 (m, 1H), 2.72-2.82 (m, 1H), 2.64-2.72 (m, 1H), 0.76-0.85 (m, 1H), 0.66-0.76 (m, 3H).
The title compound was prepared in analogy to the preparation of Example 1 by using trans-ethyl-4-cyclopropylpyrrolidine-3-carboxylate hydrochloride (Pharmablock, PBXA3214-1) and 5-bromoquinoline-8-carbonitrile instead of methyl (3R,4R)-4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (compound 1b) and bromoquinoxaline-5-carbonitrile (compound 1a). Example 14 (8 mg) was obtained as a yellow solid. MS: calc'd 404 (MH+), measured 404 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.67 (dd, J=1.4, 4.6 Hz, 1H), 8.29 (br d, J=7.2 Hz, 1H), 7.87-7.76 (m, 1H), 7.45-7.30 (m, 1H), 6.73-6.54 (m, 1H), 3.95-3.48 (m, 5H), 3.35 (br d, J=12.8 Hz, 2H), 3.00-2.87 (m, 3H), 2.86-2.68 (m, 2H), 2.13-1.81 (m, 3H), 1.74-1.46 (m, 3H), 0.70-0.52 (m, 1H), 0.32 (br d, J=8.4 Hz, 2H), 0.06-0.07 (m, 2H).
The title compound was prepared in analogy to the preparation of Example 6 by using tert-butyl 7-amino-3-azabicyclo[3.3.1]nonane-3-carboxylate instead of tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 15 (17 mg) was obtained as a yellow solid. MS: calc'd 460 (MH+), measured 460 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.81 (cd, J=1.71 Hz, 1H), 8.75 (cd, J=1.71 Hz, 1H), 7.93 (d, J=8.80 Hz, 1H), 6.74 (d, J=8.80 Hz, 1H), 4.32-4.41 (m, 1H), 4.20-4.29 (m, 1H), 4.10 (d, J=10.76 Hz, 1H), 3.89-4.00 (m, 1H), 3.72 (d, J=10.76 Hz, 1H), 3.14-3.21 (m, 2H), 3.04-3.13 (m, 2H), 2.78 (dd, J=3.79, 7.09 Hz, 1H), 2.34-2.49 (m, 4H), 1.97 (d, J=13.45 Hz, 1H), 1.47 (d, J=13.69 Hz, 1H), 1.30-1.41 (m, 2H), 0.76-0.91 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 1 by using methyl (3R,4R)-4-methylpyrrolidine-3-carboxylate HCl salt (Pharmablock, PBXA3221-1) and 5-bromoquinoline-8-carbonitrile instead of methyl (3R,4R)-4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (compound 1b) and bromoquinoxaline-5-carbonitrile (compound 1a). Example 16 (10 mg) was obtained as a yellow solid. MS: calc'd 378 (MH+), measured 378 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.86 (dd, J=1.5, 4.3 Hz, 1H), 8.77 (dd, J=1.5, 8.8 Hz, 1H), 7.95 (d, J=8.6 Hz, 1H), 7.47 (dd, J=4.2, 8.7 Hz, 1H), 6.78 (d, J=8.6 Hz, 1H), 4.06 (t, J=9.6 Hz, 1H), 3.88-3.69 (m, 2H), 3.53 (t, J=9.8 Hz, 1H), 2.95-2.83 (m, 2H), 2.80-2.65 (m, 1H), 2.31 (s, 3H), 2.26-2.08 (m, 3H), 1.99-1.84 (m, 3H), 1.77-1.51 (m, 2H), 1.24-1.16 (m, 3H).
The title compound was prepared in analogy to the preparation of Example 1 by using trans-methyl-4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (Pharmablock, PBXA3194-1) and 5-bromoquinoline-8-carbonitrile instead of methyl (3R,4R)-4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (compound 1b) and bromoquinoxaline-5-carbonitrile (compound 1a). Example 17 (8 mg) was obtained as a yellow solid. MS: calc'd 432 (MH+), measured 432 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.84 (dd, J=1.5, 4.2 Hz, 1H), 8.58 (dd, J=1.6, 8.7 Hz, 1H), 7.97 (d, J=8.2 Hz, 1H), 7.48 (dd, J=4.3, 8.7 Hz, 1H), 6.96 (d, J=8.3 Hz, 1H), 3.81-3.62 (m, 3H), 3.59-3.48 (m, 1H), 3.43-3.35 (m, 2H), 2.89 (s, 3H), 2.38-2.20 (m, 2H), 2.16-2.05 (m, 1H), 1.91-1.81 (m, 3H), 1.65-1.45 (m, 3H).
The title compound was prepared in analogy to the preparation of Example 1 by using methyl 4-(trifluoromethyl)pyrrolidine-3-carboxylate and 5-bromoquinoline-8-carbonitrile instead of methyl (3R,4R)-4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (compound 1b) and bromoquinoxaline-5-carbonitrile (compound 1a). Example 18 (6 mg) was obtained as a yellow solid. MS: calc'd 432 (MH+), measured 432 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.96 (dd, J=1.6, 4.3 Hz, 1H), 8.74-8.67 (m, 1H), 8.09 (d, J=8.3 Hz, 1H), 7.64-7.54 (m, 1H), 7.16-7.03 (m, 1H), 4.11-3.93 (m, 1H), 3.98-3.52 (m, 7H), 3.25-3.09 (m, 2H), 2.97-2.85 (m, 3H), 2.35-2.02 (m, 3H), 1.84-1.61 (m, 2H).
The title compound was prepared in analogy to the preparation of Example 1 by using trans-ethyl-4-cyclopropylpyrrolidine-3-carboxylate hydrochloride (Pharmablock, PBXA3214-1), 5-bromoquinoline-8-carbonitrile and N′,N′-dimethylethane-1,2-diamine instead of methyl (3R,4R)-4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (compound 1b), bromoquinoxaline-5-carbonitrile (compound 1a) and 1-methylpiperidin-4-amine (compound 1e). Example 19 (20 mg) was obtained as a yellow solid. MS: calc'd 378 (MH+), measured 378 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=9.03 (dd, J=1.4, 8.7 Hz, 1H), 8.90 (dd, J=1.5, 4.6 Hz, 1H), 8.03 (d, J=8.7 Hz, 1H), 7.61 (dd, J=4.6, 8.8 Hz, 1H), 6.87 (d, J=8.7 Hz, 1H), 4.15-4.01 (m, 1H), 3.98-3.85 (m, 2H), 3.83-3.62 (m, 2H), 3.58-3.41 (m, 1H), 3.11-2.89 (m, 9H), 1.97-1.84 (m, 1H), 0.94-0.79 (m, 1H), 0.62-0.50 (m, 2H), 0.24 (q, J=4.4 Hz, 2H).
The title compound was prepared in analogy to the preparation of Example 6 by using 5-methyl-5-azaspiro[2.4]heptan-7-amine instead of tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 20 (47 mg) was obtained as a yellow solid. MS: calc'd 446 (MH+), measured 446 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.69 (d, J=1.59 Hz, 1H), 8.63 (s, 1H), 7.81 (d, J=8.68 Hz, 1H), 6.61 (d, J=8.68 Hz, 1H), 4.22-4.32 (m, 1H), 4.09-4.21 (m, 1H), 3.95-4.09 (m, 2H), 3.60-3.90 (m, 1H), 3.37-3.58 (m, 3H), 3.01-3.17 (m, 1H), 2.80-2.93 (m, 3H), 2.52-2.68 (m, 1H), 0.63-0.86 (m, 8H).
The title compound was prepared in analogy to the preparation of Example 6 by using tert-butyl 5-amino-3,3a,4,5,6,6a-hexahydro-1H-cyclopenta[c]pyrrole-2-carboxylate instead of tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 21 (25 mg) was obtained as a yellow solid. MS: calc'd 446 (MH+), measured 446 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.80 (d, J=1.83 Hz, 1H), 8.75 (d, J=1.71 Hz, 1H), 7.93 (d, J=8.68 Hz, 1H), 6.74 (d, J=8.68 Hz, 1H), 4.31-4.41 (m, 1H), 4.20-4.26 (m, 1H), 4.03-4.12 (m, 2H), 3.73 (d, J=10.76 Hz, 1H), 3.35-3.44 (m, 2H), 3.19 (td, J=3.82, 11.92 Hz, 2H), 2.84-2.94 (m, 2H), 2.77 (dd, J=3.85, 7.03 Hz, 1H), 2.30-2.46 (m, 2H), 1.26-1.39 (m, 2H), 0.73-0.91 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 6 by using tert-butyl N-(6-aminospiro[3.3]heptan-2-yl)carbamate instead of tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 22 (32 mg) was obtained as a yellow solid. MS: calc'd 446 (MH+), measured 446 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.68 (d, J=1.71 Hz, 1H), 8.62 (d, J=1.71 Hz, 1H), 7.80 (d, J=8.80 Hz, 1H), 6.61 (d, J=8.80 Hz, 1H), 4.18-4.24 (m, 1H), 4.01-4.14 (m, 2H), 3.95 (d, J=10.76 Hz, 1H), 3.51-3.63 (m, 2H), 2.53-2.64 (m, 1H), 2.36-2.47 (m, 2H), 2.19-2.28 (m, 2H), 1.99-2.12 (m, 2H), 1.91 (q, J=8.40 Hz, 2H), 0.58-0.77 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 6 by using tert-butyl 3-amino-8-azabicyclo[3.2.1]octane-8-carboxylate instead of tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 23 (27 mg) was obtained as a yellow solid. MS: calc'd 446 (MH+), measured 446 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.68 (d, J=1.71 Hz, 1H), 8.63 (d, J=1.71 Hz, 1H), 7.81 (d, J=8.68 Hz, 1H), 6.62 (d, J=8.68 Hz, 1H), 4.18-4.29 (m, 1H), 4.02-4.16 (m, 2H), 3.91-4.02 (m, 3H), 3.59 (d, J=10.76 Hz, 1H), 2.63 (dd, J=3.67, 7.09 Hz, 1H), 1.91-2.06 (m, 6H), 1.58-1.74 (m, 2H), 0.60-0.75 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 6 by using tert-butyl N-(4-aminocyclohexyl)carbamate instead of tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 24 (6 mg) was obtained as a yellow solid. MS: calc'd 434 (MH+), measured 434 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.68 (d, J=1.83 Hz, 1H), 8.63 (d, J=1.83 Hz, 1H), 7.81 (d, J=8.68 Hz, 1H), 6.62 (d, J=8.80 Hz, 1H), 4.19-4.29 (m, 1H), 4.08-4.16 (m, 1H), 3.96 (d, J=10.64 Hz, 1H), 3.62 (d, J=10.64 Hz, 1H), 3.55 (dd, J=3.79, 7.70 Hz, 1H), 2.93-3.04 (m, 1H), 2.63 (dd, J=3.79, 6.97 Hz, 1H), 1.85-2.03 (m, 4H), 1.34-1.45 (m, 2H), 1.18-1.33 (m, 2H), 0.61-0.74 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 1 by using 5-bromo-8-(trifluoromethyl)quinoxaline and methyl (3S,4S)-4-methylpyrrolidine-3-carboxylate hydrochloride (Pharmablock, PBXA3220-1) instead of bromoquinoxaline-5-carbonitrile (compound 1a) and methyl (3R,4R)-4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (compound 1b). Example 25 (8 mg) was obtained as a yellow solid. MS: calc'd 422 (MH+), measured 422 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.86-8.72 (m, 2H), 7.98-7.86 (m, 1H), 6.74 (d, J=8.7 Hz, 1H), 4.22-3.96 (m, 3H), 3.67-3.49 (m, 3H), 3.23-3.09 (m, 2H), 2.98-2.87 (m, 3H), 2.77-2.55 (m, 2H), 2.38-2.07 (m, 3H), 1.90-1.74 (m, 2H), 1.21 (d, J=6.4 Hz, 3H).
The title compound was prepared in analogy to the preparation of Example 6 by using 3-[(dimethylamino)methyl]cyclobutanamine (CAS:1479049-07-4) instead of tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 26 (8 mg) was obtained as a yellow solid. MS: calc'd 448 (MH+), measured 448 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.68 (d, J=1.71 Hz, 1H), 8.63 (d, J=1.59 Hz, 1H), 7.81 (d, J=8.68 Hz, 1H), 6.61 (d, J=8.68 Hz, 1H), 4.17-4.28 (m, 2H), 4.06-4.14 (m, 1H), 3.96 (d, J=10.76 Hz, 1H), 3.57-3.66 (m, 1H), 2.84-3.02 (m, 2H), 2.38-2.69 (m, 9H), 2.11 (dt, J=3.85, 7.67 Hz, 2H), 1.64 (q, J=9.82 Hz, 1H), 0.62-0.77 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 1 by using methyl (3R,4R)-4-methylpyrrolidine-3-carboxylate HCl salt (Pharmablock, PBXA3221-1) and 5-bromo-8-nitro-quinoline instead of methyl (3R,4R)-4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (compound 1b) and bromoquinoxaline-5-carbonitrile (compound 1a). Example 27 (6 mg) was obtained as a yellow solid. MS: calc'd 398 (MH+), measured 398 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.89 (dd, J=1.5, 4.2 Hz, 1H), 8.80 (dd, J=1.6, 8.8 Hz, 1H), 8.27 (d, J=8.9 Hz, 1H), 7.51 (dd, J=4.3, 8.8 Hz, 1H), 6.78 (d, J=9.0 Hz, 1H), 4.09 (t, J=9.6 Hz, 1H), 4.01-3.92 (m, 1H), 3.84 (ddd, J=7.3, 9.9, 19.3 Hz, 2H), 3.61-3.46 (m, 1H), 3.13-2.88 (m, 3H), 2.79-2.69 (m, 3H), 2.41 (m, 1H), 2.33-1.99 (m, 3H), 1.84-1.63 (m, 2H), 1.37 (t, J=7.3 Hz, 1H), 1.22 (d, J=6.5 Hz, 3H).
The title compound was prepared in analogy to the preparation of Example 1 by using trans-methyl-4-methylpyrrolidine-3-carboxylate (Bepharm, B162777) and 5-bromoquinoline-8-carbonitrile instead of methyl (3R,4R)-4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (compound 1b) and bromoquinoxaline-5-carbonitrile (compound 1a). Example 28 (5 mg) was obtained as a yellow solid. MS: calc'd 378 (MH+), measured 378 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.88 (dd, J=1.5, 4.2 Hz, 1H), 8.79 (dd, J=1.5, 8.7 Hz, 1H), 7.98 (d, J=8.6 Hz, 1H), 7.49 (dd, J=4.2, 8.7 Hz, 1H), 6.82 (d, J=8.6 Hz, 1H), 4.07 (t, J=9.5 Hz, 1H), 3.95-3.75 (m, 3H), 3.62-3.49 (m, 1H), 3.25 (br d, J=12.5 Hz, 2H), 2.85-2.56 (m, 7H), 2.18-2.00 (m, 2H), 1.72 (br d, J=12.3 Hz, 2H), 1.21 (d, J=6.4 Hz, 3H).
The title compound was prepared in analogy to the preparation of Example 1 by using 5-bromoquinoline-8-carbonitrile and trans-ethyl-4-ethylpyrrolidine-3-carboxylate hydrochloride (Pharmablock, PBXA3209-1) instead of bromoquinoxaline-5-carbonitrile (compound 1a) and methyl (3R,4R)-4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (compound 1b). Example 29 (43 mg) was obtained as a yellow solid. MS: calc'd 392 (MH+), measured 392 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.84-8.71 (m, 2H), 7.88 (d, J=8.6 Hz, 1H), 7.48-7.37 (m, 1H), 6.74 (d, J=8.7 Hz, 1H), 3.95-3.83 (m, 2H), 3.81-3.68 (m, 2H), 3.53-3.42 (m, 3H), 3.11-2.98 (m, 2H), 2.84-2.76 (m, 3H), 2.74-2.63 (m, 1H), 2.47-2.38 (m, 1H), 2.11 (dt, J=2.6, 10.0 Hz, 2H), 2.00-1.89 (m, 1H), 1.71-1.40 (m, 3H), 0.93 (t, J=7.5 Hz, 3H).
The title compound was prepared according to the scheme below.
Compound 30c was prepared in analogy to the preparation of (3R,4R)-1-(8-cyanoquinoxalin-5-yl)-4-(trifluoromethyl)pyrrolidine-3-carboxylic acid (compound 1d) of Example 1 by using methyl 4-(trifluoromethyl)pyrrolidine-3-carboxylate and 5-bromoquinoline-8-carbonitrile instead of methyl (3R,4R)-4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (compound 1b) and bromoquinoxaline-5-carbonitrile (compound 1a).
To a solution of 1-(8-cyanoquinolin-5-yl)-4-(trifluoromethyl)pyrrolidine-3-carboxylic acid (compound 30c, 15 mg, 44.7 μmol) and tert-butyl 4-aminoazepane-1-carboxylate (compound 30d, 9.6 mg, 44.7 μmol) in DCM (10 mL) was added triethylamine (22.6 mg, 224 μmol) and HATU (51 mg, 134 μmol). After the mixture was stirred at rt for 12 hrs, the solvent was removed under vacuum. The residue was then dissolved in DCM (2.0 mL), to which was added TFA (0.5 mL). After the mixture was stirred at rt for 2 hrs, the reaction mixture was concentrated to give a crude product which was purified by prep-HPLC to afford two isomers: Example 30A (5 mg) and Example 30B (5 mg).
Example 30A: MS: calc'd 432 (MH+), measured 432 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.70 (dd, J=1.5, 8.7 Hz, 1H), 8.59 (br d, J=7.3 Hz, 1H), 8.09 (d, J=8.2 Hz, 1H), 7.60 (dd, J=4.2, 8.7 Hz, 1H), 7.08 (d, J=8.2 Hz, 1H), 4.09-3.97 (m, 1H), 3.93-3.72 (m, 4H), 3.65 (td, J=8.2, 16.4 Hz, 1H), 3.29-3.12 (m, 4H), 2.29-1.59 (m, 7H).
Example 30B: MS: calc'd 432 (MH+), measured 432 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.70 (dd, J=1.5, 8.7 Hz, 1H), 8.58 (br d, J=7.3 Hz, 1H), 8.08 (d, J=8.2 Hz, 1H), 7.60 (dd, J=4.3, 8.7 Hz, 1H), 7.08 (d, J=8.3 Hz, 1H), 4.09-3.96 (m, 1H), 3.93-3.60 (m, 5H), 3.30-3.14 (m, 4H), 2.25-1.57 (m, 7H).
The title compound was prepared in analogy to the preparation of Example 6 by using tert-butyl N-[3-(aminomethyl)cyclobutyl]carbamate instead of tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 31 (33 mg) was obtained as a yellow solid. MS: calc'd 420 (MH+), measured 420 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.68 (d, J=1.59 Hz, 1H), 8.62 (d, J=1.71 Hz, 1H), 7.80 (d, J=8.68 Hz, 1H), 6.61 (d, J=8.68 Hz, 1H), 4.16-4.28 (m, 1H), 4.10-4.16 (m, 1H), 3.93-4.02 (m, 1H), 3.65-3.76 (m, 1H), 3.47-3.63 (m, 1H), 3.24-3.31 (m, 1H), 3.05-3.16 (m, 1H), 2.64 (dd, J=3.79, 6.97 Hz, 1H), 2.28-2.54 (m, 2H), 2.04-2.26 (m, 2H), 1.64-1.78 (m, 1H), 0.59-0.77 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 30 by using trans-methyl-4-methylpyrrolidine-3-carboxylate and tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate instead of methyl 4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt and tert-butyl 4-aminoazepane-1-carboxylate (compound 30d). Example 32 (10 mg) was obtained as a yellow solid. MS: calc'd 418 (MH+), measured 418 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.90-8.76 (m, 2H), 7.98 (d, J=8.4 Hz, 1H), 7.50 (dd, J=4.3, 8.8 Hz, 1H), 6.82 (d, J=8.6 Hz, 1H), 4.00-3.79 (m, 3H), 3.69 (br t, J=5.7 Hz, 4H), 3.60-3.48 (m, 1H), 3.22 (br s, 5H), 2.80-2.67 (m, 1H), 1.87-1.74 (m, 4H), 1.71-1.56 (m, 4H), 1.22 (d, J=6.6 Hz, 3H).
The title compound was prepared in analogy to the preparation of Example 1 by using methyl (3R,4R)-4-methylpyrrolidine-3-carboxylate HCl salt (Pharmablock, PBXA3221-1) and 5-bromo-8-methyl-quinoline instead of methyl (3R,4R)-4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (compound 1b) and bromoquinoxaline-5-carbonitrile (compound 1a). Example 33 (10 mg) was obtained as a yellow solid. MS: calc'd 368 (MH+), measured 368 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=9.13-8.99 (m, 1H), 9.03-8.92 (m, 1H), 7.82 (q, J=8.0 Hz, 2H), 4.35-4.22 (m, 1H), 4.19-3.97 (m, 3H), 3.77-3.52 (m, 3H), 3.26-3.00 (m, 4H), 2.97-2.75 (m, 7H), 2.32-2.15 (m, 2H), 1.88-1.69 (m, 2H), 1.35-1.27 (m, 3H).
The title compound was prepared in analogy to the preparation of Example 6 by using tert-butyl 2-amino-7-azaspiro[3.5]nonane-7-carboxylate instead of tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 34 (26 mg) was obtained as a yellow solid. MS: calc'd 460 (MH+), measured 460 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.68 (d, J=1.71 Hz, 1H), 8.63 (d, J=1.71 Hz, 1H), 7.80 (d, J=8.68 Hz, 1H), 6.61 (d, J=8.68 Hz, 1H), 4.07-4.26 (m, 3H), 3.96 (d, J=10.76 Hz, 1H), 3.60 (d, J=10.76 Hz, 1H), 3.01-3.07 (m, 2H), 2.94-3.01 (m, 2H), 2.62 (dd, J=3.91, 7.09 Hz, 1H), 2.17-2.31 (m, 2H), 1.65-1.80 (m, 6H), 0.63-0.76 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 30 by using tert-butyl 4-(4-amino-1-piperidyl)piperidine-1-carboxylate instead of tert-butyl 4-aminoazepane-1-carboxylate (compound 30d). Example 35 (15 mg) was obtained as a yellow solid. MS: calc'd 502 (MH+), measured 502 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.89 (d, J=1.7 Hz, 1H), 8.83 (d, J=1.8 Hz, 1H), 8.04 (d, J=8.7 Hz, 1H), 6.87 (d, J=8.8 Hz, 1H), 4.46-4.20 (m, 2H), 4.13-3.99 (m, 1H), 3.81-3.52 (m, 2H), 3.24-3.11 (m, 3H), 3.00 (br d, J=11.7 Hz, 3H), 2.76-2.60 (m, 2H), 2.57-2.31 (m, 3H), 1.94 (br d, J=11.7 Hz, 4H), 1.66-1.43 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 6 by using tert-butyl N-[(1S,2R,4S,5R)-4-amino-2-bicyclo[3.1.0]hexanyl]carbamate instead of tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 36 (34 mg) was obtained as a yellow solid. MS: calc'd 432 (MH+), measured 432 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=9.02 (d, J=1.71 Hz, 1H), 8.96 (s, 1H), 8.14 (d, J=8.68 Hz, 1H), 6.95 (d, J=8.68 Hz, 1H), 4.70 (m, 1H), 4.52-4.61 (m, 1H), 4.41-4.51 (m, 1H), 4.30 (dd, J=4.28, 10.76 Hz, 1H), 4.01-4.11 (m, 1H), 3.94 (dd, J=2.81, 10.76 Hz, 1H), 3.02 (dt, J=3.91, 7.09 Hz, 1H), 2.48-2.66 (m, 1H), 1.95-2.07 (m, 1H), 1.86-1.95 (m, 1H), 1.24-1.35 (m, 1H), 0.96-1.19 (m, 5H), 0.84-0.94 (m, 1H).
The title compound was prepared in analogy to the preparation of Example 1 by using trans-methyl 4-isopropyl-pyrrolidine-3-carboxylate hydrochloride salt (CAS:1820575-33-4) and 5-bromoquinoline-8-carbonitrile instead of methyl (3R,4R)-4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (compound 1b) and bromoquinoxaline-5-carbonitrile (compound 1a). Example 37 (50 mg) was obtained as a yellow solid. MS: calc'd 406 (MH+), measured 406 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.93-8.85 (m, 2H), 8.00 (d, J=8.6 Hz, 1H), 7.56 (dd, J=4.5, 8.7 Hz, 1H), 6.87 (d, J=8.7 Hz, 1H), 4.05-3.88 (m, 2H), 3.90-3.74 (m, 2H), 3.71-3.50 (m, 3H), 3.25-3.09 (m, 2H), 2.97-2.85 (m, 4H), 2.62-2.49 (m, 1H), 2.30-2.04 (m, 2H), 1.86-1.67 (m, 3H), 1.12-0.95 (m, 6H).
The title compound was prepared in analogy to the preparation of Example 6 by using tert-butyl N-(3-aminocyclobutyl)carbamate instead of tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 38 (25 mg) was obtained as a yellow solid. MS: calc'd 406 (MH+), measured 406 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.68 (d, J=1.71 Hz, 1H), 8.62 (d, J=1.71 Hz, 1H), 7.80 (d, J=8.68 Hz, 1H), 6.61 (d, J=8.68 Hz, 1H), 4.30-4.42 (m, 1H), 4.19-4.29 (m, 1H), 4.04-4.14 (m, 1H), 3.88-4.03 (m, 1H), 3.72-3.81 (m, 1H), 3.53-3.63 (m, 1H), 2.58-2.71 (m, 2H), 2.27-2.47 (m, 3H), 0.63-0.81 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 6 by using tert-butyl N-(4-aminocyclohexyl)carbamate instead of tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 39 (33 mg) was obtained as a yellow solid. MS: calc'd 434 (MH+), measured 434 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.69 (d, J=1.71 Hz, 1H), 8.63 (d, J=1.71 Hz, 1H), 7.81 (d, J=8.68 Hz, 1H), 6.63 (d, J=8.68 Hz, 1H), 4.19-4.27 (m, 1H), 4.10-4.18 (m, 1H), 3.97 (d, J=10.76 Hz, 1H), 3.76 (s, 1H), 3.62 (d, J=10.76 Hz, 1H), 3.12 (s, 1H), 2.75 (dd, J=3.97, 7.03 Hz, 1H), 1.69-1.84 (m, 4H), 1.53-1.69 (m, 4H), 0.63-0.80 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 1 by using 5-bromoquinoline-8-carbonitrile and trans-ethyl-4-(difluoromethyl)pyrrolidine-3-carboxylate hydrochloride (Pharmablock, PBXA3200-1) instead of bromoquinoxaline-5-carbonitrile (compound 1a) and methyl (3R,4R)-4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (compound 1b). Example 40 (11 mg) was obtained as a yellow solid. MS: calc'd 414 (MH+), measured 414 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.93 (dd, J=1.5, 4.2 Hz, 1H), 8.75 (dd, J=1.6, 8.7 Hz, 1H), 8.05 (d, J=8.4 Hz, 1H), 7.56 (dd, J=4.2, 8.7 Hz, 1H), 7.00 (d, J=8.4 Hz, 1H), 6.14-5.94 (m, 1H), 3.91-3.69 (m, 5H), 3.29-3.13 (m, 2H), 3.06-2.92 (m, 2H), 2.45-2.25 (m, 5H), 1.97 (br t, J=13.1 Hz, 2H), 1.70-1.51 (m, 2H).
The title compound was prepared in analogy to the preparation of Example 6 by using 5-bromoquinoline-8-carbonitrile and 1-methylpiperidin-4-amine instead of 5-bromo-8-(trifluoromethyl)quinoxaline (Intermediate A) and tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 41 (30 mg) was obtained as a yellow solid. MS: calc'd 390 (MH+), measured 390 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.87 (dd, J=1.5, 4.2 Hz, 1H), 8.83 (dd, J=1.6, 8.8 Hz, 1H), 7.99 (d, J=8.4 Hz, 1H), 7.49 (dd, J=4.2, 8.7 Hz, 1H), 6.84 (d, J=8.6 Hz, 1H), 4.15 (dd, J=6.8, 10.0 Hz, 1H), 4.03-3.93 (m, 2H), 3.75-3.64 (m, 1H), 3.47 (d, J=9.5 Hz, 1H), 2.97-2.87 (m, 2H), 2.81 (dd, J=4.2, 6.7 Hz, 1H), 2.35 (s, 3H), 2.31-2.16 (m, 2H), 1.97-1.83 (m, 2H), 1.63-1.50 (m, 2H), 0.90-0.74 (m, 4H).
SFC-HPLC separation gave two isomers: Example 41A (5 mg) and Example 41B (5 mg).
Example 41A: MS: calc'd 390 (MH+), measured 390 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.87 (dd, J=1.5, 4.2 Hz, 1H), 8.83 (dd, J=1.5, 8.8 Hz, 1H), 7.99 (d, J=8.4 Hz, 1H), 7.49 (dd, J=4.2, 8.7 Hz, 1H), 6.84 (d, J=8.6 Hz, 1H), 4.15 (dd, J=6.9, 10.1 Hz, 1H), 4.05-3.91 (m, 2H), 3.76-3.59 (m, 1H), 3.47 (d, J=9.7 Hz, 1H), 2.95-2.74 (m, 3H), 2.31 (s, 3H), 2.24-2.08 (m, 2H), 1.96-1.77 (m, 2H), 1.65-1.44 (m, 2H), 0.95-0.68 (m, 4H).
Example 41B: MS: calc'd 390 (MH+), measured 390 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.88 (dd, J=1.5, 4.2 Hz, 1H), 8.83 (dd, J=1.5, 8.7 Hz, 1H), 7.99 (d, J=8.6 Hz, 1H), 7.49 (dd, J=4.3, 8.7 Hz, 1H), 6.84 (d, J=8.6 Hz, 1H), 4.15 (dd, J=6.9, 10.1 Hz, 1H), 4.06-3.89 (m, 2H), 3.77-3.61 (m, 1H), 3.47 (d, J=9.7 Hz, 1H), 2.95-2.74 (m, 3H), 2.31 (s, 3H), 2.23-2.07 (m, 2H), 1.97-1.80 (m, 2H), 1.65-1.46 (m, 2H), 0.92-0.71 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 6 by using tert-butyl 9-amino-3-oxa-7-azabicyclo[3.3.1]nonane-7-carboxylate instead of tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 42 (22 mg) was obtained as a yellow solid. MS: calc'd 462 (MH+), measured 462 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.81 (d, J=1.59 Hz, 1H), 8.70-8.78 (m, 1H), 7.93 (d, J=8.68 Hz, 1H), 6.75 (dd, J=2.93, 8.68 Hz, 1H), 4.34-4.44 (m, 1H), 4.24-4.34 (m, 1H), 4.06-4.24 (m, 4H), 3.96-4.06 (m, 1H), 3.91 (dd, J=5.56, 12.04 Hz, 1H), 3.62-3.74 (m, 2H), 3.41-3.55 (m, 3H), 2.92 (ddd, J=3.36, 7.03, 12.47 Hz, 1H), 2.02-2.22 (m, 2H), 0.79-0.96 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 6 by using cis-tert-butyl N-(3-aminocyclohexyl)carbamate (Pharmablock, PBZS1047, CAS:849616-22-4) instead of tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 43 (29 mg) was obtained as a yellow solid. MS: calc'd 434 (MH+), measured 434 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.69 (d, J=1.47 Hz, 1H), 8.63 (d, J=1.59 Hz, 1H), 7.81 (d, J=8.68 Hz, 1H), 6.62 (d, J=8.68 Hz, 1H), 4.19-4.29 (m, 1H), 4.07-4.17 (m, 1H), 3.96 (dd, J=3.73, 10.82 Hz, 1H), 3.57-3.69 (m, 2H), 2.99-3.10 (m, 1H), 2.63 (ddd, J=3.85, 7.00, 10.79 Hz, 1H), 2.04-2.21 (m, 1H), 1.74-1.96 (m, 3H), 1.28-1.46 (m, 1H), 1.06-1.25 (m, 3H), 0.61-0.77 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 1 by using methyl (3R,4R)-4-methylpyrrolidine-3-carboxylate HCl salt (Pharmablock, PBXA3221-1) and 4-bromopyrazolo[1,5-a]pyridine-7-carbonitrile (CAS: 1268520-74-6, Pharmablock) instead of methyl (3R,4R)-4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (compound 1b) and bromoquinoxaline-5-carbonitrile (compound 1a). Example 44 (35 mg) was obtained as a yellow solid. MS: calc'd 367 (MH+), measured 367 (MH+). 1H NMR (400 MHz, METHANOL-d4) 6=7.92 (d, J=2.6 Hz, 1H), 7.32 (d, J=8.3 Hz, 1H), 7.06 (d, J=2.6 Hz, 1H), 5.99 (d, J=8.4 Hz, 1H), 4.09-3.87 (m, 3H), 3.85-3.66 (m, 1H), 3.55-3.36 (m, 2H), 2.97 (br d, J=11.6 Hz, 2H), 2.82-2.70 (m, 1H), 2.69-2.48 (m, 1H), 2.43-2.23 (m, 3H), 2.03-1.90 (m, 2H), 1.70-1.53 (m, 2H), 1.37 (t, J=7.3 Hz, 1H), 1.20 (d, J=6.6 Hz, 3H).
The title compound was prepared in analogy to the preparation of Example 1 by using trans-methyl-4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (Pharmablock, PBXA3194-1) and 5-bromo-8-chloro-quinoline instead of methyl (3R,4R)-4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (compound 1b) and bromoquinoxaline-5-carbonitrile (compound 1a). Example 46 (27 mg) was obtained as a yellow solid. MS: calc'd 441 (MH+), measured 441 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.93-8.84 (m, 1H), 7.88-7.78 (m, 1H), 7.76-7.67 (m, 1H), 7.49 (dd, J=4.4, 8.4 Hz, 1H), 7.19-7.12 (m, 1H), 3.97-3.82 (m, 1H), 3.67-3.38 (m, 7H), 3.14-2.97 (m, 3H), 2.84-2.72 (m, 3H), 2.16-1.94 (m, 2H), 1.70-1.54 (m, 2H).
The title compound was prepared in analogy to the preparation of Example 30 and Example 1 by using trans-methyl-4-trifluoromethylpyrrolidine-3-carboxylate and (3S,4R)-tert-butyl 3-amino-4-fluoropyrrolidine-1-carboxylate and 5-bromoquinoline-8-carbonitrile instead of methyl (3R,4R)-4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (compound 1b) and bromoquinoxaline-5-carbonitrile (compound 1a) and tert-butyl 4-aminoazepane-1-carboxylate (compound 30d). Example 47 (7 mg) was obtained as a yellow solid. MS: calc'd 422 (MH+), measured 422 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.84 (dd, J=1.5, 4.2 Hz, 1H), 8.57 (dd, J=1.6, 8.7 Hz, 1H), 7.97 (d, J=8.2 Hz, 1H), 7.48 (dd, J=4.3, 8.7 Hz, 1H), 6.98 (d, J=8.3 Hz, 1H), 5.30-5.11 (m, 1H), 4.71-4.54 (m, 1H), 3.85-3.76 (m, 1H), 3.74-3.54 (m, 6H), 3.52-3.46 (m, 1H), 3.39 (q, J=7.6 Hz, 1H), 3.10 (t, J=11.4 Hz, 1H).
The title compound was prepared in analogy to the preparation of Example 6 by using tert-butyl 5-amino-2-azabicyclo[2.2.1]heptane-2-carboxylate instead of tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 48 (21 mg) was obtained as a yellow solid. MS: calc'd 432 (MH+), measured 432 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.80 (d, J=1.47 Hz, 1H), 8.75 (s, 1H), 7.93 (d, J=8.56 Hz, 1H), 6.74 (d, J=8.68 Hz, 1H), 4.34-4.43 (m, 1H), 4.26 (dt, J=4.16, 7.83 Hz, 1H), 3.89-4.19 (m, 3H), 3.63-3.75 (m, 1H), 3.36-3.41 (m, 1H), 3.09-3.23 (m, 1H), 2.90-3.06 (m, 1H), 2.84 (dd, J=3.48, 7.03 Hz, 1H), 2.21-2.39 (m, 1H), 1.90-2.00 (m, 1H), 1.73-1.87 (m, 1H), 1.46-1.71 (m, 1H), 0.69-0.93 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 30 by using trans-methyl-4-isopropylpyrrolidine-3-carboxylate and (3S,4R)-tert-butyl 3-amino-4-fluoropyrrolidine-1-carboxylate instead of methyl 4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt and tert-butyl 4-aminoazepane-1-carboxylate (compound 30d). Example 49 (12 mg) was obtained as a yellow solid. MS: calc'd 396 (MH+), measured 396 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.90 (dd, J=1.5, 4.3 Hz, 1H), 8.79 (dd, J=1.6, 8.8 Hz, 1H), 8.00 (d, J=8.6 Hz, 1H), 7.52 (dd, J=4.3, 8.8 Hz, 1H), 6.89 (d, J=8.6 Hz, 1H), 5.44-5.23 (m, 1H), 4.80-4.63 (m, 1H), 4.00-3.89 (m, 1H), 3.87-3.56 (m, 5H), 3.28-2.95 (m, 3H), 2.66-2.54 (m, 1H), 1.82 (qd, J=6.9, 14.0 Hz, 1H), 1.05 (dd, J=5.2, 6.7 Hz, 6H).
The title compound was prepared in analogy to the preparation of Example 1 by using methyl (3R,4R)-4-methylpyrrolidine-3-carboxylate HCl salt (Pharmablock, PBXA3221-1) and 5-bromo-8-chloro-quinoline instead of methyl (3R,4R)-4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (compound 1b) and bromoquinoxaline-5-carbonitrile (compound 1a). Example 50 (3 mg) was obtained as a yellow solid. MS: calc'd 387 (MH+), measured 387 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=9.03 (dd, J=1.3, 5.4 Hz, 1H), 8.04 (d, J=9.0 Hz, 1H), 7.92 (dd, J=5.5, 8.6 Hz, 1H), 7.55 (dd, J=4.4, 8.4 Hz, 1H), 7.15-7.03 (m, 1H), 4.13-3.96 (m, 2H), 3.93-3.76 (m, 2H), 3.64-3.52 (m, 3H), 3.27-3.09 (m, 3H), 2.98-2.61 (m, 4H), 2.23 (br t, J=12.5 Hz, 2H), 1.88-1.72 (m, 2H), 1.29-1.16 (m, 3H).
The title compound was prepared in analogy to the preparation of Example 6 by using 5-bromoquinoline-8-carbonitrile and (1-methyl-3-piperidyl)methanamine instead of 5-bromo-8-(trifluoromethyl)quinoxaline (Intermediate A) and tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 51 (9 mg) was obtained as a yellow solid. MS: calc'd 404 (MH+), measured 404 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.88 (d, J=4.0 Hz, 1H), 8.83 (d, J=8.8 Hz, 1H), 7.99 (d, J=8.4 Hz, 1H), 7.49 (dd, J=4.3, 8.7 Hz, 1H), 6.84 (d, J=8.6 Hz, 1H), 4.22 (dd, J=7.0, 9.9 Hz, 1H), 4.07-3.92 (m, 2H), 3.41 (d, J=9.7 Hz, 1H), 3.31-3.25 (m, 2H), 3.25-3.08 (m, 2H), 2.84-2.68 (m, 5H), 2.61-2.46 (m, 1H), 2.05-1.63 (m, 4H), 1.29-1.14 (m, 1H), 0.95-0.74 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 30 by using trans-methyl-4-methylpyrrolidine-3-carboxylate (Bepharm, B162777) instead of methyl 4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt. Prep-HPLC separation gave two isomers Example 52A (12 mg) and Example 52B (8 mg).
Example 52A: MS: calc'd 378 (MH+), measured 378 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=9.09 (dd, J=1.3, 8.8 Hz, 1H), 8.92 (dd, J=1.5, 4.8 Hz, 1H), 8.06 (d, J=8.8 Hz, 1H), 7.65 (dd, J=4.8, 8.8 Hz, 1H), 6.89 (d, J=8.8 Hz, 1H), 4.13-3.98 (m, 2H), 3.92 (ddd, J=7.3, 10.1, 14.2 Hz, 2H), 3.60 (t, J=9.8 Hz, 1H), 3.30-3.17 (m, 3H), 2.82-2.53 (m, 2H), 2.26-1.64 (m, 7H), 1.22 (d, J=6.6 Hz, 3H).
Example 52B: MS: calc'd 378 (MH+), measured 378 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=9.09 (dd, J=1.3, 8.8 Hz, 1H), 8.92 (dd, J=1.3, 4.8 Hz, 1H), 8.06 (d, J=8.7 Hz, 1H), 7.65 (dd, J=4.8, 8.8 Hz, 1H), 6.89 (d, J=8.7 Hz, 1H), 4.15-3.99 (m, 2H), 3.92 (ddd, J=7.3, 10.1, 14.2 Hz, 2H), 3.60 (t, J=9.8 Hz, 1H), 3.27-3.13 (m, 3H), 2.82-2.61 (m, 2H), 2.31-1.61 (m, 7H), 1.22 (d, J=6.5 Hz, 3H).
The title compound was prepared in analogy to the preparation of Example 30 by using trans-methyl-4-methylpyrrolidine-3-carboxylate (Bepharm, B162777) and tert-butyl 2,7-diazaspiro[4.4]nonane-2-carboxylate instead of methyl 4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt and tert-butyl 4-aminoazepane-1-carboxylate (compound 30d). Example 53 (4 mg) was obtained as a yellow solid. MS: calc'd 390 (MH+), measured 390 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=9.00-8.88 (m, 2H), 8.03 (d, J=8.6 Hz, 1H), 7.64-7.53 (m, 1H), 6.87 (d, J=8.7 Hz, 1H), 4.06-3.96 (m, 2H), 3.94-3.85 (m, 1H), 3.81 (br t, J=7.2 Hz, 1H), 3.66-3.41 (m, 7H), 3.25-3.05 (m, 1H), 2.83-2.69 (m, 1H), 2.25-1.97 (m, 5H), 1.25 (d, J=6.6 Hz, 3H).
The title compound was prepared in analogy to the preparation of Example 1 by using methyl-(3S,4S)-4-methylpyrrolidine-3-carboxylate hydrochloride (PBXA3220-1, Pharmablock) and 5-bromoquinoline-8-carbonitrile instead of methyl (3R,4R)-4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (compound 1b) and bromoquinoxaline-5-carbonitrile (compound 1a). Example 54 (50 mg) was obtained as a yellow solid. MS: calc'd 378 (MH+), measured 378 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.87-8.68 (m, 2H), 7.92 (d, J=8.6 Hz, 1H), 7.44 (dd, J=4.2, 8.7 Hz, 1H), 6.75 (d, J=8.6 Hz, 1H), 4.11-3.96 (m, 1H), 3.84-3.69 (m, 3H), 3.50 (t, J=9.7 Hz, 1H), 3.01-2.88 (m, 2H), 2.75-2.53 (m, 2H), 2.38-2.17 (m, 5H), 1.99-1.84 (m, 2H), 1.59 (q, J=11.4 Hz, 2H), 1.18 (d, J=6.4 Hz, 3H).
The title compound was prepared in analogy to the preparation of Example 1 by using trans-ethyl 4-cyclopropylpyrrolidine-3-carboxylate hydrochloride salt (Pharmablock, PBXA3214-1) and 2-morpholinoethanamine and 5-bromoquinoline-8-carbonitrile instead of methyl (3R,4R)-4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (compound 1b) and bromoquinoxaline-5-carbonitrile (compound 1a) and 1-methylpiperidin-4-amine (compound 1e). Example 55 (13 mg) was obtained as a yellow solid. MS: calc'd 420 (MH+), measured 420 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=9.16 (d, J=8.7 Hz, 1H), 8.92 (dd, J=1.2, 4.9 Hz, 1H), 8.08 (d, J=8.8 Hz, 1H), 7.68 (dd, J=4.9, 8.8 Hz, 1H), 6.92 (d, J=8.8 Hz, 1H), 4.14-4.03 (m, 3H), 3.96 (td, J=7.1, 10.1 Hz, 2H), 3.85-3.62 (m, 5H), 3.62-3.45 (m, 2H), 3.28-2.95 (m, 5H), 1.99-1.86 (m, 1H), 0.96-0.80 (m, 1H), 0.60-0.50 (m, 2H), 0.32-0.21 (m, 2H).
The title compound was prepared in analogy to the preparation of Example 30 by using trans-methyl-4-methylpyrrolidine-3-carboxylate and (3S,4R)-tert-butyl 3-amino-4-fluoropyrrolidine-1-carboxylate instead of methyl 4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt and tert-butyl 4-aminoazepane-1-carboxylate (compound 30d). Example 56 (4 mg) was obtained as a yellow solid. MS: calc'd 368 (MH+), measured 368 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.94-8.75 (m, 2H), 7.99 (d, J=8.4 Hz, 1H), 7.51 (dd, J=4.3, 8.8 Hz, 1H), 6.84 (d, J=8.6 Hz, 1H), 5.43-5.19 (m, 1H), 4.07 (t, J=9.5 Hz, 1H), 3.92-3.48 (m, 7H), 3.25 (t, J=11.3 Hz, 1H), 2.95-2.80 (m, 1H), 2.73-2.60 (m, 1H), 1.23 (d, J=6.6 Hz, 3H).
The title compound was prepared in analogy to the preparation of Example 6 by using 5-bromoquinoline-8-carbonitrile and tert-butyl 4-aminoazepane-1-carboxylate instead of 5-bromo-8-(trifluoromethyl)quinoxaline (Intermediate A) and tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Prep-HPLC separation gave two isomers Example 57A (7 mg) and Example 57B (7 mg).
Example 57A: MS: calc'd 390 (MH+), measured 390 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.88 (d, J=3.3 Hz, 1H), 8.83 (d, J=8.9 Hz, 1H), 7.99 (d, J=8.4 Hz, 1H), 7.49 (dd, J=4.3, 8.7 Hz, 1H), 6.84 (d, J=8.6 Hz, 1H), 4.18 (dd, J=7.0, 9.9 Hz, 1H), 4.02-3.83 (m, 3H), 3.45 (d, J=9.4 Hz, 1H), 3.28-3.17 (m, 2H), 3.17-3.00 (m, 2H), 2.78 (dd, J=3.7, 6.5 Hz, 1H), 2.18-1.89 (m, 3H), 1.88-1.73 (m, 2H), 1.73-1.56 (m, 1H), 0.91-0.70 (m, 4H).
Example 57B: MS: calc'd 390 (MH+), measured 390 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.88 (dd, J=1.5, 4.2 Hz, 1H), 8.83 (dd, J=1.5, 8.7 Hz, 1H), 7.99 (d, J=8.4 Hz, 1H), 7.49 (dd, J=4.3, 8.8 Hz, 1H), 6.84 (d, J=8.6 Hz, 1H), 4.18 (dd, J=6.8, 10.0 Hz, 1H), 4.02-3.87 (m, 3H), 3.45 (d, J=9.7 Hz, 1H), 3.31-3.22 (m, 2H), 3.21-3.09 (m, 2H), 2.79 (dd, J=3.7, 6.8 Hz, 1H), 2.22-1.95 (m, 3H), 1.93-1.74 (m, 2H), 1.70-1.56 (m, 1H), 0.90-0.74 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 1 by using methyl-(3S,4S)-4-(trifluoromethyl)pyrrolidine-3-carboxylate hydrochloride (Pharmablock, PBXA3228-1) and 5-bromoquinoline-8-carbonitrile instead of methyl (3R,4R)-4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (compound 1b) and bromoquinoxaline-5-carbonitrile (compound 1a). Example 58 (19 mg) was obtained as a yellow solid. MS: calc'd 432 (MH+), measured 432 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.95 (dd, J=1.5, 4.2 Hz, 1H), 8.70 (dd, J=1.6, 8.7 Hz, 1H), 8.07 (d, J=8.2 Hz, 1H), 7.59 (dd, J=4.2, 8.7 Hz, 1H), 7.07 (d, J=8.3 Hz, 1H), 3.94-3.72 (m, 3H), 3.69-3.57 (m, 1H), 3.49 (q, J=7.3 Hz, 2H), 3.06-2.90 (m, 3H), 2.43-2.27 (m, 5H), 1.97 (br t, J=12.7 Hz, 2H), 1.70-1.54 (m, 2H).
The title compound was prepared in analogy to the preparation of Example 6 by using 5-bromoquinoline-8-carbonitrile and tert-butyl N-(2-amino-1,1-dimethyl-ethyl)carbamate instead of 5-bromo-8-(trifluoromethyl)quinoxaline (Intermediate A) and tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 59 (13 mg) was obtained as a yellow solid. MS: calc'd 364 (MH+), measured 364 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.89-8.86 (m, 1H), 8.83 (d, J=8.7 Hz, 1H), 7.99 (dd, J=1.3, 8.4 Hz, 1H), 7.49 (dd, J=4.3, 8.7 Hz, 1H), 6.83 (d, J=8.6 Hz, 1H), 4.24 (dd, J=6.7, 10.0 Hz, 1H), 4.10-3.94 (m, 2H), 3.50 (d, J=14.3 Hz, 1H), 3.41 (d, J=9.5 Hz, 1H), 3.17 (d, J=14.1 Hz, 1H), 2.87-2.80 (m, 1H), 1.31 (s, 3H), 1.30 (s, 3H), 0.97-0.78 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 30 by using methyl (3R,4R)-4-methylpyrrolidine-3-carboxylate HCl salt (Pharmablock, PBXA3221-1) and tert-butyl 4-(4-amino-1-piperidyl)piperidine-1-carboxylate instead of methyl 4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt and tert-butyl 4-aminoazepane-1-carboxylate (compound 30d). Example 60 (10 mg) was obtained as a yellow solid. MS: calc'd 448 (MH+), measured 448 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.81 (d, J=1.6 Hz, 1H), 8.72 (d, J=1.5 Hz, 1H), 7.92 (d, J=8.6 Hz, 1H), 6.70 (d, J=8.7 Hz, 1H), 4.27-3.96 (m, 4H), 3.75-3.48 (m, 6H), 3.29-3.04 (m, 4H), 2.81-2.22 (m, 6H), 2.09-1.78 (m, 4H), 1.22 (d, J=6.5 Hz, 3H).
The title compound was prepared in analogy to the preparation of Example 30 by using trans-ethyl-4-difluoromethyl-pyrrolidine-3-carboxylate hydrochloride (Pharmablock, PBXA3200-1) instead of methyl 4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (compound 1b). Prep-HPLC separation gave two isomers Example 61A (14 mg) and Example 61B (8 mg).
Example 61A: MS: calc'd 414 (MH+), measured 414 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.81 (dd, J=1.5, 4.2 Hz, 1H), 8.64 (dd, J=1.6, 8.7 Hz, 1H), 7.93 (d, J=8.3 Hz, 1H), 7.44 (dd, J=4.2, 8.7 Hz, 1H), 6.88 (d, J=8.3 Hz, 1H), 6.08-5.84 (m, 1H), 3.89 (tt, J=4.6, 9.4 Hz, 1H), 3.79-3.60 (m, 5H), 3.17-2.97 (m, 5H), 2.12-1.46 (m, 6H).
Example 61B: MS: calc'd 414 (MH+), measured 414 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.81 (dd, J=1.5, 4.2 Hz, 1H), 8.64 (dd, J=1.6, 8.7 Hz, 1H), 7.93 (d, J=8.3 Hz, 1H), 7.44 (dd, J=4.2, 8.7 Hz, 1H), 6.88 (d, J=8.3 Hz, 1H), 6.08-5.84 (m, 1H), 3.89 (tt, J=4.6, 9.4 Hz, 1H), 3.79-3.60 (m, 5H), 3.17-2.97 (m, 5H), 2.12-1.46 (m, 6H).
The title compound was prepared in analogy to the preparation of Example 1 by using methyl (3S,4S)-4-methylpyrrolidine-3-carboxylate hydrochloride (Pharmablock, PBXA3220-1) and 5-bromo-8-chloro-quinoline instead of methyl (3R,4R)-4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (compound 1b) and bromoquinoxaline-5-carbonitrile (compound 1a). Example 62 (35 mg) was obtained as a yellow solid. MS: calc'd 387 (MH+), measured 387 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.75 (dd, J=1.5, 4.2 Hz, 1H), 8.63 (dd, J=1.6, 8.7 Hz, 1H), 7.60 (d, J=8.3 Hz, 1H), 7.42 (dd, J=4.3, 8.7 Hz, 1H), 6.85 (d, J=8.4 Hz, 1H), 3.74 (dd, J=8.1, 9.2 Hz, 1H), 3.66-3.57 (m, 1H), 3.45-3.28 (m, 2H), 3.18-3.09 (m, 1H), 2.91-2.74 (m, 2H), 2.62-2.47 (m, 2H), 2.23-2.19 (m, 3H), 2.14-1.99 (m, 2H), 1.89-1.75 (m, 2H), 1.54-1.38 (m, 2H), 1.10 (d, J=6.4 Hz, 3H).
The title compound was prepared in analogy to the preparation of Example 6 by using 5-bromoquinoline-8-carbonitrile and (4-methylmorpholin-2-yl)methanamine instead of 5-bromo-8-(trifluoromethyl)quinoxaline (Intermediate A) and tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 63 (14 mg) was obtained as a yellow solid. MS: calc'd 406 (MH+), measured 406 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.88 (dd, J=1.3, 4.2 Hz, 1H), 8.83 (d, J=8.7 Hz, 1H), 7.99 (d, J=8.4 Hz, 1H), 7.49 (dd, J=4.3, 8.7 Hz, 1H), 6.84 (d, J=8.6 Hz, 1H), 4.18 (ddd, J=2.9, 7.0, 10.0 Hz, 1H), 4.05-3.94 (m, 3H), 3.75-3.62 (m, 2H), 3.48-3.36 (m, 2H), 3.30-3.17 (m, 1H), 3.13-2.97 (m, 2H), 2.83 (dt, J=3.7, 6.6 Hz, 1H), 2.65-2.50 (m, 1H), 2.58 (s, 3H), 2.32 (d, J=7.9 Hz, 1H), 0.96-0.75 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 1 by using methyl-pyrrolidine-3-carboxylate and 5-bromoquinoline-8-carbonitrile instead of methyl (3R,4R)-4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt (compound 1b) and bromoquinoxaline-5-carbonitrile (compound 1a). Example 64 (3 mg) was obtained as a yellow solid. MS: calc'd 364 (MH+), measured 364 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.81-8.62 (m, 2H), 7.88 (d, J=8.3 Hz, 1H), 7.43-7.33 (m, 1H), 6.75 (d, J=8.4 Hz, 1H), 3.74-3.45 (m, 5H), 2.86 (t, J=4.2 Hz, 3H), 2.29-1.81 (m, 3H), 1.34-0.98 (m, 8H).
The title compound was prepared in analogy to the preparation of Example 6 by using 5-bromoquinoline-8-carbonitrile and tert-butyl N-(azetidin-3-yl)carbamate instead of 5-bromo-8-(trifluoromethyl)quinoxaline (Intermediate A) and tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 65 (13 mg) was obtained as a yellow solid. MS: calc'd 348 (MH+), measured 348 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.87 (d, J=4.2 Hz, 1H), 8.80 (d, J=8.8 Hz, 1H), 7.97 (d, J=8.4 Hz, 1H), 7.53-7.44 (m, 1H), 6.83 (d, J=8.6 Hz, 1H), 4.55-4.36 (m, 1H), 4.29-4.08 (m, 2H), 4.06-3.84 (m, 4H), 3.74 (dd, J=5.3, 10.8 Hz, 1H), 3.48 (dd, J=3.9, 9.5 Hz, 1H), 3.05-2.95 (m, 1H), 0.95-0.74 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 6 by using methyl (2R,4R)-4-aminopyrrolidine-2-carboxylate (Pharmablock, PB05111,CAS: 1217474-04-8) instead of tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 66 (16 mg) was obtained as a yellow solid. MS: calc'd 464 (MH+), measured 464 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.69 (d, J=1.71 Hz, 1H), 8.63 (d, J=1.34 Hz, 1H), 7.81 (d, J=8.68 Hz, 1H), 6.62 (d, J=8.68 Hz, 1H), 4.37-4.59 (m, 1H), 4.20-4.35 (m, 2H), 4.09-4.19 (m, 1H), 3.94-4.03 (m, 1H), 3.72-3.81 (m, 3H), 3.46-3.60 (m, 2H), 3.11-3.19 (m, 1H), 2.36-2.72 (m, 2H), 1.99-2.35 (m, 1H), 0.61-0.80 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 30 by using trans-ethyl-4-difluoromethyl-pyrrolidine-3-carboxylate hydrochloride (Pharmablock, PBXA3200-1) and (3S,4R)-tert-butyl 3-amino-4-fluoropyrrolidine-1-carboxylate instead of methyl 4-(trifluoromethyl)pyrrolidine-3-carboxylate HCl salt and tert-butyl 4-aminoazepane-1-carboxylate (compound 30d). Example 67 (11 mg) was obtained as a yellow solid. MS: calc'd 404 (MH+), measured 404 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.82 (dd, J=1.6, 4.3 Hz, 1H), 8.64 (dd, J=1.6, 8.8 Hz, 1H), 7.94 (d, J=8.3 Hz, 1H), 7.45 (dd, J=4.3, 8.7 Hz, 1H), 6.90 (d, J=8.4 Hz, 1H), 6.08-5.86 (m, 1H), 5.28-5.10 (m, 1H), 4.70-4.51 (m, 1H), 3.83-3.74 (m, 1H), 3.69-3.53 (m, 3H), 3.54-3.42 (m, 1H), 3.31-3.24 (m, 3H), 3.18-3.01 (m, 2H).
The title compound was prepared in analogy to the preparation of Example 6 by using 8-bromoquinoxaline-5-carbonitrile and tert-butyl (2S)-2-(aminomethyl)morpholine-4-carboxylate (CAS: 879403-42-6, Pharmablock, PBN 20121306) instead of 5-bromo-8-(trifluoromethyl)quinoxaline (Intermediate A) and tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 70 (79 mg) was obtained as a yellow solid. MS: calc'd 393 (MH+), measured 393 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.72 (d, J=1.83 Hz, 1H), 8.64 (d, J=1.71 Hz, 1H), 7.87 (d, J=8.68 Hz, 1H), 6.65 (d, J=8.80 Hz, 1H), 4.15-4.31 (m, 2H), 3.96-4.10 (m, 2H), 3.61-3.74 (m, 3H), 3.29-3.39 (m, 1H), 3.24-3.28 (m, 1H), 3.10-3.17 (m, 2H), 2.95-3.07 (m, 1H), 2.77 (dt, J=5.20, 12.07 Hz, 1H), 2.63-2.72 (m, 1H), 0.62-0.86 (m, 4H).
SFC-HPLC (40% CO2/0.5% NH3 in methanol as eluent on Daicel AD-H Column) separation gave two single isomers Example 70A (14 mg) and Example 70B (14 mg).
Example 70A: MS: calc'd 393 (MH+), measured 393 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.84 (d, J=1.71 Hz, 1H), 8.75 (d, J=1.71 Hz, 1H), 7.98 (d, J=8.68 Hz, 1H), 6.76 (d, J=8.68 Hz, 1H), 4.27-4.42 (m, 2H), 4.07-4.20 (m, 2H), 3.72-3.84 (m, 3H), 3.40-3.52 (m, 1H), 3.30 (m, 1H), 3.21-3.29 (m, 2H), 3.10-3.19 (m, 1H), 2.85-2.96 (m, 1H), 2.81 (dd, J=3.55, 6.85 Hz, 1H), 0.74-0.93 (m, 4H).
Example 70B: MS: calc'd 393 (MH+), measured 393 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.82 (d, J=1.83 Hz, 1H), 8.74 (d, J=1.71 Hz, 1H), 7.97 (d, J=8.68 Hz, 1H), 6.74 (d, J=8.80 Hz, 1H), 4.26-4.40 (m, 2H), 4.18 (d, J=11.25 Hz, 1H), 4.12 (dd, J=3.48, 13.02 Hz, 1H), 3.71-3.87 (m, 3H), 3.36-3.43 (m, 2H), 3.25-3.30 (m, 2H), 3.08-3.17 (m, 1H), 2.88 (dd, J=11.49, 12.59 Hz, 1H), 2.80 (dd, J=3.30, 6.85 Hz, 1H), 0.90-0.97 (m, 1H), 0.78-0.90 (m, 3H).
The title compound was prepared in analogy to the preparation of Example 6 by using 8-bromoquinoxaline-5-carbonitrile and tert-butyl (2R)-2-(aminomethyl)morpholine-4-carboxylate (CAS: 1174913-80-4, Pharmablock, PBN 20121305) instead of 5-bromo-8-(trifluoromethyl)quinoxaline (Intermediate A) and tert-butyl 7-amino-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (compound 6d). Example 71 (84 mg) was obtained as a yellow solid. MS: calc'd 393 (MH+), measured 393 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.72 (d, J=1.71 Hz, 1H), 8.64 (d, J=1.71 Hz, 1H), 7.87 (d, J=8.68 Hz, 1H), 6.65 (d, J=8.68 Hz, 1H), 4.15-4.32 (m, 2H), 3.95-4.12 (m, 2H), 3.59-3.73 (m, 3H), 3.30-3.41 (m, 1H), 3.24-3.29 (m, 1H), 3.07-3.16 (m, 2H), 2.95-3.07 (m, 1H), 2.77 (dt, J=5.07, 12.07 Hz, 1H), 2.63-2.72 (m, 1H), 0.65-0.84 (m, 4H).
SFC-HPLC (40% CO2/0.5% NH3 in methanol as eluent on Daicel AD-H Column) separation gave two single isomers Example 71A (14 mg) and Example 71B (18 mg).
Example 71A: MS: calc'd 393 (MH+), measured 393 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.84 (d, J=1.71 Hz, 1H), 8.76 (d, J=1.71 Hz, 1H), 7.96-8.01 (m, 1H), 6.77 (d, J=8.80 Hz, 1H), 4.35-4.43 (m, 1H), 4.27-4.35 (m, 1H), 4.19 (d, J=11.25 Hz, 1H), 4.12 (dd, J=3.36, 13.14 Hz, 1H), 3.72-3.85 (m, 3H), 3.36-3.40 (m, 2H), 3.27 (d, J=10.27 Hz, 2H), 3.08-3.17 (m, 1H), 2.84-2.93 (m, 1H), 2.79 (dd, J=3.24, 6.79 Hz, 1H), 0.87-0.96 (m, 1H), 0.77-0.87 (m, 3H).
Example 71B: MS: calc'd 393 (MH+), measured 393 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.82 (d, J=1.71 Hz, 1H), 8.74 (d, J=1.83 Hz, 1H), 7.97 (d, J=8.68 Hz, 1H), 6.74 (d, J=8.80 Hz, 1H), 4.27-4.42 (m, 2H), 4.08-4.20 (m, 2H), 3.72-3.86 (m, 3H), 3.40-3.52 (m, 1H), 3.22-3.30 (m, 3H), 3.09-3.20 (m, 1H), 2.90 (s, 1H), 2.82 (d, J=3.30 Hz, 1H), 0.88 (s, 1H), 0.76-0.86 (m, 3H).
The following tests were carried out in order to determine the activity of the compounds of formula (I) in HEK293-Blue-hTLR-7/8/9 cells assay.
HEK293-Blue-hTLR-7 Cells Assay:
A stable HEK293-Blue-hTLR-7 cell line was purchased from InvivoGen (Cat. #: hkb-htlr7, San Diego, Calif., USA). These cells were originally designed for studying the stimulation of human TLR7 by monitoring the activation of NF-κB. A SEAP (secreted embryonic alkaline phosphatase) reporter gene was placed under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. The SEAP was induced by activating NF-κB and AP-1 via stimulating HEK-Blue hTLR7 cells with TLR7 ligands. Therefore the reporter expression was declined by TLR7 antagonist under the stimulation of a ligand, such as R848 (Resiquimod), for incubation of 20 hrs. The cell culture supernatant SEAP reporter activity was determined using QUANTI-Blue™ kit (Cat. #: rep-qb1, Invivogen, San Diego, Ca, USA) at a wavelength of 640 nm, a detection medium that turns purple or blue in the presence of alkaline phosphatase.
HEK293-Blue-hTLR7 cells were incubated at a density of 250,000˜450,000 cells/mL in a volume of 170 μL in a 96-well plate in Dulbecco's Modified Eagle's medium (DMEM) containing 4.5 g/L glucose, 50 U/mL penicillin, 50 mg/mL streptomycin, 100 mg/mL Normocin, 2 mM L-glutamine, 10% (v/v) heat-inactivated fetal bovine serum with addition of 20 sL test compound in a serial dilution in the presence of final DMSO at 1% and 10 μL of 20 uM R848 in above DMEM, perform incubation under 37° C. in a CO2 incubator for 20 hrs. Then 20 μL of the supernatant from each well was incubated with 180 sL Quanti-blue substrate solution at 37° C. for 2 hrs and the absorbance was read at 620-655 nm using a spectrophotometer. The signalling pathway that TLR7 activation leads to downstream NF-κB activation has been widely accepted, and therefore similar reporter assay was modified for evaluating TLR7 antagonist.
HEK293-Blue-hTLR-8 Cells Assay:
A stable HEK293-Blue-hTLR-8 cell line was purchased from InvivoGen (Cat. #: hkb-htlr8, San Diego, Calif., USA). These cells were originally designed for studying the stimulation of human TLR8 by monitoring the activation of NF-κB. A SEAP (secreted embryonic alkaline phosphatase) reporter gene was placed under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. The SEAP was induced by activating NF-κB and AP-1 via stimulating HEK-Blue hTLR8 cells with TLR8 ligands. Therefore the reporter expression was declined by TLR8 antagonist under the stimulation of a ligand, such as R848, for incubation of 20 hrs. The cell culture supernatant SEAP reporter activity was determined using QUANTI-Blue™ kit (Cat. #: rep-qb1, Invivogen, San Diego, Ca, USA) at a wavelength of 640 nm, a detection medium that turns purple or blue in the presence of alkaline phosphatase.
HEK293-Blue-hTLR8 cells were incubated at a density of 250,000-450,000 cells/mL in a volume of 170 μL in a 96-well plate in Dulbecco's Modified Eagle's medium (DMEM) containing 4.5 g/L glucose, 50 U/mL penicillin, 50 mg/mL streptomycin, 100 mg/mL Normocin, 2 mM L-glutamine, 10% (v/v) heat-inactivated fetal bovine serum with addition of 20 μL test compound in a serial dilution in the presence of final DMSO at 1% and 10 sL of 60 uM R848 in above DMEM, perform incubation under 37° C. in a CO2 incubator for 20 hrs. Then 20 μL of the supernatant from each well was incubated with 180 sL Quanti-blue substrate solution at 37° C. for 2 hrs and the absorbance was read at 620˜655 nm using a spectrophotometer. The signalling pathway that TLR8 activation leads to downstream NF-κB activation has been widely accepted, and therefore similar reporter assay was modified for evaluating TLR8 antagonist.
HEK293-Blue-hTLR-9 Cells Assay:
A stable HEK293-Blue-hTLR-9 cell line was purchased from InvivoGen (Cat. #: hkb-htlh9, San Diego, Calif., USA). These cells were originally designed for studying the stimulation of human TLR9 by monitoring the activation of NF-κB. A SEAP (secreted embryonic alkaline phosphatase) reporter gene was placed under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. The SEAP was induced by activating NF-κB and AP-1 via stimulating HEK-Blue hTLR9 cells with TLR9 ligands. Therefore the reporter expression was declined by TLR9 antagonist under the stimulation of a ligand, such as ODN2006 (Cat. #: tlrl-2006-1, Invivogen, San Diego, Calif., USA), for incubation of 20 hrs. The cell culture supernatant SEAP reporter activity was determined using QUANTI-Blue™ kit (Cat. #: rep-qb1, Invivogen, San Diego, Calif., USA) at a wavelength of 640 nm, a detection medium that turns purple or blue in the presence of alkaline phosphatase.
HEK293-Blue-hTLR9 cells were incubated at a density of 250,000-450,000 cells/mL in a volume of 170 μL in a 96-well plate in Dulbecco's Modified Eagle's medium (DMEM) containing 4.5 g/L glucose, 50 U/mL penicillin, 50 mg/mL streptomycin, 100 mg/mL Normocin, 2 mM L-glutamine, 10% (v/v) heat-inactivated fetal bovine serum with addition of 20 sL test compound in a serial dilution in the presence of final DMSO at 1% and 10 sL of 20 uM ODN2006 in above DMEM, perform incubation under 37° C. in a CO2 incubator for 20 hrs. Then 20 sL of the supernatant from each well was incubated with 180 μL Quanti-blue substrate solution at 37° C. for 2 hrs and the absorbance was read at 620-655 nm using a spectrophotometer. The signalling pathway that TLR9 activation leads to downstream NF-x3B activation has been widely accepted, and therefore similar reporter assay was modified for evaluating TLR9 antagonist.
The compounds of formula (I) have human TLR7 and/or TLR8 inhibitory activities (IC50 value)<0.5 μM, particularly <0.020 μM. Moreover, particular compounds of this invention also have human TLR9 inhibitory activity <10 μM. Activity data of the compounds of the present invention were shown in Table 1.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/073079 | 8/28/2018 | WO | 00 |
