Radiolabeled MGL PET ligand

Abstract

The present invention relates to novel selective radiolabeled compounds with monoacylglycerol lipase (MGL) affinity, which can be used to image and quantify MGL receptor expression, distribution and enzyme occupancy in tissues using positron emission tomography (PET). The present invention also relates to compositions comprising such compounds and precursors of said compounds, and the use of such compounds and compositions for in vitro or in vivo imaging of tissues, cells or hosts.

Description

Technical Field

[0001] The present invention relates to a novel selective compound having monoacylglycerol lipase (MGL) affinity and, in one embodiment, containing a positron-emitting radioligand to enable positron emission tomography (PET); a pharmaceutical composition comprising the compound for use in assessing MGL receptor expression, distribution and enzyme occupancy, and in diagnosing diseases, disorders or conditions associated with MGL receptor activity in a subject, particularly a human. Background Art

[0002] Cannabis and Δ have been used in folk medicine for therapeutic purposes since their 9 - an analogue of tetrahydrocannabinol. The endocannabinoid system consists of two G protein-coupled receptors, the type 1 cannabinoid receptor (CB1) (Matsuda et al., Nature, 1990, 346, 561-4) and the type 2 cannabinoid receptor (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 expressed peripherally in the liver, gastrointestinal tract, pancreas, adipose tissue and skeletal muscle (DiMarzo 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) and under certain conditions (inflammation) in the brain (Benito et al., Brit J Pharmacol, 2008, 153, 277-285) as well as in skeletal muscle (Cavuoto et al., Biochem Biophys Res Commun, 2007, 364, 105-110) and cardiac muscle (Hajrasouliha et al., Eur J Pharmacol, 2008, 579, 246-252).

[0003] In 1992, N-arachidonoylethanolamine (AEA or anandamide) was found to be an endogenous ligand for cannabinoid receptors (Devane et al., Science, 1992, 258, 1946-9). Subsequently, 2-arachidonoylglycerol (2-AG) was also identified as an additional endogenous ligand for cannabinoid receptors (Mechoulam et al., Biochem Pharmacol, 1995, 50, 83-90; Sugiura et al., Biochem Biophys Res Commun, 1995, 215, 89-97). It is reported that the concentration of 2-AG is at least 100 times that of anandamide in rat brain (Buczynski and Parsons, Brit J Pharmacol, 2010, 160 (3), 423-42). Therefore, compared to anandamide, 2-AG may play a more important physiological role in the brain's endocannabinoid system (Sugiura et al., Prostaglandins Leukot Essent Fatty Acids., February-March 2002, 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 these two receptors (Suguira et al., Prog Lipid Res, 2006, 45 (5): 405-46). Unlike many classical neurotransmitters, endocannabinoids signal through a retrograde mechanism. They are synthesized in postsynaptic neurons as needed and then rapidly degraded after binding to presynaptic cannabinoid receptors (Ahn et al., Chem Rev. 2008, 108 (5): 1687-707). Monoacylglycerol lipase (MGLL, also known as MAG lipase and MGL) is a serine hydrolase responsible for the degradation 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 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 US A., 2002 Aug 6, 99(16):10819-24). In the brain, MGL is localized in 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 CB1 receptor density. Genetic ablation of MGL expression caused a 10-fold increase in brain 2-AG levels compared to wild-type controls without affecting anandamide concentrations (Schlosburg et al., Nat Neurosci., 2010 Sep, 13(9):1113-9).

[0004] Therefore, MGL modulation provides an interesting strategy for enhancing the endocannabinoid system. A major advantage of this approach is that only brain regions that efficiently produce endocannabinoids will be modulated, thereby potentially minimizing the side effects associated with exogenous CB1 agonists. Pharmacological inactivation of MGL by covalent inhibitors in animals increases 2-AG levels in the brain and peripheral tissues and has been found to produce analgesic, 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., April 26, 2018, 8(1):92). In addition to the role of MGL in terminating 2-AG signaling, MGL modulation (including MGL inhibition) also promotes CB1 / 2-independent effects on neuroinflammation (Nomura et al., Science., November 11, 2011, 334(6057):809-13).MGL modulation (including MGL inhibition) results in reduced proinflammatory prostaglandin signaling in animal models of 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), neurodegenerative diseases including 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 1 ... 334(6057), 809-13; Pasquarelli et al., Neurochem Int., November 2017, 110: 14-24), amyotrophic lateral sclerosis (Pasquarelli et al., Neuropharmacology, September 15, 2017, 124: 157-169), multiple sclerosis (Hernadez-Torres et al., Angew Chem Int Ed Engl., December 8, 2014, 53(50): 13765-70; Bernal-Chico et al., Glia., January 2015, 63(1): 163-76), Huntington's disease (Covey et al., Neuropsychopharmacology, 2018, 43, 2056- 2063), Tourette syndrome and status epilepticus (Terrone et al., Epilepsy., January 2018, 59(1), 79-91; von Ruden et al., Neurobiol Dis., May 2015, 77: 238-45.

[0005] Thus, by enhancing the cannabinoid system and attenuating the pro-inflammatory cascade, MGL modulation (including MGL inhibition) offers an attractive therapeutic approach for the treatment of a wide range of complex diseases. Importantly, MGL modulation (including MGL inhibition) in animals does not produce the same effects as Δ 9 - The full range of neurobehavioral effects observed with THC 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] Low endocannabinoid activity is a risk factor for the treatment of depression, anxiety, and post-traumatic stress disorder. The thousands of years of human use of cannabis and the brief period of human treatment with the endocannabinoid antagonist rimonabant provide support for this hypothesis. 2-AG levels are reduced in individuals with major depressive disorder (Hill et al., Pharmacopsychiatry., March 2008, 41 (2): 48-53; Hill et al., Psychoneuroendocrinology., September 2009, 34 (8): 1257-1262.). Low circulating 2-AG levels predict the incidence of depression (Hauer et al., Rev Neurosci., 2012, 23 (5-6): 681-90). Circulating 2-AG has been found to be reduced in patients with post-traumatic stress disorder (PTSD) (Hill et al., Psychoneuroendocrinology, 2013, 38 (12), 2952-2961). Healthy volunteers exposed to chronic stressors showed a gradual decrease in circulating 2-AG levels, which was associated with the onset of a decrease in positive mood measures (Yi et al., Progress in Neuro-Psychopharmacology and Biological Psychiatry, 2016, 67(3), 92-97). The CB1 receptor inverse agonist / antagonist rimonabant has been recalled from the market due to high incidences of severe depression and suicidal ideation (Christensen et al., The Lancet, 2007, 370, 1706-1713). Therefore, MGL modulators are potentially useful for the treatment of mood disorders, anxiety disorders, 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):321-31). Therefore, MGL modulators (including MGL inhibitors) may also be potentially useful for these indications. MGL exerts CB1-dependent analgesic effects in animal models of noxious chemical pain, inflammatory pain, thermal 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 touch-evoked allodynia in mice subjected to chronic compressive sciatic nerve injury (Kinsey et al., J Pharmacol Exp Ther., 2009 Sep, 330(3):902-10). MGL inhibition produces tolerance, constipation, and opioid sparing events with reduced cannabinoid side effects (Wilkerson et al., J Pharmacol Exp Ther., 2016 Apr, 357(1):145-56). MGL blockade is protective in an inflammatory bowel disease model (Alhouayek et al., FASEB J., 2011 Aug, 25(8):2711-21). MGL inhibition also reverses nociceptive behaviors and proinflammatory markers induced by paclitaxel 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 produces antiproliferative activity and reduces prostate cancer cell invasiveness (Nithipatikom et al., Cancer Res., Dec 15, 2004, 64(24):8826-30; Nithipatikom et al., Biochem Biophys Res Commun., Jul 15, 2005, 332(4):1028-33; Nithipatikom et al., Prostaglandins Other Lipid Mediat., Feb 2011, 94(1-2):34-43). MGL is upregulated in aggressive human cancer cells and primary tumors, where MGL has a unique role in providing a lipolytic source of free fatty acids for the synthesis of oncogenic signaling lipids that promote cancer aggressiveness. Therefore, in addition to the physiological role of MGL in mediated endocannabinoid signaling, MGL in cancer plays a unique role in regulating the fatty acid precursor pool for the synthesis of proto-oncogene signaling lipids in malignant human cancer cells.

[0009] MGL blockade showed antiemetic and anti-nausea effects in the lithium chloride model of shrew emesis (Sticht et al., Br J Pharmacol., 2012 Apr, 165(8):2425-35).

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

[0011] MGL modulators are also potentially useful for treating ocular disorders, including but not limited to glaucoma and disease states due to elevated intraocular pressure (Miller et al., Pharmaceuticals, 2018, 11, 50).

[0012] Positron emission tomography (PET) is a non-invasive imaging technique that offers the highest spatial and temporal resolution of all nuclear imaging techniques, with the additional advantage that it can allow true quantification of tracer concentrations in tissue. It uses positron-emitting radionuclides such as e.g. 15 O. 13 N. 11 C and 18F is detected. There is a need to provide positron emission tomography radiotracers for assessing the expression, distribution and occupancy of MGL by its inhibitors. Imaging agents play a key role in such studies and in the development of therapeutic candidates targeting MGL. Summary of the invention

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

[0014]

[0015] or a pharmaceutically acceptable salt or solvate thereof. In one embodiment, the compound of formula (I) has at least one atom that is radioactive.

[0016] In one embodiment, the compound of formula (I) is a compound of formula (IA),

[0017]

[0018] or a pharmaceutically acceptable salt or solvate thereof.

[0019] 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 a specific embodiment, the pharmaceutical composition is particularly suitable for diagnosis and can therefore be referred to as a diagnostic pharmaceutical composition. In particular, the pharmaceutical composition is a sterile solution. Therefore, an example of the present invention is a sterile solution comprising a compound of formula (I) (and a compound of formula (IA)) as described herein.

[0020] The present invention also relates to the use of a compound of formula (I) (and a compound of formula (IA)) as an imaging agent. Thus, by way of example, the present invention is the use of a compound of formula (I) (and a compound of formula (IA)) as 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)) as described herein, which is used as a contrast agent for imaging a tissue, cell or mammal in vitro, ex vivo or in vivo. The present invention also relates to a composition comprising a compound of formula (I) (and a compound of formula (IA)), which is used as a contrast agent for imaging a tissue, cell or mammal in vitro, ex vivo or in vivo.

[0021] The present invention also relates to a method for imaging a tissue, a cell or a mammal, comprising contacting the tissue, the cell or the mammal with a detectable amount of a labeled compound of formula (I) (and a compound of formula (IA)) as described herein, or providing or administering a detectable amount of a labeled compound of formula (I) (and a compound of formula (IA)) as described herein to the tissue, the cell or the mammal, and detecting the compound of formula (I) (and a compound of formula (IA)).

[0022] To further illustrate, the present invention is a method for imaging a tissue, a cell or a mammal, comprising contacting the tissue, the cell or the mammal with a compound of formula (I) (and a compound of formula (IA)) as described herein, or providing or administering a compound of formula (I) (and a compound of formula (IA)) as described herein to the tissue, the cell or the mammal, and imaging the tissue, the cell or the mammal using a positron emission tomography imaging system.

[0023] 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 a human or animal body. In one embodiment, the diagnostic method further comprises imaging a tissue, cell or mammal, the method comprising contacting the tissue, cell or mammal with a detectable amount of ... as described herein or providing or applying a detectable amount of ... as described herein to the tissue, cell or mammal, and detecting the compound of formula (I) (and a compound of formula (IA)). In one embodiment, the diagnostic method further comprises imaging a tissue, cell or mammal, comprising contacting the tissue, cell or mammal with a compound of formula (I) as described herein (and a compound of formula (IA)) or providing or applying a compound of formula (I) as described herein (and a compound of formula (IA)) to the tissue, cell or mammal, and imaging the tissue, cell or mammal with a positron emission tomography imaging system. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 . Immunohistochemistry (IHC) staining of MGL on a normal rat brain section (sagittal view) in color (Monoacylglycerol lipase (MGL) IHC on a rat sagittal brain section. 1° Ab: Monoacylglycerol lipase antibody from Novus Biologicals, catalog number NBP2-19389. Host: rabbit, reactive with: mouse, rat and human. Working solution: 1:100 dilution. Alexa Fluor TM 488 Tyramide Superboost TM Reagent test kit.)

[0025] Figure 2 . Autoradiography (ARG) of the compound of Example 3 on normal rat brain sections adjacent to the colored Figure 1 (Sagittal view). Rat brain sections were: cortex, striatum, hippocampus, thalamus, cerebellum and brainstem.

[0026] Figure 3 .Immunohistochemical (IHC) staining of MGL on normal rat brain sections (sagittal view) in black and white (Monoacylglycerol lipase (MGL) IHC on rat sagittal brain sections. 1° Ab: Monoacylglycerol lipase antibody from Novus Biologicals, catalog number NBP2-19389. Host: rabbit, reactive with: mouse, rat and human. Working solution: 1:100 dilution. Alexa Fluor TM 488 Tyramide Superboost TM Reagent test kit.)

[0027] Figure 4 . Autoradiography (ARG) of the compound of Example 3 on normal rat brain slices adjacent to the black and white Figure 1 (Sagittal view). Rat brain sections were: cortex, striatum, hippocampus, thalamus, cerebellum and brainstem. DETAILED DESCRIPTION

[0028] The present invention relates to compounds of formula (I) as defined herein before (as well as compounds of formula (IA)) and pharmaceutically acceptable salts thereof. The present invention also relates to precursor compounds of formula (IB) useful for the synthesis of compounds of formula (IA).

[0029] In one embodiment of the present invention is a compound of formula (I):

[0030]

[0031] or a pharmaceutically acceptable salt, isotope or solvate thereof.

[0032] In one embodiment of the present invention is a compound of formula (IA):

[0033]

[0034] or a pharmaceutically acceptable salt or solvate thereof.

[0035] In another embodiment, the compound of formula (I) as previously described is selected from the group consisting of:

[0036] (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

[0037] (S)-(2-chloro-6-(18F)fluorophenyl)(3-(3,5-difluorophenyl)-2,7-dimethyl-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridin-6-yl)methanone;

[0038] and pharmaceutically acceptable salts thereof.

[0039] As already mentioned, compounds of formula (I) (as well as compounds of formula (IA)) and compositions comprising compounds of formula (I) (as well as compounds of formula (IA)) can be used for in vitro or in vivo imaging of tissues, cells or hosts. In particular, the present invention relates to a method for imaging or quantifying MGL expression, distribution and occupancy by its inhibitors in tissues, cells or hosts in vitro or in vivo. The cells and tissues are preferably central nervous system cells and tissues in which the MGL enzyme is abundant.

[0040] When the method is performed in vivo, the host is a mammal. In such specific cases, the compound of formula (IA) is administered intravenously, for example by injection with a syringe or via a peripheral intravenous line (such as a short catheter).

[0041] When the host is a human, the compound of formula (IA) or a sterile solution comprising the compound of formula (IA) may be administered particularly by intravenous administration in the arm, into any identifiable vein, particularly in the back of the hand, or in the median cubital vein of the elbow.

[0042] Thus, in one embodiment, the invention relates to a method of imaging tissue or cells in a mammal, comprising intravenously administering to the mammal a compound of formula (IA) or a composition comprising a compound of formula (IA) as defined herein, and imaging the tissue or cells using a positron emission tomography imaging system.

[0043] Therefore, in another specific embodiment, the present invention relates to a method for imaging tissue or cells in a human, which comprises intravenously administering to the human a compound of formula (IA) as defined herein or a sterile formulation comprising a compound of formula (IA), and imaging the tissue or cells using a positron emission tomography imaging system.

[0044] In another embodiment, the invention relates to a method for imaging or quantifying MGL expression in a mammal, comprising intravenously administering a compound of formula (IA) or a composition comprising a compound of formula (IA) to the mammal, and imaging using a positron emission tomography imaging system.

[0045] In another embodiment, the invention relates to the use of a compound of formula (IA) for in vitro or in vivo imaging of a tissue, a cell or a host, or the invention relates to a compound of formula (IA) for use in in vitro or in vivo imaging of a tissue, a cell or a host using positron emission tomography.

[0046] In another embodiment, the invention relates to a compound of formula (IA) for use in a diagnostic method for in vitro or in vivo imaging of a tissue, cell or host. In another embodiment, the invention relates to a compound of formula (IA) for use in a diagnostic method for in vitro or in vivo imaging of a tissue, cell or host using positron emission tomography.

[0047] definition

[0048] As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

[0049] The addition salts of the compounds according to formula (I) and compounds of formula (IA) may also form stereoisomeric forms and are also intended to be encompassed within the scope of the present invention.

[0050] 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 with 18 In some embodiments, the carbon atom of the MGL inhibitor is replaced by 11 In some embodiments, the nitrogen atom of the MGL inhibitor is replaced by 13 In some embodiments, the nitrogen atom of the MGL inhibitor is replaced by 15 O alternative.

[0051] "Pharmaceutically acceptable salts" are intended to mean acid or base salts of compounds represented by Formula (I) (as well as Formula (IA)) that are non-toxic, biologically tolerable, or otherwise biologically suitable for administration to a subject. See generally: SM 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 patient tissues without undue toxicity, irritation, or allergic response.

[0052] The compounds of formula (I) (and formula (IA)) may possess sufficiently acidic groups, sufficiently basic groups, or both types of functional groups, and may therefore react with a variety of inorganic or organic bases and inorganic and organic acids to form pharmaceutically acceptable salts.

[0053] Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, octanoates, acrylates, formates, isobutyrates, hexanoates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioate, hexyne-1,6-dioate, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, gamma-hydroxybutyrates, glycolates, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, and mandelates.

[0054] The compounds of formula (I) (as well as formula (IA)) may contain at least one nitrogen with basic properties, and thus the desired pharmaceutically acceptable salts may be prepared by any suitable method available in the art, for example, by treating the free base with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, aminosulfonic acid, nitric acid, boric acid, phosphoric acid, or the like; or an organic acid 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 acid) (such as glucuronic acid or galacturonic acid), alpha-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 lauryl sulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid), any compatible mixtures of acids such as those given as examples herein, and any other acids and mixtures thereof deemed equivalent.

[0055] The compounds of formula (I) (as well as formula (IA)) may contain a carboxylic acid moiety, and the desired pharmaceutically acceptable salts may be prepared by any suitable method, for example, by treating the free acid with an inorganic or organic base such as an amine (primary, secondary or tertiary), an alkali metal hydroxide, an alkaline earth metal hydroxide, any compatible mixture of bases such as those given as examples herein, and any other bases considered by one of ordinary skill in the art to be equivalent or acceptable substitutes, and mixtures thereof. Illustrative examples of suitable salts include organic salts derived from amino acids (such as glycine and arginine), ammonia, carbonates, bicarbonates, primary, secondary, tertiary amines and cyclic amines (such as benzylamine, pyrrolidine, piperidine, morpholine, piperazine, N-methylglucamine and tromethamine), and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.

[0056] Compounds of the invention, including pharmaceutically acceptable salts thereof, either alone or in combination (collectively "active agents") are useful as MGL modulators in the methods of the invention. Such methods for modulating MGL comprise administering a therapeutically effective amount of at least one chemical compound of the invention.

[0057] In addition, some of the compounds of the present invention may form solvates with water (ie, hydrates) or with common organic solvents, and such solvates are also intended to be encompassed within the scope of the present invention.

[0058] The term "host" refers to mammals, particularly humans, mice, dogs and rats.

[0059] The term "cell" refers to a cell that expresses or contains the MGL enzyme.

[0060] The term "tissue" refers to a tissue that expresses or contains the MGL enzyme.

[0061] Any formula given herein is also intended to represent unlabeled forms of the compounds as well as isotopically labeled forms. Isotopically labeled compounds have structures depicted 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 invention in excess of natural abundance include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as 2 H (or chemical symbol D), 3 H (or 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 I. Such isotope-labeled compounds can be used for metabolic studies (preferably with 14 C) reaction kinetics studies (e.g. 2 H or 3 H), detection or imaging techniques [such as positron emission tomography (PET) or single photon emission computed tomography (SPECT)], including tissue distribution assays of drugs or substrates, or radiation therapy that may be used in patients. In particular, 18 F or 11 Compounds labeled with C are particularly preferred for use in PET or SPECT studies. 2 H or D) may provide certain therapeutic advantages resulting from greater metabolic stability, such as increased half-life in vivo or reduced dosage requirements. Isotopically labeled compounds of the invention can generally be prepared by substituting readily available isotopically labeled reagents for non-isotopically labeled reagents to carry out the procedures disclosed in the "Schemes" or in the "Examples and Preparations" described below.

[0062] The names of the compounds of the invention were generated using Advanced Chemical Development, Inc. software (ACD / Name Product Version 10.01; Build 15494, December 1, 2006) according to the nomenclature conventions agreed upon by the Chemical Abstracts Service (CAS).

[0063] Exemplary compounds that can be used in the methods of the present invention will now be described with reference to exemplary synthesis schemes for their general preparation and subsequent specific examples below. Those skilled in the art will recognize that, in order to obtain a variety of compounds herein, the raw materials can be appropriately selected so that, with or without protection as required, the final desired substituent will be carried throughout the reaction scheme to obtain the desired product. Alternatively, it may be necessary or desirable to replace the final desired substituent with a suitable group that can undergo the entire reaction scheme and be replaced with the desired substituent where appropriate. The reaction can be carried out between the melting point and reflux temperature of the solvent, and preferably between 0°C and the reflux temperature of the solvent. Conventional heating or microwave heating can be used to heat the reaction. The reaction can also be carried out in a closed pressure vessel at a normal reflux temperature higher than the solvent.

[0064] Abbreviations and acronyms used herein include the following:

[0065] Table 1 :

[0066]

[0067]

[0068] Preparation Example

[0069] Exemplary compounds useful in the methods of the present invention will now be described with reference to exemplary synthetic schemes for their general preparation below and for the specific examples that follow.

[0070] Solution 1

[0071]

[0072] According to Scheme 1, the keto-ester compound of formula (III) (wherein PG is a suitable protecting group such as BOC (tert-butyloxycarbonyl)) is prepared from a commercially available or synthetically obtainable compound of formula (II). For example, the compound of formula (II) (wherein PG is BOC) is converted to compound (III) by treating with a strong base such as lithium bis(trimethylsilyl)amide (LHMDS) in a suitable solvent such as tetrahydrofuran (THF) at a temperature of about -78°C for 30 minutes, followed by treating with ethyl cyanoformate at -78°C for a period of about 2 hours.

[0073] The compound of formula (III) is reacted with commercially available or synthetically available methylhydrazine in a suitable solvent such as toluene at a temperature of about 110° C. to provide a pyrazolone compound of formula (IV). Derivatization of the pyrazolone compound of formula (IV) with a sulfonate-based leaving group such as trifluoromethanesulfonyl (trifluoromethanesulfonate) is achieved by reaction with a trifluoromethanesulfonylating agent such as trifluoromethanesulfonic anhydride (Tf2O)), a base such as triethylamine (TEA), pyridine, N-ethyldiisopropylamine (DIEA, DIPEA), etc. in a suitable solvent such as DCM, etc. Better selectivity is achieved using a milder trifluoromethanesulfonylating agent such as N-phenylbis(trifluoromethanesulfonimide) (TF2NPh), a base such as TEA, DIEA, etc. in a suitable solvent such as DCM, etc. to provide a compound of formula (V). A mixture of compounds of formula (V) is subjected to chiral separation using methods known to those skilled in the art to provide a compound of formula (VI).

[0074] Solution 2

[0075]

[0076] According to Scheme 2, the compound of formula (VI) is reacted in a metal-mediated cross-coupling reaction with a boronic acid such as (3,5-difluorophenyl)boronic acid in the presence of a palladium catalyst such as [1,1′-bis(di-tert-butylphosphino)ferrocene]palladium(II) chloride (PdCl2(dtbpf)), tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) chloride (PdCl2(dppf)), bis(triphenylphosphine)palladium(II) chloride (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)), and the like, and a base such as K3PO4, aq. In the presence of Na2CO3, Na2CO3, Cs2CO3, etc., in a suitable solvent such as 1,2-dimethoxyethane, 1,4-dimethoxyethane The coupling is carried out in oxane, DMF, water or mixtures thereof at a temperature ranging from 60°C to 180°C (using microwave or conventional heating) for a period of about 30 minutes to 16 hours.

[0077] Cleavage of the BOC protecting group is accomplished according to procedures known to those skilled in the art and using established methods such as those described in TW Greene and PGM Wuts, "Protective Groups in Organic Synthesis", 3rd edition, John Wiley & Sons, 1999. For example, under acidic conditions such as TFA / CH2Cl2, HCl / dichlorobenzene, 1999, 1990, 1991, 1993, 1994, 1995, 1996, 1998, 1999, 1990, 1993, 1994, 1995, 1996, 1997, 1998, 19 ... The compound of formula (VII) is provided.

[0078] The compounds of formula (I) are prepared by conventional amide bond forming techniques such as coupling reactions well known to those skilled in the art (such as HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridine 3-oxide hexafluorophosphate), BOP (benzotriazol-1-yloxy-tris(dimethylamino) Hexafluorophosphate), or acid conversion to an acid chloride). For example, the compound of formula (VII) is reacted with commercially available or synthetically available 2-chloro-6-fluorobenzoic acid, wherein the acid is activated with a suitable activating agent such as a carbodiimide such as N,N′-dicyclohexylcarbodiimide (DCC) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, EDAC or EDCI), optionally in the presence of hydroxybenzotriazole (HOBt) and / or a catalyst such as 4-dimethylaminopyridine (DMAP); a halogenated triamino Salts such as (benzotriazol-1-yloxy)tris(dimethylamino) Hexafluorophosphate (BOP) or tripyrrolidinyl bromide Hexafluorophosphate Suitable pyridine Salts such as 2-chloro-1-methylpyridine chloride; or another suitable coupling agent (such as 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]pyridine 3-oxide hexafluorophosphate (HATU), 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphanin-2,4,6-trioxide The coupling reaction is carried out in a suitable solvent such as DCM, THF, DMF, etc. at a temperature ranging from about 0° C. to room temperature, optionally in the presence of a tertiary amine such as N-methylmorpholine, N-ethyldiisopropylamine (DIEA, DIPEA) or triethylamine (TEA), to provide a compound of formula (I).

[0079] 2-Chloro-6-iodobenzoic acid is converted to 2-chloro-6-iodobenzoyl chloride by reaction with oxalyl chloride in a suitable solvent such as DMC and a catalytic amount of dimethylformamide (DMF). Compounds of formula (VIII) are prepared by conventional amide bond formation techniques, such as coupling reactions with 2-chloro-6-iodobenzoyl chloride, a suitable base such as triethylamine (TEA) in a suitable solvent such as dichloromethane (DCM) at room temperature for a period of 12-24 hours.

[0080] Solution 3

[0081]

[0082] According to Scheme 23, tert-butyl (S)-(1-oxopropan-2-yl)carbamate and methylhydrazine are condensed in a suitable solvent such as THF to give tert-butyl (S,E)-(1-(2-methylhydrazinylidene)propan-2-yl)carbamate. 3,5-difluorobenzaldehyde is treated with 2-(2-nitroethyl)-1,3-dioxolane in the presence of a catalytic amount of a suitable base such as piperidine in a suitable solvent such as toluene at a temperature of 110°C to provide (E)-2-(3-(3,5-difluorophenyl)-2-nitroallyl)-1,3-dioxolane.

[0083] (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-oxoalkyl)boramine is prepared by the [3+2] cycloaddition of (S,E)-tert-butyl(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. Subsequent global deprotection and cyclization by treatment with trifluoroacetic acid and triethylsilane at 55°C affords the compound of formula (VII). In the case where a racemic mixture is obtained, a single enantiomer purified by chiral SFC may be employed.

[0084] Solution 4

[0085]

[0086] According to Scheme 4, an iodoarene compound of formula (VIII) is oxidized with chloroperoxybenzoic acid (m-CPBA) in the presence of a Bronsted acid such as p-toluenesulfonic acid or trifluorosulfonic acid, preferably trifluorosulfonic acid, and an electron-rich anisole as a directing group in a suitable solvent such as DCM or the like to provide a diaryl iodide of formula (IB) Salt compounds.

[0087] 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 under a phase change catalyst such as kryptofix 2.2.2. / Potassium carbonate (K 2.2.2. / K2CO3) in the presence of diaryl iodide. The salt precursor is unstable due to its own free radical generation under heating or alkaline conditions, so it is reacted in the presence of a free radical scavenger 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO). In a preferred method, the compound of formula (IB) is reacted in the presence of Kryptofix 2.2.2. / Potassium carbonate (K 2.2.2. The best results were obtained by reacting the reaction in acetonitrile (ACN) in the presence of 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) in the presence of K2CO3).

[0088] Compounds of formula (I) (and compounds of formula (IA)) can be converted to their corresponding salts using methods known to those of ordinary skill in the art. For example, amines of formula (I) (and compounds of formula (IA)) are treated with trifluoroacetic acid, HCl or citric acid in a solvent such as diethyl ether (Et2O), CH2Cl2, THF, methanol, chloroform or isopropanol to provide the corresponding salt forms.

[0089] Alternatively, trifluoroacetic acid or formate is obtained by reversed-phase HPLC purification conditions. The crystalline form of a pharmaceutically acceptable salt of a compound of formula (I) (and compound of formula (IA)) can be obtained by recrystallization with a polar solvent (including a mixture of polar solvents and an aqueous mixture of polar solvents) or with a non-polar solvent (including a mixture of non-polar solvents).

[0090] If the compounds according to the invention have at least one chiral center, they may exist in enantiomeric form accordingly. If the compounds have two or more chiral centers, they may exist in diastereomeric form in addition. It should be understood that all such isomers and mixtures thereof are encompassed within the scope of the present invention.

[0091] The compounds prepared according to the above schemes can be obtained as a single form, such as a single enantiomer, by form-specific synthesis or by resolution. Alternatively, the compounds prepared according to the above schemes can be obtained as mixtures of various forms, such as racemic mixtures (1: 1) or non-racemic mixtures (non-1: 1). In the case of obtaining racemic mixtures and non-racemic mixtures of enantiomers, conventional separation methods known to those of ordinary skill in the art, such as chiral chromatography, recrystallization, diastereomeric salt formation, derivatization to diastereomeric adducts, biotransformation or enzymatic transformation, can be used to separate the single enantiomers. In the case of obtaining a mixture of regioisomers or a mixture of diastereomers, conventional methods such as chromatography or crystallization can be used to separate the single isomers, as applicable.

[0092] The following specific examples are provided to further illustrate the present invention and various preferred embodiments.

[0093] Example

[0094] In obtaining the compounds and corresponding analytical data described in the Examples below, the following experimental and analytical protocols were followed unless otherwise indicated.

[0095] Unless otherwise indicated, reaction mixtures were magnetically stirred at room temperature (rt) under a nitrogen atmosphere. Where solutions were "dried", they were typically dried over a drying agent such as Na2SO4 or MgSO4. Where mixtures, solutions and extracts were "concentrated", they were typically concentrated under reduced pressure on a rotary evaporator. Reactions under microwave irradiation were performed in a Biotage Initiator or CEM (microwave reactor) Discover instrument.

[0096] Preparative reverse phase high performance liquid chromatography (RP-HPLC) was used to purify radiolabeled compounds using the following method: an internal HPLC connected to a Synthra RNPlus module (Synthra GmbH, Germany) equipped with a Waters Xbridge C18 column (5 um, 10 mm x 250 mm), a mobile phase of 10 mM NH4OAc and MeCN (50:50 v / v) at a flow rate of 4 mL / min, UV detection at 254 nm, an internal radiation gamma detector.

[0097] Analytical RP-HPLC was used as a quality control for radiolabeled compounds. The following method was used: an Agilent Eclipse XDB-C 18The column (5 μm, 4.6 mm × 150 mm) was maintained at 5% ACN / water (0.05% TFA added) for 1 minute on a Synthra (Synthra GmbH, Germany) HPLC, followed by 5%-95% ACN gradient elution for 10 minutes and then 95% ACN elution for 4 minutes at a flow rate of 1 ml / min. UV detection was performed at 254 nm. An internal gamma detector was used for radiation measurements.

[0098] Mass spectra (MS) were obtained on an Agilent 1260 infinity series system. Unless otherwise stated, electrospray ionization (ESI) was in positive mode. Calculated masses (calcd.) correspond to the exact masses.

[0099] Nuclear magnetic resonance (NMR) spectra were obtained on a Bruker DRX spectrometer. The multiplicity is defined as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad. It will be appreciated that for compounds containing exchangeable protons, the protons may or may not be visible in the NMR spectrum, depending on the choice of solvent used to perform the NMR spectrum and the concentration of the compound in solution.

[0100] Chemical names were generated using ChemDraw Ultra 12.0, ChemDraw Ultra 14.0 (CambridgeSoft Corp., Cambridge, MA) or ACD / Name version 10.01 (Advanced Chemistry).

[0101] Intermediate 1: (S)-3-(3,5-difluorophenyl)-2,7-dimethyl-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c] Pyridine hydrochloride .

[0102]

[0103] Method A :

[0104] Step A: 1-(tert-butyl)-4-ethyl 2-methyl-3-oxopiperidine-1,4-dicarboxylate. Over a 10-minute period, bis(trimethylsilyl)lithium amide (1.0M THF solution, 28.1mL, 28.1mmol) was added dropwise to a cooling (-78°C) solution of 2-methyl-3-oxopiperidine-1-formic acid tert-butyl ester (5g, 23.4mmol) in THF (35mL). Stirring was maintained at -78°C for 30 minutes, and then at -78°C, a solution of ethyl cyanoformate (3.0mL, 30.4mmol) in THF (15mL) was added dropwise over a 10-minute period. After addition, the reaction mixture was then stirred at the same temperature (-78°C) for 2h. The reaction mixture was quenched with saturated NH4Cl aqueous solution and extracted with ethyl acetate (EtOAc) (2×100mL). The combined organic extracts were dried over Na2SO4 and concentrated in vacuo. The resulting residue was purified by flash chromatography (silica gel; 0 to 30% EtOAc-hexanes) to give the title compound (3.5 g, 52% yield) as an oil. 1 H 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).

[0105] Step B: 2,7-Dimethyl-3-oxo-1,2,3,4,5,7-hexahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylic acid Tert-butyl ester To a solution of 1-(tert-butyl)4-ethyl 2-methyl-3-oxopiperidine-1,4-dicarboxylate (3.5 g, 12.1 mmol) in toluene (40.0 mL) was added methylhydrazine (0.96 mL, 18.1 mmol), and the resulting mixture was heated at 110° C. for 3 hours. After cooling to room temperature, the solvent was concentrated in vacuo; the crude residue was purified by flash chromatography (silica gel: 0 to 10% MeOH-DCM) to give the title compound (2.7 g, 83% yield) as an oil.

[0106] Step C: 2,7-dimethyl-3-(((trifluoromethyl)sulfonyl)oxy)-2,4,5,7-tetrahydro-6H-pyrazolo[3, 4-c]Pyridine-6-carboxylic acid tert-butyl ester. 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) was added N,N-diisopropylethylamine (DIEA) (1.9 mL, 11.1 mmol) at room temperature. 1,1,1-trifluoro-N-phenyl-N-((trifluoromethyl)sulfonyl)methanesulfonamide (4.0 g, 11.1 mmol) was then added. The reaction mixture was stirred at room temperature for 5 hours. The solvent was removed in vacuo; the crude residue was purified by flash chromatography (silica gel; 0-20% EtOAc-hexane) to give compound 4 (3.8 g, 86% yield) as an oil.

[0107] The resulting racemic mixture was further purified via chiral SFC to afford the desired 'S' 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, 46% yield). 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).

[0108] Step D: (S)-3-(3,5-difluorophenyl)-2,7-dimethyl-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyrrolidone Imidazolidine hydrochloride 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) was added (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-dihydro-1,4-diol. The mixture was degassed with nitrogen for 5 minutes, sealed and heated to 110°C with stirring for one hour. The heating was removed. The mixture was stirred at room temperature overnight. 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 on a combi-flash silica column with EtOAc in hexane (gradient 10% to 60%) to give an oily product (480 mg, yield 98%). MS (ESI): C 19 H 23Calculated mass of F2N3O2, 363.2; found m / z, 364.1 [M+H] + A suspension of the product from the first step (480 mg, 1.3 mmol) in DCM (8.5 mL) was treated with hydrochloric acid (4 M, 1,4-dihydrochloric acid) at room temperature. 2-Hydane, 3.3 ml, 13.2 mmol) overnight. All solvents were removed in vacuo. The residue was an off-white solid (404 mg, yield 102%). MS (ESI): C 14 H 16 Calculated mass of ClF2N3, 299.1; found m / z, 264.1 [M-HCl+H] + . 1 H NMR (DMSO-d6): δ9.74 (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).

[0109] Method B :

[0110] 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 catalytic piperidine (2 mL, 20.25 mmol) were diluted in toluene (150 mL). Refluxed overnight. The reaction mixture was cooled to room temperature and then quenched with saturated NaCl solution (150 mL). The organic layer extracted was dried over Na2SO4, filtered and concentrated into a dark oil to recover the title compound in a quantitative crude yield. The compound was used in the next step without further purification.

[0111] Step B: tert-Butyl (S,E)-(1-(2-methylhydrazinylidene)propan-2-yl)carbamate . A solution of methylhydrazine (3.04 mL, 57.73 mmol) and (S)-(1-oxopropyl-2-yl)carbamic acid tert-butyl ester (10 g, 57.73 mmol) in THF (150 mL) was stirred at room temperature for 4 hours. The reaction mixture was dried over Na2SO4, filtered and evaporated under reduced pressure overnight. The title compound was isolated as a light oil in a quantitative crude yield. The crude product was used in the next step without further purification.

[0112] Step C: (S)-(1-(4-((1,3-dioxolan-2-yl)methyl)-5-(3,5-difluorophenyl)-1-methyl- tert-Butyl (1H-pyrazol-3-yl)ethyl)carbamate. To a solution of (S, E)-(1-(2-methylhydrazine)propan-2-yl)carbamic acid tert-butyl ester (11.62 g, 57.73 mmol) in EtOH (500 mL), (E)-2-(3-(3,5-difluorophenyl)-2-nitroallyl)-1,3-dioxolane (16.27 g, 59.98 mmol) was added. The reactants were stirred overnight at room temperature and in the open air. The reaction mixture was gently heated to 40 ° C overnight to facilitate the completion of the reaction. The reactants were concentrated to an oil and 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%).

[0113] Step D: (S)-3-(3,5-difluorophenyl)-2,7-dimethyl-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyrrolidone Pyridine . A solution of (S)-(1-(4-((1,3-dioxolane-2-yl)methyl)-5-(3,5-difluorophenyl)-1-methyl-1H-pyrazol-3-yl)ethyl)carbamic acid tert-butyl ester (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 then heated to 55 ° C overnight. The reaction mixture was concentrated into an oil and then quenched with EtOAc and 1N NaOH to pH 11-12. The extracted organic layer was dried over anhydrous Na2SO4, filtered and evaporated to a light brown oil. The crude product was diluted in EtOH and 1.1 equivalents of 1N HCl (10 mL, 10 mmol) were added. The mixture was stirred over the weekend without forming any HCl salts. The mixture was concentrated to a light brown solid and then slurried in minimum 9 / 1 CH3CN / TBME overnight. The solid was filtered to recover the HCl salt of the title compound (1.92 g, 68%).

[0114] 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 .

[0115]

[0116] 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 hours. The mixture was diluted with EtOAc (15 mL) and washed with NaHCO (aqueous solution, 15 mL) and brine (15 mL). The organic layer was dried, filtered and concentrated under reduced pressure. Purification (FCC, SiO, EtOAc / hexane, 10% to 70%) gave the title compound (30 mg, 94%) as a filmy oil. MS(ESI):C 21 H 17 Mass calculated for 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).

[0117] Example 2: (S)-(3-chloro-2-(3-(3,5-difluorophenyl)-2,7-dimethyl-4,5,6,7-tetrahydro-2H-pyrazole 2-[3,4-c]pyridine-6-carbonyl)phenyl)(4-methoxyphenyl)iodide Trifluoromethanesulfonate.

[0118]

[0119] Step A: 2-Chloro-6-iodobenzoyl chloride . To a suspension of 2-chloro-6-iodobenzoic acid (687 mg, 2.43 mmol) in DCM (5 mL) was added oxalyl chloride (550 mg, 4.33 mmol) at room temperature. One drop of DMF was added to the reaction mixture to accelerate the reaction. The reaction mixture was checked after stirring at room temperature for 0.5 hours. The mixture was concentrated under vacuum to a yellow oil and used in the next step without further purification.

[0120] Step 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 step A) was mixed with a solution of (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) and triethylamine (626 μL, 4.5 mmol). The reaction mixture was stirred and allowed to stand at room temperature overnight. The mixture was further diluted with DCM (30 mL) and washed with brine (30 mL). The organic layer was dried, filtered and concentrated under reduced pressure. Purification (FCC, SiO2, EtOAc / hexane, 15% to 60%) gave the title compound (586 mg, 62%) as a light yellow oil. MS (ESI): C 21 H 17 The mass calculated value of ClF2IN3O is 527.0; the m / z found value is 528.0 [M+H]+. 1 HNMR(CDCl3): δ7.81-7.67(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=6.8Hz, 3H).

[0121] Step C: (S)-(3-chloro-2-(3-(3,5-difluorophenyl)-2,7-dimethyl-4,5,6,7-tetrahydro-2H-pyrazolo[2-(4-(4-(2-(3-(2-(4-(2 ... [3,4-c]pyridine-6-carbonyl)phenyl)(4-methoxyphenyl)iodide Triflate. 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) (-15 to 20°C in a sodium chloride-ice bath) was added trifluorosulfonic acid (94 μL, 1.06 mmol) dropwise without disturbing the temperature. After 10 minutes, a suspension of meta-chloroperbenzoic acid (m-CPBA) (max 77%, 148 mg, 0.663 mmol) in DCM (0.7 mL) was slowly added to the solution. The reaction mixture was stirred at -20°C for another 30 minutes, then the cooling was removed and the temperature was raised to room temperature. The reaction mixture was stirred at room temperature for 16. The reaction mixture was cooled in an ice bath. Water (10 μL, 0.53mmol) was added to the reaction mixture, followed by anisole (43mg, 0.398mmol). The ice bath was removed, and the reaction mixture was stirred for another 2 hours at room temperature. The reaction mixture was concentrated under reduced pressure. Ether (5mL) was added to the crude concentrated reaction mixture, and the resulting mixture was sonicated and filtered. The solid crude product was collected as a grayish yellow wax (~200mg) after drying overnight. Purification (FCC, alumina column (purged with DCM), carefully eluted with DCM and then with 10% MeOH / DCM) to obtain the title compound as an off-white solid. The title compound was redissolved in acetonitrile (20mg / mL) and filtered through a 0.45μm syringe filter, and the resulting solution was concentrated under reduced pressure. Ether was added to the resulting title compound to precipitate the title compound (140mg, 67%). MS (ESI): C 29 H 24 Mass calculated for ClF5IN3O5S, 783.0; m / z found, 633.9 [M-OTf] + . 1 H NMR (DMSO-d6): δ8.58 (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).

[0122] 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 .

[0123]

[0124] In a typical procedure, [18F]fluoride in a transport vial (obtained from a cyclotron facility) was transferred to and captured on an ion exchange column. It was then eluted with potassium carbonate (0.75 mg, 5.4 umol) and Kryptofix 222 (7.2 mg, 19.2 umol) in acetonitrile / water (0.8 mL, 6 / 2, v / v) into a Synthra The reaction vessel (RV1) of the module was added. After evaporation of the solvent at 85°C under vacuum under a stream of nitrogen, anhydrous CH3CN (0.5 mL) was added and the process was repeated, and the temperature was raised to 110°C for 3.5 minutes. The reaction vial was then cooled to 70°C and (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)iodide was added to the reaction vessel. A solution of triflate (Example 2, 15.0 mg, 19.1 umol) and TEMPO (4.4 mg, 28.1 umol) in anhydrous acetonitrile (0.7 mL). The reaction mixture was heated at 125° C. for 10 minutes. The reactor was cooled to 40° C. and diluted with water (4.3 mL), and the contents were transferred to an HPLC injection loop for purification.

[0125] 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 at 254 nm with UV detection. The purified radiotracer solution was diluted with 30 mL of water and passed through a SepPak Light C-18 column. The C-18 column was further washed with 10 mL of water before eluting the tracer with 0.5 mL of EtOH. The tracer solution was further diluted with 4.5 mL of saline. The final formulation contained an ethanol concentration of 10% and was suitable for intravenous injection (iv).

[0126] Quality control tests included identity, chemical and radiochemical purity analysis by HPLC using an Eclipse XDB C18 (5 μm, 4.6 mm×250 mm) column eluted with a mixture of 0.05% TFA solution and MeCN at a flow rate of 1 mL / min, equipped with tandem UV (254 nm) and gamma detection.

[0127] Biological data

[0128] The assay for measuring the in vitro activity of MGL was adapted from an assay 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.). The assay involves mixing endogenously expressed MGL from HeLa cells with the test compound, adding [glycerol-1,3- 3 H]-oleylglycerol, incubated for one hour, and then the cleavage of [1,3- 3 The amount of cleaved tritiated glycerol that passes through the carbon filter is proportional to the activity of the MGL enzyme in the specific well / assay conditions.

[0129] The standard conditions for this assay are 300 nM [glycerol-1,3- 3 H]-oleylglycerol was mixed with human MGL from HeLa cells and the test compound for one hour, then the reaction was filtered through activated charcoal and tritium in the flow-through was measured. The test compound concentration in the screening mode was 10 μM, and the IC 50 The highest compound concentration in the assay was determined empirically. MGL is the main hydrolase in HeLa cells / cell homogenates. The test results of the compounds prepared according to Example 1 are listed in Table 2 below:

[0130] Table 2 .

[0131]

[0132] NT means not tested.

[0133] Example 4: (S)-(2-chloro-6-(18F)fluoro-)phenyl)(3-(3,5-difluorophenyl)-2,7-dimethyl-2,4,5, Autoradiography of 7-tetrahydro-6H-pyrazolo[3,4-c]pyridin-6-yl)methanone (Example 3) and comparison with adjacent rat brain Comparison of IHC staining of MGL sections .

[0134] In vitro ARG using the compound of Example 3 was performed on frozen normal rat brain slices (sagittal section, thickness of 20 um). 15 minutes after being taken out of the refrigerator, 250 ul of a buffer solution (50 mM Tris-HCl pH = 7.4) containing the compound of Example 3 at a concentration of 400 uCi / ml was added to the rat brain slices. The brain slices were incubated with incubation buffer at room temperature for 30 minutes, and then washed with blank fresh buffer solution for 5 minutes, and repeated 3 times. The brain slices were air-dried and exposed to a fluorescent screen in a dark box for 18 hours. Then Typhoon TM The screen was scanned by a FLA 7000 image analyzer (GE) to produce an autoradiogram of the compound of Example 3 on rat brain sections ( Figure 2 , Figure 4). It was found that ARG signal was high in the hippocampus, cortex, cerebellum, and thalamus regions, and low in the brainstem. The distribution pattern matched the IHC staining pattern described below.

[0135] IHC staining was performed on frozen normal rat brain sections (sagittal section, 10 um thickness), which were adjacent to the brain sections used in the ARG above. 15 minutes after being taken out of the refrigerator, 4% paraformaldehyde was added to the rat brain sections and fixed for 20 minutes at room temperature. The sections were then washed with fresh phosphate buffer solution (PBS, 0.01 M, pH = 7.4) for 5 minutes, repeated 3 times, and then treated with hydrogen peroxide solution (3%, in PBS) for 20 minutes at room temperature. 10% goat serum (Simga # G-9023) in PBS was added to the sections to block nonspecific binding. A primary anti-MGL antibody (Novus Biologicals, catalog number NBP2-19389) (1:100 dilution) in PBS was added and incubated overnight at 4°C in a humidity chamber. The next day, the rat brain sections were washed with PBS for 10 minutes, repeated 3 times. Secondary goat anti-rabbit IgG (H+L) antibody (ThermoFisher #A-11008) was added in PBS (1:500 dilution) and incubated at room temperature for 1 hour. The sections were further washed with PBS for 10 minutes, repeated 3 times, covered with a slide with mounting medium and observed under a fluorescence microscope. Photos were taken using a fluorescence microscope (Zeiss, AXIO, Imager M2) ( Figure 1 , Figure 3 ). IHC staining indicated that MGL was highly distributed in the hippocampus, cortex, and cerebellum, a pattern that matched the above autoradiographic signals.

Claims

1. A compound according to formula (I) or a pharmaceutically acceptable salt or isotope-labeled form thereof.

2. The compound according to claim 1, which has the formula (IA) or a pharmaceutically acceptable salt thereof.

3. The compound selected from the group consisting of: (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 or pharmaceutically acceptable salts thereof.

4. A sterile solution comprising the compound of formula (IA) according to claim 2.

5. Use of a compound of formula (IA) as claimed in claim 2 in the preparation of a product for in vitro or in vivo imaging of tissues, cells or hosts.

6. The use according to claim 5, wherein the compound of formula (IA) is in a sterile solution.

7. Use of a compound of formula (IA) according to claim 2 in the preparation of a product for imaging a tissue, cell or host by the following method, the method comprising contacting the tissue, cell or host with a compound of formula (IA) or administering the compound of formula (IA) to the tissue, cell or host, and imaging the tissue, cell or host using a positron emission tomography imaging system.

8. The use according to claim 7, wherein the method is in vitro or ex vivo.

9. A compound of formula (IA) according to claim 2 or a sterile solution according to claim 4 for use in a diagnostic method.

10. A compound of formula (IA) according to claim 2 or a sterile solution according to claim 4 for use in a diagnostic method for imaging a tissue, a cell or a host.

11. A compound of formula (IA) according to claim 2 or a sterile solution according to claim 4 for use in a diagnostic method for imaging tissues, cells or hosts using positron emission tomography.

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