Bicyclononine reagent for cell imaging

Abstract

The present disclosure provides compounds and methods for preparing antibody conjugates with fluorophores, as well as methods of using these conjugates for cell imaging. In one embodiment, the conjugate can be bound to a quencher to absorb fluorescence from the fluorophore.

Description

Technical Field

[0001] Priority Claim This application claims priority to U.S. Provisional Patent Application No. 63 / 353,020, filed on June 16, 2022, the entire content of which is incorporated herein by reference.

[0002] The present invention relates to tridentate ligands containing bicyclononine (BCN) moieties and methods of using these ligands in cellular fluorescence imaging, including, for example, multiplexed cellular fluorescence imaging.

Background Art

[0003] For example, the processing of cell samples for immunostaining and image cytometry can often be very difficult. One drawback is that the number of special stains that can be performed is limited because cell samples are often scarce (often less than 1,000 cells per pass from fine needle aspiration) and lack a structural scaffold of intact tissue architecture due to their fragility. Even when treated with fluorescent antibodies, the number of different stains for cell samples is usually limited to 4 to 6, which is often insufficient for detailed cancer cell profiling for diagnosis or treatment evaluation. This limitation also extends to immunoprofiling, where it is necessary to examine more than 4 to 6 markers so that the analysis can reflect representative immune cell populations in the intratumoral microenvironment.

Summary of the Invention

Problems to be Solved by the Invention

[0004] Most fluorescence cycling methods were originally developed for paraffin-embedded tissue sections that can withstand harsh de-staining / quenching conditions. Unfortunately, these harsh conditions, which often require oxidizing agents for bleaching, are not compatible with cell samples such as those in fine needle aspiration (FNA). Furthermore, it is not uncommon for antibody-DNA cycling techniques to require sample processing for several hours to several days.

Means for Solving the Problems

[0005] This specification discloses, among other things, an ultra-fast single-cell cycling method by using a fluorophore-based linker containing a bicyclononine (BCN)-based clickable moiety in its structure.

Chemical formula

[0006] In one embodiment, a tetrazine or azide-functionalized quencher can be clicked onto the BCN moiety, thereby quenching fluorescence and turning off the fluorophore.

[0007] Advantageously and unexpectedly, BCN-based probes within the scope of the present claims exhibit excellent oxidation and photo-stability after environmental exposure (e.g., ambient light, microscope illumination, and air), as well as high quenching performance, compared to trans-cyclooctene-based probes such as rTCO, TCO, cTCO, and dTCO. The reasons for these excellent results have hitherto been unclear, and thus, the results of the BCN-based probes described in the present application could not be predicted based on TCO-based probes before testing and experimentation. All of the exemplified probes result in ligation of a quencher adjacent to the fluorophore using the same and flexible PEG4 linker, and thus, similar quenching performance could be expected. Nevertheless, and in stark contrast, as the experimental data show, the observed quenching varies significantly for BCN probes within the scope of the present claims, which exhibit far superior performance. The performance of the BCN probes is unexpectedly comparable to or better than that of the generally more stable cyclopropene (CP)-based probes, as is known, because the reactivity of the three-membered ring (including reactions with light and oxygen) is lower compared to that of the eight-membered ring. As the experimental data show, BCN-based probes within the scope of the present claims exhibit the lowest residual fluorescence after quenching following exposure to ambient light and oxygen. In other words, the BCN probes are significantly more stable compared to their TCO counterparts under normal imaging conditions (e.g., microscope illumination), comparable even to their CP counterparts, and retain their reactivity and performance characteristics during the quenching click reaction after exposure to common storage and handling protocols and bench-top atmosphere. The BCN probes also exhibit the best quenching performance among TCO-type probes (despite the potential pathways for separation / release of the quencher), avoiding the possibility of chemical decomposition and release of either the fluorophore or the quencher common to rTCO-based probes.

[0008] Furthermore, in the tetrazine / cyclooctyne reaction, the BCN-labeled antibody probes within the scope of the present claims showed a significantly improved (about 1400-fold) acceleration of the quenching reaction compared to the kinetics predicted for the Tz / BCN reaction alone, which can be interpreted as changing the time expected for a complete reaction at experimental concentrations from months to hours. In fact, as shown by the experimental data in this application, complete quenching of cells stained with BCN and fluorophore-labeled antibodies was observed within just 2 minutes at a tetrazine-based quencher concentration as low as about 1 μM. That is, the octyne / tetrazine click reaction, which is expected to take dozens of hours, can be accelerated to 2-3 minutes using the probes within the scope of the present claims in a biological context. In the reaction with azide-containing quenchers in the context of cells, the BCN-based antibody probes showed an even more dramatic (in fact, more than 5000-fold) acceleration of the reaction compared to the predicted rate with respect to the reaction kinetics of the underlying azide-alkyne. This further enables changing the reaction time frame to just a few minutes under normal imaging conditions. This dramatic improvement in the reaction rate could not have been predicted before the experiment.

[0009] Due to the improved stability and better reaction kinetics in both the tetrazine and azide click reactions, the BCN-based probes within the scope of the present claims quench fluorescence ultra-fast (less than 1 second) in clinical specimens, enabling multi-channel imaging of 20 - 30 markers within just 1 hour.

[0010] In one general aspect, the present disclosure provides a compound of formula (I)

Chemical formula

Chemical formula

[0011] In some embodiments, R 1 is H.

[0012] In some embodiments, n is an integer from 1 to 5, and each L 1 is selected from NH, O, C(=O), and C 1~6 alkylene.

[0013] In some embodiments, m is an integer from 1 to 5, and each L 2 is independently NH, C(=O), C 1~6 alkylene, -(OCH2CH2) x -, and -(CH2CH2O) x -.

[0014] In some embodiments, m is 5.

[0015] In some embodiments, x is an integer from 1 to 10.

[0016] In some embodiments, R 1 is H, n is an integer from 1 to 5, and each L 1 is selected from NH, O, C(=O), and C 1~6 alkylene, m is an integer from 1 to 5, and each L 2 is independently NH, O, C(=O), C 1~6 alkylene, -(OCH2CH2) x -, and -(CH2CH2O) x -. x is an integer from 2 to 10.

[0017] In some embodiments, p is an integer from 1 to 5, each L 3 is NR N , O, C(=O), and C 1~6 alkylene, the above C 1~6 alkylene is optionally (L 4 ) o -Y 3 substituted, R N is H, C 1~3 alkyl, and (L 4 ) o -Y 3 -.

[0018] In some embodiments, each L 3is selected from NH, O, C(=O), and optionally (L 4 ) o -Y 3 substituted C 1~6 alkylene.

[0019] In some embodiments, the compound has the formula:

Chemical formula

[0020] In some embodiments, each L 3 is selected from NR N , O, C(=O), and C 1~6 alkylene, R N is selected from H, C 1~3 alkyl, and (L 4 ) o -Y 3 .

[0021] In some embodiments, the compound has the formula:

Chemical formula

[0022] In some embodiments, o is an integer from 1 to 5, and each L 4 is independently selected from NH, O, C(=O), and C 1~6 alkylene.

[0023] In some embodiments, Y 3 is a moiety of formula (i).

Chemical formula

[0024] In some embodiments, Y 3 is a moiety of formula (ii).

Chemical formula

[0025] In some embodiments, each L 3 is selected from NR N , O, C(=O), and C 1~6 alkylene.

[0026] In some embodiments n is 1, and L 1 is C 1~6 alkylene, m is 4, and each L 2 is independently selected from NH, C(=O), C 1~6 alkylene, -(OCH2CH2) x -, and -(CH2CH2O) x -. p is 3, and each L 3 is independently selected from NH, O, and C(=O).

[0027] In some embodiments, formula (I) is the formula:

Chemical formula

[0028] In some embodiments, formula (I) is the formula:

Chemical formula

[0029] In some embodiments Y 1 is NHR 1A and Y 2 is selected from C(=O)OR a1 and a group reactive with the side chain of an amino acid of a protein.

[0030] In some embodiments, Y 1 is NH2, Y 2 is C(=O)OH.

[0031] In some embodiments, Y 1 is NHR 1A and R 1 is an amine protecting group, Y 2 is C(=O)OH.

[0032] In some embodiments, Y 1 is NHR 1A and R 1 is a fluorophore, Y 2 is C(=O)OH.

[0033] In some embodiments, Y 1 is NHR 1A and R 1 is a fluorophore, Y 2 is C(=O)OR a1 and R a1 is a carboxylic acid protecting group.

[0034] In some embodiments, Y 1 is NHR 1A and R 1 is a fluorophore, Y 2 is a group reactive with the side chain of an amino acid of a protein.

[0035] In some embodiments, the compound of formula (I) is

Chemical formula

[0036] In another general aspect, the present disclosure provides a protein conjugate of formula (II) [Chemistry] or a pharmaceutically acceptable salt thereof, wherein A is a protein, y is an integer from 1 to 10, R 1 is selected from H, C 1~3 alkyl, and C 1~3 haloalkyl, each L 1 is independently selected from N(R N ), O, C(=O), S(=O)2, C 1~6 alkylene, -(OCH2CH2) x -, and -(CH2CH2O) x -, n is an integer from 1 to 10, each L 2 is independently selected from N(R N ), O, C(=O), S(=O)2, C 1~6 alkylene, -(OCH2CH2) x -, and -(CH2CH2O) x -, m is an integer from 1 to 10, each L 3 is independently selected from N(R N ), O, C(=O), S(=O)2, C 1~6 alkylene, -(OCH2CH2) x -, and -(CH2CH2O) x -, the above L 1 , L 2 , or L3 C therein 1~6 Each of the alkylene groups is optionally (L 4 ) o -Y 3 substituted with Each R N is independently H, C 1~3 alkyl, C 1~3 haloalkyl, and (L 4 ) o -Y 3 selected from Each L 4 is independently N(R N1 )、O、C(=O)、S(=O)2、C 1~6 alkylene, -(OCH2CH2) x -, and -(CH2CH2O) x - selected from Each R N1 is independently H, C 1~3 alkyl, and C 1~3 haloalkyl selected from p is an integer from 1 to 20, o is an integer from 1 to 10, Each x is independently an integer from 1 to 2,000, Each Y 3 is independently selected from the moiety of formula (i) and the moiety of formula (ii),

Chemical formula

[0037] In some embodiments, the protein is selected from an antibody, an antibody fragment, an engineered antibody, a peptide, and an aptamer.

[0038] In some embodiments, the antibody is specific for an antigen that is a biomarker of a disease or condition.

[0039] In some embodiments, the disease or condition is cancer.

[0040] In some embodiments, y is an integer from 4 to 6.

[0041] In some embodiments, each Y 2 is C(=O), and at least one W is the NH of the lysine side chain of Protein A.

[0042] In some embodiments, each Y 2 is C(=O), and at least one W is the S of the cysteine side chain of Protein A.

[0043] In some embodiments, Y 1 is NHR 1A and.

[0044] In some embodiments, Y 1 is OR 2 and.

[0045] In some embodiments, Y 1 is C(=O)R 3 and.

[0046] In some embodiments, R 1 is H.

[0047] In some embodiments, n is an integer from 1 to 5, and each L 1 is selected from NH, O, C(=O), and C 1~6 alkylene.

[0048] In some embodiments, m is an integer from 1 to 5, and each L 2 is independently selected from NH, C(=O), C 1~6 alkylene, -(OCH2CH2) x -, and -(CH2CH2O) x -.

[0049] In some embodiments, m is 5.

[0050] In some embodiments, x is an integer from 1 to 10.

[0051] In some embodiments, R 1 is H, n is an integer from 1 to 5, and each L 1 is selected from NH, O, C(=O), and C 1~6 alkylene, m is an integer from 1 to 5, and each L 2 is independently selected from NH, O, C(=O), C 1~6 alkylene, -(OCH2CH2) x -, and -(CH2CH2O) x -, and x is an integer from 2 to 10.

[0052] In some embodiments, p is an integer from 1 to 5, each L 3 is selected from NR N , O, C(=O), and C 1~6 alkylene, the above-mentioned C 1~6 alkylene is optionally (L4 ) o -Y 3 is replaced by R N is H, C 1~3 alkyl, and optionally (L 4 ) o -Y 3 and is selected from

[0053] In some embodiments, each L 3 is NH, O, C(=O), and optionally (L 4 ) o -Y 3 and is C 1~6 alkylene substituted by and is selected from

[0054] In some embodiments, the conjugate has the formula:

Chemical formula

[0055] In some embodiments, each L 3 is NR N , O, C(=O), and C 1~6 alkylene and is selected from R N is H, C 1~3 alkyl, and optionally (L 4 ) o -Y 3 and is selected from

[0056] In some embodiments, the conjugate has the formula:

Chemical formula

[0057] In some embodiments, o is an integer from 1 to 5, and each L 4is independently selected from NH, O, C(=O), and C 1~6 alkylene.

[0058] In some embodiments, Y 3 is a moiety of formula (i).

Chemical formula

[0059] In some embodiments, Y 3 is a moiety of formula (ii).

Chemical formula

[0060] In some embodiments, each L 3 is NR N , O, C(=O), and C 1~6 alkylene.

[0061] In some embodiments, n is 1, L 1 is C 1~6 alkylene, m is 4, and each L 2 is independently selected from NH, C(=O), C 1~6 alkylene, -(OCH2CH2) x -, and -(CH2CH2O) x -, p is 3, and each L 3 is independently selected from NH, O, and C(=O).

[0062] In some embodiments, the conjugate of formula (I) has the formula:

Chemical formula

[0063] In some embodiments, the conjugate of formula (I) has the formula: [Chemistry] or a pharmaceutically acceptable salt thereof.

[0064] In yet another general aspect, the present disclosure provides a composition comprising a conjugate described herein or a pharmaceutically acceptable salt thereof, and an inert carrier.

[0065] In some embodiments, the composition is an aqueous solution.

[0066] In yet another general aspect, the present disclosure is a method for examining a cell or a component of a cell, comprising: (i) contacting a cell with a conjugate described herein comprising a fluorophore, or a pharmaceutically acceptable salt thereof, or a composition containing the conjugate described herein; (ii) imaging the cell using imaging technology; (iii) after (ii), contacting the cell with a compound of formula (III): [Mathematics] or a pharmaceutically acceptable salt thereof, wherein Y 4 is selected from N3 and the moiety of formula (iii), [Chemistry] R 6 is selected from H, C 1~6 alkyl, and C 1~6 haloalkyl, wherein said C 1~6 alkyl is optionally substituted with OH, NH2, or COOH, each L 4 is independently N(R N ), O, C(=O), S(=O)2, C 1~6 alkylene, C 6~10 arylene, C 6~10 perfluoroarylene, -(OCH2CH2) x-, and -(CH2CH2O) x selected from a is an integer from 1 to 10, each R N is selected from H and C 1~3 alkyl, x is an integer from 1 to 2,000, Q is a quencher, and contacting, Contacting in step (iii) provides a method in which the fluorescence of the fluorophore in the conjugate of formula (II), or a pharmaceutically acceptable salt thereof, is reduced.

[0067] In some embodiments, the imaging technique is fluorescence imaging.

[0068] In some embodiments, Y 4 is N3.

[0069] In some embodiments, the compound of formula (III) has the following formula,

Chemical formula

[0070] In some embodiments, R 6 is H.

[0071] In some embodiments, R 6 is C alkyl optionally substituted with OH, NH2, or COOH 1~6 alkyl.

[0072] In some embodiments, a is an integer from 1 to 7, and each L 4 is independently selected from NH, C(=O), C 1~6 alkylene, C 6~10 arylene, C 6~10 perfluoroarylene, and -(CH2CH2O) x -.

[0073] In some embodiments, the compound of formula (III) is selected from any one of the compounds of

Chemical Formula

Chemical Formula

[0074] In yet another general aspect, the present disclosure provides a method selected from profiling cells, examining cells using cytometry techniques, diagnosing a disease or condition of a subject by examining the pathology of cells obtained from the subject, monitoring the progression of a disease or condition of the subject by examining the pathology of cells obtained from the subject, and detecting disease biomarkers within cells, the method comprising: (i) obtaining cells from the subject, and (ii) examining the cells according to the methods described herein.

[0075] In some embodiments, the cells are obtained from the subject using image-guided biopsy, fine needle aspiration (FNA), surgical tissue collection, punch biopsy, liquid biopsy, brushing, swabbing, touch-prep, fluid aspiration, or blood analysis.

[0076] In some embodiments, the cytometry techniques are selected from image cytometry, holographic cytometry, Fourier ttychography cytometry, and fluorescence cytometry.

[0077] In some embodiments, the cells are selected from cancer cells, immune system cells, and host cells.

[0078] In some embodiments, the disease or condition is cancer.

[0079] In some embodiments, the cancer is selected from lymphoma, breast cancer, skin cancer, lymph nodes, head and neck cancer, and oral cancer.

[0080] 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 application belongs. The methods and materials are described herein for use in this application, and other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, this specification, including definitions, will control.

[0081] Other features and advantages of the present application will be apparent from the following detailed description of the invention, the drawings, and the claims.

Brief Description of the Drawings

[0082]

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Figure 10B

Figure 10C

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Figure 10E

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BRIEF DESCRIPTION OF THE INVENTION

[0083] Molecular analysis of cancer cells is essential for establishing a diagnosis and guiding available treatments. In an ideal world, it would be desirable to sample cancer frequently and in the least invasive way possible so that molecular information can be obtained regularly through treatment and the evolution of the cancer. "Liquid biopsies," i.e., the examination of circulating tumor cells, extracellular vesicles, or cell-free DNA in peripheral blood, offer one such option, but the detection of practical events is rare and the overall sensitivity can be low. More importantly, circulating tumor diagnostics currently cannot trace back to their anatomical origin, whether primary tumor or metastatic site. For this reason, the ability to correlate molecular events with radiological / imaging markers of cancer behavior, invasiveness, and progression is limited.

[0084] An alternative method is fine needle aspiration (FNA), which obtains cells rather than tissue from a tumor, is essentially of known location, and can be processed rapidly, i.e., without the need for embedding or sectioning. FNA is obtained using a small gauge needle (20-25G) and is generally well tolerated. Thus, image-guided FNA is ideal for repeated sample extraction and has a very low risk of procedural complications. However, as noted above, the challenges in processing these cell samples are that the number of special stains that can be performed is limited because they may be scarce (often less than 1,000 cells per pass), and they lack the structural scaffold of intact tissue architecture due to their fragility. Even when treated with fluorescent antibodies, the number of different stains is actually limited to 4-6 and is often insufficient for detailed cancer cell profiling for diagnosis or treatment evaluation. This limitation also extends to immunoprofiling, which requires examining markers that far exceed 4-6 in order for the analysis to reflect representative immune cell populations in the tumor microenvironment. In contrast, the single cell cycling method of the present disclosure enables repeated staining, destaining, and restaining of the collected cell samples for better treatment evaluation in both cancer cells and host immune cells.

[0085] Most fluorescence cycling methods were originally developed for paraffin-embedded tissue sections that can withstand harsh destaining / quenching conditions. Unfortunately, these harsh conditions typically require oxidants for bleaching at strongly alkaline pH (e.g., 4.5% H2O2, 24 mM NaOH, pH > 12) and are not well suited for cell FNA samples. Furthermore, other antibody-DNA cycling technologies require a significant investment in nucleic acid tags / technologies and it is not uncommon for sample processing, including ABCD and SCANT, to take hours to days. Other conventional methods for antibody-DNA based imaging involve similar technical hurdles, including complex chemical steps for activation of DNA barcodes and antibody-DNA bioconjugation, and / or complex fluidics required to cycle multiple sequential staining solutions.

[0086] As described in more detail below, the present disclosure provides methods of rapid and gentle reagents and single cell cycling. In one embodiment, the present disclosure provides a super-fast BCN-based clickable fluorophore (FAST probe).

[0087] Reagents and Linkers In some embodiments, the present disclosure provides a bicyclononane (BCN)-based click-reactive group capable of undergoing a click reaction with a tetrazine (Tz) or azide (N3) reagent containing a fluorescent quencher, a fluorophore detectable by fluorescence imaging, and a group reactive with the side chain of an amino acid of a protein. The three-arm reagent can be used to covalently modify the side chain of at least one amino acid of a protein. Thus, the covalently modified protein contains a fluorophore (by which the protein becomes detectable by fluorescence imaging) and a BCN-reactive group capable of undergoing a reaction with a tetrazine (Tz) or azide reagent containing a fluorescent quencher. Using the three-arm reagent, a protein can be covalently modified simultaneously with a fluorophore and a fluorescent quencher, thereby rendering the protein undetectable by fluorescence imaging (the quencher absorbs fluorescence from the fluorophore).

[0088] In some embodiments, the three-arm reagent, and synthetic intermediates useful in the preparation of the three-arm reagent, are of formula (I):

Chemical formula

[0089] In some embodiments, R 1 is H. In some embodiments, R 1 is C 1~6 alkyl.

[0090] In some embodiments, n is an integer from 1 to 7. In some embodiments, n is an integer from 1 to 5. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0091] In some embodiments, each L 1is independently NH, O, C(=O), C 1~6 alkylene, -(OCH2CH2) x -, and -(CH2CH2O) x -. In some embodiments, n is an integer from 1 to 5, and each L 1 is selected from NH, O, C(=O), and C 1~6 alkylene. In some embodiments, n is 1 and L 1 is C 1~6 alkylene. In some embodiments, at least one L 1 is N(R N ), where R N is (L 4 ) o -Y 3 ). In some embodiments, at least one L 1 is C 4 alkylene substituted with (L o ) 3 -Y 1~6 .

[0092] In some embodiments, m is an integer from 1 to 7. In some embodiments, m is an integer from 1 to 5. In some embodiments, m is at least 1. In some embodiments, m is an integer from 2 to 10. In some embodiments, m is an integer from 3 to 7.

[0093] In some embodiments, m is an integer from 1 to 5, and each L 2 is independently selected from NH, O, C(=O), C 1~6 alkylene, -(OCH2CH2) x -, and -(CH2CH2O) x -. In some embodiments, m is 4 and each L 2 is independently selected from NH, C(=O), C 1~6 alkylene, and -(OCH2CH2) x -. In some embodiments, at least one L 2 is N(R N ), where R N is (L 4 )o -Y 3 is. In some embodiments, at least one L 2 is (L 4 ) o -Y 3 is replaced by C 1~6 which is alkylene. In some embodiments, m and L 2 are (L 2 ) m wherein is long enough so that the BCN moiety in formula (I) does not interfere with the function of the protein (e.g., antibody) that can bind to Y 2 described in more detail herein.

[0094] In some embodiments, p is an integer from 1 to 7. In some embodiments, p is an integer from 1 to 5. In some embodiments, p is at least 1. In some embodiments, p is an integer from 2 to 10. In some embodiments, p is an integer from 3 to 7. In some embodiments, p is an integer from 1 to 15. In some embodiments, p is an integer from 1 to 10. In some embodiments, p is an integer from 1 to 7.

[0095] In some embodiments, each L 3 is independently selected from NH, O, C(=O), C 1~6 alkylene, -(OCH2CH2) x -, and -(CH2CH2O) x -. In some embodiments, p is an integer from 1 to 5 and each L 3 is selected from NH, O, C(=O), and C 1~6 alkylene. In some embodiments, p is 3 and each L 3 is selected from NH, O, and C(=O). In some embodiments, at least one L 3 is (L 4 ) o -Y 3 is replaced by C 1~6 which is alkylene. In some embodiments, at least one L 3 is N(R N ) where RN is (L 4 ). o -Y 3 is.

[0096] In some embodiments, o is an integer from 1 to 7. In some embodiments, o is an integer from 1 to 4. In some embodiments, o is an integer from 1 to 3. In some embodiments, o is an integer from 1 to 5. In some embodiments, each L 4 is selected from NH, O, C(=O), and C 1~6 alkylene. In some embodiments, each L 4 is independently selected from NH, O, and C(=O).

[0097] In some embodiments, R N is H. In some embodiments, R N is C 1~3 alkyl. In some embodiments, R N is (L 4 ). o -Y 3 is. In some embodiments, R N1 is H. In some embodiments, R N1 is C 1~3 alkyl.

[0098] In some embodiments, x is an integer from 2 to 10. In some embodiments, x is 3, 4, 5, or 6.

[0099] In some embodiments of formula (I), R 1 is H, n is an integer from 1 to 5, each L 1 is selected from NH, O, C(=O), and C 1~6 alkylene, m is an integer from 1 to 5, each L 2 is independently selected from NH, O, C(=O), C 1~6 alkylene, -(OCH2CH2) x -, and -(CH2CH2O) x -. x is an integer from 2 to 10, p is an integer from 1 to 5, each L 3 is independently selected from N(R N ), O, C(=O), and C 1~6 alkylene, and the above C 1~6 alkylene is optionally (L 4 ) o -Y 3 substituted, each R N is independently selected from H and (L 4 ) o -Y 3 and, o is an integer from 1 to 5, each L 4 is independently selected from NH, O, C(=O), and C 1~6 alkylene.

[0100] In some embodiments, the compound of formula (I) contains one Y 3 group. In some embodiments, p is an integer from 1 to 3, and each L 3 is independently selected from NH, O, and C(=O).

[0101] In some embodiments, the compound of formula (I) has the formula:

Chemical formula

[0102] In some embodiments, the compound of formula (I) has the formula

Chemical formula

[0103] In some embodiments, Y 3 is (when present) a moiety of formula (i). [Chemical formula]

[0104] In some embodiments, Y 3 is (when present) a moiety of formula (ii). [Chemical formula]

[0105] In some embodiments of formula (I), R 1 is H, n is 1, L 1 is C 1~6 alkylene, m is 4, and each L 2 is independently selected from NH, C(=O), C 1~6 alkylene, and -(OCH2CH2) x -. p is 3, and each L 3 is independently selected from NH, O, and C(=O), and x is an integer from 2 to 10.

[0106] In some embodiments, the compound of formula (I) has the formula: [Chemical formula] or a pharmaceutically acceptable salt thereof.

[0107] In some embodiments, the compound of formula (I) has the formula: [Chemical formula] or a pharmaceutically acceptable salt thereof.

[0108] In some embodiments, x is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, x is 4 or 5. In some embodiments, x is 4.

[0109] In some embodiments, (L 1 ) n comprises the side chain of an amino acid (e.g., lysine, serine, threonine, cysteine, tyrosine, aspartic acid, or glutamic acid), and Y 1 comprises the terminal functional group of the side chain of the amino acid (e.g., N, O, S, or C(=O)).

[0110] In some embodiments, Y 1 is NHR 1A wherein.

[0111] In some embodiments, R 1A is a fluorophore.

[0112] In some embodiments, R 1A is an amine protecting group.

[0113] In some embodiments, Y 1 is NH2.

[0114] In some embodiments, Y 1 is OR 2 wherein.

[0115] In some embodiments, Y 1 is OH.

[0116] In some embodiments, R 2 is an alcohol protecting group.

[0117] In some embodiments, R 2 is a fluorophore.

[0118] In some embodiments, Y 1 is C(=O)R 3 wherein.

[0119] In some embodiments, Y 1 is C(=O)OH.

[0120] In some embodiments, R 3 is OR a1 and R a1 is a carboxylic acid protecting group.

[0121] In some embodiments, R 3 is a fluorophore.

[0122] In some embodiments, Y 2 is C(=O)OR a1 and

[0123] In some embodiments, Y 2 is C(=O)OH.

[0124] In some embodiments, R a1 is a carboxylic acid protecting group.

[0125] In some embodiments, Y 2 is NHR 4 and

[0126] In some embodiments, Y 2 is NH2.

[0127] In some embodiments, R 4 is an amine protecting group.

[0128] In some embodiments, Y 2 is OR 5 and

[0129] In some embodiments, Y 2 is OH.

[0130] In some embodiments, R 5 is an alcohol protecting group.

[0131] In some embodiments, Y 2is a reactive group with the side chain of the amino acid of the protein. In some embodiments, the reactive group with the side chain of the amino acid of the protein is an activated ester group.

[0132] In some embodiments, Y 1 is NHR 1A and Y 2 is selected from C(=O)OR a1 and the reactive group with the side chain of the amino acid of the protein.

[0133] In some embodiments, Y 1 is NH2, Y 2 is C(=O)OH.

[0134] In some embodiments, Y 1 is NHR 1A and R 1 is an amine protecting group, Y 2 is C(=O)OH.

[0135] In some embodiments, Y 1 is NHR 1A and R 1 is a fluorophore, Y 2 is C(=O)OH.

[0136] In some embodiments, Y 1 is NHR 1A and R 1 is a fluorophore, Y 2 is C(=O)OR a1 and R a1 is a carboxylic acid protecting group.

[0137] In some embodiments, Y 1 is NHR 1A and R 1 is a fluorophore, Y 2 is a group reactive with the side chain of an amino acid of the protein.

[0138] In some embodiments, the compound of formula (I) is

Chemical formula

Chemical formula

Chemical formula

[0139] In some embodiments, a skilled chemist can select and implement any of the amine protecting groups, alcohol protecting groups, or carboxylic acid protecting groups of the present disclosure. Examples of suitable protecting groups, as well as methods for protection and deprotection, and the chemical properties of protecting groups, including the selection of appropriate protecting groups, can be found, for example, in P.G.M. Wuts and T.W. Greene, Protective Groups in Organic Synthesis, 4 th Ed., Wiley & Sons, Inc., New York (2006) (incorporated herein by reference).

[0140] Suitable examples of amine protecting groups include carbobenzyloxy (Cbz) group, p-methoxybenzylcarbonyl (Moz or MeOZ), tert-butyloxycarbonyl (BOC) group, 9-fluorenylmethyloxycarbonyl (Fmoc), acetyl (Ac), benzoyl (Bz), benzyl (Bn) group, carbamate group, p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP) group, tosyl (Ts) group, Troc (trichloroethyl chloroformate), and nosyl group.

[0141] Suitable examples of alcohol protecting groups include acetyl (Ac), benzoyl (Bz), benzyl (Bn), β-methoxyethoxymethyl ether (MEM), dimethoxytrityl, [bis-(4-methoxyphenyl)phenylmethyl] (DMT), methoxymethyl ether (MOM), methoxytrityl [(4-methoxyphenyl)diphenylmethyl] (MMT), p-methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (triphenylmethyl, Tr), silyl ether (the most common ones include trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM), triisopropylsilyl (TIPS) ether), methyl ether, and ethoxyethyl ether (EE).

[0142] Suitable examples of carboxylic acid protecting groups include methyl ester, benzyl ester, tert-butyl ester, esters of 2,6-disubstituted phenols (e.g., 2,6-dimethylphenol, 2,6-diisopropylphenol, 2,6-di-tert-butylphenol), silyl ester, orthoester, and oxazoline.

[0143] Suitable examples of groups reactive with the side chains of the amino acids of the protein are described, for example, in D. Shannon, Covalent protein modification: the current landscape of residue - specific electrophiles, Current Opinion in Chemical Biology 2015, 24, 18 - 26, which is hereby incorporated by reference in its entirety.

[0144] Suitable examples of groups reactive with the OH of serine include the following groups:

Chem.

[0145] Suitable examples of groups reactive with the SH of cysteine include the following groups:

Chem.

[0146] Suitable examples of groups reactive with the NH2 of lysine include activated esters of the following formula:

Chem.

[0147] Suitable examples of fluorophores include any fluorescent compound capable of re-emitting light upon photoexcitation. The fluorophore is excited by light having a wavelength of from about 300 nm to about 800 nm and subsequently emits light having a wavelength of from about 350 nm to about 770 nm (e.g., violet, blue, cyan, green, yellow, orange, or red light), which can be detected by a fluorescence imaging device including the ability to measure the intensity of fluorescence. Suitable examples of fluorophores include AF488, hydroxycoumarin blue, methoxycoumarin blue, Alexa fluor blue, aminocoumarin blue, Cy2 green (dark), FAM green (dark), Alexa fluor 488 green (bright), Fluorescein FITC green (bright), Alexa fluor 430 green (bright), Alexa fluor 532 green (bright), HEX green (bright), Cy3 yellow, TRITC yellow, Alexa fluor 546 yellow, Alexa fluor 555 3 yellow, R-phycoerythrin (PE) 480; yellow, Rhodamine Red-X orange, Tamara red, Cy3.5 581 red, Rox red, Alexa fluor 568 red, Red 613 red, Texas Red red, Alexa fluor 594 red, Alexa fluor 633 red, allophycocyanin red, Alexa fluor 633 red, Cy5 red, Alexa fluor 660 red, Cy5.5 red, TruRed red, Alexa fluor 680 red, and Cy7 red. The absorbance and emission wavelengths of these fluorophores are well known in the art.

[0148] In some embodiments, salts of any of the compounds disclosed herein (e.g., pharmaceutically acceptable salts) that include any compound of formula (I) are formed between an acid and a basic group such as an amino functional group of the compound, or between a base and an acidic group such as a carboxyl functional group of the compound. According to another embodiment, the compound is a pharmaceutically acceptable acid addition salt.

[0149] In some embodiments, acids commonly used for the formation of pharmaceutically acceptable salts include inorganic acids such as hydrogen bisulfide, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, and phosphoric acid, as well as organic acids such as para-toluenesulfonic acid, salicylic acid, tartaric acid, bitartaric acid, ascorbic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, lactic acid, oxalic acid, para-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, and acetic acid, and related inorganic and organic acids. Accordingly, such pharmaceutically acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and other salts. In one embodiment, pharmaceutically acceptable acid addition salts include those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and particularly those formed with organic acids such as maleic acid.

[0150] In some embodiments, bases commonly used for the formation of pharmaceutically acceptable salts include hydroxides of alkali metals including sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals such as aluminum and zinc; ammonia, organic amines such as unsubstituted or hydroxyl-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine; tributylamine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-(C1-C6)-alkylamine) such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; morpholine; thiomorpholine; piperidine; pyrrolidine; and amino acids such as arginine and lysine.

[0151] In some embodiments, the present disclosure also provides a linker of the following formula,

Chemical formula

[0152] In some embodiments, the present disclosure also provides a linker of the following formula,

Chemical formula

[0153] Protein conjugate In some embodiments, the tridentate reagent of formula (I) is reacted with a protein to obtain a protein conjugate of formula (II),

Chemical formula

Chemical formula

[0154] In some embodiments, y is an integer from 1 to 7. In some embodiments, y is an integer from 1 to 5. In some embodiments, y is selected from 1, 2, 3, 4, 5, 6, or 7. In some embodiments, y is 1.

[0155] In some embodiments, R 1 is H. In some embodiments, R 1 is C 1~6 alkyl.

[0156] In some embodiments, n is an integer from 1 to 7. In some embodiments, n is an integer from 1 to 5. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0157] In some embodiments, each L 1 is independently selected from NH, O, C(=O), C 1~6 alkylene, -(OCH2CH2) x -, and -(CH2CH2O) x -. In some embodiments, n is an integer from 1 to 5 and each L 1 is selected from NH, O, C(=O), and C 1~6 alkylene. In some embodiments, n is 1 and L 1 is C 1~6 alkylene. In some embodiments, at least one L 1 is N(R N ) where R N is (L 4 ) o -Y 3 In some embodiments, at least one L 1 is C 4 alkylene substituted with (L o ) 3 -Y 1~6 and is alkylene.

[0158] In some embodiments, m is an integer from 1 to 7. In some embodiments, m is an integer from 1 to 5. In some embodiments, m is at least 1. In some embodiments, m is an integer from 2 to 10. In some embodiments, m is an integer from 3 to 7.

[0159] In some embodiments, m is an integer from 1 to 5, and each L 2 is independently NH, O, C(=O), C 1~6 alkylene, -(OCH2CH2) x -, and -(CH2CH2O) x -. In some embodiments, m is 4, and each L 2 is independently NH, C(=O), C 1~6 alkylene, and -(OCH2CH2) x -. In some embodiments, at least one L 2 is N(R N ), where R N is (L 4 ) o -Y 3 . In some embodiments, at least one L 2 is C 4 alkylene substituted with (L o ) 3 -Y 1~6 . In some embodiments, m and L 2 are selected such that (L 2 ) m is long enough so that the BCN moiety in formula (I) does not interfere with the function of a protein (e.g., an antibody) to which Y 2 , described in more detail herein, can bind.

[0160] In some embodiments, p is an integer from 1 to 7. In some embodiments, p is an integer from 1 to 5. In some embodiments, p is at least 1. In some embodiments, p is an integer from 2 to 10. In some embodiments, p is an integer from 3 to 7. In some embodiments, p is an integer from 1 to 15. In some embodiments, p is an integer from 1 to 10. In some embodiments, p is an integer from 1 to 7.

[0161] In some embodiments, each L 3 is independently selected from NH, O, C(=O), C 1~6 alkylene, -(OCH2CH2) x -, and -(CH2CH2O) x -. In some embodiments, p is an integer from 1 to 5, and each L 3 is selected from NH, O, C(=O), and C 1~6 alkylene. In some embodiments, p is 3, and each L 3 is selected from NH, O, and C(=O). In some embodiments, at least one L 3 is C 4 alkylenereplaced by (L o ) 3 -Y 1~6 . In some embodiments, at least one L 3 is N(R N ), and R N is (L 4 ) o -Y 3 .

[0162] In some embodiments, o is an integer from 1 to 7. In some embodiments, o is an integer from 1 to 4. In some embodiments, o is an integer from 1 to 3. In some embodiments, o is an integer from 1 to 5. In some embodiments, each L 4 is selected from NH, O, C(=O), and C 1~6 alkylene. In some embodiments, each L 4 is independently selected from NH, O, and C(=O).

[0163] In some embodiments, R N is H. In some embodiments, R N is C 1~3 alkyl. In some embodiments, R N is (L 4 ) o -Y 3 In some embodiments, R N1 is H. In some embodiments, R N1 is C 1~3 alkyl.

[0164] In some embodiments, x is an integer from 2 to 10. In some embodiments, x is 3, 4, 5, or 6.

[0165] In some embodiments of formula (I), R 1 is H, n is an integer from 1 to 5, each L 1 is selected from NH, O, C(=O), and C 1~6 alkylene, m is an integer from 1 to 5, each L 2 is independently selected from NH, O, C(=O), C 1~6 alkylene, -(OCH2CH2) x -, and -(CH2CH2O) x -, x is an integer from 2 to 10, p is an integer from 1 to 5, each L 3 is independently selected from N(R N ), O, C(=O), and C 1~6 alkylene, and the above C 1~6 alkylene is optionally substituted with (L 4 ) o -Y 3 and each R N is independently selected from H and (L 4 ) o -Y 3 and o is an integer from 1 to 5, each L 4 is independently selected from NH, O, C(=O), and C 1~6 alkylene.

[0166] In some embodiments, the compound of formula (I) contains one Y 3 group. In some embodiments, p is an integer from 1 to 3, and each L 3 is independently selected from NH, O, and C(=O).

[0167] In some embodiments, the conjugate of formula (II) has the formula:[[]] [Chemical formula][[]] or a pharmaceutically acceptable salt thereof, wherein the sum of p1 and p2 is at least 1 less than p.[[]]

[0168] In some embodiments, the conjugate of formula (II) has the formula:[[]] [Chemical formula][[]] or a pharmaceutically acceptable salt thereof, wherein the sum of p1 and p2 is at least 1 less than p.[[]]

[0169] In some embodiments, Y 3 is (when present) a moiety of formula (i).[[]] [Chemical formula][[]]

[0170] In some embodiments, Y 3 is (when present) a moiety of formula (ii).[[]] [Chemical formula][[]]

[0171] In some embodiments of formula (II), R 1 is H, n is 1, and L 1 is C 1~6 alkylene, and m is 4, and each L 2 is, independently, NH, C(=O), C 1~6 alkylene, and -(OCH2CH2) x - selected from, p is 3, and each L 3 is, independently selected from NH, O, and C(=O), x is an integer from 2 to 10.

[0172] In some embodiments, the conjugate of formula (II) has the formula:

Chemical formula

[0173] In some embodiments, the conjugate of formula (II) has the formula:

Chemical formula

[0174] In some embodiments, (L 1 ) n includes the side chain of an amino acid (e.g., lysine, serine, threonine, cysteine, tyrosine, aspartic acid, or glutamic acid), and Y 1 includes the terminal functional group of the side chain of the amino acid (e.g., N, O, S, or C(=O)).

[0175] In some embodiments, y is an integer from 4 to 6. In some embodiments, y is an integer from 1 to 10. In some embodiments, y is 1. In some embodiments, y is 4. In some embodiments, y is 5. In some embodiments, y is 6. In some embodiments, y is 7.

[0176] In some embodiments, Y 1 is NHR1A is as follows.

[0177] In some embodiments, Y 1 is OR 2 is as follows.

[0178] In some embodiments, Y 1 is C(=O)R 3 is as follows.

[0179] R 1A , R 2 and R 3 Any one of the fluorophores in can be any one of the fluorophores described herein with respect to formula (I). In some embodiments, the fluorophore of formula (II) is selected from AF488, AF647, AF594, and AF555.

[0180] In some embodiments, Y before conjugating to Protein A 2 is any one of the reactive Y 2 groups described herein with respect to formula (I). Suitable examples of the Y 2 group of formula (II) include C(=O) and any one of the following moieties:

Chemical formula

[0181] In some embodiments, W is the O of the side chain of serine, threonine, or tyrosine of Protein A. In some embodiments, W is the S of the side chain of cysteine of Protein A. In some embodiments, W is the NH of the side chain of lysine of Protein A. In some embodiments, W is the C(=O) of the side chain of aspartic acid or glutamic acid of Protein A.

[0182] In some embodiments, each Y 2is C(=O), and each W is NH of the lysine side chain of protein A. In some embodiments, each Y 2 is C(=O), and at least one W is S of the cysteine side chain of protein A.

[0183] In some embodiments, R c1 is H. In some embodiments, R c1 is C 1~3 alkyl.

[0184] In some embodiments, the protein is selected from antibodies, antibody fragments, engineered antibodies, peptides, and aptamers. In some embodiments, the protein is an antibody. In some embodiments, the antibody is specific for an antigen that is a biomarker of a disease or condition. In some embodiments, the disease or condition is cancer. In some embodiments, the disease or condition is a disease of the immune system. Suitable examples of such diseases include severe combined immunodeficiency (SCID), autoimmune disorders, familial Mediterranean fever and Crohn's disease (inflammatory bowel disease), arthritis (including rheumatoid arthritis), Hashimoto's thyroiditis, type 1 diabetes mellitus, systemic lupus erythematosus, and myasthenia gravis. In some embodiments, the antigen is a biomarker of an immune system response to a viral infection or vaccine. Suitable examples of viral infections include infections caused by DNA viruses, RNA viruses, or coronaviruses. An example of a viral infection is influenza. Another example of a viral infection is a coronavirus infection such as COVID-19 (caused by SARS-CoV-2), Middle East respiratory syndrome (MERS) (caused by MERS-CoV), or severe acute respiratory syndrome (SARS) (caused by SARS-CoV). In some embodiments, the antigen is a biomarker of a cytokine storm. A cytokine storm can occur as a result of an infection (e.g., a viral infection described herein), a vaccine (e.g., a vaccine against any of the viral infections described herein), an autoimmune condition, or other disease. Suitable examples of such cytokines include inflammatory cytokines such as IL-6, IL-1, TNF-α, or interferon. In some embodiments, the antibody is specific for an antigen that exhibits an immune system response (including a cytokine storm) to COVID-19.

[0185] Suitable examples of biomarkers include CD45, CD3, CD4, CD8, PD-1, PD-L1, CD11b, F4 / 80, CD163, CD206, Ly6G, CD11c, and MHCII. Any other biomarker known in the art to indicate the severity of a disease or the presence of any disease state by its presence in a cell (e.g., on the cell surface) can be used as an antigen for antibody A of formula (B) or formula (II). Some examples of cancer biomarkers include alpha-fetoprotein (AFP), CA15-3, CA27-29, CA19-9, CA-125, calcitonin, calretinin, carcinoembryonic antigen, CD34, CD99MIC 2, CD117, chromogranin, chromosomes 3, 7, 17, and 9p21, cytokeratin (various types: TPA, TPS, Cyfra21-1), desmin, epithelial membrane antigen (EMA), factor VIII, CD31 FL1, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45, human chorionic gonadotropin (hCG), immunoglobulin, inhibin, keratin (various types), lymphocyte markers (various types), MART-1 (Melan-A), myo D1, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase (PLAP), prostate-specific antigen (PSA), PTPRC (CD45), S100 protein, smooth muscle actin (SMA), synaptophysin, thymidine kinase, thyroglobulin (Tg), thyroid transcription factor-1 (TTF-1), tumor M2-PK, and vimentin.

[0186] In some embodiments, the biomarker is selected from CD45, CD3, CD8, CD4, FoxP3, NK1.1, CD19, CD20, CD11b, F4 / 80, CD11c, Ly6G, Ly6C, MHCII, PD-1, PD-L1, granzyme B, IFNγ, CK5 / 6, p16, CD56, CD68, CD14, CD1a, CD66b, CD39, TCF1, IL-12β, and CD163. In some embodiments, the antibody is specific for PD-1 (e.g., pembrolizumab, nivolumab, or semaprimab). In some embodiments, the antibody is specific for PD-L1 (e.g., atezolizumab, avelumab, or durvalumab).

[0187] In some embodiments, the present disclosure provides a composition comprising a protein conjugate of formula (II) or a pharmaceutically acceptable salt thereof, and an inert carrier. In some embodiments, the composition is an aqueous solution (i.e., the inert carrier is water). The aqueous solution may contain a buffer, e.g., any buffer containing an inert carrier such as water, phosphate, glycine, sorbic acid, potassium sorbate, a partial glyceride mixture of saturated vegetable fatty acids, water, salts or electrolytes, e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate salts, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, or any combination thereof. Some examples of buffers include Dulbecco's phosphate buffered saline (DPBS), phosphate buffered saline, and Krebs-Henseleit buffer. The pH of the buffer may be from about 5 to about 9, e.g., the pH may be from 6 to 8. Y 2 A compound of formula (I), or a salt thereof, wherein Y is a group reactive with a protein, can be mixed with a protein (e.g., an antibody) in any of the aqueous solutions described herein to obtain a compound of formula (II).

[0188] A composition (e.g., an aqueous solution) containing the compound of formula (II) can be used to treat cells (e.g., cells containing biomarkers), and the cells can be imaged using the fluorophore of formula (II).

[0189] Method for cell analysis Accordingly, the present disclosure provides a method for examining a cell or a component of a cell (e.g., the nucleus of a cell), comprising: (i) contacting the cell with a conjugate of formula (II) containing the fluorophore, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same; (ii) imaging the cell using an imaging technique; (iii) after (ii), contacting the cell with a compound of formula (III): [Number] or a pharmaceutically acceptable salt thereof, wherein Y 4 is selected from N3 and the moiety of formula (iii), [Chemical formula] R 6 is selected from H, C 1~6 alkyl, and C 1~6 haloalkyl, wherein the above-mentioned C 1~6 alkyl is optionally substituted with OH, NH2, or COOH, each L 4 is independently selected from N(R N ), O, C(=O), S(=O)2, C 1~6 alkylene, C 6~10 arylene, C 6~10 perfluoroarylene, -(OCH2CH2) x -, and -(CH2CH2O) x -; a is an integer from 1 to 10; each R N is selected from H and C 1~3 alkyl; x is an integer from 1 to 2,000, and Q is a quencher, and contacting, The method provides that the fluorescence of the fluorophore in the conjugate of formula (II) or a pharmaceutically acceptable salt thereof decreases due to the contact in step (iii).

[0190] Without being bound by theory, when the cell is contacted with the protein conjugate of formula (II) in step (i), protein A (e.g., an antibody) binds to its antigen on the surface of the cell, in the cytoplasm of the cell (or in the nucleus of the cell), so it is considered that the cell or its components can be imaged by detecting the fluorescence of the fluorophore of formula (II).

[0191] In some embodiments, the imaging technique of step (ii) is fluorescence imaging such as microscopy, imaging probes, and spectroscopy. The fluorescence imaging device includes an excitation light source, a radiation light collection source, an optional optical filter, and a visualization means (e.g., a digital camera for taking fluorescence imaging photographs). Suitable examples of fluorescence imaging include internal reflection fluorescence microscopy, light sheet fluorescence microscopy, and fluorescence lifetime imaging microscopy. Suitable imaging techniques are described, for example, in Rao, J. et al., Fluorescence imaging in vivo: recent advances, Current Opinion in Biotechnology, 18, (1), 2007, 17 - 25, which is hereby incorporated by reference in its entirety.

[0192] In some embodiments, Y 4 is N3.

[0193] In some embodiments, the compound of formula (III) has the formula:

Chemical formula

[0194] In some embodiments, R 6 is H. In some embodiments, R 6 is CH3. In some embodiments, R 6 is C 1~6 alkyl optionally substituted with OH, NH2, or COOH.

[0195] In some embodiments, a is an integer from 4 to 10. In some embodiments, a is an integer from 3 to 7. In some embodiments, a is at least 3. In some embodiments, a is 1, 2, 3, 4, 5, 6, or 7. In some embodiments, a is an integer from 1 to 7, and each L 4 is independently selected from NH, C(=O), C 1~6 alkylene, C 6~10 arylene, C 6~10 perfluoroarylene, and -(CH2CH2O) x -. In some embodiments, each x is an integer from 1 to 10. In some embodiments, x is 1, 2, 3, 4, or 5.

[0196] In some embodiments, quencher Q is a fluorescent quencher. Suitable examples of fluorescent quenchers include aromatic azo compounds and phenazine derivatives. In some examples, the fluorescent quencher is BHQ0, BHQ1, BHQ2, BHQ3, BHQ10, or IRDye QC-1. In some embodiments, the quencher is selected from dabcyl, IowaBlack quencher, ATTO 540Q, ATTO 575Q, ATTO 580Q, ATTO 612Q, BBQ-650, QXL quencher, and TIDE quencher.

[0197] In some embodiments, the compound of formula (III) is

Chemical formula

Chemical formula

[0198] In some embodiments, upon contact in step (iii), the fluorescence of the fluorophore in the conjugate of formula (II) decreases (or the fluorescence is completely quenched). Without being bound by theory, it is believed that quencher Q can quench the fluorescence of the fluorophore of formula (II) via contact (static) quenching. Without being bound by theory, quencher Q can also quench the fluorescence of the fluorophore of formula (II) via FRET quenching, i.e., the excited fluorophore, rather than the emitted light, transfers energy to the quencher through space. In the absence of a quencher, the fluorophore would emit light and that light would be detected. In some embodiments, Q of formula (III) and the fluorophore of formula (II) are selected such that the emission spectrum of the fluorophore substantially overlaps with the absorption spectrum of quencher Q.

[0199] Without being bound by theory, in one example, as shown in Scheme 1 for example, the BCN moiety in the protein conjugate of formula (II) is thought to react with the tetrazine moiety of formula (III) to produce the protein conjugate of formula (IV). Scheme 1

Chemical Structure

[0200] Referring to Scheme 1, the BCN fragment of formula (II) participates in an inverse electron demand Diels–Alder reaction with the tetrazine of formula (III), followed by a retro-Diels–Alder reaction to remove nitrogen gas. This ligation causes the Y in the compound of formula (IV) 1 of the fluorophore and quencher Q to covalently bond and be in particularly close proximity. Without being bound by theory, Q and Y 1It is considered that the spatial proximity between the fluorophore (resulting from a covalent bond between these groups) enables efficient quenching of fluorescence.

[0201] In some embodiments, the present disclosure provides a three-arm linker of the following formula,

Chemical formula

[0202] Methods of use In some embodiments, the present disclosure provides a method of profiling cells, comprising: (i) obtaining cells from a subject; and (ii) examining the cells according to the cell analysis methods described herein.

[0203] In some embodiments, the present disclosure provides a method of examining cells using cytometry techniques, comprising: (i) obtaining cells from a subject; and (ii) examining the cells according to the cell analysis methods described herein. Suitable examples of cytometry techniques include image cytometry, holographic cytometry, Fouriertychography cytometry, and fluorescence cytometry.

[0204] In some embodiments, the present disclosure provides a method of diagnosing a disease or condition of a subject by examining the pathology of cells obtained from the subject, comprising: (i) obtaining cells from the subject; and (ii) examining the cells according to the cell analysis methods described herein.

[0205] In some embodiments, the present disclosure provides a method of monitoring the progression of a disease or condition in a subject (or monitoring the effectiveness of treatment of a disease or condition) by examining the pathology of cells obtained from the subject, the method comprising: (i) obtaining cells from the subject; and (ii) examining the cells according to a cell analysis method described herein. This method enables guiding a treatment regimen and providing individualized treatment based on the results of the examination of the cells according to the method.

[0206] In some embodiments, the present disclosure provides a method of monitoring the effectiveness of cancer treatment. Suitable examples of cancer treatment include chemotherapy, radiation therapy, and surgery, or any combination of the foregoing.Suitable examples of chemotherapy include abarelix, aldesleukin, alitretinoin, allopurinol, altretamine, anastrozole, arsenic trioxide, asparaginase, azacitidine, bexarotene, bleomycin, bortezomib, busulfan, calusterone, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, dalteparin, dasatinib, daunorubicin, decitabine, denileukin, dexrazoxane, docetaxel, doxorubicin, drostanolone propionate, emtansine, epirubicin, eribulin, erlotinib, estramustine, etoposide, everolimus, exemestane, fentanyl citrate, filgrastim, floxuridine, fludarabine, fluorouracil, fruquintinib, fulvestrant, gefitinib, gemcitabine, ozogamicin, goserelin acetate, histrelin acetate, tiuxetan, idarubicin, ifosfamide, imatinib, interferon α2a, irinotecan, ixabepilone, lapatinib, lenalidomide, letrozole, leucovorin, leuprolide acetate, levamisole, lomustine, mechlorethamine, megestrol acetate, melphalan, mercaptopurine, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone phenpropionate, nelarabine, oxaliplatin, paclitaxel, pamidronate, panitumumab, pegaspargase, pegfilgrastim, pemetrexed, pentostatin, pipobroman, plicamycin, procarbazine, quinacrine, rasburicase, sorafenib, streptozocin, sulfatinib, sunitinib, sunitinib, tamoxifen, temozolomide, teniposide, testolactone, thalidomide, thioguanine, thiotepa, topotecan, toremifene, tretinoin, uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine, volitinib, vorinostat, and zoledronic acid, or a pharmaceutically acceptable salt thereof.

[0207] In some embodiments, cancer treatment includes administering to a patient an antibody useful for treating cancer. Suitable examples of such antibodies include pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, abagovomab, adecatumumab, afutuzumab, alacizumab pegol, altumomab pentetate, amatuximab, anatumomab mafenatox, apolizumab, arcitumomab, bavituximab, bevacizumab, bivatuzumab mertansine, blinatumomab, brentuximab vedotin, canertinib mertansine, canertinib rabutansine, capromab pendetide, cetuximab, cituximab bogatox, cixutumumab, clivatuzumab tetraxetan, dacetuzumab, demcizumab, detumomab, dorzigomab, eclizumab, eculizumab, elotuzumab, enzastumab, epratuzumab, etaracizumab, farletuzumab, figitumumab, flanvotumab, galiximab, gemtuzumab ozogamicin, glembatumumab, ibritumomab tiuxetan, imiglucerase, ipilimumab, labesfamide, lexatumumab, lorvotuzumab mertansine, nimotuzumab, ofatumumab, oregovomab, panitumumab, pemtumomab, pertuzumab, takatuzumab tetraxetan, tositumomab, trastuzumab, tozummab, rituximab, alemtuzumab, durvalumab, ofatumumab, elotuzumab, and zalutumumab.

[0208] Suitable examples of cancer treatment also include immunotherapy. In some embodiments, cancer treatment includes checkpoint inhibitors. In some embodiments, the checkpoint inhibitor is selected from anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-CD20, anti-SLAMF7, and anti-CD52 (e.g., any one of the above anti-cancer antibodies).

[0209] In some embodiments, the present disclosure provides a method of detecting a disease biomarker in a cell, the method comprising: (i) obtaining a cell from a subject; and (ii) examining the cell according to the cell analysis method described herein.

[0210] In some embodiments, the cell is obtained from a subject using image-guided biopsy, fine needle aspiration (FNA), surgical tissue collection, punch biopsy, liquid biopsy, brushing, swabbing, touch-prep, fluid aspiration, or blood analysis. In some embodiments, the cell is obtained from a subject using fine needle aspiration (FNA). In some embodiments, the cell is obtained from a tissue sample such as a paraffin-embedded (FFPE) tissue sample, a fresh tissue sample, or a frozen tissue sample. In some embodiments, the cell is selected from cancer cells, immune system cells, and host cells (the methods of the present disclosure are useful for hepatocyte profiling in liver disease, etc.). In some embodiments, the cell is a cancer cell. In some embodiments, the cancer cell is infected with human papillomavirus (HPV). In some embodiments, the cancer is caused by human papillomavirus (HPV). In some embodiments, the cell sample obtained from the subject or from the subject's tissue is sparse or abundant. In some embodiments, the methods and reagents of the present disclosure are suitable for cell samples and tissue samples containing any amount of cells.

[0211] In some embodiments, the disease or condition (which can be diagnosed, monitored, or its biomarker detected using the methods of the present invention) is cancer. In some embodiments, the methods disclosed herein enable determination of the composition of the intratumoral microenvironment. Suitable examples of cancer include lymphoma, breast cancer, skin cancer, head and neck cancer, head and neck squamous cell carcinoma (HNSCC), and oral cancer. Other examples of cancer include colorectal cancer, gastric (gastrointestinal) cancer, leukemia, melanoma, and pancreatic cancer, hepatocellular carcinoma, ovarian cancer, endometrial cancer, fallopian tube cancer, lung cancer, medullary thyroid cancer, mesothelioma, sex cord-gonadal stromal tumor, adrenocortical carcinoma, synovial sarcoma, bladder cancer, leiomyosarcoma, skeletal muscle sarcoma, endometrial stromal sarcoma, glioma (astrocytoma, ependymoma), rhabdomyosarcoma, small round blue cell tumor, neuroendocrine tumor, small cell carcinoma of the lung, thyroid cancer, esophageal cancer, and gastric cancer. The present technology is useful for any cancer that is detectable by direct visualization, palpation, or image-guided biopsy and is compatible therewith.

[0212] In some embodiments, the cell is an immune cell. In some embodiments, the cell is selected from hematopoietic cells, T cells, B cells, NK cells, myeloid cells, macrophages, dendritic cells, neutrophils, and monocytes.

[0213] The applications of these methods are described in more detail below.

[0214] The linkers, reagents, compounds, and methods of the present disclosure can be used in a point-of-care environment. In developed countries, the accuracy and throughput are hampered by repeated biopsies of much smaller lesions, while in low- and middle-income countries, there is a lack of extremely limited pathology and imaging resources, large case numbers, complex and inefficient workflows, and a shortage of specialists. Advantageously, the compounds and methods described herein enable high-precision analysis of small cancer samples, particularly samples obtained by fine needle aspiration of solid lesions.

[0215] Accordingly, in some embodiments, the present disclosure provides an image cytometer that enables automated cell phenotyping of a small cell sample. Applications of various devices for the methods and compounds of the present application are described below.

[0216] The diagnosis of cell cancer is essential for clinical decision-making, i.e., establishing the correct diagnosis, selecting appropriate treatment, enrolling patients in trials, evaluating treatment efficacy, and / or re-staging the disease. Currently, cancer specimens are generally obtained by image-guided biopsy, fine needle aspiration (FNA), surgical tissue sampling, punch biopsy, brushing, swabbing, touch prep, fluid aspiration, or blood analysis (leukemia, lymphoma, liquid biopsy). Some of these methods (core biopsy and laparotomy for histopathology) provide abundant tissue for sectioning and staining, while the cell material obtained by other methods (FNA, brushing, touch prep for cytopathology) is scarce. FNA can often be obtained with minimal intervention using a small gauge needle (20-25G), has a very low complication rate, and is generally well tolerated.

[0217] The rapid on-site evaluation of cell specimens is becoming increasingly important to shorten the time from intervention to treatment initiation, ensure the quality of specimens for subsequent diagnosis, and minimize degradation and loss of samples during transportation. Current workflows are still labor-intensive and often centralized, requiring large-scale sample processing and specialized cytopathological review. Digital cytopathology and whole slide imaging are being implemented, but these also require significant time, labor, and investment. Collectively, these factors limit throughput, cost, and global deployment capabilities. A particular challenge is reliably analyzing small numbers of cells, either by manual imaging (where a skilled cytopathologist must examine the entire slide) or by automated analysis (incorporating machine learning routines for automated diagnosis).

[0218] The compounds and methods of the present invention can be used in an automated molecular imaging cytometer that uses advanced materials, engineering, and artificial intelligence (AI) for digital cell phenotyping. These novel "integrated" systems address potentially large clinical needs by enabling advanced cell diagnostics suitable for 1) the global health market, which is currently underserved, 2) repeated sample extraction at ultra-low prevalence by using smaller needles (important for repeated sample extraction in clinical trials), 3) reduced time requirements (point-of-care analysis and time savings by not core embedding or staining), 4) better, automated quality control, and 5) automated advancement that reduces both time to diagnosis and variability in interpretation. Further, the compounds and methods of the present invention can be used in low-cost flow cytometers, liquid biopsies focused on cfDNA, exosomes, circulating tumor cells (CTCs), and genomic screening tools (F1CDx, MSK-IMPACT). In some embodiments, the compounds and methods of the present invention are useful for the automated analysis of cell specimens obtained by tumor FNA (Figure 13). In some embodiments, the present disclosure provides high-throughput devices useful for the analysis of samples in centralized laboratories such as CLIA laboratories, in addition to the desktop point-of-care use small automated cytometry systems described herein.

[0219] General cell staining Conventional cytopathology mainly relied on chromogenic dyes such as hematoxylin and eosin (H&E), Papanicolaou (PAP), and Giemsa. Stained specimens are confirmed by cytopathologists who evaluate many parameters of cells, such as the nucleus / cytoplasm ratio, nuclear features, mitosis, clusters, cell uniformity, and cohesiveness. Although such analysis can be automated, it is essentially limited, so the diagnostic accuracy is not constant and molecular information is lacking. Most commercially available cell analyzers (Figure 14) use this approach for automated white blood cell (WBC) analysis rather than cancer detection. Another dye for examining nuclear morphology (aneuploidy, segmentation) includes DAPI, acridine orange, ethidium iodide, propidium iodide, or flavin. Considering the limitations of general chromogenic staining, immunostaining of cancer-related and host cell markers has emerged as an alternative and is widely used in CTC analysis.

[0220] Antibody staining Antibodies are increasingly being used in cytopathology, and it is standard to perform one staining at a time, mainly using immunocytology (measurement of the absorbance of an antibody enzyme-mediated chemical reaction) rather than immunofluorescence (radiometric measurement of a fluorescently labeled antibody). The compounds and methods of the present disclosure enable morphological evaluation of cells (e.g., cancer cells), such as HER2 immunostaining in H&E slides, and at the same time enable detection of important molecular biomarkers (e.g., cancer biomarkers).

[0221] Using multi-channel fluorescence imaging (usually 4-6 channels), more staining can be obtained on a given cell, similar to flow cytometry, although detailed cell morphological information is sacrificed. To further improve the number of channels and markers (>20), cycling techniques have been developed that can repeatedly stain, de-stain, and re-stain cancer tissues, ultimately increasing the number of markers per cell. This enables deeper per-cell profiling, pathway analysis, and immunoprofiling in a small FNA. Most cycling methods were originally developed for paraffin-embedded tissue sections that can withstand harsh de-staining conditions. However, these harsh conditions, which require oxidizing agents for bleaching, often do not suit FNA samples. Additionally, in early cycling techniques, it was not uncommon for sample processing to take several days. Several different cell-compatible cycling techniques have been developed in recent years (Figure 15). The more recent SCANT (single cell analysis for tumor phenotyping) method (Figure 15) has been shown to be robust and useful for pathway analysis in a clinical setting. However, one of the drawbacks of SCANT, like other cycling techniques, was a relatively low SNR and a relatively long de-staining time (0.5-1 hour). The methods and compounds of the present disclosure (e.g., the FAST method) avoid these drawbacks and enable extremely fast cycling (<10 seconds for >95% quenching; Figure 15).

[0222] Selection of Biomarkers Selecting appropriate molecular markers is essential for identifying cells (e.g., cancer cells), differentiating them from host cells, and profiling increasing treatment-related immune cells. Host cell markers have been fully characterized by large-scale flow cytometry tests, while epithelial cancer markers are more diverse and thus require more staining. Furthermore, tumor markers are usually expressed only in some cancer cells and cases. The compounds and methods of the present disclosure enable staining of the following combinations of biomarkers: i) EpCAM, cytokeratin (CK), CD45, and CD16; ii) combinations of multimarkers (the "Quad" marker") including, for example, EGFR, EpCAM, MUC1, and WNT2; iii) HER2, ER / PR in the case of breast cancer; iv) CD19 / 20, k, l, Ki67 in the case of lymphoma; v) EGFR, TTF1, chromogranin, synaptophysin in the case of lung cancer; vi) EpCAM, calretinin, CD45, vimentin (ATCdx), and markers of mutant proteins such as, in particular, KRASG12d, EGFRv3, IDH1132G, and BRAFV600E in the case of ovarian cancer.

[0223] Optimization of Materials for Cell Analysis Newly acquired clinical samples need to be fixed, stained, and captured on glass before they can be analyzed. All of these steps require careful optimization and, in many cases, modification of the materials. Fixation can usually be performed in paraformaldehyde, methanol / propanol, or other commercially available mixes such as CytoRich Red (CRR). The inventors have discovered through experiments that some samples are better preserved with 50% diluted CRR, while the fixation period (ideally 15 - 30 minutes) is less important.

[0224] Immunostaining is best performed by adding antibody reagents to cells in staining buffer in small plastic vials. The stability of antibody fluorochromes, quality control issues, and limitations in the use of basic tools (centrifuges, filters) are significant obstacles to using immunostaining in remote and point-of-care (POC) devices. The use of lyophilized antibodies and “cocktails” containing all necessary components can reduce variability. An alternative method is to stain cells directly on a slide glass after capture. It is also important to capture cells on the slide glass so that they can be reliably placed in the focal plane. Capture can be performed using biological “adhesives” such as dopamine, biotin / neutrAvidin, or polylysine as slide coatings. Alternatively, the slide glass may be coated with capture antibodies. Regardless of the method used, careful validation for various applications is required. Non-specific binding is usually reduced by coating the slide with a blocking material such as BSA or a PEG polymer. To simplify sample handling and processing, commercial systems may adapt cartridges to perform all of the above steps on a single platform.

[0225] Imaging cytometry system For the examination of statistically reliable heterogeneous cell populations, an imaging cytometer needs to visualize a large number of individual cells. However, conventional geometric optical elements are essentially limited by the so-called spatial bandwidth product (SBP), so that megapixel information is generated. To put this into a familiar experience, a typical microscope has either a low resolution with a wide field of view (FOV) or a high spatial resolution with a small FOV, but not both simultaneously.

[0226] Most laboratory imaging systems overcome this limitation by combining high magnification optical elements with a scanning stage to automatically scan slides and subsequently transmit information. Technologies such as whole side imaging (WSI) and digital cell pathology have advanced over the years, but still have challenges. Two major problems in digital cell pathology are i) focusing, and ii) the fact that confirmation by an expert is still required. The focus problem is generally solved by either autofocus hardware / software or 3D imaging of thick z-stacks. Autofocus software often uses either the least squares method or the average value method to identify the ideal focal plane. 3D imaging such as microscopy with optical sectioning requires confocal laser scanning microscopy (CLSM), two-photon (2P) microscopy, structured illumination microscopy (SIM), light sheet fluorescence microscopy (LSFM), or inverted selective plane illumination microscopy (iSPIM). All of these methods require expensive equipment, skilled users, and often generate / create very large datasets. Therefore, this particular approach limits deployment in remote areas with resource constraints.

[0227] Advances in new technologies are making automated molecular image cytometry increasingly possible, which is particularly useful for POC use. Electro-optical elements in which optically encoded images are digitally interpreted can extend the SBP beyond the physical limits of optical elements. Advances in optoelectronics and micro-optics are making it possible to build more compact, easy-to-control yet high-performance systems. Also, using these approaches can reduce the overall cost of the system because optoelectronic components and calculations are inexpensive. Here, the inventors highlight three new modalities that embody these new concepts, namely digital holography, Fouriertychography, and miniaturized fluorescence cytometry.

[0228] Miniaturized fluorescence cytometry As the list of known tumor markers grows, there is also an increasing need for multiplexed cell profiling, motivated mainly by the interest in improving diagnostic accuracy, enabling patient severity determination, and facilitating molecule-based treatment decisions. Conventional immunocytology based on chromogenic staining and brightfield microscopy typically probes only a few markers simultaneously. Fluorescence imaging, especially in combination with cycling techniques, is a powerful approach to in-depth multiplexing. The main technical challenge is to convert bulky and expensive microscopes into compact and inexpensive equivalents for POC applications. Fortunately, recent advances in optoelectronics have made high-quality, small-sized eight optical components available, facilitating new system engineering. For example, small LEDs can supply sufficient power to replace conventional lamps or lasers as excitation light sources, and the sensitivity of semiconductor image sensors has been significantly improved for highly reliable low-light detection. Another opportunity is the extension of manual image curation by automated analysis using machine learning approaches.

[0229] A thumb-sized fluorescence microscope (the "miniscope") integrates optical components into a single device (Figure 16). Using a gradient refractive index (GRIN) objective lens makes it possible to shorten the optical path and dramatically reduce the size of the system (2.4 cm 3 , 1.9 g). Such a small form factor enables the scope to be mounted on an animal's head with minimal interference to natural behavior to image living neuron cells. As a potential POC application, the miniscope has been used for cell profiling and bacterial detection. Furthermore, a miniscope array has been used to image large areas without scanning, taking advantage of the small lateral size of the scope (about 5 mm). System modifications and digital processing have enabled two-photon excitation, volumetric rendering, or lensless imaging.

[0230] For simultaneous multi-color (≥4) cell analysis, a Cytometry Portable Analyzer (CytoPAN) can be used. This system was originally built for operation in remote locations (Figure 17), but has additional uses in POC environments (OR, intervention suite, examination room). The excitation light source is arranged for side illumination through the slide glass, and a single emission filter with four passbands is used. No change of dichroic mirror or mechanical filter was required. Furthermore, intelligent software streamlined the entire assay, including light source calibration, sample slide detection, data acquisition, and cell analysis. CytoPAN had four different fluorescence channels (Figure 18) and brightfield imaging capabilities. An automated algorithm profiled the individual cells analyzed and generated a summary report for cancer diagnosis (Figure 19). This affordable system ($<1,000) into which the compounds and methods of this application are implemented can be operated by unskilled operators.

[0231] Since the above fluorescence system still suffers from the constraints of physical SBP limitations, there is still a trade-off between FOV and spatial resolution. Since fluorescence emission does not carry phase information, the computational methods used in coherent imaging cannot be applied. A direct approach is to combine scanning of the sample with small optical elements. An important technical requirement is to automate such operations, including stage movement and imaging stitching. Equally important is the development of tools for reliable sample preparation, for example, by connecting a fluid cartridge to a cost-effective pump system. This shortens the assay time and minimizes errors, especially in cyclic imaging where it is necessary to repeat the handling of fluids such as quenching, washing, and labeling.

[0232] Conclusion In modern clinical laboratory medicine, virtually all blood and urine tests have been automated to reduce costs, improve test quality, and accommodate the increasing volume of clinical samples. The methods disclosed herein enable the application of automation to FNA analysis of cancer samples, particularly in resource-limited settings. Suitable examples include automated POC cytometry, including the rigorous evaluation of cell markers, staining techniques, and kit development. Automated AI-based diagnostic DNA nuclear measurements are another appropriate application. Also, automated image cytometry, molecular cytology samples, and fluorescence in situ hybridization (FISH) for EGFR, KRAS, and BRAF mutations, as well as other cytogenetic abnormalities, should be feasible using appropriate amplification strategies. Finally, the compounds and methods of the present disclosure provide techniques for analyzing FNA specimens for the diagnosis and monitoring of diseases (e.g., cancer). Inexpensive automated cell analysis and molecular testing can be envisioned for organ FNAs obtained from the liver, kidney, or blood / bone marrow.

[0233] Definitions As used herein, the term "about" means "approximately" (e.g., plus or minus approximately 10% of the indicated value).

[0234] Throughout this specification, substituents of the compounds of the invention are disclosed in groups or ranges. It is specifically contemplated that the invention includes every individual sub-combination of the elements of such groups and ranges. For example, the term "C 1~6 alkyl" is specifically intended to individually disclose methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl.

[0235] Throughout various portions of this specification, various aryl rings, heteroaryl rings, cycloalkyl rings, and heterocycloalkyl rings are described. Unless otherwise specified, these rings can be attached to the remainder of the molecule at any ring member permitted by valence. For example, the term "pyridine ring" or "pyridinyl" may refer to a pyridin-2-yl ring, a pyridin-3-yl ring, or a pyridin-4-yl ring.

[0236] It is further understood that, for clarity, it is possible to provide, in a single embodiment, combinations of certain features of the invention that are described in the context of separate embodiments. Conversely, for brevity, it is also possible to provide the various features of the invention that are described in the context of a single embodiment separately or in any suitable subcombination.

[0237] As used herein, the phrase "optionally substituted" means unsubstituted or substituted. Substituents are independently selected and the substitution can be at any chemically accessible position. As used herein, the term "substituted" means that a hydrogen atom has been removed and replaced by a substituent. A single divalent substituent, for example, oxo, can replace two hydrogen atoms. It is understood that substitution at an atom is limited by valence.

[0238] Throughout this definition, the term "C n~m " indicates a range including the endpoints, where n and m are integers and indicate the number of carbons. Examples include C 1~4 、C 1~6 and the like.

[0239] As used herein, the term "C n~mThe term "alkyl" refers to a saturated hydrocarbon group that can be linear or branched and has n to m carbon atoms. Examples of alkyl moieties include chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, and higher homologues such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, etc., but are not limited thereto. In some embodiments, the alkyl group contains 1 to 6 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms.

[0240] As used herein, the term "C" employed alone or in combination with other terms n~m The term "haloalkyl" refers to an alkyl group having from 1 halogen atom to 2s + 1 halogen atoms, which may be the same or different, where "s" is the number of carbon atoms in the alkyl group, and the alkyl group has n to m carbon atoms. In some embodiments, the haloalkyl group is only fluorinated. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0241] As used herein, the term "C" employed alone or in combination with other terms n~m The term "alkylene" refers to a divalent alkyl linking group having n to m carbon atoms. Examples of alkylene groups include, but are not limited to, ethane-1,1-diyl, ethane-1,2-diyl, propane-1,1,-diyl, propane-1,3-diyl, propane-1,2-diyl, butane-1,4-diyl, butane-1,3-diyl, butane-1,2-diyl, 2-methyl-propane-1,3-diyl, etc. In some embodiments, the alkylene moiety contains 2 to 6, 2 to 4, 2 to 3, 1 to 6, 1 to 4, or 1 to 2 carbon atoms.

[0242] As used herein, the term "carboxy" refers to the -C(O)OH group.

[0243] As used herein, "halo" refers to F, Cl, Br, or I. In some embodiments, halo is F, Cl, or Br. The term "perhalo-" (such as "perfluoro-") refers to a group in which each H atom of the group has been replaced by a halogen.

[0244] As used herein, the term "aryl", employed alone or in combination with other terms, refers to an aromatic hydrocarbon group that can be monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings). The term "C n~m aryl" refers to an aryl group having n to m ring carbon atoms. Examples of aryl groups include phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and the like. In some embodiments, the aryl group has 6 to 10 carbon atoms. In some embodiments, the aryl group is phenyl or naphthyl. The term "arylene" refers to a divalent aryl group such as phenylene. The term "arylene" refers to a divalent aryl group.

[0245] As used herein, the term "compound" is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the depicted structure. A compound of the present specification identified by name or structure as a particular tautomeric form is intended to include other tautomeric forms unless otherwise specified.

[0246] The compounds described herein may be asymmetric (e.g., having one or more stereocenters). Unless otherwise specified, all stereoisomers such as enantiomers and diastereomers are contemplated. Compounds of the invention containing an asymmetrically substituted carbon atom may be isolated in optically active form, or in racemic form. Methods for preparing optically active forms from optically inactive starting materials, such as by resolution of a racemic mixture, or by stereoselective synthesis, are known in the art. Many geometric isomers, such as olefins, C=N double bonds, N=N double bonds, etc., may also be present in the compounds described herein, and all such stable isomers are contemplated in the present invention. The cis and trans geometric isomers of the compounds of the invention are described and may be isolated as mixtures of isomers, or as separated isomeric forms. In some embodiments, the compound has an (R)-configuration. In some embodiments, the compound has an (S)-configuration.

[0247] The compounds provided herein also include tautomeric forms. Tautomeric forms are obtained from the exchange of a single bond with an adjacent double bond, along with simultaneous proton transfer. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Exemplary prototropic tautomers include ketone-enol pairs, amide-imino acid pairs, lactam-lactim pairs, enamine-imine pairs, and cyclic forms, where the proton may occupy two or more positions of a heterocyclic system such as, for example, 1H- and 3H-imidazole, 1H-, 2H-, and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium, or can be sterically confined into one form by appropriate substitution.

[0248] As used herein, the term "cell" is intended to refer to cells that are in vitro, ex vivo or in vivo. In some embodiments, ex vivo cells can be part of a tissue sample removed from an organism such as a mammal. In some embodiments, in vitro cells can be cells in a cell culture. In some embodiments, in vivo cells are cells living in an organism such as a mammal.

[0249] As used herein, the terms "individual", "patient", or "subject", used interchangeably, refer to any animal including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses, or primates, most preferably humans.

[0250] As used herein, the term "treating" or "treatment" refers to: 1) inhibiting a disease, e.g., inhibiting the disease, condition, or disorder in an individual who is experiencing or displaying the pathology or symptoms of the disease, condition, or disorder (i.e., arresting further development of the pathology and / or symptoms), or 2) ameliorating a disease, e.g., ameliorating the disease, condition, or disorder in an individual who is experiencing or displaying the pathology or symptoms of the disease, condition, or disorder (i.e., reversing the pathology and / or symptoms).

Examples

[0251] Example 1 - Preparation of Fluorescent Probe Preparation of BCN-FAST linker compound (1):

Chem.

[0252] Preparation of BCN-FAST linker compound (2):

Chem.

[0253] Preparation of BCN-FAST488 compound (3):

Chem.

[0254] AF488-containing probes containing rTCO, TCO, CP, cTCO, and dTCO were prepared in a similar manner starting from compound 1 by derivatization with the corresponding activated dienophile starting materials (see Figure 1) to prepare the respective analogs of compound 2. Other fluorophore-based probes (such as AF647-based probes) were prepared using the same or similar protocols and commercially available starting materials.

[0255] Example 2 - Kinetics of Probe Stability and Quenching The stability of the fluorescent probes prepared in Example 1 under ambient light and oxygen, and the kinetics and efficiency of their quenching by BHQ3 quencher (including quenching of fluorescence on the cell surface stained with dienophile and fluorophore-labeled antibodies) are shown in Figures 2 - 12A.

[0256] General Method Measurement of FAST-AF488 Probe Stability The stock solution of the FAST-AF488 probe (prepared in Example 1) in DMSO (1 mM) was diluted in PBS to a concentration of 10 nM by adding 0 - 1000 μM of VectaCell Trolox (Vector Laboratories). 5 mL aliquots were added to 15 mL glass vials and left exposed to air under a Philips F32T8 / TL735 700 series 32 watt fluorescent lamp in a standard 6' chemical fume hood or stored in the dark. Next, the solution was transferred to a quartz cuvette for fluorescence quenching measurements. The quenching efficiency was measured using time-based fluorescence acquisition at the appropriate dye-specific wavelength. After measuring the initial fluorescence signal, Tz-BHQ3 (500 nM) was added to the cuvette and the fluorescence signal was measured again when the quenching reaction was complete (Figure 10A). The quenching performance of each probe over time was determined relative to the initial quenching efficiency at t = 0. For measurements under argon, a plastic glove bag was evacuated, filled, and purged with argon gas, after which an open glass vial containing the FAST-AF488 solution was placed inside and left under the same fluorescent lamp. See Figures 4A and 4B.

[0257] Characterization of LCMS degradation The dTCO-AF488 probe was diluted in water to a concentration of 3 μM. Two 5 mL aliquots were added to 15 mL glass vials and left exposed to air under laboratory lighting as described above for 2 hours. The vials were combined, concentrated by rotary evaporation, and redissolved to a concentration of 150 μM for LCMS injection. See Figure 5.

[0258] Modification of antibodies The FAST probe was converted to NHS ester according to the TSTU-ENBA NHS activation method published previously. Cetuximab (2 mg / mL) was buffer-exchanged to PBS-bicarbonate solution (100 mM sodium bicarbonate in PBS, pH 8.4) using a Zeba spin desalting column (40K MWCO), and then incubated with 10 - 20 equivalents of activated NHS-FAST probe at room temperature for 25 minutes. Subsequently, the excess fluorophore was removed into PBS using another Zeba spin desalting column (40K MWCO). The degree of labeling (DOL) of the conjugated antibody was determined by measuring the absorbance spectrum on a Nanodrop 1000 using the appropriate extinction coefficients and correction factors for the antibody and the dye. The conjugated antibody was stored at 4 °C in the dark until use.

[0259] Quenching kinetics of the FAST antibody The CP or BCN FAST-labeled antibody was stored at a concentration of 5 - 15 μM in PBS at 4 °C after labeling. Disposable polystyrene cuvettes were blocked with 1% BSA in 2 mL of PBS, then this was removed and replaced with a 0.01% BSA solution in PBS to reduce non-specific adsorption of the antibody. Time-based fluorescence acquisition was initiated at the appropriate dye-specific wavelength, and the baseline emission of the buffer solution was measured. The FAST-labeled antibody was diluted to a concentration of 4 - 10 nM in the blocked cuvette, and after measuring the initial fluorescence, either 10 - 20 μL of Tz-BHQ3 or Azide-BHQ3 was added via the sample addition port of the instrument, and data acquisition was continued until the quenching reaction was complete. See Figures 7A and 8B.

[0260] Kinetic fitting The data were analyzed using GraphPad Prism 9 (Graphpad Software). For the FAST-labeled antibody, the addition time of BHQ3 was set to t = 0 for fitting purposes, and the curve was fitted to a double-exponential (biphasic) decay. The rate constants are reported in the figure.

[0261] Stopped-flow click kinetics The reaction rate of BCN-PEG2-amine with benzylamino-tetrazine without acceleration was measured with a stopped-flow spectrophotometer as previously published (Carlson, J.C.T., et al., Unraveling Tetrazine-Triggered Bioorthogonal Elimination Enables Chemical Tools for Ultrafast Release and Universal Cleavage. J Am Chem Soc 140, 3603-3612 (2018)). The data were analyzed with GraphPad Prism 9 (Graphpad Software), and the second-order rate constant was calculated from the second-order rate equation (kinetics and mechanism) using a non-linear fit of the absorbance vs. time curve.

[0262] Cell culture A431 cells were purchased from the American Tissue Culture Collection (ATCC). A431 cells were passaged in DMEM (10% FBS, 1% penicillin / streptomycin) according to the specifications from ATCC. Cells were first grown in 150 mm cell culture dishes and subsequently seeded onto Millicell 8-well EZ slides (Millipore) for imaging. After 48 hours, confluency was evaluated, and the cells were fixed with 4% paraformaldehyde in PBS (for 10 minutes) and stored at 4 °C until imaging.

[0263] Immunostaining and quenching Fixed A431 cells were stained with 5 μg / ml of modified antibodies (cetuximab-TCO / rTCO-AF488, cetuximab-BCN / CP-AF488 / AF647) for 15 minutes in the dark at room temperature. For microscope illumination, the cells were exposed to microscope fluorescence light and then quenched. For quenching, 10 μM of Tz-BHQ3 was used in PBS-bicarbonate (pH 9) for different incubation times (1, 2, 4, 8 minutes), followed by three washes to remove free Tz-BHQ3. For Trolox addition, the cells were imaged in PBS with different Trolox concentrations (0, 50, 250 μM), followed by microscope illumination for 120 seconds. After irradiation, the cells were quenched and imaged to quantify the quenching efficiency.

[0264] Fluorescence Imaging and Analysis Fluorescence images were acquired using an Olympus BX-63 upright automatic epifluorescence microscope. FITC and Cy5 filter cubes were used to excite the fluorophores of AF488 and AF647, respectively. ImageJ was used to measure the fluorescence intensity of the cells. The quenching efficiency was calculated by the following formula. Residual MFI = (quenched MFI - background MFI) / (stained MFI - background MFI).

[0265] Numbered Paragraphs In some embodiments, the invention provided herein may be described by reference to the following numbered paragraphs.

[0266] Paragraph 1. A compound of formula (I):

Chemical formula

Chemical formula

[0267] Paragraph 2. R 1 is H, the compound according to Paragraph 1.

[0268] Paragraph 3. n is an integer from 1 to 5, and each L 1 is selected from NH, O, C(=O), and C 1~6 alkylene, the compound according to Paragraph 1 or 2.

[0269] Paragraph 4. m is an integer from 1 to 5, and each L 2 is independently selected from NH, C(=O), C 1~6 alkylene, -(OCH2CH2) x -, and -(CH2CH2O) x -, and the compound according to Paragraph 3.

[0270] Paragraph 5. The compound according to Paragraph 4, wherein m is 5.

[0271] Paragraph 6. The compound according to any one of Paragraphs 1 to 5, wherein x is an integer from 1 to 10.

[0272] Paragraph 7. R 1 is H, n is an integer from 1 to 5, and each L 1 is selected from NH, O, C(=O), and C 1~6 alkylene, m is an integer from 1 to 5, and each L 2 is independently selected from NH, O, C(=O), C 1~6 alkylene, -(OCH2CH2) x -, and -(CH2CH2O) x -, The compound according to Paragraph 1, wherein x is an integer from 2 to 10.

[0273] Paragraph 8. p is an integer from 1 to 5, each L 3 is selected from NR N , O, C(=O), and C 1~6 alkylene, said C 1~6 alkylene is optionally substituted with (L 4 ) o -Y 3 and R N is H, C 1~3 alkyl, and (L 4) o -Y 3 A compound according to any one of paragraphs 1 to 7 selected from

[0274] Paragraph 9. Each L 3 is NH, O, C(=O), and optionally (L 4 ) o -Y 3 substituted C 1~6 An alkylene, a compound according to any one of paragraphs 1 to 8.

[0275] Paragraph 10. Formula:

Chemical formula

[0276] Paragraph 11. Each L 3 is NR N , O, C(=O), and C 1~6 An alkylene selected from R N is H, C 1~3 An alkyl, and (L 4 ) o -Y 3 Selected from, a compound according to any one of paragraphs 1 to 8.

[0277] Paragraph 12. Formula:

Chemical formula

[0278] Paragraph 13. o is an integer from 1 to 5, and each L 4 is independently NH, O, C(=O), and C1~6 A compound according to any one of paragraphs 1 to 12, selected from alkylene.

[0279] Paragraph 14. Y 3 A compound according to any one of paragraphs 1 to 13, wherein Y is the moiety of formula (i).

Chemical formula

[0280] Paragraph 15. Y 3 A compound according to any one of paragraphs 1 to 13, wherein Y is the moiety of formula (ii).

Chemical formula

[0281] Paragraph 16. Each L 3 is NR N , O, C(=O), and C 1~6 A compound according to any one of paragraphs 1 to 8, selected from alkylene.

[0282] Paragraph 17. n is 1, and L 1 is C 1~6 alkylene, m is 4, and each L 2 is independently selected from NH, C(=O), C 1~6 alkylene, -(OCH2CH2) x -, and -(CH2CH2O) x -, p is 3, and each L 3 is independently selected from NH, O, and C(=O), a compound according to paragraph 16.

[0283] Paragraph 18. Formula (I) is the formula:

Chemical formula

Chemical formula

[0284] Paragraph 20. Y 1 is NHR 1A and Y 2 is C(=O)OR a1 and is a compound described in any one of paragraphs 1 to 19, selected from a group consisting of reactive groups with side chains of amino acids of proteins

[0285] Paragraph 21. Y 1 is NH2 Y 2 is C(=O)OH and is a compound described in any one of paragraphs 1 to 19

[0286] Paragraph 22. Y 1 is NHR 1A and R 1 is an amine protecting group Y 2 is C(=O)OH and is a compound described in any one of paragraphs 1 to 19

[0287] Paragraph 23. Y 1 is NHR 1A and R 1 is a fluorophore Y 2 is C(=O)OH and is a compound described in any one of paragraphs 1 to 19

[0288] Paragraph 24. Y 1 is NHR1A and R 1 is a fluorophore, Y 2 is C(=O)OR a1 and R a1 is a carboxylic acid protecting group, a compound according to any one of paragraphs 1 to 19.

[0289] Paragraph 25. Y 1 is NHR 1A and R 1 is a fluorophore, Y 2 is a group reactive with the side chain of an amino acid of a protein, a compound according to any one of paragraphs 1 to 19.

[0290] Paragraph 26. The compound of formula (I) is

Chemical formula

Chemical formula

Chemical formula

[0291] Paragraph 27. A protein conjugate of formula (II):

Chemical formula

[0292] Paragraph 28. The conjugate according to paragraph 27, wherein the protein is selected from an antibody, an antibody fragment, an engineered antibody, a peptide, and an aptamer.

[0293] Paragraph 29. The conjugate according to paragraph 28, wherein the antibody is specific for an antigen that is a biomarker of a disease or condition.

[0294] Paragraph 30. The conjugate according to paragraph 29, wherein the disease or condition is cancer.

[0295] Paragraph 31. The conjugate according to any one of paragraphs 27 to 30, wherein y is an integer from 4 to 6.

[0296] Paragraph 32. Each Y 2 is C(=O), and at least one W is NH of the lysine side chain of the protein A, the conjugate according to any one of paragraphs 27 to 31.

[0297] Paragraph 33. Each Y 2 is C(=O), and at least one W is S of the cysteine side chain of the protein A, the conjugate according to any one of paragraphs 27 to 31.

[0298] Paragraph 34. Y 1 is NHR 1A the conjugate according to any one of paragraphs 27 to 33.

[0299] Paragraph 35. Y 1 is OR 2 the conjugate according to any one of paragraphs 27 to 33.

[0300] Paragraph 36. Y 1 is C(=O)R 3 the conjugate according to any one of paragraphs 27 to 33.

[0301] Paragraph 37. R 1The conjugate according to any one of paragraphs 27 to 36, wherein it is H.

[0302] Paragraph 38. n is an integer from 1 to 5, and each L 1 is selected from NH, O, C(=O), and C 1~6 alkylene, the conjugate according to any one of paragraphs 27 to 37.

[0303] Paragraph 39. m is an integer from 1 to 5, and each L 2 is independently selected from NH, C(=O), C 1~6 alkylene, -(OCH2CH2) x -, and -(CH2CH2O) x -, the conjugate according to paragraph 38.

[0304] Paragraph 40. The conjugate according to paragraph 39, wherein m is 5.

[0305] Paragraph 41. The conjugate according to any one of paragraphs 27 to 40, wherein x is an integer from 1 to 10.

[0306] Paragraph 42. R 1 is H, n is an integer from 1 to 5, and each L 1 is selected from NH, O, C(=O), and C 1~6 alkylene, m is an integer from 1 to 5, and each L 2 is independently selected from NH, O, C(=O), C 1~6 alkylene, -(OCH2CH2) x -, and -(CH2CH2O) x -, The conjugate according to any one of paragraphs 27 to 36, wherein x is an integer from 2 to 10.

[0307] Paragraph 43. p is an integer from 1 to 5, each L 3 is selected from NR N O, C(=O), and C 1~6 alkylene, said C 1~6 alkylene is optionally (L 4 ) o -Y 3 substituted, R N is selected from H, C 1~3 alkyl, and (L 4 ) o -Y 3 and is a conjugate as described in any one of paragraphs 27 to 42.

[0308] Paragraph 44. Each L 3 is selected from NH, O, C(=O), and C 4 ) o alkylene optionally substituted with -Y3 1~6 and is a conjugate as described in any one of paragraphs 27 to 43.

[0309] Paragraph 45. Formula:

Chemical formula

[0310] Paragraph 46. Each L 3 is selected from NR N O, C(=O), and C 1~6 alkylene, R N is selected from H, C 1~3 alkyl, and (L 4 ) o -Y 3 and is a conjugate as described in any one of paragraphs 27 to 43.

[0311] Paragraph 47. Formula:

Chem.

[0312] Paragraph 48. o is an integer from 1 to 5, each L 4 is independently selected from NH, O, C(=O), and C 1~6 alkylene, the conjugate according to any one of Paragraphs 27 to 47.

[0313] Paragraph 49. Y 3 is the moiety of formula (i), the conjugate according to any one of Paragraphs 27 to 48.

Chem.

[0314] Paragraph 50. Y 3 is the moiety of formula (ii), the conjugate according to any one of Paragraphs 27 to 48.

Chem.

[0315] Paragraph 51. Each L 3 is selected from NR N , O, C(=O), and C 1~6 alkylene, the conjugate according to any one of Paragraphs 27 to 43.

[0316] Paragraph 52. n is 1, L 1 is C 1~6 alkylene, m is 4, each L 2 is independently NH, C(=O), C1~6 Alkylene, -(OCH2CH2) x -, and -(CH2CH2O) x selected from p is 3, and each L 3 is independently selected from NH, O, and C(=O), the conjugate according to paragraph 51.

[0317] Paragraph 53. Formula (I) is the formula:

Chemical formula

[0318] Paragraph 54. Formula (I) is the formula:

Chemical formula

[0319] Paragraph 55. A composition comprising the conjugate according to any one of paragraphs 27 to 54, or a pharmaceutically acceptable salt thereof, and an inert carrier.

[0320] Paragraph 56. The composition according to paragraph 55, which is an aqueous solution.

[0321] Paragraph 57. A method for examining cells or cell components, comprising (i) contacting the conjugate according to any one of paragraphs 27 to 55, or a pharmaceutically acceptable salt thereof, containing the fluorophore, or the composition according to paragraph 55 or paragraph 56, with the cells; (ii) imaging the cells using imaging technology; (iii) after (ii), contacting the cells with a compound of formula (III): [Number] contacting with or a pharmaceutically acceptable salt thereof, wherein in the formula, Y 4 is selected from N3 and the moiety of formula (iii), [Chemistry] R 6 is H, C 1~6 alkyl, and C 1~6 haloalkyl, and the C 1~6 alkyl is optionally substituted with OH, NH2, or COOH, each L 4 is independently N(R N ), O, C(=O), S(=O)2, C 1~6 alkylene, C 6~10 arylene, C 6~10 perfluoroarylene, -(OCH2CH2) x -, and -(CH2CH2O) x -, and is selected therefrom, a is an integer from 1 to 10, each R N is selected from H and C 1~3 alkyl, x is an integer from 1 to 2,000, Q is a quencher, and the method includes the contacting, the method wherein the fluorescence of the fluorophore in the conjugate of formula (II) or a pharmaceutically acceptable salt thereof is decreased by the contacting in step (iii).

[0322] Paragraph 58. The method according to Paragraph 57, wherein the imaging technique is fluorescence imaging.

[0323] Paragraph 59. Y 4 is N3, the method according to Paragraph 57 or Paragraph 58.

[0324] Paragraph 60. The compound of formula (III) is of the formula:

Chem.

[0325] Paragraph 61. R 6 is H, the method according to any one of paragraphs 57 to 60.

[0326] Paragraph 62. R 6 is C alkyl optionally substituted with OH, NH2, or COOH 1~6 the method according to any one of paragraphs 57 to 60.

[0327] Paragraph 63. a is an integer from 1 to 7, each L 4 is independently selected from NH, C(=O), C 1~6 alkylene, C 6~10 arylene, C 6~10 perfluoroarylene, and -(CH2CH2O) x -, the method according to any one of paragraphs 57 to 62.

[0328] Paragraph 64. The compound of formula (III) is

Chem.

Chem.

[0329] Paragraph 65. Profiling cells, Examining cells using cytometry techniques, Diagnosing a disease or condition of the subject by examining the pathology of cells obtained from the subject, Monitoring the progression of a disease or condition of the subject by examining the pathology of cells obtained from the subject, and A method selected from detecting disease biomarkers in cells, (i) Obtaining cells from the subject, and (ii) Examining the cells according to the method described in any one of paragraphs 57 to 64, the method.

[0330] Paragraph 66. The cells are obtained from the subject using image-guided biopsy, fine needle aspiration (FNA), surgical tissue sampling, punch biopsy, liquid biopsy, brushing, swab, touch prep, fluid aspiration, or blood analysis, the method described in paragraph 65.

[0331] Paragraph 67. The cytometry technique is selected from image cytometry, holographic cytometry, Fourier tycography cytometry, and fluorescence cytometry, the method described in paragraph 65 or paragraph 66.

[0332] Paragraph 68. The cells are selected from cancer cells, immune system cells, and host cells, the method described in any one of paragraphs 65 to 67.

[0333] Paragraph 69. The disease or condition is cancer, the method described in any one of paragraphs 65 to 68.

[0334] Paragraph 70. The cancer is selected from lymphoma, breast cancer, skin cancer, lymph nodes, head and neck cancer, and oral cancer, the method described in paragraph 69.

[0335] Other embodiments The present application has been described together with its detailed description. However, it should be understood that the above description is illustrative and not intended to limit the scope of the present application, and the scope of the present application is defined by the appended claims. Other aspects, advantages, and modifications are to be included within the scope of the following claims.

Claims

1. A compound of formula (I) 【Chemical 1】 or a pharmaceutically acceptable salt thereof, wherein R １ is selected from H, C １～３ alkyl, and C １～３ haloalkyl, and Each L １ is independently N(R Ｎ ), O, C(=O), S(=O) ２ , C １～６ alkylene, -(OCH ２ CH ２ ), and -(CH ｘ CH ２ CH ２ O) ｘ - and is selected from n is an integer from 1 to 10, Each L ２ is independently N(R Ｎ ), O, C(=O), S(=O) ２ , C １～６ alkylene, -(OCH ２ CH ２ ), and -(CH ｘ CH ２ CH ２ O) ｘ - and is selected from m is an integer from 1 to 10, Each L ３ is independently N(R Ｎ ), O, C(=O), S(=O) ２ , C １～６ alkylene, -(OCH ２ CH ２ ), and -(CH ｘ CH ２ CH ２ O) ｘ - and is selected from L １ 、L ２ 、 or L ３ each of the C １～６ alkylene groups within is optionally (L ４ ), ｏ -Y ３ and is substituted with Each R Ｎ is independently selected from H, C １～３ alkyl, C １～３ haloalkyl, and (L ４ ) ｏ -Y ３ and is selected from Each L ４ is independently N(R Ｎ１ ), O, C(=O), S(=O) ２ , C １～６ alkylene, -(OCH ２ CH ２ ), and -(CH ｘ CH ２ CH ２ O) ｘ - and is selected from Each R Ｎ１ is independently selected from H, C １～３ alkyl, and C １～３ haloalkyl, p is an integer from 1 to 20, o is an integer from 1 to 10, each x is independently an integer from 1 to 2,000, Each Y ３ is independently selected from the part of formula (i) and the part of formula (ii), 【Chemical 2】 Y １ is NR ｃ１ R １Ａ , OR ２ and C(=O)R ３ selected from, R １Ａ is selected from H, an amine protecting group, and a fluorophore, R ２ is selected from H, an alcohol protecting group, and a fluorophore, R ３ is selected from OR ａ１ and a fluorophore, Y ２ is C(=O)OR ａ１ NR ｃ１ R ４ OR ５ and is selected from reactive groups with the side chains of the amino acids of the protein, R ａ１ is selected from H and a carboxylic acid protecting group, R ｃ１ is selected from H, C １～３ alkyl, and C １～３ haloalkyl, and R ４ is selected from H and an amine protecting group, R ５ is selected from H and an alcohol protecting group, said compound, or a pharmaceutically acceptable salt thereof.

2. R １ The compound according to claim 1, wherein R is H.

3. n is an integer from 1 to 5, and each L １ is selected from NH, O, C(=O), and C １～６ alkylene, the compound according to claim 1.

4. m is an integer of 1 to 5, and each L ２ is independently NH, C(=O), C １～６ alkylene, -(OCH ２ CH ２ ) ｘ -, and -(CH ２ CH ２ O) ｘ -, and the compound according to claim 1, which is selected from

5. R １ is H, n is an integer from 1 to 5, and each L １ is selected from NH, O, C(=O), and C １～６ alkylene, m is an integer from 1 to 5, and each L ２ is independently selected from NH, O, C(=O), C １～６ alkylene, -(OCH ２ CH ２ ), and -(CH ｘ CH ２ CH ２ O) ｘ -, and is selected from The compound according to claim 1, wherein x is an integer from 2 to 10.

6. p is an integer from 1 to 5, Each L ３ is selected from NR Ｎ , O, C(=O), and C １～６ and alkylene said C １～６ wherein the alkylene is optionally (L ４ ), ｏ -Y ３ substituted with R Ｎ is H, C １～３ alkyl, and (L ４ ) ｏ -Y ３ The compound according to claim 1, selected from

7. Each L ３ is NH, O, C(=O), and optionally (L ４ ｏ -Y ３ substituted C １～６ alkylene selected from, the compound according to claim 1.​

8. Formula: [Chemical Formula 3] The compound according to claim 7, having the formula or a pharmaceutically acceptable salt thereof, wherein the sum of p1 and p2 is at least 1 less than p.

9. Each L ３ is NR Ｎ , O, C(=O), and C １～６ selected from alkylene, R Ｎ is H, C １～３ alkyl, and (L ４ ) ｏ -Y ３ The compound according to claim 1, selected from

10. Formula: [Chemical Formula 4] The compound according to claim 9, having the formula or a pharmaceutically acceptable salt thereof, wherein the sum of p1 and p2 is at least 1 less than p.

11. o is an integer from 1 to 5, and each L ４ is independently selected from NH, O, C(=O), and C １～６ alkylene, the compound according to claim 1.

12. Y ３ The compound according to claim 11, wherein Y is the moiety of formula (i). [Chemical Formula 5]

13. Y ３ The compound according to claim 11, wherein Y is the moiety of formula (ii). 【Chemical Formula 6】

14. Each L ３ is NR Ｎ , O, C(=O), and C １～６ The compound according to claim 1, selected from alkylene.

15. n is 1 and L １ is C １～６ is alkylene, m is 4, and each L ２ is independently NH, C(=O), C １～６ alkylene, -(OCH ２ CH ２ ) ｘ -, and -(CH ２ CH ２ O) ｘ -, and is selected from p is 3 and each L ３ is independently selected from NH, O, and C(=O), the compound according to claim 14.

16. The compound according to claim 15, wherein formula (I) is the formula: 【Chemical Formula 7】 or a pharmaceutically acceptable salt thereof.

17. The compound according to claim 15, wherein formula (I) is the formula: 【Chemical 8】 or a pharmaceutically acceptable salt thereof.

18. Y １ is NHR １Ａ and Y ２ is C(=O)OR ａ１ and a group reactive with a side chain of an amino acid of a protein, the compound according to claim 1.

19. Y １ is NH ２ and Y ２ The compound according to claim 1, wherein Y is C(=O)OH.

20. Y １ is NHR １Ａ and R １ is an amine protecting group, Y ２ The compound according to claim 1, wherein Y is C(=O)OH.

21. Y １ is NHR １Ａ and R １ is a fluorophore, Y ２ The compound according to claim 1, wherein Y is C(=O)OH.

22. Y １ is NHR １Ａ and R １ is a fluorophore, Y ２ is C(=O)OR ａ１ and R ａ１ The compound according to claim 1, wherein R is a carboxylic acid protecting group.

23. Y １ is NHR １Ａ and R １ is a fluorophore, Y ２ The compound according to claim 1, wherein Y is a reactive group with the side chain of the amino acid of the protein.

24. The compound of formula (I) is selected from any one of the following compounds: 【Chemical Formula 9-1】 【Chemical Formula 9-2】 【Chemical Formula 9-3】 or a pharmaceutically acceptable salt thereof, the compound according to claim 1.

25. A protein conjugate of formula (II): 【Chemical 10】 or a pharmaceutically acceptable salt thereof, wherein A is a protein, y is an integer from 1 to 10, R １ is selected from H, C １～３ alkyl, and C １～３ haloalkyl, Each L １ is independently N(R Ｎ ), O, C(=O), S(=O) ２ , C １～６ alkylene, -(OCH ２ CH ２ ), and -(CH ｘ CH ２ CH ２ O) ｘ -selected from n is an integer from 1 to 10, Each L ２ is independently N(R Ｎ ), O, C(=O), S(=O) ２ , C １～６ alkylene, -(OCH ２ CH ２ ), and -(CH ｘ CH ２ CH ２ O) ｘ - selected from, m is an integer from 1 to 10, Each L ３ is independently N(R Ｎ ), O, C(=O), S(=O) ２ , C １～６ alkylene, -(OCH ２ CH ２ ), and -(CH ｘ CH ２ CH ２ O) ｘ - and is selected from L １ 、L ２ 、 or L ３ each of the C １～６ alkylene groups within is optionally (L ４ ｏ -Y ３ substituted and​ Each R Ｎ is independently selected from H, C １～３ alkyl, C １～３ haloalkyl, and (L ４ ) ｏ -Y ３ and is selected from Each L ４ is independently N(R Ｎ１ ), O, C(=O), S(=O) ２ , C １～６ alkylene, -(OCH ２ CH ２ ), and -(CH ｘ CH ２ CH ２ O) ｘ - and is selected from Each R Ｎ１ is independently selected from H, C １～３ alkyl, and C １～３ haloalkyl, p is an integer from 1 to 20, o is an integer from 1 to 10, each x is independently an integer from 1 to 2,000, Each Y ３ is independently selected from the part of formula (i) and the part of formula (ii), 【Chemical 11】 Y １ is NR ｃ１ R １Ａ , OR ２ and C(=O)R ３ selected from R １Ａ , R ２ , and R ３ are each independently selected from fluorophores, R ｃ１ is selected from H, C １～３ alkyl, and C １～３ haloalkyl, and each W is (i) O of the side chain of serine, threonine, or tyrosine of said protein A, (ii) S of the side chain of cysteine of said protein A, (iii) NH of the side chain of lysine of said protein A, and (iv) C(=O) of the side chain of aspartic acid or glutamic acid of said protein A, Y ２ is a residue of a group that was a group reactive with a side chain of an amino acid of the protein A before conjugation with the protein A, said protein conjugate, or a pharmaceutically acceptable salt thereof.

26. A composition comprising the conjugate according to claim 25, or a pharmaceutically acceptable salt thereof, and an inert carrier.

27. A method for examining a cell or a component of a cell, comprising: (i) contacting the conjugate according to claim 25 containing the fluorophore, or a pharmaceutically acceptable salt thereof, or the composition according to claim 26 with the cell; (ii) imaging the cell using an imaging technique; (iii) after (ii), contacting the cell with a compound of formula (III): 【Number 1】 or a pharmaceutically acceptable salt thereof, wherein Y ４ is selected from N ３ and the moiety of formula (iii), 【Number】 R ６ is selected from H, C １～６ alkyl, and C １～６ haloalkyl, and said C １～６ alkyl is optionally substituted with OH, NH ２ , or COOH, Each L ４ is independently N(R Ｎ ), O, C(=O), S(=O) ２ , C １～６ alkylene, C ６～１０ arylene, C ６～１０ perfluoroarylene, -(OCH ２ CH ２ ), and -(CH ｘ CH ２ O) ２ -, and is selected from ｘ -. a is an integer from 1 to 10; Each R Ｎ is selected from H and C １～３ alkyl, x is an integer from 1 to 2,000; Q is a quencher, said contacting; and by the contacting in step (iii), the fluorescence of the fluorophore in the conjugate of formula (II) or a pharmaceutically acceptable salt thereof is decreased; said method.

28. The method according to claim 27, wherein the compound of formula (III) has the formula: 【Chemical 13】 or a pharmaceutically acceptable salt thereof.

29. The method according to claim 27, wherein the compound of formula (III) is selected from any one of the following compounds: 【Chemical Formula 14-1】 【Chemical Formula 14-2】 or a pharmaceutically acceptable salt thereof.

30. A method selected from profiling a cell, examining a cell using cytometry techniques, diagnosing a disease or condition of the subject by examining the pathology of a cell obtained from the subject, monitoring the progression of a disease or condition of the subject by examining the pathology of a cell obtained from the subject, and detecting an intracellular disease biomarker, said method comprising (i) obtaining a cell from the subject, and (ii) examining the cell according to the method according to claim 27. said method.

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