Selected macrolides with PDE4-inhibiting activity

Abstract

The application relates to the macrolide compound of the formula (I): wherein * indicates a stereocentre which is in (R) or (S) configuration, or a pharmaceutically acceptable salt or ester thereof and their use as PDE4 inhibitors. ##STR00001##

Claims

1. A macrolide compound of the formula (I): ##STR00031## wherein * indicates a stereocentre which is in (R) or (S) configuration, or a pharmaceutically acceptable salt or ester thereof.

2. The macrolide compound according to claim 1 having formula (I-A): ##STR00032## or a pharmaceutically acceptable salt or ester thereof.

3. The macrolide compound according to claim 1, wherein said compound is in free form.

4. A pharmaceutical composition comprising a macrolide compound according to claim 1 or a pharmaceutically acceptable salt or ester thereof and a pharmaceutically acceptable inert carrier.

5. A dosage form for oral administration comprising a macrolide compound according to claim 1 or a pharmaceutically acceptable salt or ester thereof and, optionally, a pharmaceutically acceptable inert carrier.

6. A method for treating an inflammatory, allergic or autoimmune disease in a subject in need thereof, comprising the step of administering a therapeutically effective amount of a macrolide compound according to claim 1, or a pharmaceutically acceptable salt or ester thereof, to said subject.

7. The method according to claim 6, wherein said macrolide compound, or pharmaceutically acceptable salt or ester thereof, is orally administered.

8. The method according to claim 6, wherein said inflammatory, allergic or autoimmune is chronic obstructive pulmonary disease (COPD), psoriasis, psoriatic arthritis, lupus, rheumatoid arthritis, Alzheimer, Parkinson's disease, Huntington's disease, interstitial cystitis, asthma, chronic bronchitis, emphysema, atopic dermatitis, urticaria, allergic rhinitis, allergic conjunctivitis, septic shock, ulcerative colitis, inflammatory bowel disease, adult respiratory distress syndrome, ankylosing spondylitis, uveitis, or multiple sclerosis.

9. The method according to claim 6, wherein said inflammatory, allergic or autoimmune disease is chronic obstructive pulmonary disease (COPD) or psoriasis.

10. The method according to claim 6, wherein said subject is an animal.

11. The method according to claim 6, wherein said subject is a human.

Description

EXAMPLE

(1) Abbreviations: DBU for diazabicycloundecane; DCM for dichloromethane; DIPEA for diisopropylethylamine (Huenig's base); DMF for dimethylformamide; MeOH for methanol; THF for tetrahydrofuran; MS for mass spectrometry; NMR for nuclear magnetic resonance.

(2) The numbers of the compounds referred to in the Example correspond to the numbers of the compounds mentioned in the reaction schemes above.

(3) Synthesis of Compound 6

(4) 1.8 g (2.02 mmol) of Compound 4, prepared according to WO2006084410, Example 1, A] to D], and 0.9 g (2.02 mmol) of Compound 5, prepared as described below, are dissolved in 20 ml DMF, then 0.92 g (6.06 mmol) of DBU and 121 mg (0.81 mmol) of NaI are added. The solution is stirred for 1.0 hour at room temperature. The solvent is removed in vacuo, the residue is poured into 50 ml of 0.5M aq. KH.sub.2PO.sub.4 solution and the resulting mixture is extracted twice with 50 ml of DCM. The combined organic layers are washed with water and brine, dried over anhydrous Na.sub.2SO.sub.4 and concentrated in vacuo to give the crude product, which is purified by silica gel column chromatography (eluent: DCM/MeOH=100/1 to 50/1) to give 1.7 g of the desired product as light yellow foam.

(5) MS(ESI): 640.9 [M+2H].sup.2+

(6) 1H-NMR (CDCl3): (diagnostic signals only) 8.44 (s, 2H); 6.74 (d, 1H); 6.59 (s, 1H); 6.50 (s, 1H); 6.48 (dd, 1H); 5.68 (d, 1H); 4.97 (bs, 1H); 4.66-4.72 (m, 3H); 4.60 (m, 1H); 4.48 (s, 2H); 4.35 (bs, 1H); 3.78 (bs, 4H); 3.63 (bs, 1H); 3.56 (dd, 1H); 3.20-3.33 (m, 6H); 3.16 (s, 3H); 2.66-2.76 (m, 3H); 2.40 (d, 1H); 2.26 (bs, 6H); 2.14 (s, 3H); 2.04 (s, 3H); 0.92-0.99 (m, 3H); 0.85 (t, 3H).

(7) Synthesis of Compound 7

(8) 2.0 g (1.56 mmol) of Compound 6 are dissolved under nitrogen atmosphere in 30 ml DMF, the solution is cooled to −20° C., 54 mg (1.4 mmol, 60% dispersion in oil) of NaH is added and the mixture is stirred at −20° C. until HPLC indicated no starting material remained. Then 100 ml water is added, the mixture is extracted 3 times with 50 ml DCM and the combined organic layers are washed with water and brine, dried over anhydrous Na.sub.2SO.sub.4 and concentrated in vacuo to give 2.6 g of the crude product as brown oil, which is purified by silica gel column chromatography eluting with DCM/MeOH (V/V, 60/1) to afford 1.1 g of the desired product as yellow foam.

(9) MS(ESI): 1282.5 [MH].sup.+ and 641.7 [M+2H].sup.2+

(10) 1H-NMR (CDCl3): (diagnostic signals only) 8.40 (s, 2H); 6.70 (d, 1H); 6.46 (s, 1H); 6.42 (dd, 1H); 5.38 (d, 1H); 4.93 (bs, 1H).

(11) Synthesis of Compound 8-a

(12) 600 mg (0.47 mmol) of Compound 7 is dissolved in 15 ml acetonitrile, then 24 ml 1N hydrochloride acid is added. The reaction mixture is stirred at 30° C. for 16 h. The aqueous phase is adjusted to PH=7 with 2N aq. NaHCO.sub.3 solution. The resulting mixture is extracted twice with 30 ml DCM, the combined organic layers are washed with water and brine, dried over anhydrous Na.sub.2SO.sub.4 and concentrated in vacuo to give 0.5 g of the crude product as yellow foam.

(13) MS(ESI): 1080.4 [MH].sup.+ and 540.9 [M+2H].sup.2+

(14) Synthesis of Compound 8-b

(15) 1.5 g (1.39 mml) of compound 4 is dissolved in 30 ml of MeOH and the solution is stirred for 16 h at 30° C. Then the solvent is removed in vacuo and the residue is purified by flash chromatography on silica gel (DCM/MeOH 60:1) to afford 400 mg of the desired product.

(16) MS(ESI): 1038.4 [MH].sup.+ and 519.9 [M+2H].sup.2+

(17) 1H-NMR (CDCl3): (diagnostic signals only) 8.43 (s, 2H); 6.75 (d, 1H); 6.50 (s, 1H); 6.44 (dd, 1H); 5.52 (d, 1H); 4.71 (bs, 1H); 4.58-4.61 (m, 3H); 4.40 (s, 1H); 3.77 (s, 3H); 3.71 (s, 1H); 3.47-3.60 (m, 5H); 3.37-3.42 (m, 1H); 2.83-2.88 (m, 1H); 2.47-2.49 (m, 1H); 2.06-2.08 (m, 1H); 1.44 (s, 3H); 1.25-1.38 (m, 9H); 1.08-1.14 (m, 9H); 0.83 (t, 3H).

(18) Synthesis of Compound 9

(19) 200 mg (0.15 mmol) of compound 5 is dissolved in a mixture of MeOH and THF (10 ml MeOH/2 ml THF) and 63 mg (0.77 mmol) sodium acetate is added. The mixture is stirred at 30˜35° C. or 30 minutes, then 177 mg I.sub.2 (0.70 mmol) is added. The black reaction mixture is stirred at 30-35° C. for 5 h. Saturated aq. Na.sub.2S.sub.2O.sub.3 solution is added until the color of I.sub.2 faded. The solvent is removed in vacuo, the residue is poured into 30 ml water and is extracted twice with 50 ml DCM. The combined organic layers are washed with water and brine, dried over anhydrous Na.sub.2SO.sub.4 and concentrated in vacuo to give the crude product, which is purified by flash chromatography on silica gel (DCM/MeOH 100:1-20:1) to afford 70 mg of the desired product as yellow foam.

(20) MS(ESI): 1026.4 [MH].sup.+ and 513.8 [M+2H].sup.2+

(21) Synthesis of the Compound of Formula (I) According to the Invention

(22) To a solution of 10.0 g (4.88 mmol) of Compound 9 in 150 ml THF is added 1.89 g (14.63 mmol) DIPEA at 0-5° C. under nitrogen atmosphere. The mixture is stirred for 30 minutes and 1.46 g (9.75 mmol) of 4-morpholinylcarbonyl chloride (MCC) is added. The mixture is stirred at 20° C. for 20 h. The solvent is removed under reduced pressure. The residue is dissolved in 200 ml DCM and washed with water and brine, dried over anhydrous Na.sub.2SO.sub.4 and concentrated in vacuo to give the crude product, which is purified by flash chromatography on silica gel (DCM/MeOH=200:1˜50:1) to afford 4.6 g of the desired product as a yellow foam.

(23) MS(ESI): 1137.5 [M+H].sup.+, 569.2 [M+2H].sup.2+

(24) 1H-NMR (DMSO-d6): 8.59 (s, 1H); 8.59 (s, 1H); 6.79 (d, 1H); 6.49 (d, 1H); 6.44 (dd, 1H); 5.33 (dd, 1H); 5.23 (d, 1H); 4.90 (d, 1H); 4.71 (m, 1H); 4.67 (d, 1H); 4.62 (d, 1H); 4.56 (d, 1H); 4.25 (s, 1H); 3.68 (s, 1H); 3.65 (m, 1H); 3.65 (s, 3H); 3.61 (m, 1H); 3.56 (m, 4H); 3.52 (m, 1H); 3.39 (m, 1H); 3.24 (dd, 1H); 3.20 (m, 1H); 3.12 (m, 2H); 3.08 (m, 1H); 3.05 (m, 2H); 2.93 (m, 1H); 2.88 (s, 3H); 2.78 (m, 1H); 2.71 (s, 3H); 2.59 (s, 1H); 2.56 (dd, 1H); 2.34 (m, 1H); 1.90 (m, 1H); 1.87 (m, 1H); 1.78 (m, 2H); 1.68 (1H); 1.67 (m, 2H); 1.66 (m, 2H); 1.63 (m, 1H); 1.55 (1H); 1.52 (m, 2H); 1.46 (m, 1H); 1.45 (m, 1H); 1.41 (s, 3H); 1.17 (s, 3H); 1.15 (d, 3H); 1.11 (d, 3H); 1.05 (d, 3H); 0.98 (d, 3H); 0.94 (d, 3H); 0.75 (t, 3H);

(25) Synthesis of Compound 5-C

(26) To a solution of 2.6 g (6.05 mmol) of compound 5-B, prepared according to WO2009098320, Example 15, A] and B], in 30 ml DMF is added 1.38 g (12.1 mmol) potassium thioacetate and 181 mg (1.21 mmol) sodium iodide. The reaction mixture is stirred at 60° C. for 5 h, then 100 ml water is added; the mixture is extracted twice with 100 ml ethyl acetate. The combined organic layers are washed with water and brine, dried over Na.sub.2SO.sub.4 and concentrated in vacuo to give 2.8 g of the desired product as yellow solid.

(27) MS(ESI): 469.1 [MH].sup.+

(28) Synthesis of Compound 5

(29) 8.0 g (17.0 mmol) of Compound 5-C is dissolved in 150 ml methanol, then ammonia gas is bubbled into the solution at 5° C. The resulting solution is stirred for 4 hours at this temperature under ammonia atmosphere, then evaporated under vacuum to give 7.5 g of the desired product as a yellow solid. The product is stored under an atmosphere of argon.

(30) MS(ESI): 427.2 [MH].sup.+

(31) The biological activity data of the Compound of Formula (I) according to the present invention and the comparative compounds given in Table 1 are determined as follows:

(32) Enzyme Preparations

(33) PDE4 is partially purified from undifferentiated human monocytic cells (U937) according to Thorpy et al. 1992 (J. Pharmacol. Exp. Ther. 263: 1195). Cells are grown in Iscove's modified Dulbecco's medium (GIBCO) with 5% foetal bovine serum (GIBCO) and 100 μg/mL penicillin-streptomycin (GIBCO). Cells are broken by sonication and PDE4 is purified by anion-exchange chromatography on DEAE-Sepharose CL-6B (GE Healthcare). The final preparations are specific for cAMP and do not hydrolyze cGMP above the detection limit of the assay. In addition, PDE4 preparations are validated by inhibition studies with PDE4-specific and unspecific PDE inhibitors.

(34) Enzyme Assays

(35) PDEs specifically hydrolyze cAMP and/or cGMP and release the product AMP and/or GMP. The potency of PDE inhibition by test compounds is determined with a commercially available in vitro enzymatic assay (IMAP® Fluorescence Polarization assay, Molecular Devices Corp.). Fluorescently labeled cAMP or cGMP is hydrolyzed by PDE preparations and in a second step, binding of labeled product to a large binding partner allows product detection by fluorescence polarization (FP) measurements.

(36) Stock solutions of the test compounds are made in DMSO and diluted in assay buffer (10 mM Tris-HCl, 10 mM MgCl.sub.2, 0.1% BSA 0.05% NaN.sub.3, pH 7.2) to the desired concentrations. The solutions used in the assay contain the test compound in assay buffer with 2% DMSO. 5 μl of this pre-diluted test compound solution are mixed with 10 μl of substrate (FL-cAMP or FL-cGMP) at concentrations recommended by the manufacturer and with 5 μl of appropriately diluted PDE. 5 μl of reaction buffer with 2% DMSO are used for control reactions. The final concentration of DMSO in the assay is 0.5%, which does not significantly alter the PDE activity. After incubation for 90 minutes at room temperature, 60 μl of binding reagent are added as specified by the manufacturer. Binding is allowed to proceed for 30 minutes and fluorescence polarization is measured. Dose dependence of PDE inhibition is measured by assaying dilution series of test compounds. IC.sub.50 values are determined from the measured activities by curve fitting.

(37) MIC Determination:

(38) All MIC values are determined by broth microdilution according to the guidelines by the Clinical and Laboratory Standards Institute (CLSI, Wayne Pa., USA). Staphylococcus aureus ATCC29213 is grown on Müller-Hinton agar (MHA) (Becton Dickinson) and then in cation-adjusted Müller Hinton broth (CaMHB) (Becton Dickinson) for 24 h at 37° C. Streptococcus pyogenes ATCC19615 and Moraxella catharrhalis QK34 are grown on MHA with 2.5% Laked Horse Blood (Oxoid). Liquid cultures in CaMHB+5% horse serum (Sigma) are incubated for 24 h at 35° C. in a 5% CO.sub.2 atmosphere. Haemophilus. influenzae 3168 is grown on MHA+2.5% Fildes extract (Oxoid). Liquid cultures are grown in CaMHB+5% Fildes extract at 35° C. in a 5% CO.sub.2 atmosphere. Propionibacteria are grown on Wilkins-Chalgren agar (WCA) (Oxoid) for 72 h under anaerobic conditions. Liquid cultures are grown anaerobically in Wilkins-Chalgren broth (WCB) (Oxoid) for 48 h at 35° C. MIC values are obtained by broth microdilution using WCB (Anaerobe Broth MIC, Difco). Microtitre plates are loaded into 7-L GENbox anaerobic incubation jars (BioMerieux) fitted with anaerobic atmosphere generators (BioMerieux) and a Dry Anaerobic Indicator Strip (BBL). Under these conditions, an O.sub.2 concentration <0.1% is achieved by 2.5 h, and a CO.sub.2 concentration >15% by 24 h. MIC values are read after incubation at 35-37° C. for 48 h.

(39) Oral Bioavailability:

(40) Drug concentrations in blood or plasma are determined as a function of time in pharmacokinetic studies. Mice were treated with the test compound at defined doses. 10 mg/kg is used for oral administration and 1 mg/kg is taken for intravenous administration. Blood or plasma samples are collected at defined time-points and the drug content is determined by LC-MS/MS. The drug concentration is plotted as a function of time and the non-intravenous (oral) and the intravenous area under the curve (AUC) is calculated using the linear trapezoidal rule. The oral bioavailability is then calculated using dose-normalized AUC with the following formula:
F[%]=AUC.sub.oral/AUC.sub.intravenous*100
Blocking of hERG Channel

(41) The whole-cell patch clamp technique is used to measure the effect of test compounds on hERG tail currents from stably transfected HEK 293 cells (B′SYS GmbH, CH-4108 Witterswil, Switzerland). 0.1% DMSO is used as vehicle and the system is validated with 10 nM of the selective I.sub.Kr blocker E-4031.

(42) Cells are grown in culture flasks at 37° C. in a humidified atmosphere with 5% CO.sub.2 and are passaged when 50-80% are confluent. The culture medium is a 1:1 mix of Dulbecco's modified eagle medium and nutrient mixture F-12 (D-MEM/F-12 1×, with L-Glutamine) supplemented with 9% foetal bovine serum and 0.9% penicillin/streptomycin solution. For electrophysiological measurements, cells are seeded into 35 mm sterile culture dishes containing 2 ml of culture medium with antibiotics (complete medium was supplemented with 100 μg/ml hygromicin B and 15 μg/ml blasticidin). Cells are cultivated at a density allowing single cells to be measured in order to avoid uncertainties due to electrically coupled cells (Pritchett et al. 1988, Verdoorn et al. 1990). DMSO stock solutions of test compounds are appropriately diluted with bath solution (10 mM HEPES pH 7.4, 137 mM NaCl, 4 mM KCl, 1.8 mM CaCl.sub.2, 1 mM MgCl.sub.2, 10 mM D-Glucose). Pipette solution (10 mM HEPES pH 7.2, 130 mM KCl, 1 mM MgCl.sub.2, 5 mM Mg-ATP, 5 mM EGTA.) was prepared and stored as frozen aliquots between −10° C. and −30° C.

(43) The 35 mm culture dishes are placed under the microscope and continuously perfused with bath solution at approximately 1 ml/min. All solutions applied to cells including the pipette solution are maintained at room temperature (19° C.-30° C.). After formation of a Gigaohm seal between the patch electrodes and an individual cell (pipette resistance range: 2.0 MW-7.0 MW; seal resistance range: >1 GW) the cell membrane across the pipette tip is ruptured to assure electrical access to the cell interior (whole-cell patch-configuration). As soon as a stable seal is established, hERG outward tail currents are measured upon depolarization of the cell membrane to +20 mV for 2 s (activation of channels) from a holding potential of −80 mV and upon subsequent repolarization to −40 mV for 3 s. This voltage protocol is run at least 10 times at intervals of 10 s. If the current density is too low for measurements, another cell is analyzed. Once control recordings are accomplished, cells are continuously perfused with bath solution containing a test compound. During wash-in of the test compound the voltage protocol is run continuously at 10 s intervals until the steady-state level of block is reached.

(44) Values (in pA/nA) of the peak amplitudes of outward tail currents are generated for each voltage step and printed for compilation and analysis. The recorded current amplitudes at the steady state level of current inhibition are compared to amplitudes from control conditions measured in the pre-treatment phase of the same cell. The current block is calculated as percentage of control. To determine whether the observed current inhibition is due to a test item interaction with the hERG channel or due to current rundown, these residual currents are compared to those measured in vehicle treated cells. Mean values are calculated for each compound with data from at least 2 individual cells.