Protein tyrosine phosphatase inhibitors and methods of use thereof

Abstract

The present disclose includes, among other things, compounds that inhibit PTPN1 and / or PTPN2, pharmaceutical compositions comprising the compounds, and methods of making and using the same.

Description

[0001] Attorney Docket No.: 01330-0117-00PCT

[0002] PROTEIN TYROSINE PHOSPHATASE INHIBITORS AND METHODS OF USE THEREOF

[0003] Cross Reference to Related Applications

[0004]

[0001] This application claims priority to U. S. Application No. 63 / 782,838, filed April 3, 2025, U. S. Application No. 63 / 763,634, filed February 26, 2025, and U. S. Application No. 63 / 723,775, filed November 22, 2024, the entire contents of which are incorporated by reference herein for all purposes.

[0005] Background

[0006]

[0002] The enzyme protein-tyrosine phosphatase 1B (PTP1B) is also referred to as tyrosine¬ protein phosphatase non-receptor type 1 (PTPN1). The enzyme T-cell protein tyrosine phosphatase (TC-PTP) is also referred to as tyrosine-protein phosphatase non-receptor type 2 (PTPN2). PTPN1 and PTPN2 are members of the protein tyrosine phosphatase (PTP) family, which share a highly conserved catalytic motif, essential for their tyrosine phosphatase activity.

[0007]

[0003] PTPNI / 2 phosphatases are major negative regulators of the antitumor immune response. Inhibiting or depleting PTPNI / 2 sensitizes cancer and immune cells to interferon gamma (IFNy), reshapes the tumor microenvironment and triggers a coordinated antitumor immune response involving myeloid cells, \K T cells, and cancer cells. Although protein phosphatases were for long considered undruggable due to their highly polar catalytic pocket, several small molecules inhibitors of PTPNI / 2 have recently been discovered.

[0008] [0(M| Immune checkpoint blockade therapies have shown significant effectiveness in treating a wide range of cancers, offering dramatic improvements in outcomes and durable responses especially in patients refractory' to conventional treatments (chemotherapy, radiation). However, a major limitation remains: most patients either do not respond or develop resistance to current immunotherapies, highlighting the need for new therapeutic approaches.

[0009]

[0005] This study focused on finding new' and improved PTPN1 and / or PTPN2 inhibitors useful as cancer therapeutics, potentially improving the efficacy of cancer immunotherapies. For that purpose, the Applicant herein provides certain N-phenyl-l,2,5-thiadiazolidin-3-one 1,1 -dioxide derivatives detailed below. In some embodiments, the compounds disclosed herein have improved oral bioavailability. Attorney Docket No.: 01330-0117-00PCT

[0010] Summary

[0011]

[0006] The present disclosure relates to compounds that inhibit PTPN1 and / or PTPN2.

[0012]

[0007] In some embodiments, the present disclosure includes Compound 1:

[0013]

[0014] Compound 1,

[0015] or a pharmaceutically acceptable salt thereof.

[0016]

[0008] In some embodiments, the present disclosure includes Compound 2:

[0017] O

[0018] r O< X-NH

[0019] X

[0020]

[0021] j 'I

[0022] Br""X'^'XOH

[0023] Compound 2,

[0024] or a pharmaceutically acceptable salt thereof.

[0025] In some embodiments, the present disclosure includes Compound 3:

[0026] O

[0027]

[0028] 'OH

[0029] Compound 3,

[0030] or a pharmaceutically acceptable salt thereof.

[0031]

[0010] In some embodiments, the present disclosure includes Compound 4:

[0032] O

[0033] F

[0034]

[0035] I X OH '

[0036] Compound 4, Attorney Docket No.: 01330-0117-00PCT

[0037] or a pharmaceutically acceptable salt thereof.

[0038] [Oil] In some embodiments, the present disclosure includes Compound 5:

[0039] 0

[0040] F

[0041] Cff.--I JK

[0042] Br I

[0043]

[0044] I 'OH ~

[0045] Compound 5,

[0046] or a pharmaceutically acceptable salt thereof.

[0047]

[0012] In some embodiments, the present disclosure includes Compound 6:

[0048]

[0049] Compound 6,

[0050] or a pharmaceutically acceptable salt thereof.

[0051]

[0013] In some embodiments, the present disclosure includes Compound 7:

[0052] q

[0053] p Olq-NH

[0054]

[0055] Compound 7,

[0056] or a pharmaceutically acceptable salt thereof.

[0057]

[0014] Additionally, the present disclosure includes, among other things, pharmaceutical compositions, methods of using, and methods of making Compounds 1 to 7 and / or pharmaceutically acceptable salts thereof.

[0058]

[0015] In some embodiments, the present disclosure relates to pharmaceutical compositions comprising any one of the Compounds 1 through 7 and / or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable excipient, carrier, adjuvant, or vehicle.

[0059]

[0016] In some embodiments, the present disclosure relates to methods of inhibiting PTPN1 and / or PTPN2 in a patient in need thereof, comprising administering an effective amount of any one of Compounds 1 to 7 and / or pharmaceutically acceptable salts thereof. Attorney Docket No.: 01330-0117-00PCT

[0060]

[0017] In some embodiments, the present disclosure relates to methods of treating a disease mediated by PTPNl and / or PTPN2 in a patient in need thereof, comprising administering an effective amount of any one of Compounds 1 to 7 and / or pharmaceutically acceptable salts thereof. In one embodiment, the disease mediated by PTPN1 and / or PTPN2 comprises cancer. In one embodiment, the disease mediated by PTPN1 and / or PTPN2 is cancer.

[0061] Brief Description of the Drawings

[0062]

[0018] Figure 1 shows potency of human and mouse Compound 1 compared to human and mouse ABBV-CLS-484 in phospho-STATl assay. A549-RFP or B16-F10 were stimulated for Ih with human or mouse recombinant IFNγ (100ng / mL) in HBSS buffer. pSTATl (Tyr701) was quantified by AlphaLISA® SureFire® Ultra (Revvity). The data is normalized to the maximum achieved concentration of pSTATl as 100%.

[0063]

[0019] Figure 2A-2E show’ the effect of Compound 1 on T cell function in vitro. Tolerogenic dendritic cells (DCs), generated from monocyte-derived DCs with IL-10, were treated with either / ABBV-CLS-484 or Compound 1 at various concentrations (0.3 pM, 1 pM, 3uM, or lOpM) for 2 days. After LPS stimulation for 24 hours, these DCs were co-cultured with allogeneic CD3+ T cells for 4 days to assess if PTPN1 / 2 inhibition restores T ceil functions. Activation and regulatory markers on tolerogenic DCs were assessed by FACS before coculture. Figure 2. A shows a schematic representation of the generation of tolerogenic DCs in vitro and the Mixed Lymphocyte Reaction (MLR) assay. Figure 2B and 2C show’ phenotype of tolerogenic DCs treated with ABBV-CLS-484 or Compound 1. Activation markers were assessed by FACS and found unaffected by PTPN1 / 2 inhibitors. Figure 2D and 2E show results where allogeneic CD3+ T cells were cocultured with tolerogenic DCs at a ratio of 1 / 10 Effector. Target in presence of ABBV-CLS-484 and Compound 1 at various concentrations (0.3 μM, 1μM, 3μM, or lOpM). T cell proliferation and activation were assessed by flow cytometry at endpoint. The dotted line represents the DMSO control. * p- value >0,05 ** p-value < 0.01, *** p-value < 0.001 (Two-way Anova test) (n=3 healthy donors).

[0064]

[0020] Figure 3A-3B show the effect of Compound 1 on natural killer (NK) cell activation in vitro. CD56+ NK cells were stimulated overnight with IL-2 and treated with either ABBV- CLS-484 or Compound 1 at various concentrations (0.1 uM, 0.3pM, IpM, 3pM, or lOpM). The next day, the tumor cell killing assay was performed at a ratio of 5 / 1 Effector: Target with ABBV-CLS-484 or Compound 1. The dotted line represents the DMSO control. * p- Attorney Docket No.: 01330-0117-00PCT

[0065] value < 0.05, ** p-value < 0.01 (Two-way Anova test) (n=3 healthy donors). Figure 3A shows CD69 expression after application of ABBV-CLS-484 and Compound 1 at various concentrations (O.lpM, 0.3pM, IpM, 3gM, or lOpM). Figure 3B shows NK cell killing after application of ABBV-CLS-484 and Compound 1 at various concentrations (0.1 uM, 0.3 uM, luM, 3pM, or lOpM).

[0066]

[0021] Figure 4A-4B show the effect of Compound 1 on tumor volume in vivo. The efficacy of Compound 1, 450mg / kg, a dose covering the IC10 (pSTATl assay: see Figure 1) was evaluated in an established colon cancer syngeneic tumor model, MC38. Median (Figure 4A) and individual (Figure 4B) tumor growth volume curves; n=15 mice / group). ***P-value <0.001 (Linear mixed model).

[0067]

[0022] Figure 5A-5F compares the biological activity of Compound 1 to ABBV-CLS-484 across the same cohort of patient-derived tumor samples. Figure 5A shows a schematic representation of a Patient Derived Tumor Fragment (PDTF) platform. Figure 5B shows a case study: Renal carcinoma (RCC) sample that responds to Compound 1. It is a macroscopic illustration of the tumor and a 48h~cultured tumor fragment witli the corresponding H& E, CD45, and CD8 / PanCK. Scale bar: 50 pm. Figure 5C-E show evaluation of concentration (pg / mL) of CXCL9, CXCL10, and CCL22 by MSD on RCC-derived tumor fragments treated with DMSO, ABBV-CLS-484, and Compound 1 (both at 100 uM, free concentration).

[0068] Figure 5F shows response of a PDTF cohort to Compound 1 and ABBV-CLS-484. It shows a heatmap displaying the normalized delta values between treatment (ABBV-CLS-484 or Compound 1) and untreated conditions (DMSO) for each cytokine (D z-score), with unsupervised clustering. Additionally, the concentration (pg / mL) of IFNy and CCL22 is showm 48 hours after DMSO treatment. Baseline: the heatmap also represents the cancer type, IHC analysis, immunotype, and CD45+ cells per mm2.

[0069] Detailed Description

[0070]

[0023] PTPN1 (protein phosphatase non-receptor type 1) and PTPN2 (protein phosphatase non-receptor type 2) are tyrosine phosphatases that share a significant degree of homology, especially in their catalytical domain (81% sequence similarity). They have been shown to dephosphorylate a variety of target proteins that regulate numerous cellular processes in cancer and immune cells, such as JAK1, JAK3, STAT1, STAT5, STAT6, LCK, SRC, FYN. Attorney Docket No.: 01330-0117-00PCT

[0071] By dephosphorylating their targets, PTPN1 and PTPN2 act as a negative regulators of the IFNγ-JAK-STAT1 signaling pathway in cancer and myeloid cells, and of IL-2 and TCR signaling in T cells and NK cells. In line with this, studies relying on lineage-specific genetic deletion of PTPN1 and / or PTPN2 in mouse immune or cancer cells suggest that inhibitors of PTPN1 and / or PTPN2- may be used as immunotherapy agents. Likewise, inhibition of PTPN1 and PTPN2 in preclinical models has been shown to trigger a coordinated anti-tumoral response acting both on cancer cells as well as on multiple immune cells types, and reshaping the tumor microenvironment (Baumgartner et al., Nature 622:850-862; 2023)..

[0072]

[0024] Deletion of PTPN1 in T cells increases immune infiltration and T cell function, leading to suppression of tumor growth (Wiede et al., Cancer Discovery 12(3):752-773; 2022). PTPN1 deletion in macrophages increases Ml and reduces M2 response (Traves et al. Cell Death Dis. 2014, 5(3):ell25) and increases Statl phosphorylation and IFNγ response (NO production) (Heinonen et al. Proc Natl Acad Sci USA., 2006, 103(8):2776-81).

[0073] Lineage-specific deletion of PTPN1 in dendritic cells (DC) impairs DC function due to Src activation (Martin-Granados et al., J Mol Cell Biol., 2015, 7(6):517-28). PTPN1 deletion in B cells increases B cell proliferation and activation, T-cell dependent antibody response, leading to spontaneous germinal center formation in the spleen (Medgyesi et al. J Exp Med., 2014, 211(3):427-40), and increased BCR signaling (Schwarz et al. Life Sci Alliance, 2021, 4(11):e202101084). Silencing of PTP1 in human pancreatic cancer cell lines reduces in vitro and in vivo tumor growth (Xu et al., Cell Death and Disease 10:874; 2019).

[0074]

[0025] An in vivo CRISPR screening identified PTPN2 as a novel cancer immunotherapy target (Manguso R. T. et al., Nature 547:413-418; 2017). PTPN2 deletion in cancer cells in fact results in strong tumor growth reduction, enhances the response to aPD-1, increases T cell infiltration in the tumors and enhances the antitumoral immune response in vivo (Manguso et. al., 2017, Goh et. al. Sci Adv., 2022, 8(8):eabk3338, Katkeviciute et. al. J Clin Invest., 2021, 131 (1 ):e!40281). Deletion of PTPN2 in cancer cells also sensitizes cancer cells to IFNy treatment, resulting in enhancement of IFNγ / STAT1 signaling leading to cell cycle arrest and apoptosis in a cancer cell-autologous manner.

[0075]

[0026] Studies using lineage-specific deletion of PTPN2 show that this phosphatase is an important regulator of the function several immune cells. Deletion of PTPN2 in T cells increases the expansion and survival of activated T cells (Flosbach et al. Cell Rep., 2020, Attorney Docket No.: 01330-0117-00PCT

[0076] 32(4): 107957), affects the transition from early to terminally exhausted T cells (LaFleur et al. Nat Immunol., 2019, 20(10): 1335-1347), and enhances CAR-T function (Wiede et al., 2020). PTPN2 deletion in DC ceils results in increased T cell infiltration (Katkeviciute et al., 2021), whereas PTPN2 deletion in B cells increases B cell proliferation and enhances Thl response (Wiede et al. J Autoimmun., 2017, 76:85-100).

[0077]

[0027] Beside the role in anti-tumor immunity, PTPN1 and PTPN2 also play a role in glucose homeostasis, by dephosphorylating insulin receptor, IRS1 and leptin receptor (PTPN1) (Tiganis FEBS J., 2013, 280:445-458). PTPN1 -deficient mice, in fact, exhibit improved glucose regulation and resistance to diet-induced obesity (Elchebly et al. Science, 1999, 283(5407): 1544-1548), suggesting a main role for PTPN1 in metabolic diseases.

[0078]

[0028] Despite the promise of inhibiting PTPN1 and PTPN2, targeting protein phosphatases remains a significant challenge due to the very- polar catalytic sites of these enzymes and of the molecules able to occupy it. Although several PTPNl / 2 inhibitors have recently been discovered (Baumgartner et al., Nature 622(7984):850-862; 2023), in fact, they suffer from low permeability and low bioavailability.

[0079]

[0029] In some embodiments, the N-phenyl-l,2,5-thiadiazolidin-3-one 1,1-dioxide derivatives disclosed herein as Compounds 1-7, are PTPN1 and / or PTPN2 inhibitors that benefit from high oral bioavailability, making them ideal molecules for drug development.

[0080]

[0030] In some embodiments, the present disclosure includes potent inhibitors of PTPN1, PTPN2, and dual inhibitors of PTPN1 and PTPN2, including the compounds shown in Table 1 below, pharmaceutically acceptable salts and solvates thereof, and methods of making the compounds.

[0081]

[0031] In some embodiments, the present disclosure includes the compounds described in Table 1 and / or pharmaceutically acceptable salts thereof Compound names in this table were generated using ChemDraw Professional 19.0. 1.28 naming software. Attorney Docket No.: 01330-0117-00PCT

[0082] Table 1

[0083] Cmpd# Structure Name

[0084] 0

[0085] FO=? S--NH

[0086] O 00 CD CD

[0087] 1 —, — s —y - 5-(4-chloro-2-fluoro-6-hydroxyphenyl)-l,2,5- thiadiazolidin-3-one 1,1 -di oxide JT Y CI^ YY^- °^OH

[0088] y° ° °\ O Z—a>-- °°°° O O O Z' z' Z'CcCDnD--- & D4 O ZC- 5-(4-bromo-2-fluoro-6-hydroxyphenyl)-l,2,5- 21

[0089] 11o thiadiazolidin-3-one 1,1 -di oxide1o o o

[0090] o

[0091] 0

[0092] FO^s-NH

[0093] 5 -(3 -chloro-2-fluoro-6-hy droxyphenyl)- 1,2,5- 3

[0094] thiadiazolidin-3-one 1,1 -di oxide Y Y

[0095] 5 -(3 -bromo-2-fluoro-6-hy droxyphenyl)- 1,2,5- 4

[0096] thiadiazolidin-3-one 1,1 -di oxide

[0097] 5 -(4-bromo-3 -chloro-2-fluoro-6- 5 hydroxyphenyl)-l,2,5-thiadiazolidin-3-one 1,1 -di oxide

[0098] 5-(2-fluoro-4-hydroxy-[l,l'-biphenyl]-3-yl)- 6

[0099] 1,2, 5-thiadiazolidin-3 -one 1, 1 -dioxide

[0100] 5-(2-fluoro-6-hydroxyphenyl)-l,2,5- 7

[0101] thiadiazolidin-3-one 1,1 -di oxide

[0102]

[0103] Attorney Docket No.: 01330-0117-00PCT

[0104]

[0032] Definitions

[0105]

[0033] As used herein, “a” or “an” entity refers to one or more of that entity, e.g., “a compound” refers to one or more compounds or at least one compound unless stated otherwise. As such, the terms “a” (or “an”), “one or more,” and “at least one” are used interchangeably herein.

[0106]

[0034] “About” as used herein preceding one or more specific numerical values is intended to denote that a range of values are encompassed within the specific value to the extent one of ordinary skill in the art would consider the range equivalent to the specific, recited value (e.g., having the same function or result). When “about” precedes a list of numerical values or ranges, the term modifies all the values or ranges provided in the list. In some embodiments, the term “about” refers to a range within ± 5% of the recited numerical value. In some embodiments, the term “about” refers to a range within ± 10% of the recited numerical value.

[0107]

[0035] As used herein, the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference.

[0108]

[0036] As used herein, a “therapeutically effective amount” means an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that elicits a desired biological response. In some embodiments, a therapeutically effective amount of a substance is an amount that is sufficient, when administered as part of a dosing regimen to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, the effective amount of a provided compound in a formulation to treat a disease, disorder, and / or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and / or reduces incidence of one or Attorney Docket No.: 01330-0117-00PCT

[0109] more symptoms or features of the disease, disorder, and / or condition.

[0110]

[0037] As used herein, the terms “treatment,” “treat,” and “treating” refer to partially or completely alleviating, inhibiting, delaying onset of, preventing, ameliorating and / or relieving a disorder or condition, or one or more symptoms of the disorder or condition, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed. In some embodiments, the term “treating” includes preventing or halting the progression of a disease or disorder. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence. Thus, in some embodiments, the term “treating” includes preventing relapse or recurrence of a disease or disorder.

[0111]

[0038] The term “patient,” as used herein, means an animal, preferably a mammal, and most preferably a human.

[0112]

[0039] The term “pharmaceutically acceptable carrier, adjuvant, or vehicle” refers to a nontoxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the compound(s) with which it is formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions of the compounds disclosed herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

[0113]

[0040] A “pharmaceutically acceptable derivative” means any non-toxic salt, ester, salt of an ester or other derivative of a compound of this disclosure that, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound of this disclosure or an inhibitorily active metabolite or residue thereof.

[0114]

[0041] The expression “dosage unit form” as used herein refers to a physically discrete unit Attorney Docket No.: 01330-0117-00PCT

[0115] of agent appropriate for the patient to be treated. It will be understood, however, that total daily usage of compounds and compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment. Specific effective dose level for any particular patient or organism will depend upon a variety of factors including disorder being treated and severity of the disorder; activity of specific compound employed; specific composition employed; age, body weight, general health, sex and diet of the patient; time of administration, route of administration, and rate of excretion of a specific compound employed; duration of treatment; drugs used in combination or coincidental with a specific compound employed, and like factors well known in the medical arts.

[0116]

[0042] In one embodiment, the present disclosure relates to enantiomers and isomers (including optical, geometric and tautomeric isomers) of Compounds 1 to 7. Indeed, any one of Compounds 1 to 7 may contain an asymmetric center and thus may exist as different stereoisomeric forms. Accordingly, the present disclosure includes all possible stereoisomers and includes not only racemic compounds but the individual enantiomers and their non- racemic mixtures as well. When a compound is desired as a single enantiomer, such may be obtained by stereospecific synthesis, by resolution of the final product or any convenient Intermediate compound, or by chiral chromatographic, methods as each are known in the art. Resolution of the final product, an Intermediate compound, or a starting material may be performed by any suitable method known in the art. Unless otherwise indicated, the stereochemistry of a compound has been arbitrarily assigned. Accordingly, the absolute stereochemistry of a molecule is unknown, unless otherwise indicated.

[0117]

[0043] In one embodiment, the present invention also relates to salts of Compounds I to 7. Especially, the compounds of the invention may be in the form of pharmaceutically acceptable salts. In some embodiments, the present invention also relates to solvates, including hydrates, of Compounds 1 to 7.

[0118]

[0044] In an alternative embodiment, compounds described herein may also comprise one or more isotopic substitutions. For example, hydrogen may be2H (D or deuterium) or3H (T or tritium); carbon may be, for example,13C or14C; oxygen may be, for example,18O; nitrogen may be, for example,15N, and the like. In other embodiments, a particular isotope (e.g.,3H,13C,14C,18O, or15N) can represent at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least Attorney Docket No.: 01330-0117-00PCT

[0119] 95%, at least 99%, or at least 99.9% of the total isotopic abundance of an element that occupies a specific site of the compound.

[0120]

[0045] Pharmaceutical Compositions

[0121]

[0046] In some embodiments, the present disclosure provides a composition comprising at least one of Compounds I to 7 and a pharmaceutically acceptable excipient, carrier, adjuvant, or vehicle. In some embodiments, the amount of compound in compositions contemplated herein is such that is effective to measurably treat a disease or disorder in a patient. In certain embodiments, the amount of compound in compositions of this disclosure is such that is effective to measurably treat a disease or disorder in a patient. In certain embodiments, a composition contemplated by this disclosure is formulated for administration to a patient in need of such composition. In some embodiments, a composition contemplated by this disclosure is formulated for oral administration to a patient.

[0122]

[0047] In some embodiments, compositions of the present disclosure may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. In some preferred embodiments, compositions are administered orally, intraperitoneally or intravenously. In some embodiments, sterile injectable forms of the compositions comprising one or more of Compounds I to 7 may be aqueous or oleaginous suspension. In some embodiments, suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. In some embodiments, sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3 -butanediol. In some embodiments, among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In some embodiments, additional examples include, but are not limited to, sterile, fixed oils are conventionally employed as a solvent or suspending medium.

[0123]

[0048] The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

[0124]

[0049] Pharmaceutically acceptable compositions comprising any one of Compounds 1 to 7 may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In some embodiments, carriers used Attorney Docket No.: 01330-0117-00PCT

[0125] include lactose and com starch. Lubricating agents, such as magnesium stearate, are also typically added. In some embodiments, useful diluents include lactose and dried cornstarch. In some embodiments, when aqueous suspensions are required for oral use, an active ingredient is combined with emulsifying and suspending agents. In some embodiments, certain sweetening, flavoring or coloring agents may also be added.

[0126]

[0050] Alternatively, pharmaceutically acceptable compositions comprising any one of Compounds 1 to 7 may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

[0127]

[0051] Pharmaceutically acceptable compositions comprising any one of Compounds 1 to 7 may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs. In some embodiments, pharmaceutically acceptable compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of compounds of this disclosure include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.

[0128] Alternatively, provided pharmaceutically acceptable compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyl dodecanol, benzyl alcohol and water.

[0129]

[0052] Pharmaceutically acceptable compositions comprising any one of Compounds 1 to 7 may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents.

[0130]

[0053] In some embodiments, an amount of a compound of the present disclosure that may Attorney Docket No.: 01330-0117-00PCT

[0131] be combined with the carrier materials to produce a composition in a single dosage form will vary depending upon the host treated, the particular mode of administration. Preferably, provided compositions should be formulated so that a dosage of between 0.01-100 mg / kg body weight / day of the inhibitor can be administered to a patient receiving these compositions.

[0132]

[0054] Methods of Using Compounds of the Present Disclosure

[0133]

[0055] The present disclosure is further directed to the use of the compounds of the present disclosure, or pharmaceutically acceptable salts thereof, as inhibitors of PTPN1 and / or PTPN2. Accordingly, in a particularly preferred embodiment, the present disclosure relates to the use of any one of Compounds 1 to 7, in particular those of Table I above, or pharmaceutically acceptable salts or solvates thereof, as inhibitors of PTPN1 and / or PTPN2.

[0134]

[0056] In one embodiment, the compounds of the present disclosure are inhibitors of PTPN1 and / or PTPN2. In one embodiment, the compounds of the present disclosure are inhibitors of PTPN1. In one embodiment, the compounds of the present disclosure are inhibitors of PTPN2. In one embodiment, the compounds of the present disclosure are dual inhibitors of PTPN1 and PTPN2.

[0135]

[0057] The present disclosure also provides a method for inhibiting PTPN1 and / or PTPN2 in a patient, preferably a warm-blooded animal, and even more preferably a human, in need thereof, which comprises administering to said patient an effective amount of a compound of the present disclosure, or a pharmaceutically acceptable salt or solvate thereof.

[0136]

[0058] The present disclosure is further directed to the use of the compounds of the present disclosure as a medicament, i.e. for medical use. Thus, in one embodiment, the present disclosure provides the use of the compounds of the present disclosure for the manufacturing of a medicament. Especially, the present disclosure provides the use of the compounds of the present disclosure for the manufacturing of a medicament.

[0137]

[0059] Especially, the present disclosure provides the compounds of the present disclosure, for use in the treatment and / or prevention of one or more diseases mediated by PTPN1 and / or PTPN2, Thus, in one embodiment, the present disclosure provides the use of the compounds of the present disclosure for the manufacture of a medicament for treating and / or preventing a disease mediated by PTPN1 and / or PTPN2. In one embodiment, the present disclosure provides the use of the compounds of the present disclosure for the manufacture of a Attorney Docket No.: 01330-0117-00PCT

[0138] medicament for treating and / or preventing a disease mediated by PTPNI. In one embodiment, the present disclosure provides the use of the compounds of the present disclosure for the manufacture of a medicament for treating and / or preventing a disease mediated by PTPN2. The present disclosure also provides a method of treating a disease mediated by PTPNI and / or PTPN2 in a patient in need thereof, which comprises administering to the patient a therapeutically effective amount of a compound of the present disclosure. In one embodiment, the present disclosure provides a method of treating and / or preventing a disease mediated by PTPNI. In one embodiment, the present disclosure provides a method of treating and / or preventing a disease mediated by PTPN2.

[0139]

[0060] In one embodiment, the compounds of the present disclosure are used in the treatment and / or prevention of one or more diseases mediated by PTPNI and / or PTPN2, wherein the disease is cancer. In one embodiment, the compounds of the present disclosure are used for the manufacture of a medicament for treating and / or preventing a disease mediated by PTPN I and / or PTPN2, wherein the disease is cancer. In one embodiment, the compounds of the present disclosure are used in a method of treating a disease mediated by PTPNI and / or PTPN2, the method comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present disclosure, wherein the disease is cancer. In one embodiment, the compounds of the present disclosure are used in a method of treating cancer in a patient in need thereof.

[0140]

[0061] Preferably, the patient is a warm-blooded animal, more preferably a human.

[0141]

[0062] The present disclosure also provides pharmaceutical compositions comprising any one of Compounds 1 to 7, or a pharmaceutically acceptable salt or solvate thereof, and at least one pharmaceutically acceptable carrier, diluent, excipient, and / or adjuvant.

[0142]

[0063] Another object of this present disclosure is a medicament comprising at least one compound of the present disclosure, or a pharmaceutically acceptable salt or solvate thereof, as an active ingredient.

[0143]

[0064] Generally, for pharmaceutical use, the compounds of the present disclosure may be formulated as a pharmaceutical preparation comprising at least one compound of the present disclosure and at. least, one pharmaceutically acceptable carrier, diluent, excipient, and / or adjuvant.

[0144]

[0065] By means of non-limiting examples, such a formulation may be in a form suitable for oral administration, for parenteral administration (such as by intravenous, intramuscular or Attorney Docket No.: 01330-0117-00PCT

[0145] subcutaneous injection or intravenous infusion), for topical administration (including ocular), for administration by inhalation, by a skin patch, by an implant, by a suppository, etc. Such suitable administration forms - which may be solid, semi-solid or liquid, depending on the manner of administration - as well as methods and carriers, diluents and excipients for use in the preparation thereof, will be clear to the skilled person; reference is made to the latest edition of Remington’s Pharmaceutical Sciences.

[0146]

[0066] Some preferred, but non-limiting examples of such preparations include tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, cremes, lotions, soft and hard gelatin capsules, suppositories, drops, sterile injectable solutions and sterile packaged powders (which are usually reconstituted prior to use) for administration as a bolus and / or for continuous administration, which may be formulated with carriers, excipients, and diluents that are suitable per se for such formulations, such as lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, polyethylene glycol, cellulose, (sterile) water, methylcellulose, methyl- and propyl hydroxybenzoates, talc, magnesium stearate, edible oils, vegetable oils and mineral oils or suitable mixtures thereof. The formulations can optionally contain other substances that are commonly used in pharmaceutical formulations, such as lubricating agents, wetting agents, emulsifying and suspending agents, dispersing agents, desintegrants, bulking agents, fillers, preserving agents, sweetening agents, flavoring agents, flow' regulators, release agents, etc. The compositions may also be formulated so as to provide rapid, sustained or delayed release of the active compound(s) contained therein.

[0147]

[0067] The pharmaceutical preparations of the present disclosure are preferably in a unit dosage form, and may be suitably packaged, for example in a box, blister, vial, bottle, sachet, ampoule or in any other suitable single-dose or multi-dose holder or container (which may be properly labeled); optionally with one or more leaflets containing product information and / or instructions for use.

[0148]

[0068] Depending on the condition to be prevented or treated and the route of administration, the active compound of the present disclosure may be administered as a single daily dose, divided over one or more daily doses, or essentially continuously, <?.g. using a drip infusion.

[0149]

[0069] Particularly preferred inhibitors of PTPNI and / or PTPN2 are those listed hereafter: 5-(4-chloro-2-fluoro-6-hydroxyphenyl)-l,2,5-thiadiazolidin-3-one 1,1 -dioxide; 5-(4-bromo- Attorney Docket No.: 01330-0117-00PCT

[0150] 2-fluoro-6-hydroxyphenyl)-1,2,5-thiadiazolidin-3-one 1,1-dioxide; 5-(3-chloro-2-fluoro-6-hydroxyphenyl)-1,2,5-thiadiazolidin-3-one 1,1-dioxide; 5-(3 -bromo-2-fl uoro-6- hydroxyphenyl)-1,2,5-thiadiazolidin-3-one 1,1-dioxide; 5-(4-bromo-3-chloro-2-fluoro-6- hydroxyphenyl)~l,2,5-thiadiazolidin~3-one 1, 1 -dioxide; 5-(2-fluoro-4-hydroxy-[1,1'-biphenyl]-3-yl)-1,2,5-thiadiazolidin-3-one 1,1-dioxide; and 5-(2-fluoro-6-hydroxyphenyl)- 1,2,5-thiadiazolidin-3-one 1,1-dioxide.

[0151] EXAMPLES

[0152]

[0070] Compounds described herein can be prepared as illustrated and described below. Thus, the following preparations of compounds and intermediates are given to enable those skilled in the art to more clearly understand and to practice the present disclosure. The Examples should not be considered as limiting the scope of the disclosure, but merely as being illustrative and representative thereof.

[0153]

[0071] The starting materials and reagents used in preparing these compounds are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), Bachem (Torrance, Calif ), or Sigma (St. Louis, Mo.) or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser’s Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd’s Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), March’s Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition) and Larock’s Comprehensive Organic Transformations (VCH Publishers Inc., 1989). These Examples are merely illustrative of some methods by which the compounds of this disclosure can be synthesized, and various modifications to these Examples can be made and will be suggested to one skilled in the art having referred to this disclosure. The starting materials and the intermediates, and the final products of the reaction may be isolated and purified if desired using conventional techniques, including but not limited to filtration, distillation, crystallization, chromatography and the like. Such materials may be characterized using conventional means, including physical constants and spectral data.

[0154]

[0072] Unless otherwise noted, all reagents were used without further purification.1H NMR spectra were obtained in CDCl3, DMSO-d6, or CD3OD, unless stated otherwise, at room temperature. When more than one conformer was detected, the chemical shifts for the most Attorney Docket No.: 01330-0117-00PCT

[0155] abundant one is reported. Chemical shifts of ’H NMR spectra were recorded in parts per million (ppm) on the 5 scale from an internal standard of residual solvent. Splitting patterns are designed as s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad.

[0156]

[0073] Unless otherwise indicated, the following abbreviations are defined as follows: ACN is acetonitrile; AcOH is acetic acid; DCM is dichloromethane; DiFMUP is fluorogenic SHP-2 substrate; DMEM is Dulbecco’s Modified Eagle Medium; DMA is N, N-dimethylacetamide; DMF is N,N-dimethylformamide; DMSO is dimethyl sulfoxide; dppf is 1,1’- bis(diphenylphosphino)ferrocene; DTT is Dithiothreitol; EDTA is ethylenediaminetetraacetic acid; EtOH is ethanol; EA or EtOAc is ethyl acetate; FBS is Fetal bovine serum, HESS is Hanks’ Balanced Salt Solution; IMDM is Iscove’s Modified Dulbecco’s Medium; MeOH is methanol; NBS is N-bromosuccinimide; PMBOH is / ?-methoxybenzyl alcohol; TEA is triethylamine; THF is tetrahydrofuran; TMSC1 is trimethyl silyl chloride; LCMS and LC-MS are liquid chromatography-mass spectrometry; TLC is thin layer chromatography; equiv. is equivalent; h is hours; Rf is retention factor, and rt is room temperature.

[0157]

[0074] Synthetic Examples

[0158]

[0075] Synthetic Example 1: Synthesis of 5-(4-chloro-2”fluoro-6-hydrox henyl)“l,2,5- thiadiazclidin-3~one 1,1-dioxide (Compound 1).

[0159] O F O=S-NH2

[0160] Cl 1-SM-2 Step-1 Step-2 Step-3 1-SM-1

[0161] 1-SM-3

[0162] Step-4 Step-5 Step-6

[0163]

[0164] Compound 1

[0165]

[0076] Step-1: Synthesis of 5-chloro-l-fluoro-3-((4-methoxybenzyl)oxy)-2-nitrobenzene 1- 1.

[0166]

[0077] To a stirred solution of 5-chloro-1,3-difluoro-2-nitrobenzene 1-SM-1 (50.00 g, 210.09 mmol) in DMF (200 mL) at room temperature under nitrogen atmosphere was added K2CO3 (58.07 g, 420.18 mmol) and 4-methoxybenzyl alcohol (21.75 mL, 210.09 mmol). The reaction mixture was stirred under N2 atmosphere at 75 °C for 12 h. After completion (monitored by TLC), the reaction mixture was cooled to room temperature and diluted with Attorney Docket No.: 01330-0117-00PCT

[0167] ice cold water. The precipitated solid was collected by filtration, washed with water and dried under vacuum to afford 5-chloro-l-fluoro-3-((4-methoxy benzyl alcohol))-2-nitrobenzene 1-1 (50 g, 153.317 mmol, 72.98 %) as a yellow solid. TLC: 10 % EA / Heptane (Rf 0.65). LC-MS: m / z-312 (M+l). The title compound was used for the next step without further purification.1H NMR (400 MHz, DMSO-d6) δ 7.53-7.34 (m, 4H), 6.98 - 6.96 (m, 2H), 5.27 (s, 2H), 3.76 (s, 3H).

[0168]

[0078] Step-2: Synthesis of 4-chloro-2-fluoro-6-((4-methoxybenzyl)oxy)aniline 1-2.

[0169]

[0079] To a stirred solution of 5-chloro-l-fluoro-3-((4-methoxy benzyl alcohol))-2-nitrobenzene 1-1 (25.00 g, 76.65 mmol) in EtOH (400 mL) and water (100 mL) at room temperature were added NH4CI (2.05 g, 38.32 mmol) and Fe powder (21.46 g, 383.29 mmol). The reaction mixture was stirred at 80 °C for 12 h. After completion (monitored by TLC), the reaction mixture was cooled to room temperature and filtered through celite pad. The celite pad was further washed with MeOH and die filtrate was concentrated under reduced pressure, which afforded a crude product. The crude product was diluted with ice cold water and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated under vacuum to afford 4-chloro-2-fluoro-6-((4-methoxybenzyl)oxy)aniline 1-2 (12.50 g, 42.20 mmol, 55.06 %) as an off-white semi-solid. TLC: 20% EtOAc / heptane (Rf 0.75). LC-MS: m / z 281 (M+l). The title compound was used for the next step without further purification.

[0170]

[0080] Step-3: Synthesis of 5-chloro-1-fluoro-3-[(4-methoxyphenyl)methoxy]-2-(sulfamoylamino)benzene 1-3.

[0171]

[0081] To a stirred solution of 4-chloro-2-fluoro-6-((4-methoxybenzyl)oxy)aniline 1-2 (25 g, 88.75 mmol, 100 mass%) in DCM (250 mL, 99.5 mass%) were successively added triethylamine (3 equiv., 266.20 mmol) and sulfamoyl chloride l-SM-2 (2.5 equiv., 221.9 mmol) at 0 °C. The reaction mixture was stirred for 16 h. After completion (monitored by TLC and LCMS), the reaction mixture was diluted with water and extracted with DCM, then the organic layer was separated, dried over anhydrous Na2SO4, filtered and concentrated under vacuum to get the crude compound. The desired crude material was purified by combi-flash column chromatography on silica gel using 30-35 % ethyl acetate in heptane. The desired fractions were concentrated under vacuum to obtain 5-chloro-l-fluoro-3-[(4-methoxyphenyl)methoxy]-2-(sulfamoylamino)benzene 1-3 (20 g, 55.43 mmol, 62.46 %) as a white solid. TLC: 0-35 % EtOAc / hexane. LC-MS: m / z 383 (M+23). Attorney Docket No.: 01330-0117-00PCT

[0172]

[0082] Step-4: Synthesis of tert-butyl 2-[4-chloro-2-fluoro-6-[(4-methoxyphenyl)methoxy]-N-sulfamoyl-anilino]acetate 1-4.

[0173]

[0083] To a stirred solution of 5-chloro-1-fluoro-3-[(4-methoxyphenyl)methoxy]-2-(sulfamoylamino)benzene (20 g, 55.43 mmol, 100 mass%) in acetonitrile (200 mL) was added potassium carbonate (2 equiv., 110.9 mmol) at 0 °C and stirred for 30 minutes. Then tert-butyl 2-bromoacetate l-SM-3 (1.2 equiv., 66.52 mmol) was added to the reaction mixture and stirred at 25 °C for 12 h. After completion (monitored by TLC), the reaction mixture was diluted with ice cold water, extracted with ethyl acetate, dried over anhydrous Na2SO4, filtered and concentrated under vacuum. The desired crude material was purified by combi-flash column chromatography on silica gel using 30-35 % ethyl acetate in heptane. The desired fractions were concentrated under vacuum to obtain tert-butyl 2-[4-chloro-2-fluoro-6-[(4-methoxyphenyl)methoxy]-N-sulfamoyl-anilino]acetate 1-4 (22 g, 46.33 mmol, 83.57 %) as a pale brown solid. TLC system: 30 % EtOAc / heptane (Rf = 0.4) LC-MS: m / z = 475 (M+1).1H NMR (400 MHz, DMSO-d6) d 7.43 (d, 2H, J = 8.4 Hz), 7.11 (s, 1H), 7.11 - 6.93 (m, 6 H), 5.10 (s, 2H), 4.23-4.00 (m, 2H), 3.76 (s, 3H), 1.30 (s. 9H).

[0174]

[0084] Step-5: Synthesis of 5-(4-chloro-2-fluoro-6-((4-methoxybenzyl)oxy)phenyl)- 1,2,5-thiadiazolidin-3-one 1,1 -di oxide 1-5.

[0175]

[0085] To a stirred solution tert-butyl 2-[4-chloro-2-fluoro-6-[(4-methoxyphenyl)methoxy]-N-sulfamoyl-anilino]acetate 1-4 (A, 25 g, 59.69 mmol) in dry THF (250 mL, 99.8 mass%) at 0 °C was added sodium hydride (2 equiv., 119.4 mmol) portion wise under N2 atmosphere and then stirred at 0 °C to room temperature for 6 h. After completion (monitored by TLC), the reaction was quenched with cold water, acidified with 2N HC1 and extracted with DCM (2 x 150 mL). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under vacuum. The crude product was purified by combi-flash column chromatography on silica gel using 0-5 % MeOH in DCM. The fractions containing the product were combined and evaporated to dryness to afford 5-(4-chloro-2-fluoro-6-((4-methoxybenzyl)oxy)phenyl)-1,2,5-thiadiazolidin-3-one 1,1-dioxide 1-5 (15 g, 37.43 mmol, 62.69 %) as an off-white solid. TLC: 10 % MeOH / DCM (Rf 0.5). LC-MS m / z: 399 (M-l).

[0176]

[0086] Step-6: Synthesis of 5-(4-chloro-2-fluoro-6-hydroxyphenyl)-l,2,5-thiadiazolidin-3- one 1,1 -di oxide Compound 1.

[0177]

[0087] To a solution of 5-(4-chloro-2-fluoro~6-((4-methoxybenzyl)oxy)phenyl)-1,2,5- thiadiazolidin-3-one 1,1-dioxide 1-5 (50 g, 124.8 mmol) in DCM (100 mL) at 0 °C was Attorney Docket No.: 01330-0117-00PCT

[0178] added HCl (4 mol / L) in dioxane (5 equiv., 623.8 mmol). The reaction mixture was slowly warmed to room temperature and stirred for 12 h under N2 atmosphere. After completion (monitored by TLC) of the reaction, the precipitated solid was collected by filtration, washed with DCM (3 x 100 ml..) and EtOAc (5 x 100 mL) dried under vacuum to afford product 5- (4“ChlorO“2-fluorO"6~hydroxyphenyl)-l,2,5-thiadiazoHdin~3“One 1,1 -dioxide Compound 1 (24 g, 82,092 mmol, 65.81 %) as an off-white solid. 20 % MeOH / DCM (Ry. 0.5). LC-MS m / z: 279 (M-l).1H NMR (400 MHz, DMSO-d6) δ 11.01 (br s, I H ), 6.96 - 6.93 (m, 1H) 6.89 - 6.88 (m, 1H), 4.34 (s, 2H).

[0179]

[0088] Synthetic Example 2: Synthesis of 5-(4-bromo-2-fluoro-6-hydroxyphenyl)-1,2,5-thiadiazolidin-3-one 1,1-dioxide (Compound 2).

[0180] O=S-NH2

[0181] K2CO3, CH3CN,

[0182]

[0183]

[0089] Step-1: Synthesis of 5-bromo-1-fluoro-3-[(4-methoxyphenyl)methoxy]-2-nitrobenzene 2-1

[0184]

[0090] To a stirred solution of 5-bromo-l,3-difluoro-2-nitrobenzene 2-SM-l (50 g, 205.89 mmol) in DMF (500 mL) at room temperature under nitrogen atmosphere were added K2CO3 (58.07 g, 514.73 mmol) and / 2-methoxybenzyl alcohol (31.38 mL, 247.07 mmol). The reaction mixture was stirred under N2 atmosphere at 75 °C for 16 h. After completion (monitored by TLC), the reaction mixture was cooled to room temperature, and diluted with ice cold water. The precipitated solid was collected by filtration, washed with water and dried under vacuum to afford 5-bromo-l-fluoro-3-((4-methoxybenzyl)oxy)-2-nitrobenzene 2-1 (50 g, 255.1 mmol, 68.20 %) as a yellow solid. The title compound was used tor the next step without further purification. TLC: 10 % EA / Heptane (Ry 0.7).!H NMR (400 MHz, DMSO- dd d 7.64 - 7.63 (d, 1H), 7.59 - 7.56 (m, 1H), 7.34 (d, 2H), 6.96 (d, 2H), 5.27 (s, 2H), 3.76 (s, 3H). Attorney Docket No.: 01330-0117-00PCT

[0185]

[0091] Step-2: Synthesis of 4-bromo-2-fluoro-6-[(4-methoxyphenyi)methoxy]aniline 2-2.

[0186]

[0092] To a stirred solution of 5-bromo-l -fluoro-3-((4-methoxybenzyl )oxy)-2-nitrobenzene 2-1 (50.00 g, 140.4 mmol) in ethanol (350 mL) and water (150 mL) at room temperature were added NH4CI (37.6 g, 702.0 mmol) and Fe powder (40.84 g, 702.0 mmol). The reaction mixture was stirred at 80 °C for 6 h. After completion (monitored by TLC), the reaction mixture was cooled to room temperature and filtered through a celite pad. The celite pad was further washed with MeOH and DCM, the filtrate was concentrated under reduced pressure to afford the crude product. The crude product was diluted with ice cold water and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude material was purified by combi-flash column chromatography on silica gel using 5-15 % EtOAc in heptane to afford 4- bromo-2-fluoro-6-((4-methoxybenzyl)oxy)aniline 2-2 (36 g, 110.4 mmol, 45.8 %) as an off-white solid. TLC: 20 % EtOAc / Heptane (Rf: 0.80).rH NMR (400 MHz, DMSO-4 ) δ 7.42 (d, 2H, J = 8.4 Hz), 6.98 - 6.94 (m, 4H), 5.01 (s, 2H), 4.81 (s, 2H), 3.76 (s, 3H).

[0187]

[0093] Step-3: Synthesis of 5-bromo-1-fluoro-3-[(4-methoxyphenyl)methoxy]-2-(sulfamoylamino)benzene 2-3.

[0188]

[0094] To a stirred solution of 4-bromo-2-fluoro-6-((4-methoxybenzyl)oxy)aniline 2-2 (83.0 g 254.4 mmol) in DCM (750 mL) at 0 °C under N2 atmosphere was added triethylamine (112.6 mL, 763.3 mmol). The reaction mixture was stirred for 10 min, then sulfamoyl chloride 2-SM-2 (60.0 g, 508.9 mmol) was added portion wise at 0 °C. The reaction mixture was slowly warmed up to room temperature and stirred for an additional 4 h. After completion (monitored by TLC), the reaction mixture was concentrated under reduced pressure. The crude was purified by combi-flash column chromatography on silica gel using 0-40 % EtOAc / heptane. The fractions containing the product were combined and evaporated to dryness to afford 5-bromo-1-fluoro-3-[(4-methoxyphenyl)methoxy]-2-(sulfamoylamino)benzene 2-3 (55 g, 135.73 mmol, 53.34 %) as an off-white solid. TLC: 60% EtOAc / Heptane.JH NMR (400 MHz, DMSO- ) δ 8.42 (s, 1H), 7.46 (d, 2H, J = 8.8 Hz), 7.18 - 7.16 (m, 2H), 6.94 (d, 2H, J = 8.8 Hz), 6.70 (s, 2H), 5.13 (s, 2H), 3.74 (s, 3H).

[0189]

[0095] Step-4: Synthesis of tert-butyl 2-[4-bromo-2-fluoro-6-[(4-methoxyphenyl)methoxy]-N-sulfamoyl-anilino]acetate 2-4,

[0190]

[0096] To a stirred solution of 5-bromo-l-fluoro-3-[(4-methoxyphenyl)methoxy]-2- (sulfamoylamino)benzene 2-3 (75 g, 135.1 mmol) in acetonitrile (750 mL) at 0 °C was added Attorney Docket No.: 01330-0117-00PCT

[0191] K2CO3 (65.36 g, 472.9 mmol). The reaction mixture was stirred for 10 min, and then added dropwise tert-butyl 2-bromoacetate 2-SM-3 (53.79 g, 270.2 mmol) at 0 °C. The reaction mixture was slowly warmed to room temperature and stirred for further 6 h. After completion (monitored by TLC), the reaction mixture was diluted with ice cold water, extracted with ethyl acetate, dried over Na2SO4and concentrated under reduced pressure. The resulting crude product was purified by combi-flash chromatography on silica gel using 0-40 % EtOAc in / / -heptane as the eluent. The fractions containing the product were combined and evaporated to dryness to afford tert-butyl 2-[4-bromo~2-fluoro-6-[(4- methoxyphenyl)methoxy]-N-sulfamoyl-anilino]acetate 2-4 (55 g, 105.9 mmol, 78.37 %) as an off white solid. TLC: 60 % EtOAc / Heptane (R / = 0.5).

[0192]

[0193] 1H NMR (400 MHz, DMSO-d6) δ 7.43 (d, 2H, J = 8.8 Hz), 7.22-7.16 (m, 2H), 6.96-6.93 (m, 4H), 5.11 (s, 2H), 4.23-4.00 (m, 2H), 3.74 (s, 3H), 1.30 (s, 9H).

[0194]

[0097] Step-5: Synthesis of 5-(4-bromo-2-fluoro-6-((4-methoxybenzyl)oxy)phenyl)-1,2,5- thiadiazolidin-3-one 1,1-dioxide 2-BB-14.

[0195]

[0098] To a stirred solution of tert-butyl 2~[4-bromo-2~fluoro-6-[(4~ methoxyphenyl)methoxy]-N-sulfamoyl-anilino]acetate 2-4 (60 g, 115.5 mmol) in THF (700 mL) at 0 °C was added sodium hydride (20 g, 231.0 mmol). The reaction mixture was slowly warmed to room temperature and stirred for 5 h under N2atmosphere. After completion (monitored by TLC), the reaction mixture was diluted with cold water (250 mL), extracted with 10 % MeOH in DCM, washed with ice water (250 mL), brine solution (50 mL), dried over Na2SO4and concentrated under reduced pressure. The crude product was purified by combi flash chromatography on silica gel using 0-15 % MeOH / DCM as the eluent. The fractions containing the product were combined and evaporated to dryness to afford 5-(4- bromo-2~fluoro-6-((4~methoxybenzyl)oxy)phenyl)-l,2,5-thiadiazolidin-3-one 1,1-dioxide 2- BB-14 (40 g, 89.83 mmol, 77.76%) as an off white solid. TLC: 15% MeOH / DCM (Rf: 0.3). LC-MS m / z: 443.27 (M-1).

[0196]

[0099] Step-6: Synthesis of 5-(4-bromo-2-fluoro-6-hydroxyphenyl)-1,2,5-thiadiazolidin-3-one 1,1-dioxide Compound 2.

[0197]

[0100] To a stirred solution of 5-(4-bromo-2-fluoro-6-((4-methoxybenzyl)oxy)phenyl)-l,2,5- thiadiazolidin-3-one 1,1-dioxide 2-BB-14 (35 g, 57.38 mmol) in DCM (300 mL) at 0 °C was added HCl (4 mol / L) in 1,4-dioxane (120 mL, 480 mmol). The reaction mixture was slowly warmed to room temperature and stirred for 8 h under N2atmosphere. After completion of Attorney Docket No.: 01330-0117-00PCT

[0198] the reaction (monitored by TLC), the precipitated solid was collected by filtration, washed with DCM (3 x 100 mL) and EtOAc (5 xlOO mL) and dried under vacuum to afford 5-(4- bromo-2-fluoro-6-hydroxyphenyl)-l,2,5-thiadiazolidin-3-one 1,1-dioxide Compound 2 (15 g, 44.29 mmol,77.20%) as an off-white solid. TLC: 20% MeOH / DCM (Rf: 0.2). LC-MS m / z: 323.38 (M-l) (96 %).1H NMR (400 MHz, DMSO-d6) δ 11.3 (br s, 1H), 7.17 - 7.16 (m, 1H), 7.05 - 7.03 (m, 1H), 4.36 (s, 2H).

[0199]

[0101] Synthetic Example 3: Synthesis of 5-(3~bromo-2-fluorO”6-hydroxypheuyI)~l,2,5- thiadiaxolidin-3-one 1,1-dioxide (Compound 4)and 5-(2-fIuoro~4~hydroxy-[l,r~ biphenyI]-3~yI)~l,2,5-thiadiazoIid -3-one 1,1-dioxide (Compound 6),

[0200] Step-1 Step-2 Step-3 3-SM-1

[0201] K2CO3, CH3CN, Step-5 Step-6 Step-8

[0202]

[0203] Compound 4 Compound 6

[0204]

[0102] Step-1: Synthesis of 1-(benzyloxy)-3-fluoro-2-nitrobenzene, 3-1

[0205]

[0103] To a stirred solution of l,3-difluoro-2-nitrobenzene 3-SM-l (50 g, 210.09 mmol) in DMF (200 mL) at room temperature under nitrogen atmosphere were successively added K2CO3 (58.07 g, 420.18 mmol) and benzyl alcohol (21.75 mL, 210.09 mmol). The reaction mixture was stirred under N2 atmosphere at 80 °C for 16 h. After completion (monitored by TLC), the reaction mixture was cooled to room temperature and diluted with ice cold water.

[0206] The precipitated solid was collected by filtration, washed with water and dried under vacuum afforded l-(benzyloxy)-3-tluoro-2-nitrobenzene 3-1 (50 g, 153.31 mmol, 72.98%) as a

[0207] yellow solid. TLC: 10% EtOAc / Heptane (Ry 0.65). LC-MS: m / z 248.07 (M+l). The title compound was used for the next step without further purification.

[0208]

[0104] Step-2: Synthesis of 1-(benzyloxy)-4-bromo-3-fluoro-2-nitrobenzene, 3-2.

[0209]

[0105] To a stirred solution of 1-benzyloxy-3-fluoro-2-nitrobenzene 3-1 (50 g, 202.3 mmol) in ACN (500 mL) were successively added dropwise AcOH (5 equiv., 1011 mmol) and TMSCI (2 equiv., 404.5 mmol) followed by NBS (1.5 equiv., 303.4 mmol) at 0 °C under nitrogen atmosphere. The reaction mixture was stirred at room temperature for 12 h. After completion Attorney Docket No.: 01330-0117-00PCT

[0210] (monitored by TLC), the reaction mixture was treated with ice cold water (200 mL) and the resulting solid was filtered, washed with cold water (150 mL) and dried to get the desired product l-(benzyloxy)-4-bromo-3-fluoro-2-nitrobenzene 3-2 (40 g, 121.4 mmol) as a pale yellow solid. TLC: 20% EtOAc / Heptane Rf: 0.5, LC-MS: m / z 326.12 (M+l).

[0211]

[0106] Step-3: Synthesis of 6-(benzyloxy)-3-bromo-2-fluoroaniline, 3-3.

[0212]

[0107] To a stirred solution of 1-(benzyloxy)-4-bromo-3-fluoro-2-nitrobenzene 3-2 (50 g, 151.8 mmol) in EtOH (500 mL) and water (100 mL) at room temperature were added NH4Cl (5 equiv., 759.0 mmol) and Fe powder (5 equiv., 759.0 mmol). The reaction mixture was stirred at 70 °C for 9 h. After completion (monitored by TLC), the reaction mixture was cooled to room temperature and filtered through a celite pad. The celite pad was further washed with MeOH and the filtrate was concentrated under reduced pressure. The crude product was diluted with ice cold water and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford 6-(benzyloxy)-3-bromo-2-fluoroaniline 3-3 (50 g, 116.5 mmol, 76.76%) as a brown colour liquid. TLC: 20% DCM / Hexane, Rf: 0.5. LC-MS: m / z 296.10 (M+l).

[0213]

[0108] Step-4: Synthesis of 6-bromo-l-fluoro~3”[(pheiiyI)iiiethoxy]-2~(siiIfamoyIamino) benzene, 3-4.

[0214]

[0109] To a stirred solution of 6-(benzyloxy)-3-bromo-2-fluoroaniline 3-3 (50 g, 125.0 mmol) in DCM (300 mL) at 0 °C was added triethylamine (3 equiv., 374.9 mmol) under nitrogen atmosphere followed by the addition of sulfamoyl chloride (3-SM-2) (2 equiv., 249.9 mmol,). The reaction mixture was stirred at room temperature for 4 h. After completion (monitored by TLC), the reaction mixture was concentrated under reduced pressure. The crude product was purified by combi-flash column chromatography on silica gel using 0-30% EtOAc in Heptane. The fractions containing the desired product were combined and evaporated to dryness to afford 6-bromo-1-fluoro-3-[(phenyl)methoxy]-2-(sulfamoylamino)benzene 3-4 (30.7 g, 81.06 mmol, 81.05%) as an off white solid. TLC: 50% EtOAc / Hexane. (Rf: 0.3) LC-MS: m / z 375.20 (M-l).

[0215]

[0110] Step-5: Synthesis of tert-butyl 2-[3-bromo-2-fluoro-6-[(phenyl)methoxy]-N-sulfamoyl-anilino]acetate, 3-5.

[0216] [Hl] To a stirred solution of 6-bromo-l-fluoro-3-[(phenyl)methoxy]-2- (sulfamoylamino)benzene 3-4 (33 g, 87.95 mmol) in CH2Cl2(300 mL) at room temperature Attorney Docket No.: 01330-0117-00PCT

[0217] under nitrogen atmosphere were successively added K2CO3 (3.5 equiv., 307.8 mmol) and tert-butyl 2-bromoacetate 3-SM-3 (2 equiv., 175.9 mmol). The reaction mixture was stirred at room temperature for 12 h. After completion (monitored by TLC), the reaction mixture was filtered through a celite pad. The celite pad was further washed with acetonitrile and the filtrate was concentrated under reduced pressure. The resulting crude product was purified by combi-flash column chromatography on silica gel using 0-30 % EtOAc / Heptane. The relevant fractions containing the product were combined and evaporated to dryness to afford tert-butyl 2-[3-bromo-2-fluoro-6-[(phenyl)methoxy]-N-sulfamoyl-anilino]acetate 3-5 (28 g, 57.21 mmol, 65.05%) as an off white solid. TLC: 40% EtOAc / Heptane (Rf: 0.5). LC-MS m / z:489.3 (M+l).

[0218]

[0112] Step-6: Synthesis of 5-(6-(benzyfoxy)-3"bromo-2"fiuorophenyl)-l,2,5" thiadiazoIidin-3-oiie Ll-dioxide, 3-6.

[0219]

[0113] To a stirred solution of tert-butyl 2-[3-bromo-2-fluoro-6-[(phenyl)methoxy]-N-sulfamoyl-anilino]acetate 3-5 (50 g, 82.75 mmol) in dry THF (500 mL) at 0 °C was added sodium hydride (2 equiv., 165.5 mmol). The reaction mixture was slowly warmed to room temperature and stirred for 5 h under nitrogen atmosphere. After completion (monitored by TLC), the reaction mixture was diluted with cold water (200 mL), extracted with 10% MeOH in DCM, washed with ice water (200 mL), brine solution (100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified by combi- flash column chromatography on silica gel using 0-15 % MeOH in DCM. The fractions containing the product were combined and evaporated to dryness to afford 5-(6-(benzyloxy)-3-bromo-2-fluorophenyl)-1,2,5-thiadiazolidin-3-one 1,1-dioxide 3-BB-7 (32 g, 73.22 mmol, 88.48%) as an off-white solid. TLC: 15% MeOH / DCM (Rf: 0.3). LC-MS m / z: 415.27 (M+l).

[0220]

[0114] Step-7: Synthesis of 5-(3-bromo-2-fluoro-6-hydroxyphenyl)-1,2,5-thiadiazolidin-3-one 1,1-dioxide, Compound 4.

[0221]

[0115] To stirred solution of 5-(6-(benzyloxy)-3-bromo-2-fluorophenyl)-l,2,5-thiadiazolidin- 3-one 1,1-dioxide 3-BB-7 (10 g, 24.08 mmol) in DCM (100 mL) was added BBr3in DCM (3 equiv., 72.25 mmol, 1 mol / L) under nitrogen atmosphere at 0 °C. The reaction mixture was stirred at. 25 °C for 5 h. After completion (monitored by TLC), the reaction mixture was evaporated to dryness. The crude compound was purified by flash-column chromatography on silica gel using 50-60% EtOAc / Hexane. The desired fractions were concentrated to afford 5-(3-bromO"2-fluoro-6“hydroxyphenyi)“l,2,5-thiadiazolidin-3" One 1,1-dioxide Compound 4, Attorney Docket No.: 01330-0117-00PCT

[0222] (4.50 g, 13.4 mmol, 55.7%) as an off-white solid. TLC: 15 % MeOH / DCM (Rf: 0.1). LC-MS m / z: 325.19 (M-1) (97%).1H NMR (400 MHz, DMSO-d6) δ 10.5 (br s, 1H), 7.51 - 7.46 (m, 1H), 6.74 - 6.72 (m, 1H), 4.31 (s, 2H).

[0223]

[0116] Step-8: 5-(2-flworo~4-hydroxy-[l,1 ’-biphgnyI]"3~ i)~1!>5-thiadiazondiu-3-0ne 1,1- dioxide, Com pound 6.

[0224]

[0117] To a stirred solution of 5-(3-bromo~2-fluoro-6-hydrox.yphenyl)-1,2,5~thiadiazolidin~3~ one 1,1 -dioxide Compound 4 (300 mg, 0.92 mmol) in 1,4-dioxane (16 mL) were added phenylboronic acid 3-SM-4 (1.5 equiv., 1.38 mmol), K3PO4 (3.0 equiv., 2.76 mmol), and Pd(dppf)Ch. DCM (0.15 equiv., 0.13 mmol). The reaction mixture was purged with argon for 15 min and heated at 90 °C for 16 h. After completion (monitored by TLC), the reaction mixture was filtered through a pad of celite and washed with EtOAc (100 mL). The filtrate was concentrated under reduced pressure to get the crude compound which was purified by prep-HPLC purification to afford 5-(2-fluoro-4-hydroxy-[l.r-biphenyl]-3-yl)-l,2.5- thiadiazolidin-3-one 1,1 -dioxide Compound 6 (21.0 mg, 0.06 mmol, 7.02 %) as a pale brown solid.

[0225] TLC: 15% MeOH / DCM (Rf: 0.5). LC-MS m / z: 323 (M+1, 99%).1H NMR (400 MHz, DMSO-d6) δ 9.9 (br s, 1H), 7.47 - 7.42 (m, 4H), 7.37 - 7.28 (m, 2H), 6.82 - 6.79 (m, 1H), 4.15 (s, 2H).

[0226] Prep-HPLC purification method:

[0227] Preparative Column X SELECT C18 (250*30)mm, 5μm

[0228] Mobile Phase A 0.1% TFA IN WATER

[0229] Mobile Phase B 100% Acetonitrile

[0230] Flow rate 22ml / min

[0231] Instrument ID PREP -26

[0232] Gradient (Time / %B) 0 / 10, 3 / 10, 20 / 55, 30 / 100.

[0233]

[0118] Additional Synthetic Examples: The synthesis of Compounds 3, 5, and 7 was performed in a manner similar to Compounds 1, 2, 4 and 6 as disclosed above, as would be known by those skilled in the art. Compounds 3-(3-chloro-2-fluoro-6-hydroxyphenyl)-1,2,5-thiadiazolidin-3-one 1,1-dioxide (Compound 3), 5-(4-bromo-3-chloro-2-fluoro-6- hydroxyphenyl)-l,2,5-thiadiazolidin-3-one 1,1-dioxide (Compound 5); and 5-(2-fluoro-6- hydroxyphenyl)-l,2,5-thiadiazolidin-3-one 1,1-dioxide (Compound 7) were isolated, Attorney Docket No.: 01330-0117-00PCT

[0234] characterized, and tested in the assays described below.

[0235]

[0119] Biological Assays and Examples

[0236]

[0120] Biological Example 1: PTPN1 and PTPN2 enzymatic assay

[0237]

[0121] Compound activity was determined using a human PTPN1 recombinant protein (Assay Genie, Lot #322PPTN 1, Cat# RPPB2640) or a human PTPN2 recombinant protein (Assay Genie, Lot #ER0340260211, Cat# RPES3671) in a biochemical enzymatic assay. The enzymatic reaction was carried out at 37°C in assay buffer. A containing 0.01% TWEEN-20, 150 mM NaCl, 1 mM EDTA and 2 mM DTT (Sigma Aldrich, Cat# 646563-10X.5ML) with the substrate DiFMUP (Thermo Scientific, Cat# D6567) in a Opti Plate 384-well Black Microplate (Revvity, Cat# 6007270). In each well, 12.5 pL of buffer A was added together with 12.5 μL of enzyme at 0.12 μg / mL for a final dilution of 0.03 μg / mL. Compounds resuspended in DMSO (Sigma, Cat# D8418-500ML) at 10 mM were diluted in semi-log dilutions in DMSO ranging from 10 mM to 0.001 mM and DMSO only controls were included. The compound / DMSO dilutions were further diluted 1:10000 in buffer A, and 12.5 μL of these dilutions were added in duplicates to the wells with buffer and enzyme. The final compound concentrations ranged from 1 μM to 0.00001 μM. Finally, 12.5 μL of DiFMUP at 20 μM concentration was added to all the wells for a final concentration of 5 μM. The plate was left in the incubator at 37°C for 1 hour and fluorescence was acquired using an EnVision 2105 multimode reader (Revvity®) with an excitation of 358 nm and emission at 450 nm. The data was fitted, using a 4-parameter equation, to determine the inflection point of the fit as the IC50 of the small molecule inhibitor for each specific enzyme considering 100% enzyme activity where only DMSO was added. The results of this assay are shown in Table 2 below;

[0238]

[0122] Biological Example 2: A549 PhosphoSTATI. Assay

[0239]

[0123] A549 human lung carcinoma epithelial cells (Cell Player™ A549 NucLight Red, Essen BioScience Cat#449i) wzere seeded at a density of 40000 cells per well in a sterile 384 well F-bottom white cell culture microplate (Greiner, Cat# 781080) in 80 μL total volume of IMDM + 10%FBS (Capricorn Scientific, Cat# IMDM-A) and Gibco, Cat# A5256701). Cells were allowed to adhere overnight at 37 °C + 5% CO2. The following day, compounds resuspended in DMSO (Sigma, cat# D8418-500ML) at 10 mM were diluted in semi-log dilutions in DMSO ranging from 10 mM to 0.001 mM and DMSO only controls were included. The compound / DMSO dilutions were further diluted 1:50 in HBSS, media from the Attorney Docket No.: 01330-0117-00PCT

[0240] plates was removed and 25 μL of these dilutions were added in duplicates to the cells. Final compound concentrations ranged from 100 μM to 0.001 μM with a final DMSO concentration of 1%. Cells and compounds were incubated for 1h at 37 °C + 5% CO2before the addition of 25 μL of human IFNγ (Peprotech, Cat# 300-02) at 200 ng / mL for a final concentration of 100 ng / mL. The plate was incubated for 1h at 37 °C + 5% CO2and assayed for phosphorylated STAT1 protein levels with the AlphaLISA Surefire Ultra Human and Mouse Phospho-STAT1 (Tyr701) kit (Revvity, Cat# ALSU-PST1-A10K) according to the manufacturer’s instructions. Plate signal was read using a SpectraMax i3x multiplate Spectrophotometer reader (Molecular Devices®) with an excitation of 680 nm and emission at 615 nm. The data was fitted, using a 4-parameter equation, to determine the inflection point of the fit as the EC50 of the small molecule inhibitor considering 0% phosphoSTATl where only DMSO was added. Results of this assay are shown in Table 2 below.

[0241]

[0124] Biological Example 3: B16 PhosphoSTATl Assay

[0242]

[0125] B16F10 mouse melanoma cells (ATCC Cat# CRL-6475) were seeded at a density of 40000 cells per well in a transparent F-bottom 96-well plate (Greiner, Cat# 655 180) in 150 μL total volume of DMEM + 10%FBS (Capricorn Scientific, Cat# DMEM-HPSTA and Gibco, Cat# A5256701). Cells were allowed to adhere overnight at 37 °C + 5% CO2. The following day, compounds resuspended in DMSO (Sigma, cat# D8418-500ML) at. 10 mM were diluted in semi-log dilutions in DMSO ranging from 10 mM to 0.001 mM and DMSO only controls were included. The compound / DMSO dilutions were further diluted 1:50 in HBSS, media from the plates was removed and 50 μL of these dilutions were added in duplicates to the cells. Final compound concentrations ranged from 100 μM to 0.001 μM with a final DMSO concentration of 1%. Cells and compounds were incubated for 1h at 37 °C + 5% CO2before the addition of 50 μL of mouse IFNγ (Peprotech, Cat# 315-05) at 200 ng / mL. for a final concentration of 100 ng / mL. The plate was incubated for 1h at 37 °C + 5% CO2and assayed for phosphorylated STAT1 protein levels with the AlphaLISA Surefire Ultra Human and Mouse Phospho-STATl (Tyr701) kit (Revvity, Cat# ALSU-PST1-A10K) according to the manufacturer’s instructions. Plate signal was read using a SpectraMax i3x multiplate Spectrophotometer reader (Molecular Devices®) with an excitation of 680 nm and emission at 615 nm. The data was fitted, using a 4-parameter equation, to determine the inflection point of the fit as the EC50 of the small molecule inhibitor considering 0% phosphoSTAT1 where only DMSO was added. Attorney Docket No.: 01330-0117-00PCT

[0243]

[0126] The results of this assay are shown in Table 2 below.

[0244] Table 2

[0245] ICso(nM) ECso(nM) Cmpd# Structure A549 B16

[0246] PTPN1 PTPN2

[0247] 00 STAT1 STAT1 o O TO - ■! -5 —

[0248] 1 477 687 3406 12796

[0249] O \

[0250] oOo° O o O z^ z^ z'cwcnn--- o o z*w-111

[0251] o o o

[0252] 2 o 210 345 2130 9902

[0253] 3 O o 812 1561 2995 3372 x X

[0254] u \ — — O o

[0255] 4 278 513 2083 2999 o tn

[0256] 5 254 338 2662 4156

[0257] 6 49 86 771 1236

[0258] 0

[0259] FO^s-NH

[0260] 7 683 960 3593 4694

[0261]

[0262] Attorney Docket No.: 01330-0117-00PCT

[0263]

[0127] Biological Example 4: Permeability

[0264]

[0128] Compound 6 was tested in a permeability study, and the data is summarized below:

[0265] Permeability

[0266] MDR1 A-B with PGP inh. (10-6cm / s) 0.43

[0267]

[0268]

[0129] Biological Example 5: Rat PK Study

[0269]

[0130] Compounds 1, 2 and 6 were tested in a rat PK study to determine initial pharmacokinetic and bioavailability parameters in an animal model. The data are summarized below in Table 3 below.

[0270] Table 3

[0271] Compound 1 Compound 2 Compound 6 Dose (mg / kg) 1 (IV)a3 (PO)b1 (IV) 3 (PO)d1 (IV)e3 (PO)fCL (mL / min / kg) 2.57 - 1.7 - 6.25

[0272] Total

[0273] T1 / 2 IV / PO (h) 3.94 4.64 3.88 5.24 2.25-4.99 NR

[0274] F% 76 50 73-98

[0275]

[0276] a: 0.5 mg / mL in PEG400: 10 mM PBS (pH=7.4) (70: 30). clear solution

[0277] b: 0.6 mg / mL in 10% ethanol, 30% PEG-400, 60% Phosal-50PG, clear yellowish solution

[0278] c: 0.6 mg / mL in 10% ethanol, 30% PEG-400, 60% Phosal-50PG, clear yellow solution

[0279] d: 0.5 mg / mL in PEG400: 10 mMPBS (pH=7.4) (70: 30), clear solution; clear solution pl-16.5

[0280] e: 0.5 mg / mL in PEG400: 10 mM PBS (pH=7.4) (70: 30), clear solution

[0281] f: 0.6 mg / mL in 10% ethanol. 30% PEG-400, 60% Phosal-50PG, Yellow solution

[0282] NR: Not Reported due to inadequate terminal phase

[0283]

[0131] The data in Table 3 was based on a pharmacokinetic and bioavailability study of test compounds following a single intravenous and oral dose administration in male Sprague Dawley rats. Each rat was between 6-12 weeks old, had a body weight of 300 ± 50 g, and were fed normally. A total of 3 animals were used for each dosing group. All animals were dosed through jugular vein cannula / tail vein. The dosing volume was 2 mL / kg for the IV dose group and 5 mL / kg for the PO dose group. The formulation strength was 0.5 mg / mL for the IV dose group and 0.6 mg / mL for the PO dose group.

[0284]

[0132] The sampling time points were: IV: 0.08, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 hrs, Post dose. PO: PD, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 hrs. Post dose.

[0285]

[0133] Approximately 100 pL of blood was collected from each animal through jugular vein cannula at the predetermined time points in an Eppendorf tube containing K2EDTA. An equal Attorney Docket No.: 01330-0117-00PCT

[0286] volume of heparinized saline was flushed after collection of each sample. The sample was collected through saphenous vein or retro orbital plexus in case of patency loss. Plasma was harvested by centrifugation at 10000 rpm for 10 min at set temperature of 4°C. Samples were stored at -70 ±10°C. Plasma samples were analyzed using Fit for purpose LC-MS / MS method. PO dose formulation was analyzed for accuracy by fit-for-purpose LC-MS / MS or UPLC method.

[0287]

[0134] The pharmacokinetic parameters were calculated using the non-compartmental analysis tool of Phoenix® WinNonlin software (Ver: 8.3) with Linear Trapezoidal Linear Interpolation calculation method. The pharmacokinetic parameters such as Half-life (tl / 2), clearance (CL), etc. were the outputs.

[0288]

[0135] The following formulas were used to calculate PK parameters.

[0289]

[0136] Clearance (mL / min / kg) = (Dose / AUC0-inf) / 60: Clearance is defined as the elimination rate of the compound from the plasma. AUC0-inf (ng.h / mL) is the time-averaged concentration of drug circulating in the body fluid analyzed (here, plasma), extrapolated to infinity. Here, the Dose should be converted to ng / kg.

[0290]

[0137] T1 / 2 (h)=0.693 / Ke: T1 / 2 is defined as half-life of the compound in plasma or time for plasma concentration to reduce by half. Ke is the elimination rate constant calculated from Ke = CL / Vdss. Vdss (L / kg) is the volume of distribution of the compound into the tissues at steady state.

[0291]

[0138] Bioavail ability: F% or bioavailability is defined as the % of compound reaching blood stream. AUCO-last (ng.h / mL) is the time-averaged concentration of drug circulating in the body fluid analyzed (here, plasma), up to the last measured time point.

[0292] AUC0-last PO Dose IV

[0293] %F = X X 100

[0294] AUC0-last IV Dose PO

[0295]

[0296]

[0139] Biological Example 6: Comparison study of Compound 1 and Compound 6 versus ABBV-CLS-484

[0297]

[0140] Compound 1 and Compound 6 were tested against ABBV-CLS-484 (a clinical compound currently in Phase I, which may be prepared in accordance with, for example, US Patent 10,811,073) in various studies, i.e., rat and dog PK study to determine pharmacokinetic and bioavailability parameters in animal models, in vitro photo-STAT1 Attorney Docket No.: 01330-0117-00PCT

[0298] (pSTATl) assay human and mouse studies, physical chemistry studies, and ADME profile studies.

[0299] Rat IV / PO pharmacokinetics (PK) profiles:

[0300]

[0141] Pharmacokinetics profiles were evaluated following single oral and intravenous doses to a set of fed Male Sprague Dawley Rat (3 animal s / route / dose, Hylasco India or any other provider). Compounds (Compound 1, Compound 6, and ABBV-CLS-484) were formulated in 10% ethanol, 30% PEG-400, 60% Phosal-50PG for PO administration, and PEG400: 10 mM PBS (pH 7.4) = 70: 30 for IV administration.

[0301]

[0142] For PO, all animals were administered compounds by oral gavage. For IV, all animals were dosed through jugular vein cannula / tail vein. Approximately 100 pL of blood was collected from each animal through jugular vein cannula at predetermined time points (IV / PO: 0.08, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h post dose) in Eppendorf tubes containing K2EDTA. Equal volume of heparinized saline was flushed after collection of each sample. Sample was collected through saphenous vein or retro-orbital plexus in case of patency loss. Plasma was harvested by centrifugation at 10000 rpm for 10 min at set temperature of 4°C and stored at -70°C. Plasma samples were analyzed using Fit for purpose LC-MS / MS method.

[0302]

[0143] Plasma concentration-time data was subjected to the non-compartmental analysis tool of Phoenix® WinNonline software (Version: 8.0 or higher). The peak plasma concentrations (Cmax) and time to reach peak concentrations (Tmax) were the observed values. Area Under the Curve (AUG) was estimated using linear trapezoidal rule. The other pharmacokinetic parameters reported include, but not limited to, terminal elimination plasma half-life (t½), clearance (CL), volume of distribution at steady state (Vss), back extrapolated peak concentration (Co). Absolute oral bioavailability was also calculated and reported.

[0303] Dog IV / PO PK:

[0304]

[0144] Pharmacokinetics profiles were evaluated following single oral and intravenous doses to a set of Male Beagle Dogs (3 animal s / route / dose, Vimta Labs, Hyderabad, India or any other approved vendor). Dogs were overnight fasted and feed was provided 4 h post dose.

[0305]

[0145] Compounds were formulated in 10% ethanol, 30% PEG-400. 60% Phosa1-50PG for PO administration, and PEG400: 10 mM PBS (pH=7.4) = 70: 30 for IV administration. Attorney Docket No.: 01330-0117-00PCT

[0306]

[0146] For PO, all animals were administered compounds by oral gavage. For IV, all animals were dosed through saphenous vein. Approximately 1 mL of blood was collected from each animal through cephalic or jugular vein at pre-determined time points (IV / PO: 0.08, 0.25, 0.5, 1, 2, 4, 8, 12, 24 and 48 h post dose) in K2EDTA coated vacutainer tubes. Plasma was harvested by centrifugation at 10000 rpm for 10 min at set temperature of 4°C and stored at - 70°C. Urine collection for IV group was done at 0-4, 4-8, 8-24 and 24-48 h sampling intervals. Plasma and urine samples were analyzed using Fit for purpose LC-MS / MS method.

[0307]

[0147] Plasma concentration-time data was subjected to the non-compartmental analysis tool of Phoenix® WinNonline software (Version: 8.3 or higher). Area Under the Curve (AUC) was estimated using linear trapezoidal rule. The other pharmacokinetic parameters reported include, but not. limited to, terminal elimination plasma half-life (t½), clearance (CL), volume of distribution at steady state ( Vss) and back extrapolated peak concentration (Co). Urine concentration in predetermined time intervals was presented. Percent recovery and urinary excretion rate was also calculated and reported.

[0308] Kinetic solubility:

[0309]

[0148] Reagents were prepared.

[0310]

[0149] Preparation of 100mM Phosphate buffer (pH 7.4):

[0311] Solution A: 27.2 g KH2PO4 per 200 mL (1 M final) in water.

[0312] Solution B: 34.8 g K2HPO4 per 200 mL (1 M final ) in water.

[0313] For pH 7.4, 19.8 mL of Solution A and 80.2 mL of Solution B was mixed and volume made up to 1 L and adjust pH 7.4 with HCL / NaOH.

[0314]

[0150] Preparation of test Compound and Assay Controls:

[0315] 20 mM master stock was prepared in 100% DMSO.

[0316]

[0151] Preparation of stop solution:

[0317] 1 mg / mL of internal standard of Glipizide and Albendazole were prepared in DMSO and methanol (1:1).

[0318] Acetonitrile containing internal standard (Glipizide -300 ng / mL, Albendenzole -100 ng / mL) wzas prepared using 1 mg / mL DMSO stock.

[0319]

[0152] Assay Procedure:

[0320]

[0153] Solubility analysis procedure:

[0321] Assay was performed in duplicate (n=2) in 1.1 mL 96 w'ell deep well plates. Attorney Docket No.: 01330-0117-00PCT

[0322] 5 pL of 20 rnM DMSO stock of test compound or control compound was spiked in to 495 p L of buffer solution at pH 7.4.

[0323] The plate was sealed and incubated at room temperature at 250 rpm for 4 h.

[0324] 200 uL of samples from lite incubation plate was taken and filtered through 0.22 micron filter plates and filtrate was collected in a fresh plate.

[0325] 2 pL of filtrate was added to 998 pL of acetonitrile containing internal standard and submitted for LC-MSMS analysis.

[0326]

[0154] Preparation of Calibration Curve ICC;-'

[0327] Higher standard of 300 p. M was prepared by adding 6 pL of 20 mM stock solution to 394 pL of DMSO.

[0328] Subsequent serial dilutions were prepared in 100% DMSO to produce 150, 50, 25, 10, 5 and 1 ti.

[0329] 2 pL of each calibration standard was added to 998 pL of acetonitrile containing internal standard and submitted for LC-MS / MS analysis.

[0330]

[0155] Sample analysis: all of the samples were analyzed using LC-MS / MS.

[0331]

[0156] Calculations: the concentration of test compounds and positive control in the samples were calculated from the slope generated against the calibration curve in the LC-MS / MS.

[0332]

[0157] Acceptance criteria: caffeine 156 pM -220 pM.

[0333] Measurement of Log D (lipophilicity) of test compounds by shake flask method using potassium phosphate buffer pH 7.4:

[0334]

[0158] Preparation of reagents:

[0335]

[0159] Preparation of 10mM Phosphate buffer (pH 7.4):

[0336] Solution A: 27.2 g KH2PO4 per 200 mL (I M final) in water.

[0337] Solution B: 34.8 g K2HPO4 per 200 mL (1 M final) in water.

[0338] For pH 7.4, 1.98 mL of Solution A and 8.02 mL of Solution B was mixed and volume made up to 1 L and adjust pH 7.4, if required.

[0339]

[0160] Preparation of Pre-saturated Octanol and Phosphate buffer (pH 7.4):

[0340] - Equal volumes of Octanol and buffer were mixed in a 1 L bottle and shaken at 300 rpm for 24 h. After 24 h, the mixture was transferred to a separating funnel and kept aside for saturation for 48 h without disturbing.

[0341] After 48 h, both the layers were separated and collected in a separate bottles. Attorney Docket No.: 01330-0117-00PCT

[0342]

[0161] Preparation of Test Compound and Assay Controls: 10 mM master stock was prepared in 100% DMSO.

[0343]

[0162] Preparation of stop solution:

[0344] 1 mg / ml. of internal standard of Glipizide and Albendazole were prepared in DMSO and methanol (1:1).

[0345] Acetonitrile containing internal standard (Glipizide - 300 ng / 'mL, Albendenzole -100 ng / mL) was prepared using 1 mg / mL DMSO stock.

[0346]

[0163] Assay Procedure:

[0347] 10 pL of 10 mM DMSO stock was spiked in to 2 mL 96 deep well plate.

[0348] 495 uL of presaturated octanol was added to the compound spiked in 2 mL 96 well plate and mix for 5 min at 1000 rpm on mixmate.

[0349] 495 pL presaturated phosphate buffer was added to 2 mL plate containing reference / test. compound.

[0350] The plate was kept for shaking at RT for 3 h at 1200 rpm on mixmate.

[0351] After 3 h of shaking, the plate was centrifuged at 4000 rpm for 30 min.

[0352] 200 pL of octanol (upper layer) and 200pL of buffer was separated by using Tecan instrument.

[0353]

[0164] Sample Preparation for LC-MS / MS:

[0354] - Matrix Match of Samples: 10 pL of octanol sample was added to 30 pL of blank buffer and 30 pL of buffer sample was added to 10 pL of blank octanol.

[0355] 300 uL of acetonitrile containing internal standard was added to the plate and vertexed for 5 min at 900 rpm.

[0356] The samples w7ere centrifuged at 4000 rpm for 10 min.

[0357] 200 pL supernatant was collected and submitted for LC-MS / MS analysis.

[0358] Mouse / Rat / Dog / Human Hepatocyte metabolic stability:

[0359]

[0165] Preparation of assay controls and test compounds:

[0360] Stock solution of test compounds and assay controls were prepared at 20 mM or 50 mM and 10 M in DMSO respectively. Intermediate stock of 1 mM or 3 mM were prepared in DMSO for test compound and assay controls.

[0361] 1 mL of 2 pM and 6 pM of working stock of test compound and assay control were prepared in KHB Buffer by adding 2 pL of intermediate stock to 998 p. L of KHB, Attorney Docket No.: 01330-0117-00PCT

[0362] respectively.

[0363]

[0166] Preparation of internal standard stock solution: 1 mg / mL of Cetirizine / Tolbutamide / Telmisartan / Bicalutamide was prepared in DMSO and methanol (1:1) separately.

[0364]

[0167] Preparation of quenching solution: 100 pL of Cetirizine / Telmisartan / Bicalutamide / Tolbutamide (1 mg / mL) was prepared in 1 L of acetonitrile which is used as quenching solution (lOOng / mL).

[0365]

[0168] Thawing of cryopreserved Hepatocyte Procedure:

[0366] 1. The biosafety cabinet (BSC) was sterilized by using 70 % Isopropyl alcohol (IPA) and UV light of the room and BSC was switched for 30 min.

[0367] 2. In Biosafety cabinet, 45 mL of Hepatocyte INVITROGRO H Medium and 50 mL of INVITROGRO KHB buffer were transferred to sterile tube separately and kept in the water bath at 37°C for 30 minutes.

[0368] 3. The 50 mL tube containing INVITROGRO HT Medium was wiped with 70% Isopropyl alcohol before placing in safety cabinet.

[0369] 4. Cryopreserved hepatocyte vial was quickly transferred from the liquid nitrogen storage system into the 37°C water bath.

[0370] 5. The immersed vial was shaken gently in the water bath until the vial was completely thawed. The thawing process was approximately for 2 (90-120 sec) minutes.

[0371] 6. The vial was removed from the water bath when the last ice crystal was about to disappear. The vial w7as further wiped with 70% IPA after removing the vial from the water bath. The contents of the vial were immediately transferred into a centrifuge tube containing 45 mL of pre- warmed IN VITROGRO HT Medium and the vial was rinsed twice with INVITROGRO HT Medium to retrieve if any leftover cells. The tube was inverted gently few times for proper mixing followed by centrifugation for 10 minutes at 100 x g at room temperature.

[0372] 7. After centrifugation, the tube was wiped with 70% IPA and supernatant was decanted by inverting the tube in BSC.

[0373] 8. The cells were re-suspended with 1.0 mL of INVITROGRO KHB buffer to the cell pellet. The cell pellet was gently mixed with the media until the cell pellet was dispersed and the cells were re-suspended. Attorney Docket No.: 01330-0117-00PCT

[0374]

[0169] Cell counting:

[0375] 1. 25 pL of trypan blue dye and 25 pL of cell suspension was added to 450 pL of KHB Buffer.

[0376] 2. The 10 uL of aliquot of cells was transferred to haemocytometer and counted under an inverted microscope. Based on the cell count, the cell density was adjusted to 2 x 106 cells / mL by adding appropriate volume of KHB Buffer.

[0377]

[0170] Assay Procedure:

[0378] 1. The cell suspension (2 x 106cells / mL) was placed on ice.

[0379] 2. Vibramax was placed inside the CO2 incubator after achieving appropriate temperature and CO? level in incubator.

[0380] 3. 200. L of cell suspension containing 2 x 106cells / mL and 300 pl.. of test compounds and assay controls in KHB Buffer solution (2 pM and 6 pM) were added in individual 48 well plate.

[0381] 4. The 48 well plates were placed on vibramax shaker to allow the compound solution and cell suspension to warm for approximately 10 min with gentle shaking.

[0382] 5. The reaction was initiated by adding 200 pL of KHB Buffer containing test compound and assay control to 200 uL of cell suspension containing 2 x 106cells / mL in respective well (The density of cells in the final assay was 1 x 106cells / mL, I pM of test compound and 3 pM of assay control)

[0383] 6. At each time point (0.5, 5, 10, 15, 30, 60, 90 and 120 min), 25 pL of sample was withdrawn from the 48 well plate and the reaction was terminated immediately by adding 200 pl.. of ice-cold acetonitrile containing internal standard.

[0384] 7. The samples were vortex-mixed for 5 minutes and centrifuged at 4000 rpm for 15 min 8. After centrifugation, 100 pL of supernatant was transferred to 96 well plate. The samples were analysed by LC-MS / MS.

[0385] 9. Cell viability was taken after completion of experiment.

[0386]

[0171] Data Analysis:

[0387] - Peak area ratios (analyte to internal standard) were considered for calculation of percent parent compound remaining (PCR). 0.5 min time point was considered as 100% PCR, whereas other time points %PCR was calculated against 0.5 min. The slope (k) of linear range of natural logarithmic (In) curve of percent remaining versus time was used for calculation of half-life (tm). Attorney Docket No.: 01330-0117-00PCT

[0388] The half-life and Intrinsic clearance of compounds in hepatocytes was calculated using the below formula:

[0389] Half-life (t1 / 2) (min) = -1 * 0.693 / k

[0390] V (pL / 106cells) = 1000 / number of cells incubated in million per mL

[0391] Intrinsic clearance (Clint, pL / min / million cells) = V x 0.693 / T1 / 2

[0392] In-vitro intrinsic clearance (CLint) (mL / min / kg) was calculated using the formula:

[0393]

[0394] Scaled Hepatic Clearance CLhb (mL / min / Kg) was calculated using the below formula without correcting to fraction unbound in hepatocyte and plasma:

[0395] Qh: Liver blood flow (mL / min / kg); CL’int: Scaled In-vitro intrinsic clearance (mL / min / kg)

[0396]

[0397] %Qh = CLhb*100 / hepatic blood flow; Extraction ratio = %Qh / 100

[0398] Species Cells (xlO6cells / g Liver body weight Hepatic blood flow liver) (gm / kg body weight) (mL / min / kg) Mouse 130 90 90

[0399] Rat 120 40 55

[0400] Dog 240 32 35

[0401]

[0402] Human 120 20 21

[0403] CYP inhibition:

[0404]

[0172] Preparation of 50 mM Potassium phosphate buffer (pH 7.4): 50 mM potassium phosphate buffer (pH 7.4) was prepared by dissolving 0.647 g potassium phosphate monobasic (KH2PO4) and 3.527 g potassium phosphate dibasic (K2HPO4) to 400 mL of Milli-Q water and was made up to 500 mL.

[0405]

[0173] Preparation of NADPH solution (10 mM): 10 mM NADPH working solution was prepared by dissolving appropriate amount of NADPH in 50 mM Potassium phosphate buffer (pH 7.4). Attorney Docket No.: 01330-0117-00PCT

[0406]

[0174] Preparation of microsomal mixture: Microsomes working solution of 0.125 mg / mL in 50 mM Potassium phosphate buffer (pH 7.4) was prepared.

[0407]

[0175] Substrate preparation: 10 m. M DMSO stock of each CYP substrate was prepared in DMSO and diluted with buffer to achieve the final adequate concentrations of substrates known to those skilled in the art.

[0408]

[0176] Assay procedure:

[0409] ’The human liver microsomes were taken out from -80 °C and thawed on ice bath for 30 min.

[0410] 0.125 mg / mL microsomal working solution was prepared with 50 mM potassium phosphate buffer (pH 7.4).

[0411] 159 p. L of microsomal working solution was transferred into different wells for each isoform separately (n=2) and spike 1 µL of test compound and test compounds and one vehicle control (DMSO:acetonitrile 20:80) separately.

[0412] In the above microsomal and test compound mixture, 20 µL of marker substrate (isoform specific) working solution was added. Pre-incubation was allowed for 10 min at 37° C.

[0413] The reaction was initiated by adding 20 pL of 10 mM NADPH (Note: In reaction mix, NADPH concentration will be 1 mM).

[0414] The above reaction mix was incubated as per the time mentioned in assay conditions table for each isoform at 37° C.

[0415] After incubation, the reaction was terminated by adding 200 pL of ice-cold acetonitrile containing internal standard (Albendazole / Glipizide) to the above 200 pL of reaction mixture.

[0416] The samples were mixed thoroughly and centrifuged at 4000 rpm for 10 min.

[0417] After centrifugation, 100 uL of supernatant was transferred to loading plates and submit for LC-MS / MS analysis.

[0418]

[0177] Bioanalysis: Produced metabolites were monitored using developed LC-MS / MS method.

[0419]

[0178] Data analysis:

[0420] The peak area ratios (metabolites to internal standard) should be used to calculate % inhibition

[0421] % Activity = (Peak area ratios in presence of test compound / Peak area ratios of Attorney Docket No.: 01330-0117-00PCT

[0422] DMSO control)* 100

[0423] % Inhibition:::100 - % Activity.

[0424] The IC50 values for Fluvoxamine, sulfaphenazole, ticlopidine, quinidine, ketoconazole and test compounds were calculated using GraphPad Prism Version 9.02.

[0425] In vitro investigation of CYP TDI inhibition by test compound in human liver microsomes:

[0426]

[0179] Preparation of 50 mM Potassium phosphate buffer (pH 7.4): 50 niM potassium phosphate buffer (pH 7.4) was prepared by adding 0.67531 g potassium phosphate monobasic (KH₂PO₄) and 3.54177 g potassium phosphate dibasic (K₂HPO₄) to 300 mL of Milli-Q water and volume was made up to 500 ml. with Mi lli-Q water

[0427]

[0180] Preparation of NADPH solution (40 mM): 40 mM NADPH solution was prepared by dissolving 70.29 mg of NADPH in 2108 µL of 50 potassium phosphate buffer pH 7.4.

[0428] [181 Preparation of microsomal mixture: The 83.3 µL (20 mg / mL) of human liver microsomes was diluted up to 15 mL, in 50 mM Potassium phosphate buffer to achieve 0.111 mg / mL microsomal working solution (prepared freshly on the day of compounds are screened).

[0429]

[0182] . Assay details:

[0430] Mifepristone was used as the control inhibitor and testosterone was used as the control substrate for CYP3 4 TDI assessment.

[0431]

[0183] Preparation of Primary stock of substrate:

[0432] Primary' stock of CYP substrate testosterone was prepared in methanol and further diluted with potassium phosphate buffer (pH 7.4) to achieve the final adequate concentrations of substrates known to those skilled in the art..

[0433]

[0184] Preparation of stop solution:

[0434] Internal standards, Cetirizine, Telmisartan and Tolbutamide were prepared at a concentration of 1 mg / mL in DMSO. From this stock solution, cocktail quenching solutions were prepared by adding 100 gL of Cetirizine, 100 pL of Telmisartan, 100 pL of Tolbutamide to prepare each working stock in 1000 mL of 100% acetonitrile. The final concentrations of Cetirizine, Telmisartan and Tolbutamide were 100 ng / mL in the cocktail mixtures.

[0435] [1851 Assay procedure: Attorney Docket No.: 01330-0117-00PCT

[0436] Assay were performed with three conditions:

[0437] o Condition I: 0 min t-NADPH

[0438] o Condition II: 30 min +NADPH

[0439] o Condition III. 30 min -NADPH

[0440] The human liver microsomes were removed from the freezer set at -80 °C and then thawed on an ice bath.

[0441] A microsomal working solution with a concentration of 0.111 mg / mL was prepared using a 50 mM potassium phosphate buffer pH 7.4.

[0442] 35 µL of microsomal working solution was transferred into separate wells. Then, 1 pL of the test compound / assay compound and vehicle control (DMSO: acetonitrile 40:60) were spiked separately. The mixture was then mixed for 30 seconds.

[0443] Following mixing, 90 pL of the above microsomal mix with the test compound, assay control, or vehicle control was transferred for each condition (I, II, and III) and incubated for 10 minutes at 37°C.

[0444] In condition II, 5 µL of NADPH (20 mM NADPH), while in condition III, 5 µL of incubation buffer was added using an Integra Viafl o pipette. Both conditions were incubated at 37°C for 30 min.

[0445] For condition I, 5 µL of substrate with intermediate concentration followed by 5 µL of NADPH at 20 mM was added using an Integra Viaflo pipette, then incubated for 37°C. The reaction was stopped using 200 µL ice cold acetonitrile containing internal standard, in accordance with the specified incubation time for the each isoform in the assay conditions.

[0446] Following a 30-minute incubation of Condition II and III, 5 pL of substrate (intermediate concentration) for Condition II and 5 µL of NADPH + substrate mixture for Condition III was added using an Integra Viaflo pipette then incubated for 37°C. The reaction was stopped using 200 µL ice cold acetonitrile containing internal standard, in accordance with the specified incubation time for the each isoform in the assay conditions.

[0447] The samples were thoroughly mixed using a mixmate and then centrifuged at 4000 rpm for 10 minutes.

[0448] Following centrifugation, 120 pF of supernatant from each condition were transferred to the LCMS loading plates and subsequently submitted for LC-MS / MS analysis. Attorney Docket No.: 01330-0117-00PCT

[0449]

[0186] Bioanalysis: Produced metabolites were monitored quantitatively using developed LC-MS / MS method.

[0450]

[0187] Data analysis:

[0451] The peak area ratios (metabolites to internal standard) were used to calculate % inhibition.

[0452] % Activity:::(Peak area ratios in presence of test compound / Peak area ratios of DMSO control)* 100

[0453] CYP Induction by PXR transactivation assay:

[0454]

[0188] Preparation of complete DMEM media: To DMEM high glucose with glutamax, 10% FBS and 100 UI / mL Penicillin and 100 pg / mL Streptomycin were added.

[0455]

[0189] Preparation of Assay Media: To DMEM without phenol red, 0.1% charcoal stripped serum and 100 UI / 'mL Penicillin and 100 p.g / mL Streptomycin were added.

[0456]

[0190] Transfection Media: OptiMEM with reduced serum.

[0457]

[0191] Luciferase Assay Reagent

[0458] 1 vial of SteadyLite plus luciferase reagent was reconstituted by adding 10 mL of SteadyLite buffer supplied with kit and allowing it to dissolve completely at RT for 15-20 minutes and stored at -20°C.

[0459]

[0192] Preparation of test compounds: 10 rnM DMSO stocks were prepared for all the test compounds / positive controls.

[0460]

[0193] Assay steps:

[0461] 1. HEK-293T cells were plated at a density of 5 x 104cells in 96 well white clear bottom plates using complete DMEM media and incubated overnight at 5% CO?, and 37°C.

[0462] 2. After overnight incubation, cells were co-transfected with plasmids containing PXR- LBD and pFluc reporter at a concentration of 0.1 gg / well and 0.2 ug / well for PXR-LBD and pFluc respectively using lipofectamine 2000 in OptiMEM medium.

[0463] 3. Test compounds and Rifampicin stock concentrations were made in DMSO from 10 mM - 0.1 µM (11 concentrations in half log).

[0464] 4. After 5 h of transfection, cells were incubated with positive control rifampicin and test compounds from conc. ranging from 10 µM - 0.1 nM in half log conc. (11 conc. n=2) prepared using assay media from stock cone, plate and incubated overnight for 24 hrs. 0.1% DMSO containing assay media was used as negative control. Attorney Docket No.: 01330-0117-00PCT

[0465] 5. After 24 h of compound incubation, plate containing cells was acclimatized to room temperature for 30 minutes before adding 100 pL of Luciferase assay reagent thawed previously to room temperature to each well and allowed to incubate for 20 minutes before taking the luminescence counts for each well using an Envision plate reader. 6. Percentage induction for each well is calculated by taking 10 pM Rifampicin as 100% induction and 0.1% DMSO as 0% for each concentration of positive and test compound concentration.

[0466] 7. EC50 values for positive and test compounds were calculated using GraphPad Prism by loading concentrations on X-axis and % induction on Y-axis.

[0467] hERG Patch clamp assay

[0468]

[0194] Preparation of complete MEM media:

[0469] To MEM, 10% FBS, Penicillin 100 IU / mL, Streptomycin 100 ug / mL, 100 mM sodium pyruvate and 1% non-essential amino acids were added.

[0470]

[0195] Preparation of selection media:

[0471] To Complete MEM media, Geneticin (G418) 400 pg / mL was added.

[0472]

[0196] External solution:

[0473] NaCl 130 mM, KC1 5 mM, MgCl₂·6H₂O 1 mM, CaCl₂·2H₂O 1 mM, D-Glucose 12.5 mM, HEPES 10 mM. pH: 7.4 (Adjust with NaOH); Osmolarity: 285 ± 5 mOsmol

[0474]

[0197] Internal Solution

[0475] KC1 130 mM, MgC12 1 mM, MgATP 5 mM, EGTA 5 mM, HEPES 10 mM, pH: 7.2 (Adjust with KOH); Osmolarity: 270 ± 5 mOsmol

[0476]

[0198] Preparation of test compounds:

[0477] 10 mM DMSO stocks for all the test compounds and positive controls were prepared. - Working concentrations of test / positive compounds were prepared.

[0478] 10 pM of test compounds and 10 pM Cisapride were prepared by adding 50 uL each of respective 10 mM stock concentration of test compounds and 10 mM stock concentration of positive control to 49950 pL of external solution. Vehicle (0.1% DMSO) was prepared by adding 50 µL of DMSO to 49950 pL of external solution.

[0479]

[0199] Assay steps:

[0480] 1. HEK-293 stably expressing hERG cells were plated at a density of 3X103cells on round 12 mm diameter poly-L-Lysine coated coverslips using complete MEM media Attorney Docket No.: 01330-0117-00PCT

[0481] and incubated for 48-72 h at 5% CO₂ and 37°C before using for the study.

[0482] 2. HEK 293- hERG cells plated on coverslips were taken after 48-72h and the coverslip was transferred to a bath solution containing the external solution at room temperature. Cells were selected based on the morphology and are impaled with glass pipette (1-3 µm) filled with internal solution (~ 3 MOhms).

[0483] 3. Voltage clamp recording was obtained after ensuring the prerequisite cell parameters (>200 MOhm seal resistance, <20 MOhm access resistance).

[0484] 4. For eliciting the canonical hERG tail current, the cells were held at -80 mV for 250 ms, depolarize it to +40 mV for 500 ms followed by a rapid repolarization to -50 mV for 500 ms and back to -80 mV for 250 ms.

[0485] 5. hERG tail-current is measured as the peak outward current during the -50 mV, repolarization step. Data was filtered at 2 KHz and digitized at 10 KHz.

[0486] 6. Voltage clamp readouts were obtained while incubating cells with vehicle (external solution + 0.1% DMSO) followed by addition of 10 uM concentration of test compound followed by Cisapride 10 µM each for 3-5 minutes.

[0487] 7. Steady state currents after application of vehicle (0.1% DMSO in external solution) were considered as baseline and steady state currents obtained after application of each test concentration was used to calculate % hERG currents inhibition.

[0488] 8. Percentage hERG current inhibition was calculated at 10 pM of test compound and 10 µM of Cisapride with respect to vehicle (0.1% DMSO).

[0489]

[0200] Phospho-STAT1 assay procedure: see Biological Example 6.

[0490]

[0201] The data are summarized below in Table 4 below.

[0491] Table 4

[0492] ABBV-CLS-484 Compound 1 Compound 6 Rat PK* Clp / Half-life IV 19.5 / 1.7 / 0.80 2.60 / 4.0 / 0.20 6.25 / 2.25-4.99 /

[0493] (b) / Vdss (L / kg) 0.58-1.05 Bioavailability (F%) 76 73-98 Mice PK * Clp (mL / min / kg) / Half-life IV 2.91 / 4.57 / 0.46

[0494] (b) / Vdss (L / kg)

[0495] Bioavailability (F%) 17 Dog PK* Clp (mL / min / kg) / Half-life IV 10 / 1.0 / 0.76 2.0 / 2.37 / 0.20 (h) / Vdss (L / kg)

[0496]

[0497] Attorney Docket No.: 01330-0117-00PCT

[0498] Bioavailability (F%) 36 70

[0499] In Vitro Human pSTAT1 EC50 (A549, nM) 2503 2848

[0500] UM)

[0501] Mouse pSTAT1 EC50 (B16, nM) 127 12640

[0502] ifM)

[0503] PHYSCHEM Kinetic solubility (µM), pH=5 168 180 >5

[0504] Log D, pH = 7.4 -1.25 -1.1 -0.94 ACME Hepatocyte Clint <2 / <10 / <5 <3 / <10 / 15 22.7 / 31.1 / 204

[0505] (human / dog / rat; mL / min / kg) (mice: 1038, cyno: 5.3) Microsome Clint 42 / 14 / 11 (mice / rat / human; rnL / min / kg)

[0506] PPB (Fu %) 1.32 / 0.64 / 1.33 / (mice / rat / dog / cyno / human) 0.7 / 0.56 CYP1A22C9, 2C19, 2D6. 3 Al >50 >50 >50p’vl itihib. (IC50, pM) (except for 2C9:

[0507] 19.5 uM) CYP3A4 and TDI w / o / with >50 / >50 >50 / >50 >50 µM NADPH (IC50, µM)

[0508] PXR induction (IC50, µM) >10 >10 >10 pM hERG inhibition Clean Clean 9% (b) 10µM

[0509]

[0510] * IV Img / kg; PO 3mg / kg

[0511]

[0202] Table 4 demonstrates that Compound 1 and Compound 6 show superior rat and / or dog bioavailability (F%) compared to ABBV-CLS-484. In the pSTAT1 assay, Compound 1 shows similar human cell potency compared to ABBV-CLS-484, despite modest mouse cell potency, which explains the higher dose used in mouse syngeneic model. Notably, a relevant discrepancy between human and mouse potency is also observed for ABBV-CLS-484. In addition. Compound 1 and Compound 6's clean ADME profile makes it a promising drug candidate for inhibiting PTPN1 / 2 via oral administration.

[0512]

[0203] Compound 1 and Compound 6 shows superior oral bioavailability and equivalent human potency to a clinical PTPN1 / 2 inhibitor.

[0513]

[0204] Biological Example Potency in pSTATI Assay

[0514]

[0205] A549-RFP or B16-F10 cells were stimulated for 1h with human or mouse recombinant IFNγ (100ng / mL) in HBSS buffer. pSTATI (Tyr701) was quantified by AlphaLISA® SureFire® Ultra (Revvity). Attorney Docket No.: 01330-0117-00PCT

[0515] A549 PhosphoSTATl Assay

[0516]

[0206] A549 human lung carcinoma epithelial cells (Cell Player TM A549 NucLight Red, Essen BioScience Cat#4491) were seeded at a density of 40000 cells per well in a sterile 384 well F-bottom white cell culture microplate (Greiner, Cat# 781080) in 80 pL total volume of IMDM+10 / oFBS (Capricorn Scientific, Cat# IMDM-A and Gibco, Cat# A5256701). Cells were allowed to adhere overnight at 37 °C + 5% CO?. The following day, compounds (Compound 1 and ABBV-CLS-484) resuspended in DMSO (Sigma, cat# D8418-500ML) at 10 mM were diluted in semi-log dilutions in DMSO ranging from 10 mM to 0.001 mM and DMSO only controls were included. The compound / DMSO dilutions were further diluted 1:50 in HBSS, media from the plates was removed and 25 μL of these dilutions were added in duplicates to the cells. Final compound concentrations ranged from 100 pM to 0.001 pM with a final DMSO concentration of 1%. Cells and compounds were incubated for 1h at 37 °C + 5% CO?, before the addition of 25 p L of human IFNy (Peprotech, Cat# 300-02) at 200 ng / mL for a final concentration of 100 ng / mL. The plate was incubated for 1h at 37 °C + 5% CO? and assayed for phosphorylated STAT1 protein levels with the AlphaLISA Surefire Ultra Human and Mouse Phospho-STATl (Tyr701) kit (Revvity, Cat# ALSU-PST1-A10K) according to the manufacturer’s instructions. Plate signal was read using a SpectraMax i3x multiplate Spectrophotometer reader (Molecular Devices®) with an excitation of 680 nm and emission at 615 nm. The data was fitted, using a 4-parameter equation, to determine the inflection point of the fit as the IC50 of the small molecule inhibitor considering 0% phosphoSTATl where only DMSO was added.

[0517] Bl 6 PhosphoSTATl Assay

[0518]

[0207] B16F10 mouse melanoma cells (ATCC Cat# CRL-6475) were seeded at a density of •40000 cells per well in a transparent F-bottom 96-well plate (Greiner, Cat# 655 180) in 150 pL total volume of DMEM+10%FBS (Capricorn Scientific, Cat# DMEM-HPST and Gibco, Cat# A5256701). Cells were allowed to adhere overnight at 37 °C + 5% CO2. The following day, compounds (compound 1 and ABBV-CLS-484) resuspended in DMSO (Sigma, cat# D8418-500ML) at 10 mM were diluted in semi-log dilutions in DMSO ranging from 10 mM to 0.001 mM and DMSO only controls were included. The compound / DMSO dilutions were further diluted 1:50 in HBSS, media from the plates was removed and 50 pL Attorney Docket No.: 01330-0117-00PCT

[0519] of these dilutions were added in duplicates to the cells. Final compound concentrations ranged from 100 pM to 0.001 pM with a final DMSO concentration of 1%. Cells and compounds were incubated for 1h at 37 °C + 5% CO?, before the addition of 50 pL of mouse IFNy (Peprotech, Cat# 315-05) at 200 ng / mL for a final concentration of 100 ng / mL. The plate was incubated for Ih at 37 °C + 5% CO2 and assayed for phosphorylated STAT1 protein levels with the AlphaLISA Surefire Ultra Human and Mouse Phospho-STAT1 (Tyr701) kit (Revvity, Cat# ALSU-PST1-A10K) according to the manufacturer’s instructions. Plate signal was read using a SpectraMax i3x multiplate Spectrophotometer reader (Molecular Devices®) with an excitation of 680 nm and emission at 615 nm. The data was fitted, using a 4-parameter equation, to determine the inflection point of the fit as the IC50 of the small molecule inhibitor considering 0% phosphoSTATl where only DMSO was added.

[0520]

[0208] Compound 1 is shown to have similar potency in human, but 100 times lower potency in mouse cells compared to ABBV-CLS-484. FIG. 1. Whereas Compound 1 is 4 times more potent in human vs mouse cells, ABBV-CLS-484 is 20 times less potent in human vs mouse cells. The data is normalized to the maximum achieved concentration of pSTAT1 as 100%.

[0521]

[0209] Compound 1 demonstrates human potency comparable to ABBV-CLS-484 in the phospho-STAT 1 assay.

[0522]

[0210] Biological Example 8: 1’ Ceil Functions in In Vitro Studies

[0523]

[0211] Tolerogenic dendritic cells (DCs), generated from monocyte-derived DCs with IL-10, were treated with either ABBV-CLS-484 or Compound 1 at various concentrations (0.3 µM, 1µM, 3µM, or 10µM) for 2 days. After LPS stimulation for 24 hours, these DCs were cocultured with allogeneic CD3+ T cells for 4 days to assess if PTPNl / 2 inhibition restores T cell functions.. Activation and regulatory' markers on tolerogenic DCs were assessed by FACS before co-culture. FIG. 2A-C.

[0524] Tolerogenic DCs generation & Mixed Lymphocyte Reaction (MLR) assay

[0525]

[0212] CD14+ monocytes were isolated from fresh healthy donor PBMCs and cultured with 50 ng / mL GM-CSF (R& D, #215 -GM) and 50 ng / mL IL-4 (Peprotech FAF-200-04) in RPMI Medium [RPMI-STA (Capricorn Scientific) supplemented with 10% FBS (Gibco #A5256701) and 1% P / S (Capricorn Scientific #PS~B)] for six days. Subsequently, these Attorney Docket No.: 01330-0117-00PCT

[0526] immature dendritic cells (DCs) were treated with 50 ng / mL GM-CSF, 50 ng / mL IL-4, 20 ng / mL IL-10 (Peprotech #AF-200-10-50UG), and varying concentrations of ABBV-CLS-484 & Compound 1 (0.3µM, 1µM, 3µM, or 10µM). Finally, these tolerogenic DCs were exposed to 10 ng / mL LPS (Sigma #L4391-1g) for 24 hours. The activation and regulatory markers on the tolerogenic DCs were evaluated by flow cytometry'. The antibodies utilized in this study included: PD-L1 BUV395 (BD Biosciences #740320), HLA-ABC BUV737 (BD Biosciences #752777), CD40 FITC (BioLegend #334306), CD83 PerCP-Cy5.5 (BioLegend #305320), CD80 BV421 (BD Biosciences #564160), CD86 BV711 (BioLegend #305440), HLA-DR BV785 (BioLegend #307642), CD200 APC (BioLegend #329208), CCR7 PE (BioLegend #353204), and CD209 PE-Cy7 (BioLegend #330114).

[0527]

[0213] For the MLR, allogeneic CD3 + T cells were isolated from frozen PBMCs and labelled with the cell proliferation dye eFluor450 (eBioSciences #65-0842-85). ’Then, those allogeneic CD3+ T cells were co-cultured with tolerogenic DCs at a ratio of 1: 10 Effector: Target in the presence of various concentrations of Compound 1 or ABBV-CLS-484 (0.3 µM, 1 µM, 3µM, or 10µM). The proliferation and activation of T cells were measured by flow' cytometry at the endpoint. The antibodies used in this analysis consisted of: CD8 BUV395 (BD Biosciences #563795), 4-1BB BUV737 (BD Biosciences #568348), CD69 BV510 (BioLegend #310936), CD25 BV650 (BD Biosciences #563718), CD3 BV786 (BD Biosciences #563800), CD4 APC (BD Biosciences #551980), and PD-1 PE-Vio770 (Miltenyi Biotec #130-117-698).

[0528]

[0214] Phenotype of tolerogenic DCs treated with ABBV-CLS-484 or Compound 1: activation markers were assessed by FACS and found unaffected by PTPN1 / 2 inhibitors. FIG.

[0529] 2B-C.

[0530]

[0215] Allogeneic CD3+ T cells were cocultured with tolerogenic DCs at a ratio of 1 / 10 Effector: Target in presence of ABBV-CLS-484 and Compound 1 at various concentrations. T cell proliferation and activation were assessed by flow cytometry at endpoint. FIG. 2D-E.

[0531]

[0216] Compound 1 was demonstrated to enhance T cell functions in vitro. Compound 1 was shown to not induce immunoregulatory molecules on tolerogenic DCs, and shown to inhibit tolerogenic DCs’ function by restoring T cell proliferation & activation. Attorney Docket No.: 01330-0117-00PCT

[0532]

[0217] Biological Example 9: NK Cell Cytotoxicity In Vitro Studies

[0533]

[0218] CD56+ NK cells were stimulated overnight with IL-2 and treated with either ABBV- CLS-484 or Compound 1 at various concentrations (0.1 µM, 0.3 µM, 1µM, 3µM, or 10µM). The next day, the tumor cell killing assay was performed at a ratio of 5 / 1 Effector: Target with ABBV-CLS-484 or Compound 1. FIG. 3A-B.

[0534] NK cell killing assay

[0535]

[0219] CD56+ NK cells were enriched from fresh healthy donor PBMCs and stimulated with 100ng / mL of IL-2 (Miltenyi #130-097-746) in RPMI Medium [RPMI Medium (Capricorn Scientific; RPMI- ST A) with 10% FBS (Gibco; #A5256701) and 1% P / S (Capricorn Scientific #PS-B)] with varying concentrations of ABBV-CLS-484 or Compound 1 (0.1µM, 0.3µM, 1µM, 3µM, or 10µM).

[0536]

[0220] The next day, K562 lymphoblastic cells were stained with eFluor 450 (eBioSciences: 65-0842-85) and cultured in RPMI Medium (Capricorn Scientific; RPMI-STA) with 10% FBS (Gibco, #A5256701) and 1% P / S (Capricorn Scientific #PS-B)]. Subsequently, a tumor cell killing assay was conducted at a 5 / 1 Effector / Target ratio in presence of lOOng / ml of IL- 2 and various concentrations of ABBV-CLS-484 or Compound 1 (O. IpM, 0.3 pM, IpM, 3µM or 10µM). After 4 hours of coculture, NK and K562 cells were collected and stained for FACS analysis using NKG2A FITC (Miltenyi Biotec #130-113-565), CD69 BV650 (BD Biosciences #563835), CD16 BV786 (BD Biosciences #563689), CD56 PE (BioLegend #362507), and NKG2D PE-Cy7 (BD Biosciences #562365).

[0537]

[0221] Compound 1 was demonstrated to increase NK cell cytotoxicity in vitro. FIG. 3A-B.

[0538] Compound 1 was shown to enhance NK cell activation similarly to ABBV-CLS-484, and exhibit higher cytotoxic activity.

[0539]

[0222] Biological Example 10: Anti-tumor activity in vivo in Mouse Colon Cancer tumor model

[0540]

[0223] MC38 cell line (Marc Cragg lab) were cultured and resuspended in DMEM +10% FBS medium (Capricorn Scientific, Cat# DMEM-HPSTA and Gibco, Cat# A5256701). Cells were injected subcutaneously in the right flank with a 30G syringe of adult (7-8 weeks) female mice strain C57BL / 6 (Charles River Laboratories, Italy) (500 xlO5cells / inj ection). Attorney Docket No.: 01330-0117-00PCT

[0541] Once the tumors were around 50mm3, mice were randomized into 2 groups (n=15 per group) and treated p.o. with Compound 1 (450mg / kg, BID), or vehicle (10% ethanol, 30% PEG-400, 60% Phosal-50PG, BID) during 15 days. Mice were checked daily for signs of illness and distress. The subcutaneous tumor size and mouse weight were measured with a caliper three times per week. The mice were maintained under pathogen-free conditions and with free access to standard rodent chow and water.

[0542]

[0224] Despite modest mouse potency and limited target coverage, the efficacy of Compound 1 at 450mg / kg, a dose covering the IC10 (pSTATl assay: see FIG. 1) was evaluated in an established colon cancer syngeneic tumor model, MC38. Median (FIG. 4A) and individual (FIG. 4B) tumor growth volume curves; n=15 mice / group.

[0543]

[0225] Compound 1 was shown to demonstrate anti-tumor activity in vivo despite modest mouse potency.

[0544]

[0226] Biological Example 11: Patient-derived Tumor data

[0545]

[0227] Patient Derived Tumor Fragment (PDTF) cultures have been demonstrated to be highly predictive of clinical response to immune checkpoint blockade agents (such as anti-PD-1).

[0546]

[0228] PDTF platform (schematic representation seen in FIG. 5A):

[0547] 1) Sample collection: Tumor samples were brought after surgery.

[0548] 2) Sample processing: Tumor samples were divided in several parts for baseline analysis and for slicing into tumor fragments. Tumor fragments were treated for 48h.

[0549] Patient-derived tumor fragment culture:

[0550] o Upon arrival, tumors were rinsed in PBS and divided into several parts for various analyses. Several small tumor pieces were used for baseline immunohistochemistry (THC) analysis. The remaining parts were processed into 300μm slices using a compresstome™ (Precisionary Instruments). The tumor tissues and the slices were kept on ice during the whole process.

[0551] o Tumor slices were embedded in 200 pL of collagen matrix in 48-well plate, with a 100 L layer beneath and on top of each tumor slice. The matrix was created by combining sodium bicarbonate (1.1% final concentration; Sigma- Aldrich), collagen I (1 mg / ml final concentration; Corning, # 354249) and tumor medium, which consisted of DMEM Glutamax (Sigma-Aldrich, # Attorney Docket No.: 01330-0117-00PCT

[0552] 31966-021) enriched with 1 mM sodium pyruvate (Sigma-Aldrich), 1× MEM nonessential amino acids (Thermo Fisher Scientific, # NEAA-B), 10% FBS (Gibco, #10270-106), and 1% penicillin-streptomycin (Lonza # DE17603E). All components were kept on ice to prevent premature collagen solidification. Ice-cold Matrigel (Matrix High Concentration, Phenol Red-Free, 4 mg / ml final concentration; Corning, # 354263) was then gradually added to the mixture.

[0553] o Tumor medium (600pL), containing DMSO or treatments (100 μM free fraction for ABBV-CLS-484 or Compound 1) was added on top the embedded slices for a period of 48 hours.

[0554] 3) Analysis: Baseline and on treatment tumor fragments were analysed with cutting-edge technologies.

[0555] Immunohistochemistry (IHC):

[0556] o Several pieces of tumor samples were fixed in 4% paraformaldehyde ( PF A ) upon arrival for 24 hours (Baseline). The cultured tumor slices were isolated from the collagen matrix and fixed in 4% PFAfor 3 hours (Day 2). The fixed samples were then washed in PBS and sent to Diapath (ULB, Gosselies, Belgium) for sectioning. The formalin-fixed, paraffin-embedded tumor samples were cut in serial 4 μm sections.

[0557] o As part of our baseline analyses, each received tumor sample was evaluated by a certified Pathologist (IPG, Gosselies, Belgium) for quality, tumor content and the proportion of necrosis, fibrosis and immune cell infiltration using H& E staining.

[0558] o The CD45 staining process was automated using the Ventana autostainer system by Roche. Heat-induced epitope retrieval was performed for 90 minutes at 95°C. Primary CD45 antibody (Dako Agilent, #MO701, 1 / 800) was incubated for 32 minutes at 37°C. Thereafter, an HQ-conjugated secondary antibody (Roche, #07017 782001) was added for 12 min at 37°C. Finally, an HQ-HRP (Roche, #07017936001) complex was added and the chromogen employed for visualization was DAB (3,3 '-diaminobenzidine; Roche, #05266 654001). Subsequently a counterstaining with Hematoxylin II (Roche#05277 965 001) and Bluing reagent (Roche # 05 266769001) was performed. Attorney Docket No.: 01330-0117-00PCT

[0559] o The CD8 / PanCK staining was also performed using Ventana autostainer system by Roche. Heat-induced epitope retrieval was performed for 90 minutes at 95°C. CD8a SP239 (. Abeam, #AB 17809, 1 / 100) and Pan cytokeratin (PanCK, Roche, #760-2595, 9.26 mg / mL) antibodies were used as primary antibodies and incubated for 1 hour and 4 minutes respectively at 37°C, while specific anti-Fc HRP-antibodies (Roche, #05269679001 for CD8 and #05 269652001 for PanCK) were complexed respectively to DCC and Red610 for 8 minutes at 37°C for visualization.

[0560] o After staining, the slides were mounted and scanned (20x magnification) in brightfield using the Nanozoomer scanner from Hamamatsu. Fluorescence channel were used to capture the signal from DCC (CD8) and Red610 (PanCK) and DAPI. Tumor areas were delimited by a certified pathologist. Image quantification was conducted using VisioPharm software, Al-driven pipeline. Applications were specifically designed for the analysis and certified by a Pathologist.

[0561] 4) Metadata Compilation and response to treatment: Clinical translatability of PDTF demonstrated by Voabil et al., Nature Medicine, 2021.

[0562] Case Study: renal carcinoma (RCC) sample that responds to Compound 1

[0563]

[0229] Clinical information: Female (80y) patient with well differentiated Renal Clear Cell Adenocarcinoma (grade 2) with no previous treatment.

[0564]

[0230] Cytokine and chemokines release:

[0565]

[0231] Supernatants were collected 48 hours after treatment, and centrifuged at 450g for 8 minutes for cell removal and stored at -80°C until analysis.

[0566]

[0232] On the day of the experiment, supernatants were thawed on ice and used undiluted for the analysis of several cytokines and chemokines. Each replicate was used undiluted and analyzed separately using customized U-plex MSI) kits (Meso Scale Discovery), following the manufacturer’s instructions. The concentrations (pg / μL) of CXCL9, CXCL10, CCL22 were measured for the control (DM SO) and treated (ABBV-CLS-484 and Compound 1 ) conditions and corrected for their lower and upper limits of detection (lloq and uloq).

[0567]

[0233] FIG. 5B shows macroscopic illustration of the tumor and a 48h-cultured tumor fragment with the corresponding H& E, CD45, and CD8 / PanCK. Scale bar: 50 pm. FIG. 5C- Attorney Docket No.: 01330-0117-00PCT

[0568] 5E shows concentrations (pg / mL) of CXCL9, CXCL10, and CCL22 evaluated by MSD on RCC-derived tumor fragments treated with DMSO, ABBV-CLS-484, and Compound 1 (both at 100 pM, free concentration).

[0569]

[0234] Figure 5F shows response of a PDTF cohort to Compound 1.

[0570]

[0235] Heatmap:

[0571]

[0236] A heatmap displaying analyses at baseline and day 2 was generated using R Studio (version R.4.3.0).

[0572]

[0237] 300 u -tumor slices from patient 1-11 were characterized at baseline and after 2 days of culture. For each patient-derived sample, the cancer type (NSCLC in pink and HCC in light green), the immunotype (inflamed, excluded and desert) as well as the density for CD45+ cells (number of cells / mm2) are indicated in the bottom part of the heatmap. In addition, H& E staining for each patient-derived sample was annotated by a certified pathologist for the proportion of non-tumor areas, cancer cells, immune cells, stroma and necrosis. This baseline characterization provides a comprehensive evaluation of the tumor quality and general immune content.

[0573]

[0238] Normalized delta values (A Z-score) between treatment (ABBV-CLS-484 or Compound 1) and untreated (DMSO) for each of soluble factor (CXCL9, CXCL10 and CCL22) was evaluated 2 days after treatment. The concentration (pg / uL) of IFNγ and CCL22 for DMSO conditions at day 2 were also displayed in the heatmap.

[0574]

[0239] The unsupervised clustering analysis demonstrated that patients 1-3 clustered together with elevated concentrations of CXCL9 and CXCL10, and low levels of CCL22. In addition, the levels of IFNy and CCL22 were higher in these samples treated with DMSO. Altogether, these soluble markers could be used as potential biomarkers for PTPN1 / 2 inhibitors. The characterization of samples at baseline demonstrated the quality and proportion of tumor and immune contents of patient-derived tumor upon arrival. No correlation was found between response to PTPN1 / 2 inhibitors and the immunotype, CD45+ cell density, the cancer type or the H& E annotation (IHC).

[0575]

[0240] FIG. 5F show's that Compound 1 demonstrates comparable biological activity to ABBV-CLS-484 across the same cohort of PDTF samples. Attorney Docket No.: 01330-0117-00PCT

[0576]

[0241] The various biological examples demonstrate that Compound 1 demonstrates potent inhibition of human PTPN1 / 2 with superior oral bioavailability, clean ADME profile, and equivalent human potency to a leading program in the PTPN1 / 2 landscape.

[0577]

[0242] Compound 1 was also demonstrated to have enhanced the activation and function of various immune cell subsets, promoting immune-mediated tumor killing in in vitro coculture assays.

[0578]

[0243] Compound 1 also showed efficacy in mouse models and in Patient Derived Tumor Fragments (PDTFs), an ex vivo system known for predicting clinical response to immune checkpoint inhibitors.

[0579]

[0244] Consistent with the mechanism of PTPN1 / 2 inhibition, Compound 1 was shown to significantly increase CXCL9 and CXCL10 production in RCC and NSCLC samples as a single agent in PDTF cultures, comparable to ABBV-CLS-484, highlighting its potential to drive a strong anti-tumor immune response in patients.

[0580]

[0245] Therefore, PTPN1 / 2 inhibitors represent a promising new strategy for cancer immunotherapy.

Claims

Attorney Docket No.: 01330-0117-00PCTWhat is claimed is:

1. Compound 1:or a pharmaceutically acceptable salt thereof.

2. Compound 2:o r O^-NHor a pharmaceutically acceptable salt thereof.

3. Compound 3:or a pharmaceutically acceptable salt thereof.

4. Compound 4:Attorney Docket No.: 01330-0117-00PCTor a pharmaceutically acceptable salt thereof.

5. Compound 5:or a pharmaceutically acceptable salt thereof.

6. Compound 6:or a pharmaceutically acceptable salt thereof.

7. Compound 7:07or a pharmaceutically acceptable salt thereof.

8. A compound selected from:5-(4-chloro-2-fluoro-6-hydroxyphenyl)-l,2,5-thiadiazolidin-3-one 1,1 -dioxide; 5-(4-bromo-2-fluoro-6-hydroxyphenyl)-l,2,5-thiadiazolidin-3-one 1,1 -di oxide; 5-(3-chloro-2-fluoro-6-hydroxyphenyl)-l,2,5-thiadiazolidin-3-one 1,1 -dioxide; 5-(3-bromo-2-fluoro-6-hydroxyphenyl)-l,2,5-thiadiazolidin-3-one 1,1 -di oxide;Attorney Docket No.: 01330-0117-00PCT5-(4-bromo-3 -chloro-2-fluoro-6-hydroxyphenyl)- 1,2, 5-thiadiazolidin-3 -one 1,1- dioxide;5-(2-fluoro-4-hydroxy-[ 1, 1 '-biphenyl]-3 -yl)- 1,2,5-thiadiazolidin-3 -one 1, 1 -dioxide; 5-(2-fluoro-6-hydroxyphenyl)-l,2,5-thiadiazolidin-3-one 1,1 -di oxide;and pharmaceutically acceptable salts thereof.

9. A pharmaceutical composition comprising a compound according to any of claims 1- 8 and a pharmaceutically acceptable excipient, carrier, adjuvant, or vehicle.

10. A method of inhibiting PTPN1 and / or PTPN2 in a patient in need thereof, comprising administering to said patient an effective amount of a compound according to any of claims 1-8 or a pharmaceutical composition of claim 9.

11. A method of treating a disease mediated by PTPN 1 and / or PTPN2 in a patient in need thereof, comprising administering to said patient an effective amount of a compound according to any of claims 1-8 or a pharmaceutical composition of claim 9.

12. The method according to any one of claims 10-11, wherein the compound comprises Compound 1.

13. The method according to any one of claims 10-11, wherein the compound comprises Compound 2.

14. The method according to any one of claims 10-11, wherein the compound comprises Compound 6.

15. The method according to any one of claims 11-14, wherein the disease mediated by PTPN1 and / or PTPN2 comprises cancer.

16. A process to prepare a compound according to any of claims 1-8.

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