SPAK/OSR INHIBITORS AND METHODS OF USING SAME

BACKGROUND

Many of the protein signaling networks that are critical for the emergence and progression of cancers are difficult to target due to incomplete biochemical characterization of their regulatory mechanisms. Among the numerous knowledge gaps is the role of post-translational modification, and barriers to their biochemical synthesis is a major road-block. Recently, synthetic and chemical biology based tools have provided exciting new solutions for post-translational modifications that were seemingly biochemically intractable. Several signaling pathways have been directly implicated in mechanisms of cell migration, of which, cell volume control has been recognized as an essential component.

The family of WNK (With-No-Lysine (K)) kinases and SPAK/OSR1 (STE20/SPS1-related proline/alanine-rich kinase/oxidative stress response) kinases play a key role in the maintenance of cell volume by controlling the phosphorylation of ion co-transporters, particularly NKCC1 (Na⁺—K⁺—Cl⁻ co-transporter 1). Glioblastoma multiforme (GBM), one of the most invasive and aggressive human cancers, manipulates cellular volume through alterations in the activity of ion co-transporters to facilitate migration through the extracellular matrix. In GBM, higher SPAK expression levels and/or higher OSR expression levels lead to decreased patient survival. Together, these observations suggest that inhibition of kinases in the WNK-SPAK regulatory network and subsequent reduction in ion co-transporter activity may provide an effective strategy to prevent infiltration of GBM cells. Given that the effective treatment of GBM will require new compounds and therapeutic innovations, bringing new synthetic biology-based strategies to target the WNK-SPAK kinase network is particularly compelling.

Small molecule inhibitors are essential tool compounds for drug target validation, mechanistic exploration, and inevitably serve as potential leads in discovery of clinical inhibitors. The barrier to identification of tool compounds is the capacity to conduct screens, as screens require active kinase. While bacterial over-expression systems are a gold standard for protein production, there are barriers to expression of active, post-translationally modified human kinases. Heterologous systems typically lack the signaling proteins or physiological context required to activate kinases. Often the precise mechanisms of kinase activation, mostly via phosphorylation, are poorly understood and further limit heterologous production. Acidic amino acid substitutions of aspartate or glutamate for phosphoserine, for example, often fail to recapitulate biological activity. One can enable the heterologous expression of authentically phosphorylated proteins by using a genomically recoded strain of E. coli (Isaacs, et al., 2011, Science 333:348-353; Lajoie, et al., 2013, Science 342:357-360) paired with a phosphoserine orthogonal translation system (pSerOTS) (Park, et al., 2011, Science 333:1151-1154; Pirman et al., 2015, Nature communications 6:8130). The pSerOTS uses a phosphoseryl-tRNA synthetase (pSerRS) to aminoacylate pSer onto a UAG-decoding tRNA^pSerand an engineered elongation factor Tu (EF-pSer) to deliver pSer84 tRNA^pSerto the ribosome, thus permitting recombinant expression of proteins with site-specific authentic phosphorylation (FIG. 1A).

Although the role of WNK1 in activating SPAK to modulate ion-co-transporters has been established, the upstream activators of WNK1 remain unknown. In addition, an inability to access and produce high yields of physiologically phosphorylated WNK and SPAK/OSR1 has hindered direct investigation of the underlying mechanisms.

There still remains a need in the art for small molecule inhibitors of SPAK activity. In certain embodiments, such inhibitors can be used for treating, ameliorating, and/or preventing cancer cell migration and/or metastasis. The present disclosure satisfies this need in the art.

BRIEF SUMMARY OF THE DISCLOSURE

The present disclosure provides a compound of formula (I), or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof:

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wherein R¹, R², R^b1, R^b2, R^b3, R^b4, and R^b5are described elsewhere herein. The present disclosure further provides a method of preparing a compound of the disclosure. The present disclosure further provides a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient and a compound of the disclosure.

The present disclosure provides a method of inhibiting SPAK and/or OSR activity in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of the disclosure. The present disclosure further provides a method of treating, ameliorating, and/or preventing cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of the disclosure. The present disclosure further provides a method of treating, ameliorating, and/or preventing cancer cell migration and/or invasion in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of the disclosure. The present disclosure further provides a method of reducing or reversing growth and/or viability of a cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of the disclosure. The present disclosure further provides a method of reducing volume of a cancerous tumor in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of the disclosure. The present disclosure further provides a method of extending survival in a subject afflicted with a cancer, the method comprising administering to the subject a therapeutically effective amount of a compound of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of exemplary embodiments of the disclosure will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosure, non-limiting embodiments are shown in the drawings. It should be understood, however, that the disclosure is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

FIGS. 1A-1F illustrate the finding that co-expressed phospho-activated WNK1/SPAK yield highly active kinase preparations. FIG. 1A: Phosphoserine technology. (Top) SepRS charges phosphoserine onto tRNA^pSer, which directs phosphoserine incorporation at UAG stop codons. Human phosphoprotein can be produced in E. coli by placing TAG at any position in the recombinant DNA. (Bottom) Depiction of genetically encoded phosphoserine in WNK1 yielding physiologically phospho-activated SPAK and subsequent phosphorylation of NKCC1 on its physiologically relevant sites. FIG. 1B: Schematic representation of WNK1 and SPAK displaying the domains used in this study. The T643 loop phosphorylation sites for each protein are highlighted in red. FIG. 1C: WNK1 variants containing (1-661) or lacking (1-483) the autoinhibitory domain (AID) were expressed in EcAR7 containing the Sep-OTS. The key active site residues S382 (1S^P), and S378/S382 (2S^P) of WNK1 were mutated to TAG for Sep incorporation. WT SPAK was expressed alone, or co-expressed with the S382S^PWNK1 variant (denoted in red). Samples were purified using GST-affinity chromatography and eluted with GST649 prescission protease. Phosphoserine incorporation at residue S382 in WNK1 was confirmed by immunoblotting with a phosphospecific antibody. FIG. 1D: Identified SPAK kinase motif sequence logo representing the amino acid preferences at different positions relative to the phosphorylation site. FIG. 1E: Sequence alignment of ion co-transporters regulated by phosphorylation at the highlighted red T. Residues highlighted in grey are conserved residues throughout the co-transporters that were shown to have high amino acid preference at that given position from the phosphorylation site in the motif analysis. FIG. 1F: Analysis of WNK1 and SPAK kinase activity. WNK1 variants and SPAK either singly (black) or co-expressed (red) were reacted in vitro with NKCC1 substrate. Kinase activity was monitored by immunoblotting with phosphospecific antibody recognizing phosphorylated NKCC1, TPNKCC1. Immunoblotting with Streptacin was used to verify equal loading of the strep-tagged NKCC1 substrate. Immunoblotting with Anti-SPAK, and WNK1 SP382 was used to show enzyme loading in each reaction. This data revealed that only co-expressed Sep-activated WNK1/WT SPAK samples were able to phosphorylate the NKCC1 substrate.

FIGS. 2A-2F illustrate the finding that small molecule screen against phospho-active SPAK yielded new SPAK kinase inhibitors. FIG. 2A: 320 compounds from the GSK published inhibitor set (PKISI) were evaluated in a preliminary in vitro ELISA screen, where reduction in NKCC1 phosphorylation was monitored by the phosphospecific antibody, T^PNKCC1. At least one lead compound was extracted from the screen with a reduction in NKCC1 phosphorylation at inhibitor concentrations of 27 μM compared to vehicle controls. FIG. 2B: Structures of a selected identified compound from the ELISA inhibitor screen. FIG. 2C: Dose dependent inhibition with Inh.A/Inh.B (7a) was further evaluated using both NKCC1 and KCC as substrates and immunoblotting with T^PNKCC1, and T^PKCC; respectively. FIG. 2D: mDCT15 cells were incubated with a lead inhibitor compound, and a reduction in NKCC1 phosphorylation from the cell lysates were monitored using the T^PNKCC1 antibody. Data shown is representative of duplicate experiments. FIG. 2E: mDCT15 cells were incubated with a serial dilution of (7a), and the reduction of NKCC1 phosphorylation as a function of inhibitor concentration from the cell lysates was monitored using the T^PNKCC1 antibody. Each experiment was performed in quadruplicate. The densitometry values were calculated using Bio-Rad Image Lab software and are plotted as mean±s.d. Replicate 4 was compromised and thus not included in the reported densitometry values. FIG. 2F: Co-expressed phospho-activated WNK4/SPAK and WNK1/OSR1 yield highly active kinase preparations and are inhibited by (7a). In vitro assessment of kinase activities and (7a) inhibition of the WNK1 and SPAK homologs (WNK4 and OSR1) evaluated via western blot.

FIGS. 3A-3G illustrate the finding that (7a) retards glioblastoma (GB) cell speed and persistence. FIG. 3A: Analysis of NKCC1 expression and activity in cultured GBM primary cell lines. NKCC1 expression and activity was monitored by immunoblotting with antibodies recognizing total NKCC1 and phosphorylated T^PNKCC1. SPAK expression was assessed using a SPAK specific antibody and immunoblot using GAPDH specific antibody was used as a loading control. FIG. 3B: Inhibitory effect of (7a) on primary GBM cell line 499. The phosphorylation states of NKCC1 and KCC was assessed by immunoblotting with T^PNKCC1 and T^PKCC specific antibodies, respectively. FIG. 3C: Schematic representation of migration assay employing a tissue-mimetic nanopatterned substrate coated with extracellular matrix components promoting brain cancer cell migration. FIG. 3D: Duplicate time course experiments of GB 499 cells incubated with 20 μM (7a), 40 μM (7a), or equivalent volume of DMSO vehicle for 1 hr, 2 hr, 3 hr. NKCC1 phosphorylation was monitored using the T^PNKCC1 antibody and compared to total NKCC1 and actin loading control. FIG. 3E: Histograms and box plots of distribution of number of cells with respect to average migration speed for GB 499 cells comparing cells treated with 20 μM or 40 μM (7a) and the equivalent vehicle control. FIG. 3F: Comparison of average migration speed over 10 hours of GB 499 cells treated with 20 μM or 40 μM (7a) compared to the equivalent DMSO vehicle control. FIG. 3G: Comparison of average migration speed over 10 hours of primary cell lines 499, 965, and GBM1A cells treated with 5 μM, 20 μM, or 40 μM (7a) compared to the equivalent DMSO vehicle control. Error bars represent standard error mean (SEM). Statistical significance was determined using a Wilcoxon rank-sum test (*p<0.05, **p<0.01, ***p<0.001).

FIGS. 4A-4D illustrate the finding that (7a) inhibits GBM proliferation in vitro and in vivo. FIG. 4A: Illustration of ion transporter mediated invasion of extracellular matrix. FIG. 4B: GBM1A cells were incubated with 20 μM (7a) or equivalent volume of DMSO vehicle for a 1 hr. After incubation the cells were imaged by confocal microscopy at 40× across the z-axis at increments of 0.28 microns. Cell volume was calculated using 3D reconstruction of the z-stack in ImageJ. FIG. 4C: Dose-dependent inhibition of GBM proliferation in multiple cell lines. FIG. 4D: Inhibition of GBM1A subcutaneous tumor growth in athymic nude mice (n=5 per group) in response to daily intraperitoneal injections of 0.1 mg/kg (7a).

FIGS. 5A-5B illustrate the finding that Sep-activated WNK1 variants stimulate the most SPAK activity and is further enhanced by the regulatory protein MO25α. FIG. 5A: NKCC1 phosphorylation with ³²P was monitored by autoradiography following in vitro kinase reactions with 1× and 10× WT, T233E SPAK±MO25α expressed alone or with WNK1 variants containing (1-661) or lacking (1-483) the auto inhibitory domain (AID). NKCC1 phosphorylation with ³²P was monitored by autoradiography following in vitro kinase reactions. FIG. 5B: Each sample was further analyzed by isolating the band corresponding to the NKCC1 substrate and quantifying ³²P incorporation by Cernekov counting. The reported concentration of ³²P incorporated is the average of duplicate experiments.

FIGS. 6A-6B illustrate the finding that Sep-activated WNK1 variants stimulate the most SPAK activity with or without the WNK1 AID. WNK1 variants (FIG. 6A) containing (1-661) or (FIG. 6B) lacking (1-483) the AID co-expressed with SPAK were reacted in vitro with MO25α, and NKCC1. In vitro kinase reactions were executed in intervals from 0-20 min. NKCC1 phosphorylation with ³²P was monitored by autoradiography.

FIGS. 7A-7B illustrate the finding that mass spectrometry analysis of phosphorylated SPAK. WNK1-mediated SPAK phosphorylation at (FIG. 7A) T233 and (FIG. 7B) S373 was validated by mass spec analysis.

FIGS. 8A-8B illustrate the finding that WNK1 activated SPAK is highly active and recognizes and phosphorylates its target ion cotransporters by a specific SPAK kinase motif. FIG. 8A: Illustration depicting assay conditions and observed result. FIG. 8B: SPAK kinase screen using the positional scanning peptide library (PSPL) bearing the denoted amino acid at defined positions relative to a central S/T phosphor-acceptor site.

FIG. 9 comprises a control western blot observing modulation of NKCC1 signal under hyper- and hypotonic conditions in mDCT15 cells.

FIGS. 10A-10B illustrate mass of Inh.A/Inh.B and (7a) examined by FTICR MS and MS/MS.

FIG. 11 illustrates IC₅₀for (7a) determined using ELISA based assay at 10 μM ATP.

FIGS. 12A-12F illustrate the finding that (7a) depletes phosphorylated NKCC1 and reduces cell volume in mDCT15 cells. FIG. 12A: Structures of (7a) and analog (7b). FIG. 12B: In vitro dose-dependent inhibition comparing (7a) and (7b) evaluated by western blot using T^PNKCC1. FIG. 12C: Western blot analysis of NKCC1 phospho-depletion in mDCT15 cells under various conditions. FIG. 12D: mDCT15 cells were incubated with 40 μM (7a), 40 μM Carbozantinib (XL-184), or equivalent volume of DMSO vehicle for 1 hr, 2 hr, 3 hr, and 4 hr. NKCC1 phosphorylation was monitored using the T^PNKCC1 antibody and compared to total NKCC1. FIG. 12E: HEK 293 induced to express KCC were incubated with 40 μM (7a), 40 μM Carbozantinib (XL-184), or equivalent volume of DMSO vehicle for 1 hr, and 2 hr time points. KCC phosphorylation was monitored using the T^PKCC antibody and compared to total KCC using C-Myc antibody. FIG. 12F: mDCT15 cells were incubated with 40 μM (7a), 40 μM Carbozantinib (XL-184), or equivalent volume of DMSO vehicle for a 1 hr, 2 hr, 3 hr, and 4 hr time course. After incubation the cells were imaged using 20× bright field microscopy. 1 hr and 4 hr time points are shown.

FIG. 13 comprises images of mDCT15 cells incubated with 40 μM (7a) 40 μM, Carbozantinib (XL-184), or equivalent volume of DMSO vehicle for a 1 hr, 2 hr, 3 hr, and 4 hr time course (partially shown in FIG. 12F). After incubation the cells were imaged using 20× and 80× bright field microscopy.

FIGS. 14A-14C illustrate the inhibitory effect of (7a) on primary GBM cell line GBM1A. FIG. 14A: The phosphorylation states of NKCC1 and KCC was assessed by immunoblotting with T^PNKCC1 and T^PKCC specific antibodies, respectively. FIG. 14B: Comparison of average migration speed over 10 hours of GBM1A cells treated with 20 μM or 40 μM (7a) compared to the equivalent DMSO vehicle control. FIG. 14C: Histograms and box plots of distribution of number of cells with respect to average migration speed for GBM cell line GBM1A comparing treatment with 20 μM or 40 μM (7a) and the equivalent vehicle control. Error bars represent standard error mean (SEM). Statistical significance was determined using a Wilcoxon rank-sum test (*p<0.05, **p<0.01, ***p<0.001).

FIGS. 15A-15C illustrate inhibitory effect of (7a) on primary GBM cell line 965. FIG. 15A: The phosphorylation states of NKCC1 and KCC was assessed by immunoblotting with T^PNKCC1 and T^PKCC specific antibodies, respectively. FIG. 15B: Comparison of average migration speed over 10 hours of GB 965 cells treated with 20 μM or 40 μM (7a) compared to the equivalent DMSO vehicle control. FIG. 15C: Histograms and box plots of distribution of number of cells with respect to average migration speed for GBM cell line 965 comparing treatment with 20 μM or 40 μM (7a) and the equivalent vehicle control. Error bars represent standard error mean (SEM). Statistical significance was determined using a Wilcoxon rank-sum test (*p<0.05, **p<0.01, ***p<0.001).

FIG. 16 comprises figures illustrating GBM primary cell lines incubated with 12. μM or equivalent volume of DMSO vehicle for a 1 hr. After incubation the cells were imaged using 20× bright field microscopy.

FIG. 17 comprises images illustrating H&E staining of GBM1A subcutaneous tumors treated with vehicle and (7a).

FIG. 18 illustrates primers for WNK1 constructs.

FIG. 19 illustrates a schematic view of phosphoprotein platform. SepRS charges phosphoserine onto tRNA^pSer, which directs phosphoserine incorporation at UAG stop codons. Human phosphoprotein can be produced in E. coli by placing TAG at any position in the recombinant DNA. The drawing on the left depicts genetically encoded phosphoserine in WNK1 yielding physiologically phospho-activated SPAK. and subsequent phosphorylation of NKCC1 on its physiologically relevant sites. The T-loop phosphorylation sites for each protein are highlighted in red. WNK1 variants containing (1-661) or lacking (1-483) the autoinhibitory domain (AID) were expressed with the Sep-OTS. The key active site residues S382 (1S^P), and S378/S382 (2S^P) of WNK1 were mutated to TAG for Sep incorporation.

FIG. 20 illustrates the fact that phosphoprotein platform makes active WNK/SPAK/OSR combinations.

FIG. 21 illustrates IC₅₀profiles for (7a) and (6a).

FIGS. 22A-22D illustrate in vitro validation of screen results. FIG. 22A: Western of (7a) and (6a) activity. FIG. 22B: Cell based assays validate (6a) and (7a) on target activity.

FIG. 22C: Primary human GBM cell based assays (GBM612) validate (6a) on target activity.

FIG. 22D: Human derived GBM cell based assays (GBM1A) validate (6a) on target activity.

FIGS. 23A-23B illustrate SPAK/NKCC/KCC pathway and inhibition in primary human GBM cells. FIG. 23A: Pathway expression analysis in primary human GBM cells.

FIG. 23B: Primary human GBM cell based assays (GBM612) validate (6a) on target activity.

FIG. 24 illustrates a non-limiting chemical synthesis route for (6a) and analogues thereof.

FIG. 25 comprises a section of a Western blot showing on-target activity in GBM612 cells.

FIG. 26 illustrates the finding that primary GBM experience reduced migration in response to (7a) & (6a). Note that (6a) is not active on primary human astrocytes.

FIG. 27 illustrates a migratory response of GBM to (6a), TMZ (temozolomide, and a combination thereof, demonstrating that (6a) targeting of WNK1/SPAK impedes migration whereas first-line TMZ does not.

FIG. 28 illustrates time- and dose-dependent effect of TMZ, or a combination of TMZ and (6a), on GBM cell line A172 proliferation. Cell number of each group at different time points is normalized to which at time zero. The (6a) concentration used in all groups is 1 μM. Group named A172 corresponds to regular A172 culture condition.

FIG. 29 illustrates cell number fold change after 72-hour incubation with TMZ or combination of TMZ and (6a). ns, * compared to vehicle control. # compared to TMZ_10 μM. † compared to TMZ_50 μM. § compared to TMZ_100 μM. ‡compared to TMZ_150 μM.

FIG. 30 illustrates that (6a) also impedes proliferation and viability of primary GBM cell lines.

FIGS. 31A-31B illustrate subcutaneous GBM tumor response to low-dose (6a).

FIGS. 32A-32B illustrate that (6a) reduces tumor growth in vivo in a human GMB intracranial xenograft mouse model. FIG. 32A: Schematic of the human GMB intracranial xenograft. FIG. 32B: Tumor growth profiles at two (6a) doses.

FIG. 33 illustrates the finding that (6a) extends overall survival in a GMB intracranial xenograft mouse model.

FIG. 34 illustrates the finding that (6a) reduces tumor volume in a GMB intracranial xenograft mouse model.

FIG. 35 illustrates the finding that (7a) reduces melanoma cell migration in vitro.

FIG. 36 illustrates an illustrative migratory response of pancreatic cancer cells to (6a), Gemcitabine, and a combination thereof, demonstrating that (6a) targeting of WNK1/SPAK impedes migration whereas first-line Gemcitabine does not.

FIG. 37 depicts annexin V assays performed on four GBM cell lines after 1 μM of (6a) or vehicle control.

FIGS. 38A-38B depict that (6a) induces G2-M arrest in GBM cells. FIG. 38A: Example cell cycle FACS data for 612 cells treated with 2 μM of (6a). FIG. 38B: Cell cycle analysis of GBM cell lines treated with 2 μM of (6a).

FIG. 39 depicts that the IC₅₀values for temozolomide (TMZ) decrease 2-4 fold when used in combination (6a) in two different GBM cell lines (GBM 612, GBM120).

FIGS. 40A-40C depict that SPAK/OSR1 inhibition sensitizes GBM to radiation and decreases expression of DNA damage response genes. FIG. 40A: GBM cells were pre-treated for 24 hs with 1 μM (6a) or vehicle control and irradiated. After 72 hs, cells were re-plated to study clonogenicity. 15 days after treatment clonogenic potential was the lowest in the combination treatment in vitro. FIG. 40B: Western blot shows decrease full length PARP-1 upon (6a) treatment after 24 hs. FIG. 40C: Expression of DNA damage response genes by RNAseq in cells treated with (6a) for 6 hs (dashed line delimits p<0.05). Graph bar shows mean and SEM *p=0.03 ***p<0.0002; ****p<0.0001.

FIG. 41 depicts treating healthy, tumor free, mice (ms_#) intracranially with a DMSO/PEG formulation containing vehicle or 100 μM (6a).

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure relates in part to the identification of novel SPAK inhibitors. In certain embodiments, the compounds of the disclosure can be used to inhibit, prevent, and/or minimize proliferation, viability, movement, migration, invasion, and/or cell volume change of certain cancer cells, such as but not limited to glioblastoma multiforme cancer cells and advanced stage cancer cells. In certain embodiments, the cancer comprises melanoma, pancreatic cancer, thyroid cancer, lung cancer, breast cancer, colorectal cancer, and other invasive and metastatic cancers. In certain embodiments, the advanced stage is at least grade III. In certain embodiments, the advanced stage is grade IV.

Cellular signaling and regulatory cascades often rely on post-translational modification (PTM) of proteins to efficiently modulate protein activity and transduce signals in response to environmental cues. Of all PTMs, protein phosphorylation is one of the most common and critical, governing the majority of signaling cascades in humans. Despite their important role in cellular processes, efforts to understand the function of phosphorylation sites are hindered by the apparent complexity of the phosphoproteome. Many kinases are themselves activated by phosphorylation in their kinase domain activation loops, limiting the generation of active, recombinant kinases to the relatively small pool of kinases for which the upstream activating kinase is known. These limitations are highlighted in a large-scale study that showed that the majority of human kinases expressed in bacteria are inactive. Given that these signaling networks form the basis for regulating most physiological processes, there exists a continued scientific interest in understanding these complex networks and connecting specific kinases to their downstream targets.

Ion cotransporters are involved in the invasion of healthy brain tissue by migratory GBM cells. This migratory activity is ultimately regulated through posttranslational phosphorylation, in which the family of WNK kinases have emerged as an important mediator. In addition to this observation, the identification of causal mutations in WNK kinases, which result in hereditary forms of hypertension and hyperkalemia, has guided the focus of many studies toward elucidation of the WNK signaling pathway and downstream substrates. As a result, WNK1 has been shown to regulate the STE20-related kinases SPAK/OSR1, which, in turn, phosphorylate NKCC1 to facilitate cell-volume regulation. Although these studies recognized that WNK-SPAK signaling pathway comprised potential therapeutic targets for controlling aberrant activation of ion co-transporters (as in the case of GBM and hypertension), direct methods for identifying small molecule inhibitors against the physiologically relevant forms of these kinases was technically infeasible. Despite decades of research on GBM, most treatments of this aggressive brain cancer are palliative in nature and the prognosis remains dismal. The poor prognosis is a result of a high proliferation capacity, and more importantly, a highly infiltrative behavior leading to the dissemination of tumor cells throughout the brain.

Malignant cells can modulate cell migration through the extracellular matrix by harnessing complex mechanisms controlling invasive cell spread. Upregulation of ion co-transporter activity has been shown to positively correlate with increased infiltration of migratory glioblastoma (GBM) cells. The activity of some ion co-transporters is directly regulated by the WNK/SPAK/OSR1 kinase network to maintain cellular volume homeostasis. In certain embodiments, inhibition of this signaling network can block invasive GBM migration by compromising cell volume regulation.

As shown herein, the pSerOTS system can be used to produce multiple forms of phosphorylated, active WNK1 kinase to recapitulate the WNK1-SPAK signaling cascade in recoded E. coli (without the need for the unknown, upstream activators), allowing for WNK1-dependent phospho-activation of SPAK. Biochemical characterization of the physiologically phosphorylated SPAK demonstrated that the system yields active kinase, and a physiologically active, completely heterologous WNK-SPAK-NKCC1 regulatory network. The overexpressed physiologically activated SPAK reacted with its regulatory accessory protein MO25α. This unique preparation of a druggable kinase pathway enabled a rapid in vitro drug inhibitor screen that yielded a small molecule SPAK kinase inhibitor. This compound showed robust network inhibition in vitro, in model cell lines, and in primary human GBM cell lines. It also arrested tumor growth in subcutaneous xenografts of human GBM cells in vivo. This work not only establishes the physiological basis for targeting aggressive GBM migration as a therapeutic strategy, but also paves a potential path forward toward constructing other phosphoprotein networks as scaffolds for drug discovery. In other words, the SPAK kinase motif provides insight into potential new SPAK substrates that have yet to be identified.

More broadly, the approach to use a heterologous expression system to generate an active, authentically modified human signaling network has the potential to unlock other important signaling pathways for substrate discovery and drug development. Increasing the understanding of kinase functions and their underlying network connectivity present new avenues for the development of therapeutics.

The skilled artisan will understand that the disclosure is not limited to the exemplary therapies discussed herein. Further, the skilled artisan will understand that one or more therapies can be administered alone or in any combination. Still further, the skilled artisan will understand that one or more therapies can be administered in combination with any other type of therapy.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, selected methods and materials are described.

As used herein, each of the following terms has the meaning associated with it in this section.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

“About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of 20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

The term “abnormal” when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, and so forth) from those organisms, tissues, cells or components thereof that display the “normal” (expected) respective characteristic. Characteristics that are normal or expected for one cell or tissue type might be abnormal for a different cell or tissue type.

A disease or disorder is “alleviated” if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.

As used herein, the term “specifically bind” or “specifically binds,” as used herein, is meant that a first molecule (e.g., a target protein or a phosphatase) preferentially binds to a second molecule (e.g., a target protein ligand or a phosphatase ligand, respectively), but does not necessarily bind only to that second molecule. In certain embodiments, the binding is reversible. In other embodiments, the binding is irreversible (or non-reversible).

As used herein, the term “composition” or “pharmaceutical composition” refers to a mixture of at least one compound useful within the disclosure with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient or subject. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary, and topical administration.

A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.

In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

As used herein, the terms “effective amount,” “pharmaceutically effective amount,” and “therapeutically effective amount” refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result may be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

As used herein, the term “efficacy” refers to the maximal effect (E_max) achieved within an assay.

As used herein, an “instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of a compound, composition, vector, or delivery system of the disclosure in the kit for effecting alleviation of the various diseases or disorders recited herein. Optionally, or alternately, the instructional material can describe one or more methods of alleviating the diseases or disorders in a cell or a tissue of a mammal. The instructional material of the kit of the disclosure can, for example, be affixed to a container which contains the identified compound, composition, vector, or delivery system of the disclosure or be shipped together with a container which contains the identified compound, composition, vector, or delivery system. Alternatively, the instructional material can be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.

As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

As used herein, the language “pharmaceutically acceptable salt” refers to a salt of the administered compounds prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic acids or bases, organic acids or bases, solvates, hydrates, or clathrates thereof.

Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric (including sulfate and hydrogen sulfate), and phosphoric acids (including hydrogen phosphate and dihydrogen phosphate). Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, malonic, saccharin, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2-hydroxyethanesulfonic, trifluoroacetic acid, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric, salicylic, galactaric and galacturonic acid.

Suitable pharmaceutically acceptable base addition salts of compounds of the disclosure include, for example, ammonium salts, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium, and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N′-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine. All of these salts may be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound.

As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the disclosure within or to the patient such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the disclosure, and not injurious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the disclosure, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. The “pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound useful within the disclosure. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the disclosure are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, Pa.), which is incorporated herein by reference.

The terms “patient,” “subject,” or “individual” are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In a non-limiting embodiment, the patient, subject, or individual is a human.

As used herein, the term “potency” refers to the dose needed to produce half the maximal response (ED₅₀).

A “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.

As used herein, the term “treatment” or “treating” is defined as the application or administration of a therapeutic agent, i.e., a compound of the disclosure (alone or in combination with another pharmaceutical agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnosis or ex vivo applications), who has a condition contemplated herein, a symptom of a condition contemplated herein or the potential to develop a condition contemplated herein, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect a condition contemplated herein, the symptoms of a condition contemplated herein or the potential to develop a condition contemplated herein. Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.

As used herein, the term “alkyl,” by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain hydrocarbon having the number of carbon atoms designated (i.e. C_1-6means one to six carbon atoms) and including straight, branched chain, or cyclic substituent groups. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, and cyclopropylmethyl. Most preferred is (C₁-C₆)alkyl, particularly ethyl, methyl, isopropyl, isobutyl, n-pentyl, n-hexyl and cyclopropylmethyl.

As used herein, the term “substituted alkyl” means alkyl as defined above, substituted by one, two or three substituents selected from the group consisting of halogen, —OH, alkoxy, —NH₂, —N(CH₃)₂, —C(═O)OH, trifluoromethyl, —C≡N, —C(═O)O(C₁-C₄)alkyl, —C(═O)NH₂, —SO₂NH₂, —C(═NH)NH₂, and —NO₂, preferably containing one or two substituents selected from halogen, —OH, alkoxy, —NH₂, trifluoromethyl, —N(CH₃)₂, and —C(═O)OH, more preferably selected from halogen, alkoxy and —OH. Examples of substituted alkyls include, but are not limited to, 2,2-difluoropropyl, 2-carboxycyclopentyl and 3-chloropropyl.

The term “alkylene” refers to a diradical of an alkyl group. Exemplary alkylene groups include —CH₂—, —CH₂CH₂—, and —CH₂C(H)(CH₃)CH₂—. The term “—(C₀alkylene)-” refers to a bond. Accordingly, the term “—(C_0-3alkylene)-” encompasses a bond (i.e., C₀) and a —(C_1-3alkylene) group.

As used herein, the term “haloalkyl” means alkyl as defined above, substituted by one, two or three substituents selected from the group consisting of F, Cl, Br, and I.

As used herein, the term “heteroalkyl” by itself or in combination with another term means, unless otherwise stated, a stable straight or branched chain alkyl group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized or substituted. The heteroatom(s) may be placed at any position of the heteroalkyl group, including between the rest of the heteroalkyl group and the fragment to which it is attached, as well as attached to the most distal carbon atom in the heteroalkyl group. Examples include: —OCH₂CH₂CH₃, —CH₂CH₂CH₂OH, —CH₂CH₂NHCH₃, —CH₂SCH₂CH₃, —NH—(CH₂)_m—OH (m=1-6), —N(CH₃)—(CH₂)_m—OH (m=1-6), —NH—(CH₂)_m—OCH₃(m=1-6), and —CH₂CH₂—S(═O)—CH₃. Up to two heteroatoms may be consecutive, such as, for example, —CH₂NH—OCH₃, or —CH₂CH₂—S—S—CH₃

As used herein, the term “alkoxy” employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms, as defined above, connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homologs and isomers. Preferred are (C₁-C₃) alkoxy, particularly ethoxy and methoxy.

As used herein, the term “cycloalkyl” refers to a mono cyclic or polycyclic non-aromatic radical, wherein each of the atoms forming the ring (i.e. skeletal atoms) is a carbon atom. In certain embodiments, the cycloalkyl group is saturated or partially unsaturated. In other embodiments, the cycloalkyl group is fused with an aromatic ring. Cycloalkyl groups include groups having from 3 to 10 ring atoms. Illustrative examples of cycloalkyl groups include, but are not limited to, the following moieties:

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Monocyclic cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Dicyclic cycloalkyls include, but are not limited to, tetrahydronaphthyl, indanyl, and tetrahydropentalene. Polycyclic cycloalkyls include adamantine and norbornane. The term cycloalkyl includes “unsaturated nonaromatic carbocyclyl” or “nonaromatic unsaturated carbocyclyl” groups, both of which refer to a nonaromatic carbocycle as defined herein, which contains at least one carbon carbon double bond or one carbon carbon triple bond.

As used herein, the term “aromatic” refers to a carbocycle or heterocycle with one or more polyunsaturated rings and having aromatic character, i.e. having (4n+2) delocalized π (pi) electrons, where n is an integer.

As used herein, the term “aryl,” employed alone or in combination with other terms, means, unless otherwise stated, a carbocyclic aromatic system containing one or more rings (typically one, two or three rings), wherein such rings may be attached together in a pendent manner, such as a biphenyl, or may be fused, such as naphthalene. Examples of aryl groups include phenyl, anthracyl, and naphthyl. Preferred examples are phenyl and naphthyl, most preferred is phenyl.

As used herein, the term “aryl-(C₁-C₃)alkyl” means a functional group wherein a one- to three-carbon alkylene chain is attached to an aryl group, e.g., —CH₂CH₂-phenyl. Preferred is aryl-CH₂— and aryl-CH(CH₃)—. The term “substituted aryl-(C₁-C₃)alkyl” means an aryl-(C₁-C₃)alkyl functional group in which the aryl group is substituted. Preferred is substituted aryl(CH₂)—. Similarly, the term “heteroaryl-(C₁-C₃)alkyl” means a functional group wherein a one to three carbon alkylene chain is attached to a heteroaryl group, e.g., —CH₂CH₂-pyridyl. Preferred is heteroaryl-(CH₂)—. The term “substituted heteroaryl-(C₁-C₃)alkyl” means a heteroaryl-(C₁-C₃)alkyl functional group in which the heteroaryl group is substituted. Preferred is substituted heteroaryl-(CH₂)—.

The term “carbocyclyl” refers to a saturated or unsaturated carbocyclic ring system containing one or more rings (typically one, two or three rings). In certain embodiments, the carbocyclyl is a 3-12 membered carbocyclic ring, a 3-8 membered carbocyclic ring, or a 3-6 membered carbocyclic ring.

As used herein, the term “halo” or “halogen” alone or as part of another substituent means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably, fluorine, chlorine, or bromine, more preferably, fluorine or chlorine.

The term “heteroalkylene” refers to an alkylene group in which one or more carbon atoms has been replaced by a heteroatom (e.g., N, O, or S). Exemplary heteroalkylene groups include —CH₂O—, —CH₂OCH₂—, and —CH₂CH₂O—. The heteroalkylene group may contain, for example, from 2-4, 2-6, or 2-8 atoms selected from the group consisting of carbon and a heteroatom (e.g., N, O, or S).

As used herein, the term “heterocycloalkyl” or “heterocyclyl” refers to a heteroalicyclic group containing one to four ring heteroatoms each selected from O, S and N. In certain embodiments, each heterocycloalkyl group has from 4 to 10 atoms in its ring system, with the proviso that the ring of said group does not contain two adjacent O or S atoms. In other embodiments, the heterocycloalkyl group is fused with an aromatic ring. In certain embodiments, the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen atom may be optionally quaternized. The heterocyclic system may be attached, unless otherwise stated, at any heteroatom or carbon atom that affords a stable structure. A heterocycle may be aromatic or non-aromatic in nature. In certain embodiments, the heterocycle is a heteroaryl.

An example of a 3-membered heterocycloalkyl group includes, and is not limited to, aziridine. Examples of 4-membered heterocycloalkyl groups include, and are not limited to, azetidine and a beta lactam. Examples of 5-membered heterocycloalkyl groups include, and are not limited to, pyrrolidine, oxazolidine and thiazolidinedione. Examples of 6-membered heterocycloalkyl groups include, and are not limited to, piperidine, morpholine and piperazine. Other non-limiting examples of heterocycloalkyl groups are:

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

As used herein, the term “heteroaryl” or “heteroaromatic” refers to a heterocycle having aromatic character. A polycyclic heteroaryl may include one or more rings that are partially saturated. Examples include the following moieties:

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Examples of heteroaryl groups also include pyridyl, pyrazinyl, pyrimidinyl (particularly 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl (particularly 2-pyrrolyl), imidazolyl, thiazolyl, oxazolyl, pyrazolyl (particularly 3- and 5-pyrazolyl), isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and 1,3,4-oxadiazolyl.

Examples of polycyclic heterocycles and heteroaryls include indolyl (particularly 3-, 4-, 5-, 6- and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl (particularly 1- and 5-isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (particularly 2- and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl, benzofuryl (particularly 3-, 4-, 5-, 6- and 7-benzofuryl), 2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (particularly 3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl (particularly 2-benzothiazolyl and 5-benzothiazolyl), purinyl, benzimidazolyl (particularly 2-benzimidazolyl), benzotriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, and quinolizidinyl.

The term “heteroarylene” refers to a multi-valent (e.g., di-valent or trivalent) aromatic group that comprises at least one ring heteroatom. An exemplary “heteroarylene” is pyridinylene, which is a multi-valent radical of pyridine. For example, a divalent radical of pyridine is illustrated by the formula

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In certain embodiments, the “heteroarylene” is a divalent, 5-6 membered heteroaromatic group containing 1, 2, or 3 ring heteroatoms (e.g., O, N, or S).

The term “phenylene” refers to a multivalent radical (e.g., a divalent or trivalent radical) of benzene. To illustrate, a divalent radical of benzene is illustrated by the formula

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As used herein, the term “substituted” means that an atom or group of atoms has replaced hydrogen as the substituent attached to another group. The term “substituted” further refers to any level of substitution, namely mono-, di-, tri-, tetra-, or penta-substitution, where such substitution is permitted. The substituents are independently selected, and substitution may be at any chemically accessible position. In certain embodiments, the substituents vary in number between one and four. In other embodiments, the substituents vary in number between one and three. In yet other embodiments, the substituents vary in number between one and two.

As used herein, the term “optionally substituted” means that the referenced group may be substituted or unsubstituted. In certain embodiments, the referenced group is optionally substituted with zero substituents, i.e., the referenced group is unsubstituted. In other embodiments, the referenced group is optionally substituted with one or more additional group(s) individually and independently selected from groups described herein.

In certain embodiments, the substituents are independently selected from the group consisting of oxo, halogen, —CN, —NH₂, —OH, —NH(CH₃), —N(CH₃)₂, alkyl (including straight chain, branched and/or unsaturated alkyl), substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, fluoro alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkoxy, fluoroalkoxy, —S-alkyl, S(═O)₂alkyl, —C(═O)NH[substituted or unsubstituted alkyl, or substituted or unsubstituted phenyl], —C(═O)N[H or alkyl]2, —OC(═O)N[substituted or unsubstituted alkyl]2, —NHC(═O)NH[substituted or unsubstituted alkyl, or substituted or unsubstituted phenyl], —NHC(═O)alkyl, —N[substituted or unsubstituted alkyl]C(═O)[substituted or unsubstituted alkyl], —NHC(═O)[substituted or unsubstituted alkyl], —C(OH)[substituted or unsubstituted alkyl]₂, and —C(NH₂)[substituted or unsubstituted alkyl]₂. In other embodiments, by way of example, an optional substituent is selected from oxo, fluorine, chlorine, bromine, iodine, —CN, —NH₂, —OH, —NH(CH₃), —N(CH₃)₂, —CH₃, —CH₂CH₃, —CH(CH₃)₂, —CF₃, —CH₂CF₃, —OCH₃, —OCH₂CH₃, —OCH(CH₃)₂, —OCF₃, —OCH₂CF₃, —S(═O)₂—CH₃, —C(═O)NH₂, —C(═O)—NHCH₃, —NHC(═O)NHCH₃, —C(═O)CH₃, and —C(═O)OH. In yet one embodiment, the substituents are independently selected from the group consisting of C_1-6alkyl, —OH, C_1-6alkoxy, halo, amino, acetamido, oxo and nitro. In yet other embodiments, the substituents are independently selected from the group consisting of C_1-6alkyl, C_1-6alkoxy, halo, acetamido, and nitro. As used herein, where a substituent is an alkyl or alkoxy group, the carbon chain may be branched, straight or cyclic, with straight being preferred.

In certain embodiments, an optional substituent is selected from the group consisting of C₁-C₆alkyl, C₃-C₈cycloalkyl, phenyl, C₁-C₆hydroxyalkyl, (C₁-C₆alkoxy)-C₁-C₆alkyl, C₁-C₆haloalkyl, C₁-C₆haloalkoxy, halogen, —CN, —OR^b, —N(R^b)(R^b), —NO₂, —C(═O)N(R^b)(R^b), —S(═O)₂N(R^b)(R^b), acyl, and C₁-C₆alkoxycarbonyl, wherein each occurrence of R^bis independently H, C₁-C₆alkyl, or C₃-C₈cycloalkyl, wherein in R^bthe alkyl or cycloalkyl is optionally substituted with at least one selected from the group consisting of halogen, —OH, C₁-C₆alkoxy, and heteroaryl; or substituents on two adjacent carbon atoms combine to form —O(CH₂)_1-3O—.

In certain embodiments, an optional substituent is selected from the group consisting of C₁-C₆alkyl, C₃-C₈cycloalkyl, phenyl, C₁-C₆hydroxyalkyl, (C₁-C₆alkoxy)-C₁-C₆alkyl, C₁-C₆haloalkyl, C₁-C₆haloalkoxy, halogen, —OR^b, and —C(═O)N(R^b)(R^b), wherein each occurrence of R^bis independently H, C₁-C₆alkyl, or C₃-C₈cycloalkyl, wherein in R^bthe alkyl or cycloalkyl is optionally substituted with at least one selected from the group consisting of halogen, —OH, C₁-C₆alkoxy, and heteroaryl; or substituents on two adjacent carbon atoms combine to form —O(CH₂)_1-3O—.

In certain embodiments, an optional substituent is selected from the group consisting of C₁-C₆alkyl, —OH, C₁-C₃haloalkyl, C₁-C₆alkoxy, C₃-C₈cycloalkyl, C₃-C₈cycloalkoxy, halo, and —CN.

Ranges: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Compounds and Compositions

The disclosure provides a compound of formula (I), or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof:

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wherein:

R¹is selected from the group consisting of H, F, Cl, Br, I, C₁-C₆alkyl, C₁-C₆alkoxy, CN, nitro, and CF₃;

R²is selected from the group consisting of:

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- wherein each occurrence of R^cis independently H, C₁-C₆alkyl, or C₃-C₈cycloalkyl, or two R^cbound to the same N atom combine with the N atom to form optionally substituted 3- to 8-membered heterocyclyl;

R^b1, R^b2, R^b3, R^b4, and R^b5are independently selected from the group consisting of H, F, Cl, Br, I, C₁-C₆alkyl, C₃-C₈cycloalkyl, phenyl, C₁-C₆hydroxyalkyl, C₁-C₆haloalkyl, C₁-C₆haloalkoxy, (C₁-C₆alkoxy)-C₀-C₆alkylene, —(CH₂)_0-3—NR^dR^d, —O(CH₂)_2-3—NR^dR^d, —OR^d, —C(═O)OR^d, and —C(═O)N(R^d)(R^d),

- wherein each occurrence of R^dis independently H, C₁-C₆alkyl, or C₃-C₈cycloalkyl, or two R^dbound to the same N atom combine with the N atom to form optionally substituted 3- to 8-membered heterocyclyl;

each occurrence of R^eis independently C₁-C₆alkyl; and

p is 1, 2, or 3.

In certain embodiments, R¹is H. In certain embodiments, R¹is F. In certain embodiments, R¹is Cl. In certain embodiments, R¹is Br. In certain embodiments, R¹is I. In certain embodiments, R¹is C₁-C₆alkyl (such as but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, iso-pentyl, neo-pentyl, n-hexyl, sec-hexyl, and so forth). In certain embodiments, R¹is C₁-C₆alkoxy (such as but not limited to methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentoxy, sec-pentoxy, iso-pentoxy, neo-pentoxy, n-hexoxy, sec-hexoxy, and so forth). In certain embodiments, R¹is CN. In certain embodiments, R¹is nitro. In certain embodiments, R¹is CF₃;