DUAL SRC/P38 KINASE INHIBITOR COMPOUNDS AND THEIR USE AS

THERAPEUTIC AGENTS

CROSS-REFERENCE TO RELATED APPLICATION

[0001] The benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No.

62/257,584 filed November 19, 2015, is hereby claimed and the disclosure thereof is

incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present disclosure relates to dual Src/p38 kinase inhibitor compounds and their use as therapeutic agents.

BACKGROUND OF THE INVENTION

[0003] Breast cancer is the most common potentially lethal cancer in women and the second leading cause of cancer deaths in the United States. Over the last decade and a half, multiple lines of evidence have revealed the profound genetic, gene expression, and phenotypic heterogeneity of breast cancers, leading to a current working model of approximately five subtypes.

[0004] Among individual breast cancer subtypes, those classified as triple negative breast cancer (TNBC) are especially lethal due to their high metastatic potential and propensity to recur relatively rapidly (Foulkes et al., The New England journal of medicine 2010, 363(20): 1938- 1948; Anders et al., Clinical breast cancer 2009, 9 Suppl 2:S73-81; Carey et al., Nature reviews Clinical oncology 2010, 7(12):683-692). As a group, TNBCs lack expression of the estrogen- receptor (ER) and the progesterone-receptor (PR) and also lack overexpression of human epidermal growth factor receptor 2 (ErbB2/HER-2). Thus, therapies directed against these robust targets are not effective against TNBC (Bayraktar et al., Breast cancer research and treatment 2013, 138(l):21-35). There have been tremendous advances in the treatment of breast cancer over the last three decades, particularly for patients whose tumors overexpress ErbB2 and/or have expression of a hormone receptor. In contrast, TNBC is a large (and itself heterogeneous) subset of breast cancers for which there are no FDA-approved targeted therapies, and cytotoxic agents remain the mainstay of therapy (Bayraktar et al., supra). Despite advances in chemotherapy schedules, the residual risk of recurrence in patients with non-metastatic TNBC after aggressive treatment is close to 40%, substantially higher than patients with breast cancers expressing hormone receptors or HER-2 overexpression, where the risk of recurrence is less than 25% (Dent et al., Breast cancer research and treatment 2009, 115(2):423-428). Poor clinical outcome of the patients with TNBC tumors is multifactorial, with the lack of plausible predictive markers for response to any given targeted therapy playing a substantial role.

[0005] In an attempt to identify predictive markers in response to chemotherapy, a significant relationship between TNBCs and sensitivity to inhibition of the tyrosine kinase c-Src was observed (Finn et al., Breast cancer research and treatment 2007, 105(3):319-326; Huang et al., Cancer research 2007, 67(5):2226-2238). C-Src, a ubiquitously expressed kinase, participates in a number of important signal transduction pathways involved in cell adhesion, migration, invasion, angiogenesis, and survival (Playford et al., Oncogene 2004, 23(48):7928-7946; Summy et al., Cancer metastasis reviews 2003, 22(4):337-358; Yeatman TJ, Nature reviews Cancer 2004, 4(6):470-480; Finn RS, Annals of oncology 2008, 19(8): 1379- 1386). C-Src has been shown to play a pivotal role in breast cancer progression, metastasis, and angiogenesis.

Abnormal expression of c-Src has been detected in various tumors, and c-Src is typically highly overexpressed in TNBC (Aleshin A and Finn RS, Neoplasia 2010, 12(8):599-607; Mayer EL and Krop IE, Clinical cancer research 2010, 16(14):3526-3532; Tryfonopoulos et al., Annals of oncology 2011, 22(10):2234-2240; Sanchez-Bailon et al., Cellular signalling 2012, 24(6): 1276- 1286). This overexpression of c-Src has been shown to play a role in oncogenic proliferation, migration, and invasion of TNBC cell lines (Tryfonopoulos et al., supra). Importantly, c-Src activity also regulates osteoclast function in healthy bone and enables bone metastases in metastatic TNBC (Finn, RS 2008, supra).

[0006] On the basis of these findings, c-Src is a very attractive target as a possible treatment of TNBC. Preclinical studies have validated c-Src as a therapeutic target in TNBC; however, existing c-Src inhibitors exhibited limited efficacy and high toxicity in the clinic (Finn et al., Clinical cancer research 2011, 17(21):6905-6913; Gucalp et al., Clinical breast cancer 2011, 11(5):306-311; Campone et al., Annals of ^' 2012, 23(3):610-617), which has tempered the enthusiasm for c-Src inhibition as a strategy in TNBC.

[0007] ER-negative and mutant-p53 cell lines (which comprise a majority of TNBCs) have been observed to be more sensitive to small molecule inhibition of p38a than ER-positive and wt-p53 cell lines (Chen et al., Cancer research 2009, 69(23):8853-8861). The activity of p38a activity has been demonstrated to promote oncogenesis via increased invasion, inflammation, and angiogenesis (Wagner et al., Nature reviews Cancer 2009, 9(8):537-549). The exact contribution of ρ38β to MAPK signaling has not been fully elucidated, however, there is growing evidence that ρ38β activity correlates with increased proliferation of TNBC cells. Thus, inhibiting p38a and ρ38β could lead to a potent anti-cancer effect. There is a need for therapeutic options that inhibit c-Src and/or p38 and are effective in treating cancers such as TNBC.

SUMMARY OF THE INVENTION

[0008] The present disclosure is directed to dual Src/p38 kinase inhibitors and their use as therapeutic agents, e.g., to treat cancer. The compounds of the disclosure potently inhibit both c- Src and p38 kinases. The compounds of the disclosure block growth factor mediated signaling pathways and efficiently decrease motility and invasion of cancer cells such as TNBC cells and have a high therapeutic index in vivo.

[0009] In one aspect, the present disclosure provides a compound of Formula (I), or a pharmaceutically acceptable salt thereof:

wherein: A is i_ ₃ alkyl, NR ⁶ R ⁷ , or C _3-8

heterocycloal R 2 is , , or C5_ ₆ heteroaryl; R 3 is H, OH, halo, Ci_ ₃ alkyl, or OCi_ ₂ alkyl; R ⁴ is H, halo, Ci_ ₂ alkyl, or OCH ₃ ; R ⁵ is H, Ci_ ₃ alkyl, or OCi_ ₃ alkyl; R ⁶ and

R 7 are each independently H, Ci_ ₄ alkyl, C ₃ _ ₆ cycloalkyl, or C ₃ _ ₆ heterocycloalkyl; R 8 is halo, C _1-3 alkylene-C ₃ _ ₈ heterocycloalkyl, or heteroaryl; R ⁹ is H, Ci_ ₄ alkyl, or halo; and R ¹⁰ is halo, Ci_ salkyl, OCi_ ₄ alkyl, NR ⁶ R ⁷ , or Ci_ ₃ alkylene-C ₃ _ ₈ heterocycloalkyl. In some aspects, the compound is selected from the group consisting of



C13.

[0010] In another aspect, the present disclosure provides a composition comprising a compound described herein, for example, any of compounds CI to CI 3, a compound having the structure

C14, or a combination thereof, and a pharmaceutically acceptable carrier.

[0011] In one aspect, the present disclosure provides a method of treating or preventing a neoplastic, hyperplastic, or hyperproliferative disorder in a subject in need thereof comprising administering a therapeutically effective amount of a compound disclosed herein. In another aspect, the present disclosure provides a method of inhibiting cancer growth or metastasis comprising contacting a cancer cell with an effective amount of a compound disclosed herein. In still another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof comprising administering a therapeutically effective amount of a compound disclosed herein. In any of the methods described herein, the compound is, for example, a compound of Formula (I), any one of compounds CI to C14, or a combination thereof. The methods of the present disclosure may be used to treat a cancer selected from the group consisting of group consisting of breast cancer, bone cancer, bladder cancer, brain cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, eye cancer, gastric cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, multiple myeloma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, prostate cancer, sarcoma, skin cancer, testicular cancer, throat cancer, and thyroid cancer, for example, sarcoma, pancreatic cancer, colon cancer, lung cancer, prostate cancer, or breast cancer, including TNBC.

[0012] The foregoing summary is not intended to define every aspect of the invention, and other features and advantages of the present disclosure will become apparent from the following detailed description, including the drawings. The present disclosure is intended to be related as a unified document, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features are not found together in the same sentence, paragraph, or section of this disclosure. In addition, the disclosure includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations specifically mentioned above. With respect to aspects of the disclosure described or claimed with "a" or "an," it should be understood that these terms mean "one or more" unless context unambiguously requires a more restricted meaning. With respect to elements described as one or more within a set, it should be understood that all combinations within the set are contemplated. If aspects of the disclosure are described as "comprising" a feature, embodiments also are contemplated "consisting of or "consisting essentially of the feature. Additional features and variations of the disclosure will be apparent to those skilled in the art from the entirety of this application, and all such features are intended as aspects of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 depicts the co-crystal structure of UM-164 bound to c-Src. C-Src adopts the DFG-out inactive conformation (PDB: 4YBJ).

[0014] Figure 2A depicts c-Src autophosphorylation in MDA-MB 231 cells, and Figure 2B depicts c-Src autophorphrylation in SUM 149 cells. In both cell lines, c-Src autophosphorylation was inhibited in a dose and time dependent manner by UM-164 treatment.

[0015] Figure 3A depicts Src activated signaling in SUM 149 cells treated with UM-164. Figure 3B depicts Src activated signaling in SUM 149 cells treated with dasatinib. Figure 3C depicts Src activated signaling in VARI-068 patient-derived triple negative breast cancer cells treated with UM-164.

[0016] Figure 4 depicts growth inhibition in MDA-MB-231 cells treated with dasatinib alone or in combination with a pan-p38 kinase, BIRB-796, or UM-164.

[0017] Figure 5A depicts cell cycle phase in MDA-MB 231 treated with UM- 1-64. Figure 5B depicts cell cycle phase in SUM 149 cells treated with UM-164.

[0018] Figure 6A depicts the average track area covered by cells within a 24-hour time period for MDA-MB 231 (top) and SUM 149 (bottom) cells treated with 50 nM, 100 nM, or 250 nM UM-164 for 24 hours. Figure 5B depicts the number of MDA-MB 231 (top) and SUM 149 (bottom) cells that migrated and invaded after 5 x 10 ⁴ cells were plated and treated with 50 nM, 100 nM, or 250 nM UM-164 for 24 hours. Error bars represent standard deviation of triplicate experiments. *P < 0.001 using the Student's t-test to compare DMSO treated control and UM- 164-treated cells. [0019] Figure 7 A depicts the tumor volume in MDA-MB 231 xenografted mice receiving vehicle, 10 mg/kg UM-164, or 20 mg/kg UM-164 every other day Figure 7B depicts the tumor volume in SUM 149 xenografted mice receiving vehicle, 10 mg/kg UM-164, or 20 mg/kg UM- 164 every other day. Statistical significance was determined at a threshold of 0.05 using Bonferroni multiple comparisons test.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The present disclosure provides dual Src/p38 kinase inhibitors that have antitumor, e.g., cytotoxic or cytostatic, activity against different types of hyperproliferative cells. Unlike other kinase inhibitors in clinical use, the compounds described herein are dual Src/p38 kinase inhibitors. The compounds inhibit signaling pathways involved in controlling tumor cell proliferation and survival and have anti-TNBC activity in vivo with limited toxicity.

[0021] All current FDA-approved inhibitors of c-Src inhibitors (e.g., dasatinib, bosutinib, saracatinib) act by binding the active conformation of the kinase. The compounds of the present disclosure inhibit c-Src in a specific inactive conformation (termed "DFG-out"), which results in improved efficacy against cancer cells. Preclinical target validation of c-Src has largely been performed using genetic techniques that ablate the entire c-Src gene, while pharmacological intervention with small molecules (such as dasatinib) inhibits only the kinase catalytic activity. Inhibiting a kinase in the DFG-out inactive conformation can have dramatic effects on the non- catalytic functions of the kinase in a manner similar to gene knockdown (Agius MP and Soellner MB, Chemistry & biology 2014, 21(5):569-571).

[0022] The following definitions may be useful in aiding the skilled practitioner in

understanding the disclosure. Unless otherwise defined herein, scientific and technical terms used in the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art.

[0023] As used herein, the term "alkyl" refers to straight chained and branched saturated hydrocarbon groups containing one to thirty carbon atoms, for example, one to twenty carbon atoms, or one to ten carbon atoms. The term C _n means the alkyl group has "n" carbon atoms. For example, C ₄ alkyl refers to an alkyl group that has 4 carbon atoms. C1-7 alkyl refers to an alkyl group having a number of carbon atoms encompassing the entire range (i.e., 1 to 7 carbon atoms), as well as all subgroups (e.g., 1-6, 2-7, 1-5, 3-6, 1, 2, 3, 4, 5, 6, and 7 carbon atoms). Nonlimiting examples of alkyl groups include, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec- butyl (2-methylpropyl), i-butyl (1, 1-dimethylethyl), 3,3-dimethylpentyl, and 2-ethylhexyl.

Unless otherwise indicated, an alkyl group can be an unsubstituted alkyl group or a substituted alkyl group.

[0024] As used herein, the term "alkylene" refers to an alkyl group having a substituent. For example, the term "alkylene- aryl" refers to an alkyl group substituted with an aryl group. The term C _n means the alkylene group has "n" carbon atoms. For example, C _1-6 alkylene refers to an alkylene group having a number of carbon atoms encompassing the entire range, as well as all subgroups, as previously described for "alkyl" groups.

[0025] As used herein, the term "cycloalkyl" refers to an aliphatic cyclic hydrocarbon group containing three to eight carbon atoms (e.g. , 3, 4, 5, 6, 7, or 8 carbon atoms). The term C _n means the cycloalkyl group has "n" carbon atoms. For example, C5 cycloalkyl refers to a cycloalkyl group that has 5 carbon atoms in the ring. C5-8 cycloalkyl refers to cycloalkyl groups having a number of carbon atoms encompassing the entire range (i.e., 5 to 8 carbon atoms), as well as all subgroups (e.g. , 5-6, 6-8, 7-8, 5-7, 5, 6, 7, and 8 carbon atoms). Nonlimiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Unless otherwise indicated, a cycloalkyl group can be an unsubstituted cycloalkyl group or a substituted cycloalkyl group. The cycloalkyl groups described herein can be isolated, share a carbon atom with another cycloalkyl or heterocycloalkyl group, or fused to another cycloalkyl group, a heterocycloalkyl group, an aryl group and/or a heteroaryl group. Cycloalkyl groups can be saturated or partially unsaturated ring systems optionally substituted with, for example, one to three groups, independently selected alkyl, alkylene-OH, C(0)NH ₂ , NH ₂ , oxo (=0), aryl, haloalkyl, halo, and OH.

[0026] As used herein, the term "heterocycloalkyl" or "heterocyclic" is defined similarly as cycloalkyl, except the ring contains one to three heteroatoms independently selected from oxygen, nitrogen, or sulfur. Nonlimiting examples of heterocycloalkyl groups include piperdine, tetrahydrofuran, tetrahydropyran, dihydrofuran, morpholine, and the like. Heterocycloalkyl groups can be saturated or partially unsaturated ring systems optionally substituted with, for example, one to three groups, independently selected alkyl, alkyleneOH, C(0)NH ₂ , NH ₂ , oxo (=0), aryl, haloalkyl, halo, and OH. Heterocycloalkyl groups optionally can be further N- substituted with alkyl, hydroxyalkyl, alkylene- aryl, and alkylene-heteroaryl. The heterocycloalkyl groups described herein can be isolated, share a carbon atom with another cycloalkyl or heterocycloalkyl group, or fused to another heterocycloalkyl group, a cycloalkyl group, an aryl group and/or a heteroaryl group.

[0027] As used herein, the term "aryl" refers to monocyclic or polycyclic (e.g. , fused bicyclic and fused tricyclic) carbocyclic aromatic ring systems. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, phenanthrenyl, biphenylenyl, indanyl, indenyl, anthracenyl, and fluorenyl. Unless otherwise indicated, an aryl group can be an unsubstituted aryl group or a substituted aryl group.

[0028] As used herein, the term "heteroaryl" refers to monocyclic or polycyclic (e.g. , fused bicyclic and fused tricyclic) aromatic ring systems, wherein one to four-ring atoms are selected from oxygen, nitrogen, or sulfur, and the remaining ring atoms are carbon, said ring system being joined to the remainder of the molecule by any of the ring atoms. Nonlimiting examples of heteroaryl groups include, but are not limited to, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, tetrazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, furanyl, thiophenyl, quinolinyl, isoquinolinyl, benzoxazolyl, benzimidazolyl, and benzothiazolyl. Unless otherwise indicated, a heteroaryl group can be an unsubstituted heteroaryl group or a substituted heteroaryl group.

[0029] As used herein, the term "halo" refers to a fluoro, chloro, bromo, or iodo group, the term "haloalkyl" refers to an alkyl group that is substituted with at least one halogen.

[0030] As used herein, the term "chemotherapeutic agent" refers to any compound that is toxic with respect to hyperproliferative cells (e.g., cancer cells) or inhibits the growth or proliferation of hyperproliferative cells and includes biologies, antibodies, small molecules, peptides and antisense oligonucleotides. Exemplary chemotherapeutic agents include, but are not limited to, an aromatase inhibitor, an anti-estrogen, an anti-androgen, a gonadorelin agonist, a

topoisomerase I inhibitor, a topoisomerase II inhibitor, a microtubule active agent, an alkylating agent, a retinoid, a carotenoid, a tocopherol, a cyclooxygenase inhibitor, an MMP inhibitor, a mTOR inhibitor, an antimetabolite, a platin compound, a methionine aminopeptidase inhibitor, a bisphosphonate, an antiproliferative antibody, a heparanase inhibitor, an inhibitor of Ras oncogenic isoforms, a telomerase inhibitor, a proteasome inhibitor, a compound used in the treatment of hematologic malignancies, a Flt-3 inhibitor, an Hsp90 inhibitor, a kinesin spindle protein inhibitor, a MEK inhibitor, an antitumor antibiotic, a nitrosourea, a compound targeting/decreasing protein or lipid kinase activity, a compound targeting/decreasing protein or lipid phosphatase activity, any further anti-angiogenic compound, and combinations thereof. Specific examples of antitumor agents include, but are not limited to, azacitidine, axathioprine, bevacizumab, bleomycin, capecitabine, carboplatin, chlorabucil, cisplatin, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, etoposide, fenretinide, fluorouracil, gemcitabine, herceptin, idarubicin, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, tafluposide, teniposide, tioguanine, retinoic acid, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, receptor tyrosine kinase inhibitors, and combinations thereof. Additional examples of antitumor or chemotherapeutic agents are known in the art.

[0031] The terms "therapeutically effective amount" and "effective amount" depend on the condition of a subject and the specific compound(s) administered. The terms refer to an amount effective to achieve a desired biological, e.g., clinical effect. A therapeutically effective amount varies with the nature of the disease being treated, the length of time that activity is desired, and the age and the condition of the subject. In one aspect, a therapeutically effective amount of a compound or composition of the disclosure is an amount effective to inhibit growth of hyperproliferative cells, prevent cancer cell metastasis, and/or result in cancer cell death.

[0032] In one aspect, the present disclosure provides a compound of Formula (I), or a pharmaceutically acceptable salt thereof:

wherein: A is R ¹ is H, Ci_ ₄ alkyl, OCi_ ₄ alkyl, SCi_ ₃ alkyl, NR ⁶ R ⁷ , or C _3-8

heterocycloalkyl; R , or C5- ₆ heteroaryl; R ³ is H, OH, halo, Ci_ ₃ alkyl, or OCi_ ₂ alkyl; R ⁴ is H, halo, Ci_ ₂ alkyl, or OCH ₃ ; R ⁵ is H, Ci_ ₃ alkyl, or OCi_ ₃ alkyl; R ⁶ and R 7 are each independently H, Ci_ ₄ alkyl, 8

C3_ ₆ cycloalkyl, or C3_ ₆ heterocycloalkyl; R is halo, C _1-3 alkylene-C ₃ _ ₈ heterocycloalkyl, or heteroaryl; R ⁹ is H or Ci_ ₃ alkyl; and R ¹⁰ is halo, Ci-salkyl, OCi_ ₄ alkyl, NR ⁶ R ⁷ , or Ci_ ₃ alkyle -C ₃ _ ₈ heterocycloalkyl. [0033] In one aspect A is . In another aspect, A is .

[0034] In Formula (I), R ¹ is H, Ci_ ₄ alkyl, OCi_ ₄ alkyl, SCi_ ₃ alkyl, NR ⁶ R ⁷ or C ₃ _

gheterocycloalkyl. In one aspect, R ¹ is H. In another aspect, R ¹ is Ci_ ₄ alkyl (e.g., methyl, ethyl, ^-propyl, isopropyl, n-butyl, sec-butyl, or ieri-butyl). For example, R ¹ can be CH ₃ or CH ₂ CH ₃ . In another aspect, R ¹ is OCi_ ₄ alkyl (e.g., OMe, OEt, OnPr, Oz ^' Pr, OnBu, OsecBu, OtertBu). For example, R ¹ can be OCH ₃ or OCH ₂ CH ₃ . In one aspect, R ¹ is SCi_ ₄ alkyl (e.g., SMe, SEt, SnPr, Sz ^' Pr, SnBu, SsecBu, StertBu). For example, R ¹ can be SCH ₃ or SCH ₂ CH ₃ . In one aspect, R ¹ is NR ⁶ R ⁷ and R ⁶ and R ⁷ are each independently H, CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ isopropyl, tert-

butyl, cyclopropyl, cyclobutyl, or cyclopentyl. For example, R ¹ is optionally HN^

H ₂ N X _{^ OR} ZA . in another aspect, R ¹ is C ₃ _ ₈ heterocycloalkyl, for example, aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, piperazinyl, dioxanyl, morpholinyl, thiomorpholinyl, or oxathianyl. Optionally, R ¹ is aziridinyl, piperazinyl, or morpholinyl, for example,

For example, R ¹ can be selected from the

² is , , or Cs-eheteroaryl. In one aspect, R ^z is

can be Ci_ ₃ alkylene-C3_ ₈ heterocycloalkyl, wherein the heterocycloalkyl is optionally piperazinyl or morpholinyl. For example, R can be CH ₂ -pi -piperazinyl, CH ₂ CH ₂ CH ₃ - piperazinyl, or CHCH ₃ CH ₃ -piperazinyl, such as can be heteroaryl (e.g., pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, tetrazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, furanyl, quinolinyl, isoquinolinyl, benzoxazolyl, yl, or benzothiazolyl). For example, R can be imidazolyl or

oxazolyl, such . Optionally, R ⁸ is can

be . In another aspect, R ² is . In some embodiments, R ⁹ is H. In various embodiments, R ⁹ is Ci^alkyl (e.g., CH ₃ or tBu). In some cases, R ⁹ is halo (e.g., F, CI, or Br). In some embodiments, R ¹⁰ is halo (e.g., F or CI). In various embodiments, R ¹⁰ is Ci_ ₄ alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, or tert-butyl). In some cases, R ¹⁰ is OCi_ ₂ alkyl (e.g., OMe or OEt). In various cases, R ¹⁰ is NR ⁶ R ⁷ . In some of these cases, R ⁶ and R ⁷ are each independently H or Ci_ ₄ alkyl (e.g., CH ₃ or

1 ⁰ is Cialkylene-Cs-gheterocycloalkyl. In some aspects, R ¹⁰ is F, . For example, R2 can be selected from the group consisting of . In one aspect, R ² is C5_ ₆ heteroaryl, wherein heteroaryl optionally is pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, tetrazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, furanyl, quinolinyl, isoquinolinyl, benzoxazolyl, benzimidazolyl, and benzothiazolyl. In some embodiments,

[0036] Optionally, R ² is

[0037] In Formula (I), R ³ is H, OH, halo, Ci_ ₃ alkyl, or OCi_ ₂ alkyl (e.g., OCH ₃ or OCH ₂ CH ₃ ).

In one aspect, R 3 is H. In another aspect, R 3 is OH. In some aspects, R 3 is halo (e.g., F or CI).

In one aspect, R 3 is Ci_ ₃ alkyl (e.g., methyl, ethyl, n-propyl, or isopropyl). For example, R 3 can be CH ₃ or CH ₂ CH ₃ . In some aspects, R ³ is OCi_ ₂ alkyl (e.g., OMe or OEt). Optionally, R ³ is

[0038] In Formula (I), R ⁴ is H, halo, Ci_ ₂ alkyl, or OCH _3. In one aspect, R ⁴ is H. In another aspect, R ⁴ is halo (e.g., F or Br). In still other aspects, R ⁴ is Ci_ ₂ alkyl (e.g., CH ₃ or CH ₂ CH ₃ ). In yet another aspect, R ⁴ is OCH ₃ .

[0039] In Formula (I), R ⁵ is H, Ci_ ₃ alkyl, or OCi_ ₃ alkyl. In one aspect, R ⁵ is H. In another aspect, R ⁵ is Ci_ ₃ alkyl (e.g., methyl, ethyl, n-propyl, or isopropyl). For example, R ⁵ can be CH ₃ or CH ₂ CH ₃ . In yet another aspect, R ⁵ is OCH ₃ . ; R ¹ is selected from the group consisting of CH ₃ ,

group consisting of

R is selected from the group consisting of C¾, CH ₂ CH ₃ , OCH ₃ , and OH; R is

H; and R is CH ₃ .

[0041] In other aspects, A is R ¹ is selected from the group consisting of CH ₃ ,

H; and R ⁵ is CH ₃ .

[0042] In some aspects, the compound of Formula (I) has a structure selected from the group consisting of:



C13.

[0043] The present disclosure also provides a composition comprising a compound described herein, e.g., one or more of compounds CI to C13 or a compound having the structure

C14, in combination with a pharmaceutically acceptable carrier. In one aspect, the composition is for use in the treatment of a neoplastic, hyperplastic, or hyperproliferative disease, such as cancer. Pharmaceutically acceptable carriers include, but are not limited to, water, saline, phosphate buffered saline, and buffers. Preferably, the carrier is sterile. Other excipients, including buffering agents, dispersing agents, and preservatives, are known in the art and may be included in the composition. Further examples of components that may be employed in compositions are presented in Remington's Pharmaceutical Sciences, 16 ^th Ed. (1980) and 20 ^th Ed. (2000), Mack Publishing Company, Easton, Pa. A composition may be in any suitable dosage form including, but not limited to, tablets, capsules, implants, depots, liquids, patches, lozenges, creams, ointments, lotions, aerosols, and eye drops.

[0044] A method of treating a neoplastic, hyperplastic, or hyperproliferative disorder in a subject in need thereof comprises administering a therapeutically effective amount of a compound or composition described herein to the subject. In a further aspect, a method of treating cancer in a subject in need thereof also is provided comprising administering a therapeutically effective amount of a compound or composition described herein to the subject. In one aspect, a method of inhibiting cancer growth or metastasis comprising contacting a cancer cell with an effective amount of the compound or composition described herein is provided. In one aspect, a method of the present disclosure comprises administering any one of compounds CI to C 14 or a combination thereof. In any of the foregoing methods, a compound or composition described herein may be administered in an amount effective to inhibit the kinase activity of c-Src and/or p38. The ability of the compounds and

compositions of the present disclosure to inhibit kinase activity necessary for cancer growth and metastasis provides therapeutic efficacy in treating a wide range of cancer types. In a further aspect, the cancer is selected from the group consisting of breast cancer, bone cancer, bladder cancer, brain cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, eye cancer, gastric cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, multiple myeloma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, prostate cancer, sarcoma, skin cancer, testicular cancer, throat cancer, and thyroid cancer. In one aspect, the cancer is sarcoma, pancreatic cancer, colon cancer, lung cancer, prostate cancer, or breast cancer, optionally triple negative breast cancer. One of ordinary skill will appreciate that treating a cancer does not require complete eradication of the cancer. Any beneficial physiologic response is contemplated, such as tumor stasis, tumor shrinkage, tumor cell death, reduction or halting or delay of metastasis, reduction in cancer cell markers, alleviation of symptoms and the like. In one aspect, a method of inhibiting cancer growth or metastasis comprises contacting a cancer cell with an effective amount of a compound or composition described herein.

[0045] In one aspect of the present methods, a therapeutically effective amount of a compound or composition described herein, typically formulated in accordance with pharmaceutical practice, is administered to a subject in need thereof. A particular

administration regimen for a given subject will depend, in part, upon the compound or composition, the amount administered, the route of administration, and the cause and extent of any side effects. The amount administered to a subject (e.g., a mammal, such as a human) in accordance with the disclosure should be sufficient to effect the desired response over a reasonable time frame. Dosage typically depends upon the route, timing, and frequency of administration.

[0046] Purely by way of illustration, the methods of the present disclosure comprise administering, e.g., from about 0.1 mg/kg to about 150 mg/kg or more of a compound of the disclosure based on the weight of the tumor or body weight of the subject, depending on the factors mentioned above. In some aspects, the dosage ranges from about 0.1 mg/kg to about 0.5 mg/kg, about 5 mg/kg to about 75 mg/kg, about 10 mg/kg to about 50 mg/kg, about 80 mg/kg to about 120 mg/kg, about 15 mg/kg to about 30 mg/kg, about 1 mg/kg to about 20 mg/kg, or about 10 mg/kg to about 25 mg/kg. The dosage is administered as needed, for example, continuously, one to three times daily, every other day, twice a week, weekly, every two weeks, monthly, or less frequently. The treatment period will depend on the particular condition and may last one day to several days, weeks, months, or years. The above dosages are exemplary of the average case, but there can be individual instances in which higher or lower dosages are merited, and such are within the scope of this disclosure.

[0047] Suitable methods of administering a physiologically acceptable composition, such as a composition comprising a compound described herein, are well known in the art. Although more than one route can be used to administer a compound, a particular route can provide a more immediate and more effective reaction than another route. Depending on the circumstances, a composition comprising one or more compounds described herein is introduced into tumor sites, applied or instilled into body cavities, absorbed through the skin or mucous membranes, inhaled, ingested and/or introduced into circulation. In one aspect, the compound or composition is administered orally. In another aspect, the compound or composition is injected intravenously and/or intraperitoneally. In still another aspect, the compound or composition is administered locally by directly contacting cancer cells with the compound or composition. For example, in certain circumstances, it will be desirable to deliver the composition through injection or infusion by intravenous, intratumoral, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, intralesional, intramedullary, intrathecal,

intraventricular, transdermal, subcutaneous, intranasal, transdermal, enteral, topical, sublingual, urethral, vaginal, or rectal means; by controlled, delayed, sustained or otherwise modified release systems; or by implantation devices. Alternatively, the composition is administered via implantation of a matrix, membrane, sponge, or another appropriate material onto which the compound has been absorbed or encapsulated. Where an implantation device is used, the device is, in one aspect, implanted into any suitable tissue or organ, and delivery of the desired compound is, for example, via diffusion, timed-release bolus, or continuous administration.

[0048] In one aspect, the compound may be attached to a targeting moiety specific for a tumor cell, such as an antigen binding protein including, but not limited to, antibodies, antibody fragments, antibody derivatives, antibody analogs, and fusion proteins, that bind a specific tumor cell antigen.

[0049] The compounds of the present disclosure may be used in combination with other therapeutic agents. Examples of antitumor therapies that can be used in combination with the compounds and compositions include surgery, radiotherapy (e.g., gamma-radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, and systemic radioactive isotopes), endocrine therapy, biologic response modifiers (e.g., interferon, interleukin, tumor necrosis factor (TNF), hyperthermia and cryotherapy), agents to attenuate any adverse effect (e.g., antiemetics), gene therapy, viruses, and any other chemotherapeutic agent. [0050] Tumor growth can be analyzed to determine the therapeutic activity of the compounds of the present disclosure. Tumor mass, volume, and/or length can be assessed using methods known in the art such as calipers, ultrasound imaging, computed tomography (CT) imaging, magnetic resonance imaging (MRI), optical imaging (e.g., bioluminescence and/or fluorescence imaging), digital subtraction angiography (DSA), positron emission tomography (PET) imaging and/or other imaging analysis. Tumor cell proliferation can also be analyzed using cellular assays that measure, e.g., DNA synthesis, metabolic activity, antigens associated with cell proliferation, and/or ATP. In various embodiments, the method of the present disclosure reduces the size of a tumor at least about 5% (e.g., at least about 10%, at least about 15%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80%). In other embodiments, the method of the present disclosure maintains the tumor size, i.e., prevents further growth of the tumor, for a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more.

[0051] The present disclosure will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting.

Example

[0052] The following Example describes the preparation and confirmation of compounds of the present disclosure as therapeutic agents.

[0053] Materials and Methods

[0054] Cell lines. MCF-IOA, MDA-MB 231, Hs578t, HCC1937, and MDA-MB 468 cells were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in the recommended media. Each cell line was used within 6 months of initial purchase. HME cells were obtained from Lonza and used within 6 months of purchase. The SUM149 cell line was provided by Dr. Stephen P. Ethier and authenticated via ATCC (STR # 13868). SUM149 cells were cultured in Ham's F-12 media (JRH Biosciences, Lenexa, KS) supplemented with 5% fetal bovine serum containing insulin and hydrocortisone (Sigma Chemical Co., St.

Louis, MO). VARI-068 cell line was derived from a TNBC patient-derived xenograft (PDX). The patient tumor was obtained from the Van Andel Research Institute (Grand Rapids, MI) and was propagated as a PDX. The cell line from the PDX was cultured in RPMI-1640 medium supplemented with 10% FBS. [0055] Reagents and antibodies. Dasatinib (pharmacological grade) was purchased from LC Laboratories (Boston, MA). Primary antibodies (SRC-Y416 #2101, AKT, p-AKT (S473/S308), ERK1/2, p-SRC (Y419), SRC, p-EGFR and EGFR were obtained from Cell Signaling Technologies (Cambridge, MA). Polyclonal antibodies to BAK and Bax-2 were obtained from Upstate Biotechnology (Lake Placid, NY) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively.

[0056] Synthesis of UM-164 ( C14). Unless otherwise noted, all reagents were obtained via commercial sources and used without further purification. BODIPY FL NHS ester was purchased from Lumiprobe. Tetrahydrofuran (THF) and dichloromethane (DCM) were dried over alumina under a nitrogen atmosphere. All 1 H and 13 C NMR spectra were measured with a Varian MR400 or Inova 500 spectrometer. The following abbreviations are used in the present disclosure: DIEA (diisopropylethylamine), HATU (1- [Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridi nium 3-oxid

hexaf uorophosphate, DMF (Ν,Ν-Dimethylformamide), TIPS (Triisopropylsilane), TFA (Trifluoroacetic acid), and NaH (sodium hydride).

[0057] Synthesis of S3: 4-methyl-3-nitroaniline (2.0 g, 13.1 mmol) was added to an oven- dried flask. Tetrahydrofuran (66 mL) was added. The reaction mixture was then cooled to 0 °C using an ice bath. 3-trifluoromethylbenzoyl chloride (2.742 g, 13.1 mmol) was then added, followed by diisopropylethylamine (2.039 g, 15.77 mmol). The reaction mixture was then allowed to warm to room temperature and stirred overnight. Tetrahydrofuran was then removed via rotary vaporization. The crude mixture was then suspended in water, filtered, and then rinsed with water twice. After drying, 4.1 g of S3 as a light yellow solid was obtained (96 % yield). Spectral data: 1H NMR (400 MHz, DMSO-d ₆ ) δ 10.77 (s, 1H), 8.50 (d, J = 2.3 Hz, 1H), 8.31 - 8.22 (m, 2H), 7.97 (t, J = 9.4 Hz, 2H), 7.77 (t, J = 7.8 Hz, 1H), 7.47 (d, J = 8.4 Hz, 1H), 1.23 (s, 3 H) ; ¹⁹ F NMR (376 MHz, dmso) δ -61.14.; HRMS-ESI (m/z): [M + H]+ calcd for C ₁₅ H ₁₁ F ₃ N ₂ O ₃ , 325.0795; found 325.0794.

S3

[0058] Synthesis ofS4: Compound S3 (2.0 g, 6.2 mmol) and iron (1.72 g, 30.8 mmol) were added to a round-bottom flask. Ethanol (25 mL) and water (6.2 mL) were then added, followed by the addition of several drops of concentrated hydrochloric acid. The reaction mixture was then heated to reflux for 90 minutes. The reaction mixture was then filtered through celite, and the celite was washed with additional hot ethanol. The ethanol was removed via rotary vaporization, and the crude reaction was then suspended in water and filtered. After drying, 1.4 g of S4 as an off-white solid was obtained (78 % yield). Spectral data: 1H NMR (400 MHz, DMSO-d ₆ ) δ 10.11 (s, 1H), 8.24 - 8.15 (m, 2H), 7.90 (d, J = 7.7 Hz, 1H), 7.72 (t, J = 7.9 Hz, 1H), 7.06 (s, 1H), 6.87 - 6.75 (m, 2H), 4.85 (s, 2H), 1.99 (s, 3H); ¹⁹ F NMR (376 MHz, dmso) δ -61.08; HRMS-ESI (m z): [M + H]+ calcd for C ₁₅ H ₁₃ F ₃ N ₂ O, 295.1053; found 295.1061.

S3 S4

[0059] Synthesis ofS5: ethyl 2-aminothiazole-5-carboxylate (1.0 g, 5.8 mmol) and 4,6- dichloro-2-methylpyrimidine (0.95 g, 5.8 mmol) were added to an oven dried flask.

Dimethylformamide (20 mL) was then added. The reaction mixture was then cooled to 0 °C, and sodium hydride (0.510 g, 12.8 mmol) was added. The reaction was allowed to warm to room temperature and stirred for an additional 3 hours. Excess base was quenched using ammonium chloride. The reaction was then suspended in water and filtered. After drying, the S5 was obtained as a white solid (1.4g, 81% yield). Spectral data: 1H NMR (400 MHz, DMSO-d ₆ ) δ 12.30 (s, 1H), 8.07 (s, 1H), 6.88 (s, 1H), 4.24 (q, J = 7.1 Hz, 2H), 2.54 (s, 3H), 1.26 (t, J = 7.1 Hz, 3H); ¹³ C NMR (126 MHz, dmso) δ 167.85, 162.83, 161.96, 159.07, 157.88, 145.84, 122.02, 104.11, 61.22, 25.61,14.70; HRMS-ESI (m/z): [M + H]+ calcd for CnHnClN ₄ 0 ₂ S, 299.0364; found 299.0371.

S5

[0060] Synthesis ofS6: Compound S5 (1.4 g, 4.7 mmol) and sodium hydroxide (1.5 g, 37.5 mmol) were added to an oven-dried round bottom flask. Methanol (11 mL) and water (4 mL) were then added. The reaction was stirred at room temperature for 48 hours. Methanol was then removed under reduced pressure. The crude reaction mixture was then suspended in 1 N HCI and filtered. After drying, S6 (0.95 g, 75 % yield) was obtained as a white solid.

Spectral data: 1H NMR (500 MHz, DMSO-d ₆ ) δ 8.03 (s, 1H), 6.97 (s, 1H), 2.56 (s, 3H); ₁₃ C NMR (126 MHz, dmso) δ 167.79, 163.39, 162.56, 158.94, 157.97, 145.28, 123.38, 104.03, 40.46, 40.29, 40.21, 40.13, 40.05, 39.96, 39.79, 39.62, 39.46, 25.57; HRMS-ESI (m/z): [M + H]+ calcd for C ₉ H ₇

S5 S6

[0061] Synthesis ofS9: Acid S6 (0.9 g, 3.3 mmol) was added to an oven-dried round bottom flask. Tetrahydrofuran (11 mL) was then added. The reaction was cooled to 0 °C. Oxalyl chloride (0.5 g, 4.0 mmol) was added, followed by a drop of dimethylformamide. The reaction was allowed to warm to room temperature and stirred for an additional twenty minutes. The crude mixture was then concentrated under reduced pressure. The crude reaction mixture was then again dissolved in tetrahydrofuran (1 lmL). Aniline S3 (1.0 g, 3.3 mmol) was then added followed by DIEA (0.43 g, 3.3 mmol). The reaction was then allowed to stir overnight at room temperature. The crude reaction mixture was then purified via silica gel chromatography using a Biotage Isolera One (linear gradient 40→ 100% EtOAc in hexanes) to yield 0.250 g of compound S9 as a white solid (14 % yield). Spectral data: 1H

NMR (400 MHz, DMSO-d ₆ ) δ 12.19 (s, 1H), 10.45 (s, 1H), 9.90 (s, 1H), 8.30 - 8.11 (m, 3H), 7.93 (d, J = 7.8 Hz, 1H), 7.86 - 7.71 (m, 2H), 7.56 (dd, J = 8.2, 2.3 Hz, 1H), 7.24 (d, J = 8.3 Hz, 1H), 6.91 (s, 1H), 2.56 (s, 3H), 2.19 (s, 3H); ¹⁹ F NMR (376 MHz, dmso) δ -61.10;

HRMS-ES 4.

S6 S9

[0062] Synthesis of UM-164 (C14): Compound S9 (0.250 g, 0.457 mmol) was added to an oven-dried round bottom flask. Dioxane (1.5 mL) was added. l-(2-hydroxyethyl)piperazine (1.190 g, 9.14 mmol) was then added. The reaction was heated to reflux overnight. The reaction mixture was then concentrated under reduced pressure. The crude mixture was then purified using reverse-phase Biotage C18 column to afford UM-164 (C14) as a white solid

(25 mg, 9% yield). Spectral data: 1H NMR (400 MHz, DMSO-d6) δ 11.42 (s, 1H), 10.43 (s, 1H), 9.74 (s, 1H), 8.29 - 8.20 (m, 2H), 8.17 (s, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.82 - 7.71 (m, 2H), 7.55 (dd, J = 8.3, 2.2 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 6.02 (s, 1H), 4.44 (s, 1H), 3.48 (m, 4H), 3.28 (s, 8H), 2.38 (s, 3H), 2.18 (s, 3H); ^iy F NMR (376 MHz, dmso) δ -61.09;

-ESI (m/z): [M + H]+ calcd for C ₃₀ H ₃₁ F ₃ N ₈ O ₃ S, 641.2265; found 641.2279.

S9 DAS-DFGO-II

Other compounds described herein were synthesized using the same general scheme.

[0063] 3D cell culture. Prior to plating cells, 50 Cultrex basement membrane extract (BME, Trevigen) was added to each well of a 96-well plate incubated on ice and then allowed to gel (cushion formation) over a 30-minute time period in a 37 °C incubator. Cells (MDA-MB-231 or SUM 149) were then dispersed from flasks and collected by centrifugation (200 x g for 5 minutes at room temperature). An aliquot of the resuspended cells was mixed with trypan blue solution, and the cell number was quantified using a hemacytometer. 100% DMSO compound stocks were prepared to 100X the final concentration desired in the assay. 3 of the DMSO stock solution was then added to 297 of the cell suspension

supplemented with 5% BME to give a DMSO concentration of 1%. The cells were plated at about 1.0 x 10 ⁴ cells per well (100 μΕΛνεΙΙ) in triplicate for each compound concentration.

[0064] Bead motility assay. Motility of cancer cells was measured using a blue fluorescent bead motility assay (Cellomics, Cell Motility Kit, Thermo Scientific, Rockford, IL). Briefly, both MDA-MB 231 and SUM 149 cells were grown to 70-80% confluency. The cells were trypsinized and seeded at a density of 500 to 600 cells/well onto a lawn of blue microscopic fluorescent beads plated on a collagen-I coated 96-well plate. Cells were treated with both vehicle (DMSO) and UM-164 at concentrations 50 nM, 100 nM, and 250 nM prior to seeding and allowed to migrate overnight at 37 °C. As cells moved across the lawn, they

phagocytosed and moved beads, creating clear tracks in their paths. The area tracked was proportional to the cell motility. After overnight incubation, the cells were fixed with 37% formaldehyde (Aldrich) followed by fixing with paraformaldehyde. Cells were then visualized using fluorescent microscopy and the dark tracks created by cell movement against fluorescent beads background was measured using ImageJ software. Three separate experiments with three replicates each were performed.

[0065] Cell invasion assays. Cell invasion was determined as described from the cell invasion assay kit (Chemicon International, Temecula, CA). Briefly, cells were trypsinized, washed in PBS, and resuspended in serum- free media and plated at a concentration of 25,000 to 50,000 cells/well in the upper chamber of a 24-transwell plate. The lower chamber of the transwell were filled with Dulbecco's modified Eagles medium supplemented with 10% fetal bovine serum. Cells were allowed to migrate for 24 hours. Non-migratory cells on the upper membrane surface were removed with a cotton swab, and the migratory cells attached to the bottom surface of the membrane were fixed with 100% ethanol followed by staining with 0.1% crystal violet for 40 minutes. Five fields from the well were selecting using 20x fields from all four corners and one center field for quantification. Each determination represents the average of three independent experiments and error bars represent SD.

[0066] Dose response studies: MTT assay was carried out to determine the inhibitory concentration (IC), which inhibits cell viability at 20, 40, and 50 %. To determine the effect of UM-164 on cell proliferation, each cell type was grown in the recommended medium. Cell proliferation studies were done by plating cells (5 to 8 x 10 ⁴ cells per well) in 96-well multi-well dishes (Corning, Inc., NY). After a 24-hour attachment period, the medium was aspirated and replaced with serum-free medium containing increasing concentration of drug (0.01 nmol/L to 10 μιηοΙ/L) (or vehicle control) for 72 hours. 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay reagent was used to measure mitochondrial metabolism to measure cell viability. For the MTT procedure, following incubation of cells for the above-mentioned time, 0.5 mg/mL MTT was added to each well and incubated at 37 °C for 3 hours. Following incubation, the medium was aspirated completely, with care taken not to disturb the formazan crystals. DMSO (100 was used to solubilize these crystals. After shaking for 5 minutes on a plate shaker, the plates were read at 570 nm with a SoftMax Pro 4.7.1 micro plate reader. All results represent the average of a three-experiment minimum of six wells for each experiment. An additional control consisted of medium alone with no cells.

[0067] Western blot analysis: Plated cells were washed with ice-cold PBS and then lysed with radioimmunoprecipitation (RIPA) buffer containing freshly added protease

(#1186145001) and phosphatase (#4906837001) inhibitors from Roche Applied Sciences (Indianapolis, IN). Lysed samples were sonicated and centrifuged at 14,000 rpm for

30 minutes at 4 °C. Supernatant was collected and total amount of protein in the lysate was calculated using BCA kit. 40 to 50 μg of protein was separated on 8 to 12% SDS

polyacramide gel electrophoresis (SDS-PAGE). Resolved protein was transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA). The membrane was blocked in 5 % milk in PBS-Tween for 1 hour and then probed with specific mouse or rabbit primary antibody overnight at 4 °C. After washing the membrane in PBS- Tween, it was incubated with either rabbit, mouse, or goat secondary antibody conjugated to horseradish peroxidase secondary antibodies for 1 hour at room temperature. The membrane was then washed three times in PBS -Tween and visualized with enhanced

chemiluminescence reagent, following the manufacturer's instructions (Amersham ECL Western Blotting Analysis System, GE Healthcare UK Limited, Little Chalfont, UK).

Polyclonal antibodies to fibronectin were obtained from Roche Applied Science. Propidium iodide was obtained from Boehringer Mannheim Corporation (Indianapolis, IN).

[0068] Xenograft study: NCr/nude mice 6 weeks of age were obtained from Taconic Biosciences (Germantown, NY) and housed in pathogen-free conditions. Mice were anesthetized by injecting ketamine/xylazine combination at a concentration of 100 mpk/10 mpk. Ten thousand MDA-MB 231 cells were mixed with matrigel (BD Biosciences, San Jose, CA) in 1: 1 ratio by volume and injected into both left and right fourth mammary fat gland. Mice were randomized to treatment groups when the tumors were palpable. UM-164 was dissolved in a mixture of DMSO/propylene glycol (1:9) (Sigma- Aldrich). The volume of administration was 0.05 ml/mouse. The control group received 10% DMSO, and the treatment groups received 10 mg/kg or 20 mg/kg of drug. Mice were treated every alternate day intraperitoneally for up to 52 consecutive days. The tumors were monitored twice a week, and the mice body weight was measured weekly. Tumor volume was calculated using the following formula: tumor volume = lxb x 0.5. The mean tumor volume and tumor weight for each treatment was compared to the vehicle treated group for statistical significance using two-tailed student' s t-test.

[0069] Results

[0070] UM-164 inhibited c-Src in an inactive conformation. UM-164 (C14) binds c-Src and forces a DFG-out inactive conformation. Crystallographic studies confirmed that UM- 164 bound c-Src in the DFG-out, inactive conformation (PDB code: 4YBJ) (Figure 1).

[0071] In biochemical assays, UM-164 was a highly potent inhibitor of c-Src with a binding constant comparable to dasatinib (UM-164 Kd = 2.7 nM, dasatinib Kd = 0.7 nM). To confirm that UM-164 was capable of inhibiting the activation of c-Src in cellulo, the effect of UM-164 on the c-Src autophosphorylation was examined in two TNBC cell lines (MDA-MB 23 land SUM 149). Inhibition of c-Src autophosphorylation was detected in a concentration and a time dependent manner for both MDA-MB 231 cells (Figure 2A) and SUM 149 cells (Figure 2B). At 120 minutes, complete abrogation of c-Src autophosphorylation was observed at 50 nmol/L, demonstrating that UM-164 was a potent c-Src inhibitor in vitro. [0072] UM-164 has potent anti-TNBC activity in vitro. Triple-negative breast cancers are highly diverse and heterogeneous lesions. To approximate the diversity of TNBC, a panel of cell lines was selected, and the expression of activated c-Src was determined by probing the total cell lysate with an antibody that specifically recognizes the activated form of c-Src (pY419). Three of the selected cell lines (MDA-MB 231, HCC1937, and MDA-MB 468) had large amounts of activated c-Src, and two (SUM 149, and Hs578t) had relatively lower amounts of activated c-Src. Notably, the panel included cell lines previously reported to be both sensitive and resistant to FDA-approved c-Src inhibitors (dasatinib resistant: MDA-MB 468, HCC 1937; dasatinib sensitive: Hs578t, MDA-MB 231, and SUM 149) (Finn et al., 2007, supra; Lehmann et al., The Journal of clinical investigation 2011, 121(7):2750-2767).

[0073] The inhibitory effects of UM-164 and dasatinib on cellular proliferation were tested and compared using the TNBC cell line panel. As shown in Table 1, UM-164 had potent anti-proliferative activity (average GI ₅₀ = 160 nM) in all TNBC cell lines tested.

TABLE 1

Importantly, UM-164 was significantly more potent than dasatinib (average GI ₅₀ = 2300 nM) in many cell types. UM-164 had consistent anti-proliferative activity across heterogeneous TNBC cell lines, while dasatinib only inhibited a subset of this group of cell lines. Moreover, UM-164 was active against a primary, low passage, patient-derived TNBC cell line (VARI- 062) initially grown from a patient-derived xenograft, while VARI-068 was relatively resistant to treatment with dasatinib. Together, the data was consistent with the poor response observed in clinical trials for TNBC tumors treated with dasatinib (Yeatman et al., supra; Yu et al., The AAPS journal 2011, 13(3):417-426). Other compounds of the disclosure were also confirmed to be more potent with dasatinib. For example, compounds C01, C02, and C04 exhibited IC ₅₀ values in MDA-MB-231 cells of 49 nM, 539 nM, and 546 nM, respectively.

[0074] To confirm that the anti-proliferative activity of UM-164 was not due to general cytotoxicity, its effects on the growth of primary human mammary epithelial cells (HMEC) and an immortalized nonmalignant, normal-like cell line (MCF-IOA) were examined. Both HMEC and MCF-IOA cells were relatively resistant to the anti-proliferation effect of UM- 164 (HMEC GI ₅₀ = 2.8 μΜ, MCF-IOA GI ₅₀ = 4.6 μΜ). Interestingly, TNBC cell lines with relatively little hyper-activated c-Src (SUM 149, Hs578t and SUM 190) were both highly sensitive to inhibition by UM-164.

[0075] Relative to two-dimensional substrates (such as polystyrene), three-dimensional (3D) cell growth in extracellular matrix is generally accepted as more closely resembling the natural growth environment for cells in tissues and can serve as an early predictor for in vivo activity. Thus, the anti-proliferative activities of UM-164 and dasatinib with MDA-MB 231, SUM 149, and VARI-068 cells grown in a 3D matrix of basement membrane extract (BME) were measured, and the results are shown in Table 2.

TABLE 2

The activity of UM-164 was preserved in 3D cell culture (average GI ₅₀ = 0.3μΜ), whereas in contrast, dasatinib has a striking loss of activity in 3D cell culture (average GI ₅₀ = 14 μΜ). The lack of activity for dasatinib in 3D cell culture with TNBC cell lines has previously been reported and again correlates with the poor outcomes of dasatinib in clinical trials with TNBC patients.

[0076] Comparing the anti-proliferative activities against TNBC cell lines grown in 3D cell culture to primary human cells (HMEC) provides an approximate cellular therapeutic index to estimate compound efficacy and suitability for further pre-clinical development (therapeutic index = GI ₅₀ for HMEC / avg. GI ₅₀ for TNBC in 3D). UM-164 had a promising therapeutic index (TI = 8.4), while the therapeutic index of dasatinib was very low (TI = 0.14).

[0077] UM-164 abrogated key signaling pathways in TNBC cells. c-Src regulates several key signaling transduction pathways involved in cell survival and cell proliferation.

Therefore, to probe the ability of UM-164 to inhibit c-Src mediated signaling pathways, SUM 149 cells were treated with the indicated concentrations of UM-164 or dasatinib for 24 hours and the phospho- and total protein levels of EGFR, p38MAPK, AKT, and p44/42MAPK were examined by western blot analysis. The results demonstrated that phosphorylation of EGFR (Tyrl068) was completely inhibited at 250 nM UM-164 treatment (Figure 3A), whereas dasatinib did not inhibit the phosphorylation of EGFR at this concentration (Figure 3B). Interestingly, c-Src-dependent phosphorylation of EGFR and p38MAPK, the downstream mediator of EGFR (Tyr845) signaling, were robustly inhibited by UM-164 compared to dasatinib. Similarly, UM-164 was more effective in inhibiting p44/42MAPK activation compared to dasatinib. Taken together, these data demonstrate that UM-164 inhibited diverse signaling pathways more efficiently than dasatinib. These results indicated that the increased efficacy of UM-164 against cancer and TNBCs in particular was due, at least in part, to potent inhibition of key signaling pathways (EGFR and p38MAPK) responsible for cellular proliferation.

[0078] To investigate further the activity of UM-164 and evaluate its generalizability, UM- 164 was tested against a primary, patient-derived TNBC cell line, VARI-068. Similar signaling changes were observed in the VARI-068 cell line as were observed with SUM 149 cells (Figure 3C). In addition, VARI-068 cells grown in 2D and 3D cell culture were sensitive to UM-164 (2D GI ₅₀ = 320 nM, 3D GI ₅₀ = 800 nM), demonstrating that UM-164 anti-TNBC activity is robust against a very low passage patient-derived TNBC cell line, despite the heterogeneous nature of TNBC.

[0079] Kinome-wide profiling of UM-164. A chemical genetic profiling of both UM-164 and dasatinib in MDA-MB-231 lysate was performed to identify the kinase targets for both inhibitors. UM-164 and dasatinib had nearly identical target profiles with the exception of the p38 kinases, as shown in Table 3. TABLE 3

UM-164 was a potent inhibitor of p38oc and ρ38β, whereas no FDA-approved c-Src inhibitor (e.g., dasatinib, bosutinib, saracatinib) has been identified as potently inhibiting both p38oc and ρ38β. Consistent with UM-164 being a potent p38 inhibitor, p38MAPK phosphorylation was totally absent in SUM 149 cells treated with 50 nM of UM-164.

[0080] Because p38 kinase activity was known to have an important role in the metastasis of TNBC tumors, it was hypothesized that UM-164 has superior anti-proliferative activity compared to dasatinib, due to the ability of UM-164 to potently inhibit the p38 kinases. To test this hypothesis, the effect of combining dasatinib with BIRB-796, a potent and selective inhibitor of the p38 kinase family, was examined in 3D cell culture. Together, dasatinib and BIRB-796 accurately reconstituted the targets of UM-164. The combination of dasatinib and BIRB-796 combination was synergistic in inhibiting the proliferation of MDA-MB-231 cells in 3D culture and had comparable activity to UM-164 (Figure 4). These results further indicated that the ability to potently inhibit p38 kinases aids in the potency of the anti-TNBC activity observed for UM-164.

[0081] Effect of UM-164 on cell apoptosis and cell cycle progression in TNBC cells. To further elucidate the anti-proliferative mechanisms of UM-164, cell apoptosis markers were examined, and a cell cycle analysis was performed. UM-164 exhibited a dose-dependent decrease in cell proliferation in all of the tested TNBC lines; however, no significant change in the pro-apoptotic proteins Bax-2 and Bak proteins was observed. Consistent with this finding, flow cytometry experiments demonstrated that UM-164 treatment of MDA-MB-231 and SUM-149 increased the proportion of G0/G1 cells by 25% and 28%, respectively, and concurrently decreased S cells by 16 and 19% in the cell cycle, respectively. The fraction of apoptotic cells (sub-Gl) was very low in both treated and untreated MDA-MB-231 (Figure 5 A) and SUM- 149 (Figure 5B) samples, indicating that the decrease in cellular proliferation was not due to apoptosis but to cell cycle arrest at Gl/S, demonstrating a cytostatic effect for UM-164.

[0082] UM-164 inhibited TNBC cell motility and invasion. C-Src is an important mediator of cell migration signaling pathways through its role in controlling the dynamics of focal adhesions. The ability of UM-164 to modulate cell motility and invasion using the SUM 149 cell line, which is characterized by prominent motility, was examined. UM- 164 was found to suppress both migration (Figure 6A) and invasion (Figure 6B) with an IC ₅₀ = 50 nM. To confirm that the observed effect was not due to cell death, the MDA-231 and SUM149 cell lines were pretreated with UM-164 and 24 hours after pretreatment, the viable cells were plated for invasion and similar results were obtained, confirming that the observed effect was due to inhibition in the invasive properties of the cells rather than to cell death. Enhanced migratory activity is linked to increased cross activation of c-Src and FAK activity. FAK phosphorylation was inhibited by UM-164 in SUM 149 cells. Furthermore, phosphorylation of paxillin, which serves as an adaptor protein in cell adhesion and is a substrate for the FAK-SRC complex was likewise inhibited in SUM- 149 cells.

[0083] In vivo efficacy of UM-164 in xenograft models of TNBC. On the basis of the anti- TNBC activity of UM-164 in vitro, the efficacy of UM-164 in orthotopic xenograft models of TNBC was evaluated. The maximum tolerated dose (MTD) for UM-164 was determined to be 140 mg/kg when injected intraperitoneally. A xenograft study using NCr/nude mice implanted with MDA-MB-231 and SUM- 149 cells was performed. Once the tumors reached a palpable size, the mice were randomized into control and treatment groups (10 and 20 mg/kg, n=5 for each group). Mice were injected intraperitoneally with either drug or vehicle every other day. At the selected doses (which represented 7 and 14% of the MTD for 10 and 20 mg/kg UM-164, respectively), there was no significant weight loss or gross abnormalities observed in the treated animals. However, tumor growth was significantly (p<0.024, 0.004) inhibited in both the 10 mg/kg and 20 mg/kg dose groups compared to the vehicle treated group (Figures 7A and 7B). In order to confirm the molecular basis for the antitumor effects of UM-164 in vivo, total protein from tumor lysates from both control and treated groups were analyzed for the expression of P-p38MAPk, P-EGFR, and P-Src. A significant decrease in the phosphorylation of p38MAPk and EGFR was observed at 20 mg/kg treatment group in MDA-MB 231 tumor samples, indicating a conserved mechanism of action compared to the in vitro signaling pathway analysis. [0084] The compounds of the disclosure may also be delivered via other modes of administration, such as oral or intravenous delivery. Pharmacokinetic studies of compound C01 administered intravenously at a dosage of 15 mg/kg found the compound to have an AUC(0-24) of 5807.8 hr*ng/mL, an AUC(O-infinity) of 5834.6 hr*ng/mL, a half-life (ti _/2 ) of 5.9 hr, a volume of distribution during terminal phase (Vz) of 22021.9 mL/kg and a clearance (CL) of 2570.8 mL/hr/kg.

[0085] Conclusion/Discussion

[0086] Novel c-Src inhibitors that specifically binds the DFG-out inactive conformation of target kinases were synthesized. Binding the inactive kinase conformation led to a phenotype similar to gene knockdown of c-Src. In contrast to dasatinib, the exemplary compound UM- 164 (C14) was highly active against diverse TNBC cell lines in vitro and active in 3D cell culture. Kinome profiling of UM-164 identified p38a and ρ38β as being potently inhibited by UM-164 and dose-dependent inhibition of p38MAP kinases was observed both in vitro and in vivo. A correlation between the amount of phospho-Src expression and the sensitivity to UM-164 among TNBC cell lines was not observed. Instead, UM-164 displayed potent antiproliferative activity in TNBC cell lines with varied expression of phospho-Src. The decrease in phosphorylation of Src and its downstream effectors was an immediate effect occurring within minutes of the treatment. The cell cycle changes and decreased proliferation rates were observed days post- treatment, which is consistent with the cell cycle doubling time and demonstrated the cytostatic effect of the compound. Both c-Src mediated phosphorylation of EGFR at Tyr845 and phosphorylation of EGFR at Tyr 1068 were strongly inhibited at a much lower concentration with UM-164 treatment than with dasatinib. Phosphorylation of the Tyr845 residue on EGFR has an important regulatory role in anchorage independent growth and transformation of breast cancer cells (Mueller et al., Translational oncology 2012, 5(5):327-334). UM-164 suppressed cell migration and invasion in MDA-MB 231 and SUM149 cells. The significant decrease in tumor growth in vivo correlated with a decrease in p-P38MAPK, p-EGFR and p-Src. Overall, the results demonstrated that UM-164 was active in vivo with excellent anti-TNBC activity and limited toxicity.

[0087] The foregoing Example demonstrates that UM-164 binds the inactive kinase conformation of c-Src. Kinome-wide profiling of UM-164 identified Src family and p38MAP kinases were potently inhibited by UM-164. Treatment with UM-164 resulted in a significant decrease in vivo of tumor growth for MDA-MB 231 and SUM149 xenografts. Dual c-Src/p38 inhibition was significantly more efficacious compared to mono-inhibition of c-Src or p38. The compounds of the present disclosure are kinase inhibitors that potently inhibit c-Src, p38a, and ρ38β, without potently inhibiting the majority of the rest of the kinome.

[0088] All publications, patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.