Conjugated metabolites for cancer detection and treatment

Abstract

Compounds, research tools, and methods for the detection and treatment of epithelial cell dysplasia, precancerous cells, and cancer in cells or subjects are disclosed herein. Compounds, research tools, and methods for inhibiting dysplastic cells, inhibiting the migration of metaplastic cells to dysplastic cells, and reducing the incidence of cancer in cells or subjects are disclosed herein. In some embodiments, the compounds, research tools, and methods include eicosenoic acid conjugate compounds. In some embodiments, the compounds, research tools, and methods include therapies comprising SCD1 enzyme inhibitors.

Description

Technical Field

[0001] Research funded by the federal government This invention was made with government support under grant number R37 CA244970 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0002] Reference to Sequence Listing This application was filed together with a Sequence Listing XML in ST.26 XML format in accordance with 37 C.F.R. § 1.831 and PCT Rule 13ter. The Sequence Listing XML file “093386 - 9357 - WO01_sequence_listing_xml_2 - FEB - 2024.xml” submitted to the USPTO Patent Center was created on February 2, 2024, contains 48 sequences, has a file size of 42.3 kilobytes, and is hereby incorporated by reference in its entirety into this specification.

[0003] Technical Field Compounds, research tools, and methods for the detection and treatment of epithelial cell dysplasia, pre - cancer, and cancer in cells or subjects are disclosed herein. Compounds, research tools, and methods that inhibit dysplastic cells, inhibit the transition of metaplastic cells to dysplastic cells, and reduce the incidence of cancer in cells or subjects are also disclosed herein. In some embodiments, the compounds, research tools, and methods include eicosenoic acid conjugate compounds. In some embodiments, the compounds, research tools, and methods include therapies that include SCD1 enzyme inhibitors.

Background Art

[0004] Epithelial carcinogenesis involves many steps in the carcinogenic process from precancerous metaplasia to dysplasia and adenocarcinoma, with genetic, epigenetic, and metabolic pathway changes involved. Furthermore, carcinogenesis often requires the activation of oncogenes that control signaling pathways, including the RAS signaling pathway. Gastrointestinal cancers are common, life-threatening cancers that occur along the epithelium of the gastrointestinal tract, including the esophagus, stomach, and pancreas. Metaplasia within gastrointestinal cancers can arise from mucosal damage and may be reversible. However, cellular plasticity also allows metaplastic cells to enter the carcinogenic process towards dysplasia and adenocarcinoma. In particular, it has been observed that metaplasia, arising from the plasticity of zymogen-secreting chief cells, contributes in response to severe gastric damage, and Kras activation and amplification can lead to gastric carcinogenesis. Dysplasia can be defined as a condition in which cells have abnormal cellular and structural changes; it is a localized neoplastic lesion and carries the highest risk of developing gastric cancer. Dysplasia can transform into adenocarcinoma if a carcinogenic environment persists chronically, and is therefore considered a critical transitional stage in epithelial carcinogenesis leading to adenocarcinogenesis. Dysplastic stem cells, a population of stem cells that initially exist in the dysplastic stage and transform into gastric adenocarcinoma, have recently been identified. However, the molecular and cellular mechanisms of progression from precancerous metaplasia to dysplasia and then to adenocarcinoma are still not well understood.

[0005] Metabolism includes all chemical reactions that produce energy or generate the biological building blocks of cells. Metabolic reprogramming is essential to supply the increased demand for energy when cells, particularly cancer cells, require exponential growth and proliferation. Cells stabilize metabolic pathways that control appropriate levels of key metabolites related to cell signaling pathways and epigenetics. Recent studies have reported that bioactive metabolites are important for the control of cell heterogeneity, tumor growth, and immune response. Fatty acid (FA) metabolism in cancer is involved in tumor progression, metastatic potential, and drug resistance. FAs are crucial for cellular homeostasis in various biological processes including energy supply and storage, cell signaling, transcriptional control, phospholipid synthesis, and membrane fluidity. FAs are either synthesized de novo or obtained from the diet and modified by metabolic pathways that include desaturation and elongation steps. In particular, FA desaturation generates the building blocks of membrane lipids and protects cells from the lipotoxicity of saturated fatty acids that impair membrane fluidity. Stearoyl-CoA desaturase (SCD) is a key enzyme that generates monounsaturated fatty acids (MUFAs) that contribute to lipid homeostasis. Although it is well known that FA metabolism supports cancer cell growth and progression, it remains uncertain whether FA metabolism occurs to stimulate dysplastic cells or whether any specific metabolic pathways can regulate dysplastic growth or progression.

[0006] Conjugated metabolites for cancer and precancer detection and treatment are needed. SUMMARY OF THE INVENTION

[0007] One embodiment described herein is a compound of formula (I):

Chemical formula

[0008] Another embodiment described herein relates to a compound of formula (I). [ka] (In the formula, R 1 (Selected from mertansine (DM1), taxol, calicheamicin, monomethyl auristatin E (MMAE), deruxtecan, SN-38, Pseudomonas exotoxin, diphtheria toxin, or yttrium-90). In one embodiment, the compound is [ka] That is the case.

[0009] Another embodiment described herein is a method for detecting epithelial cell dysplasia, comprising contacting epithelial cells with a compound described herein; incubating for a period of time; irradiating the epithelial cells with ultraviolet light so that the dysplastic epithelial cells fluoresce; and imaging the fluorescent dysplastic epithelial cells. In one embodiment, imaging includes positron emission tomography (PET) imaging, mass spectrometry imaging, immunofluorescence imaging, fluorescence molecular endoscopy, or fluorescence-guided intraluminal endoscopy. In another embodiment, epithelial cells include gastric epithelial cells.

[0010] Another embodiment described herein is a research tool for detecting epithelial dysplasia, comprising a compound of formula (I): [ka] (In the formula, R 1 (is a fluorophore or luminescent label selected from 7-nitrobenzo[c][1,2,5]oxadiazole-4-amine (nitrobenzoflazan), nitrobenzoselenadiazole, fluorescein, rhodamine, aminomethylcoumarin acetate (AMCA), calcein, or cyanine). In one embodiment, the compound [ka] (In the formula, X is either O or Se.)

[0011] Another embodiment described herein is a research tool comprising a compound of formula (I). [ka] (In the formula, R 1(Selected from meltansine (DM1), taxol, calichemycin, monomethyl auristatin E (MMAE), deruxtecan, SN-38, Pseudomonas exotoxin, diphtheria toxin, or yttrium-90). In one embodiment, the compound is selected from meltansine (DM1), taxol, calichemycin, monomethyl auristatin E (MMAE), deruxtecan, SN-38, Pseudomonas exotoxin, diphtheria toxin, or yttrium-90. [ka] That is the case.

[0012] Another embodiment described herein is a method for inhibiting cancer or precancerous conditions in cells or subjects requiring such inhibition, comprising administering a therapeutically effective amount of a compound of formula (I) to the cells or subjects: [ka] (In the formula, R 1 (Selected from meltansine (DM1), taxol, calichemycin, monomethyl auristatin E (MMAE), deruxtecan, SN-38, Pseudomonas exotoxin, diphtheria toxin, or yttrium-90). In one embodiment, the compound is [ka] That is the case.

[0013] Another embodiment described herein is a method for treating a subject having cancer or precancerous conditions, comprising administering to the subject a therapeutically effective amount of a compound of formula (I). [ka] (In the formula, R 1 (Selected from meltansine (DM1), taxol, calichemycin, monomethyl auristatin E (MMAE), deruxtecan, SN-38, Pseudomonas exotoxin, diphtheria toxin, or yttrium-90). In one embodiment, the compound is [ka] In another aspect, the subject has gastrointestinal cancer or precancerous disease, or is suspected of developing gastrointestinal cancer or precancerous disease.

[0014] Another embodiment described herein is a method for inhibiting the migration of metaplastic cells to dysplastic cells, comprising administering a therapeutically effective amount of 4-(2-chlorophenoxy)-N-(3-(methylcarbamoyl)phenyl)piperidine-1-carboxamide (A939572) to cells or a subject requiring it. In one embodiment, the therapeutically effective amount of A939572 is about 5 mg / kg to about 100 mg / kg. In another embodiment, the therapeutically effective amount of A939572 is administered daily for about 1 day to about 6 months. In another embodiment, the cells include gastric epithelial cells. In another embodiment, the subject has gastrointestinal cancer or precancerous disease, or is suspected of developing gastrointestinal cancer or precancerous disease.

[0015] Another embodiment described herein is a method for treating metaplastic cells to reduce the incidence of cancer, comprising administering a therapeutically effective amount of 4-(2-chlorophenoxy)-N-(3-(methylcarbamoyl)phenyl)piperidine-1-carboxamide (A939572) to cells or subjects requiring it. In one embodiment, the method inhibits the migration of metaplastic cells to dysplastic cells, thereby reducing the incidence of cancer in the cells or subjects requiring it. In another embodiment, the therapeutically effective amount of A939572 is about 5 mg / kg to about 100 mg / kg. In another embodiment, the therapeutically effective amount of A939572 is administered daily for about 1 day to about 6 months. In another embodiment, the cells include gastric epithelial cells. In another embodiment, the subjects have gastrointestinal cancer or precancerous conditions, or are suspected of developing gastrointestinal cancer or precancerous conditions.

[0016] Another embodiment described herein is a method for producing a compound comprising cis-11-eicosenoic acid conjugated to 7-nitrobenzo[c][1,2,5]oxadiazole-4-amine (nitrobenzoflazan) or nitrobenzoselenadiazole. [ka] (In the formula, X is either O or Se); The method comprises (a) mixing cis-11-eicosenoic acid with dimethylformamide (DMF) solvent to prepare a solution; (b) adding N,N-diisopropylethylamine (DIPEA) to the solution; (c) adding 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) to the solution to prepare a reaction mixture; (d) mixing the reaction mixture; (e) adding nitrobenzofurazan or nitrobenzoselenadiazole to the reaction mixture to prepare a conjugate mixture; (f) mixing the conjugate mixture; and (g) producing a compound by one or more of the conjugate mixture being diluted, washed, dried, or filtered. In one embodiment, steps (a) to (e) are carried out at approximately 0°C, and steps (f) to (g) are carried out at room temperature. In another embodiment, the conjugate mixture is diluted with ethyl acetate and washed with an aqueous citric acid solution. In another embodiment, the conjugate mixture is dried with MgSO4. In another embodiment, the method further comprises purifying the compound using column chromatography.

[0017] Another embodiment described herein is a method for producing a compound comprising cis-11-eicosenoic acid conjugated to meltansine, [ka] A method comprising: (a) preparing a solution by mixing meltansine carboxylic acid with dimethylformamide (DMF) solvent; (b) preparing a reaction mixture by adding hexafluorophosphate azabenzotriazole tetramethyluranium (HATU) and N-methylmorpholine (NMM) to the solution; (c) mixing the reaction mixture; (d) preparing a conjugate mixture by adding cis-11-eicosenoic acid conjugated to piperazine to the reaction mixture; (e) mixing the conjugate mixture; and (f) producing a compound by one or more of the conjugate mixtures being diluted, washed, dried, or filtered.

[0018] A patent or application file includes at least one drawing made in color. A copy of the published version of this patent or patent application, including the color drawing, will be provided by the Patent Office upon request and payment of the required fees. [Brief explanation of the drawing]

[0019] [Figure 1] Figure 1 shows that a Gif-rtTA mouse model was created by crossing Gif-rtTA mice with TetO-Cre and LSL-KrasG12D mouse alleles. GCK mice were administered doxycycline (DOX) in drinking water for two weeks, which resulted in the expression of Cre enzymes driven by Gif, and consequently, active Kras expression was induced. [Figure 2-1]Figures 2A–D show the major stages of gastric carcinogenesis induced by Kras activation in gastric chief cells. Figure 2A shows H&E stained sections from Gif-rtTA;TetO-Cre;KrasG12D (GCK) mouse stomachs at multiple time points after doxycycline treatment: pyloric metaplasia (PM, 2–3 weeks), incomplete intestinal metaplasia (In-IM, 5–6 weeks), and low-grade / high-grade dysplasia (LGD / HGD, 8–10 weeks). Figure 2B shows the cytological features of dysplasia observed in HGD, as indicated by the yellow arrowheads. Figure 2C shows the proportion of glandular types within the GCK stomach at multiple time points. 100 glands in the proximal region of the body were examined: 2–3 weeks (n=5), 5–6 weeks (n=8), and 10–14 weeks (n=10). Figure 2D shows measurements of glandular width within the GCK stomach. A total of 100 glands were examined in each group of 3 mice per group. All IF data represent n=3 mice in each group, and a total of 100 glands were examined for quantification in each group. Hoechst was used for nuclear staining. White dotted boxes indicate magnified areas. Scale bars: 100 μm (Figure 2A) and 25 μm (Figure 2B). Figure 2C shows mean ± SEM. Figure 2D shows mean ± SD. One-way ANOVA with Tukey's multiple comparisons. ***p<0.001, ****p<0.0001. [Figure 2-2] Same as above. [Figure 3-1]Figures 3A–E demonstrate that spatial and quantitative metabolic profiling reveals unique metabolic patterns during carcinogenesis. Figure 3A provides an illustrative overview of the MALDI-IMS workflow for visualizing the spatial distribution of metabolites in the stomach of GCK cells. Mass spectral (MS) images were obtained for each ion by plotting the m / z intensities collected for each spot. Figure 3B shows a heatmap display of the relative values ​​of the mean peak intensities of the metabolites. Figure 3C shows an overlay of the MS image of monounsaturated fatty acids (MUFA; FFA 20:1). Figure 3D provides an illustrative overview of fatty acid (FA) desaturation and elongation. Palmitates are obtained from de novo FA synthesis or diet. FAs can be converted to other FA species within cells through desaturation and elongation. Figure 3E shows an MS image of long-chain fatty acids in the stomach of GCK cells. The abundance of a given metabolite in the corresponding MS image is normalized to 100%. Scale bar = 500 μm. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 3-4] Same as above. [Figure 4-1]Figures 4A–F show the specific expression of SCD1 in GCK stomachs. Figure 4A provides an illustrative overview of key steps in the central metabolic pathway along with related enzymes. Figure 4B shows a heatmap of relative mRNA levels of genes encoding metabolic enzymes or transporters in gastric organoid lines derived from GCK stomachs. n=3 biological replicate experiments. Figure 4C shows IF staining of SCD1 (green) and Ki-67 (red) in GCK stomachs. White dotted boxes indicate enlarged areas. White dotted lines identify glandular shape. Figure 4D shows a schematic diagram of the SCD1 expression pattern in GCK stomachs. SCD1+ cells (green; cytoplasm), Ki-67+ cells (red; nucleus), and CD44v9+ cells (red; membrane). Figure 4E shows IF staining of SCD1 (green) in normal or dysplastic organoids derived from normal stomachs or GCK stomachs with high-grade dysplasia. All IF data represent n=3 mice in each group, and Hoechst was used for nuclear staining. Figure 4F shows phase-contrast images of dysplastic organoids treated for 3 days with DMSO (vehicle), BMS-303141 (ACLY inhibitor; 1 μM), C75 (FASN inhibitor; 1 μM), A939572 (SCD inhibitor; 100 nM), or SC-26196 (FADS2 inhibitor; 1 μM). Each drug treatment involved n≧3 biological replicates. Scale bars: 100 μm (Figures 4C and 4E) and 1000 μm (Figure 4F). [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 4-4] Same as above. [Figure 4-5] Same as above. [Figure 5-1]Figures 5A–J show that SCD1 regulates dysplastic cell survival both in vivo and in vitro. Figures 5A and 5D show phase-contrast images of dysplastic or normal organoids treated with DMSO (vehicle) or 100 nM A939572 (SCD inhibitor) at 0, 1, or 3 days post-treatment. Figure 5B shows quantification of the diameter of dysplastic or normal organoids before and after treatment. Figure 5C shows H&E or IF staining of cleavage caspase-3 (CC-3, green) or cell proliferation (Ki-67, red) in dysplastic or normal organoids after treatment. n≧3 biological replicate experiments for A939572 treatment. Figure 5E shows H&E stained gastric body of GCK mice 2 weeks after treatment with vehicle (n=3) or A939572 (n=4). Yellow arrowheads indicate dead cells. Figure 5F shows the percentage of F4 / 80 positive areas in the interstitial portion of glands in different regions of GCK stomachs treated with vehicle or A939572. Figure 5G shows IF staining of CC-3 (green) in GCK stomachs treated with vehicle or A939572. Figure 5H shows the quantification of CC-3+ cells per 20x field of view of the image. Figure 5I shows IF staining of terminal deoxynucleotidyltransferase (TdT) dUTP nick-end labeling (TUNEL, green) in GCK stomachs treated with vehicle or A939572. Figure 5J shows the quantification of TUNEL+ cells per 20x field of view of the image. Scale bars: 50 μm (Figure 5E, right), 100 μm (Figures 5C, 5G, and 5I), 200 μm (Figure 5E, left), and 1000 μm (Figures 5A and 5D). Hoechst was used for nuclear staining. Dotted boxes indicate enlarged areas. All panels show mean ± SD. One-way ANOVA with unpaired two-tailed Student's t-test (I) or Tukey's multiple comparisons (Figures 5B, 5H, and 5J). **p<0.01, ***p<0.001, ****p<0.0001. [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 5-4] Same as above. [Figure 5-5] Same as above. [Figure 5-6] Same as above. [Figure 5-7] Same as above. [Figure 6-1] Figures 6A–G show common features of SCD expression in dysplastic lesions across gastrointestinal carcinogenesis. Figure 6A shows immunohistochemical (IHC) staining of SCD in adjacent normal tissue (n=4), IM (n=11), LGD (n=7), HGD (n=5), and intestinal or diffuse gastric cancer (GC) tissue (n=89). Figure 6B shows IHC staining of SCD in adjacent normal tissue (n=9), Barrett's esophagus (BE, n=13), dysplasia (n=6), and esophageal adenocarcinoma (ADC, n=11). Figures 6C–D show the IHC H scores of SCD in human gastric tissue (Figure 6C) and esophageal tissue (Figure 6D). Figure 6E shows IHC staining of SCD in adjacent normal tissue (n=16), acinar-ductal metaplasia (ADM, n=14), mucinous cystic neoplasm (MCN, n=5), intraductal papillary mucinous neoplasm (IPMN, n=14), intraepithelial neoplasia (PanIN, n=18), and pancreatic adenocarcinoma (PDAC, n=10). Figure 6F shows the IHC H score of SCD in human pancreatic tissue. Figure 6G shows a schematic diagram of the proposed model. Scale bars: 50 μm (Figures 6A and 6B) and 100 μm (Figure 6C). All panels show mean ± SD. One-way ANOVA with unpaired two-sided Student's t-test (Figure 6G) or Tukey's multiple comparisons. **p<0.01, ***p<0.001, ****p<0.0001. [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 6-4] Same as above. [Figure 6-5] Same as above. [Figure 6-6] Same as above. [Figure 6-7] Same as above. [Figure 7-1]Figures 7A–J show that dysplastic cells actively use MUFA synthesized from SCD-dependent FA desaturation as an important energy source. Figure 7A provides an illustrative overview of FA elongation and desaturation of eicosenoic acid (EA; 20:1n9) or docosatetraenoic acid (DA; 22:4n6). Figure 7B shows the chemical structure of the synthesized NBD (nitrobenzoxadiazole; nitrobenzofurazan)-conjugate EA(20:1n9). Figures 7C–E show phase contrast, H&E images (Figure 7C), quantitative organoid diameter (Figure 7D), and live / dead (calcein AM / ethidium homodimer-1 [EthD-1]) cell staining (Figure 7E) of dysplastic organoids treated with A939572 for 3 days in DMSO or a medium supplemented with EA or DA. Figure 7F shows confocal images of a monolayer of dysplastic cells incubated with NBD (green) fluorescent conjugate EA (20:1n9) for 6, 24, and 48 hours, followed by staining with 100 nM MitoTracker® Red CMXRos (Mito). Hoechst was used for nuclear staining (Nuc). White dotted boxes indicate enlarged areas. Figure 7G shows the relative mRNA expression levels of fatty acid oxidation (FAO) genes in a gastric organoid line derived from GCK stomach. Figure 7H shows a schematic diagram of co-treatment with an FAO inhibitor (perhexylin 10 μM) treated with DMSO or A939572 for 3 days in EA-supplemented dysplastic organoids. Figures 7I-J show phase-contrast images (Figure 7I) and quantification of diameter (Figure 7J) of dysplastic organoids after co-treatment. n≧3 biological replicate experiments. Scale bars: 20 μm (Figure 7F), 100 μm (Figure 7C, bottom; and 7E), and 1000 μm (Figure 7C, top; and 7I). All panels show mean ± SD. One-way ANOVA with Tukey's multiple comparisons. **p<0.01, ***p<0.001, ****p<0.0001. [Figure 7-2] Same as above. [Figure 7-3] Same as above. [Figure 7-4] Same as above. [Figure 7-5] Same as above. [Figure 7-6] Same as above. [Figure 7-7] Same as above. [Figure 7-8] Same as above. [Figure 8-1] Figures 8A and 8B show the synthesis of NBD (Figure 8A) or NBD fluorescent-conjugate metabolite (Figure 8B). The chemical structure and NMR profile of the synthesized NBD (Figure 8A) or NBD fluorescent-conjugate eicosenoic acid (20:1n9) (Figure 8B) are also shown. [Figure 8-2] Same as above. [Figure 9] Figures 9A and 9B show cells treated with EA-DM1 (cis-11-eicosenoic acid conjugated to meltansine (DM1)). Figure 9A shows AGS and NCI-N87 cell lines treated with either DMSO or EA-DM1 (100 nM) for two days prior to imaging. DMSO was used as a vehicle control. Figure 9B shows NIH-3T3 and AGS cell lines treated with either DMSO or EA-DM1 (5 nM) for two days prior to imaging. The NIH-3T3 cell line was used as a negative control, and normal mouse cells and DMSO were used as vehicle controls. [Modes for carrying out the invention]

[0020] Detailed explanation Unless otherwise defined, all scientific and technical terms used herein have the same meaning as those commonly understood by those skilled in the art. For example, all nomenclature and techniques used in relation to biochemistry, molecular biology, immunology, microbiology, genetics, cell and tissue culture, and protein and nucleic acid chemistry described herein are well known and commonly used in the art. In case of any conflict, the disclosure, including definitions, shall prevail. Exemplary methods and materials are described below, but similar or equivalent methods and materials may be used in carrying out or testing the embodiments and aspects described herein.

[0021] As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” “vector,” “polypeptide,” and “protein” have their usual meanings as understood by a biochemist of the ordinary skill in the art. Standard single-letter nucleotides (A, C, G, T, U) and standard single-letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.

[0022] As used herein, terms such as “include,” “including,” “contain,” “containing,” and “having” mean “comprising.” This disclosure also intends to include other embodiments “comprising,” “basically consisting of,” and “consisting of” the embodiments or elements presented herein, whether expressly stated otherwise.

[0023] As used herein, the terms “a,” “an,” “the,” and similar terms used in the context of this disclosure (in particular, in the context of the claims) shall be construed to include both singular and plural unless otherwise specified or unless the context clearly contradicts this. Furthermore, “a,” “an,” or “the” shall mean “one or more” unless otherwise specified.

[0024] As used herein, the term "or" can refer to both binding and detaching properties.

[0025] As used herein, the terms "and / or" refer to both conjugative and separable properties.

[0026] As used herein, the term “substantially” means to a considerable or significant degree, but not entirely.

[0027] As used herein, the terms “about” or “approximately” applied to one or more values ​​under consideration refer to a value that is similar to the stated reference value or within the acceptable margin of error of a particular value as determined by those skilled in the art, where the value depends in part on the method by which it was measured or determined, such as the limits of the measurement system. In one embodiment, the term “about” refers to any value containing both integer and fractional components that is within ±10% of the value modified by the term “about.” Alternatively, “about” may mean within three or more standard deviations, according to the conventions of the art. Alternatively, in relation to a biological system or process, the term “about” may mean within one order of magnitude, within five times the value in some embodiments, and within twice the value in some embodiments. As used herein, the symbol “~” means “about” or “approximately.”

[0028] All ranges disclosed herein include both endpoints as distinct values, as well as all integers and fractions explicitly stated within those ranges. For example, the range 0.1 to 2.0 includes 0.1, 0.2, 0.3, 0.4…2.0. Where an endpoint is modified by the term “approximately”, the explicitly stated range is extended by a variation of up to ±10% of any value within that range or within three or more standard deviations, including the endpoint.

[0029] As used herein, the terms “room temperature,” “RT,” or “ambient temperature” refer to typical temperatures in a room laboratory environment. In one embodiment, the laboratory environment is temperature and humidity controlled to maintain a substantially uniform temperature or within a specific temperature range. In one embodiment, “room temperature” refers to a temperature of about 20–30°C, including all integers and endpoints within the specified range. In another embodiment, “room temperature” refers to a temperature of about 20–30°C at standard atmospheric pressure; about 22–30°C; about 25–30°C; about 27–30°C; about 20–22°C; about 20–25°C; about 20–27°C; about 22–25°C; about 22–27°C; about 25–27°C; about 20°C ± 10%; about 22°C ± 10%; about 25°C ± 10%; about 27°C ± 10%; or a temperature of about 20°C, about 22°C, about 25°C, or about 27°C.

[0030] As used herein, the terms “active ingredient” or “pharmaceutical active ingredient” refer to a drug, active ingredient, compound, or substance, composition, or mixture thereof that provides a pharmacological, often beneficial, effect.

[0031] As used herein, the terms “control” or “reference” are used interchangeably. A “reference” or “control” level may be a predetermined value or range used as a baseline or benchmark for evaluating a measurement result against it. “Control” also refers to a control experiment or control cells.

[0032] As used herein, the term “dosage” refers to any form of active ingredient preparation or composition, including cells, containing an amount sufficient to initiate or produce a therapeutic effect with at least one or more doses. The terms “preparation” and “composition” are used interchangeably herein.

[0033] As used herein, the term “prevention” means preventing or reducing the progression of a disability to a statistically significant extent or to an extent detectable by those skilled in the art.

[0034] As used herein, the terms “effective dose” or “therapeutically effective dose” mean a substantially non-toxic but sufficient amount of an action, drug, composition, or cell administered to a subject to prevent, treat, or improve to some extent one or more symptoms of a disease or condition that the subject is experiencing or is susceptible to. The result may be a reduction or mitigation of the signs, symptoms, or causes of the disease or any other desired change in the biological system. The effective dose may be based on factors that are individual to each subject, including but not limited to the subject’s age, size, type or severity of the disease, stage of the disease, route of administration, type or amount of supplemental therapy being used, ongoing disease process, and type of treatment desired.

[0035] As used herein, the term “subject” refers to an animal. Typically, the subject is a mammal. The subject may also refer to primates (e.g., humans, male or female; infant, adolescent, or adult), non-human primates, rats, mice, rabbits, pigs, cattle, sheep, goats, horses, dogs, cats, fish, birds, etc. In one embodiment, the subject is a primate. In one embodiment, the subject is a human.

[0036] As used herein, a subject is “in need of treatment” if such subject derives a biological, medical, or quality of life benefit from such treatment. Subjects in need of treatment are not necessarily symptomatic, especially in the case of preventative or prophylaxis treatment.

[0037] As used herein, the terms “inhibit,” “inhibit,” or “to inhibit” refer to the reduction or suppression of a given biological process, condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.

[0038] As used herein, “treatment” or “to treat” means the prophylaxis, preventing, suppressing, inhibiting, reversing, alleviating, improving, inhibiting the progression of, or completely eliminating a biological process, including a disorder or disease. Treatment can be acute or chronic. The term “treatment” also means reducing the severity of a disease or symptoms associated with such a disease before such a disease develops. “Suppressing” or “improving” a disease, disorder, or symptoms thereof includes administering cells, compositions, or compounds described herein to a subject after the clinical manifestation of such a disease, disorder, or symptoms thereof. “Prophylaxis” or “preventing” a disease, disorder, or symptoms thereof includes administering cells, compositions, or compounds described herein to a subject before the onset of the disease, disorder, or symptoms thereof. "Suppressing" a disease or disorder includes administering the cells, compositions, or compounds described herein to a subject after the induction of the disease or disorder but before the clinical manifestation or symptoms of the disorder appear.

[0039] As used herein, “metamorphosis” or “metamorphosis” refers to the transformation of one differentiated cell type into another differentiated cell type that is not normally present in the tissue in which it exists. The change from one type of cell to another may be part of a normal maturation process or may be caused by some kind of abnormal stimulus.

[0040] As used herein, “dysplasia” or “dysplastic” means any abnormal growth or development of any type of cell, tissue, or organ, and any abnormal tissue or anatomical structure resulting from such abnormal growth or development. As disclosed herein, dysplasia may mean a condition in which cells have abnormal cellular and structural changes and are localized neoplastic lesions with the highest risk of developing cancer.

[0041] As used herein, “cancer” or “malignant” refers to malignant and invasive epithelial adenocarcinoma characterized by rapid and uncontrolled cell growth and proliferation. Malignant adenocarcinoma cells may arise from the malignant transformation of metaplastic and dysplastic epithelial cells and may spread locally or metastasize to other parts of the body via the bloodstream and lymphatic system. In some embodiments described herein, a subject may be treated for cancer by administering one or more compounds described herein to the subject. In one embodiment, a subject may have gastrointestinal cancer or is suspected of developing gastrointestinal cancer.

[0042] As used herein, “precancerous” or “precancerous” refers to non-malignant and non-invasive metaplastic epithelial cells that have not yet undergone transformation into dysplastic or adenocarcinoma cells. Precancerous metaplasia can occur when the gastric mucosa (i.e., gastric mucosa) is replaced by intestinal epithelium. Many types of precancerous lesions exist in human epithelial cell carcinogenesis, and the terminology, order, and mutational signatures differ by organ. In pancreatic cancer, three of the most important types, including, for example, mucinous cystic neoplasms, intraductal papillary mucinous neoplasms, and pancreatic intraepithelial neoplastic lesions, are defined as precancerous lesions. In some embodiments described herein, a subject may be treated for precancerous conditions by administering one or more compounds described herein to the subject. In one embodiment, a subject may have gastrointestinal precancerous conditions or is suspected of developing gastrointestinal precancerous conditions including precancerous lesions.

[0043] Epithelial carcinogenesis occurs within a continuous oncogenic cascade from precancerous metaplasia to dysplasia and adenocarcinoma, with oncogene activation potentially driving this process. Metabolic reprogramming is considered a crucial mechanism regulating cancer cell growth and proliferation. To define the metabolic dynamics during gastric carcinogenesis, imaging mass spectrometry was performed using a mouse model of metaplasia and dysplasia, following Kras activation in zymogen-secreting chief cells. Metabolic reprogramming from glycolysis to fatty acid metabolism was confirmed to occur during precancerous cell lineage conversion to dysplastic cells. Alteration of fatty acid desaturation by stearoyl-CoA desaturase (SCD) generates novel eicosenoic acid, which stimulates dysplastic cell hyperproliferation and survival. These results suggest that oncogenic metabolic rewiring acts as a catalyst for the malignant transformation of precancerous cells in carcinogenesis.

[0044] As disclosed herein, the study focused on gastric carcinogenesis and metabolism that may be involved in the critical transition between metaplasia and dysplasia during gastric carcinogenesis, using a novel mouse model that induces Kras activation in chief cells secreting zymogen granules. Imaging mass spectrometry was used to investigate the dynamic changes in metabolite accumulation during the carcinogenic process and identified a novel fatty acid metabolic pathway that uses eicosenoic acid as the primary energy source during the progression of precancerous metaplasia to dysplasia.

[0045] Suitable small, multicolor fluorophores for use in the disclosed invention may include those described in Benson et al., SCOTfluors: small, conjugatable, orthogonal, and tunable fluorophores for in vivo imaging of cell metabolism, Angew Chem Int Ed Engl., 131(21):6985-6989 (2019), the entire contents of which are incorporated herein by reference.

[0046] One embodiment described herein is a compound of formula (I): [ka] (In the formula, R 1 (is a fluorophore or luminescent label selected from 7-nitrobenzo[c][1,2,5]oxadiazole-4-amine (nitrobenzoflazan), nitrobenzoselenadiazole, fluorescein, rhodamine, aminomethylcoumarin acetate (AMCA), calcein, or cyanine). In one embodiment, the compound is [ka] (In the formula, X is either O or Se.)

[0047] Another embodiment described herein is a compound of formula (I): [ka] (In the formula, R 1 (Selected from meltansine (DM1), taxol, calichemycin, monomethyl auristatin E (MMAE), deruxtecan, SN-38, Pseudomonas exotoxin, diphtheria toxin, or yttrium-90). In one embodiment, the compound is [ka] That is the case.

[0048] Another embodiment described herein is a method for detecting epithelial cell dysplasia, comprising contacting epithelial cells with a compound described herein; incubating for a period of time; irradiating the epithelial cells with ultraviolet light so that the dysplastic epithelial cells fluoresce; and imaging the fluorescent dysplastic epithelial cells. In one embodiment, imaging includes positron emission tomography (PET) imaging, mass spectrometry imaging, immunofluorescence imaging, fluorescence molecular endoscopy, or fluorescence-guided endoscopic endoscopy. In another embodiment, the epithelial cells include gastric epithelial cells.

[0049] Another embodiment described herein is a research tool for detecting epithelial dysplasia, comprising a compound of formula (I):

Chemical formula

Chemical formula

[0050] Another embodiment described herein is a research tool comprising a compound of formula (I):

Chemical formula

Chemical formula

[0051] Another embodiment described herein is a method of inhibiting cancer or precancer in a cell or a subject in need thereof, the method comprising administering to the cell or subject a therapeutically effective amount of a compound of formula (I):

Chemical formula

[0052] Another embodiment described herein is a method for treating a subject having cancer or precancerous conditions, comprising administering to the subject a therapeutically effective amount of a compound of formula (I). [ka] (In the formula, R 1 (Selected from meltansine (DM1), taxol, calichemycin, monomethyl auristatin E (MMAE), deruxtecan, SN-38, Pseudomonas exotoxin, diphtheria toxin, or yttrium-90). In one embodiment, the compound is [ka] In another aspect, the subject has gastrointestinal cancer or precancerous disease, or is suspected of developing gastrointestinal cancer or precancerous disease.

[0053] Another embodiment described herein is a method for inhibiting the migration of metaplastic cells to dysplastic cells, comprising administering a therapeutically effective amount of 4-(2-chlorophenoxy)-N-(3-(methylcarbamoyl)phenyl)piperidine-1-carboxamide (A939572) to cells or a subject requiring it. In one embodiment, the therapeutically effective amount of A939572 is about 5 mg / kg to about 100 mg / kg. In another embodiment, the therapeutically effective amount of A939572 is administered daily for about 1 day to about 6 months. In another embodiment, the cells include gastric epithelial cells. In another embodiment, the subject has gastrointestinal cancer or precancerous disease, or is suspected of developing gastrointestinal cancer or precancerous disease.

[0054] Another embodiment described herein is a method for treating metaplastic cells to reduce the incidence of cancer, comprising administering a therapeutically effective amount of 4-(2-chlorophenoxy)-N-(3-(methylcarbamoyl)phenyl)piperidine-1-carboxamide (A939572) to cells or subjects requiring it. In one embodiment, the method inhibits the migration of metaplastic cells to dysplastic cells, thereby reducing the incidence of cancer in the cells or subjects requiring it. In another embodiment, the therapeutically effective amount of A939572 is about 5 mg / kg to about 100 mg / kg. In another embodiment, the therapeutically effective amount of A939572 is administered daily for about 1 day to about 6 months. In another embodiment, the cells include gastric epithelial cells. In another embodiment, the subjects have gastrointestinal cancer or precancerous conditions, or are suspected of developing gastrointestinal cancer or precancerous conditions.

[0055] Another embodiment described herein is a method for producing a compound comprising cis-11-eicosenoic acid conjugated to 7-nitrobenzo[c][1,2,5]oxadiazole-4-amine (nitrobenzoflazan) or nitrobenzoselenadiazole. [ka] (In the formula, X is either O or Se); The method comprises (a) preparing a solution by mixing cis-11-eicosenoic acid with dimethylformamide (DMF) solvent; (b) adding N,N-diisopropylethylamine (DIPEA) to the solution; (c) preparing a reaction mixture by adding 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) to the solution; (d) mixing the reaction mixture; (e) preparing a conjugate mixture by adding nitrobenzofurazan or nitrobenzoselenadiazole to the reaction mixture; (f) mixing the conjugate mixture; and (g) producing a compound by one or more of the conjugate mixtures being diluted, washed, dried, or filtered. In one embodiment, steps (a) to (e) are carried out at approximately 0°C, and steps (f) to (g) are carried out at room temperature. In another embodiment, the conjugate mixture is diluted with ethyl acetate and washed with an aqueous citric acid solution. In another embodiment, the conjugate mixture is dried with MgSO4. In another embodiment, the method further comprises purifying the compound using column chromatography.

[0056] Another embodiment described herein is a method for producing a compound comprising cis-11-eicosenoic acid conjugated to meltansine, [ka] A method comprising: (a) mixing meltansine carboxylic acid with dimethylformamide (DMF) solvent to prepare a solution; (b) adding hexafluorophosphate azabenzotriazole tetramethyluronium (HATU) and N-methylmorpholine (NMM) to the solution to prepare a reaction mixture; (c) mixing the reaction mixture; (d) adding cis-11-eicosenoic acid conjugated to piperazine to the reaction mixture to prepare a conjugate mixture; (e) mixing the conjugate mixture; and (f) producing a compound by one or more of the conjugate mixtures being diluted, washed, dried, or filtered.

[0057] It will be apparent to those skilled in the art that suitable modifications and alterations of the compositions, formulations, methods, processes, and uses described herein can be made without departing from the scope of any embodiment or aspect thereof. The compositions and methods provided are illustrative and are not intended to limit the scope of any of the express embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variation or iteration. The scope of the compositions, formulations, methods, and processes described herein includes all actual or potential combinations of the embodiments, aspects, options, examples, and selections described herein. The exemplary compositions and formulations described herein may omit any components, replace any components disclosed herein, or include any components disclosed elsewhere herein. The ratio of the mass of any component of any composition or formulation disclosed herein to the mass of any other component in the formulation, or to the total mass of the other components in the formulation, is disclosed herein as if they were expressly disclosed. If the meaning of any term in any patent or publication incorporated by reference conflicts with the meaning of a term used herein, the meaning of the term or phrase herein shall prevail. Furthermore, the above discussion only discloses and describes exemplary embodiments. All patents and publications cited herein are incorporated herein by reference with respect to their specific teachings.

[0058] The various embodiments and aspects of the present invention described herein are summarized in the following sections: Item 1. Compounds of formula (I): [ka] (In the formula, R 1 (This is a fluorophore or luminescent label selected from 7-nitrobenzo[c][1,2,5]oxadiazole-4-amine (nitrobenzoflazan), nitrobenzoselenadiazole, fluorescein, rhodamine, aminomethylcoumarin acetate (AMCA), calcein, or cyanine). Section 2. [ka] The compound of item 1, where X is O or Se. Section 3. Compounds of formula (I): [ka] (In the formula, R 1 (Selected from meltansine (DM1), taxol, calichemycin, monomethyl auristatin E (MMAE), deruxtecan, SN-38, Pseudomonas exotoxin, diphtheria toxin, or yttrium-90). Section 4. [ka] The compound in item 3. Item 5. A method for detecting epithelial cell dysplasia, Bring epithelial cells into contact with the compound in item 1; Incubating for a certain period of time; Irradiating epithelial cells with ultraviolet light causes dysplastic epithelial cells to fluoresce; and Imaging fluorescent dysplastic epithelial cells A method that includes this. Item 6. The method of Item 5, wherein imaging includes positron emission tomography (PET) imaging, mass spectrometry imaging, immunofluorescence imaging, fluorescence molecular endoscopy, or fluorescence-guided intraluminal endoscopy. Item 7. The method of item 5 or 6, wherein the epithelial cells include gastric epithelial cells. Section 8. Research tools for detecting epithelial dysplasia, including compounds of formula (I): [ka] (In the formula, R 1(This is a fluorophore or luminescent label selected from 7-nitrobenzo[c][1,2,5]oxadiazole-4-amine (nitrobenzoflazan), nitrobenzoselenadiazole, fluorescein, rhodamine, aminomethylcoumarin acetate (AMCA), calcein, or cyanine). Item 9. Compounds [ka] The research tool of term 8, where X is either O or Se. Item 10. Research tools containing compounds of formula (I): [ka] (In the formula, R 1 (Selected from meltansine (DM1), taxol, calichemycin, monomethyl auristatin E (MMAE), deruxtecan, SN-38, Pseudomonas exotoxin, diphtheria toxin, or yttrium-90). Item 11. Compounds [ka] This is the research tool mentioned in item 10. Item 12. A method for inhibiting cancer or precancerous conditions in cells or subjects requiring such inhibition, comprising administering a therapeutically effective amount of a compound of formula (I) to the cells or subjects: [ka] (In the formula, R 1 (Selected from meltansine (DM1), taxol, calichemycin, monomethyl auristatin E (MMAE), deruxtecan, SN-38, Pseudomonas exotoxin, diphtheria toxin, or yttrium-90). Item 13. Compounds [ka] The method of item 12. Item 14. A method for treating a subject having cancer or precancerous conditions, comprising administering to the subject a therapeutically effective amount of a compound of formula (I): [ka] (In the formula, R 1 (Selected from meltansine (DM1), taxol, calichemycin, monomethyl auristatin E (MMAE), deruxtecan, SN-38, Pseudomonas exotoxin, diphtheria toxin, or yttrium-90). Item 15. Compounds [ka] The method of item 14. Item 16. The method of item 14 or 15, wherein the subject has gastrointestinal cancer or precancerous disease, or is suspected of developing gastrointestinal cancer or precancerous disease. Item 17. A method for inhibiting the migration of metaplastic cells to dysplastic cells, comprising administering a therapeutically effective amount of 4-(2-chlorophenoxy)-N-(3-(methylcarbamoyl)phenyl)piperidine-1-carboxamide (A939572) to cells or a subject requiring it. Item 18. The method of item 17, wherein the therapeutically effective dose of A939572 is approximately 5 mg / kg to approximately 100 mg / kg. Item 19. The method of item 17 or 18, wherein a therapeutically effective dose of A939572 is administered daily for approximately 1 day to approximately 6 months. Item 20. Any one of the methods described in items 17-19, wherein the cells include gastric epithelial cells. Item 21. Any one of the methods described in items 17-20, wherein the subject has gastrointestinal cancer or precancerous disease, or is suspected of developing gastrointestinal cancer or precancerous disease. Item 22. A method for treating metaplastic cells to reduce the incidence of cancer, comprising administering a therapeutically effective amount of 4-(2-chlorophenoxy)-N-(3-(methylcarbamoyl)phenyl)piperidine-1-carboxamide (A939572) to cells or a subject in need thereof. Item 23. The method of item 22, which inhibits the migration of metaplastic cells to dysplastic cells, thereby reducing the incidence of cancer in cells or in subjects requiring them. Item 24. The method of item 22 or 23, wherein the therapeutically effective dose of A939572 is approximately 5 mg / kg to approximately 100 mg / kg. Item 25. A therapeutically effective dose of A939572 is administered daily, for approximately 1 day to approximately 6 months, using one of the methods described in items 22 to 24. Item 26. The cells include gastric epithelial cells, in any one of the methods described in items 22-25. Item 27. Any one of the methods described in items 22-26, wherein the subject has gastrointestinal cancer or precancerous disease, or is suspected of developing gastrointestinal cancer or precancerous disease. Item 28. A method for producing a compound comprising cis-11-eicosenoic acid conjugated to 7-nitrobenzo[c][1,2,5]oxadiazole-4-amine (nitrobenzoflazan) or nitrobenzoselenadiazole. [ka] (In the formula, X is either O or Se); (a) Prepare a solution by mixing cis-11-eicosenoic acid with dimethylformamide (DMF) solvent; (b) Add N,N-diisopropylethylamine (DIPEA) to the solution; (c) Add 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) to the solution to prepare a reaction mixture; (d) Mixing the reaction mixture; (e) Adding nitrobenzoflazan or nitrobenzoserenadiazole to the reaction mixture to prepare a conjugate mixture; (f) mixing a conjugate mixture; and (g) Producing a compound by one or more of the conjugate mixtures by dilution, washing, drying, or filtration. A method that includes this. Item 29. The method of Item 28, wherein steps (a) to (e) are carried out at approximately 0°C and steps (f) to (g) are carried out at room temperature. Item 30. The method of item 28 or 29, wherein the conjugate mixture is diluted with ethyl acetate and washed with an aqueous solution of citric acid. Item 31. The conjugate mixture is dried with MgSO4, one of the methods of items 28-30. Item 32. Any one of the methods described in items 28-31, further comprising purifying the compound using column chromatography. Item 33. A method for producing a compound comprising cis-11-eicosenoic acid conjugated to meltansine, [ka] (a) Prepare a solution by mixing meltansine carboxylic acid with dimethylformamide (DMF) solvent; (b) Add hexafluorophosphate azabenzotriazole tetramethyluronium (HATU) and N-methylmorpholine (NMM) to the solution to prepare a reaction mixture; (c) Mixing the reaction mixture; (d) Add cis-11-eicosenoic acid conjugated to piperazine to the reaction mixture to prepare a conjugated mixture; (e) mixing conjugate mixtures; and (f) Producing a compound by one or more of the conjugate mixtures by dilution, washing, drying, or filtration. A method that includes this. [Examples]

[0059] Example 1 Materials and methods mouse All experiments, including those involving the mice used in this study, followed protocols approved by the Animal Experimentation Committee of Vanderbilt University Medical Center. Mice of the same litter or age were randomly assigned to either the experimental or control group. Animal body weight was recorded at the start of the experiment and at the time of euthanasia. (Gif-rtTA;TetO-Cre;Kras) G12D To generate (GCK) mice, Gif-rtTA mice were used, along with TetO-Cre mice and Lox-Stop-Lox(LSL)-Kras mice. G12D Mice (numbers 006224 and 008179, Jackson Laboratories) were crossbred. To induce Cre-mediated recombination, 6-week-old mice were administered doxycycline water at a concentration of 1 mg / mL for 2 weeks. Mice were euthanized for histological examination 2 to 14 weeks after doxycycline treatment. For in vivo A939572 treatment, A939572 (HY-50709; MedChemExpress) was dissolved in dimethyl sulfoxide (DMSO) as a 100 mg / mL stock solution, aliquot, and stored at -80°C. A939572 was administered to mice daily for 2 weeks by intraperitoneal injection (diluted with 20 mg / kg corn oil) 6 weeks after doxycycline treatment. Mice were euthanized within 1 day after the last A939572 treatment.

[0060] Human tissue acquisition and sample preparation Table 1 provides details regarding the clinicopathological characteristics of the human subjects. In total, six human tissue array sets (five gastric tissue arrays and one esophageal tissue array) were constructed from specimens derived from patients who underwent curative endoscopic or surgical resection at Seoul National University Hospital or Jeju National University Hospital in South Korea between 2010 and 2021. Briefly, representative regions were selected by histological examination from H&E stained slides. Tissue cores with a diameter of 4 mm were obtained from individual paraffin blocks and arranged in recipient paraffin blocks using a trefining device (SuperBioChips Laboratories, Seoul, Korea). The tissue array construction was approved by the Institutional Review Boards of Seoul National University Hospital and Jeju National University Hospital, respectively. Informed consent was waived by the Institutional Review Boards due to the retrospective nature of the study. The six esophageal dysplastic tissues were provided by the NCI Cooperative Human Tissue Network (CHTN) (221092). The 30 pancreatic tissue samples were obtained from 27 patients who underwent surgical resection at Vanderbilt University Medical Center.

[0061] [Table 1-1]

[0062] [Table 1-2]

[0063] Tissue structure and immunohistochemical staining Mouse tissue was isolated, fixed overnight in 4% paraformaldehyde, and embedded in paraffin. The paraffin-embedded sections were cut to a thickness of 5 micrometers and stained with hematoxylin and eosin (H&E). For immunostaining, unstained paraffin tissue sections were deparaffinized in HistoClear (HS-200, National Diagnostics) and rehydrated with serially diluted ethanol solutions. Antigen retrieval was performed in antigen retrieval solution (S1699 or S2367, Agilent) using a pressure cooker for 15 minutes. The sections were rinsed in dH2O and incubated with Serum-free Protein Block Solution (X0909, Agilent) at room temperature (RT) for 1.5 hours. The sections were further incubated with Mouse on Mouse Blocking Reagent (MKB-2213-1, Vector Laboratories) at room temperature for 30 minutes to block nonspecific staining by the primary antibody generated in mouse-on-mouse tissue. The primary antibody was diluted with Antibody Diluent (S3022, Agilent) and applied to the sections overnight at 4°C in a humidified chamber (Table 2).

[0064] [Table 2]

[0065] For immunofluorescence, paraffin sections were washed three times with 1×PBS and incubated with Alexa-conjugate secondary antibody (Life Technologies, 1:500) for 1 hour at room temperature. Sections were then washed three times with 1×PBS and incubated with Hoechst 33342 (62249, Thermo Fisher Scientific, 1:5000) for 5 minutes at room temperature. Next, sections were washed three times with 1×PBS and mounted with Prolong gold anti-bleeding reagent (P36934, Thermo Fisher Scientific). Images were acquired at 20× magnification using a Zeiss Axio Imager M2 microscope with an Axiovision digital imaging system. For immunohistochemistry, paraffin sections were washed three times with 1×PBS and incubated with HRP-conjugate secondary antibody (MP-7402, Vector Laboratories) for 15 minutes at room temperature. Sections were washed three times with 1×PBS and incubated with Envision+ Detection System Peroxidase / DAB substrate (Dako). Next, sections were washed with dH2O and counterstained with Mayer's Hematoxylin (MHS32, Sigma-Aldrich). Sections were dehydrated and mounted in aqueous mounting medium. Sections were scanned at 20× magnification using an SCN400 Slide Scanner (Leica Biosystems). TUNEL staining was performed using the Click-iT Plus TUNEL assay kit (C10618, Life Technologies) according to the manufacturer's protocol.

[0066] Histopathological analysis and quantification H&E-stained sections were scanned with an SCN400 Slide Scanner and analyzed using Aperio ImageScope software (Leica Biosystems). Glandular types were classified using the same diagnostic criteria used for human tissue sections. Briefly, glands exhibiting crypt hyperplasia without goblet cells and mucosal metaplasia at the base were diagnosed as pyloric metaplastic glands, and intestinal metaplasia was confirmed by the presence of basophilic mucin droplet-containing cells in the crypt region. Low-grade dysplasia was characterized by morphological features including crowed glandular or lumen structures composed of pseudostratified columnar cells with hyperstained nuclei (Ref). Compared to others, high-grade dysplastic glands showed structural complexity and were covered with severe cellular and nuclear atypia and depolarized cells. Mitotic figures were more frequently observed throughout the gland in high-grade dysplastic glands. Gland width was measured at both ends of the transitional region per gland using Aperio ImageScope software. A total of 100 glands from three mice at each stage were measured in each tissue section. Representative images of at least five proximal gastric bodies (≥100 glands) were obtained at 20× magnification from three mice, and the number of cells for each lineage-specific marker, including the proliferating cell marker Ki-67, in each gland was manually quantified per gland using Adobe Photoshop measurement tools. To measure the degree of macrophage infiltration after A939572 treatment, the area of ​​F4 / 80-positive cells in three representative images per mouse (n=3) was measured using ZEN 3.2 software (Zeiss) and divided by the total area of ​​each image at 20× magnification. All histological analyses were performed blinded by two independent observers. SCD expression was determined by evaluating the intensity and percentage of SCD-expressing epithelial cells for each core. The hist score (H score) was calculated by multiplying the intensity (0=negative; 1=weak; 2=moderate; 3=strong) by the percentage of positive epithelial cells (range = 0~100), and the range was 0~300.

[0067] RNA extraction and reverse transcription quantification by PCR Total RNA was extracted from mouse stomach organoids using TRIzol® reagent (15596026, Thermo Fisher Scientific) according to the manufacturer's protocol. cDNA was synthesized from 1 μg of RNA using the iScript® gDNA Clear cDNA Synthesis Kit (1725035, Bio-rad). Quantitative PCR was performed using iTaq Universal SYBR Green Supermix (1725121, Bio-rad) on a CFX96® Real-Time PCR Detection System according to the manufacturer's protocol. Expression data were normalized to Rplp0 mRNA levels. The 2-ΔΔCt method was used for relative quantification of gene expression (Table 3).

[0068] [Table 3]

[0069] imaging mass spectrometry Frozen gastric tissue from Gif-rtTA;TetO-Cre;KrasG12D(GCK) mice was excised to a thickness of 12 μm and thaw-mounted onto indium tin oxide (ITO) coated glass slides. MALDI matrix 9-aminoacridin (9AA) was spray-coated onto MALDI target plates using an automated sprayer (TM Sprayer; HTX Technologies). 9AA was prepared at 5 mg / mL in 90% methanol and subjected to four passes at a nozzle temperature of 85°C, a flow rate of 0.15 mL / min, a track spacing of 2 mm, and a stage speed of 700 mm / min. Nitrogen was used as the atomizing gas and set to 10 gauge pressure (psig). Images were acquired using a 15T Fourier transform ion cyclotron resonance mass spectrometer (FTICR MS, SolariX; Bruker Daltonics) equipped with an Apollo II dual ion source and a Smartbeam II 2kHz Nd:YAG laser frequency tripled to 355nm. Data were collected in anion mode with the laser running at 2kHz. The pixel spacing was 50μm (center-to-center distance) in both the x and y directions. Data were collected at m / z 100–1400 with a resolution of 190,000 at m / z 300. Provisional metabolite identification was typically performed by accurate mass, usually better than 1 ppm. MSiReader version 1.02 software was used for ion image visualization and data analysis. Pixel-by-pixel calibration of metabolites was exported to imzML using Mmass software and summarized in Table 4.

[0070] [Table 4]

[0071] Organoid culture and drug treatment As previously described, mouse gastric organoids were established from the stomach bodies of untreated or Gif-rtTA;TetO-Cre;KrasG12D(GCK) mice. GCK mouse gastric organoids were cultured in 48-well plates in Mouse IntestiCult medium (06005, StemCell Technology) supplemented with 1% penicillin / streptomycin (2441832, Gibco) in ECM Gel (E1270, Sigma-Aldrich) or Cultrex® Reduced Growth Factor Basement Membrane Extract, Type R1 (3433-005-R1, R&D Systems). The medium was changed every 3 days, and the organoids were divided every 3-5 days. Human gastric organoids were previously established from patients who underwent curative gastrectomy, as described above. Human gastric organoids were cultured in 48-well plates in Corning® Matrigel® Membrane Matrix (356231, Thermo Fisher Scientific) along with Human IntestiCult medium (06010, StemCell Technology) supplemented with 1% penicillin / streptomycin and 0.2% MycoZap (VZA-2031, Lonza).

[0072] For metabolic enzyme inhibitor treatment, the culture medium was switched to a metabolic enzyme inhibitor-containing medium one day after the division of mouse gastric organoids, and the organoids were cultured for 3 days. Detailed information on the inhibitors and their final concentrations are given in Table 5. For metabolite treatment, mouse gastric organoids were treated with 100 nM A939572 along with the metabolite (cis-11-eicosenoic acid or cis-7,10,13,16-docosatetraenoic acid) one day after the division of the mouse gastric organoids, and cultured for 3 days. Detailed information on the metabolites and their final concentrations are given in Table 5. Phase-contrast images of the organoids were captured or organoid growth and morphological changes were monitored in real time using an EVOS M7000 inverted microscope (Thermo Fisher Scientific) or a JuLI® stage, Real-Time Cell History Recorder (NanoEntek). Quantitative data analysis was performed using images from at least 3 wells at 4x magnification. To measure organoid growth, phase-contrast images of organoids were acquired over time and measured at the widest diameter using a ZEN 3.3 blue edition (Zeiss) measurement tool. For calcein AM / ethidium-1 (EthD-1) staining, mouse gastric organoids were cultured for 1 day after division and transferred to a microfluidic flow tip (Protein Fluidics) enabling 3D cell line assays. Medium containing 100 nM A939572 with metabolites (cis-11-eicosenoic acid or cis-7,10,13,16-docosatetraenoic acid) was added to the organoids and cultured for 3 days. The organoids were then stained with 2 μm calcein AM / 4 μm EthD-1 (L3224, Thermo Fisher Scientific) at 37°C for 30 minutes. Confocal imaging was performed using a Zeiss LSM 880 at 20× magnification. To prepare formalin-fixed, paraffin-embedded (FFPE) organoid sections, whole mouse or human gastric organoids in Matrigel® were fixed in 4% PFA at room temperature for 30 minutes and washed twice with 1×PBS.The organoids were embedded in Epredia® HistoGel® (HG-4000-012, Thermo Fisher Scientific) and processed according to a standard histological protocol for paraffin embedding.

[0073] [Table 5]

[0074] Synthesis of NBD-conjugate metabolites Figure 8 shows a schematic diagram of the synthesis of NBD (nitrobenzoxadiazole; nitrobenzofurazan)-eicosenoic acid (C20:1, n-9). To prepare the fluorescent conjugate metabolite, solvents were obtained from the MBraun MB-SPS solvent system or freshly distilled (tetrahydrofuran was distilled from sodium benzophenone; toluene was distilled from calcium hydride and used immediately; dimethyl sulfoxide was distilled from calcium hydride and stored on a 4 Å molecular sieve). Commercial reagents were used as received. Semi-preparative reverse-phase HPLC was performed using a Waters HPLC system with a Phenomenex Luna 5 μm C18(2) 100 Å Axia 250 × 10 mm column, or preparative reverse-phase HPLC (Gilson) with a Phenomenex Luna column (100 Å, 50 × 21.2 mm, 5 μm C18), by UV / Vis detection. The infrared spectrum was obtained using a Thermo Electron IR100 series instrument as a thin film on a NaCl plate, with the absorption frequency (cm²) measured. -1 The unit of measurement will be reported as ). 1 The 1H NMR spectrum is recorded using a Bruker 400, 500, or 600 MHz spectrometer and reported against the deuterated solvent signal. 1 The 1H NMR spectral data are reported as follows: chemical shift (δppm), multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, p=pentet, m=multiplet, br=broad, app=apparent), coupling constant (Hz), and integral. 13¹³C NMR spectra were recorded using a Bruker 100, 125, or 150 MHz spectrometer and reported against the deuterated solvent signal. LC / MS was performed and recorded using an Agilent Technologies 6130 Quadrupole instrument. [ka]

[0075] Compound A (NBD; 7-nitrobenzo[c][1,2,5]oxadiazole-4-amine; nitrobenzooxadiazole; nitrobenzofurazan) To a solution of 4-chloro-7-nitrobenzo[c][1,2,5]oxadiazole (1.0 g, 5.0 mmol) in MeOH (25 mL), ammonia solution (7.1 mL, 7 M in MeOH) was added at room temperature under an argon atmosphere. The solvent was evaporated under vacuum, and the crude product was then purified by ISCO column chromatography eluted with 0-40% siRNA in dichloromethane to obtain a brown solid labeled compound A (7-nitrobenzo[c][1,2,5]oxadiazole-4-amine) (0.58 g, yield 64%). 1 H NMR(400MHz,MDSO-d6)δ 8.87(s,2H,-NH2),8.49(d,J=8.8Hz 1H),6.38(d,J=8.8Hz 1H);LCMS ESI-MS(m / z)C6H4N4O3[M+H] + Calculated value: 181.03, measured value: 181.05. [ka]

[0076] NBD-cis-11-eicosenoic acid (C20:1, n-9) To a solution of cis-11-eicosenoic acid 1 (40 mg, 0.13 mmol) in DMF (1.6 mL), DIPEA (91 mL, 0.64 mmol) was added, followed by HBTU (99 mg, 0.26 mmol) at 0°C. The reaction mixture was stirred at 0°C for 30 minutes, and then compound A (31 mg, 0.17 mmol) was added to the reaction mixture at 0°C. The reaction mixture was stirred at room temperature for 16 hours, diluted with siRNA (10 mL), and washed with aqueous citric acid (1 M, 2 × 10 mL) and brine (10 mL). The combined organic phases were dried over MgSO4, filtered, and concentrated under vacuum. The crude product was purified by ISCO column chromatography eluting with 0-25% siRNA in hexane to obtain a brown solid product (34 mg, 55% yield). 1 H NMR(400MHz,CDCl3)δ 8.56(d,J=8.4Hz,1H),8.48(d,J=8.4Hz,1H),8.39(s,1H,-NH),5.38-5.30(m,2H),2.58(t,J= 7.6Hz, 2H), 2.03-1.98 (m, 4H), 1.83-1.76 (m, 2H), 1.42-1.26 (m, 24H), 0.87 (t, J=6.4Hz, 3H).

[0077] Two-dimensional monolayer culture Mouse gastric organoids were dissociated into single cells, and the dissociated cells (5,000 cells / well) were seeded on collagen-coated 8-well chamber slides (ibidi) in Mouse IntestiCult medium (06005, StemCell Technology) for 2 days until the cells reached 50% confluence. The cells were incubated with NBD (compound A) or NBD-conjugate eicosenoic acid for an extended period, and then stained with 100 nM MitoTracker® Red CMXRos (M7512, Thermo Fisher Scientific) for 20 minutes at 37°C. The cells were washed three times with pre-warmed 1× PBS and counterstained with Hoechst for 5 minutes at room temperature. Confocal imaging was performed using a Zeiss LSM 880 at 63× magnification.

[0078] statistical analysis Statistical analysis was performed using GraphPad Prism (GraphPad Software, Inc.). For two-group comparisons, an independent two-tailed Student's t-test was performed, p-values ​​were calculated, and statistically significant differences were determined. For multiple comparisons, one-way ANOVA was used, followed by Tukey's post-hoc test. All experiments were repeated at least three times. The number of independent experimental replicates, variability (mean ± SD or mean ± SEM), and statistical test results (p-values) are reported in the legend of the corresponding figures.

[0079] Example 2 Kras activation in gastric chief cells leads to the continuous development of metaplasia and high-grade dysplasia. To determine whether Kras activation in gastric chief cells alone can lead to the entire gastric carcinogenesis process, we introduced a novel gene transgenic mouse allele, Gif-rtTA;TetO-Cre;Kras. G12D (GCK) Gif-rtTA mouse, dox-inducible and chief cell-specific driver mouse alleles, TetO-Cre and Kras G12DThe mice were crossed with alleles (Figure 1). GCK mice developed metaplasia and subsequent low- and high-grade dysplasia within 10–14 weeks after doxycycline treatment (Figure 2A). These mice developed pyloric metaplasia consisting of unique cell types, such as antispasmodic polypeptide-expressing metaplastic (SPEM) cells positive for CD44v9 and AQP5, in the glandular base and foveal cells over the SPEM cell region. The pyloric metaplasia gradually progressed to intestinal metaplasia and dysplastic glands. TFF3 was expressed in the luminal cells of the intestinal metaplastic glands. TFF3 expression increased significantly within 5–6 weeks in glands with basal SPEM cells, which is typical of the glandular composition of incomplete intestinal metaplasia (In-IM). These In-IM glands progressed to low-grade dysplasia (LGD) or even high-grade dysplasia (HGD) within 10–14 weeks in approximately 25% of the mucosa (Figure 2A–C). HGD repeated the structure of dysplastic glands and exhibited all the dysplastic cell features typically seen in human patients, including hyperstained nuclei, pseudostratification, and prominent nucleoli (Figure 2B). The number of TFF3-positive IM cells was significantly reduced in HGD, but SPEM cells were present at the base of HGD, and the co-expression of CD44v9 and AQP5 was reduced. LGD glands showed a transitioning cell zone above the SPEM cell region where the cell lineage changes from metaplastic to dysplastic cells, as confirmed by upregulation of TROP2 expression, an important transition marker between metaplastic and dysplastic cells. Expression of CD133, a marker of dysplastic stem cells (DSCs) responsible for adenocarcinoma development, was present in the apical membrane of cells within the transitioning cell zone in HGD. Furthermore, CEACAM5, a marker for dysplasia and gastric adenocarcinoma in humans, was present only in the apical membrane of dysplastic cells, and its expression was significantly increased in HGD. One unique feature was the continued widening of the gastric glands induced by Kras, particularly between the LGD and HGD stages (Figure 2D). Additionally, the cell proliferation region and level expanded throughout the glands in HGD, aligned with the distribution of CEACAM5-positive dysplastic cells. CLDN3, a major structural molecule of tight junctions, was significantly reduced at the very base of the glands in LGD and HGD, suggesting loss of cell polarity and structural changes in dysplastic glands due to alteration of the tight junction structure.Therefore, these data demonstrate that Kras activation in chief cells alone is sufficient to drive the carcinogenic process to high-grade dysplasia, accompanied by dynamic changes in cell lineage development and structural changes in glandular structures.

[0080] Example 3 Metabolic reprogramming activates FA metabolism during the progression of metaplastic dysplasia. To identify metabolic pathways involved in the carcinogenic process, matrix-assisted laser desorption / ionization imaging mass spectrometry (MALDI-IMS) was performed using GCK gastric tissue (Figure 3A). A series of metabolite mass spectra (200–1400 m / z) were collected across tissue samples. Key metabolites associated with major metabolic pathways such as glycolysis, fatty acids (FAs), phospholipids, and glutathione metabolism were profiled, and metabolites abundant at the metaplastic and / or dysplastic stages were selected by in-situ visualization and quantification of their spatial distribution (Figure 3B). Hexose diphosphate, a glycolysis-related metabolite, initially increased in stomachs with pyloric dysplasia and gradually decreased during progression to dysplasia (Figure 3B). In contrast, long-chain FAs associated with FA metabolism accumulated differentially during progression. Palmitate is the most common saturated long-chain FA in the human body. Palmitate was abundant in the normal stomach, but its levels decreased significantly in pyloric metaplasia, followed by increases in in-IM and HGD. In particular, a unique form of monounsaturated fatty acid (MUFA, FFA 20:1) (Figure 3D), which was an extension of only two carbon units of FFA 18:1, showed a stepwise increase during metaplasia progression and accumulated highly in HGD (Figure 3C). Furthermore, the accumulation of MUFA (FFA 20:1) was most pronounced at the base of glands where cell lineage transitions were occurring (Figure 3E). It is important to note that oleate (18:1), a well-known form of MUFA produced from palmitate, was also abundant in the stomach (Figure 3E). However, there was no correlation between the accumulation pattern of oleate and the progression of metaplasia. One polyunsaturated fatty acid (PUFA, FFA 22:4) also showed a similar accumulation pattern to MUFA (FFA 20:1). However, this PUFA (FFA 22:4) uses linoleates obtained solely from the diet as a precursor. Therefore, these results reveal that the metabolic switch from glycolysis to fatty acid metabolism occurs during the progression to metaplastic dysplasia, producing a unique form of MUFA (FFA 20:1).

[0081] Example 4 SCD-dependent FA desaturation is necessary for the survival of dysplastic cells. To determine the role of metabolic switches in dysplastic cells, the expression levels of critical metabolic enzymes important for glycolysis or FA metabolism were investigated using gastric organoids established from GCK mice at each stage (Figure 4A). Among the genes investigated, the expression levels of glycolysis-related enzymes were relatively high in In-IM organoids compared to pyloric metaplasia, LGD, or HGD organoids. In contrast, FA desaturation pathway-related enzymes, particularly Scd1, were increased in LGD and HGD organoids (Figure 4B). Stearoyl-CoA desaturase (SCD) is the rate-limiting enzyme in MUFA production. SCD1-positive cells were often co-positive for the cell proliferation marker Ki-67 and were prominently expressed in the transitional cell regions of both LGD and HGD glands (Figures 4C-D). The functional roles of metabolic pathways were evaluated in dysplastic organoids highly expressing SCD1 using key enzyme inhibitors (Figure 4E). Dysplastic organoids were treated for 3 days with small molecule inhibitors targeting key steps in fundamental metabolic pathways, including glycolysis and FA metabolism. Inhibition of glycolysis had no effect on organoid viability or growth. Inhibition of the pentose phosphate pathway and glutamine metabolism slightly reduced organoid growth but had no effect on viability. Inhibition of de novo lipid synthesis and elongation by targeting ACLY, FASN, or ELOVL activity had no effect on the survival of dysplastic organoids (Figure 4F). However, blocking FA desaturation by inhibiting SCD activity rather than FADS2 activity resulted in 100% death of dysplastic organoids (Figure 4F).

[0082] The importance of SCD-mediated FA desaturation was investigated in dysplastic cells. SCD inhibition using A939572 rapidly inhibited dysplastic organoid growth and survival within 1 day (Figure 5A-B). Organoids treated with A939572 showed accumulation of cleavage caspase-3 positive apoptotic cells in the central lumen of dysplastic organoids and decreased cell proliferation (Figure 5C). However, normal gastric organoids showed no significant changes in response to A939572 treatment (Figure 5D). Furthermore, SCD inhibition specifically targeted dysplastic cells in vivo. In GCK mice treated with A939572 for 2 weeks 6 weeks after doxycycline treatment, SCD inhibition resulted in dramatic changes in the gastric mucosa with no effects on other organs (Figure 5E). GCK mice treated only on the vehicle showed progression to metaplasia-LGD, containing SPEM cells in the base, transitional cell regions, and IM cells in the surface region (Figure 5E). In contrast, GCK mice treated with A939572 showed loss of histological characteristics of dysplastic cells in the flattened mucosa and the transitional and surface regions of the glands. A significant increase in dead cells was observed within the transitional cell region where SCD is strongly expressed, and cell death was confirmed by both cleavage caspase-3 and terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL) (Figure 5G-J, yellow arrowheads). Furthermore, F4 / 80 + CD68 - Macrophage infiltration was significantly increased in the transitional zone and surface region, suggesting increased phagocytic activity in the mucosa (Figure 5F). F4 / 80 + CD68 + Macrophages promote metaplasia, but were only slightly increased at the base of the glands. Significant degenerative changes were observed in the gastric mucosa after A939572 treatment, but SPEM cells remained present at the base of the glands and were not co-positive for cleavage caspase-3 or TUNEL signaling (Figure 5I). Therefore, these results indicate that SCD-dependent FA desaturation is required for dysplastic cell proliferation and survival.

[0083] Example 5 SCD-dependent FA desaturation produces unsaturated long-chain fatty acids that stimulate dysplastic cells. To address how and why dysplastic cells utilize SCD-dependent FA desaturation, the functions of two different unsaturated long-chain FAs, MUFA (FFA 20:1) and PUFA (FFA 22:4), were investigated experimentally, and both were abundant in dysplasia. Two metabolites, eicosenoic acid (EA;20:1n9, MUFA) and docosatetraenoic acid (DA;22:4n6, PUFA) (Figure 7A), were selected based on increased fragment ions detected by imaging mass spectrometry (Figure 3E). Dysplastic organoids were co-treated with A939572 in combination with either EA or DA for 3 days. Organoids co-treated with A939572 and EA recovered from SCD inhibition and showed a significant increase in size (Figures 7C-E). Furthermore, the organoid structure remained undisturbed and exhibited a clear central lumen (Figure 7C). However, organoids co-treated with A939572 and DA showed no difference compared to organoids treated with A939572 alone. To confirm the intracellular localization of EA in dysplastic cells, EA was conjugated with the green fluorescent compound NBD (NBD-EA) (Figure 7B and Figures 8A-B). NBD-EA was undetectable in a two-dimensional monolayer culture of dysplastic cells 6 hours after treatment, but the fluorescence signal accumulated strongly in the mitochondria at 24 hours and was maintained weakly up to 48 hours (Figure 7F). Since EA is imported into mitochondria, experiments were conducted to investigate whether EA undergoes FA oxidation, which occurs during mitochondrial aerobic metabolism and produces energy substrates for the TCA cycle. Carnitine palmitoyltransferase (CPT) transports long-chain FA into mitochondria, and the expression of Cpt1a, an isoform of CPT1, was upregulated in dysplastic organoids (Figure 7G). Inhibition of FA oxidation using perhexylline eliminated the rescue effect of EA in dysplastic organoids (Figure 7H). Organoids co-treated with A939572, EA, and perhexylline did not show a significant increase in organoid growth (Figures 7I-J). Therefore, these data suggest that dysplastic cells actively utilize EA as the primary substrate for mitochondrial energy production.

[0084] Example 6 Metabolic rewiring patterns are observed across precancerous lesions within gastrointestinal cancers. Since epithelial cells in other gastrointestinal (GI) tract organs also undergo continuous carcinogenic progression, further testing was conducted to determine whether SCD-dependent FA desaturation is activated in the progression from precancerous stages to cancer across the human gastrointestinal tract. SCD was immunostained as an indicator of the degree of SCD-dependent FA desaturation in microarrays and sections of gastrointestinal patient tissue from numerous sets of precancerous lesions and cancers in the stomach, esophagus, and pancreas (Figure 6). In gastric cancer tissue, SCD expression was significantly increased in both LGD and HGD dysplastic lesions compared to corresponding pairs of normal and metaplastic lesions (Figure 6A). SCD was highly expressed in intestinal gastric cancers occurring within the carcinogenic cascade compared to diffuse gastric cancer (Figures 6A, 6C). Esophageal adenocarcinoma progresses from precancerous metaplasia known as Barrett's esophagus to dysplasia and then to adenocarcinoma, and SCD expression was significantly increased in esophageal dysplasia and adenocarcinoma (Figures 6B, 6D). Finally, in pancreatic cancer, the three most important types, including mucinous cystic neoplasms, intraductal papillary mucinous neoplasms, and intraepithelial neoplastic lesions of the pancreas, are defined as precancerous lesions. SCD expression was elevated in all three types of high-grade dysplasia and adenocarcinoma compared to normal or low-grade lesions (Figure 6E-F). Therefore, these results indicate that upregulation of SCD is a common feature observed during the progression of metaplasia to dysplasia in gastrointestinal carcinogenesis.

[0085] To evaluate whether SCD-dependent FA desaturation is necessary for human dysplastic cell survival, nine human gastric organoid lines derived from patient samples with metaplasia or dysplasia were treated with A939572. The organoid lines showed a broad spectrum of sensitivity to SCD inhibition. The organoid lines exhibited dysplastic tissue structure and expressed both SCD and Trop2, but did not grow and died within 6 days after treatment. However, organoids from other groups expressing low levels of SCD did not respond to treatment. Furthermore, these organoids showed a single monolayer, were negative for Trop2, and indicated metaplastic organoids. Taken together, these results suggest that SCD function is important for dysplastic cell survival and that SCD-dependent FA desaturation may be activated during the transition from precancerous metaplasia to oncogenicity.

[0086] This study, using the GCK mouse allele, focused on cellular changes during dysplasia induced by Kras activation in zymogen-secreting chief cells in the stomach. Kras activation generally induces only precancerous metaplasia in other gastrointestinal organs, with several additional oncogene activations or mutations required for progression of precancerous lesions to malignant stages. In contrast, GCK mice developed high-grade dysplasia through the progression of precancerous metaplasia. The histological phenotype in the stomach of GCK mice faithfully replicated the structure of metaplastic or dysplastic glands seen in human patients. Sequential cell lineage transitions above the SPEM cell region indicated a major event in the oncogenic transformation of precancerous cells, distinguishing transitional cell regions in both low-grade and high-grade dysplasia. Furthermore, expansion of the proliferating cell region and changes in cell adhesion and tight junction molecules such as CEACAM5 and CLDN3 gave rise to the structural progression of metaplastic glands to dysplastic glands. These changes may result in loss of cell polarity and asymmetric cell division, which are key cytological features of dysplasia.

[0087] Cancer cells often reprogram their metabolism, and metabolic switches play a central role in supporting hyperproliferation and growth in cancer development. While glycolysis is a well-known upregulated pathway in cancer cells, abnormally activated lipid metabolism can stimulate cancer cell proliferation. However, it remains unclear whether metabolic switches occur during the carcinogenic process or whether they can modulate cellular dynamics within dysplastic cells. Imaging mass spectrometry has enabled the analysis of various types of metabolites abundant in gastric mucosa at different stages of oncogenesis. Glycolysis is initially activated during metabiogenesis, but it switches to fatty acid metabolism during metaplastic progression. In particular, the long-chain monounsaturated fatty acid, eicosenoic acid (EA;20:1n9, MUFA), was observed within the transitional cell region of dysplastic glands. FA desaturation can be regulated in mammals by different enzymes such as stearoyl-CoA desaturase (SCD) and FA desaturase (FADS). SCD expression is increased in many types of cancer and can even control stem cell properties, cancer cell proliferation, and acquired resistance to chemotherapy in cancer stem cells. SCD-dependent FA desaturation occurs actively in dysplastic cells, producing EA. Multiple types of precancerous lesions exist in human epithelial cell carcinogenesis, and their terminology, order, and mutation signatures differ by organ. However, a unique but common pattern of SCD upregulation has been identified in dysplasia and adenocarcinoma across the human gastrointestinal organs. This may suggest that metaplastic cells do not require FA metabolic reprogramming, but that reprogramming occurs in dysplastic and cancer cells in gastrointestinal carcinogenesis.

[0088] Cancer-related changes in lipid metabolism include increased lipid synthesis and lipid uptake from the extracellular microenvironment, as well as increased intracellular lipid storage. In particular, FA desaturation is known to support membrane biosynthesis or prevent both lipotoxicity from excessive FA or ferroptosis caused by lipid peroxidation. However, targeting FA desaturation with the SCD inhibitor A939572 in GCK mice specifically killed dysplastic cells. Furthermore, dysplastic organoids in both mice and humans died within a few days after A939572 treatment. Tracking the intracellular localization of EA using synthesized fluorescent-conjugated EA revealed that EA can migrate into mitochondria and stimulate dysplastic cell survival and proliferation through fatty acid oxidation. Therefore, these results suggest that metabolic reprogramming within dysplastic cells is necessary to meet their high energy requirements for hyperproliferation and growth. Additionally, EA production may be a prominent feature of metabolic switches within dysplastic cells.

[0089] It is important to note that inhibition of aerobic glycolysis (Warburg effect), the most well-known metabolic pathway in cancer cells, did not affect the viability or growth of dysplastic cells. This supports the unique metabolic characteristics of dysplastic cell behavior, which utilizes FA rather than glucose as its primary energy source. Mammalian cells can utilize two main sources of free FA: de novo lipid synthesis or externally sourced free FA, but how dysplastic cells generate EA remains unclear. Rescue experiments with EA suggest that dysplastic cells can survive by taking in free FA from external sources. However, further studies are needed to evaluate the mechanism of free FA influx into dysplastic cells and its relative contribution to the generation of long-chain FA. Also, ELOVL (extremely long-chain fatty acid elongation protein) is a potential candidate for adding substrates with up to 20 carbon chains in long-chain FA. However, no significant effect on viability or growth in dysplastic organoids was observed after inhibition of ELOVL1 or 6. Little is known about the ELOVL function underlying FA elongation, and only a limited number of ELOVL inhibitors have been developed. Therefore, further testing will be necessary to elucidate the specific function of FA elongation within dysplastic cells.

[0090] This study confirms that Kras activation in chief cells is sufficient for the complete process of carcinogenesis to high-grade dysplasia in the stomach. Unlike cancer cells, which exhibit diverse cell populations with unique molecular and metabolic signatures, dysplastic cells have relatively simple characteristics. Furthermore, SCD upregulation in dysplastic cells promotes FA metabolic reprogramming, producing the energy substrate eicosenoic acid (EA;20:1n9, MUFA) through SCD1-dependent FA desaturation. Therefore, SCD-dependent FA desaturation is an essential element in tumor-fatty acid metabolism for the high energy requirements necessary for dysplastic cell overgrowth and survival in epithelial carcinogenesis.

[0091] Example 7 Synthesis of nitrobenzoselenadiazole-conjugate metabolites [ka]

[0092] Nitrobenzoselenadiazole-cis-11-eicosenoic acid (C20:1, n-9) Cis-11-eicosenoic acid was also conjugated to fluorophores and nitrobenzoselenadiazole, which are labeled with red fluorescence as described in Benson et al., Angew Chem Int Ed Engl., 131(21):6985-6989 (2019). The fluorescent eicosenoic acid conjugate compound was synthesized as follows.

[0093] To a solution of cis-11-eicosenoic acid 1 (40 mg, 0.13 mmol) in DMF (1.6 mL), DIPEA (91 mL, 0.64 mmol) was added, followed by HBTU (99 mg, 0.26 mmol) at 0°C. The reaction mixture was stirred at 0°C for 30 minutes, and then nitrobenzoselenadiazole (31 mg, 0.17 mmol) was added to the reaction mixture at 0°C. The reaction mixture was stirred at room temperature for 16 hours, diluted with siRNA (10 mL), and washed with aqueous citric acid (1 M, 2 × 10 mL) and brine (10 mL). The combined organic phase was dried over MgSO4, filtered, and concentrated under vacuum. The crude product was purified by ISCO column chromatography eluting with 0-25% siRNA in hexane to obtain a brown solid product.

[0094] Example 8 Metabolite drug conjugates (MDCs) to inhibit dysplasia Antibody-drug conjugates (ADCs) have been actively developed in recent years for targeted therapies. Cytotoxic agents typically bind to monoclonal antibodies and negatively regulate cell replication or growth. However, many ADCs have also failed during clinical development due to problems with drug specificity and cytotoxic activity. Metabolite-drug conjugates (MDCs) utilizing cis-11-eicosenoic acid offer an alternative targeted delivery platform.

[0095] Here, cis-11-eicosenoic acid was conjugated with a toxin that inhibits dysplasia. It was found that this specific eicosenoic acid metabolite is produced only in dysplastic cells, which are considered to be cancer-initiating cells. Patients with dysplasia in the gastrointestinal tract have a high risk of developing cancer.

[0096] Metabolites such as cis-11-eicosenoic acid are small molecules used as intermediates in metabolic pathways or produced from metabolic reactions. Various cytotoxicities, including drugs such as meltansine (DM1), taxol, calichemycin, monomethyl auristatin E (MMAE), deruxtecan, SN-38, Pseudomonas exotoxin, diphtheria toxin, or yttrium-90, can be conjugated to these metabolites. The small size and specificity of cis-11-eicosenoic acid are beneficial for drug conjugation and intracellular drug delivery. Therefore, some important advantages of the disclosed MDC include the fact that cis-11-eicosenoic acid may be cell-type specific, non-immunogenic, low-toxicity, and readily internalized by epithelial cells.

[0097] cis-11-eicosenoic acid was conjugated to the therapeutic toxin, meltansine (DM1, [(1S,2R,3S,5S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10,12,14(26),16,18-pentaen-6-yl](2S)-2-[methyl(3-sulfanylpropanoyl)amino]propanoate) as described below. [ka]

[0098] (Z)-1-(piperazine-1-yl)icosa-11-en-1-one tert-butyl(Z)-4-(icosa-11-enoyl)piperazine-1-carboxylate DECI (15.0 mg, 0.097 mmol), HOBt (17.5 mg, 0.130 mmol), and Et3N (17 μL, 0.194 mmol) were added to a solution of (Z)-icosa-11-enoic acid (2.00 mg, 0.064 mmol) in MeCN (2 mL) and stirred at room temperature for 45 minutes. Tert-butylpiperazine-1-carboxylate (12.0 mg, 0.064 mmol) was added and stirred at 35°C for 22 hours. The reaction mixture was quenched with water and extracted with CH2Cl2 (3 × 5 mL). The combined organic layers were dried over (MgSO4), filtered, and concentrated. The residue was purified by silica gel chromatography using Teledyne ISCO Combi-Flash, eluted with 0-35% Â in hexane, to obtain the desired product (26.0 mg, yield 84%). 1 H NMR(400MHz,CDCl3):δ 5.35-5.33(m,2H),3.58(t,J=5.3Hz,2H),3.43(s,3H),3.41-3.38(m,2H),2.34-2.30(m,2H),2.01( Quart J=5.9Hz,4H),1.67-1.58(m,4H),1.48(s,9H),1.35-1.25(m,23H),0.88(t,J=7.1Hz,3H);LC / MS 1.454 minutes, 479.0 (M+H).

[0099] (Z)-1-(piperazine-1-yl)icosa-11-en-1-one TFA (200 μL, 0.261 mmol) was added to a solution of tert-butyl(Z)-4-(icosa-11-enoyl)piperazine-1-carboxylate (26.0 mg, 0.054 mmol) in CH2Cl2 (0.5 mL) and stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in CH2Cl2 and treated with saturated NaHCO3 aqueous solution. The mixture was extracted with CH2Cl2 (3 × 5 mL). The combined organic layers were dried (MgSO4), filtered, and concentrated to obtain the desired product (20.0 mg, quantitative yield). The crude compound was used in the next step without further purification. 1H NMR(400MHz,CDCl3):δ 1.62 1 (quint J=7.8Hz, 2H), 1.35-1.27 (m, 19H), 0.88 (t, J=7.0Hz, 3H); LC / MS 0.971 min, 379.1 (M+H).

[0100] 2-((3-(((S)-1-(((1 4 S,1 6 S,3 2 S,3 3 R, 2R, 4S, 10E, 12E, 14R)-8 6 -Chloro-1 4 -Hydroxy-8 5 ,1 4 -Dimethoxy-3 3 ,2,7,10-tetramethyl-1 2 ,6-Dioxo-7-Aza-1(6,4)-Oxazinana-3(2,3)-Oxirana-8(1,3)-Benzenacyclotetradecaphane-10,12-Dien-4-yl)oxy)-1-Oxopropan-2-yl)(methyl)amino)-3-Oxopropyl)thio)acetic acid Bromoacetic acid (14.3 mg, 0.103 mmol) and saturated NaHCO3 aqueous solution (270 μL) are used in (1 4 S,1 6 S,3 2 S,3 3 R, 2R, 4S, 10E, 12E, 14R)-8 6 -Chloro-1 4 -Hydroxy-8 5 ,14-dimethoxy-3 3 ,2,7,10-tetramethyl-1 2The reaction mixture was added to a solution of 6-dioxo-7-aza-1(6,4)-oxadinana-3(2,3)-oxilana-8(1,3)-benzenacyclotetradecafan-10,12-dien-4-yl N-(3-mercaptopropanoyl)-N-methyl-L-alaninate (10.0 mg, 0.014 mmol) and stirred at room temperature for 24 hours. The reaction mixture was treated with acetic acid and extracted with CH2Cl2 (3 × 5 mL). The combined organic layers were dried in (MgSO4), filtered, and concentrated. The residue was purified by silica gel chromatography using Teledyne ISCO Combi-Flash, eluting with 0-40% MeOH in CH2Cl2, to obtain the desired product (5.2 mg, yield 49%). LC / MS 0.924 min, 777.6 (M-18).

[0101] (1 4 S,1 6 S,3 2 S,3 3 R, 2R, 4S, 10E, 12E, 14R)-8 6 -Chloro-1 4 -Hydroxy-8 5 ,14-dimethoxy-3 3 ,2,7,10-tetramethyl-1 2 ,6-Dioxo-7-Aza-1(6,4)-Oxadinana-3(2,3)-Oxirana-8(1,3)-Benzenacyclotetradecafan-10,12-Dien-4-yl N-(3-((2-(4-((Z)-Icosa-11-enoyl)piperazine-1-yl)-2-Oxoethyl)thio)propanoyl)-N-methyl-L-alaninate HATU (13.1 mg, 0.034 mmol) and N-methylmorpholine (3.8 μL, 0.035 mmol) are used in 2-((3-(((S)-1-(((1 4 S,1 6 S,3 2 S,3 3 R, 2R, 4S, 10E, 12E, 14R)-8 6 -Chloro-1 4 -Hydroxy-8 5 ,1 4 -Dimethoxy-3 3 ,2,7,10-tetramethyl-1 2,6-Dioxo-7-Aza-1(6,4)-Oxadinana-3(2,3)-Oxirana-8(1,3)-Benzenacyclotetradecafan-10,12-Dien-4-yl)oxy)-1-oxopropan-2-yl)(methyl)amino)-3-oxopropyl)thio)acetic acid (13.7 mg, 0.017 mmol) was added to a solution of DMF (1.0 mL). After 5 minutes, (Z)-1-(piperazin-1-yl)icosa-11-en-1-one (10.0 mg, 0.026 mmol) in DMF (1.0 mL) was added and the mixture was stirred at room temperature for 5 minutes. The reaction mixture was treated with water and extracted with ELISA (3 × 5 mL). The combined organic layers were washed with water (6 ×), dried (MgSO4), filtered, and concentrated. The residue was purified by silica gel chromatography using Teledyne ISCO Combi-Flash, eluting with 0-10% MeOH in CH2Cl2, to obtain the desired product (13.5 mg, 68% yield); LC / MS 1.288 min, 1137.6 (M-18).

[0102] Example 9 To evaluate whether EA-DM1 (cis-11-eicosenoic acid conjugated to meltansine (DM1)) can internalize and release free DM1 toxin into the cytosol, two different gastric cancer cell lines, AGS and NCI-N87, were treated with EA-DM1 for two days (Figures 9A-B). Gastric cancer cells died within two days after treatment with 100 nM EA-DM1 (Figure 9A). Furthermore, EA-DM1 effectively targeted gastric cancer cells at a lower concentration of 5 nM (Figure 9B). In contrast, no significant changes were observed in NIH-3T3 cells, a normal mouse fibroblast cell line, treated with 5 nM EA-DM1, indicating that EA-DM1 does not target normal mouse cells (Figure 9B). These results suggest that EA-DM1 uptake can specifically target cancer cells that utilize fatty acid metabolism.

Claims

1. Compound of formula (I): 【Chemistry 1】 (In the formula, R １ (The label is a fluorophore or luminescent label selected from 7-nitrobenzo[c][1,2,5]oxadiazole-4-amine (nitrobenzofurazan), nitrobenzoselenadiazole, fluorescein, rhodamine, aminomethylcoumarin acetate (AMCA), calcein, or cyanine.) 【Request Item 2】 【Chemistry 2】 The compound according to claim 1, wherein X is O or Se.

3. Compound of formula (I): 【Transformation 3】 (In the formula, R １ (Selected from meltansine (DM1), taxol, calichemycin, monomethyl auristatin E (MMAE), deruxtecan, SN-38, Pseudomonas exotoxin, diphtheria toxin, or yttrium-90). 【Request Item 4】 【Chemistry 4】 The compound according to claim 3.

5. A method for detecting epithelial cell dysplasia, A step of bringing epithelial cells into contact with the compound described in claim 1; A process of incubation for a certain period of time; A step of irradiating the epithelial cells with ultraviolet light so that the dysplastic epithelial cells fluoresce; and The process of imaging the fluorescent dysplastic epithelial cells. A method that includes this.

6. The method according to claim 5, wherein imaging includes positron emission tomography (PET) imaging, mass spectrometry imaging, immunofluorescence imaging, fluorescence molecular endoscopy, or fluorescence-guided intraluminal endoscopy.

7. The method according to claim 5, wherein the epithelial cells include gastric epithelial cells.

8. Research tools for detecting epithelial dysplasia, including compounds of formula (I): 【Transformation 5】 (In the formula, R １ (The label is a fluorophore or luminescent label selected from 7-nitrobenzo[c][1,2,5]oxadiazole-4-amine (nitrobenzofurazan), nitrobenzoselenadiazole, fluorescein, rhodamine, aminomethylcoumarin acetate (AMCA), calcein, or cyanine.)

9. The aforementioned compound 【Transformation 6】 The research tool according to claim 8, wherein X is O or Se in the formula.

10. Research tools containing the compound of formula (I): 【Transformation 7】 (In the formula, R １ (Selected from meltansine (DM1), taxol, calichemycin, monomethyl auristatin E (MMAE), deruxtecan, SN-38, Pseudomonas exotoxin, diphtheria toxin, or yttrium-90).

11. The aforementioned compound 【Transformation 8】 The research tool according to claim 10.

12. A method for inhibiting cancer or precancerous conditions in cells or subjects requiring such inhibition, comprising: a therapeutically effective amount of a compound of formula (I) to the cells or subjects: 【Chemistry 9】 (In the formula, R １ A method comprising administering (selected from meltansine (DM1), taxol, calichemycin, monomethyl auristatin E (MMAE), deruxtecan, SN-38, pseudomonas exotoxin, diphtheria toxin, or yttrium-90).

13. The aforementioned compound 【Chemistry 10】 The method according to claim 12.

14. A method for treating a subject having cancer or precancerous conditions, wherein the subject is given a therapeutically effective amount of a compound of formula (I): 【Chemistry 11】 (In the formula, R １ A method comprising administering (selected from meltansine (DM1), taxol, calichemycin, monomethyl auristatin E (MMAE), deruxtecan, SN-38, pseudomonas exotoxin, diphtheria toxin, or yttrium-90).

15. The aforementioned compound 【Chemistry 12】 The method according to claim 14.

16. The method according to claim 14, wherein the subject has gastrointestinal cancer or precancerous disease, or is suspected of developing gastrointestinal cancer or precancerous disease.

17. A method for inhibiting the migration of metaplastic cells to dysplastic cells, comprising administering a therapeutically effective amount of 4-(2-chlorophenoxy)-N-(3-(methylcarbamoyl)phenyl)piperidine-1-carboxamide (A939572) to cells or a subject requiring it.

18. The method according to claim 17, wherein the therapeutically effective amount of A939572 is about 5 mg / kg to about 100 mg / kg.

19. The method according to claim 17, wherein the therapeutically effective amount of A939572 is administered daily for about 1 day to about 6 months.

20. The method according to claim 17, wherein the cells include gastric epithelial cells.

21. The method according to claim 17, wherein the subject has gastrointestinal cancer or precancerous disease, or is suspected of developing gastrointestinal cancer or precancerous disease.

22. A method for treating metaplastic cells to reduce the incidence of cancer, comprising administering a therapeutically effective amount of 4-(2-chlorophenoxy)-N-(3-(methylcarbamoyl)phenyl)piperidine-1-carboxamide (A939572) to cells or a subject in need thereof.

23. The method according to claim 22, wherein the method inhibits the migration of the metaplastic cells to dysplastic cells, thereby reducing the incidence of cancer in the cells or the subject that requires them.

24. The method according to claim 22, wherein the therapeutically effective amount of A939572 is about 5 mg / kg to about 100 mg / kg.

25. The method according to claim 22, wherein the therapeutically effective amount of A939572 is administered daily for about 1 day to about 6 months.

26. The method according to claim 22, wherein the cells include gastric epithelial cells.

27. The method according to claim 22, wherein the subject has gastrointestinal cancer or precancerous disease, or is suspected of developing gastrointestinal cancer or precancerous disease.

28. 7-Nitrobenzo[c][1,2,5]oxadiazole-4-amine (nitrobenzofurazan) or nitrobenzoselenadiazole: 【Chemistry 13】 (In the formula, X is either O or Se) A method for producing a compound containing cis-11-eicosenoic acid conjugated to: (a) A step of preparing a solution by mixing cis-11-eicosenoic acid with dimethylformamide (DMF) solvent; (b) Adding N,N-diisopropylethylamine (DIPEA) to the solution; (c) A step of preparing a reaction mixture by adding 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) to the solution; (d) The step of mixing the reaction mixture; (e) A step of adding nitrobenzoflazan or nitrobenzoselenadiazole to the reaction mixture to prepare a conjugate mixture; (f) the step of mixing the conjugate mixture; and (g) A step of producing the compound by one or more of the conjugate mixtures by dilution, washing, drying, or filtration. A method that includes this.

29. The method according to claim 28, wherein steps (a) to (e) are carried out at approximately 0°C and steps (f) to (g) are carried out at room temperature.

30. The method according to claim 28, wherein the conjugate mixture is diluted with ethyl acetate and washed with an aqueous citric acid solution.

31. The conjugate mixture is MgSO ４ The method according to claim 28, wherein the method is dried by [method].

32. The method according to claim 28, further comprising the step of purifying the compound using column chromatography.

33. Meltansine: 【Chemistry 14】 A method for producing a compound containing cis-11-eicosenoic acid conjugated to: (a) A step of preparing a solution by mixing meltansine carboxylic acid with dimethylformamide (DMF) solvent; (b) A step of preparing a reaction mixture by adding hexafluorophosphate azabenzotriazole tetramethyluronium (HATU) and N-methylmorpholine (NMM) to the solution; (c) The step of mixing the reaction mixture; (d) A step of adding cis-11-eicosenoic acid conjugated to piperazine to the reaction mixture to prepare a conjugated mixture; (e) the step of mixing the conjugate mixture; and (f) A step of producing the compound by one or more of the conjugate mixtures by dilution, washing, drying, or filtration. A method that includes this.

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