Patent ID: 12195448

EXPERIMENTAL SECTION

TABLE 1AbbreviationsThe following table lists the abbreviations used herein.AbbreviationMeaningBH3· THFBorane-tetrahydrofuranBINAP2,2′-Bis(diphenylphosphino)-1,1′-binaphthylbrbroad (1H-NMR signal)CIchemical ionisationddoublet (1H-NMR signal)dday(s)DADdiode array detectordddouble-doubletDMFN,N-dimethylformamideDMSOdimethylsulfoxideESIelectrospray (ES) ionisationEtOAcEthyl acetatehhour(s)HATU1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxidehexafluorophosphate, CAS 148893-10-1HPLChigh performance liquid chromatographyLC-MSliquid chromatography mass spectrometrymmultiplet (1H-NMR signal)Mmolarminminute(s)MSmass spectrometryMTBEmethyl-tert-butyletherNaBH4Sodium borohydride, sodium tetrahydroborateNaHCO3Sodium hydrogen carbonateNa2SO4Sodium sulphateNMRnuclear magnetic resonance spectroscopy: chemicalshifts (δ) are given in ppm. The chemical shiftswere corrected by setting the DMSO signal to 2.50 ppmunless otherwise stated.PDAPhoto Diode ArrayPd2dba3Tris(dibenzylideneacetone)dipalladium (0),CAS 51364-51-3Pd(PPh3)4Tetrakis(triphenylphosphane)palladium(0),CAS 14221-01-3quant.quantitativeracracemicRt, Rtretention time (as measured either with HPLC orUPLC) in minutesRuPhos Pd G3(2-Dicyclohexylphosphino-2′,6′-diisopropoxy-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II)methanesulfonate, CAS 1445085-77-7ssinglet (1H-NMR signal)SFCSupercritical Fluid ChromatographySQDSingle-Quadrupole-Detectorttriplet (1H-NMR signal)tdtriple-doublet (1H-NMR signal)TFAtrifluoroacetic acidTHFtetrahydrofuranUPLCultra performance liquid chromatographyX-Phos2-Dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl,CAS 564483-18-7

Other abbreviations not specified herein have their meanings customary to the skilled person.

The various aspects of the invention described in this application are illustrated by the following examples which are not meant to limit the invention in any way. All publications mentioned herein are incorporated by reference in their entirety.

The example testing experiments described herein serve to illustrate the present invention and the invention is not limited to the examples given.

Experimental Section—General Part

All reagents, for which the synthesis is not described in the experimental part, are either commercially available, or are known compounds or may be formed from known compounds by known methods by a person skilled in the art.

The compounds and intermediates produced according to the methods of the invention may require purification. Purification of organic compounds is well known to the person skilled in the art and there may be several ways of purifying the same compound. In some cases, no purification may be necessary. In some cases, the compounds may be purified by crystallization. In some cases, impurities may be stirred out using a suitable solvent. In some cases, the compounds may be purified by chromatography, particularly flash column chromatography, using for example prepacked silica gel cartridges, e.g. Biotage SNAP cartridges KP-Sil® or KP-NH® in combination with a Biotage autopurifier system (SP4® or Isolera Four®) and eluents such as gradients of hexane/ethyl acetate or DCM/methanol. In some cases, the compounds may be purified by preparative HPLC using for example a Waters autopurifier equipped with a diode array detector and/or on-line electrospray ionization mass spectrometer in combination with a suitable prepacked reverse phase column and eluents such as gradients of water and acetonitrile which may contain additives such as trifluoroacetic acid, formic acid or aqueous ammonia.

In some cases, purification methods as described above can provide those compounds of the present invention which possess a sufficiently basic or acidic functionality in the form of a salt, such as, in the case of a compound of the present invention which is sufficiently basic, a trifluoroacetate or formate salt for example, or, in the case of a compound of the present invention which is sufficiently acidic, an ammonium salt for example. A salt of this type can either be transformed into its free base or free acid form, respectively, by various methods known to the person skilled in the art, or be used as salts in subsequent biological assays. It is to be understood that the specific form (e.g. salt, free base etc.) of a compound of the present invention as isolated and as described herein is not necessarily the only form in which said compound can be applied to a biological assay in order to quantify the specific biological activity.

In the case of the synthesis intermediates and working examples of the invention described hereinafter, any compound specified in the form of a salt of the corresponding base or acid is generally a salt of unknown exact stoichiometric composition, as obtained by the respective preparation and/or purification process. Unless specified in more detail, additions to names and structural formulae, such as “hydrochloride”, “trifluoroacetate”, “sodium salt” or “x HCl”, “x CF3COOH”, “x Na+” should not therefore be understood in a stoichiometric sense in the case of such salts, but have merely descriptive character with regard to the salt-forming components present therein.

This applies correspondingly if synthesis intermediates or working examples or salts thereof were obtained in the form of solvates, for example hydrates, of unknown stoichiometric composition (if they are of a defined type) by the preparation and/or purification processes described.

NMR peak forms are stated as they appear in the spectra, possible higher order effects have not been considered.

The1H-NMR data of selected compounds are listed in the form of1H-NMR peaklists. For each signal peak the δ value in ppm is given, followed by the signal intensity, reported in round brackets. The δ value-signal intensity pairs from different peaks are separated by commas. Therefore, a peaklist is described by the general form: δ1(intensity1), δ2(intensity2), . . . , δi(intensityi), . . . , δn(intensityn).

The intensity of a sharp signal correlates with the height (in cm) of the signal in a printed NMR spectrum. When compared with other signals, this data can be correlated to the real ratios of the signal intensities. In the case of broad signals, more than one peak, or the center of the signal along with their relative intensity, compared to the most intense signal displayed in the spectrum, are shown. A1H-NMR peaklist is similar to a classical1H-NMR readout, and thus usually contains all the peaks listed in a classical NMR interpretation. Moreover, similar to classical1H-NMR printouts, peaklists can show solvent signals, signals derived from stereoisomers of target compounds (also the subject of the invention), and/or peaks of impurities. The peaks of stereoisomers, and/or peaks of impurities are typically displayed with a lower intensity compared to the peaks of the target compounds (e.g., with a purity of >90%). Such stereoisomers and/or impurities may be typical for the particular manufacturing process, and therefore their peaks may help to identify the reproduction of our manufacturing process on the basis of “by-product fingerprints”. An expert who calculates the peaks of the target compounds by known methods (MestReC, ACD simulation, or by use of empirically evaluated expectation values), can isolate the peaks of target compounds as required, optionally using additional intensity filters. Such an operation would be similar to peak-picking in classical1H-NMR interpretation. A detailed description of the reporting of NMR data in the form of peaklists can be found in the publication “Citation of NMR Peaklist Data within Patent Applications” (cf. Research Disclosure Database Number 605005, 2014, 1 Aug. 2014, or researchdisclosure.com/searching-disclosures). In the peak picking routine, as described in the Research Disclosure Database Number 605005, the parameter “MinimumHeight” can be adjusted between 1% and 4%. Depending on the chemical structure and/or depending on the concentration of the measured compound it may be reasonable to set the parameter “MinimumHeight”<1%.

In NMR spectra of mixtures of stereoisomers, numbers mentioned with “/” indicate that the stereoisomers show separate signals for the respective hydrogen atom, i.e. “ . . . / . . . (2s, 1H)” means that one hydrogen atom is represented by 2 singlets, each singlet from one or more different stereoisomer(s).

IUPAC names of the following intermediates and example compounds were generated using the ACD/Name software (batch version 14.00; Advanced Chemistry Development, Inc.) or the naming tool implemented in the BIOVIA Draw software (version 4.2 SP1; Dassault Systèmes SE).

Analytical LC-MS Methods

Method 1

MS instrument type: SHIMADZU LCMS-2020, Column: Kinetex EVO C18 30*2.1 mm, 5 um, mobile phase A: 0.0375% TFA in water (v/v), B: 0.01875% TFA in Acetonitrile (v/v), gradient: 0.0 min 0% B→0.8 min 95% B→1.2 min 95% B→1.21 min 5% B→1.55 min 5% B, flow rate: 1.5 mL/min, oven temperature: 50° C.; UV detection: 220 nm & 254 nm.

Method 2

HPLC instrument type: SHIMADZU LCMS-2020, Column: Kinetex EVO C18 50*4.6 mm, 5 um, mobile phase A: 0.0375% TFA in water (v/v), B: 0.01875% TFA in Acetonitrile (v/v), gradient: 0.0 min 10% B→2.4 min 80% B→3.7 min 80% B→3.71 min 10% B→4.0 min 10% B, flow rate: 1.5 mL/min, oven temperature: 50° C.; UV detection: 220 nm & 215 nm & 254 nm.

Method 3 (LC-MS)

Instrument MS: Thermo Scientific FT-MS; Instrument type UHPLC+: Thermo Scientific UltiMate 3000; Column: Waters, HSST3, 2.1×75 mm, C18 1.8 μm; Eluent A: 1 l water+0.01% formic acid; Eluent B: 1 l Acetonitrile+0.01% formic acid; Gradient: 0.0 min 10% B→2.5 min 95% B→3.5 min 95% B; oven: 50° C.; flow rate: 0.90 ml/min; UV-Detection: 210 nm/Optimum Integration Path 210-300 nm.

Method 4 (LC-MS)

Instrument: Waters ACQUITY SQD UPLC System; Column: Waters Acquity UPLC HSS T3 1.8 μm 50×1 mm; Eluent A: 1 l water+0.25 ml formic acid, Eluent B: 1 l Acetonitrile+0.25 ml formic acid; Gradient: 0.0 min 90% A→1.2 min 5% A→2.0 min 5% A; oven: 50° C.; flow rate: 0.40 ml/min; UV-Detection: 210 nm.

Method 5 (LC-MS)

Instrument: Waters ACQUITY SQD UPLC System; Column: Waters Acquity UPLC HSS T3 1.8 μm 50×1 mm; Eluent A: 1 l water+0.25 ml formic acid, Eluent B: 1 l Acetonitrile+0.25 ml formic acid; Gradient: 0.0 min 95% A→6.0 min 5% A→7.5 min 5% A; oven: 50° C.; flow rate: 0.35 ml/min; UV-Detection: 210 nm.

Method 6 (LC-MS)

Instrument: Agilent MS Quad 6150; HPLC: Agilent 1290; Column: Waters Acquity UPLC HSS T3 1.8 μm 50×2.1 mm; Eluent A: 1 l water+0.25 ml formic acid, Eluent B: 1 l Acetonitrile+0.25 ml formic acid; Gradient: 0.0 min 90% A→0.3 min 90% A→1.7 min 5% A→3.0 min 5% A oven: 50° C.; flow rate: 1.20 ml/min; UV-Detection: 205-305 nm.

Method 7 (LC-MS)

Instrument: Waters Single Quad MS System; Instrument Waters UPLC Acquity; Column: Waters BEH C18 1.7μ50×2.1 mm; Eluent A: 1 l water+1.0 mL (25% aqueous Ammonia)/L, Eluent B: 1 l Acetonitrile; Gradient: 0.0 min 92% A→0.1 min 92% A→1.8 min 5% A→3.5 min 5% A; oven: 50° C.; flow rate: 0.45 mL/min; UV-Detection: 210 nm.

Method 8 (LC-MS)

System MS: Waters TOF instrument; System UPLC: Waters Acquity I-CLASS; Column: Waters Acquity UPLC HSS T3 1.8 μm 50×1 mm; Eluent A: 1 l Water+0.100 ml 99% ige Formic acid, Eluent B: 1 l Acetonitrile+0.100 ml 99% ige Formic acid; Gradient: 0.0 min 90% A→1.2 min 5% A→2.0 min 5% A Oven: 50° C.; Flow: 0.40 ml/min; UV-Detection: 210 nm.

Method 9 (LC-MS):

System MS: Waters TOF instrument; System UPLC: Waters Acquity I-CLASS; Column: Waters Acquity UPLC HSS T3 1.8 μm 50×1 mm; Eluent A: 1 l Water+0.100 ml 99% ige Formic acid, Eluent B: 1 l Acetonitrile+0.100 ml 99% ige Formic acid; Gradient: 0.0 min 95% A→6.0 min 5% A→7.5 min 5% A Oven: 50° C.; Flow: 0.35 ml/min; UV-Detection: 210 nm.

Preparative HPLC Methods

Instrument: Waters Prep LC/MS System, column: Phenomenex Kinetex C18 5 μm 100×30 mm, UV-detection 200-400 nm, room temperature, At-Column Injection (complete injection), eluent A:water, eluent B:acetonitrile, eluent C:2% formic acid in water, eluent D:acetonitrile/water (80 vol. %/20 vol. %); flow: 80 ml/min, gradient profile: 0 to 2 min:eluent A 47 ml/min, eluent B 23 ml/min; 2 to 10 min:eluent A from 47 ml/min to 23 ml/min, eluent B from 23 ml/min to 47 ml/min; 10 to 12 min eluent A 0 ml/min and eluent B 70 ml/min; eluent C and eluent D have a constant flow of 5 ml/min each over the whole running time.

Microwave: Reactions employing microwave irradiation may be run with a Biotage Initator® microwave oven optionally equipped with a robotic unit. The reported reaction times employing microwave heating are intended to be understood as fixed reaction times after reaching the indicated reaction temperature.

When compounds according to the invention are purified by preparative HPLC by the above-described methods in which the eluents contain additives, for example trifluoroacetic acid, formic acid or ammonia, the compounds according to the invention may be obtained in salt form, for example as trifluoroacetate, formate or ammonium salt, if the compounds according to the invention contain a sufficiently basic or acidic functionality. Such a salt can be converted to the corresponding free base or acid by various methods known to the person skilled in the art.

In the case of the synthesis intermediates and working examples of the invention described hereinafter, any compound specified in the form of a salt of the corresponding base or acid is generally a salt of unknown exact stoichiometric composition, as obtained by the respective preparation and/or purification process. Unless specified in more detail, additions to names and structural formulae, such as “hydrochloride”, “trifluoroacetate”, “sodium salt” or “x HCl”, “x CF3COOH”, “x Na+” should not therefore be understood in a stoichiometric sense in the case of such salts, but have merely descriptive character with regard to the salt-forming components present therein.

This applies correspondingly if synthesis intermediates or working examples or salts thereof were obtained in the form of solvates, for example hydrates, of unknown stoichiometric composition (if they are of a defined type) by the preparation and/or purification processes described.

Enantiomer 1 is an enantiomer which eluted first out of the column.

Enantiomer 2 is an enantiomer which eluted second out of the column.

For example 3 (enantiomer 2) the absolute configuration was determined by single crystal X-ray structure analysis to be R. Consequently all compounds annotated as enantiomer 2 should have an absolute configuration of R. The corresponding stereochemistry should survive all synthetic conditions due to its substitution pattern.

Diastereomeric mixture 1 defines a compound where its starting material is defined as Enantiomer 1 and is reacted with a building block containing at least one chiral center and where the configuration is not defined

Diastereomeric mixture 2 defines a compound where its starting material is defined as Enantiomer 2 and is reacted with a building block containing at least one chiral center and where the configuration is not defined

Diastereomer 1 and Diastereomer 2 defines the two compounds resulting from the chiral separation of the diastereomeric mixture 1 described above.

Diastereomer 3 and Diastereomer 4 defines the two compounds resulting from the chiral separation of the diastereomeric mixture 2 described above.

Stereoisomer 1 defines a compound where its starting material is defined as Enantiomer 1 and is reacted with a building block containing at least one chiral center and where the configuration is defined.

Stereoisomer 2 defines a compound where its starting material is defined as Enantiomer 2 and is reacted with a building block containing at least one chiral center and where the configuration is defined.

Starting Compounds and Intermediates

Intermediate 1A

Example 1A

Tert-butyl 3-{2-[(benzyloxy)carbonyl]hydrazino}piperidine-1-carboxylate (Racemate)

To a solution of tert-butyl 3-oxopiperidine-1-carboxylate [CAS No. 989-36-7] (300 g, 1.51 mol) in tetrahydrofuran (1.50 L) and Methanol (300 mL) was added benzyl hydrazinecarboxylate [CAS No. 5331-43-1] (250 g, 1.51 mol) at 25° C., then, the mixture was stirred at 25° C. for 1 h. Afterwards NaBH4(114 g, 3.01 mol) was added in portions to the mixture at 25° C. and stirred at 25° C. for 2 h. The reaction mixture was cooled to 10° C., and sat. NH4Cl was added dropwise to pH˜6. The mixture was extracted with EtOAc (300 mL*2) and concentrated in vacuo. The residue was dissolved in MTBE (300 mL) and petroleum ether (300 mL) was added. The mixture was filtrated off and the precipitate was washed with petroleum ether (100 mL) affording the title compound (400 g, 1.14 mol, 76.0% yield) as a white solid.

LC-MS: (Method 1) Rt=0.832 min, MS (M-100+1=250.4).

Example 2A

Tert-butyl 3-hydrazinopiperidine-1-carboxylate acetic acid (Racemate)

To a solution of tert-butyl 3-{2-[(benzyloxy)carbonyl]hydrazino}piperidine-1-carboxylate (prepared in analogy to Example 1A, 1.20 kg, 3.43 mol) in ethanol (11.0 L) was added acetic acid (415 g, 6.91 mol, 395 mL) and Pd/C (120 g, 20% purity) under H2(15 Psi). The mixture was stirred at 25° C. for 12 h. The mixture was filtrated and the precipitate was washed with ethanol (11.0 L) to give a solution of the title compound in ethanol (945 g, acetic acid salt) as a black liquid, the filtrate was used for the next step without purification.

1H-NMR (400 MHz, CDCl3) δ [ppm]: 7.52 (s, 5H), 3.59 (d, J=6.0 Hz, 12H), 3.30-3.24 (m, 2H), 2.75-2.71 (m, 2H, 1.38-1.34 (m, 1H), 1.20-1.18 (m, 1H), 1.10 (s, 9H)

LC-MS: (Method 1) Rt=0.263 min, MS (M-56+1=160.2)

Example 3A

Ethyl 2-(ethoxymethylidene)-4,4-difluoro-3-oxobutanoate

A solution of ethyl 4,4-difluoro-3-oxobutanoate [CAS No. 352-24-9] (120 g, 722 mmol) and (diethoxymethoxy)ethane (240 ml, 1.4 mol) in acetic acid anhydride (200 ml, 2.2 mol) was stirred overnight at 140° C. and evaporated to dryness affording 155 g (quant.) of the title compound which was used in the next step without further purification.

1H-NMR (600 MHz, CDCl3) δ [ppm]: 1.306 (6.05), 1.318 (16.00), 1.330 (14.48), 1.341 (4.56), 1.428 (5.99), 1.436 (5.01), 1.440 (12.20), 1.448 (9.25), 1.451 (6.31), 1.460 (4.48), 2.095 (1.59), 2.225 (1.56), 4.247 (1.97), 4.260 (5.79), 4.271 (5.85), 4.277 (1.55), 4.283 (2.00), 4.289 (4.40), 4.301 (4.37), 4.308 (2.03), 4.313 (1.64), 4.320 (5.74), 4.332 (5.78), 4.340 (1.60), 4.344 (2.01), 4.351 (4.21), 4.364 (4.20), 4.375 (1.37), 6.262 (1.79), 6.339 (1.35), 6.352 (3.56), 6.429 (2.63), 6.442 (1.72), 6.519 (1.28), 7.867 (5.48), 7.880 (7.31).

Example 4A

Tert-butyl 3-[5-(difluoromethyl)-4-(ethoxycarbonyl)-1H-pyrazol-1-yl]piperidine-1-carboxylate (Racemate)

To a mixture of tert-butyl 3-hydrazinopiperidine-1-carboxylate acetic acid (Example 2A, 945 g, 3.43 mol) in ethanol (20.0 L) was added ethyl 2-(ethoxymethylene)-4,4-difluoro-3-oxobutanoate (prepared in analogy to Example 3A, 840 g, 3.78 mol). The mixture was stirred at 25° C. for 12 h. The reaction mixture was concentrated. The residue was poured into saturated NaHCO3aqueous solution (10.0 L), and extracted with Ethyl acetate (10.0 L*2). The combined organic layer was washed with brine (10.0 L), dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography on silica gel eluted with Petroleum ether:Ethyl acetate (50:1-25:1-10:1, Rf=0.3) affording 530 g (41.4% yield) of the title compound.

1H-NMR (400 MHz, CDCl3) δ [ppm]: 7.84 (s, 1H), 7.51 (t, J=12.8 Hz, 1H), 4.47-4.41 (m, 1H), 4.30-4.10 (m, 4H), 3.19-3.13 (m, 1H), 2.69 (s, 1H), 2.15-2.10 (m, 2H), 1.83-1.78 (m, 1H), 1.60-1.55 (m, 1H), 1.40 (s, 9H), 1.32-1.29 (m, 3H) LC-MS (Method 1) Rt=0.992 min, MS (M-56+1=318.0).

Example 5A

Ethyl 5-(difluoromethyl)-1-(piperidin-3-yl)-1H-pyrazole-4-carboxylate (Racemate)

Tert-butyl 3-[5-(difluoromethyl)-4-(ethoxycarbonyl)-1H-pyrazol-1-yl]piperidine-1-carboxylate (prepared in analogy to Example 4A, 593 g, 1.59 mol) was added to a solution of hydrogen chloride in dioxane (4 M, 2.50 L), the mixture was stirred at 25° C. for 12 h. The mixture was evaporated and the residue was dissolved in 1.00 L water and extracted with MTBE 500 mL. The aqueous phase was separated and adjusted pH to 8-9 with NaHCO3. The aqueous phase was extracted with dichloromethane (1.00 L x 2), and the combined organic phases were washed with brine (1.00 L), dried over Na2SO4and concentrated to give 350 g (80.6% yield) of the title compound.

1H-NMR (400 MHz, CDCl3) δ [ppm]: 7.87 (s, 1H), 7.54 (t, J=12.8 Hz, 1H), 4.55-4.54 (m, 1H), 4.34-4.28 (m, 2H), 3.25-3.03 (m, 3H), 2.71-2.65 (m, 1H), 2.19-1.86 (m, 4H), 1.63-1.60 (m, 1H), 1.35 (t, J=7.2 Hz, 3H)

LC-MS: (Method 1) Rt=0.644 min, MS (M+1)=274.6

In analogy to Example 5A, ethyl 5-(difluoromethyl)-1-(piperidin-3-yl)-1H-pyrazole-4-carboxylate (Racemate) was prepared using different protecting groups. The two enantiomers were separated by SFC [sample preparation: 20 g were dissolved in 500 ml methanol; injection volume: 15 ml; column: Daicel AZ SCF 20 μm, 400×50 mm; eluent: carbone dioxide/methanol/aqueous ammonia (1%) 80:19:1 to 60:39:1; flow rate: 400 ml/min; temperature: 40° C.; UV detection: 220 nm]. After separation, 8.1 g of enantiomer 1 (Example 6A), which eluted first, and 8.0 g of enantiomer 2 (Example 7A), which eluted later, were isolated.

Example 6A

Ethyl 5-(difluoromethyl)-1-(piperidin-3-yl)-1H-pyrazole-4-carboxylate (Enantiomer 1)

For separation conditions see Example 5A.

Analytical SFC: Rt=0.980 min, e.e. =100% [Column Chiralpak IC-3: 50×4.6 mm; eluent: CO2/[methanol+0.2% diethyl amine]: 90:10 flow rate: 3.0 ml/min; temperature: 25° C.; UV detection: 220 nm].

1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 8.00 (s, 1H), 7.75-7.44 (m, 1H), 4.50-4.36 (m, 1H), 4.33-4.18 (m, 2H), 3.10-2.95 (m, 1H), 2.91-2.76 (m, 2H), 2.48-2.33 (m, 2H), 2.08-1.94 (m, 2H), 1.81-1.66 (m, 1H), 1.62-1.40 (m, 1H), 1.37-1.21 (m, 3H).

Example 7A

Ethyl 5-(difluoromethyl)-1-(piperidin-3-yl)-1H-pyrazole-4-carboxylate (Enantiomer 2)

For separation conditions see Example 5A.

Analytical SFC: Rt=1.227 min, e.e. =97% [Column Chiralpak IC-3: 50×4.6 mm; eluent: CO2/[methanol+0.2% diethyl amine]: 90:10 flow rate: 3.0 ml/min; temperature: 25° C.; UV detection: 220 nm].

1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 8.01 (s, 1H), 7.75-7.43 (m, 1H), 4.50-4.37 (m, 1H), 4.27 (q, 2H), 3.09-2.97 (m, 1H), 2.94-2.81 (m, 2H), 2.47-2.34 (m, 2H), 2.06-1.92 (m, 2H), 1.79-1.66 (m, 1H), 1.60-1.41 (m, 1H), 1.29 (t, 3H).

Example 8A

2-Bromo-4-chloro-1-[(4-methoxyphenyl)methoxy]benzene

A solution of 2-bromo-4-chlorophenol [CAS No. 695-96-5] (10.0 g, 48.2 mmol) in acetone (75 ml) was treated with potassium carbonate (13.3 g, 96.4 mmol) and potassium iodide (12.0 g, 72.3 mmol) and 1-(chloromethyl)-4-methoxybenzene (7.55 g, 48.2 mmol). The resulting mixture was stirred ˜19 hours at 70° C. The reaction mixture was diluted with water and extracted twice with ethyl acetate. The combined organic layers were dried over sodium sulphate and evaporated. The residue was purified by flash chromatography (silica gel, cyclohexane/ethyl acetate gradient) affording 13.8 g (86% yield) of the title compound.

LC-MS (Method 3): Rt=2.48 min; MS (ESIneg): m/z=324 [M−H]−

1H-NMR (600 MHz, DMSO-d6) δ[ppm]: 3.349 (10.98), 5.124 (16.00), 6.949 (0.87), 6.954 (8.36), 6.957 (2.68), 6.965 (2.83), 6.968 (8.92), 6.973 (1.00), 7.218 (5.23), 7.233 (6.21), 7.380 (0.90), 7.384 (7.80), 7.399 (7.44), 7.402 (4.47), 7.406 (3.89), 7.417 (3.04), 7.421 (3.07), 7.697 (6.51), 7.702 (6.34).

Example 9A

Ethyl 1-[1-{5-chloro-2-[(4-methoxyphenyl)methoxy]phenyl}piperidin-3-yl]-5-(difluoromethyl)-1H-pyrazole-4-carboxylate (Enantiomer 1)

Under argon, a solution of 2-bromo-4-chloro-1-[(4-methoxyphenyl)methoxy]benzene (prepared in analogy to Example 8A, 10.0 g, 30.5 mmol) and ethyl 5-(difluoromethyl)-1-[piperidin-3-yl]-1H-pyrazole-4-carboxylate (prepared in analogy to Example 6A, Enantiomer 1, 8.34 g, 30.5 mmol) in 1,4-dioxane (100 ml) was treated with caesium carbonate (29.8 g, 91.6 mmol), Pd2dba3(2.80 g, 3.05 mmol) and rac-BINAP (3.80 g, 6.10 mmol) and the resulting mixture was stirred overnight at 100° C. The reaction mixture was combined with a 500 mg test reaction, filtered over celite, rinsed with ethyl acetate and evaporated. The residue was retaken in water and extracted three times with ethyl acetate. The combined organic layers were washed with a saturated solution of sodium chloride, dried over sodium sulphate and evaporated. The residue was purified by flash chromatography (silica gel, cyclohexane/ethyl acetate gradient) affording 10.1 g (60% yield) of the title compound.

LC-MS (Method 4): Rt=1.44 min; MS (ESIpos): m/z=520 [M+H]+

Example 10A

Ethyl 1-[1-(5-chloro-2-hydroxyphenyl)piperidin-3-yl]-5-(difluoromethyl)-1H-pyrazole-4-carboxylate (Enantiomer 1)

A solution of ethyl 1-[1-{5-chloro-2-[(4-methoxyphenyl)methoxy]phenyl}piperidin-3-yl]-5-(difluoromethyl)-1H-pyrazole-4-carboxylate (Example 9A, Enantiomer 1, 10.1 g, 19.4 mmol) in dichloromethane (200 ml) was treated with trifluoroacetic acid and stirred over night at room temperature. The reaction mixture was evaporated. The residue was retaken in ethyl acetate and washed once with water, once with a saturated solution of sodium hydrogencarbonate and finally once with a saturated solution of sodium chloride. The organic phase was dried over sodium sulphate and evaporated. The residue was purified by flash chromatography (silica gel, cyclohexane/ethyl acetate gradient) affording 7.17 g (83% purity, 77% yield) of the title compound.

LC-MS (Method 8): Rt=1.26 min; MS (ESIpos): m/z=400 [M+H]+

Example 11A

Ethyl 1-[1-{5-chloro-2-[(trifluoromethanesulfonyl)oxy]phenyl}piperidin-3-yl]-5-(difluoromethyl)-1H-pyrazole-4-carboxylate (Enantiomer 1)

Under argon, a solution of ethyl 1-[1-(5-chloro-2-hydroxyphenyl)piperidin-3-yl]-5-(difluoromethyl)-1H-pyrazole-4-carboxylate (Example 10A, Enantiomer 1, 7.17 g, 83% purity, 14.9 mmol) in dichloromethane (160 ml) was treated with triethylamine (5.2 ml, 37 mmol) and cooled to 0° C. Trifluoromethanesulfonic anhydride was added dropwise and the resulting mixture was stirred 45 minutes at 0° C. The reaction mixture was diluted with dichloromethane (150 ml) and washed three times with water. The organic phase was dried over sodium sulphate and evaporated. The residue was purified by flash chromatography (silica gel, cyclohexane/ethyl acetate gradient) affording 7.89 g (quant.) of the title compound.

LC-MS (Method 4): Rt=1.47 min; MS (ESIpos): m/z=532 [M+H]+

Example 12A

Ethyl 1-[1-{5-chloro-2-[(4-methoxyphenyl)methoxy]phenyl}piperidin-3-yl]-5-(difluoromethyl)-1H-pyrazole-4-carboxylate (Enantiomer 2)

Under argon, a solution of ethyl 5-(difluoromethyl)-1-[piperidin-3-yl]-1H-pyrazole-4-carboxylate (prepared in analogy to Example 7A, Enantiomer 2, 43.6 g, 160 mmol) and 2-bromo-4-chloro-1-[(4-methoxyphenyl)methoxy]benzene (prepared in analogy to Example 8A, 52.3 g, 160 mmol) in 1,4-dioxane (680 ml) was treated with Pd2(dba)3(14.6 g, 16.0 mmol), rac-BINAP (19.9 g, 31.9 mmol) and freshly ground caesium carbonate (156 g, 479 mmol) and stirred 18 hours at 100° C. The reaction mixture was diluted with ethyl acetate and a 10% solution of sodium chloride, filtered over Celite and rinsed with ethyl acetate. The aqueous phase of the filtrate was extracted with ethyl acetate. The combined organic layers were washed with a 10% solution of sodium chloride, dried over sodium sulphate and evaporated. The residue was purified flash chromatography over silica gel (dichloromethane/petrol ether 4:1) affording 42 g (82% yield) of the title compound.

LC-MS (Method 3): Rt=2.78 min; MS (ESIpos): m/z=520 [M+H]+

1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.272 (3.65), 1.290 (7.68), 1.307 (3.76), 1.686 (0.44), 1.717 (0.54), 1.852 (0.73), 1.885 (0.50), 1.989 (0.47), 2.019 (0.56), 2.058 (0.99), 2.084 (0.61), 2.587 (0.51), 2.616 (0.89), 2.642 (0.45), 3.030 (0.76), 3.057 (1.51), 3.084 (0.83), 3.447 (0.72), 3.474 (0.69), 3.613 (0.74), 3.640 (0.67), 3.737 (16.00), 4.251 (1.13), 4.269 (3.48), 4.287 (3.45), 4.304 (1.12), 4.624 (0.40), 4.639 (0.48), 4.650 (0.76), 4.661 (0.51), 5.035 (6.45), 6.872 (3.47), 6.893 (5.67), 6.947 (0.98), 6.952 (0.85), 6.968 (1.72), 6.974 (1.67), 7.017 (2.84), 7.039 (1.57), 7.305 (3.66), 7.326 (3.43), 7.340 (0.56), 7.380 (0.41), 7.439 (0.93), 7.463 (0.64), 7.476 (0.48), 7.569 (1.65), 7.699 (0.76), 8.044 (3.66).

Example 13A

Ethyl 1-[1-(5-chloro-2-hydroxyphenyl)piperidin-3-yl]-5-(difluoromethyl)-1H-pyrazole-4-carboxylate (Enantiomer 2)

A solution of ethyl 1-[1-{5-chloro-2-[(4-methoxyphenyl)methoxy]phenyl}piperidin-3-yl]-5-(difluoromethyl)-1H-pyrazole-4-carboxylate (prepared in analogy to Example 12A, Enantiomer 2, 67.5 g, 130 mmol) in dichloromethane (1.0 l) was treated with trifluoroacetic acid (100 ml, 1.3 mol) and stirred overnight at room temperature. The reaction mixture was diluted with water (750 ml) and carefully treated with a 10% solution of sodium carbonate (450 ml) until no more carbon dioxide was generated. The organic phase was dried over sodium sulphate and evaporated affording 52 g (90% yield) of the title compound which was used in the next step without further purification.

LC-MS (Method 3): Rt=2.42 min; MS (ESIpos): m/z=400 [M+H]+

Example 14A

Ethyl 1-[1-{5-chloro-2-[(trifluoromethanesulfonyl)oxy]phenyl}piperidin-3-yl]-5-(difluoromethyl)-1H-pyrazole-4-carboxylate (Enantiomer 2)

A solution of ethyl 1-[1-(5-chloro-2-hydroxyphenyl)piperidin-3-yl]-5-(difluoromethyl)-1H-pyrazole-4-carboxylate (Example 13A, Enantiomer 2, 52.0 g, 117 mmol) and triethylamine (49 ml, 350 mmol) in dichloromethane (330 ml) was cooled to −50° C. Trifluoromethanesulfonic acid (28 ml, 160 mmol) was added dropwise and the resulting mixture was stirred 1 hour at −50° C. The reaction mixture was then diluted with dichloromethane (330 ml) and water (370 ml). The aqueous phase was extracted with dichloromethane (330 ml). The combined organic layers were washed with (370 ml), dried over sodium sulphate and evaporated. The resulting mixture was purified by flash chromatography (silica gel, dichloromethane/petrol ether 6:4) affording 60 g (96% yield) of the title compound.

LC-MS (Method 3): Rt=2.74 min; MS (ESIpos): m/z=532 [M+H]+

1H-NMR (600 MHz, DMSO-d6) δ[ppm]: −0.021 (0.65), 1.082 (0.51), 1.270 (7.69), 1.282 (16.00), 1.294 (7.63), 1.772 (0.48), 1.780 (0.51), 1.787 (0.63), 1.793 (0.66), 1.801 (0.62), 1.808 (0.60), 1.910 (1.25), 1.914 (0.99), 1.927 (0.67), 1.932 (0.89), 2.068 (0.72), 2.075 (1.03), 2.086 (2.45), 2.091 (2.40), 2.100 (1.41), 2.792 (0.71), 2.796 (0.83), 2.812 (1.48), 2.816 (1.50), 2.832 (0.83), 2.836 (0.72), 3.142 (1.17), 3.161 (1.04), 3.201 (1.21), 3.219 (2.80), 3.237 (1.83), 3.278 (1.37), 3.285 (1.56), 4.251 (2.26), 4.263 (7.09), 4.275 (7.06), 4.287 (2.20), 4.755 (0.50), 4.765 (0.90), 4.773 (0.89), 4.781 (0.90), 4.791 (0.49), 5.734 (2.17), 7.261 (2.19), 7.265 (2.27), 7.275 (2.69), 7.279 (2.82), 7.391 (4.65), 7.406 (3.75), 7.431 (4.73), 7.435 (4.51), 7.492 (1.26), 7.579 (2.61), 7.666 (1.07), 8.026 (6.37).

Example 15A

Tert-butyl 4-(4′-chloro-2′-{3-[5-(difluoromethyl)-4-(ethoxycarbonyl)-1H-pyrazol-1-yl]piperidin-1-yl}[1,1′-biphenyl]-4-yl)piperazine-1-carboxylate (Enantiomer 2)

Under argon, a solution of ethyl 1-[1-{5-chloro-2-[(trifluoromethanesulfonyl)oxy]phenyl}piperidin-3-yl]-5-(difluoromethyl)-1H-pyrazole-4-carboxylate (Example 14A, Enantiomer 2, 57.0 g, 107 mmol) and tert-butyl 4-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]piperazine-1-carboxylate [CAS No. 470478-90-1] (49.9 g, 129 mmol) in toluene (600 ml) and ethanol (600 ml) was treated with an aqueous solution of sodium carbonate (160 ml, 2.0 M, 320 mmol) and Tetrakis(triphenylphosphine)palladium(0) (6.19 g, 5.36 mmol). The resulting mixture was stirred 4 hours at 100° C. The reaction mixture was cooled to room temperature, filtered over Celite, washed with ethyl acetate and evaporated. The residue was purified by flash chromatography (silica gel, petrol ether/ethyl acetate 9:1 to 8:2) affording 62 g (89% yield) of the title compound.

LC-MS (Method 3): Rt=3.15 min; MS (ESIpos): m/z=644 [M+H]+

Example 16A

Ethyl 1-{1-[4-chloro-4′-(piperazin-1-yl)[1,1′-biphenyl]-2-yl]piperidin-3-yl}-5-(difluoromethyl)-1H-pyrazole-4-carboxylate hydrochloride (Enantiomer 2)

A solution of tert-butyl 4-(4′-chloro-2′-{(3-[5-(difluoromethyl)-4-(ethoxycarbonyl)-1H-pyrazol-1-yl]piperidin-1-yl}[1,1′-biphenyl]-4-yl)piperazine-1-carboxylate (Example 15A, Enantiomer 2, 60.0 g, 93.1 mmol) in dichloromethane (250 ml) was treated with a solution of hydrogen chloride in dioxane (230 ml, 4.0 M, 930 mmol). The resulting mixture was stirred 3 hours at room temperature and evaporated. The residue was co-evaporated twice with diethyl ether (250 ml×2), stirred 4 days in diisopropyl ether. The suspension was filtered, the solid was washed twice with diisopropyl ether affording 57 g (quant.) of the title compound.

LC-MS (Method 3): Rt=1.78 min; MS (ESIpos): m/z=544 [M+H]+

1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.029 (13.49), 1.044 (13.77), 1.262 (7.53), 1.280 (16.00), 1.297 (7.81), 1.496 (0.79), 1.506 (0.62), 1.527 (0.91), 1.559 (0.40), 1.716 (1.24), 1.749 (0.95), 1.888 (0.84), 1.897 (0.78), 1.918 (0.98), 1.926 (0.93), 1.966 (1.38), 1.995 (0.69), 2.580 (1.54), 2.606 (0.83), 2.992 (1.21), 3.018 (2.69), 3.044 (2.33), 3.063 (1.24), 3.435 (5.96), 3.448 (7.25), 3.460 (5.00), 3.570 (5.78), 3.586 (0.87), 3.601 (1.12), 3.616 (0.85), 4.227 (5.38), 4.238 (6.62), 4.256 (9.26), 4.273 (7.97), 4.291 (2.70), 4.444 (0.41), 4.455 (0.77), 4.470 (0.89), 4.481 (1.31), 4.491 (0.92), 4.507 (0.68), 7.045 (6.02), 7.067 (6.86), 7.074 (5.10), 7.079 (5.42), 7.099 (2.25), 7.104 (1.49), 7.120 (3.55), 7.125 (3.10), 7.164 (6.27), 7.185 (3.37), 7.383 (1.62), 7.483 (6.90), 7.505 (6.40), 7.513 (3.75), 7.643 (1.34), 8.005 (5.77), 9.399 (1.97).

Example 17A

Ethyl 1-[1-{4-chloro-4′-[4-(2-methylpropyl)piperazin-1-yl][1,1′-biphenyl]-2-yl}piperidin-3-yl]-5-(difluoromethyl)-1H-pyrazole-4-carboxylate (Enantiomer 2)

A solution of ethyl 1-{1-[4-chloro-4′-(piperazin-1-yl)[1,1′-biphenyl]-2-yl]piperidin-3-yl}-5-(difluoromethyl)-1H-pyrazole-4-carboxylate hydrogen chloride (Example 16A, Enantiomer 2, 52.0 g, 84.3 mmol) in THF was treated with N,N-diisopropylethylamine (59 ml, 340 mmol) and 2-methylpropanal [CAS No. 78-84-2] (38 ml, 420 mmol) and stirred 1 hour at room temperature. Sodium triacetoxyborohydride (71.5 g, 337 mmol) was then added and the resulting mixture was stirred 18 hours at room temperature. The reaction mixture was diluted with an aqueous solution of sodium hydrogen carbonate (10%) and ethyl acetate. The aqueous layer was extracted twice with ethyl acetate. The combined organic layers were washed with an aqueous solution of sodium chloride, dried over sodium sulphate and evaporated. The residue was purified by flash chromatography (silica gel, petrol ether/ethyl acetate 8:2) affording 47 g (93% yield) of the title compound.

LC-MS (Method 9): Rt=3.42 min; MS (ESIpos): m/z=600 [M+H]+

Example 18A

1-(2-Methylpropyl)-4-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]piperazine

1-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]piperazine (350 mg, 1.21 mmol) was placed in 7.4 ml THF and N,N-diisopropylethylamine (320 μl, 1.8 mmol) was added. Then 2-methylpropanal (440 μl, 4.9 mmol) was added and the mixture was stirred for 10 min. Then sodium triacetoxyborohydride (772 mg, 3.64 mmol) was added and the mixture was stirred at 55° C. for 4 h. The reaction mixture was cooled to room temperature, saturated aqueous sodium bicarbonate solution was added and the mixture was extracted three times with ethyl acetate. The combined organic phases were washed once with saturated, aqueous sodium chloride solution, dried over sodium sulphate, filtered and evaporated. 342 mg of the target compound (79% of theory, purity 97%) were obtained.

LC-MS (Method 3): Rt=1.23 min; MS (ESIpos): m/z=345 [M+H]+

1H-NMR (600 MHz, DMSO-d6) δ[ppm]: 0.058 (0.55), 0.927 (4.09), 0.938 (4.13), 1.316 (16.00), 2.121 (0.98), 2.133 (0.89), 2.492 (0.99), 2.508 (0.99), 2.559 (2.25), 2.599 (2.62), 3.241 (1.07), 3.249 (1.38), 3.257 (0.98), 6.935 (1.05), 6.949 (1.07), 7.552 (1.15), 7.566 (1.07).

Experimental Section—Example Compounds

Example 1

1-[1-{4-Chloro-4′-[4-(2-methylpropyl)piperazin-1-yl][1,1′-biphenyl]-2-yl}piperidin-3-yl]-5-(difluoromethyl)-1H-pyrazole-4-carboxylic acid hydrochloride (Enantiomer 1)

Ethyl 1-[1-{5-chloro-2-[(trifluoromethanesulfonyl)oxy]phenyl}piperidin-3-yl]-5-(difluoromethyl)-1H-pyrazole-4-carboxylate (prepared in analogy to Example 11A, Enantiomer 1, 80.0 mg, 147 μmol) and 1-(2-methylpropyl)-4-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]piperazine (Example 18A 62.8 mg, 97% purity, 177 μmol) were placed under argon in toluene/ethanol (820/820 μl). 2 M sodium carbonate solution (220 μl, 2.0 M, 440 μmol) and tetrakis(triphenylphosphine)palladium(0) (8.52 mg, 7.37 μmol) were added and the mixture was stirred at 100° C. overnight. The reaction mixture was diluted with ethyl acetate and 1 M hydrochloric acid was added. The aqueous phase was extracted three times with ethyl acetate. The organic phase was dried with sodium sulfate, filtered off and evaporated. The crude mixture was dissolved with THF/ethanol (2.0/0.2 ml), 1 M lithium hydroxide solution (1.5 ml, 1.5 mmol) was added and the mixture was stirred at room temperature overnight. A 1 M lithium hydroxide solution (740 μl, 740 μmol) was added again. After about 6 h the reaction mixture was evaporated at 50° C. The residue was dissolved in acetonitrile/water/0.25 ml trifluoroacetic acid and purified by preparative HPLC (RP18 column, acetonitrile/water gradient with the addition of 0.1% trifluoroacetic acid). The crude product was purified by means of thick layer chromatography (dichloromethane/methanol/formic acid: 10/1/0.1). The silica gel mixture was stirred with dichloromethane/1 M hydrochloric acid in dioxane (10/1) in ethanol, filtered off and carefully evaporated at 30° C. and lyophilized. 34 mg of the target compound (36% of theory, purity 95%) were obtained.

LC-MS (Method 6): Rt=1.23 min; MS (ESIpos): m/z=572 [M−HCl+H]+

1H-NMR (600 MHz, DMSO-d6) δ[ppm]: 1.004 (15.87), 1.015 (16.00), 1.500 (0.51), 1.521 (0.57), 1.728 (0.73), 1.750 (0.61), 1.897 (0.57), 1.917 (0.62), 1.975 (0.79), 2.122 (0.42), 2.133 (0.84), 2.144 (1.02), 2.156 (0.79), 2.571 (0.47), 2.587 (0.91), 2.610 (0.52), 3.004 (0.84), 3.022 (2.01), 3.026 (2.20), 3.038 (3.72), 3.048 (2.50), 3.065 (0.75), 3.154 (2.66), 3.161 (2.75), 3.169 (2.36), 3.177 (1.88), 3.224 (0.84), 3.237 (0.70), 3.589 (1.41), 3.602 (1.80), 3.825 (1.02), 3.841 (0.78), 3.866 (1.05), 3.882 (0.75), 4.223 (2.57), 4.445 (0.68), 4.463 (0.97), 4.481 (0.57), 7.045 (0.55), 7.055 (3.63), 7.070 (3.72), 7.084 (2.72), 7.087 (3.09), 7.110 (1.47), 7.113 (1.11), 7.123 (2.19), 7.127 (2.02), 7.163 (3.67), 7.177 (2.19), 7.215 (0.46), 7.428 (0.83), 7.495 (4.24), 7.510 (4.02), 7.515 (2.07), 7.602 (0.82), 7.959 (4.79), 9.484 (0.54).

Example 2

1-[1-{4-Chloro-4′-[4-(2-methylpropyl)piperazin-1-yl][1,1′-biphenyl]-2-yl}piperidin-3-yl]-5-(difluoromethyl)-1H-pyrazole-4-carboxylic acid (Enantiomer 2)

Method A

A solution of ethyl 1-[1-{4-chloro-4′-[4-(2-methylpropyl)piperazin-1-yl][1,1′-biphenyl]-2-yl}piperidin-3-yl]-5-(difluoromethyl)-1H-pyrazole-4-carboxylate (prepared in analogy to Example 17A, Enantiomer 2, 50.8 g, 84.6 mmol) in a THF/methanol mixture 9:1 (1.0 l) was treated with an aqueous solution of lithium hydroxide (850 ml, 1.0 M, 850 mmol) and stirred overnight at room temperature. The reaction mixture was concentrated, diluted with dichloromethane (1.5 l) and adjusted to pH=2 with an aqueous solution of hydrogen chloride (2N). The resulting suspension was stirred 45 minutes at room temperature. The solid was filtered, washed with water and dried under vacuum affording 43 g (90% yield) of the title compound.

LC-MS (Method 7): Rt=1.27 min; MS (ESIpos): m/z=572 [M+H]+

1H-NMR (600 MHz, DMSO-d6) δ[ppm]: 1.002 (15.68), 1.013 (16.00), 1.080 (0.57), 1.092 (1.18), 1.103 (0.63), 1.498 (0.74), 1.519 (0.83), 1.719 (1.03), 1.741 (0.88), 1.902 (0.78), 1.908 (0.74), 1.922 (0.88), 1.928 (0.83), 1.943 (0.45), 1.978 (1.13), 1.994 (0.74), 2.102 (0.71), 2.112 (0.85), 2.123 (0.70), 2.571 (1.40), 2.591 (0.77), 2.882 (1.10), 3.018 (1.27), 3.035 (3.01), 3.053 (2.14), 3.239 (2.40), 3.254 (2.32), 3.368 (1.13), 3.379 (1.40), 3.391 (1.33), 3.403 (0.92), 3.493 (0.76), 4.463 (0.65), 4.482 (1.12), 4.500 (0.62), 7.033 (4.22), 7.048 (4.45), 7.074 (3.47), 7.077 (4.04), 7.100 (1.85), 7.103 (1.52), 7.113 (2.53), 7.117 (2.34), 7.162 (4.18), 7.175 (2.71), 7.439 (1.03), 7.481 (4.88), 7.495 (4.57), 7.526 (2.04), 7.613 (0.91), 7.952 (5.28).

Method B

1-{1-[4-Chloro-4′-(4-isobutylpiperazin-1-yl)[biphenyl]-2-yl]piperidin-3-yl}-5-(difluoromethyl)-1H-pyrazole-4-carboxylic acid hydrochloride (prepared in analogy to Example 3, Enantiomer 2, 31.2 mg, 51.3 μmol) were dissolved in 17 ml of dichloromethane and 1 ml of methanol. The solution was shaken once with 1.5 ml of saturated, aqueous sodium bicarbonate solution. The phases were separated. 5 ml of dichloromethane and 3 ml of methanol were added to the organic phase. The organic phase was then dried over sodium sulfate, filtered, evaporated and purified by preparative HPLC (RP18 column, acetonitrile/water gradient, neutral without acid addition). Product fractions were combined and lyophilized. 22 mg of the target compound (74% of theory) were obtained.

LC-MS (Method 3): Rt=1.73 min; MS (ESIpos): m/z=572 [M+H]+

1H-NMR (600 MHz, DMSO-d6) δ[ppm]: 0.887 (15.60), 0.898 (16.00), 1.493 (0.64), 1.514 (0.70), 1.695 (0.89), 1.718 (0.74), 1.799 (0.48), 1.811 (0.88), 1.822 (1.12), 1.833 (0.92), 1.844 (0.48), 1.890 (0.68), 1.910 (0.74), 1.977 (0.93), 1.995 (0.62), 2.118 (3.91), 2.130 (3.66), 2.516 (5.14), 3.017 (1.09), 3.035 (2.76), 3.053 (1.94), 3.181 (5.03), 3.185 (5.02), 3.267 (1.53), 4.473 (0.55), 4.491 (0.96), 4.509 (0.54), 6.963 (3.96), 6.977 (4.06), 7.048 (3.13), 7.051 (3.31), 7.081 (1.60), 7.084 (1.26), 7.095 (2.21), 7.098 (1.89), 7.152 (3.52), 7.165 (2.42), 7.434 (4.45), 7.448 (4.50), 7.533 (1.51), 7.621 (0.67), 7.930 (4.14).

Example 3

1-{1-[4-Chloro-4′-(4-isobutylpiperazin-1-yl)[biphenyl]-2-yl]piperidin-3-yl}-5-(difluoromethyl)-1H-pyrazole-4-carboxylic acid hydrochloride (Enantiomer 2)

Method A

A suspension of 1-[1-{4-chloro-4′-[4-(2-methylpropyl)piperazin-1-yl][1,1′-biphenyl]-2-yl}piperidin-3-yl]-5-(difluoromethyl)-1H-pyrazole-4-carboxylic acid (prepared in analogy to Example 2, Enantiomer 2, 43.5 g, 76.0 mmol) in diethyl ether (870 ml) was treated with a solution of hydrogen chloride in diethyl ether (84 ml, 1.0 M, 84 mmol). The resulting mixture was stirred overnight at room temperature and evaporated affording 46.1 g (quant.) of the title compound.

LC-MS (Method 3): Rt=1.72 min; MS (ESIpos): m/z=572 [M+H]+

1H-NMR (600 MHz, DMSO-d6) δ[ppm]: 1.026 (15.64), 1.037 (16.00), 1.497 (0.56), 1.519 (0.61), 1.722 (0.78), 1.743 (0.65), 1.903 (0.59), 1.910 (0.53), 1.924 (0.66), 1.930 (0.61), 1.978 (0.82), 1.994 (0.50), 2.142 (0.45), 2.154 (0.91), 2.165 (1.11), 2.176 (0.89), 2.187 (0.45), 2.557 (0.64), 2.577 (1.02), 2.594 (0.55), 2.992 (1.81), 3.002 (2.77), 3.012 (1.87), 3.018 (1.15), 3.036 (2.40), 3.054 (1.60), 3.133 (1.12), 3.148 (1.19), 3.168 (0.53), 3.237 (0.88), 3.250 (0.76), 3.338 (0.81), 3.360 (1.42), 3.379 (0.88), 3.580 (1.61), 3.791 (0.89), 3.819 (1.25), 3.844 (0.81), 4.463 (0.89), 4.474 (0.97), 4.481 (1.26), 4.488 (0.99), 4.499 (0.88), 7.051 (3.56), 7.065 (3.77), 7.077 (2.72), 7.080 (3.14), 7.103 (1.42), 7.106 (1.13), 7.116 (2.00), 7.120 (1.84), 7.165 (3.40), 7.178 (2.22), 7.443 (0.84), 7.489 (4.04), 7.504 (3.79), 7.531 (1.66), 7.618 (0.72), 7.954 (4.33), 10.519 (0.49).

Method B

Ethyl 1-[1-{5-chloro-2-[(trifluoromethanesulfonyl)oxy]phenyl}piperidin-3-yl]-5-(difluoromethyl)-1H-pyrazole-4-carboxylate (prepared in analogy to Example 14A, Enantiomer 2, 80.0 mg, 150 μmol) and 1-(2-methylpropyl)-4-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]piperazine (Example 18A 64.1 mg, 97% purity, 180 μmol) were dissolved under argon in toluene/ethanol (0.83/0.83 ml). Tetrakis(triphenylphosphine)palladium(0) (8.69 mg, 7.52 μmol) and 2 M sodium carbonate solution (226 μl, 452 μmol) were added and the mixture was stirred at 100° C. overnight. The reaction mixture was diluted with ethyl acetate and water. The aqueous phase was acidified with 1 M hydrochloric acid. The phases were separated and the aqueous phase was extracted twice with ethyl acetate. The combined organic phases were dried over sodium sulfate, filtered and evaporated. The crude product was dissolved in THF/ethanol (3.9/0.39 ml), 1 M aqueous lithium hydroxide solution (1.5 ml, 1.5 mmol) was added and the mixture was stirred overnight at room temperature. The mixture was evaporated, the residue was dissolved in acetonitrile/TFA/water and purified using preparative HPLC (RP18 column, acetonitrile/water gradient with the addition of 0.1% TFA). The product fractions were combined and evaporated. The residue was mixed with 0.1 M hydrochloric acid in dioxane, carefully evaporated at 30° C. (twice) and then lyophilized. 53 mg of the target compound (55% of theory, purity 95%) were obtained.

LC-MS (Method 4): Rt=0.91 min; MS (ESIpos): m/z=572 [M−HCl+H]+

1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.004 (15.46), 1.020 (16.00), 1.491 (0.44), 1.522 (0.50), 1.722 (0.68), 1.753 (0.55), 1.890 (0.47), 1.920 (0.55), 1.967 (0.84), 2.129 (0.76), 2.146 (0.96), 2.163 (0.76), 2.582 (0.91), 2.613 (0.48), 2.999 (0.86), 3.010 (1.71), 3.025 (3.88), 3.041 (2.30), 3.131 (0.88), 3.161 (1.25), 3.177 (2.08), 3.213 (1.75), 3.242 (1.16), 3.467 (1.06), 3.496 (0.84), 3.503 (0.60), 3.519 (0.54), 3.525 (0.50), 3.549 (0.75), 3.555 (0.84), 3.572 (1.57), 3.582 (1.48), 3.589 (1.38), 3.601 (2.78), 3.608 (1.89), 3.633 (0.44), 3.640 (0.41), 3.811 (0.94), 3.847 (1.32), 3.878 (0.71), 4.329 (0.49), 4.439 (0.46), 4.466 (0.73), 4.477 (0.52), 4.839 (0.49), 7.047 (3.30), 7.070 (3.64), 7.082 (2.61), 7.087 (3.29), 7.104 (1.46), 7.109 (0.86), 7.124 (2.34), 7.129 (2.03), 7.160 (3.99), 7.181 (1.96), 7.388 (0.88), 7.490 (4.02), 7.512 (3.81), 7.519 (2.20), 7.650 (0.72), 7.959 (3.78), 9.708 (0.41).

[α]D20=−73.05°, c=0.465 g/100 cm3, trichloromethane.

Example 3 Enantiomer 2 has an absolute configuration of R as shown in example 4 below.

1-{3(R)-1-[4-Chloro-4′-(4-isobutylpiperazin-1-yl)[biphenyl]-2-yl]piperidin-3-yl}-5-(difluoromethyl)-1H-pyrazole-4-carboxylic acid hydrochloride

Example 4

1-{3(R)-1-[4-Chloro-4′-(4-isobutylpiperazin-1-yl)[biphenyl]-2-yl]piperidin-3-yl}-5-(difluoromethyl)-1H-pyrazole-4-carboxylic acid hydrochloride hemihydrate

100 mg 1-{1-[4-Chloro-4′-(4-isobutylpiperazin-1-yl)[biphenyl]-2-yl]piperidin-3-yl}-5-(difluoromethyl)-1H-pyrazole-4-carboxylic acid hydrochloride (Enantiomer 2) (example 3) were solved at 60° C. in 3.5 ml 2-propanol, wherein the 2-propanol was dosed portionwise in 100 μl-portions at 60° C. until a clear solution was obtained. Afterwards the vessel was closed with a septum and placed into a slowly cooling sand bath from 60° C. to room temperature over the weekend->small amounts of solids were detected. Thereafter the septum was provided with a cannula, in order to slowly let the solvent evaporate. After 4 weeks crystals were collected and inspected under a microscope.

Single Crystal X-Ray Structure Analysis:

The Crystal structure determination was carried out using a Bruker diffractometer (QS-no.: 02506) equipped with an Apex II-CCD area detector, an IμS-microsource with CuKa radiation, mirrors as monochromator and a Cryostream low temperature device (T=110 K). Fullsphere data collection, omega and phi scans. Programs used: Data collection and reduction Apex II v2014.11.0 (Bruker AXS, 2014), absorption correction/scaling SADABS. Crystal structure solution was achieved using direct methods as implemented in SHELXTL Version 6.14 (Bruker AXS, 2003) and visualized using XP program. Missing atoms were subsequently located from difference Fourier synthesis and added to the atom list. Least-squares refinement on F2 using all measured intensities was carried out using the program SHELXTL Version 6.14 (Bruker AXS, 2003). All non hydrogen atoms were refined including anisotropic displacement parameters.

CorrectInvertedChirality Check*structurestructureFlack Parameter (standard deviation)0.094 (0.009)0.906 (0.009)wR2-value (with Flack Parameter)0.23570.2522ChiralityR(C22)S(C22)*H. D. Flack,Acta Cryst., 1983, A39, 876-881H. D. Flack, G. Bernardinelli,J. Appl. Cryst., 2000, 33, 1143-1148S. Parsons, H. D. Flack, T. Wagner,Acta Cryst., 2013, B69, 249-259.

TABLE 1Crystal data and structure refinement for example 4Identification codeexample 4Empirical formulaC60 H76 Cl4 F4 N10 O5Formula weight1235.10Temperature110KWavelength1.54178ÅCrystal systemTrigonalSpace groupP3221Unit cell dimensionsa = 9.8693(5)Åα = 90°.b = 9.8693(5)Åβ = 90°.c = 54.159(3)Åγ = 120°.Volume4568.5(5)Å3Z3Density (calculated)1.347Mg/m3Absorption coefficient2.341mm−1F(000)1950Crystal size0.14 × 0.10 × 0.06mm3Theta range for data collection4.899 to 63.664°.Index ranges−11 ≤ h ≤ 10, −10 ≤ k ≤ 11,−62 ≤ l ≤ 61Reflections collected27868Independent reflections4640 [R(int) = 0.0378]Completeness to theta = 63.664°95.9%Absorption correctionSemi-empirical from equivalentsMax. and min. transmission0.87 and 0.74Refinement methodFull-matrix least-squares on F2Data/restraints/parameters4640/11/593Goodness-of-fit on F21.047Final R indices [I > 2sigma(I)]R1 = 0.0848, wR2 = 0.2336R indices (all data)R1 = 0.0864, wR2 = 0.2357Absolute structure parameter0.094(9)Extinction coefficientn/aLargest diff. peak and hole0.601 and −0.650 e · Å−3

TABLE 2Bond lengths [Å] and angles [°] for example 4.Cl(2)-C(3)1.767(13)N(4)-C(22)1.47(2)Cl(2′)-C(3′)1.772(13)N(5)-C(28)1.37(2)F(1)-C(30)1.341(7)N(3′)-C(1′)1.38(3)F(2)-C(30)1.339(7)N(3′)-C(21′)1.44(4)F(1′)-C(30′)1.339(7)N(3′)-C(25′)1.46(2)F(2′)-C(30′)1.38(2)N(4′)-N(5′)1.38(3)O(1)-C(29)1.22(2)N(4′)-C(26′)1.42(3)O(2)-C(29)1.30(2)N(4′)-C(22′)1.46(2)O(2)-H(2A)0.8400N(5′)-C(28′)1.32(2)O(1′)-C(29′)1.17(2)C(1)-C(6)1.35(3)O(2′)-C(29′)1.36(2)C(1)-C(2)1.42(4)O(2′)-H(2B)0.8400C(2)-C(3)1.37(3)N(1)-C(10)1.416(9)C(2)-H(2D)0.9500N(1)-C(16)1.434(12)C(3)-C(4)1.33(2)N(1)-C(13)1.470(10)C(4)-C(5)1.390(19)N(2)-C(14)1.497(9)C(4)-H(4A)0.9500N(2)-C(15)1.498(9)C(5)-C(6)1.41(2)N(2)-C(17)1.512(8)C(5)-H(5A)0.9500N(2)-H(2C)1.0000C(6)-C(7)1.506(17)N(3)-C(25)1.46(2)C(7)-C(8)1.36(2)N(3)-C(21)1.46(5)C(7)-C(12)1.382(19)N(3)-C(1)1.47(3)C(7)-C(6′)1.58(2)N(4)-C(26)1.30(3)C(8)-C(9)1.378(13)N(4)-N(5)1.32(3)C(8)-H(8A)0.9500C(9)-C(10)1.390(15)C(21)-H(21A)0.9900C(9)-H(9A)0.9500C(21)-H(21B)0.9900C(10)-C(11)1.390(16)C(22)-C(23)1.56(2)C(11)-C(12)1.391(11)C(22)-H(22A)1.0000C(11)-H(11A)0.9500C(23)-C(24)1.52(3)C(12)-H(12A)0.9500C(23)-H(23A)0.9900C(13)-C(14)1.524(10)C(23)-H(23B)0.9900C(13)-H(13A)0.9900C(24)-C(25)1.52(2)C(13)-H(13B)0.9900C(24)-H(24A)0.9900C(14)-H(14A)0.9900C(24)-H(24B)0.9900C(14)-H(14B)0.9900C(25)-H(25A)0.9900C(15)-C(16)1.519(10)C(25)-H(25B)0.9900C(15)-H(15A)0.9900C(26)-C(27)1.42(2)C(15)-H(15B)0.9900C(26)-C(30)1.500(7)C(16)-H(16A)0.9900C(27)-C(28)1.34(3)C(16)-H(16B)0.9900C(27)-C(29)1.50(3)C(17)-C(18)1.499(10)C(28)-H(28A)0.9500C(17)-H(17A)0.9900C(30)-H(30A)1.0000C(17)-H(17B)0.9900C(1′)-C(2′)1.39(3)C(18)-C(20)1.509(11)C(1′)-C(6′)1.42(2)C(18)-C(19)1.538(10)C(2′)-C(3′)1.39(3)C(18)-H(18A)1.0000C(2′)-H(2E)0.9500C(19)-H(19A)0.9800C(3′)-C(4′)1.36(2)C(19)-H(19B)0.9800C(4′)-C(5′)1.392(19)C(19)-H(19C)0.9800C(4′)-H(4B)0.9500C(20)-H(20A)0.9800C(5′)-C(6′)1.40(2)C(20)-H(20B)0.9800C(5′)-H(5B)0.9500C(20)-H(20C)0.9800C(21′)-C(22′)1.59(2)C(21)-C(22)1.541(7)C(21′)-H(21C)0.9900C(21′)-H(21D)0.9900C(25′)-H(25D)0.9900C(22′)-C(23′)1.52(2)C(26′)-C(27′)1.35(3)C(22′)-H(22B)1.0000C(26′)-C(30′)1.46(3)C(23′)-C(24′)1.52(2)C(27′)-C(28′)1.41(2)C(23′)-H(23C)0.9900C(27′)-C(29′)1.50(3)C(23′)-H(23D)0.9900C(28′)-H(28B)0.9500C(24′)-C(25′)1.55(2)C(30′)-H(30B)1.0000C(24′)-H(24C)0.9900O(1W)-H(1W)0.9010C(24′)-H(24D)0.9900O(1W)-H(1W)#10.9010C(25′)-H(25C)0.9900C(29)-O(2)-H(2A)109.5C(1′)-N(3′)-C(21′)112.1(19)C(29′)-O(2′)-H(2B)109.5C(1′)-N(3′)-C(25′)117.2(19)C(10)-N(1)-C(16)117.9(8)C(21′)-N(3′)-C(25′)119.2(19)C(10)-N(1)-C(13)113.5(6)N(5′)-N(4′)-C(26′)109(2)C(16)-N(1)-C(13)109.6(5)N(5′)-N(4′)-C(22′)118.1(15)C(14)-N(2)-C(15)109.2(5)C(26′)-N(4′)-C(22′)128(2)C(14)-N(2)-C(17)108.8(5)C(28′)-N(5′)-N(4′)106.9(15)C(15)-N(2)-C(17)113.0(5)C(6)-C(1)-C(2)119(2)C(14)-N(2)-H(2C)108.6C(6)-C(1)-N(3)120.5(18)C(15)-N(2)-H(2C)108.6C(2)-C(1)-N(3)120(2)C(17)-N(2)-H(2C)108.6C(3)-C(2)-C(1)118.4(19)C(25)-N(3)-C(21)107(2)C(3)-C(2)-H(2D)120.8C(25)-N(3)-C(1)116.5(18)C(1)-C(2)-H(2D)120.8C(21)-N(3)-C(1)112.2(18)C(4)-C(3)-C(2)123.8(15)C(26)-N(4)-N(5)113(2)C(4)-C(3)-Cl(2)120.9(12)C(26)-N(4)-C(22)127(2)C(2)-C(3)-Cl(2)115.1(14)N(5)-N(4)-C(22)120(2)C(3)-C(4)-C(5)117.5(14)N(4)-N(5)-C(28)104(2)C(3)-C(4)-H(4A)121.3C(5)-C(4)-H(4A)121.3C(14)-C(13)-H(13A)109.5C(4)-C(5)-C(6)121.0(15)N(1)-C(13)-H(13B)109.5C(4)-C(5)-H(5A)119.5C(14)-C(13)-H(13B)109.5C(6)-C(5)-H(5A)119.5H(13A)-C(13)-H(13B)108.1C(1)-C(6)-C(5)119.5(15)N(2)-C(14)-C(13)110.7(6)C(1)-C(6)-C(7)112.0(17)N(2)-C(14)-H(14A)109.5C(5)-C(6)-C(7)128.4(16)C(13)-C(14)-H(14A)109.5C(8)-C(7)-C(12)115.2(8)N(2)-C(14)-H(14B)109.5C(8)-C(7)-C(6)109.3(13)C(13)-C(14)-H(14B)109.5C(12)-C(7)-C(6)135.5(15)H(14A)-C(14)-H(14B)108.1C(8)-C(7)-C(6′)136.3(13)N(2)-C(15)-C(16)110.4(6)C(12)-C(7)-C(6′)108.4(14)N(2)-C(15)-H(15A)109.6C(7)-C(8)-C(9)124.1(12)C(16)-C(15)-H(15A)109.6C(7)-C(8)-H(8A)118.0N(2)-C(15)-H(15B)109.6C(9)-C(8)-H(8A)118.0C(16)-C(15)-H(15B)109.6C(8)-C(9)-C(10)120.2(13)H(15A)-C(15)-H(15B)108.1C(8)-C(9)-H(9A)119.9N(1)-C(16)-C(15)112.1(7)C(10)-C(9)-H(9A)119.9N(1)-C(16)-H(16A)109.2C(9)-C(10)-C(11)117.3(8)C(15)-C(16)-H(16A)109.2C(9)-C(10)-N(1)121.7(10)N(1)-C(16)-H(16B)109.2C(11)-C(10)-N(1)120.9(9)C(15)-C(16)-H(16B)109.2C(10)-C(11)-C(12)120.2(11)H(16A)-C(16)-H(16B)107.9C(10)-C(11)-H(11A)119.9C(18)-C(17)-N(2)115.7(5)C(12)-C(11)-H(11A)119.9C(18)-C(17)-H(17A)108.4C(7)-C(12)-C(11)123.0(13)N(2)-C(17)-H(17A)108.4C(7)-C(12)-H(12A)118.5C(18)-C(17)-H(17B)108.4C(11)-C(12)-H(12A)118.5N(2)-C(17)-H(17B)108.4N(1)-C(13)-C(14)110.8(6)H(17A)-C(17)-H(17B)107.4N(1)-C(13)-H(13A)109.5C(17)-C(18)-C(20)114.1(6)C(17)-C(18)-C(19)108.2(6)C(24)-C(23)-C(22)108.9(13)C(20)-C(18)-C(19)110.6(6)C(24)-C(23)-H(23A)109.9C(17)-C(18)-H(18A)107.9C(22)-C(23)-H(23A)109.9C(20)-C(18)-H(18A)107.9C(24)-C(23)-H(23B)109.9C(19)-C(18)-H(18A)107.9C(22)-C(23)-H(23B)109.9C(18)-C(19)-H(19A)109.5H(23A)-C(23)-H(23B)108.3C(18)-C(19)-H(19B)109.5C(23)-C(24)-C(25)112.6(13)H(19A)-C(19)-H(19B)109.5C(23)-C(24)-H(24A)109.1C(18)-C(19)-H(19C)109.5C(25)-C(24)-H(24A)109.1H(19A)-C(19)-H(19C)109.5C(23)-C(24)-H(24B)109.1H(19B)-C(19)-H(19C)109.5C(25)-C(24)-H(24B)109.1C(18)-C(20)-H(20A)109.5H(24A)-C(24)-H(24B)107.8C(18)-C(20)-H(20B)109.5N(3)-C(25)-C(24)107.3(15)H(20A)-C(20)-H(20B)109.5N(3)-C(25)-H(25A)110.3C(18)-C(20)-H(20C)109.5C(24)-C(25)-H(25A)110.3H(20A)-C(20)-H(20C)109.5N(3)-C(25)-H(25B)110.3H(20B)-C(20)-H(20C)109.5C(24)-C(25)-H(25B)110.3N(3)-C(21)-C(22)106(3)H(25A)-C(25)-H(25B)108.5N(3)-C(21)-H(21A)110.4N(4)-C(26)-C(27)107.8(18)C(22)-C(21)-H(21A)110.4N(4)-C(26)-C(30)124(2)N(3)-C(21)-H(21B)110.4C(27)-C(26)-C(30)127.8(16)C(22)-C(21)-H(21B)110.4C(28)-C(27)-C(26)102.7(18)H(21A)-C(21)-H(21B)108.6C(28)-C(27)-C(29)133(2)N(4)-C(22)-C(21)110(2)C(26)-C(27)-C(29)124.0(19)N(4)-C(22)-C(23)106.8(16)C(27)-C(28)-N(5)112.9(19)C(21)-C(22)-C(23)105(2)C(27)-C(28)-H(28A)123.6N(4)-C(22)-H(22A)111.7N(5)-C(28)-H(28A)123.6C(21)-C(22)-H(22A)111.7O(1)-C(29)-O(2)123(2)C(23)-C(22)-H(22A)111.7O(1)-C(29)-C(27)125.0(19)O(2)-C(29)-C(27)112(2)C(22′)-C(21′)-H(21D)109.9F(2)-C(30)-F(1)104.4(13)H(21C)-C(21′)-H(21D)108.3F(2)-C(30)-C(26)112.1(18)N(4′)-C(22′)-C(23′)108.7(16)F(1)-C(30)-C(26)110.6(17)N(4′)-C(22′)-C(21′)111.0(16)F(2)-C(30)-H(30A)109.9C(23′)-C(22′)-C(21′)117.6(19)F(1)-C(30)-H(30A)109.9N(4′)-C(22′)-H(22B)106.3C(26)-C(30)-H(30A)109.9C(23′)-C(22′)-H(22B)106.3N(3′)-C(1′)-C(2′)119.2(17)C(21′)-C(22′)-H(22B)106.3N(3′)-C(1′)-C(6′)120.3(18)C(22′)-C(23′)-C(24′)107.4(15)C(2′)-C(1′)-C(6′)120(2)C(22′)-C(23′)-H(23C)110.2C(1′)-C(2′)-C(3′)118.4(18)C(24′)-C(23′)-H(23C)110.2C(1′)-C(2′)-H(2E)120.8C(22′)-C(23′)-H(23D)110.2C(3′)-C(2′)-H(2E)120.8C(24′)-C(23′)-H(23D)110.2C(4′)-C(3′)-C(2′)125.1(15)H(23C)-C(23′)-H(23D)108.5C(4′)-C(3′)-Cl(2′)118.0(12)C(23′)-C(24′)-C(25′)114.0(14)C(2′)-C(3′)-Cl(2′)116.8(12)C(23′)-C(24′)-H(24C)108.8C(3′)-C(4′)-C(5′)114.4(13)C(25′)-C(24′)-H(24C)108.8C(3′)-C(4′)-H(4B)122.8C(23′)-C(24′)-H(24D)108.8C(5′)-C(4′)-H(4B)122.8C(25′)-C(24′)-H(24D)108.8C(4′)-C(5′)-C(6′)125.3(14)H(24C)-C(24′)-H(24D)107.7C(4′)-C(5′)-H(5B)117.3N(3′)-C(25′)-C(24′)106.9(15)C(6′)-C(5′)-H(5B)117.3N(3′)-C(25′)-H(25° C.)110.3C(5′)-C(6′)-C(1′)116.2(16)C(24′)-C(25′)-H(25° C.)110.3C(5′)-C(6′)-C(7)109.8(15)N(3′)-C(25′)-H(25D)110.3C(1′)-C(6′)-C(7)131.7(15)C(24′)-C(25′)-H(25D)110.3N(3′)-C(21′)-C(22′)109(2)H(25C)-C(25′)-H(25D)108.6N(3′)-C(21′)-H(21C)109.9C(27′)-C(26′)-N(4′)105.4(19)C(22′)-C(21′)-H(21C)109.9C(27′)-C(26′)-C(30′)134.7(19)N(3′)-C(21′)-H(21D)109.9N(4′)-C(26′)-C(30′)120(3)C(26′)-C(27′)-C(28′)108.0(15)F(1′)-C(30′)-F(2′)107.3(18)C(26′)-C(27′)-C(29′)128.4(19)F(1′)-C(30′)-C(26′)111.2(19)C(28′)-C(27′)-C(29′)123.1(17)F(2′)-C(30′)-C(26′)112.0(17)N(5′)-C(28′)-C(27′)110.3(16)F(1′)-C(30′)-H(30B)108.7N(5′)-C(28′)-H(28B)124.8F(2′)-C(30′)-H(30B)108.7C(27′)-C(28′)-H(28B)124.8C(26′)-C(30′)-H(30B)108.7O(1′)-C(29′)-O(2′)126.1(19)H(1W)-O(1W)-H(1W)#1107.2O(1′)-C(29′)-C(27′)124.4(16)O(2′)-C(29′)-C(27′)109.4(19)Symmetry transformations used to generate equivalent atoms: #1 y − 1, x + 1, −z + 1

TABLE 3Torsion angles [°] for example 4C(26)-N(4)-N(5)-C(28)4(2)N(3)-C(1)-C(6)-C(7)−5(2)C(22)-N(4)-N(5)-C(28)−173.4(17)C(4)-C(5)-C(6)-C(1)6(2)C(26′)-N(4′)-N(5′)-C(28′)0(2)C(4)-C(5)-C(6)-C(7)−171.3(14)C(22′)-N(4′)-N(5′)-C(28′)−157.8(16)C(1)-C(6)-C(7)-C(8)148.5(14)C(25)-N(3)-C(1)-C(6)148.8(17)C(5)-C(6)-C(7)-C(8)−34.4(18)C(21)-N(3)-C(1)-C(6)−87(3)C(1)-C(6)-C(7)-C(12)−33.3(19)C(25)-N(3)-C(1)-C(2)−25(3)C(5)-C(6)-C(7)-C(12)143.8(15)C(21)-N(3)-C(1)-C(2)99(3)C(12)-C(7)-C(8)-C(9)2.5(13)C(6)-C(1)-C(2)-C(3)9(3)C(6)-C(7)-C(8)-C(9)−178.9(9)N(3)-C(1)-C(2)-C(3)−177.2(18)C(6′)-C(7)-C(8)-C(9)178.8(11)C(1)-C(2)-C(3)-C(4)−7(3)C(7)-C(8)-C(9)-C(10)−1.1(13)C(1)-C(2)-C(3)-Cl(2)178.6(14)C(8)-C(9)-C(10)-C(11)−1.0(11)C(2)-C(3)-C(4)-C(5)5(3)C(8)-C(9)-C(10)-N(1)−179.4(7)Cl(2)-C(3)-C(4)-C(5)178.8(12)C(16)-N(1)-C(10)-C(9)−176.9(7)C(3)-C(4)-C(5)-C(6)−4(2)C(13)-N(1)-C(10)-C(9)−46.9(9)C(2)-C(1)-C(6)-C(5)−8(3)C(16)-N(1)-C(10)-C(11)4.8(10)N(3)-C(1)-C(6)-C(5)178.0(16)C(2)-C(1)-C(6)-C(7)169.6(16)C(13)-N(1)-C(10)-C(11)134.9(8)C(9)-C(10)-C(11)-C(12)1.5(12)C(21)-C(22)-C(23)-C(24)−57(2)N(1)-C(10)-C(11)-C(12)179.9(7)C(22)-C(23)-C(24)-C(25)53.5(18)C(8)-C(7)-C(12)-C(11)−1.9(13)C(21)-N(3)-C(25)-C(24)67(2)C(6)-C(7)-C(12)-C(11)179.9(11)C(1)-N(3)-C(25)-C(24)−166.6(17)C(6′)-C(7)-C(12)-C(11)−179.2(10)C(10)-C(11)-C(12)-C(7)0.0(14)C(23)-C(24)-C(25)-N(3)−56.8(19)C(10)-N(1)-C(13)-C(14)167.0(7)N(5)-N(4)-C(26)-C(27)−3(2)C(16)-N(1)-C(13)-C(14)−58.8(8)C(22)-N(4)-C(26)-C(27)174.2(19)C(15)-N(2)-C(14)-C(13)−55.6(7)N(5)-N(4)-C(26)-C(30)179.8(15)C(17)-N(2)-C(14)-C(13)−179.3(6)C(22)-N(4)-C(26)-C(30)−3(3)N(1)-C(13)-C(14)-N(2)57.9(8)N(4)-C(26)-C(27)-C(28)1(2)C(14)-N(2)-C(15)-C(16)55.1(8)C(30)-C(26)-C(27)-C(28)177.7(15)C(17)-N(2)-C(15)-C(16)176.4(6)N(4)-C(26)-C(27)-C(29)−175.0(16)C(10)-N(1)-C(16)-C(15)−168.9(6)C(30)-C(26)-C(27)-C(29)2(3)C(13)-N(1)-C(16)-C(15)59.3(7)C(26)-C(27)-C(28)-N(5)2(2)N(2)-C(15)-C(16)-N(1)−58.4(8)C(29)-C(27)-C(28)-N(5)176.9(18)C(14)-N(2)-C(17)-C(18)178.4(6)N(4)-N(5)-C(28)-C(27)−3.6(19)C(15)-N(2)-C(17)-C(18)56.9(8)C(28)-C(27)-C(29)-O(1)146.4(19)N(2)-C(17)-C(18)-C(20)58.0(8)C(26)-C(27)-C(29)-O(1)−39(3)N(2)-C(17)-C(18)-C(19)−178.5(6)C(28)-C(27)-C(29)-O(2)−31(3)C(25)-N(3)-C(21)-C(22)−75(3)C(26)-C(27)-C(29)-O(2)143(2)C(1)-N(3)-C(21)-C(22)156(2)N(4)-C(26)-C(30)-F(2)53(2)C(26)-N(4)-C(22)-C(21)131(3)C(27)-C(26)-C(30)-F(2)−124(2)N(5)-N(4)-C(22)-C(21)−52(3)N(4)-C(26)-C(30)-F(1)−63(2)C(26)-N(4)-C(22)-C(23)−116(2)C(27)-C(26)-C(30)-F(1)120(2)N(5)-N(4)-C(22)-C(23)61(2)C(21′)-N(3′)-C(1′)-C(2′)112(2)N(3)-C(21)-C(22)-N(4)−177(2)C(25′)-N(3′)-C(1′)-C(2′)−31(3)N(3)-C(21)-C(22)-C(23)68(3)C(21′)-N(3′)-C(1′)-C(6′)−71(2)N(4)-C(22)-C(23)-C(24)−173.8(14)C(25′)-N(3′)-C(1′)-C(6′)146.4(17)N(3′)-C(1′)-C(2′)-C(3′)180.0(19)N(4′)-C(22′)-C(23′)-C(24′)−173.4(15)C(6′)-C(1′)-C(2′)-C(3′)3(3)C(21′)-C(22′)-C(23′)-C(24′)−46(2)C(1′)-C(2′)-C(3′)-C(4′)2(3)C(22′)-C(23′)-C(24′)-C(25′)55(2)C(1′)-C(2′)-C(3′)-Cl(2′)179.1(15)C(1′)-N(3′)-C(25′)-C(24′)−161.9(18)C(2′)-C(3′)-C(4′)-C(5′)−4(3)C(21′)-N(3′)-C(25′)-C(24′)58(2)Cl(2′)-C(3′)-C(4′)-C(5′)179.0(12)C(23′)-C(24′)-C(25′)-N(3′)−59(2)C(3′)-C(4′)-C(5′)-C(6′)1(2)N(5′)-N(4′)-C(26′)-C(27′)−1(2)C(4′)-C(5′)-C(6′)-C(1′)4(3)C(22′)-N(4′)-C(26′)-C(27′)154(2)C(4′)-C(5′)-C(6′)-C(7)168.4(15)N(5′)-N(4′)-C(26′)-C(30′)−176.3(16)N(3′)-C(1′)-C(6′)-C(5′)177.6(19)C(22′)-N(4′)-C(26′)-C(30′)−21(3)C(2′)-C(1′)-C(6′)-C(5′)−5(3)N(4′)-C(26′)-C(27′)-C(28′)1.2(19)N(3′)-C(1′)-C(6′)-C(7)17(3)C(30′)-C(26′)-C(27′)-C(28′)175.6(19)C(2′)-C(1′)-C(6′)-C(7)−166.3(19)N(4′)-C(26′)-C(27′)-C(29′)−171.1(16)C(8)-C(7)-C(6′)-C(5′)−39.2(19)C(30′)-C(26′)-C(27′)-C(29′)3(3)C(12)-C(7)-C(6′)-C(5′)137.2(12)N(4′)-N(5′)-C(28′)-C(27′)1(2)C(8)-C(7)-C(6′)-C(1′)122.5(19)C(26′)-C(27′)-C(28′)-N(5′)−1(2)C(12)-C(7)-C(6′)-C(1′)−61(2)C(29′)-C(27′)-C(28′)-N(5′)171.6(15)C(1′)-N(3′)-C(21′)-C(22′)168.4(18)C(26′)-C(27′)-C(29′)-O(1′)162.9(18)C(25′)-N(3′)-C(21′)-C(22′)−49(3)C(28′)-C(27′)-C(29′)-O(1′)−8(3)N(5′)-N(4′)-C(22′)-C(23′)65(2)C(26′)-C(27′)-C(29′)-O(2′)−21(2)C(26′)-N(4′)-C(22′)-C(23′)−88(3)C(28′)-C(27′)-C(29′)-O(2′)167.6(16)N(5′)-N(4′)-C(22′)-C(21′)−66(3)C(27′)-C(26′)-C(30′)-F(1′)132(2)C(26′)-N(4′)-C(22′)-C(21′)141(2)N(4′)-C(26′)-C(30′)-F(1′)−54(2)N(3′)-C(21′)-C(22′)-N(4′)169.0(19)C(27′)-C(26′)-C(30′)-F(2′)−108(2)N(3′)-C(21′)-C(22′)-C(23′)43(3)N(4′)-C(26′)-C(30′)-F(2′)66(2)Symmetry transformations used to generate equivalent atoms: #1 y − 1, x + 1, −z + 1

TABLE 4Hydrogen bonds for example 4 [Å and °].D—Hd(D—H)d(H . . . A)<DHAd(D . . . A)AO2{circumflex over ( )}a—H2A{circumflex over ( )}a0.8402.268171.523.102Cl1 [x + 1, y − 1, z]O2′{circumflex over ( )}b—H2B{circumflex over ( )}b0.8402.219158.793.018Cl1 [x + 1, y − 1, z]N2—H2C1.0002.158162.743.128Cl1 [y, x, −z + 1]O1W—H1W0.9012.448164.203.324Cl1

FIG.1shows an Ortep-Plot (50%) with labeling scheme (without disorder), as defined in example 4.

FIG.2shows independent molecules in the asymmetric unit (with disorder), as defined in example 4.

FIG.3shows the configuration of C22, as defined in example 4.

Comparative Example 174 (WO2012/058132)

1-{1-[4-Chloro-4′-(4-cyclopropylmethylpiperazin-1-yl)[biphenyl]-2-yl]pyridin-3-yl}-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic acid

The compound was synthesized according to the procedures disclosed in WO 2012/058132 (experimental part, pages 58 to 84).

B. Assessment of Pharmacological Efficacy and Pharmacokinetic Profile

The following abbreviations are used:ATP adenosine triphosphateBrij35 polyoxyethylene(23) lauryl etherBSA bovine serum albumin:DTT dithiothreitolTEA triethanolamine
Biological Investigations

The example testing experiments described herein serve to illustrate the present invention and the invention is not limited to the examples given.

The following assays can be used to illustrate the commercial utility of the compounds according to the present invention.

Examples were tested in selected biological assays one or more times. When tested more than once, data are reported as either average values or as median values, whereinthe average value, also referred to as the arithmetic mean value, represents the sum of the values obtained divided by the number of times tested, andthe median value represents the middle number of the group of values when ranked in ascending or descending order. If the number of values in the data set is odd, the median is the middle value. If the number of values in the data set is even, the median is the arithmetic mean of the two middle values.

Examples were synthesized one or more times. When synthesized more than once, data from biological assays represent average values calculated utilizing data sets obtained from testing of one or more synthetic batch.

The in vitro activity of the compounds of the present invention can be demonstrated in the following assays.

The pharmacological action of the compounds of the invention can be demonstrated in the following assays:

B-1. Effect on a Recombinant Guanylate Cyclase Reporter Cell Line

The cellular activity of the compounds according to the invention was determined using a recombinant guanylate cyclase reporter cell line, as described in F. Wunder et al.,Anal. Biochem.339, 104-112 (2005).

Representative MEC values (MEC=minimum effective concentration) and EC50values (half maximal effective concentration) for the compounds of the invention are shown in the table below (in some cases as mean values from individual determinations):

TABLE 2ExampleMEC [nM]EC50[nM]12.39.221.08.630.62.7

B-2. Vasorelaxant Effect In Vitro

Rabbits were killed in deep anaesthesia and exsanguinated. The aorta was removed, freed from adhering tissue and divided into rings of width 1.5 mm, which were placed individually under prestress into 5 ml organ baths with carbogen-sparged Krebs-Henseleit solution at 37° C. having the following composition (each in mM): sodium chloride: 119; potassium chloride: 4.8; calcium chloride dihydrate: 1; magnesium sulfate heptahydrate: 1.4; potassium dihydrogenphosphate: 1.2; sodium bicarbonate: 25; glucose: 10. To generate a contraction, phenylephrine was added to the bath cumulatively in increasing concentration. After several control cycles, the substance to be studied was added in increasing dosage each time in every further run, and the magnitude of the contraction was compared with the magnitude of the contraction attained in the last preceding run. This was used to calculate the concentration needed to reduce the magnitude of the control value by 50% (IC50value). The standard administration volume was 5 μl; the DMSO content in the bath solution corresponds to 0.1%.

B-3. Blood Pressure Measurement on Anaesthetized Rats

Male Wistar rats having a body weight of 300-350 g were anaesthetized with thiopental (100 mg/kg i.p.). After tracheotomy, a catheter was introduced into the femoral artery to measure the blood pressure. The substances to be tested were administered as solutions, either orally by means of a gavage or intravenously via the femoral vein (Stasch et al. Br. J. Pharmacol. 2002; 135: 344-355).

B-4. Radiotelemetry Measurement of Blood Pressure in Conscious, Spontaneously Hypertensive Rats

A commercially available telemetry system from DATA SCIENCES INTERNATIONAL DSI, USA, was employed for the blood pressure measurement on conscious rats described below.

The system consists of 3 main components:implantable transmitters (Physiotel® telemetry transmitter)receivers (Physiotel® receiver) which are linked via a multiplexer (DSI Data Exchange Matrix 2.0) to adata acquisition computer.

The telemetry system makes it possible to continuously record blood pressure, heart rate and body motion of conscious animals in their usual habitat.

Animal Material

The studies were conducted on adult female spontaneously hypertensive rats (SHR Okamoto) with a body weight of >200 g. SHR/NCrl from the Okamoto Kyoto School of Medicine, 1963, were a cross of male Wistar Kyoto rats having greatly elevated blood pressure and female rats having slightly elevated blood pressure, and were handed over at F13 to the U.S. National Institutes of Health.

After transmitter implantation, the experimental animals were housed singly in type 3 Makrolon cages. They had free access to standard feed and water.

The day/night rhythm in the experimental laboratory was changed by the room lighting at 6:00 am and at 7:00 μm.

Transmitter Implantation

The HD S 10 telemetry transmitters used were surgically implanted under aseptic conditions in the experimental animals at least 14 days before the first experimental use. The animals instrumented in this way can be used repeatedly after the wound has healed and the implant has settled.

For the implantation, the fasted animals were anesthetized with isoflurane (Rimadyl analgesia) and shaved and disinfected over a large area of their abdomens. After the abdominal cavity had been opened along the linea alba, the liquid-filled measuring catheter of the system was inserted into the descending aorta in the cranial direction above the bifurcation and fixed with tissue glue (VetBonD TM, 3M). The transmitter housing was fixed intraperitoneally to the abdominal wall muscle, and the wound was closed layer by layer.

An antibiotic (Ursocyclin 10% pro inj., Serumwerk, s.c.) was administered postoperatively for prophylaxis of infection.

Substances and Solutions

Unless stated otherwise, the substances to be studied were administered orally by gavage to a group of animals in each case (n=6). In accordance with an administration volume of 2 ml/kg of body weight, the test substances were dissolved in suitable solvent mixtures or suspended in 0.5% tylose.

A solvent-treated group of animals was used as control.

Experimental Procedure

The telemetry measuring unit present was configured for 24 animals. Each experiment was recorded under an experiment number (Vyear month day).

Each of the instrumented rats living in the system was assigned a separate receiving antenna (RPC-1 Receiver, DSI).

The implanted transmitters can be activated externally by means of an incorporated magnetic switch. They were switched to transmission in the run-up to the experiment. The signals emitted could be detected online by a data acquisition system (Physio Tel HD, DSI) and processed accordingly. The data were stored in each case in a file created for this purpose and bearing the experiment number.

In the standard procedure, the following were measured for 10-second periods in each case:systolic blood pressure (SBP)diastolic blood pressure (DBP)mean arterial pressure (MAP)heart rate (HR)activity (TEMP).

The acquisition of measurements was repeated under computer control at 5-minute intervals. The source data obtained as absolute values were corrected in the diagram with the currently measured barometric pressure (Ambient Pressure Reference Monitor; APR-1) and stored as individual data. Further technical details is given in the extensive documentation from the manufacturer company (DSI).

Unless indicated otherwise, the test substances were administered at 9:00 am on the day of the experiment. Following the administration, the parameters described above were measured over 24 hours.

Evaluation

After the end of the experiment, the acquired individual data were sorted using the analysis software (Ponemah V 6.x). The blank value was assumed here to be the time 2 hours before administration, and so the selected data set encompasses the period from 7:00 am on the day of the experiment to 9:00 am on the following day.

The data were smoothed over a predefinable period by determination of the average (30-minute average) and transferred as an excel file to a storage medium. The measured values presorted and compressed in this way were transferred to Excel templates and tabulated. For each day of the experiment, the data obtained were stored in a dedicated file bearing the number of the experiment. Results and test protocols were stored in files in paper form sorted by numbers.

LITERATURE

Klaus Witte, Kai Hu, Johanna Swiatek, Claudia Müssig, Georg Ertl and Björn Lemmer: Experimental heart failure in rats: effects on cardiovascular circadian rhythms and on myocardial β-adrenergic signaling. Cardiovasc Res 47 (2): 203-405, 2000; Kozo Okamoto: Spontaneous hypertension in rats. Int Rev Exp Pathol 7: 227-270, 1969; Maarten van den Buuse: Circadian Rhythms of Blood Pressure, Heart Rate, and Locomotor Activity in Spontaneously Hypertensive Rats as Measured With Radio-Telemetry. Physiology & Behavior 55(4): 783-787, 1994.

B-5. Determination of Pharmacokinetic Parameters Following Intravenous and Oral Administration

The pharmacokinetic parameters of the compounds according to the invention were determined in male Wistar rats and and/or in female beagles and/or in cynomolgus monkeys and/or in male CD-1 mice. Intravenous administration in the case of mice and rats was carried out by means of a species-specific plasma/DMSO formulation, and in the case of dogs and monkeys by means of a water/PEG400/ethanol formulation. In all species, oral administration of the dissolved substance was performed via gavage, based on a water/PEG400/ethanol formulation.

An internal standard (which may also be a chemically unrelated substance) was added to the samples of the compounds of the invention, calibration samples and qualifiers, and there followed protein precipitation by means of acetonitrile in excess. Addition of a buffer solution matched to the LC conditions, and subsequent vortexing, was followed by centrifugation at 1000 g. The supernatant was analysed by LC-MS/MS using C18 reversed-phase columns and variable mobile phase mixtures. The substances were quantified via the peak heights or areas from extracted ion chromatograms of specific selected ion monitoring experiments.

The plasma concentration/time plots determined were used to calculate the pharmacokinetic parameters such as AUC, Cmax, t1/2(terminal half-life), F (bioavailability), MRT (mean residence time) and CL (clearance), by means of a validated pharmacokinetic calculation program.

Since the substance quantification was performed in plasma, it was necessary to determine the blood/plasma distribution of the substance in order to be able to adjust the pharmacokinetic parameters correspondingly. For this purpose, a defined amount of substance was incubated in K3 EDTA whole blood of the species in question in a rocking roller mixer for 20 min. After centrifugation at 1000 g, the plasma concentration was measured (by means of LC-MS/MS; see above) and determined by calculating the ratio of the Cblood/Cplasmavalue.

Table 3 shows data of representative compounds of the present invention following intravenous administration in rats:

TABLE 3AUCnormCLplasmat1/2MRTExample[kg · h/L][L/h/kg][h][h]11.770.561.642.2427.080.143.133.44174 (WO2012/0.771.302.332.78058132)

Table 4 shows data of representative compounds of the present invention following oral administration (p.o.) in rats:

TABLE 4AUCnormt1/2MRTFExample[kg · h/L][h][h][%]10.573.246.2831.423.773.966.2353.3174 (WO2012/0.633.608.4081.8058132)

Table 5 shows data of representative compounds of the present invention following intravenous administration in dogs:

TABLE 5AUCnormCLplasmat1/2MRTExample[kg · h/L][L/h/kg][h][h]281.70.0117.725.6174 (WO2012/5.000.2010.87.23058132)

Table 6 shows data of representative compounds of the present invention following oral administration (p.o.) in dogs:

TABLE 6AUCnormt1/2MRTFExample[kg · h/L][h][h][%]267.714.021.382.81742.087.056.1041.6(WO2012/058132)

The compounds according to the present invention show superior pharmacokinetic (PK) properties in comparison to compounds disclosed in the prior art (WO 2012/058132) (see tables 3 to 6). For instance example 2 of the present invention shows a lower plasma clearance (CLplasma) (up to 10 times) and therefore a much higher exposure in comparison to the prior art compound disclosed as example 174 in WO 2012/058132 in rats as well as in dogs. Example 2 shows also a long half-life and mean residence time (MRT) in all tested species after p.o. (per oral) application. Due to the significantly lower plasma clearance of example 2 and the resulting very high exposure (AUCnorm, exposure, area under curve normated) with good bioavailability after p.o. application in all tested species, we see a clear superiority of pharmacokinetic (PK) properties versus example 174 disclosed in WO 2012/058132.

B-6. Metabolic Study

To determine the metabolic profile of the inventive compounds, they were incubated with recombinant human cytochrome P450 (CYP) enzymes, liver microsomes or primary fresh hepatocytes from various animal species (e.g. rats, dogs), and also of human origin, in order to obtain and to compare information about a very substantially complete hepatic phase I and phase II metabolism, and about the enzymes involved in the metabolism.

The compounds of the invention were incubated with a concentration of about 0.1-10 μM. To this end, stock solutions of the compounds of the invention having a concentration of 0.01-1 mM in acetonitrile were prepared, and then pipetted with a 1:100 dilution into the incubation mixture. The liver microsomes and recombinant enzymes were incubated at 37° C. in 50 mM potassium phosphate buffer pH 7.4 with and without NADPH-generating system consisting of 1 mM NADP+, 10 mM glucose-6-phosphate and 1 unit glucose-6-phosphate dehydrogenase. Primary hepatocytes were incubated in suspension in Williams E medium, likewise at 37° C. After an incubation time of 0-4 h, the incubation mixtures were stopped with acetonitrile (final concentration about 30%) and the protein was centrifuged off at about 15 000×g. The samples thus stopped were either analyzed directly or stored at −20° C. until analysis.

The analysis was carried out by high-performance liquid chromatography with ultraviolet and mass spectrometry detection (HPLC-UV-MS/MS). To this end, the supernatants of the incubation samples were chromatographed with suitable C18 reversed-phase columns and variable mobile phase mixtures of acetonitrile and 10 mM aqueous ammonium formate solution or 0.05% formic acid. The UV chromatograms in conjunction with mass spectrometry data serve for identification, structural elucidation and quantitative estimation of the metabolites, and for quantitative metabolic reduction of the compound of the invention in the incubation mixtures.

B-7. Caco-2 Permeability Test

The permeability of a test substance was determined with the aid of the Caco-2 cell line, an established in vitro model for permeability prediction at the gastrointestinal barrier (Artursson, P. and Karlsson, J. (1991). Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells. Biochem. Biophys. 175 (3), 880-885). The Caco-2 cells (ACC No. 169, DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) were sown in 24-well plates having an insert and cultivated for 14 to 16 days. For the permeability studies, the test substance was dissolved in DMSO and diluted to the final test concentration with transport buffer (Hanks Buffered Salt Solution, Gibco/Invitrogen, with 19.9 mM glucose and 9.8 mM HEPES). In order to determine the apical to basolateral permeability (PappA-B) of the test substance, the solution comprising the test substance was applied to the apical side of the Caco-2 cell monolayer, and transport buffer to the basolateral side. In order to determine the basolateral to apical permeability (PappB-A) of the test substance, the solution comprising the test substance was applied to the basolateral side of the Caco-2 cell monolayer, and transport buffer to the apical side. At the start of the experiment, samples were taken from the respective donor compartment in order to ensure the mass balance. After an incubation time of two hours at 37° C., samples were taken from the two compartments. The samples were analyzed by means of LC-MS/MS and the apparent permeability coefficients (Papp) were calculated. For each cell monolayer, the permeability of Lucifer Yellow was determined to ensure cell layer integrity. In each test run, the permeability of atenolol (marker for low permeability) and sulfasalazine (marker for active excretion) was also determined as quality control.

B-8. Solubility Determination of Substances in Buffer pH 6.5

2-4 mg of the test compound were dissolved in DMSO to reach a concentration of 50 g/L (solution A, 515 μg/l). To 10 μl of this solution 960 μl PBS buffer pH 6.5 were added; the mixture was shaken for 24 h at rt in a 96 well plate. An aliquot was centrifuged at 42000 rpm for 30 min. The supernatant was diluted with ACN/water (8:2) 1:10 and 1:1000 resp. This diluted samples were analyzed by LC-MSMS.

Calibration: 10 μl of solution A were diluted with 823 μl DMSO (final concentration: 600 μg/ml), which was further diluted with ACN/water 8:2 by a factor of 100 (solution B).

The calibration curve was obtained from solution B by further diluting with ACN/water 8:2 with target concentrations of 1.2-12-60-600 ng/ml and injecting these four solutions for MS measurement.

MS Method Optimization:

Solution B was utilized for MS method optimization.

PBS-Puffer: 6.18 g sodium chloride and 3.96 g sodium dihydrogen phosphate were dissolved in 1 L aqua dist., the pH was adjusted to 6.5 with 1N sodium hydroxide.

LC-MSMS Optimization:

The following configurations were used for optimizationAB Sciex TRIPLE QUAD 4500, Agilent 1260 Infinity (G1312B), degasser (G4225A), column oven (G1316C or G1316A), CTC Analytics PAL injection system HTS-xt or HTC-xt.Eluent A: 0.5 ml formic acid (50% ig)/L water, Eluent B: 0.5 ml formic acid (50% ig)/L acetonitrile

timeflow[min][μl/min]% B0.00200700.08200700.0925700.6025700.65200701.1020070Autosampler: without auto inject ahead settingcolumn: stainless steel capillaryoven temperature: 22° C.flow rate: flow gradientinjected volume: 2 μl

Water Quattro Micro MS, Agilent 1100 (G1312A), degasser (G1322A), column oven (G1316A), CTC Analytics PAL injection system HTS, eluents as above

timeflow[min][μl/min]% B0.00250701.5025070Autosampler: with auto inject ahead settingcolumn: stainless steel capillaryoven temperature: 22° C.flow rate: flow gradientinjected volume: 5 μlMS method: Flow Injection Analysis (FIA) for optimization (“MS-OPTI”); Ionization mode ABSciex-MS: ESI-pos/neg, Waters-MS: ESI-pos
HPLC Method for MSMS Quantification:

Eluent A, B as above

ABSciex-MStime[min]% A% B090100.55950.845950.8590101.229010Autosampler: without auto inject ahead settingcolumn: Waters OASIS HLB, 2.1×20 mm, 25 gcolumn temperature: 30° C.flow rate: 2.5 mlinjected volume: 2 μlSplitter (before MS) 1:20

Waters-MStime[min]% A% B090100.55950.845950.8590101.59010Autosampler: with auto inject ahead settingcolumn: Waters OASIS HLB, 2.1×20 mm, 25 gcolumn temperature: 30° C.flow rate: 2.5 mlinjected volume: 5 μlSplitter (before MS) 1:20MS method: Multiple Reaction Monitoring (MRM)

B-9. Determination of Solubility from Solid

For each solvent, an Eppendorf plastic vial was charged with 0.5-1 mg of the test compound (exact weight), 2-3 μlass pearls (diameter 3 mm) and 1.0 ml of the respective solvent. The vial was closed and shaken at RT for 24 h (1400 rpm; Thermomixer, Eppendorf). Thereafter, 230 μl each of the solution/suspension was transferred into one or more centrifuge vials (Beckman Coulter) and were centrifuged at 42000 rpm for 30 min (Beckman Coulter Optima L90). At least 100 μl of the supernatant were withdrawn and further diluted with DMSO in two dilution strength: 1:5 and 1:50 (the latter obtained from the 1:5 dilution step by subsequent DMSO addition). This liquid handling was done either manually or with the help of a pipetting robot (Lissy, Zinsser Analytic).

For HPLC quantification, calibration solutions of the test compound in DMSO were prepared. Starting from an initial concentration of 600 μg/ml, three calibration solutions were prepared: 100 μg/ml, 20 μg/ml and 2.5 μg/ml (manually or via Lissy).

Both calibration solutions and the supernatant were analyzed by HPLC/UV-detection at an appropriate wave length. The solubility was determined using the linear calibration curve.

HPLC Systems:

Hewlett Packard/Agilent HPLC systems, G1311A+G1316A+G1315B as well as G1312A+G1316A+G1315Ainjector system: CTC-Analytik HTC PALor with a Agilent UPLC System (G7117C, G7116B G7167B and G7120)oven temperature: 30° C., detection: 210 and/or 254 n, injected volume: 20 μleluent A: 0.1% TFA in water, eluent B: 0.1% TFA in acetonitrilecolumn: ZORBAX Extend-C18, 3.0×50 mm, 3.5 μm
Gradient:

timeABFlow rate:[min][%][%][ml/min]0.09821.50.29821.53.310901.54.010901.54.19822.54.79822.55.09821.5
C. Working Examples of Pharmaceutical Compositions

The compounds of the invention can be converted to pharmaceutical preparations as follows:

Tablet:

Composition:

100 mg of the compound according to the invention, 50 mg of lactose (monohydrate), 50 mg of corn starch (native), 10 mg of polyvinylpyrrolidone (PVP 25) (from BASF, Ludwigshafen, Germany) and 2 mg of magnesium stearate.

Tablet weight 212 mg. Diameter 8 mm, radius of curvature 12 mm.

Production:

The mixture of compound of the invention, lactose and starch is granulated with a 5% solution (w/w) of the PVP in water. The granules are dried and then mixed with the magnesium stearate for 5 minutes. This mixture is compressed using a conventional tableting press (see above for format of the tablet). The guide value used for the pressing is a pressing force of 15 kN.

Suspension for Oral Administration:

Composition:

1000 mg of the compound of the invention, 1000 mg of ethanol (96%), 400 mg of Rhodigel® (xanthan gum from FMC, Pennsylvania, USA) and 99 g of water.

10 ml of oral suspension correspond to a single dose of 100 mg of the compound of the invention.

Production:

The Rhodigel is suspended in ethanol; the compound of the invention is added to the suspension. The water is added while stirring. The mixture is stirred for about 6 h until the swelling of the Rhodigel is complete.

Solution for Oral Administration:

Composition:

500 mg of the compound of the invention, 2.5 g of polysorbate and 97 g of polyethylene glycol 400. 20 g of oral solution correspond to a single dose of 100 mg of the compound of the invention.

Production:

The compound of the invention is suspended in the mixture of polyethylene glycol and polysorbate with stirring. The stirring process is continued until the compound according to the invention has completely dissolved.

i.v. solution:

The compound according to the invention is dissolved in a concentration below the saturation solubility in a physiologically tolerated solvent (e.g. isotonic saline, 5% glucose solution and/or 30% PEG 400 solution). The solution is sterilized by filtration and used to fill sterile and pyrogen-free injection containers.