Fluorescent probe for MAGL

Abstract

The present invention provides compounds having general formula (I) or general formula (II), or pharmaceutically acceptable salts thereof, wherein A, L, n and R1 to R3 are as defined herein; comprising the compound; methods of making the compounds; and methods of using the compounds. # imgabs0 #

Description

Technical Field

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

[0002] Fluorescence imaging probes have become high-resolution tools (LAStoddart, LEKilpatrick, SJBriddon, SJHill, Neuropharmacology 2015, 98, 48-57) for studying positioning (e.g., expression levels and protein distribution in health and disease states, structure, dynamics and function of proteins in living cells). Such probes can be applied to flow cytometry fluorescence activated cell sorting (FACS) experiments or cell transport studies, for example, using confocal live cell imaging. In addition, fluorescence imaging probes allow real-time monitoring of ligand-receptor interactions and protein visualization with high spatiotemporal precision (AJVernall, SJHill, B.Kellam, Br.J.Pharmacol. 2014, 171, 1073–1084; C.Iliopoulos-Tsoutsouvas, RNKulkarni, A.Makriyannis, SPNikas, ExpertOpin.Drug Discov. 2018, 13, 933–947). In addition, such probes provide the potential for generating equilibrium and kinetic binding data in a high-throughput manner without the need to use, for example, time-resolved fluorescence resonance energy transfer (TR-FRET) to process radioactive materials. Fluorescence imaging probes can also be used to support the conversion of preclinical pharmacology animal data into clinical pharmacology animal data, and can be applied to human dose selection. They can be used as markers for target engagement, for example, via the generation of in vitro quantitative receptor binding data in whole blood. Depending on their respective applications, fluorescence imaging probes need to match specific criteria, including affinity, selectivity and specificity to their respective targets, favorable photophysical properties, and applicability in different techniques and cell types. Summary of the invention

[0003] In a first aspect, the present invention provides a compound of formula (I) or formula (II)

[0004]

[0005] or a pharmaceutically acceptable salt thereof, wherein A, L, n and R 1 To R 3 As defined herein.

[0006] In another aspect, the present invention provides a method for producing the compound of formula (I) or formula (II).

[0007] In another aspect, the present invention provides a compound of formula (I) or formula (II) as described herein, which is manufactured according to the method described herein.

[0008] In another aspect, the present invention provides use of a compound of formula (II) as described herein for preparing a fluorescent probe of formula (I) as described herein.

[0009] In another aspect, the present invention provides a method of studying monoacylglycerol lipase (MAGL) occupancy, the method comprising contacting MAGL with a compound of formula (I) as described herein.

[0010] In another aspect, the present invention provides a method for diagnostic imaging of monoacylglycerol lipase (MAGL) in a mammal, the method comprising contacting MAGL with a compound of formula (I) described herein.

[0011] In another aspect, the invention provides a method of generating equilibrium and kinetic binding data for monoacylglycerol lipase (MAGL), the method comprising contacting MAGL with a compound of formula (I) described herein. DETAILED DESCRIPTION

[0012] definition

[0013] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All features disclosed in this specification (including any accompanying claims, abstract and drawings) and / or all steps of any method or process disclosed thereby may be combined in any combination, except for combinations in which at least some of these features and / or steps are mutually exclusive. The invention is not limited to the details of any foregoing embodiments. The invention extends to any novel feature or any novel combination of features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel step or any novel combination of steps of any method or process disclosed thereby.

[0014] The term "heteroaryl" refers to a monovalent or multivalent monocyclic, bicyclic or tricyclic, preferably bicyclic ring system having a total of 5 to 14 ring members, preferably 5 to 12 ring members and more preferably 5 to 10 ring members, wherein at least one ring in the ring system is aromatic and at least one ring in the ring system contains one or more heteroatoms. Preferably, "heteroaryl" refers to a 5- to 10-membered heteroaryl containing 1, 2, 3 or 4 heteroatoms independently selected from O, S and N. Most preferably, "heteroaryl" refers to a 5- to 10-membered heteroaryl containing 1 to 2 heteroatoms independently selected from O, S and N. Some non-limiting examples of heteroaryl groups include spiro[cyclopropane-1,3'-indolyl] (e.g., spiro[cyclopropane-1,3'-indolyl]-1'-yl), 2-pyridyl, 3-pyridyl, 4-pyridyl, pyrazin-2-yl, pyrimidin-2-yl, pyrimidin-4-yl, pyrimidin-5-yl, pyrimidin-6-yl, indol-1-yl, 1H-indol-2-yl, 1H-indol-3-yl, 1H-indol-4-yl, 1H-indol-5-yl, 1H-indol-6-yl, 1H-indol-7-yl, 1,2-benzooxazol-3-yl, 1,2-benzooxazol-4-yl, 1,2-benzooxazol-5-yl, 1,2-benzooxazol-6-yl, 1,2-benzooxazol-7-yl, 1H-indazol-3-yl, 1H-indazol-4-yl, 1H-indazol-5-yl, 1H-indazol-6-yl, 1H-indazol-7-yl, pyrazol-1-yl, 1H-pyrazol-3-yl, 1H-pyrazol-7-yl oxazol-4-yl, 1H-pyrazol-5-yl, pyrazolo[1,5-a]pyridine, 2H-pyrazolo[4,3-b]pyridine, [1,2,4]triazolo[1,5-a]pyridine, 1H-pyrrolo[2,3-b]pyridine, imidazol-1-yl, 1H-imidazol-2-yl, 1H-imidazol-4-yl, 1H-imidazol-5-yl, oxazol-2-yl, oxazol-4-yl, oxazol-5-yl, 1,2,4-oxadiazolyl, 1,3,4- oxadiazolyl, thiazol-2-yl, thiazol-4-yl, thiazol-5-yl, pyridazin-3-yl, pyridazin-4-yl, 1,2,4-triazol-4-yl, 1,2,4-triazol-1-yl, 4H-1,2,4-triazol-3-yl, 4,5,6,7-tetrahydroindazol-2-yl, 6,7-dihydro-4H-pyrano[4,3-c]pyrazol-2-yl, thiazolyl, benzofurazan-4-yl, tetrazolyl, isoxazolyl, pyrrolyl and morpholinyl.

[0015] The term "aryl" refers to a monocyclic, bicyclic or tricyclic carbocyclic ring system ("C 6 -C 10 -aryl"), wherein at least one ring in the ring system is aromatic. Some non-limiting examples of aryl include phenyl and 9H-fluorenyl (e.g., 9H-fluoren-9-yl). A particularly preferred but non-limiting example of aryl is phenyl.

[0016] The term "alkyl" refers to a monovalent or polyvalent (e.g., monovalent or divalent) straight or branched chain saturated hydrocarbon group containing 1 to 12 carbon atoms. In some preferred embodiments, the alkyl group contains 1 to 6 carbon atoms, such as 1, 2, 3, 4, 5 or 6 carbon atoms ("C 1 -C 6 -alkyl"). In other embodiments, the alkyl group contains 1 to 3 carbon atoms, such as 1, 2, or 3 carbon atoms. Some non-limiting examples of alkyl include methyl, ethyl, propyl, 2-propyl (isopropyl), n-butyl, isobutyl, sec-butyl, tert-butyl, and 2,2-dimethylpropyl. A particularly preferred but non-limiting example of an alkyl is methyl.

[0017] The term "alkoxy" refers to an alkyl group as defined above attached to the parent molecular moiety via an oxygen atom. Unless otherwise specified, an alkoxy group contains 1 to 12 carbon atoms. In some preferred embodiments, an alkoxy group contains 1 to 6 carbon atoms ("C 1 -C 6 -alkoxy"). In other embodiments, the alkoxy group contains 1 to 4 carbon atoms. In other embodiments, the 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.

[0018] The term "alkoxycarbonyl" refers to an alkoxy group as defined above attached to the parent molecular moiety through a carbonyl group. In some preferred embodiments, the alkoxy portion of the alkoxycarbonyl group contains 1 to 6 carbon atoms ("C 1 -C 6 -alkoxycarbonyl"). In other embodiments, the alkoxy group contains 1 to 4 carbon atoms. In other embodiments, the alkoxy group contains 1 to 3 carbon atoms. Some non-limiting examples of alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl, n-butoxycarbonyl, isobutoxycarbonyl, and tert-butoxycarbonyl. A particularly preferred but non-limiting example of alkoxy is tert-butoxycarbonyl.

[0019] The term "pharmaceutical salt" refers to those salts that retain the biological effect and properties of free alkali or free acid, which are not undesirable in biology or other aspects. These salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. (particularly 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, methylsulfonic acid, ethylsulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcysteine, etc. In addition, these salts can be prepared by adding inorganic bases or organic bases to free acids. Salts derived from inorganic bases include but are not limited to sodium, potassium, lithium, ammonium, calcium, magnesium salts, etc. 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, and the like). A particular pharmaceutically acceptable salt of the compound of formula (I) is the hydrochloride salt.

[0020] The compounds of formula (I) may contain several asymmetric centers and may exist as optically pure enantiomers, mixtures of enantiomers (eg racemates), optically pure diastereomers, mixtures of diastereomers, diastereomeric racemates or mixtures of diastereomeric racemates.

[0021] According to the Cahn-Ingold-Prelog Convention, an asymmetric carbon atom can be in either the "R" or "S" configuration.

[0022] The abbreviation "MAGL" refers to monoacylglycerol lipase. The terms "MAGL" and "monoacylglycerol lipase" are used interchangeably herein.

[0023] Compounds of the present invention

[0024] In a first aspect, the present invention provides a compound of formula (I) or formula (II)

[0025]

[0026] or a pharmaceutically acceptable salt thereof, wherein:

[0027] R 1 Selected from:

[0028]

[0029] The wavy line indicates R 1 The point of attachment to the rest of Formula (I) or Formula (II);

[0030] R 2 Selected from:

[0031]

[0032] in:

[0033] Wavy line indicates R 2 The point of attachment to the rest of formula (I);

[0034] p is 1 or 2;

[0035] V is selected from hydrogen and methyl;

[0036] W is selected from methyl, phenyl,

[0037] in

[0038] Ar is C 6 -C 10 - aryl or 5- to 14-membered heteroaryl;

[0039] X is selected from O, S, Si(CH 3 ) 2 and CH 2 ;and

[0040] Y is selected from OH, NH 2 、N(CH 3 ) 2 、N(CD 3 ) 2 and azetidin-1-yl;

[0041] R 3 Selected from hydrogen, C 1 -C 6 -alkoxycarbonyl and C 1 -C 6 -Alkoxycarbonyl-NH-C 1 -C 6 -alkyl-NH-C(O)–;

[0042] A is:

[0043] (i) and

[0044] L is selected from –CH 2 –, –CH 2 O–, –OCH 2 –, –CH 2 OCH 2 – and –O–; or

[0045] (ii) in

[0046] (i) X is CH; and

[0047] L is selected from –CH 2 –, –CH 2 O–, –OCH 2 –, –CH 2 OCH 2 – and –O–; or

[0048] (ii) X and L together form a group wherein the wavy line indicates the point of attachment of L to the phenyl moiety within formula (I) or formula (II), and the two asterisks indicate the points of attachment of X to the corresponding adjacent atoms within ring A;

[0049] wherein the wavy line indicates the point of attachment of A to L; and the asterisk indicates the point of attachment of A to the carbonyl group of formula (I) or formula (II); and

[0050] n is an integer selected from 1, 2, 3 and 4.

[0051] In one embodiment, the present invention provides a compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0052] In formula (I), R 1 Selected from:

[0053]

[0054] The wavy line indicates R 1 The point of attachment to the rest of formula (I);

[0055] In formula (II), R 1 Selected from:

[0056]

[0057] The wavy line indicates R 1 The point of attachment to the rest of formula (II);

[0058] R 2 Selected from:

[0059] in:

[0060] Wavy line indicates R 2 The point of attachment to the rest of formula (I);

[0061] p is 1 or 2;

[0062] V is selected from hydrogen and methyl;

[0063] W is selected from methyl, phenyl, in

[0064] Ar is C 6 -C 10 - aryl or 5- to 14-membered heteroaryl;

[0065] X is selected from O, S, Si(CH 3 ) 2 and CH 2 ;and

[0066] Y is selected from OH, NH 2 、N(CH 3 ) 2 、N(CD 3 ) 2 and azetidin-1-yl;

[0067] R 3 Selected from hydrogen, C 1 -C 6 -alkoxycarbonyl and C 1 -C 6 -Alkoxycarbonyl-NH-C 1 -C 6 -alkyl-NH-C(O)–;

[0068] A is:

[0069] (i) and L is selected from –CH 2 –, –CH 2 O–, –OCH 2 –, –CH 2 OCH 2 – and –O–; or

[0070] (ii) in

[0071] (i) X is CH; and

[0072] L is selected from –CH 2 –, –CH 2 O–, –OCH 2 –, –CH 2 OCH 2 – and –O–;

[0073] or

[0074] (ii) X and L together form a group wherein the wavy line indicates the point of attachment of L to the phenyl moiety within formula (I) or formula (II), and the two asterisks indicate the points of attachment of X to the corresponding adjacent atoms within ring A;

[0075] wherein the wavy line indicates the point of attachment of A to L; and the asterisk indicates the point of attachment of A to the carbonyl group of formula (I) or formula (II); and

[0076] n is an integer selected from 1, 2, 3 and 4.

[0077] In one aspect, the present invention provides a compound of formula (I)

[0078]

[0079] or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 , A, L and n are as defined herein.

[0080] In one aspect, the present invention provides a compound of formula (II)

[0081]

[0082] or a pharmaceutically acceptable salt thereof, wherein R 1 , R 3 , A and L are as defined herein.

[0083] In one embodiment, the present invention provides a compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof as described herein, wherein R 1 Selected from:

[0084]

[0085] The wavy line indicates R 1 The point of attachment to the rest of Formula (I).

[0086] In one embodiment, the present invention provides a compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof as described herein, wherein R 1 for

[0087]

[0088] The wavy line indicates R 1 The point of attachment to the rest of Formula (I).

[0089] In one embodiment, the present invention provides a compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof as described herein, wherein R 1 for

[0090]

[0091] The wavy line indicates R 1 The point of attachment to the rest of Formula (I).

[0092] In one embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein A is:

[0093] (i) and

[0094] L is selected from –CH 2 O–, –OCH 2 –, –CH 2 OCH 2 – and –O–; or

[0095] (ii) in

[0096] (i) X is CH; and

[0097] L is selected from –CH 2 – and –CH 2 O–; or

[0098] (ii) X and L together form a group wherein the wavy line indicates the point of attachment of L to the phenyl moiety within formula (I), and the two asterisks indicate the points of attachment of X to the corresponding adjacent atoms within ring A;

[0099] wherein the wavy line indicates the point of attachment of A to L; and the asterisk indicates the point of attachment of A to the carbonyl group of formula (I).

[0100] In one embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein

[0101] A is:

[0102] (i) and L is –CH 2 OCH 2 -;or

[0103] (ii) in

[0104] (i) X is CH and L is –CH 2 -;or

[0105] (ii) X and L together form a group wherein the wavy line indicates the point of attachment of L to the phenyl moiety within formula (I), and the two asterisks indicate the points of attachment of X to the corresponding adjacent atoms within ring A;

[0106] wherein the wavy line indicates the point of attachment of A to L; and the asterisk indicates the point of attachment of A to the carbonyl group of formula (I).

[0107] In a preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein A is:

[0108] (i) and L is –CH 2 OCH 2 -;or

[0109] (ii) and L is –CH 2 -;in

[0110] X is CH;

[0111] wherein the wavy line indicates the point of attachment of A to L; and the asterisk indicates the point of attachment of A to the carbonyl group of formula (I).

[0112] In a particularly preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein A is and L is –CH 2 OCH 2 –

[0113] wherein the wavy line indicates the point of attachment of A to L; and the asterisk indicates the point of attachment of A to the carbonyl group of formula (I).

[0114] In a particularly preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein A is and L is –CH 2 -;in

[0115] X is CH;

[0116] The wavy line indicates the point of attachment of A to L; and the asterisk indicates the point of attachment of A to the carbonyl group of formula (I).

[0117] In one embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0118] R 2 Selected from:

[0119]

[0120] in:

[0121] Wavy line indicates R 2 The point of attachment to the rest of formula (I);

[0122] p is 1;

[0123] V is a methyl group;

[0124] W is a methyl group;

[0125] X is selected from O and Si(CH 3 ) 2 ;and

[0126] Y is N(CH 3 ) 2 ;and

[0127] n is an integer selected from 1, 2 and 4.

[0128] In a preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0129] R 2 Selected from:

[0130]

[0131] in:

[0132] Wavy line indicates R 2 The point of attachment to the rest of formula (I);

[0133] p is 1;

[0134] V is a methyl group;

[0135] W is a methyl group;

[0136] X is selected from O and Si(CH 3 ) 2 ;and

[0137] Y is N(CH 3 ) 2 ;and

[0138] n is an integer selected from 1, 2 and 4.

[0139] In a preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0140] R 2 Selected from:

[0141]

[0142] in:

[0143] Wavy line indicates R 2 The point of attachment to the rest of formula (I);

[0144] p is 1;

[0145] V is a methyl group;

[0146] W is a methyl group;

[0147] X is selected from O and Si(CH 3 ) 2 ;and

[0148] Y is N(CH 3 ) 2 ;and

[0149] n is an integer selected from 1 and 4.

[0150] In a particularly preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0151] R 2 for in:

[0152] Wavy line indicates R 2 The point of attachment to the rest of formula (I); and

[0153] n is an integer selected from 1 and 4.

[0154] In a particularly preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0155] R 2 for in:

[0156] Wavy line indicates R 2 The point of attachment to the rest of formula (I);

[0157] V is a methyl group;

[0158] W is methyl; and

[0159] n is an integer selected from 1 and 4.

[0160] In a particularly preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0161] R 2 for in:

[0162] Wavy line indicates R 2 The point of attachment to the rest of formula (I);

[0163] X is selected from O and Si(CH 3 ) 2 ;

[0164] Y is N(CH 3 )2 ;and

[0165] n is an integer selected from 1 and 4.

[0166] In a particularly preferred embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein, wherein:

[0167] R 2 for in:

[0168] Wavy line indicates R 2 The point of attachment to the rest of formula (I);

[0169] p is 1; and

[0170] n is an integer selected from 1 and 4.

[0171] In one embodiment, the present invention provides a compound of formula (II) or a pharmaceutically acceptable salt thereof as described herein, wherein A is:

[0172] (i) and L is selected from –OCH 2 –, –CH 2 OCH 2 – and –O–; or

[0173] (ii) and L is –CH 2 O–; wherein X is CH;

[0174] wherein the wavy line indicates the point of attachment to L; and the asterisk indicates the point of attachment to the carbonyl group of formula (II).

[0175] In one embodiment, the present invention provides a compound of formula (I) as described herein, wherein:

[0176] R 1 Selected from:

[0177]

[0178] The wavy line indicates R 1 The point of attachment to the rest of formula (I);

[0179] R 2 Selected from:

[0180]

[0181] in:

[0182] Wavy line indicates R 2 The point of attachment to the rest of formula (I);

[0183] p is 1;

[0184] V is methyl;

[0185] W is methyl;

[0186] X is selected from O and Si(CH 3 ) 2 ;and

[0187] Y is N(CH 3 ) 2 ;

[0188] A is:

[0189] (i) and

[0190] L is selected from –CH 2 O–, –OCH 2 –, –CH 2 OCH 2 – and –O–; or

[0191] (ii) in

[0192] (i) X is CH; and

[0193] L is selected from –CH 2 – and –CH 2 O–; or

[0194] (ii) X and L together form a group wherein the wavy line indicates the point of attachment of L to the phenyl moiety within formula (I), and the two asterisks indicate the points of attachment of X to the corresponding adjacent atoms within ring A;

[0195] wherein the wavy line indicates the point of attachment to L; and the asterisk indicates the point of attachment to the carbonyl group of formula (I); and

[0196] n is an integer selected from 1, 2 and 4.

[0197] In a preferred embodiment, the present invention provides a compound of formula (I) as described herein, wherein:

[0198] R 1 Selected from:

[0199]

[0200] The wavy line indicates R 1 The point of attachment to the rest of formula (I);

[0201] R 2 Selected from:

[0202]

[0203] in:

[0204] Wavy line indicates R 2 The point of attachment to the rest of formula (I);

[0205] p is 1;

[0206] V is methyl;

[0207] W is methyl;

[0208] X is selected from O and Si(CH 3 ) 2 ;and

[0209] Y is N(CH 3 ) 2 ;

[0210] A is:

[0211] (i) and L is –CH 2 OCH 2 -;or

[0212] (ii) in

[0213] (i) X is CH and L is –CH 2 -;or

[0214] (ii) X and L together form a group wherein the wavy line indicates the point of attachment of L to the phenyl moiety within formula (I), and the two asterisks indicate the points of attachment of X to the corresponding adjacent atoms within ring A;

[0215] wherein the wavy line indicates the point of attachment of A to L; and the asterisk indicates the point of attachment of A to the carbonyl group of formula (I); and

[0216] n is an integer selected from 1, 2 and 4.

[0217] In a particularly preferred embodiment, the present invention provides a compound of formula (I) as described herein, wherein:

[0218] R 1 Selected from:

[0219]

[0220] The wavy line indicates R 1 The point of attachment to the rest of formula (I);

[0221] R 2 Selected from:

[0222]

[0223] in:

[0224] Wavy line indicates R 2 The point of attachment to the rest of formula (I);

[0225] p is 1;

[0226] V is methyl;

[0227] W is methyl;

[0228] X is selected from O and Si(CH 3 ) 2 ;and

[0229] Y is N(CH 3 ) 2 ;

[0230] A is:

[0231] (i) and L is –CH 2 OCH 2 -;or

[0232] (ii) and L is –CH 2 -;in

[0233] X is CH;

[0234] wherein the wavy line indicates the point of attachment of A to L; and the asterisk indicates the point of attachment of A to the carbonyl group of formula (I); and

[0235] n is an integer selected from 1 and 4.

[0236] In one embodiment, the present invention provides a compound of formula (I) or formula (II) as described herein, wherein the compound of formula (I) or formula (II) is selected from:

[0237] benzyl (2-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamate;

[0238] benzyl (2-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)oxy)methyl)phenoxy)ethyl)carbamate;

[0239] benzyl (2-(2-(((2-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)-2-azaspiro[3.3]heptan-6-yl)oxy)methyl)phenoxy)ethyl)carbamate;

[0240] benzyl (2-(2-(((2-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)-2-azaspiro[3.5]nonan-7-yl)oxy)methyl)phenoxy)ethyl)carbamate;

[0241] benzyl (2-(3-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamate;

[0242] benzyl (2-(3-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)oxy)methyl)phenoxy)ethyl)carbamate;

[0243] benzyl (2-(3-(((2-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)-2-azaspiro[3.3]heptan-6-yl)oxy)methyl)phenoxy)ethyl)carbamate;

[0244] benzyl (3-(3-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)propyl)carbamate;

[0245] benzyl (6-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)hexyl)carbamate;

[0246] benzyl (2-(2-(((1-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamate;

[0247] Cyclohex-1-en-1-ylmethyl (2-(2-((2-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)-2-azaspiro[3.3]heptan-6-ylidene)methyl)phenoxy)ethyl)carbamate;

[0248] benzyl (2-(3-((2-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)-2-azaspiro[3.3]heptane-6-ylidene)methyl)phenoxy)ethyl)carbamate;

[0249] benzyl (2-(2-((2-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)-2-azaspiro[3.3]heptane-6-ylidene)methyl)phenoxy)ethyl)carbamate;

[0250] benzyl (2-(2-(((7-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)-7-azaspiro[3.5]nonan-2-yl)oxy)methyl)phenoxy)ethyl)carbamate;

[0251] benzyl (5-(2-((2-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)-2-azaspiro[3.3]heptane-6-ylidene)methyl)phenoxy)pentyl)carbamate;

[0252] benzyl (2-(2-((((1R,5S)-3-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)-3-azabicyclo[3.1.0]hexan-6-yl)methoxy)methyl)phenoxy)ethyl)carbamate;

[0253] (R)-benzyl(2-(2-(((1-(3-(2-oxooxazolidin-4-yl)propanoyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamate;

[0254] Benzyl (2-(2-(((1-(3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamate;

[0255] 2-(6-(2-(2-((7-nitro-112,312-benzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)benzyl)-2-azaspiro[3.3]heptane-2-carbonyl)-7-oxa-2,5-diazaspiro[3.4]octan-6-one;

[0256] (4aR,8aS)-6-(3-(((2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)benzyl)oxy)methyl)azetidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one;

[0257] 2-(3-(((2-(2-((7-nitro-1H-312-benzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)benzyl)oxy)methyl)azetidine-1-carbonyl)-7-oxa-2,5-diazaspiro[3.4]octan-6-one;

[0258] (4aR,8aS)-6-(3-(((2-(3-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)propoxy)benzyl)oxy)methyl)azetidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one;

[0259] (4aR,8aS)-6-(3-(((2-((5-((7-nitro-1,3-dihydrobenzo[c][1,2,5]oxadiazol-4-yl)amino)pentyl)oxy)benzyl)oxy)methyl)azetidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one;

[0260] (4aR,8aS)-6-(6-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)benzyl)-2-azaspiro[3.3]heptane-2-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one;

[0261] (4aR,8aS)-6-(6-(3-(2-((7-nitro-1,3-dihydrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)benzyl)-2-azaspiro[3.3]heptane-2-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one;

[0262] 2-(7-(dimethylamino)-3-(dimethylimino)-5,5-dimethyl-3,5-dihydrodibenzo[b,e]silin-10-yl)-4-((5-(2-((2-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)-2-azaspiro[3.3]heptane-6-yl)methyl)phenoxy)pentyl)carbamoyl)benzoate;

[0263] 3,3-Dimethyl-1-(6-oxo-6-((2-(2-((2-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)-2-azaspiro[3.3]heptane-6-yl)methyl)phenoxy)ethyl)amino)hexyl)-2-((E)-3-((Z)-1,3,3-trimethylindolin-2-ylidene)prop-1-en-1-yl)-3H-indol-1-ium 2,2,2-trifluoroacetate;

[0264] 3-(5,5-difluoro-7,9-dimethyl-5H-514,614-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-3-yl)-N-(2-(2-((2-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)-2-azaspiro[3.3]heptane-6-yl)methyl)phenoxy)ethyl)propionamide;

[0265] 2,2,2-trifluoroacetic acid, 3,3-dimethyl-1-(6-oxo-6-((2-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)amino)hexyl)-2-((E)-3-((Z)-1,3,3-trimethylindolin-2-ylidene)prop-1-en-1-yl)-3H-indol-1-ium salt;

[0266] 3,3-Dimethyl-1-(6-oxo-6-((2-(2-((2-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)-2-azaspiro[3.3]heptan-6-yl)methyl)phenoxy)ethyl)amino)hexyl)-2-((E)-3-((Z)-1,3,3-trimethylindolin-2-ylidene)prop-1-en-1-yl)-3H-indol-1-ium 2,2,2-trifluoroacetate;

[0267] 3-(5,5-difluoro-7,9-dimethyl-5H-514,614-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinine-3-yl)-N-(2-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)propanamide;

[0268] 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)-4-((2-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamoyl)benzoate and 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)-5-((2-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamoyl)benzoate;

[0269] 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)-5-((2-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamoyl)benzoate;

[0270] 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthen-9-yl)-4-((3-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)propyl)carbamoyl)benzoate and 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthen-9-yl)-5-((3-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)propyl)carbamoyl)benzoate;

[0271] 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)-5-((3-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)propyl)carbamoyl)benzoate;

[0272] 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)-4-((5-(2-((2-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)-2-azaspiro[3.3]heptan-6-yl)methyl)phenoxy)pentyl)carbamoyl)benzoate compounds and 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)-5-((5-(2-((2-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)-2-azaspiro[3.3]heptan-6-yl)methyl)phenoxy)pentyl)carbamoyl)benzoate;

[0273] 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)-5-((5-(2-((2-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)-2-azaspiro[3.3]heptan-6-yl)methyl)phenoxy)pentyl)carbamoyl)benzoate;

[0274] 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthen-9-yl)-4-((5-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)pentyl)carbamoyl)benzoate and 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthen-9-yl)-5-((5-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)pentyl)carbamoyl)benzoate;

[0275] 3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)benzoic acid ethyl ester;

[0276] Ethyl 3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)oxy)benzoate;

[0277] 2-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)benzoic acid ethyl ester;

[0278] tert-Butyl (2-(3-((1-((4aR,8aS)-3-oxooctahhydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)oxy)benzamido)ethyl)carbamate;

[0279] tert-Butyl (2-(2-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)benzamido)ethyl)carbamate;

[0280] (4aR,8aS)-6-(3-((benzyloxy)methyl)azetidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one;

[0281] (4aR,8aS)-6-(6-(Benzyloxy)-2-azaspiro[3.3]heptane-2-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one; and

[0282] (4aR,8aS)-6-(3-(Phenoxymethyl)azetidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one.

[0283] In one embodiment, the present invention provides a compound of formula (I) as described herein, wherein the compound of formula (I) is selected from:

[0284] benzyl (6-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)hexyl)carbamate;

[0285] benzyl (2-(2-((2-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)-2-azaspiro[3.3]heptane-6-ylidene)methyl)phenoxy)ethyl)carbamate;

[0286] benzyl (5-(2-((2-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)-2-azaspiro[3.3]heptane-6-ylidene)methyl)phenoxy)pentyl)carbamate;

[0287] (R)-benzyl(2-(2-(((1-(3-(2-oxooxazolidin-4-yl)propanoyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamate;

[0288] Benzyl (2-(2-(((1-(3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamate;

[0289] 2-(6-(2-(2-((7-nitro-112,312-benzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)benzyl)-2-azaspiro[3.3]heptane-2-carbonyl)-7-oxa-2,5-diazaspiro[3.4]octan-6-one;

[0290] (4aR,8aS)-6-(3-(((2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)benzyl)oxy)methyl)azetidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one;

[0291] 2-(3-(((2-(2-((7-nitro-1H-312-benzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)benzyl)oxy)methyl)azetidine-1-carbonyl)-7-oxa-2,5-diazaspiro[3.4]octan-6-one;

[0292] (4aR,8aS)-6-(3-(((2-(3-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)propoxy)benzyl)oxy)methyl)azetidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one;

[0293] (4aR,8aS)-6-(3-(((2-((5-((7-nitro-1,3-dihydrobenzo[c][1,2,5]oxadiazol-4-yl)amino)pentyl)oxy)benzyl)oxy)methyl)azetidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one;

[0294] (4aR,8aS)-6-(6-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)benzyl)-2-azaspiro[3.3]heptane-2-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one;

[0295] (4aR,8aS)-6-(6-(3-(2-((7-nitro-1,3-dihydrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)benzyl)-2-azaspiro[3.3]heptane-2-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one;

[0296] 2-(7-(dimethylamino)-3-(dimethylimino)-5,5-dimethyl-3,5-dihydrodibenzo[b,e]silan-10-yl)-4-((5-(2-((2-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)-2-azaspiro[3.3]heptan-6-yl)methyl)phenoxy)pentyl)carbamoyl)benzoate;

[0297] 3,3-Dimethyl-1-(6-oxo-6-((2-(2-((2-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)-2-azaspiro[3.3]heptane-6-yl)methyl)phenoxy)ethyl)amino)hexyl)-2-((E)-3-((Z)-1,3,3-trimethylindolin-2-ylidene)prop-1-en-1-yl)-3H-indol-1-ium 2,2,2-trifluoroacetate;

[0298] 3-(5,5-difluoro-7,9-dimethyl-5H-5l4,6l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinine-3-yl)-N-(2-(2-((2-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)-2-azaspiro[3.3]heptane-6-yl)methyl)phenoxy)ethyl)propanamide;

[0299] 2,2,2-trifluoroacetic acid, 3,3-dimethyl-1-(6-oxo-6-((2-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)amino)hexyl)-2-((E)-3-((Z)-1,3,3-trimethylindolin-2-ylidene)prop-1-en-1-yl)-3H-indol-1-ium salt;

[0300] 3,3-Dimethyl-1-(6-oxo-6-((2-(2-((2-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)-2-azaspiro[3.3]heptan-6-yl)methyl)phenoxy)ethyl)amino)hexyl)-2-((E)-3-((Z)-1,3,3-trimethylindolin-2-ylidene)prop-1-en-1-yl)-3H-indol-1-ium 2,2,2-trifluoroacetate;

[0301] 3-(5,5-difluoro-7,9-dimethyl-5H-514,614-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinine-3-yl)-N-(2-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)propanamide;

[0302] 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)-4-((2-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamoyl)benzoate and 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)-5-((2-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamoyl)benzoate;

[0303] 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)-5-((2-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamoyl)benzoate;

[0304] 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthen-9-yl)-4-((3-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)propyl)carbamoyl)benzoate and 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthen-9-yl)-5-((3-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)propyl)carbamoyl)benzoate;

[0305] 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)-5-((3-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)propyl)carbamoyl)benzoate;

[0306] 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)-4-((5-(2-((2-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)-2-azaspiro[3.3]heptan-6-yl)methyl)phenoxy)pentyl)carbamoyl)benzoate compounds and 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)-5-((5-(2-((2-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)-2-azaspiro[3.3]heptan-6-yl)methyl)phenoxy)pentyl)carbamoyl)benzoate;

[0307] 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)-5-((5-(2-((2-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)-2-azaspiro[3.3]heptan-6-yl)methyl)phenoxy)pentyl)carbamoyl)benzoate; and

[0308] 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthen-9-yl)-4-((5-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)pentyl)carbamoyl)benzoate and 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthen-9-yl)-5-((5-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)pentyl)carbamoyl)benzoate.

[0309] In one embodiment, the present invention provides a compound of formula (II) as described herein, wherein the compound of formula (II) is selected from:

[0310] benzyl (2-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamate;

[0311] benzyl (2-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)oxy)methyl)phenoxy)ethyl)carbamate;

[0312] benzyl (2-(2-(((2-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)-2-azaspiro[3.3]heptan-6-yl)oxy)methyl)phenoxy)ethyl)carbamate;

[0313] benzyl (2-(2-(((2-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)-2-azaspiro[3.5]nonan-7-yl)oxy)methyl)phenoxy)ethyl)carbamate;

[0314] benzyl (2-(3-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamate;

[0315] benzyl (2-(3-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)oxy)methyl)phenoxy)ethyl)carbamate;

[0316] benzyl (2-(3-(((2-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)-2-azaspiro[3.3]heptan-6-yl)oxy)methyl)phenoxy)ethyl)carbamate;

[0317] benzyl (3-(3-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)propyl)carbamate;

[0318] benzyl (2-(2-(((1-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamate;

[0319] Cyclohex-1-en-1-ylmethyl (2-(2-((2-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)-2-azaspiro[3.3]heptan-6-ylidene)methyl)phenoxy)ethyl)carbamate;

[0320] benzyl (2-(3-((2-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)-2-azaspiro[3.3]heptane-6-ylidene)methyl)phenoxy)ethyl)carbamate;

[0321] benzyl (2-(2-(((7-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)-7-azaspiro[3.5]nonan-2-yl)oxy)methyl)phenoxy)ethyl)carbamate;

[0322] benzyl (2-(2-((((1R,5S)-3-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)-3-azabicyclo[3.1.0]hexan-6-yl)methoxy)methyl)phenoxy)ethyl)carbamate;

[0323] 3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)benzoic acid ethyl ester;

[0324] Ethyl 3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)oxy)benzoate;

[0325] 2-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)benzoic acid ethyl ester;

[0326] tert-Butyl (2-(3-((1-((4aR,8aS)-3-oxooctahhydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)oxy)benzamido)ethyl)carbamate;

[0327] tert-Butyl (2-(2-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)benzamido)ethyl)carbamate;

[0328] (4aR,8aS)-6-(3-((benzyloxy)methyl)azetidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one;

[0329] (4aR,8aS)-6-(6-(Benzyloxy)-2-azaspiro[3.3]heptane-2-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one; and

[0330] (4aR,8aS)-6-(3-(phenoxymethyl)azetidine-1-carbonyl)hexahydro-2H-pyrido[4,3-

[0331] b][1,4]oxazin-3(4H)-one.

[0332] In a particular embodiment, the invention provides a pharmaceutically acceptable salt of a compound according to formula (I) as described herein. In another particular embodiment, the invention provides a compound according to formula (I) as described herein as a free base.

[0333] Preparation process

[0334] The preparation of the compounds of formula (I) or formula (II) of the present invention can be carried out according to sequential or convergent synthetic routes. The synthesis of the present invention is shown in the following general scheme. The skills required for performing the reaction and purifying the resulting product are known to those skilled in the art. Unless otherwise indicated, the substituents and indices used in the following method descriptions have the meanings provided herein.

[0335] If one of the starting materials, intermediates or compounds of formula (I) or (II) contains one or more functional groups that are unstable or reactive under the reaction conditions of one or more reaction steps, appropriate protecting groups may be introduced before applying key steps of methods well known in the art (such as "Protective Groups in Organic Chemistry" by TW Greene and PGM Wutts, 5th edition, 2014, John Wiley & Sons, NY). Such protecting groups may be removed at a later stage of the synthesis using standard methods described in the literature.

[0336] If the starting material or intermediate contains a stereogenic center, the compound of formula (I) or formula (II) can be obtained in the form of a mixture of diastereomers or enantiomers, which can be separated by methods well known in the art (e.g., chiral HPLC, chiral SFC or chiral crystallization). Racemic compounds can be separated into corresponding enantiomers, for example, by diastereomeric salts, which are crystallized with optically pure acids, or by specific chromatography separations of enantiomers using chiral adsorbents or chiral eluents. Starting materials and intermediates containing stereogenic centers can also be separated to provide starting materials and intermediates enriched with diastereomers / enantiomers. The use of such diastereomer / enantiomer-enriched starting materials and intermediates in the synthesis of compounds of formula (I) or formula (II) generally results in corresponding diastereomers / enantiomer-enriched formula (I) or formula (II) compounds.

[0337] Those skilled in the art will recognize that in the synthesis of compounds of formula (I) or formula (II) (if not desired), an "orthogonal protecting group strategy" will be applied, which allows multiple protecting groups to be cleaved each time without affecting other protecting groups in the molecule. The principle of orthogonal protection is well known in the art and has also been reported in the literature (e.g., Barany and RB Merrifield, J. Am. Chem. Soc. 1977, 99, 7363; H. Waldmann et al., Angew. Chem. Int. Ed. Engl. 1996, 35, 2056).

[0338] Those skilled in the art will recognize that the order of reactions may vary depending on the reactivity and nature of the intermediates.

[0339] In more detail, the compound of formula (I) or formula (II) can be manufactured by the method described below, the method described in the examples or similar methods. The appropriate reaction conditions for each reaction step are known to those skilled in the art. Similarly, the reaction conditions affecting the reaction reported in the relevant literature are shown in, for example: Comprehensive Organic Transformations: A Guide to Functional Group Preparations, 2nd Edition, Richard C. Larock. John Wiley & Sons, New York, NY. 1999. It is convenient to carry out the reaction in the presence or absence of a solvent. There is no particular restriction on the nature of the solvent used, as long as it has no adverse effect on the reaction or the reagents involved and can dissolve the reagents at least to a certain extent. The described reaction can occur over a wide temperature range, and the precise reaction temperature is not critical to the present invention. The above reaction can be conveniently performed in a temperature range of -78°C to reflux. The time required for the reaction can also vary over a wide range, depending on many factors, particularly the reaction temperature and the nature of the reagents. However, it usually takes 0.5 hours to several days to obtain the intermediates and compounds. The reaction sequence is not limited to the one shown in the schemes, but, depending on the starting materials and their respective reactivity, the order of the reaction steps can be freely varied.

[0340] If starting materials or intermediates were not commercially available or their synthesis was not reported in the literature, they could be prepared by analogy to existing preparations of close analogs or as outlined in the experimental section.

[0341] The following abbreviations are used in this text:

[0342] AcOH = acetic acid, ACN = acetonitrile, Boc = tert-butyloxycarbonyl, CAS RN = Chemical Abstracts Registry Number, Cbz = benzyloxycarbonyl, Cs 2 CO 3＝ Cesium carbonate, CO = carbon monoxide, DMAP = 4-dimethylaminopyridine, DMF = N,N-dimethylformamide, DMP = Dess-Martin periodinane, DIBAL-H = diisobutylaluminum hydride, DIPEA = N,N-diisopropylethylamine, dppf = 1,1-bis(diphenylphosphino)ferrocene, EDC.HCl = N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, ESI = electrospray ionization, EtOAc = ethyl acetate, EtOH = ethanol, h = hour, FA = formic acid, H 2 O=water, H 2 SO 4= 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-urea-hexafluoro-phosphate, HCl = hydrogen chloride, HOBt = 1-hydroxy-1H-benzotriazole; HPLC = high performance liquid chromatography, K 2 CO 3 = Potassium carbonate, KHCO 3 = Potassium bicarbonate, KI = Potassium iodide, KOH = Potassium hydroxide, K 3 PO 4 = tripotassium phosphate, KOtBu = potassium tert-butoxide, LiAlH 4 or LAH = lithium aluminum hydride, LiHMDS = lithium bis(trimethylsilyl)amide, LiOH = lithium hydroxide, LiTMP = lithium tetramethylpiperidinium, MgSO 4 = magnesium sulfate, min = minute, mL = milliliter, MPLC = medium pressure liquid chromatography, MS = mass spectrometry, Ms = methanesulfonyl, NaH = sodium hydride, NaHCO 3 =Sodium bicarbonate, NaOH =Sodium hydroxide, Na 2 CO 3 =Sodium carbonate, Na 2 SO 4 =Sodium sulfate, Na 2 S 2 O 3 = sodium thiosulfate, NBS = N-bromosuccinimide, nBuLi = n-butyllithium, NEt 3 = triethylamine (TEA), NH 4 Cl = ammonium chloride, NMP = N-methyl-2-pyrrolidone, OAc = acetoxy, T 3 P = propylphosphonic anhydride, PG = protecting group, Pd-C = palladium on activated carbon, Pd(dppf)Cl 2 =1,1'-bis(diphenylphosphino)ferrocene-palladium(II) chloride, Pd 2 (dba) 3 =Tris(dibenzylideneacetone)dipalladium(0), Pd(OAc) 2 = Palladium(II) acetate, Pd(OH) 2 = Palladium hydroxide, Pd(PPh 3 ) 4=tetrakis(triphenylphosphine)palladium(0), PTSA=p-toluenesulfonic acid, Ts=p-toluenesulfonyl, R=any group, rt=room temperature, SFC=supercritical fluid chromatography, TBAI=tetrabutylammonium iodide, TEA=triethylamine, TFA=trifluoroacetic acid, THF=tetrahydrofuran, TMEDA=N,N,N',N'-tetramethylethylenediamine.

[0343] Among them, A, L, n, R 1 and R 2 The compounds of formula I as described herein can be prepared by methods analogous to those described in the literature and / or by methods such as, for example Solution 1 Synthesized as described in.

[0344]

[0345] Solution 1

[0346] Thus, the amine derivative 1 carries a suitable nitrogen protecting group "PG" at the end of the alkane chain, such as benzyloxycarbonyl (Cbz) carbamate, and an orthogonal nitrogen protecting group "PG" at the cyclic amine. 1 ", such as tert-butyloxycarbonyl (Boc) carbamate. The protecting group PG of intermediate 1 is removed by methods known in the art. 1 (e.g., removal of the Boc group using TFA in DCM solution at a temperature between 0°C and room temperature; removal of the Boc group using a suitable catalyst such as Pd or Pd(OH) supported on activated carbon in a suitable solvent such as MeOH, EtOH, EtOAc or a mixture thereof) 2 The Cbz group is removed using hydrogen in the presence of ; and as described in TW Greene and PGM Wuts, 4th edition, 2006, Wiley NY), followed by the free amine obtained being reacted with a compound of type 2a (R containing N at the point of attachment to the rest of Formula I) using a suitable base and solvent (such as sodium bicarbonate in DCM) in the presence of a urea former (such as bis(trichloromethyl) carbonate). 1 Part) reaction to provide compound 3, including compounds of formula Ia (step a1). Further urea forming agents 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 are 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 adding reagents may be important in such reactions due to the reactivity and stability of the intermediate formed carbamoyl chloride and to avoid the formation of undesirable symmetrical urea by-products in the following manner. Alternatively, R containing C at the point of attachment to the rest of Formula I1 The carboxylic acid is introduced as part of 1 by removing PG 1 The intermediate amine obtained is then coupled to form the corresponding amide, including the compound of formula Ia (step a2). This type of amide coupling is accomplished by using one of the known coupling reagents (such as DCC, HATU, EDCI, HOBt, TBTU, T3P, etc.) and a base (such as Huenig's base, triethylamine or DMAP) in a suitable solvent (such as N,N-dimethylformamide, DMA, DCM or 1-4-dioxane), preferably at 0°C to room temperature.

[0347] The protecting groups in intermediate 3 are removed using methods known in the art (e.g., removal of the Boc group using TFA in DCM solution at a temperature between 0°C and room temperature; removal of the Boc group using a suitable catalyst such as Pd or Pd(OH) supported on activated carbon in a suitable solvent such as MeOH, EtOH, EtOAc or a mixture thereof). 2 Removal of the Cbz group using hydrogen in the presence of ; and as described in, for example, TW Greene and PGM Wuts, 4th edition, 2006, Wiley NY) provides intermediate 4 (step b).

[0348] Amine 4 reacts with a fluorescent label containing a carboxylic acid moiety to give a compound of formula Ib (R 2 =fluorescent label) (step c). This type of amide coupling is accomplished by using one of the known coupling reagents (such as DCC, HATU, EDCI, HOBt, TBTU, T3P, etc.) 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 between 0°C and room temperature. Alternatively, a fluorescent label containing an N-hydroxysuccinimide (NHS) activated ester can be reacted with 4 in the presence of a suitable base (such as N,N-diisopropylethylamine (DIPEA), 2,6-lutidine, etc.) in a suitable solvent (such as DCM, THF, ACN, DMF or dioxane), preferably between 0°C and room temperature to provide a compound of formula Ib. To form the final molecule Ib bearing the nitrobenzofuran dye, amine 4 can be reacted with 4-chloro-7-nitrobenz-2-oxa-1,3-oxadiazole (CAS RN 10199-89-0) or 4-fluoro-7-nitrobenz-2-oxa-1,3-oxadiazole (CAS RN 29270-56-2), for example in the presence of a base such as triethylamine, in a solvent such as MeOH, preferably between 0° C. and ambient temperature in a nucleophilic aromatic substitution (step c).

[0349] Optionally, the amine derivative 1 wherein "PG" is a fluorescent label can be directly converted into the compound of formula Ib according to step a.

[0350] Protected amines 1 and their amine 2a or carboxylic acid 2b counterparts are commercially available or can be prepared according to literature procedures or as described below.

[0351] Intermediates of type 1, such as intermediates 1a and 1b, can be prepared by a variety of conditions, which can be exemplified by the general synthetic method outlined in Scheme 2.

[0352]

[0353] Solution 2

[0354] Intermediates 5, 6 and 11 are commercially available or can be prepared according to literature methods. The functional hydroxyl groups in 5 and 11 can be used to connect alkane chain derivatives of type 6, where "LG" is a suitable leaving group such as but not limited to mesylate (OMs), p-toluenesulfonate (OTs), bromine, iodide, in the presence of a suitable base (e.g. Cs 2 CO 3 , K 2 CO 3 ) in the presence of a suitable solvent (eg DMF), preferably at a temperature between room temperature and 80°C, via nucleophilic substitution (S N ) to produce compounds 7 and 12, respectively (step d). Intermediates of type 7 and 12 can also be prepared according to literature methods or, for example, under Mitsunobu conditions using PPh 3 and diethyl azodicarboxylate are obtained by other etherification reactions.

[0355] Benzoate esters of type 7 can be prepared by reacting with a suitable reducing agent such as LiAlH 4 , LiBH 4 , diisobutylaluminum hydride (DIBAL-H)) in a suitable solvent such as toluene, preferably at a temperature between -78°C and 0°C, to be converted to their corresponding benzyl alcohols 8 (step e).

[0356] Benzyl alcohol 8 can be prepared by reactions described in the literature, such as with CBr 4 and PPh 3 Or use PBr 3 Appel reactions carried out in a suitable solvent such as DCM, preferably at temperatures between 0 and 25 °C, convert to their benzyl bromide analogs 9 (step f).

[0357] Molecules of type 1a can be obtained by reacting benzyl bromide 9 with alcohol 10 in a nucleophilic substitution reaction in the presence of a suitable base such as KOtBu in a suitable solvent such as THF, preferably at a temperature between room temperature and 80° C. (step g). One skilled in the art will recognize that other etherification methods described in the literature and the use of analogs of bromide 9 bearing other suitable leaving groups (e.g., I, OMs, OTs) instead of bromine can be used to prepare 1a.

[0358] Boronic acids, esters and their derivatives are considered to be universal building blocks for transition metal-mediated cross-coupling reactions. Intermediates of type 1b can be synthesized by reacting with suitable building blocks such as vinyl borate ester 13 in the presence of a suitable base such as K 2 CO 3 ), a suitable aqueous solvent mixture (such as water / dioxane), using a transition metal catalyst (such as but not limited to Pd(dppf)Cl 2 ), preferably at temperatures between 25° C. and 100° C., is readily obtained (step h). Alternatively, hydrolysis of the vinyl boronate ester 13 by methods known to those skilled in the art, such as treatment with ammonium acetate-buffered water / acetone mixtures and sodium periodate, can be performed as a separate step, and the subsequent (step h) can be performed in the absence of water.

[0359] Vinyl borate esters 13 are commercially available or can be prepared according to literature methods or as described below. Vinyl borate esters 13 (e.g., vinyl boronic acid pinacol esters) can be readily obtained as described in the literature (Kovalenko et al., The Boron-Wittig Olefination of Aldehydes and Ketones with Bis[(pinacolato)boryl]methane: an Extended Reaction Scope. Eur J. Org. Chem., 2019: 5264-5635), for example by treating ketone 14 with bis((pinacolato)boryl)methane (CAS RN 78782-17-9) in the presence of a suitable base such as lithium tetramethylpiperidinium (LiTMP). The general synthesis method is described in Solution 3 (Step i).

[0360]

[0361] Solution 3

[0362] Among them, A, L, R 1 and R 3The compounds of formula II as described herein can be prepared by methods analogous to those described in the literature and / or by methods such as, for example Solution 4 Synthesized as described in.

[0363]

[0364] Solution 4

[0365] With the protecting group "PG 1 Amines of type 15 having a protecting group "LG" (such as those described for intermediate 1) and a leaving group "LG" (such as those described for intermediate 6) are commercially available or can be readily obtained in one step from alcohol derivative 10 by methods widely described in the literature (e.g., by reaction with p-toluenesulfonyl chloride in the presence of DMAP and TEA, or Comprehensive Organic Transformations: A Guide to Functional Group Preparations, 2nd edition, Richard C. Larock. John Wiley & Sons, New York, NY. 1999, step j).

[0366] Phenol ester derivative 16 can react with intermediate 15 via nucleophilic substitution to give ether intermediate 17, similar to the method for the conversion of 5 to 7 described in Scheme 2 (step d).

[0367] Compounds of type IIa can be obtained from 17, analogously to the conversion of 1 to 3 in Scheme 1 (step a).

[0368] Compounds of formula IIb can be obtained by reacting IIa with amine 18 under amide coupling conditions, for example by using well-known coupling reagents (such as DCC, HATU, EDCI, HOBt, TBTU, T3P, etc.) 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 room temperature (step k).

[0369] Treatment of benzyl bromide 19 (see above) with alcohols of type 10 under nucleophilic substitution conditions in the presence of a suitable base such as sodium hydride (NaH) and a suitable solvent such as DMF / DCM mixture, preferably at temperatures between 0°C and 25°C, affords ether intermediate 20 (step 1).

[0370] A compound of formula IIc wherein R 3 =H to the remainder of Formula II can be readily obtained from 20 similarly as described for the conversion of 1 to 3 in Scheme 1 (step a).

[0371] In one aspect, the present invention provides a method for making a compound of formula (I) as described herein, the method comprising reacting an amine 4,

[0372]

[0373] Among them, A, L, R 1 and n are as defined herein, with:

[0374] (a) a fluorescent label containing a carboxylic acid moiety in the presence of a coupling reagent and a base; or

[0375] (b) a fluorescent label containing an activated ester of N-hydroxysuccinimide (NHS) in the presence of a base; or

[0376] (c) 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole or 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole in the presence of a base;

[0377] The reaction is carried out to obtain the compound of formula (I).

[0378] In one embodiment, the fluorescent label containing a carboxylic acid moiety in reaction (a) is selected from:

[0379]

[0380] in:

[0381] X, Y, V, W, and p are as defined herein.

[0382] In one embodiment, the coupling reagent in reaction (a) is selected from DCC, HATU, EDCI, HOBt, TBTU and T3P.

[0383] In one embodiment, the base in reaction (a) is selected from Huenig's base, triethylamine and DMAP.

[0384] In one embodiment, the reaction (a) is carried out in a suitable solvent, preferably in a solvent selected from N,N-dimethylformamide, DMA, DCM and dioxane.

[0385] In one embodiment, the fluorescent label containing N-hydroxysuccinimide (NHS) activated ester in reaction (b) is selected from:

[0386]

[0387] in:

[0388] X, Y, V, W, and p are as defined herein.

[0389] In one embodiment, the base in reaction (b) is selected from N,N-diisopropylethylamine (DIPEA) and 2,6-lutidine.

[0390] In one embodiment, the reaction (b) is carried out in a suitable solvent, preferably in a solvent selected from DCM, THF, ACN, DMF and dioxane.

[0391] In one embodiment, the base in reaction (c) is triethylamine.

[0392] In one embodiment, the reaction (c) is carried out in a suitable solvent, preferably methanol.

[0393] In one aspect, the present invention provides a compound of formula (I) or formula (II) as described herein, manufactured according to any of the methods described herein.

[0394] MAGL inhibitory activity

[0395] Compounds are analyzed for their inhibitory activity against MAGL by measuring enzymatic activity following hydrolysis of the natural substrate 2-arachidonoylglycerol (2-AG) to arachidonic acid, which can then be subjected to mass spectrometry analysis. This assay is hereinafter abbreviated as the "2-AG assay".

[0396] The 2-AG assay was performed in a 384-well assay plate (PP, Greiner catalog number 784201) with a total volume of 20 μL. Compound dilutions were prepared in 100% DMSO (VWR Chemicals 23500.297) using a 3-fold dilution step in a polypropylene plate to allow the final concentration range of the assay to be 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, 03690-100 mL), 0.01% (v / v) Tween). After shaking, the plate was incubated at RT for 15 min. 10 μL of 2-arachidonoylglycerol in assay buffer was added to start the reaction. The final concentrations in this assay were 50 pM MAGL and 8 μM 2-arachidonoylglycerol. After shaking and incubation at RT for 30 min, 40 μL of ACN containing 4 μM d8-arachidonic acid was added to quench the reaction. The content of arachidonic acid was tracked using an online SPE system (Agilent Rapidfire) coupled to a triple quadrupole mass spectrometer (Agilent 6460). In the ACN / water liquid position, a C18 SPE cartridge (G9205A) was used. The mass spectrometer was operated in negative electrospray mode, with a mass ion pair of arachidonic acid of 303.1→259.1 and a mass ion pair of d8-arachidonic acid of 311.1→267.0. The activity of the compound was calculated based on the intensity ratio of [arachidonic acid / d8-arachidonic acid].

[0397] Table 1

[0398]

[0399] Use of the Compounds of the Invention

[0400] The compounds of formula (I) are fluorescent imaging probes with high affinity for MAGL. Therefore, they can be used as high-resolution tools to study localization (e.g., expression levels and protein distribution in healthy and diseased states, structure, dynamics and function of MAGL in living cells). They can also be applied, for example, to flow cytometry fluorescence activated cell sorting (FACS) experiments or cell trafficking studies using confocal live cell imaging.

[0401] In one aspect, the present invention provides a compound of formula (I) as described herein for use in monoacylglycerol lipase (MAGL) occupancy studies.

[0402] In another aspect, the present invention provides a compound such as a compound of formula (I) as described herein for use in the diagnostic imaging of monoacylglycerol lipase (MAGL) in a mammal.

[0403] In another aspect, the present invention provides a compound of formula (I) as described herein for use in generating monoacylglycerol lipase (MAGL) equilibrium and kinetic binding data.

[0404] In another aspect, the present invention provides the use of a compound of formula (I) as described herein in monoacylglycerol lipase (MAGL) occupancy studies.

[0405] In another aspect, the present invention provides for the use of a compound of formula (I) as described herein in the diagnostic imaging of monoacylglycerol lipase (MAGL) in a mammal.

[0406] In another aspect, the invention provides the use of a compound of formula (I) as described herein for generating monoacylglycerol lipase (MAGL) equilibrium and kinetic binding data.

[0407] In another aspect, the present invention provides a method of studying monoacylglycerol lipase (MAGL) occupancy, the method comprising contacting MAGL with a compound of formula (I) as described herein.

[0408] In another aspect, the present invention provides a method for diagnostic imaging of monoacylglycerol lipase (MAGL) in a mammal, the method comprising contacting MAGL with a compound of formula (I) described herein.

[0409] In another aspect, the invention provides a method of generating equilibrium and kinetic binding data for monoacylglycerol lipase (MAGL), the method comprising contacting MAGL with a compound of formula (I) described herein.

[0410] The compound of formula (II) is a useful synthetic intermediate for preparing the fluorescent probe of formula (I). Therefore, in one aspect, the present invention provides the use of the compound of formula (II) described herein for preparing the fluorescent probe of formula (I) described herein.

[0411] Examples

[0412] The present invention will be more fully understood by reference to the following examples. However, the claims should not be interpreted as being limited to the scope of the examples.

[0413] Where the preparation examples are obtained as mixtures of enantiomers, the pure enantiomers can be separated by methods described herein or by methods known to those skilled in the art such as chiral chromatography (eg chiral SFC) or crystallization.

[0414] If not stated otherwise, all reaction examples and intermediates were prepared under argon atmosphere.

[0415] Example 1

[0416] Benzyl (2-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamate

[0417]

[0418] Step d: Ethyl 2-(2-(((Benzyloxy)carbonyl)amino)ethoxy)benzoate

[0419]

[0420] Ethyl salicylate (CAS RN 118-61-6, 2.26 g, 13.6 mmol) was dissolved in DMF (8 mL) and Cs 2 CO 3 (4.43 g, 13.6 mmol) and the mixture was stirred at room temperature for 5 minutes. Next, 2-(((benzyloxy)carbonyl)amino)ethyl 4-methylbenzenesulfonate (CAS RN 93407-96-6, 2.80 g, 8.00 mmol) was added and the mixture was stirred at 55 °C for 18 h. The reaction was quenched by dilution with DCM (60 mL), extracted with sn. 10% NaOH (2 x 40 mL) aqueous solution and washed with brine (50 mL). The organic layer was washed with MgSO 4 Dry, filter and concentrate under reduced pressure.The crude product was purified by flash chromatography on silica gel (0 to 50% AcOEt in cyclohexane) to give the title compound (2.54 g, 7.40 mmol, 92%). 1 H NMR (300 MHz, CDCl 3 )δ7.82(dd,J=7.8,1.8Hz,1H),7.45(ddd,J=8.2,7.4,1.8Hz,1H),7.41–7.24(m,5H),7.07–6.90(m,2H),5.98(s,1H,NHCbz),5.12(s,2H,OCH 2 ),4.35(q,J=7.1Hz,2H,OCH 2 CH 3 ),4.14(t,J=5.0Hz,2H,OCH 2 ),3.64(m,2H,CH 2 NHCbz),1.36(t,J=7.1Hz,3H,OCH 2 CH 3 ). 13 C NMR (75MHz, CDCl 3)δ166.2,158.44,156.73(C=O CO 2 Et,C=O Cbz,C Ar -O)136.8,133.7,131.9,128.6,128.11,128.09,121.1,120.9,114.3,68.8(OCH 2 ),66.8(OCH 2 ),61.1(OCH 2 ),40.6(CH 2 NHCbz),14.4(CH 3 ). LC-HRMS(ESI)C 19 H 21 NO 5 Na[M+Na] + Calculated value: 366.1312; measured value: 366.1301.

[0421] Step e: Benzyl (2-(2-(hydroxymethyl)phenoxy)ethyl)carbamate

[0422]

[0423] At 0°C in N 2 The ester ethyl 2-(2-(((benzyloxy)carbonyl)amino)ethoxy)benzoate (1.15 g, 3.35 mmol) was dissolved in toluene (11.2 mL) under atmosphere and a 1 M DIBAL solution in DCM (15.1 mL, 15.1 mmol) was added dropwise. The reaction mixture was stirred at 0 °C until completion (1.5 h, TLC monitoring). The reaction was quenched by diluting with DCM (20 mL) and adding AcOH (0.86 mL, 15.1 mmol) dropwise. The mixture was then extracted with 10% sn.NaOH (2 x 40 mL) aqueous solution and washed with brine (40 mL). The organic layer was purified by MgSO 4 Dry, filter and concentrate under reduced pressure.The concentrate was purified by flash chromatography on silica gel (0 to 65% AcOEt in cyclohexane) to give the title compound (817 mg, 2.71 mmol, 81%). 1 HNMR (300MHz, CDCl 3 )δ7.39–7.20(m,4H),6.95(td,J=7.5,1.1Hz,1H),6.85(d,J=8.1Hz,1H),5.12(s,2H,OCH 2 ),4.66(s,2H,OCH 2 ),4.10(t,J=5.0Hz,2H,OCH 2),3.63(t,J=5.0Hz,2H,CH 2 NHCbz). 13 C NMR (75MHz, CDCl 3 )δ156.9,156.7(C=O Cbz,C Ar -O),136.5,129.5,129.4,129.3,128.7,128.3,121.3,111.6,67.6(OCH 2 ),67.1(OCH 2 ),62.1(OCH 2 ),40.8(CH 2 -NHCbz). LC-HRMS(ESI)C 17 H 20 NO 4 [M+H] + Calculated value: 302.1386; measured value: 302.1365.

[0424] Step f: Benzyl (2-(2-(bromomethyl)phenoxy)ethyl)carbamate

[0425]

[0426] Benzyl (2-(2-(hydroxymethyl)phenoxy)ethyl)carbamate (795 mg, 2.64 mmol) was dissolved in DCM (20.3 mL) and cooled to 0°C. Next, CBr was added 4 (1.00 g, 3.04 mmol) was then added PPh 3 (796 mg, 3.04 mmol, 1.5 M solution in DCM). The reaction mixture was stirred at room temperature until completion (10 min, TLC monitoring). The reaction was quenched by diluting with DCM (30 mL) and washed with saturated NaHCO 3 The organic layer was purified by centrifugation with MgSO 4 Dry, filter and concentrate under reduced pressure.The concentrate was purified by flash chromatography on silica gel (0 to 40% AcOEt in cyclohexane) to give the title compound (694 mg, 1.90 mmol, 72%). 1 H NMR (300 MHz, CDCl 3 )δ7.38–7.28(m,7H),7.04–6.81(m,2H),5.12(s,2H,OCH 2 Cbz), 4.59(d, J=27.9Hz,2H,CH 2 -Br), 4.14(t, J=4.9Hz, 2H, OCH2 CH 2 -NHCbz),3.68(q,J=5.3Hz,2H,OCH 2 CH 2 -NHCbz). 13 CNMR (75MHz, CDCl 3 )δ156.7,136.7,131.0,130.6,128.7,128.1,121.2,111.8,67.5(-OCH 2 -),66.9(-OCH 2 -),40.7(-OCH 2 CH 2 -NHCbz),29.7(CH 2 -Br). LC-MS (ESI): 386.0 [M + Na] + .

[0427] Step g tert-Butyl 3-(((2-(2-(((benzyloxy)carbonyl)amino)ethoxy)benzyl)oxy)methyl)azetidine-1-carboxylate (BB1)

[0428]

[0429] 3-(Hydroxymethyl)azetidine-1-carboxylic acid tert-butyl ester (CAS RN 142253-56-3, 196mg, 1.05mmol) was dissolved in anhydrous THF (7mL) at 0°C, KOtBu (118mg, 1.05mmol) was added, and the mixture was stirred for 5min. Next, the bromine derivative (2-(2-(bromomethyl)phenoxy)ethyl)benzyl carbamate (254.9mg, 0.70mmol) was dissolved in anhydrous THF (1.5mL) and added dropwise to the reaction mixture. The mixture was then warmed to room temperature and stirred for 4h until completion (TLC monitoring). The reaction was quenched by diluting with DCM (10mL) and adding AcOH (0.1mL, 1.05mmol) dropwise. The mixture was washed with saturated NaHCO 3 The organic layer was extracted with aqueous solution (2 x 20 mL) and washed with brine (20 mL). 4 Dry, filter and concentrate under reduced pressure.The concentrate was purified by flash chromatography on silica gel (0 to 40% AcOEt in cyclohexane) to give the title compound (186 mg, 0.40 mmol, 57%). 1 H NMR (300 MHz, CDCl 3)δ7.39–7.23(m,7H),6.96(td,J=7.4,1.0Hz,1H),6.85(d,J=8.2Hz,1H),5.50(s,1H,NHCbz),5.10(s,2H,OCH 2 ),4.54(s,2H,OCH 2 ),4.14–4.03(m,2H,OCH 2 ),3.86(t,J=8.4Hz,2H,OCH 2 ),3.67–3.51(m,6H,CH 2 NHCbz,2x N-CH 2 azetidine), 2.78–2.63 (m, 1H, CH azetidine), 1.43 (s, 9H, 3x CH 3 Boc). 13CNMR(75MHz,CDCl3)δ156.5,136.5,129.9,129.4,128.7,128.44,128.38,126.7,121.2,112.1,79.4(C(CH 3 )Boc),72.7(CH azetidine),68.8(OCH 2 ),67.6(OCH 2 ),67.0(OCH 2 ),40.8(CH 2 -NHCbz),28.5(CH 3 Boc). LC-HRMS(ESI) 26 H 35 N 2 O 6 [M+H] + Calculated value: 471.2490; measured value: 471.2475.

[0430] Step a1: Benzyl (2-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamate

[0431] 1. Boc deprotection The N-Boc deprotected derivative tert-butyl 3-(((2-(2-(((benzyloxy)carbonyl)amino)ethoxy)benzyl)oxy)methyl)azetidine-1-carboxylate (BB1, 93.0 mg, 0.20 mmol) was dissolved in DCM (1.3 mL) at 0°C and TFA (152 μL, 2.0 mmol) was added. The reaction mixture was stirred at this temperature until Boc deprotection was complete (1 h, LC-MS monitoring). The reaction was diluted with toluene (3.00 mL) and co-evaporated twice with toluene (3.00 mL) under reduced pressure. The 2,2,2-trifluoroacetate salt of the free amine 3-(((2-(2-(((benzyloxy)carbonyl)amino)ethoxy)benzyl)oxy)methyl)azetidine was used in the next step (2) without further purification.

[0432] 2. Urea coupling (4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazin-6-ium (2S,3S)-3-carboxy-2,3-bis((4-methylbenzoyl)oxy)propionate (CAS RN 2624363-49-9, 119 mg, 0.16 mmol) was dissolved in anhydrous acetonitrile (0.7 mL) and 1,1′-carbonyl-bis-(1,2,4-triazole) (CAS RN 41864-22-6, 26.3 mg, 0.16 mmol), followed by the addition of triethylamine (0.16 mL, 1.12 mmol). The reaction mixture was stirred at room temperature for 2 h. The 2,2,2-trifluoroacetate salt of the amine nucleophile 3-(((2-(2-(((benzyloxy)carbonyl)amino)ethoxy)benzyl)oxy)methyl)azetidine from step 1 was then dissolved in ACN (0.5 mL) and added dropwise to the reaction mixture. The reaction mixture was heated to 50 °C and stirred at this temperature for 2.5 h. The reaction was quenched by diluting with DCM (5.00 mL) and washed with saturated NaHCO 3 The organic layer was extracted with aqueous solution (2.00 mL) and washed with brine (2.00 mL). 4 The concentrate was purified by HPLC (25 to 75% ACN in H 2 D (containing 0.1% TFA) to give the title compound (70.0 mg, 0.13 mmol, 79%). 1 H NMR(300MHz,MeOD)δ7.37–7.16(m,7H,H-Ar),6.96–6.84(m,2H,H-Ar),5.05(s,2H,O-CH 2 -),4.51(s,2H,O-CH 2-), 4.25–4.06(m,2H),4.06–3.88(m,4H),3.82–3.62(m,3H),3.58–3.45(m,4H),3.32–3.20(m,3H),2.99–2.83(m,2H),2.80–2.66(m,1H,-CH-azetidine),1.90–1.64(m,2H.–CH 2 -HHPO). 13 C NMR (75 MHz, MeOD) δ 171.2, 163.7 (2x C=O urea, amide), 158.9, 158.0 (C=O Cbz, C ipso phenol), 138.3, 130.8, 130.3, 129.5, 129.0, 128.8, 127.8, 121.8, 112.9 (C-Ar), 73.0, 70.4, 69.18, 68.21, 68.1, 67.5, 55.2, 55.1, 50.4, 47.1, 41.5, 40.5, 30.4, 30.2. LC-HRMS (ESI) C 29 H 37 N 4 O 7 + [M+H] + Calculated value: 553.2657; measured value: 553.2665.

[0433] If not otherwise indicated, the following examples were synthesized using appropriate building blocks following synthetic methods similar to those described for Example 1.

[0434]

[0435]

[0436]

[0437]

[0438] Example 15

[0439] Benzyl (2-(2-(((7-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)-7-azaspiro[3.5]nonan-2-yl)oxy)methyl)phenoxy)ethyl)carbamate

[0440]

[0441] Step g tert-Butyl 2-((2-(2-(((benzyloxy)carbonyl)amino)ethoxy)benzyl)oxy)-7-azaspiro[3.5]nonane-7-carboxylate (BB 13)

[0442]

[0443] Analogously to the method described in Example 1g), benzyl (2-(2-(bromomethyl)phenoxy)ethyl)carbamate (see Example 1) was reacted with tert-butyl 6-hydroxy-2-azaspiro[3.3]heptane-2-carboxylate (CAS RN 240401-28-9) to give the title compound (59.6 mg, 0.114 mmol, 76%). 1 HNMR (300MHz, CDCl 3 )δ7.38–7.26(m,7H),6.95(td,J=7.5,1.0Hz,1H),6.85(d,J=8.2Hz,1H),5.57(s,1H,NHCbz),5.10(s,2H,OCH 2 ),4.41(s,2H,OCH 2 ),4.16–3.95(m,3H),3.61(q,J=5.3Hz,2H),3.23(dt,J=20.9,5.7Hz,4H),2.12(t,J=10.0Hz,2H),1.80–1.57(m,3H),1.45(m,12H). 13 C NMR (75MHz, CDCl 3 )δ156.7,155.1(C=O Cbz,C=O Boc,C Ar -O),136.6,130.1,129.4,128.7,128.4,128.3,126.9,121.2,112.2,79.4(C(CH 3 ) 3 Boc),69.1,67.7,67.0,65.7,41.0,40.9,40.8,39.6,39.4,36.7,31.0,28.6(3x CH 3 Boc), 27.1.LC-HRMS(ESI)C 30 H 40 N 2 O 6 Na[M+Na] + Calculated value: 547.2779; measured value: 547.2799.

[0444] Step a1: Benzyl (2-(2-(((7-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)-7-azaspiro[3.5]nonan-2-yl)oxy)methyl)phenoxy)ethyl)carbamate

[0445] 1. Boc deprotection. Analogously to the method described in step (1) of Example 1a1, tert-butyl 2-((2-(((benzyloxy)carbonyl)amino)ethoxy)benzyl)oxy)-7-azaspiro[3.5]nonane-7-carboxylate (BB 13) (52.5 mg, 0.10 mmol) was dissolved in DCM (0.7 mL) at 0°C and TFA (0.11 mL, 1.40 mmol) was added. The reaction mixture was stirred at this temperature until Boc deprotection was complete (30 min, LC-MS monitoring). The reaction was diluted with toluene (3.00 mL) and co-evaporated twice with toluene (3.00 mL) under reduced pressure. The trifluoroacetate salt of the free amine was used in the next step without further purification.

[0446] 2. Urea coupling. Triphosgene (CAS RN 32315-10-9, 53.8 mg, 0.10 mmol) and NaHCO were reacted at 0 °C. 3 (33.6 mg, 0.08 mmol) was suspended in DCM (1.0 mL). The deprotected amine derivative of benzyl (2-(2-(((7-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)-7-azaspiro[3.5]nonan-2-yl)oxy)methyl)phenoxy)ethyl)carbamate obtained in step a1, 1) was dissolved in anhydrous DCM (1.5 mL) and added dropwise to the triphosgene mixture. The mixture was warmed to room temperature and stirred overnight. The filtrate of the solution was then added dropwise to a solution of (4aR,8aS)-hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one (CAS RN 2377107-31-6, 15.6 mg, 0.10 mmol) and DIPEA (70.0 μL, 0.40 mmol) in DCM (1.0 mL) at 0°C. The mixture was warmed to room temperature and stirred for an additional 3 h. The mixture was concentrated in vacuo and purified by RP-HPLC (25 to 75% ACN in H2O, containing 0.1% TFA). To give the title compound (23.6 mg, 39.0 μmol, 39%). 1 H NMR(300MHz,MeOD)δ7.37–7.19(m,7H),6.91(td,J=8.3,1.4Hz,2H),5.08(s,2H,OCH 2 ),4.42(s,2H,OCH2 ),4.24–4.00(m,5H),3.96(d,J=3.0Hz,1H),3.59(dd,J=12.7,5.1Hz,1H),3.52(t,J=5.4Hz,2H),3.44–3. 32(m,2H),3.17–2.89(m,6H),2.21–2.09(m,2H),1.92–1.82(m,2H),1.77–1.64(m,2H),1.56–1.41(m,4H). 13 C NMR (75MHz, CDCl 3 )δ171.2,165.5,158.9,158.1(C=O urea, C=O amide, C=O Cbz, C Ar -O),138.4,130.9,130.3,129.5,129.0,128.7,127.9,121.8,112.8,70.6,70. 3,68.2,67.5,66.2,50.4,45.2,45.1,42.9,41.6,40.6,40.4,37.7,32.3,30.3. LC-HRMS(ESI)C 33 H 42 N 4 O 7 Na[M+Na] + Calculated value: 629.2946; measured value: 629.2961.

[0447] If not otherwise indicated, the following examples were synthesized using appropriate building blocks following synthetic methods similar to those described for Example 15.

[0448]

[0449] Example 17

[0450] (R)-Benzyl (2-(2-(((1-(3-(2-oxooxazolidin-4-yl)propanoyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamate

[0451]

[0452] Synthesis of building block: 3-[(4R)-2-oxooxazolidin-4-yl]propionic acid (3 steps)

[0453] Step 1) (S)-4-((tert-Butyloxycarbonyl)amino)-5-hydroxypentanoic acid methyl ester

[0454] To a solution of (R)-2-((tert-butoxycarbonyl)amino)-5-methoxy-5-oxopentanoic acid (1 g, 3.83 mmol, CAS: 76379-01-6) in THF (15 mL) was added N-methylmorpholine (421 μL, 3.83 mmol) followed by ethyl chloroformate (368 μL, 3.83 mmol) at −10° C. and the reaction mixture was stirred at this temperature for 10 minutes. NaBH was added in one portion. 4 (434 mg, 11.5 mmol) did not cause a temperature rise. MeOH (35 mL) was added dropwise between -1°C and 17°C over 30 min. Stirring was continued in an ice bath for 1 h. 1 M KHSO 4 Aqueous solution (40 mL) was added dropwise to the reaction mixture, and the organic solvent was then evaporated under reduced pressure. The aqueous layer was extracted twice with ethyl acetate. The combined organic layers were washed with 1 M KHSO 4 Aqueous solution and saturated NaHCO 3 Wash with aqueous solution and MgSO 4 Dry, filter, treat with silica gel and evaporate under reduced pressure. The crude compound was purified by silica gel chromatography using an MPLC system eluting with a gradient of n-heptane:ethyl acetate (100:0 to 30:70) to afford the title compound as a colorless oil (0.70 g, 66%). MS (ESI): m / z=192.1 [M+H] + .

[0455] Step 2) Methyl (R)-3-(2-oxooxazolidin-4-yl)propanoate

[0456] To a solution of (S)-methyl 4-((tert-butoxycarbonyl)amino)-5-hydroxypentanoate (690 mg, 2.79 mmol, 1.0 equiv) in THF (8.8 mL) was added sulfuryl chloride (611 μL, 8.37 mmol, 3.0 equiv) dropwise and the solution was stirred at room temperature for 3 h. Silica gel was added and the reaction mixture was evaporated under reduced pressure. The compound was purified by silica gel chromatography using an MPLC system eluting with a gradient of n-heptane:ethyl acetate (100:0 to 0:100) to give the title compound as a colorless oil (404 mg, 79%). MS (ESI): m / z=174.1 [M+H] + .

[0457] Step 3) (R)-3-(2-oxooxazolidin-4-yl)propanoic acid

[0458] To a solution of (S)-methyl 3-(2-oxooxazolidin-4-yl)propanoate (400 mg, 2.31 mmol, 1.0 equiv) in 1,4-dioxane (2 mL) and water (2 mL) was added lithium hydroxide monohydrate (107 mg, 2.54 mmol, 1.1 equiv) and the reaction mixture was stirred at room temperature for 2 h. 1,4-dioxane was evaporated and HCl (2.54 mL, 2.54 mmol, 1.1 equiv) was added dropwise to the solution. The aqueous layer was extracted five times with ethyl acetate. The combined organic layers were purified by MgSO 4 Dry, filter and evaporate to give the title compound as a colorless solid (330 mg, 86%). MS (ESI): m / z = 160.1 [M+H] + .

[0459] Step a2: (R)-benzyl (2-(2-(((1-(3-(2-oxooxazolidin-4-yl)propanoyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamate

[0460] (R)-3-(2-oxooxazolidin-4-yl)propanoic acid (12.7 mg, 0.08 mmol) was dissolved in THF / ACN 1:1 (1.60 mL), and then DIPEA (42.0 μL, 0.24 mmol) was added followed by HATU (36.5 mg, 96.0 μmol). After stirring for 10 min, the deprotected amine derivative of tert-butyl 3-(((2-(((benzyloxy)carbonyl)amino)ethoxy)benzyl)oxy)methyl)azetidine-1-carboxylate BB1 ​​(38.7 mg, 0.08 mmol, prepared as described for Example 1, Step 1, Boc deprotection) was added. The mixture was stirred at room temperature overnight, and then the mixture was directly treated with ACN / H 2 O was diluted and analyzed by RP-HPLC (15-85% ACN in H 2 O, containing 0.1% TFA) to give the title compound (16.3 mg, 0.032 mmol, 40%). LC-HRMS (ESI) 27 H 34 N 3 O 7 [M+H] + Calculated value: 512.2389; measured value: 512.2391.

[0461] Example 18

[0462] Benzyl (2-(2-(((1-(3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamate

[0463]

[0464] Analogously to the method described in Example 17, step a2, tert-butyl 3-(((2-(2-(((benzyloxy)carbonyl)amino)ethoxy)benzyl)oxy)methyl)azetidine-1-carboxylate BB1 ​​was reacted with 3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazine-6-carboxylic acid (CAS RN: 134997-87-8) to give the title compound (22.3 mg, 0.0.41 mmol, 50%). LC-HRMS (ESI) 30 H 32 N 3 O 7 [M+H] + Calculated value: 546.2235; measured value: 546.2239.

[0465] Example 19

[0466] 2-(6-(2-(2-((7-nitro-112,312-benzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)benzyl)-2-azaspiro[3.3]heptane-2-carbonyl)-7-oxa-2,5-diazaspiro[3.4]octan-6-one

[0467]

[0468] Step b 2-(6-(2-(2-aminoethoxy)benzyl)-2-azaspiro[3.3]heptane-2-carbonyl)-7-oxa-2,5-diazaspiro[3.4]octan-6-one

[0469]

[0470] A mixture of 20% tBuOH in EtOAc (HPLC grade) was washed with N 2 Purge for at least 15 min and keep in a sealed vial under N 2 In a pear-shaped flask, the starting material (benzyl 2-(2-((2-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)-2-azaspiro[3.3]heptane-6-ylidene)methyl)phenoxy)ethyl)carbamate (Example 13, 16.0 mg, 0.03 mmol) was dissolved in a pre-prepared solvent mixture and stirred at 4 ℃ for 2 hours. 2 The mixture was purged for 10 min. Next, palladium on carbon (10 mol%) was added to the mixture and further treated with N 2 Purge for another 15 min. Then use a flask filled with H 2balloon, by bubbling H 2 The reaction was carried out until completion (LC-MS monitoring). When necessary, a small amount of the previously prepared N 2 The solvent mixture was purged and the solvent mixture level was maintained at 1-2 mL. Pd on carbon was removed from the reaction mixture by filtering with a PTFE syringe filter (pore size: 0.45 μm) and washing the filter with ACN and water. The crude product was lyophilized and the obtained title compound was used in the next step (step c) without further purification. LC-MS [M+H] + :400.9.

[0471] Step c 2-(6-(2-(2-((7-nitro-112,312-benzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)benzyl)-2-azaspiro[3.3]heptane-2-carbonyl)-7-oxa-2,5-diazaspiro[3.4]octan-6-one

[0472] 2-(6-(2-(2-aminoethoxy)benzyl)-2-azaspiro[3.3]heptane-2-carbonyl)-7-oxa-2,5-diazaspiro[3.4]octan-6-one (12.0 mg, 0.03 mmol) from step c) was dissolved in MeOH (0.75 mL) and trimethylamine (13.0 μL, 0.09 mmol) was added. Next, a solution of 4-fluoro-7-nitrobenzo-2-oxa-1,3-oxadiazole (CAS RN29270-56-2, 11.0 mg, 0.06 mmol) in MeOH (0.3 mL) was added dropwise. The reaction was stirred in the dark for 3.5 h. The solvent was then removed in vacuo at 20 °C and the reaction was purified by RP-HPLC (15-85% ACN in H 2 O with 0.1% TFA) to give the title compound (3.19 mg, 6.0 μmol, 19%). 1 H NMR (600MHz, CD 3CN)δ8.53(d,J=8.8Hz,1H),7.16(td,J=7.8,1.7Hz,1H),7.06(dd,J=7.4,1.7Hz,1 H),6.92(dd,J=8.3,1.1Hz,1H),6.87(td,J=7.4,1.1Hz,1H),6.51–6.39(m,2H),4 .47(s,2H),4.27(t,J=5.0Hz,2H),4.05–3.83(m,6H),3.67(s,2H),3.60(s,2H),2 .55(d,J=7.5Hz,2H),2.26(p,J=7.8Hz,1H),2.03–1.96(m,2H),1.75–1.68(m,2H). 13 C NMR (151 MHz, CD 3 CN)δ162.19,157.44,156.29,144.81,137.33,130.22,129.00,127.24,120.80, 111.37,74.18,65.81,62.84,62.43,61.60,54.21,38.31,36.10,34.53,29.56. LC-HRMS(ESI) 27 H 30 N 7 O 7 [M+H] + Calculated value: 564.2201; measured value: 564.2192.

[0473] If not otherwise indicated, the following examples were synthesized using appropriate building blocks following synthetic methods similar to those described for Example 19.

[0474]

[0475]

[0476] Example 26

[0477] 2-(7-(Dimethylamino)-3-(dimethylimino)-5,5-dimethyl-3,5-dihydrodibenzo[b,e]silan-10-yl)-4-((5-(2-((2-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)-2-azaspiro[3.3]heptane-6-yl)methyl)phenoxy)pentyl)carbamoyl)benzoate

[0478]

[0479] Step b 2-(6-(2-((5-aminopentyl)oxy)benzyl)-2-azaspiro[3.3]heptane-2-carbonyl)-7-oxa-2,5-diazaspiro[3.4]octan-6-one

[0480]

[0481] Analogously to the method described in Example 19b), benzyl (5-(2-((2-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)-2-azaspiro[3.3]heptane-6-ylidene)methyl)phenoxy)pentyl)carbamate (Example 14) was treated with hydrogen and palladium on carbon to give the title compound which was used in the next step (step c) without further purification. LC-MS [M+H] + :442.9.

[0482] Step c 2-(7-(dimethylamino)-3-(dimethylimino)-5,5-dimethyl-3,5-dihydrodibenzo[b,e]silan-10-yl)-4-((5-(2-((2-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)-2-azaspiro[3.3]heptane-6-yl)methyl)phenoxy)pentyl)carbamoyl)benzoate

[0483] 2-(6-(2-((5-aminopentyl)oxy)benzyl)-2-azaspiro[3.3]heptane-2-carbonyl)-7-oxa-2,5-diazaspiro[3.4]octan-6-one (8.96 mg, 0.02 mmol) was dissolved in 10% DMF in ACN (0.90 mL) at 0 °C and then 2-(7-(dimethylamino)-3-(dimethylimino)-5,5-dimethyl-3,5-dihydrodibenzo[b,e]silan-10-yl)-4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)benzoate (CAS RN 1426090-09-6, 5.00 mg, 9.0 μmol) was added followed by DIPEA (9.0 μL, 7.0 μmol). The reaction was stirred at room temperature in the dark for about 4 h (LC-MS monitoring the reaction). 2 O / ACN and freeze-dried. The crude product was purified by RP-HPLC (25 to 75% H 2 O / ACN, containing 0.1% TFA) was purified within 30 min to obtain the target product (6.0 mg, 7.0 μmol, 76%). 1H NMR (600MHz, MeOD) δ8.33(d,J=8.2Hz,1H),8.14(dd,J=8.2,1.8Hz,1H),7.74(d,J=1.7Hz,1H),7.36(d,J=2.9Hz,2H),7.13(td,J=7.8,1.7Hz,1H),7 .04(dd,J=7.4,1.7Hz,1H),7.02–6.99(m,2H),6.88(dd,J=8.2,1.1Hz,1H) ,6.83(td,J=7.3,1.0Hz,1H),6.77(dd,J=9.6,2.9Hz,2H),4.53(s,2H,OCH 2 ),4.07(s,4H,OCH 2 ),4.00(t,J=6.1Hz,2H),3.93(s,2H),3.84(s,2H),3.47(t,J=7.1Hz,2H),3.32(s,12H,4x N-CH 3 ), 2.64 (d, J = 7.4 Hz, 2H), 2.44 (p, J = 7.7 Hz, 1H, CH spiro), 2.22–2.16 (m, 2H, CH-CH 2 -C spiro), 1.88 (ddd, J = 10.5, 7.9, 2.8 Hz, 4H, CH-CH 2 -C spiro ring, -CH 2 -alkyl), 1.74 (p, J = 7.2 Hz, 2H, -CH 2 -alkyl), 1.67–1.59 (m, 2H, -CH 2 -alkyl), 0.68(s,3H,Si-CH 3 ),0.62(s,3H,Si-CH 3 ). 13 C NMR (151 MHz, MeOD) δ 166.6, 162.3, 159.0, 156.8 (C=O urea, C=O carbamate, COO - ,C Ar -O),129.7,128.5,127.4,126.9,120.1,119.8,113.8,110.9,74.2(OCH 2 ),67.2(OCH 2 ), 63.0, 62.4, 61.8, 54.4 (-C-carbamate spiro), 39.8, 39.6, 38.2, 36.0, 34.7 (-C-spiro), 29.7 (CH spiro), 28.9, 28.8 (-CH 2 -alkyl), 23.5(-CH 2-alkyl), -2.20(Si-CH 3 ),-3.09(Si-CH 3 ). LC-HRMS(ESI)C 51 H 60 N 6 O 7 Si[M+H] + Calculated value: 897.4366; measured value: 897.4357.

[0484] If not otherwise indicated, the following examples were synthesized using appropriate building blocks following synthetic methods similar to those described for Example 26.

[0485] Example 32 is a regioisomeric mixture of 5- and 6-carboxytetramethylrhodamine derivatives obtained in one reaction. HPLC purification allowed the separation of a fraction of the 5-carboxytetramethylrhodamine derivative (Example 33) from the mixture. The corresponding 6-carboxytetramethylrhodamine derivative could not be obtained in pure form. The same considerations apply to Examples 34 and 35 and 36 and 37, respectively. Example 38 was obtained as a regioisomeric mixture of 5- and 6-carboxytetramethylrhodamine derivatives.

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492] Example 39

[0493] Ethyl 3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)benzoate

[0494]

[0495] Step d tert-Butyl 3-((3-(ethoxycarbonyl)phenoxy)methyl)azetidine-1-carboxylate (BB15)

[0496]

[0497] Analogously to the method described in Example 1d), ethyl 3-hydroxybenzoate (CAS RN 7781-98-8) was reacted with tert-butyl 3-(bromomethyl)azetidine-1-carboxylate (CAS RN 253176-93-1) to give the title compound (18.0 mg, 0.05 mmol, 18%). LC-MS (ESI) [M+Na] + .

[0498] [M+Na] + :338.1

[0499] Step a1 Ethyl 3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)benzoate

[0500] Analogously to the method described in Example 1, a1), (4aR,8aS)-3-oxooctahhydro-2H-pyrido[4,3-b][1,4]oxazin-6-ium (2S,3S)-3-carboxy-2,3-bis((4-methylbenzoyl)oxy)propionate (CAS RN 2624363-49-9, 66.7 mg, 0.09 mmol) was reacted with the deprotected amine derivative of tert-butyl 3-((3-(ethoxycarbonyl)phenoxy)methyl)azetidine-1-carboxylate BB15 (33.5 mg, 0.10 mmol prepared as described in Example 1, Step 1, Boc deprotection) to give the title compound (16.3 mg, 0.032 mmol, 40%). LC-MS (ESI) [M+H] + :418.2.

[0501] If not otherwise indicated, the following examples were synthesized using appropriate building blocks following synthetic methods similar to those described for Example 39.

[0502]

[0503] Example 42

[0504] tert-Butyl (2-(3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)oxy)benzamido)ethyl)carbamate

[0505]

[0506] Step k

[0507] 1. Ester hydrolysis. Ethyl 3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)oxy)benzoate (Example 40, 12.5 mg, 0.03 mmol) was dissolved in THF / MeOH / H 2 O3:1:1 (0.2 mL) and lithium hydroxide (LiOH, 2,15 mg, 0.09 mmol) was added. The reaction was stirred at room temperature until completion (about 2 h). The reaction was quenched by diluting with ethyl acetate (1.0 mL), extracted with sn.1M aqueous HCl (0.5 mL), and washed with brine (0.5 mL). The organic layer was washed with MgSO 4 Dry, filter and remove the solvent in vacuo.3-((1-((4aR,8aS)-3-oxooctanohydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)oxy)benzoic acid was used in the next step without further purification.

[0508] 2. Amide coupling. The crude 3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)oxy)benzoic acid obtained in step 1 from ethyl 3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)oxy)benzoate (Example 40) was dissolved in DMF (0.43 mL) and DIPEA (16.0 μL, 0.09 mmol) was added followed by 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, CAS RN 148893-10-1, 13.7 mg, 0.04 mmol). The mixture was stirred for 10 min, and then tert-butyl (2-aminoethyl)carbamate (CAS RN 57260-73-8, 5.28 mg, 0.03 mmol) was added. The mixture was stirred at room temperature for 18 h, then diluted with ethyl acetate (1.0 mL), washed with brine (0.5 mL), and washed with Na 2 SO 4 Dry, filter and concentrate under reduced pressure. The crude product was purified by RP-HPLC (25 to 75% H 2 O / ACN, containing 0.1% TFA) to give the title compound (5.0 mg, 0.01 mmol, 32%). LC-HRMS (ESI) C 25 H 36 N2O 7[M+H]+ calculated: 518.2609; found: 518.2601.

[0509] Example 43

[0510] tert-Butyl (2-(2-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)benzamido)ethyl)carbamate

[0511]

[0512] Analogously to the method described in Example 42, k), ethyl 2-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)benzoate (Example 41) was subjected to ester hydrolysis (step k, 1) and then reacted with tert-butyl (2-aminoethyl)carbamate (CAS RN 57260-73-8, 5.28 mg, 0.03 mmol) to give the title compound (5.0 mg, 9.0 μmol, 55%). LC-MS (ESI) [M+H] + :532.2.

[0513] Example 44

[0514] (4aR,8aS)-6-(3-((Benzyloxy)methyl)azetidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one

[0515]

[0516] Step 1 tert-Butyl 3-((Benzyloxy)methyl)azetidine-1-carboxylate

[0517]

[0518] Tert-butyl 3-(hydroxymethyl)azetidine-1-carboxylate (CAS RN 142253-56-3, 103 mg, 0.55 mmol) and sodium hydride 60% dispersion in mineral oil (CAS RN 7646-69-7, 55.0 mg, 1.38 mmol) were suspended in anhydrous DMF (2.0 mL) at 0°C for 15 min or until no gas evolution was observed. Benzyl bromide (CAS RN 100-39-0, 0.16 mL, 1.38 mmol) was then added. The reaction was allowed to warm to room temperature and stirred for 16 h. The mixture was diluted with dichloromethane (10.0 mL), neutralized with acetic acid, and washed with saturated NaHCO 3The organic layer was extracted with aqueous solution (10 mL) and washed with water (10 mL). 4 Dry, filter and concentrate in vacuo.The crude product was purified via flash chromatography on silica gel using 0 to 80% ethyl acetate in cyclohexane as eluent to afford the title compound (120 mg, 0.43 mmol, 77%). 1 H NMR (300 MHz, CDCl 3 )δ7.40–7.26(m,5H),3.99(t,J=8.5Hz,2H),3.66(dd,J=8.7,5.3Hz,2H),3.58(d,J=6.8Hz,2H),2.88–2.67(m,1H),1.43(s,9H). LC-MS[M+Na] + :300.1.

[0519] Step a1 (4aR, 8aS)-6-(3-((benzyloxy)methyl)azetidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one

[0520] Analogously to the method described in Example 1, a1), tert-butyl 3-((benzyloxy)methyl)azetidine-1-carboxylate was reacted with (4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazin-6-ium (2S,3S)-3-carboxy-2,3-bis((4-methylbenzoyl)oxy)propionate (CAS RN 2624363-49-9) to give the title compound. LC-MS (ESI) [M+H] + :360.1.

[0521] Example 45

[0522] (4aR,8aS)-6-(6-(Benzyloxy)-2-azaspiro[3.3]heptane-2-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one

[0523]

[0524] Step 1 tert-Butyl 6-(Benzyloxy)-2-azaspiro[3.3]heptane-2-carboxylate

[0525]

[0526] Analogously to the method described in Example 441), tert-butyl 6-hydroxy-2-azaspiro[3.3]heptane-2-carboxylate (CAS RN 1147557-97-8) was reacted with benzyl bromide (CAS RN 100-39-0) to give the title compound.1 H NMR (300 MHz, CDCl 3 )δ7.41–7.27(m,5H),4.39(s,2H),4.02–3.75(m,5H),2.46(ddd,J=9.8,6.8,3.0Hz,2H),2.14(ddd,J=10.1,7.3,3.0Hz,2H),1.42(s,9H).

[0527] Step a1 (4aR, 8aS)-6-(6-(benzyloxy)-2-azaspiro[3.3]heptane-2-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one

[0528] Analogously to the method described in Example 1, a1), tert-butyl 6-(benzyloxy)-2-azaspiro[3.3]heptane-2-carboxylate was reacted with (4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazin-6-ium (2S,3S)-3-carboxy-2,3-bis((4-methylbenzoyl)oxy)propionate (CAS RN 2624363-49-9) to give the title compound. LC-MS (ESI) [M+H] + :386.2.

[0529] Example 46

[0530] (4aR,8aS)-6-(3-(Phenoxymethyl)azetidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one

[0531]

[0532] Analogously to the method described in Example 1, a1), tert-butyl 3-(phenoxymethyl)azetidine-1-carboxylate (obtained from phenol, CAS RN 108-95-2) and tert-butyl 3-(bromomethyl)azetidine-1-carboxylate (CAS RN 253176-93-1), analogously to Example 1d), were reacted with (4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazin-6-ium (2S,3S)-3-carboxy-2,3-bis((4-methylbenzoyl)oxy)propionate (CAS RN 2624363-49-9) to give the title compound. LC-MS (ESI) [M+H] + :346.1

[0533] Building block synthesis

[0534] BB2

[0535] tert-Butyl 3-((2-(2-(((benzyloxy)carbonyl)amino)ethoxy)benzyl)oxy)azetidine-1-carboxylate

[0536]

[0537] Analogously to the method described in Example 1g), tert-butyl 3-hydroxy-azetidine-1-carboxylate (CAS RN141699-55-0, 125 mg, 0.72 mmol) was reacted with benzyl (2-(2-(bromomethyl)phenoxy)ethyl)carbamate (see Example 1, 131 mg, 0.36 mmol) to give the title compound (101 mg, 0.22 mmol, 61%). 1 H NMR (300 MHz, CDCl 3 )δ7.41–7.27(m,7H),6.97(t,J=7.4Hz,1H),6.86(d,J=8.1Hz,1H),5.43(s,1H,NH-Cbz),5.11(s,2H,OCH 2 -Ar),4.46(s,2H,OCH 2 Cbz), 4.31 (dq, J = 10.8, 5.1 Hz, 1H, CH2-azetidine), 4.10 (t, J = 5.0 Hz, 2H), 4.07–3.97 (m, 2H), 3.92–3.81 (m, 2H), 3.62 (q, J = 5.4 Hz, 2H, OCH 2 CH 2 -NHCbz),1.42(d,J=1.0Hz,9H,CH 3 Boc). 13 CNMR (75MHz, CDCl 3 )δ156.6,156.5(C=OCbz,C=Ourea),136.6,130.1,129.7,128.7,128.4,128.3,121.3,112.0(C-Ar),79.7(CHBoc),67.6,67.0,66.5(3xOCH 2 ),56.65,40.8(CH 2 -NHCbz),28.5(CH 3 Boc). LC-HRMS(ESI) 25 H 33 N 2 O 6 [M+H] + Calculated value: 457.2333; measured value: 457.2320.

[0538] BB3

[0539] tert-Butyl 6-((2-(2-(((benzyloxy)carbonyl)amino)ethoxy)benzyl)oxy)-2-azaspiro[3.3]heptane-2-carboxylate

[0540]

[0541] Analogously to the method described in Example 1g), benzyl (2-(2-(bromomethyl)phenoxy)ethyl)carbamate (see Example 1) was reacted with tert-butyl 6-hydroxy-2-azaspiro[3.3]heptane-2-carboxylate (CAS RN 1147557-97-8) to give the title compound (48.5 mg, 0.098 mmol, 65%). 1 HNMR (300MHz, CDCl 3 )δ7.39–7.21(m,7H),6.95(td,J=7.5,1.0Hz,1H),6.84(d,J=8.5Hz,1H),5.54(s,1H,NHCbz),5.10(s,2H,OCH 2 ),4.40(s,2H,OCH 2 ), 4.10 (t, J = 5.0 Hz, 2H), 3.93–3.72 (m, 5H, CH spiro, 2x NCH 2 Spiro), 3.61(q, J=5.3Hz,2H,CH 2 NHCbz), 2.41–2.28 (m, 2H, C-CH 2 -CH spiro), 2.14–1.95 (m, 2H, C-CH 2 -CH spiro), 1.43(s,9H,3x CH 3 Boc). 13 C NMR (75MHz, CDCl 3 )δ156.7,156.6,156.2(C=O Cbz,C=OBoc,C Ar -O),136.5,130.1,129.5,128.7,128.5,128.4,126.6,121.2,112.1,79.4(C(CH 3 ) 3 Boc),68.1,67.6,67.0,65.8,61.8,60.5,41.2,40.7(CH 2 NHCz,2x C-CH 2 -CH spiro), 30.1(-C-spiro), 28.5(3x CH 3 Boc). LC-HRMS(ESI)C28 H 36 N 2 O 6 Na[M+Na] + Calculated value: 519.2466; measured value: 519.2450.

[0542] BB4

[0543] tert-Butyl 7-((2-(2-(((benzyloxy)carbonyl)amino)ethoxy)benzyl)oxy)-2-azaspiro[3.5]nonane-2-carboxylate

[0544]

[0545] Analogously to the method described in Example 1g), benzyl (2-(2-(bromomethyl)phenoxy)ethyl)carbamate (see Example 1) was reacted with tert-butyl 7-hydroxy-2-azaspiro[3.5]nonane-2-carboxylate (CAS RN 1363383-18-9) to give the title compound (18.0 mg, 0.05 mmol, 26%). 1 HNMR (300MHz, CDCl 3 )δ7.35(s,7H),7.02–6.77(m,2H),5.41(s,1H,NHCbz),5.10(s,2H,OCH 2 ),4.53(s,2H,OCH 2 ), 4.08 (t, J = 5.0Hz, 2H), 4.04–3.93 (m, 1H), 3.69–3.41 (m, 7H), 1.94–1.72 (m, 4H), 1.54–1.32 (m, 12H). 13 C NMR (75MHz, CDCl 3 )δ156.6,156.5,156.2(C=O Cbz,C=O Boc,C Ar -O),136.4,129.5,128.9,128.6,128.5,128.2,128.1,127.9,127.4,121.1,111.7,79.2(C(CH 3 ) 3 Boc),67.3,66.9,65.2,59.0,40.7(CH 2 NHCbz),34.5,33.0,28.5,28.2(3x CH 3 Boc). LC-HRMS(ESI)C 30 H 40 N 2 O 6 Na[M+Na]+ Calculated value: 547.2779; measured value: 547.2774.

[0546] BB5

[0547] tert-Butyl 3-(((3-(2-(((benzyloxy)carbonyl)amino)ethoxy)benzyl)oxy)methyl)azetidine-1-carboxylate

[0548]

[0549] Step d Ethyl 3-(2-(((Benzyloxy)carbonyl)amino)ethoxy)benzoate

[0550]

[0551] Analogously to the method described in Example 1d), ethyl 3-hydroxybenzoate (CAS RN 778198 8) was reacted with 2-(((benzyloxy)carbonyl)amino)ethyl 4-methylbenzenesulfonate (CAS RN 93407-96-6) to give the title compound (1.05 g, 3.05 mmol, 61%). 1 H NMR (300 MHz, CDCl 3 )δ7.66(dt,J=7.7,1.3Hz,1H),7.54(dd,J=2.7,1.5Hz,1H),7.40–7.27(m,6H),7.12–7.02(m,1H),5.22(s,1H,NH-Cbz),5.12(s,2H,OCH 2 CH 3 ),4.37(q,J=7.1Hz,2H,OCH 2 ),4.09(t,J=5.1Hz,2H,OCH 2 ),3.63(q,J=5.4Hz,2H,CH 2 NHCbz),1.39(t,J=7.1Hz,3H,OCH 2 CH 3 ). 13 C NMR (75MHz, CDCl 3 )δ166.5,158.5,156.5(C=O CO 2 Et,C=O Cbz,C Ar -O),136.5,132.1,129.6,128.7,128.4,128.3,122.6,119.7,114.9,67.3(OCH 2 ),67.1(OCH 2 ),61.3(OCH 2),40.7(CH 2 NHCbz),14.5(CH 3 ). LC-HRMS(ESI)C 19 H 22 NO 5 [M+H] + Calculated value: 366.1492; measured value: 366.1508.

[0552] Step e: Benzyl (2-(3-(hydroxymethyl)phenoxy)ethyl)carbamate

[0553]

[0554] In analogy to the method described in Example 1e), ethyl 3-(2-(((benzyloxy)carbonyl)amino)ethoxy)benzoate was reacted to give the title compound (115 mg, 0.38 mmol, 66%). 1 H NMR (300 MHz, CDCl 3 )δ7.43–7.16(m,6H),7.00–6.85(m,2H),6.87–6.70(m,1H),5.11(s,2H,O-CH 2 -),4.66(s,2H,O-CH 2 -),4.04(t,J=5.1Hz,2H,-OCH 2 CH 2 NHCbz),3.60(d,J=5.3Hz,2H,-OCH 2 CH 2 NHCbz). 13 C NMR (75MHz, CDCl 3 )δ158.8,156.6(C=O Cbz,C Ar -O),142.8,136.5,129.8,128.7,128.32,128.28,119.7,113.8,112.9(C-Ar),67.01(O-CH 2 -),66.96(O-CH 2 -),65.2(O-CH 2 -),40.7(-CH 2 -NHCbz). LC-HRMS(ESI)C 17 H 20 NO 4 [M+H] + Calculated value: 302.1387; measured value: 302.1373.

[0555] Step f: Benzyl (2-(3-(bromomethyl)phenoxy)ethyl)carbamate

[0556]

[0557] In analogy to the method described in Example 1f), benzyl (2-(3-(hydroxymethyl)phenoxy)ethyl)carbamate was reacted to give the title compound (503 mg, 1.38 mmol, 63%). 1 H NMR (300 MHz, CDCl 3 )δ7.42–7.22(m,6H),7.03–6.97(m,1H),6.95–6.90(m,1H),6.88–6.79(m,1H),5.24(s,1H,NHCbz),5.13(s,2H,OCH 2 ),4.46(s,2H,CH 2 Br), 4.06(t, J=5.1Hz, 2H, OCH 2 ),3.63(q,J=5.4Hz,2H,CH 2 NHCbz). 13 C NMR (75MHz, CDCl 3 )δ158.8,156.5(C=O Cbz,C Ar -O),139.4,139.2,136.5,130.1,128.7,128.33,128.29,121.9,121.4,115.2,114.7,114.7,67.0(2xOCH 2 ),40.7(CH 2 NHCbz),33.4(CH 2 Br). LC-HRMS(ESI)C 17 H 19 BrNO 3 [M+H] + Calculated value: 366.0524; measured value: 366.0551.

[0558] Step g tert-Butyl 3-(((3-(2-(((benzyloxy)carbonyl)amino)ethoxy)benzyl)oxy)methyl)azetidine-1-carboxylate

[0559] Analogously to the method described in Example 1g), benzyl (2-(3-(bromomethyl)phenoxy)ethyl)carbamate was reacted with tert-butyl 3-(hydroxymethyl)azetidine-1-carboxylate (CAS RN 142253-56-3) to give the title compound (85.0 mg, 0.18 mmol, 55%). 1H NMR (300 MHz, CDCl 3 )δ7.40–7.20(m,6H),7.01–6.74(m,3H),5.31–5.06(m,3H,NHCbz,OCH 2 ),4.70–4.42(m,3H),4.07–3.91(m,4H),3.74–3.51(m,5H),2.88–2.69(m,1H,CH azetidine),1.43(d,J=1.2Hz,9H,3x CH 3 Boc). LC-HRMS(ESI)C 26 H 34 N 2 O 6 Na[M+Na] + Calculated value: 493.2309; Measured value: 493.2312

[0560] BB6

[0561] tert-Butyl 3-((3-(2-(((benzyloxy)carbonyl)amino)ethoxy)benzyl)oxy)azetidine-1-carboxylate

[0562]

[0563] Analogously to the method described for BB7, benzyl (2-(3-(bromomethyl)phenoxy)ethyl)carbamate was reacted with tert-butyl 3-hydroxyazetidine-1-carboxylate (CAS RN 141699-55-0) to give the title compound (55.1 mg, 0.12 mmol, 55%). LC-HRMS (ESI) 25 H 33 N 2 O 6 [M+H] + Calculated value: 457.2333; measured value: 457.2328.

[0564] BB7

[0565] tert-Butyl 6-((3-(2-(((benzyloxy)carbonyl)amino)ethoxy)benzyl)oxy)-2-azaspiro[3.3]heptane-2-carboxylate

[0566]

[0567] Benzyl (2-(3-(bromomethyl)phenoxy)ethyl)carbamate was reacted with tert-butyl 6-hydroxy-2-azaspiro[3.3]heptane-2-carboxylate (CAS RN 1147557-97-8) in analogy to the procedure described for BB7 to give the title compound (21.0 mg, 0.042 mmol, 28%). 1 H NMR (300 MHz, CDCl 3 )δ7.36(d,J=4.9Hz,5H),7.22(d,J=7.8Hz,1H),6.93–6.74(m,3H),5.11(s,2H,OCH 2 ),4.35(s,2H,OCH 2 ),4.04(t,J=5.1Hz,2H,OCH 2 ),4.01–3.78(m,5H,N-CH 2 -C spiro, CH spiro), 3.61 (q, J = 5.4 Hz, 2H, CH 2 NHCbz), 2.47 (m, 2H, C-CH 2 -CH spiro), 2.14 (m, 2H, C-CH 2 -CH spiro), 1.42(s,9H,3xCH 3 Boc). 13 C NMR (75MHz, CDCl 3 )δ158.7,156.5,156.3(C=O Cbz,C=OBoc,C Ar -O),139.9,136.5,129.7,128.7,128.34,128.30,120.6,113.8,79.5(C(CH 3 ) 3 Boc),70.1,68.1,67.0,61.9,60.6,41.2,40.7,30.3(3x CH 3 Boc), 28.5 (-C-spirocyclic). LC-HRMS (ESI) 28 H 37 N 2 O 6 [M+H] + Calculated value: 497.2646; measured value: 497.2636.

[0568] BB8

[0569] tert-Butyl 3-(((2-(3-(((benzyloxy)carbonyl)amino)propoxy)benzyl)oxy)methyl)azetidine-1-carboxylate

[0570]

[0571] Step d: Ethyl 2-(3-(((Benzyloxy)carbonyl)amino)propoxy)benzoate

[0572]

[0573] In analogy to the method described in Example 1d), ethyl salicylate (CAS RN 118-61-6) was reacted with 3-(((benzyloxy)carbonyl)amino)propyl 4-methylbenzenesulfonate (CAS RN 68076-37-9) to give the title compound (3.98 g, 11.2 mmol, 82%). 1 H NMR (300 MHz, CDCl 3 )δ7.85(dd,J=7.8,1.8Hz,1H),7.56–7.20(m,6H),7.06–6.87(m,2H),6.54(s,1H,NHCbz),5.11(s,2H,OCH 2 ),4.24(q,J=7.1Hz,2H,OCH 2 CH 3 ),4.13(t,J=5.5Hz,2H,OCH 2 ),3.57–3.40(m,2H,CH 2 NHCbz),2.06(p,J=5.6Hz,2H,-CH 2 -),1.29(t,J=7.1Hz,3H,OCH 2 CH 3 ). 13 C NMR (75MHz, CDCl 3 )δ166.0,158.7,157.0(C=O CO 2 Et,C=O Cbz,C Ar -O),137.1,133.9,132.0,128.4,128.0,127.9,120.4,119.7,112.7,68.2(OCH 2 ),66.5(OCH 2 ),61.0(OCH 2 ),39.9(CH 2 NHCbz),29.1(-CH 2 -),14.4(CH 3 ). LC-HRMS(ESI)C 20 H 23 NO 5 Calculated value for Na[M+Na]+: 380.1468; found: 380.1474.

[0574] Step e: Benzyl (3-(2-(hydroxymethyl)phenoxy)propyl)carbamate

[0575]

[0576] In analogy to the method described in Example 1e), ethyl 2-(3-(((benzyloxy)carbonyl)amino)propoxy)benzoate was reacted to give the title compound (2.35 g, 7.45 mmol, 98%). 1 H NMR (300 MHz, CDCl 3 )δ7.39–7.19(m,7H),6.94(td,J=7.4,1.1Hz,1H),6.83(d,J=8.1Hz,1H),5.08(s,2H,-OCH 2 -),4.66(s,2H,-OCH 2 -),4.03(td,J=5.9,1.7Hz,2H,-OCH 2 CH 2 CH 2 NHCbz),3.40(t,J=6.3Hz,2H,-OCH 2 CH 2 CH 2 NHCbz),1.98(p,J=5.9Hz,2H,-OCH 2 CH 2 CH 2 NHCbz). 13 C NMR (75MHz, CDCl 3 )δ156.7,156.6(C=O Cbz,C Ar -O),136.6,129.3,129.0,128.5,128.09,128.04,120.7,111.0(C-Ar),66.7(OCH 2 ),65.7(OCH 2 ),61.5(OCH 2 ),38.6(CH 2 -NHCbz),29.4(-CH 2 -connector). LC-HRMS(ESI)C 18 H 21 NO 4 Na[M+Na] + Calculated value: 338.1363; measured value: 338.1353.

[0577] Step f: Benzyl (3-(2-(bromomethyl)phenoxy)propyl)carbamate

[0578]

[0579] Benzyl (3-(2-(hydroxymethyl)phenoxy)propyl)carbamate was reacted in analogy to the method described in Example 1f) to give the title compound (2.00 g, 5.29 mmol, 80%). 1 H NMR (300 MHz, CDCl 3 )δ7.45–7.18(m,7H),6.98–6.79(m,2H),5.11(s,2H,OCH 2 ),4.54(s,2H,CH 2 -Br),4.10(t,J=5.8Hz,2H,,OCH 2 connector), 3.49 (q, J = 6.4 Hz, 2H, CH 2 -NHCbz), 2.18–1.96 (m, 2H, -CH 2 - connector). 13 C NMR (75MHz, CDCl 3 )δ156.8,156.7(C=O Cbz,C Ar -O),136.7,131.0,130.8,130.5,130.4,128.7,128.2,126.2,125.9,121.0,111.77,111.71(C-Ar),66.8(-OCH 2 -),65.85(-OCH 2 -),42.13(-CH 2 -NHCbz),38.6(CH 2 -Br),29.53(-CH 2 -connector). LC-HRMS(ESI)C 18 H 21 BrNO 3 [M+H] + Calculated value: 378.0699; Measured value: 378.0700

[0580] Step g tert-Butyl 3-(((2-(3-(((benzyloxy)carbonyl)amino)propyloxy)benzyl)oxy)methyl)azetidine-1-carboxylate

[0581] Analogously to the method described in Example 1g), benzyl (3-(2-(bromomethyl)phenoxy)propyl)carbamate was reacted with tert-butyl 3-(hydroxymethyl)azetidine-1-carboxylate (CAS RN 142253-56-3) to give the title compound (401 mg, 0.83 mmol, 59%).1 H NMR (300 MHz, CDCl 3 )δ7.41–7.28(m,7H),6.95(td,J=7.5,1.1Hz,1H),6.85(d,J=8.2Hz,1H),5.46(s,1H,NHCbz),5.10(s,2H,OCH 2 ),4.54(s,2H,OCH 2 ),4.05(t,J=5.8Hz,2H),3.92(t,J=8.4Hz,2H),3.67–3.51(m,4H),3.43(q,J=6.1Hz,2H,CH 2 NHCbz), 2.81–2.63 (m, 1H, CHazetidine), 2.02 (p, J = 6.0 Hz, 2H, CH 2 connector), 1.42(s,9H,3x CH 3 Boc). 13 C NMR (75MHz, CDCl 3 )δ156.7,156.6,156.5(C=O Cbz,C=OBoc,C Ar -O),136.8,129.6,129.2,128.6,128.2,126.4,120.8,111.4,79.4(C(CH 3 ) 3 Boc),72.4(OCH 2 ),68.7(OCH 2 ),66.7(OCH 2 ),66.4(OCH 2 ),52.0(2x N-CH 2 -azetidine), 39.1(CH 2 NHCbz), 29.6 (CH2-azetidine), 28.5 (3x CH 3 Boc),27.1(-CH 2 -connector). LC-HRMS(ESI) 27 H 37 N 2 O 6 [M+H] + Calculated value: 485.2646; measured value: 485.2646.

[0582] BB9

[0583] tert-Butyl 3-(((2-((5-(((benzyloxy)carbonyl)amino)pentyl)oxy)benzyl)oxy)methyl)azetidine-1-carboxylate

[0584]

[0585] Step d: Ethyl 2-((5-(((benzyloxy)carbonyl)amino)pentyl)oxy)benzoate

[0586]

[0587] In analogy to the method described in Example 1g), ethyl salicylate (CAS RN 118-61-6) was reacted with 5-(((benzyloxy)carbonyl)amino)pentyl 4-methylbenzenesulfonate (CAS RN 93066-51-4) to give the title compound (1.0 g, 2.59 mmol, 89%). 1 H NMR (300 MHz, CDCl 3 )δ7.77(dd,J=7.7,1.8Hz,1H),7.52–7.28(m,6H),7.04–6.84(m,2H),5.09(s,2H,O-CH 2 ),4.31(q,J=7.1Hz,2H,O-CH 2 -CH 3 ),4.02(t,J=6.2Hz,2H,O-CH 2 ),3.23(m,2H,-CH 2 -NHCbz),1.84(p,J=6.5Hz,2H,-CH 2 -alkanes), 1.64–1.53 (m, 4H, 2x-CH 2 -alkane), 1.33 (t, J = 7.1 Hz, 3H, O-CH 2 -CH 3 ). 13 C NMR (75MHz, CDCl 3 )δ166.6,158.6,156.6(C=O CO 2 Et,C=O Cbz,C Ar -O),136.8,133.4,131.66,128.6,128.22,128.19,120.8,120.3,113.2(C-Ar),68.5,66.7,60.9(3xO-CH 2 ),41.0(-CH 2 -NHCbz),31.1,28.8,23.2(3x CH 2 Alkanes), 14.44 (O-CH 2 CH 3 ). LC-HRMS(ESI)C 22 H 27NO 5 Na[M+Na] + Calculated value: 408.1781; measured value: 408.1798.

[0588] Step e: Benzyl (5-(2-(hydroxymethyl)phenoxy)pentyl)carbamate

[0589]

[0590] In analogy to the method described in Example 1e), ethyl 2-((5-(((benzyloxy)carbonyl)amino)pentyl)oxy)benzoate was reacted to give the title compound (737 mg, 2.15 mmol, 86%). 1 HNMR (300MHz, CDCl 3 )δ7.41–7.19(m,7H),6.93(td,J=7.4,1.0Hz,1H),5.10(s,2H,OCH 2 ),4.68(s,2H,OCH 2 ),4.01(t,J=6.3Hz,2H,OCH 2 ),3.23(t,J=6.6Hz,2H CH 2 NHCbz),1.84(p,J=6.5Hz,2H,-CH 2 -alkanes), 1.68–1.46 (m, 4H, 2x-CH 2 -alkanes). 13 C NMR (75MHz, CDCl 3 )δ157.0,156.6(C=O Cbz,C Ar -O),136.7,129.3,129.1,128.9,128.7,128.3,128.3,120.8,111.2,67.7(OCH 2 ),66.8(OCH 2 ),62.3(CH 2 OH),41.0(CH 2 NHCbz),29.9(-CH 2 -alkanes), 27.1(-CH 2 -alkanes), 23.5(-CH 2 -alkane). LC-HRMS(ESI)C 20 H 25 NO 4 Na[M+Na] + Calculated value: 366.1676; measured value: 366.1691.

[0591] Step f tert-Butyl 3-(((2-((5-(((benzyloxy)carbonyl)amino)pentyl)oxy)benzyl)oxy)methyl)azetidine-1-carboxylate

[0592] Analogously to the method described in Example 1f), benzyl (5-(2-(hydroxymethyl)phenoxy)pentyl)carbamate was reacted with tert-butyl 3-(hydroxymethyl)azetidine-1-carboxylate (CAS RN 142253-56-3) to give the title compound (67.6 mg, 0.132 mmol, 53%). 1 H NMR (600 MHz, CDCl 3 )δ7.38–7.28(m,5H),7.27–7.19(m,2H),6.94(t,J=7.4Hz,1H),6.84(d,J=8.3Hz,1H),5.10(s,2H,OCH 2 ),4.97(s,1H,NHCbz),4.56(s,2H,OCH 2 ),4.00–3.93(m,4H),3.70–3.64(m,2H),3.64–3.59(m,2H),3.23(q,J=6.7Hz,2H,CH 2 NHCbz), 2.82–2.73 (m, 1H, CHazetidine), 1.85–1.77 (m, 2H, CH 2 connector), 1.63–1.48 (m, 4H, CH 2 connector), 1.43(s,9H,3x CH 3 Boc). 13 C NMR (151 MHz, CDCl 3 )δ156.63,156.58,156.55(C=O Boc,C=O Cbz,C Ar -O),136.8,129.1,128.9,128.6,128.21,128.17,126.7,120.5,111.2,79.4(C(CH 3 ) 3 Boc),72.6,68.2,67.8,66.7(4x OCH 2 ),52.0(2x NCH 2 Azetidine), 41.1(CH 2 NHCbz), 29.8, 29.0, 28.8 (CHazetidine, 2x CH 2 Connector), 28.5(3xCH 3 Boc),23.5(CH 2Connector). LC-HRMS (ESI) C29H40N2O6Na[M+Na]+ calculated value: 535.2779; found value: 537.2784

[0593] BB10

[0594] tert-Butyl 6-(2-(2-(((Benzyloxy)carbonyl)amino)ethoxy)benzylidene)-2-azaspiro[3.3]heptane-2-carboxylate

[0595]

[0596] Step i Synthesis of structural unit: tert-butyl 6-((4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)methylene)-2-azaspiro[3.3]heptane-2-carboxylate

[0597]

[0598] In a drying flask, tetramethylpiperidine (CAS RN 768-66-1, 1.25 mL, 7.40 mmol) was dissolved in anhydrous THF (37.0 mL) and heated under N 2 Atmosphere was cooled to -78 ° C. A solution of nBuLi 2.5M in THF (3.00mL, 7.40mmoL) was added dropwise, and the reaction mixture was stirred at the same temperature for 30min. Next, a solution of 0.84M bis((pinacol)boryl)methane (CAS RN 78782-17-9) in THF (8.83mL, 7.40mmol) was added dropwise. The reaction was stirred for 5min, and then a 0.20M solution of ketone in anhydrous THF solution (18.5mL, 3.70mmol) was added dropwise over 5min. The reaction mixture was slowly warmed to room temperature overnight. After completion, the reaction was opened to the air and filtered through a silica plug, with Et 2 The mixture was concentrated under reduced pressure and purified by silica gel chromatography (0 to 20% EtOAc in cyclohexane) and detected by ELSD to give the title compound in quantitative yield. LC-HRMS (ESI) C 18 H 31 BNO 4 [M+H] + Calculated value: 336.2340; measured value: 336.2342.

[0599] Step d: Benzyl (2-(2-bromophenoxy)ethyl)carbamate

[0600]

[0601] Analogously to the method described in Example 1d), 2-bromophenol (CAS 95-56-7) was reacted with 2-(((benzyloxy)carbonyl)amino)ethyl 4-methylbenzenesulfonate (CAS RN 93407-96-6) to give the title compound (985 mg, 2.81 mmol, 94%). 1 H NMR (300 MHz, CDCl 3 )δ7.47(dd,J=7.9,1.6Hz,1H),7.33–7.13(m,6H),6.87–6.73(m,2H),5.31(s,1H,NHCbz),5.06(s,2H,OCH 2 ),4.03(t,J=5.0Hz,2H,OCH 2 ),3.59(q,J=5.4Hz,2H,CH 2 NHCbz). 13 C NMR (75MHz, CDCl 3 )δ156.6,154.9(C=O Cbz,C Ar -O),136.5,133.5,128.7,128.7,128.29,128.26,122.6,113.7,112.5,68.5(OCH 2 ),67.0(OCH 2 ),40.6(CH 2 NHCbz). LC-HRMS (ESI) calculated for C16H16BrNO3Na[M+Na]+: 372.0206; found: 372.0209.

[0602] Step h tert-Butyl 6-(2-(2-(((benzyloxy)carbonyl)amino)ethoxy)benzylidene)-2-azaspiro[3.3]heptane-2-carboxylate

[0603] In a sealed vial under nitrogen atmosphere, tert-butyl 6-((4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)methylene)-2-azaspiro[3.3]heptane-2-carboxylate (117 mg, 0.35 mmol), benzyl (2-(2-bromophenoxy)ethyl)carbamate (123 mg, 0.35 mmol) and Na 2 CO 3 (74.2 mg, 0.70 mmol) was suspended in a solution previously prepared by bubbling N 2 The mixture was washed with N 2 The bubbling was continued for 10 min. Then, Pd(dppf)Cl was added 2(CAS RN 72287-26-4) and the mixture was purged for another 15 min. The reaction was warmed to 55 °C and stirred at this temperature overnight. The reaction was then diluted with DCM (15 mL) and washed with saturated NaHCO 3 The organic layer was extracted with aqueous solution (20 mL) and washed with brine (20 mL). 4 Dry, concentrate under reduced pressure and purify by silica gel chromatography (0 to 60% EtOAc in cyclohexane) to give the title compound (97.0 mg, 0.20 mmol, 58%). LC-HRMS (ESI) C 28 H 34 N 2 O 5 Calculated value for Na[M+Na]+: 501.2360; found: 501.2388.

[0604] BB11

[0605] tert-Butyl 6-(3-(2-(((Benzyloxy)carbonyl)amino)ethoxy)benzylidene)-2-azaspiro[3.3]heptane-2-carboxylate

[0606]

[0607] Step d: Benzyl (2-(3-bromophenoxy)ethyl)carbamate

[0608]

[0609] Analogously to the method described for BB10, step d), 3-bromophenol (CAS RN 591-20-8) was reacted with 2-(((benzyloxy)carbonyl)amino)ethyl 4-methylbenzenesulfonate (CAS RN 93407-96-6) to give the title compound (366 mg, 0.69 mmol, 69%). LC-MS [M+Na] + :372.0.

[0610] Step h tert-Butyl 6-(3-(2-(((benzyloxy)carbonyl)amino)ethoxy)benzylidene)-2-azaspiro[3.3]heptane-2-carboxylate

[0611] In analogy to the method described for BB10, step h), benzyl (2-(3-bromophenoxy)ethyl)carbamate was reacted with tert-butyl 6-((4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)methylene)-2-azaspiro[3.3]heptane-2-carboxylate to give the title compound (181 mg, 0.38 mmol, 63%). 1 H NMR (300 MHz, CDCl3 )δ7.41–7.30(m,5H),7.21(t,J=8.1Hz,1H),6.83–6.60(m,3H),6.11(s,1H),5.22(s,1H),5. 11(s,2H),4.08–3.90(m,6H),3.60(d,J=5.5Hz,2H),3.20(s,2H),3.04(s,2H),1.42(s,9H). LC-MS(ESI):[M+H] + :479.2.

[0612] BB12

[0613] tert-Butyl 6-(2-((5-(((benzyloxy)carbonyl)amino)pentyl)oxy)benzylidene)-2-azaspiro[3.3]heptane-2-carboxylate

[0614]

[0615] Step d: Benzyl (5-(2-bromophenoxy)pentyl)carbamate

[0616]

[0617] In analogy to the method described for BB10, step d), 3-bromophenol (CAS RN 591-20-8) was reacted with 5-(((benzyloxy)carbonyl)amino)pentyl 4-methylbenzenesulfonate (CAS RN 93066-51-4) to give the title compound (403 mg, 1.03 mmol, 51%). 1 H NMR (300 MHz, CDCl 3 )δ7.52(dd,J=7.9,1.6Hz,1H),7.39–7.20(m,6H),6.94–6.71(m,2H),5.10(s,2H,OCH 2 ),4.78(s,1H,NHCbz),4.01(t,J=6.2Hz,2H,OCH 2 ),3.24(q,J=6.3Hz,2H,CH 2 NHCbz), 1.86(t, J=6.8 Hz, 2H, CH 2 connector), 1.76–1.48 (m, 4H, 2x CH 2 connector). 13 C NMR (75MHz, CDCl 3 )δ156.4,155.3(C=O Cbz,C Ar-O),136.6,133.3,128.5,128.4,128.1,121.8,113.2,112.3,68.8(OCH 2 ),66.7(OCH 2 ),41.0(CH 2 NHCbz),29.7(CH 2 Connector), 28.7(CH 2 Connector), 23.3(CH 2 Connector). LC-HRMS(ESI)C 19 H 23 BrNO 3 [M+H] + Calculated value: 392.0856; measured value: 392.0860.

[0618] Step h tert-Butyl 6-(2-((5-(((benzyloxy)carbonyl)amino)pentyl)oxy)benzylidene)-2-azaspiro[3.3]heptane-2-carboxylate

[0619] Analogously to the method described for BB10, step h), benzyl (5-(2-bromophenoxy)pentyl)carbamate was reacted with tert-butyl 6-((4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)methylene)-2-azaspiro[3.3]heptane-2-carboxylate to give the title compound (100 mg, 0.19 mmol, 25%). LC-HRMS (ESI) 31 H 41 N 2 O 5 [M+H] + Calculated value: 521.3010; measured value: 521.3000.

[0620] BB 14

[0621] tert-Butyl (1R,5S)-6-(((2-(2-(((benzyloxy)carbonyl)amino)ethoxy)benzyl)oxy)methyl)-3-azabicyclo[3.1.0]hexane-3-carboxylate

[0622]

[0623] Analogously to the method described in Example 1g), benzyl (2-(2-(bromomethyl)phenoxy)ethyl)carbamate (see Example 1) was reacted with tert-butyl (1R,5S,6r)-6-(hydroxymethyl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (CAS RN 827599-21-3) to give the title compound (32.3 mg, 0.069 mmol, 57%). 1H NMR (300 MHz, CDCl 3 )δ7.40–7.20(m,7H),6.96(td,J=7.5,1.1Hz,1H),6.85(d,J=8.3Hz,1H),5.09(s,2H,-OCH 2 -),4.52(s,2H,-OCH 2 -),4.10(t,J=5.1Hz,2H),3.65–3.39(m,5H),3.36–3.19(m,3H),1.49–1.32(m,11H,3x CH 3 Boc, 2x CH fused ring), 0.98–0.87 (m, 1H, CH propyl ring). 13 C NMR (75MHz, CDCl 3 )δ156.57,156.53,155.0(C=O Cbz,C=O Boc,C Ar -O), 136.6, 129.8, 129.2, 128.6, 128.30, 128.25, 127.0, 121.2, 112.2 (C-Ar), 79.4, 71.7, 68.0, 67.6, 66.9, 48.1 (CH cyclopropyl), 40.7 (CH 2 -NHCbz),28.6(3xCH 3 Boc), 27.0, 22.4 (CH fused ring). LC-HRMS (ESI) 28 H 37 N 2 O 6 [M+H] + Calculated value: 497.2646; measured value: 497.2658.

[0624] BB16

[0625] tert-Butyl 3-(3-(ethoxycarbonyl)phenoxy)azetidine-1-carboxylate

[0626]

[0627] Analogously to the method described in Example 39d), ethyl 3-hydroxybenzoate (CAS RN 7781-98-8) was reacted with tert-butyl 3-iodoazetidine-1-carboxylate (CAS RN 254454-54-1) to give the title compound. LC-MS (ESI) [M+Na] + :344.1.

[0628] BB17

[0629] tert-Butyl 3-((2-(ethoxycarbonyl)phenoxy)methyl)azetidine-1-carboxylate

[0630]

[0631] Analogously to the method described in Example 39d), ethyl 3-hydroxybenzoate (CAS RN 7781-98-8) was reacted with tert-butyl 3-(bromomethyl)azetidine-1-carboxylate (CAS RN 253176-93-1) to give the title compound. LC-MS (ESI) [M+Na] + :358.1.

Claims

1. A compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof, wherein: In formula (I), R 1 Selected from: The wavy line indicates R 1 The point of attachment to the rest of formula (I); In formula (II), R 1 Selected from: The wavy line indicates R 1 The point of attachment to the rest of formula (II); R 2 Selected from: in: Wavy line indicates R 2 The point of attachment to the rest of formula (I); p is 1 or 2; V is selected from hydrogen and methyl; W is selected from methyl, phenyl, in Ar is C6-C 10 - aryl or 5- to 14-membered heteroaryl; X is selected from O, S, Si(CH3)2 and CH2; and Y is selected from OH, NH2, N(CH3)2, N(CD3)2 and azetidin-1-yl; R 3 is selected from hydrogen, C1-C6-alkoxycarbonyl and C1-C6-alkoxycarbonyl-NH-C1-C6-alkyl-NH-C(O)–; A is: (i) and L is selected from –CH2–, –CH2O–, –OCH2–, –CH2OCH2–, and –O–; or (ii) in (i) X is CH; and L is selected from –CH2–, –CH2O–, –OCH2–, –CH2OCH2–, and –O–; or (ii) X and L together form a group wherein the wavy line indicates the point of attachment of L to the phenyl moiety within formula (I) or formula (II), and the two asterisks indicate the points of attachment of X to the corresponding adjacent atoms within ring A; wherein the wavy line indicates the point of attachment of A to L; and the asterisk indicates the point of attachment of A to the carbonyl group of formula (I) or formula (II); and n is an integer selected from 1, 2, 3 and 4.

2. The compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof according to claim 1, wherein R 1 Selected from: The wavy line indicates R 1 The point of attachment to the rest of Formula (I).

3. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein A is: (i) and L is selected from –CH2O–, –OCH2–, –CH2OCH2–, and –O–; or (ii) in (i) X is CH; and L is selected from –CH2– and –CH2O–; or (ii) X and L together form a group wherein the wavy line indicates the point of attachment of L to the phenyl moiety within formula (I), and the two asterisks indicate the points of attachment of X to the corresponding adjacent atoms within ring A; wherein the wavy line indicates the point of attachment of A to L; and the asterisk indicates the point of attachment of A to the carbonyl group of formula (I).

4. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 3, wherein A is: (i) and L is –CH2OCH2–; or (ii) in (i) X is CH and L is -CH2-; or (ii) X and L together form a group wherein the wavy line indicates the point of attachment of L to the phenyl moiety within formula (I), and the two asterisks indicate the points of attachment of X to the corresponding adjacent atoms within ring A; wherein the wavy line indicates the point of attachment of A to L; and the asterisk indicates the point of attachment of A to the carbonyl group of formula (I).

5. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 4, wherein A is: (i) and L is –CH2OCH2–; or (ii) and L is –CH2–; X is CH; wherein the wavy line indicates the point of attachment of A to L; and the asterisk indicates the point of attachment of A to the carbonyl group of formula (I).

6. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein: R 2 Selected from: in: Wavy line indicates R 2 The point of attachment to the rest of formula (I); p is 1; V is a methyl group; W is a methyl group; X is selected from O and Si(CH3)2; and Y is N(CH3)2; and n is an integer selected from 1, 2 and 4.

7. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 6, wherein: R 2 Selected from: in: Wavy line indicates R 2 The point of attachment to the rest of formula (I); p is 1; V is a methyl group; W is a methyl group; X is selected from O and Si(CH3)2; and Y is N(CH3)2; and n is an integer selected from 1, 2 and 4.

8. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 7, wherein: R 2 Selected from: in: Wavy line indicates R 2 The point of attachment to the rest of formula (I); p is 1; V is a methyl group; W is a methyl group; X is selected from O and Si(CH3)2; and Y is N(CH3)2; and n is an integer selected from 1 and 4.

9. The compound of formula (II) or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein A is: (i) and L is selected from -OCH2-, -CH2OCH2-, and -O-; or (ii) and L is –CH2O–; X is CH; wherein the wavy line indicates the point of attachment to L; and the asterisk indicates the point of attachment to the carbonyl group of formula (II).

10. The compound of formula (I) according to claim 1, wherein: R 1 Selected from: The wavy line indicates R 1 The point of attachment to the rest of formula (I); R 2 Selected from: in: Wavy line indicates R 2 The point of attachment to the rest of formula (I); p is 1; V is a methyl group; W is a methyl group; X is selected from O and Si(CH3)2; and Y is N(CH3)2; A is: (i) and L is selected from –CH2O–, –OCH2–, –CH2OCH2–, and –O–; or (ii) in (i) X is CH; and L is selected from –CH2– and –CH2O–; or (ii) X and L together form a group wherein the wavy line indicates the point of attachment of L to the phenyl moiety within formula (I), and the two asterisks indicate the points of attachment of X to the corresponding adjacent atoms within ring A; wherein the wavy line indicates the point of attachment to L; and the asterisk indicates the point of attachment to the carbonyl group of formula (I); and n is an integer selected from 1, 2 and 4.

11. The compound of formula (I) according to claim 10, wherein: R 1 Selected from: The wavy line indicates R 1 The point of attachment to the rest of formula (I); R 2 Selected from: in: Wavy line indicates R 2 The point of attachment to the rest of formula (I); p is 1; V is a methyl group; W is a methyl group; X is selected from O and Si(CH3)2; and Y is N(CH3)2; A is: (i) and L is –CH2OCH2–; or (ii) in (i) X is CH and L is -CH2-; or (ii) X and L together form a group wherein the wavy line indicates the point of attachment of L to the phenyl moiety within formula (I), and the two asterisks indicate the points of attachment of X to the corresponding adjacent atoms within ring A; wherein the wavy line indicates the point of attachment of A to L; and the asterisk indicates the point of attachment of A to the carbonyl group of formula (I); and n is an integer selected from 1, 2 and 4.

12. The compound of formula (I) according to claim 11, wherein: R 1 Selected from: The wavy line indicates R 1 The point of attachment to the rest of formula (I); R 2 Selected from: in: Wavy line indicates R 2 The point of attachment to the rest of formula (I); p is 1; V is a methyl group; W is a methyl group; X is selected from O and Si(CH3)2; and Y is N(CH3)2; A is: (i) and L is –CH2OCH2–; or (ii) and L is –CH2–; X is CH; wherein the wavy line indicates the point of attachment of A to L; and the asterisk indicates the point of attachment of A to the carbonyl group of formula (I); and n is an integer selected from 1 and 4.

13. The compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound of formula (I) or formula (II) is selected from: Benzyl (2-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamate; Benzyl (2-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)oxy)methyl)phenoxy)ethyl)carbamate; benzyl (2-(2-(((2-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)-2-azaspiro[3.3]heptan-6-yl)oxy)methyl)phenoxy)ethyl)carbamate; benzyl (2-(2-(((2-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)-2-azaspiro[3.5]nonan-7-yl)oxy)methyl)phenoxy)ethyl)carbamate; benzyl (2-(3-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamate; benzyl (2-(3-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)oxy)methyl)phenoxy)ethyl)carbamate; benzyl (2-(3-(((2-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)-2-azaspiro[3.3]heptan-6-yl)oxy)methyl)phenoxy)ethyl)carbamate; benzyl (3-(3-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)propyl)carbamate; benzyl (6-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)hexyl)carbamate; benzyl (2-(2-(((1-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamate; Cyclohex-1-en-1-ylmethyl (2-(2-((2-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)-2-azaspiro[3.3]heptan-6-ylidene)methyl)phenoxy)ethyl)carbamate; benzyl (2-(3-((2-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)-2-azaspiro[3.3]heptane-6-ylidene)methyl)phenoxy)ethyl)carbamate; benzyl (2-(2-((2-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)-2-azaspiro[3.3]heptane-6-ylidene)methyl)phenoxy)ethyl)carbamate; benzyl (2-(2-(((7-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)-7-azaspiro[3.5]nonan-2-yl)oxy)methyl)phenoxy)ethyl)carbamate; benzyl (5-(2-((2-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)-2-azaspiro[3.3]heptane-6-ylidene)methyl)phenoxy)pentyl)carbamate; benzyl (2-(2-((((1R,5S)-3-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)-3-azabicyclo[3.1.0]hexan-6-yl)methoxy)methyl)phenoxy)ethyl)carbamate; (R)-benzyl(2-(2-(((1-(3-(2-oxooxazolidin-4-yl)propanoyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamate; Benzyl (2-(2-(((1-(3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamate; 2-(6-(2-(2-((7-nitro-112,312-benzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)benzyl)-2-azaspiro[3.3]heptane-2-carbonyl)-7-oxa-2,5-diazaspiro[3.4]octan-6-one; (4aR,8aS)-6-(3-(((2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)benzyl)oxy)methyl)azetidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one; 2-(3-(((2-(2-((7-nitro-1H-312-benzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)benzyl)oxy)methyl)azetidine-1-carbonyl)-7-oxa-2,5-diazaspiro[3.4]octan-6-one; (4aR,8aS)-6-(3-(((2-(3-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)propoxy)benzyl)oxy)methyl)azetidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one; (4aR,8aS)-6-(3-(((2-((5-((7-nitro-1,3-dihydrobenzo[c][1,2,5]oxadiazol-4-yl)amino)pentyl)oxy)benzyl)oxy)methyl)azetidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one; (4aR,8aS)-6-(6-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)benzyl)-2-azaspiro[3.3]heptane-2-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one; (4aR,8aS)-6-(6-(3-(2-((7-nitro-1,3-dihydrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)benzyl)-2-azaspiro[3.3]heptane-2-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one; 2-(7-(dimethylamino)-3-(dimethylimino)-5,5-dimethyl-3,5-dihydrodibenzo[b,e]silan-10-yl)-4-((5-(2-((2-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)-2-azaspiro[3.3]heptan-6-yl)methyl)phenoxy)pentyl)carbamoyl)benzoate; 3,3-Dimethyl-1-(6-oxo-6-((2-(2-((2-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)-2-azaspiro[3.3]heptane-6-yl)methyl)phenoxy)ethyl)amino)hexyl)-2-((E)-3-((Z)-1,3,3-trimethylindolin-2-ylidene)prop-1-en-1-yl)-3H-indol-1-ium 2,2,2-trifluoroacetate; 3-(5,5-difluoro-7,9-dimethyl-5H-5l4,6l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinine-3-yl)-N-(2-(2-((2-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)-2-azaspiro[3.3]heptane-6-yl)methyl)phenoxy)ethyl)propanamide; 2,2,2-trifluoroacetic acid, 3,3-dimethyl-1-(6-oxo-6-((2-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)amino)hexyl)-2-((E)-3-((Z)-1,3,3-trimethylindolin-2-ylidene)prop-1-en-1-yl)-3H-indol-1-ium salt; 3,3-Dimethyl-1-(6-oxo-6-((2-(2-((2-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)-2-azaspiro[3.3]heptan-6-yl)methyl)phenoxy)ethyl)amino)hexyl)-2-((E)-3-((Z)-1,3,3-trimethylindolin-2-ylidene)prop-1-en-1-yl)-3H-indol-1-ium 2,2,2-trifluoroacetate; 3-(5,5-difluoro-7,9-dimethyl-5H-514,614-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinine-3-yl)-N-(2-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)propanamide; 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)-4-((2-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamoyl)benzoate and 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)-5-((2-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamoyl)benzoate; 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthen-9-yl)-5-((2-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamoyl)benzoate; 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthen-9-yl)-4-((3-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)ethyl)carbamoyl)benzoate octahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)propyl)carbamoyl)benzoate and 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)-5-((3-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)propyl)carbamoyl)benzoate; 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthen-9-yl)-5-((3-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)propyl)carbamoyl)benzoate; 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthen-9-yl)-4-((5-(2-((2-(6-oxo-7- 2-((6-(dimethylamino)-3-(dimethylimino)-3H-xanthen-9-yl)-5-((5-(2-((2-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)-2-azaspiro[3.3]heptan-6-yl)methyl)phenoxy)pentyl)carbamoyl)benzoate; 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)-5-((5-(2-((2-(6-oxo-7-oxa-2,5-diazaspiro[3.4]octane-2-carbonyl)-2-azaspiro[3.3]heptan-6-yl)methyl)phenoxy)pentyl)carbamoyl)benzoate; 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthen-9-yl)-4-((5-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)pentyl)carbamoyl)benzoate and 2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthen-9-yl)-5-((5-(2-(((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)methyl)phenoxy)pentyl)carbamoyl)benzoate; 3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)benzoic acid ethyl ester; Ethyl 3-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)oxy)benzoate; 2-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)benzoic acid ethyl ester; tert-Butyl (2-(3-((1-((4aR,8aS)-3-oxooctahhydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)oxy)benzamido)ethyl)carbamate; tert-Butyl (2-(2-((1-((4aR,8aS)-3-oxooctahydro-2H-pyrido[4,3-b][1,4]oxazine-6-carbonyl)azetidin-3-yl)methoxy)benzamido)ethyl)carbamate; (4aR,8aS)-6-(3-((benzyloxy)methyl)azetidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one; (4aR,8aS)-6-(6-(Benzyloxy)-2-azaspiro[3.3]heptane-2-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one; and (4aR,8aS)-6-(3-(Phenoxymethyl)azetidine-1-carbonyl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-3(4H)-one.

14. Use of a compound of formula (II) according to any one of claims 1, 2, 9 and 13 in the manufacture of a compound of formula (I) according to any one of claims 1 to 8 and 10 to 13.

15. A process for the manufacture of a compound of formula (I) according to any one of claims 1 to 8 and 10 to 13, the process comprising reacting an amine 4, Among them, A, L, R 1 and n as defined in claim 1, with: (a) a fluorescent label containing a carboxylic acid moiety in the presence of a coupling reagent and a base; or (b) a fluorescent label containing an activated ester of N-hydroxysuccinimide (NHS) in the presence of a base; or (c) 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole or 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole in the presence of a base; The reaction is carried out to obtain the compound of formula (I).

16. A compound of formula (I) according to any one of claims 1 to 8 and 10 to 13, manufactured according to the process of claim 15.

17. A compound of formula (I) according to any one of claims 1 to 8 and 10 to 13 for use in monoacylglycerol lipase (MAGL) occupancy studies.

18. A compound of formula (I) according to any one of claims 1 to 8 and 10 to 13 for use in the diagnostic imaging of monoacylglycerol lipase (MAGL) in a mammal.

19. A compound of formula (I) according to any one of claims 1 to 8 and 10 to 13 for use in the generation of monoacylglycerol lipase (MAGL) equilibrium and kinetic binding data.

20. Use of a compound of formula (I) according to any one of claims 1 to 8 and 10 to 13 in monoacylglycerol lipase (MAGL) occupancy studies.

21. Use of a compound of formula (I) according to any one of claims 1 to 8 and 10 to 13 in the diagnostic imaging of monoacylglycerol lipase (MAGL) in a mammal.

22. Use of a compound of formula (I) according to any one of claims 1 to 8 and 10 to 13 for generating equilibrium and kinetic binding data for monoacylglycerol lipase (MAGL).

23. A method of studying monoacylglycerol lipase (MAGL) occupancy, the method comprising contacting MAGL with a compound of formula (I) according to any one of claims 1 to 8 and 10 to 13.

24. A method of diagnostic imaging of monoacylglycerol lipase (MAGL) in a mammal, the method comprising contacting MAGL with a compound of formula (I) according to any one of claims 1 to 8 and 10 to 13.

25. A method of generating monoacylglycerol lipase (MAGL) equilibrium and kinetic binding data, the method comprising contacting MAGL with a compound of formula (I) according to any one of claims 1 to 8 and 10 to 13.

26. The invention as hereinbefore described.

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