Methods for modulating T cell activation using carboranes and carborane analogs

Abstract

Methods for modulating immune responses in a subject using carboranes and carborane analogs are disclosed. The carboranes and carborane analogs can selectively inhibit T cell activation and / or proliferation, reducing circulating T cell levels in a subject without significantly affecting circulating levels of neutrophils, monocytes, or B cells. As a result, the carboranes and carborane analogs can be used for therapeutic and / or prophylactic purposes, including treating or preventing chronic heart failure (CHF) in a subject following a myocardial infarction (MI), and treating or preventing graft-versus-host disease (GVHD), multiple sclerosis (MS), and / or experimental autoimmune encephalomyelitis (EAE) in a subject.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 988,239, filed March 11, 2020, which is incorporated herein by reference in its entirety.

[0002] Statement of government support This invention was made with government support under R00 HL132123 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] The activation of innate and adaptive immune cells underlies the inflammatory response in many chronic diseases. Monocytes, macrophages, and dendritic cells (DCs) mediate the innate immune response, while CD3 + CD4 + Helper T cells and CD3 + CD8 + Cytotoxic T cells mediate adaptive immunity. While innate immune cells constitute the first line of defense against acute injury, chronic inflammation often involves the activation and clonal expansion of specialized effector T cells following antigen presentation. The importance of activated monocytes, macrophages, DCs, and T cells in chronic heart failure is increasingly recognized. However, the molecular mechanisms involved in pathological immune cell activation in chronic heart failure and the specific role of such changes in the progression of adverse remodeling and inflammation remain unclear. Summary of the Invention

[0004] Disclosed herein are methods for modulating immune responses in a subject using carboranes and carborane analogs. Carboranes and carborane analogs can selectively inhibit T cell activation and / or proliferation, reducing circulating T cell levels in a subject without significantly affecting circulating levels of neutrophils, monocytes, or B cells. As a result, carboranes and carborane analogs can be used for therapeutic and / or prophylactic purposes, including the treatment or prevention of chronic heart failure (CHF) in subjects following myocardial infarction (MI), as well as the treatment or prevention of graft-versus-host disease (GVHD), multiple sclerosis (MS), and / or experimental autoimmune encephalomyelitis (EAE).

[0005] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0006] [Figure 1] Gene expression of estrogen receptor (ER) α and β (ERα and ERβ) in ovaries (positive control), hearts from both males and females (M+F), and male spleens. [Figure 2] Representative flow cytometry histograms of ERα (upper panel) and ERβ (lower panel) expression in different circulating and splenic immune cells of male mice. [Figure 3] Representative flow cytometry histograms of unstimulated or CD3 / CD28 TCR-stimulated CD4+ T cells treated with estradiol (5 nM and 50 nM) or Compound 1 (5 μM), or both, and labeled with cell tracing violet (CTV; a cell proliferation dye) are shown in Figure 3. The high-to-low peak pattern of fluorescence intensity in stimulated cells reflects the halving of dye concentration at the daughter cell membrane with each successive cell division. [Figure 4]Group quantification of cell proliferation (%) was measured as dye dilution per successive cell division in the stimulated group. Mean values ​​from three separate experiments performed in quadruplicate by isolating splenic CD4+ T cells from three male mice are reported. One-way ANOVA was used for data analysis. **P<0.01, ***p<0.001, and ****p<0.0001 indicate significance relative to the unstimulated group, while $$P<0.01, $$$p<0.001, and $$$$p<0.0001 indicate significance relative to the stimulated group. [Figure 5] Group quantification of cell proliferation (%) was measured as dye dilution per successive cell division in the unstimulated group. Mean values ​​from three separate experiments performed in quadruplicate by isolating splenic CD4+ T cells from three male mice are reported. One-way ANOVA was used for data analysis. **P<0.01, ***p<0.001, and ****p<0.0001 indicate significance relative to the unstimulated group, while $$P<0.01, $$$p<0.001, and $$$$p<0.0001 indicate significance relative to the stimulated group. [Figure 6] Cell viability of CD3 / CD28-mediated in-vitro TCR stimulation with and without Compound 1 treatment. Mean values ​​are reported from three separate experiments performed in quadruplicate by isolating splenic CD4+ T cells from three male mice. One-way ANOVA was used for data analysis. [Figure 7] Representative flow cytometry histograms of TNFα expression in unstimulated or CD3 / CD28 TCR stimulated CD4+ T cells treated with either estradiol (5 nM and 50 nM) or Compound 1 (5 μM), or both. [Figure 8]Quantification of the population of TNFα-expressing CD4+ T cells in the stimulated group. Mean values ​​from three separate experiments performed in quadruplicate by isolating splenic CD4+ T cells from three male mice are reported. One-way ANOVA was used for data analysis. **p<0.01 and ****p<0.0001 represent significance relative to the unstimulated group, while $P<0.05 and $$$p<0.001 represent significance relative to the stimulated group without any other treatment. [Figure 9] Quantification of the population of TNFα-expressing CD4+ helper T cells in the unstimulated control group. Mean values ​​from three separate experiments performed in quadruplicate by isolating splenic CD4+ T cells from three male mice are reported. One-way ANOVA was used for data analysis. **p<0.01 and ****p<0.0001 represent significance relative to the unstimulated group, while $P<0.05 and $$$p<0.001 represent significance relative to the stimulated group without any other treatment. [Figure 10] Representative flow cytometry histograms of IFNγ expression in unstimulated or CD3 / CD28 TCR stimulated CD4+ T cells treated with either estradiol (5 nM and 50 nM) or Compound 1 (5 μM), or both. [Figure 11] Quantification of the population of IFNγ-expressing CD4+ T cells in the stimulated group. Mean values ​​from three separate experiments performed in quadruplicate by isolating splenic CD4+ T cells from three male mice are reported. One-way ANOVA was used for data analysis. *p<0.05 and **p<0.01 indicate significance relative to the unstimulated group. [Figure 12] Quantification of the population of IFNγ-expressing CD4+ T cells in the unstimulated control group. Mean values ​​from three separate experiments performed in quadruplicate by isolating splenic CD4+ T cells from three male mice are reported. One-way ANOVA was used for data analysis. *p<0.05 and **p<0.01 indicate significance relative to the unstimulated group. [Figure 13]Group quantification of cell viability for unstimulated or CD3 / CD28 TCR-stimulated T cells isolated from male mice and treated with either estradiol (5 nM and 50 nM) or Compound 1 (5 μM), or both. Mean values ​​from two separate experiments performed in quadruplicate by isolating splenic CD4+ T cells from two female mice are reported. Because these experiments were repeated from only two mice, statistical analysis could not be performed on these data sets. However, T cells from female mice showed trends similar to those observed with T cells isolated from male mice. [Figure 14] Quantification of group proliferation after unstimulated or CD3 / CD28 TCR stimulation and treatment with either estradiol (5 nM and 50 nM) or Compound 1 (5 μM), or both. Mean values ​​from two separate experiments performed in quadruplicate by isolating splenic CD4+ T cells from two female mice are reported. Because these experiments were repeated from only two mice, statistical analysis could not be performed on these data sets. However, T cells from female mice showed trends similar to those observed with T cells isolated from male mice. [Figure 15] Quantification of TNFα-expressing CD4+ helper T cell populations unstimulated or CD3 / CD28 TCR-stimulated and treated with either estradiol (5 nM and 50 nM) or Compound 1 (5 μM), or both. Mean values ​​from two separate experiments performed in quadruplicate by isolating splenic CD4+ T cells from two female mice are reported. Because these experiments were repeated from only two mice, statistical analysis could not be performed on these data sets. However, T cells from female mice showed trends similar to those observed with T cells isolated from male mice. [Figure 16]Quantification of IFNγ-expressing CD4+ helper T cell populations unstimulated or CD3 / CD28 TCR-stimulated and treated with either estradiol (5 nM and 50 nM) or Compound 1 (5 μM), or both. Mean values ​​from two separate experiments performed in quadruplicate by isolating splenic CD4+ T cells from two female mice are reported. Because these experiments were repeated from only two mice, statistical analysis could not be performed on these data sets. However, T cells from female mice showed trends similar to those observed with T cells isolated from male mice. [Figure 17] Group quantification of cell viability of CD4+ T cells unstimulated or stimulated with PMA / ionomycin and treated with Compound 1 (5 μM). Mean values ​​are reported from three separate experiments performed in quadruplicate by isolating splenic CD4+ T cells from three male mice. Data were analyzed using two-way ANOVA. [Figure 18] Quantification of the population of TNFα-expressing CD4+ helper T cells unstimulated or stimulated with PMA / ionomycin and treated with compound 1 (5 μM). Mean values ​​are reported from three separate experiments performed in quadruplicate by isolating splenic CD4+ T cells from three male mice. Data were analyzed using two-way ANOVA. [Figure 19] Quantification of the population of IFNγ-expressing CD4+ helper T cells unstimulated or stimulated with PMA / ionomycin and treated with compound 1 (5 μM). Mean values ​​are reported from three separate experiments performed in quadruplicate by isolating splenic CD4+ T cells from three male mice. Data were analyzed using two-way ANOVA. [Figure 20] Quantification of the population of CD69+ CD4+ activated helper T cells unstimulated or stimulated with PMA / ionomycin and treated with Compound 1 (5 μM). Mean values ​​are reported from three separate experiments performed in quadruplicate by isolating splenic CD4+ T cells from three male mice. Data were analyzed using two-way ANOVA. [Figure 21]Body weights (g) of sham-operated and myocardial infarction (MI) mice treated with either vehicle control or Compound 1. Treatment began on day 7 after infarction (designated as day 0 on the graph). For clarity of the data, SD is shown only for the vehicle-treated heart failure group, which was comparable across all groups. [Figure 22] Kaplan-Meier curves showing mortality rates in infarcted and sham-operated mice treated with vehicle or drug. Treatment began on day 7 after myocardial infarction (designated day 0 in both graphs). [Figure 23] Body weights (g) of sham-operated and myocardial infarction (MI) mice treated with either vehicle control or Compound 1. Treatment began 28 days after infarction to inhibit immune activation in the chronic phase associated with left ventricular remodeling. For clarity of data, SD is shown only for the heart failure groups treated with either vehicle or Compound 1, which were comparable across all groups. [Figure 24] Tibia-normalized heart weight (mg / mm) of sham-operated and myocardial infarction (MI) mice treated with either vehicle control or Compound 1. Treatment was initiated 28 days after infarction to inhibit immune activation in the chronic phase associated with left ventricular remodeling. Data were analyzed using two-way ANOVA. *p<0.05, and ***p<0.001. [Figure 25] Levels of circulating CD4+ helper T cells and their subsets, namely, CD4+Foxp3+ (Treg), CD4+TNFα+ cells, CD4+IFNγ+ (Th1), CD4+IL-4+ (Th2), and CD4+IL-17+ (Th17) T cells (per μL of blood) at 8 weeks after surgery in mice treated with either vehicle or Compound 1 for 4 to 8 weeks after surgery. Data were analyzed using two-way ANOVA. [Figure 26]Quantitative group data for changes in left ventricular end-systolic volume (ESV, left panel), end-diastolic volume (EDV, middle panel), and ejection fraction (EF, right panel) in ligated mice before (4 weeks after myocardial infarction) and after (8 weeks after myocardial infarction) treatment with either vehicle or Compound 1. Student's unpaired two-tailed t-test was used for data analysis. [Figure 27] Schematic diagram showing the dynamics of CD4+ T cells in the myocardium at different time intervals after myocardial infarction. [Figure 28] Experimental design for Study #1. Considering a 10% and 40% reduction rate for the sham and heart failure groups. LAD: left anterior descending artery ligation. [Figure 29] Experimental design for Study #2. Considering attrition rates of 10% and 40% for the sham and heart failure groups. [Figure 30] Schematic diagram showing the experimental protocol. At 8 weeks post-MI, CD4+ T cells from failing hearts (150 cells) and mediastinal lymph nodes (300 cells) were flow-sorted and RNA-sequenced to identify differential gene expression changes. [Figure 31] IPA analysis confirmed that SIRT1 activation is a positive upstream regulator of cardiac CD4+ T cell migration to lymph nodes. The most potent activation node downstream of SIRT1 was found to be ESR1 (ERα). [Figure 32] 1 shows predicted ESR1-dependent gene expression changes in the dataset. [Figure 33] ERα and ERβ gene expression in female ovaries, male and female hearts, and male spleens. *P<0.05, **P<0.01, ***p<0.001, and ****p<0.0001 represent significance for the indicated groups. [Figure 34] Gene expression of ERα and ERβ in splenic T cells isolated from naive mice. *P<0.05, **P<0.01, ***p<0.001, and ****p<0.0001 represent significance for the indicated groups. [Figure 35]Representative flow cytometry histograms of ERβ expression in different circulating (left) and splenic (right) immune cells from male mice. [Figure 36] Group quantification of ERβ expression in different circulating (left) and splenic (right) immune cells of male mice. Data were analyzed using one-way ANOVA with multiple comparison correction using the two-step method of Benjamini, Krieger, and Yekutieli by controlling the false positive rate. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 represent significance for CD19+ B cells; $P<0.05, $$p<0.01, and $$$p<0.001 represent significance for CD4+ T cells; ##P<0.05 represent significance for Ly6G+ neutrophils; @P<0.05 represent significance for Ly6Clow monocytes. [Figure 37] Representative flow histograms showing ERα and ERβ expression in cardiac T cells 3 days post-MI. [Figure 38] Group quantification of ERα expression in cardiac T cells at 3 days and 8 weeks post-MI. Data were analyzed using a two-tailed Student's T-test. *P<0.05 represents significance for the indicated group. [Figure 39] Group quantification of ERβ expression in cardiac T cells at 3 days and 8 weeks post-MI. Data were analyzed using a two-tailed Student's T-test. *P<0.05 represents significance for the indicated group. [Figure 40] Representative flow histograms showing ERβ expression in different splenic (left) and cardiac (right) immune cells 3 days post-MI. [Figure 41]Group quantification of ERβ mean fluorescence intensity (MFI) in splenic CD19+ B cells, CD4+ T cells, CD11b+Ly6G+ neutrophils, and CD11b+Ly6G-Ly6C+ monocytes at 3 days (left) and 8 weeks (right) post-MI. Data were analyzed using one-way ANOVA and Tukey's post-hoc test. ****p<0.0001 indicates significance for CD19+ B cells; $$P<0.01, $$$p<0.001, and $$$$p<0.0001 indicate significance for CD4+ T cells; &&P<0.01 indicates significance for Ly6Clow monocytes; ####P<0.01 indicates significance for Ly6G+ neutrophils. [Figure 42] Group quantification of ERβ mean fluorescence intensity (MFI) in CD19+ B cells, CD4+ T cells, CD11b+Ly6G+ neutrophils, and CD11b+Ly6G-Ly6C+ monocytes in hearts at 3 days (left) and 8 weeks (right) post-MI. Data were analyzed using one-way ANOVA and Tukey's post-hoc test. ****p<0.0001 indicates significance for CD19+ B cells; $$P<0.01, $$$p<0.001, and $$$$p<0.0001 indicate significance for CD4+ T cells; &&P<0.01 indicates significance for Ly6Clow monocytes; ####P<0.01 indicates significance for Ly6G+ neutrophils. *P<0.05, **P<0.01, and ***p<0.001 represent significance for the indicated groups. [Figure 43] Group quantification of mean fluorescence intensity (MFI) of ERβ in CD4+ T cells in the spleen, blood, and heart on day 3. Data were analyzed using one-way ANOVA with Tukey's post-hoc test. *P<0.05, **P<0.01, and ***p<0.001 represent significance for the indicated group. [Figure 44] Group quantification of mean fluorescence intensity (MFI) of ERβ in CD4+ T cells in the spleen, blood, and heart 8 weeks after MI. Data were analyzed using one-way ANOVA with Tukey's post-hoc test. *P<0.05, **P<0.01, and ***p<0.001 represent significance for the indicated group. [Figure 45]ERβ expression (mean fluorescence intensity) in CD19+ B cells in the circulation, spleen, and heart 3 days after MI. [Figure 46] Representative flow cytometry histograms of cell tracing violet (CTV)-labeled CD4+ T cells unstimulated or stimulated with anti-CD3 and anti-CD28 antibodies in the absence and presence of various concentrations of compound 1. The high-to-low peak pattern of fluorescence intensity in stimulated cells represents a halving of the dye concentration at the cell membrane of daughter cells with each successive cell division. The percent cell proliferation in the presence of different drug concentrations was used to derive a dose-response curve (bottom panel, right). [Figure 47] Cell proliferation rate (%) in unstimulated CD4+ T cells treated with vehicle control or estradiol (5 nM and 50 nM) in the presence or absence of Compound 1. Mean ± SD from three separate experiments performed in triplicate by isolating splenic CD4+ T cells from three male mice is reported. Data were analyzed using one-way ANOVA with multiple comparison correction using the Benjamini, Krieger, and Yekutieli two-stage method by controlling the false positive rate. **p<0.01 and ***p<0.001 represent significance relative to the unstimulated group, while $P<0.05 represents significance relative to the stimulated group treated with vehicle. [Figure 48] Representative flow cytometry histograms of CTV-labeled CD4+ T cells unstimulated or stimulated with anti-CD3 and anti-CD28 antibodies and treated with either estradiol (5 nM and 50 nM) or Compound 1 (5 μM), or both. [Figure 49] Group quantification of cell proliferation rate (%). The average values ​​of three separate experiments (performed by isolating splenic CD4+ T cells from three male mice) performed in triplicate are reported. Data analysis used one-way ANOVA with Tukey's post-hoc test. **P<0.01, ***p<0.001, and ****p<0.0001 represent significance relative to the unstimulated group, while $$P<0.01, $$$p<0.001, and $$$$p<0.0001 represent significance relative to the stimulated group. [Figure 50] Frequency of viable cells (%CD4) in stimulated (left) or unstimulated (right) CD4+ T cells treated with vehicle control or estradiol (5 nM and 50 nM) in the presence or absence of Compound 1. Mean ± SD of three separate experiments performed in triplicate by isolating splenic CD4+ T cells from three male mice is reported. Data were analyzed using one-way ANOVA with multiple comparison correction using the Benjamini, Krieger, and Yekutieli two-stage method by controlling the false positive rate. **p<0.01 and ***p<0.001 represent significance relative to the unstimulated group, while $P<0.05 represents significance relative to the stimulated group treated with vehicle. [Figure 51] Representative flow histograms showing TNFα expression in unstimulated and stimulated CD4+ T cells treated with either estradiol or Compound 1, or both. Mean values ​​from three separate experiments performed in triplicate (performed by isolating splenic CD4+ T cells from three male mice) are reported. Data analysis used one-way ANOVA with Tukey's post-hoc test. **P<0.01, ***p<0.001, and ****p<0.0001 indicate significance relative to the unstimulated group, while $$P<0.01, $$$p<0.001, and $$$$p<0.0001 indicate significance relative to the stimulated group. [Figure 52] Group quantification of CD4+TNFα+ cell frequencies. Mean values ​​from three separate experiments performed in triplicate (performed by isolating splenic CD4+ T cells from three male mice) are reported. Data analysis used one-way ANOVA with Tukey's post-hoc test. **P<0.01, ***p<0.001, and ****p<0.0001 indicate significance relative to the unstimulated group, while $$P<0.01, $$$p<0.001, and $$$$p<0.0001 indicate significance relative to the stimulated group. [Figure 53]Group quantification of CD4+IFNγ+ cell frequencies. Mean values ​​from three separate experiments performed in triplicate (performed by isolating splenic CD4+ T cells from three male mice) are reported. Data analysis used one-way ANOVA with Tukey's post-hoc test. **P<0.01, ***p<0.001, and ****p<0.0001 indicate significance relative to the unstimulated group, while $$P<0.01, $$$p<0.001, and $$$$p<0.0001 indicate significance relative to the stimulated group. [Figure 54] Expression of TNFα in unstimulated CD4+ T cells treated with vehicle control or estradiol (5 nM and 50 nM) in the presence or absence of Compound 1. Mean ± SD of three separate experiments performed in triplicate by isolating splenic CD4+ T cells from three male mice is reported. Data were analyzed using one-way ANOVA with multiple comparison correction using the Benjamini, Krieger, and Yekutieli two-stage method by controlling the false positive rate. **p<0.01 and ***p<0.001 represent significance relative to the unstimulated group, while $P<0.05 represents significance relative to the stimulated group treated with vehicle. [Figure 55] IFNγ expression in unstimulated CD4+ T cells treated with vehicle control or estradiol (5 nM and 50 nM) in the presence or absence of Compound 1. Mean ± SD of three separate experiments performed in triplicate by isolating splenic CD4+ T cells from three male mice is reported. Data were analyzed using one-way ANOVA with multiple comparison correction using the Benjamini, Krieger, and Yekutieli two-stage method by controlling the false positive rate. **p<0.01 and ***p<0.001 represent significance relative to the unstimulated group, while $P<0.05 represents significance relative to the stimulated group treated with vehicle. [Figure 56]The frequency of CD69+ cells (% viable cells) in stimulated CD4+ T cells treated with vehicle, estradiol (5 nM and 50 nM), or Compound 1 drug (5 μM), or both. Mean ± SD values ​​are reported from three separate experiments performed in triplicate by isolating splenic CD4+ T cells from three male mice. Data were analyzed using one-way ANOVA with multiple comparison correction using the Benjamini, Krieger, and Yekutieli two-stage method by controlling the false positive rate. **p<0.01 and ***p<0.001 represent significance relative to the unstimulated group, while $P<0.05 represents significance relative to the stimulated group treated with vehicle. [Figure 57] Quantification of viable cells (%CD4) in unstimulated or CD3 / CD28 TCR-stimulated groups treated with either estradiol (5 nM and 50 nM) or Compound 1 (5 μM), or both. Mean values ​​from two separate experiments performed in quadruplicate are reported by isolating splenic CD4+ T cells from two female mice. Data were analyzed using one-way ANOVA with multiple comparison correction using the Benjamini, Krieger, and Yekutieli two-step method, controlling for false positive rates. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 represent significance relative to the vehicle-treated unstimulated group, while $P<0.05, $$P<0.01, and $$$P<0.001 represent significance relative to the vehicle-treated stimulated group. [Figure 58]Quantification of proliferation (% viable cells) of groups unstimulated or CD3 / CD28 TCR-stimulated and treated with either estradiol (5 nM and 50 nM) or Compound 1 drug (5 μM), or both. Mean values ​​from two separate experiments performed in quadruplicate are reported by isolating splenic CD4+ T cells from two female mice. Data were analyzed using one-way ANOVA with multiple comparison correction using the Benjamini, Krieger, and Yekutieli two-step method, controlling for false positive rates. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 represent significance relative to the vehicle-treated unstimulated group, while $P<0.05, $$P<0.01, and $$$P<0.001 represent significance relative to the vehicle-treated stimulated group. [Figure 59] Quantification of TNFα+ groups unstimulated or CD3 / CD28 TCR stimulated and treated with either estradiol (5 nM and 50 nM) or Compound 1 drug (5 μM), or both. Mean values ​​from two separate experiments performed in quadruplicate are reported by isolating splenic CD4+ T cells from two female mice. Data were analyzed using one-way ANOVA with multiple comparison correction using the Benjamini, Krieger, and Yekutieli two-step method, controlling for false positive rates. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 represent significance relative to the vehicle-treated unstimulated group, while $P<0.05, $$P<0.01, and $$$P<0.001 represent significance relative to the vehicle-treated stimulated group. [Figure 60]Quantification of IFNγ+ helper CD4+ T cell populations unstimulated or CD3 / CD28 TCR-stimulated and treated with either estradiol (5 nM and 50 nM) or Compound 1 drug (5 μM), or both. Mean values ​​from two separate experiments performed in quadruplicate are reported by isolating splenic CD4+ T cells from two female mice. Data were analyzed using one-way ANOVA with multiple comparison correction using the Benjamini, Krieger, and Yekutieli two-step method, controlling for false positive rates. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 represent significance relative to the vehicle-treated unstimulated group, while $P<0.05, $$P<0.01, and $$$P<0.001 represent significance relative to the vehicle-treated stimulated group. [Figure 61] Group quantification of cell viability when unstimulated or stimulated with PMA / ionomycin and treated with Compound 1 drug (5 μM). Mean values ​​are reported from three separate experiments performed in quadruplicate by isolating splenic CD4+ T cells from three male mice. [Figure 62] Quantification of TNFα+ groups unstimulated or stimulated with PMA / ionomycin and treated with Compound 1 drug (5 μM). Mean values ​​are reported from three separate experiments performed in quadruplicate by isolating splenic CD4+ T cells from three male mice. Two-way ANOVA with Tukey's post-hoc test was used for data analysis, and the respective p-values ​​are shown. *P<0.05, **P<0.01, ***p<0.001, and ****p<0.0001 represent significance for the indicated groups. [Figure 63]Quantification of IFNγ+ groups unstimulated or stimulated with PMA / ionomycin and treated with Compound 1 drug (5 μM). Mean values ​​are reported from three separate experiments performed in quadruplicate by isolating splenic CD4+ T cells from three male mice. Two-way ANOVA with Tukey's post-hoc test was used for data analysis, and the respective p-values ​​are shown. *P<0.05, **P<0.01, ***p<0.001, and ****p<0.0001 represent significance for the indicated groups. [Figure 64] Quantification of CD69+ helper T cell (CD4+) populations unstimulated or stimulated with PMA / ionomycin and treated with Compound 1 drug (5 μM). Mean values ​​are reported from three separate experiments performed in quadruplicate by isolating splenic CD4+ T cells from three male mice. Two-way ANOVA with Tukey's post-hoc test was used for data analysis, and the respective p-values ​​are shown. *P<0.05, **P<0.01, ***p<0.001, and ****p<0.0001 represent significance for the indicated groups. [Figure 65] Principal component analysis of the RNA transcriptome of naive and stimulated CD4+ T cells treated with vehicle (DMSO) or compound 1 (5 μM). [Figure 66] Volcano plot showing that several genes (marked in red) were up- or down-regulated by more than two-fold in stimulated CD4+ T cells treated with compound 1 (5 μM). Some representative genes that showed very high LogP values ​​or very high fold changes are shown. [Figure 67] Ingenuity pathway analysis of the RNA transcriptome of stimulated CD4+ T cells treated with either vehicle control (DMSO) or Compound 1 (5 μM) to demonstrate activation of ERβ and downregulation of the ERα pathway, indicating drug specificity. [Figure 68] Heatmap showing genes in the ERβ pathway that are significantly up- or down-regulated in stimulated CD4+ T cells upon treatment with Compound 1 (5 μM). [Figure 69]Heatmap showing genes in the TCR pathway that are significantly up- or down-regulated in stimulated CD4+ T cells upon treatment with compound 1 (5 μM). [Figure 70] Figure 1 shows genes that are significantly up- or down-regulated in stimulated CD4+ T cells upon treatment with Compound 1 (5 μM). [Figure 71] Schematic of the experimental design to test the efficacy of Compound 1 in the acute phase of MI and chronic HF. [Figure 72] Body weights (g) of sham-operated and myocardial infarction (MI) mice treated with either vehicle control or Compound 1 (60 mg / kg / day; gavage). For clarity of data, SD is shown only for the vehicle-treated HF group, which was comparable across all groups. [Figure 73] Kaplan-Meier curves showing mortality rates for MI and sham-operated mice treated with vehicle or drug, with treatment beginning 7 days post-MI (designated as day 0 in the graphs in Figures 72 and 73). [Figure 74] End-systolic and end-diastolic volumes (ESV and EDV) and ejection fraction (EF) of mice 4 weeks post-MI. [Figure 75] Body weight (g) of sham-operated and MI mice treated with either vehicle control or Compound 1. For clarity of data, SD is shown only for both HF groups, which were comparable across all groups. [Figure 76] Representative B-mode tracings showing systolic and diastolic heart failure at 4 weeks (at randomization) and 8 weeks post-MI after treatment with either vehicle or drug. [Figure 77] Group quantification of changes in end-systolic and end-diastolic volumes (ESV and EV), and ejection fraction (EF) 4 to 8 weeks post-MI after treatment with either vehicle or drug. Data were analyzed using an unpaired two-tailed Student's T-test. *P<0.05 and ****p<0.0001 represent significance for the indicated groups. [Figure 78]Heart rate (BPM) in mice treated with either vehicle control or Compound 1 at 4 and 8 weeks post-MI. [Figure 79] Gravimetric data of tibia-normalized heart weights from sham and HF mice treated with either vehicle or Compound 1. Data analysis used two-way ANOVA with Tukey's post-hoc test. *P<0.05, **P<0.01, ***p<0.001, and ****p<0.0001 represent significance for the indicated groups. [Figure 80] Gravimetric data of tibia-normalized LV weights from sham and HF mice treated with either vehicle or Compound 1. Data analysis used two-way ANOVA with Tukey's post-hoc test. *P<0.05, **P<0.01, ***p<0.001, and ****p<0.0001 represent significance for the indicated groups. [Figure 81] Representative images of LV sections stained with FITC-conjugated wheat germ agglutinin to demonstrate cardiac hypertrophy. Boxed areas in the top panel are shown at original magnification in the bottom panel. [Figure 82] Quantification of cardiomyocyte area groups. Data analysis was performed using a two-tailed Student's T-test. *P<0.05, **P<0.01, ***P<0.001, and ****p<0.0001 represent significance for the indicated groups. [Figure 83] Gene expression of cardiac hypertrophy markers in the remote LV of HF mice treated with either vehicle or Compound 1 at 4-8 weeks post-MI. Data analysis was performed using a two-tailed Student's T-test. *P<0.05, **P<0.01, ***P<0.001, and ****p<0.0001 represent significance for the indicated groups. [Figure 84] Heatmap showing cardiac hypertrophy genes in stimulated CD4+ T cells upon treatment with Compound 1 (5 μM). [Figure 85]Representative flow scatter plots of CD4+ and CD8+ T cells at 8 weeks post-MI in mice treated with either vehicle or Compound 1 from 4 to 8 weeks after surgery, as well as quantitative data for circulating CD4+ helper T cells ( / µL blood) and their subsets, i.e., CD4+TNFα+ cells, CD4+Foxp3+ (Treg), CD4+IFNγ+ (Th1), CD4+IL-4+ (Th2), and CD4+IL-17+ (Th17) T cells. Data analysis used an unpaired two-tailed Student's T-test, with *p<0.05, **p<0.01, and ***p<0.001, and ****p<0.0001 considered significant. [Figure 86] Representative flow scatter plots showing CD4+ and CD8+ T cells among total CD45+ cells. [Figure 87] Levels of CD4+ helper T cells and their proinflammatory subsets, i.e., CD4+TNFα+ cells and CD4+IFNγ+ (Th1) T cells, at 8 weeks post-MI in mice treated with either vehicle or Compound 1 from 4 to 8 weeks post-MI. Data analysis was performed using an unpaired two-tailed Student's T-test. *P<0.05, **P<0.01, and ***p<0.001 indicate significance for the indicated groups. [Figure 88] Representative flow scatter plots of splenic CD4+ and CD8+ T cells among CD45+ leukocytes. [Figure 89] Quantification of splenic CD4+ helper T cell populations (total cells and frequency) at 8 weeks post-MI in mice treated with either vehicle or Compound 1 drug 4-8 weeks after surgery. Data analysis was performed using an unpaired two-tailed Student's T-test. *P<0.05, **P<0.01, and ***p<0.001 indicate significance for the indicated groups. [Figure 90] Representative flow scatter plots of splenic CD4+ and FoxP3+ T cells. [Figure 91] Quantitative data of splenic CD4+FoxP3+ regulatory T cells (total cells) at 8 weeks post-MI in mice treated with either vehicle or Compound 1 4–8 weeks after surgery. Data analysis used a two-tailed Student's T-test, with *p<0.05 considered significant. [Figure 92] Quantitative data of splenic CD4+FoxP3+ regulatory T cells (frequency) at 8 weeks post-MI in mice treated with either vehicle or Compound 1 4–8 weeks after surgery. Data analysis was performed using a two-tailed Student's T-test, with *p<0.05 considered significant. [Figure 93] Quantitative data of FoxP3 MFI (protein expression) at 8 weeks post-MI in mice treated 4–8 weeks after surgery with either vehicle or compound 1. Data analysis used a two-tailed Student's T-test, with *p<0.05 considered significant. [Figure 94] ERβ MFI (protein expression) in CD4+ and CD8+ T cells (top) and in different CD4+ helper T cell subsets, namely, Treg, Th1, and Th17 T cells, in mice treated with either vehicle or Compound 1 at 4–8 weeks after surgery. Data analysis was performed using a two-tailed Student's T-test. [Figure 95] Quantitative data (frequency of occurrence) of cardiac CD11b+ myeloid cells, CD11b+Ly6G+ neutrophils, CD11b+Ly6G-Ly6C+ monocytes (Ly6Chigh pro-inflammatory and Ly6Clow patrolling), CD19+ B cells, and CD8+ T cells at 8 weeks after surgery in mice treated with either vehicle or Compound 1 at 4 to 8 weeks after surgery. Comparisons of each cell type were performed using a two-tailed Student's T-test. [Figure 96] Quantitative data (frequency of occurrence) of circulating CD11b+ myeloid cells, CD11b+Ly6G+ neutrophils, CD11b+Ly6G-Ly6C+ monocytes (Ly6Chigh proinflammatory and Ly6Clow patrolling), CD19+ B cells, and CD8+ T cells at 8 weeks after surgery in mice treated with either vehicle or Compound 1 at 4 to 8 weeks after surgery. Comparisons of each cell type were performed using a two-tailed Student's t-test. [Figure 97]Quantitative data (frequency of occurrence) of splenic CD11b+ myeloid cells, CD11b+Ly6G+ neutrophils, CD11b+Ly6G-Ly6C+ monocytes (Ly6Chigh pro-inflammatory and Ly6Clow patrolling), CD19+ B cells, and CD8+ T cells 8 weeks after surgery in mice treated with either vehicle or Compound 1 at 4 to 8 weeks after surgery. Comparisons of each cell type were performed using a two-tailed Student's t-test. [Figure 98] Tibia-normalized thymus weight at 8 weeks post-surgery in mice treated with either vehicle or Compound 1 drug at 4–8 weeks post-surgery. Comparisons were made between each cell type using a two-tailed Student's T-test. [Figure 99] Numbers (left) and frequencies (right) of single-positive double-negative (DN; CD4-CD8-), single-positive (SP; CD4+CD8- and CD4-CD8+), and double-positive (DP; CD4+CD8+) T cells in the thymus at 8 weeks after surgery in mice treated with either vehicle or Compound 1 from 4 to 8 weeks after surgery. Comparisons of each cell type were performed using a two-tailed Student's T-test. [Figure 100] At 8 weeks post-surgery in mice treated with either vehicle or Compound 1 from 4 to 8 weeks post-surgery, double-negative (DN) T cells were further separated into DN1 (CD44+CD25-), DN2 (CD44+CD25+), DN3 (CD44-CD25+), and DN4 (CD44-CD25-) T cells. A two-tailed Student's T-test was used to compare each cell type. DETAILED DESCRIPTION OF THE INVENTION

[0007] The compounds, compositions, and methods described herein may be understood more readily by reference to the following detailed description of certain aspects of the disclosed subject matter and the examples included therein.

[0008] Before the present compounds, compositions, and methods are disclosed and described, it is to be understood that the embodiments described below are not limited to particular synthetic methods or to particular reagents, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0009] Also, throughout this specification, various publications are referenced. 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 to which the subject matter of this disclosure pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

[0010] General definition In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings.

[0011] Throughout the description and claims of this specification, the word "comprise" and other forms of that word, such as "comprising" and "comprises," mean "including but not limited to" and are not intended to exclude, for example, other additives, components, integers, or steps.

[0012] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "composition" includes mixtures of two or more such compositions, reference to an "agent" includes mixtures of two or more such agents, reference to a "component" includes mixtures of two or more such components, etc.

[0013] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances in which the event or circumstance occurs and instances in which it does not occur.

[0014] Ranges can be expressed herein as from "about" one particular value and / or to "about" another particular value. "About" means within 5% of a value, for example, within 4, 3, 2, or 1% of a value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant in relation to the other endpoint, and independently of the other endpoint.

[0015] It should be understood that throughout this specification, the identifiers "first" and "second" are used merely to facilitate distinguishing between various components and steps of the disclosed subject matter. The identifiers "first" and "second" are not intended to imply a particular order, quantity, priority, or importance to the components or steps modified by these terms.

[0016] As used herein, "subject" means an individual. Thus, "subject" can include domestic animals (e.g., cats, dogs, etc.), livestock (e.g., cows, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, etc.), and birds. "Subject" can also include mammals, such as primates or humans. Thus, a subject can be a human or animal patient. The term "patient" refers to a subject receiving treatment from a clinician, e.g., an internist.

[0017] The term "inhibit" refers to a decrease in an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete elimination of the activity, response, condition, or disease. It can also include, for example, a 10% decrease in the activity, response, condition, or disease compared to native or control levels. Thus, the decrease can be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any decrease therebetween compared to native or control levels.

[0018] "Reduce" or other forms of the word, such as "reduction" or "attenuation," refer to a decrease in an event or characteristic (e.g., tumor growth). This is usually understood to be relative to some standard or expected value, in other words, a relative value, but does not necessarily refer to a standard or relative value. For example, "reducing tumor growth" means reducing the rate of tumor growth compared to a standard or control.

[0019] "Prevent" or other forms of the word, such as "inhibit" or "prevention," mean to stop a particular event or characteristic, stabilize or delay the occurrence or progression of a particular event or characteristic, or minimize the likelihood of a particular event or characteristic occurring. Prevent is usually absolute, as compared to, for example, reduce, and therefore does not require a comparison with a control. As used herein, some things can be reduced but not prevented, or some things can be prevented by reducing them. Similarly, some things can be prevented but not reduced, or some things can be reduced by preventing them. When reduce or prevent is used, it is understood that the use of other terms is also expressly disclosed unless specifically indicated otherwise. For example, the terms "prevent" or "suppress" can refer to a treatment that forestalls or delays the onset of a disease or condition or reduces the severity of a disease or condition. Thus, if a treatment can treat a disease in a subject with symptoms of the disease, it can also prevent or suppress the disease in a subject who has not yet developed some or all of the symptoms.

[0020] The term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, i.e., treatment specifically aimed at ameliorating a disease, condition, or disorder, and also includes causal treatment, i.e., treatment aimed at eliminating the cause of the associated disease, condition, or disorder. In addition, this term includes palliative treatment, i.e., treatment aimed at alleviating symptoms rather than curing the disease, pathological condition, or disorder; preventative treatment, i.e., treatment aimed at minimizing or partially or completely inhibiting the onset of the associated disease, pathological condition, or disorder; and supportive treatment, i.e., treatment employed to complement another specific therapy aimed at ameliorating the associated disease, pathological condition, or disorder. By way of example, with respect to fibrotic conditions, "treating," "treat," and "treatment" as used herein refer to partially or completely inhibiting or alleviating the fibrotic condition from which the subject is suffering. In one embodiment, the term refers to actions taken while a patient is suffering from or diagnosed with a fibrotic condition that reduce the severity of the condition or slow or delay the progression of the condition. Treatment need not result in a complete cure of the condition; partial inhibition or reduction of the fibrotic condition is encompassed by the term.

[0021] As used herein, "therapeutically effective amount" refers to the minimum amount or concentration of an ERβ agonist that, when administered alone or in combination, is sufficient to provide a therapeutic effect in treating a condition or to delay or minimize one or more symptoms associated with the condition. The term "therapeutically effective amount" can encompass an amount that improves overall therapy, reduces or avoids the symptoms or pathogenesis of a disease, or enhances the therapeutic efficacy of another therapeutic agent. A therapeutic amount need not result in a complete cure of the condition; partial inhibition or reduction of a fibrotic condition is encompassed by this term.

[0022] As used herein, the terms "prevent," "inhibit," and "prevention," unless otherwise specified, refer to an action taken before a subject begins to suffer from a condition or before such a condition recurs. Prevention need not result in a complete prevention of the condition; partial prevention or reduction of a fibrotic condition is encompassed by this term.

[0023] As used herein, unless otherwise specified, a "prophylactically effective amount" of ERβ, when administered alone or in combination, prevents a condition, or one or more symptoms associated with a condition, or prevents its recurrence. The term "prophylactically effective amount" can encompass an amount that improves overall prevention or enhances the prophylactic efficacy of another prophylactic agent. A prophylactic amount need not result in complete prevention of a condition; partial prevention or reduction of a fibrotic condition is encompassed by this term.

[0024] The term "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of safe medical judgment and commensurate with a reasonable benefit / risk ratio.

[0025] chemical definition Terms used herein have their conventional meaning in the art unless otherwise specified. The organic moieties referred to in defining variable positions within the general formulae described herein (e.g., the term "halogen") are generic terms for the individual substituents contained within the organic moiety. The prefix C before a group or moiety n -C m indicates in each case the possible number of carbon atoms in the group or moiety that follows.

[0026] As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, heteroatoms present in a compound or moiety, such as nitrogen, can have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valences of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. The terms "substituted" or "substituted with" also include the implicit proviso that such substitution is subject to the permissible valences of the substituted atom and substituent, and that the substitution results in a stable compound (e.g., a compound that does not spontaneously undergo transformation by rearrangement, cyclization, elimination, etc.).

[0027] "Z 1 "," "Z 2 "," "Z 3 " and "Z 4 " is used herein as a generic term to represent various specific substituents. These symbols can be any substituent not limited to the substituents disclosed herein, and when they are defined as specific substituents in one instance, they may be defined as several other substituents in another instance.

[0028] As used herein, the term "alkyl" refers to a saturated, straight-chain or branched saturated hydrocarbon moiety. Unless otherwise specified, C-C 24 (e.g., C1-C 22 , C1-C 20 , C1-C 18 , C1-C 16 , C1-C 14 , C1-C 12 , C1-C 10, C1-C8, C1-C6, or C1-C4 alkyl groups are contemplated. Examples of alkyl groups include methyl, ethyl, propyl, 1-methyl-ethyl, butyl, 1-methyl-propyl, 2-methyl-propyl, 1,1-dimethyl-ethyl, pentyl, 1-methyl-butyl, 2-methyl-butyl, 3-methyl-butyl, 2,2-dimethyl-propyl, 1-ethyl-propyl, hexyl, 1,1-dimethyl-propyl, 1,2-dimethyl-propyl, 1-methyl-pentyl, 2-methyl-pentyl, 3-methyl-pentyl, 4-methyl-pentyl, 1,1-dimethyl-butyl, 1,2-dimethyl-butyl, 1,3-dimethyl-butyl, 2,2-dimethyl-butyl, 2,3-dimethyl-butyl, 3,3-dimethyl-butyl, 1-ethyl-butyl, 2-ethyl-butyl, 1,1,2-trimethyl-propyl, 1,2,2-trimethyl-propyl, 1-ethyl-1-methyl-propyl, and 1-ethyl-2-methyl-propyl. Alkyl substituents may be unsubstituted or substituted with one or more chemical moieties. Alkyl groups can be substituted with one or more groups, including, but not limited to, hydroxy, halogen, acyl, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acyl, aldehyde, amino, carboxylic acid, ester, ether, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as long as the substituents are sterically compatible and chemical bonding and strain energy rules are met, as described below. Alkyl groups can also contain one or more heteroatoms (e.g., 1 to 3 heteroatoms) incorporated within the hydrocarbon portion. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus.

[0029] Although "alkyl" is used throughout this specification generally to refer to both unsubstituted and substituted alkyl groups, substituted alkyl groups are specifically referred to herein by identifying the particular substituent(s) on the alkyl group. For example, the term "halogenated alkyl" specifically refers to an alkyl group substituted with one or more halides (halogens; e.g., fluorine, chlorine, bromine, or iodine). The term "alkoxyalkyl" specifically refers to an alkyl group substituted with one or more alkoxy groups, as described below. The term "alkylamino" specifically refers to an alkyl group substituted with one or more amino groups, as described below. When "alkyl" is used in one example and a specific term such as "alkylalcohol" is used in another example, it is not intended that the term "alkyl" not also refer to the specific term such as "alkylalcohol."

[0030] This practice is also used for other groups described herein. That is, a term such as "cycloalkyl" refers to both unsubstituted and substituted cycloalkyl moieties, but the substituted moieties can be further specifically identified herein; for example, a particular substituted cycloalkyl can be referred to specifically as, for example, an "alkylcycloalkyl." Similarly, a substituted alkoxy can be specifically referred to as, for example, a "halogenated alkoxy," a particular substituted alkenyl can be, for example, an "alkenylalcohol," and the like. Again, the practice of using a general term such as "cycloalkyl" and a specific term such as "alkylcycloalkyl" is not intended to mean that the general term does not also include the specific term.

[0031] As used herein, the term "alkenyl" refers to an unsaturated, straight-chain or branched hydrocarbon moiety containing a double bond. Unless otherwise specified, C-C 24 (For example, C2-C 22 , C2-C 20 , C2-C 18 , C2-C 16 , C2-C 14 , C2-C 12 , C2-C 10, C2-C8, C2-C6, C2-C4) alkenyl groups are contemplated. The alkenyl group may contain multiple unsaturated bonds.Examples include ethenyl, 1-propenyl, 2-propenyl, 1-methylethenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-1-butenyl, 2-methyl-1-butenyl, 3-methyl-1-butenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 3-methyl-3-butenyl, 1,1-dimethyl-2-propenyl, 1,2-dimethyl-1-propenyl, 1,2-dimethyl-2-propenyl, 1-ethyl-1-propenyl, 1-ethyl-2-propenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-1-pentenyl, 2-methyl-1-pentenyl, 3-methyl-1-pentenyl, 4-methyl-1-pentenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, 1-methyl -3-pentenyl, 2-methyl-3-pentenyl, 3-methyl-3-pentenyl, 4-methyl-3-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1,1-dimethyl-2-butenyl, 1,1-dimethyl-3-butenyl, 1,2-dimethyl-1-butenyl, 1,2-dimethylyl-2-butenyl, 1,2-dimethyl-3-butenyl, 1,3-dimethyl-1-butenyl, 1,3-dimethyl-2-butenyl, 1,3-dimethyl-3-butenyl, 2,2-dimethyl-3-butenyl thenyl, 2,3-dimethyl-1-butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 3,3-dimethyl-1-butenyl, 3,3-dimethyl-2-butenyl, 1-ethyl-1-butenyl, 1-ethyl-2-butenyl, 1-ethyl-3-butenyl, 2-ethyl-1-butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, 1,1,2-trimethyl-2-propenyl, 1-ethyl-1-methyl-2-propenyl, 1-ethyl-2-methyl-1-propenyl, and 1-ethyl-2-methyl-2-propenyl.The term "vinyl" refers to a group having the structure -CH=CH; 1-propenyl refers to a group having the structure -CH=CH-CH; 2-propenyl refers to a group having the structure -CH-CH=CH. (Z. 1 Z 2 )C=C(Z 3 Z 4 Asymmetric structures, such as alkene, alkene, alkene-substituted alkenyl, alkene-substituted alkene, and alkene-substituted alkene-substituted alkenyl, are intended to include both the E and Z isomers. This can be assumed in structural formulas herein where an asymmetric alkene is present, or can be explicitly indicated by the bond symbol C═C. Alkenyl substituents can be unsubstituted or substituted with one or more chemical moieties. Examples of suitable substituents include, for example, alkyl, alkyl halide, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acyl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as long as the substituents are sterically compatible and chemical bonding and strain energy rules are satisfied, as described below.

[0032] As used herein, the term "alkynyl" refers to a straight or branched chain hydrocarbon moiety containing a triple bond. Unless otherwise specified, C-C 24 (For example, C2-C 22 , C2-C 20 , C2-C 18 , C2-C 16 , C2-C 14 , C2-C 12 , C2-C 10, C2-C8, C2-C6, C2-C4) alkynyl groups are contemplated. The alkynyl group may contain multiple unsaturated bonds. Examples include C2-C6-alkynyl, such as ethynyl, 1-propynyl, 2-propynyl (or propargyl), 1-butynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 3-methyl-1-butynyl, 1-methyl-2-butynyl, 1-methyl-3-butynyl, 2-methyl-3-butynyl, 1,1-dimethyl-2-propynyl, 1-ethyl-2-propynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 3-methyl-1-pentynyl, 4 Alkynyl substituents include 1-methyl-1-pentynyl, 1-methyl-2-pentynyl, 4-methyl-2-pentynyl, 1-methyl-3-pentynyl, 2-methyl-3-pentynyl, 1-methyl-4-pentynyl, 2-methyl-4-pentynyl, 3-methyl-4-pentynyl, 1,1-dimethyl-2-butynyl, 1,1-dimethyl-3-butynyl, 1,2-dimethyl-3-butynyl, 2,2-dimethyl-3-butynyl, 3,3-dimethyl-1-butynyl, 1-ethyl-2-butynyl, 1-ethyl-3-butynyl, 2-ethyl-3-butynyl, and 1-ethyl-1-methyl-2-propynyl. Alkynyl substituents may be unsubstituted or substituted with one or more chemical moieties. Examples of suitable substituents include, for example, alkyl, alkyl halide, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acyl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below.

[0033] As used herein, the term "aryl," and derivative terms such as aryloxy, refer to groups containing a monovalent aromatic carbocyclic group of 3 to 20 carbon atoms. An aryl group can contain a single ring or multiple fused rings. In some embodiments, an aryl group includes C6-C 10Aryl groups are included. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl, tetrahydronaphthyl, phenylcyclopropyl, and indanyl. In some embodiments, the aryl group can be a phenyl, indanyl, or naphthyl group. The term "heteroaryl" is defined as a group containing an aromatic group with at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. The term "non-heteroaryl" within the term "aryl" defines a group containing an aromatic group that does not contain a heteroatom. An aryl or heteroaryl substituent may be unsubstituted or substituted with one or more chemical moieties. Examples of suitable substituents include, for example, alkyl, alkyl halide, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acyl, aldehyde, amino, carboxylic acid, cycloalkyl, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described herein. The term "biaryl" is a special type of aryl group and is included in the definition of aryl. Biaryl refers to two aryl groups joined through a fused ring structure, as in naphthalene, or through one or more carbon-carbon bonds, as in biphenyl.

[0034] As used herein, the term "cycloalkenyl" refers to a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. The term "heterocycloalkyl" refers to a cycloalkyl group, as defined above, in which at least one of the ring carbon atoms is replaced with a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. Cycloalkyl and heterocycloalkyl groups can be substituted or unsubstituted. Cycloalkyl and heterocycloalkyl groups can be substituted with one or more groups, including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acyl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described herein.

[0035] As used herein, the term "cycloalkenyl" refers to a non-aromatic carbon-based ring consisting of at least three carbon atoms and containing at least one double bond, i.e., C=C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like. The term "heterocycloalkenyl" is a type of cycloalkenyl group defined above and is included within the meaning of the term "cycloalkenyl," in which at least one of the ring carbon atoms is replaced with a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. Cycloalkenyl and heterocycloalkenyl groups can be substituted or unsubstituted. The cycloalkenyl and heterocycloalkenyl groups may be substituted with one or more groups, including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acyl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described herein.

[0036] The term "cyclic group" is used herein to refer to aryl groups, non-aryl groups (i.e., cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups), or both. Cyclic groups have one or more ring systems that can be substituted or unsubstituted. A cyclic group can contain one or more aryl groups, one or more non-aryl groups, or one or more aryl groups and one or more non-aryl groups.

[0037] As used herein, "heteroaryl" refers to a monocyclic or polycyclic aromatic heterocycle having at least one heteroatom ring member selected from sulfur, oxygen, and nitrogen. In some embodiments, the heteroaryl ring has 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, any ring-forming N in the heteroaryl moiety can be an N-oxide. In some embodiments, the heteroaryl has 5 to 10 ring atoms and 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, the heteroaryl has 5 to 6 ring atoms and 1 or 2 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, the heteroaryl is a 5- or 6-membered heteroaryl ring. A 5-membered heteroaryl ring is a heteroaryl having a ring with 5 ring atoms, wherein one or more (e.g., 1, 2, or 3) ring atoms are independently selected from N, O, and S. Exemplary 5-membered heteroaryls are thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl. A 6-membered heteroaryl ring is a heteroaryl having a ring with 6 ring atoms, wherein one or more (e.g., 1, 2, or 3) ring atoms are independently selected from N, O, and S. Exemplary 6-membered heteroaryls are pyridyl, pyrazinyl, pyrimidinyl, triazinyl, and pyridazinyl.

[0038] As used herein, "heterocycloalkyl" refers to a non-aromatic monocyclic or polycyclic heterocycle having one or more ring-forming heteroatoms selected from O, N, or S. Heterocycloalkyl includes monocyclic 4-, 5-, 6-, and 7-membered heterocycloalkyl groups. Heterocycloalkyl groups also include spirocycles. Exemplary heterocycloalkyl groups include pyrrolidin-2-one, 1,3-isoxazolidin-2-one, pyranyl, tetrahydrofuran, oxetanyl, azetidinyl, morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, azepanyl, benzazapene, and the like. The ring-forming carbon atoms and heteroatoms of a heterocycloalkyl group can be optionally substituted with oxo or sulfido (e.g., C(O), S(O), C(S), or S(O)). A heterocycloalkyl group can be bonded through a ring-forming carbon atom or a ring-forming heteroatom. In some embodiments, a heterocycloalkyl group contains 0 to 3 double bonds. In some embodiments, a heterocycloalkyl group contains 0 to 2 double bonds. Also included within the definition of heterocycloalkyl are moieties having one or more aromatic rings fused (i.e., having a common bond) to a cycloalkyl ring, e.g., benzo or thienyl derivatives such as piperidine, morpholine, and azepine. Heterocycloalkyl groups containing fused aromatic rings can be bonded through any ring-forming atom, including a ring-forming atom of the fused aromatic ring. In some embodiments, a heterocycloalkyl has 4 to 10, 4 to 7, or 4 to 6 ring atoms, contains 1 or 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and has one or more oxidized ring members.

[0039] In certain places, definitions or embodiments refer to certain rings (e.g., azetidine ring, pyridine ring, etc.). Unless otherwise specified, these rings can be bonded to any ring member as long as the valence of the atom is not exceeded. For example, an azetidine ring can be bonded to any position on the ring, while a pyridin-3-yl ring is bonded to the 3-position.

[0040] As used herein, the term "acyl" refers to a group of the formula -C(O)Z 1 In the formula, Z 1 may be hydrogen, hydroxyl, alkoxy, alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl groups as described above. As used herein, the term "acyl" may be used interchangeably with "carbonyl." Throughout this specification, "C(O)" or "CO" is shorthand notation for C=O.

[0041] As used herein, the term "alkoxy" refers to a group of the formula Z 1 refers to the group —O—, wherein Z 1 is an unsubstituted or substituted alkyl as defined above. Unless otherwise specified, an alkoxy group (where Z 1 is C1-C 24 (e.g., C1-C 22 , C1-C 20 , C1-C 18 , C1-C 16 , C1-C 14 , C1-C 12 , C1-C 10, C1-C8, C1-C6, C1-C4) alkyl groups are contemplated. Examples include methoxy, ethoxy, propoxy, 1-methyl-ethoxy, butoxy, 1-methyl-propoxy, 2-methyl-propoxy, 1,1-dimethyl-ethoxy, pentoxy, 1-methyl-butyloxy, 2-methyl-butoxy, 3-methyl-butoxy, 2,2-dimethyl-propoxy, 1-ethyl-propoxy, hexoxy, 1,1-dimethyl-propoxy, 1,2-dimethyl-propoxy, 1-methyl-pentoxy, 2-methyl-pentoxy, 3- Examples include methyl-pentoxy, 4-methyl-penoxy, 1,1-dimethyl-butoxy, 1,2-dimethyl-butoxy, 1,3-dimethyl-butoxy, 2,2-dimethyl-butoxy, 2,3-dimethyl-butoxy, 3,3-dimethyl-butoxy, 1-ethyl-butoxy, 2-ethylbutoxy, 1,1,2-trimethyl-propoxy, 1,2,2-trimethyl-propoxy, 1-ethyl-1-methyl-propoxy, and 1-ethyl-2-methyl-propoxy.

[0042] The term "aldehyde" as used herein is represented by the formula -C(O)H.

[0043] As used herein, the term "amine" or "amino" refers to a group of the formula -NZ 1 Z 2 In the formula, Z 1 and Z 2 may each be a substituent as described herein, e.g., hydrogen, alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above. An "amide" is -C(O)NZ 1 Z 2 is.

[0044] As used herein, the term "carboxylic acid" is represented by the formula -C(O)OH. As used herein, a "carboxylate" or "carboxyl" group is represented by the formula -C(O)O - It is expressed as:

[0045] As used herein, the term "ester" refers to an ester of the formula -OC(O)Z 1 or -C(O)OZ 1 In the formula, Z 1 can be an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

[0046] As used herein, the term "ether" refers to a group of the formula Z 1 OZ 2 In the formula, Z 1 and Z 2 can independently be an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

[0047] As used herein, the term "ketone" refers to a compound of formula Z 1 C(O)Z 2 In the formula, Z 1 and Z 2 can independently be an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

[0048] The terms "halide" or "halogen" or "halo" as used herein refer to fluorine, chlorine, bromine, and iodine.

[0049] The term "hydroxyl" as used herein is represented by the formula --OH.

[0050] The term "nitro" as used herein is represented by the formula -NO2.

[0051] As used herein, the term "silyl" refers to a group of the formula -SiZ 1 Z 2 Z 3 In the formula, Z 1 , Z2 , and Z 3 may independently be hydrogen, an alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

[0052] The term "sulfonyl" refers to a group of the formula -S(O)Z 1 is used herein to refer to a sulfo-oxo group represented by the formula: 1 can be hydrogen, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

[0053] The term "sulfonylamino" or "sulfonamide" as used herein is represented by the formula -S(O)2NH-.

[0054] The term "thiol" as used herein is represented by the formula -SH.

[0055] The term "thio" as used herein is represented by the formula -S-.

[0056] As used herein, Me refers to a methyl group; OMe refers to a methoxy group; and i-Pr refers to an isopropyl group.

[0057] As used herein, "R 1 "," "R 2 "," "R 3 "," "R n " etc. (where n is any integer) may independently have one or more of the above groups. For example, R 1When is a straight-chain alkyl group, one of the hydrogen atoms of the alkyl group can optionally be replaced with a hydroxyl group, an alkoxy group, an amine group, an alkyl group, a halide, or the like. Depending on the group selected, the first group can be incorporated into the second group, or alternatively, the first group can be pendant (i.e., attached) to the second group. For example, in the phrase "an alkyl group comprising an amino group," the amino group can be incorporated within the backbone of the alkyl group. Alternatively, the amino group can be attached to the backbone of the alkyl group. The nature of the group(s) selected will determine whether the first group is embedded in or attached to the second group.

[0058] Unless stated to the contrary, formulas containing chemical bonds shown only with solid lines, rather than wedges or dashed lines, contemplate each possible stereoisomer or mixture of stereoisomers (e.g., each enantiomer, each diastereomer, each meso compound, racemic mixture, or scalemic mixture).

[0059] Reference will now be made in detail to certain aspects of the disclosed materials, compounds, compositions, articles and methods, examples of which are illustrated in the accompanying examples and drawings.

[0060] Carboranes and Carborane Analogues Dicarba-closo-dodecaborane (also referred to herein as "carborane") is an icosahedral cluster containing two carbon atoms and ten boron atoms, both of which are hexacoordinated. Carboranes exist in three isomers, depending on the carbon atom's position within the cluster: 1,2-dicarba-closo-dodecaborane (ortho-carborane), 1,7-dicarba-closo-dodecaborane (meta-carborane), and 1,12-dicarba-closo-dodecaborane (para-carborane). These structures are unique among boron compounds because they can possess high thermal stability and hydrophobicity comparable to hydrocarbons.

[0061] Carboranes are, for example, 10It can be used in boron neutron capture therapy (BNCT), which has been developed as a treatment for glioma and melanoma. 10 When B is irradiated with thermal neutrons (slow neutrons), alpha rays with an energy of 2.4 MeV are emitted, and the atoms 7 Li and 4 It is decomposed into He. The range of alpha rays is about 10 μm, which corresponds to the diameter of a cell. 10 The expected effect is to destroy only the cells that have absorbed the B atoms, without damaging other cells. The development of BNCT involves the development of a neutron beam at a concentration that can destroy cells. 10 It is important to selectively take up B atoms into cancer cells. Therefore, it is necessary to have low toxicity and 10 Other carborane scaffolds with high B content and easy synthesis have been utilized. In addition, ortho-carboranes containing nucleic acid precursors, amino acids, and porphyrins have been synthesized and evaluated.

[0062] Carborane-based ERβ agonists and carborane analogs are described, for example, in U.S. Pat. No. 6,838,574 to Endo, U.S. Patent Application Publication No. 2018 / 0264017 to Tjarks et al., and PCT / US2019 / 064228 to Coss et al., each of which is incorporated by reference in its entirety.

[0063] In some embodiments, the carborane can be defined by Formula I:

[0064] [ka] During the ceremony, R 1 represents a dicarbacloso-dodecaboran-yl group which may have one or more substituents selected from the group consisting of an alkyl group, an alkenyl group, a carboxyl group, an alkoxycarbonyl group, an amino group, a hydroxyl group, a hydroxyalkyl group, a mono- or dialkylcarbamoyl-substituted alkyl group, an alkanoyl group, an aryl group, and an aralkyl group, each of which may be substituted or unsubstituted; R2 represents a carboxyl group, an alkoxycarbonyl group, or a hydroxyl group; X represents a single bond or a linking group selected from the group consisting of groups represented by the following formulas: [ka] In the formula, Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , Y 6 , and Y 7 are independently an oxygen atom or -N(R 3 )-, where R 3 represents a hydrogen atom or an alkyl group; Y 8 is an oxygen atom, -N(R 4 )- and R 4 is a hydrogen atom or an alkyl group, -CO-, -CH2-, or -C(=CH 2 )- represents;R 5 , R 6 , and R 7 independently represent hydrogen or one or more substituents on the phenyl group; R 8 represents an optionally substituted alkyl group or aryl group; R 9 represents an alkyl group; R 10 represents a substituted or unsubstituted aryl group.

[0065] In some embodiments, the carborane can be defined by Formula II, or a pharmaceutically acceptable salt thereof: [ka] During the ceremony, Q is a substituted or unsubstituted dicarba-closo-dodecaborane cluster; [ka] and R 1 binds to Q in a para configuration; X is OH, NHR 2 , SH, or S(O)(O)NHR2 and; R 1 is a substituted or unsubstituted C4-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C3-C 20 Alkylaryl, substituted or unsubstituted C3-C 20 Alkylheteroaryl, substituted or unsubstituted C4-C 20 Alkylcycloalkyl, substituted or unsubstituted C4-C 20 Alkylheterocycloalkyl, substituted or unsubstituted C1-C 20 Acyl, or NR 3 R 4 and; R 2 is H, OH, halogen, or substituted or unsubstituted C1-C4 alkyl; R 3 and R 4 are independently substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C2-C 20 Alkylaryl, substituted or unsubstituted C4-C 20 Alkylcycloalkyl, or substituted or unsubstituted C1-C 20 selected from acyl; However, if X is OH, then R 1 is not (CH2)5CH(CH3)2 or NH2.

[0066] In some examples of Formula II, the carborane cluster can include a heteroatom. In some examples of Formula II, the carborane cluster can include an isotopically labeled atom (i.e., a radioactively labeled atom). In some examples of Formula II, the carborane cluster can include an isotopically labeled boron atom (e.g., 10 B).

[0067] In some examples of Formula II, Q is: [ka] wherein ● is a carbon atom or a boron atom; ○ is CH, C-halogen, C-alkyl, C-OH, C-NH2, BH, B-halogen, B-alkyl, B-OH, or B-NH2.

[0068] In some examples of Formula II, X is OH.

[0069] In some examples of Formula II, R 1 is a substituted or unsubstituted C 6- C 10 In some examples of Formula II, R 1 is C6-C 10 In some examples of Formula II, R 1 is a substituted or unsubstituted C3-C 16 In some examples of Formula II, R 1 is C3-C 16 In some examples of Formula II, R 1 is a substituted or unsubstituted C5-C 10 In some examples of Formula II, R 1 is a substituted or unsubstituted C4-C 10 In some examples of Formula II, R 1 is branched C4-C 10 It is a hydroxyalkyl.

[0070] In some examples of Formula II, the compound may be a compound of Formula III, or a pharmaceutically acceptable salt thereof: [ka] During the ceremony, ● is a carbon atom; ○ is BH, B-halogen, B-alkyl, B-OH, or B-NH2; X is OH, NHR 2 , SH, or S(O)(O)NHR 2 and; R 1 is a substituted or unsubstituted C4-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C3-C 20 Alkylaryl, substituted or unsubstituted C3-C 20 Alkylheteroaryl, substituted or unsubstituted C4-C 20 Alkylcycloalkyl, substituted or unsubstituted C4-C 20 Alkylheterocycloalkyl, substituted or unsubstituted C1-C 20 Acyl, or NR 3 R 4 and; R 2 is H, OH, halogen, or substituted or unsubstituted C1-C4 alkyl; R 3 and R 4 is a substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C2-C 20 Alkylaryl, substituted or unsubstituted C4-C 20 Alkylcycloalkyl, or substituted or unsubstituted C1-C 20 independently selected from acyl; However, if X is OH, then R 1 is not (CH2)5CH(CH3)2 or NH2.

[0071] In some examples of Formula III, the carborane cluster can include a heteroatom.

[0072] In some examples of Formula III, the carborane cluster can include an isotopically labeled atom (i.e., a radioactively labeled atom). In some examples of Formula III, the carborane cluster can include an isotopically labeled boron atom (e.g., 10 B).

[0073] In some examples of Formula III, X is OH.

[0074] In some examples of Formula III, R 1 is a substituted or unsubstituted C 6- C 10 In some examples of Formula III, R 1 is C6-C 10 In some examples of Formula III, R 1 is a substituted or unsubstituted C3-C 16 In some examples of Formula III, R 1 is C3-C 16 In some examples of Formula III, R 1 is a substituted or unsubstituted C5-C 10 In some examples of Formula III, R 1 is a substituted or unsubstituted C4-C 10 In some examples of Formula III, R 1 is branched C4-C 10 It is a hydroxyalkyl.

[0075] In some examples of Formula III, the compound may be a compound of Formula IV, or a pharmaceutically acceptable salt thereof: [ka] During the ceremony, ● is a carbon atom; ○ is BH, B-halogen, B-alkyl, B-OH, or B-NH2; The dotted line to the Y indicates that the bond may be single or double, valence permitting; X is OH, NHR 2, SH, or S(O)(O)NHR 2 and; Y is O, OR 2’ , NHR 2 , SH, or S(O)(O)NHR 2 and; R 5 is a substituted or unsubstituted C2-C 19 Alkyl, substituted or unsubstituted C2-C 19 Alkenyl, substituted or unsubstituted C2-C 19 Alkynyl, substituted or unsubstituted C2-C 19 Alkylaryl, substituted or unsubstituted C2-C 19 Alkylheteroaryl, substituted or unsubstituted C3-C 19 Alkylcycloalkyl, substituted or unsubstituted C3-C 19 Alkylheterocycloalkyl, or NR 3 R 4 and; R 2 is H, OH, halogen, or substituted or unsubstituted C1-C4 alkyl; R 2’ is H or substituted or unsubstituted C1-C4 alkyl; R 3 and R 4 is a substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C2-C 20 Alkylaryl, substituted or unsubstituted C4-C 20 Alkylcycloalkyl, or substituted or unsubstituted C1-C 20 acyl.

[0076] In some examples of Formula IV, the carborane cluster can include a heteroatom. In some examples of Formula IV, the carborane cluster can include an isotopically labeled atom (i.e., a radioactively labeled atom). In some examples of Formula IV, the carborane cluster can include an isotopically labeled boron atom (e.g., 10B).

[0077] In some examples of Formula IV, X is OH.

[0078] In some examples of Formula IV, Y is OH. In some examples of Formula IV, Y is O.

[0079] In some examples of Formula IV, R 5 is a substituted or unsubstituted C3-C9 alkyl. In some examples of Formula IV, R 5 is a substituted or unsubstituted C6-C9 alkyl. In some examples of Formula IV, R 5 is a substituted or unsubstituted C2-C 15 In some examples of Formula IV, R 5 is a substituted or unsubstituted branched C2-C9 alkyl.

[0080] Also disclosed herein are compounds of Formula V, and pharmaceutically acceptable salts thereof: [ka] During the ceremony, Q is a substituted or unsubstituted dicarba-closo-dodecaborane cluster; [ka] and [ka] binds to Q in a para configuration; The dotted line to the Y indicates that the bond may be single or double, valence permitting; X is OH, NHR 2 , SH, or S(O)(O)NHR 2 and; Y is O, OR 2’ , NHR 2 , SH, or S(O)(O)NHR 2 and; R 6is a substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C2-C 20 Alkylaryl, substituted or unsubstituted C2-C 20 Alkylheteroaryl, substituted or unsubstituted C4-C 20 Alkylcycloalkyl, substituted or unsubstituted C4-C 20 Alkylheterocycloalkyl, or NR 3 R 4 and; R 2 is H, OH, halogen, or substituted or unsubstituted C1-C4 alkyl; R 2 is H or substituted or unsubstituted C1-C4 alkyl; R 3 and R 4 is a substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C2-C 20 Alkylaryl, substituted or unsubstituted C4-C 20 Alkylcycloalkyl, or substituted or unsubstituted C1-C 20 independently selected from acyl; However, if X is OH, then R 6 is not CH2OH, CH(CH3)OH, CH2CH2OH, CH2CH2CH2OH, (CH2)5CH(CH3)2, or NH2.

[0081] In some examples of Formula V, the carborane cluster can include a heteroatom. In some examples of Formula V, the carborane cluster can include an isotopically labeled atom (i.e., a radiolabeled atom). In some examples of Formula V, the carborane cluster can include an isotopically labeled boron atom (e.g., 10 B).

[0082] In some examples of Formula V, Q is: [ka] wherein ● is a carbon atom or a boron atom; ○ is CH, C-halogen, C-alkyl, C-OH, C-NH2, BH, B-halogen, B-alkyl, B-OH, or B-NH2.

[0083] In some examples of Formula V, X is OH.

[0084] In some examples of Formula V, Y is OH. In some examples of Formula V, Y is O.

[0085] In some examples of Formula V, R 6 is a substituted or unsubstituted C 6- C 10 In some examples of Formula V, R 6 is a substituted or unsubstituted C2-C 15 In some examples of Formula V, R 6 is a substituted or unsubstituted branched C3-C 10 It is alkyl.

[0086] In some examples of Formula V, the compound may be a compound of Formula VI, or a pharmaceutically acceptable salt thereof: [ka] During the ceremony, ● is a carbon atom; ○ is BH, B-halogen, B-alkyl, B-OH, or B-NH2; The dotted line to the Y indicates that the bond may be single or double, valence permitting; X is OH, NHR 2 , SH, or S(O)(O)NHR 2 and; Y is O, OR 2’ , NHR 2, SH, or S(O)(O)NHR 2 and; R 6 is a substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C2-C 20 Alkylaryl, substituted or unsubstituted C2-C 20 Alkylheteroaryl, substituted or unsubstituted C4-C 20 Alkylcycloalkyl, substituted or unsubstituted C4-C 20 Alkylheterocycloalkyl, or NR 3 R 4 and; R 2 is H, OH, halogen, or substituted or unsubstituted C1-C4 alkyl; R 2 is H or substituted or unsubstituted C1-C4 alkyl; R 3 and R 4 are independently substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C2-C 20 Alkylaryl, substituted or unsubstituted C4-C 20 Alkylcycloalkyl, or substituted or unsubstituted C1-C 20 selected from acyl; However, if X is OH, then R 6 is not CH2OH, CH(CH3)OH, CH2CH2OH, CH2CH2CH2OH, (CH2)5CH(CH3)2, or NH2.

[0087] In some examples of Formula VI, the carborane cluster can include a heteroatom. In some examples of Formula VI, the carborane cluster can include an isotopically labeled atom (i.e., a radioactively labeled atom). In some examples of Formula VI, the carborane cluster can include an isotopically labeled boron atom (e.g., 10 B).

[0088] In some examples of Formula VI, X is OH.

[0089] In some examples of Formula VI, Y is OH. In some examples of Formula VI, Y is O.

[0090] In some examples of Formula VI, R 6 is a substituted or unsubstituted C6-C 10 In some examples of Formula VI, R 6 is a substituted or unsubstituted C2-C 15 In some examples of Formula VI, R 6 is a substituted or unsubstituted branched C3-C 10 It is alkyl.

[0091] Also disclosed herein are compounds of Formula VII, and pharmaceutically acceptable salts thereof: [ka] During the ceremony, Q is a substituted or unsubstituted dicarba-closo-dodecaborane cluster; [ka] and R 7 binds to Q in a para configuration; X is OH, NHR 2 , SH, or S(O)(O)NHR 2 and; R 7 is a substituted or unsubstituted C1-C 14 Alkyl, substituted or unsubstituted C2-C 14 Alkenyl, substituted or unsubstituted C2-C14 Alkynyl, substituted or unsubstituted C1-C 14 Acyl, or NR 3 R 4 and; R 8 , R 9 , R 10 , R 11 , and R 12 are independently H, OH, halogen, substituted or unsubstituted C-C 20 Alkyl, partially substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C2-C 20 Alkylaryl, substituted or unsubstituted C4-C 20 Alkylcycloalkyl, substituted or unsubstituted C1-C 20 Acyl, or NR 3 R 4 or, as far as valence allows, R 8 and R 9 , R 9 and R 10 , R 10 and R 11 , or R 11 and R 12 together with the atoms to which they are attached form a 3-10 membered substituted or unsubstituted cyclic moiety, optionally containing 1-3 heteroatoms; R 2 is H, OH, halogen, or substituted or unsubstituted C1-C4 alkyl; R 3 and R 4 are independently substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C2-C 20 Alkylaryl, substituted or unsubstituted C4-C 20 Alkylcycloalkyl, or substituted or unsubstituted C1-C 20 acyl.

[0092] In some examples of Formula VII, the carborane cluster can include a heteroatom. In some examples of Formula VII, the carborane cluster can include an isotopically labeled atom (i.e., a radioactively labeled atom). In some examples of Formula VII, the carborane cluster can include an isotopically labeled boron atom (e.g., 10 B).

[0093] In some examples of Formula VII, Q is: [ka] wherein ● is a carbon atom or a boron atom; ○ is CH, C-halogen, C-alkyl, C-OH, C-NH2, BH, B-halogen, B-alkyl, B-OH, or B-NH2.

[0094] In some examples of Formula VII, X is OH.

[0095] In some examples of Formula VII, R 7 is a substituted or unsubstituted C1-C7 alkyl. In some examples of Formula VII, R 7 is C1-C7 hydroxyalkyl.

[0096] In some examples of Formula VII, R 8 -R 12 are independently H, OH, halogen, or substituted or unsubstituted C1-C4 alkyl, or, where valences allow, R 8 and R 9 , R 9 and R 10 , R 10 and R 11 , or R 11 and R 12 taken together with the atoms to which they are attached form a 3-10 membered substituted or unsubstituted cyclic moiety, optionally containing 1-3 heteroatoms. In some examples of Formula VII, R 8 -R 12 are each H. In some examples of Formula VII, R8 , R 10 , and R 12 are H and R, respectively. 9 and R 10 together with the atoms to which they are attached form a substituted or unsubstituted 5- to 7-membered cyclic moiety.

[0097] In some examples of Formula VII, the compound may be a compound of Formula VIII, or a pharmaceutically acceptable salt thereof: [ka] During the ceremony, ● is a carbon atom; ○ is BH, B-halogen, B-alkyl, B-OH, or B-NH2; X is OH, NHR 2 , SH, or S(O)(O)NHR 2 and; R 7 is a substituted or unsubstituted C1-C 14 Alkyl, substituted or unsubstituted C2-C 14 Alkenyl, substituted or unsubstituted C2-C 14 Alkynyl, substituted or unsubstituted C1-C 14 Acyl, or NR 3 R 4 and; R 8 , R 9 , R 10 , R 11 , and R 12 are independently H, OH, halogen, substituted or unsubstituted C-C 20 Alkyl, partially substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C2-C 20 Alkylaryl, substituted or unsubstituted C4-C 20 Alkylcycloalkyl, substituted or unsubstituted C1-C 20 Acyl, or NR 3 R 4 or, as far as valence allows, R 8and R 9 , R 9 and R 10 , R 10 and R 11 , or R 11 and R 12 together with the atoms to which they are attached form a 3-10 membered substituted or unsubstituted cyclic moiety, optionally containing 1-3 heteroatoms; R 2 is H, OH, halogen, or substituted or unsubstituted C1-C4 alkyl; R 3 and R 4 are independently substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C2-C 20 Alkylaryl, substituted or unsubstituted C4-C 20 Alkylcycloalkyl, or substituted or unsubstituted C1-C 20 acyl.

[0098] In some examples of Formula VIII, the carborane cluster can include a heteroatom. In some examples of Formula VIII, the carborane cluster can include an isotopically labeled atom (i.e., a radioactively labeled atom). In some examples of Formula VIII, the carborane cluster can include an isotopically labeled boron atom (e.g., 10 B).

[0099] In some examples of Formula VIII, X is OH.

[0100] In some examples of Formula VIII, R 7 is a substituted or unsubstituted C1-C7 alkyl. In some examples of Formula VIII, R 7 is C1-C7 hydroxyalkyl.

[0101] In some examples of Formula VIII, R 8 -R 12are independently H, OH, halogen, or substituted or unsubstituted C1-C4 alkyl, or, where valences allow, R 8 and R 9 , R 9 and R 10 , R 10 and R 11 , or R 11 and R 12 taken together with the atoms to which they are attached form a 3-10 membered substituted or unsubstituted cyclic moiety, optionally containing 1-3 heteroatoms. In some examples of Formula VIII, R 8 -R 12 are each H. In some examples of Formula VIII, R 8 , R 10 , and R 12 are H and R, respectively. 9 and R 10 together with the atoms to which they are attached form a substituted or unsubstituted 5- to 7-membered cyclic moiety.

[0102] Also disclosed herein are compounds of formula IX, and pharmaceutically acceptable salts thereof: [ka] During the ceremony, Q is a substituted or unsubstituted dicarba-closo-dodecaborane cluster; [ka] and R 13 binds to Q in a para configuration; X is OH, NHR 2 , SH, or S(O)(O)NHR 2 and; R 13 is a substituted or unsubstituted C1-C 19 Alkyl, substituted or unsubstituted C2-C 19 Alkenyl, substituted or unsubstituted C2-C 19 Alkynyl, or substituted or unsubstituted C1-C 20 It is acyl; R 14 , R15 , and R 16 are independently hydrogen, halogen, hydroxyl, substituted or unsubstituted C-C 18 Alkyl, substituted or unsubstituted C2-C 18 Alkenyl, substituted or unsubstituted C1-C 18 Alkynyl, substituted or unsubstituted C2-C 18 Aryl, substituted or unsubstituted C3-C 18 Cycloalkyl, substituted or unsubstituted C1-C 20 Acyl, or NR 3 R 4 or, as far as valence allows, R 14 and R 15 , R 14 and R 16 , or R 15 and R 16 together with the atoms to which they are attached form a 3-10 membered substituted or unsubstituted cyclic moiety, optionally containing 1-3 heteroatoms; However, R 14 , R 15 and R 16 at least two of which are not hydrogen, halogen, or hydroxyl; provided that X is OH and R 13 is a C5 alkyl, R 14 、 R 15 , and R 16 is H, methyl, and not methyl.

[0103] In some examples of Formula IX, the carborane cluster can include a heteroatom. In some examples of Formula IX, the carborane cluster can include an isotopically labeled atom (i.e., a radioactively labeled atom). In some examples of Formula IX, the carborane cluster can include an isotopically labeled boron atom (e.g., 10 In some examples of Formula IX, Q can include: [ka] wherein ● is a carbon atom or a boron atom; ○ is CH, C-halogen, C-alkyl, C-OH, C-NH2, BH, B-halogen, B-alkyl, B-OH, or B-NH2.

[0104] In some examples of Formula IX, X is OH.

[0105] In some examples of Formula IX, R 13 is a substituted or unsubstituted C4-C8 alkyl. In some examples of Formula IX, R 13 is a C4-C8 hydroxyalkyl.

[0106] In some examples of Formula IX, R 14 -R 16 are independently hydrogen, halogen, hydroxyl, substituted or unsubstituted C1-C4 alkyl, provided that R 14 , R 15 and R 16 at least two of X are not hydrogen, halogen, or hydroxyl; provided that X is OH and R 13 is a C5 alkyl, R 14 、 R 15 , and R 16 is H, methyl, and not methyl.

[0107] In some examples of Formula IX, the compound may be a compound of Formula X, or a pharmaceutically acceptable salt thereof: [ka] During the ceremony, ● is a carbon atom; ○ is BH, B-halogen, B-alkyl, B-OH, or B-NH2; X is OH, NHR 2 , SH, or S(O)(O)NHR 2 and; R 13 is a substituted or unsubstituted C1-C 19 Alkyl, substituted or unsubstituted C2-C 19Alkenyl, substituted or unsubstituted C2-C 19 Alkynyl, or substituted or unsubstituted C1-C 20 It is acyl; R 14 , R 15 , and R 16 are independently hydrogen, halogen, hydroxyl, substituted or unsubstituted C-C 18 Alkyl, substituted or unsubstituted C2-C 18 Alkenyl, substituted or unsubstituted C1-C 18 Alkynyl, substituted or unsubstituted C2-C 18 Aryl, substituted or unsubstituted C3-C 18 Cycloalkyl, substituted or unsubstituted C1-C 20 Acyl, or NR 3 R 4 or, as far as valence allows, R 14 and R 15 , R 14 and R 16 , or R 15 and R 16 together with the atoms to which they are attached form a 3-10 membered substituted or unsubstituted cyclic moiety, optionally containing 1-3 heteroatoms; However, R 14 , R 15 and R 16 at least two of which are not hydrogen, halogen, or hydroxyl; provided that X is OH and R 13 is a C5 alkyl, R 14 、 R 15 , and R 16 is H, methyl, and not methyl.

[0108] In some examples of Formula X, the carborane cluster can include a heteroatom. In some examples of Formula X, the carborane cluster can include an isotopically labeled atom (i.e., a radioactively labeled atom). In some examples of Formula X, the carborane cluster can include an isotopically labeled boron atom (e.g., 10 B).

[0109] In some examples of formula X, X is OH.

[0110] In some examples of formula X, R 13 is a substituted or unsubstituted C4-C8 alkyl. In some examples of Formula X, R 13 is a C4-C8 hydroxyalkyl.

[0111] In some examples of formula X, R 14 -R 16 are independently hydrogen, halogen, hydroxyl, substituted or unsubstituted C1-C4 alkyl, provided that R 14 , R 15 and R 16 at least two of X are not hydrogen, halogen, or hydroxyl; provided that X is OH and R 13 is a C5 alkyl, R 14 、 R 15 , and R 16 is H, methyl, and not methyl.

[0112] In some instances, the compound is: [ka] [ka] [ka] and pharmaceutically acceptable salts thereof. In some examples, the carborane cluster may include a heteroatom.

[0113] Also disclosed herein are compounds of formula XI, and pharmaceutically acceptable salts thereof: [ka] During the ceremony, Q is a substituted or unsubstituted dicarba-closo-dodecaborane cluster; D is -S-, -S(O)-, -S(O)(O)-, -S(O)(NH)-, -P(O)(OH)O-, -P(O)(OH)NH-, or -O-; X is OH, NHR 2 , SH, or S(O)(O)NHR 2 and; R 6 is a substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C2-C 20 Alkylaryl, substituted or unsubstituted C2-C 20 Alkylheteroaryl, substituted or unsubstituted C4-C 20 Alkylcycloalkyl, or substituted or unsubstituted C4-C 20 alkylheterocycloalkyl; R 2 is H, OH, halogen, or substituted or unsubstituted C1-C4 alkyl.

[0114] In some examples of Formula XI, [ka] and [ka] binds to Q in a para configuration.

[0115] In some examples of Formula XI, the carborane cluster can include a heteroatom. In some examples of Formula XI, the carborane cluster can include an isotopically labeled atom (i.e., a radiolabeled atom). In some examples of Formula XI, the carborane cluster can include an isotopically labeled boron atom (e.g., 10 B).

[0116] In some examples of Formula XI, Q is: [ka] wherein ● is a carbon atom or a boron atom; ○ is CH, C-halogen, C-alkyl, C-OH, C-NH2, BH, B-halogen, B-alkyl, B-OH, or B-NH2.

[0117] In some examples of formula XI, X is OH.

[0118] In some examples of Formula XI, R 6 is a substituted or unsubstituted C6-C 10 In some examples of Formula XI, R 6 is a substituted or unsubstituted C2-C 15 In some examples of Formula XI, R 6 is a substituted or unsubstituted branched C3-C 10 It is alkyl.

[0119] In some instances, the compound is: [ka] [ka] [ka] [ka] and pharmaceutically acceptable salts thereof. In some examples, the carborane cluster may include a heteroatom.

[0120] In some embodiments, the carborane can be defined by Formula XII, or a pharmaceutically acceptable salt thereof: [ka] where Q is a substituted or unsubstituted dicarba-closo-dodecaborane cluster, and A and R 1is attached to Q in a para configuration; A is a substituted or unsubstituted heteroaryl ring; R 1 is a substituted or unsubstituted C2-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C3-C 20 Alkylaryl, substituted or unsubstituted C3-C 20 Alkylheteroaryl, substituted or unsubstituted C4-C 20 Alkylcycloalkyl, substituted or unsubstituted C4-C 20 Alkylheterocycloalkyl, substituted or unsubstituted C1-C 20 Acyl, C1-C 20 Acyl, -C(O)NR 3 R 4 , -S(O)-R 3 , -S(O2)-R 3 , substituted or unsubstituted C2-C 20 Heteroalkyl, or NR 3 R 4 and;R 3 and R 4 are independently substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C2-C 20 Alkylaryl, substituted or unsubstituted C4-C 20 Alkylcycloalkyl and substituted or unsubstituted C2-C 20 heteroalkyl.

[0121] In some embodiments, Q is [ka] where ● is a carbon atom or a boron atom, and ○ is CH, C-halogen, C-alkyl, C—OH, C—NH2, BH, B-halogen, B-alkyl, B—OH, or B—NH2.

[0122] In some embodiments, A can be a 5-membered substituted or unsubstituted heteroaryl ring. For example, A can include thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl, or 1,3,4-oxadiazolyl ring. In some embodiments, A can be a 6-membered substituted or unsubstituted heteroaryl ring. For example, A can include a pyridyl, pyrazinyl, pyrimidinyl, triazinyl, or pyridazinyl ring.

[0123] In some cases, the compound may be defined by Formula XIIA, or a pharmaceutically acceptable salt thereof: [ka] where ● is a carbon atom, ○ is BH, B-halogen, B-alkyl, B-OH, or B-NH2; X is OH, NHR 2 , SH, or S(O)(O)NHR 2 Z is, independently for each occurrence, N or CH with the proviso that at least one of the Z's is N; R 1 is a substituted or unsubstituted C2-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C3-C 20 Alkylaryl, substituted or unsubstituted C3-C 20 Alkylheteroaryl, substituted or unsubstituted C4-C 20 Alkylcycloalkyl, substituted or unsubstituted C4-C 20 Alkylheterocycloalkyl, substituted or unsubstituted C1-C 20 Acyl, C1-C 20 Acyl, -C(O)NR 3 R 4, -S(O)-R 3 , -S(O2)-R 3 , substituted or unsubstituted C2-C 20 Heteroalkyl, or NR 3 R 4 and;R 2 is H, OH, halogen, or substituted or unsubstituted C1-C4 alkyl; R 3 and R 4 are independently substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C2-C 20 Alkylaryl, substituted or unsubstituted C4-C 20 Alkylcycloalkyl and substituted or unsubstituted C2-C 20 heteroalkyl.

[0124] In some cases, one of Z can be N. In some cases, two or more of Z can be N. In some cases, three of Z can be N.

[0125] In some embodiments, the compound may be defined by one of the following formulas, or a pharmaceutically acceptable salt thereof: [ka] where ● is a carbon atom, ○ is BH, B-halogen, B-alkyl, B-OH, or B-NH2; X is OH, NHR 2 , SH, or S(O)(O)NHR 2 and;R 1 is a substituted or unsubstituted C2-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C3-C 20 Alkylaryl, substituted or unsubstituted C3-C 20 Alkylheteroaryl, substituted or unsubstituted C4-C20 Alkylcycloalkyl, substituted or unsubstituted C4-C 20 Alkylheterocycloalkyl, substituted or unsubstituted C1-C 20 Acyl, C1-C 20 Acyl, -C(O)NR 3 R 4 , -S(O)-R 3 , -S(O2)-R 3 , substituted or unsubstituted C2-C 20 Heteroalkyl, or NR 3 R 4 and;R 2 is H, OH, halogen, or substituted or unsubstituted C1-C4 alkyl; R 3 and R 4 are independently substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C2-C 20 Alkylaryl, substituted or unsubstituted C4-C 20 Alkylcycloalkyl and substituted or unsubstituted C2-C 20 heteroalkyl.

[0126] In some embodiments, the compound may be defined by one of formulas XIIB-XIIF, or a pharmaceutically acceptable salt thereof: [ka] where ● is a carbon atom, ○ is BH, B-halogen, B-alkyl, B-OH, or B-NH2; R 1 is a substituted or unsubstituted C2-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C3-C 20 Alkylaryl, substituted or unsubstituted C3-C 20 Alkylheteroaryl, substituted or unsubstituted C4-C 20Alkylcycloalkyl, substituted or unsubstituted C4-C 20 Alkylheterocycloalkyl, substituted or unsubstituted C1-C 20 Acyl, C1-C 20 Acyl, -C(O)NR 3 R 4 , -S(O)-R 3 , -S(O2)-R 3 , substituted or unsubstituted C2-C 20 Heteroalkyl, or NR 3 R 4 and;R 2 is H, OH, halogen, or substituted or unsubstituted C1-C4 alkyl; R 3 and R 4 are independently substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C2-C 20 Alkylaryl, substituted or unsubstituted C4-C 20 Alkylcycloalkyl and substituted or unsubstituted C2-C 20 heteroalkyl.

[0127] In some of the above embodiments, X can be OH.

[0128] In some of the above embodiments, R 1 is a substituted or unsubstituted C6-C 10 Alkyl (e.g., C6-C 10 hydroxyalkyl).

[0129] In some of the above embodiments, R 1 is a substituted or unsubstituted C3-C 16 Alkylaryl (e.g., C3-C 16 hydroxyalkylaryl).

[0130] In some of the above embodiments, R 1 is a substituted or unsubstituted C8-C 20Alkylaryl (e.g., C8-C 20 hydroxyalkylaryl).

[0131] In some of the above embodiments, R 1 is a substituted or unsubstituted C5-C 10 It may be acyl.

[0132] In some of the above embodiments, R 1 is a substituted or unsubstituted C4-C 10 Alkyl (e.g., branched C4-C 10 hydroxyalkyl).

[0133] In some embodiments, the compound is defined by the following formula, or a pharmaceutically acceptable salt thereof: [ka] where ● is a carbon atom, ○ is BH, B-halogen, B-alkyl, B-OH, or B-NH2; the dotted line to Y indicates that the bond may be single or double, valence permitting; A is a substituted or unsubstituted heteroaryl ring; and Y, when present, is O, halogen, OR 2’ , NHR 2 , SH, or S(O)(O)NHR 2 and;R 6 is a substituted or unsubstituted C1-C 19 Alkyl, substituted or unsubstituted C2-C 19 Alkenyl, substituted or unsubstituted C2-C 19 Alkynyl, substituted or unsubstituted C2-C 19 Alkylaryl, substituted or unsubstituted C2-C 19 Alkylheteroaryl, substituted or unsubstituted C4-C 19 Alkylcycloalkyl, substituted or unsubstituted C4-C 19 Alkylheterocycloalkyl and substituted or unsubstituted C-C 20 Heteroalkyl, or NR 3 R 4 and;R 2is H, OH, halogen, or substituted or unsubstituted C1-C4 alkyl; R 2’ is H or substituted or unsubstituted C1-C4 alkyl; R 3 and R 4 are independently substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C2-C 20 Alkylaryl, substituted or unsubstituted C4-C 20 Alkylcycloalkyl and substituted or unsubstituted C2-C 20 heteroalkyl.

[0134] In some embodiments, A can be a 5-membered substituted or unsubstituted heteroaryl ring. For example, A can include thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl, or 1,3,4-oxadiazolyl ring. In some embodiments, A can be a 6-membered substituted or unsubstituted heteroaryl ring. For example, A can include a pyridyl, pyrazinyl, pyrimidinyl, triazinyl, or pyridazinyl ring.

[0135] In some of these embodiments, Y is OH. In some of these embodiments, Y is F. In some of these embodiments, Y is O.

[0136] In some instances, R 6 is a substituted or unsubstituted C3-C 10 It can be alkyl, for example, substituted or unsubstituted C6-C9 alkyl.

[0137] In some instances, R 6is a substituted or unsubstituted C2-C 15 It may be alkylaryl.

[0138] In some instances, R 6 can be a substituted or unsubstituted branched C2-C9 alkyl.

[0139] In some instances, R 6 is a substituted or unsubstituted C3-C 10 It can be a heteroalkyl, for example, a substituted or unsubstituted C6-C9 heteroalkyl.

[0140] Also provided is a compound defined by Formula XIII, or a pharmaceutically acceptable salt thereof: [ka] where Q is a substituted or unsubstituted dicarba-closo-dodecaborane cluster, and A and R 1 is attached to Q in a para configuration; A is any substituted or unsubstituted ring or substituted or unsubstituted heteroaryl ring; R 1 is a substituted or unsubstituted C2-C 20 Heteroalkyl, -C(O)NR 3 R 4 , -S(O)-R 3 , -S(O2)-R 3 , or NR 3 R 4 and;R 3 and R 4 are independently substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C2-C 20 Alkylaryl, substituted or unsubstituted C2-C 20 Alkylheteroaryl, substituted or unsubstituted C4-C 20 Alkylcycloalkyl, substituted or unsubstituted C4-C 20 Alkylheterocycloalkyl and substituted or unsubstituted C-C 20heteroalkyl, provided that, if present, R 3 and R 4 At least one of them is C2-C 20 It is heteroalkyl.

[0141] In some embodiments, A may include a substituted or unsubstituted aryl ring (e.g., a substituted or unsubstituted phenyl ring). In some embodiments, A may include a 5-membered substituted or unsubstituted heteroaryl ring. For example, A may include thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl, or 1,3,4-oxadiazolyl ring. In some embodiments, A may include a 6-membered substituted or unsubstituted heteroaryl ring. For example, A may include a pyridyl, pyrazinyl, pyrimidinyl, triazinyl, or pyridazinyl ring.

[0142] In some embodiments, Q is [ka] where ● is a carbon atom or a boron atom, and ○ is CH, C-halogen, C-alkyl, C—OH, C—NH2, BH, B-halogen, B-alkyl, B—OH, or B—NH2.

[0143] In some embodiments, the compound may be defined by Formula XIIIA, or a pharmaceutically acceptable salt thereof: [ka] where ● is a carbon atom, ○ is BH, B-halogen, B-alkyl, B-OH, or B-NH2; X is OH, NHR 2 , SH, or S(O)(O)NHR 2Z is, independently for each occurrence, N or CH with the proviso that at least one of the Z's is N; R 1 is a substituted or unsubstituted C2-C 20 Heteroalkyl, -C(O)NR 3 R 4 , -S(O)-R 3 , -S(O2)-R 3 , or NR 3 R 4 and;R 3 and R 4 are independently substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C2-C 20 Alkylaryl, substituted or unsubstituted C2-C 20 Alkylheteroaryl, substituted or unsubstituted C4-C 20 Alkylcycloalkyl, substituted or unsubstituted C4-C 20 Alkylheterocycloalkyl and substituted or unsubstituted C-C 20 heteroalkyl, provided that, if present, R 3 and R 4 At least one of them is C2-C 20 It is heteroalkyl.

[0144] In some of these embodiments, X can be OH.

[0145] Also provided is a compound defined by any of the following formulas, or a pharmaceutically acceptable salt thereof: [ka] where ● is a carbon atom, ○ is BH, B-halogen, B-alkyl, B-OH, or B-NH2; the dotted line to Y indicates that the bond may be single or double, valence permitting; A is any substituted or unsubstituted ring or substituted or unsubstituted heteroaryl ring; and Y, when present, is O, halogen, OR 2’ , NHR2 , SH, or S(O)(O)NHR 2 and;R 6 is a substituted or unsubstituted C1-C 19 Alkyl, substituted or unsubstituted C2-C 19 Alkenyl, substituted or unsubstituted C2-C 19 Alkynyl, substituted or unsubstituted C2-C 19 Alkylaryl, substituted or unsubstituted C2-C 19 Alkylheteroaryl, substituted or unsubstituted C4-C 19 Alkylcycloalkyl, substituted or unsubstituted C4-C 19 Alkylheterocycloalkyl and substituted or unsubstituted C-C 20 Heteroalkyl, or NR 3 R 4 and;R 2 is H, OH, halogen, or substituted or unsubstituted C1-C4 alkyl; R 2’ is H or substituted or unsubstituted C1-C4 alkyl; R 3 and R 4 are independently substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C2-C 20 Alkylaryl, substituted or unsubstituted C4-C 20 Alkylcycloalkyl and substituted or unsubstituted C2-C 20 heteroalkyl.

[0146] In some embodiments, A may include a substituted or unsubstituted aryl ring (e.g., a substituted or unsubstituted phenyl ring). In some embodiments, A may include a 5-membered substituted or unsubstituted heteroaryl ring. For example, A may include thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl, or 1,3,4-oxadiazolyl ring. In some embodiments, A may include a 6-membered substituted or unsubstituted heteroaryl ring. For example, A may include a pyridyl, pyrazinyl, pyrimidinyl, triazinyl, or pyridazinyl ring.

[0147] In some of these embodiments, Y is OH. In some of these embodiments, Y is F. In some of these embodiments, Y is O.

[0148] In some instances, R 6 is a substituted or unsubstituted C3-C 10 It can be alkyl, for example, substituted or unsubstituted C6-C9 alkyl.

[0149] In some instances, R 6 is a substituted or unsubstituted C2-C 15 It may be alkylaryl.

[0150] In some instances, R 6 can be a substituted or unsubstituted branched C2-C9 alkyl.

[0151] In some instances, R 6 is a substituted or unsubstituted C3-C 10 It can be a heteroalkyl, for example, a substituted or unsubstituted C6-C9 heteroalkyl.

[0152] In some examples, carboranes are: [ka] [ka] and pharmaceutically acceptable salts thereof. In some examples, the carborane cluster may include a heteroatom.

[0153] In some embodiments, the compound can be a carborane analog, such as a dicarba-closo-dodecaborane analog of a compound described in WO 2017 / 049307 to Tjarks et al. The compound includes a spacer group substituting the carborane moiety in the compound. The resulting compound can exhibit biological activity similar to that of the compound described in WO 2017 / 049307.

[0154] For example, provided herein are compounds defined by Formula XIV, or a pharmaceutically acceptable salt thereof: [ka] wherein A is a substituted or unsubstituted aryl ring or a substituted or unsubstituted heteroaryl ring; and Q is a spacer group selected from one of the following: [ka] wherein m and n are each independently 0, 1, 2, or 3; R 1 is a substituted or unsubstituted C4-C 20 Alkyl, substituted or unsubstituted C4-C 20 Heteroalkyl, substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C3-C 20 Alkylaryl, substituted or unsubstituted C3-C 20 Alkylheteroaryl, substituted or unsubstituted C4-C 20Alkylcycloalkyl, substituted or unsubstituted C4-C 20 Alkylheterocycloalkyl, substituted or unsubstituted C1-C 20 Acyl, C1-C 20 Acyl, -C(O)NR 3 R 4 , or NR 3 R 4 and;R 3 and R 4 are independently substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C1-C 20 Heteroalkyl, substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C2-C 20 Alkylaryl or substituted or unsubstituted C4-C 20 alkylcycloalkyl.

[0155] In certain embodiments, Q may be selected from one of the following: [ka]

[0156] In some embodiments, A may include a substituted or unsubstituted aryl ring (e.g., a substituted or unsubstituted phenyl ring). In some embodiments, A may include a 5-membered substituted or unsubstituted heteroaryl ring. For example, A may include thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl, or 1,3,4-oxadiazolyl ring. In some embodiments, A may include a 6-membered substituted or unsubstituted heteroaryl ring. For example, A may include a pyridyl, pyrazinyl, pyrimidinyl, triazinyl, or pyridazinyl ring.

[0157] In some embodiments, A is [ka] wherein X is OH, NHR 2 , SH, or S(O)(O)NHR 2 and R 2 is H, OH, halogen, or substituted or unsubstituted C1-C4 alkyl. In some of these embodiments, X is OH.

[0158] In some embodiments, A is [ka] wherein X is OH, NHR 2 , SH, or S(O)(O)NHR 2 and R 2 is H, OH, halogen, or substituted or unsubstituted C1-C4 alkyl. In some of these embodiments, X is OH.

[0159] In some embodiments, A is [ka] wherein Z is, independently for each occurrence, N or CH, with the proviso that at least one Z is N; and X is OH, NHR 2 , SH, or S(O)(O)NHR 2 and;R 2 is H, OH, halogen, or substituted or unsubstituted C1-C4 alkyl. In some of these embodiments, A can be one of the following: [ka]

[0160] In some of these embodiments, X is OH.

[0161] In some embodiments, A is [ka] wherein Y is S or O; X is OH, NHR 2 , SH, or S(O)(O)NHR 2 and;R 2 is H, OH, halogen, or substituted or unsubstituted C1-C4 alkyl. In some of these embodiments, X is OH.

[0162] In some embodiments, A is [ka] wherein Y is S or O; X is OH, NHR 2 , SH, or S(O)(O)NHR 2 and;R 2 is H, OH, halogen, or substituted or unsubstituted C1-C4 alkyl. In some of these embodiments, X is OH.

[0163] In some embodiments, A is [ka] is.

[0164] In some of the above embodiments, R 1 is a substituted or unsubstituted C6-C 10 Alkyl (e.g., C6-C 10 hydroxyalkyl).

[0165] In some of the above embodiments, R 1 is a substituted or unsubstituted C3-C 16 Alkylaryl (e.g., C3-C 16 hydroxyalkylaryl).

[0166] In some of the above embodiments, R 1is a substituted or unsubstituted C8-C 20 Alkylaryl (e.g., C8-C 20 hydroxyalkylaryl).

[0167] In some of the above embodiments, R 1 is a substituted or unsubstituted C5-C 10 It may be acyl.

[0168] In some of the above embodiments, R 1 is a substituted or unsubstituted C4-C 10 Alkyl (e.g., branched C4-C 10 hydroxyalkyl).

[0169] In some embodiments, R 1 may include one of the following: [ka] where the dotted line to Y indicates that the bond may be single or double, valence permitting; Y, when present, is O, halogen, OR 2’ , NHR 2 , SH, or S(O)(O)NHR 2 and;R 6 is a substituted or unsubstituted C1-C 19 Alkyl, substituted or unsubstituted C2-C 19 Alkenyl, substituted or unsubstituted C2-C 19 Alkynyl, substituted or unsubstituted C2-C 19 Alkylaryl, substituted or unsubstituted C2-C 19 Alkylheteroaryl, substituted or unsubstituted C4-C 19 Alkylcycloalkyl, substituted or unsubstituted C4-C 19 Alkylheterocycloalkyl and substituted or unsubstituted C-C 20 Heteroalkyl, or NR 3 R 4 and;R 2 is H, OH, halogen, or substituted or unsubstituted C1-C4 alkyl; R 2’is H or substituted or unsubstituted C1-C4 alkyl; R 3 and R 4 are independently substituted or unsubstituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl, substituted or unsubstituted C2-C 20 Alkynyl, substituted or unsubstituted C2-C 20 Alkylaryl, substituted or unsubstituted C4-C 20 Alkylcycloalkyl and substituted or unsubstituted C2-C 20 heteroalkyl.

[0170] In some embodiments, A may include a substituted or unsubstituted aryl ring (e.g., a substituted or unsubstituted phenyl ring). In some embodiments, A may include a 5-membered substituted or unsubstituted heteroaryl ring. For example, A may include thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl, or 1,3,4-oxadiazolyl ring. In some embodiments, A may include a 6-membered substituted or unsubstituted heteroaryl ring. For example, A may include a pyridyl, pyrazinyl, pyrimidinyl, triazinyl, or pyridazinyl ring.

[0171] In some of these embodiments, Y is OH. In some of these embodiments, Y is F. In some of these embodiments, Y is O.

[0172] In some instances, R 6 is a substituted or unsubstituted C3-C 10 It can be alkyl, for example, substituted or unsubstituted C6-C9 alkyl.

[0173] In some instances, R6 is a substituted or unsubstituted C2-C 15 It may be alkylaryl.

[0174] In some instances, R 6 can be a substituted or unsubstituted branched C2-C9 alkyl.

[0175] In some instances, R 6 is a substituted or unsubstituted C3-C 10 It can be a heteroalkyl, for example, a substituted or unsubstituted C6-C9 heteroalkyl.

[0176] In some embodiments, the compound may include one of the following: [ka] [ka]

[0177] Also disclosed herein are pharmaceutically acceptable salts and prodrugs of the carboranes and carborane analogs described herein. Pharmaceutically acceptable salts include salts of the disclosed carboranes and carborane analogs, which are prepared using acids or bases, depending on the specific substituents found on the compound. Under conditions where the carboranes and carborane analogs disclosed herein are sufficiently basic or acidic to form stable, non-toxic acid or base salts, administration of the compounds as salts may be appropriate. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, or magnesium salts. Examples of physiologically acceptable acid addition salts include hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, carbonic acid, sulfuric acid, and organic acids such as acetic acid, propionic acid, benzoic acid, succinic acid, fumaric acid, mandelic acid, oxalic acid, citric acid, tartaric acid, malonic acid, ascorbic acid, α-ketoglutaric acid, α-glycolic acid, maleic acid, tosylate, methanesulfonic acid, etc. Thus, hydrochloride, nitrate, phosphate, carbonate, bicarbonate, sulfate, acetate, propionate, benzoate, succinate, fumarate, mandelate, oxalate, citrate, tartrate, malonate, ascorbate, α-ketoglutarate, α-glycolic acid, maleate, tosylate, and mesylate salts are disclosed herein. Pharmaceutically acceptable salts of compounds can be obtained using standard procedures well known in the art, for example, by reacting a sufficiently basic compound, such as an amine, with a suitable acid to produce a physiologically acceptable anion. Alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids can also be prepared.

[0178] In some examples, the carboranes and carborane analogs disclosed herein have a potency of 800 nM or less (e.g., 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less) at estrogen receptor β (ERβ). Bottom, 10nM or less, 9nM or less, 8nM or less, 7nM or less, 6nM or less, 5nM or less, 4.5nM or less, 4nM or less, 3.5nM or less, 3nM or less, 2.5nM or less, 2nM or less, 1.5nM EC of 1nM or less, 0.9nM or less, 0.8nM or less, 0.7nM or less, 0.6nM or less, 0.5nM or less, 0.4nM or less, 0.3nM or less, 0.2nM or less, or 0.1nM or less) 50 may have:

[0179] In some examples, the carboranes and carborane analogs disclosed herein have an ERβ activity of 1 pM or more (e.g., 0.1 nM or more, 0.2 nM or more, 0.3 nM or more, 0.4 nM or more, 0.5 nM or more, 0.6 nM or more, 0.7 nM or more, 0.8 nM or more, 0.9 nM or more, 1 nM or more, 1.5 nM or more, 2 nM or more, 2.5 nM or more, 3 nM or more, 3.5 nM or more, 4 nM or more). EC (e.g., 4.5nM or more, 5nM or more, 6nM or more, 7nM or more, 8nM or more, 9nM or more, 10nM or more, 20nM or more, 30nM or more, 40nM or more, 50nM or more, 60nM or more, 70nM or more, 80nM or more, 90nM or more, 100nM or more, 200nM or more, 300nM or more, 400nM or more, 500nM or more, 600nM or more, or 700nM or more) 50 may have:

[0180] EC of carboranes and carborane analogues on ERβ 50can range from any of the above minimum values ​​to any of the above maximum values. For example, the carboranes and carborane analogs disclosed herein have an EC at ERβ of 1 pM to 800 nM (e.g., 1 pM to 400 nM, 400 nM to 800 nM, 1 pM to 300 nM, 1 pM to 200 nM, 1 pM to 100 nM, 1 pM to 50 nM, 1 pM to 20 nM, 1 pM to 10 nM, 1 pM to 6 nM, 1 pM to 5 nM, 1 pM to 2 nM, 1 pM to 1 nM, 1 pM to 0.7 nM, 1 pM to 0.5 nM, 1 pM to 0.2 pM, or 1 pM to 0.1 nM). 50 may have:

[0181] In some instances, the carboranes and carborane analogs disclosed herein are selective ERβ agonists. In some instances, selective ERβ agonists have a lower EC agonist activity at ERβ compared to estrogen receptor α (ERα). 50 The selectivity of a compound can be measured in some instances by the EC 50 EC of the compound on ERβ 50 In some examples, the compounds disclosed herein may have an ERβ to ERα agonist ratio of 8 or greater (e.g., 10 or greater, 20 or greater, 30 or greater, 40 or greater, 50 or greater, 60 or greater, 70 or greater, 80 or greater, 90 or greater, 100 or greater, 150 or greater, 200 or greater, 250 or greater, 300 or greater, 350 or greater, 400 or greater, 450 or greater, 500 or greater, 600 or greater, 700 or greater, 800 or greater, 900 or greater, 1000 or greater, 1100 or greater, 1200 or greater, 1300 or greater, 1400 or greater, 1500 or greater, 2000 or greater, 2500 or greater).

[0182] In some examples, carboranes and carborane analogs may have an ERβ to ERα agonist ratio of 3000 or less (e.g., 2500 or less, 2000 or less, 1500 or less, 1400 or less, 1300 or less, 1200 or less, 1100 or less, 1000 or less, 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 450 or less, 400 or less, 350 or less, 300 or less, 250 or less, 200 or less, 150 or less, 100 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, or 10 or less).

[0183] The ERβ to ERα agonist ratio of carboranes and carborane analogs at ERβ can range from any of the minimum values ​​described above to any of the maximum values ​​described above. For example, carboranes and carborane analogs can have an ERβ to ERα agonist ratio of 8 to 3000 (e.g., 8 to 1500, 1500 to 3000, 400 to 3000, 500 to 3000, 600 to 3000, 700 to 3000, 800 to 3000, 900 to 3000, 1000 to 3000, or 2000 to 3000).

[0184] Manufacturing method The compounds described herein can be prepared by various methods known to those skilled in the art of organic synthesis, or by variations thereof that will be appreciated by those skilled in the art. The compounds described herein can be prepared from readily available starting materials. Optimum reaction conditions may vary depending on the particular reactants or solvents used, but such conditions can be determined by those skilled in the art.

[0185] Variations of the compounds described herein include the addition, deletion, or movement of various components described for each compound. Similarly, if one or more chiral centers are present in a molecule, the chirality of the molecule can be altered. Furthermore, the synthesis of a compound can involve the protection and deprotection of various chemical groups. The use of protection and deprotection, and the selection of appropriate protecting groups, can be determined by those skilled in the art. Protecting group chemistry is described, for example, in Wuts and Greene, Protective Groups in Organic Synthesis, 4th Ed., Wiley & Sons, 2006, which is incorporated herein by reference in its entirety.

[0186] Starting materials and reagents used in preparing the disclosed compounds and compositions may be purchased from Katchem (Prague, Czech Republic), Aldrich Chemical Co., (Milwaukee, WI), Acros Organics (Morris Plains, NJ), Fisher Scientific (Pittsburgh, PA), Sigma (St. Louis, MO), Pfizer (New York, NY), GlaxoSmithKline (Raleigh, NC), Merck (Whitehouse Station, NJ), Johnson & Johnson (New Brunswick, NJ), Aventis (Bridgewater, NJ), AstraZeneca (Wilmington, DE), Novartis (Basel, Switzerland), Wyeth (Madison, NJ), Bristol-Myers-Squibb (New York, NY), Roche (Basel, Switzerland), Lilly (Indianapolis, IN), Abbott (Abbott These reagents are available from manufacturers such as Fieser and R. Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock's Comprehensive Organic Transformations (VCH Publishers Inc.These compounds are prepared by methods known to those skilled in the art, following procedures described in references such as (Berkeley, 1989). Other materials, such as pharmaceutical excipients, disclosed herein may be obtained from commercial sources.

[0187] The reactions to produce the compounds described herein can be carried out in a solvent that can be selected by one skilled in the art of organic synthesis. The solvent can be substantially non-reactive to the starting materials (reactants), intermediates, or products under the conditions (i.e., temperature and pressure) at which the reaction is carried out. The reaction can be carried out in one solvent or a mixture of multiple solvents. The formation of the product or intermediate can be monitored according to any suitable method known in the art. For example, the formation of the product can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) It can be monitored by infrared spectroscopy, spectrophotometry (eg, UV-visible), or mass spectrometry, or chromatography, such as high performance liquid chromatography (HPLC) or thin layer chromatography.

[0188] Exemplary methods for preparing carboranes and carborane analogs are described, for example, in U.S. Pat. No. 6,838,574 to Endo, U.S. Patent Application Publication No. 2018 / 0264017 to Tjarks et al., and PCT / US2019 / 064228 to Coss et al., each of which is incorporated herein by reference in its entirety.

[0189] How to use The carboranes and carborane analogs described herein can inhibit CD4+ T cell activation and proliferation. Thus, the carboranes and carborane analogs described herein can be administered to a subject to reduce circulating CD4+ T cell levels (e.g., compared to levels prior to administration of the carborane or carborane analog, or compared to levels in a control not treated with the carborane or carborane analog). In some embodiments, the carboranes and carborane analogs described herein can selectively reduce circulating CD4+ T cell levels while leaving levels of other white blood cells substantially unchanged. For example, in some embodiments, the carboranes or carborane analogs can be administered in an amount effective to reduce circulating CD4+ T cell levels in a subject (e.g., by at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, or more) without significantly affecting circulating levels of neutrophils, monocytes, or B cells (e.g., by less than a 15%, less than a 10%, or less than a 5% change). These reductions can be measured relative to levels prior to administration of the carborane or carborane analog, or relative to levels in controls not treated with the carborane or carborane analog.

[0190] CD4+ T cells mediate wound healing after myocardial infarction (MI) but exacerbate left ventricular (LV) remodeling during chronic heart failure (HF). The mechanisms underlying this transition are unclear. However, without wishing to be bound by theory, it is believed that T cells activate specific pathological signals to promote LV remodeling during chronic HF. To identify such signals, we performed limited cellular RNA sequencing of CD4+ T cells sorted from failing hearts (8 weeks post-MI) of male mice and surprisingly observed activation of estrogen receptor (ER)-α signaling. Because ERα effects are antagonized by ERβ, ERβ agonists (including the carboranes and carborane analogs described herein) can be administered to modulate T cell activity and LV remodeling.

[0191] As detailed in the Examples below, in vitro assays demonstrated that an exemplary carborane (Compound 1) dose-dependently inhibited the activation and proliferation of T cells selected from male mice (IC 50 3.4 μM). In vivo assays (60 mg / kg / day, orally) showed no obvious toxicity and significantly reduced circulating T cells without affecting neutrophils, monocytes, or B cells, demonstrating specificity for T cells. Furthermore, this effect was specific to TCR-mediated T cell activation; the drug had no effect on PMA / ionomycin-stimulated T cells, suggesting preferential inhibition of antigen-activated T cells.

[0192] To test the therapeutic effect, male 10- to 12-week-old mice underwent coronary artery ligation or sham surgery. Four weeks after MI, they were randomized according to cardiac function and administered vehicle or Compound 1 (60 mg / kg / day, orally) for the following four weeks. Consistently, at week 8, a significant decrease in circulating and splenic T cells was observed in drug-treated mice compared with vehicle-treated mice. Furthermore, vehicle-treated HF mice exhibited progressive LV dilation accompanied by significant increases in end-diastolic and end-systolic volumes (EDV and ESV, respectively) at 4 to 8 weeks post-MI. Importantly, treatment with Compound 1 significantly inhibited these changes and blunted LV remodeling at 4 to 8 weeks post-MI. A significant decrease in tibial-normalized heart weight supported these results. These examples suggest that the carboranes and carborane analogs described herein can selectively inhibit T cell activation and blunt pathological LV remodeling during chronic HF.

[0193] Thus, provided herein are methods for treating or preventing chronic heart failure in a subject after myocardial infarction. These methods can include administering a carborane or carborane analog to the subject during the maladaptive remodeling phase after myocardial infarction. Importantly, in some embodiments, the carborane or carborane analog is not administered to the subject during the healing or repair phase preceding the maladaptive remodeling phase (i.e., administration of the carborane or carborane analog does not begin until the acute phase after MI is complete and the chronic phase begins).

[0194] In some embodiments, administration of the carborane or carborane analog begins at least 10 days after myocardial infarction, e.g., at least 14 days after myocardial infarction, at least 21 days after myocardial infarction, at least 28 days after myocardial infarction, at least 35 days after myocardial infarction, at least 42 days after myocardial infarction, at least 49 days after myocardial infarction, or at least 56 days after myocardial infarction.

[0195] These methods may further include evaluating the subject to determine whether the subject has entered a maladaptive remodeling phase. This can be done in any suitable manner. For example, in some embodiments, evaluating the subject to determine whether the subject has entered a maladaptive remodeling phase may include measuring circulating CD4+ T cell levels in the subject to determine when the subject has entered a maladaptive remodeling phase. In some embodiments, evaluating the subject to determine whether the subject has entered a maladaptive remodeling phase may include detecting one or more biomarkers in the subject to determine when the subject has entered a maladaptive remodeling phase. Such biomarkers are known in the art and include, for example, relative levels of myosin heavy chain isoforms, GLUT-1 expression levels, α-actin expression levels, natriuretic peptide expression levels, galectin expression levels, caveolin expression levels, neuronal nitric oxide synthase expression levels, angiotensin-converting enzyme expression levels, GLUT-4 expression levels, SERCA2a expression levels, and a shift from glucose oxidation to fatty acid oxidation. In some embodiments, evaluating the subject to determine whether the subject has entered a maladaptive remodeling phase may include echocardiography, ventriculography, magnetic resonance imaging, or any combination thereof. Imaging techniques such as echocardiography and / or MRI may also be used to measure left ventricular dilation (increased end-diastolic and end-systolic volumes) as an indicator of LV remodeling.

[0196] In some embodiments, the carborane or carborane analog can be administered in an amount effective to inhibit the activation and proliferation of CD4+ T cells in a subject. Thus, the carboranes and carborane analogs described herein can be administered to a subject to reduce circulating CD4+ T cell levels (e.g., compared to levels before administration of the carborane or carborane analog, or compared to levels in a control not treated with the carborane or carborane analog). In some embodiments, the carboranes and carborane analogs described herein can selectively reduce circulating CD4+ T cell levels while leaving levels of other white blood cells substantially unchanged. For example, in some embodiments, a carborane or carborane analog can be administered in an amount effective to reduce circulating CD4+ T cell levels in a subject (e.g., by at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, or more) without significantly affecting circulating levels of neutrophils, monocytes, or B cells (e.g., by less than a 15%, less than a 10%, or less than a 5% change). These reductions can be measured relative to levels before administration of the carborane or carborane analog, or relative to levels in controls not treated with the carborane or carborane analog.

[0197] In some embodiments, the carborane or carborane analog can be administered in an amount effective to alter morphological changes associated with CHF after MI. For example, in some embodiments, the carborane or carborane analog can be administered in an amount effective to reduce left ventricular (LV) remodeling in a subject. In some embodiments, the carborane or carborane analog can be administered in an amount effective to reduce changes in cardiac function associated with CHF after MI. For example, in some embodiments, the carborane or carborane analog can be administered in an amount effective to inhibit an increase in left ventricular end-diastolic volume in a subject, inhibit an increase in left ventricular end-systolic volume in a subject, or a combination thereof.

[0198] Methods for treating and preventing autoimmune disorders are also provided, in which carboranes and carborane analogs are administered to modulate T cell activity (e.g., selectively modulate T cell activity) in a subject. For example, provided herein are methods for treating or preventing graft-versus-host disease, multiple sclerosis (MS), and / or experimental autoimmune encephalomyelitis (EAE) in a subject, comprising administering a carborane or a carborane analog to the subject.

[0199] In practicing these methods, the carborane or carborane analog can be administered in an amount effective to inhibit the activation and proliferation of CD4+ T cells in a subject. For example, the carborane or carborane analog can be administered in an amount effective to reduce a decrease in circulating CD4+ T cell levels in a subject. In certain embodiments, the carborane or carborane analog can be administered in an amount effective to reduce circulating CD4+ T cell levels in a subject without significantly affecting circulating levels of neutrophils, monocytes, or B cells.

[0200] The methods and compounds described herein are useful for both prophylactic and therapeutic treatments. As used herein, the term "treatment" includes prevention; delaying onset; reducing, eradicating, or delaying the worsening of signs or symptoms after onset; and preventing recurrence. For prophylactic use, a therapeutically effective amount of the compounds and compositions described herein, or a pharmaceutically acceptable salt thereof, is administered to a subject before onset (e.g., before overt signs of a disease or disorder), during early onset (e.g., upon early signs and symptoms of a disease or disorder), or after established onset of a disease or disorder. Prophylactic administration can occur days to years before the appearance of symptoms of a disease or disorder. Therapeutic treatment includes administering a therapeutically effective amount of the compounds and compositions described herein, or a pharmaceutically acceptable salt thereof, to a subject after a disease or disorder has been diagnosed.

[0201] Compositions, Formulations and Methods of Administration In vivo application of the disclosed compounds and compositions containing them can be achieved by any suitable method and technique now or in the future known to those skilled in the art. For example, the disclosed compounds can be formulated in a physiologically or pharmaceutically acceptable form and administered by any suitable route known in the art, including, for example, oral, nasal, rectal, topical, and parenteral administration routes. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, and intrasternal administration, such as by injection. Administration of the disclosed compounds or compositions can be a single dose or continuous or at varying intervals, as can be easily determined by those skilled in the art.

[0202] The compounds disclosed herein and compositions containing them can also be administered using liposome technology, sustained-release capsules, implantable pumps, and biodegradable containers.These delivery methods can advantageously provide a uniform dose over a long period of time.The compounds can also be administered in their salt derivative form or crystalline form.

[0203] The compounds disclosed herein can be formulated according to known methods for preparing pharmaceutically acceptable compositions. Formulations are well known to those skilled in the art and are described in detail in numerous readily available sources. For example, E.W. Martin's Remington's Pharmaceutical Science (1995) describes formulations that can be used in connection with the disclosed methods. Generally, the compounds disclosed herein can be formulated so that a therapeutically effective amount of the compound is combined with suitable excipients to facilitate effective administration of the compound. The compositions used can also be in a variety of forms. These include solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions, suppositories, injectable and infusible solutions, and sprays. The preferred form depends on the intended mode of administration and therapeutic application. The compositions also preferably contain conventional pharmaceutically acceptable carriers and diluents known to those skilled in the art. Examples of carriers or diluents for use with the compounds include ethanol, dimethyl sulfoxide, glycerol, alumina, starch, saline, and equivalent carriers and diluents. To provide for administration of such dosages for the desired therapeutic treatment, the compositions disclosed herein may advantageously contain a total of about 0.1% to 100% by weight of one or more of the subject compounds, based on the weight of the total composition, including any carrier or diluent.

[0204] Suitable formulations for administration include aqueous sterile injection solutions, which may contain, for example, antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) state, requiring only the preparation of a sterile liquid carrier, e.g., water, prior to use for injection. Ready-to-use injection solutions and suspensions may be prepared from sterile powders, granules, tablets, and the like. In addition to the excipients specifically mentioned above, it should be understood that the compositions disclosed herein may contain other agents conventional in the art, taking into account the type of formulation in question.

[0205] The compounds disclosed herein and compositions containing them can be delivered to cells by direct contact with cells or via a carrier. Carrier means for delivering compounds and compositions to cells are known in the art and include, for example, encapsulating the composition in a liposome moiety. Another means for delivering the compounds and compositions disclosed herein to cells includes binding the compound to a targeted protein or nucleic acid for delivery to the target cell. U.S. Patent No. 6,960,648 and U.S. Patent Application Publication Nos. 20030032594 and 20020120100 disclose amino acid sequences that can be coupled to another composition, thereby enabling the composition to translocate across a biological membrane. U.S. Patent Application Publication No. 20020035243 also describes a composition for transporting biological moieties across a cell membrane for intracellular delivery. The compound can also be incorporated into a polymer. Examples include poly(DL lactide-co-glycolide) polymer for intracranial tumors; poly[bis(p-carboxyphenoxy)propane:sebacic acid] in a 20:80 molar ratio (as used in GLIADEL); chondroitin; chitin; and chitosan.

[0206] For the treatment of oncological disorders, the compounds disclosed herein can be administered to a patient in need of treatment in combination with other antitumor or anticancer agents, and / or radiation therapy and / or photodynamic therapy, and / or surgical treatment to remove the tumor. These other agents or treatments can be administered simultaneously or at different times with the compounds disclosed herein. For example, the compounds disclosed herein can be combined with mitotic inhibitors such as taxol or vinblastine, alkylating agents such as cyclophosamide or ifosfamide, antimetabolites such as 5-fluorouracil or hydroxyurea, DNA intercalators such as adriamycin or bleomycin, etoposide or camptothecin, antiangiogenic agents such as angiostatin, antiestrogens such as tamoxifen, and / or other anticancer agents or antibodies, such as GLEEVEC (Novartis Pharmaceuticals Corporation) and HERCEPTIN (Genentech, Inc.), respectively, or immunotherapies such as ipilimumab and bortezomib.

[0207] In certain examples, the compounds and compositions disclosed herein, optionally combined with a pharmaceutically acceptable carrier such as an inert diluent, can be administered locally to one or more anatomical sites, e.g., the site of unwanted cell proliferation (e.g., injected or topically applied to a tumor site or benign skin tumor, e.g., a tumor or skin tumor). The compounds and compositions disclosed herein can be administered systemically, e.g., intravenously or orally, optionally combined with a pharmaceutically acceptable carrier such as an inert diluent, or an assimilable edible carrier for oral delivery. They can be enclosed in hard or soft-shell gelatin capsules, compressed into tablets, or incorporated directly into the food of the patient's diet. For oral therapeutic administration, the active compounds can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, aerosol sprays, and the like.

[0208] Tablets, troches, pills, capsules, etc. may also contain binders such as tragacanth gum, acacia, cornstarch, or gelatin; diluents such as dicalcium phosphate; disintegrating agents such as cornstarch, potato starch, or alginic acid; lubricants such as magnesium stearate; and sweeteners such as sucrose, fructose, lactose, or aspartame, or flavorings such as peppermint, wintergreen oil, or cherry flavor. When the unit dosage form is a capsule, in addition to the above-mentioned materials, a liquid carrier such as vegetable oil or polyethylene glycol may be contained. Various other materials may be present as coatings or to modify the physical form of the solid unit dosage form. For example, tablets, pills, or capsules may be coated with gelatin, wax, shellac, sugar, or the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetener, methyl and propylparabens as preservatives, a dye, and a flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. Moreover, the active compounds may be incorporated into sustained-release preparations and devices.

[0209] The compounds and compositions disclosed herein, including pharmaceutically acceptable salts or prodrugs, can be administered intravenously, intramuscularly, or intraperitoneally by infusion or injection. Solutions of the active agent or its salts can be prepared in water, optionally mixed with a non-toxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, triacetin, and mixtures thereof, as well as in oils. These preparations may contain preservatives to prevent the growth of microorganisms under normal storage and use conditions.

[0210] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders containing the active ingredient, which are suitable for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The final dosage form should be sterile, fluid, and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by maintaining the required particle size in the case of dispersions, or by the use of surfactants. Optionally, the action of microorganisms can be inhibited by various other antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, such as sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the inclusion of an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0211] Sterile injectable solutions are prepared by incorporating the compounds and / or agents disclosed herein in the required amount in an appropriate solvent with various other ingredients as listed above, followed by filtered sterilization, if required. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze-drying techniques, which yield a powder that combines the active ingredient present in the previously sterile-filtered solution with any additional desired ingredients.

[0212] For topical administration, the compounds and agents disclosed herein can be applied as liquids or solids. However, it is generally desirable to combine them with a dermatologically acceptable carrier, which can be solid or liquid, and administer them topically to the skin as a composition. The compounds and agents and compositions disclosed herein can be applied topically to a subject's skin to reduce the size of malignant or benign tumors (which may include complete removal) or treat an infected site. The compounds and agents disclosed herein can be applied directly to the tumor site or infected site. Preferably, the compounds and agents are applied to the tumor site or infected site in a formulation such as an ointment, cream, lotion, solution, or tincture.

[0213] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, etc. Useful liquid carriers include water, alcohol, or glycol, or a water-alcohol / glycol mixture, optionally supplemented with a non-toxic surfactant, to dissolve or disperse the compound at an effective level. Adjuvants such as fragrances and additional antibacterial agents can be added to optimize the properties for a given application. The resulting liquid composition can be applied from absorbent pads, impregnated into bandages and other dressings, or sprayed onto the affected area using a pump-type or aerosol sprayer.

[0214] Thickening agents such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified cellulose, or modified mineral materials may also be used with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like for application directly to the user's skin.

[0215] Useful dosages of the compounds and agents and pharmaceutical compositions disclosed herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.

[0216] The dosage range for administering the composition is sufficient to produce the desired effect that the symptom or disorder is affected. The dosage should not be so large as to cause harmful side effects such as undesired cross-reactions, anaphylactic reactions, etc. Generally, the dosage varies depending on the age, condition, sex, and degree of disease of the patient, and can be determined by those skilled in the art. The dosage can be adjusted by an individual physician if there are any adverse symptoms. The dosage can vary and can be administered once or more times daily for one or more days.

[0217] Also disclosed are pharmaceutical compositions comprising the compounds disclosed herein in combination with pharmaceutically acceptable excipients. Pharmaceutical compositions suitable for oral, topical or parenteral administration, comprising a certain amount of the compound, constitute a preferred embodiment. The dose administered to a patient, particularly a human, should be sufficient to achieve a therapeutic response in the patient over a reasonable time frame, without lethal toxicity, and preferably without causing side effects or morbidity beyond an acceptable level. Those skilled in the art will recognize that the dose depends on various factors, including the subject's condition (health), the subject's weight, the type of concurrent treatment, if any, the frequency of treatment, the therapeutic ratio, and the severity and stage of the pathological condition.

[0218] Also disclosed are kits comprising a compound disclosed herein in one or more containers. The disclosed kits may optionally include a pharmaceutically acceptable carrier and / or diluent. In one embodiment, the kit includes one or more other ingredients, adjuvants, or supplements described herein. In another embodiment, the kit includes one or more anti-cancer agents, e.g., drugs described herein. In one embodiment, the kit includes instructions or packaging materials that describe how to administer the compound or composition of the kit. The containers of the kit can be made of any suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration. In one embodiment, the compound and / or drug disclosed herein is provided in the kit as a solid, such as a tablet, pill, or powder form. In another embodiment, the compound and / or drug disclosed herein is provided in the kit as a liquid or solution. In one embodiment, the kit includes an ampoule or syringe containing the compound and / or drug disclosed herein in liquid or solution form.

[0219] Numerous embodiments of the invention have been described. It will, of course, be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. [Example]

[0220] The following examples are provided to illustrate methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations that would be apparent to one skilled in the art.

[0221] Example 1. Evaluation of exemplary carboranes for the treatment of heart failure Immune and inflammatory responses contribute to left ventricular (LV) remodeling after myocardial infarction (MI). Elevated levels of proinflammatory cytokines promote cardiac remodeling and disease progression in heart failure (HF); however, clinical trials of TNF neutralization have failed to demonstrate efficacy and even demonstrated toxicity at high doses. These paradoxical results suggest that the role of inflammatory activation in HF is more complex than gleaned from cytokine levels alone. The activation of innate and adaptive immune cells underlies the inflammatory response in many chronic diseases. Monocytes, macrophages, and dendritic cells (DCs) mediate innate immune responses, while CD3+CD4+ helper T cells and CD3+CD8+ cytotoxic T cells mediate adaptive immunity. While innate immune cells constitute the first line of defense against acute injury, chronic inflammation often implies the activation and clonal expansion of specialized effector T cells following antigen presentation.

[0222] Previously published studies have shown that chronic ischemic heart failure can be characterized by global proliferation of CD4+ T cells in the blood, spleen, and heart. Furthermore, depletion of CD4+ T cells (using an anti-CD4 antibody) in mice with heart failure prevented the progression of cardiac function decline and left ventricular remodeling. These studies suggest that T lymphocyte activation during chronic heart failure may play an important role in mediating pathological left ventricular (LV) remodeling associated with progressive cardiac dysfunction (increased end-diastolic and end-systolic volumes and ejection fraction), as well as increased myocyte hypertrophy and fibrosis.

[0223] Figure 1 shows gene expression of estrogen receptor (ER) α and β (ERα and ERβ) in the ovary (positive control), both male and female hearts (M+F), and male spleen. β-actin was used as an endogenous control, and the fold change relative to the heart (left and center panels) or ERα (right panel) is shown in Figure 1. Figure 1 shows that ERα expression is similar in the heart and spleen and is almost negligible compared to the ovary. In contrast, ERβ gene expression in the spleen is approximately one-third that in the ovary and much higher than that in the heart (center panel, Figure 1). Furthermore, the ratio of ERβ to ERα is much higher in the spleen compared to the ovary or heart (right panel, Figure 1), suggesting that ERβ is preferentially expressed relative to ERα in splenocytes.

[0224] Representative flow cytometry histograms of ERα (upper panel) and ERβ (lower panel) expression in different circulating (left panel) and splenic (right panel) immune cells in male mice are shown in Figure 2. Circulating and splenic immune cells did not express ERα, whereas several immune cell subsets were found to express significant levels of ERβ protein (Figure 2). ERβ expression was highest in CD11b+ myeloid cells (Ly6G+ neutrophils and Ly6C+ monocytes / macrophages), followed by CD4+ helper T cells, and lowest in CD19+ B cells (Figure 2).

[0225] Representative flow cytometry histograms of CD4+ T cells, either unstimulated or CD3 / CD28 TCR-stimulated, treated with estradiol (5 nM and 50 nM) or Compound 1 (5 μM, structure shown below), or both, and labeled with cell tracing violet (CTV; a cell proliferation dye) are shown in Figure 3. The high-to-low peak pattern of fluorescence intensity in stimulated cells represents the halving of dye concentration at the plasma membrane of daughter cells with each successive cell division. Quantification of cell proliferation (%), measured as dye dilution with each successive cell division, for stimulated and unstimulated groups is shown in Figures 4 and 5, respectively.

[0226] [ka] The cell viability of CD3 / CD28-mediated in vitro TCR stimulation with and without treatment with Compound 1 is shown in Figure 6. The results show that treatment with Compound 1 does not affect the cell viability of T cells stimulated with CD3 / CD28 antibodies (Figure 6).

[0227] Representative flow cytometry histograms of TNFα expression in unstimulated or CD3 / CD28 TCR-stimulated CD4+ T cells treated with either estradiol (5 nM and 50 nM) or Compound 1 (5 μM), or both, are shown in Figure 7. Quantification of the stimulated groups is shown in Figure 8, and the unstimulated control group is shown in Figure 9. The results show that treatment with Compound 1 suppressed the number of TNFα+ helper T cells (CD4+).

[0228] Representative flow cytometry histograms of IFNγ expression in unstimulated or CD3 / CD28 TCR-stimulated CD4+ T cells treated with either estradiol (5 nM and 50 nM) or Compound 1 (5 μM), or both, are shown in Figure 10. Quantification of the stimulated groups is shown in Figure 11, and the unstimulated control group is shown in Figure 12. The results show that treatment with Compound 1 did not significantly affect the number of IFNγ+ helper T cells (CD4+).

[0229] The results of a CD3 / CD28 TCR-mediated in vitro T cell proliferation assay using T cells from female mice with and without Compound 1 are shown in Figures 13-16. The results show quantification of populations for cell viability (Figure 13), proliferation (Figure 14), TNFα+ helper T cells (CD4+) (Figure 15), and IFNγ+ helper T cells (CD4+) (Figure 16) following unstimulated or CD3 / CD28 TCR stimulation and treatment with either estradiol (5 nM and 50 nM) or Compound 1 (5 μM), or both. The results suggest that treatment with Compound 1 suppresses cell proliferation, the number of TNFα+ helper T cells (CD4+), and the number of IFNγ+ helper T cells without adversely affecting overall cell viability.

[0230] The results of non-specific PMA / ionomycin-mediated in vitro T cell activation with and without Compound 1 are shown in Figures 17-20.

[0231] The results show quantification of groups for cell viability (FIG. 17), TNFα+ helper T cells (CD4+) (FIG. 18), IFNγ+ helper T cells (CD4+) (FIG. 19), and CD69+ helper T cells (CD4+) (FIG. 20) after unstimulated or PMA / ionomycin-stimulated treatment with Compound 1 (5 μM). The results show that treatment with Compound 1 did not affect the levels of TNFα+, IFNγ+, or CD69+ T cells after stimulation with PMA / ionomycin, suggesting that Compound 1 specifically affects the CD3 / CD28 TCR-mediated T cell activation pathway.

[0232] Next, in vivo treatment was tested using mice. First, treatment was initiated on day 7 after infarction to inhibit immune activation in the acute phase (Figure 27). The body weights of sham-operated and myocardial infarction (MI) mice treated with either vehicle control or Compound 1 are shown in Figure 21 (day 0 corresponds to the day treatment was initiated, e.g., day 7 after MI). Kaplan-Meier curves are shown in Figure 22 for the mortality rates of vehicle- or drug-treated MI and sham-operated mice (day 0 corresponds to the day treatment was initiated, e.g., day 7 after MI). As can be seen in Figure 22, treatment with Compound 1 during the acute phase of immune activation increased mortality (reduced survival rate), indicating that an immune response during the acute phase is important for adequate healing.

[0233] Therefore, in subsequent studies, we investigated the effect of treatment 28 days after infarction to inhibit immune activation in the chronic (e.g., maladaptive remodeling) phase (Figure 27). Body weights and tibia-normalized heart weights of sham-operated and myocardial infarction (MI) mice treated with either vehicle control or Compound 1 28 days after MI are shown in Figures 23 and 24, respectively. The results show that treatment with Compound 1 28 days after MI stopped the increase in heart weight (Figure 24). Furthermore, there was no significant increase in mortality associated with Compound 1 treatment 28 days after MI.

[0234] The levels (per μL of blood) of circulating CD4+ helper T cells and their subsets, i.e., CD4+Foxp3+ (Treg), CD4+TNFα+ cells, CD4+IFNγ+ (Th1), CD4+IL-4+ (Th2), and CD4+IL-17+ (Th17) T cells, at 8 weeks post-surgery in mice treated with either vehicle or Compound 1 for 4 to 8 weeks post-surgery are shown in Figure 25. The results demonstrate that treatment with Compound 1 results in a reduction of various types of CD4+ helper T cells.

[0235] Quantitative group data on changes in left ventricular end-diastolic and end-systolic volumes (EDV and ESV) and ejection fraction (EF) in ligated mice before (4 weeks after myocardial infarction) and after (8 weeks after myocardial infarction) treatment with either vehicle or Compound 1 are shown in Figure 26. The results show that changes in end-systolic volume, end-diastolic volume, and ejection fraction were significantly less in ligated mice treated with Compound 1 than in controls.

[0236] Herein, the drug molecule Compound 1 was tested for its ability to inhibit T lymphocyte activation and proliferation during chronic heart failure. In an in vitro T cell proliferation assay, Compound 1 was found to inhibit CD3 / CD28 TCR-mediated T cell activation. Administration of Compound 1 to mice with heart failure (starting 4 weeks after myocardial infarction) reduced circulating CD4+ T lymphocyte levels and blunted progressive left ventricular remodeling measured 8 weeks after myocardial infarction.

[0237] Example 2 Sustained and inappropriate inflammatory activation contributes to disease progression in chronic heart failure (HF), as indicated by elevated levels of inflammatory cytokines such as tumor necrosis factor-α (TNF) (Ismahil et al. Circ Res. 2014;114(2);266-82). However, clinical trials of antibody-based anti-TNF therapy in HF failed to demonstrate clinical benefit (Anker et al. Int J Cardiol. 2002;86:123-30). This suggests that inflammatory mechanisms in HF, and by analogy, therapeutic immunomodulatory approaches, are more complex and nuanced than the gradual changes in cytokine responses in myocardial failure. Plasma cytokine levels are often the result of complex interactions between activated immune cells and may therefore be less sensitive indicators of underlying tissue events specifically mediated by inflammatory and immune cells. Indeed, circulatory and cardiac inflammatory cells (monocytes (Nahrendorf et al. J Exp Med. 2007;204:3037-47), macrophages (Ismahil et al. Circ Res. 2014;114(2);266-82), and T lymphocytes (Bansal et al. Circ Heart Fail. 2017;10:e003688)) increase in both the acute and chronic phases of heart failure, necessitating spatiotemporal dissection and the identification of specific molecular signatures that can be targeted to suppress pathological immune cell activation to achieve therapeutic immunomodulation.

[0238] Recent studies have established a complex interrelationship between innate immune cells (e.g., monocytes / macrophages and dendritic cells) and adaptive immune cells (e.g., T lymphocytes) in regulating tissue remodeling (Ismahil et al. Circ Res.2014;114(2);266-82). These include dendritic cells (Ismahil et al. Circ Res.2014;114(2);266-82), monocytes (Nahrendorf et al. J Exp Med.2007;204:3037-47), macrophages (Ismahil et al. Circ Res.2014;114(2);266-82), and CD4 + Helper T lymphocytes (Bansal et al. Circ Heart Fail.2017;10:e003688) influence both the acute and chronic phases of left ventricular (LV) remodeling. Importantly, in contrast to early (7 days) post-infarction cardiac remodeling, inflammation-induced iNOS in chronic HF is not regulated. + and TNF + Innate (Kingery et al. Basic Res Cardiol. 2017;112:19) and adaptive (Bansal et al. Circ Heart Fail. 2017;10:e003688) immune cells demonstrate complex pathological regulation of global immune cell networks. Furthermore, splenocytes (Ismahil et al. Circ Res. 2014;114(2);266-82) or splenic CD4+ cells from mice with heart failure (HFR) have been shown to be involved in the pathological regulation of immune cells. + Adoptive transfer of T cells (Bansal et al. Circ Heart Fail.2017;10:e003688) induced significant left ventricular remodeling and heart failure in naive mice, whereas splenectomy (Ismahil et al. Circ Res.2014;114(2);266-82) or antibody-mediated CD4 +T cell depletion (Bansal et al. Circ Heart Fail. 2017;10:e003688) inhibited left ventricular remodeling and improved cardiac function in mice with heart failure. These studies indicate that chronic ischemic heart failure is a state of global immune cell activation and proliferation in the heart, blood, spleen, and lymph nodes (LNs). Several studies have also shown that estrogen receptor (ER) α and β are expressed in myeloid (monocytes / macrophages / dendritic cells) and lymphoid (B cells and T cells) immune cells (Kovats S. Cell Immunol. 2015;294:63-9). Furthermore, ERβ activation inhibits TNFα-mediated NF-kB translocation (Xing et al. PLoS One. 2012;7:e36890), blunts IFNα and iNOS expression (Kovats S. Cell Immunol. 2015;294:63-9), and suppresses T cell-mediated autoimmunity, a key regulator of left ventricular remodeling and heart failure (Aggelakopoulou et al. J Immunol. 2016;196:4947-56). This previously unrecognized role of ERβ activation in immune cell proliferation and proinflammatory switching raises the possibility of immunomodulatory approaches that specifically reverse this pathological and tissue-damaging phenotype switching of immune cells during the progression of chronic heart failure.

[0239] Epidemiological studies have shown that premenopausal women are protected from cardiovascular disease (CVD) compared with postmenopausal women (Hayward et al. Cardiovasc Res. 2000;46:28-49). Furthermore, the incidence rate is generally much lower in women, delayed by 10 years compared with men of the same age (Wake et al. Recent Pat Cardiovasc Drug Discov. 2009;4:234-40). Men have also been shown to have significantly more pathological cardiac remodeling, accentuated by the activation of fibrotic and inflammatory genes, compared with women (Wake et al. Recent Pat Cardiovasc Drug Discov. 2009;4:234-40), suggesting a role for the estrogen receptor (ER) in mediating cardiovascular disease-related inflammation.

[0240] ERα levels are associated with maladaptive cardiac remodeling in human heart failure patients (Mahmoodzadeh et al. Faseb J.2006;20:926-34), whereas ERβ overexpression improves cardiac function and survival by reducing cardiac fibrosis (Pedram et al. Mol Cell Endocrinol.2016;434:57-68). ERα-mediated signaling regulates type I IFN production in macrophages and dendritic cells, and ERα - / -Immune cells significantly reduce the production of pro-inflammatory cytokines, including IL-6, IL-23, IL-12, and IL-1β (Kovats S. Cell Immunol. 2015;294:63-9). Notably, all of these cytokines have been shown to be elevated in patients with heart failure (Dubnika et al. Cytokine Growth Factor Rev. 2018). On the other hand, activation of ERβ has been shown to inhibit TGFβ synthesis and fibroblast-to-myofibroblast transition, blunting fibrotic events induced by angiotensin II and endothelin-1 (Pedram et al. Mol Cell Endocrinol. 2016;434:57-68). Estradiol also inhibits TNFα-mediated activation of NF-kB via ERβ stimulation (Xing et al. PLoS One. 2012;7:e36890) and inhibits iNOS production in peritoneal macrophages (Xiu-li et al. Mol Immunol. 2009;46:2413-8). Recent studies have also shown that ERβ-mediated signaling in CD4+ T cells may be important for suppressing autoimmune responses in multiple sclerosis (Aggelakopoulou et al. J Immunol. 2016;196:4947-56). These studies provide preliminary evidence for a role for ERβ in regulating immune responses, but it is unclear whether and to what extent this occurs during immune activation during heart failure. Several published studies (Ismahil et al. Circ Res. 2014;114(2);266-82; Nahrendorf et al. J Exp Med. 2007;204:3037-47; Bansal et al. Circ Heart Fail. 2017;10:e003688; Kingery et al. Basic Res Cardiol. 2017;112:19; Yang et al. Circulation. 2006;114:2056-64) have shown that acute immune activation (days 1-10) after myocardial infarction is protective in nature and mediates tissue repair.It has also been shown that chronic heart failure (4–8 weeks after myocardial infarction) is associated with a second wave of immune activation (Ismahil et al. Circ Res. 2014;114(2);266–82; Bansal et al. Circ Heart Fail. 2017;10:e003688; Kingery et al. Basic Res Cardiol. 2017;112:19). During the second wave, cardiac myocytes undergo a proinflammatory phenotypic switch associated with increased TNFα and iNOS production. This phase coincides with the highest rates of fibrosis, hypertrophy, and maladaptive left ventricular remodeling. However, the molecular mechanisms supporting this transition and the conditions that trigger immune cells to become pathological have not been clearly defined.

[0241] Therefore, it has been hypothesized that selective activation of ERβ could improve cardiac function by inhibiting TNFα-mediated NF-kB translocation, ameliorating left ventricular remodeling, and preventing pathological immune cell activation. It has further been suggested that ERβ stimulation could be a cellular target for therapeutic immunomodulation in heart failure.

[0242] The proposed study identifies the therapeutic potential of compound 1 as a selective immunomodulator to suppress persistent immune activation associated with chronic heart failure. ERβ activation in immune cells may reverse the immune cell's proinflammatory phenotype, thereby preventing pathological left ventricular remodeling and progressive cardiac dysfunction in patients with heart failure. Heart failure is widely understood to be a state of chronic inflammation; however, large-scale, safe immunomodulatory therapies for heart failure have yet to be successfully translated into clinical practice. To date, therapeutic immunomodulation attempts in heart failure have primarily focused on protein mediators, such as inflammatory cytokines. Herein, we propose an immunomodulatory paradigm that uses a selective ERβ agonist, rather than cytokine mediators, to target cardiac-primed leukocytes and reverse the proinflammatory phenotype.

[0243] Aim 1: To delineate the spatiotemporal changes of ERα and ERβ expression in immune cells during ischemic heart failure.

[0244] Rationale: Recent studies have defined a series of events leading to the activation and infiltration of systemic and tissue-resident innate and adaptive immune cells during early (Nahrendorf et al. J Exp Med. 2007;204:3037-47) and late (Ismahil et al. Circ Res. 2014;114(2);266-82; Epelman et al. Immunity. 2014;40:91-104; Heidt et al. Circ Res. 2014;115:284-95; Lavine et al. Proc Natl Acad Sci USA. 2014;111:16029-34) stages of left ventricular remodeling after myocardial infarction. Several studies have also shown that ER is constitutively expressed in immune cells of myeloid (monocytes / macrophages) and lymphoid origin (B and T lymphocytes) (Kovats S. Cell Immunol. 2015;294:63-9; Aggelakopoulou et al. J Immunol. 2016;196:4947-56). However, detailed spatiotemporal changes in ER expression on immune cell populations during ischemic heart failure have not been investigated. Therefore, our working hypothesis is that during the progression of chronic heart failure, immune cells gradually alter ER expression by decreasing ERβ levels, leading to sustained global inflammatory activation (e.g., increased systemic TNF synthesis). Because the spatiotemporal dynamics of this switch are unknown, Aim 1 aims to characterize the activation, proliferation, and proinflammatory phenotype of immune cells (monocytes / macrophages and T cells) in the PB, spleen, mediastinal LN, and heart at 1, 2, 4, and 8 weeks after myocardial infarction (Figure 28). More specifically, monocytes / macrophages and CD4+ cells in the heart, blood, spleen, and mediastinal LN were detected 1, 2, 4, and 8 weeks after coronary artery ligation in mice. +Expression of ERα and ERβ in T cells will be characterized by flow cytometry and / or immunohistochemistry compared to sham-operated controls. TNFα and nuclear transcription factor (NF)-κB p65 will be used as indicators of proinflammatory signaling and also as indicators of immune cell proliferation. Human heart failure samples will be used to characterize ER expression in circulating immune cells in outpatients with systolic heart failure and matched non-failed controls.

[0245] Aim 2: To establish the protective role of immune cell-specific ERβ against left ventricular remodeling and chronic heart failure Rationale: ERβ plays an essential role in immune cell biology by regulating immune cell activation and the expression of proinflammatory cytokines such as TNFα (Xing et al. PLoS One. 2012;7:e36890) and iNOS (Xiu-li et al. Mol Immunol. 2009;46:2413-8). Furthermore, ERβ activation has been shown to suppress immune activation in autoimmune diseases (Aggelakopoulou et al. J Immunol. 2016;196:4947-56). Because heart failure is also characterized by autoimmune responses, treatment with selective ERβ agonists may provide therapeutic benefits by inhibiting pathological left ventricular remodeling mediated by autoimmune responses. Therefore, compound 1 was injected (ip) daily into heart failure mice 4–8 weeks after myocardial infarction (Figure 29). This time point was chosen because previous studies have shown that immune cells undergo pathological phenotypic switching approximately 4 weeks after MI (Bansal et al. Circ Heart Fail. 2017;10:e003688). Cardiac function will be measured using echocardiography before (week 4 after MI) and after (week 8 after MI), and monocytes / macrophages and T cells will be characterized using flow cytometry. Cardiac function will be assessed before (week 4 after MI) and after (week 8 after MI) treatment to evaluate changes in systemic and cardiac immune cell characteristics and assess left ventricular remodeling (hypertrophy, apoptosis, fibrosis, and capillary rarefaction) during chronic heart failure. Several parameters of left ventricular remodeling will also be assessed, including muscle hypertrophy (wheat germ agglutinin staining), fibrosis (Masson's trichrome staining), myocyte apoptosis (TUNEL staining), capillary rarefaction (isolectin staining), and ERβ signaling in isolated cardiac and splenic immune cells.

[0246] Because ERβ is also expressed on muscle cells, these studies were repeated in bone marrow (BM) chimeric mice. Wild-type mice were lethally irradiated and ERβ - / -We reconstituted the mice with BM to specifically deplete ERβ from immune cells, predicting that the cardioprotective effects of ERβ activation would not be observed in these mice, clearly demonstrating the essential role of ERβ receptor signaling in immune cell activation during chronic heart failure.

[0247] These studies, investigating the importance of ERβ on immune cells in the pathogenesis of left ventricular remodeling and chronic ischemic heart failure, thereby further our understanding of the cellular basis of inflammation in this disease. Furthermore, by providing direct evidence of the protective role of ERβ in inhibiting cardiac-specific immune cell activation, we can identify the therapeutic potential of ERβ agonists for immune modulation in heart failure.

[0248] Example 3 Methods. Animal studies were approved by The Ohio State University Institutional Animal Care and Use Committee and conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals (DHHS Publication No. 85-23, revised 1996). All mice had free access to food and water, and a total of 138 mice were used in all studies.

[0249] Mouse Model, Surgical Protocol, and Drug Treatment. As previously described, male, 10-12-week-old C57BL / 6 mice (Jackson Laboratories, stock number 000664) underwent left thoracotomy followed by permanent left coronary artery ligation to induce MI and ischemic heart failure (n = 80) or sham surgery (n = 28). Eight weeks after MI, peripheral blood was collected from the facial vein, and mice were euthanized by cervical dislocation. Hearts and spleens were harvested, weighed, and processed for mononuclear cell isolation or histological analysis. Tibia length was measured to normalize all gravimetric data.

[0250] A 20x stock solution of Compound 1 was prepared in DMSO and stored at -20°C. At the time of dosing, the drug was diluted using equal volumes of Tween 20 and saline to maintain a ratio of 5:5:90 (DMSO:Tween 20:saline). All mice were weighed daily and administered the drug by oral gavage at a dose of 60 mg / kg (200 μL per 25 g body weight).

[0251] Echocardiograms. Echocardiograms were performed under 1–1.5% isoflurane anesthesia using a VisualSonics Vevo 3100, and body temperature was maintained using an adjustable heating rail system (Vevo Imaging Station) and an RMV707B scan head as previously described.

[0252] Immune cell isolation and fixation. Immune cells were isolated from the spleen, blood, and heart as previously described. Briefly, spleens were triturated using a 3 mL syringe plunger in sterile PBS supplemented with 2% BSA and 2 mM EDTA to release all splenocytes, and then filtered through a 40 μm cell strainer to remove connective tissue. Peripheral blood (100 μL) was collected from the buccal vein in a BD microtainer tube containing EDTA, and RBCs were lysed using lysis buffer (eBioScience). The heart was minced and digested with collagenase II (1 mg / mL) to obtain a single-cell suspension, which was then filtered through a 40 μm cell strainer. Cells from the tissue were pelleted by centrifugation at 500 g, resuspended in 100 μL PBS supplemented with 2% BSA and 2 mM EDTA, fixed with 100 μL of 1% w / v PFA, and stored at 4 °C for flow cytometry staining.

[0253] T cell proliferation assay. Stemcell technologies' automated RoboSep™ cell separation system and MojoSort mouse CD4 T cells were used according to the manufacturer's instructions. + Splenic CD4+ T cells were magnetically purified using a T cell isolation kit (BioLegend). Purified viable cells were counted using a trypan blue exclusion assay and diluted to 1x10 in sterile PBS.6 The dye was reconstituted to a concentration of 1 x 10 cells / mL and incubated with an equal volume of 4 μM Tag-it Violet™ (BioLegend) for 10 minutes at 37°C in the dark. Excess dye was quenched by adding 5 times the volume of the dye solution used for staining in complete RPMI medium (supplemented with 10% charcoal-stripped FBS and 1% P / S / G for endogenous hormones) (Gibco) and incubating on ice for 5 minutes. Labeled cells were pelleted by centrifugation at 500g for 5 minutes and 1 x 10 cells were used for all assays. 6 The cells were resuspended in complete RPMI medium at a concentration of 1000 cells / mL.

[0254] Flat-bottom 96-well plates (FisherSci) were incubated with 50 μL of a 4.5 mg / mL solution of anti-hamster IgG (MilliporeSigma) at room temperature. After 1 hour, the wells were washed with sterile PBS to remove excess antibody and coated with 50 μL of a 2 μg / mL rat anti-mouse CD3 antibody (BioLegend) for 1 hour at room temperature. Unstimulated control wells were incubated with sterile PBS only. All wells were washed with sterile PBS to remove excess antibody, followed by the addition of 1 × 10 antibody to each well. 5 Tag-it Violet™ dye-labeled CD4 + T cells were plated. Costimulation was performed by adding 100 μL of complete RPMI medium containing 4 μg / mL of rat anti-mouse CD28 antibody (BioLegend), while 100 μL of complete RPMI medium (without anti-CD28 antibody) was added to unstimulated wells. Cells were incubated at 37°C and 5% CO for 72 hours, and dye dilution was measured with either a Becton Dickinson LSRFortessa or NL3000 Northern Lights (Cytek®) flow cytometer after each successive cell division.

[0255] Flow cytometry staining of cells. Detailed staining protocols have been previously described. Briefly, cell pellets were resuspended, dispensed into flow tubes, and incubated extracellularly with a cocktail of rat anti-mouse antibodies on ice for 45 minutes. Cells were washed with PBS supplemented with 2% BSA and 2 mM EDTA and fixed using 1% PFA. For intracellular staining, cells were permeabilized using 0.5% v / v Tween-20 and incubated with the antibody cocktail (on ice) for 45 minutes. Anti-mouse CD4 SB600 / PE-Cy7, Foxp3-APC, IFNγ eFluor450, Ly6C eFluor450, and ERβ antibodies were obtained from ThermoFisher Scientific. CD8 APC-Cy7, CD11b APC-Cy7, and CD19 PerCP-Cy5.5 antibodies were obtained from Tonbo Biosciences. IL-17 AF700, TNFα FITC, CD11b-APC, CD69 PerCP, Ly6G PE, and CD69 AF700 antibodies were from Biolegend, and ERα PE antibody was from Abcam.

[0256] Hypertrophy (WGA) staining. Formalin-fixed, paraffin-embedded hearts were sectioned (5 μm thick), deparaffinized, rehydrated, and stained as previously described. Masson's trichrome staining was used to quantify tissue fibrosis, and Alexa Fluor 488-conjugated wheat germ agglutinin (ThermoFisher Scientific) was used to label cell membranes. Myocyte area was quantified in remote regions of the failing heart from 3–4 high-power fields per section.

[0257] Statistical analysis. All data are presented as mean ± standard deviation. Two groups were compared using an unpaired Student's "T" test for equal or unequal variances, whereas one-way or two-way analysis of variance with Tukey's post-hoc test was used for comparisons of more than two groups. In some cases, one-way analysis of variance was used with correction for multiple comparisons by controlling the false positive rate. GraphPad Prism version 9.0 was used for all statistical analyses, and a P value <0.05 was considered significant.

[0258] CD4 in ischemic HF + In T cells, ERα signaling is upregulated.

[0259] Previous studies have shown that, in contrast to wound-healing characteristics during myocardial infarction (MI), CD4+ T cells shift to a pathological phenotype during chronic heart failure (HF), accentuating left ventricular (LV) remodeling and cardiac dysfunction. Therefore, to identify potential phenotypic switches, CD4+ T cells (150–300) were sorted from failing hearts and mediastinal lymph nodes of male mice 8 weeks post-MI and subjected to limited cellular RNA sequencing (Figure 30). IPA analysis of differentially expressed genes revealed that downstream of SIRT1, ESR1 (ERα) signaling, was upregulated in cardiac CD4+ T cells compared with mediastinal lymph nodes of HF mice. + We found that several genes downstream of ESR1 signaling were significantly upregulated in T cells (Figure 31). These T cells were selected from male heart failure patients, which was very interesting. To further validate these findings, we measured gene expression of ERα and ERβ in the distant LV and splenic mononuclear cells of HF mice. As shown in Figure 32, both ERα and ERβ were increased in the heart, while only ERα was increased in splenic immune cells.

[0260] Several studies on other autoimmune diseases, such as multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), have demonstrated the role of estrogen receptors (ERs) in regulating T cell activity. However, these studies have primarily been performed using ovariectomized female mice, with very limited research in male mice. Therefore, we measured the steady-state expression of ERα and ERβ in the ovaries (as a positive control), hearts of male and female mice, and spleens of naive male mice. As shown in Figure 33, ERα expression in the heart and spleen was comparable; whereas, ERβ expression was approximately 11-fold higher in the spleen compared to the heart. Next, splenic CD4 T cells from naive male mice were analyzed. + T cells were magnetically sorted and their ER expression was measured. Interestingly, ERβ expression in splenic CD4+ T cells was 10-fold lower than that of ERα (Figure 34), suggesting that ERα signaling is dominant in CD4+ T cells and that other splenic immune cells express significantly higher levels of ERβ than CD4+ T cells. This was consistent with other studies showing that ERα expression is highest in T cells isolated from human PBMCs. Further analysis of ERβ expression in various circulating and splenic immune cells indicated this was the case, whereas cells of myeloid origin (such as Ly6G+ neutrophils and Ly6C+ monocytes) expressed much higher levels of ERβ than CD4+ T cells, whereas CD19+ B lymphocytes had comparable expression (Figures 35 and 36).

[0261] CD4 in ischemic HF + In T cells, ERβ expression varies spatially, while ERα is temporally regulated.

[0262] Epidemiological (premenopausal women) and preclinical studies have demonstrated the protective effects of ER signaling in cardiovascular disease. However, given the pathological role of T cells identified in previous studies, it is unclear whether ER signaling is related to CD4 T cell activation in MI and HF. +The role, if any, they play in regulating T cell polarization / activation is unclear. 3 days post-MI showed a peak inflammatory response, and 8 weeks post-MI showed a peak CD4 + Significant LV remodeling with pathological migration of T cells was demonstrated, suggesting that CD4 + ERa and ERβ expression in T cells was measured at these time points. As shown in Figures 37 and 38, ERa expression was significantly decreased at 3 days post-MI but significantly increased at 8 weeks, consistent with the lc-RNA sequencing data indicating activation of the ERa pathway at this time point. On the other hand, ERβ expression was unchanged at any time point when compared to sham mice (Figure 39). Further analysis of the spatial variation of ERβ expression revealed that despite low ERβ expression in the spleen (Figures 40 and 41), CD4+ T cells infiltrating the failing heart were significantly more abundant than other bone marrow cells, such as Ly6G+ neutrophils or Ly6C+ neutrophils. + Monocytes / macrophages, or cells of lymphoid origin, e.g., CD19 + We found that ERβ expression was higher in cardiac CD4 T cells compared to B cells (Figure 42). Furthermore, at 3 days post-MI, cardiac CD4 T cells + ERβ expression in T cells was much higher compared to circulating or splenic T cells and significantly lower at 8 weeks post-MI (Figures 43 and 44). These changes were not due to differences in tissue background or nonspecific tissue binding between splenic or cardiac single-cell preparations, as B lymphocytes (similar in size and ERβ expression in naive mice (Figure 36)) showed no change in ERβ expression even at day 3 post-MI (Figure 45), the peak of the acute inflammatory response after MI. These data demonstrate: 1) ERα expression is temporally regulated and its high levels are consistent with the pathological migration of T cells; 2) cardiac CD4 T cells, when compared to other immune cells after ischemic injury, show no change in ERβ expression, even at day 3 post-MI (Figure 45). + 3) ERβ expression is spatially regulated and increases significantly and persistently in T cells. + Its expression in T cells, compared with other tissues, is enhanced upon infiltration into the injured heart at 3 days post-MI but decreases at 8 weeks post-MI. Finally, and importantly, 4) ERβ activation, which suppresses ERα signaling, is associated with CD4+ This suggests that this could be a potential target for slowing down the pathological transition of T cells.

[0263] ERβ agonists inhibit anti-CD3 / CD28-mediated T cell proliferation in a dose-dependent manner ERα signaling is antagonized by ERβ agonism. Therefore, we identified compound 1, an ERβ agonist with approximately 200-fold selectivity for ERβ over ERα, a lipophilic, orally bioavailable compound. This compound was administered to CD4 cells isolated from the spleens of naive male mice. + Compound 1 was tested for its ability to inhibit TCR (anti-CD3 / CD28)-mediated proliferation of T cells. As shown in Figure 46, Compound 1 had an IC of 3.4 μM. 50 Importantly, the drug (5 μM) inhibited TCR-mediated T cell proliferation in a dose-dependent manner. + Although it had no effect on T cells (Fig. 47), it did not affect TCR-activated CD4 +T cell proliferation was significantly inhibited even in the presence of low and high concentrations of estradiol (Figures 48 and 49). Compared to unstimulated T cells, there was a trend toward increased viable cell frequency upon TCR stimulation, likely due to viable cell proliferation (Figure 50). However, TCR-stimulated T cells treated with 5 μM of the drug (Figures 50 and 54) showed no change in viable cell frequency compared to unstimulated cells, suggesting that the decrease in T cell proliferation was not due to increased cell death. This concentration was also used to test the effect of ERβ agonism on the expression of proinflammatory cytokines such as TNFα and IFN-γ. Compared to unstimulated T cells, TCR stimulation significantly increased the expression of TNFα (Figures 51 and 52) and IFN-γ (Figure 53) in both the absence and presence of estradiol, which was significantly inhibited by the drug. In unstimulated cells treated with the drug in the presence or absence of estradiol, there was no change in the expression of TNFα or IFNγ (Figures 54 and 55), suggesting that the inhibition of cytokine expression is specific to T cell activation and does not affect the constitutive expression of these cytokines. Treatment with 5 μM Compound 1 significantly increased the expression of CD69-expressing CD4 + A significant decrease in the frequency of helper T cells was also observed (Figure 56), suggesting a similar blunting of T cell activation. Similar effects were observed in CD4+ T cells isolated from female mice (Figures 57-60), suggesting that although the drug is specific for ERβ, its effect is not sex-specific, with T cells from both male and female mice being inhibited equally. This also indicates that the role of estradiol and ER in T cell activation is ubiquitous and that TCR signaling through this pathway is not sex-dependent.

[0264] To determine whether the inhibitory effect of compound 1 is directed against a specific activation mechanism or is nonspecific in nature, the drug was also tested in the presence of PMA and ionomycin (Figures 61-64). These agents inhibited intracellular Ca 2+Compound 1 activates T cells by increasing TCR-mediated T cell proliferation and PKC activity, bypassing TCR activation. PMA / ionomycin treatment significantly increased the expression of proinflammatory cytokines, TNFα and IFNγ, and the activation marker CD69 in T cells (Figures 61-64). Interestingly, these proinflammatory and activation markers were not inhibited by Compound 1. These results suggest that Compound 1 selectively inhibits TCR-mediated T cell proliferation while sparing other activation mechanisms.

[0265] Compound 1 activates ERβ but inhibits ERα signaling To further investigate the specificity of the drugs, anti-CD3 / CD28 stimulated CD4 cells cultured in the presence and absence of 5 μM of the drugs were analyzed. + RNA was isolated from T cells. Principal component analysis (Figure 65) revealed that TCR-activated CD4 + We demonstrated that the RNA transcriptome of T cells was significantly altered in the presence of 5 μM Compound 1 compared to vehicle-treated stimulator cells. Several genes were identified that were down- or up-regulated (>2-fold and p<0.01) in the presence of the drug (Figure 66). Some genes involved in the ERβ pathway were up-regulated, while others were down-regulated (Figures 67 and 68), leading to global activation of this pathway with simultaneous inhibition of ERα signaling. Down-regulation of several genes involved in TCR activation was also observed, further supporting the TCR specificity of Compound 1. We further evaluated the effects of ERβ agonism on other pathways required for immune activation. Analysis of up- and down-regulated gene transcripts indicated that several pathways involved in inflammation, immune activation, and metabolism were inhibited or adversely affected by Compound 1 (Figures 69 and 70).

[0266] Compound 1 treatment, particularly during chronic HF, ameliorates cardiac remodeling Given that CD4+ T cells are important for wound healing during the first 10-14 days after MI but are pathological and not necessarily required during chronic HF, we tested the efficacy of drugs in both phases. For acute MI, echocardiography was used to measure immobile area 7 days after MI, and mice were randomized to receive either vehicle or 60 mg / kg Compound 1 (summarized in Figure 71). Daily administration of the drug by oral gavage did not affect the body weight of infarcted or sham-operated mice (Figure 72), suggesting that the drug has no obvious side effects on rodent physiology. However, a significant increase in mortality was observed with drug treatment, as several mice (approximately 60%) died during the first week of treatment compared with vehicle controls (15-20%) (Figure 73). This, consistent with other studies, supports the importance of CD4+ T cells for proper wound healing after MI. + It highlights the important and protective role of T cells.

[0267] For chronic HF, cardiac function was measured 4 weeks after MI. All animals were randomized according to the degree of cardiac dysfunction reflected by end-systolic and end-diastolic volumes (ESV and EDV) and ejection fraction (EF) (Figure 74). Vehicle or 60 mg / kg of drug was administered daily by oral gavage for 4 weeks. No drug-related mortality or changes in body weight were observed during this period (Figure 75). Importantly, echocardiographic data at 8 weeks after MI (4 weeks after treatment) showed that cardiac dysfunction in vehicle-treated HF mice progressed during this period, with increased end-systolic and end-diastolic volumes (ESV and EDV) and decreased ejection fraction (Figures 76 and 77). In contrast, cardiac function in mice treated with Compound 1 did not deteriorate, and ESV, EDV, and EF remained unchanged over the 4-week drug treatment period. Cardiac function in drug-treated mice was significantly better than that in vehicle-treated mice 8 weeks after MI. These differences in cardiac function were not due to heart rate, as mean heart rate was above 480 BPM at both time points and was similar between both groups (Figure 78).

[0268] Compound 1 treatment ameliorates cardiac hypertrophy Gravimetric analysis of heart and LV weights showed that vehicle-treated HF mice exhibited significant increases in tibia-normalized heart and LV weights, while 4-8 weeks of drug treatment demonstrated inhibition of cardiac hypertrophy, reflected by significant decreases in heart (Figure 79) and LV (Figure 80) weights compared to vehicle-treated HF mice. To further validate these results, WGA staining was performed and LV cardiomyocyte area was measured. As shown in Figures 81 and 82, drug treatment significantly reduced cardiomyocyte area and hypertrophy compared to vehicle-treated HF mice. Significant decreases in gene expression of several hypertrophy markers (e.g., Gja1, Gja5, MyH7, and MyH6) were also observed, while some others (e.g., BNP and RyR2) showed a tendency for decrease (Figures 83 and 84). This also suggests that drug-treated CD4 + This was also supported by RNA-seq data obtained from T cells, as retrospective analysis observed that several signaling pathways in T cells that mediate cardiac hypertrophy were downregulated with compound 1 treatment.

[0269] Compound 1 treatment specifically depletes CD4+ helper T cells in HF mice Using flow cytometry, CD4+ cells were detected in the circulation, failing heart, and spleen of vehicle- and drug-treated mice at 8 weeks post-MI (4 weeks post-treatment). + T cells were measured. As shown in Figure 85, daily treatment with Compound 1 increased circulating CD4 + T cells were significantly reduced at week 8. The reduction in T cell numbers was not restricted to a specific helper T cell subset, but included both pro- and anti-inflammatory cell numbers, i.e., CD4 + TNF-alpha + , CD4 + FoxP3 + Tregs, CD4 + IFNγ + Th1 T cells, CD4 + IL-4 + Th2 T cells and CD4 + IL-17 +There was a significant decrease in cardiac CD4 T cells in Compound 1-treated HF mice compared with vehicle-treated mice. + A significant decrease in T cells was also observed (Figures 86 and 87). This decrease was due to the TNFα + Cells and IFNγ + This was reflected in a significant decrease in both CD4 + This suggests that FoxP3 inhibits cytokine production in T cells, which is consistent with the results of in vitro T cell inhibition assays. + The CD4 + A similar decrease in T cell numbers (and frequency) was observed in splenic CD4 + This was also observed in T cells (Figures 88 and 89). Interestingly, the overall frequency of Foxp3+ Tregs was increased in CD4+ T cells (Figure 92), suggesting that other Th subsets were reduced more than FoxP3+ Tregs. Because Foxp3 expression has been shown to directly correlate with the immunosuppressive capacity of Tregs, its expression was also measured in vehicle- and drug-treated mice. As shown in Figure 93, drug treatment significantly increased FoxP3 expression (reflected as MFI) in Tregs, suggesting that the FoxP3+ Tregs remaining after drug treatment were significantly more potent and immunosuppressive. This was an intriguing finding, as it has previously been shown that Tregs undergo a proinflammatory (and pathological) phenotypic switching and lose their immunosuppressive capacity during chronic HF. Therefore, the increase in FoxP3 expression by Compound 1 treatment indicates an improved ability of Tregs to suppress pathological immune activation during chronic HF.

[0270] ERβ expression is stabilized / increased in splenic T cells by Compound 1 treatment (FIG. 94).

[0271] To determine the effect of Compound 1 on other immune cells known to play important roles in LV remodeling during chronic heart failure, we measured circulating, spleen, and cardiac levels of other immune cells of myeloid and lymphoid origin. As shown in Figures 95-97, Compound 1 inhibited Ly6G in the heart (Figure 95), blood (Figure 96), or spleen (Figure 97) of HF mice. + Neutrophils or Ly6C + Myeloid cells such as monocytes / macrophages (CD11b + ), or CD19 + B cells or CD8 + This did not alter the frequency of other lymphocyte populations, such as cytotoxic T cells, but rather CD4 + This suggests a highly specific effect of Compound 1 on helper T cells.

[0272] The effect of Compound 1 on thymocytosis and T cell development was also examined (Figures 98-100).

[0273] Consideration This study demonstrated several findings. First, ERα signaling was associated with CD4+ during chronic HF. + Second, ERα expression is temporally regulated, decreasing significantly during MI and increasing during chronic HF, whereas ERβ expression is spatially regulated and specifically increased in T cells infiltrating the ischemic and failing heart compared with other cardiac immune cells. Third, ERβ agonists dose-dependently upregulated CD4 + They inhibit T cell activation, proliferation, and proinflammatory cytokine expression to a similar extent in both men and women. More importantly, this inhibition is specific to the antigenic TCR activation pathway. Fourth, ERβ agonists increase morbidity and mortality when administered early after MI, but improve cardiac function and reduce cellular hypertrophy when administered during chronic HF. This supports the role of CD4 receptors in mediating wound healing and scar formation after MI. +This highlights the important and protective, but pathological, role of T cells in chronic HF, and points to important immunological differences between the two, at least from the perspective of adaptive immunity. Fifth, ERβ agonists inhibit CD4 T cells in the failing heart, lymphoid tissue, and circulation without affecting other immune cells. + This allows ERβ agonists to selectively blunt T cell levels, potentially reducing pathological CD4 T cell responses without affecting other protective immune responses. + These findings suggest that it could be developed as a highly specific therapy for the transient modulation of T cells.

[0274] Canonical ER signaling is mediated by the binding of ligand-bound ERα to the EREs of target genes or indirectly by the binding of (un)liganded ERα to other transcription factors such as AP-1 and NF-kB, regulating T cell activation, polarization, and proliferation. ERα signaling is essential for: i) CD4+ T cell activation in response to foreign antigens, ii) polarization into IFNγ-producing Th1 T cells, and iii) trafficking and migration of activated CD4+ T cells to tissues by regulating the expression of chemokine receptors such as CCR1, CCR2, CCR3, CCR4, and CCR5. In a limited cohort of human patients, systemic estradiol levels are elevated during MI (the first 3 days of hospitalization), which positively correlates with serum creatine phosphokinase levels. Extremely high estradiol levels have been shown to be associated with increased mortality in MI patients. Similarly, the Th1 / Th2 T cell ratio is also increased in STEMI patients, which has been associated with increased adverse events. Taken together, these studies suggest a pathological role for excess estradiol-mediated ERα signaling and Th1 T cell polarization. However, preclinical studies have also shown that CD4+ T cell activation during MI is protective and necessary for proper healing and scar formation. The findings herein are consistent with these seemingly contrasting studies, demonstrating that CD4 T cell activation at day 3 post-MI reduces non-antigenic T cell activation and initiates modulation of Th1 polarization. +These findings highlight the importance of reduced ERα signaling in T cells. Nevertheless, these protective responses are significantly impaired during HF. As previously shown, ERα levels and their downstream signaling are significantly upregulated, resulting in antigen-dependent activation and CD4 T cell proliferation at 8 weeks post-MI. + The data herein also demonstrate that CD4 T cells infiltrating the ischemic and failing hearts during both acute MI and HF are involved in the pathological migration of T cells. + We also show that T cells have higher ERβ expression than other immune cells of myeloid and lymphoid origin. This is intriguing, considering that HIF1α activation is known to increase ERβ signaling, which is known to attenuate HIF1α transcriptional activity. ERβ is also known to enhance integrin α1 and β1 expression in tumor cells, enhancing vinculin-mediated adhesion, cell motility, and transmigration. Therefore, selectively increased ERβ levels in cardiac T cells may be involved in either i) regulating T cell activation by antagonizing ERα, ii) amplifying integrin-mediated T cell transmigration into the heart or attenuating their egress, or iii) regulating HIF1α activation in T cells to control ischemic injury.

[0275] Preclinical studies in pressure overload and ischemic HF models have shown that T cell activation during HF is antigen-dependent and mediated by TCR activation. Therefore, immunomodulatory strategies targeting CD4+ T cells are crucial to specifically reduce TCR-mediated T cell activation and avoid general immunosuppression and infection by opportunistic pathogens. Therefore, the ER pathway has significant therapeutic potential, as depletion of the ERα gene from CD4+ T cells results in defective TCR-mediated T cell activation by reducing NFAT1, Zap70, and STAT5 levels. This TCR signaling defect could be a direct result of lost ERα signaling or could be due to amplified ERβ signaling in the absence of ERα. Studies herein demonstrate that ERβ agonists can inhibit TCR-activated CD4+ The latter was demonstrated by the dose-dependent inhibition of T cell proliferation in a gender-independent manner. ERα also regulates the gene expression of several T cell-specific cytokines (e.g., IFNγ, TNFα, IL-4, and IL-17), chemokine receptors (e.g., CCR1, CCR2, CCR3, CCR4, and CCR5), and other transcription factors (e.g., NF-kB and NFAT), all of which are involved in autoimmune diseases, including HF. Although no significant changes in chemokine expression were observed in RNA sequencing data, compound 1 effectively suppressed the expression of inflammatory cytokines induced by TCR activation, but not when PMA / ionomycin was used as a stimulus. These findings suggest that ERα is not merely an auxiliary pathway for regulating the transcription and expression of these proinflammatory cytokines / activation markers, but rather a direct downstream regulator of TCR-specific signaling.

[0276] ER signaling is a key pathway that promotes CD4+ T cell activation in autoimmune diseases such as MS, EAE, and asthma. High concentrations of estradiol and ERα-mediated Th1 polarization exacerbate MS; whereas Th2 T cells promote EAE, suggesting that excessive polarization of T cells into pro- or anti-inflammatory subsets may disrupt the steady state and become pathological. ERα - / - Hearts from mice have also been shown to be prone to ventricular fibrillation and tachycardia, and to exhibit increased cell death and reduced contractility. Alternatively, administration of an ERα but not ERβ agonist prior to I / R reduces infarct size in female rabbits. This is due to the increased systemic ERβ activity. - / -These findings, combined with the fact that female mice with ERα exhibit increased mortality after MI, suggest that ERα activation during ischemic injury is cardioprotective and that loss of ERβ interferes with cardiac healing. Although cellular responders of protective ERα and ERβ signaling were not identified in these studies, other studies have shown that expression of ERα or ERβ in the heart is not required for estradiol-mediated cardioprotection. Similarly, chronic heart failure is reflected as a global increase in both pro- and anti-inflammatory helper T cell subsets in the circulation, spleen, mediastinal lymph nodes, and failing heart, as well as CD4 + It has previously been shown to be associated with a second wave of T cell activation. Furthermore, depletion of T cells, particularly 4–8 weeks after MI, blunts the progressive increase in ESV and EDV and improves cardiac function, suggesting that these T cells exhibit a pathological phenotype, promoting LV remodeling and progressive cardiac dysfunction. In contrast, other studies have shown that CD4 - / - Mice have been shown to exhibit enhanced atrioventricular dilation and LV remodeling after atrial fibrillation, suggesting a protective role in mediating wound healing, angiogenesis, and fibrous scar formation. These conflicting findings may be due to the CD4 receptor being activated immediately after ischemic injury in MI. + These findings suggest that T cells are immunologically and phenotypically distinct from those activated during chronic HF. However, the signaling mechanisms mediating the transition from protective during MI to pathological during HF remain unclear. These studies observed that compound 1-mediated ERβ activation and CD4+ T cell depletion at 7 days post-MI significantly increased mortality, whereas administration 4–8 weeks post-MI blunted LV remodeling and improved cardiac function. These studies suggest that ER signaling may be one of the key pathological phenotype switches in CD4+ T cells, and that selective ERβ agonists can inhibit ERα and enhance CD4+ T cell depletion during HF. +These results suggest that the balance between ERα and ERβ signaling in immune cells is important during ischemic injury, further highlighting the critical role of early immune activation after MI and the importance of identifying time-dependent changes in immune cells to determine optimal therapeutic windows for transient immune modulation.

[0277] ER expression is not limited to CD4+ T cells; other immune cells, such as macrophages, neutrophils, dendritic cells, B cells, and CD8+ T cells, also express significant levels of these transcription factors. In monocytes and macrophages, estradiol exerts receptor-dependent effects. Estradiol promotes phagocytosis and degradation via ERβ, while inducing iNOS and reducing Arg1 and IL-10 expression via ERα. In neutrophils, estradiol delays cell apoptosis and promotes nephrosis. In B cells, estradiol-mediated ERα signaling amplifies activation and inhibits apoptosis. Despite this widespread expression, compound 1 selectively inhibited CD4+ T cells without affecting any other immune cells in the circulation, spleen, or failing heart. This specificity may be due to several factors. First, it may be due to differences between steady-state and injury-mediated activation of ERβ signaling. At steady state, ERβ levels in CD4+ T cells are significantly lower than those in myeloid cells. However, during ischemic injury, significant amplification of ERβ expression was observed selectively in cardiac CD4+ T cells, even higher than in myeloid cells, suggesting an important role for this pathway in mediating T cell-dependent protective responses. Second, differences in disease pathogenesis may activate different pathways in different immune cells. Myeloid cells activate ER signaling during trauma / hemorrhage; on the other hand, CD4+ T cells likely activate this pathway specifically during autoimmune (ischemic / non-ischemic) injury. The specificity of ERβ agonists for TCR-mediated T cell activation alone further supports this. Third, ER signaling alters both CD4+ T cell activation / function and proliferation; whereas, ER signaling only regulates functional capacities, such as phagocytosis in monocytes / macrophages and nephrosis in neutrophils, without altering the cell numbers of other immune cells.

[0278] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs. Publications and the materials for which they are cited herein are specifically incorporated by reference.

[0279] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. A pharmaceutical composition for treating or preventing chronic heart failure in a subject after myocardial infarction by administering the pharmaceutical composition to the subject in a maladaptive remodeling stage after myocardial infarction, comprising: The pharmaceutical composition comprises: A pharmaceutical composition comprising a carborane and a pharmaceutically acceptable excipient, wherein the carborane comprises an ERβ agonist, the carborane comprising: 【Chemistry 1】 [In the formula, ● is a carbon atom; ○ is B—H, B-halogen, B-alkyl, B—OH, or B—NH 2 ] and pharmaceutically acceptable salts thereof.

2. 10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is not administered to the subject during the healing or repair phase that precedes the maladaptive remodeling phase.

3. 10. The pharmaceutical composition of claim 1, wherein administration of the pharmaceutical composition begins at least 10 days after the myocardial infarction.

4. The method of claim 1 , wherein the subject is evaluated to determine whether the subject has entered the maladaptive remodeling phase.

5. assessing the subject to determine whether the subject has entered the maladaptive remodeling stage includes: measuring the level of circulating CD4+ T cells in the subject to determine when the subject has entered the maladaptive remodeling phase; detecting one or more biomarkers in the subject to determine when the subject has entered the maladaptive remodeling phase; or Echocardiography, ventriculography, magnetic resonance imaging, or any combination thereof 5. The pharmaceutical composition of claim 4, comprising:

6. The pharmaceutical composition inhibiting the activation and proliferation of CD4+ T cells in said subject; reducing circulating CD4+ T cell levels in said subject; reduces circulating CD4+ T cell levels in said subject without significantly affecting circulating levels of neutrophils, monocytes, or B cells; reducing left ventricular (LV) remodeling in said subject; inhibiting an increase in left ventricular end-diastolic volume in said subject; inhibiting an increase in left ventricular end-systolic volume in said subject; or a combination thereof. The pharmaceutical composition according to any one of claims 1 to 4, wherein the composition is administered in an amount effective for the treatment of atopic dermatitis.

7. The carborane is 【Chemistry 2】 [In the formula, ● is a carbon atom; ○ is B—H, B-halogen, B-alkyl, B—OH, or B—NH 2 ] The pharmaceutical composition according to any one of claims 1 to 4, comprising:

8. The pharmaceutical composition of any one of claims 1 to 4, wherein the carborane comprises a selective ERβ agonist.

9. The carborane has an EC ５０ The pharmaceutical composition according to any one of claims 1 to 4, comprising:

10. 5. The pharmaceutical composition of claim 1, wherein the carborane has an ERβ to ERα agonist ratio of 8 or greater.

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