WO2017196544A1

WO2017196544A1 - Novel compositions for labeling biomolecules and methods using same

Info

Publication number: WO2017196544A1
Application number: PCT/US2017/029791
Authority: WO
Inventors: Jennifer M. MURPHY; Maruthi Kumar NARAYANAM; Kendall N. HOUK; Yong Liang
Original assignee: The Regents Of The University Of California
Priority date: 2016-05-13
Filing date: 2017-04-27
Publication date: 2017-11-16

Abstract

The present invention includes novel compounds useful for labeling biomolecules. The present invention further includes a novel method of labeling a biomolecule using a compound of the invention. The present invention further includes a novel method of imaging a biomolecule using a compound of the invention.

Description

TITLE OF THE INVENTION

Novel Compositions for Labeling Biomolecules and Methods Using Same CROSS-REFERENCE TO RELATED APPLICATIONS The present invention claims priority to U.S. Provisional Application No.62/335,969, filed May 13, 2016, which is hereby incorporated by reference in its entirety herein. STATEMENT REGARDING FEDERALLYSPONSORED RESEARCH OR DEVELOPMENT

This invention was made with U.S. government support under Grant No. P50 CA086306 awarded by the National Institutes of Health. The U.S.

government has certain rights in the invention. BACKGROUND OF THE INVENTION

The ability to interrogate the molecular details of a biological process in a living system demands exquisite selectivity and robustness. Chemical reactions to selectively tag a biological target and subsequently image or track that target have been a long-standing goal of organic chemists. Despite the many challenges, careful design and tuning of reaction partners has provided a myriad of bioorthogonal chemical reactions over the past 15 years. Remarkable advancement in the field of bioorthogonal“click” chemistry arose in 2004 when Bertozzi and co-workers introduced a copper-free variant, the strain-promoted azide-alkyne [3+2]

cycloaddition that has enabled cellular component labeling in living systems (Agard et al., 2004, J. Am. Chem. Soc.126:15046). This reaction exploits the strain activation inherent to cyclooctynes (Gordon et al., 2012, J. Am. Chem. Soc.

134:9199) to avoid the use of a toxic copper(I) catalysts yet retains the kinetics to proceed rapidly in a chemically complex environment, such as in living systems, and has been widely utilized in the chemical biology community (Jewwet and Bertozzi, 2010, Chem. Soc. Rev.39:1272; Baskin et al., 2007, Proc. Natl. Acad. Sci. U.S.A. 104:16793; Chang et al., 2010, Proc. Natl. Acad. Sci. U.S.A.107:1821). Since the initial report of a strain-promoted cycloaddition with a rate of 10^-3 M^-1s-1, a great deal of progress has been made towards developing chemoselective reactions for labeling biomolecules with extraordinary fast rate constants (Lang and Chin, 2014, ACS Attorney Docket No.206030-0064-00-WO.606107 Chem. Biol.9:16; Patterson et al., 2014, ACS Chem. Biol.9:592). In 2008 an alternative bioorthogonal ligation, the inverse-electron-demand Diels-Alder reaction between tetrazines and strained alkenes, was introduced (Devaraj et al., 2008, Bioconjugate Chem.19:2297; Blackman et al., 2008, J. Am. Chem. Soc.130:13518) which has reached rate constants up to 10⁴ M^-1s^-1 (Karver et al., 2011, Bioconjugate Chem.22:2263; Taylor et al., 2011, J. Am. Chem. Soc.133:9646; Lang et al., 2012, J. Am. Chem. Soc.134:10317).

To date, the most common ¹⁸F-labeling method for biomolecules utilizes ¹⁸F-SFB, a radiolabeled prosthetic group that reacts with the ε-amino group of surface-exposed lysine residues (Liu et al., 2011, Mol. Imaging 10:168; Cai et al., 2007, J. Nucl. Med.48:304; Olafsen et al., 2012, Tumor Biol.33:669). In addition, site-specific conjugation using 4-¹⁸F-fluorobenzaldehyde (¹⁸F-FBA) has also been demonstrated (Cheng et al., 2008, J. Nucl. Med.49:804). While ¹⁸F-SFB has been successfully used to generate ¹⁸F-labeled proteins and peptides, labeling with ¹⁸F-SFB is far from ideal; in addition to its unselective conjugation, its 3-step synthesis and subsequent protein conjugation results in very poor decay-corrected radiochemical yields of 1.4– 2.5%. A distinct advantage of ¹⁸F-FBA over ¹⁸F-SFB is the opportunity for site-specific conjugation; however, its synthesis and protein conjugation is only moderately improved with yields of 13– 18%.

Bioorthogonal chemical ligations involve two molecular components that selectively react with each other in high yield under physiological conditions without interference from other chemical functionalities and with fast reaction kinetics. The inherent nature of these ligations– extremely rapid, highly-selective, modular, robust– suggest their aptness for the time-sensitive and stringent conditions of ¹⁸F-radiolabeling of biomolecules. While over twenty unique bioorthogonal reaction pairs have been established for the selective tagging of biomolecules, few have demonstrated successful applications with regards to ¹⁸F-labeling. The most commonly used bioorthogonal ligation for radiolabeling biomolecules applies the azide-alkyne ligation; however, these ¹⁸F-labeled compounds are not easily accessible. For example, ¹⁸F-fluoroethylazide is highly volatile making synthesis and handling difficult and requiring a specialized reaction set-up. A simplified, one-pot protocol was recently reported for the synthesis of 4-¹⁸F-Fluorobenzyl azide, however, its precursor is generated in a modest 17% yield (Rotstein et al., 2014, Nat. Commun. Attorney Docket No.206030-0064-00-WO.606107 5:4365). The synthetic challenges associated with these and other ¹⁸F-labeled azides are a considerable limitation to their global use.

Recently, the bioorthogonal tetrazine-transcyclooctene ligation has been gaining interest for the selective introduction of fluorine-18 into biologically relevant molecules. However, preparation of an ¹⁸F-labeled prosthetic group for this ligation introduces significant synthetic challenges that impede its practicality and global utilization. For example, ¹⁸F-transcyclooctene (¹⁸F-TCO) is a five-step synthesis, one of which requires a specialized apparatus for the photochemical isomerization of the cis-cyclooctyl intermediate to yield the respective trans- cyclooctyl compound in low yield (Royzen et al., 2008, 130:3760; Li et al., 2010, Chem. Commun.46:8043). In addition, the tetrazine-TCO ligation for ¹⁸F labeling requires that the tetrazine be attached to the protein; tetrazines demonstrate poor in vivo stability in most cases, with some disubstituted derivatives showing better stability but slower reaction kinetics (Karver et al., 2011, Bioconjugate Chem.

22:2263).

There is a need in the art for novel compounds and methods for the rapid and site-specific labeling of biomolecules under physiological conditions. The present invention addresses this unmet need. SUMMARY OF THE INVENTION The present invention relates to a com ound of formula (I):

wherein in formula (I):

A¹ is selected from the group consisting of alkyl, heteroalkyl, alkylcycloalkyl, heterocyclyl, alkylheterocyclyl, aryl, alkylaryl, heteroaryl, and alkylheteroaryl, wherein the alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl group is optionally substituted with 0-5 R² groups

R¹ and each occurrence of R² are independently selected from the group consisting of H, -C₁-C₆ alkyl, -C₁-C₆ fluoroalkyl, -C₁-C₆ heteroalkyl, F, ¹⁸F, Cl, Br, I, Attorney Docket No.206030-0064-00-WO.606107 -CN, -NO₂, -OR³, -SR³, -S(=O)R³, -S(=O)₂R³, -NHS(=O)₂R³, -C(=O)R³, -OC(=O)R³, -CO₂R³, -OCO₂R³, -CH(R³)₂, -N(R³)₂, -C(=O)N(R³)₂, -OC(=O)N(R³)₂, - NHC(=O)NH(R³), -NHC(=O)R³, -NHC(=O)OR³, -C(OH)(R³)₂, and -C(NH₂)(R³)₂; each occurrence of R³ is independently selected from the group consisting of H and C₁-C₆ alkyl;

L is a linker; and

Q is an imaging moiety.

a salt thereof, and any combinations thereof.

In one embodiment, A¹ is a phenyl group, wherein the phenyl group is optionally substituted. In another embodiment, L is selected from the group consisting of a bond, an alkyl group, an amide, a poly(alkyl ether), and any combinations thereof. In another embodiment, Q is ¹⁸F. In another embodiment, Q is a fluorescent dye. In another embodiment, the fluorescent dye is BODIPY630. In another embodiment, the compound of formula (I) is a compound of formula (II):

wherein in formula (II):

n is an integer between 0 and 4;

R¹ and each occurrence of R² are each independently selected from the group consisting of H, -C₁-C₆ alkyl, -C₁-C₆ fluoroalkyl, -C₁-C₆ heteroalkyl, F, ¹⁸F, Cl, Br, I, -CN, -NO₂, -OR³, -SR³, -S(=O)R³, -S(=O)₂R³, -NHS(=O)₂R³, -C(=O)R³, -OC(=O)R³, -CO₂R³, -OCO₂R³, -CH(R³)₂, -N(R³)₂, -C(=O)N(R³)₂, -OC(=O)N(R³)₂, - NHC(=O)NH(R³), -NHC(=O)R³, -NHC(=O)OR³, -C(OH)(R³)₂, and -C(NH₂)(R³)₂; each occurrence of R³ is independently selected from the group consisting of H and C₁-C₆ alkyl;

L is a linker; and

Q is an imaging moiety.

In one embodiment, n is at least 1 and at least one R² is F. In another embodiment, the compound is selected from the group consisting of: Attorney Docket No.206030-0064-00-WO.606107

Attorney Docket No.206030-0064-00-WO.606107 any combinations thereof.

The present invention also relates to a method for labeling a biomolecule. The method includes the steps of covalently attaching or conjugating a dipolarophile to a biomolecule to form a biomolecule-dipolarophile conjugate, and contacting the biomolecule-dipolarophile conjugate with a compound, wherein the compound undergoes a cycloaddition reaction with the dipolarophile to provide a labeled biomolecule. In one embodiment, the compound is at least one compound of formula (I):

wherein in formula (I):

R¹ and each occurrence of R² are independently selected from the group consisting of H, -C₁-C₆ alkyl, -C₁-C₆ fluoroalkyl, -C₁-C₆ heteroalkyl, F, ¹⁸F, Cl, Br, I, -CN, -NO₂, -OR³, -SR³, -S(=O)R³, -S(=O)₂R³, -NHS(=O)₂R³, -C(=O)R³, -OC(=O)R³, -CO₂R³, -OCO₂R³, -CH(R³)₂, -N(R³)₂, -C(=O)N(R³)₂, -OC(=O)N(R³)₂, - NHC(=O)NH(R³), -NHC(=O)R³, -NHC(=O)OR³, -C(OH)(R³)₂, and -C(NH₂)(R³)₂; each occurrence of R³ is independently selected from the group consisting of H and C₁-C₆ alkyl;

L is a linker; and

Q is an imaging moiety.

a salt thereof, and any combinations thereof.

In one embodiment, A¹ is a phenyl group, wherein the phenyl group is optionally substituted. In another embodiment, L is selected from the group consisting of a bond, an alkyl group, an amide, a poly(alkyl ether) group, and any combinations thereof. In another embodiment, Q is ¹⁸F. In another embodiment, Q is a fluorescent dye. In another embodiment, the fluorescent dye is BODIPY630. In another embodiment, the compound of formula (I) is a compound of formula (II): Attorney Docket No.206030-0064-00-WO.606107

wherein in formula (II):

n is an integer between 0 and 4;

L is a linker; and

Q is an imaging moiety.

In one embodiment, n is at least 1 and at least one R² is F. In another embodiment the com ound is selected from the rou consistin of:

In another embodiment, the compound is selected from the group consisting of: Attorney Docket No.206030-0064-00-WO.606107

nd any combinations thereof. In another embodiment, the dipolarophile is

dibenzocyclooctyne (DBCO). In another embodiment, the dipolarophile is biarylazacyclooctynone (BARAC).

The present invention also relates to a method for imaging a biomolecule. The method includes the steps of providing a sample comprising a biomolecule, covalently attaching or conjugating a dipolarophile to a biomolecule to form a biomolecule-dipolarophile conjugate, contacting the biomolecule- dipolarophile conjugate with a compound, wherein the compound undergoes a cycloaddition reaction with the dipolarophile to provide a labeled biomolecule, and detecting the labeled biomolecule, thereby imaging the biomolecule. In one embodiment, the compound is at least one compound of formula (I): Attorney Docket No.206030-0064-00-WO.606107

(I), wherein in formula (I):

L is a linker; and

Q is an imaging moiety.

a salt thereof, and any combinations thereof.

In one embodiment, A¹ is a phenyl group, wherein the phenyl group is optionally substituted. In another embodiment, L is selected from the group consisting of a bond, an alkyl group, an amide, a poly(alkyl ether) group, and any combinations thereof. In another embodiment, Q is ¹⁸F. In another embodiment, Q is a fluorescent dye. In another embodiment, the fluorescent dye is BODIPY630. In another embodiment, the compound of formula (I) is a compound of formula (II):

wherein in formula (II):

n is an integer between 0 and 4; Attorney Docket No.206030-0064-00-WO.606107 R¹ and each occurrence of R² are each independently selected from the group consisting of H, -C₁-C₆ alkyl, -C₁-C₆ fluoroalkyl, -C₁-C₆ heteroalkyl, F, ¹⁸F, Cl, Br, I, -CN, -NO₂, -OR⁶, -SR⁶, -S(=O)R⁶, -S(=O)₂R⁶, -NHS(=O)₂R⁶, -C(=O)R⁶, -OC(=O)R⁶, -CO₂R⁶, -OCO₂R⁶, -CH(R⁶)₂, -N(R⁶)₂, -C(=O)N(R⁶)₂, -OC(=O)N(R⁶)₂, - NHC(=O)NH(R⁶), -NHC(=O)R⁶, -NHC(=O)OR⁶, -C(OH)(R⁶)₂, and -C(NH₂)(R⁶)₂; each occurrence of R⁶ is independently selected from the group consisting of H and C₁-C₆ alkyl;

L is a linker; and

Q is an imaging moiety.

Attorney Docket No.206030-0064-00-WO.606107

hereof, and any combinations thereof. In another embodiment, the dipolarophile is

dibenzocyclooctyne (DBCO). In another embodiment, the dipolarophile is biarylazacyclooctynone (BARAC). In another embodiment, the biomolecule is imaged using positron emission tomography (PET). In another embodiment, the biomolecule is imaged in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

Figure 1 is an illustration of an exemplary sydnone-DBCO bioorthogonal chemical reporter strategy.

Figure 2 is a scheme of an exemplary synthesis of a pyrazole employing a [3+2] cycloaddition of phenyl sydnone with strained alkynes.

Figure 3, comprising Figures 3A-3B, illustrates modifications of protein surfaces via bioorthogonal click reactions in tandem. Figure 3A is a scheme illustrating an exemplary synthesis of bioorthogonal click reactions yielding Attorney Docket No.206030-0064-00-WO.606107 compounds of the present invention. Dibenzocyclooctyne (DBCO) and 5-norbornene- 2-acetic acid (Nor) were appended to bovine serum albumin (BSA) and ovalbumin (OVA) respectively. The labeled proteins BSA-DBCO and OVA-Nor were simultaneously reacted with sydnone-BODIPY (Syd-630) and tetrazine-BODIPY (Tz- 504) conjugates. Figure 3B is an image of a gel analysis of DBCO-conjugated BSA and norbornene-conjugated OVA with both Syd-630 and Tz-504 simultaneously for 1-60 min or no reagent (-). The protein loading on the gels was assessed with

Coomassie stain.

Figure 4 is a scheme of an exemplary synthesis of a¹⁸F-labeled biomolecule, wherein the ¹⁸F-sydnone undergoes 3+2 cycloaddition reaction with DBCO-conjugated biomolecule to afford the ¹⁸F-labeled biomolecule.

Figure 5 is a scheme of the reaction rate constants (k).of exemplary bioorthogonal compounds of the present invention for applications in ¹⁸F- radiolabeling of biomolecules.

Figure 6, comprising Figures 6A-6C, illustrates the synthesis of exemplary compounds of the invention and experimental data. Figure 6A is a scheme of an exemplary SPAC cycloaddition of various sydnones with bicyclo-[6.1.0]- nonyne (BCN)/biarylazacyclooctynone (BARAC). Figure 6B is a series of graphs of experimental data demonstrating the exponential decay curve of phenylsydnone Abs at 310 nm with BCN and the second order rate constant plot using kobs vs. conc. of excess reagent. Figure 6C is a scheme illustrating the application of exemplary sydnones of the invention as PET probes.

Figure 7 is a scheme of an exemplary synthesis of ¹⁸F-labeled sydnones of the present invention.

Figure 8 is a scheme of an exemplary synthesis of a bioorthogonal click reaction yielding a compound of the present invention.

Figure 9 is a scheme of an exemplary mechanism for the [3+2] cycloaddition reaction of a sydnone with an alkyne.

Figure 10 is a scheme of an exemplary synthesis of a bioorthogonal click reaction yielding a compound of the present invention.

Figure 11 is a scheme of an exemplary synthesis of a poly(alkyl ether)- linked fluoro-sydnone compound of the present invention.

Figure 12 is a scheme of an exemplary synthesis of a radiolabeled poly(alkyl ether) sydnone compound of the present invention. Attorney Docket No.206030-0064-00-WO.606107 Figure 13 is a scheme of an exemplary synthesis of a compound of the present invention using a bis-sydnone precursor.

Figure 14 is a scheme of an exemplary synthesis of a compound of the present invention using an isotopic exchange reaction and exemplary compounds that can be synthesized using this procedure. If the p-NO₂ reaction is performed at 100 °C for 7 min, 88% of the isotopic exchange product (1) is obtained with 5% of the sydnone exchange product (2).

Figure 15 is a scheme of a method for radiofluorination of a biomolecule (protein, peptide, and the like): conjugation of the biomolecule to a strained alkyne (DIBAC, BCN, and the like) followed by rapid cycloaddition with the purified ¹⁸F-sydnone affords the fluorine-18 labeled biomolecule in complete conversion and >99% (radio)chemical purity.

Figure 16 is an integrated radio-TLC (thin layer chromatography) scan from the synthesis of 4-[¹⁸F]fluorophenyl sydnone, depicted in Figure 13.

Figure 17, comprising Figures 17A and 17B is a radio-HPLC (high performance liquid chromatography) trace of 4-[¹⁸F]fluorophenyl sydnone. Figure 17A is the 254 nm UV trace of the ¹⁹F reference standard. Figure 17B is the radioactivity trace of the purified 4-[¹⁸F]fluorophenyl sydnone. The HPLC conditions were 10% acetonitrile in water to 90% acetonitrile in water over 30 min; both mobile phases contained 0.1% trifluoroacetic acid.

Figure 18 is an HPLC calibration curve for cold (non-radiolabeled) 4- fluorophenyl sydnone.

Figure 19, comprising Figures 19A and 19B, is a radio-HPLC (high performance liquid chromatography) trace of the reaction mixture from the click reaction between 4-[¹⁸F]fluorophenyl sydnone and DIBAC (depicted in Figure 15). Figure 19A is the 254 nm UV trace of the reaction mixture. Figure 19B is the radioactivity trace of the reaction mixture. The HPLC conditions were 10% acetonitrile in water to 70% acetonitrile in water over 40 min, then to 95% acetonitrile at 45 min; both mobile phases contained 0.1% trifluoroacetic acid. DETAILED DESCRIPTION

The present invention relates to the unexpected discovery of novel sydnones useful for the ¹⁸F-radiolabeling of biomolecules. The compounds of the invention provide a site-selective and rapid method for labeling a biomolecule under Attorney Docket No.206030-0064-00-WO.606107 conditions which do not interfere with any biological processes. Thus, the present invention provides compositions and methods useful for labeling a biomolecule. The present invention also provides methods for imaging a biomolecule. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

As used herein, each of the following terms has the meaning associated with it in this section.

The articles“a” and“an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example,“an element” means one element or more than one element.

“About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

As used herein, the term“pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

As used herein, the language“pharmaceutically acceptable salt” refers to a salt of the administered compounds prepared from pharmaceutically acceptable non-toxic acids, including inorganic acids, organic acids, solvates, hydrates, or clathrates thereof. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, phosphoric, acetic, hexafluorophosphoric, citric, gluconic, benzoic, propionic, butyric, sulfosalicylic, maleic, lauric, malic, fumaric, succinic, tartaric, amsonic, pamoic, p-tolunenesulfonic, and mesylic. Appropriate organic acids may be selected, for example, from aliphatic, aromatic, carboxylic and sulfonic Attorney Docket No.206030-0064-00-WO.606107 classes of organic acids, examples of which are formic, acetic, propionic, succinic, camphorsulfonic, citric, fumaric, gluconic, isethionic, lactic, malic, mucic, tartaric, para-toluenesulfonic, glycolic, glucuronic, maleic, furoic, glutamic, benzoic, anthranilic, salicylic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, pantothenic, benzenesulfonic (besylate), stearic, sulfanilic, alginic, galacturonic, and the like. Furthermore, pharmaceutically acceptable salts include, by way of non-limiting example, alkaline earth metal salts (e.g., calcium or magnesium), alkali metal salts (e.g., sodium-dependent or potassium), and ammonium salts.

As used herein, the terms“imaging agent,”“imaging probe,” or “imaging compound,” means, unless otherwise stated, a molecule which can be detected by its emitted signal, such as positron emission, autofluorescence emission, or optical properties. The method of detection of the compounds may include, but are not necessarily limited to, nuclear scintigraphy, positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance imaging, magnetic resonance spectroscopy, computed tomography, or a combination thereof depending on the intended use and the imaging methodology available to the medical or research personnel.

As used herein, the term“biomolecule” refers to any molecule produced by a living organism and may be selected from the group consisting of proteins, peptides, polysaccharides, carbohydrates, lipids, as well as analogs and fragments thereof. Preferred examples of biomolecules are proteins and peptides.

As used herein, the terms“bioconjugation” and“conjugation,” unless otherwise stated, refers to the chemical derivatization of a macromolecule with another molecular entity. The molecular entity can be any molecule and can include a small molecule or another macromolecule. Examples of molecular entities include, but are not limited to, compounds of the invention, other macromolecules, polymers or resins, such as polyethylene glycol (PEG) or polystyrene, non-immunogenic high molecular weight compounds, fluorescent, chemiluminescent radioisotope and bioluminescent marker compounds, antibodies, biotin, diagnostic detector molecules, such as a maleimide derivatized fluorescein, coumarin, a metal chelator or any other modifying group. The terms bioconjugation and conjugation are used

interchangeably throughout the Specification.

As used herein, the term“alkyl,” by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain hydrocarbon Attorney Docket No.206030-0064-00-WO.606107 having the number of carbon atoms designated (i.e. C₁-₆ means one to six carbon atoms) and including straight, branched chain, or cyclic substituent groups. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, and cyclopropylmethyl. Most preferred is (C₁-C₆)alkyl, particularly ethyl, methyl, isopropyl, isobutyl, n-pentyl, n-hexyl and cyclopropylmethyl.

As used herein, the term“substituted alkyl” means alkyl as defined above, substituted by one, two or three substituents selected from the group consisting of halogen, -OH, alkoxy, -NH₂, -N(CH₃)₂, -C(=O)OH, trifluoromethyl, -C≡N, - C(=O)O(C₁-C₄)alkyl, -C(=O)NH₂, -SO₂NH₂, -C(=NH)NH₂, and -NO₂, preferably containing one or two substituents selected from halogen, -OH, alkoxy, -NH₂, trifluoromethyl, -N(CH₃)₂, and -C(=O)OH, more preferably selected from halogen, alkoxy and -OH. Examples of substituted alkyls include, but are not limited to, 2,2-difluoropropyl, 2-carboxycyclopentyl and 3-chloropropyl.

As used herein, the term“heteroalkyl” by itself or in combination with another term means, unless otherwise stated, a stable straight or branched chain alkyl group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatom(s) may be placed at any position of the heteroalkyl group, including between the rest of the heteroalkyl group and the fragment to which it is attached, as well as attached to the most distal carbon atom in the heteroalkyl group. Examples

include: -O-CH₂-CH₂-CH₃, -CH₂-CH₂-CH₂-OH, -CH₂-CH₂-NH-CH₃, -CH₂-S-CH₂-C H₃, and -CH₂CH₂-S(=O)-CH₃. Up to two heteroatoms may be consecutive, such as, for example, -CH₂-NH-OCH₃, or -CH₂-CH₂-S-S-CH₃.

As used herein, the term“alkoxy” employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms, as defined above, connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homologs and isomers. In one embodiment, the alkoxy group is (C₁-C₃) alkoxy, such as ethoxy and methoxy.

As used herein, the term“halo” or“halogen” alone or as part of another substituent means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, or isotopes thereof. Attorney Do cket No.206030-0064-00-WO.606107 As used herein, the term“cycl oalkyl” ref ers to a mon o cyclic or polycyclic n on-aromat ic radical, w herein eac h of the ato ms forming the ring (i .e. skeletal ato ms) is a car bon atom. I n one embo diment, th e cycloalky l group is saturated or partially u nsaturated. In another embodimen t, the cyclo alkyl group is fused with an aromatic ring. Cycl oalkyl grou ps include g roups havi ng from 3 t o 10 ring atoms. Illustrative examples o f cycloalky l groups in clude, but a re not limi ted to,

.

Monocy clic cycloa lkyls includ e, but are n ot limited to, cyclopro pyl, cyclobutyl, cyclopenty l, cyclohex yl, cyclohep tyl, and cy clooctyl. D icyclic cyc loalkyls include, bu t are not lim ited to, tetr ahydronap hthyl, indan yl, and tetr ahydropent alene. Polycyclic cycloalkyls include ad amantine an d norborna ne. The ter m cycloalk yl includes“u nsaturated n onaromati c carbocycl yl” or“non aromatic un saturated carbocyclyl” groups, b oth of whic h refer to a nonaromat ic carbocyc le as define d herein, whi ch contains at least one carbon ca rbon double bond or on e carbon c arbon triple bond.

As used herein, the term“hete rocycloalky l” or“hete rocyclyl” r efers to a heteroalic yclic group containing one to fou r ring heter oatoms eac h selected f rom O, Sand N. In one embod iment, each heterocycl oalkyl grou p has from 4 to 10 ato ms in its ring syst em, with th e proviso th at the ring of said gro up does not contain tw o adjacent O or S atoms. In another embodime nt, the heter ocycloalky l group is f used with an aro matic ring. In one emb odiment, th e nitrogen and sulfur h eteroatoms may be optional ly oxidized , and the nit rogen atom may be op tionally qu aternized. T he heterocycli c system m ay be attach ed, unless otherwise s tated, at any heteroatom or carbon atom that affor ds a stable s tructure. A heterocycl e may be ar omatic or n on- aromatic in nature. In o ne embodi ment, the h eterocycle is a heteroa ryl. Attorney Do cket No.206030-0064-00-WO.606107 An exam ple of a 3 -membered heterocycl oalkyl grou p includes, and is not limited to, aziridin e. Example s of 4-mem bered heter ocycloalky l groups inc lude, and are not limited to, azetidine an d a beta la ctam. Exam ples of 5-m embered heterocyclo alkyl group s include, a nd are not limited to, pyrrolidine , oxazolidin e and thiazolidine dione. Exa mples of 6- membered heterocyclo alkyl group s include, and are not limited to, piperidi ne, morpho line and pip erazine. O ther non-lim iting exam ples of heterocyclo alkyl group s are:

.

Exampl es of non-a romatic het erocycles i nclude mon ocyclic gro ups such as azir idine, oxira ne, thiirane , azetidine, oxetane, th ietane, pyr rolidine, py rroline, pyrazolidin e, imidazol ine, dioxola ne, sulfola ne, 2,3-dihy drofuran, 2 ,5-dihydro furan, tetrahydrof uran, thioph ane, piperi dine, 1,2,3, 6-tetrahydr opyridine, 1,4- dihydropyr idine, piper azine, morp holine, thio morpholin e, pyran, 2, 3-dihydrop yran, tetrahydrop yran, 1,4-d ioxane, 1,3 -dioxane, h omopiperaz ine, homop iperidine, 1,3-dioxepa ne, 4,7-dih ydro-1,3-di oxepin, and hexameth yleneoxide.

As used herein, the term“arom atic” refer s to a carbo cycle or heterocycle with one o r more poly unsaturate d rings and having arom atic chara cter, i.e. having (4n + 2) del ocalized π (pi) electro ns, where n is an integ er.

As used herein, the term“aryl ,” employe d alone or i n combinat ion with other t erms, mean s, unless ot herwise sta ted, a carbo cyclic arom atic system containing one or more rings (typ ically one, t wo or three rings), wh erein such r ings may be atta ched togeth er in a pen dent manne r, such as a biphenyl, o r may be f used, Attorney Do cket No.206030-0064-00-WO.606107 such as nap hthalene. E xamples of aryl group s include ph enyl, anthr acyl, and naphthyl.

As used herein, the term“aryl -(C₁-C₃)alk yl” means a functional group wherein a o ne- to three -carbon alk ylene chain is attached to an aryl group, e.g., -CH₂C H₂-phenyl. Preferred i s aryl-CH₂- and aryl-C H(CH₃)-. T he term “substituted aryl-(C₁-C ₃)alkyl” m eans an ary l-(C₁-C₃)alk yl function al group in which the aryl gro up is substi tuted. Prefe rred is sub stituted ary l(CH₂)-. Sim ilarly, the term “heteroaryl -(C₁-C₃)alk yl” means a functional group whe rein a one t o three carb on alkylene ch ain is attach ed to a het eroaryl gro up, e.g., -C H₂CH₂-pyri dyl. In one embodimen t, the heter oaryl-(C₁-C ₃)alkyl is h eteroaryl-(C H₂)-. The term“subst ituted heteroaryl-( C₁-C₃)alky l” means a heteroaryl- (C₁-C₃)alky l functiona l group in w hich the heteroar yl group is substituted . In one em bodiment, the substitu ted

heteroaryl-( C₁-C₃)alky l is substitu ted heteroa ryl-(CH₂)-.

As used herein, the term“hete roaryl” or“ heteroarom atic” refer s to a heterocycle having aro matic chara cter. A pol ycyclic het eroaryl may include on e or more rings that are par tially satura ted. Examp les include the follow ing moietie s:

.

Exampl es of hetero aryl group s also inclu de pyridyl, pyrazinyl, pyrimidiny l (particular ly 2- and 4 -pyrimidiny l), pyridazi nyl, thieny l, furyl, pyr rolyl (particularl y 2-pyrroly l), imidazol yl, thiazoly l, oxazolyl, pyrazolyl (particularly 3- and 5-pyrazolyl ), isothiazo lyl, 1,2,3-tr iazolyl, 1,2 ,4-triazolyl , 1,3,4-triaz olyl, tetraz olyl, 1,2,3-thiadi azolyl, 1,2, 3-oxadiazo lyl, 1,3,4-th iadiazolyl and 1,3,4-o xadiazolyl.

Examples o f polycycli c heterocyc les and het eroaryls inc lude indoly l (particula rly 3-, 4-, 5-, 6- an d 7-indolyl ), indolinyl , quinolyl, tetrahydroq uinolyl, iso quinolyl

(particularl y 1- and 5-i soquinolyl) , 1,2,3,4-te trahydroiso quinolyl, ci nnolinyl, quinoxaliny l (particula rly 2- and 5 -quinoxali nyl), quinaz olinyl, phth alazinyl, Attorney Docket No.206030-0064-00-WO.606107 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin,

1,5-naphthyridinyl, benzofuryl (particularly 3-, 4-, 5-, 6- and 7-benzofuryl),

2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (particularly 3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl (particularly 2-benzothiazolyl and 5-benzothiazolyl), purinyl, benzimidazolyl (particularly 2-benzimidazolyl), benzotriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, and quinolizidinyl.

As used herein, the term“substituted” means that an atom or group of atoms has replaced hydrogen as the substituent attached to another group. The term “substituted” further refers to any level of substitution, namely mono-, di-, tri-, tetra-, or penta-substitution, where such substitution is permitted. The substituents are independently selected, and substitution may be at any chemically accessible position. In one embodiment, the substituents vary in number between one and four. In another embodiment, the substituents vary in number between one and three. In yet another embodiment, the substituents vary in number between one and two.

As used herein, the term“optionally substituted” means that the referenced group may be substituted or unsubstituted. In one embodiment, the referenced group is optionally substituted with zero substituents, i.e., the referenced group is unsubstituted. In another embodiment, the referenced group is optionally substituted with one or more additional group(s) individually and independently selected from groups described herein.

In one embodiment, the substituents are independently selected from the group consisting of oxo, halogen, -CN, -NH₂, -OH, -NH(CH₃), -N(CH₃)₂, alkyl (including straight chain, branched and/or unsaturated alkyl), substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, fluoro alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkoxy, fluoroalkoxy, -S-alkyl, S(=O)₂alkyl, -C(=O)NH[substituted or unsubstituted alkyl, or substituted or unsubstituted phenyl], -C(=O)N[H or alkyl]₂, -OC(=O)N[substituted or unsubstituted alkyl]₂, -NHC(=O)NH[substituted or unsubstituted alkyl, or substituted or unsubstituted phenyl], -NHC(=O)alkyl, -N[substituted or unsubstituted

alkyl]C(=O)[substituted or unsubstituted alkyl], -NHC(=O)[substituted or unsubstituted alkyl], -C(OH)[substituted or unsubstituted alkyl]₂, and - C(NH₂)[substituted or unsubstituted alkyl]₂. In another embodiment, by way of example, an optional substituent is selected from oxo, fluorine, chlorine, bromine, Attorney Docket No.206030-0064-00-WO.606107 iodine, -CN, -NH₂, -OH, -NH(CH₃), -N(CH₃)₂, -CH₃, -CH₂CH₃, -CH(CH₃)₂, -CF₃, - CH₂CF₃, -OCH₃, -OCH₂CH₃, -OCH(CH₃)₂, -OCF₃, - OCH₂CF₃, -S(=O)₂-CH₃, - C(=O)NH₂, -C(=O)-NHCH₃, -NHC(=O)NHCH₃, -C(=O)CH₃, and -C(=O)OH. In yet one embodiment, the substituents are independently selected from the group consisting of C_1-6 alkyl, -OH, C_1-6 alkoxy, halo, amino, acetamido, oxo and nitro. In yet another embodiment, the substituents are independently selected from the group consisting of C_1-6 alkyl, C_1-6 alkoxy, halo, acetamido, and nitro. As used herein, where a substituent is an alkyl or alkoxy group, the carbon chain may be branched, straight or cyclic, with straight being preferred.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub- ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. Description

The present invention relates to the unexpected discovery of novel sydnones useful for the ¹⁸F-radiolabeling of biomolecules. The compounds of the invention provide a site-selective and rapid method for labeling a biomolecule under conditions which do not interfere with any biological processes. Thus, the present invention provides compositions and methods useful for labeling a biomolecule. The present invention also provides methods for imaging a biomolecule.

Until recently, the potential role of sydnones as a bioorthogonal reaction partner for labeling biomolecules via a [3+2] cycloaddition was relatively overlooked. First discovered in 1935, sydnones are remarkably stable mesoionic heterocycles that contain a 1,5-dipolar azomethinimine moiety (Earl and Mackney, 1935, J. Chem. Soc.899). Huisgen reported, upon treatment with alkynes at 170 °C, sydnones undergo a [3+2] cycloaddition to form pyrazoles, via a bicyclic intermediate which eliminates carbon dioxide (Huisgen et al., 1962, Angev. Chem. Int. Ed.1:48). Attorney Docket No.206030-0064-00-WO.606107 Using a high-throughput immunoassay technique, Taran and co-workers found that sydnones undergo 1,3-dipolar cycloaddition with alkynes at 50 °C in the presence of copper (Kolodych et al., 2013, Angew. Chem. Int. Ed.52:12056). Upon further optimization, they identified a copper phenanthroline complex that catalyzes the cycloaddition to produce 1,4-pyrazoles at room temperature in high yield. Building on precedent of the azide-alkyne cycloaddition, Chin and co-workers recently described the strain-promoted [3+2] cycloaddition reaction between phenyl sydnone and exo- ((1R,8S)-bicyclo[6.1.0]non-4-yn-9-yl)methanol (BCN) at ambient temperature to afford the corresponding pyrazole in 30 minutes, with an isolated yield of 99% (Wallace and Chin, 2014, Chem. Sci.5:1742). The pseudo-first order rate constant in 55:45 MeOH-H₂O was determined to be 0.054 M^-1s^-1.

Following this report, Taran et al. introduced a series of sydnones bearing substituents at the 3 and 4 positions of the mesoionic sydnone ring (Plougastel et al., 2014, Chem. Commun.50:9376). Their results indicated a significant effect on the rate constant of the cycloaddition reaction, with the 4-chlorinated sydnone undergoing cycloaddition with BCN 30-fold faster than the non-chlorinated analogue, yielding a rate constant of 1.593 M^-1s^-1. Rapid reactions with high intrinsic second- order rate constants, such as the sydnone-DBCO reaction, are beneficial for labeling on a short time-scale or for labeling material in low abundance. Thus, the present invention is based in part on the reactivity of sydnones and tuning their kinetics to be suitable for ¹⁸F-labeling of biomolecules (Figure 1).

Current technology used for the ¹⁸F-labeling of biomolecules includes inverse-electron-demand Diels-Alder reactions between tetrazine and strained alkenes, in addition to“click” chemistry between azides and alkynes. A key advantage of sydnones over a tetrazine ligation lies in the combination of its exceptionally easy preparation (no specialized apparatus needed) with high second- order rate constant, comparable to the fastest bioorthogonal reactions reported to date. The stability and scope to tune the reaction rates by varying the substitutions on the sydnone ring over the existing alkyl/aryl azides demonstrates that syndones may be useful as new bioorthogonal probes for imaging, such as for ¹⁸F-PET imaging.

Reliable and site-specific conjugation chemistries for coupling chemical reporters to biomolecules are critical not only for the field of molecular imaging, but also for the advancement of molecular and cellular biology. The availability of a simple, robust ¹⁸F-chemistry platform for site-selective biomolecule Attorney Docket No.206030-0064-00-WO.606107 labeling will impact molecular imaging and, therefore, clinical practice reduced to standard-of-care by advances in chemical methods. Currently, simple and efficient chemical methods for the ¹⁸F-labeling of biomolecules are severely limited. The compositions and methods described herein provide a highly selective, efficient, and rapid approach to labeling biomolecules with a chemical reporter, such as a radionuclide or fluorescent dye.

The methods of the invention include the use of biorthogonal chemistry to achieve bioconjugation using the [3+2] cycloaddition reaction between a radiolabeled compound of the present invention and a suitable dipolarophile to deliver the radiolabeled compound to the biomolecule. The compounds of the invention can be attached to the biomolecule predictably and with specificity, and are otherwise inert in biological systems. Compounds

The compounds of the present invention may be synthesized using techniques well-known in the art of organic synthesis. The starting materials and intermediates required for the synthesis may be obtained from commercial sources or synthesized according to methods known to those skilled in the art.

In one aspect, the compound of the invention is a compound of formula (I), or a salt, solv

(I),

wherein in formula (I):

R¹ and each occurrence of R² are independently selected from the group consisting of H, -C₁-C₆ alkyl, -C₁-C₆ fluoroalkyl, -C₁-C₆ heteroalkyl, F, ¹⁸F, Cl, Br, I, -CN, -NO₂, -OR³, -SR³, -S(=O)R³, -S(=O)₂R³, -NHS(=O) ³

2R³, -C(=O)R , -OC(=O)R³, Attorney Docket No.206030-0064-00-WO.606107 -CO₂R³, -OCO₂R³, -CH(R³)₂, -N(R³)₂, -C(=O)N(R³)₂, -OC(=O)N(R³)₂, - NHC(=O)NH(R³), -NHC(=O)R³, -NHC(=O)OR³, -C(OH)(R³)₂, and -C(NH₂)(R³)₂; each occurrence of R³ is independently selected from the group consisting of H and C₁-C₆ alkyl;

L is a linker; and

Q is an imaging moiety.

In one embodiment, A¹ is aryl. In one embodiment, the A¹ is a phenyl group, wherein the phenyl group is optionally substituted.

In another aspect, the compound of the invention is a compound of formula (II), or a salt, solvate, or N-oxide thereof:

wherein in formula (II):

n is an integer between 0 and 4;

L is a linker; and

Q is an imaging moiety.

In one embodiment, n is at least 1 and at least one R² is F. In another embodiment, n is at least two and at least two R² are F. In some embodiments, the aryl ring is substituted with one or more R². In one embodiment, R² is a nitro group. In another embodiment, R² is an alkyl ester. In one embodiment, n is four and all occurrences of R² are F. In one embodiment, n is four, and one or more occurrences of R² are ¹⁸F. Attorney Docket No.206030-0064-00-WO.606107 The linker L may be any suitable linker, as would be understood by one of ordinary skill the art. Examples of linkers include, but are not limited to, an alkyl group, a benzyl group, an aryl group, a heteroaryl group, a cycloalkyl group, an amide group, an ester, a sulfonamide, a carbamate, a carbonate, a sulfone, an ether, an oxime, a hydrazine, a urea, a thiourea, a phosphate, a poly(alkyl ether), a heteroatom, or combinations thereof, wherein the alkyl, benzyl, aryl, heteroaryl, cycloalkyl, amide, ester, sulfonamide, carbamate, carbonate, sulfone, ether, oxime, hydrazine, urea, thiourea, phosphate, and poly(alkyl ether) may be optionally substituted. In one embodiment, L is a bond. For example, when the L is a bond, A¹ is bonded directly to the ¹⁸F atom. In one embodiment, L is alkyl. In another embodiment, L is selected from the group consisting of a bond, an alkyl group, an amide, a poly(alkyl ether), and any combinations thereof. In another embodiment, L is comprised of an alkyl group and a poly(alkyl ether) group. In one embodiment, L wherein n is an integer from 1 to 20. In another embodiment, L is

, wherein n is an integer from 1 to 20. In some embodiments of the invention, L is bound to the aryl ring at a position para to the sydnone. In other embodiments, L is bound to the aryl ring at a position meta or ortho to the sydnone.

Q may be any suitable imaging moiety. Imaging moieties include those that are well known to those skilled in the art, and include those moieties that may be useful in the generation of diagnostic images by diagnostic techniques well known to the ordinarily skilled artisan. Non-limiting examples of imaging moieties include radioisotopes, paramagnetic species, echogenic entities, and fluorescent moiety, such as a fluorescent dye. In one embodiment, Q is a radioisotope. Examples of radioisotopes include and are not limited to ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ³⁶Cl, ¹⁸F, ¹²³I, ¹²⁵I, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³²P, and ³⁵S. In one embodiment, Q is ¹⁸F. In another embodiment, Q is a fluorescent dye. Examples of fluorescent dyes include, but are not limited to, BODIPY dyes such as BODIPY630 and BODIPY504, POPO-1, TOTO-3, TAMRA, BOXTO, BEBO, SYBR DX, SYTOX dyes, SYTO dyes, Alexa dyes, fluorescein, rhodamine, propidium idodide, Hoechst dyes, tetramethylrhodamine, R- phycoerythrin, Cy-3, Cy-5, Cy-7, Texas Red, Phar-Red, allophycocyanin (APC), fluorescein amine, eosin, dansyl, umbelliferone, 5-carboxyfluorescein (FAM), 2'7'- Attorney Docket No.206030-0064-00-WO.606107 dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), 6 carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'-dimethylaminophenylazo) benzoic acid (DABCYL), 5-(2'-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS), 8-Anilino-1-naphthalenesulfonic acid ammonium salt (ANS), 4-acetamido-4'-isothiocyanatostilbene-2,2' disulfonic acid, acridine, acridine isothiocyanate, acridine orange (N,N,N',N'-tetramethylacridine-3,6- diamine), R-amino-N-(3-vinylsulfonyl)phenylnaphthalimide-3,5, disulfonate (Lucifer Yellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide, Brilliant Yellow, coumarin, 7-amino-4-methylcoumarin, 7-amino-4-trifluoromethylcouluarin

(Coumarin 151), cyanosine, 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI), 5',5''-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red), 7- diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin diethylenetriamine pentaacetate, 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid, 4,4'- diisothiocyanatostilbene-2,2'-disulfonic acid, 4-dimethylaminophenylazophenyl-4'- isothiocyanate (DABITC), eosin isothiocyanate, erythrosin B, erythrosin

isothiocyanate, ethidium, 5-(4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF), QFITC (XRITC), fluorescamine, IR144, IR1446, Malachite Green isothiocyanate, 4- methylumbelliferone, ortho cresolphthalein, nitrotyrosine, pararosaniline, Phenol Red, B-phycoerythrin, o-phthaldialdehyde, pyrene, pyrene butyrate, succinimidyl 1-pyrene butyrate, Reactive Red 4 (Cibacron.RTM. Brilliant Red 3B-A), lissamine rhodamine B sulfonyl chloride, rhodamine B, rhodamine 123, rhodamine X, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101, tetramethyl rhodamine, thiazole orange, riboflavin, rosolic acid, and terbium chelate derivatives. In one embodiment, the fluorescent dye is BODIPY630. In another embodiment, the fluorescent dye is BODIPY540.

In one embodiment, the compound is selected from the group consisting of: Attorney Docket No.206030-0064-00-WO.606107

thereof, and any combinations thereof.

In another embodiment, the compound is selected from the group consisting of:

Attorney Docket No.206030-0064-00-WO.606107

wherein Q is BODIPY630, a salt thereof, and any combinations thereof. Preparation of the Compounds of the Invention

Compounds of Formula (I)-(III) may be prepared by the general schemes described herein, using the synthetic method known by those skilled in the art. The following examples illustrate non-limiting embodiments of the invention.

In a non-limiting embodiment, the synthesis of a compound of formula (I) may be accomplished by bis-alkylating a bis-amine with a bromoacetate and then saponifying the resulting bis-ester. Alternatively, the bis-acid can be obtained in one step from ClCH₂CO₂Na. The bis-acid can then be treated with t-BuONO and a dehydrating agent such as (CF₃CO)₂O or carbonyldiimidizole (CDI) to afford a bissydnone (Figure 13).

In one embodiment, the synthesis of a compound of formula (I) may be accomplished by the following synthetic scheme: Attorney Docket No.206030-0064-00-WO.606107

(Isolated by Prep. TLC)

m x ure o

regioisomers Compounds described herein also include isotopically-labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds described herein include and are not limited to ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ³⁶Cl, ¹⁸F, ¹²³I, ¹²⁵I, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³²P, and ³⁵S. In one embodiment, the compounds include ¹⁸F. In one embodiment, isotopically-labeled compounds are useful in drug and/or substrate tissue distribution studies. In another embodiment, substitution with heavier isotopes such as deuterium affords greater metabolic stability (for example, increased in vivo half-life or reduced dosage requirements). In yet another embodiment, substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and ¹³N, is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically-labeled compounds are prepared by any suitable method or by processes using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed.

Incorporation of ¹⁸F into a compound of the invention can be performed using any method known in the art, as would be understood by one of ordinary skill in the art. In one non-limiting embodiment, one sydnone moiety of the bissydnone can be displaced with radiolabeled ¹⁸F in the presence of an alkyl ammonium base to generate a radiolabeled sydnone of the present invention (Figure 13). In another embodiment, the bissydnone is treated with a non-radiolabeled fluoride to generate a stable intermediate which can be stored and transported if Attorney Docket No.206030-0064-00-WO.606107 necessary. In one embodiment, the non-labeled compound can be subjected to isotopic exchange with ¹⁸F to generate the radiolabeled compound (Figure 14).

The compounds of the invention may possess one or more stereocenters, and each stereocenter may exist independently in either the R or S configuration. In one embodiment, compounds described herein are present in optically active or racemic forms. It is to be understood that the compounds described herein encompass racemic, optically-active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein. Preparation of optically active forms is achieved in any suitable manner, including by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically-active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase. In one embodiment, a mixture of one or more isomer is utilized as the therapeutic compound described herein. In another embodiment, compounds described herein contain one or more chiral centers. These compounds are prepared by any means, including stereoselective synthesis, enantioselective synthesis and/or separation of a mixture of enantiomers and/ or diastereomers. Resolution of compounds and isomers thereof is achieved by any means including, by way of non-limiting example, chemical processes, enzymatic processes, fractional crystallization, distillation, and chromatography.

The methods and formulations described herein include the use of N-oxides (if appropriate), crystalline forms (also known as polymorphs), solvates, amorphous phases, and/or pharmaceutically acceptable salts of compounds having the structure of any compound of the invention, as well as metabolites and active metabolites of these compounds having the same type of activity. Solvates include water, ether (e.g., tetrahydrofuran, methyl tert-butyl ether) or alcohol (e.g., ethanol) solvates, acetates and the like. In one embodiment, the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, and Attorney Docket No.206030-0064-00-WO.606107 ethanol. In another embodiment, the compounds described herein exist in unsolvated form.

In one embodiment, the compounds of the invention may exist as tautomers. All tautomers are included within the scope of the compounds presented herein.

In one embodiment, sites on, for example, the aromatic ring portion of compounds of the invention are susceptible to various metabolic reactions.

Incorporation of appropriate substituents on the aromatic ring structures may reduce, minimize or eliminate this metabolic pathway. In one embodiment, the appropriate substituent to decrease or eliminate the susceptibility of the aromatic ring to metabolic reactions is, by way of example only, a deuterium, a halogen, or an alkyl group.

In one embodiment, the compounds described herein are labeled by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.

The compounds described herein, and other related compounds having different substituents are synthesized using techniques and materials described herein and as described, for example, in Fieser & Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon

Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), Larock's

Comprehensive Organic Transformations (VCH Publishers Inc., 1989), March, Advanced Organic Chemistry 4^th Ed., (Wiley 1992); Carey & Sundberg, Advanced Organic Chemistry 4th Ed., Vols. A and B (Plenum 2000,2001), and Green & Wuts, Protective Groups in Organic Synthesis 3rd Ed., (Wiley 1999) (all of which are incorporated by reference for such disclosure). General methods for the preparation of compound as described herein are modified by the use of appropriate reagents and conditions, for the introduction of the various moieties found in the formula as provided herein.

Compounds described herein are synthesized using any suitable procedures starting from compounds that are available from commercial sources, or are prepared using procedures described herein.

In one embodiment, reactive functional groups, such as hydroxyl, amino, imino, thio or carboxy groups, are protected in order to avoid their unwanted participation in reactions. Protecting groups are used to block some or all of the Attorney Docket No.206030-0064-00-WO.606107 reactive moieties and prevent such groups from participating in chemical reactions until the protective group is removed. In another embodiment, each protective group is removable by a different means. Protective groups that are cleaved under totally disparate reaction conditions fulfill the requirement of differential removal.

In one embodiment, protective groups are removed by acid, base, reducing conditions (such as, for example, hydrogenolysis), and/or oxidative conditions. Groups such as trityl, dimethoxytrityl, acetal and t-butyldimethylsilyl are acid labile and are used to protect carboxy and hydroxy reactive moieties in the presence of amino groups protected with Cbz groups, which are removable by hydrogenolysis, and Fmoc groups, which are base labile. Carboxylic acid and hydroxy reactive moieties are blocked with base labile groups such as, but not limited to, methyl, ethyl, and acetyl, in the presence of amines that are blocked with acid labile groups, such as t-butyl carbamate, or with carbamates that are both acid and base stable but hydrolytically removable.

In one embodiment, carboxylic acid and hydroxy reactive moieties are blocked with hydrolytically removable protective groups such as the benzyl group, while amine groups capable of hydrogen bonding with acids are blocked with base labile groups such as Fmoc. Carboxylic acid reactive moieties are protected by conversion to simple ester compounds as exemplified herein, which include conversion to alkyl esters, or are blocked with oxidatively-removable protective groups such as 2,4-dimethoxybenzyl, while co-existing amino groups are blocked with fluoride labile silyl carbamates.

Allyl blocking groups are useful in the presence of acid- and base- protecting groups since the former are stable and are subsequently removed by metal or pi-acid catalysts. For example, an allyl-blocked carboxylic acid is deprotected with a palladium-catalyzed reaction in the presence of acid labile t-butyl carbamate or base-labile acetate amine protecting groups. Yet another form of protecting group is a resin to which a compound or intermediate is attached. As long as the residue is attached to the resin, that functional group is blocked and does not react. Once released from the resin, the functional group is available to react.

Typically blocking/protecting groups may be selected from: Attorney Do cket No.206030-0064-00-WO.606107

.

Other p rotecting gr oups, plus a detailed d escription o f techniqu es applicable t o the creati on of prote cting group s and their removal are described in Greene & W uts, Protec tive Group s in Organi c Synthesis , 3rd Ed., J ohn Wiley & Sons, New York, NY, 1999, and Kocien ski, Protec tive Groups , Thieme V erlag, New York, NY, 1994, which are in corporated herein by reference fo r such disc losure. Labeling an d Imaging of Biomole cules

In one a spect, the p resent inve ntion provi des a metho d for label ing a biomolecul e. In one em bodiment, the method for labelin g a biomol ecule comp rises the steps of covalently attaching o r conjugati ng a dipola rophile to a biomolecu le to form a biom olecule-di polarophile conjugate, contacting the biomol ecule- dipolarophi le conjugat e with a com pound of the inventio n, wherein the compo und undergoes a cycloaddi tion reactio n with the d ipolarophil e to provid e a labeled biomolecul e.

In one e mbodimen t, the metho d comprise s the step o f covalentl y attaching or conjugatin g a dipolar ophile to a biomolecul e to form a biomolecu le- dipolarophi le conjugat e. Preferred examples of biomole cules are pr oteins and peptides. In one embod iment, the biomolecul e is a prote in. In anoth er embodim ent, the biomole cule is bov ine serum a lbumin (BS A). In ano ther embod iment, the biomolecul e is ovalbum in (OVA) . In another embodime nt, the biom olecule is an Attorney Docket No.206030-0064-00-WO.606107 engineered antibody fragment. A non-limiting example of an engineered antibody fragment is anti-PSCA A2 cys-diabody.

The dipolarophile can be any dipolarophile that is capable of undergoing a [3+2] cycloaddition reaction with a sydnone, as would be understood by one of ordinary skill in the art. Non-limiting examples of dipolarophiles include alkynes, such as ethynyl and propynyl. In one embodiments, the dipolarophile is an alkyne. Particularly advantageous dipolarophiles are those that are highly stable in aqueous environments, such as dibenzocyclooctyne (DBCO), biarylazacyclooctynone (BARAC), and derivatives thereof. In one embodiment, the dipolarophile is selected from the group consisting of dibenzocyclooctyne (DBCO) and a derivative thereof. In another embodiment, the dipolarophile is dibenzocyclooctyne (DBCO) In another embodiment, the dipolarophile is selected from the group consisting of

biarylazacyclooctynone (BARAC) and a derivative thereof. In another embodiment, the dipolarophile is biarylazacyclooctynone (BARAC).

The biomolecule may be attached to the dipolarophile using any method known in the art. For example, the dipolarophile may comprise a moiety capable of being conjugated to the biomolecule. Examples of such moieties include an N-hydroxysuccinimide (NHS) ester and maleimide.

In one embodiment, the method further comprises the step of contacting the biomolecule-dipolarophile conjugate with a compound of the invention, wherein the compound undergoes a cycloaddition reaction with the dipolarophile to provide a labeled biomolecule. The cycloaddition reaction that occurs between the dipolarophile and the compound of the invention results in the formation of a pyrazole moiety at the dipolarophile site (Figure 4). The pyrazole moiety is comprised of an imaging moiety, which functions as a label for imaging, thereby incorporating a label into the biomolecule. For example, when the imaging moiety is ¹⁸F, the biomolecule is labeled with ¹⁸F and can subsequently be imaged.

In another aspect, the present invention provides a method for labeling a biomolecule. The method comprises the step of contacting a dipolarophile with a compound of the invention, wherein the compound undergoes a cycloaddition reaction with the dipolarophile to provide a pyrazole moiety. The method further comprises the step of covalently attaching or conjugating the pyrazole moiety to a biomolecule to provide a labeled biomolecule. The pyrazole moiety may be attached to the biomolecule using any method known in the art. For example, the pyrazole Attorney Docket No.206030-0064-00-WO.606107 moiety may comprise a moiety capable of being conjugated to the biomolecule.

Examples of such moieties include an N-hydroxysuccinimide (NHS) ester and maleimide.

In another aspect, the present method provides a method for imaging a biomolecule. In one embodiment, the method comprises the steps of providing a sample comprising a biomolecule, covalently attaching or conjugating a dipolarophile to a biomolecule to form a biomolecule-dipolarophile conjugate, contacting the biomolecule-dipolarophile conjugate with a compound, wherein the compound undergoes a cycloaddition reaction with the dipolarophile to provide a labeled biomolecule, and detecting the labeled biomolecule, thereby imaging the biomolecule.

The biomolecule may be imaged using any method of detection known in the art. If the biomolecule is labeled with ¹⁸F, the biomolecule can be detected by its emitted signal, such as autofluorescence emission or optical properties of the labeled biomolecule. The methods of detection for ¹⁸F-labeled biomolecules may include, but are not necessarily limited to, nuclear scintigraphy, positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance imaging, magnetic resonance spectroscopy, computed tomography, or a combination thereof depending on the intended use and the imaging methodology available to the medical or research personnel. In one embodiment, the labeled biomolecule is imaged using positron emission tomography (PET). The methods of detection for biomolecules labeled with fluorescent dyes includes, but not limited to, CCD cameras, video cameras, photographic film, laser scanning devices,

fluorometers, photodiodes, quantum counters, epifluorescence microscopes, scanning microscopes, flow cytometers, fluorescence microplate readers, or by amplification devices such as photomultiplier tubes. In some embodiments, the biomolecule is imaged in vitro. In other embodiments, the biomolecule is imaged in vivo.

The sample can generally be any type of sample. For example, the sample can be a cell or group of cells, an organism, cell lysates, a cell culture medium, a bioreactor sample, and so on. In one embodiment, the sample is a cell. In one embodiment, the cell is a live cell. Alternatively, the sample can be a non- biological sample. The cells can be any type of cell, such as bacterial cells, fungal cells, insect cells, and mammalian cells. The sample can be a solid, a liquid, or a suspension. The sample can be a biological fluid such as blood, plasma, or urine. The sample can be a material immobilized in a gel, on a membrane, bound to a bead or Attorney Docket No.206030-0064-00-WO.606107 beads, arranged in an array, and so on. The sample can be a partially or fully purified preparation in a buffer or in water. Those skilled in the art recognizes, or is able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of this invention and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction times, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing/oxidizing agents, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present application.

It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present invention. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application.

The following examples further illustrate aspects of the present invention. However, they are in no way a limitation of the teachings or disclosure of the present invention as set forth herein. EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. Example 1: Rapid and Selective Cycloaddition Reaction for Applications in

Molecular Imaging

The results described herein demonstrate the reactivity of sydnones and explore the scope of this reaction and tuning its kinetics to be suitable for ¹⁸F- Attorney Docket No.206030-0064-00-WO.606107 labeling of proteins (Figure 1). The electronics of various sydnone derivatives were studied with the goal of increasing their reaction kinetics to obtain a rapid reaction that could be utilized for ¹⁸F-chemistry. The rate constant reported for the sydnone- exo-((1R,8S)-bicyclo[6.1.0]non-4-yn-9-yl)methanol (BCN) reaction is 0.054 M^-1s^-1, comparable to the well-known azide-alkyne ligation. Nearly twenty sydnone derivatives were synthesized, their electronics altered, and the second-order rate constants were studied for the reactions between these derivatives with BCN.

Depending on the substrate, rate constants from 0.003– 23.0 M^-1s^-1 were observed. Electron-poor derivatives were found to give faster rates.

During this investigation, two dipolarphiles were identified [the strained alkynes dibenzocyclooctyne (DBCO) (6) and biarylazacyclooctynone (BARAC) (8)] that undergo rapid [3+2] cycloaddition with sydnones (Figure 2). These bioorthogonal reactions possess second-order rate constants greater than 10² M- ¹s^-1 orders of magnitude faster than the previously reported sydnone-BCN ligation, and are completed in less than five seconds. Rapid reactions with high intrinsic second-order rate constants, such as the sydnone-DBCO reaction, are beneficial for labeling on a short time-scale or for labeling material in low abundance, such is the case when working with fluorine-18.⁶ Fluorine-18 has a half-life of 109 minutes and is accessible only in picomolar concentrations for labeling reactions. As a result of its extraordinary reaction kinetics and selectivity, the sydnone-DBCO ligation may be useful for applications in ¹⁸F-labeling of biomolecules.

To demonstrate the utility of this approach for protein labeling, a protein conjugation experiment was performed using bovine serum albumin (BSA) and ovalbumin (OVA) as model proteins (Figure 3). A mutually orthogonal reaction pair, the tetrazine-norbornene ligation, was identified and utilized to demonstrate dual fluorescence protein labeling in tandem. The DBCO and norbornene moieties were appended to the surface of BSA and OVA respectively, using standard coupling conditions (Figure 3). The modified proteins (BSA-DBCO, OVA-Nor) were treated with either a sydnone-BODIPY630 conjugate (Syd-630) or a tetrazine-BODIPY504 conjugate (Tz-504) or both and analyzed via in gel fluorescence imaging. When the modified proteins were jointly treated with Syd-630, only BSA-DBCO was fluorescently labeled, in agreement with observed kinetic data (lane 1, Figure 3). Likewise, in the presence of Tz-504, OVA-Nor showed selective labeling without any detection of BSA-DBCO fluorescence labeling (lane 2, Figure 3). Co-administration Attorney Docket No.206030-0064-00-WO.606107 of both fluorophore conjugates in“one pot” resulted in the concurrent labeling of both proteins (lane 6, Figure 3). In agreement with observed kinetic experiments, the protein labeling ligations demonstrate time-dependence with the BSA-DBCO labeling occurring very rapidly, exhibiting fluorescence in less than 1 min (lane 3, Figure 3). The slower tetrazine-norbornene ligation started to show protein labeling after 15 min (lane 4, Figure 3). Unmodified proteins BSA and OVA, which do not contain the dipolarphiles DBCO and norbornene, showed no fluorescence labeling after exposure to Syd-630 and Tz-504, indicating the absence of nonspecific reactivity. These experiments demonstrate the exquisite specificity of the synone and tetrazine imaging agents for DBCO- and norbornene-modified proteins respectively as well as their mutual compatibility within a single environment. Example 2: Rapid strain induced sydnone-alkyne cycloaddition reactions for bioorthogonal PET imaging

Described herein is a novel copper-free cycloaddition reaction between sydnone derivatives and the biarylazacyclooctynone (BARAC). These functionalized sydnones are used as 1,3-dipoles for metal-free cycloaddition partners with ring- strained alkynes. The highly stable sydnones display excellent reaction kinetics with BARAC. Extremely selective and high yielding bioorthogonal click chemistry reactions have been widely studied and used as tools for radiolabelling biomolecules and small molecules for positron emission tomography (PET) (Figure 6C)

(Thirumurugan et al., 2013, Chem. Rev.113:4905-4979). The novel copper-free cycloaddition reaction between sydnone derivatives and the biarylazacyclooctynone (BARAC) described herein is the fastest reported cycloaddition reaction with BARAC to date, with rates of 10¹ to 10² M^-1s^-1.

3-Arylsydnone derivatives 16 were synthesized via intramolecular cyclization of N-nitroso-N-aryl amino acids with trifluoroacetic anhydride. The rate constant for the cycloaddition between sydnone and BARAC or BCN was determined under pseudo-first order conditions and followed via the exponential decay in UV absorbance over time (Figure 6B).

The reactions of sydnones with BCN or BARAC proceed at room temperature, in aqueous media and without a transition-metal catalyst (Figure 6A). The N-3-phenyl ring and C-4 substitutions on the sydnone significantly impacted the reaction rate constants. Improved reaction rates were observed with electron deficient Attorney Do cket No.206030-0064-00-WO.606107 sydnones an d 4-chloro sydnones; t he cycload dition react ion of phen yl sydnone with BARAC is nearly 10⁴– fold faster than with B CN.

Sydnon es 21 were synthesized in two-ste ps from N- arylglycine s 19 (Figure 7).¹⁸F-labeled sydnones 24 are synth esized from precursor 23.

Second order react ion kinetics of various sydnones a nd strained alkynes we re measured on a UV-V isible spec trophotom eter (Figure 6B). The d ecay in absorbance of sydnone was monit ored over t ime by reac ting sydnon es with ex cess of strained alk ynes. Figur e 6B illustr ates the cal culation of rate consta nt of the cycloadditi on between phenylsydn one and B CN (k2= 0.019 M^-1S^-1) .

Diverse variety of arylsydnon es were syn thesized an d their reac tion kinetics we re studied w ith BCN. T he cycload dition reac tions were conducted i n methanol: w ater (55:45 ) at room t emperature (23 ºC). Ta ble 1 lists t he calculat ed second orde r rate kinet ics associat ed with the cycloaddit ion reaction between B CN and various sydnone d erivatives. Table 1: Se cond order rate kineti cs of the cy cloaddition reaction be tween BCN and various syd none deriva tives

Attorney Do cket No.206030-0064-00-WO.606107

These r esults demo nstrate that the second order rate constant of sydnones w ere improv ed by intro duction of f luorine on the phenyl ring at the C -3 position wh en compare d to their p arent comp ounds. Fur thermore, th e introduc tion of electron do nating subs tituents resu lts in an ob served dec rease in rea ction rates, while 4-nitro-2,3, 5,6-tetraflu orophenyl a t C-3 was found to dra matically i ncrease the rate of cycloadditi on. A propo sed mecha nism for the cycloaddi tion reactio n can be se en in Figure 9. T hese results demonstra te the synth esis and kin etic studie s of diverse variety of arylsydn ones with B CN, and re veal the eff ect of fluor ophenyl su bstitutions on cycloadditi on reaction rates. Othe r examples of click rea ctions betw een syndon es and alkynes can be found i n Figures 8 and 10. Example 3: Synthesis of radiolabe led sydnon es from bis -syndnone Materials a nd Method s

No-carr ier-added [ ¹⁸F]fluoride was produ ced by the ¹⁸O(p,n)¹⁸F nuclear rea ction in a S iemens RD S-112 cyclo tron at 11 M eV using a 1 mL tan talum target with havar foil. U nless othe rwise state d, reagents and solvent s were commercia lly availabl e and used w ithout furt her purific ation. HPLC grade ace tonitrile and trifluor oacetic acid were purc hased from Fisher Scie ntific. Anh ydrous Attorney Docket No.206030-0064-00-WO.606107 acetonitrile, dimethyl sulfoxide and tetraethylammonium bicarbonate were purchased from Sigma-Aldrich. Sterile product vials were purchased from Hollister-Stier. QMA- light Sep-Paks and tC18 light cartridges were purchased from Waters Corporation. HPLC purifications were performed on a Knauer Smartline HPLC system with inline Knauer UV (254 nm) detector and gamma-radiation coincidence detector and counter (Bioscan Inc.). Semi-preprative HPLC was performed using Phenomenex reverse- phase Luna column (10 × 250 mm, 5 µm) with a flow rate of 4 mL/min. Final purity and identity of compounds were determined by analytical HPLC analysis performed with a Phenomenex reverse-phase Luna column (4.6 × 250 mm, 5 µm) with a flow rate of 1 mL/min. All chromatograms were collected by a GinaStar (Raytest USA, Inc.) analog to digital converter and GinaStar software.

Dry [¹⁸F]TEAF was prepared using an ELIXYS automated radiosynthesizer (Sofie Biosciences). [¹⁸F]Fluoride was delivered to the ELIXYS in [¹⁸O]H₂O (1 mL) via nitrogen gas push and trapped on a QMA cartridge to remove the [¹⁸O]H₂O.

Trapped [¹⁸F]fluoride was subsequently eluted into the reaction vial using a solution containing Et₄NHCO₃ (9-10 mg) in acetonitrile and water (1 mL, 8:2). Contents in the reaction vial were subjected to a nitrogen stream and evaporated by heating the vial to 110 °C while applying a vacuum for 3.5 min, with stirring. Acetonitrile (1.3 mL) was passed through the QMA cartridge to wash remaining activity into the reaction vial. The combined contents in the reaction vial were dried by azeotropic distillation (heating to 110 °C under vacuum) for 2 min. Anhydrous acetonitrile (1.3 mL) was directly added to the reaction vial and azeotropic distillation was repeated once more until dryness, approximately 3-4 min. The reaction vial was cooled to 30 °C under nitrogen pressure and DMSO (1 mL) was added to provide anhydrous [¹⁸F]TEAF which was used for subsequent reactions.

General experimental procedure: Radiochemistry experiments were conducted in a 4 mL glass vial containing N-arylsydnone (4-5 mg) with 250-350 µCi of [¹⁸F]TEAF in 150-200 µL of DMSO. The contents were heated to 150 ^C for 5-20 min and progress of the reaction was observed by radio-TLC. An aliquot of the crude reaction mixture was spotted on a silica gel coated TLC plate, developed in a glass chamber with acetonitrile:water (95:5) as the eluent. The radiochemical conversion (RCC) was calculated by dividing the integrated area of the ¹⁸F-fluorinated product peak by the total integrated area of all peaks on the TLC and multiplying by 100 to Attorney Docket No.206030-0064-00-WO.606107 convert to percentage units. Analytical HPLC was used to confirm product identity and purity via UV absorbance at 254 nm, by coinjection with the ¹⁹F-reference standard. An aliquot of the crude reaction mixture (10 µL) was added to the ¹⁹F- reference standard (1 mg/mL) in acetonitrile (10 µL) and the sample was injected into the analytical HPLC. Preparation of radiolabeled sydnones

To a suspension of aryl amine (10 mmol) and sodium acetate (12 mmol) in ethanol (20 mL) was added ethyl bromoacetate (12 mmol). The mixture was refluxed for 7 h, left overnight at room temperature, and poured into crushed ice. The precipitate formed was filtrated and dried. To the intermediate ester in

water/tetrahydrofuran (1:1) (25 mL) was added sodium hydroxide (1.1 mmol), and the mixture was stirred at 100 ^C for 30 min then to room temperature for 4 hrs. The reaction mixture was acidified to pH 2 by dropwise addition of a 2 M solution of HCl. The resulting precipitate was filtered, washed with water, and purified by

recrystallization from a mixture ethanol/water to afford N-arylglycine. To a mixture of aryl glycine (1 mmol) in anhydrous THF (10 mL) was added tButyl nitrite (1.1). The mixture was stirred at room temperature for 30 min and trifluoroacetic anhydride (1.15 mmol) was added. After 1 h stirring at room temperature, ethyl acetate was added and the reaction was quenched with a saturated solution of sodium bicarbonate. The aqueous layer was extracted with ethyl acetate and the organic layers were combined, dried over MgSO4 and evaporated to obtain the bissydnone (Figure 13).

To anhydrous [¹⁸F]Et₄NF (15-45 mCi), prepared using an ELIXYS synthesis module, was added phenyl bissydnone (4-5 mg) in 0.5 mL of DMSO. The contents were stirred at 150 ^C for 15 min. The reaction mixture was cooled to room temperature and diluted with 4 mL water before purification via semi-preparative HPLC (5% Acetonitrile in water (both 0.1% TFA) 0-5 min then to 50% acetonitrile in Attorney Docket No.206030-0064-00-WO.606107 water over 40 min). The HPLC fraction was collected (retention time, 21 min) in 30 mL of water and passed through tC18 Sep-Pak, which was preactivated by sequential washing of ethanol (5 mL) and water (10 mL). The product was eluted with diethyl ether (2 mL) or acetonitrile (1.5 mL). The product identity and purity were determined by radio-HPLC and radio-TLC. Non-decay corrected radiochemical yields were calculated from the amount of activity trapped on QMA cartridge to product isolated after HPLC purification. Table 2: Base optimization in the synthesis of radiolabeled sydnones

Attorney Docket No.206030-0064-00-WO.606107

Attorney Docket No.206030-0064-00-WO.606107 The molar radioactivity of 4-[¹⁸F]fluorophenyl sydnone (21a, Figure 15) was determined by the following method. A sample of known volume of purified 21a was injected into the analytical HPLC. The absorbance (mAU*s) of UV peak corresponding to the radiofluorinated product 21a was measured. The UV area was used to calculate the concentration of the product based on linear regression analysis of 4-[¹⁹F]fluorophenyl sydnone. A calibration curve was generated from a standard solution (5 ug/mL) by measuring the UV absorbance at different concentrations. The activity injected (Ci/mL) divided by concentration of the product measured from linear regression (µmol/mL) produced the molar specific activity in Ci/µmol or GBq/ µmol.

DIBAC (1 mM) in PBS:DMSO (1:1) (200 µL) was added to purified 4-[¹⁸F]fluorophenyl sydnone (2 mCi– 5 mCi) and stirred at 50 °C for 8 min. The crude reaction mixture was injected into analytical HPLC which showed >99.5% conversion to the click product (Figure 15). The identity of the product was confirmed by co-injecting compound (23) with a cold (non-radiolabeled) click adduct. The results of the experiments are now described. Attorney Docket No.206030-0064-00-WO.606107

The desired compound 4-[¹⁸F]fluorophenyl sydnone 21a (6-10 mCi) was synthesized via the phenyl bissydnone precursor in a fully automated process using an ELIXYS radiosynthesis module, purified, and isolated within 62 min from the end of bombardment in 20 ± 3% non-decay-corrected radiochemical yield (RCY) (n=18) with a specific activity of 1.3 Ci μmol^-1. Purified 21a was stirred with 1 mM DIBAC 22 in 1:1 DMSO:PBS, a concentration typically reported for [¹⁸F]peptide labeling (Chem. Commun, 2016, 52, 6083; Nuclear Medicine and Biology, 2013, 40, 223). Clean conversion in >99% to the 3+2 cycloadduct 23 was observed after 8 mins, determined by radio-HPLC analysis of the crude product (Figure 15). Recently, Taran reported a closely related ultra fast click chemistry reaction with 4-fluorosydnones and subsequent radiofluorination of a sydnone-Pd^II complex after initial preparation of [¹⁸F]Selectfluor bis(triflate), to afford ¹⁸F-labeled 4-fluorosydnone in 7.5% RCY (Liu, et al., Angew. Chem. Int. Ed., 2016, 55, 12073-12077). The methods described herein provide significant practical advantages over previously reported methods, including synthetic ease, high RCY and full automation. The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

ttorney Docket No. 206030-0064-00-WO.606107 CLAIMS What is claimed is:

1. A com ound of formula I :

herein in formula (I):

A¹ is selected from the group consisting of alkyl, heteroalkyl, alkylcycloalkyl, eterocyclyl, alkylheterocyclyl, aryl, alkylaryl, heteroaryl, and alkylheteroaryl, wherein e alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl group and is optionally bstituted with 0-5 R² groups

R¹ and each occurrence of R² are independently selected from the group onsisting of H, -C₁-C₆ alkyl, -C₁-C₆ fluoroalkyl, -C₁-C₆ heteroalkyl, F, ¹⁸F, Cl, Br, I, - N, -NO₂, -OR³, -SR³, -S(=O)R³, -S(=O)₂R³, -NHS(=O)₂R³, -C(=O)R³, -OC(=O)R³, - O₂R³, -OCO₂R³, -CH(R³)₂, -N(R³)₂, -C(=O)N(R³)₂, -OC(=O)N(R³)₂, - HC(=O)NH(R³), -NHC(=O)R³, -NHC(=O)OR³, -C(OH)(R³)₂, and -C(NH₂)(R³)₂; each occurrence of R³ is independently selected from the group consisting of H nd C₁-C₆ alkyl;

L is a linker; and

Q is an imaging moiety.

salt thereof, and any combinations thereof.

2. The compound of claim 1, wherein A¹ is a phenyl group, wherein e phenyl group is optionally substituted. ttorney Docket No. 206030-0064-00-WO.606107

3. The compound of claim 1, wherein L is selected from the grouponsisting of a bond, an alkyl group, an amide, a poly(alkyl ether), and any combinations ereof.

4. The compound of claim 1, wherein Q is ¹⁸F.

5. The compound of claim 1, wherein Q is a fluorescent dye.

6. The compound of claim 5, wherein the fluorescent dye is

ODIPY630.

7. The compound of claim 2, wherein the compound of formula (I) is compound of formula (II):

herein in formula (II):

n is an integer between 0 and 4;

R¹ and each occurrence of R² are each independently selected from the grouponsisting of H, -C₁-C₆ alkyl, -C₁-C₆ fluoroalkyl, -C₁-C₆ heteroalkyl, F, ¹⁸F, Cl, Br, I, -N, -NO₂, -OR³, -SR³, -S(=O)R³, -S(=O)₂R³, -NHS(=O)₂R³, -C(=O)R³, -OC(=O)R³, -O₂R³, -OCO₂R³, -CH(R³)₂, -N(R³)₂, -C(=O)N(R³)₂, -OC(=O)N(R³)₂, -HC(=O)NH(R³), -NHC(=O)R³, -NHC(=O)OR³, -C(OH)(R³)₂, and -C(NH₂)(R³)₂; each occurrence of R³ is independently selected from the group consisting of Hnd C₁-C₆ alkyl;

L is a linker; and

Q is an imaging moiety. ttorney Docket No. 206030-0064-00-WO.606107

8. The compound of claim 7, wherein n is at least 1 and at least one 2 is F.

9. The compound of claim 1, wherein the compound is selected from e rou consistin of:

10. The compound of claim 1, wherein the compound is selected from e group consisting of:

ttorney Docket No. 206030-0064-00-WO.606107

ombinations thereof.

11. A method for labeling a biomolecule comprising the steps of: covalently attaching or conjugating a dipolarophile to a biomolecule to form a omolecule-dipolarophile conjugate; and

contacting the biomolecule-dipolarophile conjugate with a compound, wherein e compound undergoes a cycloaddition reaction with the dipolarophile to provide a beled biomolecule.

12. The method of claim 11, wherein the compound is at least oneompound of formula (I): ttorney Docket No. 206030-0064-00-WO.606107

(I),

herein in formula (I):

A¹ is selected from the group consisting of alkyl, heteroalkyl, alkylcycloalkyl,eterocyclyl, alkylheterocyclyl, aryl, alkylaryl, heteroaryl, and alkylheteroaryl, wherein e alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl group and is optionally bstituted with 0-5 R² groups

R¹ and each occurrence of R² are independently selected from the grouponsisting of H, -C₁-C₆ alkyl, -C₁-C₆ fluoroalkyl, -C₁-C₆ heteroalkyl, F, ¹⁸F, Cl, Br, I, -N, -NO₂, -OR³, -SR³, -S(=O)R³, -S(=O)₂R³, -NHS(=O)₂R³, -C(=O)R³, -OC(=O)R³, -O₂R³, -OCO₂R³, -CH(R³)₂, -N(R³)₂, -C(=O)N(R³)₂, -OC(=O)N(R³)₂, -HC(=O)NH(R³), -NHC(=O)R³, -NHC(=O)OR³, -C(OH)(R³)₂, and -C(NH₂)(R³)₂;

each occurrence of R³ is independently selected from the group consisting of Hnd C₁-C₆ alkyl;

L is a linker; and

Q is an imaging moiety.

salt thereof, and any combinations thereof.

13. The method of claim 12, wherein A¹ is a phenyl group, wherein thehenyl group is optionally substituted.

14. The method of claim 12, wherein L is selected from the grouponsisting of a bond, an alkyl group, an amide, a poly(alkyl ether) group, and anyombinations thereof.

15. The method of claim 12, wherein Q is ¹⁸F.

16. The method of claim 12, wherein Q is a fluorescent dye. ttorney Docket No. 206030-0064-00-WO.606107

17. The method of claim 16, wherein the fluorescent dye is

ODIPY630.

18. The method of claim 13, wherein the compound of formula (I) is aompound of formula (II):

herein in formula (II):

n is an integer between 0 and 4;

L is a linker; and

Q is an imaging moiety.

19. The method of claim 18, wherein n is at least 1 and at least one R² F.

20. The method of claim 12, wherein the compound is selected from e group consisting of: ttorney Docket No. 206030-0064-00-WO.606107

21. The method of claim 12, wherein the compound is selected from e group consisting of:

ttorney Docket No. 206030-0064-00-WO.606107

wherein Q is BODIPY630, a salt thereof, and anyombinations thereof.

22. The method of claim 11, wherein the dipolarophile is benzocyclooctyne (DBCO).

23. The method of claim 11, wherein the dipolarophile is arylazacyclooctynone (BARAC).

24. A method for imaging a biomolecule comprising the steps of: providing a sample comprising a biomolecule;

covalently attaching or conjugating a dipolarophile to a biomolecule to form a omolecule-dipolarophile conjugate;

contacting the biomolecule-dipolarophile conjugate with a compound, wherein e compound undergoes a cycloaddition reaction with the dipolarophile to provide a beled biomolecule;

and detecting the labeled biomolecule, thereby imaging the biomolecule.

25. The method of claim 24, wherein the compound is at least oneompound of formula (I):

ttorney Docket No. 206030-0064-00-WO.606107 herein in formula (I):

A¹ is selected from the group consisting of alkyl, heteroalkyl, alkylcycloalkyl,eterocyclyl, alkylheterocyclyl, aryl, alkylaryl, heteroaryl, and alkylheteroaryl, wherein e alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl group is optionally bstituted with 0-5 R² groups

L is a linker; and

Q is an imaging moiety.

salt thereof, and any combinations thereof.

26. The method of claim 25, wherein A¹ is a phenyl group, wherein thehenyl group is optionally substituted.

27. The method of claim 25, wherein L is selected from the grouponsisting of a bond, an alkyl group, an amide, a poly(alkyl ether) group, and anyombinations thereof.

28. The method of claim 25, wherein Q is ¹⁸F.

29. The method of claim 25, wherein Q is a fluorescent dye.

30. The method of claim 29, wherein the fluorescent dye isODIPY630. ttorney Docket No. 206030-0064-00-WO.606107 31. The method of claim 25, wherein the compound of formula (I) is aompound of formula (II):

herein in formula (II):

n is an integer between 0 and 4;

L is a linker; and

Q is an imaging moiety. 32. The method of claim 31, wherein n is at least 1 and at least one R² F. 33. The method of claim 25, wherein the compound is selected from e group consisting of: ttorney Docket No. 206030-0064-00-WO.606107

34. The method of claim 25, wherein the compound is selected from e group consisting of:

ttorney Docket No. 206030-0064-00-WO.606107

wherein Q is BODIPY630, a salt thereof, and any

ombinations thereof. 35. The method of claim 24, wherein the dipolarophile is benzocyclooctyne (DBCO). 36. The method of claim 24, wherein the dipolarophile is arylazacyclooctynone (BARAC). 37. The method of claim 24, wherein the biomolecule is imaged usingositron emission tomography (PET). 38. The method of claim 24, wherein the biomolecule is imaged in vo.