3-Phenylquinoazolinones as novel anticancer treatments

Abstract

The present invention relates to novel 3-phenylquinazolines, particularly for use in the treatment of cancer.

Description

[Technical Field]

[0001] The present invention relates to novel 3-phenylquinazolinone compounds, particularly for use in the treatment of cancer. [Background technology]

[0002] Ferrotosis is a type of regulated necrotic cell death that is tightly controlled by glutathione peroxidase 4 (GPX4) (Refs. 1-3). It has become increasingly clear that this pathway plays an important role in diverse physiological and pathological conditions, including antiviral immunity, neurodegeneration, ischemia / reperfusion injury, and tumor suppression (Refs. 4-7). Currently, much research is being conducted to identify genes essential for this form of cell death and develop novel ferrotosis inhibitors (Refs. 2, 8). Ferrotosis-inducing compounds targeting different nodes in the ferrotosis regulatory cascade have been reported in the past (Refs. 4, 9). However, most of these compounds do not exhibit strong ferrotosis-inducing activity in vivo. This is because some of these checkpoints can be bypassed in vivo.

[0003] Currently, there is no efficient anticancer strategy based on small molecules to induce ferrotosis. Furthermore, recombinant cysteine-degrading enzymes, next-generation system Xc -Approaches targeting the cysteine / glutathione / GPX4 axis using inhibitors (e.g., piperazine erastin, imidaketazole erastin) and next-generation GPX4 inhibitors (e.g., ML210, diacyl fluoroxanes, and masked nitrile oxide electrophiles) have been reported. However, none of these compounds have proven effective in efficiently suppressing tumor growth in vivo (Refs. 10-13). Furthermore, many known ferrotosis-inducing compounds induce ferrotosis in cell culture but do not demonstrate its efficacy in vivo. This is primarily due to the low bioavailability of these compounds or their targeting of redundant nodes in the ferrotosis regulatory network (Ref. 6). Furthermore, because some known targets (e.g., GPX4) are essential for normal physiological function, as shown in multiple genetic studies (Ref. 7), targeting these nodes may be associated with severe toxicity issues if inhibited pharmacologically. On the other hand, FSP1 knockout mice are fully viable (Refs. 14, 15), which may significantly expand the therapeutic window for newly developed FSP1 inhibitors. Indeed, the first FSP1 inhibitor (iFSP1; 1-amino-3-(4-methylphenyl)-pyrido[1,2-a]benzimidazole-2,4-dicarbonitrile) efficiently sensitizes ferrotosis-resistant tumor cells to ferrotosis (Refs. 16-18), but this class of compounds is of little use for future drug development.

[0004] Therefore, there is a need to develop more small molecule-based ferrotosis-inducing cancer therapies. Summary of the Invention

[0005] The present invention relates to a compound of formula (I) or formula (II), preferably formula (I), for use in the treatment of cancer:

[0006] [ka]

[0007] G is

[0008] [ka]

[0009] selected from the group consisting of: M is

[0010] [ka]

[0011] selected from the group consisting of: Z is selected from the group consisting of CH, C-(C1-C6) alkyl, and N, preferably CH; A is selected from the group consisting of CH, C-(C1-C6) alkyl, and N, preferably CH; Q is selected from the group consisting of CH, C-(C1-C6) alkyl, and N, preferably CH; E is selected from the group consisting of -CO- and -SO2-, preferably -CO-; L is selected from the group consisting of CH, N, CF, C—Cl, and C—(C1-C6) alkyl, preferably CH; X is selected from the group consisting of CH, N, CF, C—Cl, and C—(C1-C6) alkyl, preferably CH; Y is selected from the group consisting of CH, N, CF, C—Cl, and C—(C1-C6) alkyl, preferably CH; X and Y may be part of a ring as shown in formula (III);

[0012] [ka]

[0013] n is an integer from 1 to 3; J is selected from the group consisting of CH, N, CF, C—Cl, and C—(C1-C6) alkyl, preferably CH; D 1 , D 2 , D 3 , D 4 , D 5 are independently selected from the group consisting of C and N; R 1 is selected from the group consisting of -(C1-C6)alkyl, -(C3-C6)cycloalkyl, -CH2OH, -CH2F, -CHF2, and -CF3, preferably -(C1-C6)alkyl; R 2 is selected from the group consisting of H, —(C1-C6)alkyl, —CF3, —Cl, and —F, preferably H; R 3 is selected from the group consisting of H, —(C1-C6)alkyl, —CF3, —O(C1-C6)alkyl, —Cl, and —F; preferably —O(C1-C6)alkyl; R 4 is selected from the group consisting of H, —(C1-C6)alkyl, —CF3, —O(C1-C6)alkyl, —Cl, and —F, preferably —O(C1-C6)alkyl; R 5 is selected from the group consisting of H, —(C1-C6)alkyl, —CF3, —O(C1-C6)alkyl, —Cl, and —F, preferably —O(C1-C6)alkyl; R 6 is selected from the group consisting of H, —(C1-C6)alkyl, —CF3, —Cl, and —F, preferably H; R in adjacent position 2 , R 3 , R 4 , R 5 and R 6 Two of the groups are

[0014] [ka]

[0015] may be part of a ring selected from o is an integer from 1 to 4; p is an integer from 1 to 4; q is 1 or 0; R 7 and R 8 is independently selected from the group consisting of H, and -(C1-C6)alkyl; R 7 and R 8 may form a 3- to 5-membered cycloalkyl ring; R 9 is selected from the group consisting of H, and -(C1-C6)alkyl, or a pharmaceutically acceptable salt thereof.

[0016] The present invention further relates to a pharmaceutical composition for use in the treatment of cancer, comprising a compound as defined above and at least one pharmacologically acceptable carrier. [Brief explanation of the drawings]

[0017] [Figure 1A-B] Using AquaBluer as an indicator of cell death, we show that compound 1 (also known as icFSP1) efficiently induces ferrotosis in FSP1-overexpressing Pfa1 Gpx4KO cells (A) and in HT-1080 wild-type cells (a human fibrosarcoma cell line) (B). This form of cell death is rescued by the ferrotosis inhibitor lipostatin-1 (Lip-1). [Figure 1C-E](C) Pfa1 Gpx4KO cells overexpressing HA-tagged hFSP1 were treated with DMSO (control), 2.5 μM icFSP1, or 2.5 μM icFSP1 + 0.5 μM Lip-1 for 24 hours, after which cell death was measured by lactate dehydrogenase (LDH) release. (D,E) Gpx4KO cells stably overexpressing HA-tagged hFSP1 were treated with DMSO, 2.5 μM icFSP1 + 0.5 μM Lip-1 for 3 hours, after which lipid peroxidation was assessed by C11-BODIPY581 / 591 staining. A representative plot of one of three independent experiments (D) and the quantified median value (E) of three independent experiments are shown for the ratio of oxidized to reduced BODIPY (BODIPYox / BODIPYre). Data represent the mean ± standard error (SEM) of three independent experiments (C, D, E). [Figure 1F] Lipid peroxidation profile measured by liquid chromatography and tandem mass spectrometry (LC-MS / MS) after treatment of Gpx4KOPfa1 cells stably overexpressing HA-tagged hFSP1 with DMSO, 5 µM icFSP1, or 5 µM + 0.5 µM Lip-1 for 5 h. Heatmap shows three technical replicates from one of two independent experiments. [Figure 2A] In vitro inhibitory activity of compound 1 (icFSP1) against recombinant FSP1 enzyme activity is shown. This assay measures the difference in inhibition of resazurin reduction compared to icFSP1 at different concentrations of the known direct inhibitor compound iFSP1 (16). [Figure 2B] Representative time-lapse fluorescence images acquired immediately after treatment of Gpx4WTPfa1 cells stably overexpressing hFSP1-EGFP-Strep with 2.5 μM icFSP1. Scale bar: 10 μm. Representative result from one of three independent experiments showing the intracellular relocation of FSP1 after icFSP1 treatment. [Figure 2C]The number of aggregates per cell induced by icFSP1 was quantified from time-lapse images at different time points after treatment (0, 60, 120, 180, and 240 minutes) from one of two independent experiments. Dots represent individual cells, and n indicates the number of cells (n = 129, 124, 130, 130, and 134, respectively, from left to right). P values ​​were calculated by one-way ANOVA followed by Dunnett's multiple comparison test. [Figure 2D] Representative time-lapse fluorescence images of Gpx4KOPfa1 cells stably overexpressing hFSP1-mTagBFP after treatment with 2.5 μM icFSP1 in FluoroBrite DMEM containing PI (0.2 μg / ml). Cells were prestained with 5 μM Liperfluo for 1 hour. Scale bar is 10 μm. Representative results from three independent experiments. [Figure 3] Figure 3 shows the in vivo effect of icFSP1 (compound 1) on tumor growth inhibition. (A) Gpx4 KO / Fsp1KO double mutant mouse melanoma cells (B16F10) reconstituted with human FSP1 expression were subcutaneously implanted into C57BL6 / J mice. Treatment with vehicle (control group: n = 6) or icFSP1 (n = 7) began 6 days after implantation. Tumor volume (left) and tumor weight (right) were measured daily or at the end of the experiment. (B) GPX4KO human melanoma cells (A375) were subcutaneously implanted into nude mice. Treatment with vehicle (control group: n = 7) or icFSP1 (n = 7) began 3 days after implantation. Compound treatment significantly inhibited tumor growth in both human and mouse tumors. These results suggest that FSP1 inhibitors synergize with ferrotosis inducers (exemplified by knockout of the key ferrotosis regulator GPX4) to enhance the cell death response. P values ​​were calculated by two-way ANOVA followed by Bonferroni's multiple comparison test or unpaired t-test. [Figure 4]FSP1 aggregates are droplets. (A) Representative time-lapse fluorescence images of Gpx4WT Pfa1 cells stably overexpressing hFSP1-EGFP-Strep before and after treatment with 2.5 μM icFSP1 (compound 1). Arrowheads indicate the location of fusion of individual aggregates (left). Reversibility of hFSP1 aggregation (right). After treating cells with icFSP1 for 240 min, the medium was replaced with fresh medium without icFSP1, and recording resumed. Scale bars represent 10 μm or 2 μm in the enlarged images. Representative results from one of three independent experiments. (B) Fluorescence recovery after photobleaching (FRAP) assay of Gpx4WT Pfa1 cells overexpressing hFSP1-EGFP-Strep after treatment with 2.5 μM icFSP1 for 120 min. Grayscale images (top left) correspond to representative FRAP images immediately before and after photobleaching. Look-up table (LUT) image (bottom left) showing a zoomed-in view of the red rectangular area in the FRAP image above. Quantified FRAP rates for each aggregate (right). Data are shown as the mean ± standard deviation for five aggregates from the image on the left. Scale bar is 10 μm. Representative results from one of three independent experiments. [Figure 5A-B] Specific structural features of FSP1 are required for phase separation. (A) Representative images of Pfa1 cells overexpressing hFSP1-EGFP-Strep mutants treated with 2.5 μM icFSP1 (compound 1). Scale bar: 10 μm. (B) Representative images of Pfa1 cells overexpressing WT hFSP1-EGFP-Strep or the S187C, L217R, or Q319K mutants treated with 2.5 μM icFSP1. Scale bar: 10 μm. Data are shown as the mean ± standard deviation of three different fields from one of three independent experiments (A, B). Statistical analysis was performed by one-way ANOVA followed by Dunnett's multiple comparison test (B). [Figure 5C]Cell viability measured after 24 h of treatment with icFSP1 in Gpx4KO Pfa1 cells overexpressing WT hFSP1 or the S187C, L217R, or Q319K mutants. Data represent the mean ± standard deviation of triplicate wells from one of four independent experiments. [Figure 5D] Gpx4 KO / Fsp1KO double mutant B16F10 cells reconstituted with human WT or Q319K FSP1 expression were subcutaneously implanted into C57BL6 / J mice. At the end of the experiment, tumors were excised and stained with anti-HA (hFSP1) antibody to visualize FSP1. A representative magnified image of one of three tumor samples from one of two independent experiments is shown (D). Arrowheads indicate FSP1 aggregates (D). The scale bar is 10 µm (E). [Figure 6A] Synergistic effects of icFSP1 (compound 1) and ferrotosis inducers in various human cancer cell lines. (A) HT-1080, A375, 786-O, MDA-MB-436, and H460 cells were treated with different ferrotosis inducers (RSL3, ML210, erastin, FIN56, and FINO2 for 48 hours, and BSO for 72 hours), and then cell viability was measured. Heatmaps represent one of two independent experiments. 0.5 μM Lip-1 was used as a control. [Figure 6B-D](B) Gpx4WT cells, HT-1080, HT-29, and THP-1 cells stably overexpressing hFSP1-HAPfa1 were treated with icFSP1 (Compound 1) and their respective cell death inhibitors (ferroptosis: liproxstain-1 (Lip-1), ferrostatin-1 (Fer-1), and deferoxamine (DFO), apoptosis (zVAD-FMK), necroptosis (Nec-1s), and pyroptosis (MCC). Cell viability was measured after treatment with iFSP1 or icFSP1 (iFSP1) and Parthanatos (olaparib) or inducers (staurosporine for apoptosis, TNFα + smac mimetic (S) + z-VAD-FMK (Z) for necroptosis, and nigericin for pyroptosis) for 4 hours (pyroptosis) or 24 hours (other cases). Heatmaps show the results of one of two independent experiments. 0.5 μM Lip-1, 30 μM z-VAD-FMK, 10 μM Nec-1s, and 10 μM MCC950 were used as positive controls for each cell death mode. (C) Cell viability of HT-1080 cells treated with iFSP1 or icFSP1 and 0.5 μM Lip-1 for 72 hours. (D) Cell viability of HEK293T cells treated with iFSP1 or icFSP1 and 0.5 μM Lip-1 for 72 hours. [Figure 6E-F] (E) Cell viability of human PBMC cells treated with iFSP1 or icFSP1 for 24 hours. (F) Cell viability of FSP1 WT or FSP1 KO MDA-MB-436, 786-O, A375, and H460 cells treated with 5 μM iFSP1 or icFSP1 for 48 hours. Data are shown as mean ± standard deviation from triplicate wells in a 96-well or 384-well plate from two independent experiments (A-F) or a single experiment (E). For (E), two-way ANOVA was performed followed by Tukey's multiple comparison test. [Figure 7A-B]FSP1 forms a viscoelastic material. (A) Fluorescence recovery after photobleaching (FRAP) assay of Pfa1 Gpx4WT cells stably overexpressing hFSP1-EGFP-Strep after treatment with 2.5 μM icFSP1 (compound 1) for 240 minutes. Grayscale images show representative FRAP images immediately before photobleaching and after the indicated times. The look-up table (LUT) image shows a magnified red rectangular region of the top FRAP image. The scale bar is 5 μm. This is a representative result of one of two independent experiments. (B) Quantified FRAP rate for each aggregate. Data show the mean ± standard deviation of four aggregates. This is a representative result of one of two independent experiments. [Figure 7C] The absorbance at 600 nm was measured for non-myr-FSP1 and for different concentrations of PEG and icFSP1. Data represent the mean ± standard deviation of four wells from one 384-well plate from two independent experiments. [Figure 8] Myristoylation is required for FSP1 aggregation. (A) Confocal microscopy images of Pfa1 Gpx4WT cells overexpressing hFSP1-EGFP-Strep after pretreatment with 0.1 μM IMP-1088 for 24 hours and then treatment with 2.5 μM icFSP1 (compound 1) for the indicated times. Scale bar: 10 μm. Representative results from one of two independent experiments. (B) Time-lapse fluorescence images of Pfa1 Gpx4WT cells stably overexpressing hFSP1-EGFP-Strep;hFSP1-mTagBFP or hFSP1-G2A-EGFP-Strep;hFSP1-mTagBFP before and immediately after treatment with 2.5 μM icFSP1 for the indicated times. Scale bar: 10 μm. Representative results from one of two independent experiments. [Figure 9] Mutational analysis of human FSP1 resistant to icFSP1 (compound 1). Saturation transfer difference (STD) spectra of WT hFSP1 or its mutants S187C, L217R, and Q319K demonstrate icFSP1 binding (from bottom to top). The top spectrum shows the 1D 1H reference spectrum of icFSP1. [Figure 10A] Targeting FSP1 with icFSP1 (compound 1) as a putative anticancer therapy. (A) Pharmacokinetic (PK) parameters of icFSP1 and iFSP1. Plasma concentrations were measured after a single intraperitoneal administration (10 mg / kg). Data represent the mean ± standard deviation of three mice in one experiment. [Figure 10B] Summary of microsomal stability analysis of icFSP1 and iFSP1. [Figure 10C] Body weight changes in tumor-bearing mice during treatment of mice with icFSP1 (50 mg / kg intraperitoneally, twice daily, n=7) and control vehicle (n=6). [Figure 10D] B16F10 Gpx4 KO / Fsp1 KO cells stably overexpressing FSP1-WT or the Q319K mutant were treated with icFSP1 for 48 hours and cell viability was measured. Data are the mean ± standard deviation of triplicate wells from one of two independent experiments. [Figure 10E] icFSP1 suppresses tumor growth of cells expressing hFSP1 WT but not FSP1-Q319K in vivo. B16F10 Gpx4 KO / Fsp1 KO cells stably expressing hFSP1 WT or Q319K were subcutaneously implanted into C57BL6 / J mice (n = 33 total). Vehicle (WT: n = 10, Q319K: n = 8) or icFSP1 (50 mg / kg i.p., twice daily; WT: n = 8, Q319K: n = 7) administration was initiated 6 days after randomization. Data represent the mean ± SEM of one of two experiments. P values ​​were calculated by two-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. [Figure 11A] Targeting FSP1 with icFSP1 (compound 1) as a putative anticancer therapy in human cells. (A) At the end of the in vivo mouse experiment, tumors were excised, cryosectioned, and stained with anti-FSP1 antibody (14D7) to visualize hFSP1 and with anti-4-HNE antibody to visualize lipid peroxidation degradation products. Representative confocal microscopy images of three different samples from a single experiment are shown. [Figure 11B]Cell viability was measured in H460 WT and GPX4 KO cells after 48 hours of treatment with icFSP1. Data show the mean ± standard deviation of triplicate wells from one of two independent experiments. [Figure 11C] icFSP1 suppresses tumor growth in vivo. H460 GPX4 KO cells (a human lung cancer cell line) were subcutaneously implanted into the flanks of athymic nude mice. After tumors became palpable (day 8), mice were randomized and treatment with icFSP1 (50 mg / kg i.p., twice daily, n = 7) or vehicle (n = 7) was initiated. Data represent the mean ± standard deviation of one experiment. P values ​​were calculated by two-way ANOVA followed by Sidak's multiple comparison test. [Figure 12] FSP1 is a potential target in multiple cancer cells. Cell viability after 72-hour treatment of lymphoma cells (SUDHL5, SUDHL6, DOHH2, and OCI-Ly19) with icFSP1 (compound 1) in the presence or absence of 0.5 μM Lip-1. Data represent the mean ± standard deviation of triplicate wells from one of two independent experiments. P values ​​were calculated by two-way ANOVA followed by Tukey's multiple comparison test. [Figure 13] Plasma concentration-time curve of compound 13 in male mice after intraperitoneal administration (10 mg / kg). DETAILED DESCRIPTION OF THE INVENTION

[0018] The solution of the present invention is illustrated in the accompanying examples, drawings and reflected in the claims.

[0019] [Definition] As used herein, the singular forms "a / an" and "the" include the plural forms unless the context clearly dictates otherwise. For example, reference to "a reagent" is intended to include one or more of such different reagents, and reference to "the method" is intended to include procedures and methods that are equivalent to the methods described herein and that can be modified or substituted by one of ordinary skill in the art.

[0020] Unless specifically stated otherwise, the term "at least" preceding a series of elements is to be construed as a reference to every element of the series. Those skilled in the art will recognize, or be able to identify using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed within the scope of the present invention.

[0021] As used herein, the term "and / or" includes the meaning of "and," "or," and "all or any combination of" the elements connected by the term.

[0022] In this specification and the claims that follow, unless the context requires otherwise, the terms "comprise," and variations thereof such as "comprises," "comprise," and the like, shall be understood to include the expressly recited integer or step or group of integers or steps, and not to exclude any other integer or step or group of integers or steps. As used herein, the term "comprising" may be interchanged with the terms "containing" or "including," and in some cases, can also be interchanged with the term "having." As used herein, the term "consisting of" excludes any element, step, or ingredient not expressly recited.

[0023] The term "including" means "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0024] "Alkyl" refers to a saturated, straight- or branched-chain hydrocarbon single group. Preferably, the alkyl group consists of 1 to 10 carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 carbon atoms, more preferably 1 to 6 carbon atoms, e.g., 1 to 6 or 1 to 4 carbon atoms, more preferably 1 carbon atom. Representative alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, 1,2-dimethylpropyl, isoamyl, n-hexyl, isohexyl, sec-hexyl, and the like.

[0025] "Cycloalkyl" refers to a cyclic, non-aromatic "alkyl" having 3 to 10 carbon atoms, specifically 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, more preferably 3 to 8 carbon atoms, and even more preferably 3 to 6 carbon atoms. Representative cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, and adamantyl. Preferred examples of cycloalkyl include C3-C6-cycloalkyl, particularly cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0026] [Compound] The present invention is a compound of formula (I) or formula (II), preferably formula (I), for use in the treatment of cancer.

[0027] [ka]

[0028] G is

[0029] [ka]

[0030] selected from the group consisting of: M is [ka]

[0031] selected from the group consisting of Z is selected from the group consisting of CH, C-(C1-C6) alkyl, and N, preferably CH; A is selected from the group consisting of CH, C-(C1-C6) alkyl, and N, preferably CH; Q is selected from the group consisting of CH, C-(C1-C6) alkyl, and N, preferably CH; E is selected from the group consisting of -CO- and -SO2-, preferably -CO-; L is selected from the group consisting of CH, N, CF, C—Cl, and C—(C1-C6) alkyl, preferably CH; X is selected from the group consisting of CH, N, CF, C—Cl, and C—(C1-C6) alkyl, preferably CH; Y is selected from the group consisting of CH, N, CF, C—Cl, and C—(C1-C6) alkyl, preferably CH; X and Y may be part of a ring as shown in formula (III);

[0032] [ka]

[0033] An example of a compound containing the ring represented by formula (III) is compound 47; n is an integer from 1 to 3; J is selected from the group consisting of CH, N, CF, C—Cl, and C—(C1-C6) alkyl, preferably CH; D 1 , D 2 , D 3 , D 4 , D 5 is independently selected from the group consisting of C and N; preferably C; D 1 , D 2 , D 3 , D 4 , and / or D 5 If any one of is N, then the corresponding R 2 , R 3 , R 4 , R 5 and / or R 6 does not exist. For example, D 1 If N, then R 2 does not exist.

[0034] R 1 is selected from the group consisting of -(C1-C6)alkyl, -(C3-C6)cycloalkyl, -CH2OH, -CH2F, -CHF2, and -CF3, preferably -(C1-C6)alkyl; R 2 is selected from the group consisting of H, —(C1-C6)alkyl, —CF3, —Cl, and —F, preferably H; R 3 is selected from the group consisting of H, —(C1-C6)alkyl, —O(C1-C6)alkyl, —CF3, —Cl, and —F, preferably —O(C1-C6)alkyl; R 4 is selected from the group consisting of H, —(C1-C6)alkyl, —O(C1-C6)alkyl, —CF3, —Cl, and —F, preferably —O(C1-C6)alkyl; R 5 is selected from the group consisting of H, —(C1-C6)alkyl, —O(C1-C6)alkyl, —CF3, —Cl, and —F, preferably —O(C1-C6)alkyl; R 6is selected from the group consisting of H, —(C1-C6)alkyl, —CF3, —Cl, and —F, preferably H; R in adjacent position 2 , R 3 , R 4 , R 5 and R 6 Two of the groups are

[0035] [ka]

[0036] may be part of a ring selected from o is an integer from 1 to 4; p is an integer from 1 to 4; q is 1 or 0; R 7 and R 8 is independently selected from the group consisting of H and -(C1-C6)alkyl; R 7 and R 8 may form a 3- to 5-membered cycloalkyl ring; R 9 is selected from the group consisting of H, and -(C1-C6)alkyl, or and pharmacologically acceptable salts thereof.

[0037] In a further embodiment, Q, Z and / or A is C-CH3.

[0038] In further embodiments, the compound of formula (I) or (II) is not selected from the following compounds or pharmaceutically acceptable salts thereof:

[0039] [ka]

[0040] [ka]

[0041] [ka]

[0042] [ka]

[0043] In a further embodiment, in formula (I) or (II): G is

[0044] [ka]

[0045] and; Z is CH; A is CH; Q is CH; E is -CO-; L is CH; X is CH; Y is preferably CH; n is an integer from 1 to 3; J is CH; R 1 is -(C1-C6) alkyl; preferably methyl. 2 is H; R 3 is selected from H or —O(C1-C6)alkyl; R 4 is selected from the group consisting of H or —O(C1-C6)alkyl; R 5 is selected from the group consisting of H, —Cl, or —O(C1-C6)alkyl; R 6 is selected from the group consisting of H or -Cl; preferably H; o is an integer from 1 to 4; p is an integer from 1 to 4; q is 0 or 1; R 7 and R 8is independently selected from the group consisting of H, and -(C1-C6)alkyl; R 7 and R 8 may form a 3- to 5-membered cycloalkyl ring; R 9 is selected from the group consisting of H, and —(C1-C6)alkyl.

[0046] [Table 1-1]

[0047] [Table 1-2]

[0048] [Table 1-3]

[0049] [Table 1-4]

[0050] [Table 1-5]

[0051] In certain embodiments, the compounds of formulas (I) and (II) are selected from the group consisting of:

[0052] [ka]

[0053] In further embodiments, the compounds of formulas (I) and (II) are selected from the group consisting of:

[0054] [ka]

[0055] [Compound synthesis] The synthesis generally involves one or more of the following steps:

[0056] Step 1: In the first step, compound (IV) or (VII) is acylated.

[0057] [ka]

[0058] L is selected from the group consisting of CH, N, CF, C—Cl, and C—(C1-C6) alkyl, preferably CH; X is selected from the group consisting of CH, N, CF, C—Cl, and C—(C1-C6) alkyl, and is preferably CH. Y is selected from the group consisting of CH, N, CF, C—Cl, and C—(C1-C6) alkyl, and is preferably CH. X and Y may be part of a ring as shown in formula (III);

[0059] [ka]

[0060] n is an integer of 1 to 3. J is selected from the group consisting of CH, N, CF, C—Cl, and C—(C1-C6) alkyl, and is preferably CH. R 10 is —CO(C1-C6)alkyl, —CO(C3-C6)cycloalkyl, —COCH2F, —COCHF2, and —COCF3, preferably —CO(C1-C6)alkyl. R 11 is H or (C1-C6) alkyl, preferably (C1-C6) alkyl. R 12 is -(C1-C6) alkyl, -(C3-C6) cycloalkyl, -CH2F, -CHF2, and -CF3, preferably -(C1-C6).

[0061] Conditions for acylation of amines are well known to those skilled in the art. For example, acylation can be carried out using the corresponding acetyl halide or anhydride-based reagent. Acylation can be carried out in the presence of a base such as EtN, Hunig's base, or other suitable base. Further detailed acylation conditions can be found, for example, in "Green's Protective Groups in Organic Synthesis" by Peter G. M. Utz, 5th Edition, Wiley, 2014.

[0062] R 11 When R is (C-C) alkyl, a further hydrolysis step can be carried out, resulting in R 11 to give a compound where is H. Hydrolysis can be carried out using an inorganic base such as NaOH, KOH, LiOH, etc. in the presence of a proton donating solvent such as water and / or an alcohol (e.g., methanol, ethanol, etc.). For detailed conditions, please refer to "Green's Protecting Groups in Organic Synthesis" by Peter G.M. Utz, 5th Edition, Wiley, 2014.

[0063] [ka]

[0064] Z is selected from the group consisting of CH and N, preferably CH; A is selected from the group consisting of CH and N, preferably CH; Q is selected from the group consisting of CH and N, preferably CH; L is selected from the group consisting of CH, N, CF, C—Cl, and C—(C1-C6) alkyl, preferably CH; X is selected from the group consisting of CH, N, CF, C—Cl, and C—(C1-C6) alkyl, preferably CH; Y is selected from the group consisting of CH, N, CF, C—Cl, and C—(C1-C6) alkyl, preferably CH; X and Y may be part of a ring as shown in formula (III);

[0065] [ka]

[0066] n is an integer from 1 to 3; J is selected from the group consisting of CH, N, CF, C—Cl, and C—(C1-C6) alkyl, preferably CH; R 12 is -(C1-C6) alkyl, -(C3-C6) cycloalkyl, -CH2F, -CHF2, and -CF3, preferably -(C1-C6).

[0067] Preferably, the reaction is carried out by heating starting material compound (VI) with compound (IX) or (X) in pyridine at reflux until complete conversion of the starting material is achieved, preferably for 4 to 10 hours, more preferably 5 to 7 hours, and most preferably 6 hours.

[0068] Step 3: In step 3, compound (V) is converted to compound (Ia) or (IIa) using compound (XIII) or compound (XIV). 11 Compound (VIII) where is H can be converted to the corresponding product.

[0069] [ka]

[0070] In the above formula, M is

[0071] [ka]

[0072] selected from the group consisting of: Z is selected from the group consisting of CH, C-(C1-C6) alkyl, and N, preferably CH; A is selected from the group consisting of CH, C-(C1-C6) alkyl, and N, preferably CH; Q is selected from the group consisting of CH, C—(C1-C6) alkyl, and N, preferably CH; E is selected from the group consisting of —CO—, —SO2—, preferably —CO—; L is selected from the group consisting of CH, N, CF, C—Cl, and C—(C1-C6) alkyl, preferably CH; X is selected from the group consisting of CH, N, CF, C—Cl, and C—(C1-C6) alkyl, preferably CH; Y is selected from the group consisting of CH, N, CF, C—Cl, and C—(C1-C6) alkyl, preferably CH; X and Y may be part of a ring as shown in formula (III);

[0073] [ka]

[0074] n is an integer from 1 to 3; J is selected from the group consisting of CH, N, CF, C—Cl, and C—(C1-C6) alkyl, preferably CH; D 1 , D 2 , D 3 , D 4 , D 5 is independently selected from the group consisting of C and N; preferably C; R 1 is selected from the group consisting of -(C1-C6)alkyl, -(C3-C6)cycloalkyl, -CH2OH, -CH2F, -CHF2, and -CF3, preferably -(C1-C6)alkyl; R 2 is selected from the group consisting of H, —(C1-C6)alkyl, —CF3, —Cl, and —F, preferably H; R 3 is selected from the group consisting of H, —(C1-C6)alkyl, —O(C1-C6)alkyl, —CF3, —Cl, and —F, preferably —O(C1-C6)alkyl; R 4 is selected from the group consisting of H, —(C1-C6)alkyl, —O(C1-C6)alkyl, —CF3, —Cl, and —F, preferably —O(C1-C6)alkyl; R 5 is selected from the group consisting of H, —(C1-C6)alkyl, —O(C1-C6)alkyl, —CF3, —Cl, and —F, preferably —O(C1-C6)alkyl; R 6 is selected from the group consisting of H, —(C1-C6)alkyl, —CF3, —Cl, and —F, preferably H; R in adjacent position 2 , R 3 , R 4 , R 5 and R 6 Two of the groups are

[0075] [ka]

[0076] may be part of a ring selected from o is an integer from 1 to 4; p is an integer from 1 to 4; q is 1 or 0; R 7 and R 8 is independently selected from the group consisting of H, and -(C1-C6)alkyl; R 7 and R 8 may form a 3- to 5-membered cycloalkyl ring; R 9 is selected from the group consisting of H, and -(C1-C6)alkyl; R 10 is —CO(C1-C6)alkyl, —CO(C3-C6)cycloalkyl, —COCH2F, —COCHF2, and —COCF3, preferably —CO(C1-C6)alkyl.

[0077] The conversion in step 3 can be carried out using amide bond-forming conditions well known to those skilled in the art. For example, an amide bond-forming reagent such as EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) can be applied in the presence of an organic catalyst such as HOBt or DMAP (dimethylaminopyridine). Typically, amide bond formation is carried out in the presence of a weak organic base such as NEt3 or Et2NiPr. For detailed conditions, see Peter G. M. Utz, "Green's Protective Groups in Organic Synthesis," 5th Edition, Wiley, 2014.

[0078] Step 4: Compounds (XIII) and (XIV) can be synthesized by acylation of compound (IX) or (X) with compound (XV). Similarly, compounds (XI) or (XII) can be acylated with compound (XV).

[0079] [ka]

[0080] In the above formula, M is

[0081] [ka]

[0082] selected from the group consisting of: Z is selected from the group consisting of CH, C-(C-C) alkyl, and N, preferably CH; A is selected from the group consisting of CH, C-(C-C) alkyl, and N, preferably CH; Q is selected from the group consisting of CH, C-(C1-C6) alkyl, and N, preferably CH; E is selected from the group consisting of -CO- and -SO2-, preferably -CO-; D 1 , D 2 , D 3 , D 4 , D5 is independently selected from the group consisting of C and N; preferably C; R 1 is selected from the group consisting of -(C1-C6)alkyl, -(C3-C6)cycloalkyl, -CH2OH, -CH2F, -CHF2, and -CF3, preferably -(C1-C6)alkyl; R 2 is selected from the group consisting of H, —(C1-C6)alkyl, —CF3, —Cl, and —F, preferably H; R 3 is selected from the group consisting of H, —(C1-C6)alkyl, —O(C1-C6)alkyl, —CF3, —Cl, and —F, preferably —O(C1-C6)alkyl; R 4 is selected from the group consisting of H, —(C1-C6)alkyl, —O(C1-C6)alkyl, —CF3, —Cl, and —F, preferably —O(C1-C6)alkyl; R 5 is selected from the group consisting of H, —(C1-C6)alkyl, —O(C1-C6)alkyl, —CF3, —Cl, and —F, preferably —O(C1-C6)alkyl; R 6 is selected from the group consisting of H, —(C1-C6)alkyl, —CF3, —Cl, and —F, preferably H; R in adjacent position 2 , R 3 , R 4 , R 5 and R 6 Two of the groups are

[0083] [ka]

[0084] may be part of a ring selected from o is an integer from 1 to 4; p is an integer from 1 to 4; q is 1 or 0; R 7 and R 8is independently selected from the group consisting of H and -(C1-C6)alkyl; R 7 and R 8 may form a 3- to 5-membered cycloalkyl; R 9 is selected from the group consisting of H, and -(C1-C6)alkyl; W is -COOH, or -SO2Cl; Acylation can be carried out using known acylation conditions, such as those disclosed in "Protective Groups in Green Organic Synthesis" by Peter G. M. Utz, 5th Edition, Wiley, 2014. In particular, acylation can be carried out in the presence of HATU and a weak organic base, such as NEt3 and Et2NiPr, in a suitable solvent, such as DMF.

[0085] Step 5: Compound (XVI) and compound (XVII) can be synthesized by acylation of compound (XI) or (XII) with compound (XV).

[0086] [ka]

[0087] In the above formula, M is

[0088] [ka]

[0089] selected from the group consisting of: Z is selected from the group consisting of CH, C-(C1-C6) alkyl, and N, preferably CH; A is selected from the group consisting of CH, C-(C-C) alkyl, and N, preferably CH; Z is selected from the group consisting of CH, C-(C-C) alkyl, and N, preferably CH; E is selected from the group consisting of -CO- and -SO2-, preferably -CO-; D 1, D 2 , D 3 , D 4 , D 5 is independently selected from the group consisting of C and N; preferably C; R 1 is selected from the group consisting of -(C1-C6)alkyl, -(C3-C6)cycloalkyl, -CH2F, -CHF2, and -CF3, preferably -(C1-C6)alkyl; R 2 is selected from the group consisting of H, —(C1-C6)alkyl, —CF3, —Cl, and —F, preferably H; R 3 is selected from the group consisting of H, —(C1-C6)alkyl, —O(C1-C6)alkyl, —CF3, —Cl, and —F, preferably —O(C1-C6)alkyl; R 4 is selected from the group consisting of H, —(C1-C6)alkyl, —O(C1-C6)alkyl, —CF3, —Cl, and —F, preferably —O(C1-C6)alkyl; R 5 is selected from the group consisting of H, —(C1-C6)alkyl, —O(C1-C6)alkyl, —CF3, —Cl, and —F, preferably —O(C1-C6)alkyl; R 6 is selected from the group consisting of H, —(C1-C6)alkyl, —CF3, —Cl, and —F, and is preferably H; R 2 , R 3 , R 4 , R 5 and R 6 Two of the groups are

[0090] [ka]

[0091] may be part of a ring selected from o is an integer from 1 to 4; p is an integer from 1 to 4; q is 1 or 0; R 7 and R 8is independently selected from the group consisting of H, and -(C1-C6)alkyl; R 7 and R 8 may form a 3- to 5-membered cycloalkyl ring; R 9 is selected from the group consisting of H, and -(C1-C6)alkyl; W is -COOH or -SO2Cl.

[0092] Acylation can be carried out using known acylation conditions, such as those disclosed in "Protective Groups in Green Organic Synthesis" by Peter G. M. Utz, 5th Edition, Wiley, 2014. In particular, acylation can be carried out in the presence of HATU and a weak organic base, such as NEt3 and Et2NiPr, in a suitable solvent, such as DMF.

[0093] [Pharmaceutical composition] The present invention further provides pharmaceutical compositions comprising a compound described above in the "Compounds" section and one or more pharmaceutically acceptable excipients.

[0094] The compounds of the present invention, particularly those designated above, such as compounds of formula (I) and / or (II), and compounds in Table 1, are preferably administered to a patient in need thereof as pharmaceutical compositions. In one embodiment, the pharmaceutical composition comprises a compound described above (e.g., a compound having formula (I) and / or (II), and a compound in Table 1, or a hydrate, solvate, salt, complex, racemic mixture, diastereomer, enantiomer, tautomer, or isotopic enrichment of any of the foregoing) and one or more pharmaceutically acceptable excipients.

[0095] The pharmaceutical compositions may be administered to an individual by any route, including oral or parenteral administration.

[0096] As used herein, the terms "enteral administration" and "enterally administered" mean that the administered drug is absorbed by the stomach and / or intestinal tract. Examples of enteral administration include oral administration and rectal administration. As used herein, the terms "parenteral administration" and "parenterally administered" refer to methods of administration other than enteral administration, typically including administration by injection or topical application, and include, but are not limited to, intravenous, intramuscular, intraarterial, intraspinal, intracapsular, intraosseous, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, intrasubcutaneous, intraarticular, subcapsular, intracerebral, intraventricular, subarachnoid, intraspinal, epidural, and intrasternal administration (including by injection or infusion), and topical administration (e.g., epidermal, inhalation, or transmucosal administration (e.g., buccal, sublingual, or vaginal)).

[0097] The compounds used in the present invention are generally used in a "pharmacologically acceptable amount" and in a "pharmacologically acceptable formulation." Such compositions may include salts, buffers, preservatives, carriers, and optionally other therapeutic agents.

[0098] As used herein, the term "excipient" refers to any substance contained in a pharmaceutical composition that is not an active ingredient (e.g., a therapeutically inactive ingredient that does not exhibit a therapeutic effect in the amount / concentration used), and specifically includes, for example, a carrier, binder, lubricant, thickener, surfactant, preservative, emulsifier, buffer, flavoring agent, coloring agent, or antioxidant.

[0099] The compositions described herein may contain a pharmacologically acceptable carrier. As used herein, the term "pharmacologically acceptable carrier" includes any physiologically compatible solvents, dispersion media, coatings, isotonicity agents, absorption delaying agents, and the like. The "pharmacologically acceptable carrier" may be in any form, such as a solid, semi-solid, or liquid, or a combination thereof. Preferably, the carrier is suitable for enteral (e.g., oral) or parenteral administration (e.g., intravenous, intramuscular, subcutaneous, spinal, or epidermal administration (e.g., injection or infusion)). Depending on the route of administration, the active ingredient (i.e., the compound used in the present invention), alone or in combination with one or more additional active ingredients, may be coated with a material to protect it from acids or other natural conditions that may inactivate the active ingredient.

[0100] Examples of suitable water-soluble and water-insoluble carriers for use in the pharmaceutical compositions of the present invention include water (e.g., water for injection), ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), aqueous solutions of salts, carbohydrates, sugar alcohols, or amino acids (e.g., physiological saline or aqueous amino acid solutions), and suitable mixtures thereof and / or buffers, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0101] Examples of pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions immediately after use. The use of such media and agents for pharmacologically active substances is well known to those skilled in the art. Except insofar as conventional media or agents are incompatible with the active substance, they are contemplated for use in the pharmaceutical compositions used according to the present invention.

[0102] The additional active compound can be administered together with, before, or after the compounds used in the present invention (particularly those identified above, e.g., compounds shown in general formula (I) and / or (II), and compounds in Table 1, or compounds formulated in a composition). In one embodiment, the pharmaceutical compositions described herein comprise a compound described above (e.g., a compound having formula (I) and / or (II), and a compound in Table 1, or a hydrate, solvate, salt, complex, racemic mixture, diastereomer, enantiomer, tautomer, or isotopic enrichment of any of the above), one or more additional active ingredients, and one or more pharmaceutically acceptable excipients.

[0103] The pharmaceutical composition may contain "additional active ingredients" (excluding compounds having the structure of Formula (I) and / or Formula (II) and compounds in Table 1 specified herein), which can be selected from any compound that can be used in the treatment of cancer and / or immune diseases. The additional active ingredients can induce additive or synergistic therapeutic effects.

[0104] The pharmaceutical compositions described herein can contain at least one additional active ingredient, e.g., 1, 2, 3, 4, 5, 6, 7, or 8, in addition to the compound having the structure shown in Formula (I) and / or Formula (II) or the compound listed in Table 1 above. According to the present disclosure, the at least one additional active ingredient (e.g., an additional anticancer agent) can be combined with the compound having the structure shown in Formula (I) and / or (II) or the compound listed in Table 1 above in a single pharmaceutical composition. Alternatively, the pharmaceutical composition can be configured as a kit, where the compound having the structure shown in Formula (I) and / or (II) or the compound listed in Table 1 is provided in a first formulation, and at least one additional active ingredient (e.g., an anticancer agent) is provided in a second formulation, i.e., the second pharmaceutical composition. The first and second pharmaceutical compositions can be mixed prior to use. In other words, a formulation containing the additional active ingredient can be added to the first pharmaceutical composition containing the compound having the structure shown in Formula (I) and / or (II) or the compound listed in Table 1 above prior to administration of the pharmaceutical composition. Alternatively, the present disclosure contemplates administering a compound having the structure shown in Formula (I) and / or (II), or a compound in Table 1 above, formulated as a first pharmaceutical composition, and administering at least one additional active ingredient formulated as a second pharmaceutical composition. The pharmaceutical compositions can be administered simultaneously or sequentially. For example, the first pharmaceutical composition is administered at a first time point, and the second pharmaceutical composition is administered at a second time point, the interval between these time points being, for example, 0 minutes, or 1, 2, 3, 4, 5, or 10 minutes or less, 1, 2, 3, 4, 5, or 10 hours or less, 1, 2, 3, 4, 5, or 10 days or less, 1, 2, 3, 4, 5, or 10 weeks or less, 1, 2, 3, 4, 5, or 10 months or less, or 1, 2, 3, 4, 5, or 10 years or less.

[0105] The compositions may also contain auxiliary substances such as preservatives, wetting agents, emulsifiers, pH buffering agents, dispersing agents, etc. Prevention of the presence of microorganisms can be ensured by sterilization procedures and / or the inclusion of various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol sorbic acid, etc.). It may also be desirable to include isotonic agents such as sugars and sodium chloride in the compositions. Furthermore, the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin, can prolong the absorption of injectable pharmaceuticals.

[0106] Regardless of the route of administration selected, the active ingredient, which may have a suitable hydrated form, and / or the pharmaceutical composition used in accordance with the present invention may be administered in a conventional manner known to those skilled in the art (e.g., as described in Remington, "The Science and Practice of Pharmacy" edited by Allen, Loyd V., Jr., 2002). nd edition, Pharmaceutical Sciences, September 2012;Ansel et al., "Pharmaceutical Dosage Forms and Drug Delivery Systems", 7 th edition, Lippincott Williams & Wilkins Publishers, 1999) into a pharmacologically acceptable dosage form.

[0107] Pharmaceutical compositions can be administered in a variety of ways known in the art. As those skilled in the art will appreciate, the route and / or method of administration will vary depending on the desired results. Pharmaceutical compositions containing one or more active ingredients can be prepared in combination with a carrier that protects one or more active ingredients from rapid release, such as sustained-release formulations including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable and biocompatible polymers can be used, including, for example, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such compositions are well known to those skilled in the art (see, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978).

[0108] When the compounds used in the present invention are administered by certain routes, they may need to be coated with or co-administered with a material to prevent their inactivation. For example, the compounds may be administered to an individual in an appropriate carrier (e.g., liposomes) or diluent. Pharmacologically acceptable diluents include saline and aqueous buffers. Examples of liposomes include water-oil-water CGF emulsions and conventional liposomes (Strejan et al., J. Neuroimmunol. 7: 27 (1984)).

[0109] Pharmaceutical compositions are typically sterile and stable under the conditions of manufacture and storage. The compositions can be prepared as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium, which may contain water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it is desirable to include isotonic agents (e.g., sugars; polyalcohols such as mannitol and sorbitol; or sodium chloride) in the composition. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption, such as monostearate salts and gelatin.

[0110] Injectable compositions are sterile and fluid enough to be administered by syringe.Besides water, carriers include isotonic buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof.Sterile injectable solutions can be prepared by mixing with one or a combination of the above-listed components in a suitable solvent in the desired amount, and then sterilizing microfiltration.

[0111] Generally, dispersions are prepared by incorporating the active ingredient into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) which yield a powder containing the active ingredient plus any additional ingredient, as desired, from a previously sterile-filtered solution thereof.

[0112] Dosages are adjusted to obtain the optimum desired effect (e.g., therapeutic effect). For example, a single dose may be administered, multiple doses may be administered, or the dose may be proportionally reduced or increased depending on the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. As used herein, the term "dosage unit form" refers to a physically discrete unit suitable for single administration to an individual to be treated; each unit contains a predetermined amount of active ingredient calculated to produce the desired therapeutic effect in combination with the necessary pharmaceutical carrier. The specifications for dosage unit forms used in the present invention are directly dependent on and determined by (a) the unique characteristics of the active ingredient and the particular therapeutic effect to be achieved, and (b) the technical limitations of formulating such active ingredients for treatment of individual sensitivities.

[0113] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfate; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), lecithin, propyl gallate, and alpha-tocopherol; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0114] For therapeutic / pharmaceutical formulations, compositions used in accordance with the present invention may be suitable for enteral administration (e.g., oral or rectal) or parenteral administration (e.g., nasal, topical (including vaginal, buccal, and sublingual)). These compositions may conveniently be provided in dosage unit form and may be prepared by any method known in the art of pharmacy. The amount of active ingredient (particularly the amount of compound used in accordance with the present invention) that is combined with a carrier material to produce a pharmaceutical composition (e.g., a single dosage form) will vary depending on the individual being treated and the mode of administration. The amount of active ingredient that is combined with a carrier material to produce a single dosage form will generally be that amount of the composition that produces a therapeutic effect.

[0115] Generally, the amount of active ingredient (particularly the amount of a compound used in accordance with the present invention, if included in a pharmaceutical formulation / composition, optionally in combination with other therapeutically active ingredients) of 100% of a pharmaceutical product / pharmaceutical composition ranges from about 0.01% to about 99%, preferably from about 0.1% to about 70%, and most preferably from about 1% to about 30%, with the remainder preferably consisting of one or more pharmacologically acceptable excipients.

[0116] The amount of an active ingredient, such as a compound used in accordance with the present invention, in dosage unit form and / or when administered to an individual or used in therapy may range from about 0.1 mg to about 1000 mg (e.g., about 1 mg to about 500 mg, e.g., about 10 mg to about 200 mg) per dosage unit or per treatment unit. In certain embodiments, an appropriate amount of such an active ingredient is calculated based on the individual's body weight or body surface area, and is, for example, within the range of about 1 mg / kg to about 10 mg / kg (e.g., about 2 mg / kg to about 5 mg / kg), or about 1 mg / m 2 to approximately 400 mg / m 2 (e.g., about 3 mg / m 2 to approximately 350 mg / m 2 or approximately 10 mg / m 2 to approximately 200 mg / m 2 ) range.

[0117] The actual dosage of the active ingredient in the pharmaceutical compositions used in accordance with the present invention can be adjusted to provide an amount of the active ingredient effective to achieve the desired therapeutic effect for a particular patient, composition, and method of administration, without causing toxicity to the patient. The selected dosage will depend on pharmacokinetic factors, including the activity of the particular composition used, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health, medical history of the patient being treated, and similar factors well known in the medical field.

[0118] A physician or veterinarian of ordinary skill in the art can easily determine and prescribe an effective amount of the pharmaceutical composition. For example, the physician or veterinarian can start the dosage of the compound used in accordance with the present invention at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Generally, the appropriate daily dosage of the composition used in accordance with the present invention is the lowest effective amount to exert a therapeutic effect. Such an effective amount will depend on the factors discussed above. Administration is preferably oral, intravenous, intramuscular, intraperitoneal, or subcutaneous, and preferably administered at a site close to the target site. If desired, the effective daily dosage of the pharmaceutical composition can be administered as two, three, four, five, six, or more divided doses at appropriate intervals throughout the day, or, if desired, in a dosage unit form. While the compounds used in accordance with the present invention can be administered alone, they are preferably administered as a pharmaceutical / pharmaceutical composition.

[0119] For oral administration, pharmaceutical compositions used according to the present invention may take the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients, such as binders (e.g., pregelatinized maize starch, polyvinylpyrrolidone, hydroxypropyl methylcellulose), fillers (e.g., lactose, microcrystalline cellulose, calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, silica), disintegrants (e.g., potato starch, sodium starch glycolate), or wetting agents (e.g., sodium lauryl sulfate). Liquid preparations for oral administration may be in the form of, for example, solutions, syrups, or suspensions, or may be presented as a dry product to be reconstituted with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional methods using pharmacologically acceptable additives such as suspending agents (e.g., sorbitol, syrup, cellulose derivatives, hydrogenated edible fats), emulsifying agents (e.g., lecithin, acacia), non-aqueous solvents (vehicles) (e.g., almond oil, oily esters, ethyl alcohol, fractionated vegetable oils), preservatives (e.g., methyl or propyl-p-hydroxycarboxylic acid, sorbic acid), etc. The preparations may contain buffer salts, flavoring agents, coloring agents, and sweetening agents as needed. Preparations for oral administration can be suitably formulated to control the release of the drug composition of the present invention.

[0120] In one embodiment, the compound is administered orally at a concentration of up to 100 mg / kg body weight (e.g., up to 50 mg / kg body weight, up to 40 mg / kg body weight, up to 30 mg / kg body weight, up to 20 mg / kg body weight, up to 10 mg / kg body weight, up to 5 mg / kg body weight, up to 3 mg / kg body weight, up to 2 mg / kg body weight, up to 1 mg / kg body weight).

[0121] In one embodiment, the compound is administered parenterally (e.g., intravenously, intramuscularly, or subcutaneously) at a concentration of up to 10 mg / kg body weight (up to 5 mg / kg body weight, up to 4 mg / kg body weight, up to 3 mg / kg body weight, up to 2 mg / kg body weight, up to 1 mg / kg body weight, up to 0.5 mg / kg body weight, up to 0.4 mg / kg body weight, up to 0.3 mg / kg body weight, up to 0.2 mg / kg body weight, up to 0.1 mg / kg body weight).

[0122] The pharmaceutical composition can be prepared as a suppository using conventional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.

[0123] The pharmaceutical composition used in accordance with the present invention can be prepared for parenteral administration by injection, for example, by bolus injection or continuous intravenous infusion. In one embodiment, the compound or composition used in accordance with the present invention can be administered by slow continuous intravenous infusion over a prolonged period, such as more than 24 hours, to reduce toxic side effects. Administration can also be by continuous intravenous infusion over a period of 2 to 24 hours, for example, 2 to 12 hours. Such an administration schedule can be repeated one or more times as needed, for example, after 6 or 12 months.

[0124] In another embodiment, the compounds or compositions used according to the present invention are administered as a maintenance regimen, for example once a week, continuing for six months or more.

[0125] Injectable preparations may contain added preservatives and be provided in unit dosage form (e.g., ampoules or multi-dose containers). Pharmaceutical compositions used according to the present invention may take the form of suspensions, solutions, or emulsions in oily or aqueous vehicles and may contain formulation additives such as suspending, stabilizing, or dispersing agents. Alternatively, they may be provided in powder form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may contain a solubilizing agent and a local anesthetic such as lidocaine to alleviate pain at the injection site. Generally, the ingredients are provided as a dry lyophilized powder or water-free concentrate, either separately or mixed together in unit dosage form, for example, in a hermetically sealed container such as an ampule or sachet indicating the quantity of active ingredient. When administered intravenously, the composition can be administered using an infusion bottle containing sterile pharmaceutical-grade purified water or physiological saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0126] Compositions used according to the present invention that are suitable for vaginal administration include pessaries, tampons, creams, gels, pastes, foams, or spray formulations containing suitable carriers known in the art. Dosage forms for topical or transdermal administration of compositions used according to the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active ingredient may be mixed under sterile conditions with a pharmacologically acceptable carrier, and, if necessary, preservatives, buffers, or propellants.

[0127] The compound used according to the present invention is incorporated into liposome.In a more preferred embodiment, liposome comprises targeting moiety.In the most preferred embodiment, the compound in liposome is delivered by bolus injection to a site close to the desired area.Such liposome-based composition is fluid enough to be easily injectable, stable under the conditions of manufacture and storage, and protected from the contamination of microorganisms such as bacteria and fungi.

[0128] A "therapeutic dose" of treatment / therapy can be measured by a complete or partial objective response. A complete response (CR) is defined as the absence of clinical, radiological, or other evidence of disease, disorder, or pathology. A partial response (PR) results from a greater than 50% reduction in disease. Median time to progression is an index that characterizes the durability of objective tumor response.

[0129] A "therapeutically effective dose" for treatment / therapy can also be measured by its ability to stabilize the progression of a disease, disorder, or condition. Alternatively, the properties of the compounds described herein can be evaluated by assessing the ability of the compounds in appropriate animal model systems known to those skilled in the art. A therapeutically effective amount of a compound used in accordance with the present invention can treat, cure, alleviate, mitigate, alter, remedy, ameliorate, improve, or affect a disease, disorder, or condition; symptoms of a disease, disorder, or condition; or a propensity for a disease, disorder, or condition in an individual. Those skilled in the art can determine the dosage based on factors such as the size of the individual, the severity of the individual's symptoms, and the particular composition or route of administration selected.

[0130] Pharmaceutical compositions for use according to the present invention may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil (such as a blister pack). The pack or dispenser device may be accompanied by instructions regarding administration.

[0131] Pharmaceutical compositions used in accordance with the present invention may be administered as the sole active ingredient or in combination with other therapeutic and / or cosmetic active ingredients.

[0132] [Treatment application] The compounds of general formula (I) and / or (II), the compounds shown in Table 1, or hydrates, solvates, salts, complexes, racemates, diastereomers, enantiomers, tautomers, or isotopically enriched versions of any of the above, or pharmaceutical compositions described above, can be used to treat cancer.

[0133] The cancer is preferably one that expresses ferrotosis suppressor protein-1 (FSP1).

[0134] To elucidate the mechanisms underlying resistance to ferrotosis, a form of iron-dependent cell death, a genetic inhibitor screen was conducted, identifying mitochondria-associated apoptosis inducer 2 (AIFM2) as a novel "anti-ferrotosis" gene (Ref. 16). Despite its name, AIFM2 was shown not to play a critical role in regulating apoptosis, leading to the proposal to rename this oxidoreductase "ferrotosis suppressor protein-1 (FSP1)." Overexpression of FSP1 in glutathione peroxidase 4 (GPX4)-deficient cells (which inevitably die from rapid and extensive ferrotosis (Ref. 19)) and in wild-type tumor cells treated with a ferrotosis-inducing tool compound and the GPX4 inhibitor (1S,3R)-RSL-3 conferred unprecedented resistance to ferrotosis. FSP1 expression was further detected in a broad panel of cancer cell lines, potentially serving as a biomarker of ferrotosis resistance that predicts a ferrotosis response independent of intracellular glutathione metabolism and GPX4 activity. (Ref. 16) Initial in vivo studies using the compounds of the present invention in tumor-bearing mice provided proof-of-concept that these compounds inhibit tumor growth.

[0135] More preferably, the cancer is selected from prostate cancer, leukemia (e.g., acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia), liver cancer, breast cancer, hepatocellular carcinoma, cholangiocarcinoma, glioblastoma, uveal melanoma, adrenocortical carcinoma, thymoma, head and neck squamous cell carcinoma, kidney cancer (e.g., clear cell renal cell carcinoma, renal cell carcinoma), lymphoma (e.g., lymphoid malignancies, diffuse large B-cell lymphoma, non-Hodgkin's lymphoma), pancreatic adenocarcinoma, gallbladder cancer, myeloma, gastric cancer, brain tumors (e.g., glioblastoma, medulloblastoma, glioma), skin cancer, colon / colorectal cancer, bile duct cancer, neuroblastoma, bone tumors (e.g., Ewing's sarcoma), and lung cancer (e.g., small cell lung cancer, non-small cell lung carcinoma, mesothelioma).

[0136] Most preferably, the cancer is selected from the group consisting of prostate cancer, leukemia (e.g., acute myeloid leukemia), kidney cancer (e.g., clear cell renal cell carcinoma, renal cell carcinoma), breast cancer, hepatocellular carcinoma, cholangiocarcinoma, glioblastoma, and lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, mesothelioma).

[0137] As described in references 16, 18, 20, and 21, these cancers express FSP1.

[0138] Cancer Genome Atlas (TCGA) and Gene Expression Interactive Analysis (GEPIA2): Database analysis performed using the Cancer Genome Atlas (TCGA) and Gene Expression Interactive Analysis (GEPIA2) revealed that the following cancer types have poor prognosis and / or higher FSP1 expression levels than normal tissues: uveal melanoma, adrenocortical carcinoma, thymoma, head and neck squamous cell carcinoma, cholangiocarcinoma, clear cell renal cell carcinoma, acute myeloid leukemia, lymphoid malignancies, diffuse large B-cell lymphoma, and pancreatic adenocarcinoma.

[0139] Cancer Genome Atlas (TCGA): https: / / www.cancer.gov / about-nci / organization / ccg / research / structural-genomics / tcga The results presented herein are supported by the TCGA Research Network: 1753341098956_0 ) based on data generated by

[0140] Gene Expression Interactive Analysis (GEPIA 2): http: / / gepia2.cancer-pku.cn / #index Tang, Z., Kang, B., Li, C., Chen, T., Zhang, Z. (2019) “GEPIA2: an enhanced web server for large-scale expression profiling and interactive analysis” Nucleic Acids Res 47, 556-560

[0141] Analysis of the Cancer Dependency Map (DepMap; https: / / depmap.org / portal / , https: / / depmap.org / portal / gene / AIFM2?tab=dependency) revealed that these were cancer types with negative CRISPR Chronos (DepMap 22Q2) scores. Indicators with correlation coefficients below -0.25 were selected, indicating the potential for FSP1 dependency in each cancer cell line. The indicators shown here are: leukemia, gallbladder cancer, lymphoma, myeloma, gastric cancer, brain cancer, lung cancer, myeloma, skin cancer, colon / colorectal cancer, bile duct cancer, neuroblastoma, bone tumor, renal cancer, and prostate cancer.

[0142] For a better understanding of the present invention and its advantages, the following examples are given for reference, but are not intended to limit the scope of the present invention in any way and are merely illustrative. [Example]

[0143] 1.Synthesis 1.1 General methods and materials All reactions were performed on 0.25 mm E. Merck precoated silica gel plates (60F 254 The compounds were monitored by thin-layer chromatography (TLC) using a Waters liquid chromatography mass spectrometry (LCMS) system. LC-MS spectra were recorded on a Waters Acquity I Class UPLC system using the following system: Solvent A: acetonitrile, Solvent B: 0.1% aqueous formic acid, or Solvent A: acetonitrile, Solvent B: 0.1% aqueous ammonia, or Solvent A: acetonitrile, Solvent B: 0.1% aqueous TFA. Formic acid and ammonia or TFA were HPLC grade. All separations were performed at room temperature.

[0144] Reverse-phase HPLC was performed using a Waters HPLC system with either [solvent A: acetonitrile, solvent B: 0.1% aqueous NH3] or [solvent A: acetonitrile, solvent B: 0.1% aqueous TFA]. Ammonia was HPLC-grade. All separations were performed at room temperature. For analytical RP-HPLC analysis [Interchim: Acquity BEH C18 (2.1 × 100 mm, 1.7 μm)], the flow rate was 0.4 mL / min, injection volume was 10 μL, and detection wavelengths were 220 nm and 254 nm. The gradient was as follows: 90% B in 0.01 min, to 10% B over 8 min, and 10% B in 4 min.

[0145] Purification of reaction products was achieved by column chromatography using commercially available silica gel or by flash chromatography using a Combiflash Rf column equipped with a Teledyne Isco RediSep Rf High Performance Gold or Silicycle SiliaSep High Performance column (40, 80, or 120 g). All final compounds were >95% pure and analyzed on a Waters LCMS system.

[0146] 1 H NMR spectra were recorded on a Varion 400 MHz spectrometer and are reported in ppm using the solvent resonances as internal standards [7.26 ppm CDCl3, 2.50 ppm DMSO-d6]. Peaks are designated as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet or unresolved, br s = broad signal, coupling constants (Hz), and integrals.

[0147] [List of Abbreviations] Ac2O acetic anhydride Boc2O Di-tert-butyl dicarbonate cHex cyclohexane CV column value d Duplex (NMR) d number of days dd double doublet DAPI 4',6-diamidino-2-phenylindole DAST Diethylaminosulfur trifluoride DCM dichloromethane DIPEA N,N-Diethyldiisopropylamine DMEM Dulbecco's Modified Eagle's Medium DMF N,N-dimethylformamide DMSO dimethyl sulfoxide DMP Dess-Martin Periodinane EDC HCl 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide equiv. Et2O diethyl ether EtOAc ethyl acetate FBS fetal bovine serum FITC Fluorescein g grams h time H proton HCl Hydrochloric acid HEPES 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid H2O Water HOBT 1-Hydroxybenzotriazole Hz Hertz IPA Isopropanol J Scholar 7 1 H- 1 H-coupling constant K2CO3 Potassium Carbonate KOH Potassium hydroxide LC-MS Liquid Chromatography Mass Spectrometry LDH lactate dehydrogenase LPS lipopolysaccharide m Multiplet m Moller mAU milliabsorbance Me methyl MeCN acetonitrile MeOH Methanol mg milligram MHz Megahertz min μw microwave N2 nitrogen NADH Nicotinamide adenine dinucleotide NADPH Nicotinamide adenine dinucleotide phosphate NaH sodium hydride NaHCO3 Sodium bicarbonate NaOH Sodium hydroxide Na2SO4 Sodium Sulfate NBS N-Bromosuccinimide NCS N-chlorosuccinimide NMR nuclear magnetic resonance PBS Phosphate-buffered saline PCC Pyridinium Chlorochromate PEG polyethylene glycol PVDF Polyvinylidene Fluoride quant. R f Retention factor (TLC) rt room temperature s Singlet SiO2 Silica TCFH Tetramethylchloroformamidinium hexafluorophosphate TFA trifluoroacetic acid THF tetrahydrofuran TLC thin layer chromatography TRIS Tris(hydroxymethyl)aminomethane

[0148] Scheme 1: Synthesis of N-(4-(2-methyl-4-oxopyrido[4,3-d]pyrimidin-3(4H)-yl)phenyl)-2-(3,4,5-trimethoxyphenyl)acetamide (FS-01):

[0149] [ka]

[0150] 1.3 Synthesis of 4-acetamidoisonicotinic acid (2): A mixture of aminonicotinic acid (1) (1.0 g, 7.299 mmol, 1 equiv.) and acetic anhydride (6 mL) was stirred at room temperature for 15 minutes. The reaction mixture was refluxed for 6 hours. The reaction was monitored by TLC, and after completion, excess acetic anhydride was removed under reduced pressure. The crude product was diluted with water (20 mL) and extracted with ethyl acetate (2 × 50 mL). The organic layer was separated and washed with brine solution (20 mL). The combined organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give 4-acetamidoisonicotinic acid (2) (1.1 g, yield: 83%) as a yellow solid. This product was used in the next step without further purification. LCMS: m / z = 180.99 [M+H]+ , 75.02% (0.87 min) 1 H-NMR (400 MHz, DMSO-d6) δ ppm: 8.94 (s, 1H), 8.76 (d, J = 4.8 Hz, 1H), 7.95 (d, J = 4.8 Hz, 1H), 2.43 (s, 3H)

[0151] [ka]

[0152] 1.4 Synthesis of N-(4-aminophenyl)-2-(3,4,5-trimethoxyphenyl)acetamide (Int-1): To a stirred solution of compound 3 (500 mg, 2.252 mmol, 1 equiv.) and compound 4 (240 mg, 2.252 mmol, 1 equiv.) in DMF (5 mL) was added DIPEA (1.1 mL, 6.756 mmol) and HATU (1.02 g, 3.378 mmol, 1.5 equiv.) for 0.5 min. o C. The reaction mixture was brought to room temperature and stirred for 3 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with cold water (20 mL) and extracted with ethyl acetate (2 × 25 mL). The organic layer was separated and washed with brine solution (20 mL). The combined organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (100:200 mesh). The compound was eluted with 12% EtOAc in hexane to give Int-1 (650 mg, yield: 46%) as a brown viscous liquid. LCMS: m / z = 317.59 [M+H] + , 98.73% (0.88 min)

[0153] [ka]

[0154] 1.5 Synthesis of N-(4-(2-methyl-4-oxopyrido[4,3-d]pyrimidin-3(4H)-yl)phenyl)-2-(3,4,5-trimethoxyphenyl)acetamide (FS-01): To a stirred solution of 4-acetamidocotinic acid (2) (264 mg, 0.833 mmol, 1 equiv.) in DMF (2 mL), EDC.HCl (191 mg, 0.999 mmol, 1.2 equiv.) and HOBt (135 mg, 0.999 mmol, 1.3 equiv.) were added. o The reaction mixture was stirred for 10 min. Then, a solution of Int-1 (150 mg, 0.833 mmol, 1.0 equiv.) in DMF (1 mL) was added at 0°C. o C. The reaction mixture was allowed to warm to room temperature and stirred for 16 hours. After TLC confirmed the completion of the reaction, the reaction mixture was quenched with water (20 mL) and extracted with ethyl acetate (2 × 25 mL). The organic layer was separated and washed with brine solution (15 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified on a reverse-phase C18 column using acetonitrile / water with 0.01% HCOOH buffer to give FS-01 (40 mg, 11% yield) as a white solid after lyophilization. LCMS: m / z = 461.33 [M+H] + , 97.93% (2.11 min) 1 H NMR (400 MHz, DMSO-d6) δ ppm: 10.38 (s, 1H), 9.05 (s, 1H), 8.65 (d, J = 5.2 Hz, 1H), 7.93 (d, J = 5.2 Hz, 1H), 7.77 (d, J = 9.2 Hz, 2H), 7.38 (d, J = 9.2 Hz, 2H), 6.67 (s, 2H), 3.77 (s, 6H), 3.64 (s, 3H), 3.61 (s, 2H), 2.17 (s, 3H)

[0155] Scheme 2: Synthesis of N-(4-(2-methyl-4-oxopyrido[3,4-d]pyrimidin-3(4H)-yl)phenyl)-2-(3,4,5-trimethoxyphenyl)acetamide (FS-02):

[0156] [ka]

[0157] 1.6 Synthesis of 3-acetamidoisonicotinic acid (6): A mixture of 3-aminonicotinic acid 5 (1.0 g, 7.299 mmol, 1 equiv.) and acetic anhydride (6 mL) was stirred at room temperature for 15 minutes. The reaction mixture was refluxed for 6 hours. The reaction was monitored by TLC, and after completion, excess acetic anhydride was removed under reduced pressure. The reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (2 × 50 mL). The organic layer was separated and washed with brine solution (20 mL). The combined organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give 3-acetamidoisonicotinic acid (6) (900 mg, yield: 69%) as a white solid. This product was used in the next step without further purification. LCMS: m / z = 181.03 [M+H] + , 81.95% (0.32 min) 1 H-NMR (400 MHz, DMSO-d6) δ ppm: 12.0 (s, 1H), 11.30 (s, 1H), 9.23 (s, 1H), 8.93 (d, J = 5.6 Hz, 1H), 7.51(d, J = 5.6 Hz, 1H), 2.44 (s, 3H)

[0158] [ka]

[0159] 1.7 Synthesis of N-(4-(2-methyl-4-oxopyrido[3,4-d]pyrimidin-3(4H)-yl)phenyl)-2-(3,4,5-trimethoxyphenyl)acetamide (FS-02): To a stirred solution of 3-acetamidocotinic acid (6) (200 mg, 1.111 mmol, 1 equiv.) in DMF (2 mL), EDC.HCl (191 mg, 1.333 mmol, 1.2 equiv.) and HOBt (135 mg, 1.333 mmol, 1.2 equiv.) were added.o C. The reaction mixture was stirred for 10 min. Next, a solution of Int-1 (351 mg, 1.111 mmol, 1 equiv.) in DMF (1 mL) was added at 0°C. o C. The reaction mixture was allowed to warm to room temperature and stirred for 16 hours. After TLC confirmed the completion of the reaction, the reaction mixture was quenched with water (20 mL) and extracted with ethyl acetate (2 x 25 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified on a reverse-phase C18 column using acetonitrile / water with 0.01% HCOOH buffer to give FS-02 (40 mg, yield: 7.8%) as a white solid after lyophilization. LCMS: m / z = 461.38 [M+H] + , 95.11% (2.02 min) 1 H NMR (400 MHz, DMSO-d6) δ ppm: 10.37 (s, 1H), 9.24 (s, 1H), 8.84 (d, J = 5.6 Hz, 1H), 7.77 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 5.6 Hz, 1H), 7.39 (d, J = 8.8 Hz, 2H), 6.67 (s, 2H), 3.77 (s, 6H), 3.64 (s, 3H), 3.61 (S, 2H), 2.16 (s, 3H)

[0160] Scheme 3: Synthesis of N-(5-(2-methyl-4-oxoquinazolin-3(4H)-yl)pyrazin-2-yl)-2-(3,4,5-trimethoxyphenyl)acetamide (FS-03)

[0161] [ka]

[0162] 1.7 Synthesis of 2-methyl-4H-benzo[d][1,3]oxazin-4-one (Int-2): A mixture of 2-aminobenzoic acid (7) (5.0 g, 36.496 mmol, 1 equiv.) and acetic anhydride (30 mL) was refluxed for 4 h. The reaction mixture was cooled to room temperature, and excess acetic anhydride was removed under reduced pressure. The crude reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (2 × 100 mL). The organic layer was separated and washed with brine solution (50 mL). The combined organic layers were dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give Int-2 (3.9 g, yield: 55%) as a pale white solid. This product was used directly in the next step without further purification. LCMS: m / z = 162.04 [M+H] + , 95.75% (1.154 min)

[0163] [ka]

[0164] Synthesis of methylquinazolin-1.9 3-(5-aminopyrazin-2-yl)-2-methylquinazolin-4(3H)-one (9): Int-2 (800 mg, 4.968 mmol, 1 equiv.) and pyrazine-2,5-diamine 8 (546 mg, 4.968 mol, 1 equiv.) were added to 25 mL of anhydrous pyridine and stirred. The reaction mixture was refluxed for 6 h. The resulting mixture was cooled in an ice-water bath and treated with 10 mL of 1N HCl to give a white solid precipitate, which was washed with water and dried in air. The crude product was recrystallized from ethanol to give compound 9 (400 mg, 26% yield) as a brown solid. This product was used directly in the next step without further purification.

[0165] [ka]

[0166] 1.10 Synthesis of N-(5-(2-methyl-4-oxoquinazolin-3(4H)-yl)pyrazin-2-yl)-2-(3,4,5-trimethoxyphenyl)acetamide (FS-03): To a stirred solution of compound 3 (200 mg, 0.787 mmol, 1 equiv.) in DMF (1 mL) was added HATU (360 mg, 0.944 mmol, 1.2 equiv.) and DIPEA (0.35 mL, 1.968 mmol, 2.5 equiv.) for 0.5 min. o C. The reaction mixture was stirred for 10 min. Then, compound 9 (180 mg, 0.787 mmol, 1 equiv.) was added at 0 o C. The reaction mixture was allowed to warm to room temperature and stirred for 16 hours. After TLC confirmed the completion of the reaction, the reaction mixture was quenched with water (20 mL) and extracted with ethyl acetate (2 × 25 mL). The organic layer was separated and washed with brine solution (20 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified on a reverse-phase C18 column using acetonitrile / water with 0.01% HCOOH buffer to give FS-03 (8.0 mg, yield: 2.1%) as an off-white solid after lyophilization. LCMS: m / z = 462.40 [M+H] + , 97.89% (1.30 min) 1 H NMR (400 MHz, CDCl3) δ ppm: 9.62 (s, 1H), 8.33 (s, 1H) 8.25 (d, J = 7.6 Hz, 1H), 8.00 (brs, 1H), 7.79 (t, J = 8.4 Hz, 1H), 7.68 (d, J = 8.4 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 6.55 (s, 2H), 3.90 (s, 6H), 3.88 (s, 3H), 3.78 (S, 2H), 2.26 (s, 3H)

[0167] Scheme 4: Synthesis of N-(4-(2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)-2-(3,4,5-trimethylphenyl)acetamide (FS-04):

[0168] [ka]

[0169] 1.11 Synthesis of 3-(4-aminophenyl)-2-methylquinazolin-4(3H)-one (Int-3): Int-2 (1.5 g, 0.0108 mol, 1 equiv.) and p-phenylenediamine 4 (1.40 g, 0.0134 mol, 1.2 equiv.) were added to 15 mL of anhydrous pyridine and stirred. The reaction mixture was refluxed for 6 h. The resulting solution was cooled in an ice-water bath and acidified with 10 mL of dilute hydrochloric acid to give a white solid precipitate, which was filtered, washed with water, and dried in air. The crude product was recrystallized from ethanol to give Int-3 (920 mg, 34% yield) as an off-white solid. LCMS: m / z = 252.57 [M+H] + , 97.01% (1.04 min) 1 H NMR (400 MHz, DMSO-d6) δ ppm: 8.16 (d, J = 7.6 Hz, 1H), 7.97-7.89 (m, 2H), 7.64 (t, J = 7.2 Hz, 1H), 7.50-7.39 (m, 4H), 2.32 (s, 3H)

[0170] [ka]

[0171] 1.12 Synthesis of N-(4-(2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)-2-(3,4,5-trimethylphenyl)acetamide (FS-04): To a stirred solution of compound 10 (100 mg, 0.568 mmol, 1 equiv.) and Int-3 (171 mg, 0.681 mmol, 1.2 equiv.) in DMF (1 mL) was added HATU (323 mg, 0.852 mmol, 1.5 equiv.), followed by DIPEA (0.3 mL, 1.704 mmol, 3.0 equiv.). oC was added under a nitrogen atmosphere. The reaction mixture was allowed to warm to room temperature and stirred for 16 hours. The reaction was monitored by TLC. After completion, the reaction mixture was quenched with water (20 mL) and extracted with ethyl acetate (2 x 50 mL). The organic layer was separated and washed with brine solution (20 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by C-18 reverse-phase column chromatography to give compound FS-04 (53 mg, yield: 22%) as an off-white solid. LCMS: m / z = 410.41 [M+H] + , 99.35% (2.90 min) 1 H NMR (400 MHz, DMSO-d6) δ ppm: 10.44 (s, 1H), 8.16 (d, J = 7.6 Hz, 1H), 7.91 (t, J = 7.2 Hz, 1H), 7.83 (d, J = 8.4 Hz, 2H), 7.72 (d, J = 8.4 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.43 (d, J = 8.4 Hz, 2H), 7.29-725 (m, 2H), 7.18 (d, J = 7.6 Hz, 1H), 3.70 (s, 2H), 2.95-2.88 (m, 4H), 2.21 (s, 3H), 2.08 (qt, J = 4.0 Hz, 2H)

[0172] Scheme 5: Synthesis of N-(4-(6,7-difluoro-2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)-2-(3,4,5-trimethoxyphenyl)acetamide (FS-05):

[0173] [ka]

[0174] 1.13 Synthesis of 2-acetamido-4,5-difluorobenzoic acid (12): In a 50 mL round-bottom flask, compound 11 (500 mg, 2.89 mmol) was added to AcO (5 mL) and refluxed for 16 h. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was poured into water (30 mL) and extracted with ethyl acetate (2 × 50 mL). The organic layer was separated and washed with brine (20 mL). The combined organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (100-200 mesh). The compound was eluted with 50% EtOAc in hexane to give compound 12 (400 mg, 61% yield) as a yellow liquid. LCMS: m / z = 214.12 [M−H] - , 99.37% (1.23 min)

[0175] [ka]

[0176] 1.14 Synthesis of N-(4-(6,7-difluoro-2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)-2-(3,4,5-trimethoxyphenyl)acetamide (FS-05): To a stirred solution of compound 14 (300 mg, 1.395 mmol) and Int-1 (529 mg, 1.674 mmol) in DMF (15 mL) was added EDC.HCl (321 mg, 1.674 mmol), followed by HOBt (188 mg, 1.395 mmol) at 0 °C under a nitrogen atmosphere. The reaction mixture was allowed to warm to room temperature and stirred for 16 h. The reaction was monitored by TLC. Upon completion, the reaction mixture was quenched with water (20 mL), extracted with ethyl acetate (2 × 50 mL), and the organic layer was separated and washed with brine solution (20 mL). The combined organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The crude product was purified by preparative HPLC (Prep-HPLC) to give compound FS-05 (43 mg, yield: 61%) as a white solid. LCMS: m / z = 496.25 [M+H] +, 99.79% (2.66 min) 1 H NMR (400 MHz, DMSO-d6) δ ppm: 10.36 (s, 1H), 8.01 (t, J = 9.2 Hz, 1H), 7.78-7.73 (m, 3H), 7.36 (d, J = 8.4 Hz, 2H), 6.67 (s, 2H), 3.77 (S, 6H), 3.64 (S, 3H), 3.61 (s, 2H), 2.12 (s, 3H)

[0177] Scheme 6: Synthesis of N-(4-(2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)-2-(3,4,5-trimethylphenyl)acetamide (FS-08):

[0178] [ka]

[0179] 1.15 Synthesis of N-(4-(2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)-2-(3,4,5-trimethylphenyl)acetamide (FS-08): To a stirred solution of compound 13 (50 mg, 0.221 mmol, 1.0 equiv.) in DMF (1 mL) was added DIPEA (0.1 mL, 0.663 mmol, 3.0 equiv.) and HATU (126 mg, 0.331 mmol, 1.5 equiv.) for 0.5 min. o C. The reaction mixture was allowed to warm to room temperature and stirred for 16 hours. The reaction was monitored by TLC. After completion, the mixture was diluted with cold water (20 mL) and extracted with ethyl acetate (2 x 50 mL). The organic layer was separated and washed with brine solution (20 mL). The combined organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by C-18 reverse-phase column chromatography to give FS-08 (25 mg, yield: 27%) as a white solid. LCMS: m / z = 412.38 [M+H] + , 97.99% (2.94 min) 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.33 (s, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.83 (t, J = 7.2 Hz, 1H), 7.75 (d, J = 8.4 Hz, 2H), 7.65 (d, J = 8.0 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.34 (d, J = 8.4 Hz, 2H), 6.96 (s, 2H), 3.54 (s, 2H), 2.22 (s, 6H), 2.13 (s, 3H), 2.09 (s, 3H)

[0180] Scheme 7: Synthesis of N-(4-(2-methyl-4-oxothieno[2,3-d]pyrimidin-3(4H)-yl)phenyl)-2-(3,4,5-trimethoxyphenyl)acetamide (FS-09):

[0181] [ka]

[0182] 1.16 Synthesis of ethyl 2-acetamidothiophene-3-carboxylate (15): To a stirred solution of ethyl 2-aminothiophene-3-carboxylate (14) (1.0 g, 6.361 mmol, 1 equiv.) in dichloromethane (10 mL), EtN (1.7 mL, 12.738 mmol, 2 equiv.) and acetic anhydride (1.2 mL, 12.738 mmol, 2 equiv.) were added at room temperature and stirred for 4 h. The reaction was monitored by TLC. Upon completion, the reaction mixture was quenched with saturated NaHCO solution (30 mL), extracted with dichloromethane (2 × 50 mL), and the organic layer was washed with brine solution (20 mL). The combined organic layers were dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give compound 15 (1.0 g, 85% yield) as a yellow solid. This product was used in the next step without further purification. 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.79 (s, 1H), 7.16 (d, J = 4.0 Hz, 1H), 6.99 (d, J = 4.0 Hz, 1H), 3.83 (s, 3H), 2.26 (s, 3H)

[0183] [ka]

[0184] 1.17 Synthesis of 2-acetamidothiophene-3-carboxylic acid (16): To a stirred solution of compound 15 (2.5 g, 12.620 mmol, 1 equiv.) in methanol (50 mL) was added 0.05 mL of 2N aqueous sodium hydroxide (30 mL). o C, and the mixture was allowed to warm to room temperature and stirred for 8 hours. After completion of the reaction was confirmed by TLC, the solvent was removed in vacuo. The crude product was diluted with water (10 mL) and acidified with 3N HCl to give a white solid precipitate, which was filtered and dried in air to give compound 16 (1.9 g, yield: 82%) as an off-white solid. 1 H NMR (400 MHz, DMSO-d6) δ ppm: 13.11 (brs, 1H), 10.83 (s, 1H), 7.18 (d, J = 4.0 Hz, 1H), 6.91 (d, J = 6.0 Hz, 1H), 2.24 (s, 3H) (INT-NY-671-093-01)

[0185] [ka]

[0186] 1.18 Synthesis of N-(4-(2-methyl-4-oxothieno[2,3-d]pyrimidin-3(4H)-yl)phenyl)-2-(3,4,5-trimethoxyphenyl)acetamide (FS-09): To a stirred solution of compound 16 (150 mg, 0.810 mmol, 1 equiv.) in DMF (2 mL) was added EDC.HCl (186 mg, 0.972 mmol, 1.2 equiv.) and HOBt (131 mg, 0.972 mmol, 1.2 equiv.) for 0.5 min. o C. The reaction mixture was stirred for 10 min. Then, a solution of Int-1 (256 mg, 0.810 mmol, 1 equiv.) was added at 0. o C. The reaction mixture was allowed to warm to room temperature and stirred for 16 hours. After TLC showed the reaction was complete, the reaction mixture was quenched with water (20 mL) and extracted with ethyl acetate (2 × 25 mL). The organic layer was separated and washed with brine solution (20 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified on a reverse-phase C18 column using acetonitrile / water with 0.01% HCOOH buffer to give 100 mg of 80% purity after lyophilization. Final purification by preparative HPLC and lyophilization afforded FS-09 (21 mg, 5.5%) as an off-white solid. LCMS: m / z = 466.16 [M+H] + , 99.92% (2.36 min) 1 H NMR (400 MHz, DMSO-d6) δ ppm: 10.37 (s, 1H), 7.75 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 6.0 Hz, 1H), 7.37-7.32 (m, 3H), 6.67 (s, 2H), 3.77 (s, 6H), 3.63 (s, 3H), 3.61 (s, 2H), 2.13 (s, 3H)

[0187] Scheme 8: Synthesis of N-(4-(4-oxo-2-(trifluoromethyl)quinazolin-3(4H)-yl)phenyl)-2-(3,4,5-trimethoxyphenyl)acetamide (FS-10):

[0188] [ka]

[0189] 1.19 Synthesis of 2-(2,2,2-trifluoroacetamido)benzoic acid (18): A mixture of 2-aminobenzoic acid (7) (1.0 g, 7.296 mmol, 1 equiv.) and trifluoroacetic anhydride 17 (1.37 mL, 14.593 mmol, 2 equiv.) was stirred for 15 min. Triethylamine (3.0 mL, 21.897 mmol, 3 equiv.) was then slowly added at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 2 h. The crude product was diluted with water (20 mL) and extracted with ethyl acetate (2 × 50 mL). The organic layer was separated and washed with brine solution (20 mL). The combined organic layers were dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give compound 18 (1.1 g, 65% yield) as a white solid. This product was used in the next step without further purification.

[0190] [ka]

[0191] 1.20 Synthesis of N-(4-(4-oxo-2-(trifluoromethyl)quinazolin-3(4H)-yl)phenyl)-2-(3,4,5-trimethoxyphenyl)acetamide (FS-10): To a stirred solution of compound 18 (500 mg, 2.145 mmol, 1.0 eq) in DMF (5 mL) was added EDC.HCl (491 mg, 2.572 mmol, 1.2 eq) and HOBt (339 mg, 2.575 mmol, 1.2 eq), followed by 0 mL of DIPEA (339 mg, 2.575 mmol, 1.2 eq). o C. The reaction mixture was stirred for 10 min. Then, a solution of Int-1 (678 mg, 2.145 mmol, 1 equiv.) was added at 0. oC. The reaction mixture was allowed to warm to room temperature and stirred for 16 hours. After TLC confirmed the completion of the reaction, the reaction mixture was quenched with water (20 mL) and extracted with ethyl acetate (2 × 25 mL). The organic layer was separated and washed with brine solution (20 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified on a reverse-phase C18 column using acetonitrile / water with 0.01% HCOOH buffer, and after lyophilization, the crude compound was obtained. Final purification by preparative HPLC and lyophilization afforded white FS-10 (28 mg, yield: 3%) as a white solid. LCMS: m / z = 514.39 [M+H] + , 96.63% (2.88 min), (INT-NY-709-028-03) 1 H NMR (400 MHz, DMSO-d6) δ ppm: 10.37 (s, 1H), 8.20 (d, J = 8.0 Hz, 1H), 7.99-7.96 (m, 1H), 7.90 (d, J = 7.6 Hz, 1H), 7.77-7.72 (m, 3H), 7.43 (d, J = 8.4 Hz, 2H), 6.67 (s, 2H), 3.77 (s, 6H), 3.63 (s, 3H), 3.61 (s, 2H)

[0192] Scheme 9: Synthesis of N-(4-(2-(difluoromethyl)-4-oxoquinazolin-3(4H)-yl)phenyl)-2-(3,4,5-trimethoxyphenyl)acetamide (FS-11), N-(4-(2-(fluoromethyl)-4-oxoquinazolin-3(4H)-yl)phenyl)-2-(3,4,5-trimethoxyphenyl)acetamide (FS-12) & N-(4-(2-(hydroxymethyl)-4-oxoquinazolin-3(4H)-yl)phenyl)-2-(3,4,5-trimethoxyphenyl)acetamide (FS-15):

[0193] [ka]

[0194] 1.21 Synthesis of 2-(2-acetoxyacetamido)benzoic acid (20): To a stirred solution of 2-aminobenzoic acid (7) (5.0 g, 36.496 mmol, 1 equiv.) in THF (50 mL), triethylamine (15.2 mL, 109.48 mmol, 3 equiv.) was added, followed by ethyl 2-chloro-2-oxoacetate (19) (7.44 g, 54.744 mmol, 1.5 equiv.), and the reaction mixture was stirred at room temperature for 30 min. The reaction mixture was monitored by TLC, and upon completion, the solvent was evaporated under reduced pressure. The crude product was diluted with water (100 mL) and extracted with ethyl acetate (2 × 150 mL). The organic layer was separated and washed with brine solution (20 mL). The combined organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give compound 20 (3.2 g, 37% yield) as a yellow solid. This product was used in the next step without further purification.

[0195] [ka]

[0196] 1.21 Synthesis of (3-(4-aminophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)methyl acetate (23): To a stirred solution of compound 20 (2.0 g, 8.438 mol, 1.0 equiv.) in DMF (10 mL) was added EDC.HCl (1.9 g, 10.126 mmol, 1.2 equiv.) and HOBt (1.4 g, 0.010.126 mmol, 1.2 equiv.), followed by DIPEA (4 mL, 25.314 mmol, 3.0 equiv.). o C. The reaction mixture was stirred for 10 minutes. Then, a solution of compound 4 (908 mg, 8.438 mmol, 1.0 equiv.) was added at 0. oC. The reaction mixture was allowed to warm to room temperature and stirred for 16 hours. After TLC showed the reaction was complete, the reaction mixture was quenched with water (20 mL) and extracted with ethyl acetate (2 × 25 mL). The organic layer was separated and washed with brine solution (20 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (100:200 mesh) eluting with 20% EtOAc in hexane to give compound 21 (1.8 g, yield: 69%) as a yellow oil. LCMS: m / z = 310.08 [M+H] + , 92.75% (1.11 min)

[0197] [ka]

[0198] 1.22 Synthesis of (4-oxo-3-(4-(2-(3,4,5-trimethoxyphenyl)acetamido)phenyl)-3,4-dihydroquinazolin-2-yl)methyl acetate (22): To a stirred solution of compound 3 (300 mg, 1.327 mmol, 1.0 equiv.) and compound 21 (410 mg, 1.327 mmol, 1.0 equiv.) in DMF (3 mL), DIPEA (0.6 mL, 3.98 mmol, 3.0 equiv.) and HATU (750 mg, 1.99 mmol) were added. o C. The reaction mixture was stirred at room temperature for 16 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with cold water (20 mL), extracted with ethyl acetate (2 x 50 mL), and the organic layer was separated and washed with brine solution (20 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by reverse-phase C18 column chromatography and lyophilized to give compound 22 (210 mg, yield: 30%) as a light brown solid. LCMS: m / z = 518.12 [M+H] + , 96.93% (1.33 min)

[0199] [ka]

[0200] 1.23 Synthesis of N-(4-(2-(hydroxymethyl)-4-oxoquinazolin-3(4H)-yl)phenyl)-2-(3,4,5-trimethoxyphenyl)acetamide (FS-15): To a stirred solution of compound 22 (240 mg, 0.4642 mmol, 1.0 equiv) in methyl alcohol (4.8 mL), KCO (161 mg, 1.160 mmol, 2.5 equiv) was added at room temperature and stirred for 1 h. The reaction was monitored by TLC, and upon completion, the crude product was purified by preparative HPLC chromatography using acetonitrile / water with 0.01% HCOOH buffer to give FS-15 (7.2 mg, 3% yield) as a white solid after lyophilization. LCMS: m / z = 476.29 [M+H] + , 99.84% (2.20 min), (INT-NY-709-055-02) 1 H NMR (400 MHz, DMSO-d6) δ ppm: 10.37 (s, 1H), 8.12 (d, J = 7.6 Hz, 1H), 7.88(t, J = 8.0 Hz, 1H), 7.79-7.73 (m, 3H), 7.56 (t, J = 7.6 Hz, 1H), 7.33 (d, J = 8.4 Hz, 2H), 6.68 (s, 2H), 5.19 (t, J = 6.0 Hz, 1H), 4.05 (d, J = 6.0 Hz, 2H), 3.77 (s, 6H), 3.64 (s, 3H), 3.61 (s, 2H)

[0201] [ka]

[0202] 1.24 Synthesis of N-(4-(2-(fluoromethyl)-4-oxoquinazolin-3(4H)-yl)phenyl)-2-(3,4,5-trimethoxyphenyl)acetamide (FS-12): To a stirred solution of FS-15 (40 mg, 0.084 mmol, 1.0 equiv.) in DCM (2.4 mL) was added DAST (0.01 mL, 0.1094 mmol, 1.3 equiv.) for 0.5 min. o C. The reaction mixture was allowed to warm to room temperature and stirred for 2 hours. The reaction was monitored by TLC, and after completion, the solvent was removed in vacuo. The residue was purified on a RP C18 column using acetonitrile / water with 0.01% HCOOH buffer to give FS-12 (8.0 mg, yield: 20%) as an off-white solid after lyophilization. LCMS: m / z = 478.10 [M+H] + , 98.04% (2.39 min) 1 H NMR (400 MHz, DMSO-d6) δ ppm: 10.40 (s, 1H), 8.15 (d, J = 8.0 Hz, 1H), 7.91 (t, J = 7.6 Hz, 1H), 7.81-7.75 (m, 3H), 7.61 (t, J = 7.6 Hz, 1H), 7.39 (d, J = 8.4 Hz, 2H), 6.68 (s, 2H), 4.97 (d, J = 6.0 Hz, 2H), 3.77 (s, 6H), 3.64 (s, 3H), 3.61 (s, 2H)

[0203] [ka]

[0204] 1.25 Synthesis of N-(4-(2-(difluoromethyl)-4-oxoquinazolin-3(4H)-yl)phenyl)-2-(3,4,5-trimethoxyphenyl)acetamide (FS-11): To a stirred solution of FS-15 (500 mg, 1.05 mmol, 1.0 equiv.) in dichloromethane (5 mL), Dess-Martin reagent (530 mg, 1.26 mmol, 1.2 equiv.) was added in 0.5 mL portions. oC. After the addition was complete, the reaction mixture was allowed to warm to room temperature and stirred for 16 hours. DAST (0.1 mL, 169 mmol, 1.0 equiv) was added slowly at 0°C, and the reaction mixture was allowed to warm to room temperature and stirred for 2 hours. The reaction was monitored by TLC, and after completion, the solvent was removed in vacuo. The residue was purified by preparative HPLC to give FS-11 (8.0 mg, yield: 2%) as an off-white solid. LCMS: m / z = 518.25 [M+Na] + , 98.12% (2.64 min) 1 H NMR (400 MHz, DMSO-d6) δ ppm: 10.40 (s, 1H), 8.17 (d, J = 8.0 Hz, 1H), 7.96 (t, J = 8.4 Hz, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.75 (d, J = 8.8 Hz, 2H), 7.69 (t, J = 8.0 Hz, 1H), ), 7.40 (d, J = 8.8 Hz, 2H), 6.68 (s, 2H), 3.77 (s, 6H), 3.64 (s, 3H), 3.61 (s, 2H)

[0205] Scheme 10: N-(4-(2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)-1-phenylmethanesulfonamide (FS-13) & N-methyl-N-(4-(2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)-1-phenylmethanesulfonamide (FS-14):

[0206] [ka]

[0207] 1.26 Synthesis of N-(4-(2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)-1-phenylmethanesulfonamide (FS-13): To a stirred solution of Int-3 (650 mg, 2.589 mmol, 1.0 equiv.) in dichloromethane (10 mL), EtN (1.5 mL, 10.316 mmol, 4.0 equiv.) and compound 23 (550 mg, 18.055 mmol, 1.0 equiv.) were added. o C. The reaction mixture was allowed to warm to room temperature and stirred for 16 hours. After TLC confirmed the completion of the reaction, the reaction mixture was quenched with NaHCO3 solution (20 mL) and extracted with ethyl acetate (2 x 25 mL). The organic layer was separated and washed with brine solution (20 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified on a reverse-phase C18 column using 0.01% HCOOH buffer in acetonitrile / water to give FS-13 (100 mg, 7.2%) as a white solid after lyophilization. LCMS: m / z = 406.08 [M+H] + , 98.60% (2.48 min) 1 H NMR (400 MHz, DMSO-d6) δ ppm: 10.14 (s, 1H), 8.10 (d, J = 7.2 Hz, 1H), 7.87-7.82 (m, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.38-7.29 (m, 9H), 4.60 (s, 2H), 2.17 (s, 3H)

[0208] [ka]

[0209] 1.27 Synthesis of N-methyl-N-(4-(2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)-1-phenylmethanesulfonamide (FS-14): To a stirred solution of compound FS13 (35 mg, 0.086 mmol, 1 equiv.) was added NaH (6 mg, 0.129 mmol, 1.5 equiv.) and a 1 M solution of methyl iodide (0.12 mL, 0.129 mmol, 1.5 equiv.) in DMF (2 mL). o C. The reaction mixture was allowed to warm to room temperature and stirred for 4 hours. After TLC confirmed the completion of the reaction, the reaction mixture was quenched with water (20 mL) and extracted with ethyl acetate (2 × 25 mL). The organic layer was separated and washed with brine solution (20 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified on a reverse-phase C18 column using acetonitrile / water with 0.01% HCOOH buffer to give FS-14 (35 mg, 97%) as a white solid after lyophilization. LCMS: m / z = 420.39 [M+H] + , 99.74% (2.71 min) 1 H NMR (400 MHz, DMSO-d6) δ ppm: 8.09 (d, J = 7.6 Hz, 1H), 7.84 (t, J = 7.2 Hz, 1H), 7.65 (d, J = 7.6 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.43-7.37 (m, 9H), 4.64 (s, 2H), 3.28 (s, 3H), 2.12 (s, 3H)

[0210] [ka]

[0211] Scheme 11: N-(4-(6,7-difluoro-2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)-2-(4-(trifluoromethyl)phenyl)acetamide (FS-20):

[0212] [ka]

[0213] Synthesis of 6,7-difluoro-2-methyl-4H-benzo[d][1,3]oxazin-4-one (2): A mixture of 2-amino-4,5-difluorobenzoic acid (1) (5.0 g, 0.028 mmol, 1 equiv.) and AcO (30 mL) was stirred at room temperature for 15 minutes. The reaction mixture was refluxed for 6 hours. The reaction was monitored by TLC, and upon completion, excess acetic anhydride was removed under reduced pressure. The crude product was diluted with water (50 mL) and extracted with EtOAc (2 × 50 mL). The organic layer was separated and washed with brine solution (20 mL). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give compound (2) (4.2 g, 71% yield) as an off-white solid. This product was used in the next step without further purification. LCMS: m / z = 198.11 [M+H] + , 97.33% (1.32 min) 1 H-NMR (400 MHz, DMSO-d6) δ ppm: 8.14 (t, J = 8.8 Hz, 1H), 7.759-7.714 (m, 1H), 2.403 (s, 3H)

[0214] [ka]

[0215] Synthesis of 3-(4-aminophenyl)-6,7-difluoro-2-methylquinazolin-4(3H)-one (4): To a stirred solution of 6,7-difluoro-2-methyl-4H-benzo[d][1,3]oxazin-4-one (2) (4.0 g, 0.020 mmol, 1 equiv.) in 120 mL of methylpyridine was added benzene-1,4-diamine (3) (2.18 g, 0.020 mmol, 1 equiv.). The reaction mixture was refluxed for 6 h. The resulting mixture was cooled in an ice-water bath and treated with 50 mL of 1 N HCl to give a brown solid precipitate, which was washed with water and dried in air. The crude product was recrystallized from ethanol (20 mL) to give compound (4) (4.2 g, 72% yield) as a brown solid. This product was used directly in the next step without further purification. LCMS: m / z = 288.15 [M+H] + , 81.01% (1.24 min) 1 H-NMR (400 MHz, DMSO-d6) δ ppm: 8.0 (t, J = 8.8 Hz, 1H), 7.73 (q, J = 7.2 Hz, 1H), 6.9 (d, J = 8.8 Hz, 2H), 6.65 (d, J = 8.8 Hz, 2H), 5.41 (br, s, 2H), 2.14 (s, 3H)

[0216] [ka]

[0217] Synthesis of N-(4-(6,7-difluoro-2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)-2-(4-(trifluoromethyl)phenyl)acetamide (FS-20): To a stirred solution of 2-(4-(trifluoromethyl)phenyl)acetic acid (5) (100 mg, 0.348 mmol, 1 equiv.) and 3-(4-aminophenyl)-6,7-difluoro-2-methylquinazolin-4(3H)-one (4) (71 mg, 0.348 mmol, 1 equiv.) in DMF (1 mL), DIPEA (0.2 mL, 1.045 mmol, 4 equiv.) was added, followed by DCC (252 mg, 1.225 mmol, 2.5 equiv.). o C was added under a nitrogen atmosphere. The reaction mixture was allowed to warm to room temperature and stirred for 4 hours. The reaction was monitored by TLC. After completion, the reaction mixture was diluted with cold water (20 mL) and extracted with ethyl acetate (2 × 25 mL). The organic layer was separated and washed with brine solution (20 mL). The separated organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified on a reverse-phase C-18 column using 0.01% NH3 buffer in acetonitrile / water to give FS-20 (35 mg, 21% yield) as an off-white solid after lyophilization. LCMS: m / z = 474.24 [M+H] + , 96.59% (3.02 min) 1 H NMR (400 MHz, DMSO-d6) δ ppm: 10.49 (s, 1H), 8.01 (t, J=9.2 Hz, 1H), 7.69-7.79 (m, 5H), 7.58 (d, J=7.8 Hz, 2H), 7.37 (d, J=8.6 Hz, 2H), 3.83 (s, 2H), 2.13 (s, 3H)

[0218] FS-21: Synthesis of N-(4-(6,7-difluoro-2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)-2-(3,4,5-trimethylphenyl)acetamide (FS-21):

[0219] [ka]

[0220] Synthesis of N-(4-(6,7-difluoro-2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)-2-(3,4,5-trimethylphenyl)acetamide (FS-21): To a stirred solution of 2-(3,4,5-trimethylphenyl)acetic acid (5) (100 mg, 0.561 mmol, 1 equiv.) and 3-(4-aminophenyl)-6,7-difluoro-2-methylquinazolin-4(3H)-one (4) (193 mg, 0.674 mmol, 1.2 equiv.) in DMF (1 mL), DIPEA (0.3 mL, 0.842 mmol) and HATU (320 mg, 1.685 mmol, 1.5 equiv.) were added. o C. The reaction mixture was stirred at room temperature for 4 hours. The reaction progress was monitored by TLC, and after completion, the reaction mixture was diluted with cold water (20 mL) and extracted with ethyl acetate (2 × 25 mL). The organic layer was separated and washed with brine solution (20 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified on a reverse-phase C18 column using acetonitrile / water with 0.01% HCOOH buffer to give FS-21 (45 mg, yield: 26%) as an off-white solid after lyophilization. LCMS: m / z = 448.51 [M+H] + , 98.90% (3.21 min) 1 H NMR (400 MHz, DMSO-d6) δ ppm: 10.34 (s, 1H), 8.02 (t, J=9.2 Hz, 1H), 7.73-7.78 (m, 3H) 7.35 (d, J=8.8 Hz, 2H), 6.95 (br, s, 2H), 3.54 (s, 2H), 2.2 (br, s, 6H), 2.12 (s, 3H), 2.09 (s, 3H)

[0221] [ka]

[0222] Synthesis of N-(4-(6,7-difluoro-2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)-2-(3,4,5-trifluorophenyl)acetamide (FS-22): To a stirred solution of 2-(3,4,5-trifluorophenyl)acetic acid (5) (100 mg, 0.348 mmol, 1 equiv.) and 3-(4-aminophenyl)-6,7-difluoro-2-methylquinazolin-4(3H)-one (4) (66 mg, 0.348 mmol, 1 equiv.) in THF (1 mL) was added DIPEA (0.27 mL, 1.392 mmol) and DCC (94 mg, 0.871 mmol, 2.5 equiv.). o C. The reaction mixture was allowed to warm to room temperature and stirred for 4 hours. The reaction was monitored by TLC, and after completion, the reaction mixture was diluted with cold water (20 mL) and extracted with ethyl acetate (2 x 25 mL). The organic layer was separated and washed with brine solution (20 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by reverse-phase C18 column using acetonitrile / water with 0.01% NH3 buffer to give FS-22 (38 mg, yield: 23%) as a brown solid after lyophilization. LCMS: m / z = 460.25 [M+H] + , 97.32% (2.94 min) 1 H NMR (400 MHz, DMSO-d6) δ ppm: 10.45 (s, 1.0H), 8.01 (t, J=9.2 Hz, 1H), 7.7 (d, J=8.8 Hz, 3H), 7.29-7.386 (m, 4H), 3.69 (br, s, 2H), 2.07 (m, 3H)

[0223] [ka]

[0224] Synthesis of N-(4-(6,7-difluoro-2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)-2-(2,3-dihydro-1H-inden-5-yl)acetamide (FS-23): To a stirred solution of 2-(2,3-dihydro-1H-inden-5-yl)acetic acid (5) (100 mg, 0.568 mmol, 1 equiv.) and 3-(4-aminophenyl)-6,7-difluoro-2-methylquinazolin-4(3H)-one (4) (195 mg, 0.681 mmol, 1.2 equiv.) in DMF (1 mL), DIPEA (0.3 mL, 1.704 mmol) and HATU (323 mg, 0.852 mmol, 1.5 equiv.) were added. o C. The reaction mixture was allowed to warm to room temperature and stirred for 4 hours. The reaction was monitored by TLC. After completion, the reaction mixture was diluted with cold water (20 mL) and extracted with ethyl acetate (2 × 25 mL). The organic layer was separated and washed with brine solution (20 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified on a reverse-phase C18 column using acetonitrile / water with 0.01% HCOOH buffer to give FS-23 (40 mg, 25% yield) as a white solid after lyophilization. LCMS: m / z = 446.38 [M+H] + , 97.92% (3.16 min) 1 H NMR (400 MHz, DMSO-d6) δ ppm: 10.38 (s, 1H), 8.01 (t, J=8.8 Hz, 1H), 7.74-7.78 (m, 3.0H), 7.35 (d, J=8.8 Hz, 2H), 7.16-7.22 (m, 1H), 7.10 (d, J=7.6 Hz, 1H), 7.08 (d, J=7.6 Hz, 1H), 3.62 (s, 2H), 2.80-2.86 (m, 4H), 2.07 (s, 3H), 2.0 (t, J=7.2 Hz, 2H)

[0225] [ka]

[0226] N-(4-(6,7-difluoro-2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)-2-(2,6-difluoropyridin-4-yl)acetamide (FS-22): To a stirred solution of compound 5 (100 mg, 0.578 mmol, 1 equiv.) and 3-(4-aminophenyl)-6,7-difluoro-2-methylquinazolin-4(3H)-one 4 (165 mg, 0.348 mmol, 1 equiv.) in THF (1 mL) was added DIPEA (0.4 mL, 2.312 mmol) and DCC (269 mg, 1.308 mmol, 2.5 equiv.). o C. The reaction mixture was allowed to warm to room temperature and stirred for 4 hours. The reaction was monitored by TLC. After completion, the reaction mixture was diluted with cold water (20 mL) and extracted with ethyl acetate (2 × 25 mL). The organic layer was separated and washed with brine solution (20 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified on a reverse-phase C18 column using acetonitrile / water with 0.01% NH3 buffer to give FS-25 (23 mg, yield: 15%) as an off-white solid after lyophilization. LCMS: m / z = 443.27 [M+H] + , 99.00% (2.77min) 1 H NMR (400 MHz, DMSO-d6) δ ppm: 10.5 (s, 1.0H), 8.02 (t, J=9.2 Hz, 1H), 7.7 (d, J=9.2 Hz, 3H), 7.38 (d, J=8.8 Hz, 2H), 7.1 (s, 2H), 3.87 (br, s, 2H), 2.0 (s, 3H)

[0227] [ka]

[0228] Synthesis of N-(4-(6,7-difluoro-2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)-2-(perfluorophenyl)acetamide (FS-26): To a stirred solution of 2-(perfluorophenyl)acetic acid (5) (100 mg, 0.442 mmol, 1 equiv.) and 3-(4-aminophenyl)-6,7-difluoro-2-methylquinazolin-4(3H)-one (4) (126 mg, 0.442 mmol, 1 equiv.) in THF (1 mL) was added DIPEA (0.3 mL, 1.768 mmol) and DCC (227 mg, 1.106 mmol, 2.5 equiv.). o C. The reaction mixture was allowed to warm to room temperature and stirred for 4 hours. The reaction was monitored by TLC. After completion, the reaction mixture was diluted with cold water (20 mL), extracted with ethyl acetate (2 x 25 mL), and the organic layer was separated and washed with brine solution (20 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified on a reverse-phase C18 column using acetonitrile / water with 0.01% NH3 buffer to give FS-26 (35 mg, yield: 20%) as an off-white solid after lyophilization. LCMS: m / z = 496.23 [M+H] + , 95.21% (3.05 min) 1 H NMR (400 MHz, DMSO-d6) δ ppm: 10.63 (s, 1H), 8.02 (t, J=9.3 Hz, 1H), 7.72-7.78 (m, 3H), 7.38 (d, J=8.8 Hz, 2H), 3.95 (s, 2H), 2.13 (s, 3H)

[0229] [ka]

[0230] Synthesis of N-(4-(6,7-difluoro-2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)-2-phenylacetamide (FS-31): To a stirred solution of 2-phenylacetic acid (5) (100 mg, 0.735 mmol, 1 equiv.) and 3-(4-aminophenyl)-6,7-difluoro-2-methylquinazolin-4(3H)-one (4) (253 mg, 0.882 mmol, 1.2 equiv.) in DMF (1 mL), DIPEA (0.4 mL, 2.205 mmol) and HATU (419 mg, 1.102 mmol, 1.5 equiv.) were added. o C. The reaction mixture was allowed to warm to room temperature and stirred for 4 h. The reaction progress was monitored by TLC. Upon completion, the reaction mixture was diluted with cold water (20 mL) and extracted with ethyl acetate (2 × 25 mL). The organic layer was separated and washed with brine solution (20 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified on a reverse-phase C18 column using acetonitrile / water with 0.01% HCOOH buffer to give FS-31 (42 mg, 29% yield) as a brown solid after lyophilization. LCMS: m / z = 406.31 [M+H] + , 96.89% (2.73 min) 1 H NMR (400 MHz, DMSO-d6) δ ppm: δ = 10.43 (s, 1H), 8.02 (t, J=9.5 Hz, 1H), 7.74-7.79 (m, 3H), 7.31-7.38 (m, 6H), 7.24-7.30 (m, 1H), 3.69 (s, 2H), 2.13 (s, 3H)

[0231] [ka]

[0232] Synthesis of N-(4-(6,7-difluoro-2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)-2-(pyridin-2-yl)acetamide (FS-32): To a stirred solution of 2-(pyridin-2-yl)acetic acid hydrochloride (5) (200 mg, 1.156 mmol, 1.0 equiv.) and 3-(4-aminophenyl)-6,7-difluoro-2-methylquinazolin-4(3H)-one (4) (165 mg, 0.578 mmol, 0.5 equiv.) in THF (2 mL), DIPEA (0.72 mL, 4.624 mmol, 4.0 equiv.) and DCC (595 mg, 2.890 mmol, 2.5 equiv.) were added. o C. The reaction mixture was allowed to warm to room temperature and stirred for 3 hours. The reaction was monitored by TLC, and after completion, the reaction mixture was diluted with water (20 mL), extracted with ethyl acetate (2 × 25 mL), and the organic layer was separated and washed with brine solution (20 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified on a reverse-phase C18 column using acetonitrile / water with 0.01% NH3 buffer to give FS-32 (52 mg, 37% yield) as an off-white solid after lyophilization. LCMS: m / z = 407.29 [M+H] + , 95.46% (1.94 min) 1 H NMR (400 MHz, DMSO-d6) δppm: 10.51 (br, s, 1H), 8.52 (d, J=4.0 Hz, 1H), 8.03 (t, J= 9.6 Hz, 1H), 7.744-7.79 (m, 4H), 7.27-7.43 (m, 4H), 3.89 (s, 2H), 2.13 (br, s, 3H).

[0233] [ka]

[0234] Synthesis of N-(4-(6,7-difluoro-2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)-2-(pyridin-3-yl)acetamide (FS-33): To a stirred solution of 3-(4-aminophenyl)-6,7-difluoro-2-methylquinazolin-4(3H)-one (4) (100 mg, 0.898 mmol, 1.0 equiv.) and 2-(pyridin-3-yl)acetic acid hydrochloride (5) (150 mg, 0.898 mmol, 2.5 equiv.) in THF (1 mL) was added DIPEA (0.2 mL, 0.522 mmol) and DCC (297 mg, 1.44 mmol, 2.5 equiv.). o C. The reaction mixture was stirred at room temperature for 3 hours. The reaction was monitored by TLC, and after completion, the reaction mixture was diluted with water (20 mL), extracted with ethyl acetate (2 x 25 mL), and the organic layer was separated and washed with brine solution (20 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified on a reverse-phase C18 column using acetonitrile / water with 0.01% NH3 buffer to give a brown solid as FS-33 (52 mg, 37% yield) after lyophilization. LCMS: m / z = 407.29 [M+H] + , 96.24% (1.90 min) 1 H NMR (400 MHz, DMSO-d6) δ ppm: 10.49 (br, s, 1H), 8.54 (s, 1H), 8.47 (d, J=4.4 Hz, 1H), 8.02 (t, J=9.6 Hz, 1H), 7.75 (d, J=8.8 Hz, 4H), 7.37 (d, J=8.0 Hz, 3H), 3.75 (s, 2H), 2.12 (br, s, 3H)

[0235] [ka]

[0236] Synthesis of N-(4-(6,7-difluoro-2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)-2-(pyridin-4-yl)acetamide (FS-34): To a stirred solution of 2-(pyridin-4-yl)acetic acid (5) (100 mg, 0.729 mmol, 1 equiv.) and 3-(4-aminophenyl)-6,7-difluoro-2-methylquinazolin-4(3H)-one (4) (251 mg, 0.875 mmol, 1.2 equiv.) in DMF (1 mL) was added DIPEA (0.4 mL, 2.189 mmol) and HATU (416 mg, 1.094 mmol, 1.5 equiv.). o C. The reaction mixture was allowed to warm to room temperature and stirred for 3 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with cold water (20 mL) and extracted with ethyl acetate (2 × 25 mL). The organic layer was separated and washed with brine solution (20 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified on a reverse-phase C18 column using acetonitrile / water with 0.01% HCOOH buffer to give FS-34 (60 mg, yield: 42%) as a brown solid after lyophilization. LCMS: m / z = 407.28 [M+H] + , 99.80% (1.66 min) 1 H NMR (400 MHz, DMSO-d6) δppm: 10.53 (br, s, 1H), 8.61 (d, J=4.8 Hz, 2H), 8.02 (t, J=8.4 Hz, 1H), 7.746-7.787 (m, 3H), 7.52 (d, J=6.0 Hz, 2H), 7.38 (d, J=8.8 Hz, 2H), 3.84 (br,s, 2H), 2.12 (br, s, 3H).

[0237] [ka]

[0238] Synthesis of N-(4-(6,7-difluoro-2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)-2-(2,6-dimethylpyridin-4-yl)acetamide (FS-35): To a stirred solution of 2-(2,6-dimethylpyridin-4-yl)acetic acid (5) (50 mg, 0.248 mmol, 1 equiv.) and 3-(4-aminophenyl)-6,7-difluoro-2-methylquinazolin-4(3H)-one (4) (71 mg, 0.248 mmol, 1.0 equiv.) in THF (1 mL) was added DIPEA (0.4 mL, 2.189 mmol) and HATU (416 mg, 1.094 mmol, 1.5 equiv.). o C. The reaction mixture was allowed to warm to room temperature and stirred for 3 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with cold water (20 mL) and extracted with ethyl acetate (2 x 25 mL). The organic layer was separated and washed with brine solution (20 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by preparative HPLC to give FS-35 (8 mg, yield: 10%) as an off-white solid after lyophilization. LCMS: m / z = 435.33 [M+H] + , 96.07% (1.58 min) 1 H NMR (400 MHz, DMSO-d6) δ ppm: 10.46 (br, s, 1H), 8.02 (t, J=10 Hz, 1H), 7.77 (t, J=8.0 Hz, 3H), 7.37 (d, J=8.8 Hz, 2H), 7.01 (br, s, 2H), 3.64 (s, 2H), 2.41 (br, s, 6H), 2.07 (br, s, 3H)

[0239] [ka]

[0240] Synthesis of 2-(3,5-bis(trifluoromethyl)phenyl)-N-(4-(6,7-difluoro-2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)acetamide (FS-37): To a stirred solution of 2-(3,5-bis(trifluoromethyl)phenyl)acetic acid (5) (100 mg, 0.367 mmol, 1 equiv.) and 3-(4-aminophenyl)-6,7-difluoro-2-methylquinazolin-4(3H)-one (4) (105 mg, 0.365 mmol, 1.0 equiv.) in THF (1 mL) was added DIPEA (0.25 mL, 1.468 mmol) and DCC (99 mg, 0.917 mmol, 2.5 equiv.) at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 3 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with cold water (20 mL) and extracted with ethyl acetate (2 × 25 mL). The organic layer was separated and washed with brine solution (20 mL). The combined organic layers were dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The crude product was purified on a reverse-phase C18 column using acetonitrile / water with 0.01% HCOOH buffer to give FS-37 (48 mg, 25% yield) as a white solid after lyophilization. LCMS: m / z = 542.29 [M+H] + , 95.01% (3.42 min) 1 H NMR (400 MHz, DMSO-d6) δ ppm: 10.52 (br, s, 1H) 7.99-8.08 (m, 4H), 7.7 (d, J=4.0 Hz, 3H), 7.37 (d, J=8.8 Hz, 2H), 4.0 (br,s, 2H), 2.12 (br, s, 3H)

[0241] [ka]

[0242] Synthesis of 2-amino-3,4,5,6-tetrafluorobenzoic acid (2): A mixture of 2,3,4,5-tetrafluoro-6-nitrobenzoic acid (1) (500 mg, 2.092 mmol, 1 equiv.) in methanol (5 mL) and 10% Pd / C (50 mg) was slowly added to the reaction mixture under an H2 gas atmosphere. The reaction mixture was stirred at room temperature for 2 h. The reaction was monitored by TLC. Upon completion, the reaction mixture was filtered through a pad of Celite and washed with methyl alcohol (2 x 20 mL). The filtrate was evaporated under reduced pressure to give the target compound-2 (420 mg, 96% yield) as a white solid. LCMS: m / z = 208.05 [M−H] + , 99.60% (1.22 min)

[0243] [ka]

[0244] Synthesis of 5,6,7,8-tetrafluoro-2-methyl-4H-benzo[d][1,3]oxazin-4-one (3): A mixture of 2-amino-3,4,5,6-tetrafluorobenzoic acid (2) (400 mg, 1.913 mmol, 1 equiv.) and acetic anhydride (2.5 mL) was stirred at room temperature for 15 minutes. The reaction mixture was refluxed for 6 hours. The reaction was monitored by TLC, and upon completion, excess acetic anhydride was removed under reduced pressure. The crude product was diluted with water (50 mL) and extracted with ethyl acetate (2 × 30 mL). The organic layer was separated and washed with brine (20 mL). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give 5,6,7,8-tetrafluoro-2-methyl-4H-benzo[d][1,3]oxazin-4-one (3) (352 mg, 78% yield) as a brown solid. This product was used in the next step without further purification. LCMS: m / z = 206.08 [M−H] - , 89.22% (1.36 min)

[0245] [ka]

[0246] Synthesis of 3-(4-aminophenyl)-5,6,7,8-tetrafluoro-2-methylquinazolin-4(3H)-one (5): 5,6,7,8-tetrafluoro-2-methyl-4H-benzo[d][1,3]oxazin-4-one (3) (350 mg, 1.502 mmol, 1 equiv.) and benzene-1,4-diamine (4) (162 mg, 1.502 mmol, 1 equiv.) were added to 10 mL of anhydrous pyridine and stirred. The reaction mixture was refluxed for 6 h. The resulting mixture was cooled in an ice-water bath and treated with 10 mL of 1N HCl to give a brown solid precipitate. The precipitate was washed with water and dried in air. The crude product was recrystallized from ethanol to give compound (5) (350 mg, 72% yield) as a brown solid. This product was used directly in the next step without further purification. LCMS: m / z = 324.19 [M+H] + , 50.77% (1.32 min).

[0247] [ka]

[0248] Synthesis of N-(4-(5,6,7,8-tetrafluoro-2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)-2-(3,4,5-trifluorophenyl)acetamide (FS-27): To a stirred solution of 2-(3,4,5-trifluorophenyl)acetic acid (6) (200 mg, 1.052 mmol, 1 equiv.) and 3-(4-aminophenyl)-5,6,7,8-tetrafluoro-2-methylquinazolin-4(3H)-one (5) (339 mg, 1.052 mmol, 1 equiv.) in THF (2 mL), DIPEA (0.5 mL, 4.208 mmol) and DCC (542 mg, 2.630 mmol, 2.5 equiv.) were added. oC. The reaction mixture was allowed to warm to room temperature and stirred for 4 hours. The reaction was monitored by TLC, and after completion, the reaction mixture was diluted with cold water (20 mL) and extracted with ethyl acetate (2 × 25 mL). The reaction was monitored by TLC, and after completion, the reaction mixture was diluted with cold water (20 mL) and extracted with ethyl acetate (2 × 25 mL). The organic layer was separated and washed with brine solution (20 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by preparative HPLC, and after lyophilization, FS-27 (22 mg, yield: 12%) was obtained as an off-white solid. LCMS: m / z = 496.23 [M+H] + , 98.84% (3.06 min) 1 H NMR (400 MHz, DMSO-d6) δ ppm: 10.45 (s, 1H), 7.76 (d, J=8.0 Hz, 2H), 7.29-7.38 (m, 4H), 3.7(br, s, 2H), 2.15 (br,s, 3H).

[0249] [ka]

[0250] Synthesis of 2-methyl-4H-benzo[d][1,3]oxazin-4-one (Int-2): A mixture of 2-aminobenzoic acid (1) (1.0 g, 0.007 mmol, 1 equiv.) and acetic anhydride (6 mL) was refluxed for 4 h. The reaction mixture was cooled to room temperature, and excess acetic anhydride was removed under reduced pressure. The crude reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (2 × 50 mL). The organic layer was separated and washed with brine solution (30 mL). The combined organic layers were dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give Int-2 (620 mg, yield: 44%) as an off-white solid. This product was used directly in the next step without further purification. LCMS: m / z = 162.04 [M+H] + , 95.75% (1.154 min)

[0251] [ka]

[0252] Synthesis of 3-(4-aminophenyl)-2-methylquinazolin-4(3H)-one (Int-3): 2-methyl-4H-benzo[d][1,3]oxazin-4-one (2) (600 mg, 3.726 mmol, 1 equiv.) and p-phenylenediamine (4) (402 mg, 3.726 mmol, 1.0 equiv.) were added to 18 mL of anhydrous pyridine and stirred. The reaction mixture was refluxed for 6 h. The resulting solution was cooled in an ice-water bath and acidified with 10 mL of dilute hydrochloric acid to give a white solid precipitate, which was filtered, washed with water, and dried. The crude product was recrystallized from ethanol to give Int-3 (720 mg, 67% yield) as a brown solid. LCMS: m / z = 252.57 [M+H] + , 97.01% (1.04 min) 1 H NMR (400 MHz, DMSO-d6) δ ppm: 8.16 (d, J = 7.6 Hz, 1H), 7.97-7.89 (m, 2H), 7.64 (t, J = 7.2 Hz, 1H), 7.50-7.39 (m, 4H), 2.32 (s, 3H)

[0253] [ka]

[0254] Synthesis of N-(4-(2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)-2-phenylacetamide (FS-30): To a stirred solution of 2-phenylacetic acid (5) (100 mg, 0.735 mmol, 1 equiv.) and (4-aminophenyl)-2-methylquinazolin-4(3H)-one (4) (185 mg, 0.735 mmol, 1.0 equiv.) in DMF (1 mL) was added DIPEA (0.4 mL, 2.205 mmol), followed by 0.05 mL of HATU (419 mg, 1.102 mmol, 1.5 equiv.). oC. The reaction mixture was stirred at room temperature for 4 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with cold water (20 mL) and extracted with ethyl acetate (2 × 25 mL). The organic layer was separated and washed with brine solution (20 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified on a reverse-phase C18 column using acetonitrile / water with 0.01% HCOOH buffer to give FS-30 (38 mg, yield: 27%) as a brown solid after lyophilization. LCMS: m / z = 370.25 [M+H] + , 99.03% (2.41 min) 1 H NMR (400 MHz, DMSO-d6) δppm: δ = 10.42 (s, 1H), 8.12 (d, J=12 Hz, 1H), 7.835-7.85 (m, 1H), 7.76 (d, J=8.8 Hz, 2H), 7.65 (d, J=14 Hz, 1H), 7.51 (t, J=7.2 Hz, 1H), 7.284-7.366 (m, 6H), 7.241-7.7.270 (m, 1H), 3.69 (br, s, 2H), 2.13 (s, 3H)

[0255] 2. Biological testing 2.1: Method To identify novel small molecule inhibitors that induce ferrotosis in FSP1-overexpressing cells lacking GPX4, syngeneic fibroblasts of hFSP1 GPX4 KO and FSP1 GPX4 wild-type (WT) were screened using a high-throughput screening (HTS) approach. Specifically, both cell lines were cultured in parallel and screened against the HMGU library at a fixed concentration of 10 μM. Cell viability was assessed using the LIVE / DEAD assay Aquabluer. Cell viability (%) was measured for wild-type (WT) and knockout (KO) cell lines. Compounds that induce ferrotosis in KO cells but not in WT cells were defined as hits. The EC values ​​of each compound were then calculated. 50 To establish values, positive hits were analyzed in a dose-response study (see Table 2).

[0256] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8]

[0257] Additionally, hit compounds were evaluated for their in vitro activity in inhibiting FSP1-mediated resazurin reduction at different concentrations, following the method of Mishima et al. (2022). Assays were performed in a solution containing TBS buffer (50 mM TRIS, 150 mM NaCl (pH 8)), 100 nM resazurin, 200 μM NADH, and 50 nM recombinant FSP1 enzyme. The fluorescence emission (Ex540 / Em590) of reduced resazurin was measured every 30–60 s using a SpectraMax or SpectraMaxiD5 plate reader. Reaction rates were compared to control reactions containing DMSO.

[0258] 2.2 Subcutaneous tumor model All mice were obtained from Charles River. Syngeneic subcutaneous tumor experiments involved the use of mice stably overexpressing hFSP1-HA and Gpx4. KO / Fsp1 KO B16F10 cells (1 x 106 The cells (100 μL PBS) were subcutaneously injected into the right flank of 7-week-old female C57BL6 / J mice. The tumor size was approximately 25–50 mm. 3 After reaching 100% CI, mice were randomized and administered vehicle or icFSP1 (50 mg / kg, Inc.) intraperitoneally twice daily for 4-5 days. To generate tumor samples for staining, mice were cultured in Gpx4 mice stably expressing hFSP1-WT-HA or hFSP1-Q319K-HA. KO / Fsp1 KO B16F10 cells (1 x 10 6 The cells / 100 μL PBS) were subcutaneously injected into the right flank of 7-week-old female C57BL6 / J mice. 3 After reaching , mice were administered vehicle or icFSP1 (50 mg / kg, Intonation) by intraperitoneal injection twice daily for 3 days.

[0259] In xenograft subcutaneous tumor experiments, GPX4 KO A375 cells (5 x 10 6 The cells (100 μL / 100 μL PBS) were subcutaneously injected into the right flank of 7-week-old female atimic nude mice. The tumor size was approximately 25–100 mm. 3 After reaching , mice were randomized and administered vehicle or icFSP1 (50 mg / kg, Intonation) by intraperitoneal injection twice daily for the first 4 days and once daily thereafter.

[0260] In xenograft subcutaneous tumor experiments, GPX4 KO H460 cells (5 x 10 6 The cells (100 μL / 100 μL PBS) were subcutaneously injected into the right flank of 6-week-old female Atchimos nude mice. 3 After reaching , mice were randomized and administered vehicle or icFSP1 (50 mg / kg, Intonation) by intraperitoneal injection twice daily.

[0261] icFSP1 was dissolved in 45% PEG E 300 (Sigma, product number 91462-1KG) and 55% PBS (Gibco, product number 14190094). Tumors were measured daily with a caliper, and tumor volume was calculated using the following formula: tumor volume = length × width. 2 ×0.52. The tumor size is 1000 mm at the time of measurement. 3 If tumors exceeded the human endpoint or became necrotic, they were considered to be the human endpoint. If tumors reached the human endpoint, the experiment was stopped and no further experiments were performed.

[0262] 2.3 Cell viability assay Cells were seeded in 96-well plates and cultured overnight. The following day, the medium was replaced with medium containing the following compounds: RSL3, ML210, erastin, FIN56, FINO2, BSO, iFSP1, icFSP1, Lip-1, DFO, Fer-1, zVAD, Nec-1s, MCC950, olaparib, STS, TNFα, Smac mimetic, or nigericin at the indicated concentrations. For TAM and Dox treatment, cells were seeded simultaneously with the compounds. Cell viability was determined after treatment with nigericin at 1 hour, RSL3, ML210, erastin, FIN56, FINO2, iFSP1, icFSP1, STS, TNFα, Smac mimetic, and zVAD at 24–48 hours, or BSO, icFSP1, TAM, and Dox at 72 hours using AquaBluer (MultiTarget Pharmaceuticals, part number 6015) as an indicator of viable cells, according to the manufacturer's protocol. For apoptosis induction, HT-1080 cells were cultured with different concentrations of STS for 24 hours. For necroptosis induction, HT-29 cells were cultured with Smac mimetic (400 nM) and zVAD (30 μM) at different concentrations of TNFα (400 nM) for 24 hours. For pyroptosis induction, THP-1 cells stimulated with LPS (1 μg / mL, 2 hours) were cultured with nigericin for 1 hour. To induce ferrotosis, cells were cultured with ferrotosis inducers for 24–72 h. After 4 h of incubation under standard cell culture conditions, fluorescence intensity was measured at Ex / Em = 540 / 590 nm using a SpectraMax M5 microplate reader (SoftMax Pro v7, Molecular Devices) and SoftMax Pro v7 (Molecular Devices). Relative cell viability (%) was calculated using the following formula: (fluorescence intensity (FL) of sample – background) / (fluorescence intensity (FL) of appropriate control sample – background) × 100.

[0263] 2.4 LDH release assay Cells were seeded in 96-well plates and cultured overnight. The next day, the medium was replaced with compound-containing medium and cultured for an additional 24 hours. Cell death was measured using a Cytotoxicity Detection Kit (LDH) (Roche, part number 11644793001) according to the manufacturer's protocol. Briefly, cell culture supernatants were collected as medium samples. Cells were then lysed in PBS containing 0.1% Triton X-100 to prepare lysate samples. The medium and lysate samples were mixed with the reagent on a microplate and incubated at room temperature for 15–30 minutes. The absorbance at 492 nm was measured using a SpectraMax M5 microplate reader. The cell death rate was calculated based on the LDH release rate (%) using the following formula: [absorbance (absorbance) of medium sample – background] / [(absorbance (absorbance) of lysate sample – background) + (absorbance (absorbance) of medium sample – background)] × 100.

[0264] 2.5 Lipid peroxidation assay One day before the experiment, 100,000 cells were seeded per well in a 12-well plate. The following day, cells were treated with 2.5 μM icFSP1 for 3 hours and then incubated with 1.5 μM C11-BODIPY 581 / 591 (Invitrogen, part number D3861) for 30 minutes at 37°C in a 5% CO2 atmosphere. Cells were then washed once with PBS, trypsinized, and resuspended in 500 μL of PBS. Cells were passed through a 40 μm cell strainer and analyzed using a flow cytometer (CytoFLEX, Beckman Coulter) with a 488 nm laser for excitation. Data for the oxidized form of BODIPY were collected from the FITC detector using a 525 / 40 nm bandpass filter, and data for the reduced form of BODIPY were collected from the PE detector using a 585 / 42 nm bandpass filter using CytExpert v2.4 (Beckman Coulter). At least 10,000 events were analyzed for each sample. Data were analyzed using FlowJo Software (FlowJo LLC). The fluorescence ratio of C11-BODIPY 581 / 591 (lipid peroxidation) [FITC / PE ratio (oxidized / reduced ratio)] was calculated using the following formula (22): (median FITC-A fluorescence - median FITC-A fluorescence of unstained samples) / (median PE-A fluorescence - median PE-A fluorescence of unstained samples).

[0265] 2.6 Oxylipidomic analysis One day before the experiment, 2 million cells were seeded in a 15-cm dish. The next day, cells were treated with 5 μM icFSP1 to induce lipid peroxidation. After 5 hours, cells were harvested, frozen in liquid nitrogen, and stored at -80°C. Lipids were extracted from the cells using the methyl tert-butyl ether (MTBE) method. Briefly, cell pellets were collected in phosphate-buffered saline (PBS) containing dibutylhydroxytoluene (BHT, 100 μM) and diethylenetriaminepentaacetic acid (DTPA, 100 μM), washed, and centrifuged. 2.5 μL of SPLASH® LIPIDOMIX® (Avanti Polar Lipids) was added and the mixture was incubated on ice for 15 minutes. Then, ice-cold methanol (375 μL) and MTBE (1250 μL) were added, and the sample was vortexed and placed at 4°C for 1 hour (orbital shaker, 32 rpm). Water (375 μL) was added to induce phase separation, vortexed (orbital shaker, 32 rpm) for 10 min at 4 °C, and centrifuged (10 min, 4 °C, 1500 × g) to separate the organic and aqueous phases. The organic phase was collected, dried in a vacuum evaporator, and redissolved in 100 μL of isopropanol. The lipid extract was transferred to a glass vial and subjected to LC-MS analysis.

[0266] Reversed-phase liquid chromatography (RPLC) was performed on a Shimadzu Exion LC equipped with an Accucore C30 column (150 x 2.1 mm; 2.6 μm, 150 Å, Thermo Fisher Scientific). Lipids were separated by gradient elution using solvent A (acetonitrile / water, 1:1, by volume) and solvent B (isopropanol / acetonitrile / water, 85:15:5, by volume), both containing 5 mM NH4HCO2 and 0.1% (by volume) formic acid. Separation was performed at 50 °C with a flow rate of 0.3 mL / min using the following gradient: 0–20 min: 10–86% B (curve 4), 20–22 min: 86–95% B (curve 5), 22–26 min: 95% isocratic, and 26–26 min: 1 min: 95–10% B (curve 5), followed by a 5-min re-equilibration at 10% B (reference 23). Mass spectrometry was performed on a Sciex 7500 system equipped with an electrospray (ESI) source and operated in negative ion mode. Products were analyzed in MRM mode, monitoring the transition from parent ion to daughter ion, using the following parameters: TEM 500 °C, GS1 40, GS2 70, CUR 40, CAD 9, IS-3000V.

[0267] The area under the curve from parent ion to daughter ion was normalized and integrated by the appropriate lipid species, i.e., PC (15:0 / 18:1(d7)) or PE (15:0 / 18:1(d7)), from the SPLASH LIPIDOMIX Mass Spec Standard (Avanti). Normalized peak areas were further log-transformed and autoscaled using the MetaboAnalyst online platform v5.0 (https: / / www.metaboanalyst.ca). Zero values ​​were replaced by 0.2 times the minimum value detected for each given oxidized lipid within a sample. Oxidized lipids that showed significant differences between samples (ANOVA, adjusted P value (false discovery rate (FDR)) cutoff: 0.05) were used to generate heatmaps. Heatmaps were generated using GraphPad Prism 9. The color scheme corresponds to the autoscaled log fold change relative to the mean log value within the sample.

[0268] 2.7 In vitro saturation transfer difference experiments 600MHz on a Bruker Avance III HD spectrometer using a H / N / C triple resonance cryoprobe 1 Saturation transition difference (STD) experiments were performed at H frequencies. Spectra were recorded at 10 °C using 5 μM recombinant human FSP1 (mutant) and a 100-fold molar excess of icFSP1 in phosphate-buffered saline supplemented with 150 mM NaCl, 1% (v / v) DMSO-d6, and 10% (v / v) DO for deuterium immobilization. The saturation time was 2.5 s, and the on and off frequencies were 0.68 ppm and -17 ppm, respectively. NMR spectra were processed using Topspin 4.0.6 (Bruker).

[0269] 2.8 Immunocytochemistry Unless otherwise noted, all confocal microscopy images were captured and analyzed using an LSM880 microscope (ZEISS) equipped with a 63x objective and appropriate filter sets for the fluorescent dyes. Images were analyzed using ZEN Blue software (v3.2, ZWISS) or ImageJ / Fiji. Cells were seeded into 8-well microslides (ibidi, part number 80826) one day before the experiment. The following day, the culture medium was replaced with fresh cell culture medium supplemented with 2.5 μM icFSP1. After incubation for the indicated time, cells were fixed and stained as follows: fixed with 4% paraformaldehyde for 5–10 minutes; permeabilized and blocked with PBS containing 0.3% Triton X-100 and 10 mg / mL BSA for 15 minutes; and then treated with TBS-T or PBS containing DAPI (1:10,000) for 5 minutes at room temperature, away from light. Finally, all samples were mounted in Aqua- / poly Mount (Polysciences, part number 18606-20) and dried overnight at 4°C.

[0270] 2.9 Fluorescence recovery after photobleaching (FRAP) One day before the experiment, Pfa1 cells (20,000 cells) were seeded onto a microslide VI0.4 (Ibidi, part number 80606). The next day, the medium was replaced with DMEM-high glucose medium supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin / streptomycin, 2.5 μM icFSP1, and 10 mM HEPES. After 2–4 hours of incubation in the presence of icFSP1, two to five rectangular areas containing three or more FSP1 aggregates were selected as photobleached regions. The image before the photobleached region was designated time "0." The selected regions were photobleached at maximum laser intensity, and FRAP was observed at a minimum interval (approximately 5 seconds) using an LSM880 microscope (ZEISS).

[0271] To quantify the FRAP rate, regions of interest (ROIs) for each condensate in the photobleached region (i) and each condensate in the unphotobleached region (c) were determined using ImageJ / Fiji, and the mean fluorescence intensity f of condensate i at time t was calculated. i (t) was calculated. After obtaining each fluorescence value at each time, f i (t) to f i The relative fluorescence intensity (Rf i (t)) was obtained. To reflect the quenching effect during observation and photobleaching, each Rf i (t) is the relative value of the unbleached aggregates (f c (t) / f c (0)) and normalized as follows: Fi(t) = Rf i (t) / Rf c (t)=[f i (t) / f i (0)] / [f c (t) / f c (0)]. Finally, the FRAP rate [%] in the particle at time t is expressed as F i Calculated as the average of (t) × 100.

[0272] 2.10 Live cell imaging For co-staining or wash analysis, Pfa1 cells (15,000–30,000 cells) were seeded in a 35 mm low microdish (Ibidi, part number 80136) and cultured overnight. The following day, the cell culture medium was replaced with FluoroBrite DMEM (Gibco, part number A1896701) containing 10% FBS, 2 mM L-glutamine, and 1% penicillin / streptomycin. Live-cell microscopy was performed using 3D Cell Explorer (Nanolive) with Eve v1.8.2 software and the appropriate filter sets. During imaging, cells were maintained in a temperature-controlled culture chamber at 37°C and 5% CO2. For co-staining analysis, cells were pretreated with 5 μM Liperfluo (Dojindo, product number L248-10) for 1 hour, then the medium was changed to FluoroBrite DMEM containing 0.2 μg / mL propidium iodide (PI, Sigma, P4170), and image acquisition was initiated using Nanolive. After recording one image, icFSP1 diluted 1:100 in FluoroBrite DMEM was added to the dish (final concentration: 10 μM) while image recording continued. Images were acquired every 10 minutes for over 4 hours, and signals were acquired using GFP, BFP, and RFP filter sets. For washout experiments, the DMEM-high glucose medium was replaced with FluoroBrite DMEM medium before the experiment, and data acquisition was then performed using Nanolive. After recording several images, icFSP1 diluted 1:100 in FluoroBrite DMEM was added to the dish (final concentration: 2.5 μM), and image recording continued for 4 hours. The dishes were then carefully washed once with fresh, icFSP1-free FluoroBrite DMEM and replenished. Image acquisition was then immediately resumed. Images were recorded every 5 minutes for an additional hour, for a total data acquisition time of approximately 5 hours.

[0273] To determine the number of intracellular condensates, Pfa1 cells (15,000–20,000 cells) were seeded onto 8-well macroslides (Ibidi, part number 80826) and cultured overnight. The following day, the medium was replaced with DMEM-high glucose supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin / streptomycin, 2.5 μM icFSP1, and Hoechst. Immediately afterwards, focus adjustment was performed and Hoechst and EGFP images were recorded using a 20x air objective, a CCD camera (CoolSnap ES2, Photometrics), and corresponding filter sets on an Axio Observer Z1 imaging system equipped with VisView v4.0 (Visitron Systems, ZWISS). During imaging, cells were maintained at 37°C in a 5% CO2 atmosphere using a temperature-controlled incubation chamber. The imaging software ImageJ / Fiji was used for visualization, and CellProfiler (v4.1.3, Broad Institute) was used to count the number of aggregates per cell.

[0274] 2.11 Staining of tumor tissue Dissected tissues were fixed overnight at 4°C in 4% paraformaldehyde in PBS. For immunofluorescence (IF) staining, fixed tissues were left overnight at 4°C in 20% sucrose in PBS, then embedded in OCT mounting compound (Tissue Tek, Sakura) on dry ice and stored at -80°C. Frozen tissues were sectioned at 5 μm thickness using a Cryostat Microm HM 560 (Thermo Fisher Scientific) at -30°C. Tissue sections were postfixed in 1% paraformaldehyde in PBS for 10 minutes, followed by further fixation in 67% ethanol and 33% acetic acid for 10 minutes. The sections were incubated in blocking solution (PBS containing 5% goat serum and 0.3% Triton X-100) for 30 minutes, and then incubated overnight at 4°C in primary antibodies (anti-HA antibody (clone: ​​3F10, 1:10, developed in-house), anti-4HNE antibody (JaICA, product number: HNEJ-2, 1:50), or anti-AIFM2 antibody (FSP1, clone: ​​14D7, undiluted, developed in-house)) diluted in blocking solution. The next day, sections were incubated for 2 h at room temperature in secondary dilution buffer (PBS supplemented with 1% BSA and 0.3% Triton X-100) with appropriate fluorochrome-conjugated secondary antibodies (goat anti-rat Alexa Fluor 488 IgG (H+L) (1:500, A-11006, Invitrogen), goat anti-mouse IgG H&L Alexa Fluor 647 (1:500, ab150115, Abcam), and donkey anti-rat IgG Alexa Fluor 555 (1:500, ab150154, Abcam). DNA was visualized by DAPI staining for 5 min, and slides were mounted with Aqua- / poly Mount. Images were acquired with an LSM880 microscope (ZEISS) and analyzed with ZEN Blue or ImageJ / Fiji software.

[0275] 2.12 Lentiviral production and transduction Lentiviral particles were produced in HEK293T cells. For mouse cells, the ecotropic envelope protein of murine leukemia virus (MLV) was used, and for human cells, the amphitropic envelope protein VSV-G was used. A third-generation lentiviral packaging system consisting of a transfection plasmid, an envelope plasmid (pEcoEnv-IRES-puro or pHCMV-EcoEnv (ecotropic particles) or pMD2.G (pantropic particles)), and a packaging plasmid (pMDLg_pRRE and pRSV_Rev or psPAX2) was co-lipofected into HEK293T cells using a transfection reagent (PEI MAX (Polysciences, part number 24765) or X-tremeGENE HP Reagent (Roche, part number 06366236001)). Cell culture supernatants containing viral particles were collected 48–72 hours post-transfection and filtered through a 0.45 μm PVDF filter (Millipore, part number SLHV033RS) before being used for lentiviral transduction.

[0276] Cells were seeded in 12-well or 6-well plates in medium containing 10 μg / ml protamine sulfate, and the lentivirus was incubated with the cells overnight. The next day, the cell culture medium was replaced with fresh medium containing the appropriate antibiotic, such as puromycin (Gibco, Part Number A11138-03; 1 μg / ml), blasticidin (Invitrogen, Part Number A1113903; 10 μg / ml), or G418 (Invitrogen, Part Number 10131-035; 1 mg / ml), and the cells were cultured until non-transduced cells died.

[0277] 2.13 Gene knockout using CRISPR-Cas9 sgRNAs were designed to target critical exons of the target genes, and gene knockout was confirmed by Western blotting. The sgRNAs were cloned into BsmBI-digested lentiCRISPRv2-blast, lentiCRISPRv2-puro, and lentiGuide-neo vectors (Addgene, part numbers 98293, 98290, and 139449). To generate knockout cells, MDA-MB-436, 786-O, A375, H460, B16F10, and 4T1 cells were transiently co-transfected with the sgRNAs expressed from lentiCRISPRv2-blast and lentiCRISPRv2-puro using X-tremeGENE HP Reagent, as previously reported. One day after transfection, selection was initiated with puromycin (1 μg / ml) and blasticidin (10 μg / ml). After 2–3 days of selection, single-cell clones were isolated and verified by immunoblotting and genomic DNA sequencing. To generate Dox-inducible FSP1-EGFP-expressing cells, H460 FSP1KO cells were transduced with lentivirus (pCW-FSP1WT-EGFP-blast or pCW-FSP1Q319K-EGFP-blast). After Dox treatment, scalable FSP1 expression was confirmed by immunoblotting.

[0278] 2.14 Microsomal Stability The objective of this study was to evaluate the metabolic stability of four test substances and a reference compound in human and mouse liver microsomes at five 40-minute time points using HPLC-MS. Metabolic stability is defined as the rate of loss of parent compound over time in the presence of a metabolically active test system.

[0279] 2.14.1 Reagents and Consumables DMSO Chromasolv Plus, HPLC grade, ≥99.7% (Sigma-Aldrich, USA; Part No. 34869), acetonitrile Chromasolv, HPLC gradient grade ≥99.9% (Sigma-Aldrich, USA; Part No. 34851), methanol HiPerSolv, HPLC gradient grade, ≥99.9% (VWR Chemicals, USA; Part No. 20864.320), potassium phosphate monobasic (Bio-Basic, Canada; Lot No. N9016010), potassium phosphate dibasic (Bio-Basic, Canada; Lot No. MA7100050), magnesium chloride hexahydrate (Santa Cruz Biotechnology, USA; sc-203126A), human liver microsomes: pooled, mixed sex (XenoTech, H0630 / lot N#1210097), mouse liver microsomes: pooled, male Balb / c mice (XenoTech, M3000 / lot no. 2010026), glucose-6-phosphate dehydrogenase from baker's yeast, type XV (Sigma-Aldrich, USA; part number G6378), D-glucose-6-phosphate sodium salt (Sigma-Aldrich, USA; part number G7879-1G), NADPH tetrasodium salt (BLD Pharmatech, part number BD116582), formic acid (Sigma-Aldrich, 94318), verapamil hydrochloride (Sigma Aldrich, USA; Part Number V4629), niclosamide (Sigma-Aldrich, USA; Part Number N3510), test compound (20 mM) in DMSO stock solution (+, -), propranolol hydrochloride (Sigma-Aldrich, P0884), imipramine hydrochloride (Sigma-Aldrich, I7379), diclofenac, 96% purity (Enamine, #EN300-119509), Phenomenex Luna® C18 HPLC column, 2.1 x 50 mm, 5 μm (Part Number 5291-126), Phenomenex Luna® C18 HPLC column, 2 x 30 mm, 5 μm (Serial Number 146953-2), Matrix™ 0.75 ml empty tube (Part Number 4170), pipette tips (Thermo Scientific).

[0280] 2.14.2 Equipment Gradient HPLC system (Shimadzu), triple quadrupole mass detector API3000 with TurboIonSpray ion source (AB Sciex, Canada), nitrogen generator N2-04-L1466, nitrogen purity 99%+ (Whatman), environmental incubator shaker G24; digital refrigerated incubator / shaker Innova 4330 (New Brunswick Scientific), water purification system Millipore Milli-Q Gradient A10 (Millipore, France), multichannel pipettes 1-30 μL, 2-125 μL, and 30-850 μL (Thermo Scientific).

[0281] 2.14.3 Analysis System All measurements were performed using a Shimadzu HPLC system, including a vacuum degasser, gradient pump, reversed-phase HPLC column, column oven, and autosampler. Mass spectrometry was performed using an API3000 triple quadrupole mass detector (AB Sciex, Canada) equipped with a Turbo V ion source and a TurboIonSpray ion source with a TurboIonSpray interface. The TurboIonSpray ion source was used in both positive and negative ion modes. Data acquisition and system control were performed using AB Sciex Analyst 1.6.3 software.

[0282] 2.14.4 Method Microsome incubations were performed in five 30 μL aliquots (one per time point) in a 96-well plate. Liver microsome incubation medium contained phosphate buffer (100 mM, pH 7.4), MgCl2 (3.3 mM), NADPH (3 mM), glucose-6-phosphate (5.3 mM), glucose-6-phosphate dehydrogenase (0.67 units / ml), and 0.42 mg of liver microsomal protein per ml. In control reactions, the NADPH cofactor system was replaced with phosphate buffer. Microsomes were incubated with test compound (2 μM, final solvent concentration 1.6%) at 37°C with agitation at 100 rpm. Five time points were analyzed over a 40-minute period. The reaction was stopped by adding 5x acetonitrile containing an internal standard to the incubation aliquot, followed by centrifugation at 5500 rpm for 5 minutes to precipitate the protein. Each reaction was performed in duplicate. The supernatant was analyzed by an HPLC system coupled to a tandem mass spectrometer.

[0283] Elimination rate constant (kel), half-life (t 1 / 2 ), and intrinsic clearance (Cl int ) was determined using linear regression analysis on ln(AUC) versus time plots (Note 1):

[0284]

number

[0285] 2.14.5 Interpretation of Microsomal Stability Assay Data Test compounds can be classified into low, intermediate, or high clearance groups based on microsomal stability. Intrinsic clearance (in vitro) can be recalculated into intrinsic clearance (in vivo) using literature data on liver weight and hepatic blood flow using the following formula (24):

[0286]

number

[0287] In the above formula, In vivo CL int : Estimated in vivo intrinsic clearance, mL / min / kg In vitro CL int : In vitro microsomal clearance, mL / min / kg PBSF (physiologically based scaling factor): Mean recovery of microsomes for microsome prediction and hepatocyte cellularity for hepatocyte prediction, mg / g LW: liver weight / body weight kg, g / kg f u mic or f u heps : Unbound fraction in microsomes or stem cells (can be determined from plasma protein binding studies, tentative value if unknown)

[0288] In vivo Clint hepatic clearance can be predicted based on a “well-stirred” liver model using the following equation (25):

[0289]

number

[0290] In the above formula, CLH: predicted hepatic clearance mL / min / kg QH: liver blood flow, mL / min / kg fu: unbound fraction in blood CL int :Predicted in vivo clearance mL / min / kg is.

[0291] CL calculated using literature data on liver weight (Ref. 3) and microsomal protein concentration (Ref. 26, 27) for mouse, rat, and human species. int The classification values ​​are shown in the table below.

[0292] [Table 3]

[0293] 2.14.6 Results The results of the microsomal stability measurements are shown in Table 4.

[0294] [Table 4]

[0295] Pharmacokinetics 2.15.1 Research Subjects The objective of this study was to characterize the pharmacokinetic properties of compound 13 in male Balb / cAnN mice after intraperitoneal (IP) administration. After a single dose, the concentrations of the test compound were measured in plasma samples over time by an LC-MS / MS method.

[0296] 2.15.2 Reagents and Consumables DMSO Chromasolv DMSO Chromasolv Plus, HPLC grade, ≥99.7% (Sigma-Aldrich, USA; product number 34869); Ch acetonitrile Chromasolv, gradient grade for HPLC ≥ 99.9% (Sigma-Aldrich, USA; product number 34851); Methanol Chromasolv Plus, HPLC grade, ≥99.9% (Sigma-Aldrich, USA; product number 34860); Formic acid for mass spectrometry (Sigma-Aldrich, part number 94318); Pharmaceutical grade DMSO, ≥99.9% (pharmaceutical grade. PanReact Applichem, Germany, product number 191954.1611); (2-hydroxypropyl) β-cyclodextrin (L'eternel Worl; purity 99.5%); Water for injections (“Arterium”, Ukraine, lot number 233142); sodium hydroxide (Bio Basic Canada, Canada, product number A620617-0500); Microtina® blood collection tube K3EDTA, Henso, Lot No. 191010; 2,2,2-tribromoethanol 97% (Sigma-Aldrich; part number T48402); Amyl alcohol (UOS, Ukraine); tubes (Falcon, 5 ml, 12 × 75 mm, USA); tubes (Eppendorf, 1.5 mL); Syringe (BD, 1mL, Tuberculin slip tip, USA, REF3096). The compound propetrine was used as an internal standard (IS). Compound FS-30 (13) was used as a dry powder. The vehicle was DMSO-40% 2HPβCD in water for injection, w / v (10%:90%, v / v). The formulation was prepared under "red light." To prepare the formulation, 0.3 ml of DMSO was added to 6 mg of the compound; the mixture was vortexed for 10 seconds to obtain a clear pink solution. Next, 2 ml of 40% 2HPβCD aqueous solution was added to the formulation; the mixture was vortexed for 10 seconds to obtain a clear pink solution (pH 4.07). The formulation was then neutralized with 2 μl of 1 M NaOH, and 0.698 ml of 40% 2HPβCD aqueous solution was added to the formulation; the mixture was vortexed for 10 seconds to obtain a clear yellow solution (pH 7.29). The batch of formulation to be used was prepared 5 minutes prior to the in vivo test.

[0297] 2.15.3 Equipment gradient HPLC system (Shimadzu, Japan); MS / MS detector API3000 equipped with a TurboIonSpray electrospray module (AB Sciex, Canada); IMT-PN1280OG nitrogen generator (INMATEC Technologies GmbH, Germany); water generation system Arium mini (Sartorius, Germany); VWR Analog Vortex Mixer VM3000 (VMR, USA); Centrifuge 4-15C (Sigma, Germany)

[0298] 2.15.4 Research design Study design, animal selection, handling, and treatment were all performed in accordance with the Enamine PK study protocol and Institutional Animal Care and Use Guidelines (BACUC approval number #HC-PK-10112023). Animal procedures and sample preparation were performed by Enamine / Bienta animal facility personnel. Male Balb / cAnN mice (10 weeks old, weight range 19.6g-23.3g, mean weight 21.0g, standard deviation 0.9g) were used in this study. Animals were randomly assigned to treatment groups prior to the pharmacokinetic study; all animals fasted for 4 hours before dosing. Six blood collection time points (5, 15, 30, 60, 120, and 360 minutes) were included in this pharmacokinetic study. Each treatment group consisted of four animals, and one control group was treated with vehicle. Dosing was performed according to the treatment schedule shown in Table 5. Prior to blood collection, mice were intraperitoneally administered 2,2,2-tribromoethanol at a dose of 150 mg / kg. Blood was collected via the orbital vein using Microtina® containing K3EDTA. After blood collection, animals were euthanized by cervical dislocation. Blood samples were centrifuged at 3000 rpm for 10 minutes. All samples were processed immediately, flash-frozen, and stored at -70°C until further analysis.

[0299] [Table 5]

[0300] 2.15.5 Sample Processing Plasma samples (40 μL) were mixed with 200 μL of IS(90) solution, mixed by pipetting, and centrifuged at 6000 rpm for 4 minutes. 0.25 μL of each supernatant was injected into the LC-MS / MS system. A solution of prometryne at a concentration of 200 ng / ml in a water-methanol mixture (1:9, volume ratio) was used as an internal standard (IS(90)) for the quantification of 13 in plasma samples.

[0301] 2.15,6 Sample Analysis Analysis of plasma samples was performed by staff at Enamine / Vienta's Bioanalytical Laboratory. The concentrations of 13 compounds in the samples were measured using high-performance liquid chromatography / tandem mass spectrometry (HPLC-MS / MS). A Shimadzu HPLC system with two metering pumps, an autosampler SIL-20AC, a subcontroller FCV-14AH, and a degasser DGU-14A, was used. Mass spectrometry was performed using an AB Sciex (Canada) API3000 (Triple Quadropole) instrument with an electrospray (ESI) interface. Data acquisition and system control were performed using AB Sciex Analyst 1.6.3 software.

[0302] 2.15.7 HPLC-MS / MS conditions Chromatography conditions Column: Synergi Hydro-RP 80A, 2 x 30 mm, 4 μm Mobile phase A: acetonitrile:water:formic acid = 50:950:1 Mobile phase B: acetonitrile:formic acid = 100:0.1 Linear gradient: 0 min 10% B, 1.0 min 100% B, 1.2 min 100% B, 1.21 min 10% B, 2.4 min stop Elution rate: 400 μL / min. The flow to the detector is adjusted by a valve to 1.5-1.8 min. Column temperature: 30℃ MS / MS detection Scan type: Positive MRM, Ion source: Turbo spray, Ionization mode: ESI, Nebulization gas: 15 L / min, Curtain gas: 8 L / min, Collision gas: 4 L / min Ion spray voltage: 5000V, temperature: 400℃

[0303] [Table 6]

[0304] 2.15.8 Preparation of calibration standards Calibration standard for quantification of compound 13 in plasma samples: Compound 13 was dissolved in DMSO, and the resulting solution at a concentration of 2 mg / ml was used to prepare calibration standards (stock solution). The stock solution was serially diluted with IS(90) to prepare a series of calibration solutions with final concentrations of 10,000, 4,000, 2,000, 1,000, 400, 200, 100, 40, and 20 ng / ml. A calibration curve was generated using blank mouse plasma samples. Calibration standard solutions were prepared by mixing 40 μl of blank plasma sample with 200 μl of the corresponding calibration solution. After mixing by pipetting, the mixture was centrifuged at 6,000 rpm for 4 minutes, and 0.25 μl of each supernatant was injected into the LC-MS / MS system.

[0305] 2.15.9 Pharmacokinetic Analysis Test compound concentrations below the lower limit of quantitation (LLOQ = 100 ng / ml) were set to zero. Pharmacokinetic data analysis was performed in WinNonlin 5.2 (PharSight) using a noncompartmental, bolus injection or single-dose model, or an extravascular administration analysis model. Data below the LLOQ were treated as missing values ​​to improve the reliability of T calculations. For each dosing condition, the final concentration values ​​obtained at each time point were analyzed for outliers using Grubbs' test with the significance level set at p<0.05.

[0306] 2.15.10 Results The pharmacokinetic parameters of compound 13 were calculated by non-compartmental analysis using the WinNonlin program as shown in Table 7. The corresponding plasma concentration-time curves are shown in Figure 13. Based on the total drug concentration, the plasma concentration was calculated to be EC 50 The value was more than 10 times higher than the previous value.

[0307]

Table 7

[0308] <References> 1. Yang, W.S., SriRamaratnam, R., Welsch, M.E., Shimada, K., Skouta, R., Viswanathan, V.S., Cheah, J.H., Clemons, P.A., Shamji, A.F., Clish, C.B., et al. (2014). Regulation of ferroptotic cancer cell death by GPX4. Cell 156, 317-331. 10.1016 / j.cell.2013.12.010. 2. Friedmann Angeli, J.P., Schneider, M., Proneth, B., Tyurina, Y.Y., Tyurin, V.A., Hammond, V.J., Herbach, N., Aichler, M., Walch, A., Eggenhofer, E., et al. (2014). Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice. Nature cell biology 16, 1180-1191. 10.1038 / ncb3064. 3. Ingold, I., Berndt, C., Schmitt, S., Doll, S., Poschmann, G., Buday, K., Roveri, A., Peng, X., Porto Freitas, F., Seibt, T., et al. (2018). Selenium Utilization by GPX4 Is Required to Prevent Hydroperoxide-Induced Ferroptosis. Cell 172, 409-422 e421. 10.1016 / j.cell.2017.11.048. 4. Conrad, M., Angeli, J.P., Vandenabeele, P., and Stockwell, B.R. (2016). Regulated necrosis: disease relevance and therapeutic opportunities. Nature reviews. Drug discovery 15, 348-366. 10.1038 / nrd.2015.6. 5. Zheng, J., and Conrad, M. (2020). The Metabolic Underpinnings of Ferroptosis. Cell Metab 32, 920-937. 10.1016 / j.cmet.2020.10.011. 6. Jiang, X., Stockwell, B.R., and Conrad, M. (2021). Ferroptosis: mechanisms, biology and role in disease. Nature reviews. Molecular cell biology 22, 266-282. 10.1038 / s41580-020-00324-8. 7. Conrad, M., Lorenz, S.M., and Proneth, B. (2021). Targeting Ferroptosis: New Hope for As-Yet-Incurable Diseases. Trends Mol Med 27, 113-122. 10.1016 / j.molmed.2020.08.010. 8. Dixon, S.J., Lemberg, K.M., Lamprecht, M.R., Skouta, R., Zaitsev, E.M., Gleason, C.E., Patel, D.N., Bauer, A.J., Cantley, A.M., Yang, W.S., et al. (2012). Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149, 1060-1072. 10.1016 / j.cell.2012.03.042. 9. Angeli, J.P.F., Shah, R., Pratt, D.A., and Conrad, M. (2017). Ferroptosis Inhibition: Mechanisms and Opportunities. Trends in pharmacological sciences 38, 489-498. 10.1016 / j.tips.2017.02.005. 10. Larraufie, M.H., Yang, W.S., Jiang, E., Thomas, A.G., Slusher, B.S., and Stockwell, B.R. (2015). Incorporation of metabolically stable ketones into a small molecule probe to increase potency and water solubility. Bioorganic & medicinal chemistry letters 25, 4787-4792. 10.1016 / j.bmcl.2015.07.018. 11. Cramer, S.L., Saha, A., Liu, J., Tadi, S., Tiziani, S., Yan, W., Triplett, K., Lamb, C., Alters, S.E., Rowlinson, S., et al. (2017). Systemic depletion of L-cyst(e)ine with cyst(e)inase increases reactive oxygen species and suppresses tumor growth. Nature medicine 23, 120-127. 10.1038 / nm.4232. 12. Eaton, J.K., Ruberto, R.A., Kramm, A., Viswanathan, V.S., and Schreiber, S.L. (2019). Diacylfuroxans Are Masked Nitrile Oxides That Inhibit GPX4 Covalently. Journal of the American Chemical Society 141, 20407-20415. 10.1021 / jacs.9b10769. 13. Eaton, J.K., Furst, L., Ruberto, R.A., Moosmayer, D., Hilpmann, A., Ryan, M.J., Zimmermann, K., Cai, L.L., Niehues, M., Badock, V., et al. (2020). Selective covalent targeting of GPX4 using masked nitrile-oxide electrophiles. Nature chemical biology 16, 497-506. 10.1038 / s41589-020-0501-5. 14. Mei, J., Webb, S., Zhang, B., and Shu, HB. The p53-inducible apoptotic protein AMID is not required for normal development and tumor suppression. Oncogene 25, 849–856. doi: 10.1038 / sj.onc. 15. Tonnus, W., C. Meyer, C. Steinebach, A. Belavgeni, A. von Massenhausen, A. Gonzalez, NZ, F. Maremonti, F. Gembardt, N. Himmerkus, M. Latk, et al. (2021). Dysfunction of the key ferroptosis-surveiling systems hypersensitizes mice to tubular necrosis during acute kidney injury. Nature communications 12, 4402. 10.1038 / s41467-021-24712-6. 16. Doll, S., Freitas, FP, Shah, R., Aldrovandi, M., da Silva, MC, Ingold, I., Goya Grocin, A., Xavier da Silva, TN, Panzilius, E., Scheel, CH, et al. (2019). FSP1 is a glutathione-independent ferroptosis suppressor. Nature 575, 693–698. 10.1038 / s41586-019-1707-0 17. Zheng, J., Sato, M., Mishima, E., Sato, H., Proneth, B., and Conrad, M. (2021). Sorafenib fails to trigger ferroptosis across a wide range of cancer cell lines. Cell death & disease 12, 698. 10.1038 / s41419-021-03998-w. 18. Schmitt, A., Xu, W., Bucher, P., Grimm, M., Konantz, M., Horn, H., Zapukhlyak, M., Berning, P., Brandle, M., Jarboui, M.A., et al. (2021). Dimethyl fumarate induces ferroptosis and impairs NF-kappaB / STAT3 signaling in DLBCL. Blood 138, 871-884. 10.1182 / blood.2020009404. 19. Seiler, A., Schneider, M., Forster, H., Roth, S., Wirth, E.K., Culmsee, C., Plesnila, N., Kremmer, E., Radmark, O., Wurst, W., et al. (2008). Glutathione peroxidase 4 senses and translates oxidative stress into 12 / 15-lipoxygenase dependent- and AIF-mediated cell death. Cell Metab 8, 237-248. 10.1016 / j.cmet.2008.07.005. 20. Bersuker , K , Hendricks , JM , Li , Z , Magtanong , L , Ford , B , Tang , PH , Roberts , MA , Tong , B , Maimone , TJ , Zoncu , R , et al. (2019). The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis. Nature 575, 688–692. 10.1038 / s41586-019-1705-2 21. Pontel, LB, Good-Costa, A., Morellato, AE, Carvalho Santos, J., Roue, G., and Esteller, M. (2022). Acute lymphoblastic leukemia requires GSH-dependent ferroptosis defenses to overcome FSP1-epigenetic silencing. Redox biology 55 , 102408 . 22. T. Nakamura, M. Ogawa, K. Kojima, S. Takayanagi, S. Ishihara, K. Hattori, I. Naguro, and H. Ichijo (2021). The mitochondrial Ca2+ uptake regulator, MICU1, is involved in cold stress-induced ferroptosis. EMBO reports 22, e51532–e51532. doi: 10.15252 / embr. 23. Mishima, E., Ito, J., Wu, Z., Nakamura, T., Wahida, A., Doll, S., Tonnus, W., Nepachalovich, P., Eggenhofer, E., Aldrovandi, M., et al. (2022). A non-canonical vitamin K cycle is a potent ferroptosis suppressor. Nature. 10.1038 / s41586-022-05022-3. 24. Wood, F. L., Houston, J. B., & Hallifax, D. (2017). Clearance prediction methodology needs fundamental improvement: Trends common to rat and human hepatocytes / microsomes and implications for experimental methodology. Drug Metabolism and Disposition, 45(11), 1178-1188. https: / / doi.org / 10.1124 / dmd.117.077040 25. Lavee, T., & Funk, C. (2007). In vivo absorption, distribution, metabolism, and excretion studies in Discovery and Development. Comprehensive Medicinal Chemistry II, 31-50. https: / / doi.org / 10.1016 / b0-08-045044-x / 00118-8 26. Barter Z.E., et al., Scaling factors for the extrapolation of in vivo metabolic drug clearance from in vitro data: reaching a consensus on values of human microsomal protein and hepatocellularity per gram of liver, Current Drug Metabolism, 2007, 8, 33-45. 27. Iwatsubo T., et al., Prediction of species differences (rats, dogs, humans) in the in vivo metabolic clearance of YM796 by the liver from in vitro data, Journal of Pharmacology and Experimental Therapeutics, 1997, 283, 462-469.

Claims

1. A compound of formula (I) or formula (II), preferably formula (I), for use in the treatment of cancer, 【Chemistry 1】 G is, 【Chemistry 2】 selected from the group consisting of: M is, 【Transformation 3】 selected from the group consisting of: Z is CH, C-(C １ -C ６ ) alkyl, and N, preferably CH; A is CH, C-(C １ -C ６ ) alkyl, and N, preferably CH; Q is selected from the group consisting of CH, C—(C １ -C ６ ) alkyl, and N, preferably CH; E is -CO-, -SO ２ -, preferably -CO-; L is CH, N, C—F, C—Cl, and C—(C １ -C ６ ) alkyl, preferably CH; X is CH, N, C—F, C—Cl, and C—(C １ -C ６ ) alkyl, preferably CH; Y is CH, N, C—F, C—Cl, and C—(C １ -C ６ ) alkyl, preferably CH; X and Y may be part of a ring as shown in formula (III); 【Chemistry 4】 n is an integer from 1 to 3; J is CH, N, C—F, C—Cl, and C—(C １ -C ６ ) alkyl, preferably CH; D １ , D ２ , D ３ , D ４ , D ５ are independently selected from the group consisting of C and N; R １ is -(C １ -C ６ ) alkyl, -(C ３ -C ６ ) cycloalkyl, —CH ２ OH, -CH ２ F, -CHF ２ , and -CF ３ and preferably -(C １ -C ６ ) alkyl; R ２ is H, -(C １ -C ６ ) alkyl, —CF ３ , —Cl, and —F, preferably H; R ３ is H, -(C １ -C ６ ) alkyl, —CF ３ , -O(C １ -C ６ ) alkyl, —Cl, and —F, preferably —O(C １ -C ６ ) alkyl; R ４ is H, -(C １ -C ６ ) alkyl, —O(C １ -C ６ ) alkyl, —CF ３ , —Cl, and —F; preferably —O(C １ -C ６ ) alkyl; R ５ is H, -(C １ -C ６ ) alkyl, —O(C １ -C ６ ) alkyl, —CF ３ , —Cl, and —F, preferably —O(C １ -C ６ ) alkyl; R ６ is H, -(C １ -C ６ ) alkyl, —CF ３ , —Cl, and —F, preferably H; R in adjacent position ２ , R ３ , R ４ , R ５ and R ６ Two of the groups are 【Transformation 5】 may be part of a ring selected from o is an integer from 1 to 4; p is an integer from 1 to 4; q is 1 or 0; R ７ and R ８ is H, and -(C １ -C ６ ) alkyl; R ７ and R ８ may form a 3- to 5-membered cycloalkyl ring; R ９ is H, and -(C １ -C ６ ) alkyl; and pharmacologically acceptable salts thereof. compound.

2. 10. A method for treating cancer comprising administering to a patient a composition comprising a compound as defined in claim 1 and at least one excipient that is pharmacologically acceptable for use in the treatment of cancer. Pharmaceutical compositions.

3. The cancer is a cancer that expresses ferrotosis suppressor protein-1 (FSP1), A compound for use according to claim 1 or a pharmaceutical composition for use according to claim 2.

4. The cancer is selected from prostate cancer, leukemia, liver cancer, breast cancer, hepatocellular carcinoma, cholangiocarcinoma, glioblastoma, uveal melanoma, adrenocortical carcinoma, thymoma, head and neck squamous cell carcinoma, cholangiocarcinoma, renal cancer, lymphoid tumors, diffuse large B-cell lymphoma, pancreatic adenocarcinoma, gallbladder cancer, lymphoma, myeloma, gastric cancer, brain tumor, skin cancer, colon / colorectal cancer, bile duct cancer, neuroblastoma, bone tumor, and lung cancer; A compound for use according to claim 1 or a pharmaceutical composition for use according to claim 2.

5. a) the leukemia is selected from acute myeloid leukemia, acute lymphocytic leukemia, and chronic myeloid leukemia; and / or b) the renal cancer is selected from clear cell renal cell carcinoma and renal cell carcinoma; and / or c) the lung cancer is selected from small cell lung cancer, non-small cell lung cancer, and mesothelioma; A compound for use according to claim 4 or a pharmaceutical composition for use according to claim 4.

6. The compound is 【Chemistry 6-1】 【Chemistry 6-2】 【Transformation 6-3】 【Chemistry 6-4】 【Transformation 6-5】 A compound for use according to any one of claims 1, 3 to 5, or a pharmaceutical composition according to any one of claims 2 to 5, selected from the group consisting of:

7. The compound is 【Transformation 7】 selected from the group consisting of A compound for use according to any one of claims 1, 3 to 5, or a pharmaceutical composition according to any one of claims 2 to 5.

8. The compound is 【Transformation 8】 selected from the group consisting of A compound for use according to any one of claims 1, 3 to 5, or a pharmaceutical composition according to any one of claims 2 to 5.

9. A compound of formula (I) or (II), 【Chemistry 9】 G is, 【Chemistry 10】 selected from the group consisting of: M is, 【Chemistry 11】 selected from the group consisting of: Z is CH, C-(C １ -C ６ ) alkyl, and N, preferably CH; A is CH, C-(C １ -C ６ ) alkyl, and N, preferably CH; Q is CH, C-(C １ -C ６ ) alkyl, and N, preferably CH; E is -CO-, -SO ２ -, preferably -CO-; L is CH, N, C—F, C—Cl, and C—(C １ -C ６ ) alkyl, preferably CH; X is CH, N, C—F, C—Cl, and C—(C １ -C ６ ) alkyl, preferably CH; Y is CH, N, C—F, C—Cl, and C—(C １ -C ６ ) alkyl, preferably CH; X and Y may be part of a ring as shown in formula (III); 【Chemistry 12】 n is an integer from 1 to 3; J is CH, N, C—F, C—Cl, and C—(C １ -C ６ ) alkyl, preferably CH; D １ , D ２ , D ３ , D ４ , D ５ are independently selected from the group consisting of C and N; R １ is -(C １ -C ６ ) alkyl, -(C ３ -C ６ ) cycloalkyl, —CH ２ OH, -CH ２ F, -CHF ２ , and -CF ３ and preferably -(C １ -C ６ ) alkyl; R ２ is H, -(C １ -C ６ ) alkyl, —CF ３ , —Cl, and —F, preferably H; R ３ is H, -(C １ -C ６ ) alkyl, —O(C １ -C ６ ) alkyl, —CF ３ , —Cl, and —F, preferably —O(C １ -C ６ ) alkyl; R ４ is H, -(C １ -C ６ ) alkyl, —O(C １ -C ６ ) alkyl, —CF ３ , —Cl, and —F, preferably —O(C １ -C ６ ) alkyl; R ５ is H, -(C １ -C ６ ) alkyl, —O(C １ -C ６ ) alkyl, —CF ３ , —Cl, and —F, preferably —O(C １ -C ６ ) alkyl; R ６ is H, -(C １ -C ６ ) alkyl, —CF ３ , —Cl, and —F, preferably H; R in adjacent position ２ , R ３ , R ４ , R ５ and R ６ Two of the groups are 【Chemistry 13】 may be part of a ring selected from o is an integer from 1 to 4; p is an integer from 1 to 4; q is 0 or 1; R ７ and R ８ is H, and -(C １ -C ６ ) alkyl; R ７ and R ８ may form a 3- to 5-membered cycloalkyl ring; R ９ is H, and -(C １ -C ６ or a pharmaceutically acceptable salt thereof; does not have a structure selected from the group consisting of: 【Chemistry 14-1】 【Chemistry 14-2】 【Chemistry 14-3】 【Chemistry 14-4】 compound.

10. In formula (I) or (II), G is, 【Chemistry 15】 and Z is CH; A is CH; Q is CH; E is —CO—; L is CH; X is CH; Y is preferably CH; n is an integer from 1 to 3; J is CH; R １ Ha-(C １ -C ６ ) alkyl; preferably methyl; R ２ is H; R ３ is H or —O(C １ -C ６ ) alkyl; R ４ is H or —O(C １ -C ６ ) alkyl; R ５ is H, —Cl, or —O(C １ -C ６ ) alkyl; R ６ is selected from the group consisting of H or —Cl; preferably H; o is an integer from 1 to 4; p is an integer from 1 to 4; q is 0 or 1; R ７ and R ８ is H, and -(C １ -C ６ ) alkyl; R ７ and R ８ may form a 3- to 5-membered cycloalkyl ring; R ９ is H, and -(C １ -C ６ ) alkyl, A compound for use according to any one of claims 1, 3 to 5, a pharmaceutical composition according to any one of claims 2 to 5, or a compound according to claim 9.

Images