PYRAZOLOPYRIDINE AMINE COMPOUNDS FOR THE TREATMENT OF AUTOIMMUNE DISEASE

The present invention relates to compounds of formula (I), wherein R1, R2, R3, R4 and X are as described herein, and their pharmaceutically acceptable salt, enantiomer or diastereomer thereof, and compositions including the compounds and methods of using the compounds.

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.

FIELD OF THE INVENTION

Autoimmune connective tissue disease (CTD) include prototypical autoimmune syndromes such as Systemic Lupus Erythematosus (SEE), 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. SEE 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 immuno suppressive 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 alLancet2011, 377, 721.). Other biologies, 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. Jimenez-Dalmaroni, M. J. et alAutoimmun Rev.2016, 15, 1. Chen, J. Q., et al.Clinical Reviews in Allergy&Immunology2016, 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.

SUMMARY OF THE INVENTION

The present invention relates to novel compounds of formula (I),

wherein

X is O or CH2;

or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.

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 micro some stability and SDPK profiles, as well as low CYP inhibition.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

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 “C1-6alkyl” groups are methyl, ethyl and n-propyl.

The term “halogen” and “halo” are used interchangeably herein and denote fluoro, chloro, bromo, or iodo.

The term “haloC1-6alkyl” 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 “halopiperidinyl” denotes a piperidinyl group wherein at least one of the hydrogen atoms of the piperidinyl group has been replaced by same or different halogen atoms, particularly fluoro atoms. Examples of halopiperidinyl include fluoropyrrolidinyl and difluoropiperidinyl.

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 “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.

The present invention relates to a compound of formula (I),

wherein

X is O or CH2;

or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.

A further embodiment of present invention is (ii) a compound of formula (I) according to (i), wherein

R1is cyano;
R2is (3,4,4a,5,6,7,8,8a-octahydro-2H-naphthyridinyl;(C1-6alkyl)2amino-3,3a,4,5,6,6a-hexahydro-1H-cyclopenta[c]pyrrolyl;1,2,3,3a,4,6,7,7a-octahydropyrrolo[3,2-c]pyridinyl;1,2,3,3a,5,6,7,7a-octahydropyrrolo[3,2-b]pyridinyl;1,2,3,4,4a,5,7,7a-octahydropyrrolo[3,4-b]pyridinyl;1,3,3a,4,5,6,7,7a-octahydropyrrolo[3,4-c]pyridinyl;2,3,3a,4,6,6a-hexahydro-1H-pyrrolo[3,4-c]pyrrolyl;2,3,4,4a,5,6,7,7a-octahydropyrrolo[3,4-b]pyridinyl;3,3a,4,5,6,6a-hexahydro-2H-pyrrolo[3,4-b]pyrrolyl;aminoazabicyclo[3.2.1]octanyl;aminoazabicyclo[3.3.1]nonanyl;aminoazaspiro[3.3]heptanyl;aminooxaazabicyclo[3.3.1]nonanyl;C1-6alkyloxadiazaspiro[4.5]decanyl;C1-6alkylpiperidinylamino;diazabicyclo[2.2.2]octanyl;diazabicyclo[3.2.1]octanyl;diazabicyclo[4.2.0]octanyl;diazaspiro[2.5]octanyl;diazaspiro[3.3]heptanyl;diazaspiro[3.4]octanyl;diazaspiro[3.5]nonanyl;diazaspiro[3.6]decanyl;diazaspiro[4.4]nonanyl;diazaspiro[4.5]decanyl;diazaspiro[5.5]undecanyl;oxadiazabicyclo[3.3.1]nonanyl;oxadiazaspiro[5.5]undecanyl;oxodiazaspiro[4.4]nonanyl;piperazinyl, said piperazinyl being unsubstituted or substituted by one, two or three substituents independently selected from C1-6alkyl, phenyl, phenylC1-6alkyl, aminoC1-6alkylcarbonyl and piperidinyl;piperidinyl, said piperidinyl being unsubstituted or substituted by one, two or three substituents independently selected from amino, aminoC1-6alkyl, azepanyl, C1-6alkyl, C1-6alkylcarbonylamino, C1-6alkylpiperazinyl, carbamoyl, halogen, phenyl, piperazinyl, piperidinyl and pyrrolidinyl; orpyrrolidinyl, said pyrrolidinyl being unsubstituted or substituted by one, two or three substituents independently selected from amino and halogen;
R3is C1-6alkyl;
R4is H or halogen;

X is O;

or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.

A further embodiment of present invention is (iii) a compound of formula (I) according to (ii), wherein

R1is cyano;
R2is (3,4,4a,5,6,7,8,8a-octahydro-2H-naphthyridinyl;2,2-dimethylpropanoylamino-3,3a,4,5,6,6a-hexahydro-1H-cyclopenta[c]pyrrolyl;1,2,3,3a,4,6,7,7a-octahydropyrrolo[3,2-c]pyridinyl;1,2,3,3a,5,6,7,7a-octahydropyrrolo[3,2-b]pyridinyl;1,2,3,4,4a,5,7,7a-octahydropyrrolo[3,4-b]pyridinyl;1,3,3a,4,5,6,7,7a-octahydropyrrolo[3,4-c]pyridinyl;2,3,3a,4,6,6a-hexahydro-1H-pyrrolo[3,4-c]pyrrolyl;2,3,4,4a,5,6,7,7a-octahydropyrrolo[3,4-b]pyridinyl;3,3a,4,5,6,6a-hexahydro-2H-pyrrolo[3,4-b]pyrrolyl;aminoazabicyclo[3.2.1]octanyl;aminoazabicyclo[3.3.1]nonanyl;aminoazaspiro[3.3]heptanyl;aminooxaazabicyclo[3.3.1]nonanyl;methyloxadiazaspiro[4.5]decanyl;methylpiperidinylamino;diazabicyclo[2.2.2]octanyl;diazabicyclo[3.2.1]octanyl;diazabicyclo[4.2.0]octanyl;diazaspiro[2.5]octanyl;diazaspiro[3.3]heptanyl;diazaspiro[3.4]octanyl;diazaspiro[3.5]nonanyl;diazaspiro[3.6]decanyl;diazaspiro[4.4]nonanyl;diazaspiro[4.5]decanyl;diazaspiro[5.5]undecanyl;oxadiazabicyclo[3.3.1]nonanyl;oxadiazaspiro[5.5]undecanyl;oxodiazaspiro[4.4]nonanyl;piperazinyl, said piperazinyl being unsubstituted or substituted by one, two or three substituents independently selected from methyl, phenyl, benzyl, aminoacetyl and piperidinyl;piperidinyl, said piperidinyl being unsubstituted or substituted by one, two or three substituents independently selected from amino, aminomethyl, aminoethyl, azepanyl, methyl, 2,2-dimethylpropanoylamino, methylpiperazinyl, carbamoyl, fluoro, phenyl, piperazinyl, piperidinyl and pyrrolidinyl; orpyrrolidinyl, said pyrrolidinyl being unsubstituted or substituted by one, two or three substituents independently selected from amino and fluoro;
R3is methyl;
R4is H or fluoro;

X is O;

or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.

A further embodiment of present invention is (v) a compound of formula (I) according to (iv), wherein R2is1,2,3,3a,4,6,7,7a-octahydropyrrolo[3,2-c]pyridinyl;2,3,3a,4,6,6a-hexahydro-1H-pyrrolo[3,4-c]pyrrolyl;aminoazabicyclo[3.2.1]octanyl;aminooxaazabicyclo[3.3.1]nonanyl;diazabicyclo[2.2.2]octanyl;diazabicyclo[3.2.1]octanyl;diazabicyclo[4.2.0]octanyl;diazaspiro[4.5]decanyl;oxadiazabicyclo[3.3.1]nonanyl;oxodiazaspiro[4.4]nonanyl;piperazinyl, said piperazinyl being unsubstituted or substituted by C1-6alkyl or phenylC1-6alkyl; orpiperidinyl, said piperidinyl being unsubstituted or substituted by one, two or three substituents independently selected from amino, C1-6alkyl, C1-6alkylpiperazinyl and halogen.

A further embodiment of present invention is (viii) a compound of formula (I) according to (v) or (vi), wherein R2is C1-6alkylpiperazinylpiperidinyl; 1,2,3,3a,4,6,7,7a-octahydropyrrolo[3,2-c]pyridinyl; oxadiazaspiro[5.5]undecanyl; 2,3,3a,4,6,6a-hexahydro-1H-pyrrolo[3,4-c]pyrrolyl; diazaspiro[4.5]decanyl; diazabicyclo[4.2.0]octanyl; aminopiperidinyl; amino(C1-6alkyl)piperidinyl.

A further embodiment of present invention is (x) a compound of formula (I) according to (viii), wherein R2is diazaspiro[4.5]decanyl.

A further embodiment of present invention is (xi) a compound of formula (I) according to (x), wherein R2is 2,9-diazaspiro[4.5]decan-2-yl.

Another embodiment of present invention is that (xii) particular compounds of formula (I) are the following:4-[(2R,6S)-2-methyl-6-[[4-(4-methylpiperazin-1-yl)-1-piperidyl]methyl]morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;N-[1-[[(2S,6R)-4-(7-cyanopyrazolo[1,5-a]pyridin-4-yl)-6-methyl-morpholin-2-yl]methyl]-4-piperidyl]-2,2-dimethyl-propanamide;4-[(2S,6R)-2-(3,9-diazaspiro[5.5]undecan-3-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-[[4-(azepan-1-yl)-1-piperidyl]methyl]-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2R,6S)-2-methyl-6-(piperazin-1-ylmethyl)morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(2,7-diazaspiro[4.4]nonan-2-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2R,6S)-2-methyl-6-[[(1-methyl-4-piperidyl)amino]methyl]morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(2,6-diazaspiro[3.3]heptan-2-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(2,7-diazaspiro[3.4]octan-2-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(2,8-diazaspiro[3.5]nonan-2-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-[(6-amino-2-azaspiro[3.3]heptan-2-yl)methyl]-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-[[4-(aminomethyl)-1-piperidyl]methyl]-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-[[4-(2-aminoethyl)-1-piperidyl]methyl]-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-[[4-(2-aminoacetyl)piperazin-1-yl]methyl]-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2R,6S)-2-methyl-6-[(4-methylpiperazin-1-yl)methyl]morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2R,6S)-2-methyl-6-[[4-(1-piperidyl)-1-piperidyl]methyl]morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-[(4-amino-1-piperidyl)methyl]-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(2,8-diazaspiro[4.5]decan-2-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(2,8-diazaspiro[4.5]decan-8-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2R,6R)-2-methyl-6-[(3-oxo-2,7-diazaspiro[4.4]nonan-2-yl)methyl]morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2R,6R)-2-methyl-6-[(1-oxo-2,7-diazaspiro[4.4]nonan-2-yl)methyl]morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(2,3,3a,4,6,6a-hexahydro-1H-pyrrolo[3,4-c]pyrrol-5-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(2,8-diazaspiro[5.5]undecan-2-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(2,7-diazaspiro[3.5]nonan-2-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(2,9-diazaspiro[4.5]decan-2-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(2,7-diazaspiro[3.5]nonan-7-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(2,9-diazaspiro[5.5]undecan-9-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2R,6S)-2-methyl-6-[(3-methylpiperazin-1-yl)methyl]morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-[(4-amino-4-methyl-1-piperidyl)methyl]-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(2,5-diazabicyclo[2.2.2]octan-2-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(3,8-diazabicyclo[3.2.1]octan-3-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(2,9-diazaspiro[5.5]undecan-2-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(2,6-diazaspiro[3.5]nonan-6-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-[(4-amino-3,3-difluoro-1-piperidyl)methyl]-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-[(4-amino-3,3-difluoro-pyrrolidin-1-yl)methyl]-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-[(8-amino-3-azabicyclo[3.2.1]octan-3-yl)methyl]-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-[(3-amino-8-azabicyclo[3.2.1]octan-8-yl)methyl]-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2R,6S)-2-methyl-6-[[(3R)-3-phenylpiperazin-1-yl]methyl]morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2R,6S)-2-methyl-6-[(4-piperazin-1-yl-1-piperidyl)methyl]morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(4,7-diazaspiro[2.5]octan-7-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(1,2,3,3a,4,6,7,7a-octahydropyrrolo[3,2-c]pyridin-5-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(2,6-diazaspiro[4.5]decan-2-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(3,8-diazabicyclo[3.2.1]octan-8-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(2,7-diazaspiro[4.5]decan-7-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(1,2,3,4,4a,5,7,7a-octahydropyrrolo[3,4-b]pyridin-6-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(2,8-diazaspiro[3.6]decan-2-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(1,9-diazaspiro[5.5]undecan-9-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(1,7-diazaspiro[3.5]nonan-7-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(1,3,3a,4,5,6,7,7a-octahydropyrrolo[3,4-c]pyridin-2-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2R,6S)-2-methyl-6-[(2-methylpiperazin-1-yl)methyl]morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-[(4-amino-2-methyl-1-piperidyl)methyl]-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(3,7-diazabicyclo[4.2.0]octan-7-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(1,8-diazaspiro[4.5]decan-8-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2R,6S)-2-methyl-6-[(4-pyrrolidin-1-yl-1-piperidyl)methyl]morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(1,6-diazaspiro[3.3]heptan-1-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(3,8-diazabicyclo[4.2.0]octan-8-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2R,6S)-2-methyl-6-(3-oxa-7,9-diazabicyclo[3.3.1]nonan-9-ylmethyl)morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-[(7-amino-3-oxa-9-azabicyclo[3.3.1]nonan-9-yl)methyl]-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-[(2-benzylpiperazin-1-yl)methyl]-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2R,6S)-2-methyl-6-(l-oxa-4,9-diazaspiro[5.5]undecan-9-ylmethyl)morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(2,3,4,4a,5,6,7,7a-octahydropyrrolo[3,4-b]pyridin-1-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2R,6S)-2-methyl-6-[[cis-(3aR,6aR)-3,3a,4,5,6,6a-hexahydro-2H-pyrrolo[3,4-b]pyrrol-1-yl]methyl]morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2R,6S)-2-methyl-6-[[cis-(3aS,7aS)-1,2,3,3a,5,6,7,7a-octahydropyrrolo[3,2-b]pyridin-4-yl]methyl]morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(3,4,4a,5,6,7,8,8a-octahydro-2H-1,5-naphthyridin-1-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;Endo-4-[(2S,6R)-2-[(3-amino-9-azabicyclo[3.3.1]nonan-9-yl)methyl]-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;Exo-4-[(2S,6R)-2-[(3-amino-9-azabicyclo[3.3.1]nonan-9-yl)methyl]-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(2,9-diazaspiro[5.5]undecan-2-ylmethyl)-6-methyl-morpholin-4-yl]-3-fluoro-pyrazolo[1,5-a]pyridine-7-carbonitrile;3-fluoro-4-[(2S,6R)-2-(2,7-diazaspiro[4.5]decan-7-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;3-fluoro-4-[(2S,6R)-2-(2,9-diazaspiro[4.5]decan-2-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-(3,7-diazabicyclo[4.2.0]octan-3-ylmethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2R,6S)-2-methyl-6-(1-oxa-4,9-diazaspiro[5.5]undecan-4-ylmethyl)morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;1-[[(2S,6R)-4-(7-cyanopyrazolo[1,5-a]pyridin-4-yl)-6-methyl-morpholin-2-yl]methyl]piperidine-3-carboxamide;4-[(2S,6R)-2-[(4-amino-4-phenyl-1-piperidyl)methyl]-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-[[(3R,4S)-4-amino-3-methyl-1-piperidyl]methyl]-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2S,6R)-2-[[4-(dimethylamino)-3,3a,4,5,6,6a-hexahydro-1H-cyclopenta[c]pyrrol-2-yl]methyl]-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;4-[(2R,6S)-2-methyl-6-[(9-methyl-6-oxa-2,9-diazaspiro[4.5]decan-2-yl)methyl]morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile; and4-[(2S,6R)-2-[[2-benzyl-4-(4-piperidyl)piperazin-1-yl]methyl]-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile;

or a pharmaceutically acceptable salt, enantiomer or diastereomer thereof.

Synthesis

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, R1to R4are 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) or (II) is shown below.

wherein R is Ms, Ts or Tf. R5and R6are independently selected from H and heterocyclyl, or R5and R6together with the nitrogen they are attached to form a heterocyclyl. Y is halogen.

The coupling of compound of formula (III) with halide (IV) can be achieved by direct coupling in the presence of a base, such as DIPEA and 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). Subsequently the hydroxy group of compound of formula (V) is converted to a leaving group, such as —OTf, —OTs, or —OMs, under basic condition, such as DIPEA, TEA, K2CO3or 2,6-dimethylpyridine, with Tf2O, TsCl or MsCl. Compound of formula (VI) was further coupled with amine (VII) in the presence of base, such as K2CO3, DIPEA or Cs2CO3, to afford 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 any of the following steps:

a) the reaction of compound of formula (VI),

with amine (VII) in the presence of a base;

In step a) and d) the base can be for example K2CO3, DIPEA or Cs2CO3.

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.

Indications and Methods of Treatment

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.

EXAMPLES

Abbreviations

General Experimental Conditions

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):

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.

PREPARATIVE EXAMPLES

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:

The title compound was synthesized according to the following scheme:

To a solution of tert-butyl (2R,6R)-2-(benzyloxymethyl)-6-methyl-morpholine-4-carboxylate (Reference: US 20150105370 A1) (22.0 g, 68.4 mmol) in EtOH (500 mL) was added Pd/C (7.28 g, 10% wet) and stirred for 48 hrs at 30° C. under H2 atmosphere. The solution was then filtered, and the filtrate was concentrated to give an intermediate (15 g) as a colorless oil. To a solution of this intermediate (231 mg, 1.0 mmol) in DCM (2 mL) was added TFA (1 mL) at 0° C. The reaction mixture was stirred at rt for 2 hrs, then concentrated to give a crude compound 1a (250 mg) which was directly used in next step. MS: calc'd 132 (MH+), measured 132 (MHI).

To a solution of 4-[(2R,6R)-2-(hydroxymethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile (compound 1c, 190 mg, 0.70 mmol) and 2,6-dimethylpyridine (150 mg, 1.4 mmol) in DCM (4 mL) was added trifluoromethanesulfonic anhydride (295 mg, 1.05 mmol) dropwise at 0° C. The mixture was stirred at 0° C. for 1 h. The mixture was then diluted with DCM, washed with sat. NH4Cl and brine, dried over Na2SO4, and concentrated to give a crude product which was purified by column chromatography to give compound 1d (166 mg) as a white solid. MS: calc'd 405 (MH+), measured 405 (MH+).

To a solution of Boc-Ala-OH (398 mg, 2.3 mmol), benzyl piperazine-1-carboxylate (500 mg, 2.3 mmol) and DIPEA (587 mg, 0.79 mL, 4.5 mmol) in DCM (10 mL) was added HATU (1.29 g, 3.4 mmol). The reaction mixture was stirred at rt overnight, then diluted with DCM, washed with sat. NH4Cl and brine, dried over Na2SO4, and concentrated to give an oil which was purified by column chromatography to give a product (900 mg) as a colorless oil. To the solution of above product (200 mg, 0.53 mmol) in MeOH (20 mL) was added Pd(OH)2(20% on carbon, wet, 30 mg). The reaction mixture was charged with H2balloon and stirred at rt for 2 hrs, then filtered through celite, and the filtrated was concentrated to give compound 14a (100 mg) as an oil. MS: calc'd 244 (MH+), measured 244 (MH+).

Step 2: Preparation of 4-[(2S,6R)-2-[[4-(2-aminoacetyl)piperazin-1-yl]methyl]-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile

The title compound was prepared in analogy to the preparation of Example 3 by using tert-butyl (3S)-3-methylpiperazine-1-carboxylate instead of tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate. Example 50A (8 mg) and Example 50B (7 mg) were obtained through prep-HPLC as light yellow powders.

To a solution of 4-chloropyrazolo[1,5-a]pyridine-7-carbonitrile (compound 1b, 600 mg, 3.38 mmol) in ACN (50 mL) was added Selectfluor (2.39 g, 6.76 mmol). After the reaction mixture was stirred at rt for 24 hrs, LCMS indicated the formation of product. The reaction mixture was then concentrated to remove most ACN, diluted with water (30 mL), extracted with DCM. The organic layer was washed with sat. NH4Cl and brine, dried over Na2SO4, and concentrated to give a crude product which was purified by column chromatography to give compound 67a (419 mg) as light yellow powder. MS: calc'd 196 (MH+), measured 196 (MH+).

To a solution of 3-fluoro-4-[(2R,6R)-2-(hydroxymethyl)-6-methyl-morpholin-4-yl]pyrazolo[1,5-a]pyridine-7-carbonitrile (compound 67b, 325 mg, 1.12 mmol) in DCM (3 mL) was added 2,6-dimethylpyridine (240 mg, 258 μl, 2.24 mmol) and Tf2O (474 mg, 284 μl, 1.68 mmol) at rt. After the reaction mixture was stirred at rt for 1.5 hrs, LCMS indicated the formation of product. The mixture was then diluted with DCM, washed with Sat. NH4Cl and brine. The organic layer was dried over Na2SO4and concentrated to give the crude product which was purified by column chromatography to give compound 67c (180 mg) as a yellow solid. MS: calc'd 423 (MH+), measured 423 (MH+).

To a solution of formaldehyde (37% aqueous solution, 1.09 g, 1 mL, 13.4 mmol) and rac-tert-butyl (3aR,4R,6aS)-4-amino-3,3a,4,5,6,6a-hexahydro-1H-cyclopenta[c]pyrrole-2-carboxylate (CAS: 1251012-14-2, Wuxi AppTech) (350 mg, 1.55 mmol) in MeOH (20 mL) was added NaBH(OAc)3(1.97 g, 9.28 mmol). The reaction mixture was then stirred at 50° C. for 4 hrs. The reaction mixture was concentrated, poured into sat. NaHCO3, and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4and concentrated to give a crude product (388 mg). To a solution of this crude product (20 mg, 0.080 mmol) in DCM (2 mL) was added TFA (1 mL). The reaction mixture was stirred at rt for 1 h, then concentrated to give a crude compound 75a (24 mg) which was directly used in next step. MS: calc'd 155 (MH+), measured 155 (MH+).

To a solution of tert-butyl 3-benzylpiperazine-1-carboxylate (300 mg, 1.09 mmol) in DCM (10 mL) was added TEA (330 mg, 0.45 mL, 3.26 mmol) and CbzCl (278 mg, 0.23 mL, 1.63 mmol). After the reaction mixture was then stirred at rt for 2 hrs. The mixture was diluted with water, and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4and concentrated to give crude product which was purified by column chromatography to give a product (368 mg) as an oil. The oil was dissolved in DCM (2 ML), then TFA (124 mg, 84 μL, 1.09 mmol) was added. The reaction mixture was stirred at rt overnight, then concentrated to give a crude compound 77a (385 mg) as an oil which is directly used in next step. MS: calc'd 311 (MH+), measured 311 (MH+).

To a solution of benzyl 2-benzylpiperazine-1-carboxylate (compound 77a, 84 mg, 0.27 mmol) and tert-butyl 4-oxopiperidine-1-carboxylate (65 mg, 0.32 mmol) in ACN/MeOH (5 mL, v/v=4/1). The reaction mixture was stirred at 80° C. for 24 hrs, another portion of tert-butyl 4-oxopiperidine-1-carboxylate (65 mg, 0.32 mmol) and NaBH(OAc)3(114 mg, 0.54 mmol) were added. The reaction mixture was stirred at 80° C. for 2 d, then cooled to rt, diluted with water and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4and concentrated to give a crude product which was purified by column chromatography to give a pure product (264 mg) as an oil. The oil was then dissolved in MeOH (4 mL), Pd(OH)2/C (26 mg, 20% wet) was added. The reaction mixture was charged with H2balloon and stirred at rt for 2 hrs, then filtered through celite. The filtrate was concentrated to give crude compound 77b (100 mg) as an oil which was directly used in next step. MS: calc'd 360 (MH+), measured 360 (MH+).

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.

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 μL 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 CO2incubator for 20 hrs. Then 20 μL 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 TLR7 activation leads to downstream NF-κB activation has been widely accepted, and therefore similar reporter assay was modified for evaluating TLR7 antagonist.

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 μL of 60 uM R848 in above DMEM, perform incubation under 37° C. in a CO2incubator for 20 hrs. Then 20 μL 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 TLR8 activation leads to downstream NF-κB activation has been widely accepted, and therefore similar reporter assay was modified for evaluating TLR8 antagonist.

A stable HEK293-Blue-hTLR-9 cell line was purchased from InvivoGen (Cat. #: hkb-htlr9, 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. #: tlr1-2006-1, 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 μL test compound in a serial dilution in the presence of final DMSO at 1% and 10 μL of 20 uM ODN2006 in above DMEM, perform incubation under 37° C. in a CO2incubator for 20 hrs. Then 20 μL 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 spectrophotorneter. The signalling pathway that TLR9 activation leads to downstream NF-κB 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 (IC50value)<1 μM, particularly <0.1 μM. Moreover, some compounds also have human TLR9 inhibitory activity <1 μM, particularly <0.3 μM. Activity data of the compounds of the present invention were shown in Table 1.

TABLE 1The activity of the compounds of present inventionin HEK293-Blue-hTLR-7/8/9 cells assaysExampleTLR7 IC50(μM)TLR8 IC50(μM)TLR9 IC50(μM)10.0950.0280.1330.240.620.09040.180.0260.2850.0540.0780.8960.200.140.03170.260.320.32100.0830.130.080110.160.930.097120.240.280.20130.400.740.27140.460.383.0150.0770.1823160.100.0190.52170.0890.0580.18180.200.350.089190.130.250.092200.650.6112210.0980.03916220.100.0690.18230.180.0410.25240.100.610.10250.110.0880.088270.130.190.065280.0440.06514290.0760.0520.22300.0460.103.2310.0530.09710320.410.0910.065330.460.0540.12340.0870.06599350.830.95>100360.0380.0900.52370.190.0450.078380.120.0371.9390.200.180.096400.0950.1413410.0780.0290.14420.0980.0651.7430.0590.03110440.240.0250.11450.250.230.97460.600.150.14470.180.0310.19480.300.110.25490.180.150.1250A0.230.0641650B0.140.05224510.390.0400.23520.0920.0530.26530.230.0290.16540.190.0420.51550.540.8725560.270.480.85570.0730.06210580.0900.0580.39590.0070.0061.5600.100.0960.18610.460.306.1620.710.262.4630.270.0445.9640.250.122.2650.460.0640.14660.490.140.12670.280.0380.087680.230.0110.11690.130.0260.13700.230.120.20710.450.0550.51730.150.0470.22740.0890.0390.22750.680.600.29760.920.343.5770.160.130.15