Method for treating SHIP1-mediated diseases using Pelorol derivatives

Abstract

The present disclosure provides compounds of Formula I and their pharmaceutically acceptable salts, solvates, and / or derivatives. In addition, the present disclosure also provides methods for treating diseases, disorders, or conditions mediated by SHIP1 or treatable by SHIP1 activation, said methods comprising administering a compound of Formula I or a pharmaceutically acceptable salt, solvate, or derivative thereof. The compound of Formula I or a pharmaceutically acceptable salt, solvate, or derivative thereof can be used for treating SHIP1-mediated diseases, disorders, or conditions, including inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, multiple myeloma, liver injury, acute hepatitis, and severe sepsis.

Description

Technical Field

[0001] The present disclosure relates to compounds and their use in the treatment of SHIP1-mediated diseases, disorders or conditions such as inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, multiple myeloma, liver injury, acute hepatitis and severe sepsis. Background Art

[0002] Both Src homology 2 domain-containing inositol 5'-phosphatase 1 (SHIP1) and SHIP2 are important phosphatases in mammalian cells, which control various different cell signaling pathways including the PI3K / Akt pathway and the production of interleukin-10 (IL-10). These cell signaling pathways are involved in a large number of diseases and conditions mediated by SHIP1.

[0003] Multiple myeloma (MM) is the second most common hematological malignancy (Naymagon 2016), which is a plasma cell neoplasm characterized by an increase in malignant B cells and related monoclonal immunoglobulins in the bone marrow (BM) (Kuehl 2002). Despite the current treatment options including high-dose chemotherapy and stem cell transplantation, the vast majority of patients experience disease recurrence and remain incurable due to the development of drug resistance (Naymagon 2016, Abramson 2018, Harding 2019).

[0004] Cell-cell and cytokine-mediated interactions between MM cells and the BM microenvironment support proliferation, survival, and drug resistance by activating many signaling cascades, including the Ras / Raf / Erk, Jak2 / STAT3, and PI3K / Akt pathways (reviewed in Harding 2019), and thus there are numerous potential targets for therapeutic intervention. Signaling through the PI3K / Akt cascade is important for the survival and expansion of neoplastic plasma cell clones and the development of drug resistance (Hu 2018, Zhu 2015, Hideshima 2001, Qiang 2002, Tu 2000, Hsu 2001, Mitsiades 2002). Activation of PI3K leads to the production of phosphatidylinositol-3,4,5-trisphosphate (PIP3) in the plasma membrane, resulting in the membrane recruitment and activation of Akt and other proteins containing a pleckstrin homology (PH) domain (Zhu 2015). In MM, the phosphorylation levels of the serine / threonine kinase Akt and its downstream effectors are associated with disease progression (Hsu 2001, Alkan 2002), and inhibitors of Akt and the downstream mammalian target of rapamycin (mTOR) have been shown to induce apoptosis in vitro and in vivo (Hideshima 2006, Frost 2004, Hideshima 2007). Therefore, inhibition of PI3K / Akt signaling is a promising approach for the treatment of MM.

[0005] Under normal conditions, the cellular PIP3 level is tightly controlled by regulating the activities of both PI3K, which generates PIP3, and inositol lipid phosphatases, which hydrolyze PIP3. There are two major phosphatases that degrade PIP3: the 3'-phosphatase PTEN, which generates PI-4,5-P2, and the 5'-phosphatases SHIP1 and SHIP2, which generate PI-3,4-P2 (Vivanco 2002). PTEN and SHIP2 are expressed in all cells, while SHIP1 is expressed only in hematopoietic cells. PTEN is a known tumor suppressor (Steck 1997, Li 1997), and PTEN-deficient MM cells have higher Akt phosphorylation and are more sensitive to killing induced by Akt inhibition (Ge 2000, Shi 2002, Zhang 2003). On the other hand, SHIP1 is an important regulator of PI3K signaling in B cells (Aman 1998, Liu 1999, Helgason 2000), and reduced activity or expression of SHIP1 has been observed in hematological malignancies (Luo 2004, Fukuda 2005, Vanderwinden 2006, Liang 2006). Agents that are currently being developed to reverse the elevation of PI3K / Akt signaling include kinase inhibitors that target PI3K, Akt, or mTOR (Naymagon 2016, Abramson 2018, Harding 2019, Hu 2003, Zhu 2014). Activation of SHIP1 provides a unique approach that can be used alone or as an adjunct to existing therapies (Li 2011, Meimetis 2012, Ong 2007). In vitro studies have shown that compounds of the Pelorol family selectively activate the activity of the enzyme by binding to an allosteric activation domain within the SHIP1 phosphatase (Ong 2007). These compounds inhibit PI3K / Akt signaling in MM (but not in non-hematopoietic cancer cells) in vitro, and this is associated with reduced proliferation and increased apoptosis of MM cells (Kennah 2009).

[0006] Inflammatory bowel disease (IBD) is another example where SHIP1 contributes to the disease pathogenesis. Many factors contribute to the development of IBD, but genome-wide association studies (Verstockt 2018) and clinical data (Engelhardt 2014, Glocker 2009, Glocker 2011, Louis 2009) have shown that the anti-inflammatory effect of the cytokine interleukin-10 (IL10) (Ouyang 2011) is crucial for maintaining proper immune homeostasis. In its absence, inflammatory stimulatory pathways remain unabated, leading to inappropriate inflammation. IL10-deficient mice develop colitis similar to human IBD (Kuhn 1993, Shouval 2014). In humans, polymorphisms in the IL10 gene are associated with ulcerative colitis, and loss-of-function mutations in the homozygous IL10 receptor subunit result in early-onset colitis (Engelhardt 2014, Glocker 2009, Glocker 2011).

[0007] Studies have shown that IL10 requires the activation of SHIP1 to induce inflammation (Chan 2012, Cheung 2013). SHIP1 is a cytoplasmic protein mainly expressed in hematopoietic cells (Fernandes 2013, Huber 1999, Krystal 2000). In response to extracellular signals, SHIP1 can be recruited to the cell membrane, and one of its functions is to shut down phosphatidylinositol 3-kinase (PI3K) signaling (Brown 2010) by dephosphorylating the PI3K product PIP3 to PI-3,4-P2 (Fernandes 2013, Huber 1999, Krystal 2000, Pauls 2017). SHIP1 can also act as a docking protein for the assembly of signaling complexes (Pauls 2017). It has been shown that SHIP1 is an allosteric regulatory enzyme, and its natural agonist is its product PI-3,4-P2 (Ong 2007). Compounds of the Pelorol family are able to bind to the allosteric domain of SHIP1 to activate SHIP1 (Ong 2007). In vitro results have shown that compounds of the Pelorol family exhibit anti-inflammatory effects in a manner similar to IL-10 (Chan 2012, Cheung 2013, Ong 2007).

[0008] Activating anti-inflammatory pathways such as IL-10 may be useful in other situations. An example of enhanced inflammatory response is sepsis. Sepsis is a complex systemic disease in which a dysregulated inflammatory response to bacterial or viral infection leads to multiple organ dysfunction syndrome (MODS). The worldwide incidence data is estimated to be 31 million cases per year. Severe sepsis accounts for 2% of hospitalized patients and 10% of all intensive care unit admissions. Severe sepsis attacks both the young and the old equally, with an estimated mortality rate of 38% to 45%. In the past few decades, more than 100 clinical trials of drugs for severe sepsis have failed, highlighting the complexity and difficulty of treating this disease, and the current therapies for this severe syndrome are mainly supportive (Marshall 2014). The current pandemic COVID-19 is a type of viral sepsis. The deaths of COVID-19 patients are mainly caused by acute respiratory distress syndrome (ARDS). ARDS is caused by a dysregulated immune response of the host to viral or bacterial infection, which is a hallmark of severe sepsis. The overresponse of the host causes a "cytokine storm" (Liu 2016), leading to systemic capillary leakage, severe pulmonary edema, ARDS and patient death.

[0009] Inflammation is also common in various liver diseases such as viral hepatitis, autoimmune hepatitis, primary biliary cirrhosis and liver allograft rejection, which are associated with the activation and infiltration of T cells and the production of pro-inflammatory cytokines in the liver, leading to liver damage (Louis 2003, Asdullah 2003, Czaja 2021).

[0010] Therefore, SHIP1 provides a viable target for developing therapies targeting inflammatory diseases and tumors, and there is an urgent need in the market to develop small molecule SHIP1 agonists for treating such diseases. Summary of the Invention

[0011] It has now been shown that the compounds of the present disclosure can activate SHIP1 both in vitro and in vivo and can be used to treat SHIP1-mediated conditions described below. In addition, it has been shown that the compounds of the present disclosure can reduce the level of tumor necrosis factor α (TNFα) in LPS-induced cells and reduce the level of pro-inflammatory cytokines in a mouse model of colitis. In addition, it has been shown that in animals bearing MM tumors, the compounds of the present disclosure reduce tumor mass in vivo. In addition, it has now been shown that in a mouse IL-10 knockout model of IBD, the compounds of the present disclosure inhibit inflammation in vivo. In addition, it has been shown that the compounds of the present disclosure protect against concanavalin A (ConA)-induced liver injury in mice. In addition, in a cecal ligation and puncture (CLP) model, treating septic mice with the compounds of the present application can improve survival rate.

[0012] Since activation of SHIP1 stimulates the anti-inflammatory IL-10 signaling pathway, activation of SHIP1 can be used to inhibit the production of inflammatory cytokines regulated by the IL10 pathway for treating different inflammatory diseases or conditions. Examples include the inflammatory cytokine storm observed in sepsis and the overproduction of cytokines in various different liver injuries.

[0013] Accordingly, the present disclosure includes a compound of Formula I

[0014]

[0015] or a pharmaceutically acceptable salt, solvate, prodrug, and / or derivative thereof,

[0016] wherein R 1 is selected from H, OH, C 1-3 alkyl, OC 1-3 alkyl, NH2, NHC 1-3 alkyl, NHSO2C 1-3 alkyl, N-succinimide, and NHC(O)C 1-3 alkyl;

[0017] wherein R 2 , R 3 , R 4 and R 5 are independently selected from H, OH, OC 1-3 alkyl, NH2, NHC 1-3 alkyl, NHSO2C 1-3 alkyl, and NHC(O)C 1-3 alkyl; or R 2 and R 3 , R 3 and R 4 or R 4 and R 5 together with the atoms to which they are attached form a substituted or unsubstituted 5- or 6-membered heterocycle containing at least one NH and optionally one or more additional heteroatoms selected from N, O, and S; and

[0018] wherein when R 2 and R 3 , R 3 and R 4 or R 4 and R 5 together form the substituted or unsubstituted 5- or 6-membered heterocycle, R 4 and R 5 , R 2 and R 5 or R 2 and R 3 are each independently selected from H and C1-3 Alkyl

[0019] On the other hand, the present disclosure includes a method for treating a disease, disorder or condition mediated by SHIP1 or treatable by activation of SHIP1, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt, solvate, prodrug and / or derivative thereof.

[0020] On the other hand, the present disclosure includes a compound of the present disclosure or a pharmaceutically acceptable salt, solvate, prodrug and / or derivative thereof for treating a disease, disorder or condition mediated by SHIP1 or treatable by activation of SHIP1.

[0021] On the other hand, the present disclosure includes the use of one or more compounds of the present disclosure or pharmaceutically acceptable salts, solvates, prodrugs and / or derivatives thereof for treating a disease, disorder or condition mediated by SHIP1 or treatable by activation of SHIP1.

[0022] On the other hand, the present disclosure includes the use of one or more compounds of the present disclosure or pharmaceutically acceptable salts, solvates, prodrugs and / or derivatives thereof in the manufacture of a medicament for treating a disease, disorder or condition mediated by SHIP1 or treatable by activation of SHIP1. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings, in which:

[0024] Figure 1 Serum TNFα levels of SHIP1 in mice intraperitoneally injected with LPS, LPS + IL10 (Figure A) or LPS + Compound I-1 (ZPR-100 or ZPR-MN100 or MN-100) (Figure B) at the indicated concentrations for 1 h are shown. Data represent the mean of n≥4. *p<0.05, **p<0.01, ns = not significant when compared to mice stimulated with LPS only. Figure C shows STAT3 + / + or SHIP1 - / - bone marrow-derived macrophages (BMDM) of or SHIP1 + / + , STAT - / - 、SHIP1 + / + and SHIP1 - / - stimulated with LPS (dashed line) or LPS + IL10 (solid line) over a period of 180 min. Fractions were collected every 5 min for measurement of TNFα levels. Data represent two independent experiments.

[0025] Figure 2shows the physical association of IL-10-induced SHIP1 and STAT3. In panel A, in the LPS-stimulated TNFα production assay, the ability of J17 SHIP1 expressing His6-SHIP1 or His6-SHIP1 3PT to be inhibited by IL10 was tested. In panel B, J17 His6-SHIP1 cells were stimulated with IL6, IL10, or compound I-2 for 5 minutes. His6-SHIP1 was pulled down using nickel beads and probed with SHIP1, STAT3, and phospho-STAT3 antibodies together with the cell lysate. (Panel C) Single-cell FRET analysis of J17 SHIP1 cells expressing the FRET pair fusion constructs Clover-SHIP1 and mRuby2-STAT3, which were stimulated with “mock” or with IL6, IL10, compound I-2 for 1 minute. FRET efficiency was determined using acceptor photobleaching. Data represent the % of single-cell FRET efficiency from at least three independent experiments for each treatment (one-way ANOVA and Tukey’s correction, ****p < 0.0001). - / - shows the involvement of SHIP1 Y190 in the formation of the SHIP1 and STAT3 complex. (A) TNFα production of SHIP1 KO cells stimulated with 1 ng / ml LPS + IL10, as determined by ELISA, from which the IC50 value of IL10 was calculated (one-way ANOVA and Dunnett’s correction, ****p < 0.0001), where SHIP1 KO cells were reconstituted with WT or mutant SHIP1 or vector (none). (B) Cells expressing WT or Y190F SHIP1 were stimulated with IL10 or compound I-2 for 5 minutes. His6-SHIP1 was pulled down using nickel beads and probed with SHIP1, STAT3, and phospho-STAT3 and actin antibodies together with the cell lysate. (C) The amount of STAT3 protein pulled down with His6-SHIP1 (WT or Y190F) was quantified (two-way ANOVA and Sidak’s correction, **p < 0.01, *p < 0.05). - / -

[0026] Figure 3

[0027] Figure 4 ​​IL10-induced nuclear translocation of SHIP1 and STAT3 is shown. (A) SHIP1+ / + and STAT3+ / + perimacs were stimulated with IL10 or compound I-2 for 2 or 20 minutes and stained with CD11b, SHIP1, and STAT3 antibodies and DAPI as indicated. (B) Pearson coefficients were calculated to show the degree of overlap of SHIP1 or STAT3 with the membrane marker CD11b or the DNA marker DAPI. Data represent Pearson coefficients of single fields of cells from at least two independent experiments in each cell type (two-way ANOVA and Sidak’s correction, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05).

[0028] Figure 5 PPAC, PAC1, and PAC2 are shown to have enzyme activities similar to those of full-length SHIP1. (A) Schematic diagrams of different SHIP1 truncation constructs. PPAC consists of the PH-R domain, phosphatase, and C2 domain (aa 293–877 residues). PAC1 and PAC2 consist of the phosphatase and C2 domain (aa 402–861 and aa 402–857 residues, respectively). PAC1-cc and PAC2-cc contain surface entropy reduction mutations (E770A, E772A, E773A) in the C2 domain. This set of residues was identified using the SERp server (http: / / services.mbi.ucla.edu / SER / intro.php). (B) The initial velocity of enzyme catalysis was determined at the indicated IP4 concentrations. Kcat and Km values were calculated using GraphPad software. (C) The ability of compound I-1 to stimulate the phosphatase activities of full-length SHIP1, PPAC, and PAC (two-way ANOVA and Tukey correction for multiple comparisons, **p < 0.01, ****p < 0.0001).

[0029] Figure 6Shows the responses of PAC2 wild-type and mutant proteins to compound I-2 and PI(3,4)P2, as well as the TNFα levels in cells expressing wild-type or mutant SHIP1 or not expressing SHIP1. (A) Biolayer interferometry (BLI) data of sensors loaded with PAC2 WT and K681A exposed to 20 μM compound I-2 or PI(3,4)P2. ****p < 0.0001 when comparing WT PAC2 with K681A (unpaired Student’s t-test). (B) TNFα production in cells reconstituted with WT or K681A SHIP1 or none (SHIP1 KO) stimulated with 10 ng / ml LPS + IL10 was determined by ELISA, and the IC50 value of IL10 was calculated from it. ****p < 0.0001 when compared with cells reconstituted with WT SHIP1 (unpaired Student’s t-test).

[0030] Figure 7 Shows that the binding of AQX-1125 / Rosiptor to PAC2 is weak compared to compound I-2 and PI(3,4)P2. (A) Structures of compound I-1 and its derivative compound I-2 and AQX-1125 / Rosiptor. (B) Representative BLI curves of the binding of 20 μM compound I-2, AQX-1125, and PI(3,4)P2 to wild-type (WT) PAC2. Each data point represents data from an independent biosensor (one-way ANOVA and Tukey’s correction, ***p < 0.001, **p < 0.01).

[0031] Figure 8 Shows the effect of compound I-1 on the inflammation in IL10 - / - colitis. (A) Representative H&E-stained proximal, mid, and distal colon segments, (B) pathological scores of normal (no colitis, n = 6) and colitis IL10 mice treated with vehicle (Veh., n = 9), I-1 (3 mg / kg) (n = 8), or dexamethasone (Dex., 0.4 mg / kg) (n = 3) for 3 weeks. ****p < 0.0001 when compared with the vehicle-treated group (one-way ANOVA and Tukey’s correction, F = 34.59). (C) From normal (no colitis) and colitis IL10 treated with vehicle (Veh.), I-1 (3 mg / kg), or dexamethasone (Dex., 0.4 mg / kg) - / - mice. (unpaired Student’s t-test). - / -RT-qPCR of cDNA prepared from colon segments of mice. Data represent the mean expression of IL-17 and CCL2 relative to GAPDH. **p<0.01, ****p<0.0001 when compared to the vector-treated group (one-way ANOVA and Tukey's correction, F = 34.59).

[0032] Figure 9 IL10 and IL6 are shown to stimulate phosphorylation of STAT1 and STAT3 in BMDM. Cells were stimulated with 10 or 100 ng / mL IL10 / IL6 for 30 minutes and lysates were prepared for immunoblot analysis using antibodies against the indicated proteins and phosphorylated proteins.

[0033] Figure 10 Compound I-1 is shown to inhibit MM cell growth in vivo. MM.1S cells expressing firefly luciferase were subcutaneously injected with Matrigel basement membrane into the upper flanks of NOD / SCID mice and allowed to establish for 2 weeks. Compound I-1 or control vehicle (n = 4) was subcutaneously administered into the lower flanks in an oil depot form at a dose of 50 mg / kg body weight every 3 days. (A) Bioluminescence images of control and compound I-1-treated mice. (B) Tumor volume was quantified using bioluminescence imaging.

[0034] Figure 11 The role of compound I-1 in protecting against ConA-induced liver injury is shown. Figures A to D show the plasma enzyme levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as well as the levels of total bilirubin (TBIL) and blood urea nitrogen (BUN) in C57 mice treated with blank control, compound I-1 alone (MN-100), ConA alone, ConA and compound I-1 (MN-100) (3 mg / kg / d), or ConA and compound I-1 (MN-100) (10 mg / kg / d). Figures E and F show the plasma enzyme levels of ALT and AST in C57 mice treated with blank control, ConA alone, ConA and compound I-1 (MN-100) (10 mg / kg / d), or ConA and dexamethasone (0.5 mg / kg / d) (positive control).

[0035] Figure 12 Photographs of agar plates from blood cultures of blank / vehicle control mice (Figure A), septic mice with cecal ligation and puncture (CLP) surgery (Figure B), or sham-operated control mice (Figure C) are shown.

[0036] Figure 13It shows the dose-dependent therapeutic effect of compound I-1 (ZPR-MN100) in treating CLP sepsis mice. Mice subjected to CLP surgery were treated with I-1 at 3 mg / kg / day or 10 mg / kg / day via oral gavage, and the survival rate was compared with that of the control. Detailed Description

[0037] Other features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, although indicating embodiments of the present disclosure, are given by way of illustration only, and the scope of the claims should not be limited by these embodiments, but should be given the broadest interpretation consistent with the description as a whole.

[0038] I. Definitions

[0039] Unless otherwise specified, the definitions and embodiments described in this section and other sections are intended to apply to all aspects of the present disclosure described herein that are suitable as would be understood by those skilled in the art.

[0040] As used herein, the term "compound of the present disclosure" and the like refer to a compound of formula I or a pharmaceutically acceptable salt, solvate, prodrug, and / or derivative thereof.

[0041] As used herein, the term "composition of the present disclosure" and the like refer to a composition comprising one or more compounds of the present disclosure, such as a pharmaceutical composition.

[0042] The terms "ZPR-100", "ZPR-MN100", "AQX-MN100", or "MN-100" used herein refer to compound I-1.

[0043] The term "ZPR-151" used herein refers to compound I-2.

[0044] As used herein, the term "and / or" means that the listed items are present or used individually or in combination. In fact, this term means the use or presence of "at least one" or "one or more" of the listed items. For its pharmaceutically acceptable salts and / or solvates, the term "and / or" means that the compound of the present disclosure exists as a separate salt and hydrate and, for example, a combination of a solvate of a salt of the compound of the present disclosure.

[0045] When used in the present disclosure, a reference to no specific number includes plural referents unless the context clearly dictates otherwise. For example, an embodiment that includes "a compound" should be understood to present certain cases containing one compound or two or more additional compounds.

[0046] In embodiments that include an “additional” or “second” component, such as an additional or second compound, the second component as used herein is chemically different from the other components or the first component. A “third” component is different from the other first and second components, and further recited or “additional” components are likewise different.

[0047] As used herein, the term “suitable” means that the choice of a particular compound or condition will depend on the specific synthetic operation to be performed, the identity of the molecule to be transformed, and / or the specific use of the compound, but the choice will be entirely within the skill of one of ordinary skill in the art.

[0048] In embodiments of the present disclosure, the compounds described herein may have at least one asymmetric center. In the case where a compound has more than one asymmetric center, they may exist as diastereoisomers. It should be understood that all such isomers and mixtures thereof in any proportions are encompassed within the scope of the present disclosure. It should also be understood that although the stereochemistry of the compounds in any given compound listed herein may be as shown, such compounds may also contain a certain amount (e.g., less than 20%, suitably less than 10%, more suitably less than 5%) of the compounds of the present disclosure having alternative stereochemistry. Any optical isomers or racemic mixtures thereof as isolated, pure or partially purified optical isomers are intended to be included within the scope of the present disclosure.

[0049] The compounds of the present disclosure may also exist in different tautomeric forms, and any tautomeric forms and mixtures thereof formed by the compounds are intended to be included within the scope of the present disclosure.

[0050] The compounds of the present disclosure may also exist in different polymorphic forms, and any polymorphs or mixtures thereof formed are intended to be included within the scope of the present disclosure.

[0051] This description relates to many chemical terms and abbreviations used by those skilled in the art. However, for clarity and consistency, definitions of selected terms are provided.

[0052] When used herein, the terms “about,” “substantially,” and “approximately” mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These qualifying terms should be construed to include at least a ±5% deviation of the modified term if such deviation does not negate the meaning of the word it modifies, or unless the context suggests otherwise to one of ordinary skill in the art.

[0053] The term “alkyl” as used herein, whether alone or as part of another group, means a straight or branched chain saturated alkyl group. The possible number of carbon atoms in the alkyl group mentioned is indicated by the prefix “C n1-n2”designate. For example, the term C 1-10 alkyl means an alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.

[0054] As used herein, the term "heterocycle" refers to a substituted or unsubstituted 5- or 6-membered heterocycle, which may be aromatic or non-aromatic and contains at least one NH moiety.

[0055] As used herein, the term "substituted" refers to the situation where one or more available hydrogens on a compound are replaced by non-hydrogen functional groups.

[0056] In "available hydrogen atom" or "available atom", the term "available" refers to an atom known to those skilled in the art that can be replaced by a substituent.

[0057] When used herein, the term "amine" or "amino", whether used alone or as part of another group, means a group of the general formula NR′R″, where R′ and R″ are each independently selected from hydrogen or C 1-6 alkyl.

[0058] As used herein, the term "subject" includes all members of the animal kingdom, including mammals, and, where appropriate, refers to humans. Thus, the methods and uses of the present disclosure are applicable to both human therapy and veterinary applications.

[0059] The term "pharmaceutically acceptable" means compatible with the treatment of a subject.

[0060] The term "pharmaceutically acceptable carrier" means a non-toxic solvent, dispersant, excipient, adjuvant and other materials that are mixed with the active ingredient to allow the formation of a pharmaceutical composition, i.e., a dosage form capable of being administered to a subject.

[0061] The term "pharmaceutically acceptable salt" means an acid addition salt or a base addition salt that is suitable for or compatible with the treatment of a subject.

[0062] An acid addition salt that is suitable for or compatible with the treatment of a subject is any non-toxic organic or inorganic acid addition salt of any basic compound.

[0063] A base addition salt that is suitable for or compatible with the treatment of a subject is any non-toxic organic or inorganic base addition salt of any acidic compound.

[0064] As used herein, the term "solvate" means a compound or a salt of a compound in which suitable solvent molecules are incorporated into the lattice.

[0065] Prodrugs of the compounds of the present disclosure can be, for example, conventional esters formed with available hydroxyl, thiol, amino or carboxyl groups. Other methods of forming prodrugs are generally known to those skilled in the art and can be applied to the compounds of the present disclosure.

[0066] As used herein and as is known in the art, the term "treatment" means a method of obtaining a beneficial or desired result, including a clinical result. Beneficial or desired clinical results include, but are not limited to, alleviation or improvement of one or more symptoms or disorders, reduction in the severity of a disease, stabilization (i.e., non-worsening) of a disease state, prevention of the spread of a disease, delay or slowing of disease progression, improvement or alleviation of a disease state, reduction of disease recurrence, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" can also mean an extension of survival as compared to the expected survival in the absence of treatment. "Treatment" as used herein also includes prophylactic treatment. For example, a subject with early-stage cancer can be treated to prevent progression, or a subject in remission can be treated with a compound or composition of the present disclosure to prevent recurrence. Treatment methods include administering to a subject a therapeutically effective amount of one or more compounds of the present disclosure, and optionally consisting of or including a series of administrations by a single administration.

[0067] "Alleviating" a disease, disorder or condition means reducing the severity and / or unwanted clinical manifestations of the disease, disorder or condition as compared to not treating the disorder, and / or slowing or extending the time course of progression.

[0068] The term "prevention" as used herein refers to reducing the risk or likelihood that a patient will develop a disease, disorder or condition or exhibit symptoms associated with a disease, disorder or condition.

[0069] The term "disease, disorder or condition" as used herein refers to a disease, disorder or condition mediated by SHIP1 or treatable by activation of SHIP1, such as by a compound of the present disclosure.

[0070] The term "SHIP1" as used herein refers to Src homology 2-containing inositol 5'-phosphatase 1.

[0071] The term "mediated by SHIP1 or treatable by activation of SHIP1" as used herein means that the disease, disorder or condition to be treated is directly or indirectly affected by, regulated by, and / or has as a biological basis certain biological bases, including the presence of SHIP1 phosphatase in cells. Such biological bases include, for example, cytokines that are direct or indirect products of SHIP1 phosphatase. In an exemplary scenario, "activation of SHIP1" refers to an effect mediated in a cell or organism by a signal transduction activated by SHIP1, such as by PIP3 and / or IL-10.

[0072] As used herein, the term "effective amount" or "therapeutically effective amount" means an amount of one or more compounds of the present disclosure that is effective at the dosage and for the time period necessary to achieve the desired result. For example, in the context of treating a disease, disorder, or condition mediated by SHIP1 or treatable by activation of SHIP1, an effective amount is, for example, an amount that increases SHIP1 activity as compared to the SHIP1 activity when the one or more compounds are not administered.

[0073] As used herein, the term "administer" means to administer to a cell, tissue, organ, or subject a therapeutically effective amount of one or more compounds or compositions of the present disclosure.

[0074] As used herein, the term "neoplastic disorder" refers to a disease, disorder, or condition characterized by cells capable of autonomous growth or replication, such as an abnormal state or condition characterized by proliferative cell growth. As used herein, the term "neoplasm" refers to a mass of tissue resulting from abnormal growth and / or division of cells in a subject suffering from a neoplastic disorder.

[0075] As used herein, the term "cancer" refers to a state of a cell proliferative disease.

[0076] II. Compounds and Compositions of the Present Disclosure

[0077] The present disclosure encompasses a compound of Formula I

[0078]

[0079] or a pharmaceutically acceptable salt, solvate, prodrug, and / or derivative thereof,

[0080] wherein R 1 is selected from H, OH, OC(O)C 1-3 alkyl, OC 1-3 alkyl, NH2, NHC 1-3 alkyl, NHSO2C 1-3 alkyl, N-succinimide, and NHC(O)C 1-3 alkyl;

[0081] wherein R 2 , R 3 , R 4 and R 5 are independently selected from H, OH, C 1-3 alkyl, OC 1-3 alkyl, NH2, NHC 1-3 alkyl, NHSO2C 1-3 alkyl, and NHC(O)C 1-3 alkyl; or R 2 and R 3 , R 3 and R4 or R 4 and R 5 together with the atom to which they are attached form a substituted or unsubstituted 5- or 6-membered heterocycle, said substituted or unsubstituted 5- or 6-membered heterocycle containing at least one NH and optionally one or more additional heteroatoms selected from N, O, and S; and

[0082] wherein when R 2 and R 3 , R 3 and R 4 or R 4 and R 5 together form said substituted or unsubstituted 5- or 6-membered heterocycle, R 4 and R 5 , R 2 and R 5 or R 2 and R 3 are each independently selected from H and C 1-3 alkyl.

[0083] In certain embodiments, the compound of formula I is a compound of formula IA

[0084]

[0085] its enantiomers or pharmaceutically acceptable salts, solvates, prodrugs, and / or derivatives.

[0086] In certain embodiments, R 1 is selected from H, NH2, NHC 1-3 alkyl, NHSO2C 1-3 alkyl, N-succinimide, and NHC(O)C 1-3 alkyl.

[0087] In certain embodiments, R 2 and R 4 are H, and R 3 and R 5 are selected from OH, C 1-3 alkyl, OC 1-3 alkyl, NH2, NHC 1-3 alkyl, NHSO2C 1-3 alkyl, and NHC(O)C 1-3 alkyl.

[0088] In certain embodiments, R 3 and R 5 are selected from OH, CH3, OCH3, NHSO2CH3, and NHC(O)CH3.

[0089] In certain embodiments, R 3Selected from OH, OCH3, NHSO2CH3, and NHC(O)CH3; and R 5 is CH3.

[0090] In certain embodiments, R 2 , R 4 and R 5 are H, and R 3 is selected from OH, OC 1-3 alkyl, NH2, NHC 1-3 alkyl, NHSO2C 1-3 alkyl, and NHC(O)C 1-3 alkyl. For example, R 3 is selected from OH, OCH3, NHSO2CH3, and NHC(O)CH3.

[0091] In certain embodiments, the substituted or unsubstituted 5- or 6-membered heterocycle is aromatic or non-aromatic. In another embodiment, the 5- or 6-membered heterocycle contains an NH moiety and one or more heteroatoms selected from O and N. In other embodiments, the substituents on the 5- or 6-membered heterocycle are selected from C═O and C 1-3 alkyl.

[0092] In certain embodiments, the substituted or unsubstituted 5- or 6-membered heterocycle is selected from

[0093] In certain embodiments, R 2 and R 3 together with the atoms to which they are attached form the substituted or unsubstituted 5- or 6-membered heterocycle, and R 4 and R 5 are independently selected from H and C 1-3 alkyl.

[0094] In certain embodiments, the compounds of formula I are selected from:

[0095]

[0096]

[0097]

[0098]

[0099] and their pharmaceutically acceptable salts, solvates, prodrugs, and / or derivatives.

[0100] In certain embodiments, the compounds of Formula I of the present application do not include Compound I-1. In certain embodiments, the compounds of Formula I of the present application do not include Compound I-2. Optionally, neither Compound I-1 nor I-2 is included in the compounds of Formula I.

[0101] On the other hand, the present disclosure provides a compound of the present disclosure or a pharmaceutically acceptable salt, solvate, prodrug, and / or derivative thereof for treating a disease, disorder, or condition mediated by SHIP1 or treatable by activation of SHIP1.

[0102] In an embodiment, the pharmaceutically acceptable salt is an acid addition salt or a base addition salt. The selection of suitable salts can be made by those skilled in the art (see, for example, S.M. Berge et al., "Pharmaceutical Salts," J. Pharm. Sci. 1977, 66, 1-19).

[0103] Acid addition salts suitable for the treatment of a subject or compatible therewith are any non-toxic organic or inorganic acid addition salts of any basic compound. Basic compounds that form acid addition salts include, for example, compounds containing an amino group. Illustrative inorganic acids that form suitable salts include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid, as well as acidic metal salts such as disodium hydrogen phosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids. Examples of such organic acids are, for example, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, hydroxymaleic acid, benzoic acid, hydroxybenzoic acid, phenylacetic acid, cinnamic acid, mandelic acid, salicylic acid, 2-phenoxybenzoic acid, p-toluenesulfonic acid, and other sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, and 2-hydroxyethanesulfonic acid. In an embodiment, mono- or di-acid salts are formed, and such salts exist in hydrated, solvated, or substantially anhydrous forms. Generally, acid addition salts are more soluble in water and various hydrophilic organic solvents compared to their free base forms and generally exhibit higher melting points. The criteria for the selection of suitable salts are known to those skilled in the art. Other non-pharmaceutically acceptable salts such as, but not limited to, oxalates can be used, for example, for the isolation of the compounds of the present disclosure, for laboratory use, or for subsequent conversion to pharmaceutically acceptable acid addition salts.

[0104] A base addition salt suitable for or compatible with the treatment of a subject is any non-toxic organic or inorganic base addition salt of any acidic compound. Acidic compounds that form base addition salts include, for example, compounds containing a carboxylic acid group. Illustrative inorganic bases that form suitable salts include lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or barium hydroxide, as well as ammonia. Illustrative organic bases that form suitable salts include aliphatic, cycloaliphatic, or aromatic organic amines such as isopropylamine, methylamine, trimethylamine, methylpyridine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Exemplary organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. Selection of a suitable salt may be useful, for example, so that an ester functional group elsewhere in the compound (if any) is not hydrolyzed. Criteria for selection of suitable salts are known to those of ordinary skill in the art.

[0105] Solvates of the compounds of the present disclosure include, for example, solvates made using pharmaceutically acceptable solvents. Examples of such solvents include water (the resulting solvate is called a hydrate) and ethanol, among others. Suitable solvents are physiologically tolerable at the administered dose.

[0106] The compounds of the present disclosure are suitably formulated into a composition in a conventional manner using one or more carriers. Accordingly, the present disclosure also includes a composition comprising one or more of the compounds of the present disclosure and a carrier. The compounds of the present disclosure are suitably formulated into a pharmaceutical composition for administration to a subject in a biocompatible form suitable for in vivo administration. Accordingly, the present disclosure also includes a pharmaceutical composition comprising one or more of the compounds of the present disclosure and a pharmaceutically acceptable carrier. In embodiments of the present disclosure, the pharmaceutical composition is for the treatment of any disease, disorder, or condition described herein.

[0107] In certain embodiments, the compositions of the present disclosure consist essentially of one or more of the compounds of the present disclosure or one or more of their pharmaceutically acceptable salts, solvates, prodrugs, and / or derivatives, and a pharmaceutically acceptable carrier.

[0108] In certain embodiments, the compositions of the present disclosure consist of one or more of the compounds of the present disclosure or one or more of their pharmaceutically acceptable salts, solvates, prodrugs, and / or derivatives, and a pharmaceutically acceptable carrier.

[0109] As will be understood by those skilled in the art, depending on the selected route of administration, the compounds of the present disclosure are administered to a subject in a variety of different forms. For example, the compounds of the present disclosure are administered orally, by inhalation, parenterally, buccally, sublingually, nasally, rectally, vaginally, by patch, pump, topically or transdermally and in pharmaceutical compositions formulated therefor. In certain embodiments, administration is carried out using a pump for periodic or continuous delivery. Conventional procedures and ingredients for selecting and preparing suitable compositions are described, for example, in Remington’s Pharmaceutical Sciences (2000 - 20th Edition) and The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.

[0110] Parenteral administration includes systemic delivery routes outside the gastrointestinal (GI) tract and includes, for example, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, transcutaneous, nasal, intralung (e.g., by using an aerosol), intrathecal, rectal and topical (including the use of patches or other transdermal delivery devices) modes of administration. Parenteral administration can be carried out by continuous infusion over a selected period of time.

[0111] In certain embodiments, the compounds of the present disclosure are administered orally with an inert diluent or an assimilable edible carrier, or it is enclosed in a hard or soft shell gelatin capsule, or it is compressed into tablets, or it is directly incorporated with the food in the diet. In certain embodiments, the compound is combined with excipients and used in the form of swallowable tablets, buccal tablets, lozenges, capsules, cachets, pills, granules, troches, chewing gums, powders, syrups, elixirs, wafers, aqueous solutions and suspensions, etc. In the case of tablets, the carriers used include lactose, corn starch, sodium citrate and phosphates. Pharmaceutically acceptable excipients include binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium phosphate), lubricants (e.g., magnesium stearate, talc or silica), disintegrants (e.g., potato starch or sodium starch glycolate) or wetting agents (e.g., sodium lauryl sulfate). In an embodiment, the tablets are coated by methods known in the art. In the case of tablets, capsules, cachets, pills or granules for oral administration, a pH-sensitive enteric coating designed to control the release of the active ingredient, such as Eudragits, is optionally used. TMOral dosage forms also include modified release, such as immediate release and timed release formulations. Examples of modified release formulations include, for example, sustained release (SR), extended release (ER, XR or XL), time release or timed release, controlled release (CR) or continuous release (CR or Contin), and are used in the form of, for example, coated tablets, osmotic delivery devices, coated capsules, microencapsulated microspheres, agglomerated particles such as particles of the molecular sieve type or fine hollow permeable fiber bundles or shredded hollow permeable fibers agglomerated or held in a fiber pack. Timed release compositions are formulated, for example, as liposomes or compositions in which the active compound is protected with different degradable coatings, for example by microencapsulation, multiple coating, etc. Liposome delivery systems include, for example, small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. In certain embodiments, liposomes are formed from a variety of different phospholipids such as cholesterol, stearylamine or phosphatidylcholine. For oral administration in capsule form, useful carriers or diluents include lactose and dry corn starch.

[0112] In certain embodiments, liquid formulations for oral administration take the form of, for example, solutions, syrups or suspensions, or they suitably exist as dry products, which are constituted with water or other suitable carriers before use. When aqueous suspensions and / or emulsions are orally administered, the compounds of the present disclosure are suitably suspended or dissolved in an oily phase combined with an emulsifying and / or suspending agent. If desired, certain sweetening agents and / or flavoring agents and / or coloring agents are added. Such liquid formulations for oral administration are prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methylcellulose or hydrogenated edible fats), emulsifying agents (e.g., lecithin or gum arabic), non-aqueous carriers (e.g., almond oil, oily esters or ethanol) and preservatives (e.g., methyl or propyl parahydroxybenzoate or sorbic acid). Useful diluents include lactose and high molecular weight polyethylene glycol.

[0113] The compounds of the present disclosure can also be lyophilized, and the resulting lyophilizates are used, for example, in the preparation of injectable products.

[0114] In certain embodiments, the compounds of the present disclosure are administered parenterally. For example, solutions of the compounds of the present disclosure are prepared in water admixed with a surfactant such as hydroxypropylcellulose. In certain embodiments, dispersions are prepared in glycerol, liquid polyethylene glycol, DMSO, and mixtures thereof, with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. Those skilled in the art will know how to prepare suitable preparations. For parenteral administration, sterile solutions of the compounds of the present disclosure are generally prepared, and the pH of the solution is adjusted and buffered appropriately. For intravenous use, the total concentration of the solute should be controlled to render the preparation isotonic. For ophthalmic administration, for example, an ointment or a droppable liquid is delivered via an ophthalmic delivery system known in the art, such as an applicator or an eye dropper. In certain embodiments, such compositions contain a mucus mimetic such as hyaluronic acid, chondroitin sulfate, hydroxypropylmethylcellulose, or polyvinyl alcohol, a preservative such as sorbic acid, EDTA, or benzalkonium chloride, and a conventional amount of a diluent or a carrier. For pulmonary administration, a suitable diluent or carrier is selected to allow the formation of an aerosol.

[0115] In certain embodiments, the compounds of the present disclosure are formulated for parenteral administration by injection, including using conventional catheterization techniques or infusion. Preparations for injection are presented in unit dosage form, for example, in an ampoule or a multi-dose container, and contain a preservative. In certain embodiments, the composition takes the form of a sterile suspension, solution, or emulsion in an oily or aqueous carrier and contains formulating agents such as suspending agents, stabilizers, and / or dispersing agents. In all cases, the form must be sterile and must be fluid to the extent that easy injection is possible. Alternatively, the compounds of the present disclosure suitably take the form of a sterile powder for reconstitution with a suitable carrier such as sterile pyrogen-free water before use.

[0116] In certain embodiments, the compositions for nasal administration are conveniently formulated as aerosols, drops, gels, and powders. For intranasal or inhaled administration, the compounds of the present disclosure are conveniently delivered in the form of a body solution, dry powder formulation, or suspension from a pump spray container that is squeezed or pumped by the patient, or as an aerosol spray from a pressurized container or nebulizer. Aerosol formulations generally comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are typically present in a sealed container, e.g., in the form of a cartridge or refill for use with an atomizing device, in a sterile form in single or multiple dose amounts. Alternatively, the sealed container is an integrated dispensing device such as a single-dose nasal inhaler or an aerosol dispenser equipped with a metering valve, designed to be discarded after use. In cases where the dosage form comprises an aerosol dispenser, it will contain a propellant, which is, for example, a compressed gas such as compressed air or an organic propellant such as a chlorofluorocarbon. Suitable propellants include, but are not limited to, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, heptafluoropropane, carbon dioxide, or other suitable gases. In the case of a pressurized aerosol, the dosage unit is suitably determined by providing a valve to deliver a metered amount. In certain embodiments, the pressurized container or nebulizer contains a solution or suspension of the active compound. For example, capsules and cartridges (e.g., made of gelatin) for inhalers or insufflators are formulated to contain a powder mixture of the compounds of the present disclosure with a suitable powder base such as lactose or starch. The aerosol dosage form may also take the form of a pump nebulizer.

[0117] Compositions suitable for buccal or sublingual administration include tablets, lozenges, and troches, in which the compounds of the present disclosure are formulated with a carrier such as sugar, gum arabic, tragacanth, or gelatin and glycerin. Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base such as cocoa butter.

[0118] Suppository forms of the compounds of the present disclosure can be used for vaginal, urethral, and rectal administration. Such suppositories generally consist of a mixture of substances that are solid at room temperature but melt at body temperature. Substances commonly used to make such carriers include, but are not limited to, cocoa butter (also known as theobroma oil), glycerinated gelatin, other glycerides, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights, and fatty acid esters of polyethylene glycol. For further discussion of suppository dosage forms, see, e.g., Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing, Easton, PA, 1980, pp. 1530 - 1533.

[0119] In certain embodiments, the compounds of the present disclosure are conjugated to soluble polymers that serve as targetable drug carriers. Such polymers include, for example, polyvinylpyrrolidone, pyran copolymers, poly(hydroxypropyl methacrylamide)-phenol, poly(hydroxyethyl asparagine)-phenol, or polyethylene oxide-polylysine substituted with palmitoyl residues. In addition, in certain embodiments, the compounds of the present disclosure are conjugated to a class of biodegradable polymers that can be used to achieve controlled release of a drug, such as polylactic acid, polyglycolic acid, copolymers of polylactic acid and polyglycolic acid, poly(ε-caprolactone), polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and crosslinked or amphiphilic block copolymers of hydrogels.

[0120] The compounds of the present disclosure, including their pharmaceutically acceptable salts and / or solvates, are used per se in suitable cases, but are generally administered in the form of a pharmaceutical composition, in which the one or more compounds of the present disclosure (active ingredients) are combined with a pharmaceutically acceptable carrier. Depending on the mode of administration, the pharmaceutical composition will contain from about 0.05 wt% to about 99 wt% or from about 0.10 wt% to about 70 wt% of the active ingredient and from about 1 wt% to about 99.95 wt% or from about 30 wt% to about 99.90 wt% of the pharmaceutically acceptable carrier, all percentages being based on the weight of the total composition.

[0121] III. Methods and Uses of the Present Disclosure

[0122] On the other hand, the present disclosure includes a method of treating a disease, disorder, or condition mediated by SHIP1 or treatable by activation of SHIP1, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt, solvate, prodrug, and / or derivative thereof.

[0123] On the other hand, the present disclosure includes the use of one or more compounds of the present disclosure or pharmaceutically acceptable salts, solvates, prodrugs, and / or derivatives thereof for treating a disease, disorder, or condition mediated by SHIP1 or treatable by activation of SHIP1.

[0124] On the other hand, the present disclosure includes the use of one or more compounds of the present disclosure or pharmaceutically acceptable salts, solvates, prodrugs, and / or derivatives thereof for the preparation of a medicament for treating a disease, disorder, or condition mediated by SHIP1 or treatable by activation of SHIP1.

[0125] In certain embodiments, the disease, disorder, or condition mediated by SHIP1 or treatable by activation of SHIP1 is selected from inflammatory bowel disease (IBD), multiple myeloma, allergy, and neoplastic disorders such as colon cancer, sepsis, organ injury, trauma, cardiovascular disease, osteoporosis, and sleep disorders. In certain embodiments, the IBD is selected from Crohn's disease and ulcerative colitis.

[0126] In certain embodiments, the organ injury and trauma are mediated by IL-10 via SHIP1. In certain embodiments, the organ injury and trauma are liver injury. In certain embodiments, the liver injury is selected from viral hepatitis, autoimmune hepatitis, primary biliary cirrhosis, and hepatic allograft rejection. It is known that IL-10 administration reduces organ injury such as liver or lung inflammation and reduces neuropathy in nerve or spinal cord injury.

[0127] In certain embodiments, the cardiovascular disease includes atherosclerosis. It is known that IL-10 administration limits tissue inflammation and improves endothelial cell and macrophage function.

[0128] In certain embodiments, the osteoporosis disorder includes those in which IL-10 administration can inhibit the resorptive function of mature osteoclasts.

[0129] In certain embodiments, the disease, disorder, or condition mediated by SHIP1 or treatable by activation of SHIP1 is multiple myeloma.

[0130] In certain embodiments, the sepsis is severe sepsis. It is known that viral infections such as COVID-19 cause severe sepsis. In certain embodiments, the severe sepsis is caused by COVID-19.

[0131] In an embodiment, the treatment employs at least one symptom that effectively improves a neoplastic disorder in a subject in need thereof, such as reducing cell proliferation or shrinking a tumor mass.

[0132] Neoplasms can be benign (e.g., uterine fibroids and melanocytic nevi), potentially malignant (e.g., carcinoma in situ), or malignant (i.e., cancer). Exemplary neoplastic disorders include so-called solid tumors and liquid tumors, including but not limited to epithelial carcinomas, sarcomas, metastatic disorders (e.g., tumors arising from the prostate), hematopoietic neoplastic disorders (e.g., leukemia, lymphoma, myeloma, and other malignant plasma cell disorders), metastatic tumors, and other cancers.

[0133] The present disclosure also includes one or more compounds of the present disclosure for treating cancer. In an embodiment, the compound is administered to prevent cancer in a subject having cancer susceptibility, such as a mammal.

[0134] In an embodiment, the cancer is selected from, but not limited to: adult acute lymphoblastic leukemia, childhood acute lymphoblastic leukemia, adult acute myeloid leukemia, adrenocortical carcinoma, childhood adrenocortical carcinoma, AIDS-related lymphoma, AIDS-related malignancies, anal cancer, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, extrahepatic bile duct cancer, bladder cancer, childhood bladder cancer, bone cancer, osteosarcoma / malignant fibrous histiocytoma, childhood brainstem glioma, adult brain tumor, childhood brain tumor brainstem glioma, childhood brain tumor cerebellar astrocytoma, childhood brain tumor cerebral astrocytoma / malignant glioma, childhood brain tumor ependymoma, childhood brain tumor medulloblastoma, childhood brain tumor supratentorial primitive neuroectodermal tumor, childhood brain tumor visual pathway and hypothalamic glioma, childhood brain tumor (other), breast cancer, breast cancer and pregnancy, childhood breast cancer, male breast cancer, childhood bronchial adenoma / carcinoid, childhood carcinoid tumor, gastrointestinal carcinoid tumor, adrenocortical carcinoma, islet cell carcinoma, carcinoma of unknown primary, primary central nervous system lymphoma, childhood cerebellar astrocytoma, childhood cerebral astrocytoma / malignant glioma, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, clear cell sarcoma of tendons and aponeuroses, colon cancer, childhood colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, childhood ependymoma, ovarian epithelial cancer, esophageal cancer, childhood esophageal cancer, Ewing family of tumors, childhood extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, childhood gastric cancer, gastrointestinal carcinoid tumor, childhood extracranial germ cell tumor, extragonadal germ cell tumor, ovarian germ cell tumor, gestational trophoblastic tumor, childhood brainstem glioma, childhood visual pathway and hypothalamic glioma, hairy cell leukemia, head and neck cancer, adult (primary) hepatocellular (liver) cancer, childhood (primary) hepatocellular (liver) cancer, adult Hodgkin lymphoma, childhood Hodgkin lymphoma, Hodgkin lymphoma during pregnancy, hypopharyngeal cancer, childhood hypothalamic and visual pathway glioma, intraocular melanoma, islet cell carcinoma (endocrine pancreas), Kaposi sarcoma, kidney cancer, laryngeal cancer, childhood laryngeal cancer, adult acute lymphoblastic leukemia, childhood acute lymphoblastic leukemia, adult acute myeloid leukemia, childhood acute myeloid leukemia, chronic lymphocytic, chronic myelogenous leukemia, hairy cell leukemia, lip and oral cavity cancer, adult (primary) liver cancer, childhood (primary) liver cancer, non-small cell lung cancer, small cell lung cancer, adult acute lymphoblastic leukemia, childhood acute lymphoblastic leukemia, chronic lymphocytic leukemia, AIDS-related lymphoma, central nervous system (primary) lymphoma, cutaneous T-cell lymphoma, adult Hodgkin lymphoma, childhood Hodgkin lymphoma, Hodgkin lymphoma during pregnancy, adult non-Hodgkin lymphoma, childhood non-Hodgkin lymphoma, non-Hodgkin lymphoma during pregnancy,Primary central nervous system lymphoma, Waldenström macroglobulinemia, male breast cancer, adult malignant mesothelioma, childhood malignant mesothelioma, malignant thymoma, childhood medulloblastoma, melanoma, intraocular melanoma, Merkel cell carcinoma, malignant mesothelioma, occult primary metastatic squamous neck cancer, childhood multiple endocrine neoplasia syndrome, multiple myeloma / plasmacytoma, mycosis fungoides, myelodysplastic syndrome, chronic myelogenous leukemia, childhood acute myeloid leukemia, multiple myeloma, chronic myeloproliferative disorders, nasal and paranasal sinus cancer, nasopharyngeal cancer, childhood nasopharyngeal cancer, neuroblastoma, adult non-Hodgkin lymphoma, childhood non-Hodgkin lymphoma, non-Hodgkin lymphoma in pregnancy, non-small cell lung cancer, childhood oral cancer, oral and lip cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, childhood ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, childhood pancreatic cancer, islet cell pancreatic cancer, paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, pheochromocytoma, childhood pineal and supratentorial primitive neuroectodermal tumors, pituitary tumor, plasmacytoma / multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, pregnancy and Hodgkin lymphoma, pregnancy and non-Hodgkin lymphoma, primary central nervous system lymphoma, adult primary liver cancer, childhood primary liver cancer, prostate cancer, rectal cancer, renal cell (kidney) cancer, childhood renal cell cancer, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, childhood rhabdomyosarcoma, salivary gland cancer, childhood salivary gland cancer, Ewing's family of tumors sarcoma, Kaposi sarcoma, sarcoma (osteosarcoma) / malignant fibrous histiocytoma of bone, childhood rhabdomyosarcoma, adult soft tissue sarcoma, childhood soft tissue sarcoma, Sézary syndrome, skin cancer, childhood skin cancer, skin cancer (melanoma), Merkel cell skin cancer, small cell lung cancer, small intestine cancer, adult soft tissue sarcoma, childhood soft tissue sarcoma, occult primary metastatic squamous neck cancer, gastric cancer, childhood gastric cancer, childhood supratentorial primitive neuroectodermal tumors, cutaneous T-cell lymphoma, testicular cancer, childhood thymoma, malignant thymoma, thyroid cancer, childhood thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, gestational trophoblastic tumor, childhood cancer of unknown primary site, childhood unusual cancers, transitional cell carcinoma of the ureter and renal pelvis, urethral cancer, uterine sarcoma, vaginal cancer, childhood visual pathway and hypothalamic glioma, vulvar cancer, Waldenström macroglobulinemia and Wilms tumor. Metastases of the above cancers can also be treated by the methods described herein.,

[0135] In other embodiments, the diseases, disorders or conditions mediated by SHIP1 or treatable by activation of SHIP1 and the one or more compounds of the present disclosure are administered in combination with one or more additional cancer treatments. In another embodiment, the additional cancer treatment is selected from radiotherapy, chemotherapy, targeted therapies such as antibody therapies and small molecule therapies such as tyrosine kinase and serine-threonine kinase inhibitors, immunotherapy, hormone therapy, and anti-angiogenic therapy.

[0136] In embodiments, the effective amount varies with multiple factors such as disease state, age, sex, and / or weight of the subject. In other embodiments, the amount corresponding to the effective amount of a given one or more compounds will vary with multiple factors such as the given drug or compound, pharmaceutical formulation, route of administration, disorder, type of disease or disorder, identity of the subject being treated, etc., yet can be determined routinely by those skilled in the art.

[0137] In embodiments, the compounds of the present disclosure are administered at least once per week. However, in another embodiment, the compound is administered to the subject approximately every two weeks, three weeks, or once a month. In another embodiment, the compound is administered from approximately once a week to approximately once a day. In another embodiment, the compound is administered 2, 3, 4, 5, or 6 times per day. The length of the treatment period depends on various factors such as the severity of the disease, disorder or condition, the age of the subject, the concentration and / or activity of the compounds of the present disclosure, and / or combinations thereof. It should also be recognized that the effective dose of the compound used in the course of a particular treatment regimen may be increased or decreased. Changes in dose can be obtained and made apparent by standard diagnostic assays known in the art. In some cases, long-term administration is required. For example, the compound is administered to the subject in an amount and for a duration sufficient to treat the subject.

[0138] In embodiments, the subject is a mammal. In another embodiment, the subject is a human.

[0139] The compounds of the present disclosure are used alone or in combination with other known agents, which can be used to treat diseases, disorders or conditions mediated by SHIP1 or treatable by activation of SHIP1 and diseases, disorders or conditions treatable with a SHIP1 agonist such as the compounds disclosed herein. When used in combination with other agents that can be used to treat diseases, disorders or conditions mediated by SHIP1 or treatable by activation of SHIP1, one embodiment is to administer the compounds of the present disclosure simultaneously with those agents. As used herein, "simultaneous administration" of two substances to a subject means providing each of the two substances such that they are both active in the individual at the same time. The exact details of administration depend on the pharmacokinetics of the two substances in the presence of each other and can include administering the two substances within a few hours of each other, or even within 24 hours of administering one substance if the pharmacokinetics are appropriate. The design of suitable dosing regimens is routine for those skilled in the art. In a particular embodiment, the two substances are administered substantially simultaneously, i.e., within a few minutes of each other, or in a single composition containing both substances. Another embodiment of the present disclosure is to administer the combination of agents to the subject in a non-simultaneous manner. In an embodiment, the compounds of the present disclosure are administered simultaneously or sequentially in separate unit dosage forms with another therapeutic agent, or together in a single unit dosage form. Accordingly, the present disclosure provides a single unit dosage form comprising one or more compounds of the present disclosure, an additional therapeutic agent, and a pharmaceutically acceptable carrier.

[0140] The dosage of the compounds of the present disclosure varies with many factors, such as the pharmacodynamic properties of the compound, the mode of administration, the age, health and weight of the recipient, the nature and severity of the symptoms, the frequency of treatment and the type of co-treatment if any, and the clearance rate of the compound in the subject to be treated. A person skilled in the art can determine a suitable dosage based on the above factors. In certain embodiments, the compounds of the present disclosure are initially administered at a suitable dosage and then the dosage is adjusted as needed based on the clinical response. The dosage is generally selected to maintain the serum level of the compounds of the present disclosure at about 0.01 μg / cc to about 1000 μg / cc or about 0.1 μg / cc to about 100 μg / cc. As a representative example, for an adult, the oral dosage of one or more of the compounds of the present disclosure ranges between about 1 mg / day to about 1000 mg / day, suitably about 1 mg / day to about 500 mg / day, more suitably about 1 mg / day to about 200 mg / day. For parenteral administration, a representative amount is about 0.001 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 1 mg / kg or about 0.1 mg / kg to about 1 mg / kg. For oral administration, a representative amount is about 0.001 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 1 mg / kg or about 0.1 mg / kg to about 1 mg / kg. For administration in the form of a suppository, a representative amount is about 0.1 mg / kg to about 10 mg / kg or about 0.1 mg / kg to about 1 mg / kg.

[0141] Examples

[0142] The following non-limiting examples illustrate the present disclosure.

[0143] General Method

[0144] Mouse populations. BALB / c wild-type (+ / +) or SHIP1 knockout (- / -) mice were provided by Dr. Gerald Krystal (BC Cancer Research Centre, Vancouver, BC). Generation of STAT3- / - mice was initiated by crossing C57BL / 6 STAT3 flox / flox mice (Dr. Shizuo Akira, Hyogo College of Medicine, Nishinomiya, Japan) with C57BL / 6 LysMcre mice (Jackson Laboratory). Their offspring were then crossed with homozygous STAT3 flox / flox mice to generate STAT3 flox / flox / LysMCre+ / - mice (referred to as STAT3- / - mice) and STAT3 flox / flox mice (STAT3+ / + mice) in the same litter. All mice were maintained according to an ethical protocol approved by the Animal Care Committee of the University of British Columbia.

[0145] Constructs. Using the Gateway LR reaction, mammalian (lentiviral) expression plasmids of SHIP1 in FUGWBW were generated from pENTR1A (Invitrogen, Burlington, ON) constructs. The pENTR1A-His6-SHIP1 WT (SHIP1 Uniprot ID Q9ES52) plasmid was used as a template for standard primer-based site-directed mutagenesis to generate the K681A, Y190F, Y799F, Y659F, and Y657F mutants. The phosphatase-deficient SHIP1 construct (P671A, D675A, and R676G in the phosphatase domain) was kindly provided by Dr. KS Ravichandran (University of Virginia). Constructs were confirmed by DNA sequencing. Subsequently, a Gateway LR reaction was performed between the pENTR1A construct and FUGWBW (FUGW, in which the green fluorescent protein was replaced with a Gateway cassette and the blasticidin S resistance gene expression cassette was inserted downstream of the Gateway cassette (Peacock et al., 2009)). The success of the LR reaction was confirmed by restriction enzyme digestion. For the Clover / mRuby2-based FRET experiments (Lam et al., 2012), pENTR1A Clover-SHIP1 was constructed by inserting the Clover fragment from pcDNA3 Clover (Addgene) into the N-terminus of SHIP1 in pENTR1A-His6-SHIP1 WT to replace His6. pENTR1A STAT3-mRuby2 was constructed by cloning murine STAT3 (Uniprot ID P42227) into pENTR1A and then inserting the mRuby2 fragment from pcDNA3 mRuby2 (Addgene) into the N-terminus of STAT3. Constructs were confirmed by sequencing and transferred to FUGWBW as described above.

[0146] A bacterial expression vector was generated in the LIC-HMT vector (Van Petegem et al., 2004) by ligase-independent cloning (LIC) method to produce recombinant proteins for crystallography and biomembrane interference techniques. The plasmid contains an N-terminal tag, which consists of His6 and maltose-binding protein (MBP), followed by a TEV protease cleavage site (abbreviated as HMT-tag). The PCR products were purified and treated with T4 DNA polymerase (LIC quality) (Novagen, Madison, WI) in the presence of only dCTP. The LIC-HMT vector was digested with SspI, and the linearized plasmid was treated with T4 DNA polymerase in the presence of only dGTP. Equal volumes of the insert and the vector were mixed and incubated at room temperature for 10 minutes, then transformed into chemically competent Escherichia coli (E. coli) DH5α cells using the standard heat shock method and selected on LB agar plates containing kanamycin. Standard site-directed mutagenesis was used to generate different PAC2 mutants. The sequence information of all plasmids was confirmed by DNA sequencing.

[0147] Cell lines. The J16 and J17 cell lines, derived from SHIP1+ / + and - / - BMDM respectively, have been previously described (Ming-Lum et al., 2012) and were cultured in Mac medium (IMDM supplemented with 10% (v / v) FCS, 10 μM β-mercaptoethanol, 150 μM monothioglycolic acid, and 1 mM L-glutamine). As previously described (Cheung et al., 2013), J17 cells expressing wild-type and mutant His6-SHIP1, Clover-SHIP1, or mRuby2-STAT3 constructs were generated by lentivirus-mediated gene transfer. Transduced cells were selected with 5 μg / ml blasticidin. Clover-SHIP1 and mRuby2-STAT3 cells were further sorted for fluorescent-activated cells on a FACS Aria II cell sorter to select the brightest cells.

[0148] Isolation of mouse peritoneal macrophages. Primary peritoneal macrophages (perimacs) were isolated from mice by peritoneal lavage with 3 ml of sterile phosphate-buffered saline (PBS) (Thermo Fisher Scientific, Nepean, ON). The perimacs were collected and transferred to Mac medium.

[0149] Generation of bone marrow-derived macrophages. Bone marrow-derived macrophages (BMDMs) were generated by the following steps: First, femurs and tibias were collected from mice, and then the bone marrow was flushed out through a 26-G needle. The extracted cells were plated on 10-cm tissue culture plates in Mac medium supplemented with 5 ng / ml each of CSF-1 and GM-CSF (Stem Cell Technologies, Vancouver, BC) and incubated at 37 °C for 2 hours. Non-adherent cells were collected and replated at a density of 9×10 6 cells per 10-cm tissue culture plate. Then the cells were cultured in the presence of CSF-1 and GM-CSF. Differentiated BMDMs were used after 7 to 8 days. All cells were maintained in an incubator at 37 °C, 5% CO2, and 95% humidity.

[0150] Continuous flow culture. The continuous flow device facilitated constant stimulation and removal of cell supernatants to determine the kinetics of cytokine production over time. BMDMs were seeded at a density of 3×10 5 cells / well in 24-well tissue culture plates coated with poly-L-lysine (Thermo Fisher Scientific, Nepean, ON) and washed with PBS. After overnight incubation, the medium was removed and Leibovitz’s L-15 (L-15) medium (Invitrogen, Burlington, ON) supplemented with 3% FCS, 10 μM β-mercaptoethanol, and 150 μM monothioglycolic acid was added. The cells were allowed to equilibrate in L-15 medium for 1 hour and then placed in the continuous flow device. The stimulation solution was prepared in the same medium equilibrated at 37 °C and passed through a modified inlet mounted on the well by an infusion pump (New Era Syringe Pumps Inc., Farmingdale, NY). A flow rate of 150 μl per minute was used. At the same time, cell supernatants were removed from the wells at the same flow rate, and fractions were collected at 5-minute intervals over a 3-hour period. The levels of secreted TNFα in these fractions were analyzed by ELISA.

[0151] Real-time quantitative PCR. Total RNA was extracted using Trizol reagent (Invitrogen, Burlington, ON) according to the manufacturer's instructions. Approximately 2 - 5 μg of RNA was treated with DNAseI (Roche Diagnostics, Laval, QC) according to the product manual. For mRNA expression analysis, 120 ng of RNA was used in the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Laval, QC), and 0.1 μl to 0.2 μl of the resulting cDNA was analyzed by SYBR Green-based real-time PCR (real-time PCR) (Roche Diagnostics, Laval, QC), where real-time PCR used 300 nM gene-specific primers. The expression level of mRNA was measured using the StepOne Plus RT-PCR system (Applied Biosystems, Burlington, ON), and GAPDH was used as a normalization control to quantify the mRNA level by the comparative Ct method.

[0152] Measurement of TNFα production. Cells were seeded at a density of 50 × 10 4 cells / well in 96-well tissue culture plates and allowed to adhere overnight. The next day, the medium was changed 1 hour before stimulation. Cells were stimulated with 1 or 10 ng / ml LPS + / - different concentrations of IL10 for 1 hour. Supernatants were collected and the secreted TNFα protein level was measured using the BD OptEIA Mouse TNFα Enzyme-Linked Immunosorbent Assay (ELISA) Kit (BD Biosciences, Mississauga, ON). Each stimulation condition was performed in three parallel experimental wells. The IC50 value was calculated from three independent experiments, and the difference in the IL10 IC50 value between cells expressing the SHIP1 mutant and cells expressing the SHIP1 WT protein was analyzed by one-way ANOVA.

[0153] In vitro phosphatase assay. The phosphatase assay was performed in 96-well microtiter plates as described previously, using 10 ng of enzyme per well in a total volume of 25 μL of 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween-20, 10 mM MgCl2 (Ong et al., 2007). The enzyme was incubated with compound I-1 (dissolved in ethanol) at 23 °C for 10 minutes, and then 50 μM inositol-1,3,4,5-tetrakisphosphate (IP4) (Echelon Bioscience Inc., Salt Lake City, Utah) was added. The reaction was allowed to proceed at 37 °C for 10 minutes, and the amount of inorganic phosphate released was evaluated by adding the malachite green reagent and measuring the absorbance at 650 nm. Different concentrations of IP4 (0 - 300 μM) were used, and the reaction was terminated at different time points to determine the enzyme kinetics. The initial velocity was calculated, and K cat and K M .

[0154] In vitro pull down assay. J17 His6-SHIP1 and Y190F cells were seeded in 6-well plates at a density of 2×10 6 cells / well. After overnight incubation, fresh cell medium was added, and 30 minutes later, the cells were stimulated with 100 ng / ml of IL10, IL6, or 20 μM compound I-2 for 5 minutes. The cells were lysed in protein solubilization buffer (PSB, 50 mM Hepes, pH 7.5, 100 nM NaF, 10 mM sodium pyrophosphate, 2 mM NaVO4, 2 mM sodium molybdate, 2 mM EDTA) containing 1% octyl glucoside, 0.01 M imidazole, and protease inhibitor mixture (Roche Diagnostics, Laval, QC) at 4 °C for 30 minutes and centrifuged at 10,000 rpm for 15 minutes. Anti-EDTA Ni beads (Roche Diagnostics, Laval, QC) were added to the supernatant, and the mixture was incubated at 4 °C for 1 hour, then centrifuged, and the beads were washed three times with 0.1% octyl glucoside in PSB. The bead samples and the cell lysates of the starting materials were separated on 7.5% SDS-PAGE gels.

[0155] Immunoblotting. Protein lysates were separated on SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Etobicoke, ON). The membranes were blocked and probed overnight with the following primary antibodies where appropriate: SHIP1 (P1C1) (Santa Cruz Biotechnology), pSHIP1 (Y190) (Kinexus), STAT3 (9D8) (ThermoFisher Scientific), pSTAT3 (Y705) (ThermoFisher Scientific), STAT1 (BD Transduction Laboratories), pSTAT1 (Y701) (UpstateBiotechnology), GAPDH and actin (Sigma-Aldrich). The membranes were developed with Alexa 660 anti-mouse IgG or Alexa 680 anti-rabbit IgG antibodies (Thermo Fisher Scientific) and imaged using a LI-COR Odyssey imager.

[0156] Receptor photobleaching FRET assay. J17 cells expressing Clover-SHIP1 and / or mRuby2-STAT3 were seeded at 50×10 4Cells were seeded at a density of eff cells / well on an 8-well Ibidi μ-slide (Ibidi GmbH, Martinsried, Germany). After overnight incubation, the cells were serum-starved for 3 h in Mac medium containing 1% serum, and then the medium was replaced with Leibovitz’s (L-15) medium (Invitrogen, Burlington, ON) supplemented with 1% serum, 10 μM β-mercaptoethanol, 150 μM monothioglycolic acid, and 1 mM L-glutamine for confocal microscopy imaging. The cells were imaged on a Leica SP8X of a DMi8 confocal microscope system equipped with a 63X / 1.3 Gly HC PL APO CS2 objective lens, using a white light laser line (which uses 488 nm for donor excitation and 555 nm for acceptor excitation). Photobleaching FRET analysis was performed by measuring the Clover-SHIP1 donor fluorescence intensity of 60 frames at 100% laser intensity within the cell field of view of the "region of interest" (ROI) before and after bleaching the acceptor mRuby2-STAT3. Acceptor photobleaching was first performed in resting cells and then 1 minute (±5 s) after "simulated" stimulation with L-15 medium or L-15 medium containing 100 ng / ml IL10, IL6, or 20 μM compound I-2. The donor and acceptor fluorescence intensities of individual cells within the bleached ROI were quantified before and after bleaching. The percentage of FRET efficiency was calculated by the formula %FRET eff = 100 × (D 后 – D 前 ) / D 后 , where D 前 and D 后 represent the Clover-SHIP1 donor fluorescence intensities before and after bleaching, respectively.

[0157] Immunofluorescence. Perimacs were seeded at a density of 3 × 10 5 cells / well in an 18-well Ibidi μ-slide (Ibidi GmbH, Martinsried, Germany) and allowed to adhere for 3 h, and then washed with PBS to remove non-adherent cells. CD8+ T cells were seeded at a density of 2 × 10 6Cells were seeded at a density of [quantity of cells] in 12-well tissue culture plates. The cells were stimulated with 100 ng / ml IL10 or 20 μM Compound I-2 for 2 or 20 minutes, then washed with 3×PBS and fixed in 4% paraformaldehyde for 15 minutes at room temperature. The cells were incubated with anti-mouse CD16 / CD32 Fc Block (BD Pharmingen) for 1 hour, then incubated overnight at 4 °C with anti-SHIP1 antibody (P1C1) (Santa Cruz Biotechnology) or anti-STAT3 antibody (9D8) (ThermoFisher Scientific). The cells were then incubated with a secondary antibody, anti-mouse IgG (H+L)-Alexa Fluor 660 (ThermoFisher Scientific), for 1 hour, and then incubated with anti-CD11b-FITC antibody (BD Pharmingen) for 30 minutes for perimacs. Prior to confocal microscopy, CD8+ T cells were seeded on 18-well Ibidi μ-slides and the cells were stored in Ibidi mounting medium supplemented with ProLong Gold anti-fade reagent and DAPI (Molecular Probes, Life Technologies). The cells were imaged on a Leica SP5II confocal microscope with a 63x / 1.4-0.6 Oil PL APO objective on a DM6000 confocal microscope, using 405, 488, and 633 nm laser lines for excitation. Final images were scanned sequentially, acquiring 8 Z-stacks with an average of 4 frames. Colocalization analysis was performed using ImageJ software, first by merging the confocal images of each z-stack, and then by performing deconvolution and colocalization using the CUDA deconvolution and JACoP plugins, respectively. Pearson coefficient values were generated as a measure of the degree of overlap between SHIP1 or STAT3 and CD11b (Perimacs) or DAPI.

[0158] Mouse endotoxemia model. Groups of 6- to 8-week-old BALB / c SHIP1+ / + and SHIP1− / − mice were intraperitoneally injected with 1 or 5 mg / kg LPS, with or without co-administration of 1 mg / kg IL10. Blood was drawn by cardiac puncture 1 hour later for determination of plasma cytokine levels by ELISA. The ELISA kit for TNFα was purchased from BD Biosciences (Mississauga, ON).

[0159] Mouse colitis model. Colonic contents of conventional C57BL / 6 mice diluted 1:10 in PBS were administered via oral gavage to induce colitis in 6 - 8 week old BALB / c IL10- / - mice. Mouse body weight and fecal consistency were monitored and colitis was allowed to develop for 6 weeks. Ethanol (vehicle) and Compound I-1 (3 mg / kg) were diluted in the cage drinking water and dexamethasone (0.4 mg / kg) was administered via oral gavage every two days for a total of 3 weeks. At the end of the dosing period, proximal, mid, and distal colon segments were collected, paraffin-embedded or stored in RNALater (Invitrogen, Mississauga, ON) for RNA extraction. Slides were prepared by the UBC Pathology and Laboratory Medicine Department Histochemistry Laboratory, stained with hematoxylin and eosin and mounted. Pathological scoring of samples was performed by two independent, blinded researchers according to the method described by Madsen et al. (Madsen et al., 2001). Briefly, colonic inflammation was graded using a 4-point scale, scoring each of submucosal edema, immune cell infiltration, goblet cell ablation, and epithelial layer integrity from 0 - 3. For mRNA expression analysis, colon segments were homogenized and total RNA was extracted as described above and analyzed by real-time PCR using gene-specific primers for IL17, CCL2, and GAPDH (normalization control).

[0160] Expression of PAC2 for crystallographic analysis. The LIC-HMT-PAC2 expression vector was transformed into Escherichia coli Rosetta(DE3)pLacI cells. The overnight culture was inoculated at a 250-fold dilution to initiate the actual culture. The cells were cultured in LB medium (supplemented with 50 μg / ml kanamycin and 34 μg / ml chloramphenicol) with shaking at 225 rpm at 37 °C. When OD 600When it reaches approximately 0.6, the culture is cooled to room temperature, and then 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) is added to induce the expression of the recombinant protein. The culture is incubated overnight (usually 16 - 18 hours) at 22 °C in a shaker and then collected by centrifugation (at 4 °C, 5000 g, for 10 minutes). Subsequently, the cell pellet is resuspended in lysis buffer (20 mM Tris-HCl pH 7.4, 350 mM NaCl, 10 mM TCEP, 5 mM imidazole, supplemented with 1× EDTA-free protease inhibitor cocktail (PIC) (Roche Diagnostics, Laval, QC) and 25 μg / ml lysozyme) and lysed by sonication (2-minute pulses, for 2 cycles) on ice. Cell debris is removed by two rounds of centrifugation, the first round at 5000 g for 15 minutes at 4 °C and then at 18000 rpm for 30 minutes at 4 °C. The supernatant is filtered through a 0.45 μm filter and loaded onto a Talon Co 2+ affinity column pre-equilibrated with buffer A (20 mM Tris-HCl pH 7.4, 250 mM NaCl, 1 mM TCEP), and washed with 10 column volumes (CV) of buffer B (buffer A + 5 mM imidazole). The bound protein is eluted with 6 CV of buffer C (buffer A + 50 mM imidazole).

[0161] To remove the HMT tag, TEV protease (internally purified His6-tagged protein) was added to the eluted protein, and then it was dialyzed overnight at 4 °C with gentle stirring against buffer D (20 mM Tris-HCl pH 7.4, 250 mM NaCl, 1 mM TCEP). The dialyzed sample was loaded onto an Amylose column (New England Biolabs, Whitby, ON), and the flow-through containing the untagged protein was loaded onto a Talon column to remove the His6-TEV protease. The flow-through from the Talon column was dialyzed overnight at 4 °C with gentle stirring against buffer E (20 mM Tris-HCl pH 7.4, 25 mM NaCl, 1 mM TCEP), and then loaded onto a ResourceQ column (6 ml column volume) (GE Healthcare, Mississauga, ON) and washed with 3 CV of buffer E. The protein was eluted using buffer F (20 mM Tris-HCl pH 7.4, 1000 mM NaCl, 1 mM TCEP). The components in the protein sample were separated using a gradient from 25 mM NaCl (0% buffer G) to 200 mM NaCl (20% buffer G) in 20 CV. The fractions were analyzed by SDS-PAGE. PAC2 was typically eluted from the ResourceQ column at ~130 mM NaCl. The purified protein was concentrated to approximately 5 - 10 mg / ml using an Amicon concentrator with a 30K MWCO (Millipore, Etobicoke, ON) and exchanged into the desired buffer. The buffer required for protein crystallization contained 50 mM Tris-HCl pH 7.4, 25 mM NaCl, and 0.5 mM TCEP. For biolayer interferometry, the HMT-PAC2 protein eluted from the first Talon column was directly purified on a ResourceQ column without excision of the HMT tag.

[0162] Purification of PAC2-Avi tag for biolayer interferometry analysis. The sequence corresponding to the Avi-tag (GLNDIFEAQKIEWHE) was added to the c-terminus of PAC2 in the LIC-HMT-PAC2 expression vector by standard restriction digestion and ligation. The LIC-HMT-PAC2-Avi expression vector was then co-transformed into Escherichia coli BL21 cells with the pBirAcm expression vector at a 1:1 molar ratio. The overnight culture was inoculated at a 250-fold dilution to initiate the actual culture. The cells were grown in LB medium (supplemented with 50 μg / mL kanamycin and 10 μg / mL chloramphenicol) at 37 °C with a shaking speed of 225 rpm. When OD 600When it reached approximately 0.6, 5 mM biotin in Bicine buffer at pH 8.3 was added to the culture to obtain a final concentration of 125 μM biotin. The culture was then cooled to room temperature and then 0.4 mM isopropyl - β - D - 1 - thiogalactopyranoside (IPTG) was added to induce the expression of the recombinant protein with an Avi - tag. The rest of the method was the same as written in "Expression of PAC2 for crystallographic analysis".

[0163] Protein crystallization, data collection, phase - determination and refinement. Using commercially available crystallographic solutions (Qiagen, Toronto, ON), initial crystallization hits were obtained by sparse - matrix screening in 96 - well plates. Optimization of crystallization conditions was carried out in 24 - well plate format using the hanging - drop vapor - diffusion method. At room temperature, protein crystals of diffraction quality were obtained at a protein concentration of 4 - 7 mg / ml using 0.1 M HEPES - NaOH pH 6.7, 20% PEG1500 and 5 mM MgCl2. The PAC2 - cc protein contains surface entropy reduction mutations (E770A, E772A, E773A) and contributed to the improvement of crystal quality. A unique fragment of the protein crystal cluster was soaked in the crystallization solution containing 25% isopropanol for 5 to 10 seconds and flash - frozen in liquid nitrogen.

[0164] Diffraction data sets were collected at the Advanced Photon Source (APS) beamline 23 - ID - D - GM / CA and processed by XDSGUI 45 with XDS. In Phaser MR 47 the phase problem was solved using an unpublished structure as a search model. The initial model was refined using COOT 48 and Refmac5 49 For the final model, the occupancy of side - chains was refined for Phenix (Adams et al., 2010) and three TLS groups were defined. Data collection and refinement statistics are shown in Table 1. The model and data were deposited under Protein Data Bank ID 6DLG.

[0165] Small - angle X - ray scattering. 190 μM PAC1 samples were in 50 mM TrisHCl (pH 7.4), 150 mM NaCl, 1 mM TCEP and 2% EtOH with and without 570 μM compound I - 1 (6 - fold molar excess). Before collecting SAXS data, dynamic light scattering (DLS) data of PAC1 and PAC1 - compound I - 1 complex were collected to confirm that all samples were highly pure and suitable for data collection. Data collection was carried out on a Rigaku micro - focus sealed tube (Cu Kα radiation, ) and on a 3-pinhole camera (S-MAX3000; Rigaku Americas, The Woodlands, TX) of a confocal Max-Flux (CMF) optical system (Rigaku) operating at 40 W. Scattering data were recorded using a 200 mm multi-wire two-dimensional detector. For each sample, data of the sample and buffer in the range were collected for 3 h and processed, where s = 4πsinθ / λ (Patel et al., 2011; Patel et al., 2010; Patel et al., 2012). The normalized spatial differences (NSDs) of the PAC1 model without and with ligands were 0.6 and 1.0, respectively.

[0166] Biolayer interferometry analysis. Using a streptavidin (SSA) biosensor tip and an Octet Red 96 instrument (ForteBio, Fremont, CA), the binding affinity between PAC2 protein and small molecule allosteric regulators was detected by biolayer interferometry (BLI) experiments. Before protein immobilization, the SSA biosensor tip was hydrated in assay buffer 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10 mM MgCl2, 0.5 mM TCEP, 0.2% Tween-20. At 4 °C, 0.5 μg / mL protein was immobilized on the SSA biosensor overnight. After immobilizing the protein on the biosensor, the tip was blocked with 0.1% BSA for 90 minutes and then washed with assay buffer supplemented with 1% EtOH for 20 minutes. Kinetic measurements were performed at 30 °C using an orbital flow of 1,000 RPM. The assay buffer supplemented with 1% EtOH was held for 60 s to obtain the baseline. Binding was measured for 600 s at an analyte concentration of 20 μM, and then dissociation was measured for 300 s in the same buffer as the baseline. The raw data were analyzed using Octet Red Data Analysis Software (ver. 8.2). The raw data were aligned with the baseline and trimmed using single-reference and double-reference subtraction.

[0167] Quantitative and statistical analysis

[0168] Band quantification of all immunoblots was performed using the LI-COR Odyssey imaging system and Image Studio™ Lite software (LI-COR Biosciences, Lincoln, NE). All statistical analyses were performed using GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA). Statistical details can be found in the legends. Values are presented as mean ± standard deviation. Unpaired t-tests were used to generate two-tailed P values where appropriate. One-way or two-way ANOVA was performed using appropriate multiple comparison tests when needed. Differences were considered significant when p ≤ 0.05.

[0169] Availability of data and software

[0170] The X-ray crystallography data that support the findings of this study have been deposited in the Worldwide Protein Data Bank (wwPDB) under the ID code PDB ID 6DLG.

[0171] Compound preparation

[0172] A 50 mg / mL stock of Compound I-1 in 50% Cremophor EL / 50% ethanol v / v was prepared according to the general procedure below. A 100 mg / mL solution of Compound I-1 in Cremophore EL and an equal volume of ethanol were mixed together by pipetting (about 50 times or more to ensure only one phase was visible). Stock solutions of other compounds were prepared in a similar manner. The stock solutions were kept at 4 °C. The stock solutions could be warmed to room temperature before preparing the working solutions (see below).

[0173] A 5 mg / mL solution of Compound I-1 in 5% v / v Cremophore EL / 5% v / v ethanol and PBS (phosphate-buffered saline) solution was prepared according to the general procedure below. Calculate the volume of the 50 mg / mL stock solution of Compound I-1 in 50% Cremophor EL / 50% ethanol v / v required, excluding the volume of water. Add an equal volume of water to the required volume of the 50 mg / mL stock solution and mix well by pipetting. Rinse the pipette tip used for the stock solution several times with small remaining volumes of water until the pipette tip is clean and the desired total volume of the 5 mg / mL solution is reached. Mix the resulting solution well until homogeneous.

[0174] Administration to mice in drinking water

[0175] The mice used weighed approximately 30 g each and consumed approximately 2 mL of water per day.

[0176] Administer the compounds of the present disclosure, such as Compound I-1, to IBD model mice at 2 or 3 mg / kg. Administer the compounds of the present disclosure, such as Compound I-1, to MM model mice at about 20 to about 50 mg / kg. Dilute a suitable amount of the 5 mg / mL PBS solution containing 5% v / v Cremophore EL / 5% v / v ethanol prepared as described above in the drinking water of the mice.

[0177] Example 1. IL10 requires SHIP1 and STAT3 to inhibit TNFα production in macrophages

[0178] Takeda et al. (Takeda et al., 1999) first described the role of STAT3 in mediating IL10 inhibition of TNFα in vivo. It has been found that compared with wild-type mice, administration of LPS to STAT3 myeloid-specific knockdown mice produces more TNFα, and the conclusion is that endogenous IL10 cannot counteract STAT3 - / - LPS signaling in mice. However, a more careful examination of their data shows that although in IL10 - / - mice, TNFα levels are still higher after administration of LPS (Berg et al., 1995), but TNFα levels decline in STAT3 - / - mice with the increase in endogenous IL10 levels (Takeda et al., Figure 2 B). This implies that other proteins besides STAT3 may contribute to the action of IL10. It has been previously shown that culture-based studies have indicated that SHIP1 is involved in the action of IL10 (Chan et al., 2012; Cheung et al., 2013). Currently, in SHIP1 + / + and SHIP1 - / - mice, it is shown that IL10 can inhibit the expression of LPS-induced inflammatory cytokines / chemokines in vivo ( Figure 1 A), and it is found that IL10 inhibits TNFα in SHIP1 + / + mice, but cannot inhibit TNFα in SHIP1 - / - mice. It has been previously shown that the phosphatase activity of SHIP1 is allosterically stimulated by its product PI(3,4)P2 (Ong et al., 2007). Compound I-1 (previously known as AQX-MN100) binds to the same SHIP1 C2 domain as PI(3,4)P2 and enhances the functional activity of SHIP1 (Ong et al., 2007). Currently, it is shown that Compound I-1 can inhibit LPS-induced TNFα in SHIP1 + / + mice, but cannot inhibit LPS-induced TNFα in SHIP1 - / - mice, indicating that these compounds can mimic the anti-inflammatory properties of IL-10 and are indeed specific for SHIP1 ( Figure 1 B).

[0179] Without wishing to be bound by theory, SHIP1 and STAT3 can act independently or together in mediating the action of IL10. To help distinguish between these two possibilities, a continuous flow cell culture system was used to evaluate the kinetics of TNFα production in wild-type and knockout bone marrow-derived macrophages (BMDMs) of SHIP1 and STAT3. LPS stimulated two peaks of TNFα expression, one at around 1 hour and the other at 3 hours ( Figure 1 C). IL10 reduced the TNFα level in both SHIP1 + / + and STAT3 + / + cells, but was completely impaired in inhibiting the 1-hour peak in both STAT3 - / - and SHIP1 - / - cells and was partially impaired in inhibiting the 3-hour peak in both KO BMDMs. This consistent pattern of non-responsiveness suggests cooperation between SHIP1 and STAT3.

[0180] Figure 1 Shows the serum TNFα levels of SHIP1 + / + or SHIP1 - / - mice injected intraperitoneally with LPS, LPS + IL10 (Figure A) or LPS + compound I-1 (ZPR-100 or ZPR-MN100 or MN-100) (Figure B) at the indicated concentrations for 1 h. Data represent the mean of n≥4. *p<0.05, **p<0.01, ns = not significant when compared to mice stimulated with LPS only. Figure C shows the stimulation of STAT3 + / + , STAT - / - , SHIP1 + / + and SHIP1 - / - bone marrow-derived macrophages (BMDMs) in a continuous flow device with LPS (dashed line) or LPS + IL10 (solid line) over a period of 180 min. Fractions were collected every 5 min for measuring TNFα levels. Data represent two independent experiments.

[0181] Example 2. IL10 induces physical association of SHIP1 and STAT3 in macrophages

[0182] Both SHIP1 and STAT3 proteins are present in the cytoplasm of resting cells and are recruited to the cell membrane in response to extracellular stimuli by different mechanisms. STAT3 mainly functions as a transcription factor (Matsuda et al., 2015), while SHIP1 is best known for its lipid phosphatase activity (Pauls and Marshall, 2017). However, SHIP1 can also act as a docking or adaptor protein for the assembly of signaling complexes (Pauls and Marshall, 2017). In fact, a SHIP1 isoform with the lowest phosphatase activity (3PT) (An et al., 2005) has been found to mediate the inhibitory effect of IL10 on LPS-stimulated TNFα production ( Figure 2 A), and thus it was investigated whether SHIP1 could act as an adaptor protein in IL10 signaling and be associated with STAT3 in response to IL10. Figure 2 B shows that treating cells with IL10 led to co-precipitation of SHIP1 and STAT3. Even when STAT3 became tyrosine phosphorylated to the same extent in response to IL10, IL6 could not induce the association of STAT3 with SHIP1. Notably, treating cells with the small molecule SHIP1 allosteric modulator compound I-2 was sufficient to induce the association of SHIP1 and STAT3 ( Figure 2 B). The ability of compound I-2 to induce the association of SHIP1 and STAT3 suggests that the binding of compound I-2 may induce conformational changes that can alter the association of SHIP1 with other proteins. To observe whether the SHIP1 / STAT3 interaction occurs in intact cells, Clover-SHIP1 and mRuby2-STAT3 fusion protein constructs were generated and transduced into J17SHIP1 - / - cells for FRET analysis. Figure 2 C shows that stimulating Clover-SHIP1 / mRuby2-STAT3 cells with IL10 or compound I-2 (but not IL6) increased the Clover-mRuby2 FRET signal, indicating that SHIP1 and STAT3 interact in vivo.

[0183] Both SHIP1 and STAT3 have SH2 domains and both have been reported to be phosphorylated on tyrosine residues, so the formation of the complex may be mediated by phosphorylated tyrosine / SH2 interactions. Since Figure 2Panel B shows that STAT3 does not have to be phosphorylated to bind to SHIP1 (see lane I-2), so it was investigated whether tyrosine residues on SHIP1 might be phosphorylated to interact with the STAT3 SH2 domain. In the context of the STAT3 SH2 domain recognition sequence, there are 4 tyrosine residues in SHIP1. SHIP1 mutants were constructed in which each of these residues was replaced with phenylalanine, and they were expressed in the J17 SHIP1 - / - macrophage cell line, and the ability of IL10 to inhibit TNFα expression in these cells was tested ( Figure 3 A). Cells expressing the Y190F mutant behaved similarly to SHIP1 - / - cells ( Figure 3 A). In response to IL10 and compound I-2, the ability of the Y190F mutant to interact with STAT3 was reduced 2-fold ( Figure 3 B and 3C), indicating that phosphorylation of SHIP1 Y190 is required for a portion of the interaction of SHIP1 with STAT3.

[0184] The subcellular localization of SHIP1 and STAT3 in primary cells was also evaluated. Wild-type, SHIP1 - / - or STAT3 - / - peritoneal macrophages were stimulated with IL10 or compound I-2 and stained with antibodies against SHIP1 or STAT3. Figure 4 A and Figure 4 B show that in wild-type cells, IL10 or compound I-2 induced membrane association of SHIP1 and STAT3 at 2 min. SHIP1 did not translocate in STAT3 - / - cells, and STAT3 did not translocate in SHIP1 - / - cells ( Figure 4 B). At 20 min, both SHIP1 and STAT3 were found in the nuclei of wild-type cells, and translocation required cells to express STAT3 and SHIP1. Thus, compound I-1 can mimic IL10 with respect to SHIP1 and STAT3 translocation.

[0185] Example 3. SHIP1 undergoes conformational changes upon binding of allosteric modulators

[0186] To better understand the interaction of the small molecule allosteric regulator with SHIP1, truncated SHIP1 proteins were generated for X-ray crystallography ( Figure 5), where the truncated SHIP1 protein contains the minimal SHIP1 region required for allosterically regulated phosphatase activity. Full-length SHIP1 could not be expressed in amounts sufficient for crystallography, thus defining the minimal region of SHIP1 required for allosteric activation. The C2 domain has previously been shown to bind SHIP1 allosteric regulators (PI(3,4)P2, compound I-1) and may be involved in the PH-R domain at the N-terminus of the phosphatase domain (Ong et al., 2007). Full-length SHIP1 protein (which could only be produced in mammalian 293T cells), PPAC protein (which contains the PH-R-phosphatase-C2 domains and could be expressed in 293T cells and Escherichia coli), and PAC1 / PAC2 proteins (which contain the phosphatase-C2 domains and could be expressed in Escherichia coli) were expressed ( Figure 5 A). Their enzymatic (phosphatase) kinetic properties and the ability to be activated by compound I-1 were examined. It has been found that PPAC from 293T and Escherichia coli has the same enzymatic properties ( Figure 5 B), and all four proteins (full-length SHIP1, 293T-derived PPAC, Escherichia coli-derived PPAC, and PAC2) could be activated by compound I-1 ( Figure 5 C).

[0187] Only PAC1 and PAC2 proteins could be expressed in amounts required for structural studies, so these proteins were produced for condition screening to yield crystals of sufficient quality for structure determination. This included creating surface entropy reduction variants (Derewenda, 2004; Goldschmidt et al., 2007), designated PAC1-cc and PAC2-cc, in which three glutamate residues in PAC1 and PAC2 were replaced with alanine. The structures of several PAC1-cc and PAC2-cc crystals were solved and are available in the PDB (PDB ID 6DLG), and the data for PAC2-cc crystals diffracting at the resolution shown are presented in Table 1. PAC1 models with and without compound I-1 were generated using small-angle X-ray scattering data (SAXS). The ligand-free PAC1 solution conformation determined by SAXS confirmed the X-ray crystal structure.

[0188] Table 1. Data for PAC2-cc crystals

[0189]

[0190] *Values for the highest resolution shell are listed in parentheses

[0191] In addition, SAXS analysis showed that the binding of compound I-1 to PAC1 led to a change in its overall conformation.

[0192] Using molecular modeling (Fuqiang et al., 2018), potential binding pockets for compounds I-1 and I-2 in PAC2 were identified. The residue K681 in this pocket was predicted to be involved in binding compounds I-1 and I-2, thus generating a K681A point mutation in PAC2, and the ability of wild-type and PAC2-K681A to bind compound I-2 was tested using Biolayer Interferometry (BLI). As Figure 6 shown in A, the K681A substitution in the putative pocket impaired the ability of compound 1-2 and PI(3,4)P2 to bind to PAC2. The effect of the K681A substitution on the ability of full-length SHIP1 to mediate IL10 inhibition of macrophages was evaluated. Figure 6 B shows that in cells expressing wild-type (but not K681A) SHIP1, IL10 potently inhibits TNFα production.

[0193] Stenton et al. described a molecule called AQX-1125 (structure in Figure 7 A, later given the clinical trial name Rosiptor) as a SHIP1 agonist (Stenton et al., 2013a; Stenton et al., 2013b). However, AQX-1125 / Rosiptor has a small enhancement of SHIP1 phosphatase activity (Stenton et al., 2013b) and shows different enzyme kinetic properties from those observed when using compound I-1 (Ong et al., 2007) (Stenton et al., 2013b). Stenton et al. studied the binding of tritiated AQX-1125 / Rosiptor to the SHIP1 protein using a scintillation proximity assay, but it was difficult to assess the significance of the ~300 cpm signal they observed. Therefore, here, the ability of PI(3,4)P2, compound I-2, and AQX-1125 / Rosiptor to bind to SHIP1 was compared in a BLI assay ( Figure 7 B), and it was found that AQX-1125 / Rosiptor binds very weakly to SHIP1 compared to compound I-2 or the natural agonist of SHIP1, PI(3,4)P2.

[0194] Example 4. Remission of inflammation in the IL-10 - / - murine colitis model

[0195] To demonstrate the in vivo anti-inflammatory effect of the compounds of the present disclosure, the exemplary compound I-1 was administered to an IL-10 knockout mouse colitis model (Keubler 2015). Colitis occurs in IL10 knockout mice when colonized with normal gut microbiota because IL10 is required to temper the host immune response to intestinal commensal bacteria (Keubler 2015; Kuhn 1993). Colitis was induced in these mice by inoculating IL10− / − mice with freshly isolated colonic contents from normal, specific pathogen-free mice and allowing the inflammation to develop for 6 weeks (Sydora 2003). The mice were then treated with vehicle, 2 mg / kg of compound I-1, or 0.4 mg / kg of dexamethasone (a steroidal anti-inflammatory drug used as a positive control) for 3 weeks, and then the colon tissues were collected for analysis. Hematoxylin and eosin-stained sections were prepared from the proximal, mid, and distal colon of the mice as well as from mice that were not inoculated with microbiota (non-colitis group) ( Figure 8 A). Two researchers blinded to the treatment groups scored the sections based on submucosal edema, immune cell infiltration, presence of goblet cells, and epithelial integrity ( Figure 8 B). Among the three groups in which colitis was induced, the pathological scores of the dexamethasone and compound I-1 groups were significantly lower than those of the vehicle group ( Figure 8 B). RNA was prepared from the colon of all four groups for analysis of the expression of IL17 and CCL2 (inflammatory mediators elevated in colitis) (Lee 2007). As shown in Figure 8 C, treatment with both compound I-1 and dexamethasone significantly reduced the levels of IL17 and CCL2 mRNA. These data indicate that in colitis caused by IL10 loss, treatment with compound I-1 can alleviate inflammation. Compound I-1 was found to be as effective as dexamethasone in reducing histological and molecular markers of colonic inflammation.

[0196] Example 5. Discussion

[0197] IL6 and IL10 have opposite pro-inflammatory and anti-inflammatory effects on macrophages, respectively (Garbers et al., 2015; Yasukawa et al., 2003), but both cytokines stimulate tyrosine phosphorylation of Y705 of STAT3 in cells. It was found that IL10 (but not IL6) induces the association of STAT3 with SHIP1, and it was suggested that this difference might account for why STAT3 can mediate pro-inflammatory and anti-inflammatory responses downstream of the two cytokines. IL10-induced SHIP1 / STAT3 signaling supports the anti-inflammatory response, while IL6-induced STAT3 / STAT3 dimer supports the pro-inflammatory response. Studies by Yasukawa et al. on SOCS3 knockout cells suggested that the duration of STAT3 activation in macrophages might underlie the opposite biological effects of IL10 and IL6 (Yasukawa et al., 2003). The data of the present invention are compatible with their data, because STAT3 activation can also be prolonged by its association with SHIP1.

[0198] The question of how STAT3 can mediate opposite biological responses was also explored to understand the opposite effects of IL6 and IL27 on naive T cell differentiation. Treatment of naive T cells with IL6 inhibits Th1 and enhances Th2 and Th17 differentiation, while IL27 treatment has the opposite effect (Hirahara et al., 2015; Peters et al., 2015). Both IL6 and IL27 strongly activate STAT3, but IL27 activates STAT1 more strongly than IL6. Hirahara et al. used a genomic approach to conclude that STAT3 controls the overall intensity of transcription, but the activation level of STAT1 controls the expression of IL27-specific gene expression (Hirahara et al., 2015). Observations reported by Peters et al. are consistent with this conclusion, that is, if STAT1 is deleted, both IL6 and IL27 stimulate Th17 differentiation (Peters et al., 2015).

[0199] However, the opposite effects of IL10 and IL6 on macrophage activation are not due to different utilization of STAT3 or STAT1, because the two cytokines induce similar STAT1 and STAT3 phosphorylation ( Figure 9 ).

[0200] It has been previously shown that small molecule SHIP1 agonists have anti-inflammatory effects in vitro (Meimetis et al., 2012; Ong et al., 2007), and these effects have been attributed to stimulation of the phosphatase activity of SHIP1 to dephosphorylate the product of PI3K, PIP3, to PI(3,4)P2 (Fernandes et al., 2013; Huber et al., 1999; Krystal, 2000; Pauls and Marshall, 2017). However, the data presented in this disclosure confirm that SHIP1 protein with undetectable phosphatase activity is sufficient to mediate the anti-inflammatory effects of IL10, and thus the adaptor function of SHIP1 itself can support the action of IL10. The SAXS analysis presented in this disclosure indicates that binding of a SHIP1 agonist to SHIP1 causes a conformational change in SHIP1. This conformational change may allow SHIP1 to interact with STAT3 and allow translocation of the SHIP1 / STAT3 complex to the nucleus. The structure of the minimal SHIP1 domain (PAC1 / 2) required to mediate the allosteric effect of the SHIP1 agonist was resolved, and the drug-binding pocket was identified by molecular docking analysis. Mutations of residues predicted to be involved in the binding of Compound I-2 completely disrupted the binding of Compound I-2, as well as the ability of SHIP1 to mediate IL10 inhibition of TNFα expression in macrophages.

[0201] It was found that SHIP1 Y190 contributes to the ability of SHIP1 to associate with STAT3. The ability of the Y190F mutant to interact with STAT3 was reduced 2-fold compared to wild-type SHIP1 ( Figure 3 B). However, SHIP1 Y190F completely disrupted its ability to support IL10 inhibition of TNFα ( Figure 3 A). Without wishing to be bound by theory, one explanation is that inhibition of a fraction of the SHIP1 / STAT3 complex is physiologically important because it completely disrupts the inhibition of TNFα. Alternatively, formation of the SHIP1 / STAT3 complex is merely a function of Y190. The SHIP1 agonist Compound I-1 itself can induce the formation of the SHIP1 / STAT3 complex. Addition of Compound I-2 to perimacs can also induce translocation of SHIP1 and STAT3 to the nucleus. These data together suggest that the actions of SHIP1 agonists include both their ability to stimulate SHIP1 phosphatase activity and to induce association of SHIP1 with STAT3.

[0202] In the present and previous studies, treatment of macrophages with compound I-1 or I-2 was sufficient to elicit an anti-inflammatory effect similar to that of IL10 in vitro (Chan et al., 2012; Cheung et al., 2013; Ong et al., 2007). Therefore, compound I-1 was tested in a murine colitis model because the beneficial anti-inflammatory effect of IL10 in colitis is mediated through the action of IL10 on macrophages (Friedrich et al., 2019; Ouyang and O’Garra, 2019; Shouval et al., 2014b; Zigmond et al., 2014). Compound I-1 was found to be as effective as dexamethasone in reducing histological and molecular markers of colonic inflammation. Medzhitov's group recently reported that IL10 stimulates mitophagy and inflammasome inactivation as part of its protective role in colitis, and that this involves STAT3-dependent upregulation of the DDIT4 protein (Ip et al., 2017). It has been demonstrated here that STAT3 and SHIP1 are required for IL10 to upregulate DDIT4 in macrophages; moreover, compound I-2 alone was able to induce DDIT4 expression.

[0203] Recently, the small molecule SHIP1 allosteric modulator (AQX-1125 / Rosiptor) (Stenton et al., 2013a; Stenton et al., 2013b) was tested in a clinical trial for its effect in alleviating bladder pain experienced by patients with interstitial cystitis (IC) (Nickel et al., 2016). IC was chosen as the disease indication because AQX-1125 / Rosiptor accumulates in the bladder (Stenton et al., 2013b), two papers suggested the presence of PI3K-dependent inflammation in IC (Liang et al., 2016; Qiao et al., 2014), and the preliminary phase 2 trial appeared promising (Nickel et al., 2016). However, the phase 3 trial failed to show the efficacy of AQX-1125 / Rosiptor (AQXP, 2018). There are many reasons for the failure of small molecule drugs during the drug development process. However, it is notable that neither IL10 nor SHIP1 has been implicated in the physiology / pathophysiology of IC. In addition, AQX-1125 / Rosiptor was found to bind very weakly to SHIP1, consistent with the finding by Stenton et al. that AQX-1125 has very weak SHIP1 phosphatase activation activity (Stenton et al., 2013b).

[0204] It can be seen from this that the disease indications for which small molecule SHIP allosteric modulators are developed should be some diseases in which IL10 (or other physiological modulators of SHIP1) (Chan et al., 2012; Cheung et al., 2013; Dobranowski and Sly, 2018; Pauls and Marshall, 2017) have shown beneficial effects. These small molecules should also have binding properties to SHIP1 similar to those of the natural ligand of SHIP1, PI(3,4)P2. According to these criteria, small molecule SHIP1 agonists such as the Pelorol family can be used to treat human inflammatory bowel disease.

[0205] Example 6. Inhibiting MM in a mouse model

[0206] MM.1S cells expressing firefly luciferase were injected into the upper flanks of NOD / SCID mice together with Matrigel basement membrane and allowed to establish for 2 weeks. Compound I-1 or vehicle (n = 4) was administered via drinking water (as described in the general method). Bioluminescence images of mice treated with control and Compound I-1 were obtained and shown in Figure 10 A. Tumor volume was quantified using bioluminescence imaging and shown in Figure 10 B. Administration of Compound I-1 to mice bearing MM tumors effectively reduced the tumor mass.

[0207] Example 7. Efficacy of the compound in protecting against liver injury

[0208] The concanavalin A (ConA)-induced liver injury model is an immune-mediated liver injury model, similar to viral and autoimmune hepatitis in humans. It is known that intravenous delivery of ConA in mice can activate T cells, leading to an increase in inflammatory cytokines such as TNF-a, IFN-r, and IL-6, and a decrease in the anti-inflammatory cytokine IL-10. Infiltration of T cells in the liver results in hepatocyte apoptosis and necrosis, leading to an increase in the levels of liver enzymes ALT and AST in the plasma (Zhou 2015).

[0209] Method: Five groups (n = 5 per group) of C57 mice were treated with either a single blank control, a single Compound I-1 (10 mg / kg / d), ConA (15 mg / kg), ConA + Compound I-1 (3 mg / kg / d), or ConA + Compound I-1 (10 mg / kg / d). In each case, Compound I-1 was administered twice daily via oral gavage for 5 days before ConA injection (if applicable). One hour after the last administration, ConA (15 mg / kg) was delivered intravenously. Twelve hours after ConA injection, orbital blood was obtained from the eye and subjected to enzyme biochemistry (plasma) and blood cell type assays using an analyzer.

[0210] Results: As Figure 11 shown in A - D below, ConA at 15 mg / kg significantly increased the levels of ALT, AST, TBIL, and BUN ( Figure 11 A - D). Compound I - 1 (MN - 100) significantly decreased the levels of ALT, AST, TBIL, and BUN (*P < 0.05), confirming its anti - inflammatory effect in ConA - induced liver injury. In a repeated experiment with dexamethasone (0.5 mg / kg / d) co - administered with ConA as a positive control, the plasma levels of ALT and AST were determined ( Figure 11 E and F). It can be seen that, similar to the positive control dexamethasone, compound I - 1 significantly decreased the ConA - induced levels of ALT and AST.

[0211] Example 8. SHIP1 agonist for the treatment of severe sepsis in a mouse cecal ligation and puncture (CLP) model

[0212] The murine CLP model is a recognized clinically relevant method for anti - sepsis drug testing. The surgical procedure involves opening the abdomen of the mouse, ligating the cecum, and puncturing the ligated cecum with a needle. Moderate to high - grade experimental sepsis is induced following the method of Rittirsch, 2009, achieving a survival rate of 70 - 100% within 7 days after CLP surgery.

[0213] Methods: Five groups of mice (n = 10 per group) were designed, namely (1) blank control, (2) sham - operation control (undergoing abdominal surgery but without cecum ligation / puncture), (3) CLP group (undergoing surgery and cecum ligation / puncture), (4) CLP + compound I - 1 (MN100) (3 mg / kg / d), and (5) CLP + compound I - 1 (MN100) (10 mg / kg / d). ZPR - MN100 (compound I - 1) was administered 3 days before CLP surgery and continued until 7 days after CLP. ZPR - MN100 was delivered via oral gavage twice a day. At 24 hours after CLP, tail blood was collected under sterile conditions for blood culture to confirm septic infection after CLP surgery. The condition and survival rate of the mice were recorded daily. The experiment was terminated at 7 days after CLP, and survival curves were plotted using GraphPad Prism.

[0214] Results: As Figure 12 shown below, at 24 hours after CLP surgery, bacterial colonies formed in the blood culture from the CLP surgery group ( Figure 12 B) but not from the blank control ( Figure 12 A) or the sham - operation control ( Figure 12 C, mice only received skin incision and wound closure without cecum ligation or puncture), confirming successful establishment of the CLP model. Compound I - 1 protected mice from CLP - induced death in an oral - delivery dose - dependent mannerFigure 13 )。The 7-day survival rate of the CLP group without compound I-1 treatment was 20% ( Figure 13 A). Compound I-1 was able to increase the survival rate dose-dependently to 50% mouse survival (compared to the 20% survival rate in the CLP group without drug).

[0215] Example 9. Stimulating the IL-10 / IL-10R pathway as a treatment for allergy and asthma

[0216] It has been determined that IL-10 plays a very important role in suppressing allergic inflammation and preventing the occurrence of allergic airway diseases and asthma (Hawrylowicz et al., 2005; Coomes SM et al., 2015). Allergic rhinitis (AR) is a prevalent inflammatory airway disease for which there is no effective treatment. In a mouse model, in an OVA-induced AR model, administration of recombinant IL-10 appeared to reduce the number of eosinophils and mast cells in the nasal mucosa of AR mice (Wang et al., 2014), indicating that the IL10 / IL10R pathway is an effective target for AR treatment. Small molecule SHIP1 agonists such as compound 1-2 (ZPR-151) can be used to inhibit rhinitis inflammatory responses by activating the IL-10 / IL10R pathway.

[0217] Although the present disclosure has been described with reference to the embodiments, it should be understood that the scope of the claims should not be limited by the embodiments set forth in the examples, but should be given the broadest interpretation consistent with the entire specification.

[0218] All publications, patents, and patent applications are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, and patent application were specifically and individually indicated to be incorporated by reference in its entirety. In the event that a term in the present disclosure is found to be different from the definition in the documents incorporated by reference herein, the definition provided herein shall be used as the definition of that term.

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Claims

1. Use of a compound of formula I or a pharmaceutically acceptable salt, solvate and / or prodrug thereof in the manufacture of a medicament for the treatment of a disease, disorder or condition mediated by SHIP1 or treatable by activation of SHIP1, wherein R 1 is selected from NHSO2C 1-3 alkyl, N-succinimide and NHC(O)C 1-3 alkyl; wherein R 2 、R 3 、R 4 and R 5 are independently selected from H, OH, C 1-3 alkyl, OC 1-3 alkyl, NH2, NHC 1-3 alkyl, NHSO2C 1-3 alkyl and NHC(O)C 1-3 alkyl; or R 2 and R 3 、R 3 and R 4 or R 4 and R 5 together with the atom to which they are attached form a substituted or unsubstituted 5- or 6-membered heterocycle, said substituted or unsubstituted 5- or 6-membered heterocycle containing at least one NH and optionally one or more additional heteroatoms selected from N, O and S; and wherein when R 2 and R 3 , R 3 and R 4 or R 4 and R 5 combine together to form the substituted or unsubstituted 5- or 6-membered heterocycle, R 4 and R 5 , R 2 and R 5 or R 2 and R 3 are each independently selected from H and C 1-3 alkyl, wherein the disease, disorder or condition mediated by SHIP1 or treatable by activation of SHIP1 is selected from inflammatory bowel disease, multiple myeloma, allergic rhinitis, neoplastic disorder, sepsis, liver injury.

2. Use according to claim 1, wherein the compound of formula I is a compound of formula IA or an enantiomer thereof or a pharmaceutically acceptable salt, solvate and / or prodrug thereof.

3. Use according to claim 1, wherein R 2 and R 4 are H, and R 3 and R 5 are selected from OH, C 1-3 alkyl, OC 1-3 alkyl, NH2, NHC 1-3 alkyl, NHSO2C 1-3 alkyl and NHC(O)C 1-3 alkyl.

4. Use according to claim 3, wherein R 3 and R 5 are selected from OH, CH3, OCH3, NHSO2CH3 and NHC(O)CH3.

5. Use according to claim 3 or 4, wherein R 3 is selected from OH, OCH3, NHSO2CH3 and NHC(O)CH3; and R 5 is CH3.

6. Use according to claim 1, wherein R 2 , R 4 and R 5 are H, and R 3 is selected from OH, OC 1-3 alkyl, NH2, NHC 1-3 alkyl, NHSO2C 1-3 alkyl and NHC(O)C 1-3 alkyl.

7. Use according to claim 6, wherein R 3 is selected from OH, OCH3, NHSO2CH3 and NHC(O)CH3.

8. The use according to claim 1, wherein the substituted or unsubstituted 5- or 6-membered heterocycle is selected from and 9. Use according to claim 1, wherein R 2 and R 3 together with the atom to which they are attached form the substituted or unsubstituted 5- or 6-membered heterocycle, and R 4 and R 5 are independently selected from H and C 1-3 alkyl.

10. Use according to claim 1 or 2, wherein the compound of formula I is selected from: and pharmaceutically acceptable salts, solvates and / or prodrugs thereof.

11. Use according to claim 1, wherein the compound of formula I or a pharmaceutically acceptable salt, solvate and / or prodrug thereof is formulated in a composition, wherein the composition comprises the compound of formula I or a pharmaceutically acceptable salt, solvate and / or prodrug thereof and a pharmaceutically acceptable carrier.

12. Use according to claim 1, wherein the inflammatory bowel disease is selected from Crohn's disease and ulcerative colitis.

13. Use according to claim 1, wherein the neoplastic disorder is colon cancer.

14. Use according to claim 1, wherein the liver injury is selected from viral hepatitis, autoimmune hepatitis, primary biliary cirrhosis and liver allograft rejection.

15. Use according to claim 1, wherein the disease, disorder or condition mediated by SHIP1 or treatable by activation of SHIP1 is multiple myeloma.

16. Use according to claim 1, wherein the sepsis is severe sepsis.

17. Use according to claim 16, wherein the severe sepsis is severe sepsis caused by bacterial or viral infection.

18. Use according to claim 1, wherein the treatment of a disease, disorder or condition mediated by SHIP1 or treatable by activation of SHIP1 comprises administering to a subject in need a therapeutically effective amount of the compound of formula I or a pharmaceutically acceptable salt, solvate and / or prodrug thereof.

19. Use according to claim 18, wherein the subject is human.

20. A compound of formula I or a pharmaceutically acceptable salt, solvate and / or prodrug thereof, wherein R 1 is selected from NHSO2C 1-3 alkyl, N-succinimide and NHC(O)C 1-3 alkyl; wherein R 2 、R 3 、R 4 and R 5 are independently selected from H, OH, C 1-3 alkyl, OC 1-3 alkyl, NH2, NHC 1-3 alkyl, NHSO2C 1-3 alkyl and NHC(O)C 1-3 alkyl; or R 2 and R 3 、R 3 and R 4 or R 4 and R 5 together with the atom to which they are attached form a substituted or unsubstituted 5- or 6-membered heterocycle, said substituted or unsubstituted 5- or 6-membered heterocycle containing at least one NH and optionally one or more additional heteroatoms selected from N, O and S; and Wherein when R 2 and R 3 、R 3 and R 4 or R 4 and R 5 together form the substituted or unsubstituted 5- or 6-membered heterocycle, R 4 and R 5 、R 2 and R 5 or R 2 and R 3 are each independently selected from H and C 1-3 alkyl.

21. The compound according to claim 20, wherein the compound of formula I is a compound of formula IA or an enantiomer thereof or a pharmaceutically acceptable salt, solvate and / or prodrug thereof.

22. The compound according to claim 20, wherein R 2 and R 4 are H, and R 3 and R 5 are selected from OH, C 1-3 alkyl, OC 1-3 alkyl, NH2, NHC 1-3 alkyl, NHSO2C 1-3 alkyl, and NHC(O)C 1-3 alkyl.

23. The compound according to claim 22, wherein R 3 and R 5 are independently selected from OH, CH3, OCH3, NHSO2CH3, and NHC(O)CH3.

24. The compound according to claim 22 or 23, wherein R 3 is selected from OH, OCH3, NHSO2CH3 and NHC(O)CH3; and R 5 is CH3.

25. The compound according to claim 20, wherein R 2 , R 4 and R 5 are H, and R 3 is selected from OH, OC 1-3 alkyl, NH2, NHC 1-3 alkyl, NHSO2C 1-3 alkyl, and NHC(O)C 1-3 alkyl.

26. The compound according to claim 25, wherein R 3 is selected from OH, OCH3, NHSO2CH3 and NHC(O)CH3.

27. The compound according to claim 20, wherein the substituted or unsubstituted 5- or 6-membered heterocycle is selected from and 28. The compound according to claim 20, wherein R 2 and R 3 together with the atoms to which they are attached form the substituted or unsubstituted 5- or 6-membered heterocycle, and R 4 and R 5 are independently selected from H and C 1-3 alkyl.

29. The compound according to claim 20 or 21, wherein the compound of formula I is selected from: and pharmaceutically acceptable salts, solvates and / or prodrugs thereof.

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