Patent Application
20240254093
This application relates to compounds and their use as G protein-coupled receptor 35 (GPR35) receptor agonists. Compounds described herein may be useful in the treatment or prevention of diseases in which GPR35 receptors are involved.
GPR35 is an orphan receptor belonging to the family of seven transmembrane domain G protein-coupled receptors (GPCRs). The GPCR superfamily represents a large family of signal transducers which play a key role in regulating various aspects of human physiology. Owing to their pharmacological tractability, these receptors have been intensively studied as potential drug targets. A recent analysis has shown that over 475 drugs act at 108 unique GPCRs, representing ˜34% of FDA approved drugs. There still remains opportunity for the discovery and development of novel drugs for orphan GPCRs for which endogenous ligands have not yet been identified.
GPR35 was originally discovered as an open reading frame encoding a protein of 309 amino acids and localised to human chromosome 2q37.3 (O'Dowd et al. Genomics, 47, 310-3, 1998). The receptor was shown to be expressed in a range of tissues, with high expression reported in gastrointestinal tissues, lung and dorsal root ganglion. The receptor is also found expressed in immune cells, as well as in tissues such as the spleen, skeletal muscle and spinal cord.
Consistent with its expression pattern, there is now accumulating evidence highlighting the therapeutic role for GPR35 across various indications including airway disease, metabolic syndromes (e.g. diabetes), cardiovascular disease (e.g. hypertension) and pain. Ligands that target GPR35 may offer utility for the treatment of a wide range of human disease conditions.
The identity of the endogenous ligand for GPR35 remains a debate and to date a number of putative ligands have been described including 2-acyl lysophosphatidic acid, CXCL17 and kynurenic acid. Many of the reported ligands only show weak activity at the receptor or display lack of biological specificity, raising questions on the true identity of the endogenous ligand.
A wide range of synthetic agonists have been reported to act at GPR35 including zaprinast, pamoic acid, cromolyn, loop diuretic drugs (bumetanide, furosemide), aspirin metabolites, quercetin and dicumarol. In addition, weak agonist activity has also been reported with anti-inflammatory agents sulfasalazine and 5-aminosalicylic acid, which are widely used in the treatment of inflammatory bowel disease (EC50 ˜3 uM) (US20130316985).
Compounds of the tyrosine kinase class, tyrophstin (tyrophstin-51) as well as a catechol-O-methyltransferase (COMT) inhibitor, entacapone have also been reported to act at the receptor highlighting the diversity of the GPR35 ligand class. Owing to the potential involvement of GPR35 in human diseases, there has been an increasing interest in the development of ligands that are highly potent and show selectivity for GPR35. Elucidation of GPR35 biology has somewhat been hampered by the availability of adequate pharmacological tools. Indeed, many of the documented GPR35 agonists (endogenous and synthetic) demonstrate only weak or partial activity at GPR35 and lack target specificity, making the dissection of the pathway difficult. Furthermore, it has become clear that some compounds display species selectivity and possibly ligand bias; the putative endogenous ligand kynurenic acid is one such example where potency at the human receptor is reported to be at least 100 fold lower compared to the rat ortholog (Jenkins et al. Br J Pharmacol 162, 733-748, 2011). Potent, selective GPR35 agonists and antagonists are therefore needed to unravel the physiological role of this receptor.
More recently, genome wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) for GPR35 which have been associated with inflammatory bowel disease (IBD). Two SNPs have been described, one of which is a non-synonymous SNP (rs3749171) encoding a T108M substitution in the 3rd transmembrane domain (Ellinghaus et al. Hepatology 58, 1074-1083, 2013). This residue is not conserved throughout mammals and the impact of the polymorphism on signal transduction remains to be elucidated. A second polymorphism at the GPR35 locus (rs4676410) encodes an upstream intron variant of GPR35. A phenome wide association study using 4 large real world data cohorts demonstrated the association of this genetic variant (rs4676410) with an IBD phenotype confirming the target-disease links reported previously in earlier studies (Diogo et al. Nat Commun 9, 4285, 2018). The reported association of GPR35 polymorphism in IBD has raised interest in the potential therapeutic role of GPR35 for the treatment of gastrointestinal diseases.
Inflammatory bowel disease is a chronic relapsing inflammatory gastrointestinal disorder that commonly involves the ileum and/or colon. The pathophysiology is thought to involve an abnormal intestinal immune response, resulting in mucosal inflammation, defective intestinal barrier and increased GI permeability. Treatment strategy largely involves a stepwise approach through the combined use of agents such as aminosalicylates, corticosteroids, immunosuppressants, biologics (e.g. anti-TNF) and antibiotics, however many patients experience incomplete disease control, highlighting the high unmet need. In particular, one aspect which remains poorly treated and underrecognized is abdominal pain. Pain is a common symptom experienced by the vast majority of IBD patients during the disease course and can arise from a direct or indirect consequence of intestinal inflammation. Whatever the cause, pain negatively impacts the quality of life of IBD patients. To date, the management of IBD associated abdominal pain remains challenging. Commonly used analgesics such as non-steroidal anti-inflammatory agents (NSAIDs) can often exacerbate the condition, leaving patients with limited treatment options. There is therefore a high unmet need for agents that can provide fast onset pain relief and development of novel drugs is urgently needed.
In preclinical studies, GPR35 mutant mice display greater degrees of colonic epithelial damage following chemical injury, compared to wildtype mice. GPR35 knockout mice display elevated expression of inflammatory and remodelling cytokines, although numbers of inflammatory cell influx in the mucosa, show no overall difference (Farooq et al. Digestive Diseases and Sciences, 63, 2910-22, 2018). A role of GPR35 in barrier homeostasis has also been reported, with agents such as sodium cromoglycate demonstrating the ability to reduce GI permeability in a number of gut sensitisation models (Forbes et al. J Exp Med 205: 897-913, 2008; Yokooji et al. Int Arch Allergy Immunol. 167:193-202, 2015). Consistent with these findings, GPR35 has been shown to play a role in the regulation of tight junction proteins and promoting epithelial cell migration in in vitro studies. Finally, GPR35 is richly expressed in dorsal root ganglion (DRG) neurones where it has been shown to colocalise with nociceptive ion channels and play a role in pain processing (Ohshiro et al. Biochem. Biophys. Res. Commun. 365, 344-8, 2008). In addition to the reported effects on barrier protection, cromolyn reduces visceral hypersensitivity in a stress sensitive rat strain (Carroll et al. PLoS One.8:e84718, 2013), highlighting its potential utility in the treatment of pain.
Sodium cromoglycate is a mast cell stabiliser approved for a range of indications including systemic mastocytosis, prophylaxis of allergic rhinitis and asthma, allergic conjunctivitis and food allergy (in conjunction with dietary restriction). Use of cromoglycate in systemic mastocytosis is reported to result in improvement of diarrhoea, flushing, headaches, vomiting, urticaria and abdominal pain. Trials evaluating the effectiveness of sodium cromoglicate in food allergy have reported mixed results, with high doses generally required to offer protection. Doses of up to 2 g/day were shown to be effective in attenuating the severity of GI symptoms in patients with irritable bowel syndrome due to food allergy (Lunardi et al. Clin Exp Allergy. 21:569-72, 1991). Similar findings have been reported in children with milk allergy on gastrointestinal permeability endpoint.
In addition to gastrointestinal disorders, GPR35 has received interest as a target for the treatment of allergic disorders including asthma. In the lung, cromolyn has long been used as an effective asthma therapy with good safety and tolerability profile, but with suboptimal pharmacokinetics. It is estimated that approximately 5-12% of the drug is absorbed following deposition in the airways and more recently, an improved formulation of cromolyn has been developed (PA101) which achieves significantly higher drug deposition in the lung.
Clinically, cromolyn shows efficacy in suppressing the immediate and late onset asthmatic response following allergen challenge. In a phase II proof of concept trial, PA101 demonstrated efficacy in reducing the cough frequency in patients with idiopathic pulmonary fibrosis. GPR35 mRNA is upregulated in response to challenge with IgE antibodies and cromolyn has been reported to block inflammatory mediator release in human lung slices passively sensitised with IgE antibodies. These effects are likely to involve a number of mechanisms including regulation of mast cell stabilisation and reflex induced bronchoconstriction.
The emerging data therefore highlights a broad therapeutic potential for GPR35 agonists, ranging from mast cell disorders, treatment of acute and chronic pain conditions and diseases associated with allergic or inflammatory diseases in both the gastrointestinal system and the lung.
The present invention relates to compounds having activity as G protein-coupled receptor 35 (GPR35) receptor agonists.
Briefly, in one aspect, the invention provides compounds of the formula (1):
The compounds may be used as GPR35 receptor agonists. The compounds may be used in the manufacture of medicaments. The compounds may be for use in treating, preventing, ameliorating, controlling or reducing the risk of disorders associated with GPR35. The compounds may be used in the treatment of mast cell disorders, acute and chronic pain conditions and diseases associated with allergic or inflammatory diseases in both the gastrointestinal system and the lung.
The invention relates to novel compounds. The invention relates to the use of novel compounds as agonists of the GPR35 receptor. The invention also relates to the use of novel compounds in the treatment or prevention of diseases in which GPR35 receptors are involved. The invention further relates to the use of novel compounds in the manufacture of medicaments for use as GPR35 receptor agonists.
The invention provides compounds of the formula (1):
In the compounds herein, X can be N. X can be CH.
In the compounds herein, R1 can be H. R1 can be halo. R1 can be Cl or F. R1 can be Cl. R1 can be F. R1 can be Br.
In the compounds herein, R2 can be H. R2 can be halo. R2 can be optionally substituted C1-6 alkyl. R2 can be optionally substituted C3-6 cycloalkyl. R2 can be optionally substituted C1-6 alkoxy. R2 can be optionally substituted aryl. R2 can be optionally substituted heteroaryl. R2 can be optionally substituted monocyclic heteroaryl. R2 can be optionally substituted bicyclic heteroaryl. R2 can be optionally substituted O-aryl.
R2 can be H, C1-6 alkyl optionally substituted with 1 to 6 fluorine atoms, C3-6 cycloalkyl optionally substituted with 1 to 6 fluorine atoms or C1-6 alkoxy optionally substituted with 1 to 6 fluorine atoms. R2 can be H, trifluoromethyl, ethyl, cyclopropyl, cyclohexyl or methoxy.
R2 can be phenyl optionally substituted with R3, pyridyl optionally substituted with R3, 0-phenyl optionally substituted with R3, indazolyl optionally substituted with R3 or pyridazinyl optionally substituted with R3, wherein R3 is H, halo, C1-6 alkyl optionally substituted with 1 to 6 fluorine atoms, C3-6 cycloalkyl optionally substituted with 1 to 6 fluorine atoms, C1-6 alkoxy optionally substituted with 1 to 6 fluorine atoms, —CO2R4, —CONHCH2R4, —CONHCH2CH2OR4, —OR4, —OCH2R4, —CH2R4, —OCH2R4, —CH2CH2OR4, —OCH2CH2OR4, —CONHR4 or —CON(CH3)R4; where R4 is H, C1-6 alkyl optionally substituted with 1 to 6 fluorine atoms, or a group:
In the compounds herein, R4 can be H, methyl, or selected from the group consisting of:
In the compounds herein, R5, R6 and R7 can independently be H, CF3, CONH2 or —OCH2CH2OCH3.
In the compounds herein, R8 and R9 can independently be H or methyl. R3 can be OMe, CO2H, CO2Et, CON(CH3)2, CONHCH2CH2OCH3, or selected from the group consisting of:
In the compounds herein R2 can be selected from the group consisting of:
The compound can be a compound of formula (1a) or (1b):
or a salt or tautomer thereof, wherein R1 and R2 are as defined above.
In some embodiments, the compound can be a compound of formula (1a):
or a salt or tautomer thereof, wherein R1 and R2 are as defined above.
The compound can be a compound of formula (2a) or (2b):
or a salt or tautomer thereof, wherein R2 is as defined above.
In some embodiments, the compound can be a compound of formula (2a):
or a salt or tautomer thereof, wherein R2 is as defined above.
The compound can be a compound of formula (3a), (3b), (3c), (3d) or (3e):
or a salt or tautomer thereof; wherein X, R1 and R3 are as defined above.
In some embodiments, the compound can be a compound of formula (3a):
or a salt or tautomer thereof; wherein X, R1 and R3 are as defined above.
The compound can be a compound of formula (4a), (4b), (4c), (4d) or (4e):
or a salt or tautomer thereof; wherein R3 is as defined above.
In some embodiments, the compound can be a compound of formula (4a):
or a salt or tautomer thereof; wherein R3 is as defined above.
The compound can be selected from the group consisting of:
or a salt or tautomer thereof.
The compound can be selected from the group consisting of:
In some embodiments, the salt of the compound of the formula (1) is a pharmaceutically acceptable salt.
In some embodiments, the compound is a compound having the structure:
or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound having the structure:
The present disclosure also provides a tromethamine salt having the structure:
The present disclosure also provides crystalline forms of Compound A or a pharmaceutically acceptable salt thereof. In some embodiments, the crystalline form comprises Compound A free acid or Compound A tromethamine salt.
In some embodiments, the crystalline form comprises Compound A tromethamine salt. In some embodiments, the crystalline Compound A tromethamine salt is a hydrate. In one embodiment, the crystalline Compound A tromethamine salt is Hydrate I. In one embodiment, the crystalline Compound A tromethamine salt is characterized by an XRPD pattern substantially in accordance with
In one embodiment, the crystalline Compound A tromethamine salt is Hydrate II. In one embodiment, the crystalline Compound A tromethamine salt is characterized by an XRPD pattern substantially in accordance with
In some embodiments, the crystalline form comprises Compound A (free acid). In some embodiments, the crystalline Compound A (free acid) is a hydrate. In some embodiments, the crystalline Compound A (free acid) is Pattern 1. In some embodiments, the crystalline Compound A (free acid) is Pattern 3.
In one embodiment, the crystalline Compound A free acid is characterized by an XRPD pattern substantially in accordance with
The compounds disclosed herein can be used in therapy. The compounds disclosed herein can be used in medicine.
The compounds may be used as GPR35 receptor agonists. The compounds may be used in the manufacture of medicaments. The compounds may be for use in treating, preventing, ameliorating, controlling or reducing the risk of disorders associated with GPR35. The compounds may be used in the treatment or prevention of mast cell disorders, acute and chronic pain conditions and diseases associated with allergic or inflammatory diseases in both the gastrointestinal system and the lung.
The compounds may be used for treating gastrointestinal disorders and conditions, using agents that selectively act at GPR35 receptor. These include but are not limited to: food allergy, food intolerance and allergic disorders, celiac disease, gastrointestinal symptoms associated with systemic mastocytosis and other mast cell related disorders (mast cell activation syndrome, clonal mast cell disorder, monoclonal mast cell activation syndrome, idiopathic urticaria, idiopathic anaphylaxis), mastocytic colitis, irritable bowel syndrome (IBS), gastrointestinal motility disorders, functional gastrointestinal disorders, gastroesophageal reflux disease (GERD), duodenogastric reflux, diarrhoeal diseases, eosinophilic gastroenteritis, eosinophilic esophagitis, infectious diarrhea (such as Clostridium difficile, Salmonella, Shigella toxin), microscopic colitis, immune mediated gastrointestinal diseases, Crohn's disease, ulcerative colitis, inflammatory bowel disease, visceral abdominal pain. In some embodiments, the compounds may be used for treating irritable bowel syndrome (IBS), including IBS with constipation (IBS-C), IBS with diarrhea (IBS-D), and IBS with mixed bowel habits (IBS-M). In some embodiments, the compounds may be used for treating IBS-D.
In some embodiments, the compounds may be used for treating inflammatory bowel disease (IBD). In some embodiments, the compounds may be used for treating Crohn's disease. In some embodiments, the compounds may be used for treating ulcerative colitis.
The compounds may be used for treating the symptoms of pain associated with gastrointestinal disease and other visceral conditions including Crohn's disease, ulcerative colitis, inflammatory bowel disease, radiation colitis, radiation cystitis, celiac disease, gluten enteropathy, radiation cystitis, interstitial cystitis, painful bladder syndrome; cancer, gastroesophageal reflux disease, chemotherapy and radiotherapy mucositis, pancreatitis, prostatitis, pelvic pain, endometriosis, hepatitis; hepatic fibrosis and cirrhosis.
The compounds may be used for treating pulmonary diseases and conditions. These include but are not limited to chronic obstructive pulmonary diseases, asthma, chronic bronchitis, cystic fibrosis, emphysema, chronic idiopathic cough, hyperactive airway disorder and idiopathic pulmonary fibrosis.
The present application also provides a method of treatment according to any use of the compound of formula (1) described herein. In some embodiments, there is provided a method of treating disorders associated with GPR35 in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula (1) (e.g., Compound A) or a pharmaceutically acceptable salt thereof. In some embodiments, the subject is a human.
In some embodiments, the disorder is inflammatory bowel disease (IBD). In some embodiments, the disorder is Crohn's disease. In some embodiments, the disorder is ulcerative colitis.
In some embodiments, the disorder is irritable bowel syndrome (IBS), including IBS with constipation (IBS-C), IBS with diarrhea (IBS-D), and IBS with mixed bowel habits (IBS-M). In some embodiments, the disorder is IBS-D.
In this application, the following definitions apply, unless indicated otherwise.
The term “treatment”, in relation to the uses of any of the compounds described herein, including those of the formula (1) is used to describe any form of intervention where a compound is administered to a subject suffering from, or at risk of suffering from, or potentially at risk of suffering from the disease or disorder in question. Thus, the term “treatment” covers both preventative (prophylactic) treatment and treatment where measurable or detectable symptoms of the disease or disorder are being displayed.
The term “effective therapeutic amount” (for example in relation to methods of treatment of a disease or condition) refers to an amount of the compound which is effective to produce a desired therapeutic effect. For example, if the condition is pain, then the effective therapeutic amount is an amount sufficient to provide a desired level of pain relief. The desired level of pain relief may be, for example, complete removal of the pain or a reduction in the severity of the pain.
The terms “alkyl” as in “C1-6 alkyl”, “cycloalkyl” as in “C3-6 cycloalkyl”, “alkoxy” as in “C1-6 alkoxy”, “aryl”, “heteroaryl”, “monocyclic” and “bicyclic” are all used in their conventional sense (e.g. as defined in the IUPAC Gold Book), unless indicated otherwise.
To the extent that any of the compounds described have chiral centres, the present invention extends to all optical isomers of such compounds, whether in the form of racemates or resolved enantiomers. The invention described herein relates to all crystal forms, solvates and hydrates of any of the disclosed compounds however so prepared. To the extent that any of the compounds disclosed herein have acid or basic centres such as carboxylates or amino groups, then all salt forms of said compounds are included herein. In the case of pharmaceutical uses, the salt should be seen as being a pharmaceutically acceptable salt.
The compounds of the invention can exist in tautomeric forms. It is to be understood that any reference to a named compound or a structurally depicted compound is intended to encompass all tautomers of such compound. For example, the compound of formula (1) encompasses the tautomers shown below:
Salts or pharmaceutically acceptable salts that may be mentioned include acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.
Examples of pharmaceutically acceptable salts include acid addition salts derived from mineral acids and organic acids, and salts derived from metals such as sodium, magnesium, potassium and calcium. Representative pharmaceutically acceptable base addition salts also include, but are not limited to, aluminium, ammonium, 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS, tromethamine), arginine, benethamine (N-benzylphenethylamine), benzathine (N,N′-dibenzylethylenediamine), bis-(2-hydroxyethyl)amine, bismuth, calcium, chloroprocaine, choline, clemizole (1-p chlorobenzyl-2-pyrrolildine-1′-ylmethylbenzimidazole), cyclohexylamine, dibenzylethylenediamine, diethylamine, diethyltriamine, dimethylamine, dimethylethanolamine, dopamine, ethanolamine, ethylenediamine, L-histidine, iron, isoquinoline, lepidine, lithium, lysine, magnesium, meglumine (N-methylglucamine), piperazine, piperidine, potassium, procaine, quinine, quinoline, sodium, strontium, t-butylamine, and zinc. In one embodiment, the salts of the compounds disclosed in the present disclosure are tromethamine (TRIS) salts.
Examples of acid addition salts include acid addition salts formed with acetic, 2,2-dichloroacetic, adipic, alginic, aryl sulfonic acids (e.g. benzenesulfonic, naphthalene-2-sulfonic, naphthalene-1,5-disulfonic and p-toluenesulfonic), ascorbic (e.g. L-ascorbic), L-aspartic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulfonic, (+)-(1S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulfuric, ethane-1,2-disulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, galactaric, gentisic, glucoheptonic, gluconic (e.g. D-gluconic), glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, lactic (e.g. (+)-L-lactic and (±)-DL-lactic), lactobionic, maleic, malic (e.g. (−)-L-malic), malonic, (±)-DL-mandelic, metaphosphoric, methanesulfonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric, tannic, tartaric (e.g. (+)-L-tartaric), thiocyanic, undecylenic and valeric acids.
Also encompassed are any solvates of the compounds and their salts. Preferred solvates are solvates formed by the incorporation into the solid-state structure (e.g. crystal structure) of the compounds of the invention of molecules of a non-toxic pharmaceutically acceptable solvent (referred to below as the solvating solvent). Examples of such solvents include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulfoxide. Solvates can be prepared by recrystallising the compounds of the invention with a solvent or mixture of solvents containing the solvating solvent. Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the compound to analysis using well known and standard techniques such as thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and X-ray crystallography.
The solvates can be stoichiometric or non-stoichiometric solvates. Particular solvates may be hydrates, and examples of hydrates include hemihydrates, monohydrates and dihydrates. For a more detailed discussion of solvates and the methods used to make and characterise them, see Bryn et al, Solid-State Chemistry of Drugs, Second Edition, published by SSCI, Inc of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3.
The term “pharmaceutical composition” in the context of this invention means a composition comprising an active agent and comprising additionally one or more pharmaceutically acceptable carriers. The composition may further contain ingredients selected from, for example, diluents, adjuvants, excipients, vehicles, preserving agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavouring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents and dispersing agents, depending on the nature of the mode of administration and dosage forms. The compositions may take the form, for example, of tablets, dragees, powders, elixirs, syrups, liquid preparations including suspensions, sprays, inhalants, tablets, lozenges, emulsions, solutions, cachets, granules, capsules and suppositories, as well as liquid preparations for injections, including liposome preparations.
The compounds of the invention may contain one or more isotopic substitutions, and a reference to a particular element includes within its scope all isotopes of the element. For example, a reference to hydrogen includes within its scope 1H, 2H (D), and 3H (T). Similarly, references to carbon and oxygen include within their scope respectively 12C, 13C and 14C and 16O and 18O. In an analogous manner, a reference to a particular functional group also includes within its scope isotopic variations, unless the context indicates otherwise. For example, a reference to an alkyl group such as an ethyl group or an alkoxy group such as a methoxy group also covers variations in which one or more of the hydrogen atoms in the group is in the form of a deuterium or tritium isotope, e.g. as in an ethyl group in which all five hydrogen atoms are in the deuterium isotopic form (a perdeuteroethyl group) or a methoxy group in which all three hydrogen atoms are in the deuterium isotopic form (a trideuteromethoxy group). The isotopes may be radioactive or non-radioactive.
Therapeutic dosages may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed. Determination of the proper dosage for a particular situation is within the skill of the art. Generally, treatment is initiated with the smaller dosages which are less than the optimum dose of the compound. Thereafter the dosage is increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired.
The magnitude of an effective dose of a compound will, of course, vary with the nature of the severity of the condition to be treated and with the particular compound and its route of administration. The selection of appropriate dosages is within the ability of one of ordinary skill in this art, without undue burden. In general, the daily dose range may be from about 10 μg to about 30 mg per kg body weight of a human and non-human animal, preferably from about 50 μg to about 30 mg per kg of body weight of a human and non-human animal, for example from about 50 μg to about 10 mg per kg of body weight of a human and non-human animal, for example from about 100 μg to about 30 mg per kg of body weight of a human and non-human animal, for example from about 100 μg to about 10 mg per kg of body weight of a human and non-human animal and most preferably from about 100 μg to about 1 mg per kg of body weight of a human and non-human animal.
While it is possible for the active compound to be administered alone, it is preferable to present it as a pharmaceutical composition (e.g. formulation).
Accordingly, in some embodiments of the invention, there is provided a pharmaceutical composition comprising at least one compound of Formula (1) or a salt thereof as defined above together with at least one pharmaceutically acceptable excipient.
The pharmaceutically acceptable excipient(s) can be selected from, for example, carriers (e.g. a solid, liquid or semi-solid carrier), adjuvants, diluents (e.g solid diluents such as fillers or bulking agents; and liquid diluents such as solvents and co-solvents), granulating agents, binders, flow aids, coating agents, release-controlling agents (e.g. release retarding or delaying polymers or waxes), binding agents, disintegrants, buffering agents, lubricants, preservatives, anti-fungal and antibacterial agents, antioxidants, buffering agents, tonicity-adjusting agents, thickening agents, flavouring agents, sweeteners, pigments, plasticizers, taste masking agents, stabilisers or any other excipients conventionally used in pharmaceutical compositions.
The term “pharmaceutically acceptable” as used herein means compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (e.g. a human subject) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each excipient must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
Pharmaceutical compositions containing compounds of the Formula (1) can be formulated in accordance with known techniques, see for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, USA. The pharmaceutical compositions can be in any form suitable for oral, parenteral, intravenous, intramuscular, intrathecal, subcutaneous, topical, intranasal, intrabronchial, sublingual, buccal, ophthalmic, otic, rectal, intra-vaginal, or transdermal administration.
Pharmaceutical dosage forms suitable for oral administration include tablets (coated or uncoated), capsules (hard or soft shell), caplets, pills, lozenges, syrups, solutions, powders, granules, elixirs and suspensions, sublingual tablets, wafers or patches such as buccal patches.
The composition may be a tablet composition or a capsule composition. Tablet compositions can contain a unit dosage of active compound together with an inert diluent or carrier such as a sugar or sugar alcohol, eg; lactose, sucrose, sorbitol or mannitol; and/or a non-sugar derived diluent such as sodium carbonate, calcium phosphate, calcium carbonate, or a cellulose or derivative thereof such as microcrystalline cellulose (MCC), methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, and starches such as corn starch. Tablets may also contain such standard ingredients as binding and granulating agents such as polyvinylpyrrolidone, disintegrants (e.g. swellable crosslinked polymers such as crosslinked carboxymethylcellulose), lubricating agents (e.g. stearates), preservatives (e.g. parabens), antioxidants (e.g. BHT), buffering agents (for example phosphate or citrate buffers), and effervescent agents such as citrate/bicarbonate mixtures. Such excipients are well known and do not need to be discussed in detail here.
Tablets may be designed to release the drug either upon contact with stomach fluids (immediate release tablets) or to release in a controlled manner (controlled release tablets) over a prolonged period of time or with a specific region of the GI tract.
The pharmaceutical compositions typically comprise from approximately 1% (w/w) to approximately 95% (w/w) active ingredient and from 99% (w/w) to 5% (w/w) of a pharmaceutically acceptable excipient (for example as defined above) or combination of such excipients. Preferably, the compositions comprise from approximately 20% (w/w) to approximately 90% (w/w) active ingredient and from 80% (w/w) to 10% (w/w) of a pharmaceutically excipient or combination of excipients. The pharmaceutical compositions comprise from approximately 1% (w/w) to approximately 95% (w/w), preferably from approximately 20% (w/w) to approximately 90% (w/w), active ingredient. Pharmaceutical compositions according to the invention may be, for example, in unit dose form, such as in the form of ampoules, vials, suppositories, pre-filled syringes, dragees, powders, tablets or capsules.
Tablets and capsules may contain, for example, 0-20% (w/w) disintegrants, 0-5% (w/w) lubricants, 0-5% (w/w) flow aids and/or 0-99% (w/w) fillers/or bulking agents (depending on drug dose). They may also contain 0-10% (w/w) polymer binders, 0-5% (w/w) antioxidants, 0-5% (w/w) pigments. Slow release tablets would in addition typically contain 0-99% (w/w) release-controlling (e.g. delaying) polymers (depending on dose). The film coats of the tablet or capsule typically contain 0-10% (w/w) polymers, 0-3% (w/w) pigments, and/or 0-2% (w/w) plasticizers.
The composition may be a parenteral composition. Parenteral formulations may contain 0-20% (w/w) buffers, 0-50% (w/w) cosolvents, and/or 0-99% (w/w) Water for Injection (WFI) (depending on dose and if freeze dried). Formulations for intramuscular depots may also contain 0-99% (w/w) oils.
The composition may be in a form suitable for intranasal or intrabronchial administration. Such compositions should be suitable for atomisation, which allows inhalation through the mouth and facilitates absorption through the thin mucous membrane that lines the nasal passages.
Many drugs that are administered orally can also be administered rectally as a suppository. The composition may be in a form suitable for rectal administration. In this form, the composition may comprise a waxy substance that dissolves or liquefies after it is inserted into the rectum. Such compositions may be prescribed for people who cannot take a drug orally because they have nausea, cannot swallow, or have restrictions on eating, as is the case before and after many surgical operations.
The pharmaceutical formulations may be presented to a patient in “patient packs” containing an entire course of treatment in a single package, usually a blister pack.
The compounds of the Formula (1) will generally be presented in unit dosage form and, as such, will typically contain sufficient compound to provide a desired level of biological activity. For example, a formulation may contain from 1 nanogram to 2 grams of active ingredient, e.g. from 1 nanogram to 2 milligrams of active ingredient. Within these ranges, particular sub-ranges of compound are 0.1 milligrams to 2 grams of active ingredient (more usually from 10 milligrams to 1 gram, e.g. 50 milligrams to 500 milligrams), or 1 microgram to 20 milligrams (for example 1 microgram to 10 milligrams, e.g. 0.1 milligrams to 2 milligrams of active ingredient).
For oral compositions, a unit dosage form may contain from 1 milligram to 2 grams, more typically 10 milligrams to 1 gram, for example 50 milligrams to 1 gram, e.g. 100 milligrams to 1 gram, of active compound.
In some embodiments, the pharmaceutical compositions comprise a crystalline form of Compound A free acid or Compound A tromethamine salt. In some embodiments, the pharmaceutical compositions comprise a crystalline form of Compound A free acid. In some embodiments, the pharmaceutical compositions comprise a crystalline form of Compound A tromethamine salt.
In some embodiments, the pharmaceutical compositions comprise Compound A tromethamine salt Hydrate I. In some embodiments, the pharmaceutical compositions comprise Compound A tromethamine salt Hydrate II. In some embodiments, the pharmaceutical compositions comprise Compound A tromethamine salt Hydrate I and Compound A tromethamine salt Hydrate I.
The active compound will be administered to a patient in need thereof (for example a human or animal patient) in an amount sufficient to achieve the desired therapeutic effect (effective amount). The precise amounts of compound administered may be determined by a supervising physician in accordance with standard procedures.
Compounds of the formula (1) can be prepared in accordance with synthetic methods as described herein, some of which will be known to the skilled person. The invention provides a process for the preparation of a compound as defined in formula (1) above.
Compounds of formula (1) may be prepared as described in Scheme 1 below.
Thus, substituted amino aromatic nitriles of formula (2), which are either commercially available or readily accessible from commercial materials, are converted to tetrazole intermediates of formula (3), typically in the presence of sodium azide and ammonium chloride or zinc chloride, in solvents such as DMF, DMSO or 2-propanol, and heating to temperatures in the range 110-130° C. Alternatively, tetrazoles of formula (3) may be accessed from substituted 3-nitrobenzonitriles of formula (5), which are either commercially available or readily accessible from commercial materials. Conversion of the nitrile function to provide the corresponding tetrazoles of formula (6) is performed as above, and subsequent reduction of the nitro group is typically affected by zinc dust in the presence of ammonium chloride in 1,4-dioxane and water whilst heating to reflux. The aniline group of compounds of formula (3) is then functionalized by reaction with diethyl squarate in the presence of a base, typically triethylamine, in solvents such as EtOH or DCM. The ester function of squarates of formula (4) are then hydrolyzed, typically performed in a mixture of aqueous HCl and THF at mild temperatures, typically 60° C., to give Examples of the formula (1). Alternatively, substituted anilines of the formula (3) can be converted directly to Examples of the formula (1) by treatment with squaric acid, with conditions typically comprising water at reflux.
Alternatively, compounds of formula (1) may be prepared as described in Scheme 2 below. Aryl bromide intermediates of formula (8) can be prepared from amino anilines of the formula (7) as described in Scheme 1. Subsequent Suzuki reaction between aryl bromides of formula (8) and a suitable boronic acid or boronic ester coupling partner (R═H or alkyl) affords Examples of formula (1). The Suzuki reaction is typically carried out under microwave irradiation in the presence of a base, such as K2CO3, and a catalytic palladium source, such as [1,1′-Bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II), in a suitable solvent system, typically a combination of MeCN and H2O.
Scheme 3 shows a variation for preparation of compounds of formula (1), whereby R2 is a phenyl optionally substituted with —CONHCH2R4, —CONHCH2CH2OR4, —CONHR4 or —CON(CH3)R4. Biaryl carboxylic acid compounds of formula (9) are readily accessible from commercial 3-amino-5-bromobenzonitrile (7) and are converted to the corresponding tetrazole compounds of formula (10) as described in Scheme 1. The aniline group of intermediate compounds of formula (10) are then functionalized as described in Scheme 1. The carboxylic acid group of compounds of formula (11) are then converted to amides in the presence of a suitable amine, coupling agent such as 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate, a base such as N,N-diisopropylethylamine and a solvent such as DMF. The ester function of cyclobut-3-ene-1,2-diones of formula (12) is then hydrolyzed to afford Examples of formula (13) (corresponding to formula (1) whereby R2 is a phenyl optionally substituted with —CONHCH2R4, —CONHCH2CH2OR4, —CONHR4 or —CON(CH3)R4).
In circumstances whereby X is N, and R1 is H, compounds of formula (2b) may be prepared as described in Scheme 4. Accordingly, commercial 2,6-dichloroisonicotinonitrile (14) is subject to an SNAr reaction with (4-methoxyphenyl)methanamine in a solvent such as NMP with heating to temperatures in the range 110-120° C. The nitrile function of intermediates of formula (15) are then converted to tetrazole groups as described in Scheme 1. Where R2 is an alkoxy or phenoxy group, the chlorine group of pyridine (16) is displaced by a suitable alcohol or metal alkoxide (e.g. NaOMe) in the presence or absence of a base such as K2CO3, in a solvent such as DMSO and at temperatures varying from room temperature to 120° C. to give alkoxy or phenoxy substituted compounds (17). Alternatively, when R2 is phenyl, a Suzuki reaction between pyridyl chloride of formula (16) and the appropriate phenyl boronic acid or ester affords intermediate (17). The Suzuki reaction is typically carried out in the presence of a base such as K2CO3, and a catalytic source of palladium, typically [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II), in a solvent such as 1,4-dioxane, at temperatures of 120° C. It will be understood by the skilled person that the order of steps as laid out in Scheme 4 may be completed in a different sequence to that shown, without affecting the overall success of the synthesis of the desired compound of formula (2b). Deprotection of the amino function of compounds of formula (17) is then achieved by treatment with acid, typically TFA at 70° C. Conversion of Intermediates of formula (18) to give Examples of formula (2b) is then achieved as described in Scheme 1.
In certain reactions described above, it may be necessary to protect one or more groups to prevent reaction from taking place at an undesirable location on the molecule. Examples of protecting groups, and methods of protecting and deprotecting functional groups, can be found in Greene's Protective Groups in Organic Synthesis, Fifth Edition, Editor: Peter G. M. Wuts, John Wiley, 2014, (ISBN:9781118057483).
Compounds made by the foregoing methods may be isolated and purified by any of a variety of methods well known to those skilled in the art and examples of such methods include recrystallisation and chromatographic techniques such as column chromatography (e.g. flash chromatography), HPLC and SFC.
Where no preparative routes are included, the relevant intermediate is commercially available. Commercial reagents were utilized without further purification. Final compounds and intermediates are named using ChemDraw Professional, Version 17.0.0.206 (121). Room temperature (RT) refers to approximately 20-27° C. 1H NMR spectra were recorded at 400 or 500 MHz on either a Bruker, Varian or Jeol instrument. Chemical shift values are expressed in parts per million (ppm), i.e. (δ)—values relative to the following solvents: chloroform-d=7.26 ppm, DMSO-d6=2.50 ppm, methanol-d4=3.31 ppm. The following abbreviations are used for the multiplicity of the NMR signals: s=singlet, br=broad, d=doublet, t=triplet, q=quartet, m=multiplet. Coupling constants are listed as J values, measured in Hz. NMR and mass spectroscopy results were corrected to account for background peaks. Chromatography refers to column chromatography performed using 60-120 mesh or 40-633 μm, 60 Å silica gel and executed under nitrogen pressure (flash chromatography) conditions. Microwave-mediated reactions were performed in Biotage Initiator or CEM Discover microwave reactors.
LCMS analysis of compounds was performed under electrospray conditions using the instruments and methods given below:
Instruments: HP 1100 with G1315A DAD, Micromass ZQ; Column: Phenomenex Gemini-NX C-18, 3 micron, 2.0×30 mm; Gradient [time (min)/solvent B in A (%)]: 0.00/2, 0.10/2, 2.50/95, 3.50/95; Solvents: solvent A=2.5 L H2O+2.5 mL 28% ammonia in H2O solution; solvent B=2.5 L MeCN+135 mL H2O+2.5 mL 28% ammonia in H2O solution. Injection volume 1 μL; UV detection 230 to 400 nm; Mass detection 130 to 800 AMU; column temperature 45° C.; Flow rate 1.5 mL/min.
Instruments: Agilent 1260 Infinity LC with Diode Array Detector, Agilent 6120B Single Quadrupole MS with API-ES Source; Column: Phenomenex Gemini-NX C-18, 3 micron, 2.0×30 mm; Gradient [time (min)/solvent B in A (%)]: 0.00/5, 2.00/95, 2.50/95, 2.60/5, 3.00/5; Solvents: solvent A=2.5 L H2O+2.5 mL 28% NH3 in H2O; solvent B=2.5 L MeCN+129 mL H2O+2.7 mL of (28% NH3 in H2O); Injection volume 0.5 μL; UV detection 190 to 400 nm; Mass detection 130 to 800 AMU; column temperature 40° C.; Flow rate 1.5 mL/min.
Instruments: Agilent 1260 Infinity LC with Diode Array Detector, Agilent 6120B Single Quadrupole MS with API-ES Source; Column: Restek, Penta Fluoro Phenyl Propyl, 3 micron, 2.1×30 mm. Gradient [time (min)/solvent B in A (%)]: Method C: 0.00/5, 2.00/95, 2.50/95, 2.60/5, 3.00/5 or Method D: 0.00/2, 0.1/2, 8.4/95, 10/95, 10.1/2, 12/2; Solvents: solvent A=water (2.5 L) with 2.5 mL Formic acid; Solvent B=MeCN (2.5 L) with 125 mL water and 2.5 mL Formic acid. Injection volume 0.5 μL; UV detection 190 to 400 nm; Mass detection 130 to 800 AMU; column temperature 40° C.; Flow rate 1.5 mL/min.
Instruments: Waters Acquity H Class, Photo Diode Array, SQ Detector; Column: BEH C18, 1.7 micron, 2.1×50 mm; Gradient [time (min)/solvent B in A (%)]: 0.00/5, 0.40/5, 0.8/35, 1.20/55, 2.50/100, 3.30/100 4.00/5; Solvents: solvent A=5 mM mmmonium acetate and 0.1% formic acid in H2O; solvent B=0.1% formic acid in MeCN; Injection volume 2 μL; UV detection 200 to 400 nm; Mass detection 100 to 1200 AMU; column at ambient temperature; Flow rate 0.55 mL/min.
Instruments: Agilent 1100 Series with DAD/ELSD, Agilent LC\MSD VL (G1956A), SL (G1956B) mass-spectrometer; Column: Zorbax SB-C18, 1.8 micron, 4.6×15 mm; Gradient [time (min)/solvent B in A (%)]: 0.0/0, 1.5/100, 1.8/100, 1.81/0; Solvents: solvent A=water and 0.1% formic acid; solvent B=MeCN and 0.1% formic acid; Injection volume 1 μL; UV detection 200 to 400 nm; Mass detection 80-1000 AMU; column at ambient temperature; Flow rate 3.0 mL/min.
Instruments: Waters 2690 with PDA Detector 996, Acquity QDA mass-spectrometer; Column: X-BRIDGE C18, 5.0 micron, 4.6×100 mm; Gradient [time (min)/solvent B in A (%)]: 0.0/10, 1.0/10, 5.0/100, 7.0/100, 7.50/10, 8.0/10; Solvents: solvent A=0.1% formic acid and 10 mM ammonium bicarbonate in water; solvent B=MeCN; Injection volume 1 μL; UV detection 190 to 800 nm; Mass detection 30-1250 AMU; column temperature 35° C.; Flow rate 1.2 mL/min.
Instruments: Waters Acquity H Class with photo diode array, SQ detector mass-spectrometer; Column: BEH C18, 1.7 micron, 2.1×50 mm; Gradient [time (min)/solvent B in A (%)]: 0.0/5, 0.4/5, 0.8/35, 1.2/55, 2.5/100, 3.3/100, 3.31/5, 4.0/5; Solvents: solvent A=0.1% formic acid and 5 mM ammonium acetate in water; solvent B=0.1% formic acid in MeCN; Injection volume 1 μL; UV detection 200 to 400 nm; Mass detection 100-1200 AMU; column at ambient temperature; Flow rate 0.55 mL/min.
Instruments for Method I: Shimadzu Nexera with photo diode array, LCMS-2020 mass-spectrometer or Instruments for Method J: Agilent 1290 RRLC with photo diode array, Agilent 6120 mass-spectrometer; Column: X-BRIDGE C18, 3.5 micron, 4.6×50 mm; Gradient [time (min)/solvent B in A (%)]: 0.0/5, 5.0/90, 5.8/95, 7.20/95, 7.21/5, 10.0/5; Solvents: solvent A=0.1% ammonia in water; solvent B=0.1% ammonia in MeCN; Injection volume 1 μL; UV detection 200 to 400 nm; Mass detection 60-1000 AMU; column at ambient temperature; Flow rate 1.0 mL/min.
Instruments: Waters 2690 with PDA Detector 996, Acquity QDA mass-spectrometer; Column: X-BRIDGE C18, 5.0 micron, 4.6×100 mm; Gradient [time (min)/solvent B in A (%)]: 0.0/10, 3.0/10, 6.0/100, 7.0/100, 7.01/10, 10.0/10; Solvents: solvent A=0.1% formic acid in water; solvent B=MeOH; Injection volume 1 μL; UV detection 190 to 800 nm; Mass detection 30-1250 AMU; column temperature 35° C.; Flow rate 1.0 mL/min.
Instruments: Shimadzu LCMS-2010 EV with Shimadzu SPD-M20A PDA, single quadrupole mass-spectrometer; Column: Atlantis C18, 3.0 micron, 4.6×50 mm; Gradient [time (min)/solvent B in A (%)]: 0.0/30, 3.0/90, 6.0/90, 6.1/30; Solvents: solvent A=0.1% formic acid in water; solvent B=MeCN; Injection volume 1 μL; UV detection 254 nm; Mass detection 80-800 AMU; column temperature 40° C.; Flow rate 0.8 mL/min.
Instruments: Shimadzu LCMS-2010 EV with Shimadzu SPD-M20A PDA, single quadrupole mass-spectrometer; Column: Capcell pack C18, 3.0 micron, 4.6×150 mm; Gradient [time (min)/solvent B in A (%)]: 0.0/30, 5.0/98, 9.5/98, 11.5/3, 12/3; Solvents: solvent A=0.1% formic acid in water; solvent B=MeCN; Injection volume 1 μL; UV detection 254 nm; Mass detection 80-800 AMU; column temperature 40° C.; Flow rate 0.8 mL/min.
The invention will now be illustrated, but not limited, by reference to the following examples.
The compounds of Examples 1 to 31 shown in Table 1 below have been prepared. Their NMR and LCMS properties and the methods used to prepare them are set out in Table 3. The starting materials for each of the Examples are listed in Table 2.
1H NMR(CDCl3, 400 MHz): δ (ppm)
1H NMR
Compound A is the compound of Example 5 (structure above). 2 mL of 97.6% (THF:water 3:1): 2.4% DMSO were added to 50 mg of Compound A and the mixture was heated to 50° C. resulting in dissolution. 1 eq of 1 M aqueous tromethamine was added to the solution and it was equilibrated at 50° C. for one hour before being cooled to room temperature and left stirring overnight. The solution was then evaporated (to approx. 25% of original volume) under nitrogen until precipitation occurred. The resulting solid was filtered, washed with IPA and dried under vacuum at 45° C.
Compound A (1 wt, kg scale) was charged to a vessel under nitrogen. This was followed by the addition of THF (6.08 vol) and then aqueous tromethamine solution (0.268 wt tromethamine dissolved in 2.21 vol of water). Water (1.84 vol) was then added to the vessel. The mixture was heated to dissolution at 60° C. The solution was cooled to 50° C. Acetonitrile (1.29 vol) was added before the reaction was seeded with crystalline Hydrate I of Compound A tromethamine salt (0.0126 wt). The mixture was left to equilibrate for 1 hour. Acetonitrile (6.33 vol) was then added to the slurry over 2 hours. The mixture was then cooled to 5° C. and stirred overnight. The solids were washed with acetonitrile (3.96 vol) before being dried under vacuum at 45° C. to give Compound A tromethamine salt (Hydrate I).
The X-ray powder diffraction (XRPD) pattern of the above Hydrate I of Compound A tromethamine salt is shown in
An example differential scanning calorimetry (DSC) thermogram of the above Hydrate I of Compound A tromethamine salt is shown in
An example thermogravimetric analysis (TGA) thermogram of the above Hydrate I of Compound A tromethamine salt is shown in
2 mL of methanol were added to 100 mg of an amorphous form of Compound A tromethamine salt and the suspension was equilibrated at room temperature overnight. The resulting solid was filtered and dried under vacuum at 45° C.
60 mL of methanol were added to 3 g of Hydrate I of Compound A tromethamine salt in a vessel with overhead stirring. 60 mg (2% w/w seed loading) of the Hydrate II of Compound A tromethamine salt was added and the reaction mixture was equilibrated overnight at room temperature. The suspension thickened and became much paler overnight. The resulting solid was filtered and dried under vacuum at 45° C.
The X-ray powder diffraction (XRPD) pattern of the above Hydrate II of Compound A tromethamine salt is shown in
An example differential scanning calorimetry (DSC) thermogram of the above Hydrate II of Compound A tromethamine salt is shown in
An example thermogravimetric analysis (TGA) thermogram of the above Hydrate II of Compound A tromethamine salt is shown in
Water activity studies were conducted to determine the critical water activity at which each of Hydrate I and Hydrate II is stable. Competitive slurries of Hydrate I and Hydrate II were conducted at room temperature in a range of aqueous solvent mixtures with varying water activity. Hydrate I was isolated from all mixtures with a water activity of greater than 0.5. Equilibration of Hydrate II alone confirmed conversion to Hydrate I in solvent mixtures with water activity of 0.5 or greater. Competitive slurries of Hydrate I and Hydrate II at room temperature in solvent mixtures with a water activity of 0.2 resulted in a mixture of Hydrate I and Hydrate II solids.
Crude Compound A (free acid) was purified by reverse phase chromatography applying basic conditions (high pH) under 5-35% gradient of acetonitrile in aqueous media (0.2% of 28% ammonia hydroxide in water) on 12.5 minute method via a Gemini-NX C18 column (5 μm, 100×30 mm) on a Gilson Semi Preparative HPLC, Pumps 332 & 331, GX-271 Liquid handler, Trilution software using a flow rate of 30 mL/min and 171 Diode Array Detector at 205 nm, 210 nm and 230 nm. The desired fractions were combined then evaporated on a Biotage V10 machine to give a white solid residue (4 g), a diammonium salt.
The diammonium salt (4 g, 7.99 mmol) was dissolved in DMSO (39.96 mL) and stirred for 30 minutes. 1 N HCl (59.94 mL, 59.94 mmol) was added and the resulting precipitate was collected by filtration, washed with ice cold water (20 mL) and dried to give crude product which was re-suspended in EtOH (40 mL) and stirred for 3 h. The suspension was then filtered to give a dry white solid which was milled to a fine powder (2.94 g). NMR revealed that this powder was the desired product; however a large amount of DMSO remained in the sample. Therefore, the solid was re-suspended in EtOH (25 mL) and stirred for 18 h. The suspension was then filtered, and the solid was collected, milled to a fine powder using a pestle and mortar to give Compound A free acid (2.39 g, 5.12 mmol, 64.1% yield) as a white crystalline solid. NMR of the product shows a small amount of DMSO remained in the sample with the ratio of product to DMSO being approximately 1:0.05. The X-ray powder diffraction (XRPD) pattern of the crystalline Compound A free acid is shown in
An example thermogravimetric analysis (TGA) thermogram of the above Compound A free acid Pattern 3 is shown in
When crystalline Compound A free acid Pattern 3 was dried by heating at 45° C. under vacuum (ca. 10-15 mbar) overnight, a different crystalline solid was obtained, referred to as “Pattern 1.” The XRPD pattern of Compound A free acid Pattern 1 is shown in
An example thermogravimetric analysis (TGA) thermogram of the above Compound A free acid Pattern 1 is shown in
Overexpression of Human GPR35a Baculovirus in HEK293f cells at a cell density of 2.5×106 cells/mL and a multiplicity of infection of 2.5 over 24 h in Pro293 (Lonza)+5% FBS, 1% Glutamax and 0.4% Pen/Strep. Cells harvested and centrifuged at 2500 RPM for 10 mins at 4° C. The supernatant was then poured off and the pellet stored at −80° C. The pellet was defrosted and re-suspended in 15 mL of homogenising buffer (20 mM HEPES, 10 mM EDTA, pH 7.4). Then homogenised in mechanical homogeniser (VMR) for 10 seconds. The membrane was centrifuged in centrifuge tubes at 40,000 g for 15 mins at 4° C. The supernatant was poured away and re-suspended in 15 mL of homogenising buffer. Homogenised for 20 seconds. The membrane was centrifuged at 40,000 g for 45 mins at 4° C. The membrane was re-suspended in 3 mL of storage buffer (20 mM HEPES, 0.1 mM EDTA, pH 7.4) mixing well. The resulting membranes were then stored at −80°. GPR35 cell membrane homogenates were re-suspended in the binding buffer (50 mM TRIS+10 mM MgCl2 pH 7.4) to a final assay concentration of 5 ug/well. Test compounds were diluted in dimethylsulphoxide (DMSO (Sigma Aldrich, UK)), to form a 10 point ½ log concentration curve. Test compounds were added per plate, followed by 7 nM 3H-27966. 0.1 uM FAC Lodoxamide was added in order to allow non-specific binding to be calculated. Finally, membrane was added to each well on the plate. After 60 min incubation at room temperature, membranes were filtered onto a unifilter, 96-well white microplate with bonded GF/B filter, pre-soaked in ddH20, with a TomTec cell harvester, and washed 5 times with distilled water. Plates were dried prior to 50 ul/well scintillant added, sealed and radioactivity measured using a MicroBeta analyser. IC50 values were derived from the inhibition curve and affinity constant (Ki) values were calculated using the Cheng-Prussoff equation, where; pKi=−log 10 Ki.
HT-29 cells (ATCC HTB-38) kept in continuous culture in McCoys (Thermo 16600082) supplemented with 10% FBS. Day prior to assay, cells harvested with TrypLE (Gibco12604-013), and plated at 20 k/well in culture media in a total volume of 50 ul in Corning EPIC 384 well plates (5040) overnight 37° C. 5% CO2. On the day of assay, cell media was removed and replaced with assay buffer (HBSS+20 mM HEPES pH7.4) and reincubated for 1 h. Compounds were prepared in 100% DMSO in ECHO LDV 384 source plates. Cell plates were read on an EPIC plate reader at room temperature for 15 mins, read paused, and cell plate added to LabCyte ECHO 550 for addition of 50 nl per well compound by acoustic transfer. Plates were immediately placed back into the EPIC reader, read unpaused and Dynamic Mass Redistribution measured for 60 minutes. Raw data was analysed by EPIC Analyser software and max peak taken per concentration to enable EC50 determination. All raw DMR data was normalised to Lodoxamide and buffer corrected.
Compound A showed about 500 times higher functional potency for GPR35 than the mast cell stabiliser Cromolyn, which has been used clinically at high doses for GI disorders. Compound A also showed pharmacology across preclinical species including in PGE2-induced fluid secretion, indomethacin ileitis and barrier permeability, TNBS mouse visceral pain model and acute rat and mouse LPS challenge. Compound A further showed strong selectivity for GPR35 and no off-target effects have been observed. Compound A has a very low drug interaction potential for the major human CYPs, including CYP3A4.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2105846.6 | Apr 2021 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2022/053772 | 4/22/2022 | WO |
