Combinations of retinaldehyde dehydrogenase 1 (RALDH1) inhibitors and immunostimulants, and methods of using them.

Abstract

This disclosure relates, in part, to a method for treating solid tumors in a subject where such treatment is needed, comprising the step of administering a retinaldehyde dehydrogenase 1 (RALDH1) inhibitor and an immunostimulant to the subject. This disclosure also relates to a pharmaceutical composition comprising a RALDH1 inhibitor and an immunostimulant.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority under § 119(e) of the United States Patent Act to U.S. Provisional Patent Application No. 63 / 452,978, filed on 17 March 2023, which is incorporated herein by reference in its entirety.

[0002] Statement on federally funded research and development This invention was made with government support under W81XWH-21-1-0592 granted by the Medical Research and Development Command and CA234027 granted by the National Institutes of Health. The government has certain rights in this invention.

[0003] Sequence List An XML file named "046483_7326WO1_SequenceListing," created on March 14, 2024, containing 6,408 bytes, is incorporated into this specification in its entirety by reference. [Background technology]

[0004] Background of the Invention Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide and the third leading cause of cancer-related death. The presence of chronic liver disease is a significant risk factor. While outcomes have improved with surveillance of high-risk patients and earlier detection through better diagnostic approaches, advanced HCC continues to present significant management challenges. Depending on the grade and stage of HCC, as well as underlying liver function, surgical resection is the treatment choice in most cases. Other options include liver transplantation, image-guided tumor ablation, tyrosine kinase inhibitors, and immune checkpoint blockers, although the latter are often ineffective for patients. Despite advances in the management of early HCC, the 5-year overall survival rate for advanced cases with distal metastasis remains at approximately 2%.

[0005] Therefore, there is a need in the art for methods and compositions for the treatment of solid tumors, including but not limited to HCC. The present disclosure addresses this need. SUMMARY OF THE INVENTION

[0006] In one aspect, the present disclosure provides a method of treating, preventing, and / or ameliorating a solid tumor in a subject in need thereof. In certain embodiments, the method comprises administering to the subject a pharmaceutically effective amount of (a) at least one immunostimulant, and (b) a retinaldehyde dehydrogenase 1 (RALDH1) inhibitor wherein the RALDH1 inhibitor is a compound of formula (I): TIFF2026510946000001.tif20128 or a salt, solvate, prodrug, stereoisomer, tautomer, or isotopologue thereof, wherein R is selected from the group consisting of optionally substituted C2-C8 heterocyclyl, optionally substituted phenyl, and optionally substituted C5-C8 cycloalkenyl, wherein each optional substituent in R 1a is independently selected from the group consisting of C1-C6 alkyl, CЗ-C6 cycloalkyl, C1-C6 alkoxy, optionally substituted phenyl, optionally substituted C2-C8 heterocyclyl, halogen, OH, N(R a )(R b ), NO2, CN, C(=O)R a , C(=O)N(R a )(R b ), S(=O)2R a , S(=O)2N(R a )(R b ), and N(R a )C(=O)R b and wherein each optional substituent is optionally substituted with at least one substituent selected from the group consisting of C1-C3 alkyl, C1-C3 alkoxy, halogen, CN, and NO2, and Here, R 1a Any two vicinal or geminal substituents within may combine with the atom to which they are bonded to form a C2-C8 heterocyclyl or C3-C8 cycloalkyl; R 1b and R 1c If present, H, C1-C6 alkyl, C1-C6 alkoxy, halogen, OH, N(R) a )(R b ), NO2, and CN are each independently selected; R 2 Each occurrence is independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, C1-C3 haloalkoxy, C1-C6 hydroxyalkyl, halogen, NO2, and CN; R 3 This is a C2-C8 heterocycline which may be substituted, a phenyl which may be substituted, and N(R a ) (C3-C8 cycloalkyl which may be substituted), and N(R a Selected from the group consisting of (C2-C8 heterocyclines that may be substituted), Here, R 3 Any substituent in the group may be C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, halogen, OH, or N(R) a )(R b ), NO2, CN, C(=O)R a , C(=O)N(R a )(R b ), S(=O)2R a , S(=O)2N(R a )(R b ), and N(R a )C(=O)R b Selected independently from the group consisting of; A is C6~C even if it is not substituted. 10 Selected from the group consisting of aryls and optionally substituted C2-C8 heterocyclines, Here, each substituent in A is a C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, halogen, OH, N(R)a )(R b ), NO2, CN, C(=O)R a , C(=O)N(R a )(R b ), S(=O)2R a , S(=O)2N(R a )(R b ), and N(R a )C(=O)R b Selected independently from the group consisting of; L is selected from the group consisting of -CH2-, -C(=O)-, and -S(=O)2-; X is N and CR 1c Selected from the group consisting of; n is an integer selected from the group consisting of 0, 1, 2, 3, and 4; and R a , R b , and R c Each occurrence is independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, and C2-C8 heterocyclyl. The compound of formula (I), or its salt, solvate, prodrug, stereoisomer, tautomer, or isotopolog.

[0007] In another aspect, the present disclosure provides a method for treating, preventing, and / or improving solid tumors in subjects where such treatment is needed. In one embodiment, the method involves a pharmaceutically effective amount of (a) at least one immunostimulant, and (b) Retinaldehyde dehydrogenase 1 (RALDH1) inhibitors The process includes administering the RALDH1 inhibitor to the target, where the RALDH1 inhibitor is 1-benzylindoline-2,3-dione; 2-((4-oxo-3-(3-(pyrrolidine-1-yl)propyl)-3,4-dihydrobenzo[4,5]thieno[3,2-d]pyrimidine-2-yl)thio)ethyl acetate; 2-((2-(sec-butyl)-3-oxo-2,3-dihydroimidazo[1,2-c]quinazoline-5-yl)thio)-N-(o-tolyl)butanamide; and 8-((4-(cyclopropanecarbonyl)piperazine-1-yl)methyl)-7-isopentyl-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione; or its salts, solvates, prodrugs, stereoisomers, tautomers, or isotopologs. It is a compound selected from the group consisting of the following.

[0008] In another context, this disclosure is: (a) at least one immunostimulant, (b) A pharmaceutically acceptable carrier, and (c) Retinaldehyde dehydrogenase 1 (RALDH1) inhibitors A pharmaceutical composition comprising, wherein the RALDH1 inhibitor is of formula (I): A compound of TIFF2026510946000002.tif20128, or its salt, solvate, prodrug, stereoisomer, tautomer, or isotopolog, During the ceremony, R 1a This is selected from the group consisting of optionally substituted C2-C8 heterocyclyls, optionally substituted phenyls, and optionally substituted C5-C8 cycloalkenyls. Here, R 1a Each of the substituents in the compound may be a C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkoxy, optionally substituted phenyl, optionally substituted C2-C8 heterocyclyl, halogen, OH, or N(R) a )(R b ), NO2, CN, C(=O)R a , C(=O)N(R a )(R b ), S(=O)2R a , S(=O)2N(R a )(R b ), and N(R a )C(=O)R bIndependently selected from the group consisting of, Here, each of the substituents may be substituted with at least one substituent selected from the group consisting of C1-C3 alkyl, C1-C3 alkoxy, halogen, CN, and NO2, and Here, R 1a Any two vicinal or geminal substituents within may combine with the atom to which they are bonded to form a C2-C8 heterocyclyl or C3-C8 cycloalkyl; R 1b and R 1c If present, H, C1-C6 alkyl, C1-C6 alkoxy, halogen, OH, N(R) a )(R b ), NO2, and CN are each independently selected; R 2 Each occurrence is independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, C1-C3 haloalkoxy, C1-C6 hydroxyalkyl, halogen, NO2, and CN; R 3 This is a C2-C8 heterocycline which may be substituted, a phenyl which may be substituted, and N(R a ) (C3-C8 cycloalkyl which may be substituted), and N(R a Selected from the group consisting of (C2-C8 heterocyclines that may be substituted), Here, R 3 Any substituent in the group may be C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, halogen, OH, or N(R) a )(R b ), NO2, CN, C(=O)R a , C(=O)N(R a )(R b ), S(=O)2R a , S(=O)2N(R a )(R b ), and N(R a )C(=O)R b Selected independently from the group consisting of; A is C6~C even if it is not substituted. 10Selected from the group consisting of aryl and optionally substituted C2-C8 heterocyclyl, where each optional substituent in A is C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, halogen, OH, N(R a )(R b ), NO2, CN, C(=O)R a , C(=O)N(R a )(R b ), S(=O)2R a , S(=O)2N(R a )(R b ), and N(R a )C(=O)R b independently selected from the group consisting of; L is selected from the group consisting of -CH2-, -C(=O)-, and -S(=O)2-; X is selected from the group consisting of N and CR 1c ; n is an integer selected from the group consisting of 0, 1, 2, 3, and 4; and each occurrence of R a , R b , and R c is independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, and C2-C8 heterocyclyl, There is provided a pharmaceutical composition which is a compound of formula (I), or a salt, solvate, prodrug, stereoisomer, tautomer, or isotopologue thereof.

[0009] In another aspect, the present disclosure provides (a) at least one immunostimulant, (b) a pharmaceutically acceptable carrier, and (c) a retinol dehydrogenase 1 (RALDH1) inhibitor A pharmaceutical composition comprising, wherein the RALDH1 inhibitor is 1-benzylindoline-2,3-dione, 2-((4-oxo-3-(3-(pyrrolidine-1-yl)propyl)-3,4-dihydrobenzo[4,5]thieno[3,2-d]pyrimidine-2-yl)thio)ethyl acetate, 2-((2-(sec-butyl)-3-oxo-2,3-dihydroimidazo[1,2-c]quinazoline-5-yl)thio)-N-(o-tolyl)butanamide, and 8-((4-(cyclopropanecarbonyl)piperazine-1-yl)methyl)-7-isopentyl-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione, or its salts, solvates, prodrugs, stereoisomers, tautomers, or isotopologs. The present invention provides a pharmaceutical composition comprising a compound selected from the group consisting of the following. [Brief explanation of the drawing]

[0010] The drawings illustrate, as examples but not as limitations, various aspects of this application in general. [Figure 1] This diagram provides a schematic representation of the pathway by which tumor-derived retinoic acid (RA) promotes immune evasion. [Figure 2] This diagram provides a schematic representation of the biosynthesis of RA from vitamin A (retinol) and the regulation of gene expression by RA in tumor monocytes. [Figure 3] This provides bar graphs showing the relative expression of RALDH1, RALDH2, and RALDH3 in human cell lines HT1080 (liposarcoma), SNU398 (hepatocellular carcinoma), and PLC (hepatoma) in in vitro cultures and in vivo xenografts (xenos) in NU / J mouse hosts. [Figure 4] This provides bar graphs showing the gene expression of RALDH1, RALDH2, and RALDH3 in mouse HCC cell lines (Hepa 1-6), normal liver, and fibrosarcoma cell line (B6-PRG), normalized for Hprt expression. [Figure 5]The pharmacological profiles of the RALDH1 inhibitors C-86 and C-91 are shown. n=3; for each example, the compound was formulated as a solution in 20% HP-β-CD in physiological saline. Dosage: 2 mg / kg for intravenous (iv) administration, 10 mg / kg for oral (po) administration; plasma samples were measured for drug exposure by LC-MS / MS; CD-1 mice or Sprague-Dawley rats were used; maximum drug concentration (Cmax) was observed at t = 5 minutes, which is the first sample time after iv administration. [Figure 6] This shows the RALDH activity of mouse (Hepa 1-6) and human (SNU398) HCC cell lines, as well as mouse fibrosarcoma (B6-PRG) cell lines, as measured by fluorescence using the Aldered® (also known as Aldefluor) assay. Fluorescence reflects RALDH activity. The control (top panel) shows baseline fluorescence, and the test (bottom panel) shows fluorescence given by RALDH activity. [Figure 7] RALDH activity was suppressed in vitro in SNU398 human HCC cells by treatment with either 100 nM C-86 or C-91, as detected by the Aldered (Aldefluor) assay, with DMSO serving as a control treatment. [Figure 8] The mean fluorescence intensity (MFI) of the Aldered assay is shown, in which SNU398 cells were treated with various concentrations of C-86 or C-91 (1, 10, 100, and 1000 nM) and DMSO was used as a control. [Figure 9] The graph shows a comparison of SNU398 cell growth in the presence of various concentrations (1, 10, 100, and 1000 nM) of C-86 or C-91 compared to a DMSO control, demonstrating that neither C-86 nor C-91 has a significant effect on SNU398 cell proliferation. [Figure 10] This report provides plots showing the frequencies of myeloid antigen-presenting cells and T cells. Hepa 1-6 cells were transplanted into the flanks of C57BL / 6J mice. The tumors were dissociated into single-cell suspensions and subjected to flow cytometry using the indicated markers. [Figure 11A] This paper provides a schematic diagram of the experimental protocol used to demonstrate the restoration of activated T cell proliferation by compound C-86 in cells exposed to tumor-conditioned medium (CM); (a) Monocytes isolated from mice or humans were exposed to a vehicle (DMSO) or compound C-86, and concurrently, T cells isolated from mice or humans were labeled (CFSE), activated in vitro (anti-CD3 / 28), and their proliferation was assessed by dye dilution by flow cytometry; (b) Monocyte-derived dendritic cells (MoDCs) and activated T cells were combined in different ratios (1:0, 1:1, or 1:2) and then exposed to the following conditions: vehicle (DMSO), compound C-86 alone, SNU398 HCC cell-conditioned medium (SNU398-CM), or CM combined with C-86 (SNU398-CM + C-86). Proliferation was assessed as described in (a) and compared to unstimulated T cells. [Figure 11B] A bar graph is provided showing the restoration of activated T cell proliferation by the compound C-86 for cells exposed to tumor-conditioned medium (CM). Addition of C-86 to MoDCs and T cells exposed to SNU398-CM completely restored their proliferation rate compared to the vehicle. [Figure 12A] Figures 12A-C show that intraperitoneal C-86 inhibits in vivo growth of hepatocellular carcinoma (HCC) cell line (Huh7) and exhibits a synergistic effect with BMS493. Figure 12A: Diagram illustrating the mechanism of inhibition of retinaldehyde (RA) synthesis in HCC cells and inhibition of RA-mediated transcription in monocytes. Figures 12B-12C: Results of intraperitoneal treatment of human HCC (Huh7) with C-86, BMS493, and C-86+BMS493 compared to a control. [Figure 12B] Please refer to the explanation in Figure 12A. [Figure 12C] Please refer to the explanation in Figure 12A. [Figure 13A] Figures 13A and 13B show the tumor volume (Figure 13A) and tumor mass (Figure 13B) after intraperitoneal administration of C-86. [Figure 13B] Please refer to the explanation in Figure 13A. [Figure 14]Figures 14A-14B demonstrate that the in vivo effect of C-86 on tumors can be mediated by macrophages. Figure 14A: Macrophage depletion by liposomal clodronate (CloLipo). Figure 14B: Treatment results of human HCC (Huh7) with C-86, CloLipo, and C-86+CloLipo. [Figure 15] Pharmacokinetic (PK) data for compounds C-86 and C-97 are provided. an = 3. Formulation: 20% HP-β-CD in physiological saline for IV and PO administration. b Dosage: 2 mg / kg for intravenous (IV) administration, 10 mg / kg for oral (PO) administration. c CD-1 mice were used. d Cmax was observed at t = 5 minutes, the first sampling time after IV administration. [Figure 16] As evaluated by the Aldefluor assay, C-97 inhibits RALDH1 activity in human HCC cells (SNU398); controls: dimethyl sulfoxide (DMSO); N,N-diethylaminobenzaldehyde (DEAB). DEAB is commonly used as a selective inhibitor of aldehyde dehydrogenase. WIN 18446 is a non-selective RALDH inhibitor. [Figure 17A] Figures 17A–17B provide a comparison of orally administered C-86 and C-97 in HCC cell inhibition, as evaluated by tumor volume (Figure 17A) and tumor mass (Figure 17B). [Figure 17B] Please refer to the explanation in Figure 17A. [Figure 18-1]Figures 18A-18I: HCC overexpresses RALDH1 and produces high levels of RA. Figure 18A: Raldh1 mRNA levels (y axis, RSEM, batch normalized) in various tumor types (x axis) from the TCGA database analyzed via the cBioPortal web interface. The data show higher expression of RALDH1 in liver cancers. AdC, adrenal cortical carcinoma; BaC, bladder cancer; BrC, breast cancer; CvC, cervical cancer; CrC, colorectal cancer; EmC, endometrial cancer; EgC, esophageal and gastric cancer; GBM, glioblastoma; GL, glioma; HNC, head and neck cancer; HC, hepatobiliary tract cancer; LK, leukemia; MBN, mature B-cell tumor; ML, melanoma; MNT, other neuroepithelial tumors; SGT, non-seminoma germ cell tumor; OML, intraocular melanoma; OET, ovarian epithelial tumor; PCT, pheochromocytoma; SMN, seminoma; TC, thyroid cancer. Figure 18B: Raw sequencing counts for 183 primary HCCs previously used to identify iCluster 1-3 molecular subtypes were downloaded, and the expression levels of the three Raldh isozymes were calculated. Raldh1 levels were significantly higher than the other two isozymes in all three molecular subtypes. Figure 18C: Human tumors (header) were stained with anti-RALDH1 antibody. Arrows indicate tumor locations. Primary and metastatic HCCs show strong RALDH1 staining, while unrelated tumors such as GIST and CRCs do not. Figure 18D: Transcript levels of Raldh1, Raldh2, and Raldh3 in multiple human HCC cell lines were measured by RT-qPCR. Raldh1 is the dominant isozyme expressed in all cell lines. Figure 18E: AldeRed assay for human HCC cell lines. The "control" cell line shows AldeRed fluorescence with the aldehyde dehydrogenase inhibitor DEAB, while the "test" cell line shows AldeRed fluorescence without the inhibitor, distinguishing fluorescence due to RALDH activity from the background. The histogram is representative of three or more experiments. The numbers indicate the percentage of cells within the indicated gate. All HCC cell lines show high RALDH activity in all cells (Huh7 and Hep3B), most cells (SNU398 and SNU449), or some cells (PLC).Figure 18F: Transcript levels of Raldh1, Raldh2, and Raldh3 were measured by RT-qPCR in multiple mouse HCC cell lines. Raldh1 is the dominant isozyme expressed in all cell lines. Figure 18G: AldeRed assay in the mouse HCC strain Hepa1-6, which shows high RALDH activity in most cells. Figure 18H: Raldh1 gene deleted from Huh7 cells using CRISPR / CAS9 (RALDH1-KO cell line). AldeRed assays performed on RALDH1-KO and parental Huh7 cells show loss of AldeRed positivity in KO. Figure 18I: LC-MS-based measurement of ATRA in the indicated mouse and human HCC cell lines (x axis). [Figure 18-2] Please refer to the explanation in Figure 18-1. [Figure 18-3] Please refer to the explanation in Figure 18-1. [Figure 18-4] Please refer to the explanation in Figure 18-1. [Figure 19A] Figure 19A: Raldh1 transcript levels in human tumor formalin-fixed paraffin-embedded (FFPE) samples measured by RT-qPCR. Raldh1 is higher in primary and metastatic HCC. HCC-M: Metastatic HCC; HCC-P: Primary HCC; GIST: Gastrointestinal stromal tumor; CrC: Colorectal cancer; Lv: Normal liver. One experiment with at least three replicas for each sample type. One-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. [Figure 19B] Figure 19B: Transcript levels (Y axis, RSEM, batch normalized) of Raldh2 (upper plot) and Raldh3 (lower plot) in various tumor types (X axis) from the TCGA database analyzed via cBioPortal. [Figure 19C]Figure 19C: Transcript levels of Raldh1, Raldh2, and Raldh3 in human HCC FFPE samples measured by RT-qPCR. Raldh1 is the dominant isozyme in most samples. One experiment with at least three replicates per sample type. One-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. [Figure 19D] Figure 19D: Anonymized human tumors (archived formalin-fixed and paraffin-embedded) were sectioned and stained with anti-RALDH1 antibody. Staining intensity (Y axis) is plotted against tumor type (X axis). One experiment with at least three replicas for each sample type. One-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. Calculated significance is compared to normal liver. [Figure 19E] Figure 19E: Human HCC dataset analyzed for gene expression using a web interface. Raldh1 is the major isozyme expressed, limited to non-leukocytes within tumors. Ptprc: Protein tyrosine phosphatase (CD45), a panleukocyte marker. Raldh: Retinaldehyde dehydrogenase. [Figure 19F] Figure 19F: Transcript levels of the three Raldh isozymes in mouse HCC cell lines (Hepa 1-6) and normal mouse liver. Unlike normal liver, where all three isozymes are detected, HCC cells primarily express Raldh1. [Figure 20A]Figures 20A-20F: Raldh1-INH exhibits species-specific inhibition of RA production in HCC cells. Figure 20A: SNU398 cells (human HCC cell line) were treated with Raldh1-INH C86 or C91 for 24 hours. The representative two-color histogram shows the loss of AldeRed fluorescence by Raldh1-INH, while the bar graph shows the quantitative change in AldeRed-positive cells as a percentage of total cells when treated with different concentrations of indicated Raldh1-INH. Figure 20B: Median fluorescence intensity in AldeRed assays for various human HCC cell lines after treatment with 100 nmol / L C86 for 24 hours. Figure 20C: The representative two-color histogram shows no change in AldeRed fluorescence when mouse HCC cell lines (Hepa1-6) were treated with up to 1 mmol / L C86 or C91 for 24 hours. Figure 20D: LC-MS for ATRA on Hepa1-6 (mouse HCC cell line) or SNU398 (human HCC cell line). Cells were treated with C86 (100 nmol / L for human cells, or 1,000 nmol / L for mouse cells) or the nonspecific RALDH inhibitor WIN18446 (1,000 nmol / L) for 24 hours. WIN18446 inhibits RALDH1 in both mouse and human cells, while C86 inhibition is specific to the human isozyme. Two independent experiments were performed with at least three replicates for each experimental group. Unpaired t-tests, two-sided. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Figure 20E: SNU398 cells were treated with different concentrations of C86 or C91 for 3 days. Cell proliferation was measured by counting the number of viable cells at each time point. Figure 20F: Huh7 cells were treated with different concentrations of C86 for 3 days. Cell proliferation was measured by counting the number of viable cells at each time point. [Figure 20B] Please refer to the explanation in Figure 20A. [Figure 20C] Please refer to the explanation in Figure 20A. [Figure 20D] Please refer to the explanation in Figure 20A. [Figure 20E] Please refer to the explanation in Figure 20A. [Figure 20F]Please refer to the explanation in Figure 20A. [Figure 21A] Figure 21A: Transcript levels (RT-qPCR) of Raldh1, Raldh2, and Raldh3 in SNU398 cells treated with different concentrations of C86 or C91 for 24 hours. [Figure 21B] Figure 21B: Homology modeling identifies differences in amino acid residues between human and mouse RALDH1 at the site where RALDH1-INH is predicted to bind. [Figure 21C] Figure 21C: SNU398 cells were treated with various concentrations of C86 or C91 for 24 hours. Cell viability was measured by flow cytometry (FCM) with 7-AAD staining (the Y-axis shows the percentage of 7-AAD-negative cells). [Figure 22A]Figures 22A-22E: RALDH1 inhibition blocks the RA-mediated effect of HCC on monocyte differentiation. Figures 22A-22B: Donor-derived circulating primary human monocytes (Figure 22A) or bone marrow-derived mouse monocytes (Figure 22B) were co-cultured with SNU398 (human HCC) cells or treated with SNU398-conditioned medium (CM) in a DC differentiation system (GM-CSF and IL-4). Two-color histograms show the frequency of DCs (CD11c+CD1a+ in humans, CD11c+MHCII+ in mice). The presence of SNU398 or its CM suppresses DC differentiation, which is reversed when SNU398 cells are treated with C86. Three or more independent experiments were performed with at least three replicates for each experimental group. Unpaired t-test, two-sided. Figure 22C: Huh7 (human HCC) cells were subcutaneously injected into the flank of NU / J mice. Twelve days later, the tumor size was approximately 50 mm³, and C86 or vehicle treatment (10 mg / kg, ip, daily injection) was initiated. On day 14, 1 million primary human monocytes (obtained from donors) were injected into these tumors. Five days after monocyte injection, mice were sacrificed and tumor tissue was collected for FCS analysis. Histograms show DC (HLA-DR+CD1a+) differentiation of injected human monocytes identified by human-specific CD45 (bar graph quantification on the right). Three or more independent experiments were performed with at least three replicas for each experimental group. Unpaired t-test, two-sided. Figure 22D: Human monocytes were transplanted into Huh7 tumors using the same strategy as described in Figure 22C, and the tumors were collected six days after monocyte transplantation. The frequencies of host (mouse) DCs (CD45+F4 / 80-CD11C+MHCII-high) and macrophages (CD45+F4 / 80+) are quantified in bar graphs. Three or more independent experiments were performed with at least three replicas for each experimental group. Unpaired t-test, two-sided. Figure 22E: Circulating human primary monocytes from donors were co-cultured with CMs derived from Huh7 cells or CMs derived from Raldh1-deficient (RALDH1-KO) Huh7 cell lines in a DC differentiation system (containing GM-CSF and IL4). Representative FCM plots (histograms) and DC quantification (bar graphs) are shown. Raldh1 deletion in tumor cells enhances DC differentiation from monocytes.Two independent experiments were conducted with at least three replicates for each experimental group. Unpaired t-tests, two-tailed. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. [Figure 22B] Please refer to the explanation in Figure 22A. [Figure 22C] Please refer to the explanation in Figure 22A. [Figure 22D] Please refer to the explanation in Figure 22A. [Figure 22E] Please refer to the explanation in Figure 22A. [Figure 23A] Figures 23A-23B: Total RNA was extracted from the experiments described in Figures 22A-22B, and transcript levels of DC (Zbtb46 and Irf4) and macrophage (Mafb) related genes were measured by RT-qPCR. Figure 23A: Results for human cells corresponding to the experiment in Figure 22A. Figure 23B: Results for mouse cells corresponding to the experiment in Figure 22B. Zbtb46 expression marks all DCs, while Irf4 expression is induced during monocyte-to-DC differentiation. Mafb expression marks macrophage differentiation, and its level is low in DCs. Three or more independent experiments with at least three replicas for each sample type. Two-tailed t-test. *p < 0.05, **p < 0.01, ***p < 0.001. [Figure 23B] Please refer to the explanation in Figure 23A. [Figure 24A]Figures 24A-24E: Raldh1-INH blocks the tumor-supporting function of monocytes and macrophages. Figure 24A: Circulating primary human monocytes were harvested from a donor and cultured with MCSF (50 ng / mL) to generate macrophages. On day 3 of these cultures, DMSO or RA (100 nmol / L) was added to generate control monocyte-derived macrophages (control-MoDM) or RA monocyte-derived macrophages (RA-MoDM), respectively. On day 7 of these cultures, after washing with 1XPBS, macrophages were collected from each well and mixed with Huh7 cells in an approximately 50:50 ratio, and the mixture was injected into the flanks of NU / J mice. Huh7 tumor cells that were not mixed with macrophages before flank injection served as an additional control. Tumor volume was measured every two days (left graph). Mice were sacrificed 19 days after tumor injection, and tumor weight (right graph) was measured. Tumor cells co-transplanted with RA-treated macrophages grew significantly faster than tumors co-transplanted with control macrophages or tumors transplanted without macrophages. Three or more independent experiments were conducted with at least three replicates for each experimental group. Unpaired t-test, two-sided. Figure 24B: Huh7 cells were subcutaneously injected into NU / J mice. When the tumor size reached approximately 50 mm3, the mice were intraperitoneally injected with liposomal clodronate (CloLipo; Liposoma, #C-015) and control liposomes (CtrlLipo; Liposoma, #P-015) at 200 mL / mouse every four days. Tumor growth was monitored daily (left graph). Mice were sacrificed 13 days after tumor cell transplantation, and tumor weight was measured (right graph). Macrophage depletion slowed tumor growth. Two independent experiments were conducted with at least three replicates for each experimental group. Unpaired t-test, two-sided. Figure 24C: Macrophages were generated from primary human monocytes by culturing them with M-CSF for 7 days. Macrophages were then collected, washed, and seeded in new wells with or without tumor-conditioned medium (CM;TCM) along with the indicated compounds. After 3 days, cells were harvested, counted, and stained with PI for FCS analysis. The number of viable macrophages (y axis) under various experimental conditions (x axis) is shown.RA and tumor macrophages significantly increased macrophage numbers compared to other conditions, and this effect was reversed by reducing RA (C86-treated TCM) or blocking RA signaling (BMS493). Three or more independent experiments were conducted with at least three replicates for each experimental group. Unpaired t-test, two-sided. Figure 24D: Different human HCC lines (x-axis, first legend in each graph) were co-cultured for 3 days with macrophages pre-treated with various compounds (x-axis). Cell proliferation was measured by counting the number of viable cells (y-axis). RA-treated macrophages increased tumor cell numbers compared to control macrophages, and this effect was reversed by blocking RA signaling (BMS493). Three or more independent experiments were conducted with at least three replicates for each experimental group. Unpaired t-test, two-sided. Figure 24E: Primary human monocytes were differentiated into macrophages with M-CSF alone (control) or with M-CSF in combination with BMS493, RA, or RA + BMS493 (x axis). After 7 days, macrophages differentiated under these conditions were harvested and co-cultured with Huh7 human HCC cells labeled with carboxyfluorescein diacetate succinimimidyl ester (CFSE) (1:10 tumor cells:macrophages). After 72 hours, cells were washed, counted, and analyzed by FCS. HCC cell division is indicated by the degree of CFSE dilution (y axis). Three or more independent experiments were performed with at least three replicates for each experimental group. Unpaired t-test, two-sided. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. [Figure 24B] Please refer to the explanation in Figure 24A. [Figure 24C] Please refer to the explanation in Figure 24A. [Figure 24D] Please refer to the explanation in Figure 24A. [Figure 24E] Please refer to the explanation in Figure 24A. [Figure 25A] Figure 25A: Tumors at the endpoint of the experiment outlined in Figure 24A. [Figure 25B]Figure 25B: FCM plot of macrophages (F4 / 80+ cells) in spleen tissue from the experiment in Figure 24B, showing macrophage depletion by CloLipo. [Figure 25C] Figure 25C: Different human HCC strains were cultured for 3 days with conditioned medium (CM) (X axis, macrophage CM) from wells containing macrophages treated with different compounds. HCC cells exposed to the same compounds (X axis, control CM) without conditioned medium served as an additional control. The number of viable HCC cells was counted after 3 days (Y axis). Three or more independent experiments with at least three replicates for each sample type. Two-sided t-test. *p < 0.05, **p < 0.01, ***p < 0.001. [Figure 26A]Figures 26A-26H: Raldh1-INH inhibits HCC growth by altering macrophage function. Figure 26A: Huh7 tumor-bearing NU / J mice were treated daily with C86 (ip, 10 mg / kg) starting when the tumor size was approximately 50 mm3. Tumor volume was measured every two days. The bar graph on the right shows tumor weight at the endpoint. Three or more independent experiments were performed with at least three replicates for each experimental group. Unpaired t-test, two-sided. Figure 26B: AldeRed assay at the endpoint of Huh7 tumors treated as described in Figure 26A. The graph shows the percentage of AldeRed-positive cells in CD45+ leukocytes and CD45- cells (tumor + stromal cells). The data mostly show selective inhibition of RA in tumor cells. Three or more independent experiments were performed with at least three replicates for each experimental group. Unpaired t-test, two-sided. Figure 26C: Nude mice carrying Huh7 tumors were treated daily with different doses of C86. Tumor volume was measured every 2-3 days. The bar graph on the right shows tumor volume at the experimental endpoint. Two independent experiments were conducted with at least three replicates for each experimental group. Unpaired t-test, two-sided. Figure 26D: Body weight measured every 3-5 days for the experiments outlined in Figure 26C. Figure 26E: Parental Huh7 or RALDH1-KO Huh7 cells were subcutaneously transplanted into NU / J mice and treated daily with C86 (ip, 10 mg / kg) or vehicle (control). RALDH1-KO tumors grew significantly slower than parental Huh7 and did not respond to C86 treatment. The bar graph on the right shows tumor weight at the endpoint. Three or more independent experiments were conducted with at least three replicates for each experimental group. Unpaired t-test, two-sided. Figure 26F: Huh7 tumor-bearing NU / J mice were treated with clodronate liposomes (CloLipo) and / or C86. CloLipo-treated tumors showed slower growth and did not respond to C86 treatment. The bar graph on the right shows tumor weight at the endpoint. Two independent experiments were conducted with at least three replicas per experimental group. Unpaired t-test, two-sided.Figure 26G: Mouse Hepa 1-6 tumors were subcutaneously transplanted into C57BL6 / J WT mice or LyMCre:RosadnRAR mice. Dominant-negative RAR expression in bone marrow cells slows tumor growth. Two independent experiments were performed with at least three replicas for each experimental group. Unpaired t-test, two-sided. Figure 26H: Huh7 cells were subcutaneously transplanted into NU / J mice. When the tumor size reached approximately 50 mm3, mice were intraperitoneally injected daily with or without BMS493 treatment into the tumor every 3 days with or without a vehicle (control) or C86. BMS493 and C86 suppressed tumor growth, and combination therapy showed higher suppression than monotherapy. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. [Figure 26B] Please refer to the explanation in Figure 26A. [Figure 26C] Please refer to the explanation in Figure 26A. [Figure 26D] Please refer to the explanation in Figure 26A. [Figure 26E] Please refer to the explanation in Figure 26A. [Figure 26F] Please refer to the explanation in Figure 26A. [Figure 26G] Please refer to the explanation in Figure 26A. [Figure 26H] Please refer to the explanation in Figure 26A. [Figure 27A] Figure 27A: Nude mice carrying Huh1 tumors were treated daily with C86 (ip, 10 mg / kg). Three or more independent experiments with at least three replicates for each sample type. Two-sided t-test. *p < 0.05, **p < 0.01, ***p < 0.001. [Figure 27B] Figure 27B: The same number of Raldh1-KO Huh7 and parental Huh7 cells were seeded, and the number of surviving cells was counted over time. RALDH1 deficiency does not suppress cell proliferation in vitro. [Figure 27C]Figure 27C: NU / J mice with flank Huh7 tumors were treated with C86 daily or liposomal clodronate (CloLipo, 200 μl / mouse) every four days. Tumors were harvested 13 days after tumor induction and analyzed for macrophage frequency (left graph) and number (right graph) under different treatment conditions (X axis). Three or more independent experiments with at least three replicates for each sample type. Two-sided t-test. *p < 0.05, **p < 0.01, ***p < 0.001. [Figure 27D] Figure 27D: A photograph showing the differences in tumor size between the experiments outlined in Figure 26G. [Figure 27E] Figure 27E: FCS-based analysis of macrophage percentage (F4 / 80+ cells) in leukocytes (CD45+ cells) within tumors described in Figure 26G. Two independent experiments were performed with at least three replicates for each experimental group. Two-sided t-test. *p < 0.05, **p < 0.01, ***p < 0.001. [Figure 28A]Figures 28A-28G: C99 inhibits mouse RALDH1 and suppresses mouse HCC growth. Figure 28A: AldeRed assays were performed on Hepa1-6 (mouse HCC, left graph) and SNU398 (human HCC, right graph) cells with or without different concentrations of C99 (x axis). The percentage of AldeRed-positive cells (y axis) after 24 hours of exposure to C99 is shown. Mouse RALDH1 is sensitive to C99. Figure 28B: Hepa1-6 cells were treated in vitro with different concentrations of C99, and the number of viable cells was counted at different time points. C99 does not reduce cell viability in vitro. Figure 28C: Hepa1-6 cells were subcutaneously transplanted into C57BL6 / J mice; and tumor-bearing mice. Mice were treated with anti-CD3 to prevent spontaneous rejection of this cell line. Once the tumor reached approximately 50 mm3 in size, mice were treated daily with C86 or C99 (ip, 20 mg / kg). The graph on the right shows the tumor mass at the endpoint. C99 inhibits mouse HCC growth, while C86 does not. Two independent experiments were performed with at least three replicates for each experimental group. Unpaired t-test, two-sided. Figure 28D: Tumors from (Figure 28C) were collected at the endpoint, single-cell suspensions were prepared, and the AldeRed assay was performed along with surface staining with immunocytoplasmic markers. Percentage of AldeRed-positive cells in non-leukocytes (CD45-negative, mainly tumor cells) under different treatment conditions. Two independent experiments were performed with at least three replicates for each experimental group. Unpaired t-test, two-sided. Figure 28E: FCS-based frequency of macrophages (F4 / 80+ cells) in CD45+ leukocytes within tumor tissue from the experiments outlined in (Figures 28C-28D). Two independent experiments were conducted with at least three replicates for each experimental group. Unpaired t-test, two-sided. Figure 28F: Hepa1-6 tumor-bearing NU / J mice were treated with clodronate liposomes (CloLipo) and / or C86 / C99. CloLipo treatment inhibited tumor growth and made tumors insensitive to C99. Two independent experiments were conducted with at least three replicates for each experimental group. Unpaired t-test, two-sided.Figure 28G: Mouse fibrosarcoma (FS) cell line was subcutaneously transplanted into C57BL6 / J mice. Mice were treated daily with 25 mg / kg of C-99 or vehicle starting from 1 day after tumor cell transplantation. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. [Figure 28B] Please refer to the description of Figure 28A. [Figure 28C] Please refer to the description of Figure 28A. [Figure 28D] Please refer to the description of Figure 28A. [Figure 28E] Please refer to the description of Figure 28A. [Figure 28F] Please refer to the description of Figure 28A. [Figure 28G] Please refer to the description of Figure 28A. [Figure 29A] Figure 29A: IC50 of C-86 and C-99 against different human RALDH isozymes. [Figure 29B] Figure 29B: Cell viability was measured by flow cytometry (FCM) using 7-AAD in the experiment outlined in Figure 28B. [Figure 29C] Figure 29C: Tumors at the endpoint from the experiment in Figure 28C. [Figure 29D] Figure 29D: Tumor weight at the endpoint of Figure 28G. One experiment with at least 3 replicates per experimental group. Two-sided t-test. *p < 0.05, **p < 0.01, ***p < 0.001. [Figure 29E] Figures 29E - 29F: ALDH1 was deleted by CRISPR / Cas9 in mouse Hep55 HCC cell line (Figure 29E). Two independent clones, 2 and 19 were selected (Figure 29E). Both clones showed no difference in in vitro growth compared to parental WT cells (Figure 29E). Figure 29F: In contrast to in vitro growth, both clones showed dramatic growth inhibition in vivo in syngeneic (C57BL / 6) flank transplantation model. The pictures show tumor size at the endpoint corresponding to the experiment shown in Figure 30A. [Figure 29F]Please refer to the explanation in Figure 29E. [Figure 29G] Figure 29G: The graph shows tumor growth for the indicated genotypes and treatments syngeneically transplanted into C57BL / 6 mice. Notably, there is a small but significant synergistic effect between RALDH1 loss and anti-PD1 treatment. Clone 2 was used for the RALDH1-KO Hep55 cell line. Three or more independent experiments were conducted with at least three replicates for each experimental group. One-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. [Figure 29H] Figure 29H: Hep55 mouse HCC cells were transplanted into the flanks of syngeneic C57BL / 6 mice. Tumors were then harvested, and single-cell suspensions were subjected to multiparametric flow cytometry. A sequential (arrow) gating scheme is shown, resulting in two macrophage subsets based on CD163, a marker for immunosuppressive macrophages. The histogram overlays on the right (Mac1 subset; Mac2 subset) show the expression of anti-inflammatory (CD206 and folate receptor B) and pro-inflammatory (MHCII and CD11C) markers in the Mac1 and Mac2 subsets, supporting their pro-inflammatory and anti-inflammatory functions, respectively. [Figure 29I] Figure 29I: Flow cytometry-based comparison of macrophages in WT and RALDH1 knockout Hep55 tumors shows an increase in MHCII+ pro-inflammatory macrophages in KO. Three or more independent experiments were conducted with at least three replicates for each experimental group. Two-sided t-test. *p < 0.05, **p < 0.01, ***p < 0.001. [Figure 30A]Figures 30A-30E: Pharmacokinetics of Raldh1-INH and synergistic effects with immune checkpoint blockade. Figure 30A: RALDH1 was deleted using CRISPR / Cas9 in the mouse Hep55 HCC cell line. Based on confirmation of gene deletion, two independent clones, 2 and 19, were selected. Cell lines of the indicated genotypes were subcutaneously transplanted into immunologist syngeneic C57BL6 / J mice, and tumor size was monitored over time. Tumor size at the experimental endpoint is shown in Figure 29F. Three or more independent experiments with at least three replicas were conducted for each experimental group. One-way ANOVA. Figure 30B: Tumors from (Figure 30A) were collected at the endpoint (Figure 29F), and T cell infiltration was analyzed by flow cytometry. Three or more independent experiments with at least three replicas were conducted for each experimental group. One-way ANOVA. Figure 30C: Strategy for generating RALDH1-KO mice. Cas9 mRNA and two guide RNAs (arrows) were microinjected into single-cell zygotes. Founders were identified by a PCR screening protocol designed to detect the approximately 36kb deletion expected from the dual cut. Founders were then crossed with C57BL / 6 WT mice to "fix" the knockout allele. Figure 30D: Confirmation of RALDH1 deletion in knockout mice by quantitative PCR using a mouse RALDH1-specific TaqMan probe. Two independent experiments were performed with at least three replicates per experimental group. Unpaired t-test, two-sided. Figure 30E: Serum toxicology analysis (standard toxin panel number 62794) was performed via IDEXX Biological Analysis Services using serum from RALDH1-KO (- / -), heterozygous (+ / -), and WT mice. A small selection of analytes from a larger panel is shown. One experiment was performed with more than five replicates per genotype. One-way ANOVA. *, P < 0.05;**, P < 0.01;***, P < 0.001. [Figure 30B] Please refer to the explanation in Figure 30A. [Figure 30C] Please refer to the explanation in Figure 30A. [Figure 30D] Please refer to the explanation in Figure 30A. [Figure 30E] Please refer to the explanation in Figure 30A. [Figure 31A] Figure 31A: Complete blood counts (CBCs) were performed on whole blood samples collected from the RALDH1 genotypes (X axis) shown in IDEXX. One experiment with four replicates per experimental group. One-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. [Figure 31B] Figure 31B: Body mass of mice with the RALDH1 genotype shown. Siblings were used for all genotypes to control for confounding factors. One experiment with four replicates per experimental group. One-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. [Figure 31C] Figure 31C: Pharmacokinetics of C86 delivered orally via diet (solid feed). Diet (or PO) numbers A, B, and C (shown in the inbox of each graph) correspond to drug doses of 10, 30, and 60 mg / kg, respectively. Mice had free access to food and water during the 15-day study. The graph shows drug concentration (Y-axis) plotted against time (X-axis) in various tissues (headers). [Figure 31D] Figure 31D: Shows the body weight (Y axis) of mice measured daily (X axis) during the 15-day experiment described in Figure 31A. [Modes for carrying out the invention]

[0011] Detailed description of the invention Herein, specific aspects of the disclosed subject matter are given in detail, some examples of which are shown in the accompanying drawings. The disclosed subject matter is described in conjunction with the enumerated claims, but it should be understood that the illustrated subject matter is not intended to limit the claims to the disclosed subject matter.

[0012] Throughout this document, values ​​expressed as ranges should be interpreted flexibly to include not only the numerical values ​​explicitly stated as limits to the range, but also all individual numerical values ​​or subranges contained within that range, as if each numerical value and subrange were explicitly stated. For example, the range "approximately 0.1% to approximately 5%" or "approximately 0.1% to approximately 5%" should be interpreted to include not only approximately 0.1% to approximately 5%, but also the individual values ​​within the indicated range (e.g., 1%, 2%, 3%, and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%). Unless otherwise specified, the notation "approximately X to Y" is equivalent to "approximately X to approximately Y". Similarly, unless otherwise specified, the notation "approximately X, Y, or approximately Z" is equivalent to "approximately X, approximately Y, or approximately Z".

[0013] In this document, unless otherwise indicated by context, the terms “a,” “an,” or “the” are used to include one or more. Unless otherwise indicated, the term “or” is used to mean non-exclusive “or.” The phrases “at least one of A and B” or “at least one of A or B” are synonymous with “A, B, or A and B.” Furthermore, it should be understood that any expressions or terms used herein, unless otherwise defined, are for illustrative purposes only and not for limitation. Any use of section headings is intended to aid in the reading of this document and should not be interpreted as limiting. Information related to section headings may arise inside or outside that particular section. All publications, patents, and patent documents referenced herein are incorporated herein in whole by reference as if they were incorporated individually by reference.

[0014] In the methods described herein, acts can be performed in any order, except where a chronological or operational order is explicitly described. Further, unless the claims explicitly state that certain acts must be performed separately, they can be performed simultaneously. For example, the acts recited in a claim of performing X and the acts recited in a claim of performing Y can be performed simultaneously within one operation, and the resulting process is within the literal scope of the process recited in the claim.

[0015] Description Hepatocellular carcinoma (HCC) is a devastating disease, with an annual incidence of approximately 1 million cases predicted worldwide by 2025. In the United States alone, approximately 27,000 people die from this disease each year, and the 5-year survival rate for advanced HCC is a very poor approximately 2%. Surgical resection and liver transplantation are preferred treatments for early HCC, but local regional interventions such as radiofrequency ablation and transarterial chemoembolization are used in unresectable cases. Advanced metastatic cases present significant management challenges, with a median survival of several months. Recent advances in systemic therapies, currently including immune checkpoint inhibitors, tyrosine kinase inhibitors (TKIs), and angiogenesis inhibitors, have improved patient outcomes. Nevertheless, there is still substantial room for improvement, particularly with immunotherapy. Monotherapy immune checkpoint blockade (ICB) induces clinical responses in a minority of patients, suggesting the presence of other biological regulators of the ICB response.

[0016] Macrophages and dendritic cells (DCs) are crucial antigen-presenting cells in solid tumors, and given the limitations of current immunotherapies, there is growing interest in therapeutically targeting them. These efforts generally aim to reduce the frequency of immunosuppressive macrophages, increase the frequency of tumor-killing and pro-inflammatory macrophages, and enhance the immunostimulatory activity of DCs. Numerous approaches have been described that can achieve these effects in experimental models by targeting specific receptors and / or pathways of tumor-associated macrophages (TAMs) and DCs. In contrast, little is known about whether and how monocytes differentiate into DCs. TAMs may be targeted for cancer immunotherapy. DCs and TAMs can originate from non-monocyte progenitor cells, embryonic progenitor cells arising from the yolk sac, and myeloid progenitor cells derived from HSCs, respectively, but these progenitor cells are very rare compared to abundant circulating monocytes. Furthermore, the lifespan of DCs and TAMs within the tumor microenvironment (TME) is finite, requiring a continuous influx of progenitor cells. Therefore, targeting monocyte differentiation represents a feasible but largely unexplored therapeutic strategy in cancer immunotherapy.

[0017] As described elsewhere in this specification, some tumors produce retinoic acid (RA), which promotes the differentiation of monocytes into immunosuppressive and protumoric macrophages. Therefore, reducing RA production by tumor cells or inhibiting RA signaling in monocytes is a potential therapeutic approach in these tumors. The main obstacles to implementing this approach include identifying tumors in which this pathway is active and developing safe and effective inhibitors of the RA pathway. As described herein, HCC produces high levels of RA due to the overexpression of RALDH1, one of the three enzymes that catalyze RA production. RA production in HCC was suppressed by an exemplary RALDH1 inhibitor (Raldh1-INH). These inhibitors suppressed tumor growth in multiple mouse models of human and mouse HCC. As described herein, using genetic and pharmacological tools, the HCC-suppressive effect of Raldh1-INH is attributed to changes in macrophage number and function, as well as increased tumor infiltration by activated T cells. Pharmacological and toxicological analyses revealed a profile of Raldh1-INH favorable for potential clinical use, which was further supported by observations in newly generated RALDH1 knockout (RALDH1-KO) mice. These findings provide proof of concept for the use of Raldh1-INH in HCC and establish the scientific premise for the development of isoenzyme-specific RALDH inhibitors as a novel strategy in cancer immunotherapy.

[0018] definition As used herein, the term "about" may allow some degree of variability in a value or range, for example, within 10%, 5%, or 1% of the stated value or stated range limit, and includes the exact stated value or range.

[0019] As used herein, terms such as “administer,” “give delivery,” and “dosage” refer to methods that may be used to enable the delivery of a compound or composition to a desired site of biological action. These methods include, but are not limited to, oral, intraduodenal, parenteral injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular, or infusion), topical, and rectal administration. Those skilled in the art will be familiar with the administration techniques that may be used with the compounds and methods described herein, for example, as discussed in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition; Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, Pa. In some embodiments, the compounds and compositions described herein are administered orally.

[0020] As used herein, the term “alkenyl,” used alone or in combination with other terms, means a stable monounsaturated or diunsaturated straight-chain or branched-chain hydrocarbon group having the number of carbon atoms specified, unless otherwise stated. Examples include vinyl, propenyl (or allyl), clotyl, isopentenyl, butadienyl, 1,3-pentadienyl, 1,4-pentadienyl, and their higher homologues and isomers. Functional groups representing alkenes are exemplified by -CH2-CH=CH2.

[0021] As used herein, the term “alkoxy,” used alone or in combination with other terms, means, unless otherwise specified, an alkyl group as defined elsewhere herein, having a specified number of carbon atoms connected to the rest of the molecule by an oxygen atom, such as methoxy, ethoxy, 1-propoxy, 2-propoxy (or isopropoxy), as well as its higher homologs and isomers. Specific examples include, but are not limited to, ethoxy and methoxy (C1-C3) alkoxys.

[0022] As used herein, the term "alkyl," either alone or as part of another substituent, means having a specified number of carbon atoms (i.e., C1-C1) unless otherwise specified. 10 A C6 molecule refers to a linear or branched hydrocarbon (meaning 1 to 10 carbon atoms), and includes linear, branched, or cyclic substituents. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, and cyclopropylmethyl. Specific embodiments include, but are not limited to, alkyl molecules (C1-C6) such as ethyl, methyl, isopropyl, isobutyl, n-pentyl, n-hexyl, and cyclopropylmethyl. Linear, branched, and / or cyclic parts having a specified number of carbon atoms (e.g., C1-C6) should be interpreted flexibly to include all individual numerical values ​​or subranges encompassed within that range, as if each numerical value and subrange were explicitly stated (e.g., C1-C6 includes, in particular, the individual values ​​C1, C2, C3, C4, C5, and C6, as well as the non-limiting exemplary ranges C1-C5 and C2-C6).

[0023] As used herein, the term “alkynyl,” used alone or in combination with other terms, means a stable linear or branched hydrocarbon group with a triple carbon-carbon bond having the number of carbon atoms specified, unless otherwise stated. Non-limiting examples include ethynyl and propynyl, as well as their higher congeners and isomers. The term “propargyl” means a group exemplified by -CH2-C≡CH. The term “homopropargyl” means a group exemplified by -CH2CH2-C≡CH.

[0024] As used herein, the term “aryl,” used alone or in combination with other terms, means a carbocyclic aromatic system comprising one or more rings (usually one, two, or three rings), unless otherwise specified, where the rings may be bonded together in a pendant arrangement, such as biphenyl, or fused, such as naphthalene. Examples include phenyl, anthrasyl, and naphthyl. Examples of aryl groups include phenyl or naphthyl rings fused to one or more saturated or partially saturated carbocyclic rings (e.g., bicyclo[4.2.0]octa-1,3,5-trienyl or indanyl), which may be substituted on one or more carbon atoms of the aromatic ring and / or saturated or partially saturated ring.

[0025] As used herein, the term "cycloalkyl," either alone or as part of another substituent, means a cyclic hydrocarbon having a specified number of carbon atoms (i.e., C3-C6 means a cyclic group containing a ring group consisting of 3-6 carbon atoms), and includes linear, branched, or cyclic substituents, unless otherwise specified. Examples of (C3-C6)cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. The cycloalkyl ring may be substituted. Non-limiting examples of cycloalkyl groups include cyclopropyl, 2-methyl-cyclopropyl, cyclopropenyl, cyclobutyl, 2,3-dihydroxycyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctanyl, dekalinyl, 2,5-dimethylcyclopentyl, 3,5-dichlorocyclohexyl, 4-hydroxycyclohexyl, 3,3,5-trimethylcyclohexa-1-yl, octahydropentalenyl, octahydro-1H-indenyl, 3a,4,5,6,7,7a-hexahydro-3H-inden-4-yl, decahydroazlenyl, bicyclo[6.2.0]decanyl, decahydronaphthalenyl, and dodecahydro-1H-fluorenyl. The term "cycloalkyl" also includes bicyclic hydrocarbon rings, non-limiting examples of which include bicyclo-[2.1.1]hexanyl, bicyclo[2.2.1]heptanyl, bicyclo[3.1.1]heptanyl, 1,3-dimethyl[2.2.1]heptan-2-yl, bicyclo[2.2.2]octanyl, and bicyclo[3.3.3]undecanyl.

[0026] As described herein, the term “chimeric antigen receptor (CAR) T cell” refers to T cells that have been genetically engineered to provide artificial T cell receptors for use in immunotherapy.

[0027] As used herein, the term “co-administered” means simultaneous administration of the same formulation or of two different formulations via the same or different routes, or sequential administration via the same or different routes. “Sequential” administration means a time difference of several seconds, minutes, hours, or days between the administration of two or more distinct compounds.

[0028] The term “combination therapy” refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described herein. Such administrations include co-administration of these therapeutic agents substantially simultaneously, such as in a single formulation having a fixed ratio of active ingredients (e.g., capsules or injections) or in multiple distinct dosage forms for each active ingredient. Furthermore, such administrations also include the sequential use of each type of therapeutic agent. In any case, the treatment regimen provides the advantageous effect of drug combination in the treatment of the condition or disorder described herein.

[0029] The term "includes" is used herein to mean including a subsequent feature or act without precluding the presence of one or more additional features or acts.

[0030] As used herein, the terms “effective dose” or “therapeutic effective dose” refer to a sufficient amount of at least one compound administered to achieve a desired outcome, for example, to alleviate to some extent one or more symptoms of the disease or condition being treated. In some cases, the outcome is the reduction and / or mitigation of the signs, symptoms or causes of the disease, or other desirable changes in the biological system. In certain cases, the outcome is the reduction in growth, death, or induction of apoptosis of at least one abnormally proliferating cell, such as a cancer cell. In some cases, the “effective dose” for therapeutic use is the amount of a composition containing the compounds described herein that is necessary to produce a clinically significant reduction in the disease. The appropriate “effective” dose in each individual case is determined using any preferred technique, such as dose-escalation studies.

[0031] As used herein, the terms “halo” or “halogen,” either alone or as part of another substituent, mean a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, unless otherwise specified.

[0032] As used herein, the terms “heteroaryl” or “heteroaromatic” mean an aromatic heterocyclic compound. Polycyclic heteroaryls may contain one or more partially saturated rings. Examples include tetrahydroquinoline and 2,3-dihydrobenzofuryl.

[0033] As used herein, the terms “heterocyclic,” “heterocycline,” or “heterocyclic formula,” either alone or as part of another substituent, mean, unless otherwise specified, a stable monocyclic or polycyclic heterocyclic ring system having a specified number of carbon atoms (i.e., C2–C8 means a cyclic group containing a ring group consisting of 2–8 carbon atoms) and at least one heteroatom selected from the group consisting of N, O, and S, wherein the nitrogen and sulfur heteroatoms may be oxidized and the nitrogen atom may be quaternized. Unless otherwise specified, the heterocyclic system may be bonded at any heteroatom or carbon atom that gives a stable structure. The heterocyclic ring may be aromatic or non-aromatic. In some embodiments, the heterocyclic ring is a heteroaryl.

[0034] Examples of non-aromatic heterocycles include monocyclic groups such as aziridine, oxirane, thiirane, azetidine, oxetane, thietan, pyrrolidine, pyrroline, imidazoline, pyrazolidine, dioxolane, sulfolane, 2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophan, piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran, 1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine, 1,3-dioxepane, 4,7-dihydro-1,3-dioxepine, and hexamethylene oxide.

[0035] Examples of heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl (including but not limited to 2- and 4-pyrimidinyl), pyridadinyl, thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl.

[0036] Examples of polycyclic heterocycles include indolyl (3-, 4-, 5-, 6-, and 7-indolyl, etc., but not limited to these), indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl (1- and 5-isoquinolyl, etc., but not limited to these), 1,2,3,4-tetrahydroisoquinolyl, sinnolinyl, quinoxalinyl (2- and 5-quinoxalinyl, etc., but not limited to these), quinazolinyl, phthalazinyl, 1,8-naphthilidinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthilidinyl, benzofuryl (3 Examples include, but are not limited to, -, 4-, 5-, 6-, and 7-benzofuryl, 2,3-dihydrobenzofuryl, 1,2-benzoisoxazolyl, benzothienyl (including, but not limited to, 3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl (including, but not limited to, 2-benzothiazolyl and 5-benzothiazolyl), prinyl, benzimidazolyl, benzotriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, and quinolizidinyl.

[0037] The above list of heterocyclyl and heteroaryl moieties is intended to be representative, not restrictive.

[0038] As used herein, the term “immunostimulant” refers to a substance that can modulate an immune response (e.g., stimulate an immune response and / or participate in an immune response), and non-limiting examples include small molecules, large molecules (e.g., proteins), and biologics (e.g., T cells).

[0039] As used herein, the terms “increase” or related terms “increased,” “enhance,” or “enhanced” refer to a statistically significant increase, while the terms “decreased,” “suppressed,” or “inhibited” may refer to a statistically significant decrease. To avoid misunderstanding, increase generally refers to an increase of at least 10% of a given parameter and can include increases of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or even 100% compared to the control, baseline, or prior value. Inhibition generally refers to a decrease of at least 10% of a given parameter and can include decreases of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or even 100% compared to the control value.

[0040] As used herein, the terms “pharmaceutical composition” or “composition” mean a mixture of at least one compound useful within the scope of the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates the administration of the compound to a subject.

[0041] As used herein, the term “pharmaceutically acceptable” means a material, such as a carrier or diluent, that does not inhibit the biological activity or biological properties of a compound useful within the scope of the present invention and is relatively non-toxic; that is, the material can be administered to a subject without causing undesirable biological effects or harmful interactions with any of its components contained in the composition.

[0042] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, which is involved in transporting or delivering a compound useful within the scope of the invention into or to a subject so that it can perform its intended function. Typically, such constructs are transported or delivered from one organ or part of the body to another organ or part of the body. Each carrier must be “acceptable” in the sense that it is compatible with the other components of a formulation containing a compound useful within the scope of the invention and is not harmful to the subject. Some examples of materials that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose, and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; surfactants; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer; and other non-toxic and suitable substances used in pharmaceutical formulations. As used herein, “pharmaceutically acceptable carrier” includes any coating, antimicrobial and antifungal agent, as well as absorption retarders, etc., that are compatible with the activity of compounds useful within the scope of the present invention and are physiologically acceptable to the subject. Supplementary active compounds may be incorporated into the composition. The “pharmaceutically acceptable carrier” may further include pharmaceutically acceptable salts of compounds useful within the scope of the present invention.Further components that may be included in the pharmaceutical compositions used in the practice of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0043] The term "pharmaceutically acceptable salt" may refer to a salt prepared from a pharmaceutically acceptable, non-toxic acid or base, including inorganic acids and inorganic bases, as well as organic acids and organic bases. If the compounds disclosed herein are basic, the salt may be prepared from a pharmaceutically acceptable, non-toxic acid, including inorganic and organic acids. Suitable pharmaceutically acceptable acid addition salts for the compounds disclosed herein include acetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid (besylic acid), benzoic acid, boric acid, butyric acid, camphoric acid, camphorsulfonic acid, carbonic acid, citric acid, ethanedisulfonic acid, ethanesulfonic acid, ethylenediaminetetraacetic acid, formic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, and iodide. This includes hydrogen acids, hydroxynaphthoic acid, isethionic acid, lactic acid, lactobionic acid, lauryl sulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucoic acid, naphthylenesulfonic acid, nitric acid, oleic acid, pamoic acid, pantothenic acid, phosphoric acid, pivalic acid, polygalacturonic acid, salicylic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, tartaric acid, teoclatic acid, p-toluenesulfonic acid, and the like. When a compound contains an acidic side chain, suitable pharmaceutically acceptable base addition salts for the compounds of the present invention include, but are not limited to, metal salts made from aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc, or organic salts made from lysine, arginine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine. Further pharmaceutically acceptable salts may include, as needed, non-toxic ammonium cations bonded to alkyl groups having 1 to 20 carbon atoms, as well as carboxylic acids, sulfonic acids, and phosphonate anions.

[0044] As used herein, “pharmaceutically effective amount,” “therapeutic effective amount,” or “effective amount” of a compound means an amount of the compound sufficient to impart a beneficial effect to the subject to which the compound is administered.

[0045] As used herein, the terms “prevent,” “prevent,” or “prevention” mean avoiding or delaying the onset of symptoms associated with a disease or condition in a subject who does not have such symptoms at the time of initiation of administration of the drug or compound. In this specification, disease, condition, and disorder are used interchangeably.

[0046] As used herein, the term "retinoic acid modulator" refers to a substance that acts to increase or decrease the amount or effect of retinoic acid present in a subject by one of several mechanisms. In one embodiment, a retinoic acid modulator may decrease the amount of retinoic acid. In one embodiment, the decrease in the amount of retinoic acid may result from the inhibition of retinoic acid biosynthesis.

[0047] As used herein, the term "specifically binds" means that the first molecule preferentially binds to the second molecule (e.g., a particular receptor or enzyme), but does not necessarily bind only to that second molecule.

[0048] As used herein, the term “subject” refers to all mammals, including humans. Examples of subjects include humans, mice, primates, cattle, dogs, cats, goats, sheep, pigs, and rabbits. In some embodiments, the subject is human.

[0049] As used herein, the term “substantially” means at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%, or a majority or most.

[0050] As used herein, the term “substantially absent” may mean having nothing, or having trace amounts of the material such that the amount of the material present does not affect the material properties of the composition containing the material, such that the composition contains about 0 wt% to about 5 wt%, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than about 4.5 wt%, equal to about 4.5 wt%, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less. The term "substantially absent" may mean that the composition contains trace amounts of the material such that it is about 0 wt% to about 5 wt%, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than about 4.5 wt%, equal to about 4.5 wt%, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt%, or about 0 wt%.

[0051] As used herein, the term “substituted” means that an atom or group of atoms has replaced a hydrogen atom with a substituent bonded to another group.

[0052] As used herein, the terms “substituted alkyl,” “substituted cycloalkyl,” “substituted alkenyl,” or “substituted alkynyl” independently refer to halogen, -OH, alkoxy, tetrahydro-2-H-pyranyl, -NH2, -NH(C1~C6 alkyl), -N(C1~C6 alkyl)2, 1-methylimidazole-2-yl, pyridine-2-yl, pyridine-3-yl, pyridine-4-yl, -C(=O)OH, -C(=O)O(C1~C6)alkyl, trifluoromethyl, -C≡N, -C(=O)NH2, -C(=O)NH(C1~C6)alkyl, -C(=O)N((C1~C6) substituted with one, two, or three substituents selected from the group consisting of -SO2NH2, -SO2NH(C1~C6 alkyl), -SO2N(C1~C6 alkyl)2, -C(=NH)NH2, and -NO2, meaning alkyl, cycloalkyl, alkenyl, or alkynyl as defined elsewhere herein, and in some embodiments comprising one or two substituents independently selected from halogen, -OH, alkoxy, -NH2, trifluoromethyl, -N(CH3)2, and -C(=O)OH, or in some embodiments independently selected from halogen, alkoxy, and -OH. Examples of substituted alkyls include, but are not limited to, 2,2-difluoropropyl, 2-carboxycyclopentyl, and 3-chloropropyl.

[0053] As used herein, the terms “to treat,” “to treat,” and “treatment,” and other grammatical equivalents, include alleviating, suppressing, or reducing symptoms; reducing or suppressing severity; reducing incidence; preventive treatment; reducing or suppressing recurrence; preventing; delaying onset; delaying recurrence; alleviating, improving, or improving symptoms of a disease or condition; improving the underlying metabolic cause of symptoms; suppressing a disease or condition, for example, preventing the onset of a disease or condition; easing a disease or condition; causing regression of a disease or condition; alleviating a condition caused by a disease or condition; or cessating symptoms of a disease or condition. These terms also include achieving a therapeutic benefit. A therapeutic benefit means the elimination or improvement of an underlying disease and / or the elimination or improvement of one or more physiological symptoms associated with the underlying disease during treatment, such that an improvement is observed in the individual.

[0054] In certain embodiments, each occurrence of alkyl or cycloalkyl is independently optionally substituted with at least one substituent selected from the group consisting of C1-C6 alkyl, halo, -OR, phenyl (thus, in non-limiting examples, phenyl-(C1-C3 alkyl) which may be substituted, for example, without limitation, benzyl or substituted benzyl), and -N(R)(R), where each occurrence of R is independently H, C1-C6 alkyl, or C3-C8 cycloalkyl. In other embodiments, each occurrence of aryl or heteroaryl is independently optionally substituted with at least one substituent selected from the group consisting of C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, halo, -CN, -OR, -N(R)(R), -NO2, -S(=O)2N(R)(R), acyl, and C1-C6 alkoxycarbonyl, where each occurrence of R is independently H, C1-C6 alkyl, or C3-C8 cycloalkyl. In still other embodiments, each occurrence of aryl or heteroaryl is independently optionally substituted with at least one substituent selected from the group consisting of C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, halo, -CN, -OR, -N(R)(R), and C1-C6 alkoxycarbonyl, where each occurrence of R is independently H, C1-C6 alkyl, or C3-C8 cycloalkyl.

[0055] Unless otherwise indicated, when two substituents together form a ring having a specified number of ring atoms (e.g., when R 2 and R 3 together with the nitrogen to which they are attached form a ring having 3-7 ring members), the ring may have carbon atoms and, optionally, one or more (e.g., 1-3) additional heteroatoms independently selected from nitrogen, oxygen, or sulfur. The ring may be saturated or partially saturated and may be substituted.

[0056] Whenever a term or its prefix root appears in the name of a substituent, that name should be interpreted as including the limitations set forth herein. For example, whenever the term or its prefix root appears in the name of a substituent (e.g., arylalkyl, alkylamino), that name should be interpreted as including the limitations set forth elsewhere herein for "alkyl" and "aryl," respectively.

[0057] In some embodiments, substituents of a compound are disclosed as a group or range. The description is specifically intended to include any individual partial combination of members of the group and range. For example, "C 1~6 The term "alkyl" is specifically intended to individually disclose C1, C2, C3, C4, C5, C6, C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C2-C6, C2-C5, C2-C4, C2-C3, C3-C6, C3-C5, C3-C4, C4-C6, C4-C5, and C5-C6 alkyl groups.

[0058] method This disclosure relates to a method for treating, preventing, and / or improving solid tumors in a subject where such treatment is needed, wherein the method comprises a pharmaceutically effective amount of (a) at least one immunostimulant, and (b) Retinaldehyde dehydrogenase 1 (RALDH1) inhibitors The process includes administering the RALDH1 inhibitor to the target, where the RALDH1 inhibitor is as follows: (i) Equation (I): A compound of TIFF2026510946000003.tif20128, or its salt, solvate, prodrug, stereoisomer, tautomer, or isotopolog, During the ceremony, R 1a This is selected from the group consisting of optionally substituted C2-C8 heterocyclyls, optionally substituted phenyls, and optionally substituted C5-C8 cycloalkenyls. Here, R 1a Each of the substituents in the compound may be a C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkoxy, optionally substituted phenyl, optionally substituted C2-C8 heterocyclyl, halogen, OH, or N(R) a )(R b ), NO2, CN, C(=O)R a , C(=O)OR a , C(=O)N(R a )(R b ), S(=O)2R a , S(=O)2OR a , S(=O)2N(R a )(R b ), OCR a , N(R a )=S(=O)(R b )(R c ), S(=O)(=NR a )R b , and N(R a )C(=O)R b Independently selected from the group consisting of, Here, each of the substituents may be substituted with at least one substituent selected from the group consisting of C1-C3 alkyl, C1-C3 alkoxy, halogen, CN, and NO2, and Here, R 1a Any two vicinal or geminal substituents within may combine with the atom to which they are bonded to form a C2-C8 heterocyclyl or C3-C8 cycloalkyl; R 1b and R 1c If present, H, C1-C6 alkyl, C1-C6 alkoxy, halogen, OH, N(R) a )(R b ), NO2, and CN are each independently selected; R 2 Each occurrence is independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, C1-C3 haloalkoxy, C1-C6 hydroxyalkyl, halogen, NO2, and CN; R 3This is a C2-C8 heterocycline which may be substituted, a phenyl which may be substituted, and N(R a ) (C3-C8 cycloalkyl which may be substituted), and N(R a Selected from the group consisting of (C2-C8 heterocyclines that may be substituted), Here, R 3 Any substituent in the group may be C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, halogen, OH, or N(R) a )(R b ), NO2, CN, C(=O)R a , C(=O)OR a , C(=O)N(R a )(R b ), S(=O)2R a , S(=O)2OR a , S(=O)2N(R a )(R b ), OCR a , and N(R a )C(=O)R b Selected independently from the group consisting of; A is C6~C even if it is not substituted. 10 Selected from the group consisting of aryls and optionally substituted C2-C8 heterocyclines, Here, each substituent in A is a C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, halogen, OH, N(R) a )(R b ), NO2, CN, C(=O)R a , C(=O)OR a , C(=O)N(R a )(R b ), S(=O)2R a , S(=O)2OR a , S(=O)2N(R a )(R b ), OCR a , and N(R a )C(=O)R b Selected independently from the group consisting of; L is selected from the group consisting of -CH2-, -C(=O)-, and -S(=O)2-; X is N and CR 1c Selected from the group consisting of; n is an integer selected from the group consisting of 0, 1, 2, 3, and 4; and R a , R b , and R c Each occurrence is independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, and C2-C8 heterocyclyl. Compounds of formula (I), or their salts, solvates, prodrugs, stereoisomers, tautomers, or isotopologs; (ii) 1-benzylindoline-2,3-dione; 2-((4-oxo-3-(3-(pyrrolidine-1-yl)propyl)-3,4-dihydrobenzo[4,5]thieno[3,2-d]pyrimidine-2-yl)thio)ethyl acetate; 2-((2-(sec-butyl)-3-oxo-2,3-dihydroimidazo[1,2-c]quinazoline-5-yl)thio)-N-(o-tolyl)butanamide; and 8-((4-(cyclopropanecarbonyl)piperazine-1-yl)methyl)-7-isopentyl-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione; or its salts, solvates, prodrugs, stereoisomers, tautomers, or isotopologs. A compound selected from the group consisting of and The present invention provides a method selected from the group consisting of the following.

[0059] In one embodiment, R 1a The C2-C8 heterocyclyl in the middle is thiophene-2-yl. In one embodiment, R 1a The C2-C8 heterocyclyl groups within are thiophene-3-yl.

[0060] In one embodiment, C6~C in A may be substituted. 10The aryl is an optionally substituted phenyl. In one embodiment, the optionally substituted C2-C8 heterocyclyl is an optionally substituted C2-C5 heterocyclyl.

[0061] In one embodiment, a RALDH1 inhibitor is a compound of formula (I).

[0062] In one embodiment, the compound of formula (I) is Selected from the group consisting of TIFF2026510946000004.tif25128, Here, R 2a , R 2b , R 2c , and R 2d If present, each of the following is independently selected from the group consisting of H, C1-C6 alkyl, C1-C6 alkoxy, and halogen.

[0063] In one embodiment, R 2a , R 2b , R 2c , and R 2d If present, each element is independently selected from the group consisting of H, Me, OMe, F, and Cl.

[0064] In one embodiment, X is N.

[0065] In one embodiment, R 1a teeth, Selected from the group consisting of TIFF2026510946000005.tif15128, Here, R 5a and R 5b If present, each of the following is independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, phenyl, thiophen-2-yl, thiophen-3-yl, and CN. Here, R 5a and R 5b Each substituent in the compound may be substituted with CN, Here, R 5aand R 5b If present, they may combine with the atoms to which they are bonded to form a C2-C6 heterocyclyl or C3-C6 cycloalkyl; and R 6 is S(=O)2R a That is the case.

[0066] In one embodiment, R 5a is Me. In one embodiment, R 5a t-tBu is t. In one aspect, R 5a is 1-cyanocyclopropyl. In one embodiment, R 5a is 1-cyanocyclopentyl. In one embodiment, R 5a is phenyl. In one embodiment, R 5a is CN. In one embodiment, R 5a is 1-cyanocyclobutyl. In one embodiment, R 5a is 1-cyanocyclohexyl. In one embodiment, R 5a is thiophene-2-yl. In one embodiment, R 5a It is thiophene-3-yl.

[0067] In one embodiment, R 5b is Me. In one embodiment, R 5b t-tBu is t. In one aspect, R 5b is 1-cyanocyclopropyl. In one embodiment, R 5b is 1-cyanocyclopentyl. In one embodiment, R 5b is phenyl. In one embodiment, R 5b is CN. In one embodiment, R 5b is 1-cyanocyclobutyl. In one embodiment, R 5b is 1-cyanocyclohexyl. In one embodiment, R 5b is thiophene-2-yl. In one embodiment, R 5b It is thiophene-3-yl.

[0068] In one embodiment, R 6It is ethenylsulfonyl.

[0069] In one embodiment, R 1a teeth, This is TIFF2026510946000006.tif14128. In one embodiment, R 1a teeth, This is TIFF2026510946000007.tif12128. In one embodiment, R 1a teeth, This is TIFF2026510946000008.tif16128. In one embodiment, R 1a teeth, This is TIFF2026510946000009.tif15128. In one embodiment, R 1a teeth, This is TIFF2026510946000010.tif18128. In one embodiment, R 1a teeth, This is TIFF2026510946000011.tif18128. In one embodiment, R 1a teeth, This is TIFF2026510946000012.tif15128. In one embodiment, R 1a teeth, This is TIFF2026510946000013.tif17128. In one embodiment, R 1a teeth, The filename is TIFF2026510946000014.tif15128.

[0070] In one embodiment, R 1b H is H.

[0071] In one embodiment, R 3 teeth, Selected from the group consisting of TIFF2026510946000015.tif20128, Here, R 7 C(=O)R a and S(=O)2R a Selected from the group consisting of; and R8 The group is selected from the group consisting of C1-C6 alkoxy, C1-C6 hydroxyalkyl, and OH.

[0072] In one embodiment, R 7 is cyclopropylcarbonyl. In one embodiment, R 7 is methylsulfonyl. In one embodiment, R 7 is dimethylaminosulfonyl. In one embodiment, R 7 It is dimethylaminocarbonyl.

[0073] In one embodiment, R 8 is methoxy. In one embodiment, R 8 is 2-hydroxyethyl. In one embodiment, R 8 It is OH.

[0074] In one embodiment, R 3 teeth, This is TIFF2026510946000016.tif17128. In one embodiment, R 3 teeth, This is TIFF2026510946000017.tif16128. In one embodiment, R 3 teeth, This is TIFF2026510946000018.tif16128. In one embodiment, R 3 teeth, This is TIFF2026510946000019.tif17128. In one embodiment, R 3 teeth, This is TIFF2026510946000020.tif12128. In one embodiment, R 3 teeth, This is TIFF2026510946000021.tif16128. In one embodiment, R 3 teeth, This is TIFF2026510946000022.tif16128. In one embodiment, R 3 teeth, The filename is TIFF2026510946000023.tif14128.

[0075] In one embodiment, the compound of formula (I) is 8-(6-methoxy-3-((4-methoxyphenyl)sulfonyl)quinoline-4-yl)-1,4-dioxa-8-azaspiro[4.5]decane; 1-(3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)-6-fluoroquinoline-4-yl)-4-phenylpiperidine-4-carbonitrile; (4-(cyclopropanecarbonyl)piperazine-1-yl)(4-(4,4-dimethylcyclohexa-1-en-1-yl)-6-fluoroquinoline-3-yl)methanone; (4-(4-(tert-butyl)phenyl)-6-fluoroquinoline-3-yl)(4-(cyclopropanecarbonyl)piperazine-1-yl)methanone; 1-(4-(3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)-6-fluoroquinoline-4-yl)phenyl)cyclopropanecarbonitrile; 1-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonilicate; (4-(4,4-dimethylcyclohexa-1-en-1-yl)-6-fluoroquinoline-3-yl)(4-(methylsulfonyl)piperazine-1-yl)methanone; (4-(4-(tert-butyl)phenyl)-6-fluoroquinoline-3-yl)(4-(methylsulfonyl)piperazine-1-yl)methanone; 1-(4-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoro-N-((1r,4r)-4-hydroxycyclohexyl)quinoline-3-carboxamide; (6-Fluoro-4-(4-(vinylsulfonyl)piperazine-1-yl)quinoline-3-yl)(4-(methylsulfonyl)piperazine-1-yl)methanone; 1-(4-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclobutan-1-carbonitrile; 1-(4-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopentan-1-carbonitrile; 4-(4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-sulfonamide; 4-(4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-carboxamide; 1-(6-chloro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonilicate; 1-(6-chloro-3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonitrile; 1-(6-(4-(methylsulfonyl)piperazine-1-carbonyl)thieno[3,2-b]pyridine-7-yl)-4-phenylpiperidine-4-carbonitrile; 1-(6-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)thieno[3,2-b]pyridine-7-yl)-4-phenylpiperidine-4-carbonitrile; 1-(6-methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonilicate; 1-(7-methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonilicate; 1-(6,7-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonilicate; 1-(6,8-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonilicate; 4-(4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-sulfonamide; 4-(4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-carboxamide; 4-(6-chloro-4-(4-(1-cyanocyclopropyl)phenyl)quinoline-3-carbonyl)-N,N-dimethylpiperazine-1-sulfonamide; 1-(4-(6-chloro-3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6-(4-(methylsulfonyl)piperazine-1-carbonyl)thieno[3,2-b]pyridine-7-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)thieno[3,2-b]pyridine-7-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6-methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(7-Methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6,7-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6,8-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoro-N-((1r,4r)-4-hydroxycyclohexyl)quinoline-3-carboxamide; 4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoro-N-(1-(2-hydroxyethyl)-1H-pyrazole-4-yl)quinoline-3-carboxamide; and 4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoro-N-(1-(2-hydroxyethyl)-1H-pyrazole-4-yl)quinoline-3-carboxamide Selected from the group consisting of .

[0076] In some respects, solid tumors are carcinomas.

[0077] In one aspect, the carcinoma includes human hepatocellular carcinoma (HCC) cells.

[0078] In one embodiment, RALDH1 is overexpressed in solid tumors.

[0079] In one embodiment, the expression of Raldh1 and Raldh2 or Raldh1 and Raldh3 in solid tumors has a ratio in the range of approximately 10000:1 to approximately 2:1. In one embodiment, the expression of Raldh1 and Raldh2 or Raldh1 and Raldh3 in solid tumors has a ratio selected from the group consisting of approximately 10000:1, 9000:1, 8000:1, 7000:1, 6000:1, 5000:1, 4000:1, 3000:1, 2000:1, and 1000:1. In one embodiment, the expression of Raldh1 and Raldh2 or Raldh1 and Raldh3 in solid tumors has a ratio selected from the group consisting of approximately 900:1, 800:1, 700:1, 600:1, 500:1, 400:1, 300:1, 200:1, and 100:1. In one embodiment, the expression of Raldh1 and Raldh2 or Raldh1 and Raldh3 in solid tumors has a ratio selected from the group consisting of approximately 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 4:1, 3:1, and 2:1.

[0080] In one embodiment, the immunostimulant is at least one selected from the group consisting of immune checkpoint inhibitors, chimeric antigen receptor (CAR) T cells, T cells genetically engineered to express specific TCRs targeting tumor antigens (TCR transgenics), ex vivo expanding T cells, and bispecific T cell engagers (BiTEs).

[0081] In one aspect, immunostimulants are immune checkpoint inhibitors.

[0082] In one embodiment, the immune checkpoint inhibitor is selected from the group consisting of anti-PD1 antibodies, anti-PD-L1 antibodies, anti-CTLA4 antibodies, any fragment thereof, and any combination thereof. In one embodiment, the immune checkpoint inhibitor is an anti-PD1 antibody. In one embodiment, the immune checkpoint inhibitor is an anti-PD-L1 antibody. In one embodiment, the immune checkpoint inhibitor is an anti-CTLA4 antibody.

[0083] In one aspect, the immunostimulant is CAR T cells. In one aspect, CAR T cells are administered intravenously. In one aspect, CAR T cells are administered as CAR T cell therapy.

[0084] In one embodiment, the subjects are administered immune checkpoint inhibitors and chimeric antigen receptor (CAR) T cells.

[0085] In one embodiment, the method further includes administering at least one selected from the group consisting of retinoic acid receptor (RAR) inhibitors and retinoid X receptor (RXR) inhibitors to a target.

[0086] In one embodiment, the RAR inhibitor is selected from the group consisting of AGN 193109, BMS 195614, BMS 493, CD 2665, ER 50891, LE 135, LY 2955303, MM 11253, any salt or solvate thereof, and any combination thereof.

[0087] In one embodiment, the RXR inhibitor is selected from the group consisting of HX 531, PA 452, and UVI 3003, any salt or solvate thereof, and any combination thereof.

[0088] In one embodiment, a RALDH1 inhibitor and an immunostimulant are administered simultaneously or sequentially to the target.

[0089] In one aspect, the subject is a mammal. In one aspect, the mammal is a human.

[0090] Retinoic acid modulator The compounds of this disclosure may have one or more stereocenters, each stereocenter may exist independently in either the (R) or (S) configuration. In some embodiments, the compounds described herein exist as optically active or racemic compounds. The compounds described herein encompass racemic compounds, optically active compounds, positional isomers, and stereoisomers, or combinations thereof, having the therapeutically useful properties described herein. Preparation of optically active compounds can be achieved in any preferred manner, including, but not limited to, recrystallization techniques for racemic separation, synthesis from optically active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase. Furthermore, compounds represented by racemic formulas herein represent either one or any mixture thereof of two enantiomers, or, if two or more chiral centers are present, all diastereomers or any mixture thereof.

[0091] In one embodiment, the compounds of the present invention exist as tautomers. All tautomers are included within the range of compounds described herein.

[0092] The compounds described herein also include isotope-labeled compounds in which one or more atoms are replaced by atoms having the same number of atoms but with atomic masses or atomic mass numbers different from those commonly found in nature. Examples of isotopes suitable for inclusion in the compounds described herein include: 2 H, 3 H, 11 C, 13 C, 14 C, 36 Cl, 18 F, 123 I, 125 I, 13 N, 15 N, 15 O, 17 O, 18 O, 32 P, and 35Examples include, but are not limited to, S. In some embodiments, chemical stability is improved by substitution with heavy isotopes such as deuterium. The isotope-labeled compounds are prepared by any preferred method or by a process that uses a suitable isotope-labeled reagent instead of a separately used unlabeled reagent.

[0093] In one embodiment, the compounds described herein are labeled by means of other means, including but not limited to the use of a chromophore or fluorescent moiety, a bioluminescent label, or a chemiluminescent label.

[0094] In all embodiments described herein, examples of any suitable substituents are not intended to limit the scope of the claimed invention. The compounds of the present invention may include any substituent or combination of substituents shown herein.

[0095] The compounds described herein can be prepared from commercially available starting materials, literature-known compounds, or readily prepared intermediates by using standard synthetic methods and procedures known to those skilled in the art. Standard synthetic methods and procedures for the preparation of organic molecules, as well as the transformation and manipulation of functional groups, can be readily obtained from relevant scientific literature or standard textbooks in the art.

[0096] RALDH1 inhibitors The RALDH1 inhibitors of the present invention inhibit RALDH1 with higher selectivity than other RALDHs (i.e., RALDH2 and RALDH3) and may be chemically synthesized by methods known in the art, or they may be purchased from commercially available sources. RALDH1 inhibitors include, but are not limited to, any RALDH1 inhibitors known in the art, including Yang et al (J. Med. Chem. 2018, 61(11):4883-4903), which are incorporated herein by reference. Non-limiting examples of RALDH1 inhibitors intended for use in this disclosure include any of the compounds provided in Table 1, or their salts, solvates, prodrugs, stereoisomers, or isotopologs.

[0097] (Table 1) TIFF2026510946000024.tif183161TIFF2026510946000025.tif214161TIFF2026510946000026.tif212161TIFF2026510946000027.tif210161 TIFF2026510946000028.tif202161TIFF2026510946000029.tif213161TIFF2026510946000030.tif220161TIFF2026510946000031.tif210161

[0098] Retinoic acid receptor inhibitors or retinoid X receptor inhibitors In some embodiments, the methods and / or compositions described herein include retinoic acid receptor inhibitors and / or retinoid X receptor inhibitors. The retinoic acid receptor inhibitors or retinoid X receptor inhibitors used in the methods described herein may be chemically synthesized by methods known in the art or purchased from commercially available sources. In some embodiments, the retinoic acid receptor inhibitors are selected from the group consisting of AGN 193109, BMS 195614, BMS 493 CD 2665, ER 50891, LE 135, LY 2955303, MM 11253, any salts or solvates thereof, and any combination thereof (Table 2).

[0099] (Table 2) TIFF2026510946000032.tif132161TIFF2026510946000033.tif205161

[0100] AGN 193109 is a pan-RAR antagonist. BMS 195614 is a selective RAR-α antagonist. BMS 493 is a pan-RAR antagonist / inverse agonist. CD 2665 is a selective RAR-β and RAR-γ antagonist. ER 50891 is a selective RAR-α antagonist. LE 135 is a selective RAR-β antagonist. LY 2955303 is a selective RAR-γ antagonist. MM 11253 is a selective RAR-γ antagonist.

[0101] In some embodiments, retinoid X receptor inhibitors are selected from the group consisting of HX 531, PA 452, and UVI 3003, any salt or solvate thereof, and any combination thereof (Table 3). HX 531 is a pan-RXR antagonist. PA 452 is a pan-RXR antagonist. UVI 3003 is a pan-RXR antagonist.

[0102] (Table 3) TIFF2026510946000034.tif122161

[0103] Immunostimulants Immune checkpoint inhibitors As described herein, the term “immune checkpoint inhibitor” includes any compound or composition that can inhibit immune checkpoint molecules, which are regulatory factors of the immune system (e.g., that stimulate or inhibit the activity of the immune system). For example, some checkpoint inhibitors stimulate the function of the immune system by blocking inhibitory checkpoint molecules, for example, stimulating T cell activity against cancer cells. A non-limiting example of a checkpoint inhibitor is PD-L1 inhibitors.

[0104] As described herein, the term “PD-L1 inhibitor” includes any compound capable of directly or indirectly inhibiting the expression and / or function of protein programmed death ligand 1 (PD-L1). PD-L1, also known as surface antigen classification 274 (CD274) or B7 homolog 1 (B7-H1), is a type 1 transmembrane protein that plays a major role in suppressing the adaptive arm of the immune system. PD-L1 modulates the activation or inhibition of the adaptive arm of the immune system by binding to its receptor, the inhibitory checkpoint molecule PD-1 (found on activated T cells, B cells, and myeloid cells). In some embodiments, PD-L1 inhibitors (i.e., anti-PD-L1 antibodies) inhibit the expression and / or function of PD-L1 by at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or at least 90%.

[0105] Reported PD-L1 inhibitors include, but are not limited to, compounds described in one of the following patent application publications, all of which are incorporated herein by reference: US 2018 / 0057455; US 2018 / 0057486; WO 2017 / 106634; WO 2018 / 026971; WO 2018 / 045142; WO 2018 / 118848; WO 2018 / 119221; WO 2018 / 119236; WO 2018 / 119266; WO 2018 / 119286; WO 2018 / 121560; WO 2019 / 076343; WO 2019 / 087214.

[0106] In one embodiment, the immune checkpoint inhibitor is selected from the group consisting of anti-PD1 antibodies, anti-PD-L1 antibodies, anti-CTLA4 antibodies, any fragment thereof, and any combination thereof.

[0107] Chimeric antigen receptor (CAR) T cells As described elsewhere in this specification, the term “chimeric antigen receptor (CAR) T cell” refers to T cells genetically engineered to provide an artificial T cell receptor for use in immunotherapy. Artificial T cell receptors, including a chimeric antigen receptor (CAR) expressed on the surface of the T cell, provide both antigen-binding and T-cell-activating functions, thereby enabling activation of the host immune response via one of several mechanisms upon antigen binding (e.g., T cell proliferation and monocyte differentiation). The antigen-binding domain of the CAR is selected based on a specific protein identified as being overexpressed on the surface of tumor cells in the subject. Preferably, the antigen overexpressed on the tumor surface is specific to cancer cells and substantially absent on the surface of healthy cells.

[0108] In one embodiment, T cells are autologously collected from the subject. In another embodiment, donor T cells may be used. The collected T cells are subjected to genetic engineering to express a desired CAR on the cell surface. Furthermore, the CAR T cells are administered to the subject to induce a targeted immune response (illicit). In one embodiment, the CAR T cells are administered intravenously. CAR T cell therapy comprises the processes described herein (i.e., T cell collection, T cell modification, and CAR T cell administration) for the treatment of a disease in the subject (e.g., hepatocellular carcinoma).

[0109] TCR transgenic T cells As described elsewhere in this specification, the term “TCR transgenic T cells” refers to T cells genetically engineered to provide a native T cell receptor for use in immunotherapy. T cells recognize antigens via a T cell receptor (TCR) composed of alpha (α) and beta (β) chains. However, the αβ-TCR can only recognize and respond to antigens if they are presented as 8-11 amino acid peptide fragments by the major histocompatibility complex (MHC) on target cells. CD8 T cells recognize antigens presented by MHC class I (expressed by all nucleated cells), while CD4 T cells recognize those presented by MHC class II (expressed by specialized antigen-presenting cells). Tumor-killing T cells are typically cytotoxic (or killer) CD8 T cells that recognize tumor-associated antigens presented by MHC class I on the surface of tumor cells. The TCR transgenic approach is based on forcing the expression of a “synthetic” TCR with defined specificity for tumor antigen-MHCI complexes in T cells. The advantage of this approach is its ability to target any protein expressed by the tumor (nuclear, cytoplasmic, or membrane-bound). On the other hand, a major obstacle is the highly polymorphic nature of MHC and the need to select patients based on the co-expression of the relevant MHC-I molecule and the target antigen. Therefore, the benefits of tgTCRs are limited to patients expressing a specific MHC allele, as a synthetic TCR for an antigen on a particular MHC allele will not recognize it when presented by another MHC allele.

[0110] Adoptive transplantation of autologous antitumor T cells (ex vivo expanded T cells) As explained elsewhere, autologous antitumor T cell adoptive transplantation refers to a non-genetically modified approach in which tumor-responsive T cells are identified and isolated from the patient's own tumor, these T cells are grown ex vivo, and then reinjected into the same patient. The advantage of this approach is that, because they are autologous, the survival rate of the injected T cells is improved and rejection is minimized.

[0111] Bispecific T cell engagers (BiTEs) As described herein, bispecific T cell engagers (BiTEs) are synthetic proteins that simultaneously bind to T cells and target antigens on tumor cells, independently of MHC-TCR interactions. In one embodiment, this forces contact between T cells and tumor cells. A major advantage of BiTEs is that they are not specific to a particular patient, tumor, or T cell subtype, and therefore can be used "off the box." The main limitation is that not all T cells brought into proximity to tumor cells by this approach are activated, nor can they kill tumor cells.

[0112] compound This disclosure is based on formula (I): A compound of TIFF2026510946000035.tif20128, or its salt, solvate, prodrug, stereoisomer, tautomer, or isotopolog, During the ceremony, R 1a This is selected from the group consisting of optionally substituted C2-C8 heterocyclyls, optionally substituted phenyls, and optionally substituted C5-C8 cycloalkenyls. Here, R 1a Each of the substituents in the compound may be a C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkoxy, optionally substituted phenyl, optionally substituted C2-C8 heterocyclyl, halogen, OH, or N(R) a )(R b ), NO2, CN, C(=O)R a , C(=O)OR a , C(=O)N(R a )(R b ), S(=O)2R a , S(=O)2OR a , S(=O)2N(R a )(R b ), OCR a , N(R a )=S(=O)(R b )(R c ), S(=O)(=NRa )R b , and N(R a )C(=O)R b Independently selected from the group consisting of, Here, each of the substituents may be substituted with at least one substituent selected from the group consisting of C1-C3 alkyl, C1-C3 alkoxy, halogen, CN, and NO2, and Here, R 1a Any two vicinal or geminal substituents within may combine with the atom to which they are bonded to form a C2-C8 heterocyclyl or C3-C8 cycloalkyl; R 1b and R 1c If present, H, C1-C6 alkyl, C1-C6 alkoxy, halogen, OH, N(R) a )(R b ), NO2, and CN are each independently selected; R 2 Each occurrence is independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, C1-C3 haloalkoxy, C1-C6 hydroxyalkyl, halogen, NO2, and CN; R 3 This is a C2-C8 heterocycline which may be substituted, a phenyl which may be substituted, and N(R a ) (C3-C8 cycloalkyl which may be substituted), and N(R a Selected from the group consisting of (C2-C8 heterocyclines that may be substituted), Here, R 3 Any substituent in the group may be C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, halogen, OH, or N(R) a )(R b ), NO2, CN, C(=O)R a , C(=O)OR a , C(=O)N(R a )(R b ), S(=O)2R a , S(=O)2OR a , S(=O)2N(R a )(R b), OCR a , and N(R a )C(=O)R b Selected independently from the group consisting of; A is C6~C even if it is not substituted. 10 Selected from the group consisting of aryls and optionally substituted C2-C8 heterocyclines, Here, each substituent in A is a C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, halogen, OH, N(R) a )(R b ), NO2, CN, C(=O)R a , C(=O)OR a , C(=O)N(R a )(R b ), S(=O)2R a , S(=O)2OR a , S(=O)2N(R a )(R b ), OCR a , and N(R a )C(=O)R b Selected independently from the group consisting of; L is selected from the group consisting of -CH2-, -C(=O)-, and -S(=O)2-; X is N and CR 1c Selected from the group consisting of; n is an integer selected from the group consisting of 0, 1, 2, 3, and 4; and R a , R b , and R c Each occurrence is independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, and C2-C8 heterocyclyl. Compounds of formula (I), or their salts, solvates, prodrugs, stereoisomers, tautomers, or isotopologs; and (ii) 1-benzylindoline-2,3-dione, 2-((4-oxo-3-(3-(pyrrolidine-1-yl)propyl)-3,4-dihydrobenzo[4,5]thieno[3,2-d]pyrimidine-2-yl)thio)ethyl acetate, 2-((2-(sec-butyl)-3-oxo-2,3-dihydroimidazo[1,2-c]quinazoline-5-yl)thio)-N-(o-tolyl)butanamide, and 8-((4-(cyclopropanecarbonyl)piperazine-1-yl)methyl)-7-isopentyl-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione, or its salts, solvates, prodrugs, stereoisomers, tautomers, or isotopologs. Compounds selected from the group consisting of To provide.

[0113] In one embodiment, R 1a The C2-C8 heterocyclyl in the middle is thiophene-2-yl. In one embodiment, R 1a The C2-C8 heterocyclyl groups within are thiophene-3-yl.

[0114] In one embodiment, C6~C in A may be substituted. 10 The aryl is an optionally substituted phenyl. In one embodiment, the optionally substituted C2-C8 heterocyclyl is an optionally substituted C2-C5 heterocyclyl.

[0115] In one embodiment, a RALDH1 inhibitor is a compound of formula (I).

[0116] In one embodiment, the compound of formula (I) is Selected from the group consisting of TIFF2026510946000036.tif25139, Here, R 2a , R 2b , R 2c , and R 2d If present, each of the following is independently selected from the group consisting of H, C1-C6 alkyl, C1-C6 alkoxy, and halogen.

[0117] In one embodiment, R 2a , R 2b , R 2c , and R 2d If present, each element is independently selected from the group consisting of H, Me, OMe, F, and Cl.

[0118] In one embodiment, X is N.

[0119] In one embodiment, R 1a teeth, Selected from the group consisting of TIFF2026510946000037.tif15128, Here, R 5a and R 5b If present, each of the following is independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, phenyl, thiophen-2-yl, thiophen-3-yl, and CN. Here, R 5a and R 5b Each substituent in the compound may be substituted with CN, Here, R 5a and R 5b If present, they may combine with the atoms to which they are bonded to form a C2-C6 heterocyclyl or C3-C6 cycloalkyl; and R 6 is S(=O)2R a That is the case.

[0120] In one embodiment, R 5a is Me. In one embodiment, R 5a t-tBu is t. In one aspect, R 5a is 1-cyanocyclopropyl. In one embodiment, R 5a is 1-cyanocyclopentyl. In one embodiment, R 5a is phenyl. In one embodiment, R 5a is CN. In one embodiment, R 5ais 1-cyanocyclobutyl. In one embodiment, R 5a is 1-cyanocyclohexyl. In one embodiment, R 5a is thiophene-2-yl. In one embodiment, R 5a It is thiophene-3-yl.

[0121] In one embodiment, R 5b is Me. In one embodiment, R 5b t-tBu is t. In one aspect, R 5b is 1-cyanocyclopropyl. In one embodiment, R 5b is 1-cyanocyclopentyl. In one embodiment, R 5b is phenyl. In one embodiment, R 5b is CN. In one embodiment, R 5b is 1-cyanocyclobutyl. In one embodiment, R 5b is 1-cyanocyclohexyl. In one embodiment, R 5b is thiophene-2-yl. In one embodiment, R 5b It is thiophene-3-yl.

[0122] In one embodiment, R 6 It is ethenylsulfonyl.

[0123] In one embodiment, R 1a teeth, This is TIFF2026510946000038.tif14128. In one embodiment, R 1a teeth, This is TIFF2026510946000039.tif12128. In one embodiment, R 1a teeth, This is TIFF2026510946000040.tif16128. In one embodiment, R 1a teeth, This is TIFF2026510946000041.tif15128. In one embodiment, R 1a teeth, This is TIFF2026510946000042.tif18128. In one embodiment, R 1a teeth, This is TIFF2026510946000043.tif18128. In one embodiment, R 1a teeth, This is TIFF2026510946000044.tif15128. In one embodiment, R 1a teeth, This is TIFF2026510946000045.tif16128. In one embodiment, R 1a teeth, The filename is TIFF2026510946000046.tif15128.

[0124] In one embodiment, R 1b H is H.

[0125] In one embodiment, R 3 teeth, Selected from the group consisting of TIFF2026510946000047.tif20128, Here, R 7 C(=O)R a and S(=O)2R a Selected from the group consisting of; and R 8 The group is selected from the group consisting of C1-C6 alkoxy, C1-C6 hydroxyalkyl, and OH.

[0126] In one embodiment, R 7 is cyclopropylcarbonyl. In one embodiment, R 7 is methylsulfonyl. In one embodiment, R 7 is dimethylaminosulfonyl. In one embodiment, R 7 It is dimethylaminocarbonyl.

[0127] In one embodiment, R 8 is methoxy. In one embodiment, R 8 is 2-hydroxyethyl. In one embodiment, R8 It is OH.

[0128] In one embodiment, R 3 teeth, This is TIFF2026510946000048.tif17128. In one embodiment, R 3 teeth, This is TIFF2026510946000049.tif16128. In one embodiment, R 3 teeth, This is TIFF2026510946000050.tif16128. In one embodiment, R 3 teeth, This is TIFF2026510946000051.tif17128. In one embodiment, R 3 teeth, This is TIFF2026510946000052.tif12128. In one embodiment, R 3 teeth, This is TIFF2026510946000053.tif16128. In one embodiment, R 3 teeth, This is TIFF2026510946000054.tif16128. In one embodiment, R 3 teeth, The filename is TIFF2026510946000055.tif14128.

[0129] In one embodiment, the compound of formula (I) is 8-(6-methoxy-3-((4-methoxyphenyl)sulfonyl)56-uinoline-4-yl)-1,4-dioxa-8-azaspiro[4.5]decane; 1-(3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)-6-fluoroquinoline-4-yl)-4-phenylpiperidine-4-carbonitrile; (4-(cyclopropanecarbonyl)piperazine-1-yl)(4-(4,4-dimethylcyclohexa-1-en-1-yl)-6-fluoroquinoline-3-yl)methanone; (4-(4-(tert-butyl)phenyl)-6-fluoroquinoline-3-yl)(4-(cyclopropanecarbonyl)piperazine-1-yl)methanone; 1-(4-(3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)-6-fluoroquinoline-4-yl)phenyl)cyclopropanecarbonitrile; 1-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)57winolin-4-yl)-4-phenylpiperidine-4-carbonitrile; (4-(4,4-dimethylcyclohexa-1-en-1-yl)-6-fluoroquinoline-3-yl)(4-(methylsulfonyl)piperazine-1-yl)methanone; (4-(4-(tert-butyl)phenyl)-6-fluoroquinoline-3-yl)(4-(methylsulfonyl)piperazine-1-yl)methanone; 1-(4-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)57winolin-4-yl)phenyl)cyclopropane-1-carbonitrile; 4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoro-N-((1r,4r)-4-hydroxycyclohexyl)quinoline-3-carboxamide; (6-Fluoro-4-(4-(vinylsulfonyl)piperazine-1-yl)57winolin-3-yl)(4-(methylsulfonyl)piperazine-1-yl)methanone; 1-(4-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)57winolin-4-yl)phenyl)57winolin57e-1-carbonitrile; 1-(4-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)57winolin-4-yl)phenyl)cyclopentan-1-carbonitrile; 4-(4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-sulfonamide; 4-(4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-carboxamide; 1-(6-chloro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)57winolin-4-yl)-4-phenylpiperidine-4-carbonitrile; 1-(6-chloro-3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)57winolin-4-yl)-4-phenylpiperidine-4-carbonitrile; 1-(6-(4-(methylsulfonyl)piperazine-1-carbonyl)thieno[3,2-b]58winolin-7-yl)-4-phenylpiperidine-4-carbonitrile; 1-(6-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)thieno[3,2-b]58winolin-7-yl)-4-phenylpiperidine-4-carbonitrile; 1-(6-methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)58-winolin-4-yl)-4-phenylpiperidine-4-carbonitrile; 1-(7-Methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)58-winolin-4-yl)-4-phenylpiperidine-4-carbonitrile; 1-(6,7-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)58-winolin-4-yl)-4-phenylpiperidine-4-carbonitrile; 1-(6,8-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)58-winolin-4-yl)-4-phenylpiperidine-4-carbonitrile; 4-(4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-sulfonamide; 4-(4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-carboxamide; 4-(6-chloro-4-(4-(1-cyanocyclopropyl)phenyl)quinoline-3-carbonyl)-N,N-dimethylpiperazine-1-sulfonamide; 1-(4-(6-chloro-3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)58winolin-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6-(4-(methylsulfonyl)piperazine-1-carbonyl)thieno[3,2-b]58winolin-7-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)thieno[3,2-b]58winolin-7-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6-Methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)58-winolin-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(7-Methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)58winolin-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6,7-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)58winolin-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6,8-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)59winolin-4-yl)phenyl)cyclopropane-1-carbonitrile; 4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoro-N-((1r,4r)-4-hydroxycyclohexyl)quinoline-3-carboxamide; 4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoro-N-(1-(2-hydroxyethyl)-1H-pyrazole-4-yl)quinoline-3-carboxamide; and 4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoro-N-(1-(2-hydroxyethyl)-1H-pyrazole-4-yl)quinoline-3-carboxamide Selected from the group consisting of .

[0130] Pharmaceutical compositions and formulations This disclosure further states that (a) at least one immunostimulant; (b) pharmaceutically acceptable carriers; and I. Retinaldehyde dehydrogenase 1 (RALDH1) inhibitors A pharmaceutical composition comprising, wherein the RALDH1 inhibitor is as follows: (i) Equation (I): A compound of TIFF2026510946000056.tif20128, or its salt, solvate, prodrug, stereoisomer, tautomer, or isotopolog, During the ceremony, R 1a This is selected from the group consisting of optionally substituted C2-C8 heterocyclyls, optionally substituted phenyls, and optionally substituted C5-C8 cycloalkenyls. Here, R 1a Each of the substituents in the compound may be C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkoxy, optionally substituted phenyl, optionally substituted C2-C8, halogen, OH, or N(R) a )(R b ), NO2, CN, C(=O)R a , C(=O)OR a , C(=O)N(R a )(R b ), S(=O)2R a , S(=O)2OR a , S(=O)2N(R a )(R b ), OCR a , N(R a )=S(=O)(R b )(R c ), S(=O)(=NR a )R b , and N(R a )C(=O)R b Independently selected from the group consisting of, Here, each of the substituents may be substituted with at least one substituent selected from the group consisting of C1-C3 alkyl, C1-C3 alkoxy, halogen, CN, and NO2, and Here, R 1a Any two vicinal or geminal substituents within may combine with the atom to which they are bonded to form a C2-C8 heterocyclyl or C3-C8 cycloalkyl; R 1b and R 1c If present, H, C1-C6 alkyl, C1-C6 alkoxy, halogen, OH, N(R) a )(R b ), NO2, and CN are each independently selected; R 2 Each occurrence is independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, C1-C3 haloalkoxy, C1-C6 hydroxyalkyl, halogen, NO2, and CN; R 3 This is a C2-C8 heterocycline which may be substituted, a phenyl which may be substituted, and N(R a ) (C3-C8 cycloalkyl which may be substituted), and N(R a Selected from the group consisting of (C2-C8 heterocyclines that may be substituted), Here, R 3 Any substituent in the group may be C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, halogen, OH, or N(R) a )(R b ), NO2, CN, C(=O)R a , C(=O)OR a , C(=O)N(R a )(R b ), S(=O)2R a , S(=O)2OR a , S(=O)2N(R a )(R b ), OCR a , and N(R a )C(=O)R b Selected independently from the group consisting of; A is C6~C even if it is not substituted. 10 Selected from the group consisting of aryls and optionally substituted C2-C8 heterocyclines, Here, each substituent in A is a C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, halogen, OH, N(R) a )(R b ), NO2, CN, C(=O)R a , C(=O)OR a , C(=O)N(R a )(R b ), S(=O)2R a , S(=O)2OR a , S(=O)2N(R a )(R b ), OCR a , and N(R a )C(=O)R b Selected independently from the group consisting of; L is selected from the group consisting of -CH2-, -C(=O)-, and -S(=O)2-; X is N and CR 1c Selected from the group consisting of; n is an integer selected from the group consisting of 0, 1, 2, 3, and 4; and R a , R b , and R c Each occurrence is independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, and C2-C8 heterocyclyl. Compounds of formula (I), or their salts, solvates, prodrugs, stereoisomers, tautomers, or isotopologs; (ii) 1-benzylindoline-2,3-dione; 2-((4-oxo-3-(3-(pyrrolidine-1-yl)propyl)-3,4-dihydrobenzo[4,5]thieno[3,2-d]pyrimidine-2-yl)thio)ethyl acetate; 2-((2-(sec-butyl)-3-oxo-2,3-dihydroimidazo[1,2-c]quinazoline-5-yl)thio)-N-(o-tolyl)butanamide; and 8-((4-(cyclopropanecarbonyl)piperazine-1-yl)methyl)-7-isopentyl-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione; or its salts, solvates, prodrugs, stereoisomers, tautomers, or isotopologs. A compound selected from the group consisting of and The present invention provides a pharmaceutical composition selected from the group consisting of the following.

[0131] In one embodiment, R 1a The C2-C8 heterocyclyl in the middle is thiophene-2-yl. In one embodiment, R 1a The C2-C8 heterocyclyl groups within are thiophene-3-yl.

[0132] In one embodiment, C6~C in A may be substituted. 10 The aryl is an optionally substituted phenyl. In one embodiment, the optionally substituted C2-C8 heterocyclyl is an optionally substituted C2-C5 heterocyclyl.

[0133] In one embodiment, a RALDH1 inhibitor is a compound of formula (I).

[0134] In one embodiment, the compound of formula (I) is Selected from the group consisting of TIFF2026510946000057.tif25128, Here, R 2a , R 2b , R 2c , and R 2d If present, each of the following is independently selected from the group consisting of H, C1-C6 alkyl, C1-C6 alkoxy, and halogen.

[0135] In one embodiment, R 2a , R 2b, R 2c , and R 2d If present, each element is independently selected from the group consisting of H, Me, OMe, F, and Cl.

[0136] In one embodiment, X is N.

[0137] In one embodiment, R 1a teeth, Selected from the group consisting of TIFF2026510946000058.tif15128, Here, R 5a and R 5b If present, each of the following is independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, phenyl, thiophen-2-yl, thiophen-3-yl, and CN. Here, R 5a and R 5b Each substituent in the compound may be substituted with CN, Here, R 5a and R 5b If present, they may combine with the atoms to which they are bonded to form a C2-C6 heterocyclyl or C3-C6 cycloalkyl; and R 6 is S(=O)2R a That is the case.

[0138] In one embodiment, R 5a is Me. In one embodiment, R 5a t-tBu is t. In one aspect, R 5a is 1-cyanocyclopropyl. In one embodiment, R 5a is 1-cyanocyclopentyl. In one embodiment, R 5a is phenyl. In one embodiment, R 5a is CN. In one embodiment, R 5a is 1-cyanocyclobutyl. In one embodiment, R 5a is 1-cyanocyclohexyl. In one embodiment, R 5ais thiophene-2-yl. In one embodiment, R 5a It is thiophene-3-yl.

[0139] In one embodiment, R 5b is Me. In one embodiment, R 5b t-tBu is t. In one aspect, R 5b is 1-cyanocyclopropyl. In one embodiment, R 5b is 1-cyanocyclopentyl. In one embodiment, R 5b is phenyl. In one embodiment, R 5b is CN. In one embodiment, R 5b is 1-cyanocyclobutyl. In one embodiment, R 5b is 1-cyanocyclohexyl. In one embodiment, R 5b is thiophene-2-yl. In one embodiment, R 5b It is thiophene-3-yl.

[0140] In one embodiment, R 6 It is ethenylsulfonyl.

[0141] In one embodiment, R 1a teeth, This is TIFF2026510946000059.tif14128. In one embodiment, R 1a teeth, This is TIFF2026510946000060.tif12128. In one embodiment, R 1a teeth, This is TIFF2026510946000061.tif16128. In one embodiment, R 1a teeth, This is TIFF2026510946000062.tif15128. In one embodiment, R 1a teeth, This is TIFF2026510946000063.tif18128. In one embodiment, R 1a teeth, This is TIFF2026510946000064.tif19128. In one embodiment, R 1a teeth, This is TIFF2026510946000065.tif15128. In one embodiment, R 1a teeth, This is TIFF2026510946000066.tif16128. In one embodiment, R 1a teeth, The filename is TIFF2026510946000067.tif15128.

[0142] In one embodiment, R 1b H is H.

[0143] In one embodiment, R 3 teeth, Selected from the group consisting of TIFF2026510946000068.tif20128, Here, R 7 C(=O)R a and S(=O)2R a Selected from the group consisting of; and R 8 The group is selected from the group consisting of C1-C6 alkoxy, C1-C6 hydroxyalkyl, and OH.

[0144] In one embodiment, R 7 is cyclopropylcarbonyl. In one embodiment, R 7 is methylsulfonyl. In one embodiment, R 7 is dimethylaminosulfonyl. In one embodiment, R 7 It is dimethylaminocarbonyl.

[0145] In one embodiment, R 8 is methoxy. In one embodiment, R 8 is 2-hydroxyethyl. In one embodiment, R 8 It is OH.

[0146] In one embodiment, R 3teeth, This is TIFF2026510946000069.tif17128. In one embodiment, R 3 teeth, This is TIFF2026510946000070.tif16128. In one embodiment, R 3 teeth, This is TIFF2026510946000071.tif16128. In one embodiment, R 3 teeth, This is TIFF2026510946000072.tif16128. In one embodiment, R 3 teeth, This is TIFF2026510946000073.tif12128. In one embodiment, R 3 teeth, This is TIFF2026510946000074.tif16128. In one embodiment, R 3 teeth, This is TIFF2026510946000075.tif16128. In one embodiment, R 3 teeth, The filename is TIFF2026510946000076.tif14128.

[0147] In one embodiment, the compound of formula (I) is 8-(6-methoxy-3-((4-methoxyphenyl)sulfonyl)quinoline-4-yl)-1,4-dioxa-8-azaspiro[4.5]decane; 1-(3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)-6-fluoroquinoline-4-yl)-4-phenylpiperidine-4-carbonitrile; (4-(cyclopropanecarbonyl)piperazine-1-yl)(4-(4,4-dimethylcyclohexa-1-en-1-yl)-6-fluoroquinoline-3-yl)methanone; (4-(4-(tert-butyl)phenyl)-6-fluoroquinoline-3-yl)(4-(cyclopropanecarbonyl)piperazine-1-yl)methanone; 1-(4-(3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)-6-fluoroquinoline-4-yl)phenyl)cyclopropanecarbonitrile; 1-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonilicate; (4-(4,4-dimethylcyclohexa-1-en-1-yl)-6-fluoroquinoline-3-yl)(4-(methylsulfonyl)piperazine-1-yl)methanone; (4-(4-(tert-butyl)phenyl)-6-fluoroquinoline-3-yl)(4-(methylsulfonyl)piperazine-1-yl)methanone; 1-(4-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoro-N-((1r,4r)-4-hydroxycyclohexyl)quinoline-3-carboxamide; (6-Fluoro-4-(4-(vinylsulfonyl)piperazine-1-yl)quinoline-3-yl)(4-(methylsulfonyl)piperazine-1-yl)methanone; 1-(4-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclobutan-1-carbonitrile; 1-(4-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopentan-1-carbonitrile; 4-(4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-sulfonamide; 4-(4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-carboxamide; 1-(6-chloro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonilicate; 1-(6-chloro-3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonitrile; 1-(6-(4-(methylsulfonyl)piperazine-1-carbonyl)thieno[3,2-b]pyridine-7-yl)-4-phenylpiperidine-4-carbonitrile; 1-(6-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)thieno[3,2-b]pyridine-7-yl)-4-phenylpiperidine-4-carbonitrile; 1-(6-methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonilicate; 1-(7-methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonilicate; 1-(6,7-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonilicate; 1-(6,8-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonilicate; 4-(4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-sulfonamide; 4-(4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-carboxamide; 4-(6-chloro-4-(4-(1-cyanocyclopropyl)phenyl)quinoline-3-carbonyl)-N,N-dimethylpiperazine-1-sulfonamide; 1-(4-(6-chloro-3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6-(4-(methylsulfonyl)piperazine-1-carbonyl)thieno[3,2-b]pyridine-7-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)thieno[3,2-b]pyridine-7-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6-methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(7-Methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6,7-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6,8-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoro-N-((1r,4r)-4-hydroxycyclohexyl)quinoline-3-carboxamide; 4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoro-N-(1-(2-hydroxyethyl)-1H-pyrazole-4-yl)quinoline-3-carboxamide; and 4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoro-N-(1-(2-hydroxyethyl)-1H-pyrazole-4-yl)quinoline-3-carboxamide Selected from the group consisting of .

[0148] In one embodiment, the immunostimulant is at least one selected from the group consisting of immune checkpoint inhibitors, chimeric antigen receptor (CAR) T cells, T cells genetically engineered to express specific TCRs targeting tumor antigens (TCR transgenics), ex vivo expanding T cells, and bispecific T cell engagers (BiTEs).

[0149] In one aspect, immunostimulants are immune checkpoint inhibitors.

[0150] In one embodiment, the immune checkpoint inhibitor is selected from the group consisting of anti-PD1 antibodies, anti-PD-L1 antibodies, anti-CTLA4 antibodies, any fragment thereof, and any combination thereof.

[0151] In one aspect, the immunostimulant is CAR T cells.

[0152] In one embodiment, a pharmaceutically acceptable carrier is suitable for intravenous administration.

[0153] In one embodiment, at least one immunostimulant comprises an immune checkpoint inhibitor and CAR T cells.

[0154] In one embodiment, the pharmaceutical composition further comprises at least one selected from the group consisting of retinoic acid receptor (RAR) inhibitors and retinoid X receptor (RXR) inhibitors.

[0155] In one embodiment, the RAR inhibitor is selected from the group consisting of AGN 193109, BMS 195614, BMS 493, CD 2665, ER 50891, LE 135, LY 2955303, MM 11253, any salt or solvate thereof, and any combination thereof.

[0156] In one embodiment, the RXR inhibitor is selected from the group consisting of HX 531, PA 452, and UVI 3003, any salt or solvate thereof, and any combination thereof.

[0157] The present invention provides a pharmaceutical composition comprising at least one compound of the present invention or a salt or solvate thereof, which is useful for carrying out the method of the present invention. Such a pharmaceutical composition may consist of at least one compound of the present invention or a salt or solvate thereof in a form suitable for administration to a subject, or the pharmaceutical composition may consist of at least one compound of the present invention or a salt or solvate thereof and one or more pharmaceutically acceptable carriers, one or more further components, or some combination thereof. As is well known in the art, at least one compound of the present invention may be present in the pharmaceutical composition in the form of a physiologically acceptable salt in combination with, for example, a physiologically acceptable cation or anion.

[0158] In one embodiment, a pharmaceutical composition useful for carrying out the method of the present invention can be administered to deliver a dose of 1 ng / kg / day to 100 mg / kg / day. In another embodiment, a pharmaceutical composition useful for carrying out the present invention can be administered to deliver a dose of 1 ng / kg / day to 1,000 mg / kg / day.

[0159] The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and any further components in the pharmaceutical composition of the present invention vary depending on the uniqueness, size, and condition of the object being treated, as well as the route through which the composition is to be administered. For example, the composition may contain 0.1% to 100% (w / w) of the active ingredient.

[0160] Pharmaceutical compositions useful in the methods of the present invention can be suitably developed for nasal, inhalation, oral, rectal, vaginal, pleural, abdominal, parenteral, topical, transdermal, intrapulmonary, intranasal, buccal, intraocular, epidural, subarachnoid, intravenous, or other routes of administration. Compositions useful within the scope of the methods of the present invention can be directly administered to the brain, brainstem, or any other part of the central nervous system of mammals or birds. Other conceivable formulations include projected nanoparticles, microspheres, liposome preparations, coated particles, polymer conjugates, reencapsulated red blood cells containing active ingredients, and immunological preparations.

[0161] In one embodiment, the composition of the present invention is part of a pharmaceutical matrix, which enables the manipulation of insoluble materials and improvement of their bioavailability, the development of controlled-release or sustained-release formulations, and the production of homogeneous compositions. For example, the pharmaceutical matrix can be prepared using hot-melt extrusion, solid solutions, solid dispersions, size reduction techniques, molecular complexes (e.g., cyclodextrins), microparticles, and particle and formulation coating processes. Amorphous or crystalline phases can be used in such processes.

[0162] The route of administration is obvious to those skilled in the art and depends on any number of factors, including the type and severity of the disease being treated, and the type and age of the veterinary or human patient being treated.

[0163] Formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the fields of pharmacology and pharmaceuticals. Generally, such preparation methods include the steps of conjugating the active ingredient with a carrier or one or more other minor components, and then, if necessary or desirable, forming or packaging the product into desired single or multi-dose units.

[0164] As used herein, “unit dose” refers to an individual amount of a pharmaceutical composition containing a predetermined amount of active ingredient. The amount of active ingredient is generally equal to the dose of the active ingredient administered to a subject, or a convenient portion of said dose, for example, half or one-third of said dose. A unit dosage form may be for once-daily administration or for single-dose administration in a multi-daily administration (e.g., about 1 to 4 times or more per day). When used for multi-daily administration, the unit dosage form may be the same or different for each dose.

[0165] The descriptions of pharmaceutical compositions provided herein primarily concern pharmaceutical compositions suitable for ethical administration to humans, but those skilled in the art will understand that such compositions are generally suitable for administration to all types of animals. It is well understood that modifying pharmaceutical compositions suitable for administration to humans in order to provide compositions suitable for administration to various animals is possible, and a veterinary pharmacologist of ordinary skill can design and perform such modifications by experiment or by ordinary experiment. The pharmaceutical compositions of the present invention are intended for administration to humans and other primates, mammals including commercially suitable mammals such as cattle, pigs, horses, sheep, cats, and dogs.

[0166] In one embodiment, the compositions of the present invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In one embodiment, the pharmaceutical composition of the present invention comprises a therapeutically effective amount of at least one compound of the present invention and a pharmaceutically acceptable carrier. Useful pharmaceutically acceptable carriers include, but are not limited to, glycerin, water, saline, ethanol, recombinant human albumin (e.g., RECOMBUMIN®), solubilized gelatin (e.g., GELOFUSINE®), and solutions of other pharmaceutically acceptable salts such as phosphates and organic acid salts. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

[0167] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerin, propylene glycol, and liquid polyethylene glycol), recombinant human albumin, solubilized gelatin, a suitable mixture thereof, and vegetable oil. Appropriate fluidity can be maintained by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Inhibition of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Often, isotonic agents, such as sugars, sodium chloride, or polyhydric alcohols such as mannitol and sorbitol, are included in the composition. Long-term absorption of the injectable composition can be achieved by including absorption-delaying agents, such as aluminum monostearate or gelatin, in the composition.

[0168] The formulation can be used as a mixture with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances, suitable for oral, parenteral, nasal, inhalation, intravenous, subcutaneous, transdermal, enteral, or any other suitable mode of administration known in the art. The pharmaceutical preparations may be sterile and, if desired, may be mixed with adjuvants, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts to affect osmotic pressure, buffers, colorants, flavoring agents, and / or aromatizers. If desired, they may be combined with other effective agents, such as other analgesics, anxiolytics, or hypnotics. As used herein, “further components” include, but are not limited to, one or more components that can be used as pharmaceutical carriers.

[0169] The compositions of the present invention may contain a preservative in an amount of about 0.005% to 2.0% of the total weight of the composition. The preservative is used to prevent spoilage in the event of exposure to contaminants in the environment. Examples of preservatives useful according to the present invention include, but are not limited to, those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidourea, and any combination thereof. One such preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.

[0170] This composition may contain antioxidants and chelating agents that inhibit the degradation of compounds. Examples of antioxidants for some compounds include BHT, BHA, α-tocopherol, and ascorbic acid in an exemplary range of about 0.01% to 0.3% by weight of the total weight of the composition, or BHT in the range of 0.03% to 0.1% by weight. Chelating agents may be present in an amount of 0.01% to 0.5% by weight of the total weight of the composition. Examples of chelating agents include EDTA salts (e.g., disodium EDTA) and citric acid in a weight range of about 0.01% to 0.20% by weight, or 0.02% to 0.10% by weight of the total weight of the composition. Chelating agents are useful for chelating metal ions in the composition that may impair the shelf life of the formulation. BHT and disodium edetate are exemplary antioxidants and chelating agents, respectively, for several compounds, but they may be replaced with other suitable and equivalent antioxidants and chelating agents known to those skilled in the art.

[0171] A suspension of an active ingredient in an aqueous or oily medium can be obtained by preparing a liquid suspension using conventional methods. Examples of aqueous media include water and isotonic saline. Examples of oily media include almond oil, oily esters, ethyl alcohol, vegetable oils such as peanut oil, olive oil, sesame oil or coconut oil, rectified vegetable oils, and mineral oils such as liquid paraffin. The liquid suspension may further contain one or more further components, including but not limited to suspending agents, dispersants or wetting agents, emulsifiers, lubricants, preservatives, buffers, salts, flavorings, colorings, and sweeteners. The oily suspension may further contain thickeners. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, tragacanth gum, acacia gum, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, and hydroxypropyl methylcellulose. Known dispersants or wetting agents include, but are not limited to, natural phosphatides such as lecithin, condensates of alkylene oxides with fatty acids, condensates of long-chain aliphatic alcohols, condensates of fatty acids with partial esters derived from hexitol, or condensates of fatty acids with partial esters derived from anhydrous hexitol (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifiers include, but are not limited to, lecithin, gum arabic, and ionic or nonionic surfactants. Known preservatives include, but are not limited to, methyl p-hydroxybenzoate, ethyl or n-propyl, ascorbic acid, and sorbic acid. Known sweeteners include, for example, glycerin, propylene glycol, sorbitol, sucrose, and saccharin.

[0172] Liquid solutions of active ingredients in aqueous or oily solvents can be prepared substantially similarly to liquid suspensions, the main difference being that the active ingredient is dissolved rather than suspended in the solvent. As used herein, an "oily" liquid is a liquid containing carbon-containing liquid molecules and exhibiting lower polarity than water. Liquid solutions of the pharmaceutical compositions of the present invention may contain the components described with respect to liquid suspensions, and it will be understood that the suspending agent does not necessarily contribute to the dissolution of the active ingredient in the solvent. Examples of aqueous solvents include water and isotonic saline. Examples of oily solvents include vegetable oils such as almond oil, oily esters, ethyl alcohol, peanut oil, olive oil, sesame oil, or coconut oil, rectified vegetable oils, and mineral oils such as liquid paraffin.

[0173] Powder and granular formulations of the pharmaceutical preparations of the present invention can be prepared using known methods. Such formulations may be administered directly to a subject, or they may be used, for example, to form tablets, to fill capsules, or to prepare aqueous or oily solutions or suspensions by adding an aqueous or oily medium thereto. Each of these formulations may further contain one or more of the following: dispersants or wetting agents, suspending agents, ionic and nonionic surfactants, and preservatives. Fillers and further excipients such as sweeteners, flavorings, or colorants may also be included in these formulations.

[0174] The pharmaceutical compositions of the present invention may be prepared, packaged, or sold in the form of oil-in-water emulsions or water-in-oil emulsions. The oil phase may be a vegetable oil such as olive oil or peanut oil, a mineral oil such as liquid paraffin, or a mixture thereof. The composition may further contain one or more emulsifiers, for example, natural rubber such as gum arabic or tragacanth gum, natural phosphatides such as soy or lecithin phosphatide, esters or partial esters derived from a combination of fatty acids and anhydrous hexitol, such as sorbitan monooleate, and condensates of such partial esters and ethylene oxide, such as polyoxyethylene sorbitan monooleate. These emulsions may also contain further components, such as sweeteners or flavorings.

[0175] Methods for impregnating materials with chemical compositions or coating materials with chemical compositions are known in the art and include, but are not limited to, methods for depositing or bonding chemical compositions onto a surface, methods for incorporating chemical compositions into the structure of materials during the synthesis of materials (i.e., by physiologically biodegradable materials), and methods for absorbing aqueous or oily solutions or suspensions into absorbent materials with or without subsequent drying. Methods for mixing components include, as known to those skilled in the art, physical grinding, the use of pellets in solid and suspension formulations, and mixing in transdermal patches.

[0176] Administration / Medication The administration regimen may influence what constitutes an effective dose. The therapeutic formulation may be administered to the patient before or after the onset of the disease or disorder. Furthermore, several divided and staggered doses may be administered daily or sequentially, or the dose may be administered by continuous infusion or bolus injection. In addition, the dose of the therapeutic formulation may be increased or decreased proportionally as needed depending on the treatment or preventive situation.

[0177] The compositions of the present invention can be administered to patients, e.g., mammals, e.g., humans, using known procedures, in doses and durations effective for treating the diseases or disorders envisioned herein. The effective amount of the therapeutic compound required to obtain a therapeutic effect may vary depending on factors such as the activity of the particular compound used; the time of administration; the elimination rate of the compound; the duration of treatment; other drugs, compounds, or materials used in combination with the compound; the patient being treated's disease or disorder, age, sex, weight, condition, general health, and medical history; and similar factors well known in the medical field. The administration regimen can be adjusted to provide an optimal therapeutic response. For example, several divided doses may be administered daily, and the dose may be proportionally reduced as needed depending on the treatment situation. Non-limiting examples of the effective dose range of the therapeutic compound of the present invention are about 0.01 mg / kg to 100 mg / kg body weight / day. Those skilled in the art will be able to determine the effective dose of the therapeutic compound without excessive experimentation by considering the relevant factors.

[0178] The compound may be administered to animals several times a day, or less frequently, for example, once a day, once a week, once every two weeks, once a month, or even less frequently, for example, once every few months, or even once a year or less. It will be understood that the amount of the compound administered per day can be daily, every other day, every two days, every three days, every four days, or every five days in non-limiting examples. For example, in every-other-day administration, a dose of 5 mg per day may be started on Monday, a first subsequent dose of 5 mg per day may be administered on Wednesday, and a second subsequent dose of 5 mg per day may be administered on Friday. The frequency of administration depends on several factors, which are obvious to those skilled in the art, including but not limited to the type and severity of the disease being treated, and the type and age of the animal.

[0179] The actual dose level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient that is effective in achieving a desired therapeutic response, desired composition, and desired mode of administration for a particular patient, without causing toxicity to the patient.

[0180] A physician with ordinary skill in the art, such as an internist or veterinarian, can easily determine and prescribe the effective amount of the required pharmaceutical composition. For example, an internist or veterinarian can start the dose of the compound of the present invention used in the pharmaceutical composition at a level lower than the level required to obtain the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved.

[0181] In certain embodiments, it is particularly advantageous to formulate the compound in unit dosage forms because it is easy to administer and the dosage is uniform. As used herein, a unit dosage form means a physically separate unit suitable as a unit dosage form for a patient being treated, each unit containing a predetermined amount of the therapeutic compound calculated to produce a desired therapeutic effect, in combination with the required pharmaceutical medium. The unit dosage forms of the present invention are determined by and directly depend on (a) the unique characteristics of the therapeutic compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the field of compounding / formulating the therapeutic compound for the treatment of a disease or disorder in a patient.

[0182] In one embodiment, the compositions of the present invention are administered to a patient at a frequency ranging from one to five times or more per day. In another embodiment, the compositions of the present invention are administered to a patient at a frequency ranging from once daily, once every two days, once every three days to once a week, and once every two weeks, but not limited to these. It will be obvious to those skilled in the art that the administration frequency of the various combination compositions of the present invention will vary from subject to subject depending on many factors, including but not limited to age, the disease or disorder to be treated, sex, general health, and other factors. Therefore, the present invention should not be construed as being limited to any particular administration regime, and the exact administration frequency and composition to be administered to any patient will be determined by the attending physician, taking into account all other factors concerning the patient.

[0183] The compounds of the present invention for administration are available in doses of approximately 1 μg to 7,500 mg, approximately 20 μg to 7,000 mg, approximately 40 μg to 6,500 mg, approximately 80 μg to 6,000 mg, approximately 100 μg to 5,500 mg, approximately 200 μg to 5,000 mg, approximately 400 μg to 4,000 mg, approximately 800 μg to 3,000 mg, approximately 1 mg to 2,500 mg, and approximately 2 mg to 2, This range can be 000mg, approximately 5mg to 1,000mg, approximately 10mg to 750mg, approximately 20mg to 600mg, approximately 30mg to 500mg, approximately 40mg to 400mg, approximately 50mg to 300mg, approximately 60mg to 250mg, approximately 70mg to 200mg, approximately 80mg to 150mg, and any integer or non-integer units in between.

[0184] In some embodiments, the dose of the compound of the present invention ranges from about 0.5 μg to about 5,000 mg. In some embodiments, the dose of the compound of the present invention used in the compositions described herein is less than about 5,000 mg, or less than about 4,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, the dose of the second compound described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any integer or fractional unit in between.

[0185] In one embodiment, the present invention relates to a packaged pharmaceutical composition comprising a container for holding a therapeutically effective amount of the compound of the present invention alone or in combination with a second agent, and instructions for using the compound in a patient to treat, prevent or reduce one or more symptoms of a disease or disorder.

[0186] The term “container” includes any container for holding a pharmaceutical composition or for controlling its stability or moisture absorption. For example, in one embodiment, a container is packaging that contains a pharmaceutical composition in a double chamber, such as a liquid (solution and suspension), semi-solid, lyophilized solid, solution, and powder, or lyophilized formulation. In other embodiments, a container is not packaging that contains a pharmaceutical composition; i.e., a container is a container such as a box or vial that contains a packaged or unpackaged pharmaceutical composition and instructions for use of the pharmaceutical composition. Furthermore, packaging techniques are well known in the art. It should be understood that instructions for use of a pharmaceutical composition may be included on the packaging containing the pharmaceutical composition, thereby increasing the functional relationship between the instructions and the packaged product. However, it should be understood that the instructions may include information about the compound’s ability to perform its intended function, such as its ability to treat, prevent, or reduce a disease or disorder in a patient.

[0187] Administration Routes of administration for any composition of the present invention include inhalation, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, tongue, (trans) buccal, (trans) urethral, ​​intravaginal (e.g., transvaginal and perival), intranasal cavity (intra), and (trans) rectal), intrabladder, intrapulmonary, intraduodenal, intragastric, subarachnoid, epidural, intrapleural, intraperitoneal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and local administration.

[0188] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel capsules, lozenges, emulsions, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magma preparations, licks, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosol preparations for inhalation, and compositions and preparations for intravesical administration. It should be understood that the preparations and compositions that may be useful in the present invention are not limited to the specific preparations and compositions described herein.

[0189] Oral administration For oral administration, tablets, sugars, solutions, droplets, capsules, caplets, and gel capsules are particularly preferred. Other formulations suitable for oral administration include, but are not limited to, powders or granules, aqueous or oily suspensions, aqueous or oily solutions, pastes, gels, toothpastes, mouthwashes, coatings, oral rinses, or emulsions. Compositions intended for oral use can be prepared according to any method known in the art, and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic, and generally safe (GRAS) pharmaceutically acceptable excipients suitable for the manufacture of tablets. Examples of such excipients include inert diluents such as lactose; granulators and disintegrants such as corn starch; binders such as starch; and lubricants such as magnesium stearate.

[0190] Tablets may be uncoated or may be coated using known methods to provide sustained release and absorption of the active ingredient by enabling delayed disintegration in the target gastrointestinal tract. For example, tablets may be coated using materials such as glyceryl monostearate or glyceryl distearate. Furthermore, osmotically controlled release tablets can be formed by coating tablets using methods described in, for example, U.S. Patent Nos. 4,256,108; 4,160,452; and 4,265,874. Tablets may further contain sweeteners, flavorings, colorings, preservatives, or any combination thereof to give a pharmaceutically fine and palatable preparation. Hard capsules containing the active ingredient can be made using physiologically biodegradable compositions such as gelatin. Capsules containing the active ingredient may further contain additional components, such as an inert solid diluent, such as calcium carbonate, calcium phosphate, or kaolin.

[0191] Hard capsules containing the active ingredient can be prepared using physiologically biodegradable compositions such as gelatin. Such hard capsules contain the active ingredient and may further contain additional components, such as an inert solid diluent, including calcium carbonate, calcium phosphate, or kaolin.

[0192] Soft gelatin capsules containing an active ingredient can be prepared using physiologically degradable compositions such as gelatin from animal-derived collagen or hypromellose, a modified form of cellulose, and can be prepared using any mixture of gelatin, water, and a plasticizer such as sorbitol or glycerin. Such soft capsules contain an active ingredient, which may be mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil.

[0193] For oral administration, the compounds of the present invention may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binders; fillers; lubricants; disintegrants; or wetting agents. If desired, tablets may be coated using preferred methods and coating materials, such as the OPADRY® film coating systems available from Colorcon, West Point, Pennsylvania (e.g., OPADRY® OY, OYC, organic enteric OY-P, aqueous enteric OY-A, OY-PM, and OPADRY® White, 32K18400). It will be understood that similar types of film coatings or polymer products from other companies may be available.

[0194] Tablets containing an active ingredient can be prepared, for example, by compressing or molding the active ingredient together with one or more additional ingredients. Compressed tablets can be prepared by compressing an easily flowable form of the active ingredient, such as a powder or granular preparation, which is optionally mixed with one or more of the binders, lubricants, excipients, surfactants, and dispersants, in a suitable apparatus. Molded tablets can be prepared by molding a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least enough liquid to moisten the mixture, in a suitable apparatus. Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulators and disintegrants, binders, and lubricants. Known dispersants include, but are not limited to, potato starch and sodium starch glycolate. Known surfactants include, but are not limited to, sodium lauryl sulfate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, crystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulators and disintegrants include, but are not limited to, corn starch and alginic acid. Known binders include, but are not limited to, gelatin, gum arabic, pregelatinized corn starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricants include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.

[0195] Granulation techniques for modifying active ingredient starting powders or other particulate materials are well known in the pharmaceutical field. Typically, powders are mixed with binder materials to form larger, more permanent, and easily flowable aggregates or granules called "granules." For example, a solvent-based "wet" granulation process generally involves combining the powder with a binder material, wetting it with water or an organic solvent under conditions that form wet granules, and then ensuring that the solvent is evaporated from the granules.

[0196] Generally, melt granulation is essentially about using a material that is solid or semi-solid at room temperature (i.e., has a relatively low softening or melting point range) to facilitate the granulation of powder materials or other materials in the absence of water or other liquid solvents that are added. Low-melting-point solids liquefy when heated to a temperature within their melting point range and act as a binder or granulation medium. The liquefied solid diffuses onto the surface of the powder material it comes into contact with, and upon cooling, forms solid granules into which the initial materials are bound together. The resulting melt granules can then be subjected to a tablet press or encapsulated to prepare oral dosage forms. Melt granulation improves the dissolution rate and bioavailability of the active ingredient (i.e., drug) by forming a solid dispersion or solid solution.

[0197] U.S. Patent No. 5,169,645 discloses a directly compressible wax-containing granule having improved fluidity. The granule is obtained by mixing wax with a specific fluidity-enhancing additive in a molten material, followed by cooling and granulation of the mixture. In certain embodiments, only the wax itself melts in the molten combination of wax and additive, while in other embodiments, both the wax and additive melt.

[0198] The present invention also includes a multilayer tablet comprising a layer for delayed release of one or more compounds useful within the scope of the present invention, and a further layer for immediate release of one or more compounds useful within the scope of the present invention. A gastric insoluble composition that ensures delayed release of the active ingredient by encapsulating it can be obtained using a wax / pH-sensitive polymer mixture.

[0199] Liquid preparations for oral administration may be in the form of solutions, syrups, or suspensions. Liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methylcellulose, or hydrogenated edible fat); emulsifiers (e.g., lecithin or gum arabic); non-aqueous media (e.g., almond oil, oily esters, or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxybenzoate, or sorbic acid). Liquid formulations of the pharmaceutical compositions of the present invention, suitable for oral administration, can be prepared, packaged, and sold in liquid form or in the form of dry formulations intended to be reconstituted with water or another suitable media before use.

[0200] Parenteral administration As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physically opening the target tissue and administering the pharmaceutical composition through the opening in the tissue. Therefore, parenteral administration includes, but is not limited to, the administration of a pharmaceutical composition by injection, application of the composition through surgical incision, or application of the composition through a tissue-penetrating nonsurgical wound. In particular, parenteral administration is assumed to include, but is not limited to, subcutaneous injection, intravenous injection, intraperitoneal injection, intramuscular injection, intrasternal injection, and renal dialysis infusion techniques.

[0201] Formulations of pharmaceutical compositions suitable for parenteral administration comprise an active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations can be prepared, packaged, or sold in forms suitable for bolus or serial administration. Injectable formulations can be prepared, packaged, or sold in unit dosage forms, for example, in ampoules or in multi-dose containers containing preservatives. Injectable formulations may also be prepared, packaged, or sold as devices, such as patient-controlled analgesia (PCA) devices. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous media, pastes, and embedded sustained-release or biodegradable formulations. Such formulations may further comprise one or more further components, including, but not limited to, suspending agents, stabilizers, or dispersants. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granular) form, which is reconstituted in a suitable medium (e.g., sterile pyrogen-free water) before parenteral administration of the reconstituted composition.

[0202] Pharmaceutical compositions can be prepared, packaged, or sold in the form of sterile aqueous or oily suspensions or solutions for injection. These suspensions or solutions can be formulated according to known techniques and may contain, in addition to the active ingredient, further components such as dispersants, wetting agents, or suspending agents as described herein. Such sterile injection formulations can be prepared using, for example, water or a non-toxic, parenterally acceptable diluent or solvent such as 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and non-volatile oils such as synthetic monoglycerides or diglycerides. Other useful parenterally administered formulations include those containing the active ingredient as recombinant human albumin, in microcrystalline form in liquid gelatin, as a liposome preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or embedding may contain pharmaceutically acceptable polymer materials or hydrophobic materials such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts.

[0203] Local administration The barrier to topical administration of drugs is the stratum corneum of the epidermis. The stratum corneum is a highly resistant layer composed of proteins, cholesterol, sphingolipids, free fatty acids, and various other lipids, and contains keratinocytes and living cells. One factor limiting the penetration rate (flow rate) of compounds through the stratum corneum is the amount of active substance that can be added or applied to the skin surface. The greater the amount of active substance applied per unit area of ​​skin, the greater the concentration gradient between the skin surface and the lower layers of skin, and therefore the greater the diffusive force of the active substance through the skin. Thus, formulations containing relatively high concentrations of active substance are more likely to penetrate the skin with greater and more consistent rates than formulations with relatively low concentrations but all other things being equal.

[0204] Suitable formulations for topical administration include, but are not limited to, liquid or semi-liquid formulations, such as liniments, lotions, oil-in-water or water-in-oil emulsions, such as creams, ointments, or pastes, and solutions or suspensions. Topically administered formulations may contain, for example, about 1% to about 10% (w / w) of the active ingredient, but the concentration of the active ingredient may be the same as the solubility limit of the active ingredient in the solvent. Topically administered formulations may further contain one or more of the components described herein.

[0205] Penetration enhancers can be used. These materials increase the penetration rate of drugs through the skin. Typical enhancers in this art include ethanol, glycerol monolaurate, PGML (polyethylene glycol monolaurate), and dimethyl sulfoxide. Other enhancers include oleic acid, oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylic acids, dimethyl sulfoxide, polar lipids, or N-methyl-2-pyrrolidone.

[0206] One acceptable medium for local delivery of some compositions of the present invention may include liposomes. The composition of liposomes and their uses are known in the art (i.e., U.S. Patent No. 6,323,219).

[0207] In alternative embodiments, a locally active pharmaceutical composition may be combined with other components such as auxiliaries, antioxidants, chelating agents, surfactants, foaming agents, wetting agents, emulsifiers, thickeners, buffers, and preservatives. In other embodiments, a penetration enhancer is included in the composition, which is effective in improving the transdermal penetration of the active ingredient into and through the stratum corneum compared to compositions lacking a penetration enhancer. Various penetration enhancers, including oleic acid, oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylic acids, dimethyl sulfoxide, polar lipids, or N-methyl-2-pyrrolidone, are known to those skilled in the art. In another embodiment, the composition may further include a hydrotrope, which functions to increase the disorder of the stratum corneum structure and thus increase transport through the stratum corneum. Various hydrotropes, such as isopropyl alcohol, propylene glycol, or sodium xylene sulfonate, are known to those skilled in the art.

[0208] A topically active pharmaceutical composition should be applied in an effective amount to produce the desired change. As used herein, “effective amount” means an amount sufficient to cover the area of ​​skin surface where the change is desired. The active compound should be present in an amount of about 0.0001% to about 15% by weight of the composition. For example, it should be present in an amount of about 0.0005% to about 5% of the composition, or for example, it should be present in an amount of about 0.001% to about 1% of the composition. The compound may be of synthetic or natural origin.

[0209] buccal administration The pharmaceutical compositions of the present invention can be prepared, packaged, or sold as formulations suitable for buccal administration. Such formulations may be in the form of tablets or licks, for example, prepared using conventional methods, and may contain, for example, 0.1 to 20% (w / w) of the active ingredient, with the remainder being an orally soluble or degradable composition, and optionally one or more further ingredients as described herein. Alternatively, formulations suitable for buccal administration may include a powder, or an aerosolized or atomized solution or suspension containing the active ingredient. Such powdered formulations, aerosolized formulations, or aerosolized formulations may exhibit an average particle size or droplet diameter in the range of about 0.1 to about 200 nanometers when dispersed, and may further contain one or more further ingredients as described herein. The examples of formulations described herein are not exhaustive, and the present invention will be understood to include further modifications of these and other formulations not described herein but known to those skilled in the art.

[0210] Rectal administration The pharmaceutical compositions of the present invention can be prepared, packaged, or sold as formulations suitable for rectal administration. Such compositions may be in the form of, for example, suppositories, retained enema formulations, and rectal or colon cleansing solutions.

[0211] Suppository formulations can be prepared by combining an active ingredient with a non-irritating, pharmaceutically acceptable excipient that is solid at normal room temperature (i.e., about 20°C) and liquid at the target rectal temperature (i.e., about 37°C in healthy individuals). Suitable pharmaceutically acceptable excipients include, but are not limited to, cocoa butter, polyethylene glycol, and various glycerides. Suppository formulations may further contain a variety of additional components, including, but not limited to, antioxidants and preservatives.

[0212] Retained enema preparations, or rectal or colon cleansing solutions, can be prepared by combining an active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, enema preparations can be administered using a delivery device adapted to the anatomical morphology of the target rectum and can be packaged within the delivery device. Enema preparations may further contain a variety of additional ingredients, including but not limited to antioxidants and preservatives.

[0213] Further forms of administration Further dosage forms of the present invention include those described in U.S. Patent Nos. 6,340,475, 6,488,962, 6,451,808, 5,972,389, 5,582,837, and 5,007,790. Further dosage forms of the present invention also include those described in U.S. Patent Applications Nos. 20030147952, 20030104062, 20030104053, 20030044466, 20030039688, and 20020051820. Further dosage forms of the present invention include those described in PCT application numbers WO 03 / 35041, WO 03 / 35040, WO 03 / 35029, WO 03 / 35177, WO 03 / 35039, WO 02 / 96404, WO 02 / 32416, WO 01 / 97783, WO 01 / 56544, WO 01 / 32217, WO 98 / 55107, WO 98 / 11879, WO 97 / 47285, WO 93 / 18755, and WO 90 / 11757.

[0214] Controlled-release formulations and drug delivery systems In some embodiments, the compositions and / or formulations of the present invention may, but are not limited to, short-release formulations, rapid-release formulations, and controlled-release formulations, such as sustained-release formulations, delayed-release formulations, and pulsed-release formulations.

[0215] The term "sustained-release," as it is commonly used, refers to a drug formulation that releases the drug gradually over a long period, and can, if not necessarily, maintain a substantially constant level of the drug in the blood over that period. The duration can be longer than one month and should be longer than the release of the same amount of drug administered in bolus form.

[0216] For sustained release, the compound can be formulated using a suitable polymer or hydrophobic material that imparts sustained release properties to the compound. Therefore, the compound used in the method of the present invention can be administered, for example, by injection in the form of a particulate preparation, or by embedding in the form of a wafer or disk preparation.

[0217] In one embodiment of the present invention, a compound useful within the scope of the present invention is administered to a subject, either alone or in combination with another agent, using a sustained-release formulation.

[0218] The term "delayed release" is used herein in its usual sense and refers to a drug formulation in which the initial release of the drug occurs some time after administration, and which may, but not necessarily, include a delay of approximately 10 minutes to a maximum of approximately 12 hours.

[0219] The term pulsed release is used herein in its usual sense and refers to a drug formulation that releases the drug in such a way that it generates a pulsed plasma profile of the drug after administration.

[0220] The term "immediate release," as it is commonly used, refers to a drug formulation that releases the drug immediately after administration.

[0221] As used herein, short-term means any period of time after drug administration up to approximately 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 40 minutes, 20 minutes, or 10 minutes, and any integer or non-integer unit in between.

[0222] As used herein, rapid means any period of time up to approximately 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 40 minutes, 20 minutes, or 10 minutes after drug administration, and any integer or non-integer unit in between.

[0223] Those skilled in the art will recognize numerous equivalents to the specific procedures, embodiments, claims, and examples described herein, or will be able to verify them using mere routine experiments. Such equivalents are considered to be within the scope of the present invention and are considered to be covered by the claims appended herein. For example, it should be understood that modifying reaction conditions, including but not limited to reaction time, reaction size / volume, experimental reagents such as solvents, catalysts, pressure, atmospheric conditions such as nitrogen atmosphere, and reducing / oxidizing agents, using substitutes recognized in the art and using mere routine experiments, is within the scope of this application.

[0224] Wherever values ​​and ranges are mentioned in this specification, it should be understood that descriptions in the form of ranges are merely for convenience and brevity and should not be interpreted as rigid limitations on the scope of the invention. Therefore, all values ​​and ranges encompassed by these values ​​and ranges are intended to be included within the scope of the invention. Furthermore, all values ​​within these ranges, and any upper or lower limits on the ranges of values, are also assumed by this application. Descriptions of ranges should be considered to specifically disclose all possible subranges, the individual numerical values ​​within those ranges, and, where appropriate, the non-integers of the numerical values ​​within those ranges. For example, a description of a range such as 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and the individual numbers within those ranges, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range. [Examples]

[0225] Examples Various aspects of this application can be better understood by referring to the following exemplary examples. The scope of this application is not limited to the examples provided herein.

[0226] material and method animal Wild-type C57BL / 6 mice were purchased from Jackson Laboratories (catalog number: 000664). Immuodeficient NU / J mice were purchased from Jackson Laboratories (catalog number: 002019). dnRAR flox Mouse functionality enabled. LysM Cre I purchased a mouse from Jackson Laboratories (catalog number: 004781).

[0227] Creation of RALDH1-KO mice Aldh1A1 knockout mice were generated through the CRISPR / Cas9 Mouse Targeting Core Facility at the University of Pennsylvania. Two CRISPR RNAs were designed that encompassed approximately 36 kilobase regions within the mouse Aldh1A1 gene (Gencode gene: ENSMUSG00000053279.8. Location: mm10 chr19:20,492,715-20,643,465). TIFF2026510946000077.tif12128

[0228] A mixture of pure Cas9 mRNA and guide RNA was injected into single-cell zygotes of C57BL / 6 background mice. Founders were identified by a PCR-based genotyping protocol designed to detect gene deletions. Founders were then crossed with wild-type C57BL / 6 mice to "fix" the alleles. Heterozygotes were identified by the aforementioned PCR-based genotyping and crossed with each other to generate RALDH1-KO mice. Additional qRT-PCR assays (described in the "RNA Isolation and qPCR Analysis for Gene Expression" section) were performed to confirm the absence of Aldh1a1 transcripts.

[0229] PCR primers (sequence 5'-3') for RALDH1-KO genotyping TIFF2026510946000078.tif26134

[0230] PCR conditions 5 minutes at 94°C. 35 cycles of (1) 30 seconds at 94°C, (2) 30 seconds at 58°C, and (3) 30 seconds at 72°C. Final extension at 72°C for 7 minutes, followed by storage at 4°C. 248 base pair bands were detected in the knockout and 665 base pair bands in the WT by conventional gel electrophoresis.

[0231] tumor cells Details of the cell lines and culture media are provided herein. Upon receipt, the cell lines were initially grown (2 passages), authenticated, and then frozen in aliquots for storage. The frozen stocks were thawed and grown (average 3 passages) before experiments and discarded upon completion of each individual experiment. If necessary, the frozen stocks were regrow (average 2 passages), authenticated, and stored as frozen aliquots for additional stock. The Huh1 cell line was provided. Cells were obtained in 2022 and authenticated based on their morphological and proliferative characteristics in cell culture, as well as the histology of tumors formed upon transplantation into mice. Huh7, SNU449, SNU398, HEP3B, PLC, HEPA 1-6, HEP55, and AL458A were obtained in 2021 and authenticated based on their morphological and proliferative characteristics in cell culture, as well as the histology of tumors formed upon transplantation into mice. Fibrosarcoma (FS) cell lines are described in the literature. Tumor cell lines were cultured in DMEM (ThermosFisher, catalog no. 10567014) containing 10% FBS (GeminiBio, catalog no. 100-500), 1% Pen / Strep (ThermosFisher Scientific, catalog no. 15140122), and 2 mM glutamine (ThermosFisher Scientific, 25030081). All cells were confirmed to be negative for mycoplasma contamination, as assessed by the MycoAlert mycoplasma detection kit (Lonza, catalog no. LT07).

[0232] human sample Human FFPE (formalin-fixed, paraffin-embedded) samples of normal liver, normal kidney, HCC, GIST, and CRC were identified by pathologists from patients (anonymized) who underwent therapeutic surgical resection for diagnostic or therapeutic purposes. Fifty-six archived tissue blocks were selected and anonymized before sectioning and IHC. Normal donor human monocytes and T cells were collected by and purchased from the Human Immunology Core (HIC) at the University of Pennsylvania.

[0233] Tumor cell transplantation, tumor growth measurement, and survival analysis Cultured Huh7, Huh1, Hepa 1-6, Hep55, and FS (as shown in the legend in the figure) tumor cells were detached using 0.25% trypsin (Gibco, catalog number 25200056), washed once with 1x PBS, and counted before transplantation. 3-6 x 10 6 Tumor cells were subcutaneously transplanted (sc) into the shaved flanks of recipient mice. Tumor dimensions were measured using calipers, starting from the date shown in the legend of the attached figure, and then every 2-3 days thereafter; volume was measured as length * width. 2 The calculation was performed using the formula / 2. Tumor volume: 2,000 mm² 3 Tumor length of 2 cm or tumor ulceration was used as the endpoint for survival analysis.

[0234] Flow cytometry of tissue samples Tissue samples were collected from mouse tumors of the type shown in the legend of the corresponding figure. Single cell suspensions were prepared by digesting with collagenase B and DNase I (both Sigma Aldrich) at 37°C for 45 minutes and filtering through a 70 μM cell strainer. Erythrocytes were lysed using RBC Lysis Buffer (Biolegend). Samples were incubated with anti-mouse CD16 / 32 Fc Block (BD Biosciences) on ice for 20 minutes, followed by staining on ice with a fluorophore-conjugated primary antibody for cell population identification by flow cytometry. Flow cytometry was performed on an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (Treestar, version 10.8.1).

[0235] In vitro tumor cell proliferation assay 1 x 10 4 ~2 x 10 5 Huh7, Huh1, Hepa 1-6, and Hep55 tumor cells were plated in triplicate in 48-well or 6-well plates. Viable cell counts were counted daily for 3-4 days.

[0236] In vitro processing Cultures of tumor cells or primary monocyte-derived cells were treated with C86, C91, C99, BMS 493 (Tocris, 3509), or Win 18446 (Tocris, 4736) at the times and concentrations indicated in the legend of the corresponding figures.

[0237] AldeRed assay The AldeRed assay (EMD Millipore) was performed according to the manufacturer's instructions to identify cells with ALDH activity. In short, single-cell suspensions of Huh7, Huh1, SNU449, SNU398, HEP3B, PLC, Hepa 1-6, Hep55, and AL458A cultured cells, or single-cell suspensions generated from enzymatic digestion of tumors resulting from transplantation of these cells into mice (as shown in the legend in the figure), were incubated with a fluorescent, non-toxic ALDH substrate (AldeRed 588-A); the fluorescent product accumulated in the cells in proportion to ALDH activity. The amount of fluorescence generated was measured by flow cytometry. Diethylaminobenzaldehyde (DEAB, included in the AldeRed assay kit), an ALDH inhibitor, was used as a negative control for background fluorescence evaluation.

[0238] Cell sorting GFP-expressing Huh7 or Hep55 cells from CRISPR knockout experiments were isolated using a FACS Jazz cell sorter. Cells were identified and isolated based solely on GFP positivity in the cell sorter.

[0239] LC-MS for all-trans retinoic acid (ATRA) For the measurement of all-trans retinoic acid (ATRA), cultured cells were detached using trypsin, centrifuged, and the cell pellet was stored at -80°C. ATRA was extracted from the frozen cell pellet and quantified using liquid chromatography-tandem mass spectrometry (LC-MS) as previously described in the literature.

[0240] Isolation of mouse bone marrow monocytes Monocytes were isolated from the bone marrow of C57BL / 6 mice using the Mouse BM Monocyte Isolation Kit (Miltenyi Biotec) according to the manufacturer's instructions. Monocyte purity was assessed by flow cytometry using CD11b, Ly6C, and Ly6G.

[0241] Intratumor monocyte transplantation Monocytes were isolated from the bone marrow of C57BL / 6 mice (as described above) and counted. Subsequently, 1 x 10⁶ monocytes were counted. 6 Monocytes were resuspended in 50 μL 1x PBS and injected directly into the established tumor 12 days after tumor cell transplantation. The tumor was harvested at the specified time and analyzed by flow cytometry. For human monocytes, primary human monocytes were purchased from the HIC Core Facility at the University of Pennsylvania and 1 x 10⁶ cells as described elsewhere in this specification. 6 Monocytes were injected into the tumor. This method of intratumor monocyte transplantation is described in the literature.

[0242] In vitro and ex vivo mouse and human monocyte differentiation assays Mouse monocytes were isolated from bone marrow as described herein and then cultured in RPMI 1640 (ThermoFisher Scientific, catalog number 11875085) containing 10% fetal bovine serum (GeminiBio, catalog numbers 100-500). GM-CSF (20 ng / mL, peprotech 315-03) and IL4 (20 ng / mL, peprotech 214-14) were added for dendritic cell differentiation, while M-CSF (20 ng / mL, peprotech 315-02) was added for macrophage differentiation. Human monocytes were purchased from the Human Immunology Core Facility at the University of Pennsylvania and cultured in RPMI 1640 (ThermoFisher Scientific, catalog number 11875085) containing 10% fetal bovine serum (GeminiBio, catalog numbers 100-500). GM-CSF (50 ng / mL, peprotech 300-03) and IL4 (50 ng / mL, peprotech 200-04) were added to the cultures for dendritic cell differentiation, while M-CSF (50 ng / mL, peprotech 300-25) was added for macrophage differentiation. RA (200 nM; Sigma Aldrich), C86 (100 nM), or tumor-conditioned medium (TCM) was added at the specified time points for the differentiation assays shown. Cell identity and function of differentiated monocytes were evaluated by flow cytometry and quantitative PCR (qPCR).

[0243] Cell depletion in vivo To deplete T cells, 200 μg of mouse CD3-specific antibody (clone 17A2) was administered intravenously starting 3 days before tumor transplantation and repeated every 3-4 days until the mice were sacrificed. To deplete macrophages, 200 μL of clodronate liposomes (CloLipo) or PBS-liposomes (CtrlLipo) (both Liposoma) were administered intravenously starting 3 days before tumor transplantation and repeated every 4 days until the mice were sacrificed. The macrophage depletion effect in the spleen and tumors was confirmed by flow cytometry using the standard macrophage marker F4 / 80.

[0244] In vivo drug treatment Compounds 86, 97, and 99 powders were dissolved in 20% HPβCD saline (2-hydroxypropyl)-β-cyclodextrin). The drug was administered to tumors with a volume of 50-150 mm². 3 When the condition reached a certain level, IP or PO was administered and repeated daily.

[0245] 200 μg of PD1-specific monoclonal blocking antibody (clone RMP 1-14) was administered to tumors with a volume of 50-150 mm². 3 When the blood pressure reached a certain level, IP administration was started and repeated every two days.

[0246] Pharmacokinetic (PK) and toxicology research PK studies were performed at the NIH or commercial CROs (Pharmacon) using their in-house standard protocols. For C91, IV, and PO, the studies were conducted at the NIH using CD-1 mice; formulation: 20% HPbCD in saline. For C86, IV, and PO, the studies were conducted at Pharmaron using CD-1 mice; formulation: 20% HPbCD in saline. For C99, IV, and PO, the studies were conducted at the NIH using CD-1 mice; formulation: 20% HPbCD in saline. IP PK of C86 at 10 mpk and 30 mpk was conducted at the NIH using C57BL / 6J mice; formulation: 60% PEG400 in DI water. Solid feed PK of C86 was conducted at Pharmaron at 10, 30, and 60 mpk using CD-1 mice, with dose concentrations in solid feed of 0.05, 0.15, and 0.3 mg / g, respectively, based on a calculation of 5 g food consumption / mouse / day.

[0247] Pharmacokinetic (PK) studies at the NIH were conducted by the DMPK group. Male CD1 or C57BL / 6J mice, 6–8 weeks old and weighing approximately 20–30 grams, were administered compounds 86, 91, and 99 at doses of 2 mg / kg (IV), 10 mg / kg (PO), and 10 or 30 mg / kg (IP). The compounds were formulated using a 20% hydroxypropyl-β-cyclodextrin (HPbCD) solution in physiological saline and prepared on the day of administration or immediately before administration. Each treatment group consisted of three mice. Plasma was collected at 5, 15, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours post-administration for IV administration, and at 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours post-administration for PO administration. Approximately 0.025 mL of blood was collected from the dorsal metatarsal vein at each time point. The collected blood samples were then transferred to plastic microcentrifuge tubes containing heparin sodium as an anticoagulant. The samples were then centrifuged at 4000 g for 5 minutes at 4°C to obtain plasma. The plasma samples were then stored in polypropylene tubes, rapidly frozen, and kept at -75°C until analyzed by LC / MS / MS. The following pharmacokinetic parameters were measured: terminal phase half-life (T1 / 2), concentration immediately after injection (C0), maximum concentration (Cmax), time to maximum concentration (Tmax), clearance (CL), volume of distribution (Vd), area under the curve (AUClast), and bioavailability (%F). Animals were also monitored once daily by cage-side observation during the survival stage; adverse clinical signs were recorded as part of the PK report.

[0248] Serum toxicological assays were performed by IDEXX Bioanalytics (Standard Tox Panel 62794). In short, peripheral blood was collected by tail snip into a standard Eppendorf tube according to an approved protocol. Serum was prepared by clotting the blood and tested to ensure no hemolysis. Serum samples were stored at -80°C until sent to IDEXX.

[0249] Hematological studies, including complete blood counts, were performed at IDEXX Bioanalytics. In short, peripheral blood was collected by tail snips into heparin-coated microhematocrit capillaries (VWR, 15401-560) and stored overnight at 4°C before being sent to IDEXX.

[0250] RNA isolation and qPCR analysis of gene expression Total RNA from mouse tissue samples and in vitro cultured cells was isolated using the GenElute Mammalian Total RNA Miniprep Kit (Sigma) according to the manufacturer's protocol. Total RNA from human FFPE samples was isolated using the Quick-RNA FFPE Miniprep Kit (Zymo Research) according to the manufacturer's protocol. 1000 ng of RNA was used for reverse transcription using the High Capacity RNA to cDNA Kit (Life Technologies). The cDNA product was diluted 10×. 2.5 μL of this cDNA was used for qPCR for each sample. Three or more replicas were used for each reaction. Target gene expression was normalized to the appropriate housekeeping gene shown in the legend of the figure showing the RT-qPCR data. The expression fold was calculated as 2^-(CT target gene - CT housekeeping gene). Quantitative PCR was performed on a Viia 7 real-time PCR system (ThermoFisher). The following target genes and housekeeping genes were measured using a commercially available TaqMan probe (ThermoFisher): Mouse HPRT (Mm03024075_m1), Mouse Irf4 (Mm00516431_m1), Mouse Zbtb46 (Mm00511327_m1), Mouse Mafb (Mm00627481_s1), Mouse Aldh1a1 (Mm00657317_m1), Mouse Aldh1a2 (Mm00501306_m1), Mouse Aldh1a3 (Mm00474049_m1), Human HPRT (4333768F), Human IRF4 (Hs01056533_m1), Human ZBTB46 (Hs01008168_m1), Human MAFB (Hs00271378_s1), Human ALDH1A1 (Hs00946916_m1), human ALDH1A2 (Hs00180254_m1), and human ALDH1A3 (Hs00167476_m1).

[0251] Western blotting 1 × 10 7Cells were collected and washed three times with PBS. 50 μL of RIPA lysis buffer (Thermo Scientific, catalog number: 89901) containing a proteinase inhibitor (Thermo Scientific, catalog number: 78442) was added to the cell pellet and thoroughly mixed. The cell lysates were kept on ice for 30 minutes and centrifuged at 14000 × g for 15 minutes. The supernatant was collected and the protein concentration was measured using a BCA protein assay kit (Thermo Scientific, 23227). 30 μg of cell lysates from each sample were mixed with loading buffer (Thermo Scientific, catalog number: NP0007) to a final volume of 20 μL and incubated at 95°C for 5 minutes. Samples were loaded onto precast 4–15% SDS polyacrylamide gels (catalog number 4561084, BIO-RAD) and electrophoresis was performed at 120 V (constant voltage) for 40–60 minutes until the dye reached the bottom of the gel. The sample was transferred from the gel to a PVDF membrane in Tris-glycine transfer buffer at 100 V for 1.5 hours with a constant current (not exceeding 0.4 A). The PVDF membrane was removed from the blotting cassette and rinsed three times at room temperature for 5 minutes with TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20). Nonspecific binding on the membrane was blocked at room temperature for 1 hour on a shaker with freshly prepared 5% skim milk powder (Labscientific, catalog no. M0841). Aldh1a1 rabbit polychron primary antibody (Invitrogen, catalog no. PA5-32127) and GAPDH(14C10) rabbit mAb (Cell Signaling Technology, catalog no. 2118S) were diluted in 5% BSA at a ratio of 1:1000 and incubated with the PVDF membrane overnight at 4°C. Each membrane was washed three times with TBST for 5 minutes and incubated with HRP-conjugated secondary antibody (Cell Signaling Technology, catalog number 7074) at room temperature for 1 hour. Each membrane was washed three times with TBST for 5 minutes and incubated with ECL substrate (PerkinElmer, catalog number NEL104001EA) for 1 minute.Images were captured using the ChemiDoc imaging system (BioRad).

[0252] Establishment of CRISPR-mediated gene deletion tumor cell lines Nonviral delivery of Cas9-RNP has been previously described. Briefly, crRNA and tracrRNA (both from Integrated DNA Technologies) were mixed at equimolar concentrations, heated at 95°C for 5 minutes, and then annealed by direct hybridization at room temperature for 15 minutes. The annealed crRNA / XT-tracrRNA double strands were complexed by mixing with Cas9 in a 3:1 molar ratio and incubating at room temperature for at least 20 minutes. Nucleofection of Cas9-RNP was performed using Nucleofector® 2 (Lonza) along with a GFP expression plasmid vector (to identify cells in which nucleofection was successful). For Huh7-RALDH1 knockout cell lines, GFP-positive cells were sorted by FACS Jazz, and loss of RALDH1 expression was confirmed by Western blotting. For Hep55-RALDH1 knockout cell lines, GFP-positive cells were sorted by FACS Jazz, and single-cell clones were established from the sorted cells. Sanger sequencing performed at the University of Pennsylvania's core facility and in-house Western blotting confirmed the loss of ALDH1A1 in individual clones. The GFP expression plasmid used in the aforementioned nucleofection was supplied as part of the Lonza nucleofection kit.

[0253] Computer analysis of RNA sequencing (RNA-Seq) data from human tumors To compare RALDH isozyme expression in HCC with that in other human tumors in the TCGA dataset, the cBioPortal website interface was used for gene expression queries. To examine RALDH isozyme expression across different HCC molecular subtypes, raw sequence counts were downloaded for 371 primary tumor samples in TCGA-LIHC from the Genomic Data Commons Data Portal, filtered, and retained 183 samples contained in iCluster 1-3 as described in the literature. Several Bioconductor packages in R were used for subsequent steps on a local workstation. The count data was annotated with biomaRt. Principal component analysis (PCA) and plots were created with PCAtools. Normalization and statistical analysis were performed with DESeq2. Exploratory GSEA pathway analysis was performed using fgsea against Hallmark pathways configured from the Molecular Signatures Database (MsigDB), with DESeq2 statistics as the ranking metric. Clustering was performed using the degPatterns function from the DEGreport package.

[0254] To investigate the expression of RALDH isozymes in single-cell RNA sequencing data of human HCC, we utilized previously published and publicly available datasets. ALDH1A1, ALDH1A2, and ALDH1A3 were used as gene query terms, with all samples in the dataset used for scatter plot output.

[0255] Homology Modeling To construct the homology model, the MOE software was used with default settings. The MOE (Molecular Operating Environment) software is a set of various software tools developed by Chemical Computing Group Inc. The homology modeling algorithm within MOE consists of the following steps: First, initial partial geometry specification: The initial partial geometry of each target sequence is copied from a region of one or more template strands. If residue identity is preserved between the target sequence and its template, all heavy atom coordinates are copied; otherwise, only the main chain coordinates are copied. Second, insertions and deletions for correcting unassigned main chain coordinates. These are modeled from high-resolution strand fragments from the Protein Data Bank, and these are well superimposed on anchor residues on both sides of the insertion region. Third, loop selection and side-chain packing: After indel data collection is complete, a set of independent models is created. Loops are initially modeled in a random order. For each loop, the contact energy function analyzes a list of candidates collected in the segment search phase, considering all atoms already modeled and any atoms designated by the user as belonging to the model environment (e.g., ligands bound to the template, or structural water molecules). These energies are then used to perform a Boltzmann-weighted selection from the candidates, whose coordinates are then copied to the model. Once all loops are selected, the side chains are modeled. Side chain data are assembled from an extensive rotational isomer library created by systematic clustering of conformations from the rotational isomer library. A deterministic procedure based on unary quadratic optimization is then performed to select the optimal packing. After all main chain segments and side chain conformations have been selected for the intermediate model, hydrogen is added to the full valence requirement, and the model is submitted to a series of minimizations designed to first mitigate severe steric strain, and then to prepare the model for scoring.Next, it is written to the output database along with numerous quality assessment measurements that can flag significant geometric problems. The fourth stage is the selection and refinement of the final model. The final model is based on the highest-scoring intermediate model. In this study, electrostatic solvation energy was used and calculated using the generalized Born / volume integral (GB / VI) method. After the homology modeling procedure was completed, the final model was examined using MOE's protein geometry stereochemical quality assessment tools, including Ramachandran Maps.

[0256] Immunohistochemical staining of mouse HCC tumor slides Huh 7 xenograft tumor slides were dewaxed with CitriSolv for 20 minutes, and then with 100%, 95%, 85%, and 75% ethanol and dH2O for 3 minutes each. Peroxidase was blocked with 3% H2O2 at RT for 10 minutes. The slides were then washed three times with PBS and the target was restored at high temperature for 15 minutes. The slides were then blocked with an avidin / biotin blocking kit (Vector Laboratories) according to the manufacturer's instructions. Rabbit anti-mouse CD163 (Abcam, Ab6720) was diluted 1:200 and incubated with the slides overnight at 4°C. Anti-rabbit secondary (Abcam) was diluted 1:200 and incubated with the slides for 1 hour at RT. The signals were amplified with a VECSTAIN ABC kit (Vector Laboratories) and stained with a DAB substrate kit (Vector Laboratories) according to the manufacturer's instructions. The stained slides were scanned using a Leica Aperio Slide Scanner, as previously described, and analyzed with QuPath.

[0257] ALDH1A1 (D9Q8E) IHC on human tissue. 5-micron sections of formalin-fixed, paraffin-embedded tissue were stained with an antibody against ALDH1A1 (D9Q8E, Cell Signaling 54135S, 1:400). Staining was performed using a Bond Polymer Refine Detection System (Leica Microsystems DS9800) on a Leica Bond-III™ instrument. Heat-induced epitope recovery was performed with ER1 solution (Leica Microsystems AR9961) for 20 minutes. All experiments were performed at room temperature. Slides were washed three times with binding wash buffer or water between each step. Slides were scanned with a Hamamatsu NannoZoomer S360.

[0258] Quantification and statistical analysis Statistical significance between two groups was calculated using an unpaired Student's t-test. Statistical significance between multiple groups was calculated using a one-way ANOVA with Tukey's HSD post-hoc test. Survival significance was calculated using Kaplan-Meier with log-rank analysis. The analyses were performed using GraphPad Prism 8. Error bars represent the SEM, and p < 0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001).

[0259] Example 1: HCC expresses high levels of RALDH1 and RA. Recent studies have identified an immune evasion pathway in solid tumors in which RA produced by tumor cells acts on tumor monocytes to promote their differentiation into immunosuppressive tumor-associated monocytes (TAMs) (Figure 1). Therefore, blocking RA production by tumor cells and / or blocking RA signaling in monocytes may alleviate immunosuppression and induce an anti-tumor immune response. However, RA production and signaling exhibit significant genetic redundancy. RALDH1, RALDH2, and RALDH3 each catalyzed the same rate-limiting step in RA production (Figure 2). Because RA modulates many important developmental processes, nonspecific inhibition of all RALDH isoforms is not a viable therapeutic approach. Therefore, to identify tumors that may exhibit selective overexpression of specific RALDH isoforms, gene expression profiles in publicly available databases were examined, and as a result, HCC was identified as selectively overexpressing RALDH1. RALDH1 overexpression was investigated in human and mouse cell lines (Figures 3-4). In particular, unlike liver tissue, HCC appears to be almost exclusively dependent on RALDH1 for RA production (Figure 4). Therefore, RALDH1 inhibition may reduce RA production in HCC without significantly affecting normal liver function.

[0260] To investigate whether other types of cancer exhibit similar RA-dependent immune evasion, we analyzed publicly available TCGA RNA-seq data from human tumors for RALDH isozyme expression and found high RALDH1 transcript levels in HCC (Figure 18A). This was also confirmed by qRT-PCR in archived formalin-fixed paraffin-embedded (FFPE) samples from human patients (Figure 19A). In contrast, the other two RALDH isozymes were not highly expressed in HCC compared to other tumors in the TCGA RNA-seq database (Figures 19B-19C). Next, we investigated whether high RALDH1 expression was associated with specific subtypes of HCC. Previous reports have described three distinct molecular subtypes of HCC based on DNA copy number, DNA methylation, mRNA expression, miRNA expression, and proteomics. Computer analysis of RNA-seq data downloaded from the aforementioned study showed high levels of RALDH1 rather than RALDH2 or RALDH3 in all subtypes, suggesting that RALDH1 overexpression is characteristic of HCC (Figure 18B). This was further confirmed by performing RALDH1 immunohistochemical testing on primary and metastatic HCC, as well as unrelated tumors, revealing strong RALDH1 staining in HCC (Figures 18C and 19D).

[0261] High levels of RALDH1 expression in HCC tumors may originate from tumor cells, immunoinfiltrates, or other stromal components. To identify the primary source, we examined publicly available single-cell RNA-seq (scRNA-seq) datasets of human HCC. In this dataset, tumor cells and hepatocytes were the primary RALDH1-producing cells, expressing small amounts of RALDH2 and 3 (Figure 19E). Next, we measured transcript levels of the three RALDH isozymes in five different human HCC cell lines, all of which showed high levels of RALDH1 (Figure 18D). To measure RALDH enzyme activity, we performed the AldeRed assay. Consistent with elevated RALDH1 transcripts, AldeRed positivity was detected in all human HCC cell lines tested (Figure 18E). Similarly, mouse HCC also showed high Raldh1 and AldeRed positivity (Figures 18F-18G). Notably, while normal livers express all three Raldh isozymes, mouse HCCs appeared to loosen / inhibit Raldh2 and 3 (Figure 18F). Therefore, HCCs are likely dependent on RALDH1 for RA production. To confirm this, deletion of RALDH1 in human HCC cells using CRISPR / Cas9 resulted in a dramatic decrease in AldeRed activity (Figure 18H). Finally, LC / MS-based measurements of all-trans isomers (ATRA) of retinoic acid, the dominant biologically active isomer of RA formed by RALDH1-catalyzed oxidation of retinaldehyde, confirmed high levels of RA in HCC cells (Figure 18I). In summary, the results in this section demonstrate that HCCs produce high levels of RA via RALDH1.

[0262] Example 2: C-86 and C-91 inhibit RALDH1 activity. The human aldehyde dehydrogenase (ALDH) family comprises 19 isozymes, including three isoforms (RALDH1, RALDH2, and RALDH3) that catalyze the conversion of retinaldehyde to RA. Given their role in RA production, there is considerable interest in developing isozyme-specific inhibitors of RALDH. Recently, two compounds, compound 86 (C-86) and compound 91 (C-91), have been identified as potent RALDH1-specific inhibitors, both possessing particularly favorable profiles (Figure 5 and Table 6). Therefore, C-86 and C-91 are promising as agents that reduce RA and promote antitumor immune responses in HCC.

[0263] Consistent with high Raldh1 transcription (Figures 3-4), human and mouse HCC cell lines showed clear evidence of high RaldhH activity, as determined by the Aldered assay (Figure 6). Importantly, inhibition of Raldh1 with C-86 or C-91 resulted in a significant decrease in RaldhH activity in HCC cells, as determined by the Aldered assay (Figures 7-8). Furthermore, inhibition with either compound did not inhibit HCC cell growth in vitro (Figure 9). In summary, these findings indicate that HCC produces high levels of RA via Raldh1 overexpression, which can be inhibited by C-86 and C-91.

[0264] Mouse HCC tumors generated by Hepa 1-6 cells showed significant infiltration by myeloid antigen-presenting cells and T cells (Figure 10). A considerable number of T cells were present. Without being bound by theory, the data suggest that additional immunosuppressive pathways, such as RA-mediated immunosuppression, may be preventing these T cells from acting in HCC.

[0265] Example 3: RALDH1 inhibitor completely restores dendritic cell (DC) differentiation from monocytes in HCC cell lines. Many solid tumors produce high levels of retinoic acid (RA) receptors, inhibiting the formation of tumor-suppressive dendritic cells (DCs) through activation of retinoic acid receptors (RAR) and retinoid X receptors (RXR). This, in turn, promotes the differentiation of tumor-tolerant macrophages, thereby enabling tumor survival and growth. When RA synthesis and / or signaling are inhibited, respectively, through inhibition of RALDH1 and / or RAR / RXR, monocytes differentiate into DCs, DCs activate T cells, and T cells then kill tumors such as hepatocellular carcinoma (HCC). Therefore, altering the tumor microenvironment balance through inhibition of RA production is desirable from a therapeutic standpoint. Furthermore, this approach can be combined with checkpoint inhibitors to enhance immune checkpoint blockade therapy.

[0266] Using the strategy described herein (Figure 11A), we achieved the restoration of activated T cell proliferation from monocytes exposed to HCC-conditioned medium using C-86. The addition of C-86 to MoDCs and T cells exposed to SNU398-CM completely restored the proliferation rate compared to the vehicle (Figure 11B). Therefore, this represents the first use of a RALDH1 inhibitor in cancer immunotherapy.

[0267] Example 4: C-86 inhibits in vivo growth of hepatocellular carcinoma cell line (Huh7). In one aspect, as described elsewhere in this specification, the compounds of this disclosure act to interfere with retinaldehyde (RA) synthesis by inhibiting RALDH1 (Figure 12A). RA produced by tumor cells acts on tumor monocytes to promote differentiation into immunosuppressive tumor-associated monocytes (TAMs). Therefore, blocking RA production by tumor cells and / or blocking RA signaling in monocytes may alleviate immunosuppression and induce an antitumor immune response.

[0268] The tumor suppressor activity of compound C-86 was evaluated against the Huh7 HCC cell line. Intraperitoneal administration of C-86, BMS 493, and C-86 + BMS 493 each showed a significant reduction in tumor mass and volume compared to the control, and the combination of C-86 and BMS 493 showed a synergistic effect (Figures 12B-12C). Furthermore, dose-dependent effects on tumor volume and tumor mass were observed for C-86 (Figures 13A-13B).

[0269] Example 5: The in vivo effect of C-86 on tumors can be mediated by macrophages. Without being constrained by theory, we hypothesized that the in vivo effect of C-86 on tumors could be mediated by macrophages (Figure 14A). Therefore, we conducted tumor studies using liposomal clodronate (CloLipo) alone or in combination with C-86. Liposomal encapsulated clodronate (CloLipo) has previously been shown to be a potent anti-macrophage agent. By inducing apoptosis in these cells, it selectively depletes macrophages from animals within 24 hours after administration.

[0270] Intraperitoneal administration of C-86, CloLipo, and C-86+CloLipo each resulted in a significant reduction in tumor mass compared to the control (Figure 14B). Therefore, macrophage depletion may contribute to the in vivo effects of C-86, and / or more broadly, RALDH1 inhibitors, on tumors.

[0271] Example 6: C-97 inhibits ALDH activity in SNU98 cells. The Aldefluor assay is used to identify and isolate cells with high ALDH activity. This assay is based on the principle that ALDH can convert its substrate, Bodipy-aminoacetaldehyde (BAAA), into Bodipy-aminoacetic acid (BAA), which is retained within the cell. BAAA is uncharged and can freely diffuse into intact viable cells, while BAA, due to its net negative charge, cannot cross the membrane and remains inside the cell. The assay buffer prevents the outflow of BAA from the cell. Therefore, the amount of intracellular BAA fluorescence is proportional to ALDH activity and can be measured using a flow cytometer. Diethylaminobenzaldehyde (DEAB), a specific inhibitor of ALDH, is used as a background fluorescence control.

[0272] Administration of C-97 at 1 nM resulted in a decrease in ALDH1 activity compared to the DMSO control group (i.e., partial inhibition of ALDH activity at 1 nM C-97), and essentially complete inhibition of ALDH occurred at concentrations of 10 nM or higher (Figure 16). Therefore, C-97 potently inhibits ALDH activity.

[0273] Example 7: C-97 inhibits in vivo growth of hepatocellular carcinoma cell line (Huh7). The relative tumor suppressor activity of compound C-97 compared to C-86 was evaluated against the Huh7 HCC cell line. Intraperitoneal administration of C-86 and C-97, respectively, resulted in significant reductions in tumor mass and volume compared to the control (Figures 17A-17B). Furthermore, compound C-97 showed enhanced tumor suppressor activity compared to C-86.

[0274] Example 8: Activity of compounds selected as RALDH1 inhibitors The ALDH inhibitory effects of the compounds C-9125, C-9163, and C-9175 were evaluated in several cell lines, including MiaPaCa2 and OV90 cell lines, using the AldeFlour assay described elsewhere in this specification (Table 4).

[0275] (Table 4) TIFF2026510946000079.tif30161

[0276] Example 9: RALDH1 inhibitors suppress RA production in HCC cells. RA can drive autocrine or paracrine signaling by binding to the RAR / RXR transcription factor heterodimer and regulating gene expression. Therefore, reducing RA production by inhibiting the RALDH enzyme and / or blocking RA signaling by RAR / RXR may suppress the aforementioned RA-mediated tumor immunity evasion. However, RA is an important morphogen and signaling molecule, which for this reason excludes overall RA blockade as a therapeutic strategy. RAR and RXR have several isoforms that give rise to a diverse repertoire of RAR / RXR heterodimers. Isoform-specific inhibitors of RAR and RXR have been developed, but they are used as tool compounds because they are toxic and have no approved clinical indications.

[0277] In contrast, RALDH isozyme-specific inhibitors as a strategy for RA blockade have not been sufficiently investigated. Two best-in-class RALDH1 inhibitors (Raldh1-INH), compounds 86 (C86) and 91 (C91), also known as NCT-505 and NCT-506, respectively, exhibited PK and pharmacodynamic profiles favorable to potential clinical applications. Therefore, we investigated whether C86 and / or C91 could inhibit RA production in HCC cells.

[0278] Both inhibitors reduced AldeRed fluorescence in human HCC, but C86 showed greater potency (Figures 20A-20B and Figure 5). Therefore, C86 was primarily used in subsequent experiments. Neither C86 nor C91 resulted in compensatory increases in the transcription of other RALDH isozymes, which is consistent with the AldeRed data and demonstrates the efficacy of these inhibitors in suppressing RA production in HCC (Figure 21A).

[0279] In contrast to human HCC, neither inhibitor was able to reduce AldeRed fluorescence in mouse HCC cells (Figure 20C). LC / MS-based RA measurement confirmed the lack of C86-mediated RA suppression in mouse HCC (Figure 20D). Therefore, C86 and C91 activity exhibit species specificity. Homology modeling based on the partial crystal structures of these Raldh1-INH cells suggests that differences in key drug-interacting amino acids between mouse and human cells may underlie this observation (Figure 21B). Finally, it was confirmed that the reduction in RA and AldeRed activity in human HCC cell lines by C86 or C91 was not due to increased cell death or decreased cell viability (Figures 20E-20F and 21C). Thus, the results in this section demonstrate the efficacy of Raldh1-INH in suppressing RA production in human HCC cell lines.

[0280] Example 10: HCC-derived RA regulates monocyte differentiation To investigate whether HCC regulates monocyte differentiation, human monocytes were co-cultured with either human HCC cell lines or cell culture supernatants (conditioned medium, CM) from these cell lines. Flow cytometry analysis showed that DC differentiation was suppressed in the presence of HCC cells or CM (Figure 22A). qRT-PCR analysis confirmed this, showing repression of DC-related genes and increased expression of macrophage-related genes by CM (Figure 22A). These findings were replicated in mouse monocytes cultured with HCC cells or CM (Figures 22B and 23B). Pretreatment of HCC cells with Raldh1-INH C86 reversed this effect (Figures 22A-22B and 23A-23B). Therefore, HCC-derived RA regulates monocyte differentiation in vitro, and this can be "rescue" by blocking RA production via Raldh1-INH.

[0281] Next, this was tested in vivo by transplanting the human HCC cell line Huh7 into immunodeficient (NU / J) mice and, once tumors were established, by intratumoral injection of primary human monocytes. Mice were treated with either a vehicle (control) or C86. In this setting, C86 selectively inhibited RALDH1 in transplanted human cells (HCC and human monocytes) due to the species specificity of this inhibitor as described elsewhere herein. Five days after monocyte transplantation, tumors were analyzed by flow cytometry, where human leukocytes and mouse leukocytes were distinguished using standard species-specific anti-CD45. Raldh1-INH treatment increased DC differentiation from transplanted human monocytes (Figure 22C). Without wishing to be bound by any theory, this may reflect an effect of reducing RA production in HCC cells, which is consistent with the results of co-culture experiments described elsewhere herein. While a direct effect of inhibitors on transplanted human monocytes could explain this observation, this is unlikely given that host (mouse) immune cells in TME that are not sensitive to C86 also showed an increased frequency of DCs and a decreased frequency of macrophages (Figure 22D). Furthermore, C86 treatment of monocytes in vitro did not alter their potential to differentiate into DCs (Figure 22A). Finally, RA was confirmed to be a key mediator of the aforementioned effects of HCC on monocyte differentiation by using RALDH1-KO HCC cells, where CM from knockout cells did not suppress DC differentiation (Figure 22E). In summary, the data presented in this section indicate that Raldh1-INH suppresses HCC RA production and its associated effects on monocyte differentiation.

[0282] Example 11: RA induces tumor-promoting properties in monocyte-derived macrophages. Monocytes can differentiate into macrophages or dendritic cells (DCs), and the adaptive immunization consequences of RA-mediated suppression of monocyte-to-DC differentiation in tumor immunity are described elsewhere in this specification. However, it remained unclear whether and how tumors are affected by RA-induced macrophages. As described elsewhere in this specification, HCC-derived RA can increase macrophage frequency in TMEs (Figure 22D). To investigate whether RA also alters macrophage function, we utilized a macrophage-tumor co-transplantation approach. Primary human monocytes were differentiated into macrophages in or without RA, mixed 50:50 with human HCC cell line (Huh7), and transplanted into immunodeficient NU / J mice. HCC cells transplanted without macrophages served as an additional control. RA-treated macrophages promoted tumor growth compared to HCC cells transplanted alone or with control macrophages (Figures 24A and 25A). Therefore, the overall impact of macrophages in HCC TMEs was next evaluated by depleting TAMs via ip liposomal clodronate (CloLipo), a common method for macrophage depletion. CloLipo-induced TAM reduction was confirmed, and a decrease in tumor growth was observed under these conditions (Figures 24B and 25B). Thus, HCC-associated TAMs support tumor growth, which is a characteristic that can be induced by high RA in HCC TMEs.

[0283] To further investigate this, a reductionist approach was employed, co-culturing RA-induced macrophages with HCC cells. First, RA exposure was observed to increase macrophage numbers, consistent with in vivo observations in TME, suggesting that RA may increase macrophage proliferation and / or survival (Figure 24C). RA-pretreated macrophages significantly increased tumor cell numbers compared to untreated control macrophages; this effect was reversed when macrophages were exposed to the RAR signaling inhibitor BMS493 (Figure 24D). Consistent with the increase in tumor cell numbers, CFSE labeling suggested increased tumor cell proliferation in the presence of RA-treated macrophages compared to untreated control macrophages (Figure 24E). These effects were repeated when HCC cells were grown with CM from RA-treated or control macrophages, suggesting that RA exposure may lead to the production of soluble "mitogenic" factors by macrophages (Figure 25C). Notably, when tumor cells were cultured with control (untreated) monocytes / macrophages, they showed reduced proliferation, likely due to nutrient competition (Figure 24D). Pretreatment with RA eliminated this inhibitory effect, demonstrating the tumor-supporting effect of RA exposure. Experiments using CM (Figure 25C) further clarified this, as the absence of monocytes / macrophages eliminated this nutrient competition and "unmasked" the promitotic effect of soluble factors.

[0284] In summary, the results described herein indicate that HCC-derived RA induces TAM to produce factors that support HCC growth.

[0285] Example 12: RALDH1 inhibitors suppress HCC growth Data described elsewhere in this specification indicate that Raldh1-INH can alter monocyte differentiation and macrophage function in the tumor mesenter by blocking RA production in HCC cells. To investigate the therapeutic implications, huh1 and huh7 human HCC cell lines were transplanted into immunodeficient mice, and the mice were treated with C86, resulting in significant tumor inhibition (Figures 26A and 27A). C86 treatment reduced RADLH activity and therefore RA production in tumor cells but not in infiltrating leukocytes (Figure 26B). C86 showed a dose-response effect, exhibiting tumor suppression at doses of ≥10 mg / kg once daily, with weight loss observed only at high doses of 40 mg / kg (Figures 26C-26D). In response to this, CRISPR / Cas9-mediated deletion of RALDH1 in HCC cells (RALDH1-KO) slowed tumor growth in vivo but not in vitro (Figures 26E and 27B). RALDH1-KO HCC tumors did not respond to C86 therapy, demonstrating that the tumor-suppressive effect of Ralddh1-INH was primarily due to an on-target effect on RALDH1 (Figure 26E). Next, the role of TAM in mediating the therapeutic effect of C86 was investigated by depleting TAM through CloLipo treatment. TAM depletion suppressed HCC growth, and C86 treatment did not further suppress tumors in the absence of TAM (Figures 26F and 27C). Therefore, the therapeutic effect of reducing HCC-derived RA requires the presence of TAM.

[0286] Next, we tested whether HCC-derived RA directly acts on TAM to promote the tumor growth observed above. To this end, we used mice that conditionally express the dominant-negative isoform of RAR from the Rosa26 locus (dnRAR). flox We obtained mice. dnRAR expression leads to inhibition of the RAR-mediated effect of RA. dnRAR flox This is a Lysozyme 2-Cre mouse (LysM) that expresses Cre recombinase in bone marrow cells, including macrophages. CreHuman HCC cells (Huh7) were crossed with control mice and Lysm after T cell depletion. Cre dnRAR flox When transplanted into mice, bone marrow-specific dnRAR expression resulted in a significant slowdown of tumor growth and a reduction in TAM (Figures 26G and 27D-27E). This suggests that tumor-derived RA induces tumor-promoting TAM, which is consistent with data provided elsewhere in this specification. Nevertheless, dnRAR-mediated repression of RA signaling is partial, as it inhibits only the RAR-mediated pathway and not other RAR isoforms or RXR, and very high levels of RA can still overcome dnRAR-mediated RAR inhibition.

[0287] To further investigate the effects of blocking RAR signaling, particularly in a therapeutic context, Huh7-carrying mice were treated with the pan-RAR blocker BMS493 alone or in combination with Raldh1-INH. Monotherapy with BMS493 or Raldh1-INH slowed tumor growth, but combination therapy showed the greatest effect (Figure 26H). Therefore, blocking RA production with Raldh1-INH and / or blocking RA signaling with a RAR inhibitor can suppress HCC growth.

[0288] Example 13: Reduction of tumor-derived RA is a major mechanism of tumor suppression by Raldh1-INH. As described elsewhere in this specification, C86 and C91 exhibit species specificity and do not inhibit mouse RALDH1 (Figures 20C-20D). Therefore, the aforementioned therapeutic effects on xenograft-based tumor models represent a scenario in which the drug acts only on transplanted tumor cells and not on host cells, and there are no T cells to initiate an anti-tumor immune response. To overcome these limitations, compound 99 (C99), a potential inhibitor of mouse RALDH1, was selected from a previous chemistry series for further evaluation. In vitro, C99 suppressed RALDH1 activity in both mouse and human HCC cell lines, albeit at much higher concentrations (micromolar, Figure 28A) compared to C86 (nanomolar range, Figure 20A) (Figure 28A). Correspondingly, the IC50 of C99 50 This is the IC for C86 50 It was found to be significantly larger than (Figure 29A). Despite its lower potency, C99 allowed us to circumvent the limitations of the aforementioned interspecific xenograft model and provided an opportunity to further investigate the biological response to Raldh1-INH.

[0289] C99 did not reduce the proliferation or survival rate of the mouse HCC cell line Hepa 1-6 in vitro, but significantly suppressed tumor growth in vivo (Figures 28B-28C and 29B-29C). Human-specific C86 did not suppress mouse Hepa 1-6 growth in vivo (Figure 28C). C99 treatment reduced AldeRed activity and the frequency of TAMs in TME in tumor cells, but C86 treatment did not (Figures 28D-28E). Furthermore, TAM depletion by CloLipo suppressed Hepa 1-6 tumor growth and made tumors numb to C99 treatment (Figure 28F). These findings reflect the effects of C86 on human HCC described elsewhere in this specification and suggest that the tumor suppressor activity of RALDH1 inhibitors depends on their ability to block RA production in tumor cells. To further confirm this, we tested C99 in an FS mouse model of fibrosarcoma that expressed high levels of both Raldh1 and Raldh3 and whose RA production was not solely dependent on Raldh1; C99 did not suppress fibrosarcoma growth (Figures 28G and 29D). In summary, the data in this section demonstrate the efficacy of Raldh1-INH in suppressing HCC growth through inhibition of RA production.

[0290] Example 14: Raldh1-INH for HCC immunotherapy As described elsewhere in this specification, C86 monotherapy demonstrated therapeutic efficacy in xenograft tumor models even without T cells. C99 also suppressed tumor growth in syngeneic tumor models with intact adaptive immunity, but had two main limitations: (1) C99 is not as potent as C86 or C91; and (2) mouse Hepa 1-6 tumor cells tend to produce a significant T cell response after subcutaneous transplantation into C57BL6 / J mice, which can sometimes lead to spontaneous delayed tumor rejection. These limitations make it difficult to investigate the true therapeutic potential of C99 in Hepa 1-6. Nevertheless, we considered it important to investigate the effects of complete RALDH1 inhibition in the presence of T cells and to test combinations with ICB. To this goal, Hep55 was identified as a mouse HCC cell line that exhibits little spontaneous T cell response and does not show rejection. As shown in Figure 18F, Hep55 cells also have high Raldh1 expression.

[0291] To overcome the limited efficacy of C99, a genetic approach was used to create a RALDH1 deletion in Hep55 cells using CRISPR / Cas9. While Raldh1 gene loss was compatible with normal growth of Hep55 cells in vitro, tumor suppression was significantly observed when cells were transplanted in vivo (Figures 30A and 29E-29F). Tumor suppression was accompanied by a significant enhancement of RALDH1-KO tumor infiltration by activated T cells (Figure 30B). Anti-PD1 treatment further significantly suppressed growth by RALDH1-KO Hep55 tumors (Figure 29G). Hep55 tumors showed infiltration by both pro-inflammatory and anti-inflammatory macrophages, with the frequency of pro-inflammatory macrophages significantly increasing with the loss of RALDH1 activity in tumor cells (Figures 29H-29I). These findings are consistent with both macrophages and T cells driving the therapeutic effect of RALDH1 inhibition. In this regard, it is noteworthy that the growth of RALDH1-KO Hep55 tumors was more significantly inhibited than that of xenografted human HCCs suppressed by C86 treatment. Without wishing to be bound by any theory, this may be due to a greater degree of RALDH1 inhibition by gene knockout, but a more likely explanation is the presence of T cells in the syngeneic Hep55 model.

[0292] A key concern regarding the use of Raldh1-INH is on-target toxicity, particularly considering RALDH1 expression in normal livers. While no signs of toxicity were observed in C86 and C91, the main concern is the species specificity of these compounds. To investigate the potential on-target toxicity of RADH1 inhibition, RALDH1 gene deletions were created in mice (Figures 30C-30D). RALDH1-KO mice showed no apparent toxicity compared to their wild-type siblings and developed normally. Basic toxicological analyses of serum and complete blood counts also showed no abnormalities in RALDH1-KO mice, and there were no significant differences in body weight between genotypes (Figures 30E, 31A-31B). For off-target assays (Eurofins Cereps Panlabs 85), a standard panel of in vitro assays was performed, and no significant concerns regarding off-target effects at expected therapeutic concentrations were raised (Table 5). Therefore, the RALDH1-INH tested here is unlikely to cause serious toxicity.

[0293] (Table 5) Safety Screen Assay for C86 TIFF2026510946000080.tif212161TIFF2026510946000081.tif187161

[0294] Given the superior efficacy and toxicity profiles of RALDH1-INH, the PK and pharmacodynamic (PD) properties of three exemplary RALDH1 inhibitors (i.e., C86, C91, and C99) were investigated. Here, the half-life of C99 was also found to be significantly shorter than that of the other two exemplary Raldh1-INHs (Table 6). The superior IC50 of C86 compared to C91 (Figure 5) was the rationale for using C86 in all experiments, but when the compound was administered via oral (PO) or intravenous (IV) routes, the half-life of C86 was found to be lower than that of C91 (Table 6). Nevertheless, ip delivery was used in all the in vivo experiments described above, which is associated with a superior pharmacokinetic profile compared to the PO or IV routes (Tables 6-7).

[0295] (Table 6) Exemplary pharmacokinetic and pharmacodynamic data for specific compounds a TIFF2026510946000082.tif40161 a Research using mouse plasma; b Intravenous administration; c Oral administration.

[0296] (Table 7) Exemplary pharmacokinetic and pharmacodynamic data for C-86 in mice TIFF2026510946000083.tif59161 a Mesenteric

[0297] C86 also showed good in vivo distribution to various tissues upon ip delivery (Table 7). Meanwhile, C86 was incorporated into solid feed, and the PK / PD of the inhibitor was examined over 15 days (Figure 31C). This approach demonstrated good stability of C86 in solid feed and consistent long-term drug exposure without affecting body weight (Figures 31C-31D). Therefore, Raldh1-INH exhibits superior efficacy and PK / PD profile.

[0298] In summary, the data described herein establish RALDH1 as a genuine therapeutic target for HCC immunotherapy. Its efficacy as a monotherapy has been demonstrated. Furthermore, given its unique mechanism of action, there are opportunities for combination with other therapeutic approaches. As proof of concept, an additive effect with ICB has been demonstrated. Finally, this disclosure establishes RALDH inhibition as a viable therapeutic approach in other cancers.

[0299] The terms and expressions used herein are for illustrative purposes only, not limiting purposes, and the use of such terms and expressions is not intended to exclude equivalents or parts thereof of the characteristics shown and described, however it is recognized that various modifications are possible within the scope of the embodiments of this application. Accordingly, while this application describes certain embodiments and any characteristics, it should be understood that modifications and variations of the compositions, methods and concepts disclosed herein are conceivable to those skilled in the art, and that such modifications and variations are considered to fall within the scope of the embodiments of this application.

[0300] Table of arrays TIFF2026510946000084.tif115128

[0301] Enumerated aspects The following illustrative embodiments are provided, but the numbering is not intended to be interpreted as indicating a level of importance. Embodiment 1 provides the following: A method for treating, preventing, and / or improving solid tumors in a subject in need thereof, wherein the method comprises a pharmaceutically effective amount of (a) at least one immunostimulant, and (b) Retinaldehyde dehydrogenase 1 (RALDH1) inhibitors The process includes administering the RALDH1 inhibitor to the subject, wherein the RALDH1 inhibitor is as follows: (i) Equation (I): A compound of TIFF2026510946000085.tif20128, or its salt, solvate, prodrug, stereoisomer, tautomer, or isotopolog, During the ceremony, R 1a This is selected from the group consisting of optionally substituted C2-C8 heterocyclyls, optionally substituted phenyls, and optionally substituted C5-C8 cycloalkenyls. Here, R 1aEach of the substituents in the compound may be a C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkoxy, optionally substituted phenyl, optionally substituted C2-C8 heterocyclyl, halogen, OH, or N(R) a )(R b ), NO2, CN, C(=O)R a , C(=O)N(R a )(R b ), S(=O)2R a , S(=O)2N(R a )(R b ), and N(R a )C(=O)R b Independently selected from the group consisting of, Here, each of the substituents may be substituted with at least one substituent selected from the group consisting of C1-C3 alkyl, C1-C3 alkoxy, halogen, CN, and NO2, and Here, R 1a Any two vicinal or geminal substituents within may combine with the atom to which they are bonded to form a C2-C8 heterocyclyl or C3-C8 cycloalkyl; R 1b and R 1c If present, H, C1-C6 alkyl, C1-C6 alkoxy, halogen, OH, N(R) a )(R b ), NO2, and CN are each independently selected; R 2 Each occurrence is independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, C1-C3 haloalkoxy, C1-C6 hydroxyalkyl, halogen, NO2, and CN; R 3 This is a C2-C8 heterocycline which may be substituted, a phenyl which may be substituted, and N(R a ) (C3-C8 cycloalkyl which may be substituted), and N(R a Selected from the group consisting of (C2-C8 heterocyclines that may be substituted), Here, R 3Any substituent in the group may be C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, halogen, OH, or N(R) a )(R b ), NO2, CN, C(=O)R a , C(=O)N(R a )(R b ), S(=O)2R a , S(=O)2N(R a )(R b ), and N(R a )C(=O)R b Selected independently from the group consisting of; A is C6~C even if it is not substituted. 10 Selected from the group consisting of aryls and optionally substituted C2-C8 heterocyclines, Here, each substituent in A is a C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, halogen, OH, N(R) a )(R b ), NO2, CN, C(=O)R a , C(=O)N(R a )(R b ), S(=O)2R a , S(=O)2N(R a )(R b ), and N(R a )C(=O)R b Selected independently from the group consisting of; L is selected from the group consisting of -CH2-, -C(=O)-, and -S(=O)2-; X is N and CR 1c Selected from the group consisting of; n is an integer selected from the group consisting of 0, 1, 2, 3, and 4; and R a , R b , and R c Each occurrence is independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, and C2-C8 heterocyclyl. Compounds of formula (I), or their salts, solvates, prodrugs, stereoisomers, tautomers, or isotopologs; (ii) 1-benzylindoline-2,3-dione, 2-((4-oxo-3-(3-(pyrrolidine-1-yl)propyl)-3,4-dihydrobenzo[4,5]thieno[3,2-d]pyrimidine-2-yl)thio)ethyl acetate, 2-((2-(sec-butyl)-3-oxo-2,3-dihydroimidazo[1,2-c]quinazoline-5-yl)thio)-N-(o-tolyl)butanamide, and 8-((4-(cyclopropanecarbonyl)piperazine-1-yl)methyl)-7-isopentyl-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione, or its salts, solvates, prodrugs, stereoisomers, tautomers, or isotopologs. A compound selected from the group consisting of and The method selected from the group consisting of the above. Embodiment 2 provides the following: The method according to embodiment 1, wherein the RALDH1 inhibitor is a compound of formula (I). Embodiment 3 provides the following: The compound of formula (I) is Selected from the group consisting of TIFF2026510946000086.tif25140, R 2a , R 2b , R 2c , and R 2d However, if present, each is independently selected from the group consisting of H, C1-C6 alkyl, C1-C6 alkoxy, and halogen. The method of embodiment 2. Embodiment 4 provides the following: R 2a , R 2b , R 2c , and R 2d The method of embodiment 3, wherein, if present, each is independently selected from the group consisting of H, Me, OMe, F, and Cl. Embodiment 5 provides the following: Any of embodiments 2 to 4, wherein X is N. Embodiment 6 provides the following: R 1a but, Selected from the group consisting of TIFF2026510946000087.tif15128, Here, R 5a and R 5b If present, each of the following is independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, phenyl, thiophen-2-yl, thiophen-3-yl, and CN. Here, R 5a and R 5b Each substituent in the compound may be substituted with CN, Here, R 5a and R 5b If present, they may combine with the atoms to which they are bonded to form a C2-C6 heterocyclyl or C3-C6 cycloalkyl; and R 6 is S(=O)2R a That is, Any method according to embodiment 2 to 5. Embodiment 7 provides the following: R 5a and R 5b The method according to embodiment 6, wherein each of the following is independently selected from the group consisting of Me, t-Bu, 1-cyanocyclopropyl, 1-cyanocyclobutyl, 1-cyanocyclopentyl, 1-cyanocyclohexyl, phenyl, thiophen-2-yl, thiophen-3-yl, and CN. Embodiment 8 provides the following: R 6 The method of embodiment 6 or 7, wherein is ethenylsulfonyl. Embodiment 9 provides the following: R 1a but, A method according to any of embodiments 2 to 8, selected from the group consisting of TIFF2026510946000088.tif40153. Embodiment 10 provides the following: R 1bA method in any of embodiments 2 to 9, wherein H is present. Embodiment 11 provides the following: R 3 but, Selected from the group consisting of TIFF2026510946000089.tif20128, R 7 C(=O)R a and S(=O)2R a Selected from the group consisting of, R 8 This is selected from the group consisting of C1-C6 alkoxy, C1-C6 hydroxyalkyl, and OH. Any method of embodiment 2 to 10. Embodiment 12 provides the following: R 7 The method according to embodiment 11, wherein the substance is selected from the group consisting of cyclopropylcarbonyl, methylsulfonyl, dimethylaminosulfonyl, and dimethylaminocarbonyl. Embodiment 13 provides the following: R 8 The method according to embodiment 11 or 12, wherein the group is selected from methoxy, 2-hydroxyethyl, and OH. Embodiment 14 provides the following: R 3 but, A method selected from the group consisting of TIFF2026510946000090.tif38138, according to any of embodiments 2 to 10. Embodiment 15 provides the following: The compound of formula (I) is 8-(6-methoxy-3-((4-methoxyphenyl)sulfonyl)quinoline-4-yl)-1,4-dioxa-8-azaspiro[4.5]decane; 1-(3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)-6-fluoroquinoline-4-yl)-4-phenylpiperidine-4-carbonitrile; (4-(cyclopropanecarbonyl)piperazine-1-yl)(4-(4,4-dimethylcyclohexa-1-en-1-yl)-6-fluoroquinoline-3-yl)methanone; (4-(4-(tert-butyl)phenyl)-6-fluoroquinoline-3-yl)(4-(cyclopropanecarbonyl)piperazine-1-yl)methanone; 1-(4-(3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)-6-fluoroquinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonilicate; (4-(4,4-dimethylcyclohexa-1-en-1-yl)-6-fluoroquinoline-3-yl)(4-(methylsulfonyl)piperazine-1-yl)methanone; (4-(4-(tert-butyl)phenyl)-6-fluoroquinoline-3-yl)(4-(methylsulfonyl)piperazine-1-yl)methanone; 1-(4-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoro-N-((1r,4r)-4-hydroxycyclohexyl)quinoline-3-carboxamide; (6-Fluoro-4-(4-(vinylsulfonyl)piperazine-1-yl)quinoline-3-yl)(4-(methylsulfonyl)piperazine-1-yl)methanone; 1-(4-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclobutan-1-carbonitrile; 1-(4-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopentan-1-carbonitrile; 4-(4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-sulfonamide; 4-(4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-carboxamide; 1-(6-chloro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonilicate; 1-(6-chloro-3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonitrile; 1-(6-(4-(methylsulfonyl)piperazine-1-carbonyl)thieno[3,2-b]pyridine-7-yl)-4-phenylpiperidine-4-carbonitrile; 1-(6-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)thieno[3,2-b]pyridine-7-yl)-4-phenylpiperidine-4-carbonitrile; 1-(6-methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonilicate; 1-(7-methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonilicate; 1-(6,7-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonilicate; 1-(6,8-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonilicate; 4-(4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-sulfonamide; 4-(4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-carboxamide; 4-(6-chloro-4-(4-(1-cyanocyclopropyl)phenyl)quinoline-3-carbonyl)-N,N-dimethylpiperazine-1-sulfonamide; 1-(4-(6-chloro-3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6-(4-(methylsulfonyl)piperazine-1-carbonyl)thieno[3,2-b]pyridine-7-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)thieno[3,2-b]pyridine-7-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6-methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(7-Methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6,7-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6,8-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoro-N-((1r,4r)-4-hydroxycyclohexyl)quinoline-3-carboxamide; 4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoro-N-(1-(2-hydroxyethyl)-1H-pyrazole-4-yl)quinoline-3-carboxamide; and 4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoro-N-(1-(2-hydroxyethyl)-1H-pyrazole-4-yl)quinoline-3-carboxamide A method selected from the group consisting of the following, one of embodiments 2 to 14. Embodiment 16 provides the following: The method according to any of embodiments 1 to 15, wherein the solid tumor is a carcinoma. Embodiment 17 provides the following: The method according to embodiment 16, wherein the carcinoma comprises human hepatocellular carcinoma (HCC) cells. Embodiment 18 provides the following: A method in which RALDH1 is overexpressed in the solid tumor, as described in any of embodiments 1 to 17. Embodiment 19 provides the following: The method according to embodiment 18, wherein the expression of Raldh1 and Raldh2 or Raldh1 and Raldh3 in the solid tumor has a ratio selected from the group consisting of approximately 1000:1, 500:1, 250:1, 100:1, 50:1, 25:1, 10:1, and 5:1. Embodiment 20 provides the following: The method according to any one of embodiments 1 to 19, wherein the immunostimulant is at least one selected from the group consisting of immune checkpoint inhibitors, chimeric antigen receptor (CAR) T cells, T cells genetically engineered to express specific TCRs targeting tumor antigens (TCR transgenic), ex vivo expanding T cells, and bispecific T cell engagers (BiTEs). Embodiment 21 provides the following: The method according to embodiment 20, wherein the immunostimulant is an immune checkpoint inhibitor. Embodiment 22 provides the following: The method according to embodiment 21, wherein the immune checkpoint inhibitor is selected from the group consisting of anti-PD1 antibody, anti-PD-L1 antibody, anti-CTLA4 antibody, any fragment thereof, and any combination thereof. Embodiment 23 provides the following: The method according to embodiment 20, wherein the immunostimulant is CAR T cells. Embodiment 24 provides the following: The method according to embodiment 23, wherein the CAR T cells are administered intravenously. Embodiment 25 provides the following: The method according to embodiment 23 or 24, wherein the CAR T cells are administered as CAR T cell therapy. Embodiment 26 provides the following: The method according to any of embodiments 1 to 25, wherein the subject is administered an immune checkpoint inhibitor and chimeric antigen receptor (CAR) T cells. Embodiment 27 provides the following: A method in any of embodiments 1 to 26, further comprising the step of administering to the subject at least one selected from the group consisting of retinoic acid receptor (RAR) inhibitors and retinoid X receptor (RXR) inhibitors. Embodiment 28 provides the following: The method according to embodiment 27, wherein the RAR inhibitor is selected from the group consisting of AGN 193109, BMS 195614, BMS 493, CD 2665, ER 50891, LE 135, LY 2955303, MM 11253, any salt or solvate thereof, and any combination thereof. Embodiment 29 provides the following: The method according to embodiment 27 or 28, wherein the RXR inhibitor is selected from the group consisting of HX 531, PA 452, and UVI 3003, any salt or solvate thereof, and any combination thereof. Embodiment 30 provides the following: A method in which the RALDH1 inhibitor and the immunostimulant are administered to the subject simultaneously or sequentially, as described in any of embodiments 1 to 29. Embodiment 31 provides the following: The method according to any of embodiments 1 to 30, wherein the subject is a mammal. Embodiment 32 provides the following: The method according to embodiment 31, wherein the mammal is a human. Embodiment 33 provides the following: (a) at least one immunostimulant; (b) pharmaceutically acceptable carriers; and (c) Retinaldehyde dehydrogenase 1 (RALDH1) inhibitors A pharmaceutical composition comprising, wherein the RALDH1 inhibitor is as follows: (i) Equation (I): A compound of TIFF2026510946000091.tif20128, or its salt, solvate, prodrug, stereoisomer, tautomer, or isotopolog, During the ceremony, R 1a This is selected from the group consisting of optionally substituted C2-C8 heterocyclyls, optionally substituted phenyls, and optionally substituted C5-C8 cycloalkenyls. Here, R 1a Each of the substituents in the compound may be a C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkoxy, optionally substituted phenyl, optionally substituted C2-C8 heterocyclyl, halogen, OH, or N(R) a )(R b ), NO2, CN, C(=O)R a , C(=O)N(R a )(R b ), S(=O)2R a , S(=O)2N(R a )(R b ), and N(R a )C(=O)R b Independently selected from the group consisting of, Here, each of the substituents may be substituted with at least one substituent selected from the group consisting of C1-C3 alkyl, C1-C3 alkoxy, halogen, CN, and NO2, and Here, R 1a Any two vicinal or geminal substituents within may combine with the atom to which they are bonded to form a C2-C8 heterocyclyl or C3-C8 cycloalkyl; R 1b and R 1c If present, H, C1-C6 alkyl, C1-C6 alkoxy, halogen, OH, N(R) a )(R b ), NO2, and CN are each independently selected; R 2Each occurrence is independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, C1-C3 haloalkoxy, C1-C6 hydroxyalkyl, halogen, NO2, and CN; R 3 This is a C2-C8 heterocycline which may be substituted, a phenyl which may be substituted, and N(R a ) (C3-C8 cycloalkyl which may be substituted), and N(R a Selected from the group consisting of (C2-C8 heterocyclines that may be substituted), Here, R 3 Any substituent in the group may be C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, halogen, OH, or N(R) a )(R b ), NO2, CN, C(=O)R a , C(=O)N(R a )(R b ), S(=O)2R a , S(=O)2N(R a )(R b ), and N(R a )C(=O)R b Selected independently from the group consisting of; A is C6~C even if it is not substituted. 10 Selected from the group consisting of aryls and optionally substituted C2-C8 heterocyclines, Here, each substituent in A is a C1-C6 alkyl, C1-C6 alkoxy, C1-C6 hydroxyalkyl, halogen, OH, N(R) a )(R b ), NO2, CN, C(=O)R a , C(=O)N(R a )(R b ), S(=O)2R a , S(=O)2N(R a )(R b ), and N(R a )C(=O)R b Selected independently from the group consisting of; L is selected from the group consisting of -CH2-, -C(=O)-, and -S(=O)2-; X is N and CR1c Selected from the group consisting of; n is an integer selected from the group consisting of 0, 1, 2, 3, and 4; and R a , R b , and R c Each occurrence is independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, and C2-C8 heterocyclyl. Compounds of formula (I), or their salts, solvates, prodrugs, stereoisomers, tautomers, or isotopologs; (ii) 1-benzylindoline-2,3-dione, 2-((4-oxo-3-(3-(pyrrolidine-1-yl)propyl)-3,4-dihydrobenzo[4,5]thieno[3,2-d]pyrimidine-2-yl)thio)ethyl acetate, 2-((2-(sec-butyl)-3-oxo-2,3-dihydroimidazo[1,2-c]quinazoline-5-yl)thio)-N-(o-tolyl)butanamide, and 8-((4-(cyclopropanecarbonyl)piperazine-1-yl)methyl)-7-isopentyl-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione, or its salts, solvates, prodrugs, stereoisomers, tautomers, or isotopologs. A compound selected from the group consisting of and A pharmaceutical composition selected from the group consisting of the following. Embodiment 34 provides the following: The pharmaceutical composition according to embodiment 33, wherein the RALDH1 inhibitor is a compound of formula (I). Embodiment 35 provides the following: The compound of formula (I) is 8-(6-methoxy-3-((4-methoxyphenyl)sulfonyl)quinoline-4-yl)-1,4-dioxa-8-azaspiro[4.5]decane; 1-(3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)-6-fluoroquinoline-4-yl)-4-phenylpiperidine-4-carbonitrile; (4-(cyclopropanecarbonyl)piperazine-1-yl)(4-(4,4-dimethylcyclohexa-1-en-1-yl)-6-fluoroquinoline-3-yl)methanone; (4-(4-(tert-butyl)phenyl)-6-fluoroquinoline-3-yl)(4-(cyclopropanecarbonyl)piperazine-1-yl)methanone; 1-(4-(3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)-6-fluoroquinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonilicate; (4-(4,4-dimethylcyclohexa-1-en-1-yl)-6-fluoroquinoline-3-yl)(4-(methylsulfonyl)piperazine-1-yl)methanone; (4-(4-(tert-butyl)phenyl)-6-fluoroquinoline-3-yl)(4-(methylsulfonyl)piperazine-1-yl)methanone; 1-(4-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoro-N-((1r,4r)-4-hydroxycyclohexyl)quinoline-3-carboxamide; (6-Fluoro-4-(4-(vinylsulfonyl)piperazine-1-yl)quinoline-3-yl)(4-(methylsulfonyl)piperazine-1-yl)methanone; 1-(4-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclobutan-1-carbonitrile; 1-(4-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopentan-1-carbonitrile; 4-(4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-sulfonamide; 4-(4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-carboxamide; 1-(6-chloro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonilicate; 1-(6-chloro-3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonitrile; 1-(6-(4-(methylsulfonyl)piperazine-1-carbonyl)thieno[3,2-b]pyridine-7-yl)-4-phenylpiperidine-4-carbonitrile; 1-(6-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)thieno[3,2-b]pyridine-7-yl)-4-phenylpiperidine-4-carbonitrile; 1-(6-methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonilicate; 1-(7-methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonilicate; 1-(6,7-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonilicate; 1-(6,8-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonilicate; 4-(4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-sulfonamide; 4-(4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-carboxamide; 4-(6-chloro-4-(4-(1-cyanocyclopropyl)phenyl)quinoline-3-carbonyl)-N,N-dimethylpiperazine-1-sulfonamide; 1-(4-(6-chloro-3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6-(4-(methylsulfonyl)piperazine-1-carbonyl)thieno[3,2-b]pyridine-7-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)thieno[3,2-b]pyridine-7-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6-methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(7-Methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6,7-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6,8-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoro-N-((1r,4r)-4-hydroxycyclohexyl)quinoline-3-carboxamide; 4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoro-N-(1-(2-hydroxyethyl)-1H-pyrazole-4-yl)quinoline-3-carboxamide; and 4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoro-N-(1-(2-hydroxyethyl)-1H-pyrazole-4-yl)quinoline-3-carboxamide A pharmaceutical composition according to embodiment 34, selected from the group consisting of the following. Embodiment 36 provides the following: A pharmaceutical composition according to any one of embodiments 33 to 35, wherein the immunostimulant is at least one selected from the group consisting of immune checkpoint inhibitors, chimeric antigen receptor (CAR) T cells, T cells genetically engineered to express specific TCRs targeting tumor antigens (TCR transgenic), ex vivo expanding T cells, and bispecific T cell engagers (BiTEs). Embodiment 37 provides the following: The pharmaceutical composition according to embodiment 36, wherein the immunostimulant is an immune checkpoint inhibitor. Embodiment 38 provides the following: The pharmaceutical composition according to embodiment 37, wherein the immune checkpoint inhibitor is selected from the group consisting of anti-PD1 antibody, anti-PD-L1 antibody, anti-CTLA4 antibody, any fragment thereof, and any combination thereof. Embodiment 39 provides the following: A pharmaceutical composition according to any one of embodiments 33 to 35, wherein the immunostimulant is CAR T cells. Embodiment 40 provides the following: A pharmaceutical composition according to any one of embodiments 33 to 39, wherein the at least one immunostimulant comprises an immune checkpoint inhibitor and CAR T cells. Embodiment 41 provides the following: A pharmaceutical composition according to any one of embodiments 33 to 40, further comprising at least one selected from the group consisting of retinoic acid receptor (RAR) inhibitors and retinoid X receptor (RXR) inhibitors. Embodiment 42 provides the following: A pharmaceutical composition according to embodiment 41, wherein the RAR inhibitor is selected from the group consisting of AGN 193109, BMS 195614, BMS 493, CD 2665, ER 50891, LE 135, LY 2955303, MM 11253, any salt or solvate thereof, and any combination thereof. Embodiment 43 provides the following: A pharmaceutical composition according to embodiment 41 or 42, wherein the RXR inhibitor is selected from the group consisting of HX 531, PA 452, and UVI 3003, any salt or solvate thereof, and any combination thereof. Embodiment 44 provides the following: A pharmaceutical composition according to any one of embodiments 33 to 43, wherein the pharmaceutically acceptable carrier is suitable for intravenous administration.

[0302] Each and all disclosures of patents, patent applications and publications cited herein are incorporated herein by reference in their entirety. While the present invention has been disclosed with reference to specific embodiments, it will be apparent that other embodiments and variations of the present invention can be devised by those skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed as encompassing all such embodiments and equivalent variations.

Claims

1. A method for treating, preventing, and / or improving solid tumors in a subject in need thereof, wherein the method comprises a pharmaceutically effective amount of (a) at least one immunostimulant, and (b) Retinaldehyde dehydrogenase 1 (RALDH1) inhibitors The process includes administering the RALDH1 inhibitor to the subject, wherein the RALDH1 inhibitor is as follows: (i) Equation (I): A compound of, or a salt thereof, solvate, prodrug, stereoisomer, tautomer, or isotopolog thereof, During the ceremony, R 1a C may be substituted. 2 ~C 8 Heterocyclyl, optionally substituted phenyl, and optionally substituted C 5 ~C 8 Selected from the group consisting of cycloalkenyls, Here, R 1a each of any substituents in 1 is independently selected from the group consisting of C 6 -C 3 alkyl, C 6 -C 1 cycloalkyl, C 6 -C 2 alkoxy, optionally substituted phenyl, optionally substituted C 8 -C a heterocyclyl, halogen, OH, N(R b )(R 2 ), NO a , CN, C(=O)R a (R b ), S(=O) 2 R a , S(=O) 2 N(R a (R b ), and N(R a )C(=O)R b ; Here, each arbitrary substituent is C 1 ~C 3 Alkyl, C 1 ~C 3 Alkoxy, halogens, CN, and NO 2 It may be substituted with at least one substituent selected from the group consisting of and Here, R 1a Any two vicinal or geminal substituents in a molecule combine with the atom they are bonded to to form a C molecule. 2 ~C 8 Heterocyclyl or C 3 ~C 8 A cycloalkyl group may also be formed; R 1b and R 1c If present, H, C 1 ~C 6 Alkyl, C 1 ~C 6 Alkoxy, halogen, OH, N(R) a )(R b ), NO 2 Each is independently selected from the group consisting of , and CN; R 2 Each occurrence is C 1 ~C 6 Alkyl, C 1 ~C 6 Alkoxy, C 1 ~C 3 Haloalkoxy, C 1 ~C 6 Hydroxyalkyl, halogen, NO 2 Independently selected from the group consisting of , and CN; R 3 C may be substituted. 2 ~C 8 Heterocyclyl, optionally substituted phenyl, N(R a )(C may be replaced) 3 ~C 8 Cycloalkyl), and N(R a )(C may be replaced) 2 ~C 8 Selected from a group consisting of heterocyclines, Here, R 3 Each substituent in the middle is C 1 ~C 6 Alkyl, C 1 ~C 6 Alkoxy, C 1 ~C 6 Hydroxyalkyl, halogen, OH, N(R a )(R b ), NO 2 , CN, C(=O)R a , C(=O)N(R a )(R b ), S(=O) 2 R a , S(=O) 2 N(R a )(R b ), and N(R a )C(=O)R b Selected independently from the group consisting of; A may be replaced by C 6 ~C 10 Aryl and possibly substituted C 2 ~C 8 Selected from a group consisting of heterocyclines, Here, each optional substituent in A is C 1 to C 6 alkyl, C 1 to C 6 alkoxy, C 1 to C 6 hydroxyalkyl, halogen, OH, N(R a )(R b ), NO 2 , CN, C(=O)R a , C(=O)N(R a )(R b ), S(=O) 2 R a , S(=O) 2 N(R a )(R b ), and N(R a )C(=O)R b and is independently selected from the group consisting of; L is -CH 2 -, -C(=O)-, and -S(=O) 2 - Selected from the group consisting of; X is N and CR 1c Selected from the group consisting of; n is an integer selected from the group consisting of 0, 1, 2, 3, and 4; and R a , R b , and R c Each occurrence is H, C 1 ~C 6 Alkyl, C 2 ~C 6 Alkenil, C 2 ~C 6 Alkinyl, C 3 ~C 8 Cycloalkyl, and C 2 ~C 8 Independently selected from the group consisting of heterocyclines, Compounds of formula (I), or their salts, solvates, prodrugs, stereoisomers, tautomers, or isotopologs; (ii) 1-benzylindoline-2,3-dione, 2-((4-oxo-3-(3-(pyrrolidine-1-yl)propyl)-3,4-dihydrobenzo[4,5]thieno[3,2-d]pyrimidine-2-yl)thio)ethyl acetate, 2-((2-(sec-butyl)-3-oxo-2,3-dihydroimidazo[1,2-c]quinazoline-5-yl)thio)-N-(o-tolyl)butanamide, and 8-((4-(cyclopropanecarbonyl)piperazine-1-yl)methyl)-7-isopentyl-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione, or its salts, solvates, prodrugs, stereoisomers, tautomers, or isotopologs. A compound selected from the group consisting of and The method selected from the group consisting of the above.

2. The method according to claim 1, wherein the RALDH1 inhibitor is a compound of formula (I).

3. The compound of formula (I) is Selected from the group consisting of, R 2a , R 2b , R 2c , and R 2d However, if present, H, C 1 ~C 6 Alkyl, C 1 ~C 6 A compound independently selected from the group consisting of alkoxys and halogens, The method according to claim 2.

4. R 2a , R 2b , R 2c , and R 2d The method according to claim 3, wherein, if present, each is independently selected from the group consisting of H, Me, OMe, F, and Cl.

5. The method according to any one of claims 2 to 4, wherein X is N.

6. R 1a but, Selected from the group consisting of, Here, R 5a and R 5b If it exists, C 1 ~C 6 Alkyl, C 1 ~C 6 Alkoxy, C 3 ~C 6 Each is independently selected from the group consisting of cycloalkyl, phenyl, thiophen-2-yl, thiophen-3-yl, and CN. Here, R 5a and R 5b Each substituent in the compound may be substituted with CN, Here, R 5a and R 5b If present, they combine with the atoms they are bonded to to form C 2 ~C 6 Heterocyclyl or C 3 ~C 6 A cycloalkyl group may be formed; and R 6 is S(=O) 2 R a That is, The method according to any one of claims 2 to 5.

7. R 5a and R 5b The method according to claim 6, wherein each of the following is independently selected from the group consisting of Me, t-Bu, 1-cyanocyclopropyl, 1-cyanocyclobutyl, 1-cyanocyclopentyl, 1-cyanocyclohexyl, phenyl, thiophen-2-yl, thiophen-3-yl, and CN.

8. R 6 The method according to claim 6 or 7, wherein is ethenylsulfonyl.

9. R 1a but, A method according to any one of claims 2 to 8, selected from the group consisting of the following.

10. R 1b The method according to any one of claims 2 to 9, wherein is H.

11. R 3 but, Selected from the group consisting of, R 7 C(=O)R a and S(=O) 2 R a Selected from the group consisting of, R 8 C 1 ~C 6 Alkoxy, C 1 ~C 6 Selected from the group consisting of hydroxyalkyl and OH, The method according to any one of claims 2 to 10.

12. R 7 The method according to claim 11, wherein the substance is selected from the group consisting of cyclopropylcarbonyl, methylsulfonyl, dimethylaminosulfonyl, and dimethylaminocarbonyl.

13. R 8 The method according to claim 11 or 12, wherein the group is selected from methoxy, 2-hydroxyethyl, and OH.

14. R 3 but, A method according to any one of claims 2 to 10, selected from the group consisting of the following.

15. The compound of formula (I) is 8-(6-methoxy-3-((4-methoxyphenyl)sulfonyl)quinoline-4-yl)-1,4-dioxa-8-azaspiro[4.5]decane; 1-(3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)-6-fluoroquinoline-4-yl)-4-phenylpiperidine-4-carbonitrile; (4-(cyclopropanecarbonyl)piperazine-1-yl)(4-(4,4-dimethylcyclohexa-1-en-1-yl)-6-fluoroquinoline-3-yl)methanone; (4-(4-(tert-butyl)phenyl)-6-fluoroquinoline-3-yl)(4-(cyclopropanecarbonyl)piperazine-1-yl)methanone; 1-(4-(3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)-6-fluoroquinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonil; (4-(4,4-dimethylcyclohexa-1-en-1-yl)-6-fluoroquinoline-3-yl)(4-(methylsulfonyl)piperazine-1-yl)methanone; (4-(4-(tert-butyl)phenyl)-6-fluoroquinoline-3-yl)(4-(methylsulfonyl)piperazine-1-yl)methanone; 1-(4-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoro-N-((1r,4r)-4-hydroxycyclohexyl)quinoline-3-carboxamide; (6-Fluoro-4-(4-(vinylsulfonyl)piperazine-1-yl)quinoline-3-yl)(4-(methylsulfonyl)piperazine-1-yl)methanone; 1-(4-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclobutan-1-carbonitrile; 1-(4-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopentan-1-carbonitrile; 4-(4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-sulfonamide; 4-(4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-carboxamide; 1-(6-chloro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonil; 1-(6-chloro-3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonitrile; 1-(6-(4-(methylsulfonyl)piperazine-1-carbonyl)thieno[3,2-b]pyridine-7-yl)-4-phenylpiperidine-4-carbonitrile; 1-(6-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)thieno[3,2-b]pyridine-7-yl)-4-phenylpiperidine-4-carbonitrile; 1-(6-methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonil; 1-(7-methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonil; 1-(6,7-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonilicate; 1-(6,8-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonil; 4-(4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-sulfonamide; 4-(4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-carboxamide; 4-(6-chloro-4-(4-(1-cyanocyclopropyl)phenyl)quinoline-3-carbonyl)-N,N-dimethylpiperazine-1-sulfonamide; 1-(4-(6-chloro-3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6-(4-(methylsulfonyl)piperazine-1-carbonyl)thieno[3,2-b]pyridine-7-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)thieno[3,2-b]pyridine-7-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6-methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(7-Methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6,7-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6,8-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoro-N-((1r,4r)-4-hydroxycyclohexyl)quinoline-3-carboxamide; 4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoro-N-(1-(2-hydroxyethyl)-1H-pyrazole-4-yl)quinoline-3-carboxamide; and 4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoro-N-(1-(2-hydroxyethyl)-1H-pyrazole-4-yl)quinoline-3-carboxamide A method according to any one of claims 2 to 14, selected from the group consisting of the following.

16. The method according to any one of claims 1 to 15, wherein the solid tumor is a carcinoma.

17. The method according to claim 16, wherein the carcinoma comprises human hepatocellular carcinoma (HCC) cells.

18. The method according to any one of claims 1 to 17, wherein RALDH1 is overexpressed in the solid tumor.

19. The method according to claim 18, wherein the expression of Raldh1 and Raldh2 or Raldh1 and Raldh3 in the solid tumor has a ratio selected from the group consisting of approximately 1000:1, 500:1, 250:1, 100:1, 50:1, 25:1, 10:1, and 5:

1.

20. The method according to any one of claims 1 to 19, wherein the immunostimulant is at least one selected from the group consisting of immune checkpoint inhibitors, chimeric antigen receptor (CAR) T cells, T cells genetically engineered to express specific TCRs targeting tumor antigens (TCR transgenic), ex vivo expanding T cells, and bispecific T cell engagers (BiTEs).

21. The method according to claim 20, wherein the immunostimulant is an immune checkpoint inhibitor.

22. The method according to claim 21, wherein the immune checkpoint inhibitor is selected from the group consisting of an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, any fragment thereof, and any combination thereof.

23. The method according to claim 20, wherein the immunostimulant is CAR T cells.

24. The method according to claim 23, wherein the CAR T cells are administered intravenously.

25. The method according to claim 23 or 24, wherein the CAR T cells are administered as CAR T cell therapy.

26. The method according to any one of claims 1 to 25, wherein the subject is administered an immune checkpoint inhibitor and chimeric antigen receptor (CAR) T cells.

27. The method according to any one of claims 1 to 26, further comprising the step of administering to the subject at least one selected from the group consisting of retinoic acid receptor (RAR) inhibitors and retinoid X receptor (RXR) inhibitors.

28. The method according to claim 27, wherein the RAR inhibitor is selected from the group consisting of AGN 193109, BMS 195614, BMS 493, CD 2665, ER 50891, LE 135, LY 2955303, MM 11253, any salt or solvate thereof, and any combination thereof.

29. The method according to claim 27 or 28, wherein the RXR inhibitor is selected from the group consisting of HX 531, PA 452, and UVI 3003, any salt or solvate thereof, and any combination thereof.

30. The method according to any one of claims 1 to 29, wherein the RALDH1 inhibitor and the immunostimulant are administered to the subject simultaneously or sequentially.

31. The method according to any one of claims 1 to 30, wherein the subject is a mammal.

32. The method according to claim 31, wherein the mammal is a human.

33. (a) at least one immunostimulant; (b) pharmaceutically acceptable carriers; and (c) Retinaldehyde dehydrogenase 1 (RALDH1) inhibitors A pharmaceutical composition comprising, wherein the RALDH1 inhibitor is as follows: (i) Equation (I): A compound of, or a salt thereof, solvate, prodrug, stereoisomer, tautomer, or isotopolog thereof, During the ceremony, R 1a C may be substituted. 2 ~C 8 Heterocyclyl, optionally substituted phenyl, and optionally substituted C 5 ~C 8 Selected from the group consisting of cycloalkenyls, Here, R 1a Each substituent in the middle is C 1 ~C 6 Alkyl, C 3 ~C 6 Cycloalkyl, C 1 ~C 6 Alkoxy, optionally substituted phenyl, optionally substituted C 2 ~C 8 Heterocyclyl, halogen, OH, N(R) a )(R b ), NO 2 , CN, C(=O)R a , C(=O)N(R a )(R b ), S(=O) 2 R a , S(=O) 2 N(R a )(R b ), and N(R a )C(=O)R b Independently selected from the group consisting of, Here, each arbitrary substituent is C 1 ~C 3 Alkyl, C 1 ~C 3 Alkoxy, halogens, CN, and NO 2 It may be substituted with at least one substituent selected from the group consisting of and Here, R 1a Any two vicinal or geminal substituents in a molecule combine with the atom they are bonded to to form a C molecule. 2 ~C 8 Heterocyclyl or C 3 ~C 8 A cycloalkyl group may also be formed; R 1b and R 1c If present, H, C 1 ~C 6 Alkyl, C 1 ~C 6 Alkoxy, halogen, OH, N(R) a )(R b ), NO 2 Each is independently selected from the group consisting of , and CN; R 2 Each occurrence is C 1 ~C 6 Alkyl, C 1 ~C 6 Alkoxy, C 1 ~C 3 Haloalkoxy, C 1 ~C 6 Hydroxyalkyl, halogen, NO 2 Independently selected from the group consisting of , and CN; R 3 C may be substituted. 2 ~C 8 Heterocyclyl, optionally substituted phenyl, N(R a )(C may be replaced) 3 ~C 8 Cycloalkyl), and N(R a )(C may be replaced) 2 ~C 8 Selected from a group consisting of heterocyclines, Here, R 3 Each substituent in the middle is C 1 ~C 6 Alkyl, C 1 ~C 6 Alkoxy, C 1 ~C 6 Hydroxyalkyl, halogen, OH, N(R a )(R b ), NO 2 , CN, C(=O)R a , C(=O)N(R a )(R b ), S(=O) 2 R a , S(=O) 2 N(R a )(R b ), and N(R a )C(=O)R b Selected independently from the group consisting of; A may be replaced by C 6 ~C 10 Aryl and possibly substituted C 2 ~C 8 Selected from a group consisting of heterocyclines, Here, each substituent in A is C 1 ~C 6 Alkyl, C 1 ~C 6 Alkoxy, C 1 ~C 6 Hydroxyalkyl, halogen, OH, N(R a )(R b ), NO 2 , CN, C(=O)R a , C(=O)N(R a )(R b ), S(=O) 2 R a , S(=O) 2 N(R a )(R b ), and N(R a )C(=O)R b Selected independently from the group consisting of; L is -CH 2 -, -C(=O)-, and -S(=O) 2 - Selected from the group consisting of; X is N and CR 1c Selected from the group consisting of; n is an integer selected from the group consisting of 0, 1, 2, 3, and 4; and R a , R b , and R c Each occurrence is H, C 1 ~C 6 Alkyl, C 2 ~C 6 Alkenil, C 2 ~C 6 Alkinyl, C 3 ~C 8 Cycloalkyl, and C 2 ~C 8 Independently selected from the group consisting of heterocyclines, Compounds of formula (I), or their salts, solvates, prodrugs, stereoisomers, tautomers, or isotopologs; (ii) 1-benzylindoline-2,3-dione, 2-((4-oxo-3-(3-(pyrrolidine-1-yl)propyl)-3,4-dihydrobenzo[4,5]thieno[3,2-d]pyrimidine-2-yl)thio)ethyl acetate, 2-((2-(sec-butyl)-3-oxo-2,3-dihydroimidazo[1,2-c]quinazoline-5-yl)thio)-N-(o-tolyl)butanamide, and 8-((4-(cyclopropanecarbonyl)piperazine-1-yl)methyl)-7-isopentyl-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione, or its salts, solvates, prodrugs, stereoisomers, tautomers, or isotopologs. A compound selected from the group consisting of and A pharmaceutical composition selected from the group consisting of the following.

34. The pharmaceutical composition according to claim 33, wherein the RALDH1 inhibitor is a compound of formula (I).

35. The compound of formula (I) is 8-(6-methoxy-3-((4-methoxyphenyl)sulfonyl)quinoline-4-yl)-1,4-dioxa-8-azaspiro[4.5]decane; 1-(3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)-6-fluoroquinoline-4-yl)-4-phenylpiperidine-4-carbonitrile; (4-(cyclopropanecarbonyl)piperazine-1-yl)(4-(4,4-dimethylcyclohexa-1-en-1-yl)-6-fluoroquinoline-3-yl)methanone; (4-(4-(tert-butyl)phenyl)-6-fluoroquinoline-3-yl)(4-(cyclopropanecarbonyl)piperazine-1-yl)methanone; 1-(4-(3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)-6-fluoroquinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonil; (4-(4,4-dimethylcyclohexa-1-en-1-yl)-6-fluoroquinoline-3-yl)(4-(methylsulfonyl)piperazine-1-yl)methanone; (4-(4-(tert-butyl)phenyl)-6-fluoroquinoline-3-yl)(4-(methylsulfonyl)piperazine-1-yl)methanone; 1-(4-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoro-N-((1r,4r)-4-hydroxycyclohexyl)quinoline-3-carboxamide; (6-Fluoro-4-(4-(vinylsulfonyl)piperazine-1-yl)quinoline-3-yl)(4-(methylsulfonyl)piperazine-1-yl)methanone; 1-(4-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclobutan-1-carbonitrile; 1-(4-(6-fluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopentan-1-carbonitrile; 4-(4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-sulfonamide; 4-(4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-carboxamide; 1-(6-chloro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonil; 1-(6-chloro-3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonitrile; 1-(6-(4-(methylsulfonyl)piperazine-1-carbonyl)thieno[3,2-b]pyridine-7-yl)-4-phenylpiperidine-4-carbonitrile; 1-(6-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)thieno[3,2-b]pyridine-7-yl)-4-phenylpiperidine-4-carbonitrile; 1-(6-methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonil; 1-(7-methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonil; 1-(6,7-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonilicate; 1-(6,8-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)-4-phenylpiperidine-4-carbonil; 4-(4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-sulfonamide; 4-(4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoroquinoline-3-carbonyl)-N,N-dimethylpiperazine-1-carboxamide; 4-(6-chloro-4-(4-(1-cyanocyclopropyl)phenyl)quinoline-3-carbonyl)-N,N-dimethylpiperazine-1-sulfonamide; 1-(4-(6-chloro-3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6-(4-(methylsulfonyl)piperazine-1-carbonyl)thieno[3,2-b]pyridine-7-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)thieno[3,2-b]pyridine-7-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6-methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(7-Methoxy-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6,7-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 1-(4-(6,8-difluoro-3-(4-(methylsulfonyl)piperazine-1-carbonyl)quinoline-4-yl)phenyl)cyclopropane-1-carbonitrile; 4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoro-N-((1r,4r)-4-hydroxycyclohexyl)quinoline-3-carboxamide; 4-(4-(1-cyanocyclopropyl)phenyl)-6-fluoro-N-(1-(2-hydroxyethyl)-1H-pyrazole-4-yl)quinoline-3-carboxamide; and 4-(4-cyano-4-phenylpiperidine-1-yl)-6-fluoro-N-(1-(2-hydroxyethyl)-1H-pyrazole-4-yl)quinoline-3-carboxamide A pharmaceutical composition according to claim 34, selected from the group consisting of the following.

36. The pharmaceutical composition according to any one of claims 33 to 35, wherein the immunostimulant is at least one selected from the group consisting of immune checkpoint inhibitors, chimeric antigen receptor (CAR) T cells, T cells genetically engineered to express a specific TCR targeting a tumor antigen (TCR transgenic), ex vivo expanding T cells, and bispecific T cell engagers (BiTEs).

37. The pharmaceutical composition according to claim 36, wherein the immunostimulant is an immune checkpoint inhibitor.

38. The pharmaceutical composition according to claim 37, wherein the immune checkpoint inhibitor is selected from the group consisting of an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, any fragment thereof, and any combination thereof.

39. The pharmaceutical composition according to any one of claims 33 to 35, wherein the immunostimulant is CAR T cells.

40. The pharmaceutical composition according to any one of claims 33 to 39, wherein the at least one immunostimulant comprises an immune checkpoint inhibitor and CAR T cells.

41. The pharmaceutical composition according to any one of claims 33 to 40, further comprising at least one selected from the group consisting of retinoic acid receptor (RAR) inhibitors and retinoid X receptor (RXR) inhibitors.

42. The pharmaceutical composition according to claim 41, wherein the RAR inhibitor is selected from the group consisting of AGN 193109, BMS 195614, BMS 493, CD 2665, ER 50891, LE 135, LY 2955303, MM 11253, any salt or solvate thereof, and any combination thereof.

43. The pharmaceutical composition according to claim 41 or 42, wherein the RXR inhibitor is selected from the group consisting of HX 531, PA 452, and UVI 3003, any salt or solvate thereof, and any combination thereof.

44. The pharmaceutical composition according to any one of claims 33 to 43, wherein the pharmaceutically acceptable carrier is suitable for intravenous administration.

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