AMINO-SUBSTITUTED ANNULATED PYRIMIDINES AND USE THEREOF
20170240566 · 2017-08-24
Assignee
Inventors
- Alexandros Vakalopoulos (Hilden, DE)
- Gaelle Valot (Wuppertal, DE)
- Markus Follmann (Köln, DE)
- Damian Brockschnieder (Haan, DE)
- Johannes-Peter STASCH (Grottaferrata (RM), IT)
- Tobias Marquardt (Wuppertal, DE)
- Adrian Tersteegen (Wuppertal, DE)
- Frank Wunder (Wuppertal, DE)
- Lisa Dietz (Wuppertal, DE)
- Dieter Lang (Velbert, DE)
- Ursula Krenz (Leichlingen, DE)
Cpc classification
A61P9/04
HUMAN NECESSITIES
A61K31/519
HUMAN NECESSITIES
A61P25/28
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61P9/14
HUMAN NECESSITIES
A61P9/10
HUMAN NECESSITIES
C07D519/00
CHEMISTRY; METALLURGY
International classification
C07D519/00
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
A61K31/519
HUMAN NECESSITIES
Abstract
The present application relates to novel amino-substituted fused pyrimidines, to processes for their preparation, to their use alone or in combinations for the treatment and/or prophylaxis of diseases, and to their use for producing medicaments for the treatment and/or prophylaxis of diseases, in particular for the treatment and/or prophylaxis of cardiovascular disorders.
Claims
1. A compound of the general formula (I) ##STR00083## in which A represents nitrogen or carbon, R.sup.1 represents phenyl, pyridyl, 3,3,3-trifluoroprop-1-yl, 4,4,4-trifluorobut-1-yl or 3,3,4,4,4-pentafluorobut-1-yl, where phenyl is substituted by 1 to 3 substituents independently of one another selected from the group consisting of fluorine, chlorine, (C.sub.1-C.sub.4)-alkyl, cyclopropyl and (C.sub.1-C.sub.4)-alkoxy, and where pyridyl is substituted by 1 or 2 substituents independently of one another selected from the group consisting of fluorine, (C.sub.1-C.sub.4)-alkyl, cyclopropyl and (C.sub.1-C.sub.4)-alkoxy, R.sup.2 represents hydrogen or (C.sub.1-C.sub.4)-alkyl, R.sup.3 represents (C.sub.1-C.sub.6)-alkyl, where (C.sub.1-C.sub.6)-alkyl is substituted by amino and up to five times by fluorine, R.sup.4 represents (C.sub.1-C.sub.4)-alkyl, where (C.sub.1-C.sub.4)-alkyl may be substituted up to five times by fluorine, R.sup.5 represents (C.sub.1-C.sub.4)-alkyl, where (C.sub.1-C.sub.4)-alkyl may be substituted up to five times by fluorine, or R.sup.4 and R.sup.5 together with the carbon atom to which they are attached form a 3- to 6-membered carbocycle, R.sup.6 represents hydrogen, R.sup.7 represents hydrogen or fluorine, R.sup.8 represents hydrogen, chlorine, fluorine or (C.sub.1-C.sub.4)-alkyl, and the N-oxides, salts, solvates, salts of the N-oxides and solvates of the N-oxides and salts thereof.
2. The compound of the formula (I) as claimed in claim 1 in which A represents nitrogen or carbon, R.sup.1 represents phenyl or pyridyl, where phenyl is substituted by 1 to 3 substituents independently of one another s elected from the group consisting of fluorine and methyl, and where pyridyl is substituted by 1 or 2 substituents independently of one another selected from the group consisting of fluorine and methyl, R.sup.2 represents hydrogen or methyl, R.sup.3 represents ##STR00084## where ## represents the point of attachment to the nitrogen atom, R.sup.4 represents methyl or ethyl, where methyl and ethyl may be substituted up to three times by fluorine, R.sup.5 represents methyl or ethyl, where methyl and ethyl may be substituted up to three times by fluorine, R.sup.6 represents hydrogen, R.sup.7 represents hydrogen or fluorine, R.sup.8 represents hydrogen, chlorine, methyl or ethyl, and the N-oxides, salts, solvates, salts of the N-oxides and solvates of the N-oxides and salts thereof.
3. The compound of the formula (I) as claimed in claim 1 in which A represents nitrogen, R.sup.1 represents phenyl or pyridyl, where phenyl is substituted by 1 to 3 fluorine substituents, and where pyridyl is substituted by fluorine, R.sup.2 represents hydrogen, R.sup.3 represents ##STR00085## where ## represents the point of attachment to the nitrogen atom, R.sup.4 represents methyl or trifluoromethyl, R.sup.5 represents methyl or trifluoromethyl, R.sup.6 represents hydrogen, R.sup.7 represents hydrogen or fluorine, R.sup.8 represents hydrogen, methyl or ethyl, and the N-oxides, salts, solvates, salts of the N-oxides and solvates of the N-oxides and salts thereof.
4. The compound of the formula (I) as claimed in claim 1, in which A represents nitrogen, R.sup.1 represents a phenyl group of the formula ##STR00086## where # represents the point of attachment to the methylene group, and R.sup.9 represents hydrogen or fluorine, R.sup.10 represents fluorine, R.sup.11 represents hydrogen or fluorine, or represents 3-fluoropyridin-2-yl, R.sup.2 represents hydrogen, R.sup.3 represents ##STR00087## where ## represents the point of attachment to the nitrogen atom, R.sup.4 represents methyl, R.sup.5 represents methyl or trifluoromethyl, R.sup.6 represents hydrogen, R.sup.7 represents hydrogen or fluorine, R.sup.8 represents hydrogen or methyl, and the N-oxides, salts, solvates, salts of the N-oxides and solvates of the N-oxides and salts thereof.
5. A process for preparing compounds of the formula (I) as defined in claim 1, comprising reacting a compound of the formula (II) ##STR00088## in which R.sup.1, R.sup.6, R.sup.7 and R.sup.8 each have the meanings given above, in an inert solvent, optionally in the presence of a suitable base, with a compound of the formula (III) ##STR00089## in which R.sup.4 and R.sup.5 each have the meanings given above and T.sup.1 represents (C.sub.1-C.sub.4)-alkyl, to give a compound of formula (IV) ##STR00090## in which R.sup.1, R.sup.4, R.sup.5, R.sup.6, R.sup.7 and R.sup.8 each have the meanings given above, then converting the compound of formula (IV) with isopentyl nitrite and an iodine equivalent into a compound of the formula (V) ##STR00091## in which R.sup.1, R.sup.4, R.sup.5, R.sup.6, R.sup.7 and R.sup.8 each have the meanings given above, and subsequently converting the compound of formula (V) in an inert solvent with a compound of the formula (VI) ##STR00092## in which R.sup.2 and R.sup.3 each have the meanings given above, and optionally converting the resulting compounds of the formula (I), with the appropriate (i) solvents and/or (ii) bases or acids, into the solvates, salts and/or solvates of the salts thereof.
6. (canceled)
7. (canceled)
8. A medicament comprising a compound as defined in claim 1 in combination with one or more inert, nontoxic, pharmaceutically suitable excipients.
9. A medicament comprising a compound as defined in claim 1 in combination with a further active compound selected from the group consisting of organic nitrates, NO donors, cGMP-PDE inhibitors, antithrombotic agents, hypotensive agents and lipid metabolism modifiers.
10. (canceled)
11. A method for the treatment and/or prophylaxis of heart failure, angina pectoris, hypertension, pulmonary hypertension, ischemias, vascular disorders, renal insufficiency, thromboembolic disorders, arteriosclerosis, dementia disorders and erectile dysfunction in humans and animals comprising administering an effective amount of at least one compound of the formula (I) as defined in claim 1 to a human or animal in need thereof.
12. A method for the treatment and/or prophylaxis of heart failure, angina pectoris, hypertension, pulmonary hypertension, ischemias, vascular disorders, renal insufficiency, thromboembolic disorders, arteriosclerosis, dementia disorders and erectile dysfunction in humans and animals comprising administering an effective amount of the medicament of claim 8 to a human or animal in need thereof.
13. A method for the treatment and/or prophylaxis of heart failure, angina pectoris, hypertension, pulmonary hypertension, ischemias, vascular disorders, renal insufficiency, thromboembolic disorders, arteriosclerosis, dementia disorders and erectile dysfunction in humans and animals comprising administering an effective amount of the medicament of claim 9 to a human or animal in need thereof.
14. A method for the treatment and/or prophylaxis of heart failure, angina pectoris, hypertension, pulmonary hypertension, ischemias, vascular disorders, renal insufficiency, thromboembolic disorders, arteriosclerosis, dementia disorders and erectile dysfunction in humans and animals comprising administering an effective amount of the compound of claim 2 to a human or animal in need thereof.
15. A method for the treatment and/or prophylaxis of heart failure, angina pectoris, hypertension, pulmonary hypertension, ischemias, vascular disorders, renal insufficiency, thromboembolic disorders, arteriosclerosis, dementia disorders and erectile dysfunction in humans and animals comprising administering an effective amount of the compound of claim 3 to a human or animal in need thereof.
16. A method for the treatment and/or prophylaxis of heart failure, angina pectoris, hypertension, pulmonary hypertension, ischemias, vascular disorders, renal insufficiency, thromboembolic disorders, arteriosclerosis, dementia disorders and erectile dysfunction in humans and animals comprising administering an effective amount of the compound of claim 4 to a human or animal in need thereof.
Description
A. EXAMPLES
[0253] Abbreviations: [0254] abs. absolute [0255] aq. aqueous solution [0256] calc. calculated [0257] Boc tert-butyloxycarbonyl [0258] br. s broad singlet (in NMR) [0259] Cbz benzyloxycarbonyl [0260] δ shift in the NMR spectrum (stated in ppm) [0261] d doublet (NMR coupling pattern) [0262] TLC thin-layer chromatography [0263] DCI direct chemical ionization (in MS) [0264] dd doublet of doublet (NMR coupling pattern) [0265] ddt doublet of doublet of triplet (NMR coupling pattern) [0266] DMF dimethylformamide [0267] DMSO dimethyl sulfoxide [0268] ent enantiomerically pure [0269] eq. equivalent(s) [0270] ESI electrospray ionization (in MS) [0271] Et ethyl [0272] h hour(s) [0273] HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) [0274] HPLC high-pressure, high-performance liquid chromatography [0275] HRMS high-resolution mass spectrometry [0276] conc. concentrated [0277] LC-MS liquid chromatography-coupled mass spectrometry [0278] m multiplet [0279] Me methyl [0280] min minute(s) [0281] MS mass spectrometry [0282] NMR nuclear magnetic resonance spectrometry [0283] PdCl.sub.2(dppf)CH.sub.2Cl.sub.2 1,1′-bis(diphenylphosphino)ferrocenepalladium(II) dichloride/dichloromethane complex [0284] Ph phenyl [0285] q quartet (NMR coupling pattern) [0286] quint. quintet (NMR coupling pattern) [0287] rac racemic [0288] rel relative stereochemistry [0289] RT room temperature [0290] R.sub.t retention time (in HPLC) [0291] s singlet (NMR coupling pattern) [0292] SFC supercritical fluid chromatography [0293] t triplet (NMR coupling pattern) [0294] TBTU (benzotriazol-1-yloxy)bisdimethylaminomethylium fluoroborate [0295] TFA trifluoroacetic acid [0296] THF tetrahydrofuran [0297] UV ultraviolet spectrometry [0298] v/v volume to volume ratio (of a solution)
[0299] HPLC, GCMS and LC-MS Methods:
[0300] Method 1 (LC-MS):
[0301] Instrument: Waters ACQUITY SQD UPLC System; column: Waters Acquity UPLC HSS T3 1.8μ, 50×1 mm; mobile phase A: 1 l of water+0.25 ml of 99% strength formic acid, mobile phase B: 1 l of acetonitrile+0.25 ml of 99% strength formic acid; gradient: 0.0 min 90% A.fwdarw.1.2 min 5% A.fwdarw.2.0 min 5% A; oven: 50° C.; flow rate: 0.40 ml/min; UV detection: 208-400 nm.
[0302] Method 2 (LC-MS):
[0303] Instrument: Waters ACQUITY SQD UPLC System; column: Waters Acquity UPLC HSS T3 1.8μ, 50×1 mm; mobile phase A: 1 l of water+0.25 ml of 99% strength formic acid, mobile phase B: 1 l of acetonitrile +0.25 ml of 99% strength formic acid; gradient: 0.0 min 95% A.fwdarw.6.0 min 5% A.fwdarw.7.5 min 5% A; oven: 50° C.; flow rate: 0.35 ml/min; UV detection: 210-400 nm.
[0304] Method 3 (LC-MS):
[0305] Instrument: Micromass Quattro Premier with Waters UPLC Acquity; column: Thermo Hypersil GOLD 1.9μ 50×1 mm; mobile phase A: 1 l of water+0.5 ml of 50% strength formic acid, mobile phase B: 1 l of acetonitrile+0.5 ml of 50% strength formic acid; gradient: 0.0 min 97% A.fwdarw.0.5 min 97% A.fwdarw.3.2 min 5% A.fwdarw.4.0 min 5% A oven: 50° C.; flow rate: 0.3 ml/min; UV detection: 210 nm.
[0306] Method 4 (LC-MS):
[0307] MS instrument: Waters (Micromass) Quattro Micro; HPLC instrument: Agilent 1100 series; column: YMC-Triart C18 3μ 50×3 mm; mobile phase A: 1 l of water+0.01 mol of ammonium carbonate, mobile phase B: 1 l of acetonitrile; gradient: 0.0 min 100% A.fwdarw.2.75 min 5% A.fwdarw.4.5 min 5% A; oven: 40° C.; flow rate: 1.25 ml/min; UV detection: 210 nm.
[0308] Method 5 (LC-MS):
[0309] MS instrument: Waters (Micromass) QM; HPLC instrument: Agilent 1100 series; column: Agilent ZORBAX Extend-C18 3.0×50 mm 3.5 micron; mobile phase A: 1 l of water+0.01 mol of ammonium carbonate, mobile phase B: 1 l of acetonitrile; gradient: 0.0 min 98% A.fwdarw.0.2 min 98% A.fwdarw.3.0 min 5% A.fwdarw.4.5 min 5% A; oven: 40° C.; flow rate: 1.75 ml/min; UV detection: 210 nm.
[0310] Method 6 (GC-MS):
[0311] Instrument: Micromass GCT, GC6890; column: Restek RTX-35, 15 m×200 μm×0.33 μm; constant helium flow rate: 0.88 ml/min; oven: 70° C.; inlet: 250° C.; gradient: 70° C., 30° C./min.fwdarw.310° C. (maintain for 3 min).
[0312] Method 7 (LC-MS):
[0313] Instrument: Agilent MS Quad 6150; HPLC: Agilent 1290; column: Waters Acquity UPLC HSS T3 1.8μ 50×2.1 mm; mobile phase A: 1 l of water+0.25 ml of 99% strength formic acid, mobile phase B: 1 l of acetonitrile+0.25 ml of 99% strength formic acid; gradient: 0.0 min 90% A.fwdarw.0.3 min 90% A.fwdarw.1.7 min 5% A.fwdarw.3.0 min 5% A oven: 50° C.; flow rate: 1.20 ml/min; UV detection: 205-305 nm.
[0314] Method 8 (GC-MS):
[0315] Instrument: Thermo Scientific DSQII, Thermo Scientific Trace GC Ultra; column: Restek RTX-35MS, 15 m×200 μm×0.33 μm; constant flow rate with helium: 1.20 ml/min; oven: 60° C.; inlet: 220° C.; gradient: 60° C., 30° C./min.fwdarw.300° C. (maintain for 3.33 min).
[0316] Method 9 (LC-MS):
[0317] MS instrument: Waters SQD; HPLC instrument: Waters UPLC; column: Zorbax SB-Aq (Agilent), 50 mm×2.1 mm, 1.8 μm; mobile phase A: water+0.025% formic acid, mobile phase B: acetonitrile (ULC)+0.025% formic acid; gradient: 0.0 min 98% A-0.9 min 25% A-1.0 min 5% A-1.4 min 5% A-1.41 min 98% A-1.5 min 98% A; oven: 40° C.; flow rate: 0.600 ml/min; UV detection: DAD; 210 nm.
[0318] Method 10 (Preparative HPLC):
[0319] MS instrument: Waters, HPLC instrument: Waters; column: Waters X-Bridge C18, 19 mm×50 mm, 5 μm, mobile phase A: water+0.05% ammonia, mobile phase B: acetonitrile (ULC) with gradient; flow rate: 40 ml/min; UV detection: DAD; 210-400 nm).
or:
[0320] MS instrument: Waters, HPLC instrument: Waters (column Phenomenex Luna 5μ C18(2) 100A, AXIA Tech. 50×21.2 mm, mobile phase A: water+0.05% formic acid, mobile phase B: acetonitrile (ULC) with gradient; flow rate: 40 ml/min; UV detection: DAD; 210-400 nm).
[0321] Method 11 (LC-MS):
[0322] MS instrument: ThermoFisherScientific LTQ-Orbitrap-XL; HPLC instrument type: Agilent 1200SL; column: Agilent, POROSHELL 120, 3×150 mm, SB-C18 2.7 μm; mobile phase A: 1 l of water+0.1% trifluoroacetic acid; mobile phase B: 1 l of acetonitrile+0.1% trifluoroacetic acid; gradient: 0.0 min 2% B.fwdarw.1.5 min 2% B.fwdarw.15.5 min 95% B.fwdarw.18.0 min 95% B; oven: 40° C.; flow rate: 0.75 ml/min; UV detection: 210 nm.
[0323] Further Details:
[0324] In the case of purifications of compounds of the invention by preparative HPLC by the above-described methods in which the eluents contain additives, for example trifluoroacetic acid, formic acid or ammonia, the compounds of the invention can be obtained in salt form, for example as trifluoroacetate, formate or ammonium salt, if the compounds of 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.
[0325] Furthermore, amidines can be present as free compounds or partially (depending on the preparation if acetic acid is involved) as acetate salts or acetate solvates.
[0326] 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 CF.sub.3COOH”, “x Na.sup.+” 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.
[0327] to 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.
[0328] Furthermore, the secondary amides according to the invention may be present as rotational isomers/isomer mixtures, in particular in NMR studies. Purity figures are generally based on corresponding peak integrations in the LC/MS chromatogram, but may additionally also have been determined with the aid of the .sup.1H NMR spectrum. If no purity is indicated, the purity is generally 100% according to automated peak integration in the LC/MS chromatogram, or the purity has not been determined explicitly.
[0329] Stated yields in % of theory are generally corrected for purity if a purity of <100% is indicated. In solvent-containing or contaminated batches, the formal yield may be “>100%”; in these cases the yield is not corrected for solvent or purity.
[0330] In all .sup.1H NMR spectra data, the chemical shifts δ are stated in ppm.
[0331] The multiplicities of proton signals in .sup.1H NMR spectra reported in the paragraphs which follow represent the signal form observed in each case and do not take account of any higher-order signal phenomena. In general, the stated chemical shift refers to the center of the signal in question. In the case of broad multiplets, an interval is given. Signals obscured by solvent or water were either tentatively assigned or have not been listed. Significantly broadened signals—caused, for example, by rapid rotation of molecular moieties or because of exchanging protons—were likewise assigned tentatively (often referred to as a broad multiplet or broad singlet) or are not listed.
[0332] Melting points and melting-point ranges, if stated, are uncorrected.
[0333] All reactants or reagents whose preparation is not described explicitly hereinafter were purchased commercially from generally accessible sources. For all other reactants or reagents whose preparation likewise is not described hereinafter and which were not commercially obtainable or were obtained from sources which are not generally accessible, a reference is given to the published literature in which their preparation is described.
[0334] Starting Compounds and Intermediates:
Example 1A
5-Fluoro-6-methyl-1H-pyrazolo[3,4-b]pyridine-3-amine
[0335] ##STR00018##
[0336] 58 g (340.03 mmol) of 2-chloro-5-fluoro-6-methylnicotinonitrile (preparation described in WO2007/041052, Example U-2, page 80) were initially charged in 1,2-ethanediol (580 ml), and hydrazine hydrate (24.81 ml) and 56.09 ml (340.03 mmol) of N,N-diisopropylethylamine were then added. The mixture was stirred at 80° C. for 16 h and then at 120° C. for 6 h. After cooling to RT, water (2.5 l) and ethyl acetate (2.5 l) were added and the resulting solid was filtered off with suction. The solid obtained was dried under reduced pressure. This gave 28.4 g (47% of theory) of the target compound.
[0337] LC-MS (Method 4): R.sub.t=1.77 min
[0338] MS (ESIpos): m/z=167 [M+H].sup.+
Example 2A
5-Fluoro-3-iodo-6-methyl-1H-pyrazolo[3,4-b]pyridine
[0339] ##STR00019##
[0340] 28 g (168.5 mmol) of 5-fluoro-6-methyl-1H-pyrazolo[3,4-b]pyridine-3-amine from Example 1A were initially charged in 1.32 l of THF, and the mixture was cooled to 0° C. 41.45 ml (337.03 mmol) of boron trifluoride diethyl ether complex were then added slowly. The reaction mixture was cooled to −10° C. A solution of 25.66 g (219.07 mmol) of isopentyl nitrite in 166 ml of THF was then added slowly, and the mixture was subsequently stirred for a further 30 min. The reaction solution was then concentrated to about a third of its volume. 988 ml of acetone were then added, and the solution was cooled to 0° C. A solution of 32.84 g (219.07 mmol) of sodium iodide in 412 ml of acetone was added dropwise to this solution, and the mixture was then stirred at RT for 2 h. The reaction mixture was poured into 5 l of ice-water and extracted three times with in each case 750 ml of ethyl acetate. The combined organic phases were washed with 750 ml of saturated aqueous sodium chloride solution, dried and then concentrated under reduced pressure. The crude product was purified using silica gel (silica gel, mobile phase: cyclohexane/ethyl acetate, gradient 9:1 to 1:1). This gave 14.90 g (32% of theory) of the title compound.
[0341] LC-MS (Method 1): R.sub.t=0.84 min
[0342] MS (ESIpos): m/z=278 [M+H].sup.+
Example 3A
1-(2,3-Difluorobenzyl)-5-fluoro-3-iodo-6-methyl-1H-pyrazolo[3,4-b]pyridine
[0343] ##STR00020##
[0344] 2.60 g (9.37 mmol) of 5-fluoro-3-iodo-6-methyl-1H-pyrazolo[3,4-b]pyridine from Example 2A were initially charged in 35 ml of DMF. A solution of 3.67 g (11.26 mmol) of cesium carbonate and 1.94 g (9.37 mmol) of 1-(bromomethyl)-2,3-difluorobenzene in 10 ml of DMF was then added, and the mixture was subsequently stirred at RT overnight. The reaction mixture was added to 200 ml of water and extracted twice with ethyl acetate. The collected organic phases were dried over sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (silica gel, mobile phase: petroleum ether/ethyl acetate=10/1) and the product fractions were concentrated. Further purification was carried out by preparative HPLC (column: Sunfire C18, 5 μm, 250×20 mm; mobile phase: 12% water+85% methanol+3% 1% strength aqueous TFA solution; flow rate: 25 ml/min; temperature: 40° C.; wavelength: 210 nm). This gave 2.67 g (71% of theory) of the title compound.
[0345] LC-MS (Method 1): R.sub.t=1.29 min
[0346] MS (ESIpos): m/z=404 [M+H].sup.+
[0347] Analogously to Example 3A, the exemplary compounds shown in Table 1A were prepared by reacting 5-fluoro-3-iodo-6-methyl-1H-pyrazolo[3,4-b]pyridine from Example 2A with 1-(bromomethyl)-2-fluorobenzene, 2-(bromomethyl)-1,3,4-trifluorobenzene or 2-(chloromethyl)-3-fluoropyridine hydrochloride (1.1-1.5 equivalents) and cesium carbonate (1.2-2 equivalents) under the reaction conditions described (reaction time: 2-72 h; temperature: RT to 60° C.) in DMF.
[0348] Exemplary Work-Up of the Reaction Mixture:
[0349] Method A: The reaction mixture was added to water and then stirred at room temperature for about 1 h. The solid formed was filtered off, washed with water and dried under high vacuum.
[0350] Method B: Alternatively, the reaction mixture was added to water and extracted with ethyl acetate. The collected organic phases were dried over sodium sulfate, filtered and concentrated. The residue was purified by column chromatography on silica gel (mobile phase: petroleum ether/ethyl acetate or dichloromethane/methanol).
[0351] Method C: Alternatively, the reaction mixture was diluted with acetonitrile and purified by preparative HPLC (RP18 column, mobile phase: acetonitrile/water gradient with addition of 0.1% TFA or 0.05% formic acid).
TABLE-US-00001 TABLE 1A Ex- IUPAC name/structure ample (Yield) Analytical data 4A 5-fluoro-1-(2-fluorobenzyl)-3-iodo-6-methyl-1H- .sup.1H-NMR (400 MHz, DMSO-d.sub.6) δ = pyrazolo[3,4-b]pyridine 2.60 (d, 3H), 5.68 (s, 2H), 7.13- 7.25 (m, 3H), 7.33-7.40 (m, 1H),
Example 7A
1-(2,3-Difluorobenzyl)-5-fluoro-6-methyl-1H-pyrazolo[3,4-b]pyridine-3 -carbonitrile
[0352] ##STR00024##
[0353] A mixture of 2.47 g (6.13 mmol) of 1-(2,3-difluorobenzyl)-5-fluoro-3-iodo-6-methyl-1H-pyrazolo[3,4-b]pyridine from Example 3A and 0.576 g (6.43 mmol) of copper(I) cyanide was initially charged in 12.1 ml of abs. DMSO in a flask which had been dried by heating, and the mixture was stirred at 150° C. for 3 h. Ethyl acetate was added to the cooled reaction solution, and the mixture was washed three times with a mixture of semisaturated aqueous ammonium chloride solution and aqueous concentrated ammonia solution (3/1). The organic phase was dried over sodium sulfate, filtered and concentrated by evaporation. The crude product was purified by flash chromatography (silica gel, mobile phase: cyclohexane/ethyl acetate gradient: 15/1 to 10/1; then dichloromethane/methanol: 10/1). This gave 780 mg of the target compound (42% of theory).
[0354] LC-MS (Method 1): R.sub.t=1.19 min
[0355] MS (ESIpos): m/z=303 [M+H].sup.+
[0356] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ [ppm]=2.65 (d, 3H), 5.87 (s, 2H), 7.10-7.25 (m, 2H), 7.39-7.48 (m, 1H), 8.41 (d, 1H).
[0357] The exemplary compounds shown in Table 2A were prepared analogously to Example 7A by reacting the appropriate iodides with copper(I) cyanide (1.1-1.5 equivalents) under the reaction conditions described (reaction time: 1-5 h; temperature: 150° C.) in DMSO.
[0358] Exemplary Work-Up of the Reaction Mixture:
[0359] Method A: After cooling, ethyl acetate was added to the reaction mixture, and the mixture was washed three times with a mixture of semisaturated aqueous ammonium chloride solution and aqueous concentrated ammonia solution (3/1). The organic phase was dried over sodium sulfate and filtered and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (silica gel, mobile phase: cyclohexane/ethyl acetate gradient: or dichloromethane/methanol gradient).
[0360] Method B: Alternatively, the reaction mixture was diluted with acetonitrile and purified by preparative HPLC (RP18 column, mobile phase: acetonitrile/water gradient with addition of 0.1% TFA or 0.05% formic acid).
TABLE-US-00002 TABLE 2A Ex- IUPAC name/structure ample (Yield) Analytical data 8A 5-fluoro-1-(2-fluorobenzyl)-6-methyl-1H-pyrazolo[3,4- .sup.1H-NMR (400 MHz, DMSO-d.sub.6) b]pyridine-3-carbonitrile δ = 2.65 (d, 3H), 5.82 (s, 2H), 7.18 (dt, 1H), 7.21-7.27 (m,
Example 11A
1-(2,3-Difluorobenzyl)-5-fluoro-6-methyl-1H-pyrazolo[3,4-d]pyridine-3-carboximidamide
[0361] ##STR00028##
[0362] 960 mg (3.18 mmol) of 1-(2,3-difluorobenzyl)-5-fluoro-6-methyl-1H-pyrazolo[3,4-b]pyridine-3-carbonitrile from Example 7A were initially charged in 9.47 ml of methanol. 0.69 ml (3.18 mmol) of sodium methoxide in methanol was added, and the mixture was subsequently stirred at RT for 1 h. Another 10 ml of methanol were then added, and the reaction mixture was subsequently stirred at 60° C. for 1 h. 204 mg (3.81 mmol) of ammonium chloride and 0.71 ml (12.39 mmol) of acetic acid were added and the reaction mixture was stirred under reflux for 7 h. The solvent was removed under reduced pressure and the residue was stirred with 38 ml of 1 N aqueous sodium hydroxide solution at room temperature for 1 h. The precipitate was then filtered off and washed with water. This gave 1.0 g of the target compound (90% of theory, purity 90%).
[0363] LC-MS (Method 1): R.sub.t=0.68 min
[0364] MS (ESIpos): m/z=320 [M+H].sup.+
[0365] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ [ppm]=2.60 (d, 3H), 5.77 (s, 2H), 6.62 (br. s, 3H), 6.91-6.98 (m, 1H), 7.11-7.20 (m, 1H), 7.34-7.44 (m, 1H), 8.29 (d, 1H).
[0366] The exemplary compounds shown in Table 3A were prepared analogously to Example 11A by reacting the appropriate nitriles with sodium methoxide (1.0-1.2 equivalents) in methanol and subsequently with ammonium chloride (1.2-1.5 equivalents) and acetic acid (3.5-5 equivalents) under the reaction conditions described (reaction time after addition of ammonium chloride and acetic acid: 5-24 h; temperature: reflux).
[0367] Exemplary Work-Up of the Reaction Mixture:
[0368] The solvent was evaporated and the residue was stirred with 1 N aqueous sodium hydroxide solution at room temperature for 0.5-2 h. The precipitate was then filtered off and washed with water and subsequently dried.
[0369] The target compounds obtained may, if appropriate partially, be present as acetate salt or acetate solvate.
TABLE-US-00003 TABLE 3A Ex- IUPAC name/structure ample (Yield) Analytical data 12A 5-fluoro-1-(2-fluorobenzyl)-6-methyl-1H-pyrazolo[3,4- .sup.1H-NMR (400 MHz, DMSO-d.sub.6) δ = b]pyridine-3-carboximidamide 2.59 (d, 3H), 5.73 (s, 2H), 6.51 (br. s, 3H), 7.07-7.17 (m, 2H), 7.20-
Example 15A
5-Fluoro-1-[(3-fluoropyridin-2-yl)methyl]-1H-pyrazolo[3,4-b]pyridine-3-carboximidamide acetate
[0370] ##STR00032##
[0371] The preparation of the compound is described in WO 2013/004785, example 14A, pp. 69-70.
Example 16A
6-Chloro-1-(2-fluorobenzyl)-1H-indazole-3-carboximidamide acetate
[0372] ##STR00033##
[0373] The preparation of the compound is described in WO2013/104598, example 54A, pp. 97-98.
Example 17A
4-Amino-2-[1-(2,3-difluorobenzyl)-5-fluoro-6-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl]-5,5-dimethyl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
[0374] ##STR00034##
[0375] 2.34 g (6.67 mmol; purity 90%) of 1-(2,3-difluorobenzyl)-5-fluoro-6-methyl-1H-pyrazolo[3,4-b]pyridine-3-carboximidamide from Example 11A were initially charged in 50.5 ml of tert-butanol. 1.33 g (8.00 mmol) of methyl 3,3-dicyanopivalate were then added, and the mixture was subsequently stirred under reflux for 6 h. Another 8 ml of tert-butanol were added and the mixture was then heated under reflux overnight. After cooling to RT, water was added and the reaction mixture was stirred at room temperature for 30 min. The precipitate formed was filtered off and washed with water. The solid was dried under high vacuum. This gave 3.25 g (99% of theory; purity: 92%) of the title compound.
[0376] LC-MS (Method 1): R.sub.t=1.03 min
[0377] MS (ESIpos): m/z=454 [M+H].sup.+
[0378] The exemplary compounds shown in Table 4A were prepared analogously to Example 17A by reacting the appropriate carboximidamides (amidines) with methyl 3,3-dicyanopivalate (1.1-1.5 equivalents) in tert-butanol [0.2-1.4 equivalents of potassium tert-butoxide were added to amidines present as acetate salt or acetate solvate] under the reaction conditions described (reaction time: 4-24 h).
[0379] Exemplary Work-Up of the Reaction Mixture:
[0380] Water was added to the reaction mixture and the mixture was stirred at room temperature for 30 min. The precipitate formed was filtered off and washed with water.
TABLE-US-00004 TABLE 4A Ex- IUPAC name/structure ample (Yield) Analytical data 18A 4-amino-2-[5-fluoro-1-(2-fluorobenzyl)-6-methyl-1H- LC-MS (Method 1): R.sub.t = 1.01 min pyrazolo[3,4-b]pyridin-3-yl]-5,5-dimethyl-5,7-dihydro-6H- MS(ESIpos): m/z = 436 [M + H].sup.+ pyrrolo[2,3-d]pyrimidin-6-one
Example 23A
Methyl 3,3-dicyano-2-(trifluoromethyl)acrylate
[0381] ##STR00040##
[0382] The synthesis of this compound is described in Journal of Fluorine Chemistry 1991, vol. 51, 3, pp. 323-334.
Example 24A
Methyl 2-(dicyanomethyl)-3,3,3-trifluoro-2-methylpropanoate
[0383] ##STR00041##
[0384] 3.00 g (14.70 mmol) of Example 23A were dissolved in tetrahydrofuran (30 ml) and the solution was cooled to 0° C. 7.35 ml (22.05 mmol) of methylmagnesium chloride (3 M in THF) were then added dropwise such that the temperature did not exceed 5° C. After the addition had ended, the mixture was stirred for another 10 min. 1 N aqueous hydrochloric acid was then added to the mixture, and the mixture was subsequently extracted with ethyl acetate. The phases were separated and the aqueous phase was extracted twice more with ethyl acetate. The combined organic phases were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered and concentrated. The crude product was then purified by column chromatography (silica gel, mobile phase: cyclohexane, then cyclohexane:ethyl acetate 9:1 (v:v)). Concentration gave 3.24 g (63% of theory) of the title compound.
[0385] .sup.1H-NMR (400 MHz, CDCl.sub.3): δ [ppm]=1.81 (s, 3H), 3.95 (s, 3H), 4.48 (s, 1H).
Example 25A
rac-4-Amino-2-{5-fluoro-1-[(3-fluoropyridin-2-yl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl}-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
[0386] ##STR00042##
[0387] 23.0 g (66.02 mmol) of 5-fluoro-1-[(3-fluoropyridin-2-yl)methyl]-1H-pyrazolo[3,4-b]pyridine-3-carboximidamide acetate from Example 15A were initially charged in tert-butanol (400 ml), and 13.43 g (119.68 mmol) of potassium tert-butoxide were added. Subsequently, 21.08 g (95.75 mmol) of methyl 2-(dicyanomethyl)-3,3,3-trifluoro-2-methylpropanoate from Example 24A in tert-butanol (100 ml) were added, and the mixture was heated under reflux overnight. After cooling to RT, water was added and the reaction mixture was stirred at room temperature for a further 30 min. The precipitate formed was filtered off and washed with water and a little diethyl ether. The solid was dried under high vacuum. This gave 16.1 g of the title compound (51% of theory).
[0388] LC-MS (Method 1): R.sub.t=0.95 min;
[0389] MS (ESIpos): m/z=477 [M+H].sup.+
[0390] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ [ppm]=1.72 (s, 3H), 5.96 (s, 2H), 7.10 (br. s, 2H), 7.42-7.48 (m, 1H), 7.75-7.80 (m, 1H), 8.27 (d, 1H), 8.68 (dd, 1H), 8.86 (dd, 1H), 11.60 (br. s, 1H).
[0391] The exemplary compounds shown in Table 5A were prepared analogously to Example 25A by reacting the appropriate carboximidamides (amidines) with methyl 2-(dicyanomethyl)-3,3,3-trifluoro-2-methylpropanoate (1.1-1.5 equivalents) in tert-butanol [0.2-1.4 equivalents of potassium tert-butoxide were added to amidines present as acetate salt or acetate solvate] under the reaction conditions described (reaction time: 0.5-24 h).
[0392] Alternatively, the reactions can be carried out in the microwave [0.5-10 h, 100° C.]
[0393] Exemplary Work-Up of the Reaction Mixture:
[0394] Water was added, and the reaction mixture was stirred at room temperature for 30 min. The precipitate formed was filtered off and washed with water.
TABLE-US-00005 TABLE 5A Ex- IUPAC name/structure ample (Yield) Analytical data 26A rac-4-amino-2-[5-fluoro-1-(2-fluorobenzyl)-6-methyl-1H- .sup.1H-NMR (400 MHz, DMSO-d.sub.6) δ = pyrazolo[3,4-b]pyridin-3-yl]-5-methyl-5-(trifluoromethyl)- 1.72 (s, 3H), 2.63 (d, 3H), 5.78 (s, 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one 2H), 7.07 (br. m, 2H), 7.12-7.27 (m, 3H), 7.33-7.40 (m, 1H), 8.77
Example 30A
2-[1-(2,3-Difluorobenzyl)-5-fluoro-6-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl]-4-iodo-5,5-dimethyl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
[0395] ##STR00047##
[0396] 3.25 g (6.61 mmol; purity 92%) of 4-amino-2-[1-(2,3-difluorobenzyl)-5-fluoro-6-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl]-5,5-dimethyl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one from Example 17A were initially charged in 64 ml of dioxane, 4.42 ml (33.04 mmol) of isopentyl nitrite and 2.66 ml (33.04 mmol) of diiodomethane were added and the mixture was then heated at 85° C. for 3 h. After cooling, the mixture was concentrated under reduced pressure and the residue was chromatographed on silica gel (mobile phase: dichloromethane/methanol gradient). Removal of the solvent under reduced pressure gave 2.32 g (51% of theory, purity 82%) of the title compound.
[0397] LC-MS (Method 1): R.sub.t=1.34 min
[0398] MS (ESIpos): m/z=565 [M+H].sup.+
[0399] The exemplary compounds shown in Table 6A were prepared analogously to Example 30A by reacting the appropriate anilines with diiodomethane (3-18 equivalents) and isopentyl nitrite (3-10 equivalents) in dioxane under the reaction conditions described (temperature: 85° C.; reaction time: 2-10 h).
[0400] Exemplary Work-Up of the Reaction Mixture:
[0401] The reaction mixture was concentrated [if appropriate partitioned between water and an organic solvent and then concentrated] and the residue was chromatographed on silica gel (mobile phase: dichloromethane/methanol or cyclohexane/ethyl acetate gradient]. Optionally, further purification was carried out by preparative HPLC [column: Sunfire C18, 5 μM, 100×30 mm; mobile phase: water/acetonitrile+0.2% strength formic acid].
TABLE-US-00006 TABLE 6A Ex- IUPAC name/structure ample (Yield) Analytical data 31A 2-[5-fluoro-1-(2-fluorobenzyl)-6-methyl-1H-pyrazolo[3,4- .sup.1H-NMR (400 MHz, DMSO-d.sub.6) δ = b]pyridin-3-yl]-4-iodo-5,5-dimethyl-5,7-dihydro-6H- 1.42 (s, 6H), 2.64 (d, 3H), 5.82 (s, pyrrolo[2,3-d]pyrimidin-6-one 1) 2H), 7.12-7.20 (m, 2H), 7.20- 7.27 (m, 1H), 7.34-7.41 (m, 1H),
Example 36A
rac-2-{5-Fluoro-1-[(3-fluoropyridin-2-yl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl}-4-iodo-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
[0402] ##STR00053##
[0403] 798 μl (5.93 mmol) of isopentyl nitrite and 286 μl (3.56 mmol) of diiodomethane were added to 565 mg (1.19 mmol) of rac-4-amino-2-{5-fluoro-1-[(3 -fluoropyridin-2-yl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl}-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one from Example 25A in 15 ml of dioxane, and the mixture was heated to 85° C. for 4 h. After cooling, the mixture was concentrated under reduced pressure, the residue was taken up in dichloromethane, kieselguhr was added and the mixture was then concentrated under reduced pressure. The crude compound adsorbed on kieselguhr was then purified by column chromatography (silica gel, mobile phase: cyclohexane/ethyl acetate gradient). Concentration gave 297 mg (42% of theory) of the title compound.
[0404] LC-MS (Method 1): R.sub.t=1.19 min;
[0405] MS (ESIpos): m/z=588 [M+H].sup.+
[0406] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ [ppm]=1.81 (s, 3H), 6.04 (s, 2H), 7.43-7.47 (m, 1H), 7.77-7.82 (m, 1H), 8.26 (d, 1H), 8.47 (dd, 1H), 8.76 (dd, 1H), 12.41 (br. s, 1H).
[0407] The exemplary compounds shown in Table 7A were prepared analogously to Example 36A by reacting the appropriate anilines with diiodomethane (4-18 equivalents) and isopentyl nitrite (4-12 equivalents) in dioxane under the reaction conditions described (temperature: 85° C.; reaction time: 2-10 h).
[0408] Exemplary Work-Up of the Reaction Mixture:
[0409] The reaction mixture was concentrated and the residue was chromatographed on silica gel (mobile phase: dichloromethane/methanol gradient). Optionally, further purification was carried out by preparative HPLC [column: Kinetex C18, 5 μM, 100×300 mm; mobile phase: water/acetonitrile 35:65].
TABLE-US-00007 TABLE 7A Ex- IUPAC name/structure ample (Yield) Analytical data 37A rac-2-[5-fluoro-1-(2-fluorobenzyl)-6-methyl-1H- .sup.1H-NMR (400 MHz, DMSO-d.sub.6) δ = pyrazolo[3,4-b]pyridin-3-yl]-4-iodo-5-methyl-5- 1.81 (s, 3H), 2.64 (d, 3H), 5.84 (s, (trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin- 2H), 7.13-7.27 (m, 3H), 7.34-7.41 6-one (m, 1H), 8.37 (d, 1H), 12.39 (s, 1H). LC-MS (Method 7): R.sub.t = 1.64 min
Example 41A
2-Amino-3 -fluoro-2-(fluoromethyl)propanonitrile
[0410] ##STR00058##
[0411] 8.75 g (178.6 mmol) of sodium cyanide were initially charged in 132 ml of 2N ammonia solution in methanol. 15.0 g (159.5 mmol) of 1,3-difluoroacetone and 9.55 g (178.6 mmol) of ammonium chloride were added at RT. The suspension was stirred at oil bath temperature of 70° C. for 2 h. 300 ml of diethyl ether were added to the cooled reaction mixture, the mixture was stirred for 10 min and the solid was filtered off. The filtrate was concentrated under reduced pressure (50° C., 70 mbar). This gave 19.2 g (100% of theory) of the target compound. The product was reacted further without further purification.
[0412] GC-MS (Method 8): R.sub.t=1.78 min
[0413] MS (ESpos): m/z=121 (M+H).sup.+
Example 42A
Benzyl (2-cyano-1,3-difluoropropan-2-yl)carbamate
[0414] ##STR00059##
[0415] 5.0 g (41.6 mmol) of 2-amino-3-fluoro-2-(fluoromethyl)propanonitrile from Example 41A were initially charged in 14.5 ml (83.3 mmol) of N,N-diisopropylethylamine. 10.65 g (62.5 mmol) of benzyl chloroformate was slowly added dropwise at RT, and the mixture was stirred at RT for 3 days. The reaction mixture was diluted with 25 ml of dichloromethane and, at 0° C., added dropwise to a solution of 12.9 g (124.9 mmol) of N-(2-aminoethyl)ethane-1,2-diamine in 225 ml of dichloromethane, and the mixture was stirred for 10 min. 200 ml of saturated ammonium chloride solution were then added dropwise at RT. The phases were separated and the aqueous phase was extracted three times with dichloromethane. The combined organic phases were concentrated. The crude product was then chromatographed in silica gel (mobile phase: cyclohexane: ethyl acetate gradient). This gave 4.40 g (42% of theory) of the title compound.
[0416] LC-MS (Method 1): R.sub.t=0.92 min
[0417] MS (ESpos): m/z=255 (M+H).sup.+
Example 43A
Benzyl [1-amino-3-fluoro-2-(fluoromethyl)propan-2-yl]carbamate
[0418] ##STR00060##
[0419] AT RT, 5.00 g (16.32 mmol) of benzyl [1-amino-3-fluoro-2-(fluoromethyl)propan-2-yl]carbamate from Example 42A were initially charged in 80 ml of abs. ethanol. 3.09 g (81.62 mmol) of sodium borohydride were added at RT, and the mixture was stirred at RT for 2 h. With ice cooling, 250 ml of saturated ammonium chloride solution were very slowly added dropwise. 1 N aqueous hydrochloric acid was then added until a pH of 4 had been established (about 100 ml). The reaction mixture was then saturated with saturated aqueous sodium chloride solution and extracted six times with ethyl acetate. The combined organic phases were washed once with saturated aqueous sodium bicarbonate solution, dried over sodium sulfate, filtered and concentrated. The starting compound was stored at −18° C. This gave 4.16 g (83% of theory; purity 84%) of the title compound.
[0420] LC-MS (Method 5): R.sub.t=1.99 min
[0421] MS (ESpos): m/z=259 (M+H).sup.+
Example 44A
3-Fluoro-2-(fluoromethyl)propane-1,2-diamine
[0422] ##STR00061##
[0423] 4.16 g (13.53 mmol) of benzyl [1-amino-3-fluoro-2-(fluoromethyl)propan-2-yl]carbamate from Example 43A were initially charged in 12 ml of 1-methyl-2-pyrrolidone, and 216 mg (0.20 mmol) of 10% palladium on activated carbon were added under argon. The reaction mixture was hydrogenated at RT and standard pressure overnight. The reaction mixture was filtered over Celite and the filter was then washed with 2.5 ml of 1-methyl-2-pyrrolidone. The combined solutions were employed directly for the next reaction.
[0424] A concentration of 1.07 mol/l (133 mg/ml) of the target compound was proceeded from.
WORKING EXAMPLES
Example 1
4-{[2-Amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2[1-(2,3-difluorobenzyl)-5-fluoro-6-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl]-5,5-dimethyl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
[0425] ##STR00062##
[0426] A solution of 144 mg (1.16 mmol) of 3-fluoro-2-(fluoromethyl)propane-1,2-diamine in 1-methyl-2-pyrrolidone (NMP) from Example 44A [assumed concentration: 1.07 mol/l] was added to 200 mg (0.29 mmol; purity 82%) of 2-[1-(2,3-difluorobenzyl)-5-fluoro-6-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl]-4-iodo-5,5-dimethyl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one from Example 30A, and the mixture was diluted with 0.5 ml of 1-methyl-2-pyrrolidone (NMP). The mixture was stirred at 130° C. in the microwave for 3.5 h. Water/acetonitrile/TFA was added and the reaction solution was purified by preparative HPLC (RP18 column, mobile phase: acetonitrile/water gradient with addition of 0.1% TFA). The product fractions were concentrated by evaporation. The residue obtained was taken up in dichloromethane and a little methanol and washed twice with saturated aqueous sodium bicarbonate solution. The combined aqueous phases were extracted twice with dichloromethane. The combined organic phases were dried over sodium sulfate, filtered and concentrated by evaporation. This gave 22 mg (12% of theory; purity 92%) of the title compound.
[0427] LC-MS (Method 1): R.sub.t=0.82 min
[0428] MS (ESIpos): m/z=561 [M+H].sup.+
[0429] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ [ppm]=1.36 (s, 6H), 1.84 (br. s, 2H), 2.62 (d, 3H), 3.73 (d, 2H), 4.18-4.48 (m, 4H), 5.82 (s, 2H), 6.49 (br. s, 1H), 6.99-7.06 (m, 1H), 7.11-7.20 (m, 1H), 7.34-7.43 (m, 1H), 8.55 (d, 1H), 11.08 (br. s, 1H).
Example 2
4-{[2-Amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2-[5-fluoro-1-(2-fluorobenzyl)-6-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl]-5,5-dimethyl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
[0430] ##STR00063##
[0431] A solution of 273 mg (2.20 mmol) of 3-fluoro-2-(fluoromethyl)propane-1,2-diamine in 1-methyl-2-pyrrolidone from Example 44A [assumed concentration: 1.07 mol/1] was added to 300 mg (0.55 mmol) of 2-[5-fluoro-1-(2-fluorobenzyl)-6-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl]-4-iodo-5,5-dimethyl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one, and the mixture was diluted with 2.7 ml of 1-methyl-2-pyrrolidone. The mixture was stirred at 130° C. for 7 h. The reaction solution was purified directly by preparative HPLC (RP18 column, mobile phase: methanol/water gradient with addition of 0.1% TFA). The product fractions were concentrated by evaporation. Further purification was carried out by preparative HPLC [Kinetex C18, 5 μm, 100×21.2 mm; mobile phase: water/acetonitrile/1% formic acid in water=60/35/5, isocratic]. This gave 40 mg (12% of theory; purity 86%) of the title compound.
[0432] LC-MS (Method 7): R.sub.t=1.06 min
[0433] MS (ESIpos): m/z=543 [M+H].sup.+
[0434] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ [ppm]=1.38 (s, 6H), 2.62 (d, 3H), 3.76 (d, 2H), 4.21-4.31 (m, 1H), 4.36 (d, 2H), 4.47 (d, 1H), 5.78 (s, 2H), 6.56 (t, 1H), 7.11-7.27 (m, 314), 7.32-7.40 (m, 1H), 8.53 (d, 1H), 11.07 (s, 1H).
Example 3
4-{[2-Amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2-[5-fluoro-6-methyl-1-(2,3,6-trifluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5,5-dimethyl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
[0435] ##STR00064##
[0436] A solution of 256 mg (2.06 mmol) of 3-fluoro-2-(fluoromethyl)propane-1,2-diamine in 1-methyl-2-pyrrolidone from Example 44A [assumed concentration: 1.07 mol/1] was added to 300 mg (0.51 mmol) of 2-[5-fluoro-6-methyl-1-(2,3,6-trifluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-4-iodo-5,5-dimethyl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one from Example 32A, and the mixture was diluted with 2.5 ml of 1-methyl-2-pyrrolidone. The mixture was stirred at 130° C. for 4 h. The reaction solution was purified directly by preparative HPLC (RP18 column, mobile phase:
[0437] methanol/water gradient with addition of 0.1% TFA). The production fractions were concentrated by evaporation. The residue obtained was taken up in dichloromethane and a little methanol and washed twice with saturated aqueous sodium bicarbonate solution. The combined aqueous phases were extracted twice with dichloromethane. The combined organic phases were dried over sodium sulfate, filtered and concentrated by evaporation. This gave 20 mg (6% of theory; purity 86%) of the title compound.
[0438] LC-MS (Method 5): R.sub.t=2.55 min
[0439] MS (ESIpos): m/z=579 [M+H].sup.+
[0440] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ [ppm]=1.37 (s, 6H), 1.85 (br. s., 2H), 2.63 (d, 3H), 3.73 (d, 2H), 4.19 (d, 1H), 4.31 (d, 2H), 4.43 (d, 1H), 5.80 (s, 2H), 6.55 (t, 1H), 7.18 (ddt, 1H), 7.54 (ddt, 1H), 8.52 (d, 1H), 11.05 (s, 1H).
Example 4
4-{[2-Amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2-{5-fluoro-1-[(3-fluoropyridin-2-yl)methyl]-6-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl}-5,5-dimethyl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
[0441] ##STR00065##
[0442] A solution of 169 mg (1.36 mmol) of 3-fluoro-2-(fluoromethyl)propane-1,2-diamine in 1-methyl-2-pyrrolidone from Example 44A [assumed concentration: 1.07 mol/l] was added to 200 mg (0.34 mmol) of 2-{5-fluoro-1-[(3-fluoropyridin-2-yl)methyl]-6-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl}-4-iodo-5,5-dimethyl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one from Example 33A, and the mixture was diluted with 0.4 ml of 1-methyl-2-pyrrolidone (NMP). The mixture was stirred at 130° C. in the microwave for 5 h. Water/acetonitrile/TFA was added, and the reaction solution was purified by preparative HPLC (RP18 column, mobile phase: acetonitrile/water gradient with addition of 0.1% TFA). The product fractions were concentrated by evaporation. The residue obtained was taken up in dichloromethane and a little methanol and washed twice with saturated aqueous sodium bicarbonate solution. The combined aqueous phases were extracted twice with dichloromethane. The combined organic phases were dried over sodium sulfate, filtered and concentrated by evaporation. This gave 19 mg (10% of theory) of the title compound.
[0443] LC-MS (Method 1): R.sub.t=0.72 min
[0444] MS (ESIpos): m/z=544 [M+H].sup.+
[0445] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ [ppm]=1.37 (s, 6H), 1.83 (br. s, 2H), 2.60 (d, 3H), 3.75 (d, 2H), 4.17-4.47 (m, 4H), 5.91 (s, 2H), 6.51 (t, 1H), 7.40-7.47 (m, 1H), 7.72-7.80 (m, 1H), 8.28 (d, 1H), 8.53 (d, 1H), 11.02 (br. s, 1H).
Example 5
4-{[2-Amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2-{5-fluoro-1-[(3-fluoropyridin-2-yl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl}-5,5-dimethyl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
[0446] ##STR00066##
[0447] A solution of 348 mg (2.80 mmol) of 3-fluoro-2-(fluoromethyl)propane-1,2-diamine in 1-methyl-2-pyrrolidone (NMP) from Example 44A [assumed concentration: 1.07 mol/l] was added to 450 mg (0.70 mmol; purity 83%) of 2-{5-fluoro-1-[(3-fluoropyridin-2-ypmethyl]-1H-pyrazolo[3,4-b]pyridin-3-yl}-4-iodo-5,5-dimethyl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one from Example 34A, and the mixture was diluted with 1.2 ml of 1-methyl-2-pyrrolidone (NMP). The mixture was stirred at 130° C. in the microwave for 4 h. Water/acetonitrile/TFA was added and the reaction solution was purified by preparative HPLC (RP18 column, mobile phase: acetonitrile/water gradient with addition of 0.1% TFA). The product fractions were concentrated by evaporation. The residue obtained was taken up in dichloromethane and a little methanol and washed twice with saturated aqueous sodium bicarbonate solution. The combined aqueous phases were extracted twice with dichloromethane. The combined organic phases were dried over sodium sulfate, filtered and concentrated by evaporation. This gave 43 mg (12% of theory; purity 97%) of the title compound.
[0448] LC-MS (Method 1): R.sub.t=0.66 min
[0449] MS (ESIpos): m/z=530 [M+H].sup.+
[0450] .sup.1H-NMR (500 MHz, DMSO-d.sub.6): δ [ppm]=1.39 (s, 6H), 1.85 (br. s, 2H), 3.76 (d, 2H), 4.20-4.46 (m, 4H), 5.96 (s, 2H), 6.53 (t, 1H), 7.40-7.46 (m, 1H), 7.73-7.80 (m, 1H), 8.25-9.29 (m, 1H), 8.63-8.69 (m, 2H), 11.05 (br. s, 1H).
Example 6
rac-4-{[2-Amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2-[1-(2,3-difluorobenzyl)-5-fluoro-6-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
[0451] ##STR00067##
[0452] A solution of 141 mg (1.14 mmol) of 3-fluoro-2-(fluoromethyl)propane-1,2-diamine in 1-methyl-2-pyrrolidone from Example 44A [assumed concentration: 1.07 mol/l] was added to 200 mg (0.29 mmol; purity 88%) of rac-2-[1-(2,3-difluorobenzyl)-5-fluoro-6-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl]-4-iodo-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one from Example 39A, and the mixture was diluted with 0.4 ml of 1-methyl-2-pyrrolidone (NMP). The mixture was stirred at 130° C. in the microwave for 4.5 h. Water/acetonitrile/TFA was added and the reaction solution was purified by preparative HPLC (RP18 column, mobile phase: acetonitrile/water gradient with addition of 0.1% TFA). The product fractions were concentrated by evaporation. The residue obtained was taken up in dichloromethane and a little methanol and washed twice with saturated aqueous sodium bicarbonate solution. The combined aqueous phases were extracted twice with dichloromethane. The combined organic phases were dried over sodium sulfate, filtered and concentrated by evaporation. This gave 57 mg (31% of theory; purity 95%) of the title compound.
[0453] LC-MS (Method 1): R.sub.t=0.91 min
[0454] MS (ESIpos): m/z=615 [M+H].sup.+
[0455] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ [ppm]=1.66 (s, 3H), 1.87 (br. s, 2H), 2.61 (d, 3H), 3.59-3.68 (m, 1H), 3.73-3.84 (m, 1H), 4.21 (d, 1H), 4.28-4.36 (m, 2H), 4.40-4.47 (m, 1H), 5.83 (s, 2H), 6.24 (br. s, 1H), 6.97-7.05 (m, 1H), 7.09-7.22 (m, 1H), 7.32-7.44 (m, 1H), 8.51 (d, 1H), 11.69 (br. s, 1H).
Example 7
ent-4-{[2-Amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2-[1-(2,3-difluorobenzyl)-5-fluoro-6-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (Enantiomer A)
[0456] ##STR00068##
[0457] 50 mg of rac-4-{[2-amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2-[1-(2,3 -difluorobenzyl)-5-fluoro-6-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (Example 6) were separated into the enantiomers on a chiral phase [column: Daicel Chiralcel OX-H, 5 μm, 250×20 mm, mobile phase: 80% isohexane, 20% ethanol+0.2% diethylamine, flow rate 15 ml/min; 40° C., detection: 220 nm]. The product fractions were collected on dry ice, concentrated by evaporation and then lyophilized.
[0458] Enantiomer A: 14 mg (99% purity, 99% ee)
[0459] R.sub.t=5.36 min [Daicel Chiralcel OX-H, 5 μm, 250×4.6 mm; mobile phase: 80% isohexane, 20% ethanol +0.2% diethylamine; flow rate 1.0 ml/min; 40° C.; detection: 220 nm].
Example 8
ent-4-{[2-Amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2-[1-(2,3-difluorobenzyl)-5-fluoro-6-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (Enantiomer B)
[0460] ##STR00069##
[0461] 50 mg of rac-4-{[2-amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2-[1-(2,3-difluorobenzyl)-5-fluoro-6-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (Example 6) were separated into the enantiomers on a chiral phase [column: Daicel Chiralcel OX-H, 5 μm, 250×20 mm, mobile phase: 80% isohexane, 20% ethanol+0.2% diethylamine, flow rate 15 ml/min; 40° C., detection: 220 nm]. The product fractions were collected on dry ice, concentrated by evaporation and then lyophilized.
[0462] Enantiomer B: 16 mg (99% purity, 99% ee)
[0463] R.sub.t=8.31 min [Daicel Chiralcel OX-H, 5 μm, 250×4.6 mm; mobile phase: 80% isohexane, 20% ethanol+0.2% diethylamine; flow rate 1.0 ml/min; 40° C.; detection: 220 nm].
Example 9
rac-4-{[2-Amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2-[5-fluoro-1-(2-fluorobenzyl)-6-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
[0464] ##STR00070##
[0465] A solution of 165 mg (1.33 mmol) of 3-fluoro-2-(fluoromethyl)propane-1,2-diamine in 1-methyl-2-pyrrolidone from Example 44A [assumed concentration: 1.07 mol/l] was added to 200 mg (0.33 mmol) of rac-2-[5-fluoro-1-(2-fluorobenzyl)-6-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl]-4-iodo-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one from Example 37A, and the mixture was diluted with 1.5 ml of 1-methyl-2-pyrrolidone. The mixture was stirred at 130° C. in the microwave for 5 h. The reaction solution was purified directly by preparative HPLC (RP18 column, mobile phase: methanol/water gradient with addition of 0.1% TFA). The product fractions were freed from methanol and extracted repeatedly with a mixture of dichloromethane/methanol (10/1). The combined organic phases were washed with saturated aqueous sodium bicarbonate solution and sodium chloride, dried over sodium sulfate, filtered and concentrated by evaporation. The residue was purified again by preparative HPLC (RP18 column, mobile phase: methanol/water gradient with addition of 0.1% TFA). The product fractions were concentrated. This gave 37 mg (16% of theory; purity 85%) of the title compound.
[0466] LC-MS (Method 7): R.sub.t=1.11 min
[0467] MS (ESIpos): m/z=597 [M+H].sup.+
[0468] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ [ppm]=1.75 (s, 3H), 1.95 (br. s, 2H), 2.63 (d, 3H), 3.66 (dd, 1H), 3.86 (dd, 1H), 4.22 (d, 1H), 4.30-4.36 (m, 2H), 4.44 (dd, 1H), 5.80 (s, 2H), 6.52 (t, 1H), 7.09-7.27 (m, 3H), 7.32-7.41 (m, 1H), 8.50 (d, 1H), 11.72 (br. s, 1 H).
Example 10
ent-4-{[2-Amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2-[5-fluoro-1-(2-fluorobenzyl)-6-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (Enantiomer A)
[0469] ##STR00071##
[0470] 36 mg of rac-4-{[2-amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2-[5-fluoro-1-(2-fluorobenzyl)-6-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (Example 9) were separated into the enantiomers on a chiral phase [column: Daicel Chiralcel OZ-H, 5 μm, 250×20 mm, mobile phase: 80% isohexane, 20% ethanol, flow rate 15 ml/min; 35° C., detection: 220 nm].
[0471] Enantiomer A: 7 mg (>99% purity, >99% ee)
[0472] R.sub.t=4.15 min [Daicel Chiralcel OZ-H, 5 μm, 250×4.6 mm; mobile phase: 70% isohexane, 30% ethanol +0.2% diethylamine; flow rate 1.0 ml/min; 30° C.; detection: 270 nm].
Example 11
ent-4-{[2-Amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2-[5-fluoro-1-(2-fluorobenzyl)-6-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (Enantiomer B)
[0473] ##STR00072##
[0474] 36 mg of rac-4-{[2-amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2-[5-fluoro-1-(2-fluorobenzyl)-6-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (Example 9) were separated into the enantiomers on a chiral phase [column: Daicel Chiralcel OZ-H, 5 μm, 250×20 mm, mobile phase: 80% isohexane, 20% ethanol, flow rate 15 ml/min; 35° C., detection: 220 nm].
[0475] Enantiomer B: 11 mg (95% purity, >99% ee)
[0476] R.sub.t=5.60 min [Daicel Chiralcel OZ-H, 5 μm, 250×4.6 mm; mobile phase: 70% isohexane, 30% ethanol+0.2% diethylamine; flow rate 1.0 ml/min; 30° C.; detection: 270 nm].
Example 12
rac-4-{[2-Amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2-[5-fluoro-6-methyl-1-(2,3,6-trifluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
[0477] ##STR00073##
[0478] A solution of 156 mg (1.26 mmol) of 3-fluoro-2-(fluoromethyl)propane-1,2-diamine in 1-methyl-2-pyrrolidone from Example 44A [assumed concentration: 1.07 mol/l] was added to 200 mg (0.31 mmol) of rac-2-[5-fluoro-6-methyl-1-(2,3,6-trifluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-4-iodo-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one from Example 38A, and the mixture was diluted with 1.5 ml of 1-methyl-2-pyrrolidone. The mixture was stirred at 130° C. in the microwave for 5 h. The reaction solution was purified directly by preparative HPLC (RP18 column, mobile phase: methanol/water gradient with addition of 0.1% TFA). The product fractions were concentrated, diluted with a mixture of dichloromethane/methanol (10/1), washed with saturated aqueous sodium bicarbonate solution and sodium chloride, dried over sodium sulfate, filtered and concentrated by evaporation. This gave 84 mg (35% of theory; purity 82%) of the title compound.
[0479] LC-MS (Method 1): R.sub.t=0.86 min
[0480] MS (ESIpos): m/z=633 [M+H].sup.+
[0481] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ [ppm]=1.74 (s, 3H), 1.86 (br. s, 2H), 2.64 (d, 3H), 3.63 (dd, 1H), 3.83 (dd, 1H), 4.20 (d, 1H), 4.27-4.35 (m, 2H), 4.42 (dd, 1H), 5.82 (s, 2H), 6.53 (t, 1H), 7.19 (ddt, 1H), 7.54 (ddt, 1H), 8.48 (d, 1H), 11.71 (br. s, 1H).
Example 13
ent-4-{[2-Amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2-[5-fluoro-6-methyl-1-(2,3,6-trifluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (Enantiomer A)
[0482] ##STR00074##
[0483] 84 mg of rac-4-{[2-amino-3 -fluoro-2-(fluoromethyl)propyl]amino}-2-[5-fluoro-6-methyl-1-(2,3,6-trifluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3 -yl]-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (Example 12) were separated into the enantiomers on a chiral phase [column: Daicel Chiralcel OZ-H, 5 μm, 250×20 mm, mobile phase: 80% isohexane, 20% ethanol, flow rate 15 ml/min; 35° C., detection: 220 nm].
[0484] Enantiomer A: 18 mg (>99% purity, >99% ee)
[0485] R.sub.t=4.27 min [Daicel Chiralcel OZ-H, 5 μm, 250×4.6 mm; mobile phase: 70% isohexane, 30% ethanol+0.2% diethylamine; flow rate 1.0 ml/min; 30° C.; detection: 270 nm].
Example 14
ent-4-{[2-Amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2-[5-fluoro-6-methyl-1-(2,3,6-trifluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (Enantiomer B)
[0486] ##STR00075##
[0487] 84 mg of rac-4-{[2-amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2-[5-fluoro-6-methyl-1-(2,3,6-trifluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (Example 12) were separated into the enantiomers on a chiral phase [column: Daicel Chiralcel OZ-H, 5 μm, 250×20 mm, mobile phase: 80% isohexane, 20% ethanol, flow rate 15 ml/min; 35° C., detection: 220 nm].
[0488] Enantiomer B: 19 mg (>99% purity, about 98% ee)
[0489] R.sub.t=4.99 min [Daicel Chiralcel OZ-H, 5 μm, 250×4.6 mm; mobile phase: 70% isohexane, 30% ethanol+0.2% diethylamine; flow rate 1.0 ml/min; 30° C.; detection: 270 nm].
Example 15
rac-4-{[2-Amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2-{5-fluoro-1-[(3-fluoropyridin-2-yl)methyl]-6-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl}-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
[0490] ##STR00076##
[0491] A solution of 132 mg (1.06 mmol) of 3-fluoro-2-(fluoromethyl)propane-1,2-diamine in 1-methyl-2-pyrrolidone from Example 44A [assumed concentration: 1.07 mol/l] was added to 200 mg (0.27 mmol; purity 80%) of rac-2-{5-fluoro-1-[(3-fluoropyridin-2-yl)methyl]-6-methyl-1H-pyrazolo[3,4-b]pyridin-3 -yl}-4-iodo-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one from Example 40A, and the mixture was diluted with 0.4 ml of 1-methyl-2-pyrrolidone (NMP). The mixture was stirred in the microwave at 130° C. for 4.5 h. Water/acetonitrile/TFA was added and the reaction solution was purified by preparative HPLC (RP18 column, mobile phase: acetonitrile/water gradient with addition of 0.1% TFA). The product fractions were concentrated by evaporation. The residue obtained was taken up in dichloromethane and a little methanol and washed twice with saturated aqueous sodium bicarbonate solution. The combined aqueous phases were extracted twice with dichloromethane. The combined organic phases were dried over sodium sulfate, filtered and concentrated by evaporation. The residue was re-purified by thick-layer chromatography (mobile phase: dichloromethane/2N ammonia in methanol=10/1). This gave 37 mg (23% of theory) of the title compound.
[0492] LC-MS (Method 1): R.sub.t=0.81 min
[0493] MS (ESIpos): m/z=598 [M+H].sup.+
[0494] .sup.1H-NMR (500 MHz, DMSO-d.sub.6): δ [ppm]=1.73 (s, 3H), 1.88 (br. s, 2H), 2.61 (d, 31-1), 3.63-3.70 (m, 1H), 3.82-3.88 (m, 1H), 4.22 (d, 1H), 4.30-4.36 (m, 2H), 4.40-4.46 (m, 1H), 5.94 (s, 2H), 6.49 (t, 1H), 7.40-7.46 (m, 1H), 7.73-7.79 (m, 1H), 8.28 (d, 1H), 8.50 (d, 1H), 11.68 (br. s, 1H).
Example 16
ent-4-{[2-Amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2-{5-fluoro-1-[(3-fluoropyridin-2-yl)methyl]-6-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl}-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (Enantiomer A)
[0495] ##STR00077##
[0496] 32 mg of rac-4-{[2-amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2-{5-fluoro-1-[(3-fluoropyridin-2-yl)methyl]-6-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl}-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (Example 15) were separated into the enantiomers on a chiral phase [column: Daicel Chiralpak AZ-H, 5 μm, 250×30 mm, mobile phase: 70% isohexane, 30% ethanol +0.2% diethylamine, flow rate 30 ml/min; 40° C., detection: 220 nm]. The product fractions were collected on dry ice, concentrated by evaporation and then lyophilized.
[0497] Enantiomer A: 14 mg (99% purity, 99% ee)
[0498] R.sub.t=3.97 min [Daicel Chiralpak AZ-H, 5 μm, 250×4.6 mm; mobile phase: 30% isohexane, 70% ethanol+0.2% diethylamine; flow rate 1.0 ml/min; 40° C.; detection: 220 nm].
Example 17
ent-4-{[2-Amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2-{5-fluoro-1-[(3-fluoropyridin-2-yl)methyl]-6-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl}-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (Enantiomer B)
[0499] ##STR00078##
[0500] 32 mg of rac-4-{[2-amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2-{5-fluoro-1-[(3-fluoropyridin-2-yl)methyl]-6-methyl-1H-pyrazolo[3,4-b]pyridin-3-yl}-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (Example 15) were separated into the enantiomers on a chiral phase [column: Daicel Chiralpak AZ-H, 5 μm, 250×30 mm, mobile phase: 70% isohexane, 30% ethanol+0.2% diethylamine, flow rate 30 ml/min; 40° C., detection: 220 nm]. The product fractions were collected on dry ice, concentrated by evaporation and then lyophilized.
[0501] Enantiomer B: 15 mg (95% purity, 98% ee)
[0502] R.sub.t=6.33 min [Daicel Chiralpak AZ-H, 5 μm, 250×4.6 mm; mobile phase: 30% isohexane, 70% ethanol+0.2% diethylamine; flow rate 1.0 ml/min; 40° C.; detection: 220 nm].
Example 18
rac-4-{[2-Amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2-{5-fluoro-1-[(3-fluoropyridin-2-yl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl}-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
[0503] ##STR00079##
[0504] A solution of 148 mg (1.20 mmol) of 3-fluoro-2-(fluoromethyl)propane-1,2-diamine in 1-methyl-2-pyrrolidone from Example 44A [assumed concentration: 1.07 mol/l] was added to 200 mg (0.34 mmol) of rac-2-{5-fluoro-1-[(3-fluoropyridin-2-yl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl}-4-iodo-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one from Example 36A, and the mixture was diluted with 0.4 ml of 1-methyl-2-pyrrolidone (NMP). The mixture was stirred in the microwave at 130° C. for 4.5 h. Water/acetonitrile/TFA was added and the reaction solution was purified by preparative HPLC (RP18 column, mobile phase: acetonitrile/water gradient with addition of 0.1% TFA). The product fractions were concentrated by evaporation. The residue obtained was taken up in dichloromethane and a little methanol and washed twice with saturated aqueous sodium bicarbonate solution. The combined aqueous phases were extracted twice with dichloromethane. The combined organic phases were dried over sodium sulfate, filtered and concentrated by evaporation. This gave 49 mg (25% of theory; purity about 92%) of the title compound.
[0505] LC-MS (Method 1): R.sub.t=0.77 min
[0506] MS (ESIpos): m/z=584 [M+H].sup.+
[0507] .sup.1H-NMR (500 MHz, DMSO-d.sub.6): δ [ppm]=1.74 (s, 3H), 1.89 (br. s, 2H), 3.64-3.71 (m, 1H), 3.81-3.88 (m, 1H), 4.22 (d, 1H), 4.30-4.36 (m, 2H), 4.40-4.46 (m, 1H), 5.98 (s, 2H), 6.52 (t, 1H), 7.40-7.46 (m, 1H), 7.73-7.79 (m, 1H), 8.26 (d, 1H), 8.59-8.63 (m, 1H), 8.68-8.71 (m, 1H), 11.70 (br. s, 1H).
Example 19
ent-4-{[2-Amino-3 -fluoro-2-(fluoromethyl)propyl]amino}-2-{5-fluoro-1-[(3-fluoropyridin-2-yl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl}-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (Enantiomer A)
[0508] ##STR00080##
[0509] 36 mg of rac-4-{[2-amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2-{5-fluoro-1-[(3-fluoropyridin-2-yl(methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl}-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (Example 18) were separated into the enantiomers on a chiral phase [column: Daicel Chiralpak AZ-H, 5 μm, 250×30 mm, mobile phase: 40% isohexane, 60% ethanol+0.2% diethylamine, flow rate 30 ml/min; 40° C., detection: 220 nm]. The product fractions were collected on dry ice, concentrated by evaporation and then lyophilized
[0510] Enantiomer A: 13 mg (99% purity, 99% ee)
[0511] R.sub.t=4.05 min [Daicel Chiralpak AZ-H, 5 μm, 250×4.6 mm; mobile phase: 30% isohexane, 70% ethanol+0.2% diethylamine; flow rate 1.0 ml/min; 40° C.; detection: 220 nm].
Example 20
ent-4-{[2-Amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2-{5-fluoro-1-[(3-fluoropyridin-2-yl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl}-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (Enantiomer B)
[0512] ##STR00081##
[0513] 36 mg of rac-4-{[2-amino-3-fluoro-2-(fluoromethyl)propyl]amino}-2-{5-fluoro-1-[(3-fluoropyridin-2-yl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl}-5-methyl-5-(trifluoromethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (Example 18) were separated into the enantiomers on a chiral phase [column: Daicel Chiralpak AZ-H, 5 μm, 250×30 mm, mobile phase: 40% isohexane, 60% ethanol+0.2% diethylamine, flow rate 30 ml/min; 40° C., detection: 220 nm]. The product fractions were collected on dry ice, concentrated by evaporation and then lyophilized.
[0514] Enantiomer B: 17 mg (about 92% purity, 97% ee)
[0515] R.sub.t=5.56 min [Daicel Chiralpak AZ-H, 5 μm, 250×4.6 mm; mobile phase: 30% isohexane, 70% ethanol+0.2% diethylamine; flow rate 1.0 ml/min; 40° C.; detection: 220 nm].
Example 21
4-{[2-Amino-3 -fluoro-2-(fluoromethyl)propyl]amino}-2-[6-chloro-1-(2-fluorobenzyl)-1H-indazol-3-yl]-5,5-dimethyl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one trifluoroacetate
[0516] ##STR00082##
[0517] A solution of 70 mg (0.57 mmol) of 3-fluoro-2-(fluoromethyl)propane-1,2-diamine in 1-methyl-2-pyrrolidone from Example 44A [assumed concentration: 1.07 mol/l] was added to 80 mg (0.14 mmol) of 2-[6-chloro-1-(2-fluorobenzyl)-1H-indazol-3-yl]-4-iodo-5,5-dimethyl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one from Example 35A, and the mixture was diluted with 0.2 ml of 1-methyl-2-pyrrolidone (NMP). The mixture was stirred in the microwave at 130° C. for 4 h. Another solution of 35 mg (0.28 mmol) of 3-fluoro-2-(fluoromethyl)propane-1,2-diamine in 1-methyl-2-pyrrolidone from Example 44A [assumed concentration: 1.07 mol/l] was then added, and the mixture was stirred in the microwave at 130° C. for 2 h. Water/acetonitrile/TFA was added and the reaction solution was purified by preparative HPLC (RP18 column, mobile phase: acetonitrile/water gradient with addition of 0.1% TFA). This gave 3 mg (3% of theory; purity about 93%) of the title compound.
[0518] LC-MS (Method 1): R.sub.t=0.84 min
[0519] MS (ESIpos): m/z=544.4 [M+H].sup.+
[0520] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ [ppm]=1.38 (s, 6H), 3.75-3.81 (m, 2H), 4.58-4.69 (m, 2H), 4.71-4.82 (m, 2H), 5.83 (s, 2H), 6.90 (t, 1H), 7.06-7.11 (m, 1H), 7.12-7.18 (m, 1H), 7.21-7.28 (m, 1H), 7.32-7.42 (m, 2H), 7.94 (s, 1H), 8.50 (d, 1H), 8.98 (br. s, 3H), 11.22 (s, 1H).
B. ASSESSMENT OF PHARMACOLOGICAL EFFICACY
[0521] The following abbreviations are used hereinbelow: [0522] BSA bovine serum albumin [0523] EDTA ethylenediaminetetraacetic acid [0524] μCi micro Curie [0525] Tris tris(hydroxymethyl)aminomethane
[0526] The pharmacological activity of the compounds of the invention can be demonstrated in the following assays:
B-1. Vasorelaxant Effect in Vitro
[0527] The determination of the relaxant activity of the compounds of the invention on isolated vessels was carried out as described in J P Stasch et al., Br J Pharmacol. 2002; 135, 333-343. Rabbits are stunned by a blow to the neck and exsanguinated. The aorta is removed, freed from adhering tissue and divided into rings of width 1.5 mm, which are 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. The contractile force is determined with Statham UC2 cells, amplified and digitalized using A/D transducers (DAS-1802 HC, Keithley Instruments Munich), and recorded in parallel on linear recorders.
[0528] To generate a contraction, phenylephrine is added to the bath cumulatively in increasing concentration. After several control cycles, the substance to be studied is added in increasing dosage each time in every further run, and the magnitude of the contraction is compared with the magnitude of the contraction attained in the last preceding run. This is used to calculate the concentration needed to reduce the magnitude of the control value by 50% (IC.sub.50 value). The standard administration volume is 5 μl; the DMSO content in the bath solution corresponds to 0.1%.
B-2. Effect on a Recombinant Guanylate Cyclase Reporter Cell Line
[0529] The cellular activity of the compounds of the invention is determined using a recombinant guanylate cyclase reporter cell line, as described in F. Wunder et al., Anal. Biochem. 339, 104-112 (2005). Representative values (MEC=minimum effective concentration) for the compounds of the invention are shown in the table below (Table 1; in some cases as means of individual determinations):
TABLE-US-00008 TABLE 1 Example MEC no. [μM] 1 0.3 2 0.1 3 0.03 4 1 5 1 6 0.1 7 0.3 8 0.1 10 0.2 11 0.1 13 0.3 14 0.1 15 0.1 16 0.07 17 0.65 18 0.1 19 0.07 20 0.65 21 0.3
B-3. Inhibition of human phosphodiesterase 5 (PDE 5)
[0530] PDE 5 preparations are obtained from human platelets by disruption (Microfluidizer®, 800 bar, 3 passes), followed by centrifugation (75 000 g, 60 min, 4° C.) and ion exchange chromatography of the supernatant on a Mono Q 10/10 column (linear sodium chloride gradient, elution with a 0.2-0.3M solution of sodium chloride in buffer (20 mM Hepes pH 7.2, 2 mM magnesium chloride). Fractions having PDE 5 activity are combined (PDE 5 preparation) and stored at −80° C.
[0531] To determine their in vitro action on human PDE 5, the test substances are dissolved in 100% DMSO and serially diluted. Typically, dilution series (1:3) from 200 μM to 0.091 μM are prepared (resulting final concentrations in the test: 4 μM to 0.0018 μM). In each case 2 μl of the diluted substance solutions are placed into the wells of microtitre plates (Isoplate-96/200W; Perkin Elmer). Subsequently, 50 μl of a dilution of the above-described PDE 5 preparation are added. The dilution of the PDE 5 preparation is chosen such that during the later incubation less than 70% of the substrate are converted (typical dilution: 1:100; dilution buffer: 50 mM tris/hydrochloric acid pH 7.5, 8.3 mM magnesium chloride, 1.7 mM EDTA, 0.2% BSA). The substrate, [8-.sup.3H] cyclic guanosine-3′,5′-monophosphate (1 μCi/μl; Perkin Elmer), is diluted 1:2000 with assay buffer (50 mM tris/hydrochloric acid pH 7.5, 8.3 mM magnesium chloride, 1.7 mM EDTA) to a concentration of 0.0005 μμCi/μl. By addition of 50 μl (0.025 μCi) of the diluted substrate, the enzyme reaction is finally started. The test mixtures are incubated at room temperature for 60 min and the reaction is stopped by adding 25 μl of a suspension of 18 mg/ml yttrium scintillation proximity beads in water (phosphodiesterase beads for SPA assays, RPNQ 0150, Perkin Elmer). The microtitre plates are sealed with a film and left to stand at room temperature for 60 min. Subsequently, the plates are analysed for 30 s per well in a Microbeta scintillation counter (Perkin Elmer). IC.sub.50 values are determined using the graphic plot of the substance concentration against percentage PDE 5 inhibition.
[0532] Representative IC.sub.50 values for the compounds of the invention are shown in the table below (Table 2; in some cases as means of individual determinations):
TABLE-US-00009 TABLE 2 Example IC.sub.50 no. [nM] 1 52 2 160 3 194 4 180 5 70 6 32 7 120 8 120 10 290 11 390 13 300 14 100 15 100 16 57 18 100 19 53 20 120
B-4. Radiotelemetry Measurement of Blood Pressure in Conscious, Spontaneously Hypertensive Rats
[0533] A commercially available telemetry system from DATA SCIENCES INTERNATIONAL DSI, USA, is employed for the blood pressure measurement on conscious rats described below.
[0534] The system consists of 3 main components: [0535] implantable transmitters (Physiotel® telemetry transmitter) [0536] receivers (Physiotel® receiver) which are linked via a multiplexer (DSI Data Exchange Matrix) to a data acquisition computer. [0537] the telemetry system makes it possible to continuously record blood pressure, heart rate and body motion of conscious animals in their usual habitat.
[0538] Animal Material
[0539] The studies are 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.
[0540] After transmitter implantation, the experimental animals are housed singly in type 3 Makrolon cages. They have free access to standard feed and water.
[0541] The day/night rhythm in the experimental laboratory is changed by the room lighting at 6:00 am and at 7:00 pm.
[0542] Transmitter Implantation
[0543] The TA11 PA-C40 telemetry transmitters used are 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.
[0544] For the implantation, the fasted animals are anesthetized with pentobarbital (Nembutal, Sanofi: 50 mg/kg i.p.) and shaved and disinfected over a large area of their abdomens. After the abdominal cavity has been opened along the linea alba, the liquid-filled measuring catheter of the system is inserted into the descending aorta in the cranial direction above the bifurcation and fixed with tissue glue (VetBonD™, 3M). The transmitter housing is fixed intraperitoneally to the abdominal wall muscle, and the wound is closed layer by layer.
[0545] An antibiotic (Tardomyocel COMP, Bayer, 1 ml/kg s.c.) is administered postoperatively for prophylaxis of infection.
[0546] Substances and Solutions
[0547] Unless stated otherwise, the substances to be studied are administered orally by gavage to a group of animals in each case (n=6). In accordance with an administration volume of 5 ml/kg of body weight, the test substances are dissolved in suitable solvent mixtures or suspended in 0.5% tylose.
[0548] A solvent-treated group of animals is used as control.
[0549] Experimental Procedure
[0550] The telemetry measuring unit present is configured for 24 animals. Each experiment is recorded under an experiment number (Vyear month day).
[0551] Each of the instrumented rats living in the system is assigned a separate receiving antenna (1010 Receiver, DSI).
[0552] The implanted transmitters can be activated externally by means of an incorporated magnetic switch. They are switched to transmission in the run-up to the experiment. The signals emitted can be detected online by a data acquisition system (Dataquest™ A.R.T. for WINDOWS, DSI) and processed accordingly. The data are stored in each case in a file created for this purpose and bearing the experiment number.
[0553] In the standard procedure, the following are measured for 10-second periods in each case: [0554] systolic blood pressure (SBP) [0555] diastolic blood pressure (DBP) [0556] mean arterial pressure (MAP) [0557] heart rate (HR) [0558] activity (ACT).
[0559] The acquisition of measurements is repeated under computer control at 5-minute intervals. The source data obtained as absolute values are corrected in the diagram with the currently measured barometric pressure (Ambient Pressure Reference Monitor; APR-1) and stored as individual data. Further technical details are given in the extensive documentation from the manufacturer company (DSI).
[0560] Unless indicated otherwise, the test substances are administered at 9:00 am on the day of the experiment.
[0561] Following the administration, the parameters described above are measured over 24 hours.
[0562] Evaluation
[0563] After the end of the experiment, the acquired individual data are sorted using the analysis software (DATAQUEST™ A.R.T.™ ANALYSIS). The blank value is 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.
[0564] The data are smoothed over a predefinable period by determination of the average (15-minute average) and transferred as a text file to a storage medium. The measured values presorted and compressed in this way are transferred to Excel templates and tabulated. For each day of the experiment, the data obtained are stored in a dedicated file bearing the number of the experiment. Results and test protocols are stored in files in paper form sorted by numbers.
REFERENCES
[0565] 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;
[0566] Kozo Okamoto: Spontaneous hypertension in rats. Int Rev Exp Pathol 7: 227-270, 1969;
[0567] 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 Organ-Protective Effects in a Long-Term Experiment on Rats
[0568] The organ-protective effects of the compounds of the invention are shown in a therapeutically relevant “low nitric oxide (NO)/high renin” hypertension model in rats. The study was carried out analogously to the recently published article (Sharkovska Y, et al. J Hypertension 2010; 28: 1666-1675). This involves treating renin-transgenic rats (TGR(mRen2)27) to which the NO synthase inhibitor L-NAME had been administered via drinking water simultaneously with the compound according to the invention or vehicle over several weeks. Hemodynamic and renal parameters are determined during the treatment period. At the end of the long-term study, organ protection (kidney, lung, heart, aorta) is shown by histopathological studies, biomarkers, expression analyses and cardiovascular plasma parameters.
B-6. Measurements of the Pulmonary Artery Pressure (PAP) in Conscious Dogs Under Hypoxia Conditions
[0569] A telemetry system from DATA SCIENCES INTERNATIONAL DSI, USA, for example, is employed for the blood pressure measurement on conscious dogs described below. The system consists of implantable pressure transmitters, receiver and a data acquisition computer. The telemetry system makes it possible to continuously monitor blood pressures and heart rate of conscious animals. The telemetry transmitters used are surgically implanted under aseptic conditions in the experimental animals 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. The tests are carried out using adult male beagles. Technical details can be found in the documentation from the manufacturing company (DSI).
[0570] Substances and Solutions
[0571] The substances to be tested are each administered to a group of dogs (n=3-6), orally via a gelatine capsule or intravenously in suitable solvent mixtures. A vehicle-treated group of animals is employed as control.
[0572] Experimental Procedure
[0573] For the measurements under hypoxia conditions, the animals are transferred to a chamber with a hypoxic atmosphere (oxygen content about 10%). This is established using commercially available hypoxia generators (from Hoehenbalance, Cologne, Germany). In a standard experiment, for example, one hour and five hours after substance administration the dogs are transferred to the hypoxia chamber for 30 min About 10 min before and after entering the hypoxia chamber, as well as during the stay in the hypoxia chamber, pressures and heart rate are measured by telemetry.
[0574] Evaluation
[0575] In healthy dogs, under hypoxia there is a rapid increase in PAP. By substance administration, this increase can be reduced. To quantify the PAP increase and the differences in heart rate and systemic blood pressure, the data before and during the hypoxia period, smoothed by determination of means, are compared. The courses of the measured parameters are presented graphically using the Prism software (GraphPad, USA).
B-7. Determination of Pharmacokinetic Parameters Following Intravenous and Oral Administration
[0576] The pharmacokinetic parameters of the compounds of the invention are determined in male CD-1 mice, male Wistar rats, female beagles and female cynomolgus monkeys. Intravenous administration in the case of mice and rats is effected 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 is performed via gavage, based on a water/PEG400/ethanol formulation. The removal of blood from rats is simplified by inserting a silicone catheter into the right Vena jugularis externa prior to substance administration. The operation is carried out at least one day prior to the experiment with isofluran anaesthesia and administration of an analgesic (atropine/rimadyl (3/1) 0.1 ml s.c.). The blood is taken (generally more than 10 time points) within a time window including terminal time points of at least 24 to a maximum of 72 hours after substance administration. The blood is removed into heparinized tubes. The blood plasma is then obtained by centrifugation; if required, it is stored at −20° C. until further processing.
[0577] An internal standard (which may also be a chemically unrelated substance) is added to the samples of the compounds of the invention, calibration samples and qualifiers, and there follows protein precipitation by means of acetonitrile in excess. Addition of a buffer solution matched to the LC conditions, and subsequent vortexing, is followed by centrifugation at 1000 g. The supernatant is analysed by LC-MS(/MS) using C18 reversed-phase columns and variable mobile phase mixtures. The substances are quantified via the peak heights or areas from extracted ion chromatograms of specific selected ion monitoring experiments or high-resolution LC-MS experiments.
[0578] The plasma concentration/time plots determined are used to calculate the pharmacokinetic parameters such as AUC, C.sub.max, F (bioavailability), t.sub.1/2 (terminal half life), MRT (mean residence time) and CL (clearance), using a validated pharmacokinetic calculation program.
[0579] Since the substance quantification is performed in plasma, it is 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 is incubated in heparinized whole blood of the species in question in a rocking roller mixer for 20 min. Plasma is obtained by centrifugation at 1000 g. After measurement of the concentrations in plasma and blood (by LC-MS(/MS); see above), the C.sub.blood/C.sub.plasma value is determined by quotient formation.
B-8. Metabolic Study
[0580] To determine the metabolic profile of the compounds of the invention, they are 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.
[0581] 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.sup.+, 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 is 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 are 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-9. Caco-2 Permeability Test
[0582] 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 (Cato-2) cells. Biochem. Biophys. 175 (3), 880-885]. The Caco-2 cells (ACC No. 169, DSMZ, Deutsche Sammlung von Mikroorganismen and Zellkulturen, Braunschweig, Germany) were sown in 24-well plates having an insert and cultivated for 15 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 deter mine the apical to basolateral permeability (P.sub.appA-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 (P.sub.appB-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 (P.sub.app) 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-10. Determination of the Solubility of Substances in Buffer pH 6.5
[0583] 40 μl of DMSO are added to 2 mg of a test substance [50 g/l]. 10 μl of this solution are removed and introduced into 990 μl of PBS buffer pH 6.5 (c=500 μg/ml). This solution/suspension is shaken at room temperature for 24 h. After 30 min of ultracentrifugation at 114 000 g, the supernatant is removed, diluted with ACN/water 8:2 and analyzed by LC-MSMS.
[0584] For calibration, likewise 10 pi are removed from the DMSO stock solution and introduced into 823 μl of DMSO (c=600 μg/ml). Quantification takes place using a five-point calibration curve.
[0585] Instrument for LC-MSMS Quantification:
[0586] AB Sciex TRIPLE QUAD 4500; Agilent 1260 with primary pump (G1312B Infinity), degasser (G4225a Infinity), column thermostat (G1316C Infinity); CTC Analytics PAL injection system THC-xt
[0587] HPLC Method:
[0588] Mobile Phase:
[0589] A: 0.5 ml of formic acid (50% strength)/l of water
[0590] B: 0.5 ml of formic acid (50% strength)/l of acetonitrile
[0591] Gradient:
TABLE-US-00010 Time [min] % A % B 0 90 10 0.5 5 95 0.84 5 95 0.85 90 10 1.50 90 10
[0592] Flow rate: 2.5 ml
[0593] Injection volume: 5 μl
[0594] Column: Waters OASIS HLB, 2.1×20 mm, 25μ
[0595] Column temperature: 30° C.
[0596] Splitter (upstream of MS) 1:20
[0597] MS Methods:
[0598] Flow Injection Analysis (FIA) for optimization
[0599] Multiple Reaction Monitoring (MRM) for quantification
[0600] Mobile Phase:
[0601] A: 0.5 ml of formic acid (50% strength)/l of water
[0602] B: 0.5 ml of formic acid (50% strength)/l of acetonitrile
[0603] Flow rate: 0.25 ml
[0604] Injection volume: 5 μl
[0605] Column: Stainless steel capillary
[0606] Capillary temperature: 22° C.
[0607] Representative solubilities of the compounds according to the invention are listed in Table 3.
TABLE-US-00011 TABLE 3 Solubility Example [mg/l] 1 7.6 2 5.1 4 260.5 5 278.4
B-11. Determination of the Solubility of Substances in Buffers Having Different pH Values
[0608] Per substance and buffer, in each case 0.5-0.6 mg are weighed out exactly (8 weighed portions). A further weighed portion of 0.5-0.6 mg is required for the DMSO calibration solution (weighed portion 9). In each case, buffer is added to the sample such that a concentration of c=500 μg/ml is obtained. This sample solution is shaken at RT and 1400 rpm for 24 h.
[0609] The Eppendorf vessel with the weighed portion for calibration is filled with DMSO to a concentration of c=600 μg/ml. From this stock solution, 2 calibration solutions are prepared. 1000 μl of DMSO are initially charged in a 2 ml Eppendorf vessel, and 34.4 μl of the stock solution are pipetted in (c=20 μg/ml). Of this solution (c=20 μg/ml), 71.4 μl are added to 500 μl of DMSO in a further 2 ml Eppendorf vessel (c=2.5 μg/ml). Both calibration solutions are transferred into HPLC vials. After shaking of the sample solutions, in each case 200 μl of the supernatant are transferred into a centrifuge tube and centrifuged at 114 000 g for 30 min. 150 μl of the supernatant are then removed, diluted with DMSO 1:5 and 1:100 and transferred into HPLC vials. The two calibration solutions and the diluted sample solutions are analyzed by HPLC. Quantification is carried out using the respective peak areas.
[0610] Solvents and Buffers
[0611] Distilled water
[0612] Perchloric acid (Fluka; 77227)
[0613] Acetonitrile (tap quality)
[0614] DMSO (Merck; 8.02912.2500)
[0615] Buffer
TABLE-US-00012 pH 1 HCl buffer (Fluka) pH 2 Citrate buffer (Fluka) pH 4 Citrate buffer (Fluka) pH 5 Citrate buffer (Fluka) pH 6 PBS buffer (Fluka) pH 7 PBS buffer (Fluka) pH 8 Boric acid buffer (Fluka) pH 10 Boric acid buffer (Fluka)
[0616] Instruments
[0617] Agilent 1100 or comparable instrument with UV detection, variable wavelength (e.g. diode array)
[0618] Ultrasonic bath
[0619] Janke & Kunkel Vibromix
[0620] Eppendorf Thermomixer
[0621] HPLC Method:
[0622] Mobile phase A: 5 ml of HClO.sub.4/l of water
[0623] Mobile phase B: acetonitrile
[0624] Gradient:
TABLE-US-00013 Time A B [mm] [%] [%] 0.0 98 2 0.5 98 2 4.5 10 90 6.5 10 90 6.7 98 2 7.5 98 2
[0625] Column: Kromasil 100 C18, 60×2.1 mm, 3.5 μm
[0626] Column oven: 30° C.
[0627] Flow rate: 0.75 ml/min
[0628] Detector: 210 nm Injection volume: 60 μl
C. WORKING EXAMPLES OF PHARMACEUTICAL COMPOSITIONS
[0629] The compounds of the invention can be converted to pharmaceutical preparations as follows:
[0630] Tablet:
[0631] Composition:
[0632] 100 mg of the compound of the invention, 50 mg of lactose (monohydrate), 50 mg of corn starch (native),
[0633] 10 mg of polyvinylpyrrolidone (PVP 25) (BASF, Ludwigshafen, Germany) and 2 mg of magnesium stearate.
[0634] Tablet weight 212 mg. Diameter 8 mm, radius of curvature 12 mm.
[0635] Production:
[0636] 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.
[0637] Suspension for Oral Administration:
[0638] Composition:
[0639] 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.
[0640] 10 mL of oral suspension correspond to a single dose of 100 mg of the compound of the invention.
[0641] Production:
[0642] 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.
[0643] Solution for Oral Administration:
[0644] Composition:
[0645] 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.
[0646] Production:
[0647] The compound of the invention is suspended in the mixture of polyethylene glycol and polysorbate with stirring. The stirring operation is continued until dissolution of the compound of the invention is complete.
[0648] I.V. Solution:
[0649] The compound of the invention is dissolved in a concentration below the saturation solubility in a physiologically acceptable solvent (e.g. isotonic saline solution, glucose solution 5% and/or PEG 400 solution 30%). The solution is subjected to sterile filtration and dispensed into sterile and pyrogen-free injection vessels.