Methods for assessing small molecule distribution using tellurophene analogues

Abstract

The present disclosure relates to small molecule tellurophene analogs and methods of detecting small molecules using those tellurophene analogs. The present disclosure further relates to compositions and kits that include the disclosed tellurophene analogs. The present disclosure also relates to methods of determining dosages of small molecules.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 335,926, filed April 28, 2022, entitled "METHOD FOR ASSESSING SMALL MOLECULE DISTRIBUTION USING TELLUROPHEN ANALOGS," which is incorporated by reference in its entirety.

[0002] Field The present disclosure relates to tellurofen containing small molecule analogs and methods for detecting small molecules in samples obtained from subjects or cell / tissue cultures using tellurofen as a reporter. The present disclosure also relates to tellurofen teniposide analogs and tellurofen carfilzomib analogs. The present disclosure further includes the use of the analogs of the present disclosure in assessing the distribution of small molecules. Additionally, the present disclosure relates to methods for determining the dosage of small molecules. [Background technology]

[0003] Introduction The biodistribution of exogenously administered small molecule therapeutics is rarely homogenous in vivo. Furthermore, target engagement at the desired location is key to translating promising in vitro results into in vivo responses. Currently, methods to monitor biodistribution and target engagement are primarily focused on assays performed in bulk, with few methods offering cellular resolution, especially in whole biological models. Early approaches to monitor the cellular biodistribution of small reversible inhibitors used microautoradiography, but the high specific activity, long exposure times and technical challenges required have prevented this method from being widely adopted. Advances in fluorescence-based histological techniques have allowed for the visualization and quantification of target engagement in processed tissue samples, but these assays are limited in their ability to simultaneously identify and characterize phenotypes associated with the cell types present. Due to the inherent challenges associated with multiplexed fluorescence-based microscopy, the combination of biodistribution and cellular characterization is best suited for IMAGING MASS CYTOMETRY™ (IMC™).

[0004] IMC™ is a high-dimensional tissue imaging platform that builds on the CYTOF™ Mass Cytometry (MC) technology and is designed for epitope measurements on tissue sections. An earlier version of the platform was introduced by the Bodenmiller group in 2014, using a 32-antibody panel to examine underlying tumor heterogeneity in human breast cancer samples. More recent examples have examined over 40 markers that can be quantified at subcellular resolution using current IMC™ reagents and workflows, thus enabling deep profiling of individual cells in their native microenvironment. The spatially resolved information acquired by IMC™ can be analyzed to study cell phenotypes with respect to spatial organization. Furthermore, a library of appropriate probes can be used to track temporal changes in tissue, as was done with hypoxia. Coupling this information with the biodistribution of small molecule therapeutics will facilitate the design of dosing regimens and help understand perturbed cellular biochemistry.

[0005] Despite these advances, the potential of IMC™ has not been explored in drug discovery and development. To track the localization of a drug in tissues with IMC™, the molecule must have a heavy isotope (>80 amu) that is compatible with mass cytometers. Qing et al. exploited the capabilities of IMC™ to detect platinum isotopes resulting from dosing of the chemotherapy drug cisplatin, allowing visualization of its biodistribution in xenografts derived from pancreatic cancer patients. This groundbreaking study revealed unexpected drug distribution, as extensive and unexpected binding of platinum to collagen fibers in tumor stroma and normal tissues was observed. These findings provide insight into the long clearance time of cisplatin and some of the findings on the long-term toxicity of the treatment. Although this study demonstrated the applicability of IMC™ to track the localization of cisplatin, most therapeutic agents do not have MC-compatible elements present to enable their detection. Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, there is a need to develop methods that allow the application of mass cytometry to small molecules in general, without the need for MC compatible elements. [Means for solving the problem]

[0007] Any of the embodiments disclosed herein may be used in combination with one or more other embodiments.

[0008] IMC Visualization of Small Molecules It has been shown that tellurophene analogs can be used to detect biologically active small molecules other than metallodrugs by IMC™. It has also been shown that analogs can be accessed by isosteric substitution of 5- or 6-membered aromatic rings with tellurophene. Tellurophene is a stable and biocompatible mass label for MC and IMC™. As an example, this bioisostere design strategy has been successfully implemented to develop tellurophene teniposide analogs and tellurophene carfilzomib analogs. The exemplary analogs retain similar biological activity compared to the original small molecule and are detectable and quantifiable by MC. Effect of the Invention

[0009] Thus, in one aspect, the disclosure provides a method for detecting a small molecule in a sample, comprising: Providing a sample obtained from a subject or cell / tissue culture administered a tellurophene small molecule analog, including tellurophene containing a tellurium atom; and performing mass spectrometry on the sample to determine the level of tellurium atoms; Including, wherein the small molecule has a structure including 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 small molecule's structure is replaced with tellurophene, the level of tellurium atoms corresponds to the level of the analog, and detection of the analog indicates detection of the small molecule in a sample.

[0010] In another aspect, the disclosure includes the use of tellurophene analogs in the detection of small molecules by mass spectrometry in subjects administered the analogs or cell / tissue cultures administered the analogs, the small molecules having a structure that includes one or more monocyclic or bicyclic aromatic rings, the analogs having a structure in which at least one of the one or more monocyclic or bicyclic aromatic rings of the small molecule's structure is replaced with tellurophene.

[0011] In another aspect, the present disclosure provides a compound of formula I [ka] or a salt or solvate thereof.

[0012] In another aspect, the present disclosure provides a compound of formula II [ka] or a salt or solvate thereof.

[0013] In another aspect, the disclosure includes a composition comprising a compound of the disclosure and a carrier or excipient.

[0014] In another aspect, the present disclosure includes the use of a compound of formula I of the present disclosure, or a composition comprising a compound of formula I of the present disclosure, in the detection of teniposide by mass spectrometry in a subject administered a compound of formula I, or a salt or solvate thereof, or in a cell / tissue culture administered a compound of formula I, or a salt or solvate thereof.

[0015] In another aspect, the disclosure includes the use of a compound of formula II of the disclosure or a composition comprising a compound of formula II of the disclosure in the detection of carfilzomib by mass spectrometry in a subject administered a compound of formula II or a salt or solvate thereof or in a cell / tissue culture administered a compound of formula II or a salt or solvate thereof.

[0016] In another aspect, the present disclosure provides a compound of formula I [ka] or a salt or solvate thereof; and A kit for mass cytometry analysis that includes a tellurium standard or a plurality of tellurium standards is included.

[0017] In another aspect, the present disclosure provides a compound of formula II [ka] or a salt or solvate thereof; and A kit for mass spectrometry, and optionally mass cytometry analysis, including a tellurium standard or a plurality of tellurium standards is included.

[0018] In another aspect, the disclosure provides a method for determining a dosage of a small molecule to achieve a 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 on the target; detecting the small molecule in a subject by the methods of the disclosure; Measuring the effect produced by the small molecule; and Determining an appropriate dose of tellurophene analog to achieve a desired distribution of the small molecule. wherein the dosage of the tellurophene analog indicates the dosage of the small molecule.

[0019] In one aspect, a method for detecting a small molecule compound in a sample includes providing either a subject or a cell / tissue culture specimen; providing a small molecule analog of a small molecule compound, the small molecule compound having a structure that includes one or more monocyclic or bicyclic aromatic rings, the small molecule analog having a structure in which at least one of the one or more monocyclic or bicyclic aromatic rings of the small molecule compound's structure is substituted with a tellurophene that includes a tellurium atom, thereby forming a tellurophene small molecule analog; administering the tellurophene small molecule analog to the subject or cell / tissue culture specimen; providing a sample taken from either the subject or the cell / tissue culture specimen after administration of the tellurophene small molecule analog; and 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.

[0020] In various embodiments, such as those described above, the method further comprises quantifying the amount of tellurium atoms in the sample, where the tellurium atom level indicates the amount of small molecule compounds in the sample. In various embodiments, such as those described above, the method further comprises quantifying one or more other analytes in the sample, where the method further comprises labeling the sample with one or more mass-labeled analyte binding agents and determining the level of the one or more analyte binding agents prior to performing mass spectrometry. In various embodiments, such as those described above, the one or more mass-labeled analyte binding agents 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. In various embodiments, such as those described above, the subject is a mammal, optionally a mouse, a rat, or a human.

[0021] In various embodiments such as those described above, mass spectrometry is performed at a plurality of separate locations, and the level of tellurium atoms is determined at each of the plurality of separate locations to provide a distribution of the small molecule analog in the sample, the distribution of the analog in the sample being indicative of the distribution of the small molecule compound in the sample. In various embodiments such as those described above, the sample is a single cell, and the distribution of the small molecule analog in the single cell is indicative of the distribution of the small molecule compound at a subcellular level. In various embodiments such as those described above, a plurality of samples is provided, the samples comprising different tissues, cells or secretions of a subject, where detection of the small molecule analog in the plurality of samples provides a distribution of the small molecule analog in the subject, indicative of the distribution of the small molecule compound in the subject, such as tissue or organ distribution. In various embodiments such as those described above, the sample is or comprises urine, stool, 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. In various embodiments such as those described above, the sample is a frozen tissue section. In various embodiments such as those described above, the sample is or includes cells from a cell culture or medium from a cell culture. In various embodiments such as those described above, the sample is a single cell, and the distribution of the analog within the single cell indicates the distribution of the small molecule compound at the subcellular level. In various embodiments such as those described 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 described above, the tellurium atoms are 120 Te, 122 Te, 123 Te, 124 Te, 125 Te, 126 Te, 128 Te, 130Te, Te, and combinations thereof. In various embodiments such as those described above, the tellurium atom comprises multiple tellurium isotopes, and the mass cytometry is multichannel mass spectrometry, optionally multichannel mass cytometry. In various embodiments such as those described above, the one or more monocyclic aromatic rings are 5- or 6-membered aromatic rings, and optionally the 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 those described above, the one or more bicyclic aromatic rings are independently selected from naphthyl, indole, benzothiophene, or benzofuran.

[0022] In various embodiments such as those described above, the structure of the small molecule compound includes one or more monocyclic 5- or 6-membered aromatic rings, and at least one of the monocyclic 5- or 6-membered aromatic rings is substituted with tellurophene. In various embodiments such as those described above, the structure of the small molecule compound includes one or more bicyclic aromatic rings, and at least one of the bicyclic aromatic rings is substituted with benzo[b]tellurophene or 4H-telluropheno[3,2-b]pyrrole. In various embodiments such as those described above, the structure of the small molecule compound includes 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. In various embodiments such as those described above, the one or more 5-membered aromatic rings are thiophene. In various embodiments such as those described above, the small molecule compound is teniposide and the tellurophene small molecule analog is represented by formula I [ka] or a salt or solvate thereof.

[0023] In various embodiments such as those described above, the structure of the small molecule compound includes one or more six-membered aromatic rings, optionally each independently selected from phenyl or pyridine, and optionally the one or more six-membered aromatic rings are phenyl. In various embodiments such as those described above, the small molecule compound is carfilzomib and the tellurophene small molecule analog is represented by Formula II [ka] or a salt or solvate thereof.

[0024] In various embodiments, such as those described above, the small molecule analog interacts irreversibly, optionally covalently, with the target in the sample. In various embodiments, such as those described above, the subject or cell / tissue culture specimen is administered a tellurophene small molecule analog in combination with a small molecule compound, and the method further includes directly detecting the level of the small molecule compound, and a comparison of the level of the small molecule compound to the level of the analog indicates putative competitive binding of the analog.

[0025] In another aspect, the use of a tellurophene small molecule analog in a method for detecting a small molecule compound in a sample, comprising the steps of providing either a subject or a cell / tissue culture specimen, providing a small molecule analog of a small molecule compound, the small molecule compound having a structure that includes one or more monocyclic or bicyclic aromatic rings, the small molecule analog having a structure in which at least one of the aromatic rings of the small molecule compound is replaced with a 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 specimen, providing a sample taken from either the subject or the cell / tissue culture specimen after administration of the tellurophene small molecule analog, and 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 described above, mass spectrometry is performed on a sample, optionally multiple samples, obtained from the subject or cell / tissue culture specimen. In various embodiments such as those described above, the subject is a mammal, optionally a mouse, a rat or a human. In various embodiments such as those described above, the sample is or comprises urine, stool, 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 stomach cells or secretions. In various embodiments such as those described above, the sample is a frozen tissue section. In various embodiments such as those described above, the sample is or comprises cells from a cell culture or medium from a cell culture. In various embodiments such as those described 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.

[0026] In various embodiments such as those described above, the one or more aromatic rings are 5- or 6-membered aromatic rings, and optionally the 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 those described above, the one or more bicyclic aromatic rings are independently selected from naphthyl, indole, benzothiophene, or benzofuran. In various embodiments such as those described above, the structure of the small molecule compound includes 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. In various embodiments such as those described above, the one or more 5-membered aromatic rings are thiophene. In various embodiments such as those described above, the small molecule compound is teniposide and the tellurophene small molecule analog is represented by formula I [ka] or a salt or solvate thereof.

[0027] In various embodiments such as those described above, the structure of the small molecule compound includes one or more six-membered aromatic rings, optionally each independently selected from phenyl or pyridine, and optionally the one or more six-membered aromatic rings are phenyl. In various embodiments such as those described above, the small molecule compound is carfilzomib and the tellurophene small molecule analog is represented by Formula II [ka] or a salt or solvate thereof.

[0028] In another aspect, the present disclosure provides a compound of formula I [ka] or a salt or solvate thereof.

[0029] In various embodiments, such as those described above, the Te atom is an isotope, and optionally the Te atom is 120 Te, 122Te, 123 Te, 124 Te, 125 Te, 126 Te, 128 Te, 130 Te, and combinations thereof. In another embodiment, the composition is a composition in which the small molecule compound is carfilzomib and the tellurophene small molecule analog is selected from the group consisting of formula II [ka] or a salt or solvate thereof and the carrier or excipient.

[0030] In another embodiment, the composition comprises a compound of formula I [ka] or a salt or solvate thereof and a carrier or excipient.

[0031] In various embodiments, such as those described above, the Te atom is 120 Te, 122 Te, 123 Te, 124 Te, 125 Te, 126 Te, 128 Te, 130 In yet another aspect, the present disclosure provides a compound of formula II [ka] or a salt or solvate thereof.

[0032] In various embodiments as described above, the Te atoms are isotopically enriched, and optionally the Te atoms are 120 Te, 122 Te, 123 Te, 124 Te, 125 Te, 126 Te, 128 Te, 130 In another embodiment, the composition is selected from Formula I [ka] or a salt or solvate thereof and a carrier or excipient.

[0033] In another embodiment, the composition comprises a compound of formula II [ka] or a salt or solvate thereof and a carrier or excipient.

[0034] In another aspect, the disclosure provides a method for detecting teniposide by mass spectrometry, comprising the steps of providing a teniposide analog, the teniposide analog being represented by formula I [ka] or a salt or solvate thereof, optionally in which the Te atom is an isotope, and optionally in which the Te atom is an isotopic form, optionally in a composition comprising at least one carrier or excipient. 120 Te, 122 Te, 123 Te, 124 Te, 125 Te, 126 Te, 128 Te, 130 and combinations thereof, comprising the steps of providing either a subject or a cell / tissue culture specimen, administering a teniposide analog to either the subject or the cell / tissue culture specimen, providing a sample taken from either the subject or the cell / tissue culture specimen after administration of the teniposide analog, and 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 detection of the teniposide analog indicates detection of teniposide in the sample.

[0035] In another aspect, the disclosure provides a method for detecting carfilzomib by mass spectrometry, comprising the steps of: providing a carfilzomib analog having a structure represented by formula II; [ka] or a salt or solvate thereof, optionally in which the Te atom is isotopically enriched, and optionally in which the Te atom is present in a composition optionally comprising at least one carrier or excipient. 120 Te, 122 Te, 123 Te, 124 Te, 125 Te, 126 Te, 128 Te, 130 and combinations thereof, comprising the steps of providing either a subject or a cell / tissue culture specimen, administering a carfilzomib analog to either the subject or the cell / tissue culture specimen, providing a sample taken from either 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 tellurium atoms present in the sample, wherein the level of tellurium atoms corresponds to the level of the carfilzomib analog, and detection of the carfilzomib small molecule analog indicates detection of carfilzomib in the sample.

[0036] In various embodiments such as those described above, mass spectrometry is performed on a sample, optionally multiple samples, obtained from a subject or cell culture. In various embodiments such as those described above, the subject is a mammal, optionally a mouse, rat or human. In various embodiments such as those described above, the sample is or comprises urine, stool, 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. In various embodiments such as those described above, the sample is a frozen tissue section. In various embodiments such as those described above, the sample is or comprises cells from a cell culture or medium from a cell culture. In various embodiments such as those described 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 described above, the tellurium atoms include multiple isotopes and the mass spectrometry is multi-channel mass spectrometry, optionally multi-channel mass cytometry.

[0037] In another embodiment, the kit for mass cytometry analysis comprises a compound of formula I [ka] or a salt or solvate thereof, and a tellurium standard or standards.

[0038] In another embodiment, a kit for mass spectrometry, and optionally mass cytometry analysis, comprises a compound represented by formula II [ka] or a salt or solvate thereof, and a tellurium standard or multiple tellurium standards.

[0039] In various embodiments, such as those described above, the tellurium in the analog and the tellurium in the standard are 120 Te, 122 Te, 123 Te, 124 Te, 125 Te, 126 Te, 128 Te, 130 Te, and combinations thereof.

[0040] In yet another aspect, a method for determining a dosage of a small molecule compound to achieve a desired target engagement of the small molecule compound in a subject or cell / tissue culture comprises the steps of providing either 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, the small molecule analog having a structure in which at least one of the one or more aromatic rings of the small molecule compound's structure is replaced with a tellurophene comprising a tellurium atom, thereby forming a tellurophene small molecule analog; providing a small molecule analog of the small molecule compound having a structure comprising one or more monocyclic or bicyclic aromatic rings, the small molecule analog having a structure in which at least one of the one or more aromatic rings of the small molecule compound is replaced with a tellurophene comprising a tellurium atom, thereby forming a tellurophene small molecule analog; The method includes the steps of administering a small molecule analog to a subject or a cell / tissue culture specimen, providing a sample taken from either the subject or the cell / tissue culture specimen after administration of the tellurophene small molecule analog, detecting the tellurophene small molecule analog in the sample to which the tellurophene small molecule analog was administered, using mass spectrometry by a method defined in any of the embodiments described herein, measuring the effect produced by the tellurophene small molecule analog, and determining a dosage of the tellurophene small molecule analog suitable for achieving a desired distribution of the small molecule compound, wherein the dosage of the tellurophene small molecule analog indicates the dosage of the small molecule compound.

[0041] Embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. [Brief description of the drawings]

[0042] [Figure 1]Figure 1 shows the effect of teniposide and Te-teniposide analog 1 on Top2-catalyzed decatenation of kinetoplast DNA. Panel A shows a photograph of an agarose gel of a Top2 decatenation assay. Panel B shows a graph of relative Top2 inhibition based on image density of the kDNA band in lane 3. 1: linear DNA; 2: decatenated 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. [Diagram 2] FIG. 2 shows a graph of relative cell proliferation of HL-60 cells treated with teniposide or Te-teniposide analog 1. [Diagram 3] FIG. 3 shows a graph of dose-dependent cytotoxicity of teniposide and analog 1 in PANC-1 cells as determined by alamar blue assay. [Figure 4] Figure 4 shows the proliferation curves of PANC-1 cells incubated with various concentrations of teniposide and 1 as determined by imaging cell confluency. [Diagram 5] Figure 5 shows a photograph of a Western blot of pH2AXSer139, a marker of DNA DSBs, in PANC-1 cells treated with 1 μM of the drugs. Teniposide and 1 exposure induced extendible DNA damage at both 24 and 48 hours relative to the control. [Figure 6] Figure 6 shows 130Te histograms of cell competitive binding assays of teniposide and compound 1 analyzed by CYTOF®. a) HL-60 cells treated with teniposide (red); compound 1 equally labels HL-60 cells at 2 μM and 10 μM (blue and green), despite presaturation of intracellular drug binding sites by equivalent amounts of teniposide (orange and purple). b) Co-incubation of 0 (red), 2 (blue), 10 (orange and 20 μM (green) teniposide with 2 μM teniposide. [Figure 7]Figure 7 shows representative images of immunostained FFPE tumor tissue sections in normal (a) saline, (b) teniposide (20 mg / kg, IP) or (c) compound 1 (20 mg / kg, IP) treated PANC-1 xenograft mice. Brown staining was performed to visualize pH2AXSer139 puncta, followed by hematoxylin counterstaining. [Figure 8] Figure 8 shows images of IMC™ analysis of Te-teniposide in PANC-1 xenografts. A) Teniposide-treated, B) Te-teniposide-treated, C) image with the average signal in A subtracted from B, and a region enlarged to show the Te-teniposide specific signal. D) Histograms of 125Te pixel counts from teniposide and Te-teniposide-treated mice; E) Standard curve of 125Te counts vs. Te atoms / pixel. [Figure 9] FIG. 9 shows a graph of the percent decrease in apparent enzyme velocity upon inhibition with carfilzomib, 7b, or 8b versus untreated. [Figure 10] FIG. 10 shows a graph of the median 128Te counts per cell obtained from MC analysis of cells treated with TeCar7b (500 μM), carfilzomib (500 μM), control 8b, or a combination of carfilzomib with either 7b or 8b. [Figure 11] FIG. 11 shows the standard curve for the in vitro binding assay. [Figure 12] FIG. 12 shows a mass cytometry system and controller. [Figure 13] FIG. 13 shows a mass cytometry system. [Figure 14] FIG. 14 illustrates an exemplary computer system that can function as the controller. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] Other features and advantages of the present disclosure will become apparent from the following detailed description, but it should be understood that the detailed description and specific examples, while indicating embodiments of the present disclosure, are given by way of example only, and that the claims should not be limited by these embodiments, but should be given the broadest interpretation consistent with the description as a whole.

[0044] Description of Various Embodiments I. Definition Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the disclosure described herein as appropriate, as understood by one of skill in the art.

[0045] Terms such as "disclosed compound(s)" or "disclosed compound(s)" as used herein refer to the tellurophene small molecule analogs 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, where the analogs include tellurophene and structures in which at least one of the monocyclic or bicyclic aromatic rings is replaced with tellurophene. Thus, it can be understood that tellurophene small molecule analogs have a structure that mimics the structure of the small molecule. For example, the compounds of the present disclosure include compounds of formula I or a pharma- ceutically acceptable salt and / or solvate thereof, and compounds of formula II or a pharma- ceutically acceptable salt and / or solvate thereof.

[0046] The terms "disclosed composition(s)" or "composition(s) of the disclosure" as used herein refer to a composition, such as a pharmaceutical composition, that includes one or more compounds of the disclosure.

[0047] The term "and / or" as used herein means that the listed items are present or used individually or in combination. In practice, the term means that "at least one" or "one or more" of the listed items are used or present. The term "and / or" with respect to pharma- ceutically 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, for example, of salts of the compounds of the present disclosure.

[0048] As used in this disclosure, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, an embodiment including "a compound" should be understood to present one compound, or two or more additional compounds in a particular embodiment.

[0049] In embodiments that include an "additional" or "second" component, such as an additional or second compound, the second component, as used herein, is chemically distinct from the other component or the first component. A "third" component is distinct from the other component, the first component, and the second component, and further recited or "additional" components are similarly distinct.

[0050] As used in this disclosure and claim(s), "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "include" and "includes") or "containing" (and any form of containing, such as "contain" and "contains") are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.

[0051] The term "consisting of" and its derivatives, as used herein, are intended to be closed term specifying the presence of stated features, elements, components, groups, integers, and / or steps, and excluding the presence of other unrecited features, elements, components, groups, integers, and / or steps.

[0052] The term "consisting essentially of," as used herein, is intended to specify the presence of the described features, elements, components, groups, integers, and / or steps, as well as those that do not materially affect the basic and novel characteristic(s) of those features, elements, components, groups, integers, and / or steps.

[0053] The term "suitable" as used herein means that the selection of a particular compound or conditions will depend on the particular synthetic operation being performed, the identity of the molecule(s) being converted, and / or the particular use of the compound, but the selection will be within the purview of a person trained in the art.

[0054] In an embodiment of the present disclosure, the compounds described herein may have at least one asymmetric center. When compounds possess two or more asymmetric centers, they may exist as diastereomers. It is to be understood that all such isomers and mixtures thereof in any proportion are encompassed within the scope of the present disclosure. It is further understood that the stereochemistry of a compound may be as shown in any given compound listed herein, but such a compound may also contain a certain amount (e.g., less than 20%, suitably less than 10%, more suitably less than 5%) of a compound of the present disclosure having an alternative stereochemistry. Any optical isomers, as separated, pure or partially purified optical isomers or racemic mixtures thereof, are intended to be encompassed within the scope of the present disclosure.

[0055] Compounds of the present disclosure may also exist in different tautomeric forms, and any tautomeric forms that the compounds form, as well as mixtures thereof, are intended to be included within the scope of the present disclosure.

[0056] The compounds of the present disclosure may further exist in different polymorphic forms, and it is contemplated that any polymorphs or mixtures thereof are within the scope of the present disclosure.

[0057] This specification refers to several chemical terms and abbreviations used by those of ordinary skill in the art. Nonetheless, definitions of selected terms are provided for clarity and consistency.

[0058] The terms "about," "substantially," and "approximately," as used herein, refer to a reasonable amount of deviation of the modified term so that the end result is not materially altered. These terms of degree should be interpreted as including at least ±5% deviation of the modified term, unless this deviation negates the meaning of the modified term or the context would suggest otherwise to one of ordinary skill in the art.

[0059] The term "alkyl," as used herein, whether used alone or as part of another group, means a straight or branched chain saturated alkyl group. The number of carbon atoms possible in the referenced alkyl group is determined by the prefix "C n1~n2 For example, C 1~10 The term alkyl refers to alkyl groups having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.

[0060] The term "alkylene," whether used alone or as part of another group, means a straight or branched chain saturated alkylene group, i.e., a saturated carbon chain containing substituents at both ends. The number of carbon atoms possible in the referenced alkylene group is indicated by the prefix "C n1~n2 For example, C 2~6 The term alkylene refers to alkylene groups having 2, 3, 4, 5 or 6 carbon atoms.

[0061] The term "available," as in "available hydrogen atom" or "available atom," refers to an atom known to one of ordinary skill in the art to be substitutable by a substituent.

[0062] The term "amine" or "amino", as used herein, whether used alone or as part of another group, refers to a group of general formula NR'R", where R' and R" are each independently hydrogen or C. 1~6 is selected from alkyl.

[0063] The term "atm" as used herein refers to atmospheric pressure.

[0064] The term "MS," as used herein, refers to mass spectrometry.

[0065] The term "aq." as used herein refers to aqueous.

[0066] As used herein, terms such as "protecting group" or "PG" refer to a chemical moiety that protects or masks a reactive portion of a molecule to prevent side reactions at the reactive portion of the molecule while a different portion of the molecule is being manipulated or reacted. After the manipulation or reaction is complete, the protecting group is removed under conditions that do not degrade or decompose the remaining portion of the molecule. Selection of an appropriate protecting group can be performed by one of ordinary skill in the art. Many conventional protecting groups are known in the art, for example, as described in Greene, TW and Wuts, PGM, "Protective Groups in Organic Synthesis", John Wiley & Sons, 3rd Edition, 1999, in "Protective Groups in Organic Chemistry", McOmie, JFW (ed.), Plenum Press, 1973, and Kocienski, P. Protecting Groups, 3rd Edition, 2003, Georg Thieme Verlag (The Americas).

[0067] The term "small molecule" as used herein refers to an organic compound with a 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.

[0068] As used herein, the term "subject" includes all members of the animal kingdom, including mammals, and suitably refers to rats, mice and humans, such that the methods and uses of the present disclosure are applicable to both human therapy and veterinary applications.

[0069] The term "pharmaceutical acceptable" means compatible with the treatment of a subject.

[0070] The term "pharmaceutical acceptable carrier" means a non-toxic solvent, dispersant, excipient, adjuvant or other material that is mixed with an active ingredient to enable the formation of a pharmaceutical composition, i.e., a dosage form that can be administered to a subject.

[0071] The term "pharmaceutical acceptable salt" means either an acid addition salt or a base addition salt which is suitable or compatible with the treatment of a subject.

[0072] An acid addition salt suitable or compatible for the treatment of a subject is any non-toxic organic or inorganic acid addition salt of any basic compound.

[0073] A base addition salt suitable or compatible for the treatment of a subject is any non-toxic organic or inorganic base addition salt of any acidic compound.

[0074] The term "solvate" as used herein means a compound or a salt of a compound, wherein molecules of a suitable solvent are incorporated into the crystal lattice.

[0075] The term "treat" or "treatment" as used herein means an approach for obtaining beneficial or desired results, including clinical results, as is well understood in the art. Beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms or pathology, whether detectable or not, reduction in the extent of disease, a stabilized (i.e., not worsening) state of disease, prevention of disease spread, delay or slowing of disease progression, improvement or palliation of the disease state, reduction in recurrence of disease, and remission (whether partial or total). "Treat" and "treatment" can also mean prolonging survival compared to expected survival in the absence of treatment. "Treat" and "treatment" as used herein also include prophylactic treatment. For example, a subject with early stage cancer can be treated to prevent progression, or a subject in remission can be treated with a compound or composition of the present disclosure to prevent recurrence. A method of treatment includes administering to a subject a therapeutically effective amount of one or more compounds of the present disclosure, optionally consisting of a single dose or including a series of doses.

[0076] 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.

[0077] The term "administration," as used herein, means administration of a therapeutically effective amount of one or more compounds or compositions of the present disclosure to a cell, tissue, organ, or subject.

[0078] The term "neoplastic disorder" as used herein refers to a disease, disorder, or condition characterized by cells capable of autonomous growth or replication, e.g., an abnormal state or condition characterized by proliferative cell growth. The term "neoplasm" as used herein refers to a mass of tissue resulting from the abnormal growth and / or division of cells in a subject with a neoplastic disorder.

[0079] The term "cancer," as used herein, refers to a cell proliferative disease state.

[0080] II. Compounds and Compositions of the Disclosure In one aspect, the disclosure includes small molecule tellurophene analogs, which include tellurophene analogs that contain a tellurium atom, where the small molecule has a structure that includes 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 small molecule's structure is replaced with tellurophene.

[0081] In some embodiments, the small molecule is any organic compound that produces a physiological effect in the subject that is administered the organic compound.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.

[0082] In some embodiments, the one or more monocyclic aromatic rings are 5- or 6-membered aromatic rings, and optionally the one or more monocyclic 5- or 6-membered aromatic rings are independently selected from thiophene, furan, pyrrole, phenyl, or pyridine.

[0083] In some embodiments, the one or more bicyclic aromatic rings are independently selected from naphthyl, indole, benzothiophene, or benzofuran.

[0084] 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 substituted with tellurophene.

[0085] 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 substituted with benzo[b]tellurophene or 4H-telluropheno[3,2-b]pyrrole.

[0086] In some embodiments, the structure of the small molecule comprises one or more five-membered aromatic rings, optionally each of which is independently selected from thiophene, furan, or pyrrole.

[0087] In some embodiments, one or more of the five-membered aromatic rings is a thiophene.

[0088] In some embodiments, the structure of the small molecule comprises one or more 6-membered aromatic rings, optionally each of which is independently selected from phenyl or pyridine, and optionally wherein one or more of the 6-membered aromatic rings is phenyl.

[0089] It is contemplated that a small molecule tellurophene analog, such as a therapeutic agent, that contains one or more aromatic rings 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 as the small molecule. Thus, detection of the analog indicates detection of the small molecule. Some non-limiting examples are provided in Scheme A below. [ka]

[0090] In some embodiments, the small molecule is teniposide and the tellurophene small molecule analog is of formula I [ka] or a salt or solvate thereof.

[0091] In some embodiments, the small molecule is carfilzomib and the analog is of formula II [ka] or a salt or solvate thereof.

[0092] In another aspect, the present disclosure provides a compound of formula I [ka] or a salt or solvate thereof.

[0093] In another aspect, the present disclosure provides a compound of formula II [ka] or a salt or solvate thereof.

[0094] In another aspect, the present disclosure includes a composition comprising a compound of the present disclosure and a carrier or excipient.

[0095] In embodiments, the pharma- ceutically acceptable salt is an acid or base addition salt. The selection of an appropriate salt can be made by one skilled in the art (see, for example, S. M. Berge et al., "Pharmaceutical Salts," J. Pharm. Sci. 1977, 66, 1-19).

[0096] The acid addition salt suitable or compatible for the treatment of the subject is any non-toxic organic or inorganic acid addition salt of any basic compound. Basic compounds that form acid addition salts include, for example, compounds that contain an amine group. Exemplary inorganic acids that form suitable salts include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid, as well as acid metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Exemplary organic acids that form 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 embodiments, mono- or di-acid salts are formed, and such salts exist in either hydrated, solvated, or substantially anhydrous form. In general, acid addition salts are more soluble in water and various hydrophilic organic solvents, and generally exhibit higher melting points compared to their free base forms. Those skilled in the art will know the criteria for selecting a suitable salt. For example, in the isolation of compounds of the present disclosure for laboratory use or for subsequent conversion to a pharma- ceutical acceptable acid addition salt, other pharma- ceutical unacceptable salts, such as, but not limited to, oxalates, may be used.

[0097] The base addition salt suitable or compatible for the treatment of the subject is any non-toxic organic or inorganic base addition salt of any acidic compound. Acidic compounds that form base addition salts include, for example, compounds that contain a carboxylic acid group. Exemplary inorganic bases that form suitable salts include lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide or barium hydroxide as well as ammonia. Exemplary organic bases that form suitable salts include aliphatic, alicyclic or aromatic organic amines such as isopropylamine, methylamine, trimethylamine, picoline, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Exemplary organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. For example, it may be useful to select an appropriate salt so that any ester functional groups in the compound, if any, are not hydrolyzed. The criteria for selecting an appropriate salt will be known to those skilled in the art.

[0098] Solvates of the compounds of the present disclosure include, for example, those made with pharma- ceutically acceptable solvents. Examples of such solvents include water (the resulting solvates are called hydrates) and ethanol, etc. Suitable solvents are physiologically acceptable at the dosage administered.

[0099] The compounds of the present disclosure are suitably formulated into compositions using one or more carriers in a conventional manner.Accordingly, the present disclosure also includes compositions comprising one or more compounds of the present disclosure and a carrier.The compounds of the present disclosure are suitably formulated into pharmaceutical compositions for administration to a subject in a biologically compatible form suitable for administration in vivo.Accordingly, the present disclosure further includes pharmaceutical compositions comprising one or more compounds of the present disclosure and a pharma- ceutical acceptable carrier.In an embodiment of the present disclosure, the pharmaceutical compositions are used for the treatment of any of the diseases, disorders or conditions described herein.

[0100] The compounds of the present disclosure are administered to a subject in various forms depending on the route of administration selected, as will be understood by those skilled in the art. For example, the compounds of the present disclosure are administered by oral, inhalation, parenteral, buccal, sublingual, nasal, rectal, vaginal, patch, pump, topical or transdermal administration, and the pharmaceutical composition is formulated accordingly. In some embodiments, administration is by pump for periodic or continuous delivery. Conventional procedures and ingredients for the selection and preparation of suitable compositions are described, for example, in Remington's Pharmaceutical Sciences (2000-20th edition) and The National Formulary (USP 24 NF19), published in 1999.

[0101] Parenteral administration includes systemic delivery routes other than the gastrointestinal (GI) tract, and includes, for example, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, transepithelial, intranasal, 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 may be by continuous infusion over a selected period of time.

[0102] In some embodiments, the compounds of the present disclosure are administered orally, for example, using an inert diluent or an assimilable edible carrier, or are enclosed in hard or soft shell gelatin capsules, or are compressed into tablets, or are directly incorporated with dietary food. In some embodiments, the compounds are incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, caplets, pellets, granules, lozenges, chewing gum, powders, syrups, elixirs, wafers, aqueous solutions and suspensions, and the like. For tablets, carriers used include lactose, corn starch, sodium citrate, and phosphate salts. Pharmaceutically acceptable excipients include binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). In an embodiment, the tablet is coated by methods well known in the art. For tablets, capsules, caplets, pellets or granules for oral administration, pH-sensitive enteric coatings such as Eudragits™ designed to control the release of the active ingredient are optionally used. Oral dosage forms also include modified release, e.g., immediate release and timed release formulations. Examples of modified release formulations include, e.g., sustained release (SR), extended release (ER, XR, or XL), timed or timed release, controlled release (CR), or continuous release (CR or continence), employed, e.g., in the form of coated tablets, osmotic delivery devices, coated capsules, microencapsulated microspheres, agglomerated particles, e.g., agglomerated particles such as molecular sieve type particles, or fine hollow permeable fiber bundles, or chopped hollow permeable fibers aggregated or held in a fibrous packet. Timed release compositions are formulated, e.g., as liposomes or where the active compound is protected by a differentially degradable coating, 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 from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.For oral administration in a capsule form, useful carriers or diluents include lactose and dried corn starch.

[0103] In some embodiments, liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or may be suitably presented as a dry product for constitution with water or other suitable vehicle before use. When aqueous suspensions and / or emulsions are administered orally, the compounds of the present disclosure are suitably suspended or dissolved in an oily phase combined with an emulsifying and / or suspending agent. If necessary, certain sweeteners and / or flavorings and / or colorings are added. Such liquid preparations for oral administration are prepared by conventional means using pharma-ceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methylcellulose or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid). Useful diluents include lactose and high molecular weight polyethylene glycols.

[0104] For example, for the preparation of injectable products, it is also possible to lyophilize the compounds of the present disclosure and use the resulting lyophilizate.

[0105] In some embodiments, the compounds of the present disclosure are administered parenterally. For example, a solution of the compounds of the present disclosure is prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. In some embodiments, dispersions are prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof, and in oils, with or without alcohol. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. Those skilled in the art know how to prepare suitable formulations. For parenteral administration, a sterile solution of the compounds of the present disclosure is usually prepared, and the pH of the solution is appropriately adjusted and buffered. For intravenous use, the total concentration of solutes should be controlled to make the preparation isotonic. For ocular administration, an ointment or a droppable liquid is delivered by an ocular delivery system known in the art, such as an applicator or eye dropper. In some embodiments, such compositions include a mucus mimetic such as hyaluronic acid, chondroitin sulfate, hydroxypropylmethylcellulose or polyvinyl alcohol, a preservative such as sorbic acid, EDTA or benzyl chromium chloride, and a diluent or carrier in the usual amount. For pulmonary administration, the diluent or carrier is selected to be appropriate to allow the formation of an aerosol.

[0106] In some embodiments, the compounds of the present disclosure are formulated for parenteral administration by injection, including the use of conventional catheterization or infusion. Preparations for injection are presented, for example, in unit dosage form, such as ampoules or multi-dose containers, with the addition of preservatives. In some embodiments, the compositions take the form of, for example, a sterile suspension, solution or emulsion in an oily or aqueous vehicle, and contain formulating agents such as suspending agents, stabilizing agents and / or dispersing agents. In all cases, the form must be sterile and must be fluid to the extent that it can be easily injected. Alternatively, the compounds of the present disclosure are suitably in sterile powder form for reconstitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.

[0107] In some embodiments, compositions for nasal administration are conveniently formulated as aerosols, drops, gels and powders.For intranasal administration or administration by inhalation, the compounds of the present disclosure are conveniently delivered in the form of a solution, in the form of a dry powder formulation or suspension from a pump spray container that the patient presses or pumps, or as an aerosol spray presentation from a pressurized container or nebulizer.Aerosol formulations typically comprise a solution or fine suspension of an active substance in a physiologically acceptable aqueous or non-aqueous solvent, and are usually presented in single-dose or multi-dose amounts in a sterile form in a sealed container, usually in the form of a cartridge or refill for use with a nebulizer device.Alternatively, the sealed container is an integrated dispensing device, such as a single-dose nasal inhaler, or an aerosol dispenser fitted with a metering valve intended to be discarded after use.When the dosage form comprises an aerosol dispenser, it contains a propellant, which is, for example, a compressed gas, such as compressed air, or an organic propellant, such as fluorochlorohydrocarbon. Suitable propellants include, but are not limited to, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, heptafluoroalkane, carbon dioxide or another suitable gas. In the case of a pressurized aerosol, the dosage unit is suitably 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. Capsules and cartridges (e.g., made from gelatin) for use in an inhaler or insufflator are formulated to contain, for example, a powder mix of the disclosed compound and a suitable powder base, such as lactose or starch. Aerosol dosage forms can also take the form of a pump-atomiser.

[0108] Compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, wherein the compounds of the present disclosure 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 a conventional suppository base such as cocoa butter.

[0109] Suppository forms of the compounds of the present disclosure are useful for vaginal, urethral and rectal administration. Such suppositories will generally be composed of a mixture of materials that are solid at room temperature but melt at body temperature. Materials commonly used to produce such vehicles include, but are not limited to, theobroma oil (also known as cocoa butter), glycerinated gelatin, other glycerides, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycols. For further description of suppository dosage forms, see, for example, Remington's Pharmaceutical Sciences, 16th Edition, Mack Publishing, Easton, PA, 1980, pp.1530-1533.

[0110] In some embodiments, the compounds of the present disclosure are coupled to soluble polymers as targetable drug carriers. Such polymers include, for example, polyvinylpyrrolidone, pyran copolymers, polyhydroxypropylmethacrylamidephenol, polyhydroxyethylaspartamidephenol, or polyethylene oxide polylysine substituted with palmitoyl residues. Furthermore, in some embodiments, the compounds of the present disclosure are coupled to classes of biodegradable polymers useful for achieving controlled release of drugs, such as polylactic acid, polyglycolic acid, copolymers of polylactic acid and polyglycolic acid, polyepsiloncaprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and crosslinked hydrogels or amphiphilic block copolymers.

[0111] Although the compounds of the present disclosure, including pharma- ceutically acceptable salts and / or solvates thereof, are suitably used per se, they will generally be administered in the form of a pharmaceutical composition in which one or more compounds of the present disclosure (active ingredients) are associated with a pharma- ceutically acceptable carrier. Depending on the mode of administration, the pharmaceutical composition contains about 0.05% by weight to about 99% by weight or about 0.10% by weight to about 70% by weight of the active ingredient, and about 1% by weight to about 99.95% by weight or about 30% by weight to about 99.90% by weight of the pharma- ceutically acceptable carrier, based on the total weight percentage of the total composition.

[0112] III. Methods and Uses of the Disclosure In one aspect, the disclosure provides a method for detecting a small molecule in a sample, comprising: Providing a sample obtained from a subject or cell / tissue culture administered a tellurophene small molecule analog, including tellurophene containing a tellurium atom; and Mass spectrometry of the sample to determine the level of tellurium atoms Including, wherein the small molecule has a structure that includes 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 small molecule's structure is replaced with tellurophene, 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.

[0113] In some embodiments, the method further comprises quantifying the amount of tellurium atoms in the sample, where the tellurium atom level is indicative of low molecular weight molecules in the sample.

[0114] In some embodiments, the method further comprises quantifying one or more other analytes in the sample, and the method further comprises labeling the sample with one or more mass-labeled analyte binding agents prior to performing mass spectrometry to determine the level of the one or more analyte binding agents.

[0115] In some embodiments, the one or more mass-labeled analyte binding agents 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 viability indicators such as cisplatin, barcoding reagents such as Cd-labeled CD45 and Pt-labeled CD45. In some embodiments, the polymer-labeled antibody comprises a metal. For example, the polymer can be bound to a metal.

[0116] In some embodiments, the subject is a mammal, optionally a mouse, rat or human.

[0117] In some embodiments, mass spectrometry is performed at a plurality of separate locations and the level of tellurium atoms is determined at each of the plurality of separate locations to provide a distribution of analogs in the sample, which distribution of analogs in the sample is indicative of the distribution of small molecules in the sample.

[0118] In some embodiments, the sample is a single cell, and the distribution of the analog within the single cell indicates the distribution of the small molecule at the subcellular level.

[0119] In some embodiments, multiple samples are provided, the samples comprising different tissues, cells or secretions of a subject, where detection of the analog in the multiple samples provides a distribution of the analog within the subject, indicative of the distribution of the small molecule within the subject, such as tissue or organ distribution.

[0120] In some embodiments, the sample is or comprises urine, stool, 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, cardiac cells, lung cells or secretions, pancreatic cells or secretions, and / or gastric cells or secretions.

[0121] In some embodiments, the sample is a frozen tissue section.

[0122] In some embodiments, the sample is or comprises cells from a cell culture or medium from a cell culture.

[0123] In some embodiments, the sample is a single cell, and the distribution of the analog within the single cell indicates the distribution of the small molecule at the subcellular level.

[0124] In some embodiments, the analog interacts irreversibly, optionally covalently, with a target in a sample.

[0125] In some embodiments, the subject or cell / tissue culture is administered a tellurophene small molecule analog in combination with the small molecule, and the method further comprises directly detecting the level of the small molecule, and a comparison of the level of the small molecule to the level of the analog indicates putative competitive binding of the analog.

[0126] In another aspect, the disclosure includes the use of tellurophene analogs in the detection of small molecules by mass spectrometry in subjects administered the analogs or cell / tissue cultures administered the analogs, the small molecules having a structure that includes one or more monocyclic or bicyclic aromatic rings, the analogs having a structure in which at least one of the one or more monocyclic or bicyclic aromatic rings of the small molecule's structure is replaced with tellurophene.

[0127] In another aspect, the present disclosure includes the use of a compound of formula I of the present disclosure, or a composition comprising a compound of formula I of the present disclosure, in the detection of teniposide by mass spectrometry in a subject administered a compound of formula I, or a salt or solvate thereof, or in a cell / tissue culture administered a compound of formula I, or a salt or solvate thereof.

[0128] In another aspect, the disclosure includes the use of a compound of formula II of the disclosure or a composition comprising a compound of formula II of the disclosure in the detection of carfilzomib by mass spectrometry in a subject administered a compound of formula II or a salt or solvate thereof or in a cell / tissue culture administered a compound of formula II or a salt or solvate thereof.

[0129] In another aspect, the present disclosure provides a compound of formula I [ka] or a salt or solvate thereof; and A kit for mass cytometry analysis that includes a tellurium standard or a plurality of tellurium standards is included.

[0130] In another aspect, the present disclosure provides a compound of formula II [ka] or a salt or solvate thereof; and A kit for mass spectrometry, and optionally mass cytometry analysis, including a tellurium standard or a plurality of tellurium standards is included.

[0131] In another aspect, the disclosure provides a method for determining a dosage of a small molecule to achieve a 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 on the target; detecting the small molecule in a subject by the methods of the disclosure; Measuring the effect produced by the small molecule; and Determining an appropriate dose of tellurophene analog to achieve a desired distribution of the small molecule. wherein the dosage of the tellurophene analog indicates the dosage of the small molecule.

[0132] In addition to enabling single cell analysis, mass cytometry can include mass cytometry imaging methods, such as those described in (Giesen et al. 2014, incorporated herein by reference). In such methods, tissues or cell populations are in vitro labeled with mass-labeled substances, such as analyte binding agents and / or compounds that contain atoms suitable for mass cytometry, the tissues or cell populations are subjected to laser ablation coupled with 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.

[0133] In some embodiments, the mass spectrometry is mass cytometry or multiplexed ion beam imaging, and optionally the mass cytometry is mass cytometry imaging or mass cytometry suspension.

[0134] In some embodiments, the tellurium atom is 120 Te, 122 Te, 123 Te, 124 Te, 125 Te, 126 Te, 128 Te, 130 Te, and combinations thereof.

[0135] In some embodiments, the tellurium atoms include multiple tellurium isotopes and the mass cytometry is multi-channel mass spectrometry, optionally multi-channel mass cytometry.

[0136] It can be appreciated that the disclosed methods and the disclosed tellurophene analogs can be used to track drug distribution in organs or tissues in general. For example, the disclosed methods can be used to assess small molecule distribution when the small molecule is administered through different routes of administration (e.g., inhalation, injection, etc.).

[0137] It is contemplated that the disclosed tellurophene analogs and the disclosed methods can be used in combination with other analytical methods and reagents, either simultaneously or sequentially. For example, the disclosed tellurophene analogs and the disclosed methods can be used in combination with labeled antibodies (e.g., metal-labeled antibodies and polymer-labeled antibodies) such as MAXPAR® reagents, metal-labeled oligonucleotides, metal-containing intercalators, and viability indicators (e.g., metal viability indicators) such as CELL-ID™ reagents, barcoding reagents such as Cell-ID™ 20-Plex Pd Barcoding Kit; Cd-CD45; Pt-CD45, and / or calibration beads such as EQ™ Four Element Calibration Beads and EQ™ Six Element Calibration Beads.

[0138] It is also contemplated that the disclosed tellurophene analogs and methods of the present disclosure can be used in different applications, including diagnostics, preclinical studies, including animal studies, pharmacokinetic (eg, pulse-chase studies), and pharmacodynamic studies.

[0139] Depending on the nature of the small molecule and its analogs, it is contemplated that the methods of the present disclosure and the analogs of the present disclosure can be used to study a variety of conditions, including neoplastic disorders such as leukocytosis, neuroblastoma, and non-Hodgkin's lymphoma.

[0140] IV. Methods for Preparing the Compounds of the Disclosure The compounds of the present disclosure can be prepared by various synthetic processes. The selection of certain structural features and / or substituents may influence the selection of one process over another. The selection of a particular process for preparing a given compound of the present disclosure is within the scope of one skilled in the art. Some starting materials for preparing the compounds of the present disclosure are available from commercial chemical suppliers. For example, other starting materials described below are easily prepared from available precursors using straightforward transformations well known in the art. In the following schemes showing the preparation of the compounds of the present disclosure, all variables are as defined herein unless otherwise indicated.

[0141] The formation of a desired compound salt is accomplished using standard techniques, for example, a neutral compound is treated with an acid or base in a suitable solvent and the formed salt is isolated by filtration, extraction or any other suitable method.

[0142] The formation of solvates varies depending on the compound and the solvate. In general, solvates are formed by dissolving the compound in a suitable solvent and isolating by cooling the solvate or using an anti-solvent. The solvate is typically dried or azeotroped under ambient conditions. The selection of suitable conditions for forming a particular solvate can be made by one of ordinary skill in the art. Examples of suitable solvents are ethanol, water, etc. When water is the solvent, the molecule is called a "hydrate". The formation of solvates of the compounds of the present disclosure varies depending on the compound and the solvate. In general, solvates are formed by dissolving the compound in a suitable solvent and isolating by cooling the solvate or using an anti-solvent. The solvate is typically dried or azeotroped under ambient conditions. The selection of suitable conditions for forming a particular solvate can be made by one of ordinary skill in the art.

[0143] It is to be understood that suitable protecting groups are added and subsequently removed from various reactants and intermediates as necessary throughout the processes described herein, as will be readily understood by those skilled in the art. Conventional procedures for the use of such protecting groups and examples of suitable protecting groups are described, for example, in Protective Groups in Organic Synthesis, TW Green, PG M Huts, Wiley-Interscience, New York, (1999). It should also be understood that the conversion of a group or substituent to another group or substituent by chemical manipulation may be performed on any intermediate or final product on the synthetic route to the final product, with the types of conversions possible being limited only by the inherent incompatibility of other functional groups possessed by the molecule at that stage with the conditions or reagents used for the conversion. Such inherent incompatibilities, and how to avoid them by carrying out the appropriate conversions and synthetic steps in the appropriate order, will be readily understood by those skilled in the art. Examples of conversions are given herein, and it should be understood that the conversions described are not limited to merely the general groups or substituents for which the conversions are exemplified. References and descriptions of other suitable transformations are provided in "Comprehensive Organic Transformations-A Guide to Functional Group Preparations" RC Larock, VHC Publishers, Inc. (1989). References and descriptions of other suitable reactions can be found in organic chemistry textbooks such as "Advanced Organic Chemistry", March, 4th Edition, McGraw Hill (1992) or "Organic Synthesis", Smith, McGraw Hill, (1994). Purification techniques for intermediates and final products include, for example, normal and reverse phase chromatography on columns or spinning plates, recrystallization, distillation and liquid-liquid or solid-liquid extraction, as will be readily understood by those skilled in the art.

[0144] Thus, in some embodiments, compounds of the present disclosure can be prepared as described below. For example, compounds of formula I can be prepared as shown in Scheme 1. For example, compounds of formula II can be prepared as shown in Scheme 2. It can be understood that tellurophene moieties can be incorporated into small molecules to replace one or more aromatic rings by other suitable synthetic approaches. Some suitable examples are provided in the following schemes.

[0145] Tellurophene Addition Method In some embodiments, tellurophene can be incorporated via an amine group. For example, tellurophenylamine can be obtained from tellurophene aldehyde by reductive amination, as shown in Scheme B. [ka]

[0146] In some embodiments, tellurophene can be incorporated into small molecules via the carboxyl group. For example, tellurophene carboxylic acid can be obtained from tellurophene aldehyde via oxidation. The resulting tellurophene carboxylic acid can be coupled to small molecules via an amide or ester bond. (Scheme C) [ka]

[0147] In some embodiments, tellurophene can be converted by halogenation to a tellurophene halide, which can be incorporated into a small molecule via a metal-catalyzed cross-coupling reaction. (Scheme D) [ka]

[0148] It is also contemplated that other tellurophene-containing heterocycles (e.g., bicyclic heterocycles) can be used to replace aromatic rings in small molecules. For example, analogs of small molecules containing bicyclic aromatic rings can be designed and prepared by replacing the bicyclic aromatic ring with a bicyclic tellurophene containing heterocycle. In some embodiments, benzofuran or indole can be replaced with benzo[b]tellurophene. Benzo[b]tellurophene can be prepared by cyclization of an alkynylaryl halide in the presence of tellurium. (Scheme E) [ka]

[0149] In some embodiments, thieno[3,2-b]pyrrole and / or pyrrolo[3,2-b]pyrrole can be replaced with telluropheno[3,2-b]pyrrole. For example, telluropheno[3,2-b]pyrrole can be obtained from cyclization of an alkynylpyrrolo halide. (Scheme F) [ka]

[0150] Working Example The following non-limiting examples are illustrative of the present disclosure.

[0151] Example 1 Tellurofenteniposide Analogues: Synthesis and Evaluation Tellurium-substituted analogs of the anticancer xenobiotic teniposide (compound of formula I) have been developed. Teniposide is a semisynthetic podophyllototoxin that inhibits topoisomerase II (Top2) enzyme activity by binding to the Top2-DNA complex. Top2 alters DNA topology by introducing transient double-strand breaks via the covalent Top2-DNA complex. Upon binding, teniposide stabilizes the DNA-Top2 complex and prevents DNA from religating, resulting in the accumulation of double-strand breaks (DSBs). The cytotoxic effect of teniposide is associated with apoptosis, leading to excessive DSBs. Teniposide is a clinically approved chemotherapeutic agent primarily used to treat acute lymphoblastic leukemia (ALL), Hodgkin's lymphoma, and neuroblastoma. Teniposide inhibits the proliferation of cells with high copy number (1 × 10 in transformed cell lines). 6 ) Top2, suggesting that tellurium-labeled teniposide analogs bound to Top2 should be detectable by MC.

[0152] Teniposide possesses a thiophene ring that was hypothesized to be amenable to synthetic substitution. Synthesis of teniposide derivatives in which the thienyl group was replaced with a tellurophene ring readily afforded MC visible analogs. Te-teniposide analogs were demonstrated to be indistinguishable from teniposide in cell-based assays. Furthermore, it was shown that MC can be used to track the localization of the compound in cells. In vivo Te-teniposide behaved similarly to teniposide and was directly detected in tissue sections.

[0153] Results and Discussion Synthesis of Te-teniposide Using a semisynthetic strategy, the Te-teniposide analogue (1) was prepared starting from 4'-demethylepipodophyllotoxin α-D-glucopyranoside, which could be conveniently obtained by mild acid hydrolysis of etoposide. As is often done for the formation of benzylidene acetals with the corresponding dimethyl acetals, the formation of the desired tellurophenylidene acetal was initially envisaged to proceed directly from the corresponding diethyl acetal 3, which was generated from propargylaldehyde diethyl acetal and (bromoethynyl)triisopropylsilane under Cadiu-Chodkiewicz conditions. The resulting diyne 2 was cyclized to tellurophene 3 by treatment with sodium hydrogen telluride, which was generated in situ from tellurium metal and sodium borohydride.

[0154] Tellurophene 3 was used to investigate a series of model condensation reactions with methyl-α-D-glucopyranoside. 3 Common conditions used for the introduction of ZnCl (CN or DMF) failed, resulting in recovery of tellurophene aldehyde 4. Literature methods for the generation of teniposide and similar acetals use neat aldehydes and freshly fused ZnCl 2 We proposed conditions using the tellurophene diethyl acetal 3, which could be cleanly converted to the aldehyde 4 by mild acid hydrolysis. Condensation of 4'-demethylepipodophyllotoxin with neat tellurophene aldehyde 4 afforded the desired teniposide analog 1 in moderate yield (Scheme 1). [ka] Synthesis of tellurophene-substituted teniposide analogue 1. (a) Bromoethynyl)triisopropylsilane, 5 mol% CuCl, NH 2 OH HCl, 30% BuNH 2 Aqueous solution, 0°C, 4 hours (57%); (b) Te metal (1.1 equivalent), NaBH 4(8 equiv.), EtOH:HO (1:1), 50 °C, 12 h (52%); (c) 1.0 M HCl:THF (1:1), rt, 4 h (quantitative); (D) 20% aqueous acetic acid, 75 °C, 20 h (66%); (e) 2-tellurophenecarboxaldehyde (4) (neat), anhydrous ZnCl 2 (2.0 equivalents) (16%).

[0155] In vitro analysis of Te-teniposide The relative Top2 inhibitory activity of 1 compared to teniposide was assessed in a decatenation assay using kinetoplast DNA (kDNA). DNA decatenation catalyzed by Top2 can be readily resolved using agarose gel electrophoresis. Both teniposide and 1 demonstrated dose-dependent inhibition of the decatenation reaction (Figure 1). The calculated IC of 1 50 The value (5.6 μM) is similar to that of teniposide calculated from fluorescence polarization measurements and DNA relaxing gel assays in the literature (1.3-9.8 μM).

[0156] Cytotoxicity of Te-teniposide To understand the spatial and temporal distribution of Top2 expression and its associated drug resistance mechanisms in cells, the HL-60 promyelocytic leukemia cell line was used as a model system. After 24 h of incubation, the toxicity of 1 was compared to teniposide in HL-60 cells using a WST-1 viability assay. The dose-response curves for teniposide and 1 were observed to be identical within experimental variability (Figure 2), consistent with the literature, with both Top2 toxins inhibiting HL-60 cell proliferation by 60% at a maximum concentration of 50 μM.

[0157] Similarly, PANC-1 cells were characterized for subsequent in vivo experiments. PANC-1 cells were incubated with 1 or teniposide using Alamar Blue viability and proliferation assays. Both drug forms exhibited concentration-dependent cytotoxicity in PANC-1 cells with indistinguishable dose responses, with near complete loss of cell viability at the highest dose of 25 μM (Figure 3). In the proliferation assay, time course analysis of over 100 hours of incubation revealed that concentrations of teniposide or 1 ≥ 0.1 μM substantially reduced PANC-1 cell proliferation for both compounds (Figure 4, panels A and B). Taken together, these data suggest that 1 effectively mimics the antiproliferative activity of teniposide in PANC-1 cells.

[0158] Te-teniposide induces DNA damage We next examined the ability of 1 to induce DSBs, the main type of DNA damage resulting from Top2 inhibition. In response to DSBs, a cascade of DNA repair mechanisms is initiated, including phosphorylation of the histone variant H2AX at Ser139. Therefore, the level of pH2AXSer139 serves as a reliable marker of Top2-bound DSBs. Western blotting was used to measure pH2AXSer139 in PANC-1 cells treated with 1 μM of 1 or teniposide for 24 and 48 h. Increased levels of pH2AXSer139 were clearly observed for both inhibitors compared to DMSO-treated controls, indicating that 1 is comparable to teniposide in its ability to induce DSBs (Figure 5).

[0159] Cellular uptake of Te-teniposide To determine whether cell labeling with 1 depends 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 2 h to saturate intracellular Top2-DNA ternary complexes with unlabeled ligand, and then 1 (2 or 10 μM) was introduced into the cell medium and the suspension was gently mixed. After 2 h, cells were harvested and washed three times with PBS. Cells were then fixed and permeabilized, followed by DNA staining with Ir intercalator. 130 Cell pellets were analyzed by MC for Te content (Fig. 6).

[0160] Cells labeled with 1 were readily detected at both concentrations (2 and 10 μM), whereas in cells pretreated with unlabeled teniposide, 130 The expected decrease in Te signal was not observed (Figure 6A). Additionally, we performed co-incubation experiments in which HL-60 cells were treated with a cocktail of either 1 (2 μM) and 2, 10, or 20 μM teniposide (Figure 6B). The population histograms for all three treatment groups were identical, suggesting that under these conditions, no competition for Top2 could be observed. Without wishing to be bound by theory, it is hypothesized that the lack of competitive labeling observed in these experiments could be due to a combination of inhibition of Top2 and a significant degree of nonspecific binding by other cellular components. Several reports have demonstrated nonspecific accumulation of etoposide and teniposide via oxidation catalyzed by cytochrome P450-dependent monooxygenase, peroxidase, and tyrosinase enzymes. These redox transformations produce catechol and quinone metabolites, which significantly impair the DNA-binding ability of Top2. It is also possible that these reactive species modify other intracellular proteins and thus contribute to drug accumulation in cells, but this is accidental and irreversible. These results illustrate the utility of MC visual probes to follow the specificity of pharma- ceutical active compounds, where nonspecific binding may dominate target binding of teniposide and possibly its analog Te-teniposide 1.

[0161] In vivo evaluation of tellurium-labeled teniposide Despite the inability to demonstrate selective reversible binding in cell suspensions by MC, the ability to easily detect compound 1 in cell culture and the promising activity of compound 1 were demonstrated. Visualizing the nonspecific accumulation and lack of target specificity in tissue sections was not observed with cisplatin. 8 This will improve our understanding of the distribution of such therapeutic agents.

[0162] IMC™ can be used to image tissue localization and quantify the concentration of 1 in tissue samples. For in vivo studies, a PANC-1 tumor xenograft-bearing mouse model was selected. The tumor microenvironment and general tissue morphology in sections derived from this human pancreatic cancer model can be characterized.

[0163] PANC-1 xenograft-bearing mice were injected IP with either teniposide or Te-teniposide analog 1. A dose of 20 mg / kg was selected based on literature reports detailing teniposide drug delivery schedules. 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 Ser139 Expression levels were first assessed by immunohistochemistry. Serial sections from PANC-1 xenograft tumors were stained with primary antibodies followed by standard brown staining with HRP-conjugated secondary antibodies. DSBs were characterized by large, discrete pH2AX Ser139 Representative images of immunostained slides show increased levels of pH2AXSer139 in treated mice compared to saline-injected controls with clear punctate staining (Figure 7). These results indicate that 1 was distributed throughout tumor tissue and maintained the ability to induce DSBs in vivo similar to teniposide.

[0164] For IMC™ analysis, the first objective was to visualize tellurium signal distribution 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 intercalator to visualize cell nuclei, and then imaged by IMC™. 125 Te isotopes were visualized. Unfortunately, no Te signal above background could be detected in tumor sections. Efforts to combine intensities from multiple tellurium channels to improve the signal-to-noise ratio were also unsuccessful. Without wishing to be bound by theory, it was hypothesized that processing of FFPE tissues contributed to the loss of tellurium signal as teniposide interactions with cells are primarily non-covalent, and therefore 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 tumors were sectioned and analyzed by IMC™. Using this technique, we found that 100% Te was detected in tissue samples exposed to Te-teniposide 2 hours prior to sacrifice. 125 A Te signal was observed (Figure 8). In samples taken at later time points after administration, no Te signal was observed in strains where the half-life of teniposide in mice ranged from 1 to 5 hours, depending on the route of administration and dosing regimen. 24 .

[0165] A set of standards containing increasing concentrations of Te was analyzed by IMC™ to determine the corresponding concentrations of Te present in the samples. The counts / Te atomic constant was calculated based on the total counts present in each standard Te spot (Figure 8E). The counts observed in the frozen samples suggest that the average Te-teniposide concentration in the tissue is in the low tens of micromolar range at the time of analysis. The observed signal was low, so an exact value could not be calculated. The observed signal, which is slightly above background, is consistent with the expected sensitivity of the IMC™ instrument to tellurium.

[0166] conclusion The ability to directly quantify the cellular distribution of pharmacologically active small molecules in tissues using IMC™ will provide cell-specific information on target engagement and biodistribution. With antitumor agents, teniposide can generate analogs with indistinguishable biological activity to the native compound through the replacement of the tellurenyl functional group with thienyl, as shown. Using Te-teniposide analogs, a high level of non-specific binding of the compound to cells was demonstrated by MC, suggesting that Top2, a known target of teniposide, is only a small portion of the binding sites for the compound. This is consistent with the high level of protein binding of teniposide. In vivo, the activity of Te-teniposide generated the expected DSBs in PANC-1 xenografts, and Te signals could be observed in tissues.

[0167] Experimental Department chemical synthesis General Materials and Methods: Etoposide (CarboSynth), propargylaldehyde diethyl acetal (TCI America) and glacial acetic acid (Caledon Laboratories Ltd) were purchased from the vendors indicated. All other reagents were purchased from Sigma Aldrich. All reactions were carried out under a dry argon atmosphere using oven-dried glassware. Absolute ethanol was obtained from Green Field Specialty Alcohols, Inc., while all other anhydrous solvents were purchased from Sigma Aldrich and dried over 4 Å molecular sieves before use. Purification by flash column chromatography (SiliCycle Silica-P Flash Silica Gel, 40-60 μm, 60 Å pore size) used silica gel and the gradient solvent mixtures listed. Compounds were purified using a BIOTAGE ISOLERA™ system. The purity of the final compound 1 was confirmed using RP-HPLC coupled with low resolution mass spectra (ESI) collected on an AGILENT™ Technologies 1200 series HPLC coupled to a 6130 mass spectrometer. The final compound 1 was resolved on a Phenomenex's KINETEX™ 2.6 μm C18 50×4.6 mm column at room temperature with a flow rate of 1 mL / min. The gradient consisted of eluents A (0.1% formic acid in double distilled water) and B (0.1% formic acid in HPLC grade acetonitrile). A linear gradient starting at 5% to 95% over 7 min with a flow rate of 1.0 mL / min. 1 H and 13 C NMR spectra were obtained using CDCl 3 , CD 3 O.D. and C. 2 D 6 All data were recorded on Bruker 400 MHz and Agilent 500 MHz spectrometers in SO. Chemical shifts (δ) were expressed as a function of the CDCl internal standard solvent peak. 3 : 7.26 ppm and C 2 D 6 SO: 2.49 ppm, CD 3Coupling constants (J) are reported in Hz. High resolution mass spectra were obtained on a VG70-250S (double focusing) mass spectrometer at 70 eV or on an ABI / SCIEX QSTAR™ mass spectrometer with an ESI source and accurate mass capability.

[0168] (Bromoethynyl)triisopropylsilane: To a solution of (triisopropylsilyl)acetylene (2.0 mL, 8.9 mmol, 1.0 equiv.) in acetone (0.15 M), N-bromosuccinimide (1.75 g, 9.8 mmol, 1.1 equiv.) and silver nitrate (0.15 g, 0.89 mmol, 0.1 equiv.) were added successively. The reaction mixture was stirred at room temperature for 5 h, then washed with saturated NH 4 Cl (30 mL) was added. The resulting mixture was then extracted with diethyl ether (3×30 mL). The organic layers were combined, washed with water (30 mL), brine (30 mL), and MgSO 4 The oil was dried under high vacuum to give the corresponding bromoalkyne as a pale yellow oil which was carried on to the next step. 1 H NMR (500 MHz, CDCl 3 ) 1.11 (m; no alkyne proton).13C NMR (101MHz, CDCl3) = 83.50, 61.72, 18.49, 11.29.

[0169] (5,5-Diethoxypenta-1,3-diyn-1-yl)triisopropylsilane (2): In a round-bottom flask containing CuCl (4 mg, 0.38 mmol, 0.05 equiv.), 30% n-BuNH 2 Aqueous solution (0.5M) was added to produce a blue solution. The reaction was cooled to 0° C. A small amount of NH 2OH·HCl was added until the reaction mixture became colorless. Propargylaldehyde diethyl acetal alkyne (0.99 mL, 7.59 mmol, 1.1 equiv) was added to the flask at 0 °C, causing the solution to turn slightly yellow. After 5 min, (bromoethynyl)triisopropylsilane (2.18 g, 8.34 mmol, 1.0 equiv) 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 washed with MgSO 4 It was dried at 40° C., filtered and concentrated under reduced pressure. Silica gel column chromatography using a gradient of 100% pentane to 5% EtOAc / 95% pentane afforded the diyne 2 as a yellow oil (2.57 g, 57% yield). 1 H NMR (500 MHz, CDCl 3 ) 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 (126 MHz, CDCl 3 = 91.41, 88.43, 85.88, 71.31, 70.44, 61.23, 18.47, 15.01, 11.17. [C 18 H 33 O 2 Si] + [M+H 2 O+H] + ESI LRMS m / z calculated for 309.54, found 309.17.

[0170] 2-(Diethoxymethyl)tellurophene (3): To a round-bottom flask was added crushed tellurium metal (0.323 g, 2.53 mmol, 1.2 equiv.) and sodium borohydride (0.384 g, 10.1 mmol, 4.8 equiv.). The flask was purged with argon multiple times. Degassed water (10 mL) was added to the flask containing tellurium and sodium borohydride and allowed to stir under argon at 50° C. Over the course of 1 h, the metal suspension turned deep purple and eventually became a homogeneous colorless solution. Concurrently, a 20 mL scintillation vial charged with diyne 2 (0.650 g, 2.10 mmol, 1 equiv.) 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 stirred for approximately 1 h. The diyne solution was allowed to warm to room temperature and slowly concentrated using a rotary evaporator. The crude mixture was diluted with Et 2 The flask was diluted with 200 mL of 1000 sulphate (30 mL) and brine (30 mL). The organic layer was separated, washed twice with brine (2 x 25 mL) and concentrated under reduced pressure without heating to yield a deep red oil that decomposed on standing. The oil was taken up in degassed EtOH (10 mL) and purged with an argon balloon using sonication. This solution was added dropwise via syringe to the sodium hydrogen telluride solution. After 12 h, TLC indicated that the diyne raw material had been consumed and a new UV active spot had appeared. The flask was cooled to room temperature and then exposed to air for at least 1 h. The crude mixture was filtered through a Celite pad. A small amount of EtOH (~5 mL) was used to wash the Celite pad. The filtered orange-red solution was diluted with DCM (50 mL) and brine (50 mL) and extracted with DCM (5 x 30 mL). The separate organic layers were collected and washed with MgSO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure. Tellurophene was purified using silica gel column chromatography with a gradient of 100% pentane to 10% EtOAc / 90% pentane to give tellurophene 3 as a deep red-orange oil (52% yield). 1 H NMR (400 MHz, CDCl 3) = 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). [C 4 H 3 Te]-[MC 5 H 11 O 2 ] - ESI LRMS m / z calculated for 180.93, found 181.14.

[0171] 2-Tellurophenecarboxylaldehyde (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 allowed to stir. HCl (1.0 M, 5 mL) was added to the vial and the solution was stirred at room temperature for 6 h. 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 x 20 mL). The DCM portions were combined and diluted with MgSO 4 The mixture was dried at 40° C., filtered, and concentrated under reduced pressure to give the corresponding aldehyde 4 as a red liquid. 1 H NMR (400 MHz, CDCl 3 ) = 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, CDCl 3 ) = 187.82, 150.60, 147.45, 138.72, 138.41. [C 4 H 3 ESI LRMS m / z calculated for Te]-[M-CHO-H]- 180.93, found 181.14.

[0172] 4-Demethylepipodophyllotoxin: In a round-bottom flask (50 mL), 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. 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, adsorbed onto silica, and subsequently purified using silica gel column chromatography with a gradient from 100% DCM to 20% MeOH / 80% DCM. The hydrolyzed etoposide derivative was obtained as a white powder (66% yield). 1 H NMR (500 MHz, CD 3 OD) = 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;CH 3 OH resonance), 2.94 (m, 1H). [C 27 H 31 O 13 ESI LRMS m / z calculated for [M+H] 563.52, found 563.18. 13 C NMR (126 MHz, 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.

[0173] Tellurium-labeled teniposide (1): To a 1-dram vial was added zinc chloride (12 mg, 0.89 mmol, 2.0 equiv) and a stir bar and flame dried for 2 min. The flask was immediately placed under vacuum for at least 30 min. 4-Demethylepipodophyllotoxin (25 mg, 0.44 mmol, 1.0 equiv) was quickly added and the vial was purged multiple times with argon. Aldehyde 4 (approximately 0.250 g) was added dropwise and the vial was carefully sonicated to ensure all solid material was in suspension. The reaction was allowed to stir gently at room temperature for 16 h. 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 combined organic fractions were pooled and diluted with MgSO 4 The mixture was dried at 400 rpm and concentrated. The residue was purified by silica gel column chromatography using a gradient of 10% to 50% ethyl acetate in pentane to give 1 (16%) as a pale yellow waxy solid. 1H NMR (500 MHz, C 2 D 6 SO) = 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 = 13C NMR (126 MHz, C 2 D 6SO) = 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, [C 32 H 32 O 13 130 TeNa] + [M+Na] + ESI HRMS m / z calculated for 777.081, found 777.079.

[0174] In vitro experiments Top2 Decatenation Assay. The assay was performed according to the manufacturer's protocol (TopoGEN, Inc.). The total reaction volume was 20 pL of 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 of kinetoplast 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 minutes. The reaction was stopped by adding 5 pL of stop buffer (5% sarkosyl, 0.025% bromophenol blue, and 50% glycerol). Samples were then analyzed by electrophoresis using a 1% agarose gel in Tris-borate-EDTA buffer and stained with 0.5 pg / mL ethidium bromide. Gels were imaged using a Syngene G:Box gel imager (Chemi-XT4 GENESys software preset for ethidium bromide stained agarose gels). Band intensities were analyzed using ImageJ software.

[0175] WST-1 metabolic cytotoxicity assay: HL-60 cells (ATCC® CCL-240™) were maintained in DMEM medium with 2 mM L-glutamine (Gibco) and supplemented with 20% bovine calf serum and 1:100 diluted penicillin-streptomycin solution (Gibco). 2 The cells were maintained at 37°C in a humidified atmosphere. HL-60 cells (100 pL) were diluted at 5 × 10 5 Cells were seeded into clear 96-well plates at a density of 1000 cells / mL. Cells were treated with either DMSO stocks or appropriate inhibitors from DMSO stocks. Dilutions were made carefully to ensure that DMSO concentrations did not exceed 1%. Cells were incubated at 37 °C for 24 h in 5% CO 2 The cells were incubated for 24 hours at 37°C in a 5% CO atmosphere. WST-1 reagent (10 pL; Roche Diagnostics, 05015944001) was added to each well and mixed gently by pipetting. 2 The plates were incubated at 37°C for 1 hour under ambient conditions. UV-vis absorbance readings at 450 nm for each well were recorded using a TECAN Safire 2 plate reader. Data were background corrected against wells containing cells and cell growth medium without WST-1. Experiments were performed in triplicate.

[0176] IncuCyte ZOOM™ Cell Proliferation Assay: Human pancreatic ductal carcinoma (PANC-1) cell line was purchased from ATCC (CRL-1469). Cells were cultured in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% FBS. Cell maintenance and experiments were performed at 37 °C in 5% CO 2The experiments were carried out in a humidified 37 °C incubator with 5% CO. Cells were routinely tested for mycoplasma contamination. PANC-1 cells (5000) were seeded in a 96-well plate and incubated for 18 h. The medium was then removed and replaced with fresh medium containing drugs (0–25 pM). Cells were then transferred to an IncuCyte ZOOM system (ESSEN BioScience, Ann Arbor, MI, USA) and live-cell phase-contrast images were obtained using a 10x objective. Cell confluence was analyzed using IncuCyte ZOOM 2016B software.

[0177] pH2AX blotting: PANC-1 cells (0.5 × 10 6 ) cells were seeded in 60 mm plastic Petri dishes (Corning Inc. NY) and incubated for 18 h. Spent medium was removed and replaced with fresh medium containing drug (1 pM) and incubated for either 24 or 48 h. Control cells were treated with DMSO only. Cells were then lysed in RIPA buffer (25 mM Tris·HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) (ThermoFisher Scientific) containing Halt protease inhibitor cocktail (cat. no. 78410) and Halt phosphatase inhibitor cocktail (cat. no. 78420) according to the manufacturer's protocol. Extracted protein samples were subjected to electrophoresis using Bolt 4–12% Bis-Tris, 1.0 mm gels (ThermoFisher Scientific) and immunoblotted as suggested. Antibodies (Abcam, Cambridge, MA) targeting the following proteins were used: H2AX (S139) (ab11174, polyclonal) and β-tubulin (ab6046, polyclonal).

[0178] Alamar Blue assay: PANC-1 cells were seeded into black clear-bottom 96-well assay plates (Corning; CLS3603) and incubated for 24 h. The medium was replaced with 0.01–25 pM teniposide or compound 1. After 72 h exposure to drugs, Alamar Blue (Thermo Fisher Scientific; DAL1100) cell viability reagent was added and incubated for 4 h at 37 °C. Fluorescence intensity was measured using an excitation wavelength of 560 nm and emission of 590 nm. Cell viability was calculated by normalizing to untreated controls.

[0179] CYTOF® labeling: for pre-saturation experiments, 3 x 10 6 HL-60 cells (5 × 10 5 cells / mL) in 5% CO in air 2 The cells were incubated with the appropriate teniposide concentration for 2 hours at 37°C in a humidified atmosphere at 10°C. Compound 1 was pipetted into the cell suspension and mixed gently. The cells were incubated for another 2 hours, then centrifuged at 300×g for 6 minutes and the medium was aspirated. For controls, HL-60 cells were treated consecutively with teniposide or compound 1 for 4 hours. For co-incubation experiments, cells were incubated with the drug cocktail for 4 hours. Drug-treated cells were washed with medium and then with PBS. Cells were fixed with 3.7% formaldehyde (Sigma Aldrich F1635) diluted in PBS for 10 minutes. Fixed cells were centrifuged at 800×g for 5 minutes and washed with PBS. The cell pellet was then stained with Ir intercalator (diluted 1:1000 in PBS) for 1 hour at room temperature. Cells were centrifuged at 800×g for 5 minutes, followed by washing twice with PBS and finally with ddH 2 The cells were washed with O. The cell pellet was taken up in 10% EQ™ 4 element calibration bead solution prepared by CAS and filtered into a polystyrene tube through a 35 pm cell strainer cap. The sample was then injected into the CYTOF® Helios™ and analyzed.

[0180] In vivo experiments Generation of mouse 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 Princesss Margaret Cancer Centre (PMCC) facility. All animal experiments in this study were performed in accordance with the University Health Network Institutional Animal Research Committee guidelines. Subcutaneous PANC-1 xenograft mice were generated as previously described in the Telox study. Vehicle was prepared as follows: 30 mg benzyl alcohol, 60 mg N,N-dimethylacetamide, 500 mg purified Cremophor EL (Kolliphor EL) and 100 mg ethanol. EL) and 2.1 mL of absolute alcohol were combined in a vial. To this solution, distilled water was added to a final volume of 5 mL. The pH of the clear solution was adjusted to a pH of approximately 5 with maleic acid. For injection, the drug was diluted in vehicle to a final concentration of 10 mg / mL and administered IP at 20 mg / kg. Mice were maintained in three groups, with mice in group 1 injected with teniposide, group 2 given teniposide 1, and group 3 given normal saline. After 2 hours, mice were sacrificed and tumors were extracted. Half of the tumor was fixed and proceeded to paraffin embedding, and the remaining xenograft tissue was extracted using OCT (Tissue-Tek The tumor samples were embedded in 100% PBS (Sakura-Finetek) and flash frozen in liquid nitrogen. Flash frozen tumor samples were stored at -80°C. Cryostat sections were cut (5 pm) using a microtome and mounted on microscope slides. These sections were stored at -80°C until IMC™ analysis or histochemical staining. Sections were subjected to H&E staining to evaluate tissue morphology and pH2AX IHC staining to visualize pH2AX speckles. Adjacent sections were stained for IMC™ 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 using a ScanScope AT2 (Aperio) at 20x magnification (approximately 0.5 pm / pixel) and viewed using ImageScope software.

[0181] IMC™ Analysis: Frozen tissue sections (5 pm) were thawed from -80°C to room temperature and subjected to IMC™ analysis directly on a HYPERION™ Imaging System (Fluidigm) without DNA staining or washing steps. To assess the tellurium sensitivity of IMC™, serial dilutions of tellurium standard solution (Sigma Aldrich Cat. No. 92027) were prepared in trypan blue. A volume of 2 pL was arrayed on air-dried 2% agarose-coated microscope slides to give a final concentration range of 0-50 μM.

[0182] IMC™ Data Acquisition and Analysis: Slides were ablated at 200 Hz using a HYPERION™ Imaging System (Fluidigm) and images were acquired as per the text files. Each image was analyzed using the method described by Bassan and Nitz. 25 The mass channels were extracted into separate numpy arrays for each channel using the teimc package by . These numpy arrays were used to prepare raw images, which were then analyzed using Numpy, pandas, Matplotlib, and the Scikit image library. For the tellurium standard curve, the entire spot was ablated and the entire cross-sectional area was 125 The Te counts were summed ( 125 For the blank spot ([Te] = 0 μM), 125 The average per pixel is calculated by dividing the Te signal by the number of pixels in the ablation cross-sectional area. 125 The Te intensity was calculated. This value represents the background signal per pixel in the 125 amu channel corresponding to the particular HYPERION™ imaging system ( 125To account for the contribution of this background across tellurium-containing images, the following correction was performed for each concentration: 125 Te 補正 (i)= 125 Te 総数 (i)-( 125 Te バックグラウンド × area (i)) The resulting series 125 The Te corrected values ​​were plotted against the total number of Te atoms calculated from the volume of dispensed standard solution. A linear regression model was fitted to the data points within the linearity range to calculate the number of tellurium atoms in the xenograft sections. Finally, to calculate [Te] in the tissue sections, the tissue volume was estimated by multiplying the cross-sectional area (1 mm × 1 mm) by the tissue section thickness (5 μm).

[0183] Example 2: Tellurophene-labeled carfilzomib analogues: synthesis and evaluation Monitoring target engagement in vivo is a challenging multidimensional problem. Several methods are available to quantify engagement at the cellular level. The use of tellurium in combination with mass cytometry has allowed engagement to be characterized at the cellular level in heterogeneous samples.

[0184] L-2-Tellurienylalanine (TePhe) has been demonstrated to act as a bioisostere for phenylalanine (Phe). A peptide drug version of carfilzomib containing TePhe was synthesized. Carfilzomib is an irreversible inhibitor of the β5 chymotrypsin-like site of the proteasome and an FDA-approved drug for multiple myeloma.

[0185] The TePhe-substituted carfilzomib analog (TeCar, compound of formula II, 7b) was synthesized according to Scheme 2. Coupling of the final peptide at the epoxide tip led to isomerization of the penultimate residue. This epimer (8b) was used as a control in biological studies. [ka] [Table 1]

[0186] Scheme 2 - Synthesis of TeCar Analogue 7b (Compound of Formula II) In vitro experiments with purified proteasomes led to the reduced activity expected with a covalent inhibitor: the activity observed was indistinguishable from that of carfilzomib (Figure 9).

[0187] A concentration-dependent decrease in overall cell viability was observed in Jurkat cells treated with increasing concentrations of TeCar7b (Table 1). Compared to carfilzomib, similar concentrations of 7b (Te-Car) were required to cause a 50% decrease in Jurkat cell viability.

[0188] [Table 2]

[0189] Analysis of TeCar-treated Jurkat cells by mass cytometry showed activity-dependent accumulation of tellurium within the cells. Co-administration of Te-Car7b and Car to cells led to an approximately 50% reduction in Te labeling, confirming on-target labeling of the modified compound (Figure 10). This evidence supports that TeCar can be used to monitor target engagement of carfilzomib in more complex disease-relevant samples.

[0190] method General method of synthesis All reagents were purchased from Sigma Aldrich unless otherwise stated. N 2(g) Before the reaction in solution, (g) or prior to reversed-phase high-performance liquid chromatography (RP-HPLC) purification. (g)Solvents were degassed by bubbling with 1000 ml of ... 18 RP-HPLC was performed using a 10 x 250 mm column and a Waters 1525 binary HPLC pump. Lyophilization was performed with a THERMO MODULYO™ freeze dryer. Low- and high-resolution mass spectra were acquired using a Bruker AUTOFLEX SPEED™ matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) or an Agilent 6538 Q-TOF mass spectrometer interfaced with an electrospray ionization (ESI) source, respectively. Nuclear magnetic resonance (NMR) spectra were acquired on either a 500 MHz Agilent DD2 spectrometer or a 700 MHz Agilent DD2 spectrometer using an XSENS™ C13 cold probe.

[0191] The synthesis of 5b was carried out according to 10. It was isolated by crystallization and 1 H NMR (CDCl 3 After verifying sufficient purity using a 500 MHz NMR spectrometer in 400 MHz NMR (FM Hz), 5b was used in the following SPPS step without further purification.

[0192] Synthesis, purification, and characterization of 6a and 6b 6a and 6b were synthesized using SPPS. Briefly, Fmoc-L-Phe-OH (5a) (1 equiv., 77 mg) or Fmoc-L-TePhe-OH (5b) (1.25 equiv., 100 mg) was dissolved in anhydrous (anh)DCM (2–2.5 mL). To this solution, diisopropylethylamine (DiPEA) (4 equiv., 110–139 μL) was added and the solution was loaded onto dried 2-chlorotrityl resin (163–200 mg, 1 mmol / g) in a fritted polypropylene tube (10 mL). The tube was then inverted for 2 h at room temperature (rt). The reaction mixture was drained from the column using vacuum filtration and the resin was washed with N in a capping mixture (17:2:1 anh.DCM / MeOH / DiPEA). 2(g) The resin was washed three times by bubbling with 1 mL of anh.DMF for 1 min and then evacuated using vacuum filtration. The resin was then washed repeatedly with alternating DCM and DMF in a similar manner. The resin was then inverted in a 20% solution of piperidine in anh.DMF (3 mL) for 20 min at rt to deprotect the Fmoc group. Again, the solvent was evacuated and the resin was washed repeatedly with DMF and DCM. Next, the resin was inverted in a 20% solution of piperidine in anh.DMF ([resin]=0.04 M) for 1 h at rt. 2 Fmoc-L-Leu-OH (5 eq.) was coupled using 0 (5 eq.), pyBOP (Fluka) (5 eq.), and collidine (10 eq.), 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-morpholinylacetic acid HCl salt (Alfa Aesar) (5 eq.) and excess collidine (15 eq.). For each of these three couplings, the carboxylic acid was first activated with HOBt / pyBOP in the presence of collidine for 8 min at rt before loading onto the resin.

[0193] The resin was then washed ten times with 1% trifluoroacetic acid (TFA) in anh.DCM (1 mL), collecting each wash fraction in a round-bottom flask. The solution was evaporated and rediluted multiple times with toluene, then thoroughly dried under vacuum to give a yellow oil.

[0194] The resulting oil was then evaluated by MALDI-TOF-MS to confirm the presence of the desired peptide (6a or 6b). 2 The crude peptide dissolved in O / ACN was cocrystallized with an α-cyano-4-hydroxycinnamic acid (CHCA) matrix. A peak at 567 m / z ([M+H]) confirmed the presence of 6a, whereas peaks at 667, 669, 671 and 689, 691, 693 m / z ([M+H] and [M+Na] of the three most abundant Te isotopic species, respectively) confirmed the presence of 6b.

[0195] 6a and 6b were then purified using flash silica chromatography or RP-HPLC.

[0196] For flash silica chromatography, a solvent system of 9:1 DCM / MeOH + 1% AcOH was used, and product elution was assessed using thin layer chromatography, with a shortwave UV lamp and KMnO 4 Visualization was performed using staining.

[0197] For RP-HPLC, use 98% to 2% solvent A (MQ H 2 A 90 min gradient of 254 nm (6a) or 213 and 280 nm (6b) was used and product elution was assessed by monitoring at wavelengths of 213 and 254 nm (6a) or 213 and 280 nm (6b).

[0198] Similar fractions from either method were combined, concentrated and the purity assessed using MS. Similar levels of purity were obtained using both methods, with flash silica chromatography being more rapid.

[0199] Later, D 6 -Acquired at 500MHz at a concentration of approximately 0.02M in DMSO 1 The structure of 6b was analyzed using 1 H NMR. 1H NMR (700 MHz, dmso) δ 10.21 (bs, 1H), 8.83 (bs, 1H), 8.61 (dd, J = 6.9, 1.3 Hz, 1H), 8.32 (d, J = 8.0 Hz, 1H), 8.18 (d, J = 8.5 Hz, 1H), 7.50 (dd, J = 6.9, 3.8 Hz, 1H), 7.40 (dq, J = 3.8, 1.3 Hz, 1H), 7.33 - 7.26 (m, 2H), 7.22 - 7.14 (m, 3H), 4.43 (dtd, J = 11.4, 8.3, 5.5 Hz, 2H), 4.33 (ddd, J = 9.1, 8.0, 4.4 Hz, 1H), 3.98 (bs, 2H), 3.90 (bs, 2H), 3.77 (bs, 3H), 3.34 (ddd, J = 15.0, 4.4, 1.1 Hz, 1H), 3.18 (bs, 4H), 3.11 (ddd, J = 15.0, 9.1, 1.3 Hz, 1H), 2.61 (ddd, J = 13.5, 11.3, 5.2 Hz, 1H), 2.55 (ddd, J = 13.7, 11.0, 5.9 Hz, 1H), 1.95 (ddt, J = 13.4, 11.0, 5.4 Hz, 1H), 1.89 - 1.76 (m, 1H), 1.61 (ddt, J = 14.8, 13.0, 6.4 Hz, 1H), 1.52 - 1.40 (m, 2H), 0.87 (dd, J = 26.9, 6.6 Hz, 6H). HRMS:C 29 H 40 N 4 O 6 130 The calculated value of Te(M+H)+のm / Z is 671.5636; the measured value is 671.21.

[0200] Solution synthesis of 7a / 8a (carfilzomib and its epimer) and 7b / 8b (Te-carfilzomib analogue and its epimer) One equivalent of either 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 equiv.), pyBOP (2 equiv.), and DiPEA (5 equiv.) in degassed anh.DMF (2 mL) was added. The reaction was incubated at rt for 5 min under Ar (g) After stirring under 100° C., (2S)-2-amino-4-methyl-1-[(2R)-2-methyloxiranyl]-1-pentanone trifluoroacetate (1.3 equiv.) (Ontario Chemicals) dissolved in degassed anh.DMF (1.5 mL) was added. The final concentration of peptide 6a or 6b was approximately 0.01 M. The reaction was incubated at rt for up to 5 h under Ar (g) The mixture was stirred under reduced pressure. The reaction was monitored hourly using MALDI-TOF-MS with the same parameters as above. Once complete disappearance of the starting material was observed, the reaction was taken up in 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 of brine and powdered MgSO 4(s) After drying over 100° C., 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 product was stored at −20° C. until purification.

[0201] Purification and characterization of 7a / 8a and 7b / 8b The crude material was dissolved in a minimum amount of ACN (approximately 2 mL) and divided into fractions (100 μL). 2 The mixture was diluted with 200 mL (approximately 0.9 mL) and filtered using a 0.22 μm syringe before injection into the RP-HPLC. The solvent systems used were: MQ 200 for solvent A; 2 For solvent B it was 0.1% TFA in O and for solvent B it was 0.1% TFA in ACN. The following gradient method was used: Maintains 98%A for 0-10 minutes 10-40 minutes 98% to 50%A Maintain 50%A for 40-60 minutes 60-100 minutes 50% to 2%A Maintain 2% A for 100-110 minutes.

[0202] Peaks were monitored at 213 nm and 254 nm (7a / 8a) or 213 nm and 280 nm (7b / 8b). 213 Only peaks with both 213 nm and either 254 or 280 nm features > 0.5 were collected and evaluated using MALDI-TOF-MS with the parameters specified above. The 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.

[0203] 7b: 1 H NMR (700 MHz, dmso) δ 8.60 (d, J = 6.8 Hz, 1H), 8.24 (s, 1H), 8.13 (d, J = 8.3 Hz, 2H), 7.50 (dd, J = 6.9, 3.8 Hz, 1H), 7.37 (d, J = 5.0 Hz, 1H), 7.28 (t, J = 7.6 Hz, 2H), 7.18 (dd, J = 16.5, 7.7 Hz, 3H), 4.37 (s, 4H), 4.03 (bs, 1H), 3.91 (bs, 1H), 3.74 (bs, 3H), 3.22 (dd, J = 15.3, 3.7 Hz, 3H), 3.14 (d, J = 5.2 Hz, 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 = 7.4 HRMS:C 38 H 55 N 5 O 7 130 Te(M+H) + Calculated m / z 824.7845; measured value 824.32.

[0204] 8b: 1 1H NMR (700 MHz, dmso) δ 8.69 (dd, J = 6.9, 1.3 Hz, 1H), 8.25 (bs, 1H), 8.18 (bs, 1H), 8.12 (d, J = 8.0 Hz, 1H), 7.49 (dd, J = 6.9, 3.8 Hz, 1H), 7.36 (dd, J = 3.8, 1.3 Hz, 1H), 7.27 (t, J = 7.6 Hz, 2H), 7.21 - 7.14 (m, 3H), 4.44 (td, J = 9.0, 4.6 Hz, 1H), 4.38 (dtd, J = 21.8, 8.1, 4.3 Hz, 2H), 4.30 (bs, 1H), 3.88 (bs, 2H), 3.74 (bs, 3H), 3.20 (dd, J = 14.2, 4.4 Hz, 3H), 3.12 (d, J = 5.3 Hz, 2H), 2.99 (dd, J = 14.5, 9.6 Hz, 1H), 2.91 (d, J = 5.2 Hz, 1H), 2.59 - 2.50 (m, 2H), 1.92 (ddt, J = 13.5, 10.9, 5.5 Hz, 1H), 1.80 (dddd, J = 13.7, 10.9, 8.7, 5.3 Hz, 1H), 1.54 (dtd, J = 9.5, 6.8, 4.8 Hz, 1H), 1.39 (d, J = 6.7 Hz, 1H), 1.37 (s, 3H), 1.33 (dd, J = 9.5, 3.6 Hz, 1H), 1.23 (bs, 1H), 1.29 (t, J = 7.3 Hz, 3H), 0.84 (d, J = 6.6 Hz, 3H), 0.79 (d, J = 6.6 Hz, 6H), 0.77 (d, J = 6.5 Hz, 3H).HRMS:C 38 H 55 N 5 O 7 130 Te(M+H) + Calculated m / z for Te(M+H) 824.7845; found 824.32.

[0205] Subsequent runs had consistently reproducible HPLC spectra, therefore the subsequent HPLC run was stopped at the 90 min mark and the column was flushed briskly with 100% B for 10 min before commencing the following purification.

[0206] Both compounds, carfilzomib ("a") and Te-carfilzomib ("b"), had two split elution peaks of the correct mass and maintained separation, so similar fractions were designated peak 1 (7a) and peak 2 (8a) for carfilzomib and peak 1 (7b) and 2 (8b) for Te-carfilzomib (TeCar). All four combined fractions were lyophilized to give a fluffy white powder and evaluated using ESI-MS to further confirm mass and purity.

[0207] 7b and 8b also 1 H and 13 C NMR (1D, gCOSY, and HSQC) was used to characterize and confirm that the fractions were epimeric. Due to the small amount of product isolated after HPLC, D 6 These NMR spectra were obtained in -DMSO.

[0208] 7a and 8a were not evaluated using NMR due to the low combined yields isolated from HPLC. Carfilzomib (Focus Biomolecules) was later purchased for further use in biological assays and for comparative purposes using NMR.

[0209] Stock solutions of 7b (232 μM), 8b (384 μM), and purchased carfilzomib (8800 μM) were made in DMSO and stored at −80° C. until needed. The concentration of carfilzomib stock was determined based on weighed mass, while the concentrations of 7b and 8b solutions were confirmed using amino acid analysis, utilizing carfilzomib stock solution as a standard to ensure accuracy.

[0210] Obtained within 8 months after storage at -80°C 11 H NMR confirmed that no degradation occurred under these storage conditions.

[0211] General Methods in Biology Purified human 26S proteasome was purchased from Novus™ biologicals. The β5 site activity probe succinate-leucine-leucine-valine-tyrosine-7-amino-4-methylcoumarin (Succ-LLVY-AMC) was purchased from Enzo Life Sciences™ and 7-amino-4-methylcoumarin (AMC) was purchased from Sigma-Aldrich™. WST-1 cell viability reagent was purchased from Roche™. 96-well plates were purchased from Corning or Starstedt and all 96-well plate measurements were performed on a CLARIOSTAR® plate reader. Data were processed using Microsoft Excel and GraphPad Prism.

[0212] Jurkat cells (CRL-2899) were purchased from ATCC and grown in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37 °C for 24 h at 4 °C in a cool, dark room temperature at 2 °C. 2(g) The cells were maintained in a humidified 37° C. incubator with 5% CO and cultured according to ATCC guidelines in culture flasks purchased from Starstedt.

[0213] Cell-ID™ Intercalator-Ir was purchased from Fluidigm. Cytometric time-of-flight (CyTOF) measurements were recorded on a CYTOF2® instrument. CyTOF data were processed and analyzed using FlowJo and GraphPad Prism software.

[0214] In vitro binding assay using purified proteasomes In a 96-well plate, buffer (50 mM Tris-HCl buffer + 1 mM DTT + 5 mM MgCl 226S proteasome (2 nM, 25 μL) and various concentrations of carfilzomib 7b, or 8b (1-100 nM, 25 μL) or blank control (1% v / v DMSO, 25 μL) in 100 mM NaCl + 40 mM KCl + 2 mM ATP + 0.5 mg / mL BSA were mixed and incubated at room temperature for 15 min. The enzyme reaction was then initiated by adding the substrate peptide Suc-LLVY-AMC (200 μM, 50 μL) in buffer. The plate was placed in an incubator set at 37 °C for the remainder of the experiment, except when it was removed to perform fluorescence measurements. The final well volume was 100 μL, and the final concentrations of all reactions immediately after addition of substrate were 0.5 nM (1 μg / mL) proteasome, 0.25-25 nM inhibitor, and 100 μM Succ-LLVY-AMC substrate.

[0215] In addition to a blank control ([inhibitor] = 0 nM), the following controls were also tested as part of this experiment: Substrate autohydrolysis was assessed by incubating the substrate in buffer (100 μM) alone. Any chemical interaction between 7b or 8b and the substrate was assessed by incubating the substrate (200 μM, 50 μL) with either drug (50 nM, 50 μL) in buffer. The substrate for each of these controls was added to each well at the same time as the experimental wells described above.

[0216] A standard curve of AMC (0-5 μM, 100 μL) was also prepared and run with the assay. Standards were plated during the last 5 min of the 15 min incubation, immediately prior to addition of substrate to the experimental wells.

[0217] Fluorescence measurements were then taken at λ of 350 nm using a microplate reader. ex and λ of 440 nm em Recordings were taken at time "0" (approximately 3 min after addition of substrate) and then every 15 min for a total of 75 min.

[0218] The standard curve served to demonstrate the linear time dependence of [AMC] in solution over the range of the experiment and its corresponding fluorescence readout (see FIG. 11).

[0219] Before further analysis, each experimental and control well was corrected for autohydrolysis by subtracting the fluorescence value for the autohydrolysis control well at the corresponding time point.

[0220] First, the apparent initial velocity of the uninhibited enzyme (v) was determined by plotting the average corrected fluorescence readings (FU, arbitrary fluorescence units) versus time as follows: 0 ) was decided: FU=v 0 * t+constant In the formula, the slope of the plot is v 0 (μM substrate / min).

[0221] Define the remaining active enzyme in each well after dosing with either inhibitor over a concentration range of 1.15 nM to 1.15 μM v 0 The kinetic parameters were determined similarly. 0 Loss in ([E] 合計 decreases, so [E] in the solution 活性 (due to the decrease in the enzyme activity) is compared to the uninhibited enzyme in terms of the percentage decrease. Decrease rate v 0 =(v 0 (non-inhibitory)-v 0 (inhibition)) / v 0 (Non-inhibitory) x 100% was used to calculate.

[0222] This assay was repeated a total of three times, and the average percent change between these three biological replicates is shown in Figure 10.

[0223] Cell viability in Jurkat cells Jurkat cells were seeded at a concentration of approximately 12500 cells / mL in complete medium dosed with increasing concentrations (0-200 nM) of carfilzomib or 7b (final volume of 100 μL) in 96-well plates and incubated for 44 h. Each concentration was replicated three times. 10 μL of WST-1 was then added to each well and the plate was incubated for 2 h before measuring absorbance at 440 nm. Absorbance signal vs. log(concentration) was plotted after first subtracting the absorbance signal in 100 μL complete medium + 10 μL WST-1 from each well. The concentration required to observe a 50% decrease in absorbance signal was then determined using GraphPad Prism software. These values ​​are presented in Table 1.

[0224] Uptake of 7b and 8b into live Jurkat cells with and without carfilzomib Jurkat cells were cultured at 75 mm at a concentration of approximately 5 million cells / mL for 24 h prior to dosing. 2 The cells were seeded into 100 mL plastic culture flasks and dosed for 1 hour in complete medium with either carfilzomib, 7b, or 8b at a final concentration of 500 nM, or with combinations of carfilzomib / 7b (500 nM each), carfilzomib / 8b (500 nM each), or DMSO control (final percentage 0.5%).

[0225] The medium was then immediately removed and the cells were resuspended in 3 mL of cold phosphate buffered saline (PBS) and then dispensed into three 1 mL aliquots per dosing regimen. These samples were then prepared for CYTOF® analysis.

[0226] Briefly, each sample aliquot was washed three times with cold PBS (1 mL × 3). Cells were then fixed, permeabilized, and stained with Cell-ID™ Intercalator-Ir stain in PBS (1 mL) for 1 h at rt. Cell pellets were then washed 2-3 times with cold PBS (1 mL × 2-3) and then stained with cold MQ H 2 The pellet was then stored at 4° C. overnight.

[0227] CYTOF® analysis was performed the following day. Cell pellets were resuspended in 250-500 μL of bead solution in PBS and filtered immediately prior to injection into the device. Approximately 40,000 events were collected for each sample. The cell populations ( 191 Ir and 193 Average of events positive for Ir signal 128 The Te signal was determined.

[0228] Exemplary System 12, in some embodiments, a system for imaging tissues or cells includes an imaging mass cytometry system 100 and a controller (e.g., a computer system) 200 connected to and in communication with the imaging mass cytometry system 100 via a wired or wireless connection. As described in further detail herein, the controller 100 controls the operation of the imaging mass cytometry system 100, receives images from the imaging mass cytometry system 100, and displays the received images.

[0229] 13 illustrates an exemplary imaging mass cytometry system 100 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 disposed within a cell (e.g., a flow cell) 108. In some embodiments, the sample 106 may be disposed on a movable stage (e.g., an XYZ stage) 110 disposed within the cell 108.

[0230] The radiation source 102 is configured to transmit radiation for ablation and / or fluorescence through the sample 106. In some embodiments, the radiation source 102 is a UV laser, and the radiation source 102 may operate at a wavelength of 213 nm. Irradiation of various spot 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 establish the spot size, or multiple laser shots can be scanned over the ablation area corresponding to one pixel by rapidly dithering the optics.

[0231] The system 100 may further include a shutter (e.g., a rastering 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 continuous operation of the radiation source 102 while ceasing delivery to the sample 106 during movement of the stage 110. The shutter 112 may also function as a safety mechanism that activates when a safety interlock is triggered within the system 100.

[0232] The system 100 also includes an attenuator 114, beam shaping optics 116. The attenuator 114 is disposed along the path 104 between the shutter 112 and the sample 106. The attenuator 114 provides the ability to vary the energy of the radiation emitted by the radiation source 102 for precise ablation conditions of a given sample 106. In some embodiments, the attenuator 114 operates based on polarization rotation and a polarizer to filter the radiation. The optics 116 are configured to shape the emitted radiation to generate a focused spot that is directed to the sample 106. The optics 116 may include one or more objective lenses and / or apertures to focus the emitted radiation.

[0233] The system 100 further 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 half mirror, etc.) 122 disposed along the first path 104 between the optical system 116 and the sample 106 and along a second path between the light source 118 and the sample 106. 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 lens 126 disposed along the third path 124 between the first beam splitter 122 and the sample 106. The microscope objective lens 126 focuses the radiation from the radiation source 102 and the light from the light source 118 onto the sample 106. Thus, in some embodiments, the system 100 may have a common convergent path for imaging the light emitted by the radiation source 102 and the light emitted by the light source 118 .

[0234] The system 100 also includes a tube lens 128, a second beam splitter 130, and a camera (e.g., a charge-coupled device image sensor-based (CCD) camera, an active pixel sensor-based camera, etc.) 132. The tube lens 128 is disposed along the second path 120 between the first beam splitter 122 and the second beam splitter 130. The tube lens enables focusing of the optical image onto the camera 132. In some embodiments, the system 100 may use an infinite conjugate setup using the tube lens 128. This setup may also reduce some types of aberrations and improve the quality of the image on the camera 132. The second beam splitter 130 may include, but is not limited to, a half mirror.

[0235] A second beam splitter 130 is disposed 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 towards a camera 132.

[0236] The system 100 further includes a mass cytometer 136 coupled to the cell 108. The mass cytometer includes an injector 138, an inductively coupled plasma (ICP) ion source 140, and a detector 142. The injector 138 is configured to receive the ablated sample 106 from the chamber 108 and transfer the ablated sample 106 to the ICP ion source 140. The ICP ion source 140 receives the ablated sample 106 and generates a plurality of ions. A detector (e.g., a time-of-flight (TOF) analyzer) 142 is disposed downstream of the ICP ion source 140 and receives the plurality of ions. The detector 142 provides a mass analysis of the ions exiting the ICP ion source 140 according to the mass-to-charge ratio (m / z) of the ions.

[0237] The images provided by 132 and the mass spectrometry provided by detector 142 may be output to a controller 200. The controller 200 may output the images and mass spectrometry to a display. This output may allow a user to visually determine the location and level of a given molecule (e.g., an analog of a small molecule as discussed herein) within the sample 106.

[0238] 14, a computer system 200 is shown according to an exemplary embodiment. The computer system 200 may function 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 transmitting data. Computer systems include, but are not limited to, microprocessor-based systems, personal computers, servers, handheld computing devices, tablets, smartphones, multiprocessor-based systems, mainframe computer systems, and the like.

[0239] As shown in FIG. 14, computer system 200 includes one or more processors or processing units 202, a system memory 204, and a bus 206 that connects various components of computer system 200, including system memory 204, to processor 202. System memory 204 includes computer-readable storage medium 208 and volatile memory 210 (e.g., random access memory, cache, etc.). As used herein, computer-readable storage medium includes any medium capable of storing computer-readable, program instructions and accessible by a processor. Computer-readable storage medium 208 includes non-volatile and non-transitory storage media (e.g., flash memory, read-only memory (ROM), hard disk drive, etc.). Computer program instructions described herein include program modules (e.g., routines, programs, objects, components, logic, data structures, etc.) that are executable by a processor. Furthermore, computer-readable program instructions, when executed by a processor, can cause the computer system to function in a particular manner such that the computer-readable storage medium includes an article of manufacture. In particular, the computer readable program instructions, when executed by a processor, can create means for performing at least a portion of the steps of the methods disclosed herein.

[0240] Bus 206 may be any type of one or more bus structures capable of transmitting data between the constituent elements of computer system 200 (eg, a memory bus, a memory controller, a peripheral bus, an accelerated graphics port, etc.).

[0241] Computer system 200 may further include a communications adapter 212 that enables computer system 200 to communicate with one or more other computer systems / devices via one or more communications protocols (e.g., Wi-Fi, BTLE, etc.) and, in some embodiments, may enable 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.).

[0242] In some embodiments, 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 a computer system (e.g., a mouse, a keyboard, a touch screen, etc.). External devices 214 and display 216 may communicate with processor 202 and system memory 204 via input / output (I / O) interface 218.

[0243] The display 216 may display a graphical user interface (GUI), which may include a number of selectable icons and / or editable fields. A user may use the external device 214 (e.g., a mouse) to select one or more icons and / or edit one or more editable fields. The selection of an icon and / or the editing of a field may cause the processor 202 to execute computer readable program instructions stored in the computer readable storage medium 208. In one example, a user may use the 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 some of the steps of the methods disclosed herein.

[0244] While the present disclosure has been described with reference to examples, it should be understood that the claims should not be limited to the embodiments described in the examples, but should be accorded the broadest interpretation consistent with the description as a whole.

[0245] All publications, patents, and patent applications are herein incorporated 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. If a term in this disclosure is found to be otherwise defined in a document incorporated herein by reference, the definition provided herein shall serve as the definition of that term.

Claims

1. A method for detecting low molecular weight compounds in a sample, A step of providing either a target or a cell / tissue culture sample; A step of providing a low molecular weight analog of a low molecular weight compound, wherein the low molecular weight analog is of formula I 【Chemistry 1】 Or equation II 【Chemistry 2】 A step of providing low molecular weight analogs of low molecular weight compounds having the structure of salts or solvates thereof; A step of administering a terolofen low molecular weight analog to the subject or the cell / tissue culture sample; A step of providing a sample taken from either the subject or the cell / tissue culture sample after administration of the terolofen low molecular weight analog; A step of performing mass spectrometry on the sample to determine the level of tellurium atoms present in the sample. A method comprising, where the level of the tellurium atom corresponds to the level of the low molecular weight analog of the tellurium, and the detection of the low molecular weight analog of the tellurium indicates the detection of a low molecular weight compound in the sample.

2. Equation I 【Transformation 3】 A compound of or a salt or solvate thereof.

3. The compound according to claim 2, wherein the Te atom is an isotope.

4. A composition comprising the compound described in claim 2, or a salt or solvate thereof, or a salt or solvate thereof, and a carrier or excipient.

5. Formula II 【Chemistry 4】 A compound of or a salt or solvate thereof.

6. The compound according to claim 5, wherein the Te atom is isotope enriched.

7. The compound according to claim 3 or 6, wherein 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.

8. A composition comprising the compound described in claim 5, or a salt or solvate thereof, and a carrier or excipient.

9. Equation I 【Transformation 5】 A tellophene analog of teniposide, or Formula II 【Transformation 6】 tellophene analogs of carfilzomib, or salts or solvates thereof, and A kit for mass spectrometry containing a tellurium standard or multiple tellurium standards.

10. The kit according to claim 9, wherein the mass spectrometry is mass cytometry.

11. The tellurium in the analog and the tellurium in the standard material １２０ Te, １２２ Te, １２３ Te, １２４ Te, １２５ Te, １２６ Te, １２８ Te, １３０ A kit according to claim 9 or 10, independently selected from Te and combinations thereof.

12. The compound according to claim 2 or 5, wherein the solvate is DMSO solvate.

13. The kit according to claim 9 or 10, wherein the tellophene analog is in the form of a DMSO solvate.

14. The kit according to claim 9 or 10, wherein the tellurium standard material comprises a single tellurium isotope.

15. The kit according to claim 9 or 10, wherein the plurality of tellurium standard materials each contain a different tellurium isotope.

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