6-chromanol derivatives for use as a medicament

Abstract

The present invention relates to the (S) enantiomeric form of certain 6-chromanol derivatives for use as a medicament. Especially, the present invention relates to the (S) enantiomeric form of a 6-chromanol derivative for use as a medicament wherein said 6-chromanol derivative is chosen from the group consisting of (6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(piperazin-1-yl)methanone; N-(benzyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide; N-(phenyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide; methyl 4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamido)benzoate; (6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(morpholino)methanone, and pharmaceutically acceptable salts or bases thereof and pharmaceutically acceptable salts thereof.

Claims

1. Method for treating a disorder requiring long systemic treatment in a patient, comprising administering an (S) enantiomeric form of a 6-chromanol derivative, wherein said derivative is (2S)(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(piperazin-1-yl)methanone or a pharmaceutically acceptable salt thereof, wherein the disorder is lung disorders, Cushing's syndrome, metabolic syndrome or organ damage that occur as secondary cause of the disorder, wherein said treatment does not have an effect on the hemodynamic behaviour of a patient.

2. The method for treating a disorder requiring long systemic treatment according to claim 1, wherein the 6-chromanol derivative is administered in an amount sufficient to achieve a concentration of 0.5 μM or higher.

3. The method for treating a disorder requiring long systemic treatment according to claim 2, wherein the 6-chromanol derivative is administered in an amount sufficient to achieve a concentration of about 5 μM or higher.

4. The method for treating a disorder requiring long systemic treatment according to claim 1, wherein said long treatment is lifelong treatment.

Description

(1) The present invention will be further illustrated using the examples and figures below. In the examples, reference is made to figures wherein:

(2) FIG. 1: Shows the constriction responses to phenylephrine (PE) in the presence of different doses of SUL-150 (B) and SUL-151 (C). Data is obtained from 2-3 experiments (n=4-6 per group). Constriction in graphics B and C is expressed as percentage of response to final PE addition.

(3) FIG. 2: Shows intracellular calcium measurements in transgenic CHO cells overexpressing α.sub.1 adrenoceptor subtypes A (A), B (B) and D (C) after stimulation with phenylephrine in the presence of vehicle (10% DMSO), 10 μM, 30 μM or 100 μM SUL-150. Figure D shows the effect of SUL-151 on the α.sub.1A receptor.

(4) FIG. 3: Shows displacement of tritium-labelled prazosin by SUL-150 and SUL-151 in α.sub.1A adrenoceptor transgenic CHO cells.

(5) FIG. 4: Shows the structural formula of the SUL-121 ((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)-(piperaziniyl)-methanone) compound. SUL-121 is a racemic 1:1 mixture of (R)-enantiomer SUL-150 and (S)-enantiomer SUL-151. The*indicates the chiral centre of SUL-121 in the structural formula, which is at the 2 position in the chromanol group. Therefore, generally, the group of enantiomers is named the “2-S enantiomer of”.

EXAMPLES

Example 1

(6) Tissue Preparation and Myography in Isolated Porcine Renal Arteries

(7) Porcine kidneys were obtained from a local slaughterhouse (Kroon Vlees, Gotenburgweg 30, 9723 ™ Groningen, The Netherlands) and transported on ice in normal physiological Krebs buffer containing 120 mM NaCl, 6 mM KCl, 2.5 mM CaCl.sub.2×2H.sub.2O, 1.2 mM MgCl.sub.2×6H.sub.2O, 1.2 mM NaH.sub.2PO.sub.4×H.sub.2O, 25 mM NaHCO.sub.3 and 11.4 mM D-(+)-Glucose monohydrate (all ingredients were purchased from Merck) in ultrapure water.

(8) The renal artery tree was dissected from the kidney, cleaned of surrounding connective tissue and cut into equally-sized ring segments (2 mm in length). In some rings, endothelium denudation was performed by gentle rubbing of the intimal surface with a paper clip. Rings were mounted in organ baths as described previously (Buikema et al., 2000).

(9) Arterial rings were washed thoroughly by replacing Krebs buffer and allowed to equilibrate for a period of 60 min under 1.4 g of resting tension before they were assessed for viability by inducing 2 subsequent constrictions with KCl (60 mM). Rings that failed to produce a threshold increase in diameter of 100 μm were excluded. After washout and stabilization, rings were treated for 30 minutes by incubation with vehicle (0.1% DMSO), SUL-121, SUL-150 or SUL-151, followed by subsequent incubation with cumulative doses of phenylephrine.

(10) Dose-dependent constriction responses to phenylephrine (10.sup.−8M-10.sup.−4M) were recorded in isolated porcine intrarenal arteries. Buffer was warmed to 37° C. and aerated with 95% O.sub.2 and 5% CO.sub.2 before use.

Example 2

(11) Cell Culture

(12) CHO—K1 cells were stably transfected with a plasmid containing human α.sub.1-AR subtypes A, B and D in separate cell lines in DMEM-F12 medium with 10% FBS, 1% penicillin-streptomycin and 200 μg/mL Geneticin (G418, Invitrogen, Carlsbad, Calif.).

(13) HeLa cells endogenously expressing histamine and TP receptors were grown in DMEM-F12 medium enriched with 10% FBS and 1% penicillin-streptomycin. Cells were kept in a tissue culture incubator at 37° C. in 5% O.sub.2/95% CO.sub.2 atmosphere and grown in 75 cm.sup.2 non-treated cell culture flasks. Plating was performed 24 hours before measurement on black transparent-bottom 96-well plates at 20,000 cells per well density.

(14) Intracellular Calcium Assays

(15) On the next day, CHO cells were treated with either vehicle (0.1% DMSO) or SUL-150 resp. SUL-151 for 30 min at 37° C. and stimulated with a 3-fold dilution series of PE (20 μM-100 μM). [Ca.sup.2+].sub.i was measured using the fluorescent FLIPR Calcium 6 assay kit (Molecular Devices) in immortalized CHO cells stably expressing the α.sub.1 adrenoceptor.

(16) Initially, calcium responses induced by non-cumulative concentration series of phenylephrine were investigated using fluorescent measurements in α.sub.1A adrenoceptor-overexpressing CHO cells treated with SUL-150 and SUL-151 (FIG. 4). Fluorescent measurement data was processed and analyzed in SoftMax Pro 7 and expressed as % of baseline AUC with a 3-fold multiplier using an average of first 10 measurement points as baseline.

(17) The “Vehicle” used in all experiments is 0.1% DMSO solution.

(18) Data and Statistical Analysis

(19) Vascular constriction responses are expressed as percentage of final response to KCl. Data are expressed as mean±SEM. *p<0.05.

Example 3

(20) Induced Fit Molecular Docking Simulation

(21) The binding of SUL-150 and SUL-151 to the antagonist binding site on the α.sub.1A-AR, induced fit molecular docking simulation was performed, using prazosin as a reference as follows.

(22) The primary sequence of α.sub.1A adrenoceptor was obtained from UniProt database (The UniProt Consortium, 2017) using reference code P35348 and uploaded to SWISS-MODEL in order to build a homology model, resulting in 373 templates. Subsequently, template ligand codes were used to query the PDB database to obtain structural data in SMILES format, which were processed by Chemmine to detect similarities with SUL-150. The SWISS-MODEL template that contained a ligand with the highest similarity score (a D3 dopamine receptor in complex with, Eticlopride, ETQ) was used to align the α.sub.1A-AR sequence onto a modelled backbone. The resulting homology model was validated by Ramachandran plot and prepared with Protein Preparation Wizard by the addition of hydrogens, bond order assignment, generation of partial charges to heteroatoms and disulfide bonds. Final refinement was performed by hydrogen bond assignment at pH 7.4 and restrained minimization at 0.3 RMSD.

(23) Prazosin, SUL-150 and SUL-151 structural files were converted from SMILES to 3D structures using LigPrep. Protonation states were generated with Epik at pH 7.4 and small molecule energy parameters were computed using OPLS3 forcefield.

(24) Flexible molecular docking simulation was performed using Induced Fit, part of the Schrödinger Small-Molecule Drug Discovery suite. A binding centroid was defined between residues involved in antagonist binding confirmed by mutagenesis (PHE312, PHE308 and ASP106) and ligands were docked within 15 Å, using an extended sampling protocol without constrains. Residues within 5 Å of resulting ligand poses were refined using Prime to improve ligand conformational sampling. Finally, the Scorpion server was used for the assessment and classification of small molecule-protein interactions and the final results were rendered using PyMOL 2.0.

(25) Results

(26) Effects of SUL-150 and SUL-151 on α.sub.1 Adrenoceptor Mediated Vasoconstriction

(27) To explore the effects of 2R and 2S-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(piperazin-1-yl)methanone (resp. SUL-150 and SUL-151) on constriction of isolated porcine intrarenal arteries, cumulative dose response curves to the α.sub.1 adrenoceptor agonist phenylephrine (PE) were constructed in the presence and absence of SUL-150 resp SUL-151 (FIG. 1). Whereas SUL-150 exerted a dose-dependent increase of EC.sub.50 similar to SUL-121 (FIG. 1A, Table 1), we observed no significant effects after treatment with SUL-151 (FIG. 1B, Table 1).

(28) TABLE-US-00001 TABLE 1 LogEC.sub.50 values of phenylephrine-induced constriction responses in the presence of SUL-150 and SUL-151. phenylephrine logEC50 SUL-150 SUL-151 vehicle −5.77 ± 0.04 10 μM −5.11 ± 0.09* −5.82 ± 0.05 30 μM −5.06 ± 0.06*  −5.89 ± 0.04# 100 μM  −4.44 ± 0.09* −5.77 ± 0.04 #p < 0.05 vs vehicle, *p < 0.0001 vs vehicle
Effects of SUL-150 and SUL-151 on PE-Induced Intracellular Calcium Signalling

(29) To further investigate the mechanisms through which SUL-150 inhibits α.sub.1 adrenoceptor mediated contractions, PE-induced calcium transients were studied in CHO cells stably overexpressing the human α.sub.1 adrenoceptor subtypes A, B and D. SUL-150 shifted dose response curves rightwards for all three α.sub.1 adrenoceptor subtypes (FIGS. 2A, B and C, Table 2). SUL-151 did not affect calcium transients in any of the investigated α.sub.1 adrenoceptor subtypes (only shown for the α.sub.1 adrenoceptor A subtype in FIG. 2D).

(30) TABLE-US-00002 TABLE 2 LogEC.sub.50 values of phenylephrine-induced calcium influx after treatment with SUL-150. SUL-150 SUL-151 logEC50 α1A α1B α1D α1A vehicle −8.07 ± 0.03  −7.99 ± 0.08  −7.68 ± 0.13  −8.10 ± 0.11 10 μM −7.62 ± 0.03* −7.51 ± 0.08* −7.37 ± 0.10  −8.09 ± 0.10 30 μM −7.28 ± 0.03* −7.24 ± 0.07* −7.04 ± 0.06* −8.09 ± 0.11 100 μM  −6.88 ± 0.03* −6.72 ± 0.08* −6.80 ± 0.10* −7.98 ± 0.10 *p < 0.0001 versus vehicle
Radioligand Binding Assay in α.sub.1A Adrenoceptor Overexpressing CHO Cells

(31) SUL-150 affected PE-induced calcium transients, indicating that the effects of SUL-150 are upstream of calcium. We therefore explored whether SUL-150 could directly interact with the α.sub.1 adrenoceptor as a receptor antagonist. For this, a displacement binding assay was performed on the α.sub.1A adrenoceptor transgenic CHO cells using radiolabeled prazosin, an established α.sub.1A adrenoceptor antagonist. SUL-150 was significantly more potent in displacing the radioligand compared to SUL-151, which displaced [7-Methoxy-3H]-prazosin only at concentrations higher than 10 μM (FIG. 3).

(32) Induced Fit Molecular Modelling

(33) Prazosin coexists in two protonation forms at pH 7.4. In the protonated form, the N1 assumes a positive charge, subsequently forming a salt bridge with the negatively charged side chain of ASP106, ultimately causing this form to assume an inverted orientation relative to its non-protonated form. The quinazoline scaffold of non-protonated prazosin was docked close to TM5 to form a confocal hydrogen bond between the 6,7-methoxy groups and SER188, a hydrogen bond between furan oxygen and SER83, and between prazosin carboxamide and GLN177 side chain; van der Waals interactions with side chains of PHE86, VAL107, ILE178, PHE289, MET292, and PHE312; π-π interactions with PHE288 and PHE312; and a π-hydrogen bond interaction with the side chain carboxy group and backbone peptide carboxamide of ASP106. The proposed binding mode of non-protonated prazosin indicated interactions which were in accord with those described in the literature.

(34) An induced fit of SUL-150 and SUL-151 demonstrated alignment of the chromane scaffold with prazosin quinazoline, 6-hydroxy groups (SUL) and 6-metoxy (prazosin) as well as over their common piperazine moiety. Residues which were involved in forming contacts with all three compounds were VAL107, ILE178, SER188, PHE288, PHE289.

(35) Glide scores computed using the Schrödinger Small-Drug Discovery Suite were −10.8 kcal×mol.sup.−1, −10.2 kcal×mol.sup.−1 and −9.4 kcal×mol.sup.−1 for prazosin, SUL-150 and SUL-151 respectively. Prazosin and SUL-150 formed contacts with PHE312 and ASP106 confirmed in prazosin binding by mutagenesis, whereas SUL-151 did not show interactions with these residues. Additionally, the chirality of SUL-150 enables the orientation of its carboxamide towards ASN179, effectively forming a hydrogen bond. Additional hydrogen bond was formed between its protonated N-terminal and TYR316.

(36) Comparison of the binding site-interactions of the several SUL compounds with the binding SUL-150, and the non-binding of SUL-151 and SUL-132 and SUL-138, allowed the prediction that the now claimed SUL-compounds indeed lack binding properties, whereas the R enantiomers would bind. Thus, the now claimed compounds do not show unwanted side effects of vascular restriction.