Radiolabeled MGL PET Ligand

Abstract

The present invention relates to novel, selectively radiolabeled compounds with monoacylglycerol lipase (MGL) affinity that are useful for imaging and quantitating MGL receptor expression, distribution, and enzyme occupancy in tissues using positron emission tomography (PET). The present invention is also directed to compositions comprising such compounds, the use of such compounds and compositions for imaging tissues, cells, or hosts in vitro or in vivo, and precursors of such compounds.

Description

Technical Field

[0001] The present invention relates to novel selective compounds having monoacylglycerol lipase (MGL) affinity. In one embodiment, the compounds contain a positron-emitting radioligand and enable positron-emission tomography (PET). The invention also relates to pharmaceutical compositions containing such compounds, to the use of such compounds for assessing the expression, distribution, and enzyme occupancy of the MGL receptor, and to the diagnosis of diseases, disorders, or conditions associated with MGL receptor activity in a subject, particularly a human.

Background Art

[0002] Asa and Δ 9-Analogs of tetrahydrocannabinol have been used for therapeutic purposes since the era of folk medicine. The endocannabinoid system consists of two G protein-coupled receptors, cannabinoid receptor type 1 (CB1) (Matsuda et al., Nature, 1990, 346, 561-4) and cannabinoid receptor type 2 (CB2) (Munro et al., Nature, 1993, 365, 61-5). The CB1 receptor is one of the most abundant G protein-coupled receptors expressed in the brain (Herkenam et al., Proc. Nat. Acad. Sci., 1990, 87(5), 1932-1936). CB1 is also peripherally expressed in the liver, gastrointestinal tract, pancreas, adipose tissue, and skeletal muscle (Di Marzo et al., Curr Opin Lipidol, 2007, 18, 129~140). CB2 is mainly expressed in immune cells such as monocytes (Pacher et al., Amer J Physiol, 2008, 294, H1133-H1134), as well as in the brain under certain conditions (inflammation) (Benito et al., Brit J Pharmacol, 2008, 153, 277-285), and in skeletal muscle (Cavuoto et al., Biochem Biophys Res Commun, 2007, 364, 105-110), and in cardiomyocytes (Hajrasouliha et al., Eur J Pharmacol, 2008, 579, 246-252).

[0003] In 1992, it was found that N-arachidonoylethanolamine (arachidonoylethanolamine, AEA or anandamide) is an endogenous ligand of cannabinoid receptors (Devane et al., Science, 1992, 258, 1946-9). Subsequently, 2-arachidonoylglycerol (2-AG) was also identified as an additional endogenous ligand of cannabinoid receptors (Mechoulam et al., Biochem Pharmacol, 1995, 50, 83-90; Sugiura et al., Biochem Biophys Res Commun, 1995, 215, 89-97). In the rat brain, the concentration of 2-AG has been reported to be at least 100-fold higher than that of anandamide (Buczynski and Parsons, Brit J Pharmacol, 2010, 160(3), 423-42). Therefore, 2-AG may play a more important physiological role than anandamide in the brain endocannabinoid system (Sugiura et al. Prostaglandins Leukot Essent Fatty Acids., 2002, Feb-Mar, 66(2~3):173-92). The endocannabinoid 2-AG is a full agonist of CB1 and CB2 receptors, while anandamide is a partial agonist of both receptors (Suguira et al., Prog Lipid Res, 2006, 45(5):405-46). Unlike many classical neurotransmitters, endocannabinoids signal via a retrograde mechanism. They are synthesized in response to demands in postsynaptic neurons and are then rapidly degraded after binding to presynaptic cannabinoid receptors (Ahn et al., Chem Rev., 2008, 108(5):1687-707).Monoacylglycerol lipase (also known as MGLL, MAG lipase, and MGL) is a serine hydrolase involved in the breakdown of 2-AG to arachidonic acid and glycerol in the central nervous system (Mechoulam et al., Biochem Pharmacol, 1995, 50, 83-90; Sugiura et al., Biochem Biophys Res Commun, 1995, 215, 89-97; Long et al., Nat Chem Biol., 2009 Jan, 5(1):37-44; Schlosburg et al., Nat Neurosci., 2010, Sep;13(9):1113-9) and also in peripheral tissues (Long et al., Chem Biol., 2009 Jul 31;16(7):744-53). Anandamide is hydrolyzed by fatty acid amide hydrolase (FAAH) (Piomelli, Nat Rev Neurosci, 2003, 4, 873-884). MGL exists in both soluble and membrane-bound forms (Dinh et al., Proc Natl Acad Sci U S A., 2002, Aug 6;99(16):10819-24). In the brain, MGL is located within presynaptic neurons (Straiker et al., Mol Pharmacol., 2009, Dec;76(6):1220-7) and astrocytes (Walter et al., J Neurosci., 2004, Sep 15;24(37):8068-74) in regions associated with high densities of CB1 receptors. Gene disruption of MGL expression results in a 10-fold increase in brain 2-AG levels without affecting anandamide concentrations compared to wild-type controls (Schlosburg et al., Nat Neurosci., 2010, Sep;13(9):1113-9).

[0004] Therefore, MGL modulation offers an interesting strategy for enhancing the endocannabinoid system. The main advantage of this approach is that only the brain regions where endocannabinoids are actively produced are modulated, potentially minimizing the side effects associated with exogenous CB1 agonists. Pharmacological inactivation of MGL by a covalent inhibitor in animals has been found to increase 2-AG content in the brain and peripheral tissues and produce anti-nociceptive, anxiolytic, and anti-inflammatory effects that are dependent on CB1 and / or CB2 receptors (Long et al., Nat Chem Biol., 2009, Jan, 5(1):37-44; Ghosh et al., Life Sci., 2013, Mar 19, 92(8-9):498~505; Bedse et al., Biol Psychiatry., 2017, Oct 1, 82(7):488-499; Bernal-Chico et al., Glia., 2015, Jan, 63(1):163-76; Patel et al. Neurosci Biobehav Rev., 2017, May, 76(Pt A):56-66; Betse et al., Transl Psychiatry., 2018, Apr 26, 8(1):92). In addition to its role in terminating 2-AG signaling, MGL modulation, including MGL inhibition, also promotes CB1 / 2-independent effects on neuroinflammation (Nomura et al., Science., 2011, Nov 11;334(6057):809-13).MGL regulation, including MGL inhibition, results in a decrease in inflammatory prostaglandin signaling in animal models of neurodegeneration, including traumatic brain injury (Katz et al., J Neurotrauma., 2015, Mar 1;32(5):297-306, Zhang et al., J Cereb Blood Flow Metab., 2015, Mar 31;35(4):706), Alzheimer's disease (Piro et al., Cell Rep., 2012, Jun 28,1(6):617-23; Wenzel et al., Life Sci., 2018, Aug 15,207:314-322; Chen et al., Cell Rep., 2012, Nov 29,2(5):1329-39), Parkinson's disease (Nomura et al., Science, 2011, Nov 11,334(6057),809-13; Pasquarelli et al., Neurochem Int., 2017, Nov,110:14-24), amyotrophic lateral sclerosis (Pasquarelli et al., Neuropharmacology, 2017, Sep 15,124:157-169), multiple sclerosis (Hernadez-Torres et al., Angew Chem Int Ed Engl., 2014, Dec 8,53(50):13765-70; Bernal-Chico et al., Glia., 2015, Jan,63(1):163-76), Huntington's disease (Covey et al., Neuropsychopharmacology, 2018,43,2056-2063), Tourette syndrome and status epilepticus (Terrone et al., Epilepsia., 2018, Jan,59(1),79-91, von Ruden et al., Neurobiol Dis., 2015, May;77:238-45).

[0005] Therefore, by enhancing the cannabinoid system and attenuating the inflammatory cascade, MGL regulation, including MGL inhibition, provides a compelling therapeutic approach for the treatment of a vast number of complex diseases. Importantly, MGL regulation, including MGL inhibition, in animals results in a Δ 9-It does not produce the full range of neurobehavioral effects observed with tetrahydrocannabinol and other CB1 agonists (Tuo et al., J Med Chem., 2017, Jan 12, 60(1), 4-46; Mulvihill et al., Life Sci., 2013, Mar 19, 92(8-9), 492-7).

[0006] Reduced endocannabinoid activity is a risk factor for the treatment of depression, anxiety, and post-traumatic stress disorder. The fact that humans have used cannabis for thousands of years and, albeit briefly, have been treated with rimonabant, an endocannabinoid antagonist, supports this hypothesis. In individuals with major depression, 2-AG levels are decreased (Hill et al., Pharmacopsychiatry., 2008, Mar;41(2):48-53; Hill et al., Psychoneuroendocrinology., 2009, Sep;34(8):1257-1262). The prevalence of depression can be predicted from low circulating 2-AG concentrations (Hauer et al., Rev Neurosci., 2012, 23(5-6):681-90). A decrease in the circulating concentration of 2-AG has previously been found in patients with post-traumatic stress disorder (PTSD) (Hill et al., Psychoneuroendocrinology, 2013, 38(12), 2952~2961). Healthy volunteers exposed to chronic stress factors showed a progressive decrease in the circulating concentration of 2-AG, which correlated with the onset of a decrease in positive affect (Yi et al., Progress in Neuro-Psychopharmacology and Biological Psychiatry, 2016, 67(3), 92~97). Rimonabant, a CB1 receptor inverse agonist / antagonist, was withdrawn from the market due to a high incidence of severe depression and suicidal ideation (Christensen et al., The Lancet, 2007, 370, 1706-1713). Therefore, MGL regulators are potentially useful for the treatment of mood disorders, anxiety, and PTSD.

[0007] Cannabinoid receptor agonists are clinically used to treat pain, spasticity, vomiting, and anorexia (Di Marzo, et al., Annu Rev Med., 2006, 57:553-74; Ligresti et al., Curr Opin Chem Biol., 2009, Jun;13(3):32131). Therefore, MGL regulators, including MGL inhibitors, are also useful for these indications. MGL exerts a CB1-dependent anti-nociceptive effect in animal models of noxious chemical pain, inflammatory pain, heat pain, and neuropathic pain (Guindon et al., Br J Pharmacol., 2011, Aug;163(7):1464-78; Kinsey et al., J Pharmacol Exp Ther., 2009, Sep;330(3):902-10; Long et al., Nat Chem Biol., 2009, Jan;5(1):37-44). MGL blockade reduces mechanical and acetone-induced cold allodynia in mice with chronic constriction injury of the sciatic nerve (Kinsey et al., J Pharmacol Exp Ther., 2009, Sep;330(3):902-10). MGL inhibition results in an opioid-sparing event with reduced tolerance, constipation, and cannabis-like side effects (Wilkerson et al., J Pharmacol Exp Ther., 2016, Apr;357(1):145-56). MGL blockade serves as a protective agent in a model of inflammatory bowel disease (Alhouayek et al., FASEB J., 2011, Aug;25(8):2711-21). MGL inhibition also reverses paclitaxel-induced nociceptive behavior and inflammatory markers in a mouse model of chemotherapy-induced neuropathy (Curry et al., J Pharmacol Exp Ther., 2018, Jul;366(1):169-18).

[0008] Inhibition of 2-AG hydrolysis exerts anti-proliferative activity and a decrease in the invasiveness of prostate cancer cells (Nithipatikom et al., Cancer Res., 2004, Dec 15, 64(24):8826-30, Nithipatikom et al., Biochem Biophys Res Commun., 2005, Jul 15, 332(4):1028-33, Nithipatikom et al., Prostaglandins Other Lipid Mediat., 2011, Feb, 94(1-2):34-43). MGL is upregulated in invasive human cancer cells and primary tumors and has a unique role in providing a lipolytic source of free fatty acids for the synthesis of tumorigenic signaling lipids that promote cancer invasiveness. Thus, beyond its physiological role in mediating endocannabinoid signaling, MGL in cancer plays a distinct role in the regulation of the fatty acid precursor pool for the synthesis of tumor-promoting signaling lipids in human malignant cancer cells.

[0009] MGL blockade shows anti-emetic and anti-vomiting effects in the lithium chloride model of vomiting in ferrets (Sticht et al., Br J Pharmacol., 2012, Apr, 165(8):2425-35).

[0010] MGL modulators, including MGL inhibitors, may be useful in modulating drug dependence on opioids. MGL blockade reduces the intensity of naloxone-induced morphine withdrawal symptoms in mice. MGL blockade also attenuates the symptoms during spontaneous withdrawal in morphine-dependent mice (Ramesh et al., J Pharmacol Exp Ther., 2011, Oct, 339(1):173-85).

[0011] MGL modulators may also be potentially useful in the treatment of eye conditions including, but not limited to, glaucoma and disease states resulting from elevated intraocular pressure (Miller et al., Pharmaceuticals, 2018, 11, 50).

[0012] Positron Emission Tomography (PET) is a non-invasive imaging technique that provides the highest spatial and temporal resolution among all nuclear imaging methods and has the further advantage of enabling true quantification of tracer concentration in tissues. Positron Emission Tomography (PET) uses radionuclides that emit positrons, such as, for example, 15 O, 13 N, 11 C, and 18 F, for detection. There is a need to provide positron emission tomography radiotracers for the evaluation of MGL expression, distribution, and occupancy by its inhibitors. Contrast agents play an important role in such studies and in the development of therapeutic candidates targeting MGL. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0013] The present invention relates to a compound having the formula (I)

[0014]

Chemical formula

[0015] In certain embodiments, the compound of formula (I) is a compound of formula (IA)

[0016]

Chemical formula

[0017] The present invention also relates to a pharmaceutical composition comprising a compound of formula (I) (and a compound of formula (IA)) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent. In certain embodiments, the pharmaceutical composition is particularly suitable for diagnosis and may thus be referred to as a diagnostic pharmaceutical composition. In particular, the pharmaceutical composition is a sterile solution. Thus, an example of the present invention is a sterile solution comprising a compound of formula (I) (and a compound of formula (IA)) described herein.

[0018] The present invention further relates to the use of a compound of formula (I) (and a compound of formula (IA)) as a contrast agent. Thus, examples of the present invention are the use of a compound of formula (I) (and a compound of formula (IA)) described herein for imaging a tissue, cell, or mammal in vitro or in vivo, or a method for imaging a tissue, cell, or mammal in vitro or in vivo. In particular, the present invention relates to a compound of formula (I) (and a compound of formula (IA)) described herein for use as a contrast agent for imaging a tissue, cell, or mammal in vitro, ex vivo, or in vivo. The present invention further relates to a composition comprising a compound of formula (I) (and a compound of formula (IA)) for use as a contrast agent for imaging a tissue, cell, or mammal in vitro, ex vivo, or in vivo.

[0019] The present invention also relates to a method for imaging a tissue, cell, or mammal, comprising contacting, providing, or administering to the tissue, cell, or mammal a detectable amount of a labeled compound of formula (I) (and a compound of formula (IA)) described herein and detecting the compound of formula (I) (and a compound of formula (IA)).

[0020] A further example of the present invention is a method for imaging a tissue, cell, or mammal, comprising contacting, providing, or administering a compound of formula (I) (and a compound of formula (IA)) described herein to the tissue, cell, or mammal and imaging the tissue, cell, or mammal with a positron emission tomography system.

[0021] The present invention also relates to a labeled compound of formula (I) (and a compound of formula (IA)) for use in a diagnostic method performed on the body of a human or animal. In one embodiment, the diagnostic method further comprises imaging a tissue, cell, or mammal, and the method comprises contacting, providing, or administering to the tissue, cell, or mammal a detectable amount of a as described herein, and detecting the compound of formula (I) (and the compound of formula (IA)). In one embodiment, the diagnostic method further comprises imaging a tissue, cell or mammal, and the method comprises contacting, providing, or administering to the tissue, cell, or mammal the compound of formula (I) (and the compound of formula (IA)) as described herein, and imaging the tissue, cell, or mammal with a positron emission tomography system. BRIEF DESCRIPTION OF THE DRAWINGS

[0022]

Figure 1

Figure 2

Figure 3

Figure 4

[0023] The present invention is directed to compounds of formula (I) (and compounds of formula (IA)) as already defined herein, and pharmaceutically acceptable salts thereof. The present invention is also directed to precursor compounds of formula (IB) used in the synthesis of compounds of formula (IA).

[0024] In one embodiment of the present invention, formula (I)

[0025] CHEMICAL STRUCTURE a compound of, or a pharmaceutically acceptable salt, isotope, or solvate thereof.

[0026] In one embodiment of the present invention, formula (IA)

[0027] CHEMICAL STRUCTURE a compound of, or a pharmaceutically acceptable salt or solvate thereof.

[0028] In a further embodiment, the aforementioned compound of formula (I) is (S)-(2-Chloro-6-fluorophenyl)(3-(3,5-difluorophenyl)-2,7-dimethyl-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridin-6-yl)methanone, and (S)-(2-Chloro-6-( 18 F)fluorophenyl)(3-(3,5-difluorophenyl)-2,7-dimethyl-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridin-6-yl)methanone, and is selected from the group consisting of its pharmaceutically acceptable salts.

[0029] As already described, the compounds of formula (I) (and the compounds of formula (IA)) and the compositions containing the compounds of formula (I) (and the compounds of formula (IA)) can be used to image tissues, cells, or hosts, in vitro or in vivo. In particular, the present invention relates to a method for imaging or quantifying the expression, distribution, and occupancy by its inhibitors of MGL in tissues, cells, or hosts, in vitro or in vivo. Such cells and tissues are preferably central nervous system cells and tissues rich in MGL enzyme.

[0030] When the method is carried out in vivo, the host is a mammal. In such a particular case, the compound of formula (IA) is administered intravenously, for example, by injection using a syringe or by means of a peripheral venous line such as a short catheter.

[0031] When the host is a human, the compound of formula (IA) or a sterile solution containing the compound of formula (IA) can be administered, in particular, by administration into any distinguishable vein of the arm, especially the veins on the back of the hand, or the median cubital vein at the elbow.

[0032] Accordingly, in certain embodiments, the present invention relates to a method for imaging mammalian tissues or cells, comprising intravenous administration to a mammal of a compound of formula (IA) or a composition containing the compound of formula (IA) as defined herein, and imaging tissues or cells with a positron emission tomography imaging system.

[0033] Thus, in a further specific embodiment, the present invention relates to a method for imaging human tissue or cells, comprising intravenous administration to a human of a compound of formula (IA) as defined herein or a sterile formulation comprising a compound of formula (IA), and imaging tissue or cells with a positron emission tomography system.

[0034] In a further embodiment, the present invention relates to a method for imaging or quantifying the expression of MGL in a mammal, comprising intravenous administration to a mammal of a compound of formula (IA) or a composition comprising a compound of formula (IA), and imaging with a positron emission tomography imaging system.

[0035] In another embodiment, the present invention relates to the use of a compound of formula (IA) for imaging a tissue, cell, or host in vitro or in vivo, or the present invention relates to a compound of formula (IA) for use in imaging a tissue, cell, or host in vitro or in vivo using positron emission tomography.

[0036] In another embodiment, the present invention relates to a compound of formula (IA) for use in a diagnostic method for imaging a tissue, cell, or host in vitro or in vivo. In another embodiment, the present invention relates to a compound of formula (IA) for use in a diagnostic method for imaging a tissue, cell, or host in vitro or in vivo using positron emission tomography.

[0037] Definitions As used herein, the term "composition" is intended to encompass a product containing specific components in specific amounts, as well as any product resulting directly or indirectly from the combination of specific amounts of the specific components.

[0038] The addition salts of the compounds described by formula (I) and the compounds of formula (IA) can also form stereoisomeric forms and are intended to be encompassed within the scope of the present invention.

[0039] The term "MGL inhibitor containing a positron emission tomography ("PET") tracer radionuclide" means that one or more atoms of the MGL inhibitor are replaced with a PET tracer radionuclide. In some embodiments, the fluorine atoms of the MGL inhibitor are replaced by 18 F. In some embodiments, the carbon atoms of the MGL inhibitor are replaced by 11 C. In some embodiments, the nitrogen atoms of the MGL inhibitor are replaced by 13 N. In some embodiments, the nitrogen atoms of the MGL inhibitor are replaced by 15 O.

[0040] "Pharmaceutically acceptable salts" are intended to mean salts of the acids or bases of the compounds represented by formula (I) (and formula (IA)) that are either non-toxic, biologically acceptable, or biologically suitable for administration to a patient. Generally, see S.M. Berge, et al., "Pharmaceutical Salts", J Pharm Sci., 1977, 66:1-19, and Handbook of Pharmaceutical Salts, Properties, Selection, and Use, Stahl and Wermuth, Eds., Wiley-VCH and VHCA, Zurich, 2002. Preferred pharmaceutically acceptable salts are those that are pharmacologically effective and suitable for contact with the tissues of a patient without undue toxicity, irritation, or allergic response.

[0041] The compounds of formula (I) (and formula (IA)) have sufficiently acidic groups, sufficiently basic groups, or both types of functional groups, and can thus react with many inorganic bases or organic bases, as well as inorganic acids and organic acids, to form pharmaceutically acceptable salts.

[0042] Examples of pharmaceutically acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, γ-hydroxybutyrate, glycolate, tartrate, methane-sulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, and mandelate.

[0043] Since the compounds of formula (I) (and formula (IA)) may contain at least one nitrogen having basic characteristics, the desired pharmaceutically acceptable salts can be prepared by any suitable method available in the art, for example, with inorganic acids (such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, nitric acid, boric acid, phosphoric acid, etc.), or organic acids (such as acetic acid, phenylacetic acid, propionic acid, stearic acid, lactic acid, ascorbic acid, maleic acid, hydroxymaleic acid, isethionic acid, succinic acid, valeric acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, oleic acid, palmitic acid, lauric acid, pyranosidyl acids (such as glucuronic acid or galacturonic acid), α-hydroxy acids (such as mandelic acid, citric acid, or tartaric acid), amino acids (such as aspartic acid or glutamic acid), aromatic acids (such as benzoic acid, 2-acetoxybenzoic acid, naphthoic acid, or cinnamic acid), sulfonic acids (such as laurylsulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid)), any suitable mixture of acids such as those given as examples herein, and any other acids and mixtures thereof considered as equivalents, by treating the free base.

[0044] The compounds of formula (I) (and formula (IA)) may contain a carboxylic acid moiety, and the desired pharmaceutically acceptable salts can be prepared by any suitable method, for example, treating the free acid with an inorganic or organic base, such as an amine (primary, secondary, tertiary), an alkali metal hydroxide, an alkaline earth metal hydroxide, any suitable mixture of bases as exemplified herein, and other bases and mixtures thereof that are regarded as equivalents or acceptable alternatives in light of the ordinary level of skill in the art. Exemplary examples of suitable salts include amino acids such as glycine and arginine, ammonia, carbonates, bicarbonates, primary, secondary, and tertiary amines, and organic salts derived from cyclic amines such as benzylamine, pyrrolidine, piperidine, morpholine, piperazine, N-methyl-glucamine, and tromethamine, and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum, and lithium.

[0045] Whether alone or in combination, the compounds of the invention comprising the pharmaceutically acceptable salts of the invention (collectively "active agent(s)") are useful as MGL regulators in the methods of the invention. Such methods of modulating MGL include the use of a therapeutically effective amount of at least one chemical substance of the invention.

[0046] In addition, some of the compounds of the invention may form solvates (i.e., hydrates) with water or with common organic solvents, and such solvates are also intended to be encompassed within the scope of the invention.

[0047] The term "host" refers to mammals, particularly humans, mice, dogs, and rats.

[0048] The term "cell" refers to cells that express or incorporate the MGL enzyme.

[0049] The term "tissue" refers to tissues that express or incorporate the MGL enzyme.

[0050] In addition, any formula given herein is intended to represent both the unlabeled form and the isotopically labeled form of the compound. Isotopically labeled compounds have the structures described by the formulas given herein, except that one or more atoms are replaced with atoms having a selected atomic mass or mass number in enriched form. Examples of isotopes that can be incorporated into the compounds of the present invention in forms that exceed natural abundance ratios are, respectively, 2 H (or the chemical symbol D), 3 H (or the chemical symbol T), 11 C, 13 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F, 36 Cl, and 125 isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as I. Such isotopically labeled compounds are useful in metabolic studies (preferably using 14 C), kinetic studies (e.g., using 2 H or 3 H), detection or imaging techniques [such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT)] (including drug or substrate tissue distribution assays), or radiotherapy of patients. In particular, 18 F or 11 C-labeled compounds may be particularly preferred for PET or SPECT examinations. Further, heavier isotopes, such as deuterium (i.e., 2Substitution with, for example, H, or D, etc. can result in higher metabolic stability, such as a longer in vivo half-life or a lower required dosage, thereby obtaining certain therapeutic advantages. The isotope-labeled compounds of the present invention can generally be prepared by using readily available isotope-labeled reagents in place of non-isotope-labeled reagents and implementing the procedures disclosed in the schemes, examples, and preparation methods described below.

[0051] The names of the compounds of the present invention are generated according to the nomenclature agreed upon by the Chemical Abstracts Service (CAS) using software manufactured by Advanced Chemical Development, Inc. (ACD / Name product version 10.01, build 15494, 1 Dec 2006).

[0052] Next, exemplary compounds useful in the methods of the present invention will be described by referring to the following exemplary synthetic schemes for their general preparation and the specific examples following them. Those skilled in the art will understand that in order to obtain the various compounds herein, the starting materials can be suitably selected such that the ultimately desired substituents are retained through the reaction scheme, with or without appropriate protection, so as to obtain the desired product. Alternatively, it may be necessary or desirable to use suitable groups that are retained through the reaction scheme and can be appropriately substituted with the ultimately desired substituents. The reaction can be carried out between the melting point and the reflux temperature of the solvent, preferably between 0 °C and the reflux temperature of the solvent. The reaction may also be carried out under heating using conventional heating or microwave heating. The reaction may also be carried out in a sealed pressure vessel at a temperature higher than the normal reflux temperature of the solvent.

[0053] The abbreviations and acronyms used herein are as follows.

[0054]

Table 1

[0055] Preparation Example Next, exemplary compounds useful in the method of the present invention will be described by reference to the following exemplary synthetic schemes for their general preparation and the following specific examples.

[0056] [Chemical formula]

[0057] According to Scheme 1, the ketoester compound of formula (III) wherein PG is a suitable protecting group such as BOC (tert-butyloxycarbonyl) is prepared from a commercially available or synthetically available compound of formula (II). For example, a compound of formula (II) wherein PG is BOC is treated with a strong base such as lithium bis(trimethylsilyl)amide (LHMDS) at -78 °C for 30 minutes in a suitable solvent such as tetrahydrofuran (THF), followed by treatment with ethyl cyanoformate at -78 °C for 2 hours to be converted to compound (III).

[0058] The compound of formula (III) is reacted with commercially available or synthetically accessible methylhydrazine in a suitable solvent such as toluene at a temperature of about 110 °C to obtain the pyrazolone compound of formula (IV). The formation of derivatives of the compound of formula (VI) having a sulfonic acid-based leaving group such as trifluoromethanesulfonyl (triflate) is achieved by reacting with a triflating agent such as trifluoromethanesulfonic anhydride (Tf2O) and a base such as triethylamine (TEA), pyridine, N-ethyldiisopropylamine (N-ethyldiisopropylamine, DIEA, DIPEA) in a suitable solvent such as DCM. A more gentle triflating agent such as N-phenylbis(trifluoromethanesulfonimide) (TF2MPh) and a base such as TEA, DIEA are used in a suitable solvent such as DCM for better selectivity to obtain the compound of formula (V). The compound of formula (VI) is obtained by chiral separation of the mixture of the compound of formula (V) using methods known to those skilled in the art.

[0059]

Chemical formula

[0060] According to Scheme 2, the compound of formula (VI) is coupled in a metal-mediated cross-coupling reaction with a boronic acid such as (3,5-difluorophenyl)boronic acid and a palladium catalyst such as [1,1'-bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) (PdCl2(dtbpf)), tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (PdCl2(dppf)), palladium(II) bis(triphenylphosphine) dichloride (Pd(PPh3)2Cl2), XPhos-Pd-G2 precatalyst (chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II)) in the presence of a base such as K3PO4, aqueous Na2CO3 solution, Na2CO3, Cs2CO3 in a suitable solvent such as 1,2-dimethoxyethane, 1,4-dioxane, DMF, water, or a mixture thereof at a temperature in the range of 60 to 180 °C using microwave or conventional heating methods for about 30 minutes to 16 hours.

[0061] Cleavage of the BOC protecting group is achieved according to procedures known to those skilled in the art using established methodologies such as those described in T.W. Greene and P.G.M. Wuts, "Protective Groups in Organic Synthesis", 3rd ed., John Wiley & Sons, 1999. For example, the compound of formula (VII) is obtained under acidic conditions such as TFA / CH2Cl2, HCl / dioxane.

[0062] The compound of formula (I) is prepared by conventional amide bond formation techniques such as coupling reactions well known to those skilled in the art (HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate), BOP (benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate), or conversion to the acid chloride of the acid, etc.).For example, in the reaction of a compound of formula (VII) with commercially available or synthetically accessible 2-chloro-6-fluorobenzoic acid, this acid is activated in the presence of a suitable activating reagent, for example, optionally in the presence of a catalyst such as hydroxybenzotriazole (HOBt) and / or 4-dimethylaminopyridine (DMAP), with N,N'-dicyclohexylcarbodiimide (DCC) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, EDAC, or EDCI), etc. carbodiimides, (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), or a halotrisaminophosphonium salt such as bromotripyrrolidinophosphonium hexafluorophosphate (PyBroP®), a suitable pyridinium salt such as 2-chloro-1-methylpyridinium chloride, or N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide (T3P®), etc. in the presence of other suitable coupling agents.The coupling reaction is carried out in a suitable solvent such as DCM, THF, DMF, etc., optionally in the presence of a tertiary amine such as N-methylmorpholine, N-ethyldiisopropylamine (DIEA, DIPEA), or triethylamine (TEA), at a temperature in the range of about 0 °C to room temperature to obtain the compound of formula (I).

[0063] 2-Chloro-6-iodobenzoic acid is converted to 2-chloro-6-iodobenzoyl chloride by a reaction using oxalyl chloride and a catalytic amount of dimethylformamide (DMF) in a suitable solvent such as DMC. The compound of formula (VIII) is prepared by a conventional amide bond formation technique such as a coupling reaction at room temperature for 12 to 24 hours in a suitable solvent such as dichloromethane (DCM) between 2-chloro-6-iodobenzoyl chloride and a suitable base such as triethylamine (TEA).

[0064] [Chemical formula]

[0065] According to Scheme 23, tert-butyl (S)-(1-oxopropan-2-yl)carbamate and methylhydrazine are condensed in a suitable solvent such as THF to obtain tert-butyl (S,E)-(1-(2-methylhydrazinylidene)propan-2-yl)carbamate. 3,5-Difluorobenzaldehyde is treated with 2-(2-nitroethyl)-1,3-dioxolane at a temperature of 110 °C in a suitable solvent such as toluene in the presence of a catalytic amount of a suitable base such as piperidine to obtain (E)-2-(3-(3,5-difluorophenyl)-2-nitroallyl)-1,3-dioxolane.

[0066] (S)-N-(1-(4-((1,3-dioxolan-2-yl)methyl)-5-(3,5-difluorophenyl)-1-methyl-1H-pyrazol-3-yl)ethyl)-1-(11-methyl)-1-(11-oxidanyl)boranamine is prepared by [3+2] cycloaddition of tert-butyl (S,E)-(1-(2-methylhydrazinylidene)propan-2-yl)carbamate and (E)-2-(3-(3,5-difluorophenyl)-2-nitroallyl)-1,3-dioxolane at a temperature of 40 °C. Subsequently, the compound of formula (VII) is obtained by complete deprotection and cyclization by treatment at 55 °C with trifluoroacetic acid and triethylsilane. When a racemic mixture is obtained, a single enantiomer can be used by chiral SFC purification.

[0067] [Chemical formula]

[0068] According to Scheme 4, the iodoarene compound of formula (VIII) is oxidized with m-chloroperoxybenzoic acid (m-CPBA) in a suitable solvent such as DCM in the presence of a Brønsted acid such as tosylic acid or trifluoromethanesulfonic acid, preferably trifluoromethanesulfonic acid, and electron-rich anisole as an orienting group to obtain the diaryliodonium salt compound of formula (IB).

[0069] The labeling reaction is carried out in a solvent such as acetonitrile (CH3CN), water (H2O), N,N-dimethylformamide (DMF), or dimethyl sulfoxide (DMSO), or a mixture thereof, with cryptand 2.2.2. / potassium carbonate (K 2.2.2.It is carried out in the presence of a phase transfer catalyst such as / K2CO3). Since diaryliodonium salt precursors are unstable due to their own radical generation under heating or alkaline conditions, the reaction was carried out in the presence of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), a radical scavenger. In a preferred method, the best results are obtained when the compound of formula (IB) is cryptofix 2.2.2. / potassium carbonate (K 2.2.2. / K2CO3) in acetonitrile (ACN) in the presence of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO).

[0070] Using methods known to those skilled in the art, the compounds of formula (I) (and the compounds of formula (IA)) can be converted to their corresponding salts. For example, the amines of formula (I) (and the compounds of formula (II)) are treated with trifluoroacetic acid, HCl, or citric acid in a solvent such as diethyl ether (Et2O), CH2Cl2, THF, methanol, chloroform, or isopropanol to obtain the corresponding salt form.

[0071] Alternatively, trifluoroacetate or formate salts are obtained as a result of reverse phase HPLC purification conditions. The crystalline forms of the pharmaceutically acceptable salts of the compounds of formula (I) (and the compounds of formula (IA)) can be obtained in crystalline form by recrystallization from polar solvents (including mixtures of polar solvents and aqueous mixtures of polar solvents) or from non-polar solvents (including mixtures of non-polar solvents).

[0072] If the compounds described in the present invention have at least one chiral center, as a result, they may exist as enantiomers. If the compounds have two or more chiral centers, they may additionally exist as diastereomers. It is understood that all such isomers and mixtures thereof are included within the scope of the present invention.

[0073] Compounds prepared according to the above schemes can be obtained as a single form, such as a single enantiomer, by morphological specific synthesis or by resolution. Alternatively, compounds prepared according to the above schemes may be obtained as mixtures in various forms, such as a racemic (1:1) mixture or a non-racemic (not 1:1) mixture. When racemic and non-racemic mixtures of enantiomers are obtained, conventional isolation methods known to those skilled in the art, such as chiral chromatography, recrystallization, diastereomer salt formation, derivatization to diastereomeric adducts, in vivo conversion, or enzymatic conversion, etc., can be used to isolate a single enantiomer. When a mixture of positional isomers or a mixture of diastereomers is obtained, if applicable, a single isomer may be separated using conventional methods, such as chromatography or crystallization, etc.

[0074] The following specific examples are provided to further illustrate the present invention and various preferred embodiments.

Example

[0075] When obtaining the compounds and corresponding analytical data described in the following examples, unless otherwise indicated, the following experimental and analytical protocols were followed.

[0076] Unless otherwise specified, the reaction mixture was magnetically stirred at room temperature (rt) under a nitrogen atmosphere. When the solution was "dried", it was generally dried with a desiccant such as Na2SO4 or MgSO4. When the mixture, solution, and extract were "concentrated", they were typically concentrated on a rotary evaporator under reduced pressure. Reactions under microwave irradiation conditions were carried out on a Biotage Initiator or a CEM (Microwave Reactor) Discover instrument.

[0077] Preparative reverse-phase high-performance liquid chromatography (RP-HPLC) was used to purify radioisotope-labeled compounds using the following method. Built-in HPLC equipped with a Waters Xbridge C18 column (5um, 10×250mm), attached to a Synthra RNPlus module (Synthra GmbH, Germany), mobile phase of 10 mM NH4OAc and MeCN (50:50 v / v), flow rate of 4 mL / min, UV detection at 254 nm, built-in radioactive gamma-ray detector.

[0078] Analytical RP-HPLC was used for the quality control of radioisotope-labeled compounds. The following method was used. Agilent Eclipse XDB-C 18 With a Synthra (Synthra GmbH, Germany) HPLC equipped with a column (5μm, 4.6mm×150mm), using a mobile phase of 5% ACN in water (added with 0.05% TFA) for 1 minute, then a gradient of 5 - 95% CAN over 10 minutes, then 95% CAN over 4 minutes, and held at a flow rate of 1 mL / min. UV detection was carried out at 254 nm. The built-in gamma-ray detector was used for radiation measurement.

[0079] Mass spectra (MS) were obtained on an Agilent series 1260 Infinity system. Electrospray ionization (ESI) was carried out in the positive mode unless otherwise specified. The calculated mass (calcd.) corresponds to the exact mass.

[0080] Nuclear magnetic resonance (NMR) spectra were obtained on a Bruker DRX spectrometer. The definitions of multiplicities are as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad. For compounds containing exchangeable protons, it will be understood that such protons may or may not be visible in the NMR spectrum depending on the choice of solvent used for the NMR spectrum and the concentration of the compound in solution.

[0081] The chemical name was generated using ChemDraw Ultra 12.0, ChemDraw Ultra 14.0 (CambridgeSoft Corp., Cambridge, MA), or ACD / Name Version 10.01 (Advanced Chemistry).

[0082] Intermediate 1: (S)-3-(3,5-Difluorophenyl)-2,7-dimethyl-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine hydrochloride

[0083] [Chemical formula]

[0084] Method A: Step A: 2-Methyl-3-oxopiperidine-1,4-dicarboxylic acid = 1-(tert-butyl) = 4-ethyl. To a cooled (-78 °C) solution of tert-butyl 2-methyl-3-oxopiperidine-1-carboxylate (5 g, 23.4 mmol) in THF (35 mL) was added dropwise lithium bis(trimethylsilyl)amide (1.0 M in THF, 28.1 mL, 28.1 mmol) over 10 minutes. After stirring was maintained at -78 °C for 30 minutes, a solution of ethyl cyanoformate (3.0 mL, 30.4 mmol) in THF (15 mL) was added dropwise over 10 minutes at -78 °C. After addition, the reaction mixture was stirred at the same temperature (-78 °C) for 2 hours. The reaction mixture was quenched with saturated aqueous NH4Cl and extracted with ethyl acetate (EtOAc) (100 mL × 2). The combined organic extracts were dried over Na2SO4 and concentrated under vacuum. The resulting residue was purified by flash chromatography (silica gel, 0 - 30% EtOAc - hexane) to give the title compound as an oil (3.5 g, 52% yield). 1 1H NMR (500 MHz, chloroform-d): δ: 4.26 - 4.10 (m, 2H), 2.79 (s, 1H), 2.34 - 2.14 (m, 2H), 1.47 (d, J = 27.0 Hz, 2H), 1.40 (s, 9H), 1.36 (s, 1H), 1.29 (d, J = 6.9 Hz, 3H), 1.23 (t, J = 7.1 Hz, 3H).

[0085] Step B: tert-Butyl 2,7-dimethyl-3-oxo-1,2,3,4,5,7-hexahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylate. Methylhydrazine (0.96 mL, 18.1 mmol) was added to a solution of 2-methyl-3-oxopiperidine-1,4-dicarboxylic acid 1-(tert-butyl) 4-ethyl (3.5 g, 12.1 mmol) in toluene (40.0 mL), and the resulting mixture was heated at 110 °C for 3 h. After cooling to room temperature, the solvent was concentrated under vacuum. The crude residue was purified by flash chromatography (silica gel: 0–10% MeOH-DCM) to afford the title compound as an oil (2.7 g, 83% yield).

[0086] Step C: tert-Butyl 2,7-dimethyl-3-(((trifluoromethyl)sulfonyl)oxy)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylate. N,N-Diisopropylethylamine (DIEA) (1.9 mL, 11.1 mmol) was added to a solution of tert-butyl 2,7-dimethyl-3-oxo-1,2,3,4,5,7-hexahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylate (2.7 g, 10.1 mmol) in DCM (45.0 mL) at room temperature. Subsequently, 1,1,1-trifluoro-N-phenyl-N-((trifluoromethyl)sulfonyl)methanesulfonamide (4.0 g, 11.1 mmol) was added. The reaction mixture was stirred at room temperature for 5 h. The solvent was removed under vacuum. The crude residue was purified by flash chromatography (silica gel, 0–20% EtOAc-hexane) to afford Title compound A thing it as an oil (3.8 g, 86% yield).

[0087] The resulting racemic mixture was further purified via chiral SFC to obtain the desired enantiomer ((S)-tert-butyl 2,7-dimethyl-3-(((trifluoromethyl)sulfonyl)oxy)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylate) (1.84 g, yield 46%). The procedure for chiral separation is described in the following section. 1 H NMR (500 MHz, chloroform-d) δ: 5.23 (s, 1H), 4.25 (s, 1H), 3.70 (s, 3H), 2.85 (s, 1H), 2.47 (dtd, J = 30.7, 15.4, 4.0 Hz, 2H), 1.41 (s, 9H), 1.34 (d, J = 6.8 Hz, 3H).

[0088] Step D: (S)-3-(3,5-Difluorophenyl)-2,7-dimethyl-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine hydrochloride. (3,5-Difluorophenyl)boronic acid (256 mg, 1.62 mmol), Xphos-Pd-G2 (106 mg, 0.135 mmol), sodium carbonate (1 M aqueous solution, 4 mL, 4 mmol) and 1,4-dioxane (5 mL) were added to a solution of (S)-tert-butyl 2,7-dimethyl-3-(((trifluoromethyl)sulfonyl)oxy)-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylate (540 mg, 1.35 mmol). The mixture was degassed with nitrogen for 5 minutes, sealed and heated to 110 °C for 1 hour with stirring. The heating was stopped. The mixture was stirred overnight at room temperature. The mixture was diluted with EtOAc (20 mL) and washed with brine (30 mL). The organic layer was dried and concentrated. The residue was purified by combiflash silica gel column using EtOAc in hexane (gradient 10% - 60%) to obtain an oily product (480 mg, yield 98%). MS (ESI): mass calculated for C 19 H 23 F2N3O2, 363.2, m / z found, 364.1 [M+H] +。A suspension of the product (480 mg, 1.3 mmol) from the first step in DCM (8.5 mL) was treated with hydrochloric acid (4 M, 1,4-dioxane, 3.3 mL, 13.2 mmol) at room temperature overnight. All solvents were removed under vacuum. The residue was an off-white solid (404 mg, 102% yield) MS (ESI): mass calculated, C 14 H 16 ClF2N3, 299.1, m / z found, 264.1 [M - HCl + H] + 。 1 H NMR (DMSO-d6): δ 9.74 (s, 1H), 9.17 (br, 1H), 7.41 (m, 1H), 7.29 - 7.25 (m, 2H), 4.53 (s, 1H), 3.84 (s, 3H), 3.70 (m, 1H), 3.20 (s, 1H), 2.88 - 2.77 (m, 2H), 1.58 (d, J = 4.0 Hz, 3H).

[0089] Method B: Step A: (E)-2-(3-(3,5-difluorophenyl)-2-nitroallyl)-1,3-dioxolane. 2-(2-Nitroethyl)-1,3-dioxolane (20.26 g, 137.72 mmol), 3,5-difluorobenzaldehyde (19.57 g, 137.72 mmol), and the catalyst piperidine (2 mL, 20.25 mmol) were diluted with toluene (150 mL). Heated and refluxed overnight. The reaction mixture was cooled to room temperature and then quenched with saturated NaCl solution (150 mL). The extracted organic layer was dried over Na2SO4, filtered, and concentrated to a dark-colored oil to recover a quantitative crude yield of the title compound. The compound was used in the next step without further purification.

[0090] Step B: (S,E)-(1-(2-Methylhydrazinylidene)propan-2-yl)carbamic acid tert-butyl. A solution of methylhydrazine (3.04 mL, 57.73 mmol) and (S)-(1-oxopropan-2-yl)carbamic acid tert-butyl (10 g, 57.73 mmol) in THF (150 mL) was stirred at room temperature for 4 h. The reaction mixture was dried over Na2SO4, filtered, and evaporated overnight under reduced pressure. The title compound was isolated as a light-colored oil in quantitative crude yield. The crude product was used in the next reaction without further purification.

[0091] Step C: (S)-(1-(4-((1,3-Dioxolan-2-yl)methyl)-5-(3,5-difluorophenyl)-1-methyl-1H-pyrazol-3-yl)ethyl)carbamic acid tert-butyl. To a solution of (S,E)-(1-(2-methylhydrazinylidene)propan-2-yl)carbamic acid tert-butyl (11.62 g, 57.73 mmol) in EtOH (500 mL) was added (E)-2-(3-(3,5-difluorophenyl)-2-nitroallyl)-1,3-dioxolane (16.27 g, 59.98 mmol). The reaction was stirred overnight at room temperature under open air. The reaction mixture was gently heated to 40 °C overnight to complete the reaction. The reaction was concentrated to an oil, then quenched with EtOAc (250 mL) and NaCl solution (250 mL). The extracted organic layer was washed with water, then dried over Na2SO4, filtered, and concentrated to a dark orange oil. Purification (FCC, SiO2, 7 / 3 hexane / EtOAc) gave the title compound (13.32 g, 54.5%).

[0092] Project D: (S)-3-(3,5-difluorophenyl)-2,7-dimethyl-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine. A solution of tert-butyl (S)-(1-(4-((1,3-dioxolan-2-yl)methyl)-5-(3,5-difluorophenyl)-1-methyl-1H-pyrazol-3-yl)ethyl)carbamate (4 g, 9.446 mmol) in CH2Cl2 (30 mL), TFA (8 mL, 104.54 mmol) and triethylsilane (23 mL, 144.0 mmol) was stirred for 30 minutes and heated at 55 °C overnight. The reaction mixture was concentrated to an oil, then quenched with EtOAc and 1N NaOH to pH 11 - 12. The extracted organic layer was dried over Na2SO4, filtered and evaporated to a pale brown oil. The crude product was diluted with EtOH and 1.1 equivalents of 1N HCl (10 mL, 10 mmol) was added. The mixture was stirred over the weekend without forming the HCl salt. The mixture was concentrated to a pale brown solid, then slurried overnight in a minimum amount of 9 / 1 CH3CN / TBME. The solid was filtered to recover the HCl salt of the title compound (1.92 g, 68%).

[0093] Example 1: (S)-(2-chloro-6-fluorophenyl)(3-(3,5-difluorophenyl)-2,7-dimethyl-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridin-6-yl)methanone.

[0094] [Chemical Structure]

[0095] 2-Chloro-6-fluorobenzoic acid (20 mg, 0.12 mmol) and (S)-3-(3,5-difluorophenyl)-2,7-dimethyl-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine hydrochloride (Intermediate 1, 20 mg, 0.076 mmol) were mixed in a solution of HATU (43.6 mg, 0.12 mmol) in DMF (0.6 mL). Triethylamine (32 μL, 0.23 mmol) was added dropwise to the mixture. The solution was stirred at room temperature for 0.5 h. The mixture was diluted with EtOAc (100 mL) and washed with NaHCO3 (aqueous solution, 15 mL) and brine (15 mL). The organic layer was dried, filtered, and concentrated under reduced pressure. Purification (FCC, SiO2, EtOAc in hexane, 10% - 70%) gave the title compound as a thin film-like oil (30 mg, 94%). MS (ESI): mass calculated, C 21 H 17 ClF3N3O, 419.1, m / z found, 420.1 [M + H] + 。 1 H NMR (CDCl3): δ 7.34 - 7.18 (m, 2H), 7.12 - 6.98 (m, 1H), 6.90 - 6.84 (m, 3H), 5.95 - 5.91 and 5.01 - 4.98 (m, 1H), 4.76 - 4.69 and 3.57 - 3.50 (m, 1H), 3.86 and 3.80 (s, 3H), 3.39 - 3.30 and 3.11 - 3.03 (m, 1H), 2.86 - 2.73 and 2.69 - 2.63 (m, 1H), 2.53 - 2.49 and 2.39 - 2.35 (m, 1H), 1.63 - 1.56, 1.49 and 1.45 (m, 3H).

[0096] Example 2: (S)-(3-Chloro-2-(3-(3,5-difluorophenyl)-2,7-dimethyl-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine-6-carbonyl)phenyl)(4-methoxyphenyl)iodonium trifluoromethanesulfonate.

[0097]

Chemical Structure

[0098] Process A: 2-Chloro-6-iodobenzoyl chloride. Oxalyl chloride (550 mg, 4.33 mmol) was added to a suspension of 2-chloro-6-iodobenzoic acid (687 mg, 2.43 mmol) in DCM (5 mL) at room temperature. One drop of DMF was added to the reaction mixture to accelerate the reaction. The reaction mixture was stirred at room temperature for 0.5 h and then checked. The mixture was concentrated under vacuum to give a yellow oil, which was used in the next step without further purification.

[0099] Process B: (S)-(2-Chloro-6-iodophenyl)(3-(3,5-difluorophenyl)-2,7-dimethyl-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridin-6-yl)methanone. 2-Chloro-6-iodobenzoyl chloride (from Process A) was mixed with (S)-3-(3,5-difluorophenyl)-2,7-dimethyl-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine hydrochloride (540 mg, 1.801 mmol) in DCM (10 mL) containing triethylamine (626 μL, 4.5 mmol). The reaction mixture was stirred and left at room temperature overnight. The mixture was further diluted with DCM (30 mL) and washed with brine (50 mL). The organic layer was dried, filtered, and concentrated under reduced pressure. Purification (FCC, SiO2, EtOAc in hexane, 15% - 60%) gave the title compound as a light yellow oil (586 mg, 62%). MS (ESI): calculated mass, C 21 H 17 ClF2IN3O, 527.0, measured m / z, 528.0 [M + H]+. 1 H NMR (CDCl3): δ 7.81 - 7.67 (m, 1H), 7.44 - 7.32 (m, 1H), 6.97 - 6.92 (m, 1H), 6.83 - 6.77 (m, 3H), 5.87 - 5.80 (m, 1H), 3.79 (s, 3H), 3.43 - 3.36 (m, 1H), 3.33 - 3.24 (m, 1H), 2.91 - 2.70 (m, 1H), 2.32 - 2.26 (m, 1H), 1.61 and 1.57 (d, J = 6.8 Hz, 3H).

[0100] Project C: (S)-(3-chloro-2-(3-(3,5-difluorophenyl)-2,7-dimethyl-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine-6-carbonyl)phenyl)(4-methoxyphenyl)iodonium trifluoromethanesulfonate. To a cooled solution of (S)-(2-chloro-6-iodophenyl)(3-(3,5-difluorophenyl)-2,7-dimethyl-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridin-6-yl)methanone (140 mg, 0.265 mmol) in DCM (anhydrous, 1 mL) in a sodium chloride-ice bath (-15 to -20 °C) was added trifluoromethanesulfonic acid (94 μL, 1.06 mmol) dropwise so as not to disturb the temperature. After 10 minutes, a suspension of meta-chloroperbenzoic acid (m-CPBA) (up to 77%, 148 mg, 0.663 mmol) in DCM (0.7 mL) was added slowly to the solution. The reaction mixture was stirred at -20 °C for an additional 30 minutes, then cooling was stopped and the temperature was allowed to rise to room temperature. The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was cooled in an ice bath. Water (10 μL, 0.53 mmol) was added to the reaction mixture, followed by anisole (43 mg, 0.398 mmol). The ice bath was removed and the reaction mixture was stirred at room temperature for an additional 2 h. The reaction mixture was concentrated under reduced pressure. Diethyl ether (5 mL) was added to the crude concentrated reaction mixture, the resulting mixture was sonicated and filtered. The solid crude product was dried overnight and collected as an off-yellow wax (ca. 200 mg). Purification (FCC, alumina column (purged with DCM), eluted carefully with DCM then 10% MeOH / DCM) afforded the title compound as an off-white solid. The title compound was redissolved in acetonitrile (20 mg / mL), filtered through a 0.45 μm syringe filter and the resulting solution was concentrated under reduced pressure. Diethyl ether was added to the resulting title compound to precipitate the title compound (140 mg, 67%). MS (ESI): mass calculated, C 29 H 24 ClF5IN3O5S, 783.0, m / z found, 633.9 [M-OTf] + . 11H NMR (DMSO-d6): δ 8.58 (m, 1H), 8.13 - 8.07 (m, 2H), 7.90 - 7.86 (m, 1H), 7.67 - 7.58 (m, 1H), 7.38 - 7.17 (m, 3H), 7.10 (m, 1H), 7.02 (m, 1H), 5.73 - 5.58 (m, 1H), 3.87 - 3.74 (m, 6H), 3.45 - 3.19 (m, 2H), 2.92 - 2.63 (m, 1H), 2.43 - 2.29 (m, 1H), 1.63 - 1.51 (m, 3H).

[0101] Example 3: (S)-(2-chloro-6-( 18 F)fluoro-)phenyl)(3-(3,5-difluorophenyl)-2,7-dimethyl-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridin-6-yl)methanone.

[0102]

Chemical formula

[0103] In a typical procedure, the [18F] fluoride in the shipping vial (obtained from the cyclotron facility) is transferred to and trapped on an ion exchange cartridge. Next, a solution of potassium carbonate (0.75 mg) and Kryptofix 222 (7.2 mg, 19.2 μmol) in 0.8 mL of acetonitrile / water (6 / 2, v / v) is used to elute it into the reaction vessel (RV1) of the Synthra RNPlus® module. After the solvent is evaporated under vacuum at 85 °C under a nitrogen stream, anhydrous CH3CN (0.5 mL) is added and this process is repeated, raising the temperature to 110 °C for 3.5 minutes. Next, the reaction vial is cooled to 70 °C and then a solution of (S)-(3-chloro)-2-(3-(3,5-difluorophenyl)-2,7-dimethyl-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine-6-carbonyl)phenyl)(4-methoxyphenyl)iodonium trifluoromethanesulfonate (Example 2, 15.0 mg, 19.1 μmol) and TEMPO (4.4 mg, 28.1 μmol) in anhydrous acetonitrile (0.7 mL) is added to the reaction vessel. The reaction mixture is heated at 125 °C for 10 minutes. The reactor is cooled to 40 °C, diluted with water (4.3 mL), and the contents are transferred to the HPLC injector loop for purification.

[0104] Purification was performed by HPLC using a semi-preparative Eclipse XDB-C18 column (5 μm, 9.4 mm × 250 mm), with a mixture of 10 mM NH4OAc and MeCN (50:50 v / v) at a flow rate of 4 mL / min and UV detection at 254 nm. The purified radioactive tracer solution was diluted with 30 mL of water and passed through a SepPak Light C-18 cartridge. After the C-18 cartridge was further washed with 10 mL of water, the tracer was eluted using 0.5 mL of EtOH. The tracer solution was further diluted with 4.5 mL of saline. The final formulation contains 10% ethanol, which is suitable for intravenous (i.v.) injection.

[0105] The quality control tests include identification by HPLC using an Eclipse XDB C18 (5 μm, 4.6×250 mm) column eluted with a mixture of 0.05% TFA solution and MeCN at a flow rate of 1 mL / min, with serial UV (254 nm) detection and gamma ray detection, as well as chemical and radiochemical purity tests.

[0106] Biological data The assay used to measure the in vitro activity of MGL employs the assay used for another serine hydrolase (FAAH) described in Wilson et al., 2003 (A high-throughput-compatible assay for determining the activity of fatty acid amide hydrolase. Wilson SJ, Lovenberg TW, Barbier AJ. Anal Biochem. 2003 Jul 15;318(2):270-5). This assay combines endogenously expressed MGL from HeLa cells with a test compound and 3 [glycerol-1,3- 3 H]-oleyl glycerol is added and incubated for 1 hour, and then the amount of cleaved [1,3-

[0107] H]-glycerol passing through an activated carbon filter is measured. The amount of cleaved tritium-labeled glycerol passing through the carbon filter is proportional to the activity of the MGL enzyme under specific well / test conditions. 3 Under the standard conditions of this assay, 300 nM [glycerol-1,3- 50 H]-oleyl glycerol is mixed with human MGL from HeLa cells and a test compound for 1 hour, and then the reaction mixture is filtered through activated carbon and the tritium in the passing fraction is measured. The test compound concentration in the screening mode is 10 μM, and the maximum concentration of the compound in the

[0108]

Table 2

[0109] Example 4: Comparison of autoradiography of (S)-(2-chloro-6-(18F)fluoro)phenyl)(3-(3,5-difluorophenyl)-2,7-dimethyl-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridin-6-yl)methanone (Example 3) and IHC staining of adjacent rat brain sections for MGL. In vitro ARG using the compound of Example 3 was performed on frozen normal rat brain sections (sagittal section, 20 μm thick). Fifteen minutes after removal from the freezer, 250 μL of buffer (50 mM Tris-HCl, pH = 7.4) containing the compound of Example 3 at a concentration of 400 μCi / mL was added to the rat brain sections. The brain sections were incubated with the incubation buffer at room temperature for 30 minutes and then washed with fresh blank buffer for 5 minutes. This washing was repeated three times. The brain sections were air-dried and exposed to a phosphor screen in a cassette in a dark environment for 18 hours. The screen was then scanned with a Typhoon™ FLA7000 Image Analyzer (GE) to generate autoradiography of the compound of Example 3 for the rat brain sections (Figures 2 and 4). The ARG signal was high in the hippocampus, cortex, cerebellum, and thalamus regions and low in the brainstem. The distribution pattern was consistent with the IHC staining pattern described below.

[0110] IHC staining was performed on frozen normal rat brain sections (sagittal section, 10 μm thick) adjacent to the brain sections used for the above ARG. Fifteen minutes after removal from the freezer, 4% paraformaldehyde was added to the rat brain sections and fixed at room temperature for 20 minutes. Subsequently, the sections were washed with fresh phosphate-buffered saline (PBS, 0.01 M, pH = 7.4) for 5 minutes. This washing was repeated three times. Subsequently, treatment with a hydrogen peroxide solution (3% in PBS) was performed at room temperature for 20 minutes. 10% goat serum in PBS (Sigma, catalog number G-9023) was added to the sections to block non-specific binding. The primary antibody, MGL antibody (Novus Biologicals, catalog number NBP2-19389) in PBS (1:100 dilution), was added and incubated overnight at 4 °C in a humid chamber. On the second day, the rat brain sections were washed with PBS for 10 minutes. This washing was repeated three times. The secondary antibody, goat anti-rabbit IgG (H+L) antibody (Thermo Fisher, catalog number A-11008), was added to PBS (1:500 dilution) and incubated at room temperature for 1 hour. The sections were further washed with PBS for 10 minutes. This washing was repeated three times. The sections were placed on a mounting medium and covered with a glass slide and observed under a fluorescence microscope. Photographs (Figure 1, Figure 3) were taken using a fluorescence microscope (Zeiss, AXIO, Imager M2). IHC staining showed that MGL was distributed at high concentrations in the hippocampus, cortex, and cerebellum, and this pattern was consistent with the above autoradiography signal. The invention described in the original claims of the present application is appended below. [Claim 1] The compound according to formula (I), or a pharmaceutically acceptable salt, isotope, or solvate thereof.

Chem.

Chem.

Claims

1. The compound according to formula (VIII) 【Chemical 10】 .

2. The compound according to formula (IB) 【Chemical 20】 .

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