Methods of assessing small molecule distribution using tellurophen analogs

Abstract

The present disclosure relates to tellurophen analogs of small molecules, and methods of detecting small molecules using the tellurophen analogs thereof. The present disclosure also relates to compositions comprising the tellurophen analogs of the present disclosure and kits comprising the tellurophen analogs of the present disclosure. The present disclosure also relates to methods of determining the dose of small molecules.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 335,926, filed on April 28, 2022, entitled “Method of Evaluating Small Molecule Distribution Using Tellurophene Analogues,” which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to tellurophene containing small molecule analogs, and methods for detecting small molecules in samples obtained from subjects or cell / tissue cultures using tellurophene as reporters. The present disclosure also relates to a tellurophene teniposide analog and a tellurophene carfilzomib analog. The present disclosure also includes the use of the analogs of the present disclosure in assessing the distribution of small molecules. In addition, the present disclosure relates to methods for determining the dosage of small molecules. Background Art

[0004] The in vivo biodistribution of exogenously administered small molecule therapeutics is rarely uniform. However, target engagement at the desired location is key to translating promising in vitro results into in vivo responses. Currently, methods for monitoring biodistribution and target engagement focus primarily on assays performed in large numbers, and few methods provide cellular resolution, especially in whole organism models. Early methods for monitoring the cellular biodistribution of small reversible inhibitors used microautoradiography, but this approach has not been widely adopted due to the high specific activities required, long exposure times, and technical challenges. While advances in fluorescence-based tissue histology techniques have enabled visualization and quantification of target engagement in processed tissue samples, these assays are limited in their ability to simultaneously identify and characterize phenotypes associated with the cell types present. Due to the inherent challenges of multiplexed fluorescence-based microscopy, combining biodistribution with cellular characterization is best suited for IMAGING MASS CYTOMETRY TM (IMC TM ).

[0005] IMC TM For A high-dimensional tissue imaging platform based on mass spectrometry (MC) technology and designed for epitope measurement on tissue sections. An earlier version of the platform was introduced by the Bodenmiller group in 2014, which used a panel of 32 antibodies to examine potential tumor heterogeneity in human breast cancer samples. More recent examples have explored more than 40 markers that can be identified using current IMC TMThe reagents and workflows quantify at a subcellular resolution, thus enabling in-depth analysis of individual cells in their native microenvironment. TM The spatially resolved information obtained can be analyzed to study cellular phenotypes associated with spatial organization. In addition, using libraries of appropriate probes, temporal changes in tissues can be followed, as is done under hypoxia. Overlaying this information with small molecule therapeutic biodistribution will facilitate the design of dosing regimens and help understand the perturbed cellular biochemistry.

[0006] Despite this progress, the IMC TM The potential of IMC in drug discovery and drug development remains largely untapped. TM To track the localization of drugs in tissues, molecules must carry heavy isotopes (>80 amu) that are compatible with mass spectrometry flow cytometry. TM The ability to detect platinum isotopes generated by dosing chemotherapeutic cisplatin enabled visualization of biodistribution in xenografts derived from pancreatic cancer patients. This landmark study revealed unexpected drug distribution, as unexpectedly extensive binding of platinum to collagen fibers in both tumor stroma and normal tissue was observed. These findings provided insight into the long clearance time of cisplatin and some observations regarding the long-term toxicity of therapy. While this work demonstrated that IMC TM The applicability of cisplatin localization has been tracked, but few therapeutic agents possess MC-compatible elements that enable their detection.

[0007] Therefore, it is necessary to develop a method to apply mass cytometry to general small molecules without the need for MC-compatible elements. Summary of the invention

[0008] Any embodiment disclosed herein may be used in combination with one or more other embodiments.

[0009] The use of IMC to visualize small molecule tellurium analogs has been demonstrated. TM It is possible to detect bioactive small molecules other than metallodrugs. It has also been demonstrated that analogs can be obtained by replacing five- or six-membered aromatic rings with tellurophene isosteric. Tellurophene is a MC and IMC TM Stable, biocompatible mass tags are designed to be used for the production of MCs. For example, this bioisostere design strategy has been successfully implemented to develop tellurofen-teniposide analogs and tellurofen-carfilzomib analogs. The exemplary analogs retain similar biological activities compared to the original small molecules and can be detected and quantified by MC.

[0010] Thus, in one aspect, the present disclosure includes a method of detecting a small molecule in a sample, the method comprising:

[0011] providing a sample obtained from a subject or cell / tissue culture to which a tellurphene small molecule analog comprising tellurphene has been administered, the tellurphene comprising a tellurium atom; and

[0012] The samples were subjected to mass spectrometry to determine the levels of tellurium atoms;

[0013] wherein the small molecule has a structure comprising one or more monocyclic or bicyclic aromatic rings, and the analog has a structure in which at least one of the one or more aromatic rings of the structure of the small molecule is replaced by tellurophene, and

[0014] Therein the level of tellurium atoms corresponds to the level of the analog, and detection of the analog indicates detection of the small molecule in the sample.

[0015] In another aspect, the present disclosure includes the use of tellurphene analogs for detecting small molecules by mass spectrometry in a subject to which the analogs have been administered or in a cell / tissue culture species to which the analogs have been administered, wherein the small molecule has a structure comprising one or more monocyclic or bicyclic aromatic rings, and the analog has a structure in which at least one of the one or more monocyclic or bicyclic aromatic rings of the structure of the small molecule is replaced by tellurphene.

[0016] In another aspect, the present disclosure includes a compound of Formula I:

[0017]

[0018] or a salt or solvate thereof.

[0019] In another aspect, the present disclosure includes a compound of Formula II:

[0020]

[0021] or a salt or solvate thereof.

[0022] In another aspect, the present disclosure includes a composition comprising a compound of the present disclosure, and a carrier or excipient.

[0023] In another aspect, the present disclosure includes the use of a compound of Formula I or a composition comprising a compound of Formula I of the present disclosure for detecting teniposide by mass spectrometry in a subject to which a compound of Formula I or a salt or solvate thereof has been administered, or in a cell / tissue culture to which a compound of Formula I or a salt or solvate thereof has been administered.

[0024] In another aspect, the present disclosure includes the use of Formula II or a composition comprising a compound of Formula II of the present disclosure for detecting carfilzomib by mass spectrometry in a subject to which a compound of Formula I or a salt or solvate thereof has been administered, or in a cell / tissue culture to which a compound of Formula II or a salt or solvate thereof has been administered.

[0025] In another aspect, the present disclosure includes a kit for mass spectrometry flow cytometry analysis, comprising a teniposide tellurophene analog of Formula I:

[0026]

[0027] or a salt or solvate thereof, and

[0028] One or more tellurium standards.

[0029] In another aspect, the present disclosure includes a kit for mass spectrometry analysis, optionally mass cytometry analysis, comprising a carfilzomib tellurophene analog of Formula II:

[0030]

[0031] or a salt or solvate thereof, and

[0032] One or more tellurium standards.

[0033] In another aspect, the disclosure includes a method of achieving a dosage of a small molecule for desired target engagement of the small molecule in a subject or cell / tissue culture, wherein the small molecule engages a target in the subject or cell / tissue culture and produces a measurable effect at the target, the method comprising

[0034] detecting a small molecule in a subject by the methods of the present disclosure;

[0035] Measuring the effects of small molecules; and

[0036] A dosage of the tellurophene analog is determined that is appropriate to achieve the desired distribution of small molecules, wherein the dosage of the tellurophene analog is indicative of the dosage of the small molecules.

[0037] In one aspect, the present invention includes a method for detecting a small molecule compound in a sample, the method comprising the following steps: providing a subject or a cell / tissue culture sample; providing a small molecule analog of the small molecule compound, wherein the small molecule compound has a structure comprising one or more monocyclic or bicyclic aromatic rings, and the small molecule analog has a structure in which at least one of the one or more monocyclic or bicyclic aromatic rings of the structure of the small molecule compound is replaced by tellurophene comprising a tellurium atom, thereby forming a tellurophene small molecule analog; administering the tellurophene small molecule analog to the subject or cell / tissue culture sample; providing a sample obtained from the subject or cell / tissue culture sample after administering the tellurophene small molecule analog; performing mass spectrometry on the sample to determine the level of tellurium atoms present in the sample, wherein the level of tellurium atoms corresponds to the level of the tellurophene small molecule analog, and detection of the tellurophene small molecule analog indicates detection of the small molecule compound in the sample.

[0038] In various embodiments, such as the above embodiments, the method further comprises quantifying the amount of tellurium atoms in the sample, wherein the level of tellurium atoms indicates the number of small molecule compounds in the sample. In various embodiments, such as the above embodiments, the method further comprises quantifying one or more other analytes in the sample, the method further comprises labeling the sample with one or more analyte binders with mass labels before performing mass spectrometry, and determining the level of one or more analyte binders. In various embodiments, such as the above embodiments, one or more analyte binders with mass labels are selected from the group consisting of: metal-labeled antibodies, optionally polymer-labeled antibodies; metal-labeled oligonucleotides; polymer-labeled oligonucleotides; intercalators, such as 5-iodo-2'-deoxyuridine (IdU) and metal-containing intercalators (e.g., Rh-containing intercalators and Ir-containing intercalators); metal-containing activity indicators, such as cisplatin; barcode reagents, such as Cd-labeled CD45 and Pt-labeled CD45; and combinations thereof. In various embodiments, such as the above embodiments, the subject is a mammal, optionally a mouse, rat or human.

[0039] In various embodiments, such as the above embodiments, mass spectrometry is performed at multiple discrete locations, and the level of tellurium atoms is determined at each of the multiple discrete locations to provide a distribution of small molecule analogs in the sample, and wherein the distribution of analogs in the sample indicates the distribution of small molecule compounds in the sample. In various embodiments, such as the above embodiments, the sample is a single cell, and the distribution of small molecule analogs in a single cell indicates the distribution of small molecule compounds at the subcellular level. In various embodiments, such as the above embodiments, multiple samples are provided, and the sample includes different tissues, cells or secretions of a subject, and wherein the detection of small molecule analogs in multiple samples provides the distribution of small molecule analogs in the subject, and the distribution indicates the distribution of small molecule compounds in the subject, such as tissue or organ distribution. In various embodiments, such as the above embodiments, the sample is or contains urine, feces, blood or fractions thereof, cerebrospinal fluid (CSF), saliva, muscle cells, fat cells, bone cells, hair, nails, skin cells, tumor cells or secretions, liver cells or secretions, heart cells, lung cells or secretions, pancreatic cells or secretions and / or gastric cells or secretions. In various embodiments, such as those discussed above, the sample is a frozen tissue section. In various embodiments, such as those discussed above, the sample is or comprises a cell from a cell culture or a medium from a cell culture. In various embodiments, such as those discussed above, the sample is a single cell, and the distribution of analogs within a single cell is indicative of the distribution of the small molecule compound at the subcellular level. In various embodiments, such as those discussed above, the mass spectrometry is mass cytometry or multiplexed ion beam imaging, and optionally the mass cytometry is mass cytometry imaging or mass cytometry suspension. In various embodiments, such as those discussed above, the tellurium atom is selected from 120 Te, 122 Te, 123 Te, 124 Te, 125 Te, 126 Te, 128 Te, 130 Te and combinations thereof. In various embodiments, such as the embodiments discussed above, the tellurium atom comprises a plurality of tellurium isotopes, and the mass cytometry is a multi-channel mass spectrometry, optionally a multi-channel mass cytometry. In various embodiments, such as the embodiments discussed above, one or more monocyclic aromatic rings are 5- or 6-membered aromatic rings, optionally one or more monocyclic 5- or 6-membered aromatic rings are independently selected from thiophene, furan, pyrrole, phenyl or pyridine. In various embodiments, such as the embodiments discussed above, one or more bicyclic aromatic rings are independently selected from naphthyl, indole, benzothiophene or benzofuran.

[0040] In various embodiments, such as the embodiments discussed above, the structure of the small molecule compound comprises one or more monocyclic 5 or 6-membered aromatic rings, and at least one of the monocyclic 5 or 6-membered aromatic rings is replaced by tellurophene. In various embodiments, such as the embodiments discussed above, the structure of the small molecule compound comprises one or more bicyclic aromatic rings, and at least one of the bicyclic aromatic rings is replaced by benzo[b]tellurophene or 4H-telluropheno[3,2-b]pyrrole. In various embodiments, such as the embodiments discussed above, the structure of the small molecule compound comprises one or more 5-membered aromatic rings, optionally one or more 5-membered aromatic rings are each independently selected from thiophene, furan or pyrrole. In various embodiments, such as the embodiments discussed above, one or more 5-membered aromatic rings are thiophene. In various embodiments, such as the embodiments discussed above, the small molecule compound is teniposide, and the tellurophene small molecule analog is as shown in Formula I:

[0041]

[0042] or a salt or solvate thereof.

[0043] In various embodiments, such as those discussed above, the structure of the small molecule compound comprises one or more 6-membered aromatic rings, optionally one or more 6-membered aromatic rings are each independently selected from phenyl or pyridine, optionally one or more 6-membered aromatic rings are phenyl. In various embodiments, such as those discussed above, the small molecule compound is carfilzomib, and the tellurophene small molecule analog is as shown in Formula II:

[0044]

[0045] or a salt or solvate thereof.

[0046] In various embodiments, such as those discussed above, the small molecule analog interacts irreversibly, optionally covalently, with a target in a sample. In various embodiments, such as those discussed above, a subject or cell / tissue culture sample has been administered a combination of a tellurophene small molecule analog and a small molecule compound, wherein the method further comprises directly detecting the level of the small molecule compound, and wherein a comparison of the level of the small molecule compound to the level of the analog indicates putative competitive binding of the analog.

[0047] In another aspect, the present invention includes a use of a tellurophene small molecule analog in a method for detecting a small molecule compound in a sample, the method comprising the following steps: providing a subject or a cell / tissue culture sample; providing a small molecule analog of a small molecule compound, wherein the small molecule compound has a structure comprising one or more monocyclic or bicyclic aromatic rings, and the small molecule analog has a structure in which at least one of the one or more aromatic rings of the structure of the small molecule compound is replaced by tellurophene containing a tellurium atom, thereby forming a tellurophene small molecule analog; administering the tellurophene small molecule analog to the subject or cell / tissue culture sample; providing a sample obtained from the subject or cell / tissue culture sample after administration of the tellurophene small molecule analog; performing mass spectrometry on the sample to determine the level of tellurium atoms present in the tellurophene small molecule analog. In various embodiments, such as those discussed above, mass spectrometry is performed on a sample, optionally multiple samples, obtained from a subject or cell / tissue culture sample. In various embodiments, such as those discussed above, the subject is a mammal, optionally a mouse, a rat, or a human. In various embodiments, such as the above-mentioned embodiments, the sample is or comprises urine, feces, blood or its fraction, cerebrospinal fluid (CSF), saliva, muscle cells, fat cells, bone cells, hair, nails, skin cells, tumor cells or secretions, liver cells or secretions, heart cells, lung cells or secretions, pancreatic cells or secretions and / or gastric cells or secretions. In various embodiments, such as the embodiments discussed above, the sample is a frozen tissue section. In various embodiments, such as the embodiments discussed above, the sample is or comprises cells from cell culture or culture medium from cell culture. In various embodiments, such as the embodiments discussed above, mass spectrometry is mass spectrometry flow cytometry or multiple ion beam imaging, and optionally mass spectrometry flow cytometry is mass spectrometry flow cytometry imaging or mass spectrometry flow cytometry suspension.

[0048] In various embodiments, such as the embodiments discussed above, one or more aromatic rings are 5 or 6-membered aromatic rings, and optionally one or more monocyclic 5 or 6-membered aromatic rings are independently selected from thiophene, furan, pyrrole, phenyl or pyridine. In various embodiments, such as the embodiments discussed above, one or more bicyclic aromatic rings are independently selected from naphthyl, indole, benzothiophene or benzofuran. In various embodiments, such as the embodiments discussed above, the structure of the small molecule compound comprises one or more 5-membered aromatic rings, and optionally one or more 5-membered aromatic rings are each independently selected from thiophene, furan or pyrrole. In various embodiments, such as the embodiments discussed above, one or more 5-membered aromatic rings are thiophene. In various embodiments, such as the embodiments discussed above, the small molecule compound is teniposide, and the tellurophene small molecule analog is as shown in Formula I:

[0049]

[0050] or a salt or solvate thereof.

[0051] In various embodiments, such as those discussed above, the structure of the small molecule compound comprises one or more 6-membered aromatic rings, optionally one or more 6-membered aromatic rings are each independently selected from phenyl or pyridine, optionally one or more 6-membered aromatic rings are phenyl. In various embodiments, such as those discussed above, the small molecule compound is carfilzomib, and the tellurophene small molecule analog is as shown in Formula II:

[0052]

[0053] or a salt or solvate thereof.

[0054] In another aspect, the present disclosure relates to a compound of formula I:

[0055]

[0056] or a salt or solvate thereof.

[0057] In various embodiments, such as those discussed above, the Te atom is an isotope, optionally the Te atom is selected from 120 Te, 122 Te, 123 Te, 124 Te, 125 Te, 126 Te, 128 Te, 130 Te and combinations thereof. In another aspect, the composition comprises a small molecule compound carfilzomib and a tellurophene small molecule analogue as shown in Formula II:

[0058]

[0059] or a salt or solvate thereof, and a carrier or excipient.

[0060] In another aspect, the composition comprises a compound of formula I:

[0061]

[0062] or a salt or solvate thereof, and a carrier or excipient.

[0063] In various embodiments, such as those discussed above, the Te atoms are selected from 120 Te, 122 Te, 123 Te, 124 Te, 125 Te, 126 Te, 128 Te,130 Te and combinations thereof. In another aspect, the present disclosure relates to a compound of formula II:

[0064]

[0065]

[0066] or a salt or solvate thereof.

[0067] In various embodiments, such as those discussed above, the Te atoms are isotopically enriched, optionally the Te atoms are selected from 120 Te, 122 Te, 123 Te, 124 Te, 125 Te, 126 Te, 128 Te, 130 Te and combinations thereof. In another aspect, the composition comprises a compound of formula I:

[0068]

[0069] or a salt or solvate thereof, and a carrier or excipient.

[0070] In another aspect, the composition comprises a compound of formula II:

[0071]

[0072] or a salt or solvate thereof, and a carrier or excipient.

[0073] In another aspect, the present disclosure relates to the use of a teniposide analog in a method for detecting teniposide by mass spectrometry, the method comprising the steps of: providing a teniposide analog, wherein the teniposide analog is a compound of formula I:

[0074]

[0075] or a salt or solvate thereof, optionally wherein the Te atom is an isotope, optionally the Te atom is selected from 120 Te, 122 Te, 123 Te, 124 Te, 125 Te, 126 Te, 128 Te, 130Te and combinations thereof, optionally in a composition comprising at least one carrier or excipient; providing a subject or a cell / tissue culture sample; administering a teniposide analog to the subject or the cell / tissue culture sample; providing a sample taken from the subject or the cell / tissue culture sample after administration of the teniposide analog; performing mass spectrometry on the sample to determine the level of tellurium atoms present in the sample, wherein the level of tellurium atoms corresponds to the level of the teniposide analog, and the detection of the teniposide analog indicates the detection of teniposide in the sample.

[0076] In another aspect, the present disclosure relates to the use of a carfilzomib analog in a method for detecting carfilzomib by mass spectrometry, the method comprising the steps of: providing a carfilzomib analog, wherein the carfilzomib analog is a compound of formula II:

[0077]

[0078] or a salt or solvate thereof, optionally wherein the Te atom is isotopically enriched, optionally the Te atom is selected from 120 Te, 122 Te, 123 Te, 124 Te, 125 Te, 126 Te, 128 Te, 130 Te and combinations thereof, optionally in a composition comprising at least one carrier or excipient; providing a subject or a cell / tissue culture sample; administering a carfilzomib analog to the subject or the cell / tissue culture sample; providing a sample taken from the subject or the cell / tissue culture sample after administration of the carfilzomib analog; performing mass spectrometry on the sample to determine the level of tellurium atoms present in the sample, wherein the level of tellurium atoms corresponds to the level of the carfilzomib analog, and the detection of the carfilzomib small molecule analog indicates the detection of carfilzomib in the sample.

[0079] In various embodiments, such as the above-mentioned embodiments, mass spectrometry is carried out on a sample obtained from a subject or a cell culture, optionally a plurality of samples. In various embodiments, such as the embodiments discussed above, the subject is a mammal, optionally a mouse, a rat or a human. In various embodiments, such as the above-mentioned embodiments, the sample is or comprises urine, feces, blood or its fraction, cerebrospinal fluid (CSF), saliva, muscle cells, fat cells, bone cells, hair, nails, skin cells, tumor cells or secretions, liver cells or secretions, heart cells, lung cells or secretions, pancreatic cells or secretions and / or gastric cells or secretions. In various embodiments, such as the above-mentioned embodiments, the sample is a frozen tissue section. In various embodiments, such as the above-mentioned embodiments, the sample is or comprises a cell from a cell culture or a culture medium from a cell culture. In various embodiments, such as the above-mentioned embodiments, mass spectrometry is mass spectrometry flow cytometry or multiple ion beam imaging, and optionally mass spectrometry flow cytometry is mass spectrometry flow cytometry imaging or mass spectrometry flow cytometry suspension. In various embodiments, such as those described above, the tellurium atom comprises multiple isotopes and the mass spectrometry is multi-channel mass spectrometry, optionally multi-channel mass cytometry.

[0080] In another aspect, a kit for mass spectrometry flow cytometry analysis comprises a teniposide tellurophene analog of Formula I:

[0081]

[0082] or a salt or solvate thereof and one or more tellurium standards.

[0083] In another aspect, a kit for mass spectrometry analysis, optionally mass cytometry analysis, comprises a carfilzomib tellurophene analog of Formula II:

[0084]

[0085] or a salt or solvate thereof and one or more tellurium standards.

[0086] In various embodiments, such as the above embodiments, the tellurium in the analog and the tellurium in the standard are selected from 120 Te, 122 Te, 123 Te, 124 Te, 125 Te, 126 Te, 128 Te, 130 Te and combinations thereof.

[0087] In another aspect, the present disclosure includes a method for determining a dose of a small molecule compound to achieve a desired target binding of the small molecule compound in a subject or a cell / tissue culture, the steps comprising: providing a subject or a cell / tissue culture sample; providing a small molecule analog of the small molecule compound, wherein the small molecule compound has a structure comprising one or more monocyclic or bicyclic aromatic rings, and the small molecule analog has a structure in which at least one of the one or more aromatic rings of the structure of the small molecule compound is replaced by a tellurium-containing tellurium atom, thereby forming a tellurium-containing tellurium-containing small molecule analog; administering a tellurophene small molecule analog to a subject or a cell / tissue culture sample; providing a sample obtained from the subject or the cell / tissue culture sample after administration of the tellurophene small molecule analog; detecting the tellurophene small molecule analog in the sample to which the tellurophene small molecule analog has been administered using mass spectrometry by a method as defined in any of the embodiments discussed herein; measuring the effect produced by the tellurophene small molecule analog; and determining a dose of the tellurophene analog suitable for achieving a desired distribution of the small molecule compound, wherein the dose of the tellurophene small molecule analog is indicative of the dose of the small molecule compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings, in which:

[0089] Figure 1 The effects of teniposide and Te-teniposide analog 1 on Top2-catalyzed decatenation of kinesiomal DNA are shown. Panel A shows a picture of an agarose gel of the Top2 decatenation assay. Panel B shows a graph of relative Top2 inhibition based on the image density of the kDNA band in lane 3. 1: linear DNA; 2: decatenation kDNA; 3: control kDNA; 4: kDNA incubated with Top2; 5-8: kDNA incubated with 5, 10, 25 and 50 μM teniposide; 9-12: kDNA incubated with 5, 10, 25 and 50 μM 1.

[0090] Figure 2 Graph showing relative cell proliferation of HL-60 cells treated with teniposide or Te-teniposide analog 1.

[0091] Figure 3 Graph showing the dose-dependent cytotoxicity of teniposide and Analog 1 in PANC-1 cells as determined by alamarBlue assay.

[0092] Figure 4 Shown are proliferation curves of PANC-1 cells incubated with varying concentrations of teniposide and 1 as determined by imaging cell confluence.

[0093] Figure 5pH2AX in PANC-1 cells treated with 1 μM drug Ser139 (a marker of DNA DSBs). Teniposide and 1 both induced extended DNA damage at 24 and 48 hours of exposure relative to control.

[0094] Figure 6 Display by Analysis of Cellular Competitive Binding Assays of Teniposide and Compound 1 130 Te histogram. a) HL-60 cells treated with teniposide (red); despite presaturation of drug binding sites in the cells with equal amounts of teniposide (orange and purple), compound 1 still labeled HL-60 cells equally at 2 μM and 10 μM (blue and green). b) 2 μM teniposide was co-incubated with 0 (red), 2 (blue), 10 (orange), and 20 μM (green) teniposide.

[0095] Figure 7 Representative images of immunostained FFPE tumor tissue sections from PANC-1 xenograft mice dosed with (a) saline, (b) teniposide (20 mg / kg, IP), or (c) compound 1 (20 mg / kg, IP). Brown staining was performed to detect pH2AX. Ser139 The spots were imaged and then counterstained with hematoxylin.

[0096] Figure 8 Demonstrating IMC of Te-teniposide in PANC-1 xenografts TM A) Teniposide administration, B) Te-teniposide administration, C) Image of the average signal of A subtracted from B, where the area is magnified to show the Te-teniposide specific signal. D) Teniposide and Te-teniposide administration in mice 125 Histogram of Te pixel counts; E) 125 Standard curve of Te counts versus Te atoms / pixel.

[0097] Fig. 9 Graph showing the percent reduction in apparent enzyme velocity upon inhibition with carfilzomib, 7b or 8b relative to no treatment.

[0098] Fig.10 Graph showing median 128Te counts per cell obtained from MC analysis of cells treated with TeCar 7b (500 μM), carfilzomib (500 μM), control 8b, or a combination of carfilzomib with 7b or 8b.

[0099] Fig.11 Standard curves for in vitro binding assays are shown.

[0100] Fig.12 The mass cytometry system and controller are shown.

[0101] Fig.13 Shown is a mass cytometry system.

[0102] Fig.14 An exemplary computer system that may be used as a controller is shown.

[0103] Other features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that although the detailed description and specific examples indicate embodiments of the present disclosure, they are only given in an illustrative manner, and the scope of the claims should not be limited by these embodiments, but should be given the broadest interpretation consistent with the entire description. DETAILED DESCRIPTION

[0104] I. Definitions

[0105] Unless otherwise indicated, the definitions and embodiments described here and elsewhere are intended to apply herein to all embodiments and aspects of the present disclosure as understood by those skilled in the art to be appropriate.

[0106] As used herein, the terms "compounds of the present disclosure" or "compounds disclosed herein" and the like refer to tellurophene small molecule analogs as described herein. As used herein, tellurophene small molecule analogs refer to structural analogs of small molecules containing one or more monocyclic or bicyclic aromatic rings, wherein the analogs contain tellurophene and a structure in which at least one of the monocyclic or bicyclic aromatic rings is replaced by tellurophene. Therefore, it can be understood that tellurophene small molecule analogs have a structure that mimics a small molecule structure. For example, the compounds of the present disclosure include compounds of formula I or pharmaceutically acceptable salts and / or solvates thereof, and compounds of formula II or pharmaceutically acceptable salts and / or solvates thereof.

[0107] As used herein, the terms "composition of the present disclosure" or "compound disclosed herein" and the like refer to a composition, such as a pharmaceutical composition, comprising one or more compounds of the present disclosure.

[0108] As used herein, the term "and / or" means that the listed items are present or used alone or in combination. In practice, this term means "at least one" or "one or more" of the listed items are used or present. The term "and / or" with respect to pharmaceutically acceptable salts and / or solvates thereof means that the compounds of the present disclosure are present as individual salts and hydrates, as well as combinations of solvates of salts of the compounds of the present disclosure, for example.

[0109] As used in this disclosure, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise. For example, embodiments including "a compound" should be understood to present certain aspects having one compound or two or more additional compounds.

[0110] In embodiments comprising an "additional" or "second" component (such as an additional or second compound), the second component as used herein is chemically different from the other components or the first component. The "third" component is different from the other components, the first component and the second component, and further listed or "additional" components are similarly different.

[0111] As used in this disclosure and claims, the words "comprising" (and any form of comprising, such as "comprising" and "including"), "having" (and any form of having, such as "having" and "having"), "including" (and any form of including, such as "including" and "including"), or "containing" (and any nature of containing, such as "containing" and "containing") are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.

[0112] As used herein, the term "consisting of" and its derivatives are intended to serve as closed terms that specify the presence of stated features, elements, components, groups, integers and / or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0113] As used herein, the term "consisting essentially of" is intended to specify the presence of stated features, elements, components, groups, integers, and / or steps, as well as the presence of features, elements, components, groups, integers, and / or steps that do not materially affect the basic and novel characteristics of such features, elements, components, groups, integers, and / or steps.

[0114] The term "suitable" as used herein means that the selection of particular compounds or conditions depends on the specific synthetic operation to be performed, the identity of the molecule to be transformed, and / or the specific use of the compound, but the selection is well within the skills of one skilled in the art.

[0115] In embodiments of the present disclosure, the compounds described herein may have at least one asymmetric center. When compounds have more than one asymmetric center, they may exist as diastereomers. It should be understood that all such isomers and mixtures thereof in any proportion are encompassed within the scope of the present disclosure. It should also be understood that, although the stereochemistry of the compound may be as shown in any given compound listed herein, such compounds may also contain a certain amount (e.g., less than 20%, suitably less than 10%, more suitably less than 5%) of the disclosed compounds with alternative stereochemistry. Any optical isomer intended to be separated, pure or partially purified optical isomers or their racemic mixtures are included within the scope of the present disclosure.

[0116] The compounds of the present disclosure may also exist in different tautomeric forms, and it is intended that any tautomeric forms formed by the compounds as well as mixtures thereof are included within the scope of the present disclosure.

[0117] The compounds of the present disclosure may also exist in different polymorphic forms, and any polymorphs formed or mixtures thereof are contemplated to be included within the scope of the present disclosure.

[0118] The description of the present invention refers to many chemical terms and abbreviations used by those skilled in the art. However, for the sake of clarity and consistency, definitions of selected terms are provided.

[0119] As used herein, the terms "about", "substantially" and "approximately" mean a reasonable amount of deviation of the modified term so that the end result is not significantly changed. In the case where such deviation does not negate the meaning of the word it modifies, or unless the context suggests otherwise to a person skilled in the art, these degree terms should be interpreted as including a deviation of at least ±5% of the modified term.

[0120] As used herein, the term "alkyl", whether used alone or as part of another group, means a straight or branched chain saturated alkyl group. The possible number of carbon atoms in the alkyl group is indicated by the prefix "C n1-n2 " indicates. For example, the term C 1-10 Alkyl means an alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.

[0121] The term "alkylene", whether used alone or as part of another group, means a straight or branched saturated alkylene group, i.e. a saturated carbon chain containing substituents at both ends. The possible number of carbon atoms in the alkylene group mentioned is indicated by the prefix "C n1-n2 " indicates. For example, the term C 2-6 Alkylene means an alkylene group having 2, 3, 4, 5 or 6 carbon atoms.

[0122] The term "available" as in "available hydrogen atom" or "available atom" refers to an atom known by those skilled in the art to be replaceable with a substituent.

[0123] As used herein, the term "amine" or "amino", whether used alone or as part of another group, refers to a group of the general formula NR'R", wherein R' and R" are each independently selected from hydrogen or C 1-6 alkyl.

[0124] As used herein, the term "atm" refers to atmospheric air.

[0125] The term "MS" as used herein refers to mass spectrometry.

[0126] As used herein, the term "aq." refers to an aqueous solution.

[0127] As used herein, the term "protecting group" or "PG" or the like refers to protecting or masking the reactive portion of a molecule to prevent side reactions of those reactive portions of the molecule while manipulating different portions of the molecule or the chemical portions that react it. After the manipulation or reaction is complete, the protecting group is removed without degrading or decomposing the remainder of the molecule. A person skilled in the art can select a suitable protecting group. Many conventional protecting groups are known in the art, for example, as described in "Protective Groups in Organic Chemistry" McOmie, JFW, ed., Plenum Press, 1973; Greene, TW and Wuts, PGM, "Protective Groups in Organic Synthesis", John Wiley & Sons, 3rd edition, 1999; and Kocienski, P. Protecting Groups, 3rd edition, 2003, Georg Thieme Verlag (The Americas).

[0128] As used herein, the term "small molecule" refers to an organic compound of low molecular weight. For example, a small molecule can have a molecular weight of less than about 1500 g / mol, less than about 1300 g / mol, or less than about 1000 g / mol.

[0129] The term "subject" as used herein includes all members of the animal kingdom, including mammals, and suitably refers to rats, mice and humans. Thus, the methods and uses of the present disclosure are applicable to both human therapy and veterinary applications.

[0130] The term "pharmaceutically acceptable" means compatible with the treatment of the subject.

[0131] The term "pharmaceutically acceptable carrier" means a non-toxic solvent, dispersant, excipient, adjuvant or other material that is mixed with the active ingredient to allow for formation of a pharmaceutical composition (ie, a dosage form that can be administered to a subject).

[0132] The term "pharmaceutically acceptable salt" means an acid addition salt or a base addition salt that is suitable or compatible with the treatment of a subject.

[0133] Acid addition salts suitable for or compatible with the treatment of a subject are any non-toxic organic or inorganic acid addition salt of any basic compound.

[0134] Base addition salts suitable for or compatible with the treatment of a subject are any non-toxic organic or inorganic base addition salt of any acidic compound.

[0135] The term "solvate" as used herein means a compound or a salt of a compound in which molecules of a suitable solvent are bound in a crystal lattice.

[0136] As used herein and as is well known in the art, the term "treatment" or "therapy" means a method for obtaining a beneficial or desired result (including a clinical result). Beneficial or desired clinical results include, but are not limited to, alleviation or improvement of one or more symptoms or conditions, reduction of the extent of the disease, stabilization of the disease state (i.e., no deterioration), prevention of disease spread, delay or slowing of disease progression, improvement or alleviation of the disease state, reduction of disease recurrence, and alleviation (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonged survival compared to the survival expected when not receiving treatment. "Treatment" as used herein also includes preventive treatment. For example, a subject with early-stage cancer can be treated to prevent progression, or alternatively, a subject in remission can be treated with a compound or composition of the present disclosure to prevent recurrence. The method of treatment includes administering a therapeutically effective amount of one or more compounds of the present disclosure to the subject, and optionally consisting of a single administration, or alternatively, including a series of administrations.

[0137] As used herein, the term "effective amount" or "therapeutically effective amount" means an amount of one or more compounds of the present disclosure effective, at dosages and for periods of time necessary to achieve the desired result.

[0138] As used herein, the term "administering" means applying a therapeutically effective amount of one or more compounds or compositions of the present disclosure to a cell, tissue, organ, or subject.

[0139] As used herein, the term "neoplastic disorder" refers to a disease, disorder or condition characterized by the ability of cells to grow or replicate autonomously, such as an abnormal state or condition characterized by proliferative cell growth. As used herein, the term "neoplasm" refers to a mass of tissue caused by the abnormal growth and / or division of cells in a subject with a neoplastic disorder.

[0140] As used herein, the term "cancer" refers to a cell proliferative disease state.

[0141] II. Compounds and Compositions of the Disclosure

[0142] In one aspect, the present disclosure includes a small molecule tellurophene small molecule analog, wherein the tellurophene small molecule analog includes tellurophene, the tellurophene comprising a tellurium atom, wherein the small molecule has a structure comprising one or more monocyclic or bicyclic aromatic rings, and the analog has a structure in which at least one of the one or more aromatic rings of the structure of the small molecule is replaced by tellurophene.

[0143] In some embodiments, the small molecule is any organic compound that produces a physiological effect in a subject to which the organic compound is administered. For example, the small molecule is a drug. In some embodiments, the small molecule is not phenylalanine, and the tellurophene small molecule analog is not TePhe.

[0144] In some embodiments, the one or more monocyclic aromatic rings are 5- or 6-membered aromatic rings, optionally the one or more monocyclic 5- or 6-membered aromatic rings are independently selected from thiophene, furan, pyrrole, phenyl or pyridine.

[0145] In some embodiments, one or more bicyclic aromatic rings are independently selected from naphthyl, indole, benzothiophene, or benzofuran.

[0146] In some embodiments, the structure of the small molecule comprises one or more monocyclic 5- or 6-membered aromatic rings, and at least one of the monocyclic 5- or 6-membered aromatic rings is replaced by tellurophene.

[0147] In some embodiments, the structure of the small molecule comprises one or more bicyclic aromatic rings, and at least one of the bicyclic aromatic rings is replaced by benzo[b]tellurophene or 4H-telluro[3,2-b]pyrrole.

[0148] In some embodiments, the structure of the small molecule comprises one or more 5-membered aromatic rings, optionally the one or more 5-membered aromatic rings are each independently selected from thiophene, furan or pyrrole.

[0149] In some embodiments, the one or more 5-membered aromatic rings are thiophene.

[0150] In some embodiments, the structure of the small molecule comprises one or more 6-membered aromatic rings, optionally one or more 6-membered aromatic rings are each independently selected from phenyl or pyridine, optionally one or more 6-membered aromatic rings are phenyl.

[0151] It is contemplated that small molecule (such as therapeutic agents comprising one or more aromatic rings) tellurophene analogs can be designed and prepared by replacing at least one of the one or more aromatic rings. Thus, it is contemplated that the structure of the analog mimics the structure of the small molecule such that the analog exhibits substantially similar biological activity to the small molecule. Thus, detection of the analog is indicative of detection of the small molecule. Some non-limiting examples are provided in Scheme A below.

[0152]

[0153] In some embodiments, the small molecule is teniposide, and the tellurophene small molecule analog is as shown in Formula I

[0154]

[0155] or a salt or solvate thereof.

[0156] In some embodiments, the small molecule is carfilzomib, and the analog is as shown in Formula II

[0157]

[0158] or a salt or solvate thereof.

[0159] In another aspect, the present disclosure includes a compound of formula I

[0160]

[0161] or a salt or solvate thereof.

[0162] In another aspect, the present disclosure includes a compound of formula II

[0163]

[0164] or a salt or solvate thereof.

[0165] In another aspect, the present disclosure includes a composition comprising a compound of the present disclosure, and a carrier or excipient.

[0166] In one embodiment, the pharmaceutically acceptable salt is an acid addition salt or a base addition salt. Suitable salts can be selected by those skilled in the art (see, for example, SM Berge et al., "Pharmaceutical Salts," J. Pharm. Sci. 1977, 66, 1-19).

[0167] Acid addition salts suitable for the treatment of a subject or compatible with the treatment of a subject are any nontoxic organic or inorganic acid addition salts of any basic compound. The basic compound forming the acid addition salt includes, for example, a compound comprising an amine group. Exemplary inorganic acids forming suitable salts include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid and phosphoric acid, and acidic metal salts such as sodium monohydrogen phosphate and potassium hydrogen sulfate. Exemplary organic acids forming suitable salts include monocarboxylic acids, dicarboxylic acids and tricarboxylic acids. Examples of such organic acids are, for example, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, hydroxymaleic acid, benzoic acid, hydroxybenzoic acid, phenylacetic acid, cinnamic acid, mandelic acid, salicylic acid, 2-phenoxybenzoic acid, p-toluenesulfonic acid and other sulfonic acids such as methanesulfonic acid, ethanesulfonic acid and 2-hydroxyethanesulfonic acid. In one embodiment, a monoacid salt or a diacid salt is formed, and such salt exists in a hydrated form, a solvated form, or a substantially anhydrous form. In general, acid addition salts are more soluble in water and various hydrophilic organic solvents, and typically exhibit a higher melting point compared to their free base forms. The selection criteria for suitable salts will be known to those skilled in the art. Other non-pharmaceutically acceptable salts may be used, such as, but not limited to, oxalates, for example, in separating compounds of the present disclosure for laboratory use, or for subsequent conversion to pharmaceutically acceptable acid addition salts.

[0168] Base addition salts suitable for the treatment of a subject or compatible with the treatment of a subject are any nontoxic organic or inorganic base addition salts of any acidic compound. The acidic compound forming the base addition salt includes, for example, a compound comprising a carboxylic acid group. The exemplary inorganic bases forming suitable salts include lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide or barium hydroxide and ammonia. The exemplary organic bases forming suitable salts include aliphatic, alicyclic or aromatic organic amines, such as isopropylamine, methylamine, trimethylamine, picolin, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hyaluronic acid, choline, betaine, ethylenediamine, aminoglucose, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Exemplary organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. It may be useful to select an appropriate salt, for example, so that ester functional groups (if any) elsewhere in the compound are not hydrolyzed. The selection criteria for appropriate salts will be known to those skilled in the art.

[0169] Solvates of the disclosed compounds include, for example, solvates made with pharmaceutically acceptable solvents. Examples of such solvents include water (the resulting solvates are referred to as hydrates) and ethanol, etc. Suitable solvents are physiologically tolerable at the doses administered.

[0170] The disclosed compounds are suitably formulated into compositions using one or more carriers in a conventional manner. Therefore, the present disclosure also includes a composition comprising one or more disclosed compounds and a carrier. The disclosed compounds are suitably formulated into pharmaceutical compositions so as to be administered to subjects in a biocompatible form suitable for in vivo administration. Therefore, the present disclosure also includes a pharmaceutical composition comprising one or more disclosed compounds and a pharmaceutically acceptable carrier. In an embodiment of the present disclosure, the pharmaceutical composition is used to treat any disease, disorder or condition described herein.

[0171] As will be appreciated by those skilled in the art, the disclosed compounds are administered to subjects in various forms, depending on the selected route of administration. For example, the disclosed compounds are administered orally, by inhalation, parenterally, buccal, sublingually, nasally, rectally, vaginally, by patch, pump, topical or transdermal administration, and the pharmaceutical compositions thus formulated. In some embodiments, administration is achieved by means of a pump for regular or continuous delivery. For example, Remington describes conventional procedures and ingredients for selecting and preparing suitable compositions in Pharmaceutical Sciences (2000-20th Edition) and The United States Pharmacopeia: The National Formulary (USP24NF19) published in 1999.

[0172] Parenteral administration includes systemic routes of delivery other than the gastrointestinal tract (GI), and includes, for example, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary (e.g., by use of an aerosol), intrathecal, rectal, and topical (including use of a patch or other transdermal delivery device) modes of administration. Parenteral administration can be continuous infusion over a selected period of time.

[0173] In some embodiments, the disclosed compounds are administered orally, for example, together with an inert diluent or with an absorbable edible carrier, or they are enclosed in a hard or soft shell gelatin capsule, or they are compressed into tablets, or they are directly combined with food of a meal. In some embodiments, the compound is combined with an excipient and is used in the form of edible tablets, buccal tablets, lozenges, capsules, caplets, pills, granules, lozenges, chewing gum, powders, syrups, elixirs, discs, aqueous solutions and suspensions. For tablets, the carriers used include lactose, corn starch, sodium citrate and phosphate. Pharmaceutically acceptable excipients include adhesives (e.g., pregelatinized corn starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc or silicon dioxide); disintegrants (e.g., potato starch or sodium carboxymethyl starch); or wetting agents (e.g., sodium lauryl sulfate). In an embodiment, the tablets are coated by methods well known in the art. For tablets, capsules, caplets, pills or granules for oral administration, a pH sensitive enteric coating such as Eudragits designed to control the release of the active ingredient is optionally used. TM Oral dosage forms also include modified release (e.g., immediate release and timed release) formulations. Examples of modified release formulations include, for example, sustained release (SR) formulations, extended release (ER, XR or XL) formulations, time release formulations or timed release formulations, controlled release (CR) formulations or continuous release (CR or Contin) formulations, which are in the form of, for example, coated tablets, osmotic delivery devices, coated capsules, microencapsulated microspheres, agglomerated particles, for example, agglomerated particles of molecular sieve-type particles, or fine hollow permeable fiber bundles, or chopped hollow permeable fibers, agglomerated or retained in fiber bags. Timed release compositions are formulated into, for example, liposomes or those in which active compounds are protected by differentially degradable coatings such as by microencapsulation, multiple coatings, etc. Liposomal delivery systems include, for example, small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. In some embodiments, liposomes are formed by a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholine. For oral administration in a capsule form, useful carriers or diluents include lactose and dried corn starch.

[0174] In some embodiments, the liquid preparation for oral administration takes the form of, for example, a solution, syrup or suspension, or they are suitably presented as a dry product so as to be constituted with water or other suitable vehicles before use. When an aqueous suspension and / or emulsion is administered orally, the disclosed compound is suitably suspended or dissolved in an oil phase combined with an emulsifier and / or a suspending agent. If necessary, certain sweeteners and / or flavoring agents and / or coloring agents are added. Such liquid preparations for oral administration are prepared by conventional means with pharmaceutically acceptable additives, such as suspending agents (e.g., sorbitol syrup, methylcellulose or hydrogenated edible fats); emulsifiers (e.g., lecithin or gum arabic); non-aqueous vehicles (e.g., almond oil, oily esters or ethanol); and preservatives (e.g., methyl or propyl paraben or sorbic acid). Useful diluents include lactose and high molecular weight polyethylene glycols.

[0175] It is also possible to freeze-dry the compounds of the disclosure and use the lyophilisate obtained, for example, in order to prepare an injectable product.

[0176] In some embodiments, the compounds of the present disclosure are parenterally administered. For example, a solution of the compounds of the present disclosure is prepared in water, which is suitably mixed with a surfactant (such as hydroxypropyl cellulose). In some embodiments, dispersions are prepared in glycerol, liquid polyethylene glycol, DMSO and mixtures thereof (with or without alcohol) and in oil. Under general storage and use conditions, these preparations contain a preservative to prevent microbial growth. Those skilled in the art will know how to prepare suitable preparations. For parenteral administration, a sterile solution of the compounds of the present disclosure is usually prepared, and the pH of the solution is suitably adjusted and buffered. For intravenous use, the total concentration of the solute should be controlled to make the preparation isotonic. For ocular administration, ointments or dropperable liquids are delivered, for example, by an ocular delivery system known in the art (such as an applicator or an eye dropper). In some embodiments, such compositions include a mucus mimetic (such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or polyvinyl alcohol), a preservative (such as sorbic acid, EDTA or benzyl chromium chloride) and a conventional amount of a diluent or carrier. For pulmonary administration, an appropriate diluent or carrier will be selected to allow aerosol formation.

[0177] In some embodiments, the disclosed compounds are formulated for parenteral administration by injection, including using conventional catheter insertion techniques or infusions. For example, injection preparations are provided in unit dosage forms with the addition of preservatives, for example, in ampoules or in multi-dose containers. In some embodiments, the composition takes the form of a sterile suspension, solution or emulsion such as in an oily or aqueous vehicle, and contains a preparatant such as a suspending agent, a stabilizer and / or a dispersant. In all cases, the form must be sterile and must be a fluid of a degree that is easy to inject. Alternatively, the disclosed compounds are suitably in a sterile powder form, so as to be reconstituted with a suitable vehicle (e.g., sterile pyrogen-free water) before use.

[0178] In some embodiments, the composition for nasal administration is conveniently formulated as an aerosol, drops, gel and powder. For intranasal administration or inhalation administration, the disclosed compounds are conveniently delivered in the form of a solution, dry powder preparation or suspension from a pump spray container squeezed or pumped by the patient, or delivered in the form of an aerosol spray from a pressurized container or atomizer. Aerosol preparations generally include a solution or fine suspension of an active substance in a physiologically acceptable aqueous or non-aqueous solvent, and are generally provided in a single dose or multiple doses in a sterile form in a sealed container, the container for example taking the form of a cartridge or a replacement for use with a spray device. Alternatively, the sealed container is an integrated dispensing device, such as a single dose nasal inhaler or an aerosol dispenser equipped with a metering valve, and the metering valve is intended for use in the disposal after use. When the dosage form includes an aerosol dispenser, it will contain a propellant, and the propellant is, for example, a compressed gas, such as compressed air, or an organic propellant, such as a fluorochlorocarbon. Suitable propellants include, but are not limited to, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, sevoflurane, carbon dioxide or another suitable gas. In the case of a pressurized aerosol, the dosage unit is determined by providing a valve to deliver a metered amount. In some embodiments, the pressurized container or nebulizer contains a solution or suspension of the active compound. For example, capsules and cartridges (e.g., made of gelatin) for use in an inhaler or insufflator contain a powder mixture of the disclosed compounds and a suitable powder base (such as lactose or starch). Aerosol dosage forms can also take the form of a pump-sprayer.

[0179] Compositions suitable for buccal or sublingual administration include tablets, lozenges and pastilles, in which the disclosed compounds are formulated with a carrier such as sugar, acacia, tragacanth or gelatin and glycerin. Compositions for rectal administration are conveniently in the form of suppositories containing conventional suppository bases such as cocoa butter.

[0180] The suppository form of the disclosed compounds can be used for vaginal, urethral and rectal administration. Such suppositories are generally composed of a mixture of substances that are solid at room temperature but melt at body temperature. The materials commonly used to produce such vehicles include, but are not limited to, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol. For further discussion of suppository dosage forms, see, for example, Remington's Pharmaceutical Sciences, 16th edition, Mack Publishing, Easton, PA, 1980, pp. 1530-1533.

[0181] In some embodiments, the disclosed compounds are coupled to soluble polymers as targetable drug carriers. Such polymers include, for example, polyvinylpyrrolidone, pyran copolymers, polyhydroxypropylmethacrylamide-phenol, polyhydroxyethylaspartamide-phenol, or polyethylene oxide-polylysine substituted with palmitoyl residues. In addition, in some embodiments, the disclosed compounds are coupled to a class of biodegradable polymers that can be used to achieve controlled release of drugs, such as polylactic acid, polyglycolic acid, copolymers of polylactic acid and polyglycolic acid, poly-ε-caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and crosslinked or amphiphilic hydrogel block copolymers.

[0182] The disclosed compounds (including pharmaceutically acceptable salts and / or solvates thereof) are suitable for use alone, but are generally administered in the form of a pharmaceutical composition of one or more disclosed compounds (active ingredients) combined with a pharmaceutically acceptable carrier. Depending on the mode of administration, the pharmaceutical composition will contain from about 0.05% to about 99% by weight or from about 0.10% to about 70% by weight of the active ingredient and from about 1% to about 99.95% by weight or from about 30% to about 99.90% by weight of a pharmaceutically acceptable carrier, all weight percentages being based on the total composition.

[0183] III. Methods and Uses of the Disclosure

[0184] In one aspect, the present disclosure includes a method of detecting a small molecule in a sample, the method comprising:

[0185] providing a sample obtained from a subject or cell / tissue culture to which a tellurphene small molecule analog comprising tellurphene has been administered, the tellurphene comprising a tellurium atom; and

[0186] The samples were subjected to mass spectrometry to determine the levels of tellurium atoms;

[0187] wherein the small molecule has a structure comprising one or more monocyclic or bicyclic aromatic rings, and the analog has a structure in which at least one of the one or more aromatic rings of the structure of the small molecule is replaced by tellurophene, and

[0188] Therein the level of tellurium atoms corresponds to the level of the analog, and detection of the analog indicates detection of the small molecule in the sample.

[0189] In some embodiments, the method further comprises quantifying the amount of tellurium atoms in the sample, wherein the level of tellurium atoms is indicative of the number of small molecules in the sample.

[0190] In some embodiments, the method further comprises quantifying one or more other analytes in the sample, the method further comprising labeling the sample with one or more analyte binders having a mass tag prior to performing mass spectrometry, and determining the level of the one or more analyte binders.

[0191] In some embodiments, one or more analyte binders with mass labels are selected from metal-labeled antibodies, polymer-labeled antibodies, metal-labeled oligonucleotides, polymer-labeled oligonucleotides, intercalators (such as 5-iodo-2'-deoxyuridine (IdU) and metal-containing intercalators (e.g., Rh-containing intercalators and Ir-containing intercalators)), metal-containing activity indicators (such as cisplatin), barcode reagents (such as Cd-labeled CD45 and Pt-labeled CD45). In some embodiments, the polymer-labeled antibody contains a metal. For example, the polymer can be metal-bound.

[0192] In some embodiments, the subject is a mammal, optionally a mouse, rat, or human.

[0193] In some embodiments, mass spectrometry is performed at a plurality of discrete locations, and the level of tellurium atoms is determined at each of the plurality of discrete locations to provide a distribution of analogs in the sample, and wherein the distribution of analogs in the sample is indicative of a distribution of small molecules in the sample.

[0194] In some embodiments, the sample is a single cell, and the distribution of the analog within the single cell is indicative of the distribution of the small molecule at the subcellular level.

[0195] In some embodiments, multiple samples are provided and the samples include different tissues, cells or secretions of a subject, and wherein detection of the analog in the multiple samples provides a distribution of the analog within the subject, which is indicative of the distribution of the small molecule within the subject, such as tissue or organ distribution.

[0196] In some embodiments, the sample is or comprises urine, feces, blood or a fraction thereof, cerebrospinal fluid (CSF), saliva, muscle cells, fat cells, bone cells, hair, nails, skin cells, tumor cells or secretions, liver cells or secretions, heart cells, lung cells or secretions, pancreatic cells or secretions, and / or stomach cells or secretions.

[0197] In some embodiments, the sample is a frozen tissue section.

[0198] In some embodiments, the sample is or comprises cells from a cell culture or culture medium from a cell culture.

[0199] In some embodiments, the sample is a single cell, and the distribution of the analog within the single cell is indicative of the distribution of the small molecule at the subcellular level.

[0200] In some embodiments, the analog interacts irreversibly, optionally covalently, with a target in a sample.

[0201] In some embodiments, the subject or cell / tissue culture has been administered a tellurophene small molecule analog in combination with a small molecule compound, wherein the method further comprises directly detecting the level of the small molecule compound, and wherein a comparison of the level of the small molecule to the level of the analog indicates putative competitive binding of the analog.

[0202] In another aspect, the present disclosure includes the use of tellurphene analogs for detecting small molecules by mass spectrometry in a subject to which the analogs have been administered or in a cell / tissue culture species to which the analogs have been administered, wherein the small molecule has a structure comprising one or more monocyclic or bicyclic aromatic rings, and the analog has a structure in which at least one of the one or more monocyclic or bicyclic aromatic rings of the structure of the small molecule is replaced by tellurphene.

[0203] In another aspect, the present disclosure includes the use of a compound of Formula I or a composition comprising a compound of Formula I of the present disclosure for detecting teniposide by mass spectrometry in a subject to which a compound of Formula I or a salt or solvate thereof has been administered, or in a cell / tissue culture to which a compound of Formula I or a salt or solvate thereof has been administered.

[0204] In another aspect, the present disclosure includes the use of Formula II or a composition comprising a compound of Formula II of the present disclosure for detecting carfilzomib by mass spectrometry in a subject to which a compound of Formula I or a salt or solvate thereof has been administered, or in a cell / tissue culture to which a compound of Formula II or a salt or solvate thereof has been administered.

[0205] In another aspect, the present disclosure includes a kit for mass spectrometry flow cytometry analysis, comprising a teniposide tellurophene analog of formula I

[0206]

[0207] or a salt or solvate thereof, and

[0208] One or more tellurium standards.

[0209] In another aspect, the present disclosure includes a kit for mass spectrometry analysis, optionally mass cytometry analysis, comprising a carfilzomib tellurophene analog of Formula II

[0210]

[0211] or a salt or solvate thereof, and

[0212] One or more tellurium standards.

[0213] In another aspect, the disclosure includes a method of achieving a dosage of a small molecule for desired target engagement of the small molecule in a subject or cell / tissue culture, wherein the small molecule engages a target in the subject or cell / tissue culture and produces a measurable effect at the target, the method comprising

[0214] detecting a small molecule in a subject by the methods of the present disclosure;

[0215] Measuring the effects of small molecules; and

[0216] A dosage of the tellurophene analog is determined that is appropriate to achieve the desired distribution of small molecules, wherein the dosage of the tellurophene analog is indicative of the dosage of the small molecules.

[0217] In addition to enabling single cell analysis, mass cytometry can also include mass cytometry imaging methods, such as those described in Giesen et al. 2014, which is incorporated herein by reference. In such methods, tissues or cell populations are labeled in vitro with mass-labeled entities (such as analyte binders and / or compounds containing atoms suitable for mass cytometry), the tissues or cell populations are subjected to laser ablation coupled to mass cytometry, and the tellurium signals are processed to provide images showing single cell segmentation. Different tissue preparations can be used, including, for example, formalin-fixed tissues and fresh tissues.

[0218] In some embodiments, the mass spectrometry is mass cytometry or multiplexed ion beam imaging, and optionally the mass spectrometry is mass cytometry imaging or mass cytometry suspension.

[0219] In some embodiments, the tellurium atom is selected from 120 Te, 122 Te, 123 Te, 124 Te, 125 Te, 126 Te, 128 Te,130 Te and combinations thereof.

[0220] In some embodiments, the tellurium atoms include multiple tellurium isotopes, and the mass cytometry is multi-channel mass spectrometry, optionally multi-channel mass cytometry.

[0221] It is understood that the disclosed methods and the disclosed tellurophene analogs can be used to track the distribution of drugs in organs or tissues in general. For example, when the small molecule is administered by different routes of administration (e.g., inhalation, injection, etc.), the method can be used to evaluate the distribution of the small molecule.

[0222] It is contemplated that the tellurophene analogs and methods of the present disclosure may be used in combination with other analytical methods and reagents, either simultaneously or sequentially. For example, the tellurophene analogs and methods of the present disclosure may be used with labeled antibodies (e.g., metal-labeled antibodies and polymer-labeled antibodies) such as reagents, metal-labeled oligonucleotides, metal-containing intercalators, and activity indicators (e.g., metal activity indicators) such as CELL-ID TM Reagents, barcoded reagents such as Cell-ID TM 20-PlexPd barcode kit; Cd-CD45; Pt-CD45, and / or calibration beads such as EQ TM Four-element calibration beads and EQ TM Six element calibration beads are used in combination, simultaneously or sequentially.

[0223] It is also contemplated that the disclosed tellurophene analogs and disclosed methods can be used in a variety of applications, including diagnostics, preclinical studies (including animal studies), pharmacokinetic (eg, pulse-chase studies), and pharmacodynamic studies.

[0224] It is contemplated that, depending on the properties of the small molecules and their analogs, the disclosed methods and disclosed analogs may be used to study a variety of conditions, including neoplastic disorders such as leukemia, neuroblastoma, and non-Hodgkin's lymphoma.

[0225] IV. Methods of Preparing the Disclosed Compounds

[0226] The disclosed compounds can be prepared by various synthetic processes. The selection of specific structural features and / or substituents may affect the selection of one process relative to another process. It is within the knowledge of those skilled in the art to select a specific process to prepare a given disclosed compound. Some starting materials for preparing the disclosed compounds can be obtained from commercial chemical sources. Other starting materials, such as those described below, are easily prepared from available precursors using direct conversions well known in the art. Unless otherwise stated, in the following schemes showing the preparation of the disclosed compounds, all variables are as defined herein.

[0227] The formation of salts of the desired compounds is achieved using standard techniques. For example, by treating the neutral compound with an acid or base in a suitable solvent and isolating the formed salt by filtration, extraction or any other suitable method.

[0228] The formation of solvates will vary depending on the compound and the solvate. Generally, solvates are formed by dissolving the compound in an appropriate solvent and separating the solvate by cooling or using an antisolvent. Solvates are typically dried or azeotroped at ambient conditions. One skilled in the art can select appropriate conditions to form a particular solvate. Examples of suitable solvents are ethanol, water, and the like. When water is the solvent, the molecule is referred to as a "hydrate." The formation of solvates of the disclosed compounds will vary depending on the compound and the solvate. Generally, solvates are formed by dissolving the compound in an appropriate solvent and separating the solvate by cooling or using an antisolvent. Solvates are typically dried or azeotroped at ambient conditions. One skilled in the art can select appropriate conditions to form a particular solvate. ""

[0229] Throughout the process described herein, it should be understood that in appropriate cases, suitable protecting groups are added to various reactants and intermediates and subsequently removed therefrom in a manner that is readily understood by those skilled in the art. Conventional procedures for using such protecting groups and examples of suitable protecting groups are described in, for example, "Protective Groups in Organic Synthesis", TW Green, PGM Wuts, Wiley-Interscience, New York, (1999). It should also be understood that a group or substituent can be converted to another group or substituent by chemical operations on any intermediate or final product on the synthetic pathway toward the final product, wherein possible conversion types are limited only to the inherent incompatibility of other functional groups carried by the molecule at the stage with the conditions or reagents used in the conversion. Such inherent incompatibility and the mode of avoiding such inherent incompatibility by performing appropriate conversion and appropriate order of synthetic steps will be readily understood by those skilled in the art. Conversion examples are given herein, and it should be understood that the conversion described is limited only to the universal groups or substituents that illustrate the conversion. References and descriptions of other suitable transformations are given in "Comprehensive Organic Transformations-A Guide to Functional Group Preparations" RC Larock, VHC Publishers, Inc. (1989). References and descriptions of other suitable reactions are described in organic chemistry textbooks, such as "Advanced Organic Chemistry", March, 4th Edition. McGraw Hill (1992) or "Organic Synthesis", Smith, McGraw Hill, (1994). Techniques for purifying intermediates and final products include direct and reverse phase chromatography, recrystallization, distillation and liquid-liquid or solid-liquid extraction on columns or rotating plates, which will be readily understood by those skilled in the art.

[0230] Therefore, in some embodiments, the disclosed compounds can be prepared as described below. For example, the compound of formula I can be prepared as shown in Scheme 1. For example, the compound of formula II can be prepared as shown in Scheme 2. It is understood that the tellurophene moiety can be incorporated into a small molecule to replace one or more aromatic rings by other suitable synthetic methods. Some suitable examples are provided in the following schemes.

[0231] Method of adding tellurium

[0232] In some embodiments, tellurophene can be introduced via an amine group. For example, tellurophene amine can be obtained from tellurophene aldehyde by reductive amination, as shown in Scheme B.

[0233]

[0234] Plan B

[0235] In some embodiments, tellurophene can be incorporated into a small molecule via a carboxyl group. For example, tellurophene carboxylic acid can be obtained from tellurophene aldehyde by oxidation. The resulting tellurophene carboxylic acid can be coupled to a small molecule via an amide or ester linkage. (Scheme C)

[0236]

[0237] Plan C

[0238] In some embodiments, tellurophene can be converted to tellurophene halides by halogenation, which can then be incorporated into small molecules by metal-catalyzed cross-coupling reactions. (Scheme D)

[0239]

[0240] Scheme DX = halo

[0241] It is also contemplated that other tellurophene-containing heterocycles (e.g., bicyclic heterocycles) may be used to replace aromatic rings in small molecules. For example, small molecule analogs containing bicyclic aromatic rings may be designed and prepared by replacing the bicyclic aromatic rings with bicyclic tellurophene containing heterocycles. In some embodiments, benzo[b]tellurophene may be used to replace benzofuran or indole. Benzo[b]tellurophene may be prepared by cyclization of alkynylaryl halides in the presence of tellurium. (Scheme E)

[0242]

[0243] Plan E

[0244] In some embodiments, telluro[3,2-b]pyrrole may be used in place of thieno[3,2-b]pyrrole and / or pyrrolo[3,2-b]pyrrole. For example, telluro[3,2-b]pyrrole may be obtained by cyclization of an alkynylpyrrole halide.

[0245] (Scheme F)

[0246]

[0247] Plan F

[0248] Example

[0249] The following non-limiting examples are illustrative of the present disclosure.

[0250] Example 1 Tellurofentiniposide Analogs: Synthesis and Evaluation

[0251] Tellurium-substituted analogs of the anticancer xenobiotic teniposide (compounds of formula I) have been developed. Teniposide is a semisynthetic podophyllotoxin that inhibits the enzymatic activity of topoisomerase II (Top2) by binding to the Top2-DNA complex. Top2 modifies DNA topology by introducing transient double-strand breaks via covalent Top2-DNA complexes. Upon binding, teniposide stabilizes the DNA-Top2 complex and prevents rejoining of DNA, leading to accumulation of double-strand breaks (DSBs). The cytotoxic effects of teniposide are associated with apoptosis caused by excessive DSBs. Teniposide is a clinically approved chemotherapeutic agent, primarily used to treat acute lymphoblastic leukemia (ALL), Hodgkin's lymphoma, and neuroblastoma. The copy number of Top2 is high (1 x 10 in transformed cell lines). 6 ) indicated that tellurium-labeled teniposide analogs bound to Top2 should be detected by MC.

[0252] Teniposide bears a thiophene ring, which is presumably amenable to synthetic substitution. Analogs that are visible to MC can be readily obtained by synthesizing teniposide derivatives by substituting the thiophene group with a tellurophene ring. In cell-based assays, Te-teniposide analogs have proven indistinguishable from teniposide. In addition, it has been shown that MC can be used to track the localization of compounds in cells. In vivo Te-teniposide behaves similarly to teniposide and is directly detected in tissue sections.

[0253] Results and Discussion

[0254] Synthesis of Te-teniposide

[0255] Using a semisynthetic strategy, Te-teniposide analog (1) was prepared starting from 4'-demethylepipodophyllotoxin α-D-glucopyranoside, which can be conveniently obtained by mild acidic hydrolysis of etoposide. It was initially envisioned that the desired tellurophenyl acetal would be formed directly from the corresponding diethyl acetal 3, as the formation of benzylidene acetals is often accomplished with the corresponding dimethyl acetals. The diethyl acetal 3 was generated from propionaldehyde diethyl acetal and (bromoethynyl)triisopropylsilane under Cadoit-Chodkiewicz conditions. The resulting diyne 2 was cyclized to the tellurophene 3 by treatment with sodium tellurhydride generated in situ from tellurium metal and sodium borohydride.

[0256] Using tellurophene 3, a series of model condensation reactions with methyl-α-D-pyranoglucoside were investigated. Common conditions for the assembly of benzylidene acetals (TsOH, CH3CN or DMF) failed and resulted in the recovery of tellurophene aldehyde 4. The conditions suggested in the literature for the generation of teniposide and similar acetals use neat aldehydes and freshly molten ZnCl2. Tellurophene diethyl acetal 3 can be cleanly converted to aldehyde 4 under mild acidic hydrolysis. Condensation of 4'-demethylepipodophyllotoxin with neat tellurophene aldehyde 4 gave the desired teniposide analog 1 in moderate yield (Scheme 1).

[0257]

[0258] Scheme 1 - Synthesis of tellurophene mass-tagged teniposide 1 Synthesis of tellurophene-substituted teniposide analogs 1. (a) Bromoethynyltriisopropylsilane, 5 mol% CuCl, NH2OH·HCl, 30% aq.BuNH2, 0°C, 4h (57%); (b) Te metal (1.1 eq), NaBH4 (8 eq), EtOH:H2O (1:1), 50°C, 12h (52%); (c) 1.0 M HCl:THF (1:1), rt, 4h (quantitative); (d) 20% aqueous acetic acid, 75°C, 20h (66%); (e) 2-tellurophene carboxaldehyde (4) (neat), anhydrous ZnCl2 (2.0 eq) (16%).

[0259] In vitro analysis of Te-teniposide

[0260] The relative Top2 inhibitory activity of 1 compared to teniposide was evaluated in a debonding assay using kinetosomal DNA (kDNA). DNA debonding catalyzed by Top2 can be easily resolved using agarose gel electrophoresis. Both teniposide and 1 showed dose-dependent inhibition of the debonding reaction ( Figure 1 ) Calculation IC of 1 50 The value (5.6 μM) is similar to that calculated in the literature for teniposide based on fluorescence anisotropy and DNA relaxation gel assays (1.3-9.8 μM).

[0261] Cytotoxicity of Te-teniposide

[0262] The HL-60 promyelocytic leukemia cell line has been used as a model system to understand the spatial and temporal distribution of Top2 expression in cells and its associated resistance mechanisms. The toxicity of 1 and teniposide in HL-60 cells was compared after 24 h of incubation using the WST-1 viability assay. The dose-response curves of teniposide and 1 were observed to be identical in terms of experimental variation ( Figure 2); consistent with the literature, both Top2 poisons inhibited the proliferation of HL-60 cells by up to 60% at the highest concentration of 50 μM.

[0263] Similarly, PANC-1 cells were characterized for later in vivo experiments. PANC-1 cells were incubated with 1 or teniposide using alamarBlue viability assay and proliferation assay. Both drug forms exhibited concentration-dependent cytotoxicity in PANC-1 cells with indistinguishable dose responses and almost complete loss of cell viability at the maximum dose of 25 μM ( Figure 3 In the proliferation assay, analysis of the time course over a 100 h incubation indicated that concentrations above 0.1 μM of teniposide or 1 resulted in a significant reduction in PANC-1 cell proliferation for both compounds ( Figure 4 Taken together, these data indicate that 1 effectively mimics the antiproliferative activity of teniposide in PANC-1 cells.

[0264] Te-teniposide induces DNA damage

[0265] Next, the ability of 1 to induce DSBs, the major type of DNA damage caused by Top2 inhibition, was investigated. In response to DSBs, a range of DNA repair mechanisms are triggered, including phosphorylation of the histone variant H2AX at Ser139. Therefore, levels of pH2AXSer139 serve as a reliable marker for Top2-associated DSBs. Western blotting was used to measure pH2AXSer139 in PANC-1 cells treated with 1 μM 1 or teniposide for 24 and 48 hours. A significant increase in pH2AXSer139 levels was observed for both inhibitors relative to DMSO-treated controls, indicating that 1 is comparable to teniposide in its ability to induce DSBs ( Figure 5 ).

[0266] Cellular uptake of Te-teniposide

[0267] To determine whether cell labeling with 1 is dependent on the presence of Top2-DNA binding sites, competitive binding MC experiments were performed using teniposide. HL-60 cells were treated with teniposide (10 μM) for two hours to saturate the Top2-DNA ternary complex with unlabeled ligand within the cells, and then 1 (2 or 10 μM) was introduced into the cell culture medium and the suspension was gently mixed. After two hours, the cells were harvested and washed three times with PBS. The cells were then fixed and permeabilized, followed by DNA staining with Ir intercalators. Cell pellets were analyzed by MC. 130 Te content ( Figure 6 ).

[0268] While cell labeling with 1 was readily detected at both concentrations (2 and 10 μM), the expected labeling was not observed in cells pretreated with unlabeled teniposide. 130 The Te signal decreases ( Figure 6 A). In addition, co-incubation experiments were performed in which HL-60 cells were treated with a mixture of 1 (2 μM) and 2, 10 or 20 μM teniposide ( Figure 6 B). The population histograms of all three treatment groups are identical, indicating that no competition of Top2 is observed under these conditions. Without being bound by theory, it is assumed that the lack of competitive labeling observed in these experiments may be due to the inhibition of Top2 and the degree of nonspecific binding to other cellular components. Multiple reports have demonstrated the nonspecific accumulation of etoposide and teniposide via oxidation catalyzed by cytochrome P450-dependent monooxygenases, peroxidases, and tyrosinases. These redox conversions generate catechol and quinone metabolites, thereby significantly impairing the DNA binding ability of Top2. It is also possible that these reactive species modify other intracellular proteins, thus causing intracellular drug accumulation, but in a messy and irreversible manner. These results illustrate the practicality of MC visible probes in tracking the specificity of pharmaceutically active compounds. In this case, nonspecific binding may dominate the target binding of teniposide, and may be its analog Te-teniposide 1.

[0269] In vivo evaluation of tellurium-labeled teniposide

[0270] Although selective reversible binding in cell suspension could not be demonstrated by MC, the ease with which Compound 1 could be detected and the promising activity of Compound 1 in cell culture were demonstrated. Visualizing nonspecific accumulation and lack of targeting specificity in tissue sections, as observed in the case of cisplatin, will improve the understanding of the distribution of therapeutic agents. 8

[0271] IMC TM It can be used to image tissue localization and quantify the concentration of 1 in tissue samples. For in vivo studies, a mouse model bearing PANC-1 tumor xenografts was selected. The tumor microenvironment and general tissue morphology in sections derived from this human pancreatic cancer model can be characterized.

[0272] Mice bearing PANC-1 xenografts were injected IP with teniposide or Te-teniposide analog 1. A dose of 20 mg / kg was selected based on literature detailing teniposide drug delivery strategies. After 2 hours, mice were sacrificed and tissues of interest were harvested, formalin fixed and paraffin embedded. Tissue sections (5 μm) were cut and mounted on microscope slides. pH2AX was first evaluated by immunohistochemistry Ser139Expression levels. Serial sections from PANC-1 xenograft tumors were stained with primary antibody followed by HRP-conjugated secondary antibody to achieve standard brown staining. DSBs are identified by large, discrete pH2AX Ser139 Representative images of immunostained slides show increased levels of pH2AXSer139 in treated mice relative to saline-injected controls, with clear punctate staining ( Figure 7 ). These results indicate that 1 distributes across tumor tissue and maintains its ability to induce DSBs in vivo in a manner similar to teniposide.

[0273] For IMC TM The initial goal of the analysis was to visualize the distribution of tellurium signals in tumor sections and subsequently measure the relative tellurium content in individual cells. Tumor sections were dewaxed, rehydrated, and washed. Slides were then stained with Ir embedding agent to visualize cell nuclei and then analyzed by IMC. TM Proceed to imaging. 125 Te isotopes were visualized. Unfortunately, no Te signal above background was detected in tumor sections. Efforts to combine the intensities from multiple tellurium channels to improve the signal-to-noise ratio were also unsuccessful. Without being bound by theory, it is hypothesized that processing of FFPE tissues results in a loss of tellurium signal because the interaction of teniposide with cells is largely non-covalent, and thus the multiple washing steps required for dewaxing (xylene and ethanol washes) may have removed bound 1. To reduce the number of tissue processing steps, snap-frozen samples prepared from the same tumor were sectioned and analyzed by IMC. TM Using this method, we observed in tissue samples exposed to Te-teniposide 2 h before sacrifice. 125 Te signal ( Figure 8 In samples collected at later time points after dosing, no Te signal was observed, consistent with the half-life of teniposide in mice of 1-5 hours, depending on the route of administration and dosing regimen. 24

[0274] In order to determine the corresponding Te concentration present in the sample, IMC TM A set of standards containing increasing Te concentrations were analyzed. Based on the total counts present in each standard Te patch, the count / Te atom constant ( Figure 8 E) Counts observed in frozen samples indicate that the average concentration of Te-teniposide in tissue at the time of analysis was in the low tens of micromolar range. The low signal observed made it impossible to calculate an exact value. The observed signal was slightly above background, consistent with the IMC TM The instrument's sensitivity to tellurium was consistent with expectations.

[0275] in conclusion

[0276] Using IMC TM The ability to directly quantify the cellular distribution of pharmacologically active small molecules in tissues will provide cell-specific information about target engagement and biodistribution. As shown, using the anti-neoplastic agent teniposide, analogs whose biological activity is indistinguishable from the natural compound can be generated by replacing the tellurenyl functional group with a thienyl group. Using Te-teniposide analogs, MC demonstrated a high level of nonspecific binding of the compound to cells, indicating that Top2, a known target of teniposide, represents only a small part of the compound binding site. This is consistent with the high protein binding level of teniposide. In vivo, the activity of Te-teniposide produced the expected DSB in PANC-1 xenografts, and Te signals could be observed within the tissue.

[0277] Experimental Section

[0278] Chemical synthesis

[0279] General Materials and Methods: Etoposide (CarboSynth), propionaldehyde diethyl acetal (TCI America), and glacial acetic acid (Caledon Laboratories Ltd) were purchased from the indicated suppliers. All other reagents were purchased from Sigma Aldrich. All reactions were performed under a dry argon atmosphere using oven-dried glassware. Anhydrous ethanol was obtained from Green Field Speciality Alcohols Inc., while all other anhydrous solvents were purchased from Sigma Aldrich and purified by HPLC before use. Molecular sieves were dried. Purification by flash column chromatography was performed using silica gel (SiliCycle Silica-P Flash SilicaGel, 40-60 μm, pore size) and gradient solvent mixtures, as described above. TM The purity of the final compound 1 was confirmed by RP-HPLC coupled with low-resolution mass spectrometry (ESI), which was coupled with an AGILENT 6130 mass spectrometer. TM The samples were collected on a Phenomenex Technologies 1200 series HPLC. The flow rate was 1 mL / min at room temperature on a Phenomenex KINETEX TMFinal compound 1 was resolved on a 2.6 μm C18 50×4.6 mm column. The gradient consisted of eluent A (0.1% formic acid in deionized water) and B (0.1% formic acid in HPLC grade acetonitrile). At a flow rate of 1.0 mL / min, a linear gradient was started from 5% B to 95% in 7 min. The concentrations of CDCl3, CD3OD and C2D6SO were recorded on Bruker 400 MHz and Agilent 500 MHz spectrometers. 1 H and 13 C NMR spectra. Chemical shifts (δ) are reported in parts per million (ppm) after calibration to internal reference solvent peaks CDCl3 7.26 ppm and C2D6SO 2.49 ppm, CD3OD 3.31 ppm. Coupling constants (J) are reported in Hz. Spectra were acquired on a VG 70-250S (double focusing) mass spectrometer at 70 eV or on an ABI / SCIEX QSTAR with ESI source and accurate mass capability. TM High-resolution mass spectra were obtained on a mass spectrometer.

[0280] (Bromoethynyl)triisopropylsilane: To a solution of (triisopropylsilyl)acetylene (2.0 mL, 8.9 mmol, 1.0 equiv) in acetone (0.15 M) were added N-bromosuccinimide (1.75 g, 9.8 mmol, 1.1 equiv) and silver nitrate (0.15 g, 0.89 mmol, 0.1 equiv) successively. The reaction mixture was stirred at room temperature for 5 hours, after which saturated NH4Cl (30 mL) was added. The resulting mixture was then extracted with ether (3×30 mL). The organic layers were combined, washed with water (30 mL), brine (30 mL), dried over MgSO4, and concentrated in vacuo. The oil was dried under high vacuum to give the corresponding bromoalkyne as a light yellow oil, which was used in the next step. 1 H NMR (500 MHz, CDCl3) 1.11 (m; no alkyne protons). 13C NMR (101 MHz, CDCl3) = 83.50, 61.72, 18.49, 11.29.

[0281] (5,5-Diethoxypenta-1,3-diyn-1-yl)triisopropylsilane (2): In a round-bottom flask containing CuCl (4 mg, 0.38 mmol, 0.05 eq.) was added 30% n-BuNH2 aqueous solution (0.5 M) to form a blue solution. The reaction was cooled to 0°C. A small amount of NH2OH·HCl was added until the reaction mixture became colorless. Propionaldehyde diethyl acetal (0.99 mL, 7.59 mmol, 1.1 eq.) was added to the flask at 0°C and the solution developed a slight yellow color. After 5 minutes, (bromoethynyl)triisopropylsilane (2.18 g, 8.34 mmol, 1.0 eq.) was added dropwise at 0°C. The reaction mixture was allowed to warm to room temperature and monitored by TLC. Once the alkyne was consumed, the solution was exposed to air and diluted with brine (100 mL). The resulting mixture was extracted with DCM (3×100 mL) and the pooled organic fractions were dried over MgSO 4 , filtered and concentrated under reduced pressure. Silica gel column chromatography using a gradient of 100% pentane to 5% EtOAc / 95% pentane gave diyne 2 (2.57 g, 57% yield) as a yellow oil. 1 H NMR (500MHz, CDCl3) 5.30 (s, 1H), 3.75 (dd, J = 9.4, 7.1, 2H), 3.60 (dd, J = 9.4, 7.1, 2H), 1.24 (t, J = 7.1, 6H), 1.07 (m, J = 1.0, 21H). 13 C NMR (126MHz, CDCl3=91.41,88.43,85.88,71.31,70.44,61.23,18.47,15.01,11.17. [C 18 H 33 O2Si] + [M+H2O+H] + ESI LRMS m / z calcd: 309.54; found: 309.17.

[0282] 2-(Diethoxymethyl)tellurophene (3): To a round bottom flask were added ground tellurium metal (0.323 g, 2.53 mmol, 1.2 eq) and sodium borohydride (0.384 g, 10.1 mmol, 4.8 eq). The flask was purged with argon several times. Degassed water (10 mL) was added to the flask containing tellurium and sodium borohydride and set to stir at 50 °C under argon. After one hour, the metal suspension turned dark purple and eventually became a uniform colorless solution. At the same time, a 20 mL scintillation vial containing diyne 2 (0.650 g, 2.10 mmol, 1 eq) was purged with argon. THF (10 mL) was added to the vial and the yellow solution was cooled to 0 °C using an ice-water bath. TBAF (1.0 M in THF, 8.43 mL, 8.43 mmol) was added dropwise to the vial and allowed to stir for about one hour. The diyne solution is warmed to room temperature and slowly concentrated using a rotary evaporator. The crude mixture is diluted with Et2O (30mL) and brine (30mL). The organic layer is separated and washed twice with brine (2×25mL), and concentrated under reduced pressure without heating to produce a dark red oily substance, which will decompose after standing. The oily substance is absorbed in degassed EtOH (10mL) and purged with an argon balloon in the case of ultrasonic treatment. This solution is added dropwise to a sodium telluride solution via a syringe. After 12 hours, TLC indicates that the diyne starting material is consumed, and new UV active patches have appeared. The flask is cooled to room temperature and then exposed to air for at least one hour. The crude mixture is filtered through a diatomaceous earth pad. A small amount of EtOH (about 5mL) is used to wash the diatomaceous earth pad. The filtered orange-red solution is diluted with DCM (50mL) and brine (50mL), and extracted with DCM (5×30mL). A single organic layer is collected, dried over MgSO4, filtered and concentrated under reduced pressure. The tellurophene was purified using silica gel column chromatography with a gas gradient of 100% pentane to 10% EtOAc / 90% pentane to afford tellurophene 3 as an orange-red oil (52% yield). 1 HNMR (400MHz, CDCl3) = 8.89 (dt, J = 6.8, 1.3, 1H), 7.81 (ddd, J = 6.8, 4.0, 1.3, 1H), 7.71 –7.65(m,1H),5.62(s,1H),3.75(m,2H),3.69–3.57(m,2H),1.27(td,J=7.1,1.3,6H). [C4H3Te]–[M–C5H 11 O2] – ESI LRMS m / z calculated: 180.93; found: 181.14.

[0283] 2-Tellurophenecarboxaldehyde (4): A scintillation vial (20 mL) was charged with diethyl acetal protected tellurophene 3 (0.300 g, 1.06 mmol). THF (5 mL) was added and the reaction was set to stirring. HCl (1.0 M, 5 mL) was added to the vial, and the solution was stirred at room temperature for 6 hours. The reaction mixture was transferred to a separatory funnel and diluted with DCM (20 mL) and brine (20 mL). The layers were separated and the aqueous layer was washed with DCM (3×20 mL). The DCM portions were combined, dried over MgSO4 and filtered. Concentration under reduced pressure gave the corresponding aldehyde 4 as a red liquid. 1 H NMR (400MHz, CDCl3) = 9.59–9.57 (m, 1H), 9.49 (d, J = 6.7, 1H), 8.53 (dd, J = 4.1, 1.3, 1H), 8.06 (dd, J = 6.6, 4.1, 1H). 13 C NMR (101 MHz, CDCl3) = 187.82, 150.60, 147.45, 138.72, 138.41. ESI LRMS m / z calculated for [C4H3Te]-[M–CHO–H]–: 180.93; found: 181.14.

[0284] 4-Demethyl epipodophyllotoxin: A suspension of etoposide (0.200 g, 0.358 mmol) in 20% aqueous acetic acid (2.5 mL) was heated at 75 °C for 20 h in a round-bottom flask (50 mL). The resulting solution was concentrated to dryness under high vacuum. The resulting white crude material was dissolved in a 1:1 mixture of DCM:MeOH and adsorbed onto silica gel before purification using silica gel column chromatography with a gradient of 100% DCM to 20% MeOH / 80% DCM. The hydrolyzed etoposide derivative was obtained as a white powder (66% yield). 1 H NMR (500 MHz, CD3OD) = 6.98 (s, 1H), 6.50 (s, 1H), 6.26 (s, 2H), 5.94 (dd, J = 7.2, 1.2, 2H), 5.10 (d, J = 3.2, 1H), 4.55 (d, J = 5.4, 1H), 4.54-4.45 (m, 2H), 4.32 (dd, J = 8.7, 7.7, 1H), 3.94 (dd, J = 11.8, 2.0, 1H), 3.70 (s, 6H), 3.68-3.65 (m, 1H), 3.49 (dd, J = 14.0, 5.4, 1H), 3.38-3.19 (m, 3H; overlapped with CH3OH resonance), 2.94 (m, 1H). [C 27 H 31 O 13ESILRMS m / z calcd for ][M+H]: 563.52; found: 563.18. 13 C NMR (126MHz, CD3OD)=176.65,148.53,147.10,146.86,134.30,133.01,130.69,128.44,110.14,109.67 ,107.90,101.40,100.39,76.51,73.62,71.62,70.39,68.50,61.62,55.60,43.69,41.03,37.90,19.84.

[0285] Tellurium-labeled teniposide (1): In a 1 dram vial, zinc chloride (12 mg, 0.89 mmol, 2.0 equiv) and a stirrer were added and flame dried for 2 minutes. The flask was immediately placed under vacuum for at least 30 minutes. 4-Demethyl epipodophyllotoxin (25 mg, 0.44 mmol, 1.0 equiv) was added quickly and the vial was purged several times with argon. Aldehyde 4 (about 0.250 g) was added dropwise and the vial was sonicated carefully to ensure that all solid materials were in suspension. The reaction was set to stir gently at room temperature for 16 hours. The solution was diluted with DCM (10 mL) and washed with brine (10 mL). The aqueous layer was separated and washed twice with DCM (10 mL). The organic portions were pooled together, dried over MgSO4 and concentrated. The residue was purified by silica gel column chromatography using a gradient of 10% to 50% ethyl acetate in pentane to afford 1 (16%) as a light yellow waxy solid. 1H NMR (500 MHz, C2D6SO) = 8.89 (dd, J = 6.6, 1.4, 1H), 8.23 ​​(s, 1H), 7.87-7.58 (m, 2H), 7.01 (s, 1H), 6.52 (s, 1H), 6.17 (s, 2H), 6.10-5.93 (m, 2H), 5.64 (s, 1H), 5.24 (s, 2H), 4.93 (d, J = 3.4, 1H), 4.60 (d ,J=7.6,1H),4.48(d,J=5.4,1H),4.33–4.21(m,2H),4.20(dd,J=10.2,4.8,1H),3.74(t,J=10.1, 1H), 3.60 (s, 6H), 3.48–3.23 (m, 5H), 3.09 (t, J = 8.0, 1H), 2.88 (dddd, J = 13.7, 10.0, 8.2, 3.4, 1H). 13C NMR (126MHz, C2D6SO)=175.18,148.18,147.71,147.57,146.59,136.67,135.50,135.09,133.27,130.67,129.31, 129.16,110.35,108.77,101.78,101.76,101.73,80.76,74.88,73.15,72.13,68.18,68.09,66.14,55.50,40.89. [C 32 H 32 O 13 130 TeNa] + [M+Na] + ESI HRMS m / z calcd: 777.081; found: 777.079.

[0286] In vitro experiments

[0287] Top2 off-chain assay: The assay was performed according to the manufacturer's protocol (TopoGEN, Inc.). The total reaction volume was 20 pL assay buffer (120 mM KCl, 50 mM Tris-HCl, 10 mM MgCl2, 0.5 mM DTT, 0.5 mM ATP, and 30 pg / mL BSA) and 120 ng kinesiosome DNA (kDNA) substrate. One unit of TopoII enzyme was added to the reaction microcentrifuge tube in the presence or absence of inhibitors. The reaction was incubated at 37°C for 30 min. The reaction was terminated by adding 5 pL of stop buffer (5% sarkosyl, 0.025% bromophenol blue, and 50% glycerol). The samples were then analyzed using electrophoresis in Tris-borate-EDTA buffer with 0.5 pg / mL ethidium bromide stain using 1% agarose gel. The gel was imaged using a Syngene G:Box gel imager (Chemi-XT4GENESys software, pre-set ethidium bromide-stained agarose gel). The band intensity was analyzed using ImageJ software.

[0288] WST-1 metabolic cytotoxicity assay: HL-60 cells ( CCL-240TM) were maintained in DMEM medium (Gibco) with 2 mM L-glutamine supplemented with 20% calf serum and 100× diluted penicillin-streptomycin solution (Gibco). The cells were maintained at 37°C in an atmosphere of 5% CO2 in humidified air. HL-60 cells (100 pL) were cultured at 5×10 5 The density of cells / mL was inoculated into a 96-well transparent plate. The cells were treated with DMSO or an appropriate inhibitor from a DMSO stock solution. Dilute carefully so that the DMSO concentration does not exceed 1%. The cells were incubated for 24 hours at 37°C, 5% CO2 atmosphere. WST-1 reagent (10pL; Roche Diagnostics, 05015944001) was added to each well and gently mixed by pipetting. The cells were incubated for 1 hour at 37°C, 5% CO2 atmosphere. The UV-vis absorbance measurement results of each well at 450nm were recorded using a TECAN Safire 2 plate reader. The data were background corrected for wells containing cell growth medium without cells and WST-1. The experiment was performed in triplicate.

[0289] IncuCyte ZOOM TMCell proliferation assay: Human pancreatic ductal carcinoma (PANC-1) cell line was purchased from ATCC (CRL-1469). Cells were cultured in RPMI culture medium supplemented with 10% FBS. Cell maintenance and experiments were performed in a humid 37C incubator with 5% CO2. Mycoplasma contamination of cells was routinely tested. PANC-1 cells (5000) were inoculated in 96-well plates and incubated for 18h. The culture medium was then removed and replaced with fresh culture medium containing drugs (0-25pM). The cells were then transferred to the IncuCyte ZOOM system (ESSEN BioScience, Ann Arbor, MI, USA), and live cell phase contrast images were obtained using a 10× objective lens. Cell confluence was analyzed using IncuCyte ZOOM 2016B software.

[0290] pH2AX blotting: PANC-1 cells (0.5×10 6 Cells) were seeded in 60 mm plastic culture dishes (Corning Inc. NY) and incubated for 18 h. The spent medium was removed and replaced with fresh medium containing the drug (1 pM) and incubated for 24 or 48 h. Control cells were treated with DMSO only. The cells were then lysed with RIPA buffer (25 mM Tris·HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) (ThermoFisherScientific) containing Halt protease inhibitor cocktail (Catalog No. 78410) and Halt phosphatase inhibitor cocktail (Catalog No. 78420) according to the manufacturer's protocol. The extracted protein samples were electrophoresed using Bolt 4–12% Bis-Tris, 1.0 mm gels (ThermoFisherScientific) and immunoblotted as recommended. Antibodies targeting the following proteins were used (Abcam, Cambridge, MA): H2AX (S139) (ab11174, polyclonal) and β-tubulin (ab6046, polyclonal).

[0291] AlamarBlue assay: PANC-1 cells were seeded into black, clear-bottomed 96-well assay plates (Corning; CLS3603) and incubated for 24 hours. The medium was replaced with 0.01-25 pM teniposide or compound 1. After 72 hours of drug exposure, alamarBlue (Thermo Fisher Scientific; DAL1100) cell viability reagent was added and incubated at 37°C for 4 hours. Fluorescence intensity was measured using an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Cell viability was calculated by normalization to untreated controls.

[0292] Marking: For presaturation experiments, 3 × 10 6 HL-60 cells (5×10 5 Cells / mL) were incubated at 37°C in a 5% CO2 atmosphere in humidified air for 2 hours with the appropriate teniposide concentration. Compound 1 was pipetted into the cell suspension and gently mixed. The cells were further incubated for 2 hours, then centrifuged at 300×g for 6 minutes, and the culture medium was aspirated. For the control, HL-60 cells were treated with teniposide or compound 1 for 4 hours continuously. For the co-incubation experiment, the cells were incubated with the drug mixture for 4 hours. The drug-treated cells were washed with culture medium and then washed with PBS. The cells were fixed with 3.7% formaldehyde (Sigma Aldrich F1635) diluted in PBS for 10 minutes. The fixed cells were centrifuged at 800×g for 5 minutes and washed with PBS. The cell pellet was then stained with Ir embedding agent (diluted 1:1000 in PBS) for 1 hour at room temperature. The cells were centrifuged at 800×g for 5 minutes, then washed twice with PBS, and finally washed with ddH2O. The cell pellet was taken up in a 10% solution of EQTM four-element calibration beads prepared in CAS and filtered through 35 pm cell cap strainer caps into polystyrene tubes. The sample was then injected into The samples were analyzed in HeliosTM.

[0293] In vivo experiments

[0294] Generation of murine PANC-1 xenograft model: All 6-week-old female NOD SCID mice (Charles River (Wilmington, MA)) were maintained under specific pathogen-free (SPF) conditions at the Princess Margaret Cancer Centre (PMCC) facility. All animal experiments in this study were performed according to the guidelines of the Animal Research Committee of the University of Toronto Health Network. As described in the previous Telox study, subcutaneous PANC-1 xenograft mice were produced. The vehicle was prepared as follows: 30 mg benzyl alcohol, 60 mg N, N-dimethylacetamide, 500 mg purified Cremophor EL (Kolliphor EL) and 2.1 mL of anhydrous alcohol were combined in a vial. Distilled water was added to the solution, and the final volume was 5 mL. The pH of the clear solution was adjusted to pH about 5 using maleic acid. For injection, the drug was diluted with the vehicle, the final concentration was 10 mg / mL, and it was administered with 20 mg / kg IP. The mice were maintained in three groups; group 1 mice were injected with teniposide, group 2 mice were injected with 1, and group 3 mice were injected with saline. After 2 hours, the mice were sacrificed and the tumors were extracted. Half of the tumors were fixed and embedded in paraffin, while the remaining xenograft tissue was embedded in OCT (Tissue-Tek Sakura-Finetek) and snap-frozen in liquid nitrogen. The snap-frozen tumor samples were stored at -80°C. Cryostat sections (5 pm) were cut using a microtome and mounted on microscope slides. These sections were stored at -80°C until IMC was performed. TM Analysis or histochemical staining. Sections were stained with H&E to assess tissue morphology and pH2AX IHC to visualize pH2AX puncta. Adjacent sections were stained for IMC TM Analysis. Optical imaging of tissue sections: Tissue sections were fixed and blocked as previously described. Unconjugated rabbit anti-mouse H2AX (pSer139) antibody (Abcam, ab11174) and HRP-conjugated anti-rabbit IgG were used for color development with 3,3-diaminobenzidine chromogen. The entire stained section was then scanned at 20× magnification (approximately 0.5 pm / pixel) using ScanScope AT2 (Aperio) and viewed using ImageScope software.

[0295] IMC TM Analysis: Frozen tissue sections (5 μm) were thawed from -80°C to room temperature and directly analyzed in HYPERION TM IMC was performed on an imaging system (Fluidigm) TM No DNA staining or washing steps are required for analysis.TM For tellurium sensitivity, serial dilutions of a tellurium standard solution (Sigma Aldrich Cat. No. 92027) were prepared in trypan blue. Volumes of 2 pL were arrayed on air-dried 2% agarose-coated slides, with final concentrations ranging from 0-50 μM.

[0296] IMC TM Data acquisition and analysis: using HYPERION TM The imaging system (Fluidigm) ablated the slides at 200 Hz and acquired images as .txt files. Each image was unpacked into a separate numpy array for each mass channel using the teimc package by Bassan and Nitz. 25 These numpy arrays were used to prepare raw images and subsequently analyzed using Numpy, pandas, Matplotlib, and Scikit-image libraries. For the tellurium standard curve, the entire point was ablated and the entire cross-sectional area was plotted. 125 Te counts are summed up ( 125 For blank white patches ([Te] = 0 μM), the total 125 The Te signal was divided by the number of pixels in the ablation cross-sectional area to calculate the average value for each pixel. 125 Te intensity. This value indicates the intensity corresponding to a specific HYPERION TM The background signal per pixel in the 125amu channel of the imaging system ( 125 Te background). To calculate the contribution of this background in the tellurium-containing images, we applied the following correction for each concentration, 125 Te 校正 (i)= 125 Te 总计 (i)–( 125 Te 背景 x Area(i)

[0297] The resulting series 125 The corrected values ​​were plotted against the total number of Te atoms calculated from the volume of the dispensed standard solution. A linear regression model was fitted to the data points within the linear limit to calculate the number of tellurium atoms in the xenograft sections. Finally, to calculate [Te] in tissue sections, the tissue volume was extrapolated by multiplying the cross-sectional area (1 mm × 1 mm) by the tissue section thickness (5 μm).

[0298] Example 2: Tellurophene-tagged Carfilzomib analogs: Synthesis and evaluation

[0299] Monitoring target engagement in vivo is a challenging, multidimensional problem. Few methods are available to quantify engagement at the cellular level. The use of tellurium in combination with mass cytometry enables characterization of engagement at the cellular level in heterogeneous samples.

[0300] L-2-Tellurophenylalanine (TePhe) has been shown to act as a bioisostere of phenylalanine (Phe). A TePhe-containing version of the peptide drug carfilzomib has been synthesized. Carfilzomib is an irreversible inhibitor of the β5 chymotrypsin-like site of the proteasome and is an FDA-approved drug for multiple myeloma.

[0301] TePhe substituted carfilzomib analogs (TeCar, compound of formula II, 7b) were synthesized according to Scheme 2. The final peptide coupling of the epoxide warhead resulted in isomerization of the penultimate residue. This epimer (8b) was used as a control in biological studies.

[0302]

[0303]

[0304] Scheme 2 - Synthesis of TeCar analog 7b (compound of formula II)

[0305] In vitro experiments using purified proteasomes resulted in the expected reduction in activity in the presence of a covalent inhibitor. The activity observed was indistinguishable from that of carfilzomib ( Fig. 9 ).

[0306] In Jurkat cells treated with increasing concentrations of TeCar 7b, a concentration-dependent decrease in overall cell viability was observed (Table 1). Compared to carfilzomib, similar concentrations of 7b (Te-Car) were required to produce a 50% decrease in Jurkat cell viability.

[0307] Table 1 - Viability of Jurkat cells treated with inhibitors

[0308] Biological replicates# <![CDATA[IC 50 Carfilzomib (nM)]]> <h2 style=";text-align:left;direction:ltr"><![CDATA[IC <h2 style=";text-align:left;direction:ltr"> 50 <h2 style=";text-align:left;direction:ltr"> 7b(nM)]]><h2 style=";text-align:left;direction:ltr"> 1 9.127 9.481 2 9.992 9.869 3 9.412 9.777 Mean + / - SD 9.510+ / -0.441 9.709+ / -0.293

[0309] Analysis of TeCar-treated Jurkat cells by mass cytometry showed that intracellular activity was dependent on tellurium accumulation. Co-administration of cells with Te-Car 7b and Car resulted in a 50% reduction in Te labeling, confirming on-target labeling of the modified compound ( Fig.10 ). This evidence supports that TeCar can be used to monitor target engagement of carfilzomib in more complex and disease-relevant samples.

[0310] method

[0311] General method of synthesis

[0312] Unless otherwise specified, all reagents were purchased from Sigma-Aldrich. 2(g) , before the reaction in solution with Ar (g) , or with He before purification by reverse phase high performance liquid chromatography (RP-HPLC) (g) The solvent was degassed by bubbling. Resin swelling and SPPS were performed in a sintered polypropylene column purchased from Bio-rad. SILIAFLASH from Silicycle was used. TM P60 for flash column chromatography. TM The solvent was evaporated on a rotary evaporator. The spectrophotometer was used with a Waters XBRIDGE TM Prep BEH130C 18 RP-HPLC was performed using a Waters 1525 binary HPLC pump with a 10×250 mm column. TM Freeze drying was performed on a freeze dryer. Bruker AUTOFLEX SPEED TM Low-resolution and high-resolution mass spectra were collected using a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) or an Agilent 6538Q-TOF mass spectrometer coupled to an electrospray ionization (ESI) source. TM Nuclear magnetic resonance (NMR) spectra were collected on a 500 MHz Agilent DD2 spectrometer with a C13 cryoprobe or on a 700 MHz Agilent DD2 spectrometer.

[0313] The synthesis of 5b was carried out according to 10. Once isolated via crystallization and used 1 H NMR (500 MHz in CDCl 3 ) confirmed sufficient purity and 5b was used in the subsequent SPPS step without further purification.

[0314] Synthesis, purification and characterization of 6a and 6b

[0315] SPPS was used to synthesize 6a and 6b. Briefly, Fmoc-L-Phe-OH (5a) (1 equivalent, 77 mg) or Fmoc-L-TePhe-OH (5b) (1.25 equivalents, 100 mg) was dissolved in anhydrous (anh.) DCM (2-2.5 mL). Diisopropylethylamine (DiPEA) (4 equivalents, 110-139 μL) was added to the solution, and the solution was added to dry 2-chlorotrityl resin (163-200 mg, 1 mmol / g) in a sintered polypropylene tube (10 mL). The tube was then inverted at room temperature (rt) for 2 hours. The reaction mixture was evacuated from the column using vacuum filtration, and the column was evacuated by N ion exchange before vacuum filtration. 2(g) The resin in the capping mixture (17:2:1anh.DCM / MeOH / DiPEA) was bubbled through for one minute to wash the resin three times. The resin was then washed repeatedly with DCM and DMF in a similar manner. The resin was then inverted for 20 min in a 20% piperidine solution (3 mL) in anh.DMF at rt to deprotect the Fmoc group. Similarly, the solvent was emptied and the resin was washed repeatedly with DMF and DCM. Next, Fmoc-L-Leu-OH (5 equivalents) was coupled at rt for 1 h using HOBt.H2O (5 equivalents), pyBOP (Fluka) (5 equivalents) and collidine (10 equivalents) in anh.DMF ([resin] = 0.04M), then the resin was washed, and the Fmoc group was deprotected as above. These steps were then repeated using Fmoc-L-homophenylalanine-OH (Alfa Aesar) (5 eq.), followed by 4-morpholineacetic acid hydrochloride (Alfa Aesar) (5 eq.) and additional collidine (15 eq.). For each of the three couplings, the carboxylic acid was first activated with HOBt / pyBOP in the presence of collidine at rt for 8 min before being added to the resin.

[0316] The resin was then washed 10x with 1% trifluoroacetic acid (TFA) in anh.DCM (1 mL) and fractions from each wash were collected into a round bottom flask. The solution was evaporated and rediluted multiple times with toluene before being thoroughly dried under vacuum to yield a yellow oil.

[0317] The resulting oil was then evaluated by MALDI-TOF-MS to confirm the presence of the desired peptide (6a or 6b). The crude peptide dissolved in 1:1 MQ H2O / ACN was co-crystallized with an α-cyano-4-hydroxycinnamic acid (CHCA) matrix. The 567 m / z peak ([M+H]) confirmed the presence of 6a, while the peaks at 667, 669, 671 and 689, 691, 693 m / z ([M+H] and [M+Na] of the 3 most abundant Te isotopes, respectively) confirmed the presence of 6b.

[0318] 6a and 6b were then purified using flash silica gel chromatography or RP-HPLC.

[0319] For flash silica gel chromatography, a solvent system of 9:1 DCM / MeOH+1% AcOH was used, and product elution was assessed using thin layer chromatography and visualized using short wave UV light and KMnO4 staining.

[0320] For RP-HPLC, a 90 min gradient of 98% to 2% solvent A (MQ H2O + 0.1% TFA) in solvent B (ACN + 0.1% TFA) was used and product elution was assessed by monitoring at 213 and 254 nm (6a) or 213 and 280 nm (6b) wavelengths.

[0321] Similar fractions from either method were combined, concentrated and assessed for purity using MS. Similar purity levels were obtained using both methods, with flash silica gel chromatography being more convenient.

[0322] Later, the 500 MHz acquisition was performed at a concentration of about 0.02 M in D6-DMSO. 1 The structure of 6b was analyzed by H NMR. 1HNMR(700MHz,dmso)δ10.21(bs,1H),8.83(bs,1H),8.61(dd,J=6.9,1.3Hz,1H) ,8.32(d,J=8.0Hz,1H),8.18(d,J=8.5Hz,1H),7.50(dd,J=6.9,3.8Hz,1H),7.40 (dq,J=3.8,1.3Hz,1H),7.33-7.26(m,2H),7.22-7.14(m,3H),4.43(dtd,J=11. 4,8.3,5.5Hz,2H),4.33(ddd,J=9.1,8.0,4.4Hz,1H),3.98(bs,2H),3.90(bs,2H ),3.77(bs,3H),3.34(ddd,J=15.0,4.4,1.1Hz,1H),3.18(bs,4H),3.11(ddd,J =15.0,9.1,1.3Hz,1H),2.61(ddd,J=13.5,11.3,5.2Hz,1H),2.55(ddd,J=13.7, 11.0,5.9Hz,1H),1.95(ddt,J=13.4,11.0,5.4Hz,1H),1.89-1.76(m,1H),1.61( ddt,J=14.8,13.0,6.4Hz,1H),1.52-1.40(m,2H),0.87(dd,J=26.9,6.6Hz,6H). HRMS:C 29 H 40 N4O6 130 m / z calculated for Te(M+H)+: 671.5636; found: 671.21.

[0323] 7a / 8a (carfilzomib and its diastereomers) and 7b / 8b (Te-carfilzomib analogs and its diastereomers) Synthesized in solution

[0324] One equivalent of 6a (30 mg) or 6b (45 mg) (both white solids) was dissolved in degassed anh.DMF (1.5 mL), and then a solution of HOBt anh. (2 equivalents), pyBOP (2 equivalents) and DiPEA (5 equivalents) in degassed anh.DMF (2 mL) was added. The reaction was allowed to proceed at rt, Ar (g) The mixture was stirred for five minutes at RT, followed by the addition of (2S)-2-amino-4-methyl-1-[(2R)-2-methyloxiranyl]-1-pentanone trifluoroacetate (1.3 eq.) dissolved in degassed anh.DMF (1.5 mL). The final concentration of peptide 6a or 6b was approximately 0.01 M. The reaction was stirred at rt, Ar (g)The reaction mixture was stirred for up to 5 h at 4 °C. MALDI-TOF-MS was used to monitor the reaction per hour using the same parameters as previously described. Once the complete loss of the starting material was observed, the reactant was absorbed into ethyl acetate (10 mL) and washed twice with deionized water (10 mL x 2). The ethyl acetate layer was then washed with an equal-volume saline solution and the mixture was concentrated to a volume of 100 mL. The reaction mixture was concentrated to a volume of 100 mL. 4(s) Drying, filtration and evaporation of the solvent gave a crude yellow oil which was then further dried under high vacuum to give a yellow oil with white crystals. The crude was stored at -20°C until purification.

[0325] Purification and characterization of 7a / 8a and 7b / 8b

[0326] The crude product was dissolved in a minimum volume of ACN (about 2 mL) and aliquoted into fractions (100 uL). Each fraction was purified by MQ TM HO (about 0.9 mL) and filtered using a 0.22 um syringe before injection into RP-HPLC. The solvent system used was: solvent A, 0.1% TFA in MQ HO; and solvent B, 0.1% TFA in ACN. The following gradient method was used:

[0327] 0-10min, maintain 98% A

[0328] 10-40min 98%-50% A

[0329] 40-60min, maintain 50% A

[0330] 60-100min 50-2% A

[0331] 100-110min, maintain 2% A

[0332] Monitor peaks at 213nm and 254nm (7a / 8a) or 213nm and 280nm (7b / 8b). Collect only peaks at at least A 213 ≥ 0.5 with characteristic peaks at 213 nm and 254 or 280 nm and were evaluated using MALDI-TOF-MS using the parameters specified above. Peaks eluting at about 60 min (7a or 8a) and about 64 min (7b or 8b) were found to contain masses consistent with the desired product.

[0333] 7b: 1H NMR(700MHz,dmso)δ8.60(d,J=6.8Hz,1H),8.24(s,1H),8.13(d,J=8.3Hz,2H),7.50(dd,J=6.9,3.8Hz,1H),7.37(d,J=5.0Hz,1 H),7.28(t,J=7.6Hz,2H),7.18(dd,J=16.5,7.7Hz,3H),4.37(s,4H),4.03(bs,1H),3.91(bs,1H),3.74(bs,3H),3.22(dd,J=15 .3,3.7Hz,3H),3.14(d,J=5.2Hz,2H),3.00-2.95(m,3H),2.53(bs,4H),1.94(s,1H),1.82(s,1H),1.62(s,1H),1.57(s,1H),1. 42(d,J=7.4Hz,1H),1.40(s,3H),1.37-1.32(m,1H),1.31-1.26(m,1H),1.24(bs,1H),0.92-0.85(m,6H),0.82(t,J=7.0Hz,6H). HRMS:C 38 H 55 N5O7 130 m / z calculated for Te(M+H)+: 824.7845; found: 824.32.

[0334] 8b: 1H NMR(700MHz,dmso)δ8.69(dd,J=6.9,1.3Hz,1H),8.25(bs,1H),8.18(bs,1H),8.12(d,J= 8.0Hz,1H),7.49(dd,J=6.9,3.8Hz,1H),7.36(dd,J=3.8,1.3Hz,1H),7.27(t,J=7.6Hz,2H ),7.21-7.14(m,3H),4.44(td,J=9.0,4.6Hz,1H),4.38(dtd,J=21.8,8.1,4.3Hz,2H),4.3 0(bs,1H),3.88(bs,2H),3.74(bs,3H),3.20(dd,J=14.2,4.4Hz,3H),3.12(d,J=5.3Hz,2H ),2.99(dd,J=14.5,9.6Hz,1H),2.91(d,J=5.2Hz,1H),2.59-2.50(m,2H),1.92(ddt,J=13 .5,10.9,5.5Hz,1H),1.80(dddd,J=13.7,10.9,8.7,5.3Hz,1H),1.54(dtd,J=9.5,6.8,4. 8Hz,1H),1.39(d,J=6.7Hz,1H),1.37(s,3H),1.33(dd,J=9.5,3.6Hz,1H),1.23(bs,1H),1 .29(t,J=7.3Hz,3H), 0.84(d,J=6.6Hz,3H), 0.79(d,J=6.6Hz,6H), 0.77(d,J=6.5Hz,3H). HRMS:C 38 H 55 N5O7 130 m / z calculated for Te(M+H)+: 824.7845; found: 824.32.

[0335] Subsequent runs had consistent and reproducible HPLC profiles, so the later HPLC run was stopped at the 90 min mark and the column was flushed with 100% B for 10 min before starting the next purification.

[0336] Since the carfilzomib ("a") and Te-carfilzomib ("b") compounds had two separate peaks eluting at the correct mass, they were kept separate and similar fractions were referred to as carfilzomib peak 1 (7a) and peak 2 (8a) and Te-carfilzomib (TeCar) peak 1 (7b) and 2 (8b). All four pooled fractions were lyophilized to give a fluffy white powder and evaluated using ESI-MS to further confirm quality and purity.

[0337] 7b and 8b also use1 H and 13 C NMR (1D, gCOSY and HSQC) were evaluated to confirm the fractions were diastereomers.Due to the small amount of product isolated after HPLC, these NMRs were collected on a 700 MHz instrument in D6-DMSO with sample volumes of 190-200 uL.

[0338] Due to low combined yields from HPLC isolation, 7a and 8a were not evaluated using NMR.Carfilzomib (Focus Biomolecules) was subsequently purchased for further use in bioassays and NMR was used for comparative purposes.

[0339] Stock solutions of 7b (232 uM), 8b (384 uM), and purchased carfilzomib (8800 uM) were prepared in DMSO and stored at -80°C until needed. The concentration of the carfilzomib stock solution was determined based on weighed mass, while the concentrations of the 7b and 8b solutions were confirmed using amino acid analysis, using the carfilzomib stock solution as a standard to confirm accuracy.

[0340] Store at -80°C for up to 8 months 1 H NMR confirmed that no degradation occurred under these storage conditions.

[0341] General Methods in Biology

[0342] Purified human 26S proteasome was purchased from Novus TM The β5 site active probe succinate-leucine-leucine-valine-tyrosine-7-amino-4-methylcoumarin (Succ-LLVY-AMC) was purchased from Enzo LifeSciences TM , and 7-amino-4-methylcoumarin (AMC) was purchased from Sigma-Aldrich TM WST-1 cell viability reagent was purchased from Roche TM 96-well plates were purchased from Corning or Starstedt, and all 96-well plate measurements were performed in Data were processed using Microsoft Excel and GraphPad Prism.

[0343] Jurkat cells (CRL-2899) purchased from ATCC were cultured in an atmosphere of 5% CO 2(g)The cells were maintained in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin in a humidified 37°C incubator and cultured in culture flasks purchased from Starstedt according to ATCC guidelines.

[0344] Cell-ID TM The intercalating agent Ir was purchased from Fluidigm. Cytometry time-of-flight (CyTOF) measurements were recorded on the instrument. CyTOF data were processed and analyzed using FlowJo and GraphPad Prism software.

[0345] In vitro binding assay using purified proteasomes

[0346] 26S proteasome (2nM, 25uL) and different concentrations of carfilzomib, 7b or 8b (1-100nM, 25uL) or blank control (1% v / v DMSO, 25uL) in buffer (50mM Tris-HCl buffer + 1mM DTT + 5mM MgCl2 + 40mM KCl + 2mM ATP + 0.5mg / mL BSA) were mixed in a 96-well plate and incubated at room temperature for 15min. The substrate peptide Suc-LLVY-AMC (200uM, 50uL) in buffer was then added to initiate the enzymatic reaction. The plate was placed in an incubator set at 37°C to complete the remainder of the experiment, except when taken out to obtain fluorescence measurements. The final well volume was 100 uL and the final concentrations of all reactions immediately after addition of substrate were: 0.5 nM (1 ug / mL) proteasome, 0.25-25 nM inhibitor, and 100 uM Succ-LLVY-AMC substrate.

[0347] In addition to the blank control ([inhibitor] = 0 nM), the following controls were tested as part of this experiment. The autohydrolysis of the substrate was evaluated by incubating the substrate in buffer (100 uM) alone. Any chemical interaction between 7b or 8b and the substrate was evaluated by incubating the drug (50 nM, 50 uL) with the substrate (200 uM, 50 uL) in buffer. The substrate for each of these controls was added to the corresponding wells simultaneously with the above experimental wells.

[0348] A standard curve of AMC (0-5uM, 100uL) was also prepared and run simultaneously with the assay.The standards were plated during the last 5min of the 15min incubation, just before the addition of substrate to the experimental wells.

[0349] Then at time "zero" (about 3 min after adding substrate), the microplate reader reads ex350nm and λ em Fluorescence measurements were recorded at 440 nm and then every 15 min for a total of 75 min.

[0350] The standard curve demonstrates the linear time dependence of [AMC] in solution and its corresponding fluorescence reading over the experimental range. (See Fig.11 )

[0351] Each experimental and control well was corrected for autolysis by subtracting the fluorescence value of the autolysis control well at the corresponding time point before further analysis.

[0352] The apparent initial velocity (v) of the uninhibited enzyme was first determined by plotting the mean of the corrected fluorescence readings (FU, arbitrary fluorescence units) against time. o ), such that:

[0353] FU=v o *t+constant

[0354] The slope of the curve is v o (uM substrate / min).

[0355] The remaining active enzyme v in each well after dosing with either inhibitor in the concentration range of 1.15 nM to 1.15 uM was determined in a similar manner. o Kinetic parameters. Then v o Loss (due to [E] 总计 decrease, and therefore [E] 活性 Reduction) is compared to the uninhibited enzyme as a percentage reduction and is calculated using:

[0356] Percent reduction v o =(v o(未抑制) -v o(抑制) ) / v o(未抑制) *100%

[0357] This assay was repeated three times in total. The average percent change between these three biological replicates is shown in Fig.10 .

[0358] Cell viability of Jurkat cells

[0359] Jurkat cells with a concentration of about 12500 cells / mL are inoculated into complete medium in 96-well plates, wherein carfilzomib or 7b (final volume 100uL) of increasing concentration (0-200nM) are dosed, and incubated for 44 hours. Each concentration is repeated in triplicate. Then 10uL WST-1 is added to each well, and the plate is incubated for 2 hours, and then the absorbance at 440nm is measured. First, the absorbance signal of 100uL complete medium+10uL WST-1 is subtracted from each well, and then the absorbance signal is plotted against the logarithm (concentration). Then GraphPad Prism software is used to determine the concentration required for seeing a 50% reduction in absorbance signal. These values ​​are shown in Table 1.

[0360] Incorporation of 7b and 8b into living Jurkat cells with or without carfilzomib

[0361] 24 hours before drug administration, Jurkat cells were seeded in 75 mm 2 In plastic culture flasks, the concentration was about 5 million cells / mL. In complete medium, cells were dosed with carfilzomib, 7b or 8b at a final concentration of 500 nM, or the combination carfilzomib / 7b (500 nM each), carfilzomib / 8b (500 nM each), or DMSO control (final percentage 0.5%) for 1 hour.

[0362] The culture medium was then immediately removed and the cells were suspended in 3 mL of cold phosphate buffered saline (PBS) before being aliquoted into three 1 mL aliquots according to each dosing regimen. The samples were then prepared for analyze.

[0363] Briefly, each sample aliquot was washed three times with cold PBS (1 mL x 3). The cells were then fixed, permeabilized and treated with Cell-ID TM Intercalator Ir dye staining was performed at rt for 1 hour. The cell pellet was then washed two to three times with cold PBS (1 mL x 2-3) and then washed once with cold MQ H2O (1 mL). The pellet was then stored at 4°C overnight.

[0364] The next day Analysis. Cell pellets were suspended in 250-500 uL of bead solution in PBS and filtered immediately before injection into the instrument. Approximately 40,000 events were collected for each sample. The average 128 Te signal ( 191 Ir and 193 events with positive Ir signal).

[0365] Exemplary Systems

[0366] Reference now Fig.12 In some embodiments, a system for imaging tissue or cells includes an imaging mass spectrometry flow cytometry system 100 and a controller (e.g., a computer system) 200 that is connected and communicates with the imaging mass spectrometry flow cytometry system 100 via a wired or wireless connection. As will be discussed in further detail herein, the controller 100 controls the operation of the imaging mass spectrometry flow cytometry system 100, receives images from the imaging mass spectrometry flow cytometry system 100, and displays the received images.

[0367] Fig.13 An exemplary imaging mass spectrometry flow cytometry system 100 is depicted according to some embodiments of the present disclosure. The system 100 includes a radiation source (e.g., a UV laser, a femtosecond laser, an excimer laser, etc.) configured to emit radiation (also referred to as "imaging light") along a first path 104 toward a sample 106 located within a cell (e.g., a flow cell) 108. In some embodiments, the sample 106 can be positioned on a movable stage (e.g., an XYZ stage) 110 disposed within the cell 108.

[0368] The radiation source 102 is configured to deliver radiation for ablating and / or causing the sample 106 to fluoresce. In some embodiments, where the radiation source 102 is a UV laser, the radiation source 102 can operate at a wavelength of 213 nm. The irradiation of various patch sizes can be achieved using a mechanically controlled aperture or an array of interchangeable apertures and / or an objective lens (e.g., along the beam path 104) with appropriate magnification to determine the patch size, or alternatively, multiple laser shots can be scanned over the ablation area corresponding to one pixel by rapidly dithering the optics.

[0369] The system 100 may also include a shutter (e.g., a grating shutter) 112 disposed along the path 104 between the radiation source 102 and the sample 106. The shutter 112 provides energy stability to the system 100 by allowing the radiation source 102 to continue to operate while shutting off delivery to the sample 106 during movement of the stage 110. The shutter 112 may also serve as a safety feature that is activated when a safety interlock in the system 100 is triggered.

[0370] The system 100 also includes an attenuator 114, a beam shaping optical device 116. The attenuator 114 is disposed along the path 104 between the shutter 112 and the sample 106. The attenuator 114 is capable of changing the energy of the radiation emitted by the radiation source 102 so as to achieve accurate ablation conditions for a given sample 106. In some embodiments, the attenuator 114 operates based on polarization rotation and polarizer filtering of the radiation. The optical device 116 is configured to shape the emitted radiation to produce a focused spot directed toward the sample 106. The optical device 116 may include one or more objective lenses and / or apertures for focusing the emitted radiation.

[0371] The system 100 also includes a light source (e.g., an LED) 118 configured to emit light along a second path 120 toward the sample 106. The system 100 also includes a first beam splitter (e.g., a dichroic mirror, a semi-reflective mirror, etc.) 122 positioned between the optics 116 and the sample 106 along the first path 104 and between the light source 118 and the sample 106 along the second path. The first beam splitter 122 is configured to direct radiation received along the first beam path 104 and light emitted by the light source 118 along the second path 120 toward the sample 106 along a third path 124. The system 100 also includes a microscope objective 126 positioned between the first beam splitter 122 and the sample 106 along the third path 124. The microscope objective 126 focuses radiation from the radiation source 102 and light from the light source 118 onto the sample 106. Thus, in some embodiments, system 100 can have a common focusing path for imaging light emitted by radiation source 102 and light emitted by light source 118 .

[0372] The system 100 also includes a tube lens 128, a second beam splitter 130, and a camera (e.g., a camera based on a charge coupled device image sensor (CCD), a camera based on an active pixel sensor, etc.) 132. The tube lens 128 is positioned between the first beam splitter 122 and the second beam splitter 130 along the second path 120. The tube lens allows the optical image to be focused onto the camera 132. In some embodiments, the system 100 can employ an infinite conjugate setup using the tube lens 128. Such a setup can reduce some types of aberrations and can also improve image quality on the camera 132. The second beam splitter 130 can include, but is not limited to, a half-reflective mirror.

[0373] A second beam splitter 130 is positioned along the second path 120 between the tube lens 128 and the light source 118. The beam splitter directs light reflected from the sample 106 along a fourth path 134 to the camera 132.

[0374] The system 100 also includes a mass spectrometer 136 coupled to the cell 108. The mass spectrometer includes an injector 138, an inductively coupled plasma (ICP) ion source 140, and a detector 142. The injector 138 is configured to receive the ablation sample 106 from the chamber 108 and transfer the ablation sample 106 to the ICP ion source 140. The ICP ion source 140 receives the ablation sample 106 and generates a plurality of ions. A detector (e.g., a time-of-flight (TOF) analyzer) 142 is positioned downstream of the ICP ion source 140 and receives a plurality of ions. The detector 142 performs mass analysis on ions leaving the ICP ion source 140 based on the mass-to-charge ratio (m / z) of the ions.

[0375] The image provided by 132 and the mass analysis provided by detector 142 can be output to controller 200. Controller 200 can output the image and mass analysis to a display. This output can allow a user to visually determine the location and level of a given molecule (e.g., a small molecule analog discussed herein) within sample 106.

[0376] Reference now Fig.14 , a computer system 200 is shown according to an exemplary embodiment. The computer system 200 can be used as any computer system (e.g., controller 200) disclosed herein. As used herein, a computer system (or device) is any system / device capable of receiving, processing and / or sending data. Computer systems include, but are not limited to, microprocessor-based systems, personal computers, servers, handheld computing devices, tablet computers, smart phones, multiprocessor-based systems, mainframe computer systems, etc.

[0377] like Fig.14As shown, the computer system 200 includes one or more processors or processing units 202, a system memory 204, and a bus 206 that connects the various components of the computer system 200 (including the system memory 204) to the processor 202. The system memory 204 includes a computer-readable storage medium 208 and a volatile memory 210 (e.g., a random access memory, a cache, etc.). As used herein, a computer-readable storage medium includes any medium that can store computer-readable program instructions and can be accessed by a processor. The computer-readable storage medium 208 includes non-volatile and non-transient storage media (e.g., flash memory, read-only memory (ROM), hard drive, etc.). The computer program instructions as described herein include program modules (e.g., routines, programs, objects, components, logic, data structures, etc.) that can be executed by a processor. In addition, when the computer-readable program instructions are executed by a processor, the computer-readable program instructions can direct the computer system to operate in a specific manner so that the computer-readable storage medium constitutes an article of manufacture. Specifically, when the computer-readable program instructions are executed by a processor, the computer-readable program instructions can create a means for performing at least a portion of the steps of the method disclosed herein.

[0378] The bus 206 may be one or more of any type of bus structures capable of transferring data between components of the computer system 200 (eg, a memory bus, memory controller, peripheral bus, accelerated graphics port, etc.).

[0379] The computer system 200 may also include a communication adapter 212, which allows the computer system 200 to communicate with one or more other computer systems / devices via one or more communication protocols (e.g., Wi-Fi, BTLE, etc.), and in some embodiments, may allow the computer system 200 to communicate with one or more other computer systems / devices via one or more networks (e.g., a local area network (LAN), a wide area network (WAN), a public network (Internet), etc.).

[0380] In some implementations, the computer system 200 may be connected to one or more external devices 214 and a display 216. As used herein, an external device includes any device that allows a user to interact with the computer system (e.g., a mouse, keyboard, touch screen, etc.). The external devices 214 and the display 216 may communicate with the processor 202 and the system memory 204 via an input / output (I / O) interface 218.

[0381] The display 216 can display a graphical user interface (GUI) that can include a plurality of selectable icons and / or editable fields. A user can use an external device 214 (e.g., a mouse) to select one or more icons and / or edit one or more editable fields. Selecting an icon and / or editing a field may cause the processor 202 to execute computer-readable program instructions stored in the computer-readable storage medium 208. In one embodiment, a user can use an external device 214 to interact with the computer system 200 and cause the processor 202 to execute computer-readable program instructions associated with at least a portion of the steps of the method disclosed herein.

[0382] Although the present disclosure has been described with reference to the examples, it should be understood that the scope of the claims should not be limited by the embodiments set forth in the examples, but should be given the broadest interpretation consistent with the specification as a whole.

[0383] All publications, patents, and patent applications are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. When a term in the present disclosure is found to have a different definition in a document incorporated herein by reference, the definition provided herein serves as the definition of that term.

Claims

1. A method for detecting small molecule compounds in a sample, the method comprising the following steps: Providing subjects or cell / tissue culture samples; Providing a small molecule analog of a small molecule compound, wherein the small molecule compound has a structure comprising one or more monocyclic or bicyclic aromatic rings, and the small molecule analog has a structure in which at least one of the one or more monocyclic or bicyclic aromatic rings of the structure of the small molecule compound is replaced by tellurophene comprising a tellurium atom, thereby forming a tellurophene small molecule analog; administering the tellurophene small molecule analog to the subject or cell / tissue culture sample; providing a sample obtained from the subject or the cell / tissue culture specimen after administration of the tellurophene small molecule analog; performing mass spectrometry on the sample to determine the level of the tellurium atoms present in the sample, wherein the level of the tellurium atoms corresponds to the level of the tellurophene small molecule analog, and detection of the tellurophene small molecule analog indicates detection of the small molecule compound in the sample. 2 . The method of claim 1 , wherein the method further comprises quantifying the amount of the tellurium atoms in the sample, wherein the level of the tellurium atoms is indicative of the amount of small molecule compounds in the sample.

3. The method of claim 1, wherein the method further comprises quantifying one or more other analytes in the sample, the method further comprising labeling the sample with one or more analyte binders having a mass tag prior to performing mass spectrometry, and determining the levels of the one or more analyte binders.

4. The method of claim 1 , wherein the one or more analyte binders having a mass tag are selected from the group consisting of: metal-labeled antibodies, optionally polymer-labeled antibodies; metal-labeled oligonucleotides; polymer-labeled oligonucleotides; intercalators, such as 5-iodo-2′-deoxyuridine (IdU) and metal-containing intercalators (e.g., Rh-containing intercalators and Ir-containing intercalators); metal-containing viability indicators, such as cisplatin; barcode reagents, such as Cd-labeled CD45 and Pt-labeled CD45; and combinations thereof.

5. The method of claim 1, wherein the subject is a mammal, optionally a mouse, a rat or a human.

6. The method of claim 1 , wherein the mass spectrometry is performed at a plurality of discrete locations, and the level of the tellurium atoms is determined at each of the plurality of discrete locations to provide a distribution of the small molecule analogs in the sample, and wherein the distribution of the analogs in the sample indicates the distribution of the small molecule compounds in the sample. 7 . The method according to claim 1 , wherein the sample is a single cell, and the distribution of the small molecule analogs within the single cell indicates the distribution of the small molecule compound at the subcellular level.

8. The method of claim 1, wherein a plurality of samples are provided and the samples include different tissues, cells or secretions of the subject, and wherein the detection of the small molecule analogs in the plurality of samples provides the distribution of the small molecule analogs within the subject, the distribution indicating the distribution of the small molecule compound within the subject, such as tissue or organ distribution.

9. The method of claim 1, wherein the sample is or comprises urine, feces, blood or a fraction thereof, cerebrospinal fluid (CSF), saliva, muscle cells, fat cells, bone cells, hair, nails, skin cells, tumor cells or secretions, liver cells or secretions, heart cells, lung cells or secretions, pancreatic cells or secretions, and / or gastric cells or secretions.

10. The method of claim 1, wherein the sample is a frozen tissue section.

11. The method of claim 1, wherein the sample is or comprises cells from the cell culture or culture medium from the cell culture. 12 . The method according to claim 1 , wherein the sample is a single cell, and the distribution of the analogs within the single cell indicates the distribution of the small molecule compound at the subcellular level.

13. The method of claim 1, wherein the mass spectrometry is mass cytometry or multiplexed ion beam imaging, optionally the mass cytometry is mass cytometry imaging or mass cytometry suspension.

14. The method according to claim 1, wherein the tellurium atom is selected from 120 Te, 122 Te, 123 Te, 124 Te, 125 Te, 126 Te, 128 Te, 130 Te and combinations thereof.

15. The method of claim 1, wherein the tellurium atoms include a plurality of tellurium isotopes, and the mass cytometry is multi-channel mass spectrometry, optionally multi-channel mass cytometry.

16. The method of claim 1, wherein the one or more monocyclic aromatic rings are 5- or 6-membered aromatic rings, optionally the one or more monocyclic 5- or 6-membered aromatic rings are independently selected from thiophene, furan, pyrrole, phenyl or pyridine.

17. The method of claim 1, wherein the one or more bicyclic aromatic rings are independently selected from naphthyl, indole, benzothiophene, or benzofuran.

18. The method according to claim 1, wherein the structure of the small molecule compound comprises one or more monocyclic 5- or 6-membered aromatic rings, and at least one of the monocyclic 5- or 6-membered aromatic rings is replaced by tellurophene.

19. The method of claim 1, wherein the structure of the small molecule compound comprises one or more bicyclic aromatic rings, and at least one of the bicyclic aromatic rings is replaced by benzo[b]tellurophene or 4H-telluro[3,2-b]pyrrole.

20. The method according to claim 1, wherein the structure of the small molecule compound comprises one or more 5-membered aromatic rings, optionally, the one or more 5-membered aromatic rings are each independently selected from thiophene, furan or pyrrole.

21. The method of claim 1, wherein the one or more 5-membered aromatic rings are thiophene.

22. The method according to claim 1, wherein the small molecule compound is teniposide, and the tellurophene small molecule analog is as shown in Formula I: or a salt or solvate thereof.

23. The method according to claim 1, wherein the structure of the small molecule compound comprises one or more 6-membered aromatic rings, optionally the one or more 6-membered aromatic rings are each independently selected from phenyl or pyridine, optionally the one or more 6-membered aromatic rings are phenyl.

24. The method according to claim 1, wherein the small molecule compound is carfilzomib, and the tellurophene small molecule analog is as shown in Formula II: or a salt or solvate thereof.

25. The method of claim 1, wherein the small molecule analog interacts with a target in the sample irreversibly, optionally covalently.

26. The method of claim 1, wherein the subject or the cell / tissue culture sample has been administered a combination of the tellurophene small molecule analog and the small molecule compound, wherein the method further comprises directly detecting the level of the small molecule compound, and wherein a comparison of the level of the small molecule compound to the level of the analog indicates putative competitive binding of the analog.

27. Use of a tellurophene small molecule analog in a method for detecting a small molecule compound in a sample, comprising the following steps: Providing subjects or cell / tissue culture samples; Providing a small molecule analog of a small molecule compound, wherein the small molecule compound has a structure comprising one or more monocyclic or bicyclic aromatic rings, and the small molecule analog has a structure in which at least one of the one or more aromatic rings of the structure of the small molecule compound is replaced by a tellurium-containing tellurium atom, thereby forming a tellurium-containing tellurium-containing small molecule analog; administering the tellurophene small molecule analog to the subject or cell / tissue culture sample; providing a sample obtained from the subject or the cell / tissue culture specimen after administration of the tellurophene small molecule analog; Mass spectrometry is performed on the sample to determine the level of the tellurium atoms in the tellurophene small molecule analog.

28. Use according to claim 27, wherein said mass spectrometry is performed on a sample, optionally a plurality of samples, obtained from said subject or said cell / tissue culture specimen.

29. The use according to claim 27, wherein the subject is a mammal, optionally a mouse, a rat or a human.

30. The use according to claim 27, wherein the sample is as defined in claim 9.

31. The use according to claim 27, wherein the sample is as defined in claim 10.

32. The use according to claim 27, wherein the sample is as defined in claim 11.

33. The use according to claim 27, wherein the mass spectrometry is mass cytometry or multiplexed ion beam imaging, optionally the mass cytometry is mass cytometry imaging or mass cytometry suspension.

34. The use according to claim 27, wherein the one or more aromatic rings are as defined in claim 16.

35. The use according to claim 27, wherein the one or more aromatic rings are as defined in claim 17.

36. The use according to claim 27, wherein the structure of the small molecule compound comprises one or more 5-membered aromatic rings, and optionally, the one or more 5-membered aromatic rings are each independently selected from thiophene, furan or pyrrole.

37. The use according to claim 27, wherein the one or more 5-membered aromatic rings are thiophene.

38. The use according to claim 27, wherein the small molecule compound is teniposide, and the tellurphene small molecule analog is as shown in Formula I: or a salt or solvate thereof.

39. The use according to claim 27, wherein the structure of the small molecule compound comprises one or more 6-membered aromatic rings, optionally the one or more 6-membered aromatic rings are each independently selected from phenyl or pyridine, optionally the one or more 6-membered aromatic rings are phenyl.

40. The use according to claim 27, wherein the small molecule compound is carfilzomib, and the tellurophene small molecule analog is as shown in Formula II: or a salt or solvate thereof.

41. A compound of formula I or a salt or solvate thereof.

42. The compound according to claim 41, wherein the Te atom is an isotope, optionally the Te atom is selected from 120Te, 122Te, 123 Te, 124 Te, 125 Te, 126 Te, 128 Te, 130 Te and combinations thereof.

43. A composition comprising a compound according to claim 40, and a carrier or excipient.

44. A compound of formula II or a salt or solvate thereof.

45. The compound according to claim 44, wherein the Te atom is isotopically enriched, optionally the Te atom is selected from 120 Te, 122 Te, 123 Te, 124 Te, 125 Te, 126 Te, 128 Te, 130 Te and combinations thereof.

46. ​​A composition comprising a compound according to claim 42, and a carrier or excipient.

47. Use of a teniposide analog in a method for detecting teniposide by mass spectrometry, comprising the following steps: Providing a teniposide analog, wherein the teniposide analog is a compound according to claim 41, optionally in a composition comprising at least one carrier or excipient; Providing subjects or cell / tissue culture samples; administering the teniposide analog to a subject or a cell / tissue culture sample; providing a sample obtained from the subject or the cell / tissue culture specimen after administration of the teniposide analog; performing mass spectrometry on the sample to determine the level of the tellurium atoms present in the sample, wherein the level of the tellurium atoms corresponds to the level of the teniposide analog, and detection of the teniposide analog indicates detection of teniposide in the sample.

48. Use of a teniposide analog in a method for detecting teniposide by mass spectrometry, comprising the following steps: Provided is a teniposide analog, wherein the teniposide analog is The compound of claim 42, optionally in a composition comprising at least one carrier or excipient; Providing subjects or cell / tissue culture samples; administering the teniposide analog to a subject or a cell / tissue culture sample; providing a sample obtained from the subject or the cell / tissue culture specimen after administration of the teniposide analog; performing mass spectrometry on the sample to determine the level of the tellurium atoms present in the sample, wherein the level of the tellurium atoms corresponds to the level of the teniposide analog, and detection of the teniposide analog indicates detection of teniposide in the sample.

49. Use of a carfilzomib analog in a method for detecting carfilzomib by mass spectrometry, comprising the following steps: Provided is a carfilzomib analog, wherein the carfilzomib analog is The compound of claim 44, optionally in a composition comprising at least one carrier or excipient; Providing subjects or cell / tissue culture samples; administering the carfilzomib analog to a subject or a cell / tissue culture sample; providing a sample obtained from the subject or the cell / tissue culture specimen after administration of the carfilzomib analog; performing mass spectrometry on the sample to determine the level of the tellurium atoms present in the sample, wherein the level of the tellurium atoms corresponds to the level of the carfilzomib analog, and detection of the carfilzomib analog indicates detection of carfilzomib in the sample.

50. Use of a carfilzomib analog in a method for detecting carfilzomib by mass spectrometry, comprising the following steps: Provided is a carfilzomib analog, wherein the carfilzomib analog is The compound of claim 45, optionally in a composition comprising at least one carrier or excipient; Providing subjects or cell / tissue culture samples; administering the carfilzomib analog to a subject or a cell / tissue culture sample; providing a sample obtained from the subject or the cell / tissue culture specimen after administration of the carfilzomib analog; performing mass spectrometry on the sample to determine the level of the tellurium atoms present in the sample, wherein the level of the tellurium atoms corresponds to the level of the carfilzomib analog, and detection of the carfilzomib analog indicates detection of carfilzomib in the sample.

51. The use according to claim 47, wherein the mass spectrometry is performed on a sample, optionally a plurality of samples, obtained from the subject or the cell culture.

52. The use according to claim 47, wherein the subject is a mammal, optionally a mouse, a rat or a human.

53. The use according to claim 47, wherein the sample is as defined in claim 9.

54. The use according to claim 47, wherein the sample is as defined in claim 10.

55. The use according to claim 47, wherein the sample is as defined in claim 11.

56. The use according to claim 47, wherein the mass spectrometry is mass cytometry or multiplexed ion beam imaging, optionally the mass cytometry is mass cytometry imaging or mass cytometry suspension.

57. The use according to claim 47, wherein the tellurium atom comprises multiple isotopes and the mass spectrometry is multiplex mass spectrometry, optionally multiplex mass flow cytometry.

58. A kit for mass spectrometry flow cytometry analysis, comprising a teniposide tellurophene analog of formula I: or a salt or solvate thereof, and One or more tellurium standards.

59. A kit for mass spectrometry analysis, optionally mass cytometry analysis, comprising a carfilzomib tellurophene analog of Formula II: or a salt or solvate thereof, and One or more tellurium standards.

60. The kit of claim 58, wherein the tellurium in the analog and the tellurium in the standard are selected from 120 Te, 122 Te, 123 Te, 124 Te, 125 Te, 126 Te, 128 Te, 130 Te and combinations thereof.

61. A method for determining the dosage of a small molecule compound to achieve a desired target engagement of the small molecule compound in a subject or cell / tissue culture, the steps comprising: Providing subjects or cell / tissue culture samples; Providing a small molecule analog of a small molecule compound, wherein the small molecule compound has a structure comprising one or more monocyclic or bicyclic aromatic rings, and the small molecule analog has a structure in which at least one of the one or more monocyclic or bicyclic aromatic rings of the structure of the small molecule compound is replaced by a tellurium-containing tellurium atom, thereby forming a tellurium-containing tellurium-containing small molecule analog; administering the tellurophene small molecule analog to the subject or cell / tissue culture sample; providing a sample obtained from the subject or the cell / tissue culture specimen after administration of the tellurophene small molecule analog; Detecting the tellurphene small molecule analog in the sample to which the tellurphene small molecule analog has been administered using mass spectrometry according to the method of claim 1; measuring the effect produced by the tellurophene small molecule analog; as well as determining a dosage of the tellurophene analog suitable for achieving a desired distribution of the small molecule compound, The dosage of the tellurphene small molecule analog indicates the dosage of the small molecule compound.

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