Compositions and methods for treating cancer

Abstract

The present invention relates to the compound N-(2-methylpropyl)-4,5-dihydro-1,3-oxazol-2-amine or a pharmaceutically acceptable salt thereof, for treatment of integrin alpha5beta1 overexpressing cancers, such as triple-negative breast cancer. Further disclosed are methods of making and using the compound, as well as methods of formulating the compound in a pharmaceutical composition, and methods of utilization of the pharmaceutical composition for cancer treatment.

Description

Docket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025 COMPOSITIONS AND METHODS FOR TREATING CANCER

[0001] This application claims priority from U.S. Provisional Application No.63 / 687,441, filed on August 27, 2024, the entire contents of which are incorporated herein by reference.

[0002] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records but otherwise reserves any and all copyright rights. FIELD OF THE INVENTION

[0004] The present invention relates to a composition and method thereof for treatment of triple-negative breast cancer. BACKGROUND OF THE INVENTION

[0005] Breast cancer is an umbrella term for a highly heterogenous disease with several subtypes, each with distinct histology and pathologies. It remains the most common cancer among women in the US. Triple-negative breast cancer (TNBC) is the most aggressive and challenging subtype of breast cancer to treat, representing approximately 10-20% of all new breast cancer cases and contributing significantly to breast cancer mortality. TNBC does not rely on hormones for tumor progression or nor does it have over-expressed histological features to exploit for treatment. As a result, traditional therapies are ineffective against TNBC. SUMMARY OF THE INVENTION

[0006] Aspects of the disclosure are drawn towards a compound of formula (I):(I), or a stereoisomer, a tautomer, a solvate, a hydrate, or a salt thereof. In embodiments, the salt is selected from the group consisting of an a hydrochloride salt of formula (I), a sodium salt ofDocket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025 formula (I), a sulfate salt of formula (I), an acetate salt of formula (I), a phosphate salt of formula (I), a fumarate salt of formula (I), a potassium salt of formula (I), a calcium salt of formula (I), a citrate salt of formula (I), a maleate salt of formula (I), a mesylate salt of formula (I), a tartrate salt of formula (I), a gluconate salt of formula (I), a succinate salt of formula (I), or any combination thereof.

[0007] Aspects of the disclosure are drawn towards a pharmaceutical composition comprising a compound of formula (I):(I), or a stereoisomer, a tautomer, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable excipient. In embodiments, the pharmaceutically acceptable salt is selected from the group consisting of an a hydrochloride salt of formula (I), a sodium salt of formula (I), a sulfate salt of formula (I), an acetate salt of formula (I), a phosphate salt of formula (I), a fumarate salt of formula (I), a potassium salt of formula (I), a calcium salt of formula (I), a citrate salt of formula (I), a maleate salt of formula (I), a mesylate salt of formula (I), a tartrate salt of formula (I), a gluconate salt of formula (I), a succinate salt of formula (I), or any combination thereof. In embodiments, the compound of formula (I) is formulated for oral administration, parenteral administration, intravenous administration, or intramuscular administration.

[0008] Aspects of the disclosure are drawn towards a method of treating a subject afflicted with cancer, the method comprising administering a therapeutically effective amount of a compound of formula (I) or a pharmaceutical composition comprising a compound of formula(I), or a stereoisomer, a tautomer, a hydrate, or a pharmaceutically acceptable salt thereof, to a subject in need thereof. In embodiments, the cancer is an integrin α5β1 overexpressing cancer. In embodiments, administering the compound activates nischarin expression. In embodiments, the integrin α5β1 overexpressing cancer comprises breast cancer, cervical cancer, pancreatic cancer, ovarian cancer, renal cell carcinoma, prostate cancer, bladder cancer, gastric cancer,Docket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025 melanoma, colorectal cancer, lung cancer, brain cancer or any combination thereof. In embodiments, the breast cancer is triple-negative breast cancer. In embodiments, treating cancer comprises preventing cancer metastasis, reducing tumor size, or a combination thereof. In embodiments, the pharmaceutically acceptable salt is selected from the group consisting a hydrochloride salt of formula (I), a sodium salt of formula (I), a sulfate salt of formula (I), an acetate salt of formula (I), a phosphate salt of formula (I), a fumarate salt of formula (I), a potassium salt of formula (I), a calcium salt of formula (I), a citrate salt of formula (I), a maleate salt of formula (I), a mesylate salt of formula (I), a tartrate salt of formula (I), a gluconate salt of formula (I), a succinate salt of formula (I), or any combination thereof. In embodiments, the compound is administered orally, parenterally, intravenously, or intramuscularly. In embodiments, the compound is administered to a subject in need thereof in a dosage of about 1 mg / kg to about 50 mg / kg. In embodiments, the dosage is administered about once daily, about twice daily, about three times daily, about four times daily, or about five times daily.

[0009] Aspects of the disclosure are drawn towards the use of a therapeutically effective amount of a compound of formula (I)(I), or a stereoisomer, a tautomer, a hydrate, or a pharmaceutically acceptable salt thereof, to treat a subject afflicted with cancer. In embodiments, the cancer is an integrin α5β1 overexpressing cancer. In embodiments, administering the compound activates nischarin expression. In embodiments, the integrin α5β1 overexpressing cancer comprises breast cancer, cervical cancer, pancreatic cancer, ovarian cancer, renal cell carcinoma, prostate cancer, bladder cancer, gastric cancer, melanoma, colorectal cancer, lung cancer, brain cancer or any combination thereof. In embodiments, the breast cancer is triple-negative breast cancer. In embodiments, treating cancer comprises preventing cancer metastasis, reducing tumor size, or a combination thereof. In embodiments, the pharmaceutically acceptable salt is selected from the group consisting a hydrochloride salt of formula (I), a sodium salt of formula (I), a sulfate salt of formula (I), an acetate salt of formula (I), a phosphate salt of formula (I), a fumarate salt of formula (I), a potassium salt of formula (I), a calcium salt of formula (I), a citrate salt of formula (I), a maleate salt of formula (I), a mesylate salt of formula (I), a tartrate salt of formula (I), a gluconate salt of formula (I), a succinate salt of formula (I), or any combination thereof. In embodiments, the compound is administered orally, parenterally, intravenously, orDocket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025 intramuscularly. In embodiments, the therapeutically effective amount comprises a dosage of about 1 mg / kg to about 50 mg / kg. In embodiments, the dosage is administered about once daily, about twice daily, about three times daily, about four times daily, or about five times daily.

[0010] Aspects of the disclosure are drawn towards the use of a therapeutically effective amount of a compound of formula (I)(I), or a stereoisomer, a tautomer, a hydrate, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament to treat a subject afflicted with cancer.

[0011] Other objects and advantages of this invention will become readily apparent from the ensuing description. BRIEF DESCRIPTION OF THE FIGURES

[0012] FIG. 1 shows non-limiting, exemplary graphs of NISCH expression and the effects thereof. Panel A shows a non-limiting, exemplary graph showing coding mRNA data for NISCH expression across BC patient tissues from TCGA. Panel B shows non-limiting, exemplary TCGA data grouped relating Overal Survival (OS) in months (mo) to either high NISCH expression (red) or low NISCH expression (blue).

[0013] FIG.2 shows non-limiting, exemplary graphs and images of the effects of Rilmenidine and DP-120 on NISCH expression Panel A shows non-limiting, exemplary data comparing Nischarin and Vinculin expression for various cell lines and tissue types, indicating the decreased NISCH expression in cancer cells. Panel B shows a non-limiting, exemplary graph indicating cell viability (%) as a function of drug concentration [µM] for both Rilmenidine and the analog DP-120, indicating a decreased cancer cell viability in the presence of DP-120 as opposed to Rilmenidine at all concentrations tested. Panel C shows non-limiting, exemplary data comparing Nischarin and Vinculin expression at varying concentrations of Rilmenidine and DP-120. Panel D shows non-limiting, exemplary images of mice xenograft models implanted with MDA-MB cells in both the mammary fat pad and the flank.

[0014] FIG.3 shows non-limiting, exemplary images of molecular docking models. Panel A shows a non-limiting, exemplary image of a close-up molecular docking model of DP-120 inDocket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025 the NISCH binding pocket, with the purple ribbon indicating leucine-rich repeats (amino acids:286-410). Amino acids in the binding pocket and nearby important amino acids are shown in stick model and hydrogen bon interaction are shown with yellow dashed lines. Panel B shows a non-limiting, exemplary image of a close-up molecular docking model of Rilmenidine in the NISCH binding pocket, with the purple ribbon indicating leucine-rich repeats (amino acids:286-410). Amino acids in the binding pocket and nearby important amino acids are shown in stick model and hydrogen bon interaction are shown with yellow dashed lines. Panel C shows a non-limiting, exemplary image of a close-up molecular docking model of Moxonidine in the NISCH binding pocket, with the purple ribbon indicating leucine-rich repeats (amino acids:286-410). Amino acids in the binding pocket and nearby important amino acids are shown in stick model and hydrogen bon interaction are shown with yellow dashed lines.

[0015] FIG. 4 shows non-limiting, exemplary images of space-filling models and skeletal structures. Panel A shows a non-limiting, exemplary image of a space-filling model (top) and skeletal structure (bottom) of moxonidine with calculated cLogP and tPSA. Panel B shows a non-limiting, exemplary image of a space-filling model (top) and skeletal structure (bottom) of rilmenidine with calculated cLogP and tPSA. Panel C shows a non-limiting, exemplary image of a space-filling model (top) and skeletal structure (bottom) of DP-120 with calculated cLogP and tPSA.

[0016] FIG. 5 shows non-limiting, exemplary data indicating the dose response of lab synthesized Rilmenidine (DP-144) in the presence of a three triple-negative-breast-cancer cell lines (MDAMB231, MDAMB468, and SUM159PT), and Human Mammary Epithelial Cells (HMLE) with test performed with an n=3.

[0017] FIG.6 shows non-limiting, exemplary data indicating the dose response of DP-120 in the presence of four different triple-negative breast cancer cell lines (MDAMB231, LM2-4175, MDAMB468, and SUM159PT), and Human Mammary Epithelial Cells (HMLE) with tests performed with an n=3.

[0018] FIG. 7 shows non-limiting, exemplary images indicating the results of a clonogenic assay conducted comparing Rilmenidine and DP-120.

[0019] FIG. 8 shows a non-limiting, exemplary graph showing the results of an MTT assay indicating the ability of Rilmenidine, Moxonidine, DP-144 (lab synthesized Rilmenidine), DP- 120, and DP-93 to impact the viability of MDAMB468 cells.

[0020] FIG. 9 shows a non-limiting exemplary1HNMR spectrum of N-2-chloroethyl-N- isobutyl urea.Docket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025

[0021] FIG.10 shows a non-limiting exemplary1HNMR spectrum of DP-120.

[0022] FIG.11 shows a non-limiting exemplary FTIR spectrum of DP-120.

[0023] FIG.12 shows non-limiting exemplary mass spectra of DP-120. DETAILED DESCRIPTION OF THE INVENTION

[0024] Detailed descriptions of one or more embodiments are provided herein. It is to be understood, however, that the invention can be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the invention in any appropriate manner.

[0025] The singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0026] Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly, “an example,” “exemplary” and the like are understood to be nonlimiting.

[0027] The term “substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited.

[0028] The terms “comprising” and “including” and “having” and “involving” (and similarly “comprises”, “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a, b, and c” means that the process includes at least steps a, b and c. Wherever the terms “a” or “an” are used, “one or more” is understood, unless such interpretation is nonsensical in context.

[0029] As used herein, the term “about” can refer to approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower). In embodiments, the term “about” can be denoted by “~”.Docket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025

[0030] As used herein, the term “substantially the same” or “substantially” can refer to variability typical for a particular method is taken into account.

[0031] The terms “sufficient” and “effective”, as used interchangeably herein, can refer to an amount (e.g., mass, volume, dosage, concentration, and / or time period) needed to achieve one or more desired result(s).

[0032] Before explaining at least one embodiment of the disclosure in detail, it is to be understood that the disclosure is not necessarily limited in its application to the details set forth in the following description or exemplified by the examples. The disclosure can be used for other embodiments or of being practiced or carried out in various ways. Other compositions, compounds, methods, features, and advantages of the disclosure will be or become apparent to one having ordinary skill in the art upon examination of the following drawings, detailed description, and examples. All such additional compositions, compounds, methods, features, and advantages can be included within this description, and be within the scope of the disclosure.

[0033] The term "alkyl" refers to the radical of saturated aliphatic groups, including straight- chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl- substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups.

[0034] In some embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30for straight chains, C3-C30for branched chains), 20 or fewer, 12 or fewer, or 7 or fewer. Likewise, in some embodiments cycloalkyls have from 3- 10 carbon atoms in their ring structure, e.g., have 5, 6 or 7 carbons in the ring structure. The term "alkyl" (or "lower alkyl") as used throughout the specification, examples, and claims can include both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to alkyl moieties having one or more substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents include, but are not limited to, halogen, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, a hosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety.

[0035] Unless the number of carbons is otherwise specified, "lower alkyl" as used herein can refer to an alkyl group, as defined herein, but having from one to ten carbons, or from one to six carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl"Docket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025 have similar chain lengths. In some embodiments, alkyl groups are lower alkyls. In some embodiments, a substituent described herein as alkyl can be a lower alkyl.

[0036] It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For instance, the substituents of a substituted alkyl can include halogen, hydroxy, nitro, thiols, amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN and the like. Cycloalkyls can be substituted in the same manner.

[0037] The term “heteroalkyl”, as used herein, can refer to straight or branched chain, or cyclic carbon-containing radicals, or combinations thereof, containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P, Se, B, and S, wherein the phosphorous and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized. Heteroalkyls can be substituted as defined herein for alkyl groups.

[0038] The term "alkylthio" can refer to an alkyl group, as defined herein, having a sulfur radical attached thereto. In some embodiments, the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, and -S-alkynyl. Representative alkylthio groups include methylthio, and ethylthio. The term “alkylthio” also encompasses cycloalkyl groups, alkene and cycloalkene groups, and alkyne groups. “Arylthio” refers to aryl or heteroaryl groups. Alkylthio groups can be substituted as defined herein for alkyl groups.

[0039] The terms "alkenyl" and "alkynyl", refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described herein, but that contain at least one double or triple bond respectively. As used herein, the term “alkenyl” can refer to an unsaturated branched, straight-chain, or cyclic alkyl radical having at least one carbon-carbon double bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkene. The group can be in either the cis or trans conformation about the double bond(s). In embodiments described herein, the alkenyl group can be C2-C13 alkenyl. Non- limiting examples of alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, etc.

[0040] The terms "alkoxyl" or "alkoxy" as used herein can refer to an alkyl group, as defined herein, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, and tert-butoxy. An "ether," for example, can be two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -O-Docket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025 alkyl, -O-alkenyl, and -O-alkynyl. Aroxy can be represented by -O-aryl or O-heteroaryl, wherein aryl and heteroaryl are as defined herein. The alkoxy and aroxy groups can be substituted as described herein for alkyl.

[0041] As used herein, the term “halogen” can refer to -F, -Cl, -Br or -I; the term "sulfhydryl" can refer to -SH; the term "hydroxyl" can refer to -OH; and the term "sulfonyl" can refer to -SO2-.

[0042] The term “substituted” as used herein, can refer to permissible substituents of the compounds described herein. In the broadest sense, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, but are not limited to, halogens, hydroxyl groups, or any other organic groupings containing any number of carbon atoms, for example 1-14 carbon atoms, and optionally include one or more heteroatoms such as oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclic structural formats. Representative substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl, C3-C20 cyclic, substituted C3-C20cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, and polypeptide groups. As used herein in reference to an “R” group, the name used to describe said “R” group can be the chemical name prior to the removal of a hydrogen. For example, wherein “R” is described as an “alkane” can refer to an “alkyl” group.

[0043] Heteroatoms such as nitrogen can have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. It is understood that “substitution” or “substituted” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

[0044] In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents ofDocket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025 organic compounds. Illustrative substituents include, for example, those described herein. The permissible substituents can be one or more and the same or different for appropriate organic compounds. Heteroatoms such as nitrogen can have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms.

[0045] In various aspects, the substituent can be selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone, each of which optionally is substituted with one or more suitable substituents. In some embodiments, the substituent is selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone, wherein each of the alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone can be further substituted with one or more suitable substituents.

[0046] Examples of substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, thioketone, ester, heterocyclyl, -CN, aryl, aryloxy, perhaloalkoxy, aralkoxy, heteroaryl, heteroaryloxy, heteroarylalkyl, heteroaralkoxy, azido, alkylthio, oxo, acylalkyl, carboxy esters, carboxamido, acyloxy, aminoalkyl, alkylaminoaryl, alkylaryl, alkylaminoalkyl, alkoxyaryl, arylamino, aralkylamino, alkylsulfonyl, carboxamidoalkylaryl, carboxamidoaryl, hydroxyalkyl, haloalkyl, alkylaminoalkylcarboxy, aminocarboxamidoalkyl, cyano, alkoxyalkyl, perhaloalkyl, arylalkyloxyalkyl, and the like.

[0047] As used herein, the term hydroxyalkyl can refer to a hydroxy terminated alkyl. For example, the hydroxyalkyl can be any hydroxyalkyl known in the art. In embodiments the hydroxyalkyl can be C1-C12hydroxyalkyl. For example, the hydroxyalkyl can be hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, hydroxypentyl, hydroxyhexyl, etc.

[0048] Aspects of the disclosure are drawn towards a compound of formula (I):Docket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025(I), or a stereoisomer, a tautomer, a hydrate, a solvate, or a salt thereof.

[0049] Salts of the compound of the disclosure can comprise those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange.

[0050] Other non-limiting salts comprise adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from comprise alkali metal, alkaline earth metal, and ammonium.

[0051] Non-limiting, exemplary alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Additional salts can comprise nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

[0052] In embodiments, the salt can be selected from the group consisting of a hydrochloride salt of formula (I), a sodium salt of formula (I), a sulfate salt of formula (I), an acetate salt of formula (I), a phosphate salt of formula (I), a fumarate salt of formula (I), a potassium salt of formula (I), a calcium salt of formula (I), a citrate salt of formula (I), a maleate salt of formula (I), a mesylate salt of formula (I), a tartrate salt of formula (I), a gluconate salt of formula (I), or any combination thereof.Docket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025

[0053] The term “solvate” can refer to forms of the compound that are associated with a solvent, for example by a solvolysis reaction. This physical association can include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like. The compounds of Formula (I) can be prepared, e.g., in crystalline form, and can be solvated. Suitable solvates comprise pharmaceutically acceptable solvates and further include both stoichiometric solvates and non-stoichiometric solvates. In some instances, the solvate can be capable of isolation, for example, when one or more solvent molecules are incorporated in the crystal lattice of a crystalline solid. “Solvate” encompasses both solution-phase and isolable solvates. Representative solvates can comprise hydrates, ethanolates, and methanolates.

[0054] The term “hydrate” can refer to a compound that is associated with water. For example, the number of water molecules contained in a hydrate of a compound can be in a definite ratio to the number of the compound molecules in the hydrate. Therefore, a hydrate of a compound can be represented, for example, by the general formula R x H2O, wherein R is the compound and wherein x is a number greater than 0. A given compound can form more than one type of hydrates, including, e.g., monohydrates (x is 1), lower hydrates (x is a number greater than 0 and smaller than 1, e.g., hemihydrates (R 0.5 H2O)), and polyhydrates (x is a number greater than 1, e.g., dihydrates (R-2 H2O) and hexahydrates (R- 6 H2O)).

[0055] The term “tautomers” can refer to compounds that are interchangeable forms of a particular compound structure, and that vary in the displacement of hydrogen atoms and electrons. Thus, two structures can be in equilibrium through the movement of n electrons and an atom (e.g., H). For example, enols and ketones are tautomers because they are rapidly interconverted by treatment with either acid or base. Another example of tautomerism is the aci- and nitro- forms of phenylnitromethane, that are likewise formed by treatment with acid or base. Tautomeric forms can be used to attain the optimal chemical reactivity and biological activity of a compound of interest.

[0056] It is also to be understood that compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed “isomers.” Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers.”

[0057] Stereoisomers that are not mirror images of one another can refer to “diastereomers” and those that are non- superimposable mirror images of each other can refer to “enantiomers.” When a compound has an asymmetric center, for example, it is bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterized by the absoluteDocket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025 configuration of its asymmetric center and is described by the R- and S- sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (z.e., as (+) or (-)- isomers respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a “racemic mixture.”

[0058] Aspects of the disclosure are drawn towards a pharmaceutical composition comprising the compound of formula (I):or a stereoisomer, a tautomer, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof.

[0059] The phrase "pharmaceutical composition" or a “pharmaceutical formulation” can refer to a composition or pharmaceutical composition suitable for administration to a subject, such as a mammal, especially a human and that can refer to the combination of an active agent(s), or ingredient (i.e., a compound of Formula I) with a pharmaceutically acceptable carrier or excipient, making the composition suitable for diagnostic, therapeutic, or preventive use in vitro, in vivo, or ex vivo.

[0060] In embodiments, pharmaceutically acceptable salts can include, but are not limited to, amine salts, such as but not limited to N,N'-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N- methylglucamine, procaine, N-benzylphenethylamine, 1-para-chlorobenzyl-2-pyrrolidin-1'- ylmethylbenzimidazole, diethylamineand other alkylamines, piperazine and tris(hydroxymethyl) aminomethane; alkali metal salts, such as but not limited to lithium, potassium and sodium; alkali earth metal salts, such as but not limited to barium, calcium and magnesium; transition metal salts, such as but not limited to zinc; and other metal salts, such as but not limited to sodium hydrogen phosphate and disodium phosphate; and also including, but not limited to, salts of mineral acids, such as but not limited to hydrochlorides and sulfates; and salts of organic acids, such as but not limited to acetates, lactates, malates, tartrates, citrates, ascorbates, succinates, butyrates, valerates and fumarates.

[0061] In embodiments, the pharmaceutically acceptable salt can be selected from the group consisting of an a hydrochloride salt of formula (I), a sodium salt of formula (I), a sulfate salt of formula (I), an acetate salt of formula (I), a phosphate salt of formula (I), a fumarate salt of formula (I), a potassium salt of formula (I), a calcium salt of formula (I), a citrate salt of formulaDocket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025 (I), a maleate salt of formula (I), a mesylate salt of formula (I), a tartrate salt of formula (I), a gluconate salt of formula (I), a succinate salt of formula (I), or any combination thereof.

[0062] In embodiments, the pharmaceutical composition can comprise one or more pharmaceutically acceptable excipients. A variety of pharmaceutically acceptable excipients are known in the art. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy,” 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., eds., 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., 3rd ed. Amer. Pharmaceutical Assoc. The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

[0063] A "pharmaceutically acceptable excipient," "pharmaceutically acceptable diluent," "pharmaceutically acceptable carrier," or "pharmaceutically acceptable adjuvant" can refer to an excipient, diluent, carrier, and / or adjuvant that are useful in preparing a pharmaceutical composition that are safe, non-toxic and neither biologically nor otherwise undesirable, and can include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary use and / or human pharmaceutical use. A pharmaceutically acceptable excipient, diluent, carrier and / or adjuvant" as used herein can include one and more such excipients, diluents, carriers, and adjuvants.

[0064] In an embodiment of the disclosure, the composition or pharmaceutical composition can be administered to the subject using any means capable of resulting in the desired effect. Thus, the composition or pharmaceutical composition can be incorporated into a variety of formulations for therapeutic administration. For example, the composition or pharmaceutical composition can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and can be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, creams, and aerosols. Moreover, the compositions of the disclosure can include those formed by impregnation of the composition or pharmaceutical composition described herein into absorptive materials, such as sutures, bandages, and gauze, or coated onto the surface of solid phase materials, such asDocket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025 surgical staples, zippers and catheters to deliver the compositions. Other delivery systems of this type will be readily apparent to those skilled in the art in view of the disclosure.

[0065] Suitable excipient vehicles for the composition or pharmaceutical composition are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, antioxidants or pH buffering agents. Methods of preparing such dosage forms are known, or will be apparent upon consideration of this disclosure, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985. The composition or formulation to be administered will, in any event, contain a quantity of the composition or pharmaceutical composition adequate to achieve the desired state in the subject being treated.

[0066] Compositions of the present disclosure can include those that comprise a sustained release or controlled release matrix. In addition, embodiments of the present disclosure can be used in conjunction with other treatments that use sustained-release formulations. As used herein, a sustained-release matrix is a matrix made of materials, for example polymers, which are degradable by enzymatic or acid-based hydrolysis or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids. A sustained-release matrix desirably is chosen from biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (copolymers of lactic acid and glycolic acid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone. Illustrative biodegradable matrices can include a polylactide matrix, a polyglycolide matrix, and a polylactide co-glycolide (co-polymers of lactic acid and glycolic acid) matrix. In another embodiment, the pharmaceutical composition of the present disclosure (as well as combination compositions) can be delivered in a controlled release system.

[0067] For example, the compound or pharmaceutical composition can be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump can be used (Sefton (1987). CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al. (1980). Surgery 88:507; Saudek et al. (1989). N. Engl. J. Med.321:574). In another embodiment, polymeric materials are used. In yet another embodiment a controlled release system is placed in proximity of the therapeutic target thus requiring only a fraction of the systemic dose. In yet another embodiment, a controlled releaseDocket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025 system is placed in the proximity of the therapeutic target, thus requiring only a fraction of the systemic. Other controlled release systems are discussed in the review by Langer (1990). Science 249:1527-1533.

[0068] In embodiments, the pharmaceutical composition can be incorporated into a formulation. As used herein, the term “formulation" can refer to any collection of components of a compound, mixture, or solution selected to provide optimal properties for a specified end use, including product specifications and / or service conditions. The term formulation can include liquids, semi-liquids, colloidal solutions, dispersions, emulsions, microemulsions, and nanoemulsions, including oil-in-water emulsions and water-in-oil emulsions, pastes, powders, and suspensions. The formulations of the present invention can also be included in, or packaged with, other non-toxic compounds, such as cosmetic carriers, excipients, binders and fillers, and the like. For example, the acceptable cosmetic carriers, excipients, binders, and fillers for use in the practice of the present disclosure are those which render the compounds amenable to delivery and / or provide stability such that the formulations of the present invention exhibit a commercially acceptable storage shelf life.

[0069] The formulations or pharmaceutical composition can also be included, or packaged, with other non-toxic compounds, such as pharmaceutically acceptable carriers, excipients, binders and fillers including, but not limited to, glucose, lactose, gum acacia, gelatin, mannitol, xanthan gum, locust bean gum, galactose, oligosaccharides and / or polysaccharides, starch paste, magnesium trisilicate, talc, corn starch, starch fragments, keratin, colloidal silica, potato starch, urea, dextrans, dextrins, and the like. For example, the pharmaceutically acceptable carriers, excipients, binders, and fillers for use in the present disclsoure are those which render the compounds of the invention amenable to any method of administration described herein.. Moreover, the packaging material can be biologically inert or lack bioactivity, such as plastic polymers or silicone, and can be processed internally by the subject without affecting the effectiveness of the composition / formulation packaged and / or delivered therewith.

[0070] In another embodiment, the compositions or pharmaceutical compositions of the present disclosure (as well as combination compositions separately or together) can be part of a delayed-release formulation. Delayed-release dosage formulations can be prepared as described in standard references such as “Pharmaceutical dosage form tablets,” eds. Liberman et. al. (New York, Marcel Dekker, Inc., 1989), “Remington-The science and practice of pharmacy,” 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and “Pharmaceutical dosage forms and drug delivery systems,” 6th Edition, Ansel et al., (Media, PA: Williams and Wilkins, 1995). These references provide information on excipients,Docket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025 materials, equipment and processes for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules.

[0071] Different forms of the formulation described herein can be calibrated in order to adapt both to different individuals and to the different needs of a single individual.

[0072] Aspects of the disclosure are drawn towards methods of treating cancer. In embodiments, the disclosure is drawn towards methods of treating a subject afflicted with cancer. For example, conditions that can be treated by administration of the compound or pharmaceutical composition of the disclosure include comprise integrin α5β1 overexpressing cancers. For example, the cancers that can be treated with the compounds and compositions described herein can comprise breast cancer, cervical cancer, pancreatic cancer, ovarian cancer, renal cell carcinoma, prostate cancer, bladder cancer, gastric cancer, melanoma, colorectal cancer, lung cancer, brain cancer or any combination thereof. In embodiments, the breast cancer is triple-negative breast cancer. In embodiments, the brain cancer can be glioblastoma.

[0073] In embodiments, the disclosure is drawn towards methods of treating a subject afflicted with cancer. In embodiments, the methods described herein comprise administering a therapeutically effective amount of the compound or a pharmaceutical composition comprising the compound of formula (I):(I), or a stereoisomer, a tautomer, a hydrate, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

[0074] As used herein, the term "therapeutically effective amount" can refer to that amount of an embodiment of the compound or pharmaceutical composition being administered that will relieve to some extent one or more of the symptoms of the disease or condition being treated (e.g., cancer), and / or that amount that will prevent, to some extent, one or more of the symptoms of the condition or disease that the subject being treated has or is at risk of developing.

[0075] For example, the therapeutically effective amount of the compound or pharmaceutical composition described herein can comprise less than about 0.005 mg / kg, about 0.005 mg / kg, about 0.0075 mg / kg, about 0.01 mg / kg, about 0.0125 mg / kg, about 0.025 mg / kg, about 0.05 mg / kg, about 0.075 mg / kg, about 0.1 mg / kg, about 0.25 mg / kg, about 0.5 mg / kg, about 0.75Docket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025 mg / kg, about 1.0 mg / kg, about 2.5 mg / kg, about 5 mg / kg, about 7.5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, about 100 mg / kg, or greater than about 100 mg / kg.

[0076] As used herein, the term “subject” or “patient” can refer to any organism to which aspects of the invention can be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. The term " subject " can include a mammal, for example, a human at any age suffering from pathology. In another embodiment, the term encompasses a subject at risk of developing pathology. Subjects to which compounds or compositions of the present disclosure can be administered will be animals, for example mammals, such as primates, especially humans. For veterinary applications, a wide variety of subjects will be suitable, e.g., livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals for example pets such as dogs and cats. For diagnostic or research applications, a wide variety of mammals will be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine such as inbred pigs and the like. The term “living subject” can refer to a subject noted herein or another organism that is alive. The term “living subject” can refer to the entire subject or organism and not just a part excised (e.g., a liver or other organ) from the living subject.

[0077] As used herein, the term “administration” or “administering” can refer to introducing a composition of the present disclosure into a subject. For example, administration of the compound or composition described herein can comprise oral administration, parenteral administration, intravenous administration, or intramuscular administration. However, any route of administration, such as topical, intranasal, subcutaneous, peritoneal, intra-arterial, inhalation, vaginal, rectal, introduction into the cerebrospinal fluid, intravascular either veins or arteries, or instillation into body compartments can be used.

[0078] As used herein, "treatment" and "treating" can refer to the management and care of a subject for the purpose of combating a condition, disease or disorder, in any manner in which one or more of the symptoms of a disease or disorder are ameliorated or otherwise beneficially altered. For example, the condition, disease, or disorder can comprise cancer. The term “treating” can comprise the full spectrum of treatments for a given condition from which the patient is suffering, such as administration of the active compound for the purpose of: alleviating or relieving symptoms or complications; delaying the progression of the condition,Docket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025 disease or disorder; curing or eliminating the condition, disease or disorder; and / or preventing the condition, disease or disorder.

[0079] In embodiments, "preventing" or "prevention" can refer to the management and care of a patient for the purpose of hindering the development of the condition, disease or disorder (e.g., cancer), and can include the administration of the compounds or compositions described herein to prevent or reduce the risk of the onset of symptoms or complications. Skilled artisans will appreciate a variety of methodologies and assays can be used to assess the development of pathology, and similarly, a variety of methodologies and assays can be used to reduce pathology, driveway, or regression.

[0080] Integrin α5β1 is implicated promoting cancer metastasis. In embodiments, the cancer can comprise an integrin α5β1 overexpressing cancer. In non-limiting, exemplary embodiments, the overexpression can comprise an overexpression of about 50% or greater than about 50%. In embodiments, the integrin α5β1 overexpressing cancer can comprise breast cancer, cervical cancer, pancreatic cancer, ovarian cancer, renal cell carcinoma, prostate cancer, bladder cancer, gastric cancer, melanoma, colorectal cancer, lung cancer, brain cancer or any combination thereof. In embodiments, the breast cancer is triple-negative breast cancer.

[0081] In embodiments, treating cancer can comprise preventing cancer metastasis, reducing tumor size, or a combination thereof. As used herein, the term “metastatic,” or “metastasize” can refer to the spread or migration of cancerous cells from a primary original tumor to another organ or tissue. In embodiments, this can be identifiable by the presence of a “secondary tumor” or “secondary cell mass” of the tissue type of the primary original tumor and not of that of the organ or tissue in which the secondary (metastatic) tumor is located. For example, a triple- negative breast cancer that has migrated to the lymph nodes is said to be metastasized triple- negative breast cancer and includes cancerous triple-negative breast cancer cells growing in lymphatic tissue.

[0082] As used herein, the term “tumor" as it applies to a subject diagnosed with, or suspected of having, a cancer can refer to a malignant neoplasm or tissue mass of any size and includes primary tumors and secondary neoplasms.

[0083] In embodiments, the compound or pharmaceutical composition is administered to a subject in need thereof in therapeutically effective amount. For example, the therapeutically effective amount can comprise a dosage of about 0.005 mg / kg to about 100 mg / kg. While individual needs vary, determination of optimal dosage ranges for a particular disease or condition is within the skill of the art. For example, the dosage administrated will be dependentDocket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025 upon the age, health and weight of the recipient, disease to be treated, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired.

[0084] The frequency of administration of the compound or pharmaceutical composition described herein can vary depending on any of a variety of factors, e.g., severity of the symptoms, and the like. For example, in an embodiment, the composition or pharmaceutical composition can be administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (qd), twice a day (qid), three times a day (tid), or four times a day. In embodiments, the composition or pharmaceutical composition is administered 1 to 4 times a day over a 1 to 10-day time period. In embodiments, the dosage is administered about once daily, about twice daily, about three times daily, about four times daily, or about five times daily. EXAMPLES

[0085] Examples are provided herein to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results. EXAMPLE 1

[0086] Targeted therapeutic treatment of cancer

[0087] Breast cancers (BC) are the most common cancers worldwide. BC is an umbrella term for a highly heterogeneous disease with several subtypes, each with distinct histology and pathologies. It remains the most common cancer among women in the US, accounting for 15.5% of all new cancer cases and causing 19.3 deaths per 100,000 women annually (Christiana Neophytou et al., 2018).

[0088] Triple-negative breast cancer (TNBC) is a highly aggressive subtype of BC that accounts for approximately 10-15% of new BC cases and despite admirable progress being made in the treatment of BC, TNBC will still claim the lives of approximately 34% of patients with regional disease that has spread to nearby lymph nodes and 88% of patients with distalDocket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025 metastatic disease (seer.cancer.gov / statfacts / html / breast.html; Mitchell et al. 2024; www.cancer.org / cancer / types / breast-cancer / about / types-of-brest-cancer / triple-negative.html; Xupeng Bai et al.2021; Gallagher et al.2021).

[0089] To further compound this issue, those diagnosed with TBNC will likely have progressed disease at the time of diagnosis and thus, poorer prognosis. These statistics result from the availability of fewer treatment options for TNBC versus other forms of BC. TNBC is characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and HER2 / neu expression, thus rendering ineffective the ER-targeting hormone therapies and PR- or HER2-targeting small molecule drugs and immunotherapies that are commonly used to treat other forms of the disease.

[0090] Current Treatment Regimens Can Be Lacking

[0091] Current treatment for TNBC depends upon the state of the disease and comprises various combinations of surgery, radiotherapy, chemotherapy, and immunotherapy. The limited success of current therapeutic approaches for TNBC, particularly in advanced disease, creates an urgent imperative to develop targeted therapies to increase survival and enhance the quality of life for those diagnosed. TNBC patients primarily receive cytotoxic chemotherapies as a standard of care (Obidiro et al. 2023; www.cancer.org / cancer / types / breast- cancer / treatment / treatment-of-triple- negative.html#:~:text=Common%20chemo%20drugs%20used%20include,used%20alone%2 0or%20in%20combination ).

[0092] These medications are associated with adverse side effects, including persistent brain fog, nausea, and vomiting; all of which can negatively impact patient compliance, morale, and overall health (Gallagher et al.2023). Furthermore, some chemotherapies are linked to the risk of developing secondary cancers (www.canecr.org / cancer / survivorship / long-term-health- concerns / second-cancers-in-adults / treatment-risks.html). Despite their use, these therapies focus on non-targeted tumor shrinkage and achieve a pathological complete response in only 30-50% of patients after neoadjuvant chemotherapy (Spring et al. 2020). Once the disease metastasizes, treatment effectiveness declines, leading to regimens that combine various chemotherapies, immunotherapies, or targeted therapies, and these combinations achieve variable success (Rose et al.2020). Immunotherapies, such as checkpoint blockade and anti- PD1 treatments, show promise but yield inconsistent results, with an overall patient response rate of around 20% (Parvez et al.2023). PARP inhibitors can enhance overall response rates (OPR) and progression-free survival (PFS) in TNBC patients and are used alongside traditional therapies. However, they are often associated with hematological toxicity and require regularDocket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025 monitoring. Moreover, resistance to PARP inhibitors frequently develops over time, rendering the treatment useless. These therapies often fail in the treatment of TNBC because they inadequately manage the risk of metastasis while utilizing an often non-targeted and cytotoxic approach that puts clonal pressure on the tumor to mutate while negatively impacting patient health.

[0093] Integrin protein targeting through Nischarin

[0094] One way to target cell migration and its associated pathways involves the integrin family of proteins. Integrins are involved in cell motility, adhesion, differentiation, migration, and proliferation, and several cancers are characterized by integrin overexpression or dysregulation (Georgakopoulous-Soares et al.2020). Among these cancer-associated integrins is integrin α5β1 is closely linked to cancer progression, particularly by promoting metastasis (Mierke et al.2011; Wang et al.2013; Hou et al.2020). In vivo studies on BC have indicate that integrin α5β1 facilitates cell invasion and promotes resistance to systemic treatments commonly used for BC, such as doxorubicin (Morozevich et al.2017). Beyond BC, a study of 149 cervical cancer specimens revealed that 84% overexpressed integrin α5β1. This overexpression was closely associated with lymph node metastasis, poor chemotherapy response, and recurrence (Morozevich et al. 2011; Zhu et al. 2017). Without wishing to be bound by theory, the tumor suppressor protein Nischarin (NISCH) is a possible target that can be used to overcome this overexpression in the α5β1 integrin.

[0095] NISCH, a tumor suppressor and an α5β1 integrin regulator, is found at low levels in uncontrolled metastatic breast cancer cells compared to surrounding healthy epithelial tissues. NISCH is expressed both in tumor cells and in cancer-associated fibroblasts (CAFs) and has been repeatedly reported as a positive prognostic matter in BC (Okpechi et al.2022). Analyzing datasets from the Molecular Taxonomy of Breast Cancer International Consortium and The Caner Genome Atlas, the NISCH gene expression is higher in normal breast tissues than in BC tissues and is greatly reduced in TNBC tissues (Baranwal et al.2011).

[0096] Additionally, NISCH expression is inversely correlated with genes regulating epithelial-mesenchymal transition (EMT), a biological program often hijacked by cancer cells, to acquire tissue invasiveness and metastatic progression and migration (Alahari et al. 2000; Jain et al. 2013; Alahar et al. 2004; Ding et al. 2008). As TNBC progresses to metastatic disease, NISCH expressions drop to levels below detection (Ding et al. 2008; Ostojié et al. 2024; Qui et al.2015; Cai et al.2020). Targeting the upregulation of NISCH expression and activity in early TNBC disease, before NISCH levels drop below treatable levels, could be an avenue to present TNBC cellular migration / metastasis and keep disease localized to the breastDocket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025 (Grahovac et al.2023). Given this inhibitory interaction of NISCH with the α5β1 integrin and its role in preventing cell migration, NISCH represents an attractive early target for systemic small molecule therapy to treat metastatic resistance TNBC.

[0097] Small molecule targeting of NISCH in the inhibition of integrin proteins.

[0098] NISCH binds to the cytoplasmic domain of the alpha5 subunit of integrin α5β1, to inhibit its function and expression, and to significantly reduce α5β1 integrin-dependent migration (Alahari et al. 2000). NISCH is an imidazoline receptor for which two main drug agonists have been studied. The anti-hypertensive agents moxonidine, and rilmenidine have been shown to target the NISCH. Those two compounds are allosteric agonists to NISCH, meaning they bind to a defined imidazoline receptor site separate from the NISCH active binding site to induce a conformation change in NISCH. This conformational change can increase the binding ability of NISCH to the integrin protein. Rilmenidine has been repurposed and considered as a cancer therapeutic, in addition to its use as a hypertension medication (Morozevich et al. 2017; Vucicevic et al. 2016). Studies have shown that rilmenidine can increase the susceptibility of cells to doxorubicin (Morozecivh et al. 2017). Additionally, in vivo studies have demonstrated that rilmenidine inhibits the migration and invasion of pancreatic ductal adenocarcinoma cells by acting as a NISCH agonist (Grahovac et al.2023).

[0099] Since BC often forms in the duct and must invade the surrounding breast tissue to become metastatic, without wishing to be bound by theory, optimization of NISCH agonists through the development of rilmenidine analogs, can have the potential to treat and control TNBC. This can keep the disease localized, for which the 5-year survival rate of patients is 91% (seer.cancer.gov / statfacts / html / breast.html; www.webmd.com / brest-cancer / triple- negative-breast-cancer; www.cancer.org / cancer / types / breast-cancer / about / types-of-brest- cancer / triple-negative.html).

[0100] Development of rilmenidine analog DP-120

[0101] Approach

[0102] There is a correlation to NISCH under expression and increased cellular migration and metastasis in BC. Moreover, publicly available data from TCGA was utilized to examine NISCH expression at the transcription level in invasive carcinoma BC patient tissues (Fig. 1 Panel A) and related these data as a prognostic marker to patient survival (Fig. 1 Panel B) (Baranwal et al.2011).

[0103] It was found that normal, healthy breast tissue had relatively high NISCH transcription levels compared to basal cells, with around an 11.5-fold concentration of coding mRNA. In contrast, Luminal A and Luminal B tissues showed a significant (P<0.0001)Docket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025 decrease in transcriptional NISCH expression, dropping to an average of under 11-fold for Luminal A subtype BC and to near basal levels in Luminal B subtype BC. Some outliers had less coding mRNA expression of NISCH than basal cells (Fig. 1 Panel A). This decrease in NISCH expression correlated with a decrease in the overall survival of these patients (Fig. 1 Panel B). BC patients with higher tissue levels of NISCH coding mRNA had a higher overall survival, with half of these patients surviving past 132 months and 40% surviving past 220 months (red line). Conversely, BC patients with low tissue levels of NISCH coding mRNA had significantly (P=0.0003) reduced overall survival, with over half of the patients surviving less that 88 months and only around 10% living past 176 months (blue line).

[0104] Conducting in vivo studies in human BC patient tissue, high levels of NISCH protein expression in normal epithelial cells were observed. A noticeable reduction in NISCH protein levels was seen as cancer progressed from the primary tumor to invasive metastasis tissue sample. Breast tumors retained high protein levels of NISCH, while invasive ductal carcinoma had significantly reduced NISCH levels. NISCH expression became essentially insignificant once BC progressed to invasive lobular carcinoma (Fig.2 Panel A). NISCH protein expression between MCF-7 cells, commonly used to study hormone-positive BC, and MDA-MB cells, used to model TNBC, was compared (Fig.2 Panel A). Given the enhanced migratory pattern of gene expression in TNBC versus hormone-positive BC, the MDA-MB cells exhibited almost no protein expression of NISCH, indicating their increased potential for invasive metastasis compared to MCF-7 cells.

[0105] Mouse xenograft models implanted with MDA-MB cells in both the mammary fat pad and the flank were also studied. Mice were treated intratumorally with GFP or Nischarin protein. Preliminary data show that NISCH inhibits tumor growth and metastasis in vivo (Fig. 2 Panel D). Based on these findings, without wishing to be bound by theory, NISCH demonstrates an importance as a protein in the regulation of BC progression and metastasis. As discussed, rilmenidine increases NISCH protein activation as an agonist but requires a high dose to increase the actual transcription and expression of this vital regulator. Therefore, an analog of rilmenidine, referred to herein as DP-120, was created and found to promote NISCH expression in MDA-MB cells. Additionally, it can do so as effectively as rilmenidine at smaller doses (Fig.2 Panel C, Fig.2 Panel B).

[0106] DP-120 as a superior NISCH agonist compared to rilmenidine.

[0107] To demonstrate that DP-120 is a superior NISCH agonist compared to rilmenidine, we conducted molecular docking studies to compare the binding affinities of NISCH-activating compounds: moxonidine, rilmenidine, and DP-120. As mentioned previously, theseDocket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025 compounds are allosteric agonists to NISCH. They act on NISCH such that the target binding ligand, Phoshpatidylinositol-3-phosphate (PI3P), is placed in a more favorable conformational position (Okpechi et al.2022). The binding poses of these compounds in the proposed ligand binging site are demonstrated herein (Fig. 3 Panel A-C). Docking models show that DP-120 and rilmenidine share identical binding interactions, with the 1,3-oxazole nitrogen forming hydrogen bonds with Thr107’s side chain hydroxyl group and backbone amine, and the central amine interacting with the backbone carbonyl of Thr107. Rilmenidine’s weaker binding is attributed to the exposure of one cyclopropyl group to the solvent. In contrast, moxinidine binds less effectively and differently, with its 1,3-imidazole forming a hydrogen bond with the side chain carbonyl of Asn123.

[0108] Docking scores indicate that DP-120 (-6.13 kcal / mol) exhibits a stronger binding affinity for NISCH than rilmenidine (-5.37 kcal / mol) and moxinidine (-4.96 kcal / mol), which aligns with the experimentally observed IC50 values. Comparing the functional groups on the compounds DP-120 and rilmenidine indicates that the lipophilicity (cLogP) and the topological polar surface area (tPSA) of the two molecules are similar (Figure 4 Panel A-C) (Comer et al. 2001; Lipinski et al.2000). However, the isobutyl sidechain of DP-120 is much smaller that the dicyclopropylmethyl side chain of rilmenidine. The resulting decreased molecular volume of DP-120 can allow for deep penetration of the compound into the allosteric binding site.This leads to a larger conformational change and a more potent activation of the NISCH by making the PI3P binding sire more accessible than it is for either rilmenidine or moxonidine. Without wishing to be bound by theory, the reduced steric bulk around the amino group of DP-120 can allow for stronger H-bonding interaction between the drug and the allosteric site, leading to a more potent activation of the protein.

[0109] Conclusion

[0110] The research described herein indicates that DP-120 achieves the anti-cancer effects of rilmenidine at lower doses, exhibiting enhanced cytotoxicity to TNBC cells while sparing healthy cells, and binding with greater affinity to NISCH. This underscores the role of NISCH in inhibiting breast cancer progression and highlights DP-120’s ability to improve therapeutic outcomes. These findings indicate that DP-120 can be used to treat, not only breast cancer, but other cancers. Without wishing to be bound by theory, DP-120 can be used to treat cancers by targeting the NISCH:integrin α5β1 pathway to impede metastasis. Non-limiting, exemplary cancers which can be treated by targeting the NISCH:integrin α5β1 comprise cervical and pancreatic cancers.

[0111] References Cited HereinDocket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025

[0112] Christiana Neophytou, P.B., Panagiotis Papageorgis, Molecular Mechanisms and Emerging Therapeutic Targets of Triple-Negative Breast Cancer Metastasis. Frontiers in Oncology, 2018.8(31).

[0113] Institute, N.C. Cancer State Facts: Female Brest Cancer. 2022; Available from: https: / / seer.cancer.gov / statfacts / html / breast.html

[0114] Kristin Mitchell, P.S. Triple-Negative Breast Cancer (TNBC).2024; Available from: www.webmd.com / brest-cancer / triple-negative-breast-cancer

[0115] Society, A.C. Triple Negative Breast cancer. 2023; Available from: www.cancer.org / cancer / types / breast-cancer / about / types-of-brest-cancer / triple-negative.html

[0116] Xupeng Bai, J.N., Julia Beretov, Peter Graham, Yong Li, Triple-negative breast cancer therapeutic resistance: Where is the Achille's heel? Cancer Letters, 2021.497: p.100- 111.

[0117] Gallagher, C.M. Research Reveals New Avenues for Triple-Negative Breast Cancer Treatment. MedStar Health 2023

[0118] Onyinyechi Obidiro, G.B., and Emmanuel O. Akala, Triple Negative Breast Cancer Treatment Options and Limitations: Future Outlook. Pharmaceutics, 2023.15(7).

[0119] C. Treatment of Triple-negative Breast Cancer. All About Cancer; Available from: www.cancer.org / cancer / types / breast-cancer / treatment / treatment-of-triple- negative.html#:~:text=Common%20chemo%20drugs%20used%20include,used%20alone%2 0or%20in%20combination

[0120] Society, A.C. Second Cancers Related to Treatment. All About Cancer; Available from: www.canecr.org / cancer / survivorship / long-term-health-concerns / second-cancers-in- adults / treatment-risks.html

[0121] Laura M. Spring, G.F., Andrea Arfe, Chandni Sharma, Rachel Greenup, Kerry L. Reynolds, Barbara L. Smith, Brian Alexander, Beverly Moy, Steven J. Isakoff, Giovanni Parmigiani, Lorenzo Trippa and Aditya Bardia, Pathological complete response after neoadjuvant chemotherapy and impact on breast cancer recurrence and survival: a comprehensive meta-analysis. Clin Cancer Res., 2020.26(12): p.2838-2848.

[0122] Maddison Rose, J.T.B., Kenneth CYByrne, Derek J. Richard and Emma Bolderson, PARP Inhibitors: Clinical Relevance, Mechanisms of Action and Tumor Resistance. Frontiers in Cell and Developmental Biology, 2020.

[0123] Adil Parvez, F.C., Priyal Mudgal, Rahila Khan, Kamal A. Qureshi, Humaira Farooqi and Ashok Aspatwar, PD-I and PD-L 1: architects of immune symphony and immunotherapy breakthroughs in cancer treatment. Front. Immunol, 2023.14.Docket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025

[0124] LLias Georgakopoulous-Soares, D.V.C., Venetsana Kyriazopoulou, Apostolos Zaravinos, EMT Factors and Metabolic Pathways in Cancer. Frontiers in Oncology, 2020.10.

[0125] Claudia Tanja Mierke, B.F., Martina Fellner, Martin Herrmann, Ben Fabry, Integrin a5ß1 facilitates cancer cell invasion through enhanced contractile forces. J Cell Sci, 2011. 124(3): p.369-383.

[0126] Hua-Yi Wang, Z.C., Zhu-Hui Wang, Hong Wang, Li-Ming Huang, Prognostic significance of a5ß1integrin expression in cervical cancer. Asian Pac J Cancer Prev, 2013. 14(6): p.3890-3895.

[0127] Jianbing Hou, D.Y., Yudong Liu, Pan Huang, Hongjuan Cui, The Roles of Integrin a5ß1 in Human Cancer.2020.13: p.13329-13344.

[0128] G E Morozevich, N.I.K., O Y Susova, A Y Lupatov, A E Berman, Hyperexpression of Integrin a5ß1 promotes Resistance of MCF-7 Human Breast Carcinoma Cells to Doxorubicin via ERK Protein Kinase Down-regulation. Biochemistry (Mosc), 2017.82(9): p. 1017-1024.

[0129] G E Morozevich, N.I.K., N A Ushakova, M E Preobrazhenskaia, A E Berman, Implication of integrin alpha5beta1 in human breast carcinoma apoptosis and drug resistance. Biomed Khim, 2011.57(1): p.77-84.

[0130] Haiyan Zhu, Aixue Chen, Saisai Li, Xuejiao Tao, Bo Sheng, Mandika Chetry, and Xueqiong Zhu, Predictive role of galectin-l and integrin a5ß1 in cisplatin-based neoadjuvant chemotherapy of bulky squamous cervical cancer. Biosci Rep, 2017.37(5).

[0131] Samuel C Okpechi, H.Y., Khoa Nguyen, Thomas Cheng, Nikhilesh V Alahari, Bridgette CollinsBurow, Matthew E Burow, Suresh K Alahari Role of Nischarin in the pathology of diseases: a special emphasis on breast cancer. Oncogene, 2022.8(41): p.1079- 1086.

[0132] Somesh Baranwal, Y.W., Rajamani Rathinam, Jason Lee, Lianjin Jin, Robin McGoey, Yuliya Pylayeva, Filippo Giancotti, Gerard C. Blobe, and Suresh K. Alahari, Molecular Characterization of the TumorSuppressive Function of Nischarin in Breast Cancer. J Natl Cancer Inst., 2011.103(20): p.1513-1528.

[0133] Suresh K. Alahari, J.W.L., Rudy L. Juliano, Nischarin, a Novel Protein That Interacts with the Integrin a5 Subunit and Inhibits Cell Migration. Journal of Cell Biology, 2000.6(151): p.1141-1154.

[0134] Prachi Jain, S.B., Shengli Dong, Amanda P. Struckhoff, Rebecca A. Worthylake, Suresh K. Alahari, Integrin-binding Protein Nischarin Interacts with Tumor Suppressor LiverDocket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025 Kinase Bl (LKBI) to Regulate Cell Migration of Breast Epithelial Cells. Journal of Biological Chemistry, 2013.288(22): p.15495-15509.

[0135] Suresh K Alahari, P.J.R., Rudy L Juliano, The integrin-binding protein Nischarin regulates cell migration by inhibiting PAK. EMBO J., 2004.23(14).

[0136] Yuemin Ding, T.M., Suresh K Alahari, Nischarin inhibits LIM kinase to regulate cofilin phosphorylation and cell invasion. Mol Cell Biol., 2008.28(11 ): p.3742-3756.

[0137] Marija Ostojié, A.D., Kristina iivié,Jelena Grahovac, Analysis of the nischarin expression across human tumor types reveals its context-dependent role and a potential as a target for drug repurposing in oncology. PLOS ONE, 2024.

[0138] Qui, H. requent Loss of NISCH Promotes Tumor Proliferation and Invasion in Ovarian Cancer via Inhibiting the FAKSignal Pathway. Molecular Cancer Therapeutics, 2015. 14(5):p.1202-1212.

[0139] Yuan-Jie Cai, B.M., Mei-Li Wang, Jie Chen, Fu-Guang Zhao, Juan-Di Zhou, Xu Guo, Lei Zheng, ChunJing Xu, Yi Wang, Yi-Bo He, Jian Liu, and Shang-Nao Xie, Impact of Nischarin on EMT regulators in breast cancer cell lines. Oncology Letters, 2020.20(6): p.291.

[0140] J. Grahovac, K.Z., D. Galun, Nischarin can be a target for stromal normalisation in pancreatic ductal adenocarcinoma. 2023, Institute for Oncology and Radiology of Serbia, Cllinical Center of Serbia: Annals of Oncology.

[0141] Jelica Vucicevic, T.S.-R., Marco Pieroni, Jonne M.M. Laurila, Vladimir Perovic, Sabrina Tassini, Elisa Azzali, Gabriele Costantino, Sanja Glisic, Danica Agbaba, and K.N. Mika Scheinin, Marco Radi, Nevena Veljkovic A combined ligand- and structure-based approach for the identification of rilmenidine-derived compounds which synergize the antitumor effects of doxorubicin. Bioorganic and Medicinal Chemistry, 2016.24(14).

[0142] Okpechi, S.C., Yousefi, H., Nguyen, K. et al. Role of Nischarin in the pathology of diseases: a special emphasis on breast cancer. Oncogene 41, 1079-1086 (2022)

[0143] Comer J, T.K.L.P.T.a.M.l.T.B., van de Waterbed H, Folkers G, Guy R. Pharmacokinetic Optimization in Drug Research: Biological, Physicochemical, and Computational Strategies. (secondary). Weinheim: Wiley-VCH.2001, pp.275-304.

[0144] Lipinski CA, L.F., Dominy BW, Feeney PJ. Experimental and Computational Approaches to Estimate Solubility and Permeability in Drug Discovery and Development Settings. Adv. Drug Deliv. Rev., 1997, 23, 3-25, (b) Lipinski, CA Drug-like properties and the causes of poor solubility and poor permeability. J Pharmacol Toxicol Methods 2000, 44, 235- 249.Docket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025 EXAMPLE 2

[0145] Therapeutic Agent for the Treatment of Breast Cancer

[0146] The amino-oxazolidine rilmenidine can activate the expression of Nischarin which in turn can reduce breast cancer cell proliferation. Structural modification of rilmenidine can lead to potent activation of Nischarin expression and ultimately to greater efficacy in breast cancer cells.

[0147] Nischarin is large cytosolic protein that selectively binds to the cytoplasmic tail of the a5 subunit of integrin receptors. Over-expression of Nischarin inhibits cell migration and reorganization of the actin cytoskeleton. Nischarin also associates with and inhibits PAK kinases involved in cell growth.3Rilmenidine can treat hypertension and can also exhibit moderate activation of Nischarin toward inhibition of breast cancer cell proliferation.3

[0148] Rilmenidine, is a 2-amino-2-oxazoline derivative which can bind to an allosteric binding site on the Nischarin protein which, without wishing to be bound by theory, can lead to a protein conformation for kinase inactivation. Without wishing to be bound by theory, rilmenidine analogs can be an approach for the development of chemotherapeutics for eradication of cancerous cells in breast tissue.2

[0149] References Cited Herein

[0150] 1. Hron, R. J.; Jursic, B. S.; Neumann, D. M. Synthesis of N-Aryl and N- arylcarbamolyamino derivatives of 1,3-diazinane-5-carboxamide and their activity against glioblastoma LN-229 cell line. Bioorg. Med.Chem.2016, 24, 6183-6193.

[0151] 2. Wong, W.C.; Wang, D.; Forray, C.; Vaysse, P. J.-J.; Branchek, T. A.; Gluchowski, C. A Convenient Synthesis of 2-Amino-2-Oxazolines and Their Pharmocological Evaluation at Cloned Human ^^^^Adrenergic Receptors. Biooorg. Med. Chem.1994, 4, 2317-2322.

[0152] 3. Maziveyi, M.; Alahari, S. K.; Breast Cancer Tumor Suppressors: A Special Emphasis on Novel Protein Nischarin. Cancer Res.2015.75, 4252-4259. EXAMPLE 3

[0153] Synthesis, Characterization, and Comparative Studies of Rilmenidine Analogs

[0154] Synthesis and Characterization of Rilmenidine Analog DP-120 via Two-Part Synthetic RouteDocket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025

[0155] Scheme I.: Non-limiting exemplary synthesis of DP-120.

[0156] Step (a): N-(2-chloroethyl)-N'-isobutylurea

[0157] A solution of isobutylamine (4.4 g, 0.060mol) in tetrahydrofuran (15 mL) was added dropwise over 30 minutes to a stirred solution of 2-chloroethyl isocyanate (5.0 mL, 0.059 mol) in tetrahydrofuran (8 mL) while maintaining the mixture temperature at 0 to 5 °C. Then, the reaction mixture was allowed to warm to room temperature and was stirred for 18 hours. The solution was evaporated to dryness and the dry solid residue, weighing 10.6 g, was sufficiently pure to be used for the next step of the synthesis. A pure sample of the N-(2-chloroethyl)-N'- isobutylurea was obtained via flash column chromatography as mixture of rotomers (SiO2, CH2Cl2:CH3OH:NH4OH(conc); 90:10:1).

[0158] Step (b): 2-isobutylamino-oxazoline (DP-120)

[0159] The crude N-(2-chloroethyl)-N'-isobutylurea (10.6 g) obtained by step (a) was suspended in deionized water (50 mL) and heated at reflux for 2 hours. After allowing the temperature of the mixture to return to room temperature, the aqueous solution is extracted with t-butyl methyl ether. The aqueous phase was separated and made alkaline by adding concentrated ammonium hydroxide. The mixture was extracted with dichloromethane (3 x 50 mL) and the combined extracts were washed with brine (50 mL) and dried over sodium sulfate. The solvent was evaporated under reduced pressure to give an oil. The crude oxazoline was purified by flash column chromatography (SiO2, CH2Cl2:CH3OH:NH4OH(conc); 90:10:1) . to yield a light yellow wax that exists as a mixture of rotomers (2.5 g, 30% yield). MS (70 ev) m / z (int): 142 (100). FTIR: 3285, 2956, 1622, 1549 cm-1:1H NMR (400 MHz, CDCl3: δ 3.46- 3.25 (m, 4H), 3.02-2.93 (m, 2H), 2.07(br s, 1H), 1.80-1.72 (m, 1H), 0.90 (d, J = 8.0 Hz, 6H).

[0160] Characterization of Step (a) ProductDocket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025

[0161] 1H NMR (400 MHz, CDCl3: δ 9.81 (br s, 1H), 9.47 (br s, 1H) 3.56 (m, 2H), 3.52 (m, 2H), 2.98 (d, J = 8.0 Hz, 2H), 1.88-1.72(m, 1 H), 0.90 (d, J = 8.0 Hz, 6H).

[0162] Characterization of Step (b) Product

[0163] MS (70 ev) m / z (int): 142 (100). FTIR: 3285, 2956, 1622, 1549 cm-1:1H NMR (400 MHz, CDCl3: δ 3.46-3.25 (m, 4H), 3.02-2.93 (m, 2H), 2.07(br s, 1H), 1.80-1.72 (m, 1H), 0.90 (d, J = 8.0 Hz, 6H).

[0164] Comparative Studies of Rilmenidine and New Analog (DP-120)

[0165] Dose Response of DP-120 vs. Rilmenidine

[0166] Three different triple-negative breast cancer cell lines (MDAB231, MDAMB468, and SUM159PT), and human mammary epithelial cells (HMLE) were exposed to various doses of lab synthesized Rilmenidine (DP-144). Data show that DP-144 is not that effective compared to normal HMLE cells (Figure 5). The same procedure was carried out for DP-120 with the addition of the triple-negative breast cancer cell line LM2-4175. The data show that DP120 has a more pronounced effect on cell viability of cancer cell lines as compared to normal HMLE cells at the higher doses (Figure 6).

[0167] Clonogenic Assay Comparing Rilmenidine and DP-120

[0168] A clonogenic assay was conducted to compare the ability of Rilmenidine and DP-120 to inhibit cell proliferation.The results of this assay are shown in Figure 7.

[0169] MTT Assay Comparing Moxonidine, Rilmenidine, DP-93, and DP-120Structure IIDocket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025Structure IV

[0170] Breast cancer cells of cell line MDAMB468, were treated with different doses of Moxonidine (Structure I), Rilmenidine (Structure II), DP144 (lab synthesized Rilmenidine), DP-120 (Structure IV), and DP-93 (Structure III) over a 48-hour period. DMSO was used as a control. After treatment, an MTT assay was done, and data was normalized to DMSO and plotted (Figure 8).

[0171] The data show that at the lowest dose of 1 [µM] all target drugs have similar behavior. Once the doses are increased by one and two orders of magnitude to 10, and 100 µM respectively, the effects of DP-120 can be seen. ***** EQUIVALENTS

[0172] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

Claims

Docket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025 CLAIMS What is claimed is:or a stereoisomer, a tautomer, a solvate, a hydrate, or a salt thereof.

2. The compound of claim 1 wherein the salt is selected from the group consisting of an a hydrochloride salt of formula (I), a sodium salt of formula (I), a sulfate salt of formula (I), an acetate salt of formula (I), a phosphate salt of formula (I), a fumarate salt of formula (I), a potassium salt of formula (I), a calcium salt of formula (I), a citrate salt of formula (I), a maleate salt of formula (I), a mesylate salt of formula (I), a tartrate salt of formula (I), a gluconate salt of formula (I), a succinate salt of formula (I), or any combination thereof.

3. A pharmaceutical composition comprising a compound of formula (I):or a stereoisomer, a tautomer, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable excipient.

4. The pharmaceutical composition of claim 3, wherein the pharmaceutically acceptable salt is selected from the group consisting of an a hydrochloride salt of formula (I), a sodium salt of formula (I), a sulfate salt of formula (I), an acetate salt of formula (I), a phosphate salt of formula (I), a fumarate salt of formula (I), a potassium salt of formulaDocket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025 (I), a calcium salt of formula (I), a citrate salt of formula (I), a maleate salt of formula (I), a mesylate salt of formula (I), a tartrate salt of formula (I), a gluconate salt of formula (I), a succinate salt of formula (I), or any combination thereof.

5. The pharmaceutical composition of claim 3, wherein the compound of formula (I) is formulated for oral administration, parenteral administration, intravenous administration, or intramuscular administration.

6. A method of treating a subject afflicted with cancer, the method comprising administering a therapeutically effective amount of a compound of formula (I):or a stereoisomer, a tautomer, a hydrate, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

7. The method of claim 6, wherein the cancer is an integrin α5β1 overexpressing cancer.

8. The method of claim 7, wherein the integrin α5β1 overexpressing cancer comprises breast cancer, cervical cancer, pancreatic cancer, ovarian cancer, renal cell carcinoma, prostate cancer, bladder cancer, gastric cancer, melanoma, colorectal cancer, lung cancer, brain cancer or any combination thereof.

9. The method of claim 8, wherein the breast cancer is triple-negative breast cancer.

10. The method of claim 6, wherein treating cancer comprises preventing cancer metastasis, reducing tumor size, or a combination thereof.

11. The method of claim 6, wherein the pharmaceutically acceptable salt is selected from the group consisting a hydrochloride salt of formula (I), a sodium salt of formula (I), a sulfate salt of formula (I), an acetate salt of formula (I), a phosphate salt of formula (I), a fumarate salt of formula (I), a potassium salt of formula (I), a calcium salt of formulaDocket No.: 2932719-000278-WO1 Date of Filing: August 27, 2025 (I), a citrate salt of formula (I), a maleate salt of formula (I), a mesylate salt of formula (I), a tartrate salt of formula (I), a gluconate salt of formula (I), a succinate salt of formula (I), or any combination thereof.

12. The method of claim 6, wherein the compound is administered orally, parenterally, intravenously, or intramuscularly.

13. The method of claim 6, wherein the compound is administered to a subject in need thereof in a dosage of about 1 mg / kg to about 50 mg / kg.

14. The method of claim 13, wherein the dosage is administered about once daily, about twice daily, about three times daily, about four times daily, or about five times daily.

15. Use of a therapeutically effective amount of a compound of formula (I)or a stereoisomer, a tautomer, a hydrate, or a pharmaceutically acceptable salt thereof, to treat a subject afflicted with cancer.

16. Use of a therapeutically effective amount of a compound of formula (I)or a stereoisomer, a tautomer, a hydrate, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament to treat a subject afflicted with cancer.

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