Patent Application
20070105920
The present invention relates to 2-(benzimidazol-1-yl)-N-(4-phenylthiazol-2-yl)acetamide derivatives, to pharmaceutical compositions comprising the same and to the use of these 2-(benzimidazol-1-yl)-N-(4-phenylthiazol-2-yl)acetamide derivatives in the treatment of TRPV1 related disorders.
The vanilloid receptor (VR1 or TRPV1), a non-selective ligand-gated cation channel belonging to the Transient Receptor Channel family (TRP family) of cation channels, is highly expressed on the peripheral termini of small diameter sensory neurones innervating many tissues including skin, bladder, airway and gastrointestinal tract. More specifically TRPV1 receptors are located on a subset Aδ and C fibres, the afferents commonly associated with nociception (Mezey et al., Proc. Natl. Acad. Sci. 97, 3655-3660, 2000). Characterisation of this channel at the molecular level identified it as the target of the vanilloid capsaicin, the main pungent constituent of hot chili peppers (Caterina et al., Nature 389, 816-824, 1997). Indeed, sensitivity to capsaicin has been used for many years as a marker of nociceptor activity. These, polymodal nociceptors are activated by multiple noxious stimuli including chemical, mechanical and thermal. Study of the functional properties of TRPV1 demonstrated that this receptor shares many properties common to nociceptors including activation by thermal stimuli (>43° C.) and chemicals (including capsaicin and endovanilloids such as N-arachidonoyl-dopamine (NADA) and lipoxygenase metabolites), as well as sensitisation and activation by acidification. Furthermore, inflammatory mediators (including ATP and bradykinin) have been shown to functionally sensitise TRPV1 in vitro. This evidence suggests that TRPV1 has an integral role in the polymodal detection of noxious stimuli and contributes to the transduction of inflammatory pain responses and potentially also peripheral tissue injury (reviewed in Di Marzo et al., Curr. Opin. Neurobiol. 12, 372-379, 2002).
A role for TRPV1 in the detection of painful stimuli is also inferred from data in gene knockout mice. Mice null for TRPV1 show attenuated development of behavioural thermal hyperalgesia after an inflammatory insult (Caterina et al., Science 288, 306-313, 2000, Davis et al., Nature 405, 183-187, 2000). Small diameter sensory neurones from these animals also show altered responses to thermal and acid stimuli. Moreover, altered expression and/or functional activity of TRPV1 has been demonstrated following inflammation and nerve injury in animals models (Amaya et al., Brian Res. 963, 190-196, 2003, Rashid et al., J. Pharm. Exp. Ther. 304, 940-948, 2003, Hong & Wiley, J. Biol. Chem. 280, 618-627, 2005).
In humans, intradermal exposure to capsaicin leads at first to the sensation of burning pain due to neuronal excitation, followed by a long lasting period of analgesia which is believed to be a consequence of functional desensitisation (reviewed in Bley, Exp. Opin Investig Drugs. 13, 1445-1456, 2004). This led to the development of TRPV1 agonists as potential analgesic compounds. However, these compounds suffer from a number of issues including pain and a burning sensation on initial application. More recently, TRPV1 antagonists including capsazepine (Walker et al., J. Pharm. Exp. Ther. 304, 56-62, 2003) and BCTC (Pomonis et al., J. Phar. Exp. Ther. 306, 387-393, 2004) have been shown to be active in a variety of preclinical animal models of inflammatory and neuropathic pain.
In addition to a role in pain transduction there is also growing evidence for a role for TRPV1 in regulating afferent and efferent function of sensory nerves and the function of non-neuronal cells. Indeed, altered bladder function, with a higher frequency of low amplitude, non-voiding bladder contractions and an increase in bladder capacity has been observed by in TRPV1 KO mice (Birder et al., Nat. Neurosci. 5, 856-860, 2002). This may involve neuronal TRPV1 and TRPV1 expressed on uroepithelial cells. Thus, there is clear evidence to suggest that agents modulating TRPV1 activity will have utility not only in pain states and other diseases involving inflammation but also in conditions involving hyperactivity of primary sensory fibres (e.g. bladder overactivity and urge incontinence).
2-(Benzimidazol-1-yl)acetamide derivatives have been disclosed in the International Patent Applications WO 2004/100865 and WO 2006/033620 (AstraZeneca AB) as inhibitors of the TRPV1 receptor and useful in the treatment of TRPV1 mediated disorders, such as in the treatment of acute and chronic pain disorders, acute and chronic neuropathic pain, acute and chronic inflammatory pain, and respiratory diseases. There remains a need for additional compounds that are useful in the treatment of TRPV1 mediated disorders.
To this aim the present invention provides 2-(benzimidazol-1-yl)-N-(4-phenylthiazol-2-yl)acetamide derivatives having the general Formula I
wherein
R1 is H, (C1-4)alkyl, (C1-4)alkyloxy or halogen;
R2 represents 1-3 substituents selected from H, (C1-4)alkyl (optionally substituted with 1 or more halogens), (C1-4)alkyloxy (optionally substituted with 1 or more halogens), halogen, CF3 or cyano; or a pharmaceutically acceptable salt thereof;
with the exclusion of
The excluded compounds relate to the disclosure thereof by S. C. Sharma (Indian J. Chem 4, 33-36, 1966) as local anaesthetics.
The term (C1-4)alkyl as used in the definition of Formula I means a branched or unbranched alkyl group having 1-4 carbon atoms, like butyl, isobutyl, tertiary butyl, propyl, isopropyl, ethyl and methyl.
In the terms (C1-4)alkyloxy, (C1-4)alkyl has the meaning as defined above.
The term halogen means F, Cl, Br or I. Preferred halogens are Cl and F.
There is a preference for the 2-(benzimidazol-1-yl)-N-(4-phenylthiazol-2-yl)acetamide derivatives of formula I, wherein R1 is H.
Specifically preferred compounds of the invention are:
The 2-(benzimidazol-1-yl)-N-(4-phenylthiazol-2-yl)acetamide derivatives of the invention can be prepared by methods well known in the art of organic chemistry.
In an illustrative general route to compounds of the present invention, as depicted in Scheme I, the intermediate (1H-benzimidazol-1-yl)acetic acid 2 can be prepared from benzimidazole, a suitable deprotonating base such as potassium tert-butoxide and alkylating with the appropriate nitrile such as bromoacetonitrile in a suitable solvent such as ethanol (J. Das et al. Bioorganic and Medicinal Chemistry Letters 15(2), 337-343, 2005). The nitrile 1 can then be hydrolysed to the desired acid with 18% hydrochloric acid and is well known to someone skilled in the art. Various salt forms of this intermediate can be formed such as the hydrochloride and triethylamine salt. The carboxylic acid of formula 2 or its salt forms (such as hydrochloride or triethylamine) can be converted to amide of formula 3 via its conversion into an activated form i.e. an acyl azide by treatment with diphenylphosphorylazide (DPPA), an acyl chloride by treatment with thionyl chloride or the activated ester by treatment with O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) and further treatment with the appropriate amine H2N—Ar1—Ar2 of formula 4 (J. Am. Chem. Soc., Vol. 108, No. 22, 6950-6960, 1986), wherein —Ar1—Ar2 represents appropriately substituted 4-phenylthiazol-2-yl.
Alternative methods of coupling amines of formula 4 to the acid 2 include, but are not limited to the use of peptide coupling reagents such as 1,3-dicylohexylcarbodiimide (DCC), 1,3-diisopropylcarbodiimide (DIC) or bromotripyrrolidinophosphonium hexafluoro-phosphate (PyBroP). Suitable solvents are aprotic polar solvents such as dimethylformamide (DMF) or acetonitrile although other solvents may be used. Bases such as tertiary amines e.g. triethylamine can be used as well as heteroaromatic bases e.g pyridine. The temperature may be between 0 to 100° C. using either conventional or microwave heating and the reaction time between 1 h and 30 h. The target compounds of formula 3 can exist in various salt forms such as hydrochloride and trifluoroacetic acid salts.
The amine intermediates represented by formula 4 can be prepared using a variety of methods known to those skilled in the art, one of which is outlined in Scheme II. Alpha bromo ketones of formula 5 can be converted to the aminothiazole 6 with thiourea using standard chemistry outlined by J. Brienholt et al., J. Heterocyclic Chemistry 38, 569, 2001.
Pharmaceutically acceptable salts of the 2-(benzimidazol-1-yl)-N-(4-phenylthiazol-2-yl)acetamide derivatives of the invention may be obtained by treating a free base of a compound of Formula I with a mineral acid such as hydrochloric acid, hydrobromic acid, phosphoric acid and sulfuric acid, or an organic acid such as for example ascorbic acid, citric acid, tartaric acid, lactic acid, maleic acid, malonic acid, fumaric acid, oxalic acid, glycolic acid, succinic acid, propionic acid, acetic acid and methane sulfonic acid.
The compounds of the invention may exist in unsolvated as well as in solvated forms with pharmaceutically acceptable solvents such as water, ethanol and the like. In general, the solvated forms are considered equivalent to the unsolvated forms for the purpose of the invention.
Some of the 2-(benzimidazol-1-yl)-N-(4-phenylthiazol-2-yl)acetamide derivatives of Formula I and their salts may contain at least one centre of chirality, and exist therefore as stereoisomers, including enantiomers and diastereomers. The present invention includes the aforementioned stereoisomers within its scope and each of the individual R and S enantiomers of the compounds of Formula I and their salts, substantially free, i.e. associated with less than 5%, preferably less than 2%, in particular less than 1% of the other enantiomer, and mixtures of such enantiomers in any proportions including the racemic mixtures containing substantially equal amounts of the two enantiomers. Methods for asymmetric synthesis or chiral separation whereby the pure stereoisomers are obtained are well known in the art, e.g. synthesis with chiral induction or starting from commercially available chiral substrates, or separation of stereoisomers, for example using chromatography on chiral media or by crystallisation with a chiral counter-ion.
The present invention further provides pharmaceutical compositions comprising a 2-(benzimidazol-1-yl)-N-(4-phenylthiazol-2-yl)acetamide derivative according to general Formula I, or a pharmaceutically acceptable salt thereof, in admixture with pharmaceutically acceptable auxiliaries, and optionally other therapeutic agents. The term “acceptable” means being compatible with the other ingredients of the composition and not deleterious to the recipients thereof. Compositions include e.g. those suitable for oral, sublingual, subcutaneous, intravenous, epidural, intrathecal, intramuscular, transdermal, pulmonary, local, or rectal administration, and the like, all in unit dosage forms for administration.
For oral administration, the active ingredient may be presented as discrete units, such as tablets, capsules, powders, granulates, solutions, suspensions, and the like. For parenteral administration, the pharmaceutical composition of the invention may be presented in unit-dose or multi-dose containers, e.g. injection liquids in predetermined amounts, for example in sealed vials and ampoules, and may also be stored in a freeze dried (lyophilized) condition requiring only the addition of sterile liquid carrier, e.g. water, prior to use.
Mixed with such pharmaceutically acceptable auxiliaries, e.g. as described in the standard reference, Gennaro, A. R. et al., Remington: The Science and Practice of Pharmacy (20th Edition, Lippincott Williams & Wilkins, 2000, see especially Part 5: Pharmaceutical Manufacturing), the active agent may be compressed into solid dosage units, such as pills, tablets, or be processed into capsules, suppositories or patches. By means of pharmaceutically acceptable liquids the active agent can be applied as a fluid composition, e.g. as an injection preparation, in the form of a solution, suspension, emulsion, or as a spray, e.g. a nasal spray.
For making solid dosage units, the use of conventional additives such as fillers, colorants, polymeric binders and the like is contemplated. In general any pharmaceutically acceptable additive which does not interfere with the function of the active compounds can be used. Suitable carriers with which the active agent of the invention can be administered as solid compositions include lactose, starch, cellulose derivatives and the like, or mixtures thereof, used in suitable amounts. For parenteral administration, aqueous suspensions, isotonic saline solutions and sterile injectable solutions may be used, containing pharmaceutically acceptable dispersing agents and/or wetting agents, such as propylene glycol or butylene glycol.
The invention further includes a pharmaceutical composition, as hereinbefore described, in combination with packaging material suitable for said composition, said packaging material including instructions for the use of the composition for the use as hereinbefore described.
The 2-(benzimidazol-1-yl)-N-(4-phenylthiazol-2-yl)acetamide derivatives of the invention were found to have antagonistic properties at the vanilloid receptor as measured by a functional calcium influx assay using a Chinese Hamster Ovary cell line in which a human recombinant VR1 receptor had been stably expressed. Methods to construct such recombinant cell lines are well known in the art (Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, latest edition).
The compounds of the invention are thus useful in the treatment of TRPV1 mediated disorders, such as in the treatment of acute and chronic pain disorders, acute and chronic neuropathic pain, acute and chronic inflammatory pain, and respiratory diseases. The compounds of the invention may be administered to humans in a sufficient amount and for a sufficient amount of time to alleviate the symptoms. Illustratively, dosage levels for humans can be in the range of 0.001-50 mg per kg body weight, preferably in a dosage of 0.01-20 mg per kg body weight.
The invention is illustrated by the following Examples.
To an ice cooled solution of benzimidazole (10 g, 0.085 mol) in dry N,N-dimethylformamide (500 mL) was added potassium tert-butoxide (9.6 g, 0.085 mol) portionwise. The mixture was stirred at room temperature for 1 h then bromoacetonitrile (6 mL, 0.086 mol) was added in one portion and stirred for 3 h. The mixture was then quenched with solid carbon dioxide followed by water and the organics separated. The organics were washed further with water (100 mL×5), and brine (100 mL×1) combined and dried (Na2SO4), filtered and evaporated to dryness. The residue was passed through a silica gel column eluting with dichloromethane:ethanol (1% up to 6% ethanol) to give a yellow solid (12 g, 89%). 1H NMR (400 MHz, CDCl3) δ ppm 5.08 (2H, s), 7.36-7.43 (2H, m), 7.47 (1H, d, J=7.4 Hz), 7.85 (1H, d, J=7.2 Hz), 7.93 (1H, s).
(1H-Benzimidazol-1-yl)acetonitrile (35 g, 0.23 mol) (Example 1A) was dissolved in 18% hydrochloric acid (500 mL) and heated to reflux for 5 h. The solution was then evaporated to dryness under reduced pressure using acetonitrile as a co-solvent to azeptropically remove all the solvent. Acetone was added and the solid (NH4Cl) filtered and washed with acetone. The filtrate was then left to stand cool for 24 h and the light brown crystals collected and dried (45 g, 100%). 1H NMR (400 MHz, CD3OD) δ ppm 5.47 (2H, s), 7.68-7.70 (2H, m), 7.88-7.92 (2H, m), 9.51 (1H, s). MS (ES) m/z 177.4 [M+H]+.
To a solution of (1H-benzimidazol-1-yl)acetic acid (1 g, 5.68 mmol) (Example 1B) in N,N-dimethylformamide (25 mL) was added thionyl chloride (0.4 mL, 5.56 mmol) dropwise and the reaction mixture stirred at room temperature for 1 h. 2-Amino-4-(4-chlorophenyl)thiazole (1.15 g, 5.46 mmol) and pyridine (4 mL) were then added to the reaction mixture and stirred at room temperature for 17 h. The reaction mixture was then concentrated in vacuo and dichloromethane was added and transferred to a separating funnel where the organics were washed with 0.1 M hydrochloric acid and 10% ammonium hydroxide solution. The organic layers were combined and washed with saturated aqueous sodium chloride, dried (Mg2SO4), filtered and concentrated in vacuo. The residue was then purified by column chromatography using silica and eluting with 0-10% methanol in dichloromethane, affording the title compound (93.7 mg, 4.5%).
1H NMR (400 MHz, DMSO-d6) δ ppm 5.34 (2H, s), 7.19-7.27 (2H, m), 7.51 (1H, d, J=8.5 Hz), 7.59 (1H, d, J=7.2 Hz), 7.68 (1H, d, J=7.1 Hz), 7.71 (1H, s), 7.93 (2H, d, J=8.5 Hz), 8.25 (1H, s), 12.79 (1H, s). MS (ES) m/z: 369.0 [M+H].
2-Bromo-1-(3,5-dichloro-2-methoxy-phenyl)-ethanone (200 mg, 0.746 mmol), thio-urea (100 mg, 1.3 mmol) in ethanol (2 mL) and heated to 165° C. for 300 s in a Emrys optimizer EXP microwave. The volatile solvent was removed and the residue dissolved in water and adjusted to pH 7 with sodium hydroxide. The aqueous mixture was extracted with ethyl acetate (3×50 mL), and evaporated to dryness to give a oily residue. This was then used without further purification in the next step.
(1H-Benzimidazol-1-yl)acetic acid (50 mg, 0.28 mmol) (Example 1B) was dissolved in toluene and N,N-dimethylformamide. Triethylamine (0.03 mL, 0.42 mmol) then added followed by diphenyl phosphoryl azide (DPPA) (0.07 mL, 0.36 mmol) and stirred at room temperature for 24 h. 4-(3,5-dichloro-2-methoxy-phenyl)-thiazol-2-ylamine (57 mg, 0.21 mmol) (Example 2A) was then added in one portion and the mixture stirred for 24 h at room temperature. Mixture was evaporated to dryness and DMSO (1 mL) added before purification by preparative LCMS to give an oily residue (8.9 mg, 7%). 1H NMR (400 MHz, CD3OD) δ ppm 3.81 (3H, s), 5.34 (2H, s), 7.29-7.36 (3H, m), 7.42 (1H, d, J=2.5 Hz), 7.53 (1H, d, J=7.2 Hz), 7.72 (1H, d, J=7.1 Hz), 8.06 (1H, d, J=2.6 Hz), 8.25 (1H, s). MS (ES) m/z: 434.3 [M+H].
Prepared following the method in Example 2A using 2-bromo-3,4-difluoroacetophenone (4.4 g, 0.018 mol) in place of 2-bromo-1-(3,5-dichloro-2-methoxy-phenyl)-ethanone. Product was isolated as a white solid (3.7 g, 93%) 1H NMR (400 MHz, CDCl3) δ ppm 5.01 (2H, brs), 6.67 (1H, s), 7.15 (1H, dd, J=8.4 and 18.5 Hz), 7.47-7.50 (1H, m), 7.57-7.62 (1H, m). MS (ES) m/z: 213.3 [M+H].
(1H-Benzimidazol-1-yl)acetic acid (3.1 g, 0.017 mol) (Example 1B) was dissolved in ethyl acetate. Triethylamine (9 mL, 0.064 mmol) then added followed by 4-(3,4-difluorophenyl)-thiazol-2-ylamine (Example 3A) (3.7 g, 0.017 mol) and stirred at room temperature for 1 h. 50% wt. propylphosphonic anhydride solution in ethyl acetate (PPA) (10.8 mL, 0.017 mol) was then added dropwise and the mixture stirred for 24 h at room temperature. The mixture was then partitioned between ethyl acetate and sodium carbonate solution and washed with sodium carbonate (3×100 mL). The organics were combined, dried (MgSO4), filtered and evaporated to dryness. The solid was then triturated with methanol to afford a white solid (3.1 g, 48%). 1H NMR (400 MHz, CD3OD) δ ppm 5.65 (2H, s), 7.28 (1H, dd, J=8.4 and 18.9 Hz), 7.47 (1H, s), 7.66-7.74 (3H, m), 7.79-7.84 (1H, m), 7.88-7.94 (2H, m), 9.49 (1H, s). MS (ES) m/z: 371.2 [M+H].
The functional activity of compounds at the TRPV1 receptor was determined using a Molecular Devices Flexstation II based Ca2+ influx assay, employing a Ca2+ sensitive fluorescent dye and a CHO cell line stably expressing human TRPV1 (VR1).
Test compounds were prepared as stock solution in DMSO and tested for activity over several log units (ranging 100 μM-100 pM). Compounds were further diluted in assay buffer as necessary for IC50 determination.
CHO-K1 cells, stably expressing recombinant human VR1, under the control of a CMV promoter, were seeded (30,000 cells/well) in black clear-bottomed 96-well plates assay plates (Costar) 24 hr prior to assay. Cells were maintained at 37° C./5% CO2 in normal growth medium (Dulbecco's Modified Eagles medium (DMEM/NUT.MIX.F-12 GLUTAMAX-1 (1:1) with PYRIDOXINE) supplemented with 10% fetalclone II serum and 0.4 mg/ml G418, all Invitrogen). Prior to assay, cells were washed once with assay buffer (150 μl, Hepes-buffered saline pH7.4, supplemented with 10 mM Glucose, 2 mM CaCl2, 1 mM MgCl2 and 0.5 mM Probenicid). Cells were then incubated in the dark with 100 μl 5 μM Fluo-3AM (Calbiochem) prepared in assay buffer for 1 hr at 37° C./5% CO2. Excess dye was removed by washing the cells twice more with buffer, prior to pre-incubation (10 min, RT) with an appropriate cocentration of test compound or buffer alone. VR1 responses were assessed following addition, in the Flexstation II, of agonist (capsaicin) at an EC80 concentration and Ca2+ influx assessed by measurement of fluorescence emission (488 nm/525 nm). Baseline fluorescence responses were measured for approximately 20 s (16 reads at 1.28 s intervals) prior to addition of capsaicin. Increases in fluorescence emission following capsaicin addition were measured for a further 40 s (31 reads at 1.28 s intervals). Responses were recorded as Max-Min fluorescence. Antagonist induced inhibition of TRPV1 mediated increases in intracellular [Ca2+] was assessed relative to wells on the same plate to which capsaicin was added in the absence of antagonist (i.e pre-incubation in buffer alone). Typical IC50 values measured in the in vitro assay described above are 3 μM or less. For several embodiments of the invention the IC50 was found to be below 100 nM.
The antinociceptive effects of test compounds were determined in the formalin paw test in mice. This model assesses behavioural responses to continuous, noxious stimulation generated by injured tissue. The injection of dilute solution of formalin into one hind paw of the mouse produces two distinct phases of nociceptive behaviour in several species (Dubuisson and Dennis, Pain, 4 (2), 161-174, 1977). The first period begins immediately after formalin injection and lasts for 4-5 minutes. This early phase is followed by a period of 10-15 minutes of quiescent behaviour, after which a second phase of nociceptive behaviour occurs. This phase continues for a further 20-30 minutes. In mice, recording the time spent licking or biting the injected paw is the most common method of behavioural assessment.
Male ICR mice (22-30 g; n=6-10 per dose) were habituated to their test environment by placing them, singly, into clear Perspex observation boxes for 1 hour prior to drug administration on the day of the experiment. Formalin solution, 0.3% in sterile saline, was prepared as a fresh solution daily. Test compounds, dissolved in 5% solutol in water and were administered intravenously (i.v.), 10 ml.kg−1, 5 minutes prior to the subcutaneous injection into the dorsal surface of one hind paw of 20 μl of formalin solution. The number of counts of nociceptive behaviour exhibited for each animal was then measured using an automated system. Nociceptive behaviour was measured during two time periods after formalin injection; 0-5 minutes (Phase 1) and 20-30 minutes (Phase 2). ED50 values were calculated for each compound for each of the two phases of licking using a non-linear regression fit, sigmodal dose-response curve (Xlfit, IDDBs).
A typical ED50 in phase II of the Formalin test is 50 μmol/Kg or less. For several 2-(benzimidazol-1-yl)-N-(4-phenylthiazol-2-yl)acetamide derivatives of the invention the ED50 was found to be below 15 μmol/Kg.
| Number | Date | Country | |
|---|---|---|---|
| 60734446 | Nov 2005 | US |
