Compounds targeting PBX1 transcriptional complex and their applications

Abstract

The present disclosure provides novel compounds of formula (A) or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomer thereof. The compounds may be used as inhibitors of PBX1-DNA interaction and for treatment of disease associated with inhibition of PBX1-DNA interaction.

Description

T54267 / PC0704COMPOUNDS TARGETING PBX1 TRANSCRIPTIONAL COMPLEX AND THEIR APPLICATIONSCross-Reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional .Application No. 63 / 691 ,452, filed on 6 September 2024, the entire contents of which are incorporated herein by reference.Field of the Disclosure

[0002] I he present disclosure relates to small-molecule PBX1 inhibitors. Particularly, the present disclosure provides novel compounds used as inhibitors of PBX1-DN A interaction and for treatment of disease associated with inhibition of PBX1-DNA interaction.Description of the Related Art

[0003] I 'he targeting of oncogenic transcription factors (TFs) has long been challenging due to the limited binding sites available for small molecule inhibitors, leading to their classification as "undruggable targets." Efforts have been made to produce pharmaceutical inhibitors that specifically target oncogenic TFs like MYC, [3-Catenm. HIF, and PBX1 .

[0004] These TFs play a crucial role in regulating the expression of several genes that facilitate tumor formation. The roles of PBX1 transcription in cancer, such as breast cancer, ovarian cancer, prostate cancer, non-small cell lung adenocarcinoma, melanoma, and pre-B-cell acute lymphoblastoid leukemia were documented.

[0005] However, despite these endeavors, no inhibitors with both effectiveness and selectivity have been successfully translated into clinical applications. Therefore, there is still a need for small molecules which directly interfere with PBXJ -DNA interaction, and thereby inhibit cancer growth.SUMMARY

[0006] The present disclosure provides a compound having formula (A)or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomer thereof, whereinXrxY is selected from the group consisting of -CH2-CH2-, -CH=CH-, -O-CH2-, -NH-CH2-,Ri is independently selected from the group consisting of H, halogen, nitro, cyano, Ci-Ck alkyl, C2-C& alkenyl, C2-C6 alkynyl, and Ci-Ce alkoxyl, wherein Ci-Ct, alkyl, C2-C6 alkenyl, C2-C6 alkynyl, or Ci-Cs alkoxyl is independently unsubstituted or substituted with cyano, nitro, or halogen;R2 is independently selected from the group consisting of H, halogen, nitro, cyano. C1-C.5 alkyl, Ce-Ce alkenyl. C2-C6 alkynyl. and Ci-Ce alkoxyl, wherein Ci-Cs alkyl. C2-C0 alkenyl. C2-C6 alkynyl, or Ci-Ce alkoxyl is independently unsubstituted or substituted with cyano, nitro, or halogen;Rs is independently selected from the group consisting of H, halogen, nitro, cyano, Ci-Cs alkyl, hydroxyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C.5 alkoxyl, carboxylic acid, ester, carbonate, phosphate, sulfonate, amino, amide, imine, and carbamate, wherein Ci-Ce alkyl, C2-C0 alkenyl, Ci-Cs alkynyl , or C i-C& alkoxyl is independently unsubstituted or substituted with cyano, nitro, or halogen, b is independently 0. 1, 2. 3 or 4; c is independently 0. 1, 2, 3, 4 or 5; and d is independently 0, 1 , 2, 3 or 4.

[0007] In some embodiments, the compound of the present disclosure has formula (B):wherein X, Y, Ri, R3, b and d are defined as above, or a pharmaceutically acceptable salt, solvate. prodrug or stereoisomer thereof

[0008] In some embodiments, the compound of the present disclosure has formula (C):wherein X, ¥, Rj, R2, c and d are defined as above, or a pharmaceutically acceptable salt, solvate. prodrug or stereoisomer thereof

[0009] In some embodiments, the compound of the present disclosure has formula (D):wherein Ri and d are defined as above, or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomer thereof.[00W| In some embodiments, the compound of the present disclosure is selected from the group consisting of:and 94 , or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomer thereof.

[0011] The present disclosure also provides a pharmaceutical composition comprising the compound of the present disclosure or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomer thereof, and a pharmaceutically acceptable excipient In one embodiment, the pharmaceutical composition further comprises an additional anticancer drag.

[0012] The present disclosure also provides a nanocarrier comprising the compound of the present disclosure or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomer thereof. In some embodiments, the nanocarrier is a liposome or a micelle. In one embodiment, the nanocarrier further comprises an additional anticancer drug.

[0013] The present disclosure also provides a method for inhibiting PBXl -DNA interaction in a cell, comprising contacting the cell with the compound of the present disclosure.

[0014] The present disclosure further provides a method for treating a disease associated with inhibition of PBXl-DNA interaction in a subject, which comprises administering an effective amount of a compound of the present disclosure.

[0015] In some embodiments, the disease includes, but is not limited to, a cancer, cancer invasion, cancer metastasis and chemotherapy drug-resistant cancers (such as gemcitabine- resistant cancers). In some embodiments, the cancer is a multi -drug resistant cancer, recurrent cancer, refractory cancer or relapsed cancer.

[0016] In some embodiments, the cancer includes but is not limited to pancreatic cancer, esophageal cancer, glioma, ovarian cancer, multiple myeloma, colorectal cancer, lung cancer, liver cancer, breast cancer, gastric cancer, head and neck cancer, prostate cancer, testicular, intestinal cancer, bladder cancer, or urothelial cancer

[0017] In one embodiment, the method described herein further comprises an additional therapeutic agent In one embodiment, the additional therapeutic agent is an additional anticancer drug.

[0018] In one embodiment, the additional anticancer drug is a chemotherapy drug. In some embodiments, the chemotherapy drug is platinum compounds (such as cisplatin, carboplatin), antimetabolite drugs (such as 5 -fluorouracil, cytarabine, gemcitabine, pentostatin and methotrexate), anthracychne drug which targets DNA (such as doxorubicin and epirubicin), drugs which target DNA or drugs which target topoisomerases or other chemotherapy drugs. In a further embodiment, the additional anticancer drug is cisplatin, docetaxel, paclitaxel, gemcitabine, navelbine, doxorubicin, irinotecan, gemcitabine, regorafenib, olaparib, lenalidomide, sorafenib, tagrisso or 5-fluorodeoxyuridine.BRIEF DESCRIPTION OF THE DR.AWING

[0019] Figure 1 A shows a schematic diagram illustrating the drug screening pipeline.

[0020] Figure IB shows a display of chemical structures and molecular weights of candidate compounds.

[0021] Figures 1 C to IE show survival analysis conducted on A2780 cells treated with a series of concentrations of indicated drugs. Cell viability was assessed using the PrestoBlue CellViability assay and normalized to the viability of untreated ceils (C and D), with IC50 values calculated using GraphPad Prism software (E).

[0022] Figure IF shows a heatmap illustrating the differential expression profiles of canonical PBX1 downstream genes in A2780 cells 24 hours after treatment with 2.5 pM of the indicated compounds.

[0023] Figure 2A shows a heatmap illustrating the dynamic gene expression profiles of gemcitabine-resistant MIA PaCa-2 (MIA PaCa-2 CR) cell lines at various timepoints subsequent to gemcitabine treatment.

[0024] Figure 2B shows a quantitative PCR (qPCR) analysis revealing the expression levels ofPBX1. metastasis-related genes, sternness-related genes, and drug-resistant genes in MIA PaCa-2 CR cell lines at different time intervals post-gemcitabine treatment.

[0025] Figure 2C shows a qPCR analysis depicting the expression levels of PBXl, metastasis- related genes, sternness-related genes, and drug-resistant genes in MIA PaCa-2 parental (PT) cells, CR cells, CR cells with PBX1 knockdown, and CR cells treated with 2.5 pM of T417,A409, B004, or gemcitabine.[0026 J Figure 2D shows a heatmap illustrating the differential gene expression profiles of MIAPaCa-2 PT cells, CR cells, CR cells with PBX1 knockdown, and CR cells treated with 2.5 uM of indicated drugs.

[0027] Figure 2E shows representative immunofluorescent images depicting the expression and localization of Pbxl and Nanog proteins in MIA PaCa-2 CR cells treated with DMSO (control).2.5 uM gemcitabine. 2.5 pM B004, or 2.5 pM gemcitabine + 2.5 pM B004.

[0028] Figure 2F shows representative immunofluorescent images showing the expression and distribution of Zebl and N-cadherin proteins in MIA PaCa-2 CR cells treated with DMSO (control), 2.5 pM gemcitabine, 2.5 pM B004. or 2.5 pM gemcitabine + 2,5 uM B004,

[0029] Figure 2G shows a heatmap illustrating the altered gene expression profiles of MIA PaCa-2 CR cells upon treatment with the indicated drugs and doses.

[0030] Figure 2H shows a qPCR analysis delineating the expression levels of PBX1, metastasis - related genes, and sternness-related genes in MIA PaCa-2 CR cells treated with the indicated drugs and doses.

[0031] Figure 3A shows micrographs of wound healing assay with DMSO (ctrl), 0.625 pM gemcitabine, or 1.25 uM gemcitabine in MIA PaCa-2 CR and PANC-1 CR cells (left). Quantification of gap covered percentage in indicated groups with Image.) software (right).

[0032] Figure 3B shows crystal violet-stained invaded cells from MIA PaCa-2 CR and PANC- 1 CR cells treated with DMSO (Ctrl), 0.625 uM gemcitabine, or 1.25 pM gemcitabine (left). Bar charts demonstrate the relative invasion in each group (right).

[0033] Figure 3C shows tumor sphere formation assays conducted with MIA PaCa-2 CR and PANC-I CR cells treated with DMSO (ctrl), 0.625 pM gemcitabine, or 1.25 pM gemcitabine (left). Bar charts exhibit the number of tumor spheres and area of tumor spheres in each field (5 random fields / group). Tumor spheres are defined as tumor cell aggregations larger than 800am2(right).

[0034] Figures 3D and 3E show micrographs of wound healing assays conducted with DMSO(ctrl) or 2.5 iiM of indicated drugs in PANC-1 CR cells (D) and MIA PaCa-2 CR cells (E).

[0035] Figure 3F show's a quantification of gap covered percentage in indicated groups usingImage! software.

[0036] Figure 3 G-S 1 shows the differential expression profiles of canonical P BX1 downstream genes in A2780 cells 24 hours after treatment with 2.5 pM of the indicated compounds. Figures 3 G-Sl-B to G-Sl -D show representative micrographs of wound healing assays conducted with PT cells (ctrl), metastatic cancer stem cells (MCSC), and MCSC treated with 2.5 pM of T417, A409, or B004 in DLD- 1 (G-S 1 -B), MIA PaCa-2 (G-S 1 -C), and PC-9 (G-S 1 -D) cells. Figures 3 G-Sl-E to G-Sl -G show quantification of the gap covered percentage in the indicated groups of wound healing assays in DLD-1 cells (G-Sl-E), MIA PaCa-2 cells (G-Sl-F), and PC-9 cells (G-SI-G). Figure 3 G-Sl-H shows colony formation assays of AsPC-1 cells treated with 0, 2, or 4 pM of gemcitabine in the indicated groups (PT cells, CR cells, CR cells + 2 gM B004) (left), and quantification of colony counts per well in each group (right).

[0037] Figure 3G shows crystal violet-stained invaded cells from MIA PaCa-2 CR and PANC-I CR cells treated with DMSO (ctrl) or 2.5 pM of indicated drugs.

[0038] Figure 3H shows bar charts of the relative invasion in each group.

[0039] Figure 31 shows tumor sphere formation assays conducted with AsPC-1, MIA PaCa-2, and PANC-1 parental wild type (PT) or CR cells treated with DMSO (ctrl) or 2.5 pM of indicated drugs.

[0040] Figures 3J to 31, show bar charts showing the number of tumors and area of tumor spheres in each field in AsPC-1 (J), MIA PaCa-2 (K), and PANC-1 (L) cells. Each group is quantified for 5 random fields per experiment. Tumor spheres are defined as tumor cell aggregations larger than 800 pm2.

[0041] Figure 3M shows soft agar assays conducted with MIA PaCa-2 and PANC-1 parental wild type (PT) or CR cells treated with DMSO (ctrl) or 2.5 pM of indicated drugs.

[0042] Figures 3N and 30 show bar charts showing the number of tumors and area of tumor spheres formed in soft agar in each field in MIA PaCa-2 (N) and PANC-1 (O) cells. Each group is quantified for 5 random fields per experiment. Tumor spheres are defined as tumor cellaggregations larger than 800 gm2

[0043] Figure 4A presents survival analysis conducted on MIA PaCa-2 and PANC-l cells, which included parental (PT), chemo-resistant (CR), and CR cells with PBX1 knockdown. These cells were treated with serial concentrations of the specified drugs. Cell viability was assessed using the PrestoBlue Cell Viability7assay and normalized to the viability7of untreated cells. Non-linear regression trends were determined using GraphPad Prism software.

[0044] Figure 4B shows representative microscopic images of MIA PaCa-2 cell lines at indicated doses for each group. Scale bar = 100 pm.

[0045] Figure 4C shows a colony forming assay of PANC-l cells treated with 0, 2, or 4 pM of gemcitabine in the indicated groups (Left). Colony counts per well in each group (Right).

[0046] Figure 4D shows a survival analysis of MIA PaCa-2 CR and AsPC-1 CR cells treated with serial concentrations of indicated drugs. Cell viability was assessed using the PrestoBlueCell Viability assay and normalized to the viability of untreated cells.

[0047] Figure 4E show-s the IC50 (tiM) values of MIA PaCa-2 CR and AsPC-1 CR cells under treatment with T417, A409. B004, and gemcitabine. The IC50 values and non-linear regression of trends were calculated using GraphPad Prism software.

[0048] Figure 4F show s a 3D relapse model of MIA PaCa-2 CR and PANC-l CR cells treated with low doses of gemcitabine (50nM) and / or B004 (lOOnM). Left: Schematic diagram of the dosing schedule Middle: Changes in tumor volume from day 5 to day716. MIA PaCa-2 CR and PANC-l CR cells were cultured as 3D colonies in 96-well ultra-low7attachment plates, and photos of each well were captured every other day. Tumor volume was estimated using the formula: Tumor volume = (length x width2) / 2. Length represents the longest diameter, and width represents the perpendicular diameter of length. Right: Cell viability measured using a CellTiter-Glo 3D assay on day 16, and survival was normalized to the viability of cells in the control group.

[0049] Figure 4G shows a combination index (CI) plot of combined treatments of T417, A409, or B004 with chemotherapeutic agents in chemo-resistant cancer cell lines. The combination index was calculated using CompuSyn software.

[0050] Figures 4H to 4M show7that a combination index analysis w7as performed to assess the effectiveness of combining B004 with chemotherapeutic agents, each corresponding to the inherent resistance profile of CR cell lines at selected doses. CKO.l ; very7strong synergism;T54267 / PC0704CI=O. 1-0.3: strong synergism; CI=0.3-0.7: synergism; CI=0.7-0.85: moderate synergism; 0=0.85-0.9: slight synergism; CI=0.9-l . l: nearly additive, 0=1. 1-1.2: slight antagonism; 0=1.2-1.45: moderate antagonism; 0=1.45-3.3: antagonism.

[0051] Figures 5 A, 5F. 5M, 5R, and 5W show that dil -labeled chemo-resistant tumor cells were injected into the perivitelline space of 48-hour post-fertilization (2dpf) zebrafish embryos. The tumor-bearing zebrafish were individually cultured in E3 medium within a well of a 24-well plate and treated with indicated drugs, which were renewed daily. Tumor metastases were detected using fluorescent microscopy at day 6 post-injection, with white arrowheads indicating disseminated tumor foci.

[0052] Figures 5B, 51. 5N. 5S and 5X show quantification of the number of disseminated tumor foci at day 6 post-tumor implantation following the indicated treatments

[0053] Figures 5C. 5J, 50, 5T and 5Y show a measurement of maximal distances of metastatic foci at day 6 post-tumor implantation using ImageJ software after the indicated treatments.

[0054] Figures 5D, 5K, 5P, 5U and 5Z show a relative quantification of tumor load at day 6 post-tumor implan tation presented as the ratio of tumor size to total body area.

[0055] Figures 5E, 5L, 5Q, 5V and 5AA show Kaplan-Meier survival curves of tumor-bearing zebrafish embry os after the indicated treatments.

[0056] Figure 6A shows tumor growth curves of subcutaneous DLD-1 CR tumors in NOD-scid IL2rynull (NSG) mice following treatment with either control vehicle, regorafenib 5mg / kg / inj ection, T4I7 5mg / kg / injection, A409 5mg / kg / injeclion, B004 5mg / kg / injection, or regorafenib 5mg / kg / inj ection +- B004 5mg / kg / inj ection. Tumor size was measured twice a week with a caliper, and tumor volume was estimated using the formula: Tumor volume = (length x widthA2) / 2. { t s 7 tumors per group)

[0057] Figure 6B shows bar charts demonstrating the tumor volume of subcutaneous DLD-1 CR tumors at day 23 in NSG mice post-tumor implantation, (n = 7 tumors per group)

[0058] Figure 6C shows gross images of subcutaneous DLD-1 CR tumors from indicated groups.

[0059] Figures 6D and 6E show immunofluorescent staining of TUNEL and cleaved caspase 3 from subcutaneous DLD-1 CR tumor sections from indicated groups Representative images (D) and relative fluorescent intensity (E) are presented (n = 7 tumors analyzed per group).

[0060] Figures 6F and 6G show orthotopic chemo-resistant pancreatic cancer model in NSG mice. (F) Image of tumor growth and metastasis of AsPC-1 CR-Luc captured with IVIS® Lumina XRMS imaging system after receiving the indicated treatments. (G) Total flux of the respective regions of interest (ROI) (photons / sec) of each group over time, (n::::3 mice per group)

[0061] Figures 6H and 61 show representative IHC staining images I with cleaved caspase 3 of orthotopic chemo-resistant pancreatic tumor sections from indicated groups (n::::3 mice per group). Bar charts H represent the H score and apoptotic index of indicated groups (2 random fields analyzed per mouse).

[0062] Figure 6J show’s the hematological and biochemistry profiles of orthotopic chemoresistant pancreatic cancer model obtained at the experiment endpoint.

[0063] Figure 7A show’s a survival analysis of fibroblast cells treated with serial concentrations of indicated drugs. Cell viability was assessed using the PrestoBlue Cell Viability assay and normalized to the viability of untreated cells (Left), with IC50 values calculated using GraphPadPrism software (Right).

[0064] Figure 7B shows a survival analysis of 5 days post-fertilization (dpf) zebrafish after 24 hours of incubation with 0 to 512 pM of indicated drugs (n 10 / group). Percentages of survived (Left) and grossly unaffected (Right) zebrafish are presented

[0065] Figure 7C shows an indication and maximum tolerated dose of each drug in 5dpf zebrafish.

[0066] Figure 7D shows representative images demonstrating gross morphologies of zebrafish after 24 hours of incubation with indicated drugs and doses. White arrows indicate areas of edema.

[0067] Figure 7E shows gross images of major organs of NOD-scid IL2iynull (NSG) mice 3 weeks after receiving control vehicle or 500mg / kg of T417, A409, B004, regorafenib, gemcitabine, lenalidomide, or olaparib (Control: n=2 / group; T417, A409, and B004: n = 3 / group; regorafenib (Reg), gemcitabine (Gem), lenalidomide (Len), and olaparib (Ola): n = 1 / group).

[0068] Figure 7F shows a weight change of mice between baseline and 3 weeks after receiving the indicated treatments.T54267 / PC0704

[0069] Figures 7G and 7H show the biochemistry (G) and hematological CH) profiles of mice obtained 3 weeks after receiving the indicated treatments.

[0070] Figure 71 shows representative histologic sections of major organs of mice at 3 weeks after receiving control vehicle, A409 500 mg / kg, or B004 500 mg / kg.

[0071] Figure 7J shows representative histologic sections of the liver of mice at 3 weeks after receiving lenalidomide 500rng / kg, regorafenib 500mg / kg, or gemcitabine 500 mg / kg.DETAILED DESCRIPTION

[0072] The present disclosure is described with reference to the following embodiments.Aside from the following embodiments, the present disclosure may be performed according to another method without departing from the spirit of the present disclosure.

[0073] For ease of understanding of the disclosure of this specification, a plurality of terms are defined as follows. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present disclosure. Tire singular forms are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "include." "have" or "comprise" when used in this specification, specify the presence of stated features, numbers, steps, components, or combinations thereof, but do not preclude the presence or addition of one or more other features, numbers, steps, components, or combinations thereof.

[0074] All numerical values expressing the contents, proportions, and physical characteristics, etc. used in the specification and claims are to be construed as being modified by the term "about." Herein, the term “about” refers to an acceptable error of a specific value determined by those skilled in the art, depending in part on how the value is measured or determined.

[0075] The term "Ci-Ce alkyl" used herein includes straight chain and branched alkyl groups having from 1 to 6 carbon atoms or, in some embodiments, from 1 to 6, or 1 to 4 carbon atoms. Alkyl groups further include cycloalkyl groups Examples of straight chain alkyl groups include those with from 1 to 6 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl. groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, tert-butyl, neopentyl, isopentyl, and 2,2-dirnethylpropyl groups. Representative substituted alkyl groups may be substituted one or more times with substituents such as those listed above.[0076 j The terms "alkenyl" and "alkynyl" indicate an alkyl group that further includes a doubleT54267 / PC0704 bond or a triple bond, respectively.

[0077] The term "alkylene", as used herein, means a straight or branched chain divalent hydrocarbon group of formula -CnHja-. Non-limiting examples include ethylene, and propylene.

[0078] The terms "alkenylene" and "alkynylene" indicate an alkylene group that further includes a double bond or a triple bond, respectively.

[0079] The term "cycloalkyl" used herein refers to cyclic alkyl groups such as. but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl groups.

[0080] The term "halogen", as used herein, refers to fluoride, chloride, bromide, or iodide.

[0081] The term ' pharmaceutically acceptable salt" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids.

[0082] The compounds of the present disclosure, and pharmaceutically acceptable salts thereof, may exist in unsolvated and solvated forms. The term "solvate" is used herein to describe a molecular complex comprising the compound of formula (A), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term "hydrate" is employed when said solvent is water.

[0083] The term "subject" includes living organisms such as humans, monkeys, cows, sheep, horses, pigs, cattle, goats, dogs, cats, mice, rats, cultured cells, and transgenic species thereof. In a preferred embodiment, the subject is a human.

[0084] The term "administering" includes routes of administration which allow the active ingredients of the present disclosure to perform their intended function,

[0085] The term "treat" or "treatment" refers to a method of reducing the effects of a disease or condition. Treatment, can also refer to a method of reducing the underlying cause of the disease or condition itself rather than just the symptoms. The treatment can be any reduction from native levels and can be, but is not limited to, the complete ablation of the disease, condition, or the symptoms of the disease or condition.

[0086] The phrase "therapeutically effective amount" refers to that amount of a compound, material, or composition comprising a compound of the present disclosure which is effective for producing a desired therapeutic effect, at a reasonable benefit / nsk ratio applicable to anyT54267 / PC0704 medical treatment

[0087] It has been shown that the DNA binding domains of PBX1 are located in closer proximity to a specific region compared to the DNA binding domains of other transcription factors. Several small-molecule PBX1 inhibitors were synthesized by in silico prediction, and they have demonstrated the ability7to selectively and significantly disrupt the connection between PBX1 and downstream genes, therefore atenuating the PBX1 pathway. Consequently, the inhibition of the Notch3 / PBX1 sternness pathway by this small molecular inhibitor has the potential to overcome the resistance exhibited by cancer stem cells (CSCs).

[0088] Accordingly the present disclosure provides compounds as described herein and their derivatives that can inliibit the interaction between PBX1 and DNA, thereby providing a potential treatment for diseases associated with this inhibition.

[0089] The pharmaceutically acceptable salt of the present disclosure includes salts of basic compounds that refer to non-toxic salts of the compounds of this invention which are generally- prepared by reacting the free base with a suitable organic or inorganic acid. Representative salts of basic compounds of the present disclosure include, but are not limited to, the following: acetate, ascorbate, adipate, alginate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, clavulanate, citrate, cyclopentane propionate, diethylacetic, digluconate, dihydrochloride, dodecylsulfanate, edelate, edisylate, estolate, esylate, ethanesulfonate, formic, fumarate, gluceptate, glucoheptanoate, gluconate, glutamate, glycerophosphate, glycollylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, 2-hydroxyethanesulfonate, hydroxynaphthoate, iodide, isonicotinic, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate. methanesulfonate, mucate. 2- naphthalenesulfonate. napsylate, nicotinate, nitrate. N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate), palmitate, pantothenate. pectinate, persulfate, phosphate / diphosphate, pimehc, phenylpropionic, polygalacturonate, propionate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate, thiocyanate, tosylate, triethiodide, trifluoroacetate, undeconate, valerate and the like. Furthermore, where the compounds of the present disclosure carry' an acidic moiety, suitable pharmaceutically acceptable salts thereof include, but are not limited to, salts derived from inorganic bases including aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, mangamous, potassium, sodium, zinc, and the like Also included are the ammonium, calcium, magnesium, potassium, and sodium salts. Salts derived from pharmaceuticallyacceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, cyclic amines, dicyclohexyl amines and basic ion-exchange resins, such as arginine. betaine, caffeine, choline, N.N-dibenzylethylenediamine, diethylannne, 2-diethylaminoethanoL 2- dimethylaminoethanol, ethanolamine, ethylamine, ethyl enedi amine, N-ethylmorphoiine, N- ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, tn ethyl amine, trimethylamine, tripropylamine, tromethamine, and the like. Also, included are the basic nitrogen-containing groups that may be quatemized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides and others.[0090j Certain derivatives of a compound of the present disclosure which may have little or no pharmacological activity themselves can, when administered into or onto the body, be converted into a compound of the present disclosure having the desired activity, for example by hydrolytic cleavage, particularly hy drolytic cleavage promoted by an esterase or peptidase enzyme. Such derivatives are referred to as "prodrugs."

[0091] Prodrugs in accordance with the present disclosure can, for example, be produced by replacing appropriate functionalities present in the compounds of the present disclosure with certain moieties known to those skilled in the art as "pro-raoieties" as described. For example, a prodrug in accordance with the present disclosure is (a) an ester or amide derivative of a carboxylic acid in a compound of the present disclosure; (b) an ester, carbonate, carbamate, phosphate or ether derivative of a hydroxyl group in a compound of the present disclosure; (c) an amide, imine, carbamate or amine derivative of an amino group in a compound of the present disclosure; (d) an oxime or imine derivative of a carbonyl group in a compound of the present disclosure, or (e) a methyl, primary alcohol or aldehyde group that can be metabolically oxidized to a carboxylic acid in a compound of the present disclosure.

[0092] Compounds of the present disclosure containing one or more asymmetric carbon atoms can exist as two or more stereoisomers. Where a compound of the present disclosure contains an alkenyl or alkenylene group, geometric cis / trans (or Z / E) isomers are possible. Where structural isomers are interconvertible via a low energy' barrier, tautomeric isomerism ("tautomerism") can occur. This can take the form of proton tautomerism in compounds of the present disclosure containing, for example, an imino, keto, or oxime group, or so-called valence tautomerism in compounds which contain an aromatic moiety. It follows that a singlecompound may exhibit more than one type of isomerism.

[0093] The compounds of the present disclosure can be prepared according to general chemical synthetic procedures. The preparation of the embodiments of the compounds of the present disclosure is illustrated below.

[0094] Scheme 1Reagents and conditions: (a) 3-Fluorobenzoic acid. EDCI’HCl, HOBt, NMM, rt, overnight; (b) Methyl bromoacetate. K2CO3, ACN, reflux, 3h: (c) IN LiOH(aq), p- dioxane, 40°C, overnight;(d) 4-aminobenzene-l,2-diol, HBTU, DIPEA, 11, overnight.13: 2-C1 14: 2-C119: 2-F 20: 2-F25: 2-OMe 26: 2-OMe41: 3-OMe 42: 3-OMe Reagents and conditions: (a) Methyl bromoacetate, K.-COj. ACN, reflux. 3h; (b) Iron powder. NH4CI, IPA / HzO, reflux, Ih; (c) 3-Fluorobenzoic acid. EDC1HC1, HOBt, NMM, rt. overnight; (d) IN LiOH(aq), p- dioxane, 40°C, overnight: (e) 4-aminobenzene-l ,2-diol, HBTU, DIPEA, rt, overnight

[0097] Scheme 4Reagents and conditions: (a) Methyl (triphenyl phosphoranylidine) acetate. Toluene, reflux, 5h; (b) Iron powder, NH4CI, TPA / H2O, reflux, Ih, (c) 3-Fluorobenzoic acid, IIBTU, DIPEA, rt, overnight; (d) IN LiOH(aq), p- dioxane, 40°C. overnight; (e) 4-aminobenzene-l,2-diol, EDCIHCI, HOBt, NMM, rt, overnight.

[0098] Scheme 5eagents and conditions: (a) Methyl (triphenyl phosphoranylidine) acetate. Toluene, reflux, 5h; (b) IN LiOH(aq), p- dioxane, 40°C, overnight; (c) 3,4-bis((tert-butyldimethylsilyl)oxy)aniline, HBTU, DIPEA, it, overnight; (e) Iron powder, NH4CI, IPA / H2O, reflux, Ih; (f) 3-Fluorobenzoic acid.HBTU, D1PEA, rt, overnight; (g) TBAF, THE 0°C to it, Ih.

[0099] Scheme 6T54267 / PC0704Reagents and conditions: (a) Methyl (triphenyl phospho ranyli dine) acetate, Toluene, reflux, 5h; (b) Pd / C, H'2, MeOH, it, overnight, (c) 3 -Fluorobenzoic acid, EDCIHC1, HOBt, NMM, it, overnight; (d) IN LiOH(aq), p- dioxane, 40°C, overnight; (e) 4-aminobenzene-l,2.-diol, EDCI HC1, HOBL NMM, rt, overnight.

[0100] The present disclosure also provides applications or uses of the compounds described herein in inhibiting PBX1-DNA interaction in a cell, hereby treating or preventing a disease associated with inhibition of PBXl -DNA interaction in a subject.

[0101] The disease associated with PBX1 -DNA interaction may be a cancer, cancer invasion, cancer metastasis and chemotherapy drug-resistant cancer (such as a cancer resistant to cisplatin, docetaxel, paclitaxel, gemcitabine, navelbine, doxorubicin, irinotecan, gemcitabine, regorafenib, olaparib, lenalidomide, sorafenib, tagrisso or 5-fluorodeoxyuridine), multi-drug resistant cancer, recurrent cancer, refractory cancer or relapsed cancer. The cancer includes, but is not limited to. pancreatic cancer, esophageal cancer, glioma, ovarian cancer, multiple myeloma, colorectal cancer, lung cancer, liver cancer, breast cancer, gastric cancer, lung cancer, head and neck cancer, prostate cancer, testicular, intestinal cancer, bladder cancer, or urothelial cancer.

[0102] The compounds as described herein can be formulated as a pharmaceutical composition or pharmaceutical combination. The pharmaceutical composition, Combination comprises theT54267 / PC0704 aforesaid compound or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomer thereof, and a pharmaceutically acceptable excipient.

[0103] The term "pharmaceutically acceptable excipient" includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.

[0104] As a solid compos! tion / combinati on for the oral administration, a tablet, powder, a granule, and the like is used. In such solid composite on / com bination, one or two or more kinds of active ingredients are mixed with at least one inert excipient. The composition may contain an inert additive, for example, a lubricant, a disintegrant. a stabilizer, a solubilizer, and the like by commonly used methods.[OIOS] A liquid composition / combination for the oral administration includes an emulsion, a solution preparation, a suspension, a syrup or an elixir, and the like which is pharmaceutically acceptable, and includes a generally used inert diluent, for example, purified water or ethanol. The liquid composition may contain adjuvants such as a solubilizing agent, a wetting agent, and a suspension, a sweetener, a flavor, an aromatic, or a preservative in addition to the inert diluent.[lit 06] The solid composition / combination or the liquid composition / combination may also be encapsulated to form capsules, including but not being limited to soli capsules or hard capsules.

[0107] The pharmaceutical composition / combination may also be used via injection to a subject. The injection for the parenteral administration includes a sterile aqueous or non-aqueous solution preparation, a suspension, a microemulsion or an emulsion. As the aqueous solvent, for example, distilled water for injection or physiological saline is included. As the non-aqueous solvent, for example, alcohols such as ethanol are included. Such a composition may further include a tonicity' agent, a preservative, a wetting agent, an emulsifier, a dispersant, a stabilizer, or a solubilizer. These are sterilized by, for example, filtration through a bacteria-retaining filter, mixing of a germicide, or irradiation. In addition, these can also be used in a manner in which a sterile solid composition is prepared, and is dissolved or suspended in sterile water or a sterile solvent for injection before being used.[0W8] The compound / pharmaceutical composition / combination as described herein also can be formulated in a drug delivery' system. A drug delivery' system is defined as a formulation or a device that enables the introduction of the therapeutically active substance in the body.Examples of the drug delivery system include, but are not limited to, nanoparticle drug delivery system and self-microemulsifying mrug delivery system (SMEDDS). SMEDDS formulation system consists of the drug dissolved or suspended m an oil phase along with a surfactant and a co-surfactant or solubilizer. The basic principle of this system is that when such system is diluted with an aqueous phase under gentle agitation, a fine oil-in-water (o / w) emulsion is formed instantaneously.

[0109] The compound / pharmaceutical composition / combination as described herein also can be formulated as a nanocarrier for ding delivery by encapsulating the compound into the nanocarrier. The nanocarrier may be a liposome or micelle

[0110] Micelles are nanocarrier systems that consist of an amphiphilic copolymer or surfactant and contain a lipophilic core and a hydrophilic shell. The core is an advantage for targeting high lipophilic drugs to hydrophilic environments. Particle sizes of micelles are in the range of 5- 200 nm. By physical encapsulation and / or chemical covalent attachment drug can be loaded into polymeric micelles find the drug release from micelles may be controlled by an external stimulus such as pH, temperature, enzymes, and ultrasound.

[0111] Liposomes are self-assembled (phospho)lipid-based drug vesicles that form a bilayer (uni-lamellar) and / or a concentric series of multiple bilayers (multilameilar) enclosing a central aqueous compartment. Liposomes can be classified as unilamellar vesicles (ULVs), oligolamellar vesicles (OLVs), multilameilar vesicles (MLVs), and multi vesicular liposomes (MVLs) depending on the compartment structure and iamellarity. OLVs and MLVs show an onion-like structure but present 2-5 and >5 concentric lipid bilayers, respectively. Different from MLVs. MVLs include hundreds of non -concentric aqueous chambers bounded by a single bilayer lipid membrane and display a honeycomb-like structure. Based on the particle size, ULVs can be further divided into small unilamellar vesicles (SUVs, 30-100 nm), large unilamellar vesicles (LUVs, >100 nm), and grant unilamellar vesicles (GUVs, >1000 nm). Different size range of ULVs was reported, i.e., SUVs with a size of less than 200 nm and LUV s with a size of 200-500 nm.

[0112] An additional therapeutic agent can be used in combination with the compound of the present disclosure. The additional therapeutic agent can be simultaneously or separately combined with the compound described herein. The additional anticancer drug can be an additional anticancer agent; preferably a chemotherapy drug. The chemotherapy drug includes, but is not limited to, platinum compounds (such as cisplatin, carboplatin), antimetabolite drugs (such as 5-fluorouracil, cytarabine, gemcitabine, pentostatin and methotrexate), anthracycline drug which targets DNA (such as doxorubicin and epirubicin), drugs which target DNA ordrugs which target topoisomerases or other chemotherapy drugs. In a preferred embodiment, the additional anti cancer drug is selected from cisplatin, docetaxel, paclitaxel, gemcitabine, navel bine, doxorubicin, irinotecan, gemcitabine and 5-fluorodeoxyuridine.

[0113] The compound of the present disclosure can be liposomally or micellally encapsulated in combination with the additional anticancer drug. The administration of a combination of different drugs encapsulated in the same liposome or micelles makes it possible to use lower dosages for attaining efficacy thus avoiding or reducing the toxicity of the drugs.

[0114] Non-limiting Exemplary' Embodiments

[0115] Embodiment 1 : A compound having formula (A)or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomer thereof, wherein is selected from the group consisting of -CH2-CH2-, -CH=CH-, -O-CH2-, -NH-CH2-,Rt is independently selected from the group consisting of H, halogen, nitro, cyano, C i-Cs alkyl, C2-C6 alkenyl. C?-Ce alkynyl, and Ci-C,6 alkoxyl, wherein Cj-Ce alkyl. C?-Ce alkenyl, Cz-C<5 alkynyl, or Ci-Ck, alkoxyl is independently unsubstituted or substituted with cyano, nitro, or halogen;R2 is independently selected from the group consisting of H, halogen, nitro, cyano, C j-Cr, alky], C2-C6 alkenyl, Cz-Ce alkynyl, and Ci-Ce alkoxyl, wherein Ci-Ce alkyl, Cz-CV, alkenyl, (T-Ce alkynyl, or Ci-Cg alkoxyl is independently unsubstituted or substituted with cyano, nitro, or halogen;R?, is independently selected from the group consisting of H, halogen, nitro, cyano, Cj-Ce alky], hydroxyl, C2-C6 alkenyl, C2-C.6 alkynyl, Ci-Ce alkoxyl, carboxylic acid, ester, carbonate,phosphate, sulfonate, amino, amide, imine, and carbamate, wherein Cj-Ce alkyl, C?-Ce alkenyl, C2-C6 alkynyl, or Ci-Cg alkoxyl is independently unsubstituted or substituted with cyano, nitro, or halogen: b is independently 0, 1, 2, 3 or 4: c is independently 0, 1 , 2, 3, 4 or 5; and d is independently 0, 1, 2. 3 or 4.

[0116] Embodiment 2: The compound of Embodiment 1, wherein the compound has formula (B):wherein X, Y, Ri, R3, b and d are defined as above, or a pharmaceutically acceptable salt, solvate. prodrag or stereoisomer thereof

[0117] Embodiment 3: The compound of Embodiment 1 , wherein the compound has formula (C):T54267 / PC0704 wherein X, Y, Ri, R?, c and d are defined as above, or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomer thereof.

[0118] Embodiment 4: The compound of Embodiment 1, wherein the compound of the present disclosure has formula (D):wherein Ri and d are defined as above, or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomer thereof

[0119] Embodiment 5: The compound of any one of Embodiments 1 to 4, whereinis --O-CH2-; Ri is H. halogen, C i-6alkyl. or Ci-Cs alkoxyl. wherein Ci-Ce alkoxyl is unsubstituted or substituted with cyano, nitro, or halogen; and d is 0. 1 or 2.

[0120] Embodiment 6: The compound of any one of Embodiments 1 to 5, wherein XY is --O-CH?-; Rj is H, halogen, or C1-C4 alkoxyl, wherein Cj-Ct alkoxyl is unsubstituted; and d is 0 or 1.

[0121] Embodiment 7: The compound of any one of Embodiments 1 to 6, wherein X miY is --O-CH2-; R2 is H or halogen; and c is 1 or 2.

[0122] Embodiment 8: The compound of any one of Embodiments 1 to 7, whereinis --O-CH2-; Rs is hydroxyl; and b is 1 or 2.

[0123] Embodiment 9: The compound of any one of Embodiments 1 to 8, wherein Xis - -O-CH2-; Ri is H, halogen, or G-C4 alkoxyl, wherein Cj-C4 alkoxyl is unsubstituted; d is 0 or 1; R? is H or halogen; c is 1 or 2; R3 is hydroxyl; and b is 1 or 2.

[0124] Embodiment 10: The compound of any one of Embodiments I to 9, wherein Xx^xY is -CH=CH-; Ri is H. halogen, Ci-ealkyl, or Ci-Ce alkoxyl, wherein Ci-Ce alkoxyl is unsubstitutedT54267 / PC0704 or substituted with cyano, nitro, or halogen; and d is 0, 1 or 2.

[0125] Embodiment 1 1: The compound of any one of Embodiments 1 to 10, wherein Xis -CH=CH~; Ri is H, halogen, or C1-C4 alkoxyl, wherein C1-C4 alkoxyl is unsubstituted: and d is O or 1.

[0126] Embodiment 12: The compound of any one of Embodiments 1 to 11, wherein X ;™.V ishalogen; and c is 1 or 2.

[0127] Embodiment 13: The compound of any one of Embodiments 1 to 12, wherein XiiiiY is -('! I CH-: R? is hydroxyl: and b is 1 or 2.

[0128] Embodiment 14: The compound of any one of Embodiments 1 to 13, wherein XY is -CH:::CTI-: Ri is H, halogen, or C1-C4 alkoxyl, wherein Cj-CU alkoxyl is unsubstituted; d is 0 or 1 ; R2 is H or halogen; c is 1 or 2; R? is hydroxyl; and b is 1 or 2.

[0129] Embodiment 15: The compound of any one of Embodiments I to 14, wherein X=Y is -CH2-CH2-; Ri is H, halogen, Ci-galkyl, or Ci-Ce alkoxyl, wherein Ci-CY alkoxyl is unsubstituted or substituted with cyano, nitro, or halogen; and d is 0. 1 or 2.

[0130] Embodiment 16: The compound of any one of Embodiments 1 to 15, wherein X "-Y is -CH2-CH2-; Ri is H, halogen, or C1-C4 alkoxyl, -wherein C1-C4 alkoxyl is unsubstituted; and d is 0 or 1

[0131] Embodiment 17 : The compound of any one of Embodiments 1 to 16, wherein X xixxY is -CH2-CH2-; i< is H or halogen; and c is 1 or 2.

[0132] Embodiment 18: The compound of any one of Embodiments 1 to 17, wherein X^^Y is -CH2-CH2-; R3 is hydroxyl; and b is 1 or 2.

[0133] Embodiment 19: The compound of any one of Embodiments I to 18, wherein X "."Y is -CH2-CH2-; Ri is H, halogen, or C1-C4 alkoxyl, wherein C1-C.4 alkoxyl is unsubstituted; d is 0 or 1 , R2 is H or halogen; c is 1 or 2; R3 is hydroxyl, and b is 1 or 2.

[0134] Embodiment 20: The compound of any one of Embodiments 1 to 19, selected from the group consisting of:or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomer thereof.

[0135] Embodiment 21: The compound of any one of Embodiments 1 to 19. which is, or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomer thereof.

[0136] Embodiment 22: The compound of any one of Embodiments 1 to 19, which is, or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomer thereof.

[0137] Embodiment 23: A pharmaceutical combination comprising the compound of any one of Embodiments 1 to 21 or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomer thereof, and a pharmaceutically acceptable excipient.

[0138] Embodiment 24: The pharmaceutical combination of Embodiment 23. which further comprises an additional therapeutic agent drug.

[0139] Embodiment 25: The pharmaceutical combination of any one of Embodiments 23 and 24, wherein the additional therapeutic agent is an additional anticancer drug; preferably, a chemotherapy drug.

[0140] Embodiment 26: The pharmaceutical combination of any one of Embodiments 2.3 to 25, wherein the chemotherapy drug is platinum compounds (such as cisplatin, carboplatin), antimetabolite drugs (such as 5-fluorouracil. cytarabine, gemcitabine, pentostatin and methotrexate), anthracycline drug which targets DNA (such as doxorubicin and epirubicin), drugs which target DMA or drugs which target topoisomerases or other chemotherapy drugs.T54267 / PC0704

[0141] Embodiment 27: The pharmaceutical combination of any one of Embodiments 23 to 26, wherein the chemotherapy drug is cisplatin, docetaxel, paclitaxel, gemcitabine, navelbine, doxorubicin, innotecan, gemcitabine, regorafenib. olaparib. lenalidomide, sorafenib, tagrisso or 5-fluorodeoxyuridine.

[0142] Embodiment 28: A nanocarrier comprising the compound of any one of Embodiments1 to 21 or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomer thereof

[0143] Embodiment 29: The nanocarrier of Embodiment 28, which is a liposome or a micelle.

[0144] Embodiment 30: The nanocarrier of any one of Embodiments 28 and 29, which further comprises an additional therapeutic agent.

[0145] Embodiment 31: The nanocarrier of any one of Embodiments 28 to 30, which further comprises an additional therapeutic agent drug.

[0146] Embodiment 32: The nanocarrier of any one of Embodiments 28 to 31, wherein the additional therapeutic agent is an additional anti cancer drug; preferably, a chemotherapy drug.

[0147] Embodiment 33: The nanocarrier of any one of Embodiments 28 to 32, wherein the chemotherapy drug is platinum compounds (such as cisplatin, carbopl atin), antimetabolite drugs (such as 5-fluorouracil, cytarabine, gemcitabine, pentostatm and methotrexate), anthracycline drug which targets DNA (such as doxorubicin and epirubicin), drugs which target DNA or drugs which target topoisomerases or other chemotherapy drugs.

[0148] Embodiment 34: The nanocarrier of any one of Embodiments 28 to 33, wherein the chemotherapy drug is cisplatin, docetaxel, paclitaxel, gemcitabine, navelbine, doxorubicin, irinotecan, gemcitabine, regorafenib, olaparib, lenalidomide, sorafenib, tagrisso or 5- fl uorodeoxyuridine.

[0149] Embodiment 35: A method for inhibiting PBXl-DNA interaction in a cell, comprising contacting the cell with the compound of any one of Embodiments 1 to 21 .

[0150] Embodiment 36: A method for treating or preventing a disease associated with inhibition of PBXl -DNA interaction in a subject, which comprises administrating a therapeutically effective amount of a compound of any one of Embodiments 1 to 21 to the subject.

[0151] Embodiment 37: The method according to Embodiment 36, wherein the disease is a cancer, cancer invasion, cancer metastasis and chemotherapy drug-resistant cancer.T54267 / PC0704

[0152] Embodiment 38: The method according to Embodiment 37, wherein the chemotherapy drug-resistant cancer is a cancer resistant to cisplatin, docetaxel, paclitaxel, gemcitabine, navelbine, doxorubicin, irinotecan, gemcitabine, regorafenib. olaparib, lenalidomide, sorafenib, tagrisso or 5-fluorodeoxyuridine.

[0153] Embodiment 39: The method according to Embodiment 37 or 38. wherein the chemotherapy drug-resistant cancer is a cancer resistant to gemcitabine, regorafenib, olaparib, lenalidomide, sorafenib or tagrisso.

[0154] Embodiment 40: The method according to any one of Embodiments 36 to 39, wherein the cancer is a multi -drug resistant cancer, recurrent cancer, refractory cancer or relapsed cancer.

[0155] Embodiment 41 : The method according to any one of Embodiments 36 to 40, wherein the cancer is a pancreatic cancer, esophageal cancer, glioma, ovarian cancer, multiple myeloma, colorectal cancer, lung cancer, liver cancer, breast cancer, gastric cancer, lung cancer, head and neck cancer, prostate cancer, testicular, intestinal cancer, bladder cancer, or urothelial cancer.

[0156] Embodiment 41 ; The method according to any one of Embodiments 36 to 41, wherein the cancer is pancreatic cancer, colorectal cancer, breast cancer, multiple myeloma, lung cancer or liver cancer.

[0157] Embodiment 42: A drug delivery' system, comprising a compound of any one of Embodiments 1 to 21 or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomer thereof, or a pharmaceutical combination of any one of Embodiments 23 to 27, or a nanocarrier of any one of Embodiments 28 to 34[015S] Embodiment 43: The drug delivery system, which is SMEDDS.

[0159] The examples disclosed herein are for illustrating the embodiments of the present disclosure and explaining the technical features of the present disclosure, rather than limiting the scope of protection of the present disclosure. It would be obvious to persons of ordinary skill in the art that various other changes and modifications can be made without departing from the concept of the present disclosure.EXAMPLES

[0160] I. Preparation Examples of CompoundsT54267 / PC0704

[0161] Example 1: Compound 8 (MPT1A407)

[0162] A mixture of 1 (1.0 g, 6.45mmol), DIPEA (2,471 mL) and DCM (12 mL) was stirred at 0°C for 10 mins and then added TBDMSCl (2. 14 g, 14. 18 mmol). After stirred for a while, the reaction was stirred at room temperature for Hi. TLC was performed to verify the completion of the reaction. The mixture was extracted with dichloromethane and water. The organic layer was collected and concentrated in vacuo to afford 2 (2.2 g, 89.1%) as a white solid without more purification.

[0163] The compound 2 (2.2 g, 5 73 mmol) was dissolved in MeOH (30 mL) and added 10% palladium on activated carbon as the catalyst at room temperature and stirred under Hz overnight. TLC was performed to verify the completion of the reaction. The 10% palladium on activated carbon was removed by methanol wash through celite packing. The organic layer was collected and concentrated in vacuo to afford 3 (2.0 g, 98.9%) as a brown solid without more purification.

[0164] A mixture of 3 -Fluorobenzoic acid (0.5 g, 4.58 mmol), EDCI HC1 (1.42 g, 9.16 mmol), HOBt (0.93 g. 6.87mmol), NMM (1.01 mL. 9.16 mmol) and DMF (3 mL) was stirred for a while then added compound 4 (0.64 g, 4.58mmol) at room temperature and stirred overnight.TLC was performed to verify the completion of the reaction. The reaction mixture was extractedby ethyl acetate and water. The organic layer collected and concentrated tn vacuo. The crude product was purified via column chromatography on silica gel with EtOAc / n -Hexane to afford 5 (0.5 g. 47.2%).

[0165] A mixture of 5 (0.5 g. 2. 16 mmol), potassium carbonate (0.6 g, 4,32 mmol) and ACN(22 mb) was added methyl bromoacetate (0.22 mL, 2.38 mmol) at room temperature and then stirred and refluxed for 3h. TLC was performed to verify the completion of the reaction. Then, acetonitrile was removed in vacuo. The residue was extracted by ethyl acetate and water The organic layer collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford 6 (0.65 g, 99.2%).

[0166] A mixture of 6 (0.65 g, 2. 14 mmol) and 1,4- dioxane (18 mL) was added 1 N LiOH(aq) (6 mL) and stirred at 40 °C overnight. The solvent was removed and residue was dissolved in water. The water layer was added 3N HCl(aq) and filtered by suction to afford 7 (0.5 g, 80.7%).

[0167] A mixture of 7 (0.5 g, 1.73 mmol), EDCI HC1 (0.54 g, 3.46 mmol), HOBt (0.35 g, 2.59mmol), NMM (0.380 mL, 3,46 mmol) and DMF (3 mL) was stirred for a while then added 4- aminobenzene-l,2-diol (0.22 g. 1.73 mmol) at room temperature and stirred overnight. TLC was performed to verify the completion of the reaction. The reaction mixture was extracted by ethyl acetate and water. The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EtOAc / 'n-Hexane to afford pink solid 8 (0.24 g, 35 0%). m.p.: 216 1 °C § : fl-i NMR (300 MHz, DMSO-d6) 10.21 (s, 1H), 9.69 (s, 1H), 8.96 (s, 1H), 8.64 (s, 1H), 7.82-7.71 (m, 2H), 7.67 (d, J = 3 Hz, 2H), 7.61- 7.53 (m, 1H), 7.46-7.39 (m, 1 H), 7. 16 (d, J - 3 Hz, 1H), 7.00 (d, J = 9 Hz, 1 H), 6.82 (dd, J = 3 Hz, 9 Hz, 1H), 6.64 (d, J - 9 Hz, 1H), 4.60 (s, 2H). HRMS (ESI, MH; ) calculated folC 21H17FN 2O5: 396.1121, found 396.1123.8 : “C NMR ( 125 MHz, CD3OD)5 167.6. 165.8, 163.6, 161.7, 154.9. 144.8, 142.4, 137.1, 132.2, 130.2, 129.5, 123.0, i22.7, 118.2, 118.1, 114,7. 114.2, 114.0, 112.6, 109.2, 67.4.

[0168] Example 2: Compound 14 (MPT1B010)29 10

[0169] A mixture of 9 (0.5 g, 2.88mmoi), potassium carbonate (0.5 g, 5.76 mmol) and ACN (30 mL) was added methyl bromoacetate (0 294 mL, 3 17 mmol) at room temperature and then stirred and refluxed for 3h. TLC test was performed to verify the completion of the reaction. Then, acetonitrile was removed in vacuo. The residue was extracted by ethyl acetate and water. The organic layer collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EtOAc / n -Hexane to afford 10 (0.65 g, 91.9%).

[0170] The compound 10 (0.65 g, 2.65 mmol) in IPA (24 mL) and water (6 ml,) was added ironpowder (0.59 g. 10.59 mmol) and ammonium chloride (0.14 g, 2.65 mmol). The mixture was heated to reflux and was stirred for 1 hour. TLC test was performed to verify the completion of the reaction. The mixture was filtered with celite to remove solid residue. The filtrate was extracted with ethyl acetate and water. The organic layer was collected and concentrated to obtain a yellow residue. The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford yellow solid 11 (0.63 g. 99.2%).

[0171] A mixture of 3 -Fluorobenzoic acid (0.63 g, 2,92 mmol), EDCTHCl (0.91 g, 5.84 mmol), HOBt (0.59 g. 4.38 mmol), NMM (0.642. mL, 5.84 mmol) and DMF (3 mL) was stirred for a while then added compound 11 (0,5 g, 2,82 mmol) at room temperature and stirred overnight.TLC test was performed to verify the completion of the reaction. The reaction mixture was extracted by ethyl acetate and water. The organic layer collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford yellow- solid 12 (0.21 g, 21.3%).

[0172] A mixture of 12 (0.21 g, 0.62 mmol) and 1 ,4- dioxane (6 mL) was added 1 N LiOH(aq) (2 mL) and stirred at 40°C overnight. Tire solvent was removed, and the residue was dissolved in water. The water layer was added 3N HCI(aq) and filtered by suction to afford yellow- solid 13 (0.11 g, 54.7%).

[0173] A mixture of 13 (0.11 g, 0.34 mmol). EDCTHC1 (0.11 g, 0.68 mmol), HOBt (0.07 g, 0.51 mmol), NMM (0.074 mL, 0.68 mmol) and DMF (3 mL) was stirred for a while then added 4-aminobenzene-l,2-diol (0.04 g, 0.34 mmol) at room temperature and stirred overnight. TLC test was performed to verify the completion of the reaction. The reaction mixture was extracted by ethyl acetate and water. The organic layer collected and concentrated in vacuo. The crude product was purified via. column chromatography on silica gel with EtOAc / n-Hexane to afford pink solid 14 (0.03 g, 20.5%). m.p.: 231.7 °C 14 : ’H NMR (300 MHz, DMSO-de) 10.33 (s, 1H), 9.73 (s, 1H), 8.99 (s, 1H). 8.65 (s, 1 H), 7.95 (d. J - 3 Hz. 1H), 7.82-7.73 (m, 3H), 7.64-7.42 (m, 3H), 7.14-7.08 (m, 1H), 6.83-6.79 (m. 1H), 6.65 (d. J = 9 Hz. 1H), 4.73 (s. 2H).HRMS (ESI. MH1) calculated for C M l h CH-.MV 430.0732. found 430.073114 :13C NMR ( 125 MHz, CDsOD)6 166.7, 165.7, 163.6, 161.7, 150.3, 145.0, 142.4. 133.4. 130.2, 129.5, 123.0, 122.8, 118.4, 118.2, 114.7, 114.2, 114.0, 113.8, 111.9, 108.6, 68.6.

[0174] Example 3: Compound 20 (MPT1B012)

[0175] A mixture of 15 (0 5 g, 3.18 mmol), potassium carbonate (0.88 g, 6.37 mmol) and ACN(32 mL) was added methyl bromoacetate (0.325 ml., 3.50 mmol) at room temperature and then stirred and refluxed for 3h. TLC was performed to verify the completion of the reaction. Then, acetonitrile was removed in vacuo. The residue was extracted by ethyl acetate and water. The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford yellow solid 16 (0.29 g.

[0176] The compound 16 (0.29 g, 1.27 mmol) in IPA(12 mb) and water (3 niL) was added iron powder (0.28 g, 5.06 mmol) and ammonium chloride (0.07 g, 1.27 mmol). The mixture was heated to reflux and was stirred for 1 hour. TLC was performed to verify the completion of the reaction. The mixture was filtered with cehte to remove solid residue. The filtrate was extracted with ethyl acetate and water. The organic layer was collected and concentrated to obtain a yellow residue. The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford yellow solid 17 (0.25 g, 99.2%).

[0177] A mixture of 3 -Fluorobenzoic acid (0.38 g, 1,91 mmol), EDCI HC1 (0.59 g, 3.82 mmol), HOBt (0.39 g. 2.86 mmol), NMM (0.420 ml,, 3.82 mmol) and DMF (3 ml) was stirred for a while then added compound 17 (0.27 g, 1.91mmol) at room temperature and stirred overnight. TLC was performed to verify the completion of the reaction. The reaction mixture was extracted by ethyl acetate and water. The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford yellow' solid 18 (0. 12 g, 20.0%).

[0178] A mixture of 18 (0.26 g, 0.81mmol) and 1 ,4- dioxane (6 mL) was added 1 N LiOH<aq)(2 mL) and stirred at 40 °C overnight. The solvent was removed, and the residue was dissolved in water. The water layer was added 3N HCIoq) and filtered by suction to afford yellow solid 19 (0. 17 g, 68.4%).

[0179] A mixture of 19 (0.17 g, 0.55 mmol), EDCIHC1 (0.17 g, 1.1 1 mmol), HOBi (0.1 1 g, 0.83 mmol), NMM (0.074 mL, 0.68 mmol) and DMT (3 mL) was stirred for a while then added 4-aminobenzene-J,2-diol (0.07 g. 0.55 mmol) at room temperature and stirred overnight. TLC was performed to verify the completion of the reaction. The reaction mixture was extracted by ethyl acetate and water. The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford pink solid 20 (0.04 g, 17.5%). m.p : 193.7 °C 20 :lH N MR (300 MHz, DMSO-d6) 10.35 (s, 1H), 9.77 (s, 1H), 8.98 (s, 1H), 8.67 (s, 1H), 7.80-7.69 (rn, 3H), 7.62-7.55 (m, 1H), 7.47-7.41 (m, 3H), 7.15-7.08 (m, 1H), 6.81 (dd, J = 3 Hz, 9 Hz, 1H), 6.64 (d, J = 9 Hz, 1H), 4.71 (d, J = 12 Hz, 2H). HRMS (ESI, MH+) calculated for C21HJ6F2N2O5: 414.1027, found414.1022.20 :nC NMR (125 MHz, CD3OD)6 167.1, 165.7, 163.6, 161.7, 144.9, 142.6, 142.4. 136.9, 130.1, 129.5, 118.4, 116.6, 116.3, 114.7, 114.2, 114.0, 112.3, 109.7, 108.9, 69.0,

[0180] Example 4; Compound 26 (MPT1B014)21 22

[0181] A mixture of 21 (0 5 g. 2.96 mmol), potassium carbonate (0.82 g. 5 91 mmol) and ACN (30 mL) was added methyl bromoacetate (0.301 mL, 3.25 mmol) at room temperature and then stirred and refluxed for 3h. TLC was performed to verify the completion of the reaction. Then, acetonitrile was removed in vacuo. The residue was extracted by ethyl acetate and water. 'The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford yellow solid 22 (0.68 g. 95.7%).

[0182] The compound 22 (0.68 g, 2.82 mmol) in 1PA (24 mL) and water (6 mL) was added iron powder (0.63 g, 11.28 mmol) and ammonium chloride (0.15 g, 2.82 mmol). The mixture was heated to reflux and was stirred for 1 hour. TLC w as performed to verify the completion of the reaction. The mixture was filtered with celite to remove solid residue. The filtrate was extracted with ethyl acetate and water. The organic layer was collected and concentrated to obtain a yellow residue The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford yellow solid 23 (0.59 g, 99.2%).23 24

[0183] A mixture of 3-Fluorobenzoic acid (0.7 g, 3.31 mmol). EDCIHC1 (1.03 g. 6.63mmol), HOBt (0.67 g. 4.97 mmol). NMM (0.729 mL, 6.63 mmol) and DMF (5 mL) was stirred for a while then added compound 23 (0.46 g. 3.31 mmol) at room temperature and stirred overnight. TLC was performed to verify the completion of the reaction The reaction mixture -was extracted by ethyl acetate and water. The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford yellow' solid 24 (0.75 g, 68.0%).

[0184] A mixture of 24 (0.75 g, 2.25 mmol) and 1 ,4- dioxane (18 mL) was added I N LiOH(aq) (6 mL) and stirred at 40 °C overnight. The solvent was removed, and the residue was dissolved in water. The water layer was added 3N HCl(aq> and filtered by suction to afford yellow solid 25 (0.51 g, 71 0%).

[0185] A mixture of 25 (0.51 g, 1.60 mmol), EDCIHCI (0.50 g, 3.19 mmol), HOBt (0.32 g, 2.40 mmol), NMM (0.352 mL, 3. 19 mmol) and DMF (3 mL) was stirred for a while then added 4-aminobenzene- 1,2-diol (0.20 g. 1.60 mmol) at room temperature and stirred overnight. TLC was performed to verify the completion of the reaction The reaction mixture was extracted byethyl acetate and water. The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford white solid 26 (0.16 g, 23.5%) m.p. : 22.2.8 °C 26 :!H NMR (300 MHz, DMSO-de ) 10.20 (s, i l l ). 9.61 (s, i l l ). 8.98 (s, 1H), 8.64 (s, i l l ). 7.81-7.72 (m, 2H), 7.61-7.54 (m, 1H), 7.52 i d. J === 3 Hz, 1H), 7.46-7.39 (m, 1 H ). 7.29 (dd, J == 3 Hz, 9 Hz, 1H), 7. 16 (d, J =- 3 Hz, 1 H ),6.97 (d, J = 9 Hz, 1H), 6.80 (dd, J = 3 Hz, 9 Hz, 1H), 6.64 (d, J = 9 Hz, 1H), 4.57 (s, 2H), 3.81 (s, 3H). HRMS (ESI, MIT) calculated for C22H19FN2O6: 426. 1227, found 426. 1223.26 :13C NMR (125 MHz. CD3OD)5 167.7, 163.7, 149.9, 145.0, 144.4, 142.4, 134.0, 130.2, 129.5, 123.0, 118.3, 118.1, 116.5, 114.7, 114.2, 114.0, 113.4, 112.0, 108.6, 70,0, 55.2.

[0186] Example 5: Compound 31 (MPT1B011)

[0187] A mixture of 3-Fluorobenzoic acid (0.49 g, 3.48 mmol), EDCI HCl (1.08 g, 6.97 mmol), HOBt (0.71 g, 5.22 mmol), NMM (0.767 mL, 6.97 mmol) and DMF (3 mL) was stirred for a while then added compound 27 (0.5 g, 2.82mmol) at room temperature and stirred overnight. TLC was performed to verify the completion of the reaction. The reaction mixture was extracted by ethyl acetate and water. The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford yellow solid 28 (0.56 g, 60.5%).

[0188] A mixture of 28 (0.56 g, 2. 11 mmol), potassium carbonate (0.58 g. 4.22 mmol) and ACN(21 mL) was added methyl bronioacetate (0.215 mL, 2.32 mmol) at room temperature and then stirred and refluxed for 3h. TLC was performed to verify the completion of the reaction. Then, acetonitrile was removed in vacuo. The residue was extracted by ethyl acetate and water. The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford yellow solid 29 (0.70 g, 99.0%).

[0189] A mixture of 29 (0.70 g. 2.07mmol) and 1,4- dioxane (15 ml.) was added 1 N LiOH(aq) (5 mL) and stirred at 40 °C overnight. The solvent was removed, and the residue was dissolved in water. The water layer was added 3N HCLaq) and filtered by suction to afford yellow solid 30 (0.37 g, 55.2%).

[0190] A mixture of (0.37 g, 1.14mmol), HBTU (0.43 g, 1 , 14mmol ), DIPEA (0.199 mi . 1.14 mmol) and DMF (3 ml) was stirred for a while then added 4-aminobenzene-l,2-diol (0.11 g, 0.88 mmol) at room temperature and stirred overnight. TLC was performed to verify the completion of the reaction. The reaction mixture was extracted by ethyl acetate and water. The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford pink solid 31 (0.06 g, 12.2%). m.p.: 199 1 °C31 : »C NMR (125 MHz, CD3OD)5 166.9, 166.3, 161.7, 156.8, 144.9, 142.4, 136.2, 130.8, 130.2, 129.5, 128.7, 128.1, 126.9, 123.1, 118.4, 115.8, 114.6, 113.6. 112.5, 109.1, 67.4.

[0191] Example 6: Compound 36 (MPT1B013)

[0192] A mixture of 3-Fluorobenzoic acid (0.55 g, 3.93 mmol), EDCIHCl (1 .22 g, 7 87 mmol), HOBt (0.80 g, 5.90 mmol), NMM (0.867 mL, 7.87 mmol) and DMF (3 mL) was stirred for a while then added compound 32 (0.5 g, 3.93 mmol) at room temperature and stirred overnight. TLC was performed to verify the completion of the reaction. The reaction mixture was extracted4!by ethyl acetate and water. The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford yellow solid 33 (0.44 g, 44.8%).

[0193] A mixture of 33 (0.44 g, 1.77 mmol), potassium carbonate (0.49 g, 3.53 mmol) and ACN(18 mL) was added methyl bromoacetate (0. 180 mL, 1 .94 mmol) at room temperature and then stirred and refluxed for 3h. TLC was performed to verify the completion of the reaction. Then, acetonitrile was removed m vacuo. The residue was extracted by ethyl acetate and water. The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford yellow solid 34 (0.53 g, 93.5%).

[0194] A mixture of 34 (0.53 g. 1.65 mmol) and 1,4- dioxane (15 mL) was added 1 N LiOH(aq) (5 mL) and stirred at 40°C overnight. The solvent was removed, and the residue was dissolved in water. The water layer was added 3N HCl(aq> and filtered by suction to afford y ellow' solid 35 (0.45 g, 88.9%).

[0195] A mixture of 35 (0.45 g, 1.46 mmol), HBTU (0.56 g, 1 .46 mmol). DIPEA (0.255 mL, 1 .46 mmol) and DMF (3 mL) was stirred for a while then added 4-aminobenzene-l,2-diol (0. 14 g, 1.13 mmol) at room temperature and stirred overnight. TLC was performed to verify the completion of the reaction. The reaction mixture was extracted by ethyl acetate and water. The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford pink solid 36 (0.06 g, 9.9%). m.p.: 204.8 °C 36 :lH NMR (300 MHz. DMSO-ds) 10.08 (s. IH), 9.74 (s, 1H), 8.97 (s, 1H), 8.66 (s, IH), 7.84-7.70 (m, 2H), 7.63-7.54 Cm, 1H). 7.49-7.40 (m, 2H), 7.16 (d, J = 3 Hz, IH), 6.99 (dd, J = 3 Hz, 12.0 Hz, IH), 6.89-6.80 (m, 2H), 6.65 (d, J - 9 Hz, IH), 4.67 (s, 2H). HRMS (ESI, MHfy calculated for C21H16F2N2O5: 414. 102.7, found 414. 1028.36 :!3C NMR (125 MHz. CD jOD)8 167.0, 166.2. 161.7, 157.0, 155.8. 144.9, 142.4, 136.3. 130.3, 129.5, 127.5. 123.2, 118.5. 114.7, 114.3, 112.5. 110.1, 109.1. 103.0. 102.8, 67.4.

[0196] Example 7: Compotmd 42 (MPT1B015)

[0197] A mixture of 37 (0.5 g. 2.96 mmol), potassium carbonate (0.82 g. 5.91 mmol) and ACN (30 mL) was added methyl bromoacetate (0.301 mL, 3.25 mmol) at room temperature and then stirred and refluxed for 3h. TLC was performed to verify the completion of the reaction. Then, acetonitrile was removed in vacuo. The residue was extracted by ethyl acetate and water. The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford yellow solid 38 (0 62 g.[0198 J The compound 38 (0.62 g, 2.57 mmol) in IPA (20 mL) and water (5 mL) was added iron powder (0.57 g, 10.28 mmol) and ammonium chloride (0.14 g, 2.57 mmol). The mixture was heated io reflux and was stirred for 1 hour. TLC was performed to verify the completion of the reaction. The mixture was filtered with celite to remove solid residue. The filtrate was extracted with ethyl acetate and water. The organic layer was collected and concentrated to obtain a yellow residue. The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford yellow solid 39 (0.39 g, 72.2%).

[0199] A mixture of 3 -Fluorobenzoic acid (0.34 g, 2,40 mmol), HBTU (0.91 g, 2.41mrnol), DIPEA (0.418 mL, 2.40 mmol) and DMF (3 mL) was stirred for a while then added compound 39 (0.39 g, 1 ,85 mmol) at room temperature and stirred overnight. TLC was performed to verify the completion of the reaction. The reaction mixture was extracted by ethyl acetate and water. The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford yellow solid 40 (0.23 g, 37.4%).

[0200] A mixture of 40 (0.23 g. 0.72 mmol) and 1.4- dioxane (6 mL) was added 1 N(2 mL) and stirred at 40 °C overnight. The solvent was removed, and the residue was dissolved in water. The water layer was added 3N HCl(aq) and filtered by suction to afford yellow solid 41(0.2 g. 90 9%).

[0201] A mixture of 41 (0.2 g, 0.63 mmol), HBTU (0.24 g, 0 63 mmol), DIPEA (0.109 ml.. 0.63 mmol) and DMF (3 mL) was stirred for a while then added 4-aminobenzene-l ,2-diol (0.06 g, 0.48 mmol) at room temperature and stirred overnight. TLC was performed to verify- the completion of the reaction The reaction mixture was extracted by ethyl acetate and water. The organic layer was collected and concentrated in vacuo The crude product was purified via column chromatography on silica gel with EtOAc / n -Hexane to afford white solid 42 (0.02 g, 7.49%). m.p.: 187.3 °C 42 :rH NMR (300 MHz, DMSO-d6) 9.71 (s, IH), 9.54 (s, IH), 8.97 (s, IH), 8.66 (s, 1H), 7.82-7.70 (m, 2H), 7.60-7.52 (m, 1H), 7.47-7.38 (m, 2H), 7.17 (d, J = 3 Hz, IH), 6.83 (dd, J = 3 Hz. 9 Hz, IH), 6.78 (d, J = 3 Hz, IH), 6.65 (d, J = 9 Hz, IH), 6.58 (dd, J = 3 Hz, 9 Hz, IH), 4.63 (s, 2H). FIRMS (ESI, MH1) calculated for C22H19FN2O6: 426.1227, found 426.1223.

[0202] Example 8: Compound 48 (MPT1A409; A409) l (triphesiylphos- nylidine) acetateToluene reflux, 5hNO244

[0203] The compound 43 (2,0 g, 13.23 mmol) in Toluene (65 mL) was added Methyl(triphenylphosphoranylidine) acetate (6,56 g, 15.88 mmol). The mixture was heated to reflux and was stirred for 5h. TLC was performed to verify the completion of the reaction. Then, Toluene was removed in vacuo. Tire residue was extracted with ethyl acetate and water. Theorganic layer was collected and concentrated in vacuo to afford 44 (2.70 g, 98.5%) as a white solid without more purification.

[0204] The compound 44 (1.0 g. 4.83 mmol) in II’A (40 mL) and water (10 mL) was added iron powder (1.08 g, 19.30 mmol) and ammonium chloride (0.26 g, 4.83 mmol). The mixture was heated to reflux and was stirred for 1 hour. TLC was performed to verify the completion of the reaction. The mixture was filtered with celite to remove solid residue. The filtrate was extracted with ethyl acetate and water. The organic layer was collected and concentrated to obtain a yellow residue. The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford brown solid 45 (0.85 g, 99.4%).

[0205] A mixture of 3-Fluorobenzoic acid (0.67 g, 4.80 mmol). EDCIHC1 (1.49 g. 9.59 mmol). HOBt (0.97 g, 7.20 mmol). NMM (1.05 mL. 9.59 mmol) and DMF (5 mL) w*as stirred for a while then added compound 45 (0.85 g, 4.80 mmol) at room temperature and stirred overnight. TLC was performed to verify the completion of the reaction. The reaction mixture was extracted by ethyl acetate and water The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford yellow solid 46 (0.83 g, 57.8'%).

[0206] A mixture of 46 (0.83 g, 2.77 mmol) and 1 ,4- dioxane (24 mL) w as added 1 N LiOH(aq) (6 mL) and stirred at 40 °C overnight. The solvent was removed, and the residue was dissolved in w ater. 'The water layer was added 3N HCl<aq) and filtered by suction to afford yellow solid 47 (0.32 g, 40.5%).

[0207] A mixture of 47 (0.32 g, 1.12 mmol), EDCIHC1 (0.35 g, 2.24 mmol), HOBt (0.23 g, 1.68 mmol), NMM (0.25 mL, 2.24 mmol) and DMF (3 mL) was stirred for a while then added 4-aminobenzene-l,2-diol (0.14 g. 1.12mmol) at room temperature and stirred overnight. TLC was performed to verily the completion of the reaction. The reaction mixture was extracted by ethyl acetate and water. The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EtOAc / n -Hexane to afford yellow solid 48 (0.04 g. 9.1%). m.p.: 272.8 °C 48 :]H NMR (300 MHz, CD3OD) 7.82- 7.77 (m, 3H), 7.71-7.67 (m, 1H), 7.54-7.51 (m, 4H), 7.38-7.34 (m, 1H), 7.26 (d, J - 3 Hz, 1H), 6.90 (dd, J - 3 Hz, 9 Hz, 1H). 6.74 (d, J - 1.5 Hz, 1H), 6.71 (d, J = 6 Hz, 1H). FIRMS (ESI,MH’) calculated for C22H17FN2O4: 392.1172, found 392.1171.48 :I3C NMR ( 125 MHz, CD3OD)3 165.0, 163.7. 161.7, 144.9, 142.0, 140.0, 139.9, 131.1, 130.9, 130.2, 128.1, 123.1, 120.7, 120.1, 114.7, 114.3, 114.1, 111.6, 108.3.

[0208] Example 9: Compound 55 (MPT1B004: B004)

[0209] The compound 49 (0.5 g, 2.69 mmol) in Toluene (16.5 mL) was added Methyl(triphenylphosphoranylidine) acetate (0.99 g, 2 96 mmol) The mixture was heated to reflux and was stirred for 5 hours. TLC was performed to verify the completion of the reaction. Then, Toluene was removed in vacuo. The residue was extracted with ethyl acetate and water. The organic layer was collected and concentrated in vacuo to afford 50 (0.63 g, 96.8%) as a white solid 50 without more purification.

[0210] A mixture of 50 (0.63 g, 2 61 mmol) and 1,4- dioxane (21 mL) was added 1 N LiOH(aq) (7 mL) and stirred at 40 °C overnight. The solvent was removed, and the residue was dissolved in water. 'The water layer was added 3N HCl(aq) and filtered by suction to afford yellow solid 51 (0.57 g, 96. 1 %).SI 52

[0211] A mixture of 51 (0.57 g, 2.50mmol). HBTU (0.95 g, 2.50mmol), DIPEA (0.436 mL, 2.50 mmol) and DMF (5 mL) was stirred for a while then added 3.4-bis((tert- butyldimethylsilyl)oxy)aniline (0.68 g. 1.90 mmol) at room temperature and stirred overnight.TLC was performed to verify the completion of the reaction. The reaction mixture was extracted by ethyl acetate and water. The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford yellow solid 52 (0.50 g, 35.5%).

[0001] The compound 52 (0.50 g, 0.89 mmol) in IPA (8 mL) and water (2 mL) was added iron powder (0.20 g, 3.55 mmol) and ammonium chloride (0.05 g. 0.89 mmol) The mixture was heated to reflux and was stirred for 1 hour. TLC was performed to verify the completion of the reaction. The mixture was filtered with celite to remove solid residue. The filtrate was extracted with ethyl acetate and water. The organic layer was collected and concentrated to obtain a yellow residue. The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford brown solid 53 (0.43 g, 90.9%).

[0212] A mixture of 3-Fluorobenzoic acid (0.15 g, 1.05 mmol), HBTU (0.40 g, 1.05 mmol), DIPEA (0. 183 mL, 1.05 mmol) and DMF (3 mL) was stirred for a while then added compound 53 (0.43 g, 0 81 mmol) at room temperature and stirred overnight. TLC was performed to verify the completion of the reaction The reaction mixture was extracted by ethyl acetate and water. The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford yellow solid 54 (0.20 g, 37.9%).54 55

[0213] A mixture of 54 (0.20 g, 0.31 mmol) and THF (3 ml.) was stirred at 0 °C for 10 mins find then added TBAF (0. 177 mL, 0 61 mmol). After stirred for a while, the reaction was stirred at room temperature for Ih. TLC was performed to verify the completion of the reaction. The mixture was extracted with di chloromethane and water. The organic layer was collected and concentrated in vacuo to afford 55 (0.05 g, 38.5%) as a yellow' solid without more purification, m.p.: 242.2 °C 55:!H NMR (300 MHz, CD3OD) 8.02 (d, J - 15 Hz, IH), 7.99 (s, IH), 7.79-7.66 (m, 4H). 7.59-7 51 (m, IH), 7.39-7.31 (m, IH), 7.27 (d, J = 3 Hz, IH), 6.91 (dd, J = 3 Hz, 9 Hz, IH), 6.76 (d, J = 9 Hz, IH), 6 72 (d, J = 3 Hz, 1 H). HRMS (ESI, MH } calculated for C22HI6C1FN2O4: 426.0783, found 426.0783.55 :13C NMR (125 MHz, CD3OD)8 165.9, 164.4, 161.7, 144.9, 142.1, 140.7, 136.7, 135.7, 134.6, 130.8, 130.2, 128.6. 127.4, 123.2, 122.7, 121.0, 119.0. 118.6, 114.7, 114.3. 111.7, 108.3.

[0214] Example 10: Compound 62 (MPT1B006)Methyl (triphenylphos- phoranylidine) acetateToiuetiereflux, 5hNO2

[0215] The compound 56 (0.5 g, 2.96 mmol) in Toluene (16.5 mL) was added Methyl (triphenylphosphoranylidine) acetate (1.09 g, 3.25 mmol). The mixture was heated to reflux and was stirred for 5h. TLC was performed to verify the completion of the reaction. Then, toluene was removed in vacuo. The residue was extracted wdth ethyl acetate and water. The organic layer was collected and concentrated in vacuo to afford 57 (0.51 g, 76.7%) as a yellow'solid without more purification57 58

[0216] A mixture of 57 (0.97 g, 4.31 mmol) and 1,4- dioxane (15 mL) was added 1 N LiOH(aq) (5 mL) and stirred at 40 °C overnight. The solvent was removed, and the residue was dissolved in water. The water layer was added 3N HCl(aq) and filtered by suction to afford yellow solid 58 (0.90 g, 99.0%).

[0217] A mixture of 58 (0.8 g, 3.80mmol), HBTU (1.44 g, 3.80 mmol), DIPEA (0.660 mL, 3.80 mmol) and DMF (10 mL) was stirred for a. while then added 3,4-bis((tert- butyldimethylsilyl)oxy)aniline (1.04 g, 2.92mmol) at room temperature and stirred overnight.TLC was performed to verify the completion of the reaction. The reaction mixture was extracted by ethyl acetate and water. The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with ElOAc / n-Hexane to afford yellow solid S9 (1.40 g, 67.7%).

[0218] The compound 59 (0.34 g. 0.62 mmol) in IPA (8 mL) and water (2 mL) was added iron powder (0.14 g, 2.49 mmol) and ammonium chloride (0.03 g. 0.62 mmol). The mixture was heated to reflux and was stirred for 1 hour. TLC was performed to verify the completion of the reaction. The mixture was filtered with celite to remove solid residue. The filtrate was extracted with ethyl acetate. The organic layer was collected and concentrated to obtain a yellow residue. The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford brown solid 60 (0.32 g, 99.0%).

[0219] A mixture of 3-Fluorobenzoic acid (0.44 g, 3.17 mmol). HBTU (1.20 g, 3.17 mmol), DIPEA (0.552 mL, 3.17 mmol) and DMF (3 mL) was stirred for a while then added compound 60 (1.26 g, 2.44 mmol) at room temperature and stirred overnight. TLC was performed to verify the completion of the reaction. The reaction mixture was extracted by ethyl acetate and w ater. The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford yellow7solid 61 (0.53 g, 34.0%).61 62

[0220] A mixture of 61 (0.55 g, 0.86 mmol) and THF (9 mL) was stirred at 0 °C for 10 mins and then added TBAF (0.499 mL. 1 .72 mmol). After stirred for a while, the reaction was stirred at room temperature for Ih. TLC was performed to verify the completion of the reaction. The mixture was extracted with dichloromethane and water. The organic layer was collected and concentrated in vacuo to afford 62 (0.07 g, 20.0%) as a yellow solid without more purification.m.p.: 241.4 °C 62 :;H NMR (300 MHz, CD3OD) 7.82-7.52 (m, 8H), 7.39-7.32 (m, IH), 7.27 (d, J = 3 Hz, IH), 6.91 (dd, 1 3 1 ? / . 9 Hz. 1 H), 6.83 (d, J = 16.0 Hz, IH), 6.73 (d, J = 9 Hz, IH). FIRMS (ESI, MH1) calculated for Cd i TAA: 410 1078. found 410.1079.62 :BC NMR (125 MHz, DMSO-dd5 165.0. 163.2. 161.4, 160.0, 145.4, 142.0, 137.2, 131.9, 131.8, 131.2, 131.1, 130.1. 124.5, 118.2, 116.8, 115.8, 115.2, 115.0, 110.9, 108 2, 107.8, 107.6.

[0221] Example 11: Compound 69 (MPT1B008)Methyl (triphenyiphos- phoranylidine) acetate ................................... jga,.Toluenereflux, 5 hrs6c 7c

[0222] The compound 6c (1.00 g. 5.52 mmol) in toluene (50 mL) was added Methyl- (triphenylphosphoranylidine) acetate (2.03 g. 6.07 mmol). The mixture was heated to reflux and was stirred for 5 hours. TLC was performed to verify the completion of the reaction. Then, toluene was removed in vacuo. The residue was purified via column chromatography on silica gel with EA / n-Hexane (1 : 5, Rf == 0.45) to afford 7c (1 .26 g. 96. 18 %) as a white solid. IH NMR (300 MHz, CDCI3) : 37.96 (d, J - 16.2 Hz, IH), 7.85 (dd, J - 8.4 Hz, 2.1 Hz, IH),7.77 (d, J = 2.1 Hz, IH), 7.63 (d, J = 8.4 Hz, IH). 6.63 (d, J = 16.5 Hz, IH), 4.00 (s, 3H), 3.83 (s, 3H).7c 8c

[0223] A mixture of 7c (1.00 g, 4.22 mmol) and 1.4- dioxane (27 mL) was added 1 N LiOH(aq) (9 mL) and stirred at 40 °C overnight. The reaction was removed and the residue was dissolved in water. The aqueous layer was added the 3N HCl (aq) and filtered by suction to afford 8c as a white solid in quantitative yield. IH NMR (300 MHz, MeOD) ■ 3 7.96 (d, J =16.2 Hz. IH), 7.88 - 7.84 (m. 3H), 6.68 (d, J = 16.2 Hz, IH). 4.03 (s, 3H).8c 9c

[0224] A mixture of 8c (0.61 g, 2.74 mmol), HBTU (1 ,35 g, 3.56 mmol), DIPEA (0,62 mL, 3.56 mmol) and DMF (5 mL) was stirred for 15 minutes then added compound 3 (1.26 g, 3.56 mmol) and stirred at 40°C overnight. TLC was performed to verify the completion of the reaction. The reaction mixture was extracted with ethyl acetate and water The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica using with EA / n-Hexane (1 : 5. Rf = 0.45) to afford 9c (0.59 g, 38.56 %) as an orange solid!H NMR (300 MHz. CDCh ) : J7.96 id. J = 15.6 Hz, IH), 7,82 (s. IH).7.76 (dd, J - 8.4 Hz, 2.1 Hz, IH), 7.70 (d, J - 2.1 Hz, IH). 7.53 (d, J - 8.4 Hz, IH), 7.45 (d, J = 2.1 Hz, IH), 6.98 (dd, J = 8.7 Hz, 2.4 Hz. IH), 6.83 - 6.76 (m, 2H), 3 92 (s, 3H). 0.98 (s, 18H), 0.23 (s, 6H), 0.18 (s, 6H).

[0225] The compound 9e (0.71 g. 1.2.1 mmol) m IPA (10 mb) and water (2 mL) was added iron powder (0.2 g, 3.62 mmol) and ammonium chloride (0.13 g, 2.41 mmol). The mixture was heated to reflux and was stirred for 3 hours. TLC was performed to verify the completion of the reaction The mixture was filtered to remove solid residue. The aqueous filtrate was purified via column chromatography on silica gel using EA / n-Hexane (2 : 3, Rf = 0.3) to afford orange solid 10c (0.42 g, 62.50 %).*H NMR (300 MHz, CDCh) : 0'7.85 (d, J - 15.6 Hz, IH), 7.38 (s. IH). 7.29 (s, IH), 7.12 (s, IH), 6.89 (d. J = 8.1 Hz, IH), 6.76 (d. J = 8.7 Hz, 1H), 6.47 (d, J - 15.6 Hz, IH), 6.25 (dd, J - 8.1 Hz, 2.1 Hz, IH), 6.19 (d, J - 2.1 Hz, IH), 3.83 (s, 3H), 0.99 (s, 9H), 0.98 (s, 9H), 0.24 (s, 6H), 0.19 (s, 6H).

[0226] A mixture of 10c (0.34 g, 0.64 mmol), TEA (0. 14 ml, 0.96 mmol) and DCM (6 mL) was added 3-Fluorobenzoyl chloride (0 10 ml, 1.77 mmol) at 0°C and then stirred at room temperature overnight. When the reaction was completed, the mixture was added NaHCO? (aq) at 0°C and extracted with ethyl acetate and water. Tire organic layer collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel using EA / n-Hexane (I : 2, Rf = 0.3) to afford 11c (0.34 g, 80.95%) as a yellow solid.!H NMR (300 MHz, CDCh) : 33. 14 (s, IH), 7.88 (d, J - 15.6 Hz, IH), 7 66 - 7.59 (rn, 311), 7.49 - 7.35 (m, 4H), 7.28 - 7.22 (m, 1H). 6.99 (d, J = 8.1 Hz, 1H), 6.92 (d, J = 7.8 Hz, 1H). 6.76 (d, J = 8.4 Hz, IH). 6.62 (d. J = 15.6 Hz, I H), 3.88 (s, 3H). 0.99 (s, 9H). 0.98 (s. 9H), 0.23 (s. 611 ). 0.19 (s, 6H).11C 69[0227 j A mixture of 11c (0.34 g, 0.52 mmol) and THF (10 mL) was stirred at 0 °C for 10 min and then added TBAF (1 mL. 1.04 mmol). After stirring for a while, the reaction was stirred al room temperature for Ih. TLC was performed to verify the completion of the reaction (DCM : MeOH = 10 : 1, Rf = 0.45). The mixture was purified via column chromatography on silica gel using DCM : MeOH = 15 : 1 ~ 10 : 1 to afford 69 ( 0.09 g, 47.37 %) as a brown solid!H NMR (300 MHz, DMSO) : 3 10 46 (s, IH), 9.78 (s, IH), 8 97 (s, IH), 8.60 (s, IH), 7.84 - 7.76 (m, 2H), 7.69 (d, J - 15.6 Hz, IH). 7.65 - 7.44 (m, 5H), 7.27 (d, J == 2,4 Hz, IH),6.90 (dd, J = 8.6, 2.4 Hz, IH). 6.78 (d, J = 15.9 Hz. IH), 6.65 (d, J = 8.7 Hz. I H), 3.89 (s, 3H).[02280 C to rt, Ih1 2

[0229] A mixture of 1 (2.0 g, 12.90 mmol), imidazole (1.11g, 12.38 mmol), DMF (2 mL) and DCM (12 mL) was stirred at 0 °C for 20 mins and then added TBDMSC1 (2.43 g, 16.13 mmol). After stirred for a while, the reaction was stirred al room temperature for Ih. TLC was performed to verify the completion of the reaction. The mixture was purified via column chromatography on silica gel with EA / n-Hexane (1 : 30, Rf 0.4) to afford 2 (4.05 g, 81 .82%) as a white solid without more purification. ‘H NMR (300 MHz, CDCI3) 5 7.75 (dd. J = 3 Hz, 9Hz, IH). 7.71 (d. J = 3 Hz. IH). 6.87 (d, J = 9 Hz. IH), 1.00 (s. 9H), 0.99 (s, 9H), 0.25 (s, 12H).

[0230] The compound 2 (2.2 g, 5.73 mmol) was dissolved in MeOH (30 mb) and added 10% palladium on activated carbon as the catalyst at room temperature and stirred under H2 overnight. TLC was performed to verify the completion of the reaction. The 10% palladium on activated carbon was removed through celite and washed by methanol. The organic layer was collected and concentrated in vacuo to afford 3 (2.0 g, 98.9%) as a brown solid without further purification.4a 5a

[0231] The compound 4a (2.00 g, 9.92 mmol) in THF (50 mL) was added 1,1’- carbonyldiimidazole (CD!) (2.00 g. 1 1.91 mmol). The mixture was stirred for 1 hour at room temperature. TLC was performed to verify the completion of the reaction. Then, NaBlk (1.13g, 29.76 mmol) dissolving in 3.5 ml HzO was dropwise into the said mixture at 0 °C. The mixture was stirred at the room temperature overnight. The crude product was purified via column chromatography on silica gel with EA / n-Hexane (1 : 1, Rf= 0.45) to afford 5a (1.70 g, 91.40 %) as a yellow' solid. 41 NMR (300 MHz, DMSO-d6) : <58.05 (d, J = 8 4 Hz. 1H), 7 66 (s, 1H), 7.51 (d, J - 8. 1 Hz, 1H), 4.60 (s, 2H).

[0232] The compound Sa (0.68 g. 3.63 mmol) in THF (35 mL) w’as added manganese oxide (1 57 g, 18. 13 mmol). The mixture was stirred at the room temperature overnight. TLC was performed to verify the completion of the reaction. Then, the mixture was filtered with celite to remove solid residue. The filtrate was purified via column chromatography on silica gel with EA / n-Hexane (1 : 3, Rf ” 0.5) to afford 6a (0.37 g, 54.73 %) as a yellow' solid.(300 MHz, DMSO-d6) : J 10.07 (s, 1H), 8.27 (d, J - 8.4 Hz. 1H), 8.26 (d, J - 1.5 Hz, 1H), 8.08 (dd, J = 8.4Hz, 1.5 Hz. 1H).Methyl (triphenylphos- phoranylldine) acetate-Toluenereflux, 5 hrs6a 7a

[0233] The compound 6a (0.48 g, 2.57 mmol) in toluene (25 mL) was added methyl (triphenylphosphoranylidine) acetate (0.95 g. 2.83 mmol). The mixture was heated to reflux and was stirred for 5 hours TLC was performed to verify the completion of the reaction Then, toluene was removed in vacuo. The residue was purified via column chromatography on silica gel with EA / n-Hexane (I : 5. Rf = 0.45) to afford 7a (0.55 g, 88.71 %) as a white solid.!H NMR (300 MHz, DMSO-d6) : (58.21 (d. J = 1.5 Hz, 1H), 8.1 1 (d, J = 8.4 Hz, 1H), 7.96 (dd, J - 8.7 Hz, 1.8 Hz, 1H), 7.72 (d, J = 16.2 Hz. 1H), 6.93 (d, J = 15.9 Hz, 1H), 3.76 (s, 3H).7a 8a

[0234] The compound 7a (0.87 g, 3.60 mmol) and 1,4 - dioxane (30 niL) was added 1 N LiOH(aq) (7.8 niL) and stirred at 40 °C overnight. The solvent was removed and the residue was dissolved in water. The aqueous solution was added the 3N HCl(aq) and filtered by suction to afford Sa (0 72 g, 87.80 %) as a white solid.NMR (300 MHz. DMSO-d6) : 5 12.73 (s, 1H), 8.17 id. J - 1.5 Hz, 1H), 8.10 (d, J - 8.7 Hz, 1H), 7.92 (dd, J - 8.4Hz. 1.5 Hz, 1H), 7.63 (d, J = 15.9 Hz, 1H), 6.80 (d, J = 15.9 Hz, 1H).

[0235] A mixture of Ba (0.72 g, 3.15 mmol), HBTU (1.55 g, 4.09 mmol), DIPEA (0.71 mL. 4.09 mmol) and DMF (7 mL) was stirred for 15 minutes then added compound 3 (1 .46 g, 4.09 mmol) and stirred at 40°C overnight. TLC was performed to verify the completion of the reaction. The reaction mixture was extracted by ethyl acetate and water. The organic layer was collected and concentrated in vacuo The crude product was purified via column chromatography on silica gel with EA / n-Hexane (1 : 4, Rf:::0.55) to afford 9a (0.66 g, 37.29 %) as an orange solid. 'H NMR (300 MHz, DMSO-d6) '■ d 10.17 (s, 1H), 8.15 (d, J = 8.4 Hz, 1H). 8 01 (d, J = 1.5 Hz, 1H), 7.81 (dd, J = 8.4 Hz, 1.5 Hz, 1H), 7.63 (d, J = 15.6 Hz, 1H), 7.51 (d. J - 2.4 Hz, 1H), 7. 10 (dd, J - 8.7 Hz. 2.7 Hz, 1H). 6.95 ( d , J - 15.9 Hz, H l). 6.81 (d, J - 8.4 Hz, 1H), 0.96 (s, 9H). 0.95 (s. 9H), 0.22 (s. 6H), 0.17 (s, 6H).

[0236] The compound 9a (1.04 g, 1 85 mmol) in IP A (9 mL) and water (3 mL) was added iron powder (0.30 g, 5.54 mmol) and ammonium chloride (0.20 g, 3.69 mmol) The mixture was heated to reflux and was stirred for 3 hours. TLC was performed to verify the completion of the reaction. The mixture was filtered to remove solid residue. The filtrate was purified via column chromatography on silica gel with EA / n-Hexane (1 : 4, Rf = 0.3) to afford orange solid 10a (0.63 g, 63.86 %).!H NMR (300 MHz, CDC13) : £7.58 (d, J = 15.3 Hz, 1H), 7.45 (s, IH), 7.38 (s, IH). 7.23 (d, J = 7.2 Hz, 1H), 7.14 (s, IH), 6.89 (d, J = 7.5 Hz, IH), 6.77 (d, J = 8.7 Hz, IH). 6.73 (d, J - 8.1 Hz, IH), 6.31 (d. J === 15.6 Hz, IH), 0.99 (s, 9H), 0.98 (s. 9H), 0.24 (s, 6H), 0.19 (s, 6H).

[0237] A mixture of 10a (0 61 g, 1.14 mmol), TEA (0.24 ml, 1.72 mmol) and DCM (1 1 mL) was added 3-fluorobenzoyl chloride (0.17 ml, 1.37 mmol) at 0°C and then stirred at room temperature overnight When the reaction was completed, the mixture was added NaHCO3 (aq) at 0°C and extracted by ethyl acetate and water. The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EA / n-Hexane (1 : 4, Rf = 0.4) to afford Ila (0,50 g, 66.43%) as a yellow solid,rH NMR (300 MHz, CDC13) : £8.60 (d, J - 8.4 Hz, IH), 8 48 (s. IH), 7.69 ~ 7.63 (m, 2H), 7.66 (d. J = 15.6 Hz, IH), 7.59 (s, IH). 7.55 - 7.48 (m. 2H), 7.39 (s, IH), 7.31 (t, J = 8.4 Hz, IH),7 18 (s, 1H). 6.90 (d, J = 7.8 Hz. IH), 6.79 id. J = 8.4 Hz, IH), 6.48 (d, J = 15.3 Hz, IH), 1.00 (s. 9H), 0.99 (s, 9H), 0.25 (s, 6H), 0.20 (s, 6H).11a 76

[0238] A mixture of I la (0.4 g, 0.61 mmol) and THF (6 mL) was stirred at 0‘ C for 10 mins and then added TBAF (1.8 mL, 1 .83 mmol). After stirred for a while, the reaction was stirred at room temperature for th TLC was performed to verify the completion of the reaction. The mixture was concentrated in vacuo and purified via column chromatography on silica gel with EA / MeOH (25 : 1 , Rf = 0.8). The combined organic layer was concentrated in vacuo to give solid crude compound, and then it was washed with EA to afford 76 (0.07 g, 26.92 %) as a yellow solid. 1H NMR (300 MHz, DMSO-d6) ■ <5 10.22 (s, i l l ). 9.88 (s, H i ). 9.01 (s, 1H), 8.66 (s, 1H), 7.86 (d, J - 7.8 Hz, 1H), 7.81 7.77 (m. 2H), 7.70 - 7.58 (m, 3H). 7 51 (d, J - 15.3 Hz. 1H), 7.54 - 7.46 (m. 1H), 7.26 (d. J = 2.4 Hz. 1H), 6.91 (dd, J = 8.7 Hz, 2.4 Hz, 1H). 6.83 (d, J = 15 6 Hz, FH). 6 67 (d, J = 8.4 Hz, 1H).

[0239] Example 13: Compound 83 (MPMethyl (trlphenylphos- phoranylidine) acetateToluene reflux, 5h6d

[0240] The compound 6d (1.00 g, 5.91 mmol) in toluene (50 mL) was added methyl (triphenylphosphoranylidme) acetate (2. 18 g. 6.50 mmol). The mixture was heated to reflux and was stirred for 5 hours. TLC was performed to verify the completion of the reaction. Then, toluene was removed in vacuo. The residue was purified via column chromatography on silica gel with EA / n-Hexane (1 : 8. Rf = 0.3) to afford 7d (1.25 g. 82.78 %) as an orange solid. *H NMR (300 MHz, CDCh) : <58.10 (t, J - 8 1 Hz, 1H), 7.64 (d, J - 15.9 Hz, IH) , 7 43 (s, 1H),7.40 (d, J = 1 .5 Hz, IH), 6.54 (d, J = 16.2 Hz. IH), 3.84 (s. 3H)7d 8d

[0241] The compound 7d (1 00 g, 4.44 mmol) and 1,4-dioxane (30 mb) was added 1 N LiOH(aq) (10 mb) and stirred at 40 ° C overnight. The solvent was removed and the residue was dissolved in water. The water layer was added the 3N HCl(aq) and filtered by suction to afford8d as a pink solid in a quantitative yield.

[0242] A mixture of 8d (0.41 g, 1.93 mmol), HBTU (0.95 g, 2.51 mmol), DIPEA (0.44 mb,2.51 mmol) and DMF (7 raL) was stirred for 15 minutes then added compound 3 (0.90 g, 2.51 mmol) and stirred at 40°C overnight. TLC was performed to verify the completion of the reaction. The reaction mixture was extracted by ethyl acetate and water. The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EA / n-Hexane (1 : 5, Rf ~= 0.35) to afford 9d (0.50 g, 48.1 1 %) as an orange solid.!H NMR (300 MHz, CDC13) : <78.10 (t, J ==:7.8 Hz, 3H). 7.71 (d, J - 15.6 Hz, IH), 7.44 - 7.38 (m, I H). 6 93 - 6.89 (m. 3H), 6 80 (d. J = 8.7 Hz. IH), 6.62 (d, J = 15 6 Hz, IH), 1.00 (s, 10H), 0.99 (s, 10H), 0.25 (s, 7H)„ 0.20 (s, 7H).[0243 j The compound 94 (0.50 g, 0 91 mmol) in IP A (8 mL) and water (2 mL) was added iron powder (0.1 (5 g, 2.74 mmol) and ammonium chloride (0.10 g, 1.83 mmol) The mixture was heated to reflux and was stirred for 3 hours TLC was performed to verify the completion of the reaction The mixture was filtered to remove solid residue. The filtrate was purified via column chromatography on silica gel with EA / n-Hexane (1 : 3, Rf ~ 0.25) to afford orange solid lOd (0.33 g, 70.21 %).’HNMR (300 MHz, CDCh) : c> 8.60 (s, IH), 7.55 - 7.50 (m, 2H), 7.08 (d. J = 8 / 4 Hz. 1H), 6.99 (dd, J = 12 Hz, 1.5 Hz, IH), 6.90 (d, J = 8.4 Hz, IH), 6.76 (d, J = 8.7 Hz, IH), 6.56 (t, J - 8.7 Hz. IH), 6.43 (d. J - 15.3 Hz, IH), 0.98 (s, 9H), 0.96 (s, 9H), 0.21 (s, 6H), 0.17 (s, 6H).

[0244] A mixture of lOd (0.34 g, 0.66 mmol), TEA (0 14 ml, 0.99 mmol) and DCM (5 mL) was added 3 -fluorobenzoyl chloride (0.10 ml, 0.79 mmol) at 0°C and then stirred at room temperature overnight TLC was performed to verify the completion of the reaction (EA / n- hexane ~ 1 : 4. Rf " 0.3). Hie mixture was added NaHCOs(aq) at 0°C and extracted by ethyl acetate and water. The precipitated in organic layer was collected and filtered by suction filtration to afford lid as a while solid (0.37g, 88.10 %). lH NMR (300 MHz, CDCh):£8.51 (I. J - 8.4 Hz. IH), 8.10 (d. J - 3 0 Hz. 1 H ), 7.70 - 7.60 (m. 3H), 7.55 - 7.48 (m. 2H), 7.54 - 7.47 (m. IH), 7.39 -- 7.29 (m. 4H), 7.2.1 (s, IH). 6.92 -- 6.89 (m, IH). 6.79 (d, J = 8.4 Hz, IH),6.46 (d, J = 15 3 Hz, IH). 1 00 (s. 9H), 0.99 (s. 9H), 0.25 (s, 6H). 0.20 (s, 6H)

[0245] A mixture of lid (0.51 g, 0.8 mmol) and THF (16 mL) was stirred at 0 °C for 10 min and then added TBAF (1.6 mL, 1.6 mmol). After stirred for a while, the reaction was stirred at room temperature for ihr. TLC was performed io verify the completion of the reaction (DCM : MeOH = 10 : 1, Rf = 0.35). The mixture was extracted by DCM / LPA (3 : 1) and water. The precipitated in organic layer was collected and filtered by suction filtration to afford 69 as a white solid (0.05 g, 15.15%). ‘H NMR (300 MHz, DMSO) : 610.34 (s, IH), 9.90 (s, I H), 7 86 (d, J = 7.8 Hz, IH), 7.80 (d, J - 9.6 Hz, 1 H), 7.74 (t, J - 8.1 Hz, 1 H), 7.66 - 7.47 (m. 5H), 7.29 (d, J = 2.4 Hz, IH), 6.93 (dd, J = 8.4, 2.4 Hz, IH), 6.82 (d, J = 15.6 Hz, IH), 6.69 (d, J = 8.7 Hz, I H)

[0246] Example 14: Compound 90 (MPT1B009)4b 5b

[0247] The compound 4b (2.00 g, 10.14 mmol) in THF (50 mL) was added 1 ,1'- carbonyl diimidazole (CDI) (2,00 g, 12. 17 mmol). The mixture was stirred for 1 hour at room temperature. TLC was performed to verify’ the completion of the reaction. Then, NaBH?, (1 15 g, 30.42 mmol) dissolving in 3.5 ml H2O was dropwise into the said mixture at 0°C. The mixture was stirred at the room temperature overnight. The crude product was purified via column chromatography on silica gel with EA / n-Hexane (1 : 1. Rf = 0.3) to afford 5b (1.12 g, 60.34 %) as a yellow solid ’H NMR (300 MHz, DMSO-d6) : S 7 85 (d. J = 8.1 Hz, IH). 7.28 (s, IH), 7.04 (d, J === 8.4 Hz, IH), 5 49 (t, J === 6.0 Hz, IH), 4.58 (d, J - 5.7 Hz, 2H), 3.91 (s,3H).6.35b 6b10248 j The compound 5b (0.60 g, 3.28 mmol) in THF (30 mb) was added manganese oxide (1.99 g, 22.93 mmol). The mixture was stirred at the room temperature overnight. TLC was performed to verify the completion of the reaction. The mixture was filtered with celite to remove solid residue. The filtrate was purified via column chromatography on silica gel with EA / n-Hexane (1 : 2. Rf = 0.3) to afford 6b (0.56 g, 94.37 %) as a yellow solid. 'H NMR (300 MHz, DMSO-d6) : 010.08 (s. J Hl 8.07 (d, J = 8.4 Hz. I HL 7.83 (d, J = I .5 Hz. 1 H). 7.66 (dd. J - 8.4 Hz, 1.5 Hz, 1H), 4.01 Is. 3H).Methyl (triphenylphos- phoranylidine) acetateToluene reflux, 5 hrs7b

[0249] The compound 6b (0.72 g, 3.97 mmol) in toluene (40 mL) was added methyl (triphenylphosphoranylidine) acetate (1.46 g, 4.37 mmol). The mixture was heated to reflux and w as stirred for 5 hours. TLC was perforated to verify the completion of the reaction. Then, toluene was removed in vacuo. The residue was purified via column chromatography on silica gel with EA / n-Hexane (1 : 3. Rf = 0.45) to afford 7b (0.91 g, 96.09 %) as a white solid.JH NMR (300 MHz, DMSO-d6) : 0'7.90 (d. J ==:8.4 Hz. H i ). 7.73 (d, J == 1.5 Hz, 1H), 7.72 (d, J = 16.2 Hz, IH), 7.47 (dd, J = 8.4 Hz, 1.5 Hz, IH), 6.91 (d, J = 15.9 Hz, 1 H), 3 97 (s, 3H). 3.76 (s, 3H).

[0250] The compound 7b (0.91 g, 3.82 mmol) and 1,4-dioxane (30 mL) was added 1 N LiOH(8.3 ml.) and stirred at 40 ° C overnight. The solvent was removed and the residue was dissolved m water. The water layer was added 3N HCl (aq) and filtered by suction to afford 8b (0 81 g, 94.96 %) as a white solid. 1H NMR (300 MHz, DMSO-d6) : A 7 89 (d. J - 8.4 Hz, 1H), 7.68 (d, J - 1.2 Hz, 1H), 7.63 (d, J - 16.2 Hz, 1H), 7.43 (dd, J = 8.4 Hz, 1.5 Hz. 1H), 6.78 (d, J - 15.9 Hz, 1H), 3.91 (s, 3H).

[0251] A mixture of 8b (0.5 g, 2.24 mmol), HBTU (1 10 g, 2.91 mmol), DIPEA (0.51 ml.,, 2.91 mmol) and DMF (6 mL) was stirred for 15 minutes then added compound 3 (1.03 g, 2.91 mmol ) and stirred at room temperature for overnight. TLC test was performed to verify the completion of the reaction. The reaction mixture was extracted by ethyl acetate and water. The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EA / n-Hexane (1 : 4, Rf = 0.45) to afford 9b (0.77 g. 61.60 %) as an orange solid ’H NMR (300 MHz, DMSO-d6) : 8 10.17 (s, 1H), 7.95 (d, J = 8.7 Hz, 1H). 7.64 (d. J - 15.9 Hz. 1H), 7.60 (s, 1H), 7.50 (d, J - 2.4 Hz, 1H), 7.35 (d, J - 8.4 Hz, 1H), 7.12 (dd, J = 8.7 Hz, 2.1 Hz, 1H), 6.92 (d, J = 15.6 Hz, 1H). 6.81 (d, J = 8.7 Hz, 1H), 3.98 (s, 3H). 0 97 ( s. 9H). 0 95 (s. 9H), 0.22 (s. 6H), 0.17 (s, 6H)[0252 j The compound 9b (0 6 g, 1 .07 mmol) in IP A (8 mL) and water (2 mL) was added iron powder (0.18 g, 3.21 mmol) and ammonium chloride (0.11 g, 2.14 mmol) The mixture was heated to reflux and was stirred for 3 hours TLC was performed to verify the completion of the reaction The mixture was filtered to remove solid residue. The filtrate was purified via column chromatography on silica gel with EA / n-Hexane (1 : 4, Rf = 0. 15) to afford orange solid 10b (0.44 g, 77.19 %).fH NMR (300 MHz, DMSO-d6) : 39.80 (s, 1H), 7.47 (d, J = 2.4 Hz, IH). 7 41 (d, J = 1.5.6 Hz, IH), 7.09 (dd, J = 8.7 Hz, 2.4 Hz, IH), 7.03 (d, J = 1.5 Hz, IH), 6.95 (dd, J == 8. 1 Hz, 1.5 Hz, IH), 6.77 (d, J - 8.7 Hz, IH), 6.63 (d, J - 7.8 Hz, IH), 6.45 (d. J - 15.6 Hz, IH), 5.28 (s, 2H). 3.82 (s. 3H), 0.96 (s, 9H), 0.95 (s, 9H), 0.21 (s, 6H), 0. 16 (s, 6H).

[0253] A mixture of 10b (0.44 g, 0.83 mmol), TEA (0 17 ml, 1 .25 mmol) and DCM (8 mL) was added 3 -fluorobenzoyl chloride (0.12 ml, 1.00 mmol) at 0°C and then stirred at room temperature overnight. When the reaction was completed, the mixture was added NaHCCh(aq) at 0°C and extracted by ethyl acetate and water. The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EA / n-Hexane (1 : 4, Rf = 0.25) to afford lib (0.41 g, 75.93%).lH NMR (300 MHz, DMSO-d6) : o 10.03 (s, IH), 9.63 (s, IH), 7.88 (d, J - 8.1 Hz, IH), 7.82 (d, J - 7.8 Hz, IH), 7.77 (d, J = 9.6 Hz, IH), 7.58 (m, IH), 7.57 (d, J = 15.9 Hz, IH). 7.49 (d, J = 2.4 Hz, IH), 7.45(t, J = 9.0 Hz). 7.35 (d, J = 1.5 Hz, IH). 7 25 (dd. J = 8.4 Hz, 1.5 Hz. IH), 7. 13 (dd, J = 8.7 Hz, 2.7 Hz, IH). 6.80 (d, J =- 8.7 Hz, I H). 6.76 (d, J - 15.6 Hz, IH), 3.91 (s, 3H). 0.97 (s, 9H). 0.95 (s. 9H), 0.22 (s. 6H), 0.17 (s. 6H).

[0254] A mixture of 11b (0.39 g, 0.60 mmol) and THF (5 mL) was stirred at O' C for 10 min and then added TBAF (1 .80 mL, 1.80 mmol). After stirred for a while, the reaction was stirred at room temperature for Ih. TLC was performed to verify the completion of the reaction. The mixture was concentrated in vacuo and purified via column chromatography on silica gel with EA / MeOH (20 : 1, Rf = 0.8). The combined organic layer was concentrated in vacuo to give solid crude compound, and then it was washed with EA to afford 90 (0.09 g, 36.00 %) as a yellow solid. IH NMR (300 MHz. DMSO-d6) : 0'9.85 (s, IH). 9.63 (s. IH). 8.99 (s, IH), 8.63 (s, IH), 7.87 (d, J = 8.4 Hz, IH), 7.82 (d, J = 7.8 Hz, IH), 7.79 - 7.75 (m, I H). 7.63 - 7.55 (m,IH), 7.52 i d. J - 15.6 Hz, I H), 7.49 - 7.43 (m, IH), 7.33 (d. J - 1.5 Hz, IH), 7.28 (d, J - 2.4 Hz, IH), 7.24 (dd, J = 8.4 Hz, 1.5 Hz, IH), 6.91 (dd, J = 8.4 Hz, 2.4 Hz, IH), 6.78 (d, J = 15.6 Hz, IH), 6.66 (d, J - 8.4 Hz, IH), 3.91 (s, 3H).

[0255] Example 15: Compound 94 (MPT1A408)

[0256] The compound 44 (0.5 g, 2.41 mmol) was dissolved in MeOH (25 mL) and added 10% palladium on activated carbon as the catalyst at room temperature and stirred under H2 overnight. TLC was performed to verily the completion of the reaction. The 10% palladium on activated carbon was removed by methanol wash through celite packing. The organic layer was collected and concentrated in vacuo to afford 91 (0.41 g, 94.9%) as a brown solid without more

[0257] A mixture of 3-Fluorobenzoic acid (0.32 g, 2.29 mmol), EDCI'HCl (0.71 g, 4.58 mmol), HOBt (0.46 g, 3.43mmol), NMM (0.50 mL, 4.58 mmol) and DMF (3 mL) was stirred for a while then added compound 91 (0.41 g, 2.29 mmol) at room temperature and stirred overnight. TLC was performed to verify the completion of the reaction. The reaction mixture was extracted by ethyl acetate and water. The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EtOAc / n -Hexane to afford 92 (0.49 g, 71.1 %).

[0258] A mixture of 92 (0.49 g, 1.63 mmol) and 1.4- dioxane ( 15 mL) was added 1 N LiOH(aqi(5 mL) and stirred at 40°C overnight. The solvent was removed, and the residue was dissolved in water. The water layer was added 3N HCl(aq) and filtered by suction to afford yellow solid 93(0 38 g, 81.4%).93 94

[0259] A mixture of 93 (0.38 g, 1.32 mmol), EDCIHC1 (0.41 g, 2.65 mmol), HOBt (0.27 g, 1.98 mmol ). NMM (0.293 mL, 2.65 mmol) and DMF (3 mL) was stirred for a while then added 4-aminobenzene-l,2-diol (0.17 g. 1.32 mmol) at room temperature and stirred overnight. TLC was performed to verify the completion of the reaction. The reaction mixture was extracted by ethyl acetate and water. The organic layer was collected and concentrated in vacuo. The crude product was purified via column chromatography on silica gel with EtOAc / n-Hexane to afford white solid 94 (0.03 g, 5.8%) m.p.: 232 1 °C 94 :!H NMR (500 MHz, CD3OD) 7 75 (d, J = 7.5 Hz, 1H), 7.65 (d, J = 10 Hz, 1H), 7.59 (d, J - 10 Hz, 2H), 7.54-7.49 (m, 1 H), 7.33- 7.29 (m, 1H), 7.25 (d, J - 10 Hz, 2H), 7.06 (d, J - 2.5 Hz, 1H), 6.73 (dd, J - 2.5 Hz. 8.5 Hz, 1H), 6.67 (d, J = 8.5 Hz, 1H), 2.97 (t, J = 10 Hz, 2H), 2.60 (t, J = 10 Hz, 2H). HRMS (ESI, Mff) calculated for C ■ I fo / rN'394.1329, found 394.1326.94 :13C NMR (125 MHz, CDSOD.)8 171.7, 165.8, 163.6, 161.7, 144.8, 141.9, 137.4, 136.3, 130.6, 130.2, 128.4, 123.0, 121.1, 118.2, 118.0, 114.6, 114.2, 114.0, 111.9, 108.7, 38.2, 38.0.

[0260] II. Biological Assays

[0261] Evaluation of the characteristics of CSCs following the administration of B004 derivatives

[0262] The objective of this study is to investigate the potential of new B004 derivatives, which involve prodrug and benzamide alteration as well as dual target drugs, in addressing the characteristics of CSCs. Specifically, the inventors aim to examine the impact of these derivatives on CSC properties such as sternness regulation, drug resistance, tumorigenicity, spherogenesis, and metastatic potential, considering the known role of NOTCH3 / PBX1 in these processes. In order to investigate this inquiry, the identification of CSCs is achieved by the detection of OCT4 activity utilizing an OCT4-GFP reporter.

[0263] CSC percentage analysis: The OCT4-GFP reporter assay utilizes either a flow cytometer or an ELISA reader to measure the quantity of fluorescent reaction products. This measurement is directly proportional to the level of OCT4-GFP activity' present in the cells.

[0264] TOF-S1MS analysis: The nuclear localization test is conducted to determine the ability of B004 derivatives to enter the nucleus and target the PBX1 transcription factor. Time-of-flight secondary7-ion mass spectrometry7(TOF-SIMS) is employed to quantify- the nuclear accumulation of B004 derivatives. The aforementioned machine has the capability to provide visual illustrations of drug distribution inside cellular compartments.

[0265] Soft agar assay: A tumorigenicity experiment is conducted using a soft agar assay to evaluate the potential of tumorigenicity in vitro. In order to prevent cellular adhesion, a 1.2% soft agar solution is used as the foundational layer on a 6-well plate. Subsequently. CSCs are introduced as a second layer using a 0.4% soft agar solution. Once the soft agar has undergone solidification, a further layer of medium devoid of serum is introduced over the surface. The impact of B004 derivatives on the growth of soft agar colonies is assessed. The quantification of clonogenic development is conducted after two weeks.

[0266] Tumor sphere assay: The spherogenesis evaluation is conducted to investigate the enduring self-renewal capacity and anti-anoikis properties. This involves the implementation of a tumor sphere formation assay A 1.2% concentration of soft agar is used in the experimental procedure in order to facilitate the detachment of cells from dish bottoms Following this, the seeded CSCs are cultured using a serum-free medium. The suspension milieu induces the 3D proliferation of cells possessing sustained self-renewal capabilities and resistance to anoikis, resulting in the formation of spheroids. The addition of B004 derivatives is conducted in order to investigate their impact on the process of spherogenesis. The enumeration of the tumor sphere is conducted over a period of 10 days.

[0267] Cell viability assay: The drug sensitivity experiment utilizes first-line chemotherapy regimens, including gemcitabine, regorafenib, olaparib, and paclitaxel, to investigate if B004 derivatives might reduce the drug resistance phenotype observed in CSCs. The utilization of the PrestoBlueTM Cell Viability Reagent is intended tor the quantitative assessment of cell viability7.[026S] Migration ability assay: The ibidi wound healing migratory- assay is employed to investigate the impact of the PBX1 inhibitor on the migration capacities of CSCs. CSCs are introduced into a silicone insert, resulting in the creation of a predetermined 500 pm cell-free region upon removal of the insert. The monitoring of cell motility is conducted by the utilizationT54267 / PC0704 of time-lapse microscopy subsequent to the introduction of the B004 derivatives.

[0269] Transwell invasion assay: The transwell invasion test is employed to examine if the inhibition of PBX1 has an impact on the invasion capacity of CSCs. In order to create a thin gel layer, the application of matrigel onto a 24-well transwell insert is conducted, followed by solidification at a temperature of 37 °C for a duration of 15 -30 minutes. In order to replicate penetration through the extracellular matrix, CSCs, both with and without B004 derivatives, are positioned on the surface of the Matrigel covering. The cells are immobilized in a solution of 70% ethanol and thereafter subjected to staining using a 0.2% concentration of crystal violet. This staining procedure aims to ascertain the number of cells that possess invasive characteristics.

[0270] Analysis of alterations in the transcriptome triggered by B004 derivativesIn order to conduct a more comprehensive assessment of the impact of B004 derivatives on transcriptome levels, the inventors utilize RNA sequencing (RNA-seq) methodology to determine the specific genes and pathways that are influenced by PBXli. B004 derivatives are employed for the purpose of ascertaining the specificity of targeting the PBX1 pathway and finding new mechanisms for the PBX1 network.

[0271] Analysis of the effects of B004 derivatives in moose models

[0272] Subcutaneous model (36 ms de mice):

[0273] Once tumors reached an average volume of 100 mm3, mice (n = 4) were randomized into 9 arms and treated with vehicle DMSO (1%) (i.p. injection), DMSO (1 %) (oral gavage), B004 (i.p. injection), prodrug#! (oral gavage), prodrug#2, prodrug#3, benzamide modified compound (i.p. injection), dual target compound (i.p. injection), or gemcitabine (i.p. injection) for 3-4 weeks. All the drugs are administered at 5 mg / kg and by i.p. injection or oral gavage three times a week. Upon reaching the designated endpoint, the width and length of each tumor burden are assessed, along with the body weight. The tumor dimension was measured by a Vernier caliper every' other day. Tumor burden is calculated as volume [mm3] = 0.52 x width2x length The endpoints are statistically evaluated for the significant differences between each group m (i) tumor weight and volume at sacrifice, (ii) PBXI signaling activities in tumors harvested at the end point assessed by qRT-PCR or western blot, and (iii) CSC percentage in tumors harvested at the end point assessed by identification of OCT4-GFP activity through flow cytometry. The inventors compare the hematologic or clinical chemistry profiles of mice treated with the indicated drugs with those of animals treated with DMSO. The inventors also check to see if the mice have any unusual medical conditions, such as lethargy, weight loss, or seriousT54267 / PC0704 physical problems. Necropsies are also conducted to assess tissue damage or histological abnormalities in the brain, heart, lungs, liver, spleen, kidney, and intestine.

[0274] Orthotopic metastatic model (42 nude mice):

[0275] Given PBXl ’s capacity to regulate genes associated with epithelial-mesenchymal transition (EMT), it is plausible to hypothesize that the inhibition of PBX1 might potentially mitigate the metastatic potential of cancer. As delineated in previous literature8, the inventors want to establish an orthotopic murine model of liver metastasis by orthotopically injecting pancreatic CSCs. This enables the inventors to examine the efficacy of the medicine in inhibiting the occurrence of metastasis. The inventors select the most promising candidate prodrugs by subcutaneous model. Starting from the second week after tumor injection, mice (n = 6) were randomized into 7 arms and treated with vehicle DMSO (1%) (i.p. injection), DMSO (1%) (oral gavage), B004 (i.p. injection), prodrug (oral gavage), benzamide modified compound (i.p. injection), dual target compound (i.p. injection), or gemcitabine (i.p. injection) for 3-4 weeks. All the drugs are administered at 5 mg / kg three times a week Bioluminescence imaging using IV IS Spectrum was employed to monitor the development and metastasis of the tumor cells. The inventors compare the hematologic or clinical chemistry7profiles of mice treated with the indicated drugs with those of animals treated with DMSO The inventors also check to see if the mice have any unusual medical conditions, such as lethargy ; weight loss, or serious physical problems. Necropsies are also conducted to assess tissue damage or histological abnormalities in the brain, heart, lungs, liver, spleen, kidney, and intestine.

[0276] Maximum tolerated dose (MTD) analysis of B004 derivatives (64 C57BL / 6 mice):

[0277] The inventors perform the MTD and assess the toxicity of B004 derivatives to infer future human toxicity7. The inventors found that B004 did not show’ toxicity of 500 mg / kg (100X of effective dose) in Fig. 9. Escalating doses (0, 600, 800, 1000 mg / kg) of 8004 (i.p injection), prodrug (oral gavage), benzamide modified compound (i.p injection), and dual target compound (i.p. injection) are intraperitoneally injected into C57BL / 6 mice (n = 4), and the drug-induced toxicities (morbidity, weight loss, diarrhea, etc.) are monitored for up to 4 weeks. There are 4 C57BL / 6 mice per group and dose, totaling 64 mice across 4 groups and 4 dosages. At the end, animals are sacrificed for necropsy and histopathology7. Blood cell counts and clinical chemistry profiles are performed by7the core facility at Taipei Medical University.

[0278] Investigation of the complex stnicftire of PBX1 and BG04 by using structural approachesT54267 / PC0704

[0279] Purification of N-terminal His tagged PBX1 DB

[0280] The expression construct of the DNA binding region of PBX1 is designed by referring previous study9. Briefly, the DNA binding region of PBX1 (PBX 1 DB; amino acids 233-319) is constructed into pET2l expression vector. The recombinant PBX1 DB proteins with C- terminal His tag are expressed in Escherichia coli BL21 (DE3) in LB (Luria-Bertani) medium in the presence of 100 ug / 'ral of Ampicillin. Cells are grown at 37 °C to an OD600 of about 0.4 and induced by 1 mM IPTG, and incubated overnight at 25 °C. The purification steps for this C -terminal His tagged PBX1 DB are as follows:(1) The cells were suspended by Lysis buffer (20 mM Tris HC1, pH8.0. lOOmM NaCl) and lysed by French pressure cell press.(2) After centrifugation, the supernatant was loaded onto 5 ml Ni NT A excel column. The column was washed with 40 CV of W ash buff er (20mM Tris HCl, pH8.0, 100 mM NaCl, 20mM imidazole), and the bound protein was eluted with Elution buffer (20mM TrisHCl pH8.0, 100 mM NaCl. 500mM imidazole).(3) The 24 ml enrich SEC 650 gel filtration was used to further improve the purity of PBX1 DB (Gel filtration buffer: 20mM TrisHCl. pl IS o. 100 mM NaCL 1 mM DTT.

[0281] Crystallization, data collection and structure determination of recombinant PBX1 DB / B004 complex

[0282] For crystallization, purified PBX1 DB proteins (apo form or with compound) are screened by reagents from commercialized kits (Hampton crystal screen T&H; Molecular Dimensions structure screen I&II and Qiagen JCSG core I--IV). The obtained crystals are used to collect X-ray diffraction data using beamlines in the National Synchrotron Radiation Research Center in Hsinchu, Taiwan. The data is processed using HKL2OOO10. The molecular replacements are used to determine the structures. The programs BUCCANEER11, COOT12and Refniac13are used in the subsequent model building and refinement.

[0283] Development of preformulation for PBX1-B004

[0284] Solubility studies

[0285] The solubility of PBX1-B004 in oils, surfactants and selected excipient for topical use is determined. An excess of PBX1-B004 is placed in 2 ml of the vehicle in screw-capped glass vials, and the mixture is heated at 60 °C in a water-bath to facilitate solubilisation using a vortex mixer. Mixtures are equilibrated at 25 °C for 3 days in a water bath and then centrifuged at 12,000 rpm (Sigma-3K18, Sigma) for 10 min to separate the nndissolved drug. Aliquots ofsupernatant are diluted with methanol and quantified by UV-visible spectroscopy A.=400 nm (DU800 Beckman Coulter).

[0286] Preparation of SMEDDS

[0287] Based on the phase diagrams, microemulsion (ME) was selected for the incorporation of PBX1-B004. Firstly, ME is added to the oily phase (lecithin + essential oil) pri or to addition of the aqueous phase (containing cationic surfactant). The formulations are then vortexed at 2500 rpm for 3 mm at 25 °C. The formulated ME are equilibrated at 25CC for 3 days.

[0288] Optimization and physiochemkai characterizations of SMEDDS as oral drag delivery system

[0289] Dynamic laser scattering (DLS) and zeta potential

[0290] DES and zeta potential measurement are performed on a commercial laser light scattering instrument (Nano-ZS90, Malvern, UK). The apparent Z-average hydrodynamic diameter and polydispersity index (PDI) are obtained at 25 °C using a fixed scattering angle of 90°. The diameter of the nanoparticles is determined after dispersion in ultrapure water (1: 10) and is measured at 25 °C with a scattering angle of 90° The zeta potential is measured after dispersion of the dried nanoparticles in 1 mM KC1 solution.

[0291] Drag loading and entrapment efficiency

[0292] The concentration of PBX1-B004 is determined by HPLC. The entrapment efficiency(EE) and drug-loading (DL) of API in the SMEDDSs are calculated from the following equations: the EE and DL are calculated according to Equations 1 and 2, respectively: 100(1) (2) where WM is the weight of drug in the microemulsions. Wi is the weight of initial feeding drug, and Wp is the weight of initial feeding polymers.

[0293] Transmission electron microscopy (TEM)[02941 The shape of formed particles is analyzed by taking TEM photographs using a Technai transmission electron microscope (FEI Co , The Netherlands) The particles are dispersed in medium (i.e., Milli-Q water at appropriate pH), and one drop of the diluted dispersion is placed on a 200-niesh carbon-coated copper grid The photographs are taken at various magnificationsand 100 kV voltage.

[0295] Stability in the GIT

[0296] Lipid formulation should be able to keep the drug in the solubilized form in the gastrointestinal tract (GIT). Precipitation of the drug from the system nullifies the advantage offered by the lipid-based formulation system, API loaded SMEDDS is evaluated for their stability in different simulated GIT fluids (SGF, pH 1.2 and SIF, pH 6.8) to assess the stability of formulations under various pH and enzymatic, conditions. Freeze dried formulations were reconstituted pnor to use. The simulated gastric fluid (SGF) comprised of 0.2% NaCI, pepsin, 0.7% HC1 with pH 1.2 while simulated intestinal fluid (SIF) comprised of 0.685% monobasic potassium phosphate, 1% NaOH and 1% pancreatin with pH 6.8. In order to simulate the effect of bile salts, 3 mM sodium taurocholate is added in the SIF. Each ml of the formulations is added to 9 ml of gastric simulated GI fluids. The samples are incubated for 2h and 6h in SGF and SIF. respectively. The formulations are then evaluated for particle size, PDI, zeta potential and % encapsulation efficiency

[0297] Experimental design mathematic model

[0298] A four-factor Doehleri matrix design is undertaken to investigate the main effects and the interactions of the four factors (ratio of lecithin and tween 80, amount of API and oil, and Surfactant and Co-surfactant) on the three responses (particle size, entrapment efficiency, and drug loading). The data obtained for the three responses in each trial is fitted to the classical non-linear quadratic model. The mathematical model is expressed as follows:where Xi-X4correspond to the studied factors, Y represents the response associated with each factor level combination, po is an intercept and pi-psr are the regression coefficients. Data are analyzed by non-linear estimation using STATISTICA 6.0 software.

[0299] In vitro drag release

[0300] In vitro drug release from the micelles is studied using dialysis membrane bags (MWCO=60(X)~8000 Da) performed in simulated gastric fluid (SGF) without pepsin, simulated intestinal fluid (SIF) without trypsin and phosphate-buffered saline (PBS, pH 7.4) as release media, respectively at 37CC with continuous orbital mixing (50 rpm). At predetermined intervals, triplicate suspensions (0.5 ml) are withdrawn and replaced with equal volume of freshT54267 / PC0704 release medium. The amount of API released is evaluated by the HPLC analytical method. Release profiles are expressed in terms of cumulative release of API in percentage and plotted versus time.

[0301] In-vivo pharmacokinetic study on PBX1 DB / B004

[0302] Development and validation of an analysis method in plasma

[0303] Sample preparation procedures are optimized, encompassing extraction, purification, and, if necessary, derivatization methods. The selection and configuration of appropriate analytical instrumentation, such as high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS). or other relevant techniques, are undertaken. The development of the method involves establishing key analytical conditions, including the choice of columns, mobile phases, gradient programs, and detector settings. Following method development, a comprehensive validation study is conducted, covering parameters such as accuracy, precision, linearity, specificity, robustness, and stability The obtained data is rigorously analyzed to assess the method’s sensitivity, accuracy, and reliability for the intended application.

[0304] Rat pharmacokinetic study

[0002] Sprague-Dawley (SD) rats (weighing 180-200 g) are separated randomly into several groups six rats each, and fasted overnight for at least 12 h in each experiment, although free access to water is allowed. After iv and oral administration of the formulations. 0.5 ml blood samples are collected at predetermined time intervals. Plasma is immediately separated by centrifugation of the blood samples at 8000 rpm for 10 min. Ail plasma samples are immediately frozen at -20°C until analysis. Methodological studies, such as linearity, specificity, recovery, precision ofwith-in and between days, can satisfy the requirements of the methodology.

[0305] Pharmacokinetic data analysis

[0306] Pharmacokinetic parameters, ie, maximum plasma concentration (Cmax), time point of maximum plasma concentration, elimination rate constant (Ke), half-life (Ti 2), mean residence time, and area under the plasma concentration-time curve (AUC) are calculated based on serum API concentrations.

[0307] Results

[0308] Research findings indicate that gemcitabine administration triggers EMT alterations in chemoresistant pancreatic cancer ceil lines by activating the ERK / ZEB1 axis. Conversely, literature reports indicate that PBX1 recruits its transcriptional target FoxCl to the ZEB2 promoter, leading to the transcriptional suppression of E-cadherin This mechanism induces EMT and promotes cancer metastasis in esophageal cancer and glioma. Given the structural resemblance between ZEB1 and ZEB2, the inventors are intrigued by whether PBXl also influences ZEB1 expression and whether this modulation contributes to gemcitabine induced EMT alterations in chemoresistant pancreatic cancer

[0309] As known in the art, a small molecule inhibitor, T417 (N-(4-(2-((3,4- dihydroxyphenyl)amino)-2-oxoethoxy)naphthalen-l-yl)-3-fluorobenzamide; CAS#: 2032123-28-5), exhibits the ability to dock at the DNA-PBX1 interaction site, thereby inhibiting the activity of PBXl (Figure 1A). The administration of T417 significantly suppresses the selfrenewal and proliferation of ovarian and multiple myeloma cancer cells, while also resensitizing platinum-resistant ovarian tumors to carboplatin, While ensuring the preservation of the central structure crucial for T417's hydrogen bond formation with the PBXl protein's contact interface, the inventors made modifications to the hydrophobic sidechains to augment their hydrophilic properties (Figure IB). Preliminary' testing on the A2780 ovarian cell line revealed that structural modifications led to enhanced efficacy and potency in suppressing cell proliferation. Notably, A409 and B004 emerged as the strongest candidates and were consequently chosen for further experimentation (Figures 1C, I D, IE, and IF). Furthermore. the inventors investigated the drugs' capacity to inhibit PBXl downstream targets. Both compounds A409 and B004 exhibited significant inhibition of the expression of the five preselected PBXl targets at the mRNA level, surpassing the effectiveness of T417 and other compounds (Figures 1 G and III).

[0310] Up-regulated metastasis and sternness-related genes in chemo-resistant cancer cells are effectively inhibited by PBXl inhibitors A409 and B004.

[0311] Gemcitabine resistance has long been associated with the EMT process. While various pathways have been implicated, it remains unclear whether gemcitabine directly induces EMT in gemcitabine-resistant (CR) cancer and whether PBXl plays a role in this process. To gain a comprehensive understanding of this phenomenon, the inventors' first step is to explore the effects of administering gemcitabine in CR cancer cells. Therefore, the inventors initially generated a CR MIA PaCa-2 cell line by subjecting the cells to long-term exposure to escalating doses of Gem. Survival analysis indicated that the CR cell line exhibited a 7.46-fold increase in IC50 to gemcitabine compared to its parental wild-type (PT) cell line (Fig. 5A). Subsequently,reverse transcription polymerase chain reaction (RT-PCR) was performed, revealing a significant increase in multiple EMT-inducing transcription factors, including TWIST, ZEBI, SNAIL, and SLUG (Fig. 2A and 2.B). in a time-dependent manner following gemcitabine administration. Moreover, gemcitabine administration significantly enhanced the expression of multiple sternness-regulating genes, such as OCT4. SOX2, MYC, KLF4, BMI1, and CTNNB1, as well as drag resistance-related genes including ABCB1, ABCA1, ABCA3, and ABC A7 (Fig. 2A and 2B). Additionally, there was a notable increase in the expression of PBX1 , reaching approximately 165-fold at 36 hours post-administration, along with canonical PBX1 downstream genes JAK2. STAT3, and mTOR (Fig. 2A and 2B). These results suggest that administration of gemcitabine in CR cells induces a series of genetic changes, which includes the upregulation of EMT, sternness genes, and PBX1 -related pathways, potentially as a protective mechanism.

[0312] To verify the correlation between PBX1 and the upregulation of EMT as well as sternness markers, further RT-PCR was performed (Fig. 2F and 2G). The results demonstrated that all 13 EMT and sternness genes were upregulated in CR cells compared to their parental cell line, while inhibition of PBX1 via shPBXl. ,4409. or B004 abolished this effect. Administration of T417 also partially reversed the effect, albeit less significantly compared to A409 and B004. Consistent with previous experiments, gemcitabine administration induced significant upregulation of PBX1, SLUG. ZEBI. ZEB2, and KLF4 in CR compared to CR without gemcitabine stimulation. However, the levels of TWIST, SNAIL, OCT4, SOX2, MYC, NANOG and CTNNB1 were not significantly increased in the CR+ gemcitabine group compared to CR only (Fig. 2F and 2G). This indicates a positive association between PBX1 and the upregulated EMT and sternness genes in CR cancer cells, and also suggests that SLUG, ZEBI, ZEB2, and KLF4 might serve as major factors in this phenomenon. These findings were further validated through immunofluorescence staining of MIA PaCa-2 CR cells. Treatment with gemcitabine led to an increase in the expression of Pbxl and Nanog, which were effectively inhibited by co-administration of B004. Notably, Nanog, besides its role as a transcription factor regulating sternness properties, also serves as an upstream factor of PBX1 (Fig. 2H). The simultaneous upregulation of Pbxl and Nanog suggests activation of the PBX1 axis, which can be effectively blocked by B004 co-administration. Additionally, gemcitabine induced elevated levels of Zebl and N-cadherin proteins, both vital mesenchymal markers, indicating an augmented EMT response. This response was significantly attenuated by the administration of B004. The ability of B004 to inhibit the gemcitabine-induced increase in Zebl and N-cadherin further underscores the regulatory' relationship between PBXl, Zebl, and N- cadherin (Fig. 21).

[0313] Following this, the inventors investigated the impact of escalating doses of gemcitabine and B004 on CR MIA PaCa-2 cell lines, the inventors' findings revealed a dose-dependent elevation in PBX1, ZEB1, ZEB2. as well as KLF4 and MYC levels subsequent to gemcitabine administration. Consistently, downstream factors of EMT also changed accordingly. showing a dose-dependent increase in mesenchymal markers MMP9 and LEF-1, as well as a dosedependent decrease in epithelial markers COL4A1, SDC1. and NIDI . Moreover, exposure to escalating doses of B004 inhibited the changes induced by gemcitabine in a dose-dependent manner (Fig. 2J and 2K). Altogether, the observations suggest that gemcitabine-resistant pancreatic cancer cells develop an enhanced expression of EMT and sternness genes, which further escalate upon exposure to gemcitabine. This change is regulated by PBXl, which can be significantly inhibited by the administration of PBX1 inhibitors A409 or B004.

[0314] PBXl is functionally linked to the chemo-resistance, metastatic ability, invasiveness, and spherogenic capacity in gemcitabine-resistant pancreatic cancer cells.

[0315] Following the identification of molecular-level changes in CR cells in the above experiments, the inventors aimed to examine their impact on the cellular level. To further confirm the role of PBXl in mediating metastasis and sternness properties, a series of functional assays were conducted in gemcitabine-resistant pancreatic cancer cells. Initially, gemcitabine was administered in increasing doses to MIA PaCa-2 CR and PANC-1 CR cells. The results revealed that gemcitabine administration enhanced the migration speed of the CR cells compared to the control (Fig. 3A). Furthermore, gemcitabine significantly increased the invasiveness (Fig. 3B) and tumor sphere formation ability (Fig. 3C) of CR tumor cells.

[0316] Next. the inventors investigated the effect of PBXl inhibition in CR cells. The inventors' results revealed that PBXl inhibitors T417, A409. and B004 effectively suppressed the migration and invasion of CR pancreatic cancer cells. Notably. A409 and B004 exhibited a stronger capacity to inhibit CR ceils compared to T417 (Fig 3D-F). To further validate the effect of PBXl inhibitors on the inhibition of sternness properties, wound healing assays were conducted on highly metastatic cancer stem cells (MCSC) from three different cancer types: colorectal cancer (DLD-1 ), pancreatic cancer (MIA PaCa-2), and lung cancer (PC9). The results showed that MCSC cells displayed enhanced migration ability compared to their respective parental wild-type cell lines Conversely, A409 and B004 significantly suppressed the migration of MCSC ceils in all three cancer models, reducing their migration capacity ev en lower than that of the wild-type cells (Figs. 3G-S1-B to 3G-S1-G). Finally, the inventors assessed tumorigenic capacity using tumor sphere formation and soft agar assays. The inventors observed that CR cells exhibited an enhanced capacity to form tumor spheres compared to theirT54267 / PC0704 parental wild-type cell lines. In contrast, inhibition of PBX1 via PBXl inhibitors T417. A409, and B004 successfully suppressed tumori genesis in CR ceils in both tumor sphere (Fig. 3I-L) and soft agar assays (Fig. 3M-O).

[0317] PBX1 Inhibitors A409 and B004 repress the survival of chemo-resistant cancer cells while demonstrating a synergistic effect with multiple chemotherapy drugs.

[0318] While PBX1 is recognized for its involvement in the survival and proliferation of various cancer types, its role in maintaining CR cells within the tumor population remains unexplored. Therefore, the inventors aimed to determine whether targeting PBX1, either as a single drug or in combination with existing chemotherapy agents, holds the key to reversing chemoresistance, The inventors initially conducted survival analysis on PT, CR. and CR cells with PBX1 knockdown in MIA PaCa-2 and AsPC-1 cells. The results revealed that CR cells exhibited greater resistance to gemcitabine compared to their parental counterparts. Conversely, targeting PBX1 in CR cells, either through knockdown with shPBXl RNA or via treatment with B004, effectively sensitized the CR cells to a survi val response similar to that of the parental cells (Fig 4A and 4B). Under microscopic examination, the inventors found that administration of gemcitabine induces the cells to acquire a more elongated spindle shaped morphology, while the cells administered B004 did not experience this change (Fig. 4B). The transition from an epithelial to mesenchymal phenotype is consistent with the upregulation of EMT characteristics induced by gemcitabine, as observed in the aforementioned experiments. These results were further validated by colony-forming assays, w hich revealed an increased colony -forming capacity in CR cells resistant to gemcitabine treatment. PBX1 knockdown or B004 treatment effectively overcame the resistance in CR cells and sensitized them to gemcitabine (Fig. 4C and Fig. 3G-S1-H). The efficacy of PBX1 inhibitors in suppressing CR cancer was then compared. In both MIA PaCa-2 CR and AsPC-1 CR cell lines, gemcitabine exhibited minimal effect in suppressing tumor growth, with IC50 values of 249.7 pM and 648.9 uM. respectively, while PBX1 inhibitors demonstrated the ability to suppress tumor survival at much lower doses. Specifically, the IC50 values of T417, A409. and B004 were 102.7, 68.63, and 30.47 in MIA PaCa-2 CR, and 84.83, 17.67, and 17.93 in AsPC-I CR. respectively (Fig, 4D and 4E). Notably, A409 and B004 showed greater efficacy than T417, highlighting their superior effectiveness in overcoming chemoresistance m pancreatic cancer.

[0319] To evaluate the influence of gemcitabine and B004 treatment over time, a tumor recurrence model of CR pancreatic cancer cell lines was employed using 3D colony culture on ultra-low attachment plates. MIA PaCa-2 CR and PANC-1 CR cells were initially treated with low doses of gemcitabine (50nM), gemcitabine (50nM) + B004 (lOOnM), or control DMSO forfive consecutive days. Subsequently, after a two-day drug withdrawal period, one group originally treated with gemcitabine alone and gemcitabine + B004 received DMSO for another five days. Meanwhile, another group initially treated with gemcitabine alone was given a low dose of' B004 (l OOnM) for five days (Fig. 4F) The results revealed a notable pattern: while tumors initially reduced in size with gemcitabine treatment for 5 days, they eventually relapsed and significantly increased in size after drug withdrawal. However, administering B004 for an additional 5 days after drug withdrawal effectively prevented this relapse. Furthermore, initial treatment with gemcitabine combined with B004 resulted in continued reduction in tumor size even after drug withdrawal, suggesting a potential residual tumor inhibiting effect with combination therapy. Survival analysis, measured using a luminescent ATP-based cell viability assay at experiment endpoint, demonstrated that post-treatment with B004 after drag withdrawal or initial treatment with gemcitabine + B004 significantly prevented relapse occurrence (Fig. 4F). Overall, these findings underscore the potential of B004 in preventing tumor relapse after gemcitabine treatment withdrawal, both as a standalone post -treatment and in combination therapy.

[0320] To assess the combination effect of PBXI inhibitors with existing chemotherapy drugs and determine the optimal dosage for combination therapy, a series of drug combination assays were conducted These experiments were performed on chemotherapy-resistant (CR) cell lines from six cancer types: pancreatic (MIA PaCa-2 CR and AsPC-1 CR), colorectal (DLD-1 CR), breast (MDA-MB-231 CR), multiple myeloma (RPMI-8226 CR), liver (HepG2 / C3a CR), and lung (HI299 CR) cancer. The objective was to evaluate whether administering PBXI inhibitors would sensitize the CR cancer cells to the chemotherapeutic agents, including gemcitabine, regorafenib, olaparib, lenalidomide, sorafenib, and tagrisso. The combination index plots of T417, A409, and B004 were exhibited in Fig. 4G. It was found that T417, A409, and B004 exhibited synergism with most tested agents (Fig 4G). Particularly, B004 demonstrated the strongest synergism with gemcitabine and olaparib at a range of low doses, with a combination index below 0.1, indicating "very strong synergism." Several doses contributing to this very strong synergism were at a nanomolar scale, suggesting the potential to reduce the effective dose of both drugs while generating a very strong effect when administered simultaneously (Fig. 4H-M).

[0321] PBXI Inhibitors A409 and B004 suppress tumor growth and metastasis in xenograft chemo-resistant zebrafish models.

[0322] To further explore the role of PBX1 in promoting tumor cel! invasion, dissemination, and metastasis in chemo-resistant cancers, the inventors established a zebrafish tumor model ofmetastasis. In this model, chemo-resistant human tumor cells labeled with CM-Dil dye were implanted into 48-hour post-fertilization Tg(flil :EGFP) zebrafish embryos and monitored for a duration of 6 days. Five human cancer cell lines from four different cancer types were cultured in vitro to develop resistance to commonly used chemotherapy drugs. These included two pancreatic cancer ceil lines (AsPC-1 and MIA PaCa-2), one breast cancer cell tine (MDA-MB- 231). one liver cancer cell line (Huh7), and one colorectal cancer cell line (DLD-1). The chemotherapy drugs used for inducing resistance were gemcitabine, olaparib, sorafenib, and regorafeni b, respectively Remarkably, foci of metastases were observable in the CR control group on day 6 post-tumor injection across all five tumor models Consistent with the findings of the in vitro experiments, administration of chemotherapy drugs exacerbated metastases in all five zebrafish CR tumor models, as evidenced by a significantly increased number of disseminated tumor foci and maximal distance of metastasis on day 6 (Fig 5A-C, 5F-J, 5M-O, 5R-T, and 5W-Y). Notably, the total tumor load, as indicated by the ratio of tumor size to body size, did not exhibit significant differences between the gemcitabine group and the control group (Fig. 5D, 5K, 5P, 5U, and 5Z). It is noteworthy that the toxicity of certain chemotherapy drugs, specifically sorafenib and regorafenib, was notably high, leading to the inability of zebrafish embryos implanted with tumor cells to sun w c until day 6. Consequently, the results pertaining to DLD-1 CR treated with regorafenib were omitted, while the findings related to Huh7 CR treated with sorafenib were only presented for doses of lOOnM, 50nM. and 25nM, as these were the highest tolerated doses in the inventors' model. Notably, administration of sorafenib in sorafenib-resistant Huh? cells exacerbated metastasis compared to control conditions. Interestingly, a dose-dependent trend was observed, indicating that increasing doses of sorafenib resulted in more significant metastasis (Fig. 5R-T).

[0323] In contrast, PBXl inhibitors effectively suppressed metastasis in CR tumor cells and led to a marked reduction in the average size of primary' tumors (Fig. 5A-D, 5F-K, 5M-P, 5R-Q, and 5W-Z). Particularly noteworthy, among the PBXl inhibitors evaluated, B004 exhibited the strongest anti -metastatic and anti-tumor effects, nearly eradicating tumor signals in the MIA PaCa-2, AsPC-l, Huh7, and DLD-1 CR models. The effect of combining B004 with a chemotherapeutic drug was assessed in the CR MDA-MB-231 model, given the favorable combination index observed between B004 and Olaparib in previous in vitro testing. 5 pM B004 was co-administered with 5 pM Olaparib, resulting in the near-complete elimination of both the primary tumor and metastatic cells (Fig. 5M-Q). The survival of the zebrafish was monitored daily. In four out of the five CR cancer models, the group treated with B004 demonstrated the highest survival rate at the end of the experiments, which terminated on day 6 post-implantation of each tumor cell (Fig. 5E, 5L, 5V. and 5AA). In the MDA-MB-231 model, the T417 group. A409 group, and B004+01aparib group all achieved a survival rate of 100%,while the B004 group exhibited a survival rate of 80% (Fig, 5Q). These findings present compelling evidence that targeting PBX1 with A409 and B004 effectively reverses the chemotherapy-induced EMI' in chemo-resistant cancer cells.

[0324] PBX1 Inhibitors A409 and B004 suppress tumor growth and metastasis in xenograft chemo-resistant murine models.

[0325] To investigate PBXl 's role in mediating chemoresistance in vivo, the inventors first established a subcutaneous tumor model of regorafenib-resistant DLD-1 (DLD-1 CR) in six- week-old NOD-scid ILlrynull (NSG) mice Subsequently, mice were treated with various agents, including control vehicle, regorafenib at 5mg / kg, T417 at 5mg / kg. A409 at 5mg / kg, B004 at 5mg / kg, or a combination of regorafenib at 5mg / kg with B004 at 5mg / kg, administered three times a week for 5-6 weeks. Consistent with the inventors' in vitro findings, the DLD- 1 CR cell line exhibited resistance to regorafenib treatment, as evidenced by minimal differences in tumor growth between the control group and the regorafenib-treated group (p>0.05) (Fig. 6A-C). In contrast, treatment with T417, A409, B004, or regorafenib+B004 significantly suppressed tumor growth (Fig. 6A-C). Immunofluorescence staining of tumor tissues revealed no significant differences in TUNEL and cleaved caspase 3 signals between the control and regorafenib-treated groups (p > 0.05). However, targeting PBX1 with T417, A409. B004, or regorafenib+B004 led to significant increases in both TUNEL and cleaved caspase 3 signals, indicating heightened DNA fragmentation and activation of caspase-mediated apoptotic pathways, respectively (Fig. 6D and E). These findings validate the increased apoptosis in tumors treated with PBX1 inhibitors and underscore their potential to overcome chemotherapy- induced apoptosis resistance in resistant cancer cell lines.10326 ] The inventors performed the following experiments to assess whether treatment with I’BXl inhibitors alone or in combination with gemcitabine affects metastasis in CR tumorbearing mice. To enable in vivo imaging of AsPC-1 CR cells, the inventors transfected the cells with a luciferase (Luc) expression vector and established stable clones. These AsPC-1 CR-Luc cells were then orthotopically injected into the pancreases of NSG mice. One-week post-tumor cell inoculation, the tumor-bearing mice received treatment with either control vehicle, T417, A409, B004, or B004 + gemcitabine three times a week for 5-6 cycles. Monitoring tumor growth and metastasis using the IVIS® Lumina XRMS imaging system revealed that T417 did not effectively decrease the metastasis of AsPC-1 CR-Luc cells compared to the control group. IVIS images and autopsy findings demonstrated the presence of metastatic tumor cells in the colon, liver, and peritoneum of mice from both groups at the fifth week post-tumor inoculation (Fig. 6F and G). In contrast, targeting PBX1 with A409, B004, or B004 -1- gemcitabinesignificantly inhibited tumor growth and metastasis, with B004 and B004 + gemcitabine exhibiting the strongest effect in suppressing the ROI value on 1 VIS (Fig. 6F and G). Consistent with the aforementioned findings, IHC staining of primary tumor samples also revealed that cleaved caspase 3 was significantly upregulated in the groups treated with A409, B004, or B004 + gemcitabine (Fig. 6H and I). In contrast, the control and T4l7-treated groups exhibited a less prominent level of cleaved caspase 3, indicating a lower degree of apoptosis in the primary tumors (Fig 6H and I). Furthermore, blood samples were collected from the mice during sacrifice at week 5 No significant changes in the biochemistry profile were observed among the mice treated with the drug However, a non-significant increase in free liver enzymes, AST and ALT, was observed in the control and T417 groups, likely attributed to liver metastasis in these groups (Fig. 6J). Regarding the hematological profile, an increased platelet count was found in the B004 + gemcitabine treated group compared to the control (Fig. 6J); however, the values remained within normal limits (10).

[0327] In summary, the inventors’ findings demonstrate that pharmaceutical inhibition of PBXI with A409 and B004 effectively hinders the growth of CR colorectal cancer cells and the metastasis of CR pancreatic cancer cells. Among the currently available agents that directly target PBXI . B004 exhibits the most potent anti-tumor and anti-metastasis effects, demonstrating efficacy whether administered as a single drug or in combination with gemcitabine.

[0328] PBXI inhibitors .4409 and B004 exhibit exceptional safety profiles in vitro and in vivo.

[0329] A series of examinations were conducted to test the safety of A409 and B004. First, the inventors evaluated the toxicity of T417, A409, and B004 on a normal human fibroblast cell line, comparing them to other commonly used chemotherapy drugs such as regorafenib, sorafenib, iagrisso, lenalidomide, and gemcitabine. The IC50 values for T417, A409, and B004 were 31.10, 44.26, and 98.58 pM, respectively, which were notably higher than the IC50 values of the tested chemotherapy agents, ranging from 5.928 to 19.44 uM Additionally, the IC50 values ofT417, A409, and B004 exceeded the therapeutic doses (5 pM) at which tumor invasion, migration, and spherogenesis were effectively inhibited in previous in vitro experiments. Interestingly, the inventors observed that at relatively low doses, T417, A409, and B004 paradoxically stimulated the growth of fibroblasts, suggesting a potential protective effect of PBX1 inhibitors on benign cells at therapeutic doses (Fig. 7 A). To ascertain the maximum tolerated doses of A409 and B004 in vivo, the inventors proceeded to assess the tolerability- of zebrafish embryo to T417. A409, B004, and chemotherapy drugs, including regorafenib,sorafenib, tagrisso, lenalidomide, and gemcitabine. Zebrafish at 5 days post-fertilization (dpf) were exposed to escalating concentrations, ranging from 0 to 512 pM, of the aforementioned drugs, with each concentration tested on 10 zebrafish. Subsequently, their morphology and survival were monitored after 24 hours of exposure. The results revealed that T4 I 7, A409, and B004 exhibited the highest survival rates at high concentrations compared to all tested chemotherapy drugs (Fig. 7B and C). The PBX1 inhibitors achieved a 100% survival rate at 256 pM, equivalent to 102 times the effective dose (Fig. 7B and C) In contrast, most chemotherapy drugs displayed decreased survival rates starting from 8 uM. with a high proportion of exposed zebrafish embryos exhibiting various morphological abnormalities (Fig. 7D). Subsequently, the safety ofPBXl inhibitors at high doses was assessed in murine models. Six-week-old NSG mice were administered 100 times the effective doses of T417, A409. and B004, along with regorafenib, gemcitabine, lenalidomide, or olaparib via intraperitoneal injection. The weight of each mouse was recorded at both baseline and endpoint, which was 3 weeks post-treatment. Additionally, autopsy results of vital organs, hematological profiles, and biochemistry profiles were obtained at the experiment endpoint. The results revealed no identifiable change in the gross appearance of vital organs compared to the control group (Fig. 7E). Most tested mice erhibiied increased body weight over the course of 3 weeks, except those treated with gemcitabine, which experienced a weight loss of about 2g (Fig. 7F). The biochemistry profile showed no notable abnormalities in most groups, except for those treated with gemcitabine and lenalidomide, which demonstrated a significantly increased liver enzyme AST (Fig. 7G). Hematological profiles displayed no notable abnormalities in all groups (Fig. 7H). Upon microscopic examination, organs from mice treated with high dose T417, A409, and B004 showed no notable changes compared to the control (Fig. 71). In contrast, liver biopsies from mice treated with lenalidomide, regorafenib, and gemcitabine exhibited patched areas of degeneration, consistent with the increased liver enzyme levels observed in blood examinations (Fig. 7J). Collectively, A409 and B004 exhibited an exceptional safety profile in both in vitro and in vivo examinations. The inventors' results indicated that A409 and B004 have a wide safety margin, as they did not cause identifiable damage to normal organs even at 100 times the therapeutic doses.

Claims

What is claimed is: (A):or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomer thereof, whereinX rmY is selected from the group consisting of -CH2-CH2-, -CH:::CH-, -O-CH2-, -NH-CH2-,Ri is independently selected from the group consisting of H, halogen, nitro, cyano, Ci-Cs alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and Cj-Ce alkoxyl, wherein Ci-Ce alkyl, Cz-Ce alkenyl, C2-C6 alkynyl, or Ci-Cs alkoxyl is independently unsubstituted or substituted with cyano, nitro, or halogen;R2 is independently selected from the group consisting of H, halogen, nitro, cyano, Ci-Ct, alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and Ci-C« alkoxyl, wherein Cj-Cc alkyl, Cs-Ce alkenyl, C2-C6 alkynyl, or Ci-Ce alkoxyl is independently unsubstituted or substituted with cyano, nitro, or halogen;Rs is independently selected from the group consisting of H, halogen, nitro, cyano, Ci-Cs alkyl, hydroxyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-Ck, alkoxyl, carboxylic acid, ester, carbonate, phosphate, sulfonate, amino, amide, imine, and carbamate, wherein Ci-Ce alkyl, C2-C6 alkenyl, C2-C6 alkynyl, or Ci-Ce alkoxyl is independently unsubstituted or substituted with cyano, nitro, or halogen; b is independently 0, 1, 2, 3 or 4, c is independently 0, 1, 2, 3, 4 or 5: and d is independently 0, 1 , 2, 3 or 4.

2. The compound according to claim 1. wherein the compound has formula (B):wherein X, Y. R], R?„ b and d are defined as Claim 1, or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomer thereof. g to claim 1. wherein the compound has formula (C):wherein X, Y, Ri, Re, c and d are defined as Claim 1, or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomer thereof4. The compound according to claim 1 . wherein the compound has formula (D):T54267 / PC0704wherein R.i and d are defined as Claim 1, or a pharmaceutically acceptable salt, solvate. prodrug or stereoisomer thereof.

5. A compound according to claim 1 , selected from the group consisting of:or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomer thereof.

6. A pharmaceutical combination comprising the compound of any of claims I to 5 or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomer thereof, and a pharmaceutically acceptable excipient.

7. The pharmaceutical combination of claim 6, which further comprises an additional therapeutic agent drug.8 The pharmaceutical combination of claim 7, wherein the additional therapeutic agent is an additional anti cancer drug: preferably, a chemotherapy drag.

9. The pharmaceutical combination of claim 8, wherein the chemotherapy drug is platinum compounds (such as cisplatin, carboplatin), antimetabolite drugs (such as 5 -fluorouracil, cytarabine, gemcitabine, pentostatin and methotrexate), anthracycline drug which targets DNA (such as doxorubicin and epirubicm), drugs which target DNA or drugs which target topoisomerases or other chemotherapy drugs.

10. The pharmaceutical combination of claim 8, wherein the chemotherapy drug is cisplatin, docetaxel, paclitaxel, gemcitabine, navelbine, doxorubicin, irinotecan, gemcitabine, regorafenib, olaparib, lenalidomide, sorafenib, tagrisso or 5-11 uorodeoxyuri dine1 1. A nanocarrier comprising the compound of any of claims 1 to 5 or a pharmaceuticallyT54267 / PC0704 acceptable salt, solvate, prodrag or stereoisomer thereof.12 The nanocarrier of claim 11, which is a liposome or a micelle.

13. The nanocanier of claim 11 or 12, which further comprises an additional therapeutic agent.

14. The nanocamer of claim 13. wherein the additional therapeutic agent is an additional anticancer drug; preferably, a chemotherapy drug.

15. The nanocarrier of claim 14. wherein the chemotherapy drag is platinum compounds (such as cisplatin, carboplatin), antimetabolite drugs (such as 5 -fluorouracil, cytarabine, gemcitabine, pentostatin and methotrexate), anthracycline drug which targets DNA (such as doxorubicin and epirubicin), drugs which target DNA or drugs which target topoisomerases or other chemotherapy drugs.

16. The nanocarrier of claim 14, wherein the chemotherapy drug is cisplatin, docetaxel, paclitaxel, gemcitabine, navelbine, doxorubicin, irinotecan, gemcitabine, regorafenib, olaparib, lenalidomide, sorafenib, tagrisso or 5-fluorodeoxyuridme.

17. A method for inhibiting PBX1-DNA interaction in a cell, comprising contacting the cell with the compound of any of claims 1 to 5.

18. A method for treating or preventing a disease associated with inhibition of PBX1-DNA interaction m a subject, which comprises administrating a therapeutically effective amount of a compound of any one of Claims 1 to 5 to the subject.

19. The method according to claim 18, wherein the disease is a cancer, cancer invasion, cancer metastasis and chemotherapy drug-resistant cancer20. The method according to claim 19, wherein the cancer is a multi -drug resistant cancer, recurrent cancer, refractory cancer or relapsed cancer.

21. The method according to claim 18 or 19, wherein the cancer is a pancreatic cancer, esophageal cancer, glioma, ovarian cancer, multiple myeloma, colorectal cancer, lung cancer, liver cancer, breast cancer, gastric cancer, lung cancer, head and neck cancer, prostate cancer, testicular, intestinal cancer, bladder cancer, or urothelial cancer.

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