Fluorescent probe for monoacylglycerol lipase (MAGL)

Abstract

The present invention relates to a compound represented by general formula (I) TIFF2022549306000104.tif38169 (Wherein X, Y, L and R 1 ~R 4 The present invention provides a fluorescent probe having a fluorophore having a fluorophore group (wherein the fluorophore group is as described herein), compositions containing the compound, methods for making the compound, and methods for using the compound. The compound is useful as a fluorescent probe for monoacylglycerol lipase (MAGL).

Description

[Technical Field]

[0001] The present invention relates to organic compounds useful as fluorescent probes for monoacylglycerol lipase (MAGL). [Background technology]

[0002] Fluorescence imaging probes have emerged as high-resolution tools for investigating the localization (e.g., expression levels and protein distribution in health and disease), structure, dynamics, and function of proteins in live cells (LAStoddart, LEKilpatrick, SJBriddon, SJHill, Neuropharmacology, 2015, 98, 48-57). Such probes can be applied, for example, to cellular trafficking studies using flow cytometry fluorescence-activated cell sorting (FACS) experiments or confocal live-cell imaging. Furthermore, fluorescent imaging probes enable real-time monitoring of ligand-receptor interactions and visualization of proteins with high spatiotemporal precision (A.J. Vernall, S.J. Hill, B. Kellam, Br. J. Pharmacol., 2014, 171, 1073-1084; C. Iliopoulos-Tsoutsouvas, R.N. Kulkarni, A. Makriyannis, S.P. Nikas, Expert Opin. Drug Discov., 2018, 13, 933-947). Furthermore, such probes offer the possibility of generating equilibrium and kinetic binding data in a high-throughput manner without handling radioactive materials, for example, using time-resolved fluorescence resonance energy transfer (TR-FRET). Fluorescent imaging probes can also be useful in supporting the translation of preclinical pharmacology animal data to the clinic and can be applied to dose selection in humans. They can be used, for example, as markers of target engagement via the generation of ex vivo quantitative receptor binding data in whole blood. Depending on the respective application, fluorescent imaging probes must meet certain criteria, including affinity, selectivity and specificity for the respective target, favorable photophysical properties, and applicability across different techniques and cell types. Summary of the Invention

[0003] In a first aspect (A1), the present invention provides a compound of formula (I): [ka] or a pharmaceutically acceptable salt thereof, wherein X, Y, L and R 1 ~R 4 is as described herein.

[0004] In a further aspect, the present invention provides a process for preparing a compound of formula (I) as described herein, comprising: (a) reacting a first amine 4a,5,6,7,8,8a-hexahydro-4H-pyrido[4,3-b][1,4]oxazin-3-one (2) with [ka] Second Amine 1 [ka] (In the formula, R 2 , R 3 , R 4 , L and PG are as described herein) in the presence of a base and a urea-forming reagent, formula 3 [ka] (In the formula, R 2 , R 3 , R 4 , L and PG are as described herein; or (b) reacting a first amine 4a,5,6,7,8,8a-hexahydro-4H-pyrido[4,3-b][1,4]oxazin-3-one (2) with [ka] Second Amine 1a [ka] (In the formula, R 1 , R 2 , R 3 , R 4and L is as described herein) in the presence of a base and a urea-forming reagent, forming a compound of formula (I) above The process includes:

[0005] In a further aspect, the present invention provides a compound of formula (I) as described herein when prepared according to the process described herein.

[0006] In a further aspect, the present invention provides a compound selected from: N-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-(2-aminoethoxy)propanamide; tert-Butyl N-[2-[3-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethyl]carbamate; N-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-(2-aminoethoxy)ethoxy]propanamide; tert-Butyl N-[2-[2-[3-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethoxy]ethyl]carbamate; (4aR,8aS)-6-[4-[[3-(2-aminoethoxy)phenyl]-phenyl-methyl]piperidine-1-carbonyl]-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazin-3-one; tert-Butyl N-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]carbamate; N-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propanamide; and tert-Butyl N-[2-[2-[2-[3-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethyl]carbamate; and the use thereof for preparing the fluorescent probe of the present invention.

[0007] In a further aspect, the present invention provides a method of studying monoacylglycerol lipase (MAGL) occupancy, comprising contacting MAGL with a compound of formula (I) as described herein.

[0008] In a further aspect, the present invention provides a method for diagnostic imaging of monoacylglycerol lipase (MAGL) in a mammal, comprising contacting the MAGL with a compound of formula (I) described herein.

[0009] In a further aspect, the present invention provides a method for generating equilibrium and kinetic binding data for monoacylglycerol lipase (MAGL), comprising contacting MAGL with a compound of formula (I) described herein. DETAILED DESCRIPTION OF THE INVENTION

[0010] definition It should be understood that any feature, integer, property, compound, chemical moiety, or group described in connection with a particular aspect, embodiment, or example of the invention is applicable to any other aspect, embodiment, or example described herein, except where incompatible. All features disclosed in this specification (including any accompanying claims, abstract, and drawings), and / or all steps of any method or process so disclosed, may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of any foregoing embodiment. The invention extends to any novel one or any novel combination of features disclosed in this specification (including any accompanying claims, abstract, and drawings), or any novel one or any novel combination of steps of any method or process so disclosed.

[0011] The term "alkyl" refers to a monovalent or polyvalent, e.g., monovalent or divalent, straight-chain or branched saturated hydrocarbon group having 1 to 12 carbon atoms. In some preferred embodiments, the alkyl group contains 1 to 6 carbon atoms, e.g., 1, 2, 3, 4, 5, or 6 carbon atoms ("C1-C6-alkyl"). In other embodiments, the alkyl group contains 1 to 3 carbon atoms, e.g., 1, 2, or 3 carbon atoms. Some non-limiting examples of alkyl include methyl, ethyl, propyl, 2-propyl (isopropyl), n-butyl, iso-butyl, sec-butyl, tert-butyl, and 2,2-dimethylpropyl. A particularly preferred, but non-limiting, example of alkyl is methyl.

[0012] The term "alkoxy" refers to an alkyl group, as defined above, attached to the parent molecular moiety through an oxygen atom. An alkoxy group contains 1 to 12 carbon atoms, unless otherwise specified. In some preferred embodiments, an alkoxy group contains 1 to 6 carbon atoms ("C1-C6-alkoxy"). In other embodiments, an alkoxy group contains 1 to 4 carbon atoms. In still other embodiments, an alkoxy group contains 1 to 3 carbon atoms. Some non-limiting examples of alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, and tert-butoxy. A particularly preferred, but non-limiting example of alkoxy is methoxy.

[0013] The term "halogen" or "halo" refers to fluoro (F), chloro (Cl), bromo (Br), or iodo (I). Preferably, the term "halogen" or "halo" refers to fluoro (F), chloro (Cl), or bromo (Br). Particularly preferred, but non-limiting, examples of "halogen" or "halo" are fluoro (F) and chloro (Cl).

[0014] The term "haloalkyl" refers to an alkyl group in which at least one of the alkyl group's hydrogen atoms has been replaced with a halogen atom, preferably fluoro. Preferably, "haloalkyl" refers to an alkyl group in which one, two, or three of the alkyl group's hydrogen atoms have been replaced with a halogen atom, most preferably fluoro. Particularly preferred, but non-limiting, examples of haloalkyl are trifluoromethyl and trifluoroethyl.

[0015] The term "haloalkoxy" refers to an alkoxy group in which at least one of the alkoxy group's hydrogen atoms has been replaced with a halogen atom, preferably fluoro. Preferably, "haloalkoxy" refers to an alkoxy group in which one, two, or three of the alkoxy group's hydrogen atoms have been replaced with halogen atoms, most preferably fluoro. A particularly preferred, but non-limiting example of a haloalkoxy is trifluoromethoxy (-OCF).

[0016] The term "pharmaceutically acceptable salt" refers to salts that retain the biological effectiveness and properties of the free base or free acid, without being biologically or otherwise undesirable. Salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid (especially hydrochloric acid), and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and N-acetylcysteine. These salts may also be prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyimine resins, etc. A particular pharmaceutically acceptable salt of the compound of formula (I) is the hydrochloride salt.

[0017] The term "protecting group" (PG) refers to a group that selectively blocks a reactive site in a polyfunctional compound so that a chemical reaction, in the sense conventionally associated with synthetic chemistry, can be selectively carried out at an otherwise unprotected reactive site. The protecting group can be removed at an appropriate time. Exemplary protecting groups are an amino-protecting group, a carboxy-protecting group, or a hydroxy-protecting group. Specific protecting groups are tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), fluorenylmethoxycarbonyl (Fmoc), and benzyl (Bn). Further specific protecting groups are tert-butoxycarbonyl (Boc) and fluorenylmethoxycarbonyl (Fmoc). An even more specific protecting group is tert-butoxycarbonyl (Boc). Exemplary protecting groups and their use in organic synthesis are described, for example, in Protective Groups in Organic Chemistry, T.W. Greene and P.G.M. Hutts, 5th Edition, 2014, John Wiley & Sons, NY.

[0018] The term "urea-forming reagent" refers to a compound that can react a first amine with a second amine to form a urea derivative. Non-limiting examples of urea-forming reagents include bis(trichloromethyl)carbonate, phosgene, trichloromethyl chloroformate, (4-nitrophenyl)carbonate, and 1,1'-carbonyldiimidazole. The urea-forming reagents described in G. Sartori et al., Green Chemistry, 2000, 2, 140 are incorporated herein by reference.

[0019] The compounds of formula (I) may contain several asymmetric centers and may exist in the form of optically pure enantiomers, mixtures of enantiomers, e.g. racemates, optically pure diastereoisomers, mixtures of diastereoisomers, racemates of diastereoisomers or racemic mixtures of diastereoisomers.

[0020] According to the Cahn-Ingold-Prelog rules, the asymmetric carbon atom can be of the "R" or "S" configuration.

[0021] The abbreviation "MAGL" refers to the enzyme monoacylglycerol lipase. The terms "MAGL" and "monoacylglycerol lipase" are used interchangeably herein.

[0022] Compounds of the Invention In a first aspect (A1), the present invention provides a compound of formula (I): [ka] or a pharmaceutically acceptable salt thereof, wherein: L is a linker; R 1 is a fluorescent label; R 2 , R 3 and R 4 are each independently selected from hydrogen, halogen, C-C-alkyl, halo-C-C-alkyl, C-C-alkoxy and halo-C-C-alkoxy; X and Y are both CH; or X and Y together form a double bond (C=C).

[0023] In one embodiment, the compound of formula (I) is a compound of formula (Ia): [ka] In the formula, L and R 1 ~R 4 is as described herein.

[0024] The present invention also provides the following enumerated embodiments (E) of the first aspect (A1) of the invention:

[0025] E1.A1, a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein: L is [ka] is a linker selected from: where: each occurrence of n is independently an integer selected from 1, 2, 3, 4, 5, 6, and 7; The wavy line is R 1 Shows the point of attachment; The asterisk indicates the point of attachment to the remainder of formula (I); R 1 teeth, [ka] TIFF0007736677000011.tif217169 TIFF0007736677000012.tif221169 a fluorescent label selected from: where the wavy line indicates the point of attachment to the linker L; R 2 , R 3 and R 4 are each independently selected from hydrogen, halogen, C-C-alkyl, halo-C-C-alkyl, C-C-alkoxy and halo-C-C-alkoxy; X and Y are both CH; or A compound or a pharmaceutically acceptable salt thereof, wherein X and Y together form a double bond (C=C).

[0026] E2.A1, a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the linker L is [ka] Selected from; each occurrence of n is independently an integer selected from 1, 2, 3, 4, 5, 6, and 7; The wavy line indicates the fluorescently labeled R 1 Shows the point of attachment; The asterisk indicates the point of attachment to the remainder of formula (I), or a pharmaceutically acceptable salt thereof.

[0027] E3. Compounds of formula (I) according to A1 and E1 or pharmaceutically acceptable salts thereof, wherein the linker L is [ka] Selected from; each occurrence of n is independently an integer selected from 1, 2, 3, 4, 5, 6, and 7; The wavy line indicates the fluorescently labeled R 1 Shows the point of attachment; The asterisk indicates the point of attachment to the remainder of formula (I), or a pharmaceutically acceptable salt thereof.

[0028] E4. Compounds of formula (I) according to A1 and E1 or pharmaceutically acceptable salts thereof, wherein the linker L is [ka] is selected from each occurrence of n is independently an integer selected from 1, 2, and 3; The wavy line indicates the fluorescently labeled R 1 Shows the point of attachment; The asterisk indicates the point of attachment to the remainder of formula (I), or a pharmaceutically acceptable salt thereof.

[0029] E5. Compounds of formula (I) according to A1 and E1 or pharmaceutically acceptable salts thereof, wherein the linker L is [ka] is selected from each occurrence of n is independently an integer selected from 1, 2, and 3; The wavy line indicates the fluorescently labeled R 1 Shows the point of attachment; The asterisk indicates the point of attachment to the remainder of formula (I), or a pharmaceutically acceptable salt thereof.

[0030] E6. Compounds of formula (I) according to A1 and E1 or pharmaceutically acceptable salts thereof, wherein the linker L is [ka] is selected from The wavy line indicates the fluorescently labeled R 1 Shows the point of attachment; The asterisk indicates the point of attachment to the remainder of formula (I), or a pharmaceutically acceptable salt thereof.

[0031] E7. Compounds of formula (I) according to A1 and E1 or pharmaceutically acceptable salts thereof, wherein the linker L is [ka] is selected from The wavy line indicates the fluorescently labeled R 1 Shows the point of attachment; The asterisk indicates the point of attachment to the remainder of formula (I), or a pharmaceutically acceptable salt thereof.

[0032] E8. A compound of formula (I) according to any one of A1 and E2 to E7 or a pharmaceutically acceptable salt thereof, wherein the compound is fluorescently labeled R 1 but, [ka] TIFF0007736677000020.tif216169 TIFF0007736677000021.tif221169 is selected from The wavy line indicates the point of attachment to the linker L, or a pharmaceutically acceptable salt thereof.

[0033] E9. A compound of formula (I) according to any one of A1 and E1 to E7 or a pharmaceutically acceptable salt thereof, wherein the compound is fluorescently labeled R 1 but, [ka] wherein the wavy line indicates the point of attachment to the linker L, or a pharmaceutically acceptable salt thereof.

[0034] E10. A compound of formula (I) according to any one of A1 and E1 to E7 or a pharmaceutically acceptable salt thereof, wherein the compound is fluorescently labeled R 1 but, [ka] wherein the wavy line indicates the point of attachment to the linker L, or a pharmaceutically acceptable salt thereof.

[0035] E11.R 2 A compound of formula (I) according to any one of A1 and E1 to E10, or a pharmaceutically acceptable salt thereof, wherein is hydrogen.

[0036] E12.R 3 A compound of formula (I) according to any one of A1 and E1 to E11, or a pharmaceutically acceptable salt thereof, wherein is hydrogen.

[0037] E13.R 4 A compound of formula (I) according to any one of A1 and E1 to E12, or a pharmaceutically acceptable salt thereof, wherein is hydrogen.

[0038] A compound of formula (I) according to E14.A1 or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) is a compound of formula (II): [ka] The linker L is [ka] is selected from where: each occurrence of n is independently an integer selected from 1, 2, and 3; The wavy line indicates the fluorescently labeled R 1 Shows the point of attachment; The asterisk indicates the point of attachment to the remainder of formula (I); Fluorescent Label R 1 teeth, [ka] is selected from wherein the wavy line indicates the point of attachment to the linker L, a compound or a pharmaceutically acceptable salt thereof.

[0039] A compound of formula (I) according to E15.A1 or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) is a compound of formula (II) or (III), [ka] The linker L is [ka] is selected from where: each occurrence of n is independently an integer selected from 1, 2, and 3; The wavy line indicates the fluorescently labeled R 1 Shows the point of attachment; The asterisk indicates the point of attachment to the remainder of formula (I); Fluorescent Labeling R 1 teeth, [ka] is selected from wherein the wavy line indicates the point of attachment to the linker L, a compound or a pharmaceutically acceptable salt thereof.

[0040] A compound of formula (I) according to E16.A1 or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) is a compound of formula (II): [ka] The linker L is [ka] is selected from where: each occurrence of n is independently an integer selected from 1, 2, and 3; The wavy line indicates the fluorescently labeled R 1 Shows the point of attachment; The asterisk indicates the point of attachment to the remainder of formula (I); Fluorescent Labeling R 1 teeth, [ka] is selected from wherein the wavy line indicates the point of attachment to the linker L, a compound or a pharmaceutically acceptable salt thereof.

[0041] A compound of formula (I) according to E17.A1 or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) is a compound of formula (II) or (III), [ka] The linker L is [ka] is selected from where: The wavy line indicates the fluorescently labeled R 1 Shows the point of attachment; The asterisk indicates the point of attachment to the remainder of formula (I); Fluorescent Labeling R 1 teeth, [ka] is selected from wherein the wavy line indicates the point of attachment to the linker L, a compound or a pharmaceutically acceptable salt thereof.

[0042] E18. The compound of formula (I) is: N-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethoxy]propanamide; N-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethoxy]propanamide; N-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethoxy]ethoxy]propanamide; N-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethoxy]ethoxy]propanamide; N-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethoxy]ethoxy]propanamide; N-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethoxy]propanamide; N-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-[2-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethoxy]ethoxy]ethoxy]propanamide; N-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-[2-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethoxy]ethoxy]ethoxy]propanamide; N-[2-[3-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethyl]-3-[2,2-difluoro-12-(1H-pyrrol-2-yl)-3-aza-1-azonia-2-boranuidatricyclo[7.3.0.03,7]dodeca-1(12),4,6,8,10-pentaen-4-yl]propanamide; and (4aR,8aS)-6-(4-((3-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)phenyl)(phenyl)methylene)piperidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one A compound of formula (I) according to any one of A1 and E1 to E17, or a pharmaceutically acceptable salt thereof, selected from:

[0043] E19. The compound of formula (I) is: N-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethoxy]ethoxy]propanamide; N-[2-[3-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethyl]-3-[2,2-difluoro-12-(1H-pyrrol-2-yl)-3-aza-1-azonia-2-boranuidatricyclo[7.3.0.03,7]dodeca-1(12),4,6,8,10-pentaen-4-yl]propanamide A compound of formula (I) according to any one of A1 and E1 to E17, or a pharmaceutically acceptable salt thereof, selected from:

[0044] E20. The compound of formula (I) is: N-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethoxy]ethoxy]propanamide; N-[2-[3-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethyl]-3-[2,2-difluoro-12-(1H-pyrrol-2-yl)-3-aza-1-azonia-2-boranuidatricyclo[7.3.0.03,7]dodeca-1(12),4,6,8,10-pentaen-4-yl]propanamide; and (4aR,8aS)-6-(4-((3-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)phenyl)(phenyl)methylene)piperidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one A compound of formula (I) according to any one of A1 and E1 to E17, or a pharmaceutically acceptable salt thereof, selected from:

[0045] The present invention also relates to synthetic intermediates (ie, unlabeled compounds) useful in making the fluorescent probes of the present invention.

[0046] Enumerated embodiments 21-23 provide preferred examples of such synthetic intermediates.

[0047] E21. Below: N-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-(2-aminoethoxy)propanamide; tert-Butyl N-[2-[3-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethyl]carbamate; tert-Butyl N-[2-[2-[3-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethoxy]ethyl]carbamate; (4aR,8aS)-6-[4-[[3-(2-aminoethoxy)phenyl]-phenyl-methyl]piperidine-1-carbonyl]-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazin-3-one; tert-Butyl N-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]carbamate; N-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propanamide; tert-Butyl N-[2-[2-[2-[3-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethyl]carbamate; and N-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-(2-aminoethoxy)ethoxy]propanamide A compound selected from:

[0048] E22. Below: N-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-(2-aminoethoxy)propanamide; tert-Butyl N-[2-[3-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethyl]carbamate; N-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-(2-aminoethoxy)ethoxy]propanamide; tert-Butyl N-[2-[2-[3-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethoxy]ethyl]carbamate; (4aR,8aS)-6-[4-[[3-(2-aminoethoxy)phenyl]-phenyl-methyl]piperidine-1-carbonyl]-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazin-3-one; tert-Butyl N-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]carbamate; N-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propanamide; tert-Butyl N-[2-[2-[2-[3-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethyl]carbamate; tert-Butyl (2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-ylidene)(phenyl)methyl)phenoxy)ethyl)carbamate; tert-Butyl (2-(4-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-ylidene)(phenyl)methyl)phenoxy)ethyl)carbamate; tert-Butyl N-[3-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidylidene]-phenyl-methyl]phenyl]prop-2-ynyl]carbamate; tert-Butyl N-[3-[4-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidylidene]-phenyl-methyl]phenyl]prop-2-ynyl]carbamate; tert-Butyl N-[(E)-3-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidylidene]-phenyl-methyl]phenyl]allyl]carbamate; tert-Butyl N-[(E)-3-[4-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidylidene]-phenyl-methyl]phenyl]allyl]carbamate; tert-Butyl N-[2-[3-[(SR)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]carbamate; tert-Butyl N-[2-[4-[(SR)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]carbamate; tert-Butyl N-[3-[3-[(SR)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenyl]propyl]carbamate; tert-Butyl N-[3-[4-[(SR)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenyl]propyl]carbamate; tert-Butyl N-[3-[4-[(SR)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenyl]prop-2-ynyl]carbamate; tert-Butyl N-[3-[3-[(SR)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenyl]prop-2-ynyl]carbamate; tert-Butyl N-[(E)-3-[4-[(SR)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenyl]allyl]carbamate; tert-Butyl N-[(E)-3-[3-[(SR)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenyl]allyl]carbamate; tert-Butyl N-[2-[3-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidylidene]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethyl]carbamate; tert-Butyl N-[3-[4-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidylidene]-phenyl-methyl]phenyl]propyl]carbamate; tert-Butyl N-[3-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidylidene]-phenyl-methyl]phenyl]propyl]carbamate A compound selected from:

[0049] E23. Below: N-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-(2-aminoethoxy)propanamide; tert-Butyl N-[2-[3-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethyl]carbamate; N-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-(2-aminoethoxy)ethoxy]propanamide; tert-Butyl N-[2-[2-[3-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethoxy]ethyl]carbamate; (4aR,8aS)-6-[4-[[3-(2-aminoethoxy)phenyl]-phenyl-methyl]piperidine-1-carbonyl]-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazin-3-one; tert-Butyl N-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]carbamate; N-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propanamide; tert-Butyl N-[2-[2-[2-[3-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethyl]carbamate; tert-butyl(2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-ylidene)(phenyl)methyl)phenoxy)ethyl)carbamate; and tert-Butyl (2-(4-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-ylidene)(phenyl)methyl)phenoxy)ethyl)carbamate A compound selected from:

[0050] E24. Use of a compound according to any one of E21 to E23 for the preparation of a fluorescent probe of formula (I) according to any one of A1 and E1 to E20.

[0051] In certain embodiments, the present invention provides pharmaceutically acceptable salts of compounds according to formula (I) described herein. In more particular embodiments, the present invention provides compounds according to formula (I) described herein as the free base.

[0052] Manufacturing Process The preparation of the compounds of formula (I) of the present invention can be carried out by sequential or convergent synthetic routes. The synthesis of the present invention is shown in the following general scheme. The techniques required to carry out the reactions and purification of the resulting products are known to those skilled in the art. The substituents and indices used in the description of the following processes have the meanings given herein unless indicated to the contrary.

[0053] If one of the starting materials, intermediates, or compounds of formula (I) contains one or more functional groups that are not stable or reactive under the reaction conditions of one or more reaction steps, suitable protecting groups (e.g., as described in Protective Groups in Organic Chemistry, T.W. Greene and P.G.M. Hutts, 5th Edition, 2014, John Wiley & Sons, NY) can be introduced prior to a critical step by applying methods well known in the art. Such protecting groups can be removed at a later stage of the synthesis using standard methods described in the literature.

[0054] When the starting material or intermediate contains a stereocenter, the compound of formula (I) may be obtained as a mixture of diastereomers or enantiomers, which can be separated by methods well known in the art, such as chiral HPLC, chiral SFC, or chiral crystallization. Racemates can be separated into their antipodes via diastereomeric salts, for example, by crystallization with an optically pure acid, or by separating the antipodes by specific chromatographic methods using either a chiral adsorbent or a chiral eluent. Starting materials and intermediates containing a stereocenter can also be separated to obtain diastereomerically / enantiomerically enriched starting materials and intermediates. The use of such diastereomerically / enantiomerically enriched starting materials and intermediates in the synthesis of the compound of formula (I) typically results in the respective diastereomerically / enantiomerically enriched compound of formula (I).

[0055] Those skilled in the art will recognize that in the synthesis of compounds of formula (I) (unless otherwise desired), an "orthogonal protecting group strategy" can be applied to cleave several protecting groups one at a time without affecting other protecting groups in the molecule. The principle of orthogonal protection is well known in the art and has been described in the literature (e.g., Barany and R.B. Merrifield, J. Am. Chem. Soc., 1977, 99, 7363; H. Waldmann et al., Angew. Chem. Int. Ed. Engl., 1996, 35, 2056).

[0056] Those skilled in the art will recognize that the reaction sequence may vary depending on the reactivity and nature of the intermediates.

[0057] More specifically, compounds of formula (I) can be prepared by the methods described below, the methods described in the Examples, or similar methods. Appropriate reaction conditions for the individual reaction steps are known to those skilled in the art. For literature-described reaction conditions affecting the described reactions, see, for example, Comprehensive Organic Transformations: A Guide to Functional Group Preparations, 2nd Edition, Richard C. Larock, John Wiley & Sons, New York, NY, 1999. The reactions could be easily carried out with or without a solvent. There are no particular restrictions on the nature of the solvent used, so long as it does not adversely affect the reaction or the reagents involved and is capable of dissolving the reagents to at least some extent. The described reactions can occur over a wide range of temperatures, and the exact reaction temperature is not critical to the present invention. It is convenient to carry out the described reactions at temperatures ranging from -78°C to reflux. The reaction time required can also vary widely, depending on many factors, particularly the reaction temperature and the nature of the reagents. However, a period of 0.5 hours to several days is usually sufficient to produce the described intermediates and compounds. The reaction sequence is not limited to the sequence shown in the scheme, but the order of the reaction steps can be freely changed depending on the starting materials and their respective reactivities.

[0058] If starting materials or intermediates are not commercially available or their synthesis is not described in the literature, they can be prepared by analogy with existing procedures for analogous materials or as outlined in the experimental section.

[0059] The following abbreviations are used in the text: AcOH = acetic acid, ACN = acetonitrile, Boc = tert-butyloxycarbonyl, CAS RN = Chemical Abstracts Registry Number, Cbz = benzyloxycarbonyl, Cs2CO3 = cesium carbonate, CO = carbon monoxide, CuCl = copper(I) chloride, CuCN = copper(I) cyanide, CuI = copper(I) iodide, DMAP = 4-dimethylaminopyridine, DME = dimethoxyethane, DMEDA = N,N'-dimethylethylenediamine, DMF = N,N-dimethylformamide, DIPEA = N,N-diisopropylethylamine, dppf = 1,1 bis(diphenylphosphino)ferrocene, EDC.HCl = N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, EI = electron impact, ESI = electrospray ionization, EtOAc = ethyl acetate, EtOH = ethanol, h = hour, FA = formic acid. H2O = water, H2SO4 = sulfuric acid, Hal = halogen, HATU = 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate, HBTU = O-benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluorophosphate, HCl = hydrogen chloride, HOBt = 1-hydroxy-1H-benzotriazole; HPLC = high-performance liquid chromatography, iPrMgCl = isopropylmagnesium chloride, I2 = iodine, IPA = 2-propanol, (Ir[dF(CF3)ppy]2(dtbpy))PF6 = [4,4'-bis(1,1-dimethylethyl)-2,2'-bipyridine-N1,N1']bis[3,5-difluoro-2-[5-(trifluoromethyl)-2-pyridinyl-N]phenyl-C]iridium(III) hexafluorophosphate, ISP = positive ion spray (mode), ISN = negative ion spray (mode), K2CO3 = potassium carbonate, KHCO3 = potassium bicarbonate, KI = potassium iodide, KOH = potassium hydroxide, K3PO4 = tripotassium phosphate, LiAlH4 or LAH = lithium aluminum hydride, LiHMDS = lithium bis(trimethylsilyl)amide, LiOH = lithium hydroxide, MgSO4 = magnesium sulfate, min = minute, mL = milliliter, MPLC = medium pressure liquid chromatography, MS = mass spectrum, NaH = sodium hydride, NaHCO3 = sodium bicarbonate, NaNO2 = sodium nitrite, NaOH = sodium hydroxide, Na2CO3 = sodium carbonate, Na2SO4 = sodium sulfate, Na2S2O3 = sodium thiosulfate, NBS = N-bromosuccinimide, nBuLi = n-butyllithium, NEt3 = triethylamine (TEA), NH4Cl = ammonium chloride, NiCl2 glyme = nickel(II) chloride ethylene glycol dichloride Methyl ether complex, NMP = N-methyl-2-pyrrolidone, OAc = acetoxy, T3P = propylphosphonic anhydride, P2O5 = phosphorus pentoxide, PE = petroleum ether, PG = protecting group, Pd-C = palladium on activated carbon, PdCl2(dppf)-CH2Cl2 = 1,1'-bis(diphenylphosphino)ferrocene-palladium(II) dichloride dichloromethane complex, Pd2(dba)3 = tris(dibenzylideneacetone)dipalladium(0), Pd(OAc)2 = palladium(II) acetate, Pd(OH)2 = palladium hydroxide, Pd(PPh3) 4 = tetrakis(triphenylphosphine)palladium(0), PTSA = p-toluenesulfonic acid, R = any group, RT = room temperature, SFC = supercritical fluid chromatography, S-PHOS = 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl, T3P = propylphosphonic anhydride, TBAI = tetrabutylammonium iodide, TEA = triethylamine, TFA = trifluoroacetic acid, THF = tetrahydrofuran, TMEDA = N,N,N',N'-tetramethylethylenediamine, ZnCl2 = zinc chloride, Xantphos = 4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene.

[0060] L,n,R 1 ,R 2 ,R 3 and R 4 Compounds of formula I described herein can be synthesized analogously to literature procedures and / or as shown, for example, in Scheme 1. [ka]

[0061] Scheme 1

[0062] Thus, 4a,5,6,7,8,8a-hexahydro-4H-pyrido[4,3-b][1,4]oxazin-3-one 2 is reacted with intermediate 1 in the presence of a urea-forming reagent, such as bis(trichloromethyl)carbonate, using a suitable base and a solvent, such as sodium bicarbonate in DCM, to give a compound of formula 3 (Step a). Additional urea-forming reagents include, but are not limited to, phosgene, trichloromethyl chloroformate, (4-nitrophenyl)carbonate, or 1,1'-carbonyldiimidazole. This type of reaction and the use of these reagents have been widely described in the literature (e.g., G. Sartori et al., Green Chemistry, 2000, 2, 140). Those skilled in the art will recognize that the order of addition of reagents can be important in this type of reaction due to the reactivity and stability of the carbamoyl chloride formed as an intermediate and to avoid the formation of undesired symmetrical urea by-products. Piperidine derivative 1 bears a suitable protecting group "PG", such as a Cbz or Boc protecting group. Piperidine 1 and 4a,5,6,7,8,8a-hexahydro-4H-pyrido[4,3-b][1,4]oxazin-3-one 2 are either commercially available or can be prepared according to literature methods or as shown below.

[0063] Removal of the protecting groups in intermediate 3, applying methods known in the art (e.g., Boc group using TFA in DCM at temperatures between 0° C. and room temperature, Cbz group using hydrogen in the presence of a suitable catalyst such as Pd on charcoal or Pd(OH) in a suitable solvent such as MeOH, EtOH, EtOAc, or mixtures thereof, and groups as described, for example, in Protective Groups in Organic Chemistry, T.W. Greene and P.G.M. Buts, 4th Edition, 2006, Wiley, NY) provides molecules of formula Ia (step b).

[0064] Reaction of amine Ia with a fluorescent label containing a carboxylic acid moiety provides compounds of formula Ib (R 1 = fluorescent label) is obtained (step c). This type of amide coupling can be achieved by using one of the well-known coupling reagents, such as DCC, HATU, EDCI, HOBt, TBTU, or T3P, and a base, such as Huenig's base, triethylamine, or DMAP, in a suitable solvent, such as N,N-dimethylformamide, DMA, DCM, or dioxane, preferably at 0 °C to room temperature. To form the final molecule bearing the nitrobenzofurazan dye Ib, the amine Ia can be reacted in a nucleophilic aromatic substitution with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (CAS RN 10199-89-0) in a solvent such as MeOH, preferably at ambient temperature, in the presence of a base, such as N,N-diisopropylethylamine (step c).

[0065] Optionally, piperidine derivative 1, in which "PG" is a fluorescent label or tert-butoxycarbonyl, can be directly converted to a compound of formula (Ib) according to step a. When "PG" is tert-butoxycarbonyl, R 1 is equal to tert-butoxycarbonyl to obtain the final molecule of formula (Ib).

[0066] 4a,5,6,7,8,8a-Hexahydro-4H-pyrido[4,3-b][1,4]oxazin-3-one 2 can be synthesized, for example, as shown in Scheme 2 and / or analogously to methods described in the literature. [ka]

[0067] Scheme 2

[0068] Thus, 3-aminopiperidin-4-ol derivatives 3, where "PG" represents a suitable protecting group such as a Cbz or Boc protecting group, can be acylated with, for example, acyl chlorides 4, where "LG" represents a suitable leaving group (e.g., Cl or Br), using a suitable base such as sodium or potassium carbonate, sodium hydroxide or sodium acetate in a suitable solvent such as THF, water, acetone, or a mixture thereof, to provide intermediates 5 (step a). Intermediate 4 is commercially available or can be prepared according to literature methods.

[0069] Intermediate 5 can be cyclized to intermediate 6 (step b) using methods well known in the art, for example, by treating 5 with sodium hydride in THF or potassium tert-butoxide in IPA and water. Reactions of this type are described in the literature (e.g., Z. Rafinski et al., J. Org. Chem., 2015, 80, 7468; S. Dugar et al., Synthesis, 2015, 47(5), 712; WO 2005 / 066187).

[0070] Removal of the protecting groups in intermediate 6 applying methods known in the art (e.g., Boc group using TFA in DCM at temperatures between 0° C. and room temperature, Cbz group using hydrogen in the presence of a suitable catalyst such as Pd on charcoal or Pd(OH) in a suitable solvent such as MeOH, EtOH, EtOAc, or mixtures thereof, and groups as described, for example, in Protective Groups in Organic Chemistry, T.W. Greene and P.G.M. Buts, 4th Edition, 2006, Wiley, NY) gives intermediate 2 (step c).

[0071] Intermediate 2 can be obtained as a mixture of diastereomers and enantiomers, respectively, or as a single stereoisomer, depending on whether a racemic mixture or an enantiomerically pure form of cis- or trans-3-aminopiperidin-4-ol derivative 3 is used in their synthesis. Intermediate 3 is commercially available, and its synthesis is also described in the literature (e.g., WO 2005 / 066187, WO 2011 / 0059118, WO 2016 / 185279). The optically pure cis-configured intermediate 2 can be obtained, for example, by chiral separation of commercially available rac-(4aR,8aS)-4a,5,6,7,8,8a-hexahydro-4H-pyrido[4,3-b][1,4]oxazin-3-one (optionally in the form of a salt, for example the hydrochloride salt) using methods known in the art, such as diastereomeric salt crystallization or chiral chromatography.

[0072] Intermediates of type 1 can be prepared by a variety of conditions, which can be exemplified by the general synthetic procedure outlined in Scheme 3. [ka]

[0073] Scheme 3

[0074] The ketone of formula 7 can be reacted to N-tosylhydrazone 8 (step a) using, for example, NHNHT in a solvent such as 1,4-dioxane, preferably by heating to 80° C. In a further step, the N-tosylhydrazone of formula 8 can be converted to an aryl halide 9 (wherein X 1 is selected from the group consisting of Cl, Br, and I) in the presence of a catalyst system to give intermediates of structure 10 (step b). A suitable catalyst system for such a transformation consists, for example, but is not limited to, [Pd(PPPh3)2Cl2] in the presence of LiOtBu in a solvent such as 1,4-dioxane at a temperature of 80° C. Heterogeneous catalytic hydrogenation of olefin 10 using a catalyst such as Pd(OH)2 or Pd / C, preferably Pd / C, in a solvent such as THF, MeOH, EtOH, EtOAc, or mixtures thereof, preferably THF, at about room temperature and under atmospheric pressure of, for example, hydrogen, gives intermediates of type 11 (step c). Cleavage of the BOC group of intermediate 11 gives intermediates of type 1 using acidic conditions, for example, 4 M HCl in dioxane in a solvent such as MeOH, or preferably TFA in DCM, at about room temperature (step d).

[0075] Ketones 7 and aryl halides 9 are commercially available or can be synthesized according to methods known to those skilled in the art.

[0076] In one aspect, the present invention provides a process for preparing a compound of formula (I) as described herein, comprising: (a) reacting a first amine 4a,5,6,7,8,8a-hexahydro-4H-pyrido[4,3-b][1,4]oxazin-3-one (2) with [ka] Second Amine 1 [ka] (In the formula, R 2 , R 3 , R 4, L and PG are as described herein) in the presence of a base and a urea-forming reagent, formula 3 [ka] (In the formula, R 2 , R 3 , R 4 , L and PG are as described herein; or (b) reacting a first amine 4a,5,6,7,8,8a-hexahydro-4H-pyrido[4,3-b][1,4]oxazin-3-one (2) with [ka] Second Amine 1a [ka] (In the formula, R 1 , R 2 , R 3 , R 4 and L is as described herein) in the presence of a base and a urea-forming reagent, forming a compound of formula (I) above The present invention provides a process including:

[0077] In one embodiment there is provided a process according to the present invention wherein the base is sodium bicarbonate.

[0078] In one embodiment, there is provided a process according to the present invention wherein the urea-forming reagent is selected from bis(trichloromethyl)carbonate, phosgene, trichloromethyl chloroformate, (4-nitrophenyl)carbonate, and 1,1′-carbonyldiimidazole, preferably the urea-forming reagent is bis(trichloromethyl)carbonate.

[0079] In one embodiment, there is provided a process according to the present invention, wherein said protecting group "PG" is a Cbz or Boc protecting group.

[0080] In one aspect, the present invention provides a compound of formula (I) as described herein when prepared according to any one of the processes described herein.

[0081] MAGL inhibitory activity Compounds were profiled for MAGL inhibitory activity by determining the enzymatic activity following the hydrolysis of the natural substrate, 2-arachidonoylglycerol (2-AG), to generate arachidonic acid. Mass spectrometry may then be performed. This assay is hereinafter abbreviated as the "2-AG assay."

[0082] The 2-AG assay was performed in a 384-well assay plate (PP, Greiner, catalog no. 784201) in a total volume of 20 μL. Compound dilutions were made in 100% DMSO (VWR Chemicals 23500.297) in 3-fold dilution steps in the polypropylene plate to give a final concentration range in the assay of 12.5 μM to 0.8 pM. 0.25 μL of compound dilution (100% DMSO) was added to 9 μL of MAGL in assay buffer (50 mM TRIS (GIBCO, 15567-027), 1 mM EDTA (Fluka, 03690-100ml), 0.01% (v / v) Tween). After shaking, the plate was incubated at room temperature for 15 minutes. 10 μL of 2-arachidonoylglycerol in assay buffer was added to initiate the reaction. The final concentrations in the assay were 50 pM MAGL and 8 μM 2-arachidonoylglycerol. After shaking and incubation at room temperature for 30 min, the reaction was quenched by adding 40 μL of ACN containing 4 μM d8-arachidonic acid. The amount of arachidonic acid was monitored using an online SPE system (Agilent Rapidfire) coupled to a triple quadrupole mass spectrometer (Agilent 6460). A C18 SPE cartridge (G9205A) was used with an ACN / water liquid setup. The mass spectrometer was operated in negative electrospray mode, following the mass transitions of 303.1 → 259.1 for arachidonic acid and 311.1 → 267.0 for d8-arachidonic acid. Compound activity was calculated based on the intensity ratio (arachidonic acid / d8-arachidonic acid). [Table 1]

[0083] In one aspect, the present invention provides compounds of formula (I), and pharmaceutically acceptable salts or esters thereof as described herein, wherein the compounds of formula (I), and pharmaceutically acceptable salts or esters thereof, have an IC50 activity for MAGL inhibition as measured in the MAGL assay described herein. 50 is less than 25 μM, preferably less than 10 μM, more preferably less than 5 μM.

[0084] In one embodiment, the compounds of formula (I) and their pharmaceutically acceptable salts or esters described herein have an IC 50 (MAGL inhibition) values ​​range from 0.000001 μM to 25 μM, and certain compounds have IC 50 The IC value is 0.000005 μM to 10 μM, and certain compounds have IC 50 The value is 0.00005 μM to 5 μM.

[0085] Uses of the Compounds of the Invention The compounds of formula (I) are fluorescent imaging probes with high affinity for MAGL. Therefore, they can be used as high-resolution tools to investigate the localization (e.g., expression levels and protein distribution in health and disease), structure, dynamics, and function of MAGL in live cells. They can also be applied to cellular trafficking studies, for example, using flow cytometry fluorescence-activated cell sorting (FACS) experiments or confocal live-cell imaging.

[0086] In one aspect, the present invention provides a compound of formula (I) as described herein for use in monoacylglycerol lipase (MAGL) occupancy studies.

[0087] In a further aspect, the present invention provides a compound of formula (I) as described herein for use in diagnostic imaging of monoacylglycerol lipase (MAGL) in a mammal.

[0088] In a further aspect, the present invention provides a compound of formula (I) as described herein for use in generating equilibrium and kinetic binding data for monoacylglycerol lipase (MAGL).

[0089] In a further aspect, the present invention provides the use of compounds of formula (I) as described herein in monoacylglycerol lipase (MAGL) occupancy studies.

[0090] In a further aspect, the present invention provides the use of a compound of formula (I) as described herein in the diagnostic imaging of monoacylglycerol lipase (MAGL) in a mammal.

[0091] In a further aspect, the present invention provides the use of a compound of formula (I) as described herein for the generation of equilibrium and kinetic binding data for monoacylglycerol lipase (MAGL).

[0092] In a further aspect, the present invention provides a method of studying monoacylglycerol lipase (MAGL) occupancy, comprising contacting MAGL with a compound of formula (I) as described herein.

[0093] In a further aspect, the present invention provides a method for diagnostic imaging of monoacylglycerol lipase (MAGL) in a mammal, comprising contacting the MAGL with a compound of formula (I) described herein.

[0094] In a further aspect, the present invention provides a method for generating equilibrium and kinetic binding data for monoacylglycerol lipase (MAGL), comprising contacting MAGL with a compound of formula (I) described herein. [Example]

[0095] The present invention will be more fully understood by reference to the following examples, which, however, should not be construed as limiting the scope of the claims to the examples.

[0096] Where preparations are obtained as mixtures of enantiomers, the pure enantiomers can be separated by the methods described herein or by methods known to those skilled in the art, such as chiral chromatography (e.g., chiral SFC) or crystallization.

[0097] Unless otherwise stated, all reactions and intermediates were prepared under an argon atmosphere.

[0098] Example 1 3-(2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethoxy)ethoxy)-N-(2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-yl)(phenyl)methyl)phenoxy)ethyl)propanamide [ka]

[0099] Step a) Benzyl-(Z)-4-(phenyl(2-tosylhydrazineylidene)methyl)piperidine-1-carboxylate

[0100] A solution of 4-methylbenzenesulfonhydrazide (1.54 g, 8.29 mmol; CAS RN 1576-35-8) and tert-butyl 4-benzoylpiperidine-1-carboxylate (2.0 g, 6.91 mmol; CAS RN 922504-27-6) in 1,4-dioxane (150 mL) was stirred at 100 °C for 16 h. EtOAc and aqueous HO were added. The layers were separated and the organic layer was dried over NaSO. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography (ISCO silica gel, 25 g cartridge, Hept AcOEt 0-30) to give the title compound (3.55 g, quantitative) as a light brown oil. LC-HRMS (ESI): 492.1945 ([M+H] + ).

[0101] Step b) Benzyl 4-((3-(2-((tert-butoxycarbonyl)amino)ethoxy)phenyl)(phenyl)methylene)piperidine-1-carboxylate

[0102] A mixture of benzyl (Z)-4-(phenyl(2-tosylhydrazinylidene)methyl)piperidine-1-carboxylate (1 g, 2.03 mmol), bis(triphenylphosphine)palladium(II) chloride (143 mg, 203 μmol), lithium tert-butoxide (246 mg, 3.05 mmol), and N-Boc-2-(3-bromophenoxy)ethylamine (772 mg, 2.44 mmol; CAS RN 1098107-26-6) in 1,4-dioxane (3 mL) was stirred at 80 °C for 14 h. EtOAc and aqueous HO were added. The layers were separated, and the organic layer was dried over NaSO. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography (ISCO silica gel, 80 g cartridge, Hept AcOEt 0-30) to give the title compound (280 mg, 22%) as a pale yellow foam. LC-HRMS (ESI): 443.2309 ([M-Boc+H] + ).

[0103] Step c) tert-butyl (2-(3-(phenyl(piperidin-4-yl)methyl)phenoxy)ethyl)carbamate In an autoclave, Pd+Pt / C (2.5%+2.5%, 7.01 g) was added to a solution of benzyl 4-((3-(2-((tert-butoxycarbonyl)amino)ethoxy)phenyl)(phenyl)methylene)piperidine-1-carboxylate (4.8 g, 8.85 mmol) in methanol (240 mL) and AcOH (4.8 mL). The reaction vessel was purged with hydrogen gas three times. The mixture was stirred under 50 bar hydrogen gas pressure at 50° C. for 18 hours. The catalyst was filtered off and washed with methanol. The filtrate was concentrated in vacuo to give the title compound (3.386 g, 93%) as a colorless solid. LC-HRMS (ESI): 411.2644 ([M+H] + ).

[0104] Step d) (+)-cis-4a,5,6,7,8,8a-hexahydro-4H-pyrido[4,3-b][1,4]oxazin-3-one The enantiomers of rac-(4aR,8aS)-hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one dihydrochloride (500 mg, 2.18 mmol, ChemBridge Corporation) were separated by preparative chiral HPLC (ReprosilChiral NR column) using an isocratic mixture of EtOH (containing 0.05% NHOAc):n-heptane (30:70).

[0105] First eluting enantiomer: (+)-cis-4a,5,6,7,8,8a-hexahydro-4H-pyrido[4,3-b][1,4]oxazin-3-one. Yellow solid (0.150 g; 44.0%). MS (ESI): m / z = 157.1 [M+H] + .

[0106] Second eluting enantiomer: (-)-cis-4a,5,6,7,8,8a-hexahydro-4H-pyrido[4,3-b][1,4]oxazin-3-one. Yellow solid (0.152 g; 44.6%). MS (ESI): m / z = 157.1 [M+H] + .

[0107] Step e) tert-butyl (2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-yl)(phenyl)methyl)phenoxy)ethyl)carbamate A mixture of (+)-cis-4a,5,6,7,8,8a-hexahydro-4H-pyrido[4,3-b][1,4]oxazin-3-one (295 mg, 1.1 mmol) and sodium bicarbonate (368 mg, 4.38 mmol) in DCM (13 mL) was cooled to 0 °C. Triphosgene (228 mg, 767 μmol) was added, and the mixture was stirred at ambient temperature for 14 h and cooled to 0 °C again. tert-Butyl (2-(3-(phenyl(piperidin-4-yl)methyl)phenoxy)ethyl)carbamate (450 mg, 1.1 mmol) and DIPEA (567 mg, 766 μL, 4.38 mmol) were added. The suspension was stirred at ambient temperature for 2 h. EtOAc and aqueous H2O were added. The layers were separated, and the organic layer was dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by preparative HPLC to give the title compound (392 mg, 60%) as an off-white foam. MS (ESI): m / z = 493.4 ([M-Boc+H] + ).

[0108] Step f) (4aR,8aS)-6-(4-((3-(2-aminoethoxy)phenyl)-(phenyl)methyl)piperidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one TFA (274 mg, 185 μL, 2.4 mmol) was added to a solution of tert-butyl (2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-yl)(phenyl)methyl)phenoxy)ethyl)carbamate (178 mg, 300 μmol) in DCM (2.49 mL). The reaction mixture was stirred at ambient temperature for 3 h. Ethyl acetate and aqueous KHCO3 were added. The layers were separated. The aqueous layer was extracted with ethyl acetate. The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure to give the title compound (178 mg, quantitative) as a pale yellow foam, which was used in the next step without further purification. LC-HRMS (ESI): 493.2813 ([M+H] + ).

[0109] Step g) tert-butyl (12-oxo-15-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-yl)(phenyl)methyl)phenoxy)-3,6,9-trioxa-13-azapentadecyl)carbamate DIPEA (52.5 mg, 70.9 μL, 406 μmol) was dissolved in ((4aR,8aS)-6-(4-((3-(2-aminoethoxy)phenyl)(phenyl)methyl)piperidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one (50 mg, 101 μmol), 3-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]propanoic acid (33 mg, 101 μmol; CAS RN 0.1100-1)-2-(2-(tert-butoxycarbonylamino)ethoxy]ethoxy) ... To a mixture of 1347750-75-7) and HATU (43 mg, 112 μmol). The mixture was stirred at ambient temperature for 18 h. Ethyl acetate and aqueous KHCO3 were added. The layers were separated. The organic layer was washed with water and dried over sodium sulfate. Removal of the solvent gave the crude product, which was purified by flash chromatography (ISCO silica gel, 12 g cartridge, Hept EtOAc 0-70% and DCM MeOH 0-10%) to give the title compound (36 mg, 48%) as a colorless solid. LC-HRMS (ESI): 796.4494 ([M+H] + ).

[0110] Step h) 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-N-(2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-yl)(phenyl)methyl)phenoxy)ethyl)propanamide TFA (51 mg, 34.4 μL, 446 μmol) was added to a solution of tert-butyl (12-oxo-15-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-yl)(phenyl)methyl)phenoxy)-3,6,9-trioxa-13-azapentadecyl)carbamate (74 mg, 56 μmol) in CHCl (21 μL). The reaction mixture was stirred at ambient temperature for 3 h. Ethyl acetate and aqueous KHCO were added. The layers were separated. The aqueous layer was extracted with ethyl acetate. The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure to give the title compound (36 mg, 48%) as a pale yellow foam. LC-HRMS(ESI): 696.3963 ([M+H] + ).

[0111] Step i) 3-(2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethoxy)ethoxy)-N-(2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-yl)(phenyl)methyl)phenoxy)ethyl)propanamide 4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole (2.8 mg, 14 μmol; CAS RN 10199-89-0) and N,N-diisopropylethylamine (1.9 mg, 2.5 μL, 14 μmol) were added to a solution of 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-N-(2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-yl)(phenyl)methyl)phenoxy)ethyl)propanamide (10 mg, 14.4 μmol) in MeOH (240 μL). The reaction mixture was stirred at ambient temperature for 14 hours and poured into ethyl acetate / water. The layers were separated. The organic layer was dried over sodium sulfate and evaporated to dryness. The crude material was purified by flash chromatography (ISCO silica gel, 25 g cartridge, DCM MeOH 0-40%) to give the title compound (5 mg, 34%) as an orange solid. LC-HRMS (ESI): 859.3985 ([M+H] + ).

[0112] Example 2 3-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)-N-(2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-yl)(phenyl)methyl)phenoxy)ethyl)propanamide" [ka]

[0113] Step a) tert-butyl (2-(3-oxo-3-((2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-yl)(phenyl)methyl)phenoxy)ethyl)amino)propoxy)ethyl)carbamate Analogously to the procedure described in Example 1g), (4aR,8aS)-6-(4-((3-(2-aminoethoxy)phenyl)(phenyl)methyl)piperidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one (Example 1f) was condensed with 3-[2-(tert-butoxycarbonylamino)ethoxy]propanoic acid (CAS RN 1260092-44-1) to give the title compound as a colorless oil.

[0114] Step b) 3-(2-aminoethoxy)-N-(2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-yl)(phenyl)methyl)phenoxy)ethyl)propanamide In a similar manner to the procedure described in Example 1h), tert-butyl (2-(3-oxo-3-((2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-yl)(phenyl)methyl)phenoxy)ethyl)amino)propoxy)ethyl)carbamate was deprotected with TFA to give the title compound as an off-white solid. LC-HRMS (ESI): 608.3437 ([M+H] + ).

[0115] Step c) 3-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)-N-(2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-yl)(phenyl)methyl)phenoxy)ethyl)propanamide In a similar manner to the procedure described in Example 1 i), 3-(2-aminoethoxy)-N-(2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-yl)(phenyl)methyl)phenoxy)ethyl)propanamide was reacted with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole to give the title compound as an orange solid. LC-HRMS (ESI): 770.3461 ([M+H] + ).

[0116] Example 3 3-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethoxy)-N-(2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-yl)(phenyl)methyl)phenoxy)ethyl)propanamide [ka]

[0117] Step a) tert-butyl (2-(2-(3-oxo-3-((2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-yl)(phenyl)methyl)phenoxy)ethyl)amino)propoxy)ethoxy)ethyl)carbamate Analogously to the procedure described in Example 1g), (4aR,8aS)-6-(4-((3-(2-aminoethoxy)phenyl)(phenyl)methyl)piperidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one (Example 1f) was condensed with 3-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]propanoic acid (CAS RN 1365655-91-9) to give the title compound as a colorless oil. LC-HRMS (ESI): 774.4065 ([M+Na] + ).

[0118] Step b) 3-(2-(2-aminoethoxy)ethoxy)-N-(2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-yl)(phenyl)methyl)phenoxy)ethyl)propanamide In a similar manner to the procedure described in Example 1h), tert-butyl (2-(2-(3-oxo-3-((2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-yl)(phenyl)methyl)phenoxy)ethyl)amino)propoxy)ethoxy)ethyl)carbamate was deprotected with TFA to give the title compound as an off-white solid. LC-HRMS (ESI): 652.3702 ([M+H] + ).

[0119] Step c) 3-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethoxy)-N-(2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-yl)(phenyl)methyl)phenoxy)ethyl)propanamide In a similar manner to the procedure described in Example 1 i), 3-(2-(2-aminoethoxy)ethoxy)-N-(2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-yl)(phenyl)methyl)phenoxy)ethyl)propanamide was reacted with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole to give the title compound as an orange solid. LC-HRMS (ESI): 814.3638 ([M+H] + ).

[0120] Example 4 tert-Butyl N-[2-[3-[(R or S)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]carbamate [ka]

[0121] Step a) tert-butyl N-[2-[3-[(R or S)-phenyl(4-piperidyl)methyl]phenoxy]ethyl]carbamate

[0122] The enantiomers of rac-tert-butyl (2-(3-(phenyl(piperidin-4-yl)methyl)phenoxy)ethyl)carbamate (Example 1c) were separated by preparative chiral HPLC. First eluting enantiomer: (-)-tert-butyl N-[2-[3-[(R or S)-phenyl(4-piperidyl)methyl]phenoxy]ethyl]carbamate. White foam LC-HRMS (ESI): 411.2648 ([M+H] + ). Second eluting enantiomer: (+)-tert-butyl N-[2-[3-[(S or R)-phenyl(4-piperidyl)methyl]phenoxy]ethyl]carbamate. White foam LC-HRMS (ESI): 411.2649 ([M+H] + ).

[0123] Step b) tert-butyl N-[2-[3-[(R or S)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]carbamate In a similar manner to the procedure described in Example 1(e), (+)-cis-4a,5,6,7,8,8a-hexahydro-4H-pyrido[4,3-b][1,4]oxazin-3-one (Example 1(d)) was reacted with tert-butyl N-[2-[3-[(R or S)-phenyl(4-piperidyl)methyl]phenoxy]ethyl]carbamate to give the title compound as a colorless oil. LC-HRMS (ESI): 593.3335 ([M+H] + ).

[0124] Example 5 tert-Butyl N-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]carbamate [ka] Step a) tert-butyl N-[2-[3-[(S or R)-phenyl(4-piperidyl)methyl]phenoxy]ethyl]carbamate The enantiomers of rac-tert-butyl (2-(3-(phenyl(piperidin-4-yl)methyl)phenoxy)ethyl)carbamate (Example 1c) were separated by preparative chiral HPLC. First eluting enantiomer: (-)-tert-butyl N-[2-[3-[(R or S)-phenyl(4-piperidyl)methyl]phenoxy]ethyl]carbamate. White foam LC-HRMS (ESI): 411.2648 ([M+H] + ). Second eluting enantiomer: (+)-tert-butyl N-[2-[3-[(S or R)-phenyl(4-piperidyl)methyl]phenoxy]ethyl]carbamate. White foam LC-HRMS (ESI): 411.2649 ([M+H] + ).

[0125] Step b) tert-butyl N-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]carbamate In a similar manner to the procedure described in Example 1(e), (+)-cis-4a,5,6,7,8,8a-hexahydro-4H-pyrido[4,3-b][1,4]oxazin-3-one (Example 1(d)) was reacted with tert-butyl N-[2-[3-[(S or R)-phenyl(4-piperidyl)methyl]phenoxy]ethyl]carbamate to give the title compound as an off-white foam. LC-HRMS (ESI): 593.3336 ([M+H] + ).

[0126] Example 6 (4aR,8aS)-6-[4-[(S or R)-[3-(2-aminoethoxy)phenyl]-phenyl-methyl]piperidine-1-carbonyl]-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazin-3-one [ka]

[0127] In a similar manner to the procedure described in Example 1f), tert-butyl N-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]carbamate (Example 5b) was deprotected with TFA to give the title compound as a yellow oil. LC-HRMS (ESI): 493.2815 ([M+H] + ).

[0128] Example 7 (4aR,8aS)-6-[4-[(R or S)-[3-(2-aminoethoxy)phenyl]-phenyl-methyl]piperidine-1-carbonyl]-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazin-3-one [ka]

[0129] In a similar manner to the procedure described in Example 1f), tert-butyl N-[2-[3-[(R or S)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]carbamate (Example 4b) was deprotected with TFA to give the title compound as a yellow oil. LC-HRMS (ESI): 493.2815 ([M+H] + ).

[0130] Example 8 tert-Butyl N-[2-[2-[3-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethoxy]ethyl]carbamate [ka]

[0131] Analogously to the procedure described in Example 1 g), (4aR,8aS)-6-[4-[(S or R)-[3-(2-aminoethoxy)phenyl]-phenyl-methyl]piperidine-1-carbonyl]-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazin-3-one (Example 6) was condensed with 3-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]propanoic acid (CAS RN 1365655-91-9) to give the title compound as a white powder. LC-HRMS (ESI): 752.4237 ([M+H] + ).

[0132] Example 9 tert-Butyl N-[2-[2-[3-[2-[3-[(R or S)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethoxy]ethyl]carbamate [ka]

[0133] Analogously to the procedure described in Example 1 g), (4aR,8aS)-6-[4-[(R or S)-[3-(2-aminoethoxy)phenyl]-phenyl-methyl]piperidine-1-carbonyl]-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazin-3-one (Example 7) was condensed with 3-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]propanoic acid (CAS RN 1365655-91-9) to give the title compound as a white powder. LC-HRMS (ESI): 752.4236 ([M+H] + ).

[0134] Example 10 N-[2-[3-[(R or S)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-(2-aminoethoxy)ethoxy]propanamide [ka]

[0135] In a similar manner to the procedure described in Example 1h), tert-butyl N-[2-[2-[3-[2-[3-[(R or S)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethoxy]ethyl]carbamate (Example 9) was deprotected with TFA to give the title compound as a pale yellow oil. LC-HRMS (ESI): 652.3703 ([M+H] + ).

[0136] Example 11 N-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-(2-aminoethoxy)ethoxy]propanamide [ka]

[0137] In a similar manner to the procedure described in Example 1h), tert-butyl N-[2-[2-[3-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethoxy]ethyl]carbamate (Example 8) was deprotected with TFA to give the title compound as a pale yellow oil. LC-HRMS (ESI): 652.3705 ([M+H] + ).

[0138] Example 12 N-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethoxy]ethoxy]propanamide [ka]

[0139] In a similar manner to the procedure described in Example 1 i), N-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-(2-aminoethoxy)ethoxy]propanamide (Example 11) was reacted with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole to give the title compound as a yellow solid. LC-HRMS (ESI): 813.3573 ([M-H] - ).

[0140] Example 13 N-[2-[3-[(R or S)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethoxy]ethoxy]propanamide [ka]

[0141] In a similar manner to the procedure described in Example 1 i), N-[2-[3-[(R or S)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-(2-aminoethoxy)ethoxy]propanamide (Example 10) was reacted with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole to give the title compound as a yellow solid. LC-HRMS (ESI): 813.3571 ([M-H] - ).

[0142] Example 14 tert-Butyl N-[2-[3-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethyl]carbamate [ka]

[0143] Analogously to the procedure described in Example 1 g), (4aR,8aS)-6-[4-[(S or R)-[3-(2-aminoethoxy)phenyl]-phenyl-methyl]piperidine-1-carbonyl]-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazin-3-one (Example 6) was condensed with 3-[2-(tert-butoxycarbonylamino)ethoxy]propanoic acid (CAS RN 1260092-44-1) to give the title compound as a colorless oil. LC-HRMS (ESI): 708.3957 ([M+H] + ).

[0144] Example 15 tert-Butyl N-[2-[3-[2-[3-[(R or S)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethyl]carbamate [ka]

[0145] Analogously to the procedure described in Example 1 g), (4aR,8aS)-6-[4-[(R or S)-[3-(2-aminoethoxy)phenyl]-phenyl-methyl]piperidine-1-carbonyl]-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazin-3-one (Example 7) was condensed with 3-[2-(tert-butoxycarbonylamino)ethoxy]propanoic acid (CAS RN 1260092-44-1) to give the title compound as a colorless oil. LC-HRMS (ESI): 708.3957 ([M+H] + ).

[0146] Example 16 N-[2-[3-[(R or S)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-(2-aminoethoxy)propanamide [ka]

[0147] In a similar manner to the procedure described in Example 1h), tert-butyl N-[2-[3-[2-[3-[(R or S)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethyl]carbamate (Example 15) was deprotected with TFA to give the title compound as a pale yellow oil. LC-HRMS (ESI): 608.3442 ([M+H] + ).

[0148] Example 17 N-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethoxy]propanamide [ka]

[0149] Step a) N-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-(2-aminoethoxy)propanamide In a similar manner to the procedure described in Example 1h), tert-butyl N-[2-[3-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethyl]carbamate (Example 14) was deprotected with TFA to give the title compound as a pale yellow oil. LC-HRMS (ESI): 608.3441 ([M+H] + ).

[0150] Step b) N-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethoxy]propanamide In a similar manner to the procedure described in Example 1 i), N-[2-[3-[(R or S)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-(2-aminoethoxy)propanamide was reacted with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole to give the title compound as a yellow solid. LC-HRMS (ESI): 771.3464 ([M+H] + ).

[0151] Example 18 N-[2-[3-[(R or S)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethoxy]propanamide [ka]

[0152] In a similar manner to the procedure described in Example 1 i), N-[2-[3-[(R or S)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-(2-aminoethoxy)propanamide (Example 16) was reacted with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole to give the title compound as a yellow solid. LC-HRMS (ESI): 771.3464 ([M+H] + ).

[0153] Example 19 tert-Butyl N-[2-[2-[2-[3-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethyl]carbamate [ka]

[0154] Analogously to the procedure described in Example 1 g), (4aR,8aS)-6-[4-[(S or R)-[3-(2-aminoethoxy)phenyl]-phenyl-methyl]piperidine-1-carbonyl]-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazin-3-one (Example 7) was condensed with 3-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]propanoic acid (CAS RN 1347750-75-7) to give the title compound as a brown oil. LC-HRMS (ESI): 796.4466 ([M+H] + ).

[0155] Example 20 N-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propanamide [ka]

[0156] In a similar manner to the procedure described in Example 1h), tert-butyl N-[2-[2-[2-[3-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethyl]carbamate (Example 19) was deprotected with TFA to give the title compound as a yellow oil. LC-HRMS (ESI): 696.3961 ([M+H] + ).

[0157] Example 21 N-[2-[3-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethyl]-3-[2,2-difluoro-12-(1H-pyrrol-2-yl)-3-aza-1-azonia-2-boranuidatricyclo[7.3.0.03,7]dodeca-1(12),4,6,8,10-pentaen-4-yl]propanamide [ka]

[0158] To a stirred solution of 3-(2-aminoethoxy)-N-(2-(3-((S)-(1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-yl)(phenyl)methyl)phenoxy)ethyl)propanamide (Example 17a, 3.4 mg, 5.6 μmol) in DMF (0.5 mL) at room temperature was added 7-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)-5,5-difluoro-3-(1H-pyrrol-2-yl)-5H-514-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborin-4-ium (2 mg, 4.7 μmol; CAS RN 201998-61-0) was added, followed by DIPEA (1.8 mg, 2.4 μL, 14.1 μmol). After 14 h, DIPEA (3.0 mg, 4.1 μL, 23.5 μmol) was added, and the mixture was stirred at room temperature for an additional 24 h. Acetonitrile (0.1 mL) was added, and the crude reaction mixture was purified by preparative HPLC to give the title compound as a purple foam. LC-HRMS (ESI): 919.452 ([M+H] + ).

[0159] Example 22 N-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-[2-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethoxy]ethoxy]ethoxy]propanamide [ka]

[0160] In a similar manner to the procedure described in Example 1 i), N-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propanamide (Example 20) was reacted with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole to give the title compound as a yellow foam. LC-HRMS (ESI): 859.3996 ([M+H] + )

[0161] Example 23 tert-Butyl (2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-ylidene)(phenyl)methyl)phenoxy)ethyl)carbamate [ka]

[0162] Step a) tert-butyl (2-(3-(phenyl(piperidin-4-ylidene)methyl)phenoxy)ethyl)carbamate Pd / C 10% (10%, 10 mg) was added to a solution of benzyl 4-((3-(2-((tert-butoxycarbonyl)amino)ethoxy)phenyl)(phenyl)methylene)piperidine-1-carboxylate (110 mg, 0.2 mmol) in ethanol (10 mL). The reaction vessel was purged with hydrogen gas. The mixture was stirred under 1 atmosphere of hydrogen gas pressure at room temperature for 5 hours. The catalyst was filtered off through a short plug of Celite and washed with ethanol. The filtrate was concentrated in vacuo to give the title compound (81 mg, quantitative) as a yellow oil. NMR of the crude product revealed the desired product.

[0163] Step b) tert-butyl (2-(3-((1-(3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-ylidene)(phenyl)methyl)phenoxy)ethyl)carbamate A mixture of (+)-cis-4a,5,6,7,8,8a-hexahydro-4H-pyrido[4,3-b][1,4]oxazin-3-one as a mixture with chiral auxiliary (MW=744 g / mol, 201 mg, 0.27 mmol) and sodium bicarbonate (92 mg, 1.1 mmol) in DCM (4 mL) was cooled to 0 °C. Triphosgene (57 mg, 191 μmol) was added, and the mixture was stirred at ambient temperature for 14 h and cooled again to 0 °C. tert-Butyl (2-(3-(phenyl(piperidin-4-ylidene)methyl)phenoxy)ethyl)carbamate (115 mg, 0.27 mmol) and DIPEA (142 mg, 191 μL, 1.1 mmol) were added. The suspension was stirred at ambient temperature for 2 h. EtOAc and aqueous HO were added. The layers were separated and the organic layer was dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by column chromatography (SiO2, 30-100% EtOAc in cyclohexane, then 0-10% MeOH in DCM) to give the title compound (50 mg, 32%) as a white oil. LC-HRMS (ESI): m / z = 591.3172 ([M+H] + ).

[0164] Example 24 (4aR,8aS)-6-(4-((3-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)phenyl)(phenyl)methylene)piperidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one [ka]

[0165] Step a) (4aR,8aS)-6-(4-((3-(2-aminoethoxy)phenyl)-(phenyl)methylene)piperidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one In a similar manner to the procedure described in Example 1h), tert-butyl (2-(3-((1-(3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-ylidene)(phenyl)methyl)phenoxy)ethyl)carbamate was deprotected with TFA to give the title compound as an off-white solid. LCMS (ESI): 491.1 ([M+H] + ).

[0166] Step b) 3-(2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethoxy)ethoxy)-N-(2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-yl)(phenyl)methyl)phenoxy)ethyl)propanamide In a similar manner to the procedure described in Example 1 i), 3-(2-aminoethoxy)-N-(2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-yl)(phenyl)methyl)phenoxy)ethyl)propanamide was reacted with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole to give the title compound as an orange solid. LC-HRMS (ESI): 654.2675 ([M+H] + ).

[0167] Example 25 tert-Butyl (2-(4-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-ylidene)(phenyl)methyl)phenoxy)ethyl)carbamate [ka]

[0168] Step a) Benzyl 4-((4-(2-((tert-butoxycarbonyl)amino)ethoxy)phenyl)(phenyl)methylene)piperidine-1-carboxylate A mixture of benzyl (Z)-4-(phenyl(2-tosylhydrazinylidene)methyl)piperidine-1-carboxylate (200 mg, 0.4 mmol), tetrakis(triphenylphosphine)palladium(0) (46 mg, 40 μmol), lithium tert-butoxide (48 mg, 0.6 mmol), and N-Boc-2-(4-bromophenoxy)ethylamine (200 mg, 0.6 mmol) in 1,4-dioxane (8 mL) was stirred at 80 °C for 14 h. EtOAc and aqueous HO were added. The layers were separated, and the organic layer was dried over NaSO. The solvent was removed under reduced pressure, and the crude product was purified by flash chromatography (SiO, 15 g cartridge, 0–30% EtOAc in cyclohexane) to give the title compound (52 mg, 25%) as a pale yellow foam. LCMS(ESI): 565.1 ([M+Na] + ).

[0169] Step b) (4aR,8aS)-6-(4-((4-(2-aminoethoxy)phenyl)-(phenyl)methylene)piperidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one In a similar manner to the procedure described in Example 23 a), benzyl 4-((4-(2-((tert-butoxycarbonyl)amino)ethoxy)phenyl)(phenyl)methylene)piperidine-1-carboxylate was deprotected with Pd / C and hydrogen to give the title compound as a brown oil. LCMS (ESI): 409.1 ([M+H] + ).

[0170] Step c) tert-butyl (2-(4-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-ylidene)(phenyl)methyl)phenoxy)ethyl)carbamate In a similar manner to the procedure described in Example 23, b), (4aR,8aS)-6-(4-((4-(2-aminoethoxy)phenyl)(phenyl)methylene)piperidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one was reacted with (+)-cis-4a,5,6,7,8,8a-hexahydro-4H-pyrido[4,3-b][1,4]oxazin-3-one to form a urea using triphosgene to give the title compound as a clear oil. LC-HRMS (ESI): 591.3168 ([M+H] + ).

[0171] The following examples can be prepared similarly to Examples 1-25.

[0172] Example 26 tert-Butyl N-[3-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidylidene]-phenyl-methyl]phenyl]prop-2-ynyl]carbamate [ka]

[0173] Example 27 tert-Butyl N-[3-[4-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidylidene]-phenyl-methyl]phenyl]prop-2-ynyl]carbamate [ka]

[0174] Example 28 tert-Butyl N-[(E)-3-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidylidene]-phenyl-methyl]phenyl]allyl]carbamate [ka]

[0175] Example 29 tert-Butyl N-[(E)-3-[4-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidylidene]-phenyl-methyl]phenyl]allyl]carbamate [ka]

[0176] Example 30 tert-Butyl N-[2-[3-[(SR)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]carbamate [ka]

[0177] Example 31 tert-Butyl N-[2-[4-[(SR)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]carbamate [ka]

[0178] Example 32 tert-Butyl N-[3-[3-[(SR)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenyl]propyl]carbamate [ka]

[0179] Example 33 tert-Butyl N-[3-[4-[(SR)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenyl]propyl]carbamate [ka]

[0180] Example 34 tert-Butyl N-[3-[4-[(SR)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenyl]prop-2-ynyl]carbamate [ka]

[0181] Example 35 tert-Butyl N-[3-[3-[(SR)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenyl]prop-2-ynyl]carbamate [ka]

[0182] Example 36 tert-Butyl N-[(E)-3-[4-[(SR)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenyl]allyl]carbamate [ka]

[0183] Example 37 tert-Butyl N-[(E)-3-[3-[(SR)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenyl]allyl]carbamate [ka]

[0184] Example 38 tert-Butyl N-[2-[3-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidylidene]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethyl]carbamate [ka]

[0185] Example 39 tert-Butyl N-[3-[4-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidylidene]-phenyl-methyl]phenyl]propyl]carbamate [ka]

[0186] Example 40 tert-Butyl N-[3-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidylidene]-phenyl-methyl]phenyl]propyl]carbamate [ka]

Claims

1. Formula (I) 【Chemical 1】 or a pharmaceutically acceptable salt thereof, wherein L is, 【Chemistry 2】 is a linker selected from During the ceremony, each occurrence of n is independently an integer selected from 1, 2, 3, 4, 5, 6, and 7; The wavy line is R １ indicates the point of attachment to The asterisk indicates the point of attachment to the remainder of formula (I); R １ teeth, 【Chemistry 3】 【change】 【change】 is a fluorescent label selected from In the formula, the wavy line indicates the point of attachment to the linker L; R ２ , R ３ , and R ４ are each independently hydrogen, halogen, or C １ -C ６ -Alkyl, halo-C １ -C ６ -Alkyl, C １ -C ６ -alkoxy and halo-C １ -C ６ - selected from alkoxy; X and Y are both CH; or X and Y together form a double bond (C=C); The compound or a pharmaceutically acceptable salt thereof.

2. 2. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the linker L is 【Chemistry 4】 is selected from During the ceremony, each occurrence of n is independently an integer selected from 1, 2, and 3; The wavy line indicates the fluorescent label R １ indicates the point of attachment to The asterisk indicates the point of attachment to the remainder of formula (I). The compound or a pharmaceutically acceptable salt thereof.

3. 3. A compound of formula (I) according to claim 2 or a pharmaceutically acceptable salt thereof, wherein the linker L is 【Chemistry 5】 is selected from During the ceremony, The wavy line indicates the fluorescent label R １ indicates the point of attachment to The asterisk indicates the point of attachment to the remainder of formula (I). The compound or a pharmaceutically acceptable salt thereof.

4. A compound of formula (I) according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein the fluorescent label R １ but, 【Chemistry 6】 is selected from wherein the wavy line indicates the point of attachment to the linker L, or a pharmaceutically acceptable salt thereof.

5. R ２ 5. A compound of formula (I) according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein is hydrogen.

6. R ３ 6. A compound of formula (I) according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, wherein is hydrogen.

7. R ４ 7. A compound of formula (I) according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, wherein is hydrogen.

8. 2. The compound of formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) is a compound of formula (II) or (III): 【Chemistry 7】 During the ceremony, The linker L is 【Chemistry 8】 is selected from During the ceremony, each occurrence of n is independently an integer selected from 1, 2, and 3; The wavy line indicates the fluorescent label R １ indicates the point of attachment to The asterisk indicates the point of attachment to the remainder of formula (I); The fluorescent label R １ teeth, 【Chemistry 9】 is selected from In the formula, the wavy line indicates the point of attachment to the linker L. The compound or a pharmaceutically acceptable salt thereof.

9. 9. A compound of formula (I) according to claim 8 or a pharmaceutically acceptable salt thereof, The linker L is 【Chemistry 10】 is selected from During the ceremony, The wavy line indicates the fluorescent label R １ indicates the point of attachment to The asterisk indicates the point of attachment to the remainder of formula (I); The fluorescent label R １ teeth, 【Chemistry 11】 is selected from In the formula, the wavy line indicates the point of attachment to the linker L. The compound or a pharmaceutically acceptable salt thereof.

10. The compound of formula (I) 3-(2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethoxy)ethoxy)-N-(2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-yl)(phenyl)methyl)phenoxy)ethyl)propanamide; 3-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)-N-(2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-yl)(phenyl)methyl)phenoxy)ethyl)propanamide; 3-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethoxy)-N-(2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-yl)(phenyl)methyl)phenoxy)ethyl)propanamide; N-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethoxy]ethoxy]propanamide; N-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethoxy]propanamide; N-[2-[3-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethyl]-3-[2,2-difluoro-12-(1H-pyrrol-2-yl)-3-aza-1-azonia-2-boranuidatricyclo[7.3.0.03,7]dodeca-1(12),4,6,8,10-pentaen-4-yl]propanamide; N-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-[2-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethoxy]ethoxy]ethoxy]propanamide; and (4aR,8aS)-6-(4-((3-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)phenyl)(phenyl)methylene)piperidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one, 10. A compound of formula (I) according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, selected from:

11. The compound of formula (I) N-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethoxy]ethoxy]propanamide; N-[2-[3-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethyl]-3-[2,2-difluoro-12-(1H-pyrrol-2-yl)-3-aza-1-azonia-2-boranuidatricyclo[7.3.0.03,7]dodeca-1(12),4,6,8,10-pentaen-4-yl]propanamide; and (4aR,8aS)-6-(4-((3-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)phenyl)(phenyl)methylene)piperidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one, 11. The compound of formula (I) according to claim 10, selected from: or a pharmaceutically acceptable salt thereof.

12. tert-butyl N-[2-[3-[(R or S)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]carbamate; (4aR,8aS)-6-[4-[(R or S)-[3-(2-aminoethoxy)phenyl]-phenyl-methyl]piperidine-1-carbonyl]-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazin-3-one; tert-butyl N-[2-[2-[3-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethoxy]ethyl]carbamate; N-[2-[3-[(R or S)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-(2-aminoethoxy)ethoxy]propanamide; tert-butyl N-[2-[3-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethyl]carbamate; N-[2-[3-[(R or S)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-(2-aminoethoxy)propanamide; tert-butyl N-[2-[2-[2-[3-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethyl]carbamate; N-[2-[3-[(S or R)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]-3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propanamide; tert-butyl(2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-ylidene)(phenyl)methyl)phenoxy)ethyl)carbamate; tert-butyl(2-(4-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)piperidin-4-ylidene)(phenyl)methyl)phenoxy)ethyl)carbamate; tert-butyl N-[3-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidylidene]-phenyl-methyl]phenyl]prop-2-ynyl]carbamate; tert-butyl N-[3-[4-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidylidene]-phenyl-methyl]phenyl]prop-2-ynyl]carbamate; tert-butyl N-[(E)-3-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidylidene]-phenyl-methyl]phenyl]allyl]carbamate; tert-butyl N-[(E)-3-[4-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidylidene]-phenyl-methyl]phenyl]allyl]carbamate; tert-butyl N-[2-[3-[(SR)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]carbamate; tert-butyl N-[2-[4-[(SR)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenoxy]ethyl]carbamate; tert-butyl N-[3-[3-[(SR)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenyl]propyl]carbamate; tert-butyl N-[3-[4-[(SR)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenyl]propyl]carbamate; tert-butyl N-[3-[4-[(SR)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenyl]prop-2-ynyl]carbamate; tert-butyl N-[3-[3-[(SR)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenyl]prop-2-ynyl]carbamate; tert-butyl N-[(E)-3-[4-[(SR)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenyl]allyl]carbamate; tert-butyl N-[(E)-3-[3-[(SR)-[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidyl]-phenyl-methyl]phenyl]allyl]carbamate; tert-butyl N-[2-[3-[2-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidylidene]-phenyl-methyl]phenoxy]ethylamino]-3-oxo-propoxy]ethyl]carbamate; tert-butyl N-[3-[4-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidylidene]-phenyl-methyl]phenyl]propyl]carbamate; and tert-butyl N-[3-[3-[[1-[(4aR,8aS)-3-oxo-4,4a,5,7,8,8a-hexahydropyrido[4,3-b][1,4]oxazine-6-carbonyl]-4-piperidylidene]-phenyl-methyl]phenyl]propyl]carbamate, A compound selected from:

13. 13. Use of a compound according to claim 12 for the preparation of a compound according to any one of claims 1 to 11.

14. A process for preparing a compound of formula (I) according to any one of claims 1 to 11, comprising: (a) reacting a first amine 4a,5,6,7,8,8a-hexahydro-4H-pyrido[4,3-b][1,4]oxazin-3-one (2) with 【Chemistry 12】 A second amine 1 (wherein R ２ , R ３ , R ４ and L is as defined in any one of claims 1 to 11, and PG is a suitable protecting group; 【Chemistry 13】 reacting in the presence of a base and a urea-forming reagent, Formula 3 (wherein, R ２ , R ３ , R ４ and L is as defined in any one of claims 1 to 11, and PG is a suitable protecting group; 【Chemistry 14】 or (b) reacting a first amine 4a,5,6,7,8,8a-hexahydro-4H-pyrido[4,3-b][1,4]oxazin-3-one (2) with 【Chemistry 15】 A second amine 1a (wherein R １ , R ２ , R ３ , R ４ and L is as defined in any one of claims 1 to 11), 【Chemistry 16】 reacting in the presence of a base and a urea-forming reagent, forming a compound of formula (I) The process includes:

15. 12. A compound of formula (I) according to any one of claims 1 to 11 for use in monoacylglycerol lipase (MAGL) occupancy studies.

16. 12. A compound of formula (I) according to any one of claims 1 to 11 for use in diagnostic imaging of monoacylglycerol lipase (MAGL) in mammals.

17. 12. A compound of formula (I) according to any one of claims 1 to 11 for use in generating equilibrium and kinetic binding data for monoacylglycerol lipase (MAGL).

18. 12. Use of a compound of formula (I) according to any one of claims 1 to 11 in monoacylglycerol lipase (MAGL) occupancy studies.

19. A pharmaceutical composition for use in diagnostic imaging of monoacylglycerol lipase (MAGL) in a mammal, comprising a compound of formula (I) according to any one of claims 1 to 11.

20. 12. Use of a compound of formula (I) according to any one of claims 1 to 11 in generating equilibrium and kinetic binding data for monoacylglycerol lipase (MAGL).

21. 12. A method for studying monoacylglycerol lipase (MAGL) occupancy, comprising contacting MAGL with a compound of formula (I) according to any one of claims 1 to 11.

22. 12. A pharmaceutical composition comprising a compound of formula (I) as defined in any one of claims 1 to 11, for use in a method for diagnostic imaging of monoacylglycerol lipase (MAGL) in a mammal, the method comprising contacting MAGL with a compound of formula (I) as defined in any one of claims 1 to 11.

23. 12. A method for generating equilibrium and kinetic binding data for monoacylglycerol lipase (MAGL), comprising contacting MAGL with a compound of formula (I) according to any one of claims 1 to 11.

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