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WO2021077031A1 - Compounds and methods targeting qsox1 - Google Patents

Compounds and methods targeting qsox1 Download PDF

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Publication number
WO2021077031A1
WO2021077031A1 PCT/US2020/056174 US2020056174W WO2021077031A1 WO 2021077031 A1 WO2021077031 A1 WO 2021077031A1 US 2020056174 W US2020056174 W US 2020056174W WO 2021077031 A1 WO2021077031 A1 WO 2021077031A1
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cancer
carcinoma
cell
lymphoma
tumor
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Sergei Svarovsky
Alim SEIT-NEBI
Catalina VALENCIA
Douglas Lake
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Sapphire Biotech Inc
Arizona State University ASU
Arizona State University Downtown Phoenix campus
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Sapphire Biotech Inc
Arizona State University ASU
Arizona State University Downtown Phoenix campus
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/12—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
    • C07D295/135—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms separated by carbocyclic rings or by carbon chains interrupted by carbocyclic rings
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/42—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/44—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton with carbon atoms of carboxamide groups and singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring
    • C07C235/58—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton with carbon atoms of carboxamide groups and singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring with carbon atoms of carboxamide groups and singly-bound oxygen atoms, bound in ortho-position to carbon atoms of the same non-condensed six-membered aromatic ring
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/18—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member
    • C07D207/22—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/24—Oxygen or sulfur atoms
    • C07D207/26—2-Pyrrolidones
    • C07D207/263—2-Pyrrolidones with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms
    • C07D207/27—2-Pyrrolidones with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms with substituted hydrocarbon radicals directly attached to the ring nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/38—Nitrogen atoms
    • C07D215/40—Nitrogen atoms attached in position 8
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/16—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms
    • C07D295/18—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms by radicals derived from carboxylic acids, or sulfur or nitrogen analogues thereof
    • C07D295/182—Radicals derived from carboxylic acids
    • C07D295/185—Radicals derived from carboxylic acids from aliphatic carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/68—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/50—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom condensed with carbocyclic rings or ring systems
    • C07D333/52—Benzo[b]thiophenes; Hydrogenated benzo[b]thiophenes
    • C07D333/62—Benzo[b]thiophenes; Hydrogenated benzo[b]thiophenes with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the hetero ring
    • C07D333/68—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • C07D333/70—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen attached in position 2
    • C—CHEMISTRY; METALLURGY
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • This disclosure relates to the field of anti-neoplastic compounds and methods, and more particularly to such compounds and methods that modulate QSOX1.
  • Cancer and other hyperproliferative diseases is characterized by uncontrolled cell proliferation.
  • the desmoplasia fibrosis
  • the desmoplasia that forms around many solid tumors is a highly complex mixture of stellate cells, cancer associated fibroblasts, myofibroblasts, macrophages, and even endothelial cells and adipocytes.
  • Each of these cell types secretes ECM proteins that contribute to the complex fibrotic barrier around the tumor.
  • Components of this fibrotic barrier include fibronectin, laminin, decorin, tenascin, periostin, multiple types of collagen and various stroma and tumor-derived enzymes.
  • QSOX1 Quiescin Sulfhydryl Oxidase 1
  • PDAC pancreatic ductal adenocarcinoma
  • luminal B breast cancer a FAD-dependent sulfhydryl oxidase that is over-produced by many types of tumor cells and is undetectable by antibody staining in adjacent non-malignant tissues.
  • QSOX1 catalyzes disulfide bond formation during protein folding. It is the only known enzyme that both generates and shuttles disulfides in proteins.
  • protein disulfide isomerase (PD I) requires QSOX1 for protein folding but QSOX1 does not require PDI.
  • QSOX1 shuttles disulfides using its CxxC motifs, ultimately using FAD to transfer electrons to 02, producing H202 as a byproduct. Without QSOX1 to form disulfide bonds, cysteines have free thiols which can indiscriminately cross link as they are secreted from the cell. This leads to improper disulfide cross-linking and incorrect folding of proteins, aggregate formation and a disorganized, disrupted ECM.
  • QSOX1 Quiescin sulfhydryl oxidase 1
  • Two transcript variants encoding two different isoforms of QSOX1 have been found.
  • Recombinant QSOX1 has been expressed and used to screen a library of chemical compounds to identify any compounds that inhibit the enzymatic activity of QSOX1.
  • AI- 6 are each independently selected from C ORN;
  • Z is independently selected from C OR S;
  • W is independently selected from O, O2 or NH; and wherein Rs can be further independently selected from
  • the compound is set forth in Table 1A-F.
  • the compounds correspond to structures 1-9 and 64-89 in Table 1A-F.
  • the compound comprises the structure corresponding to SPX-9230.
  • compositions comprising the compounds, or an analogs thereof; and a pharmaceutically acceptable carrier.
  • said composition is a stereoisomer, prodrug form, or pharmaceutically acceptable salt of an SBI- 183 -analog compound, or an analogue thereof.
  • Also provided herein is a method for neoplastic cell growth inhibition, comprising the step of contacting said neoplastic cell with an effective amount of an SBI-183-analog compound or an analogue thereof or a pharmaceutically acceptable salt thereof. Also provided herein is an SBI-183-analog compound, or an analogue thereof or a pharmaceutically acceptable salt thereof for use in the treatment of a tumor or a cancer.
  • a method for treating cancer comprising administering to a subject having a tumor an amount effective of an SBI- 183 -analog compound, or an analogue thereof or a pharmaceutically acceptable salt thereof.
  • the cancer is selected from the group consisting of: carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.
  • cancers include, but are not limited to, breast cancer (Luminal A, TNBC, Ductal), prostate cancer, squamous cell tumors, squamous cell carcinoma (e.g., squamous cell lung cancer or squamous cell head and neck cancer), neuroendocrine tumors, urothelial cancer, vulvar cancer, mesothelioma, liver cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, lung cancer, small cell lung cancer, non-small cell lung cancer, cutaneous or intraocular malignant melanoma, renal cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, non-Hodgkin's lymphoma, cancer of the eso
  • this disclosure relates to analogs of a compound corresponding to, 3-methoxy-N-[4-(l- pyrrolidinyl)phenyl]benzamide (SBI-183- analogs for short):
  • this disclosure relates to stereoisomers, prodrugs and pharmaceutically acceptable salts of SBI-183-analogs.
  • this disclosure relates to the use of SBI-183-analogs and related compounds to modulate QSOX1, e.g., either inhibit (antagonize) or activate (agonize) QSOX1.
  • this disclosure relates to the use of SBI-183-analogs and related compounds to inhibit growth and invasive properties of neoplastic cells.
  • this disclosure relates to the use of SBI-183-analogs and related compounds to inhibit neoplastic cells that over-express the QSOX1 enzyme, such as in, for example, myeloma and pancreatic, renal, and breast cancer.
  • An SB-183-analog compound of the present disclosure or a pharmaceutically acceptable salt thereof can be administered as such to a human patient or can be administered in pharmaceutical compositions in which the foregoing materials are mixed with suitable carriers or excipient(s).
  • suitable carriers or excipient(s) include REMINGTON'S PHARMACOLOGICAL SCIENCES, Mack Publishing Co., Easton, PA, latest edition.
  • FIG. 1A Suppression of xenograft growth in mice of human pancreatic tumor cell line MIAPaCa2 transduced with shQSOXl constructs sh742 and sh528. Average tumor weight is shown in the y-axis.
  • FIG. 1B Sh742 -transduced BT549 tumor cells did not invade through Matrigel- coated trans-well inserts (bar 3) compared to controls (bars 1&2). Invasion was rescued by exogenous addition of functional rhQSOXl (bar 4), but not mutated inactive rhQSOXl (bar 5). Number of cells invading through Matrigel is shown in the Y-axis.
  • FIG. 1C Diagram of 16 hour 2-D invasion assay.
  • Figure 2 An in vitro assay to screen for QSOX1 inhibitors; Fluorogenic assay (also can be HTS). Enzymatically active recombinant QSOX1 is incubated with individual compounds in individual wells of a 384 well plate. If compounds inhibit the activity of the enzyme, no H202 will be made by the enzyme because it can't fold RNAse A (surrogate substrate). Therefore, no fluorescence is observed. If compounds do not inhibit QSOX1, the enzyme with re-fold RNAse A, produce H202 and the fluorescent indicator will fluoresce.
  • Fluorogenic assay also can be HTS.
  • FIG. 5A Treatment of human tumor xenograft-bearing mice with SBI-183 suppresses tumor growth. Treatment was started on day 7 after tumor implantation. Experiment was terminated on day 41. Tumor volume, shown on the Y -axis was calculated using length and width measurements.
  • FIG. 5B Treatment of mice bearing MDA-MB-231 -Luc tumors with lOOmg/kg SBI-183, daily resulted in decreased lung metastasis in treated mice. Mice were injected with luciferin at the end of the experiment followed by ISIS 2000 imaging of lung mets and calculation of mean lung radiance.
  • Figure 6A 3D Spheroid Invasion Assay. Graphs are shown on the left as area of MDA-MB-231 as in Matrigel at 0, 1 and 3 days. Replicates of 6 spheroids for each concentration of compound was performed. Different color lines show 2-fold serial dilutions of SPX-009 compound compared to parent compound SBI-183 at 1250nM.Photographs on the right show invasive growth of spheroids on Day 3 in either 0.01% DMSO vehicle.
  • FIG. 6B 3D Spheroid Invasion Assay. Graphs are shown on the left as area of RCJ-41T2 spheroids as in Matrigel at 0, 1 and 3 days. Replicates of 6 spheroids for each concentration of compound was performed. Different color lines show 2-fold serial dilutions of SPX-009 compound compared to parent compound SBI-183 at 1250nM. Photographs on the right show invasive growth of spheroids on Day 3 in either 0.01% DMSO vehicle.
  • FIG. 7 SBI-183-analog structure scaffold; sample structures; and representative examples of structural elements to be used for SAR optimization. The exact nature of these elements will be adjusted based on the outcome of each round of screening.
  • Figure 8 Shows results from a 3D tumor cell spheroid assay in MDA-MB-231 cells for SPX-52091.
  • Figure 9 Shows results from a 3D tumor cell spheroid assay in MDA-MB-231 cells for SPX-52092.
  • Figure 10 Shows results from a 3D tumor cell spheroid assay in MIA PaCa2 cells for SPX-52091.
  • Figure 11 Shows results from a 3D tumor cell spheroid assay in MDA-MB-231 cells for SPX-52092.
  • Figure 12 Shows results from a 3D tumor cell spheroid assay in RCJ-41T2 cells for SPX-52091.
  • Figure 13 Shows results from a 3D tumor cell spheroid assay in RCJ-41T2 cells for SPX-52092.
  • Figure 14 Shows results from a 3D tumor cell spheroid assay in MDA-MB-231 cells for SPX-5229.1,-5229.2, and -5229.3.
  • Figure 15. Shows results from a 3D tumor cell spheroid assay in MIA PaCa2 cells for SPX-5229.1,-5229.2, and -5229.3.
  • Figure 16 Shows results from a 3D tumor cell spheroid assay in MIA PaCa2 cells for comparing SPX-5226 and SPX-5226R.
  • SPX-5226 is chromatography purified, whereas SPX-5226R is the same structure that was also recrystallized.
  • Figure 17. Shows results from a 3D tumor cell spheroid assay in RCJ-41T2 cells for SPX-5229.1,-5229.2, and -5229.3.
  • Figure 18 Shows results from a 3D tumor cell spheroid assay in RCJ-41T2 cells for comparing SPX-5226 and SPX-5226R.
  • QSOX1 is Quiescin Sulfhydryl Oxidase 1, also called QSCN6.
  • the protein accession number for the long variant of QSOX1 on the NCBI database is NP — 002817, and the accession number for the short form is NP — 001004128.
  • QSOX1 refers to both the long and short variants of QSOX1.
  • a compound derived from a chemical library in the Sanford Burnham collection of compounds 3-methoxy-N-[4-(l-pyrrolidinyl)phenyl]benzamide was discovered to be an inhibitor (designated SBI-0143183 or SBI-183 for short) of QSOX1.
  • a peptide was identified in plasma from pancreatic cancer patients that mapped back to the QSOX1 parent protein.
  • QSOX1 was shown to be over-expressed in many human and murine tumor types.
  • a high throughput screening assay (HTS) was employed to screen a library of chemical compounds (50,000 compounds). The assay detects H202 produced by QSOX1 when it is enzymatically active ( Figure 2). Inhibition of the enzymatic activity of QSOX1 (with a chemical compound) suppresses H202 production, decreasing fluorescent or bioluminescent signal in the assay.
  • a secondary screening of 20 lead compounds identified as promising hits in HTS. The secondary screen consisted of incubating pancreatic (MIAPaCa2) and kidney (786-0) tumor cell lines with 20, 10, and 5 uM SBI-183 (0143183) for 48 hours followed by MTT assay (measure of mitochondrial respiration).
  • the QSOX1 enzyme is over-expressed in every histological type of tumor tested so far including blood cancers.
  • SBI-183 slows the growth and invasion of pancreatic, renal and breast cancer cells.
  • SBI-183 has been tested in-vivo in a murine model of cancer. SBI-183 suppresses the growth of UOK 1 17 renal cell carcinoma xenografts in nude mice.
  • MTT assay results from exposure of myeloma cell lines to SBI-183 shows decreased viability at higher does.
  • results disclosed herein indicate that the use of SBI-183 and related compounds inhibit neoplastic cells that over-express the QSOX1 enzyme, such as in, for example, myeloma and pancreatic, renal, and breast cancer.
  • methods for neoplastic cell inhibition, tumor inhibition, and cancer treatment involving contacting a cell, a tumor, or a subject having cancer with an effective amount of SBI-183 or a pharmaceutically acceptable salt thereof.
  • the subject can be any mammal, preferably a human.
  • an amount effective refers to the amount of inhibitor that provides a suitable inhibition and/or treatment effect.
  • treating tumors means accomplishing one or more of the following: (a) reducing tumor mass; (b) slowing the increase in tumor mass; (c) reducing tumor metastases; (d) slowing the incidence of tumor metastases; (e) limiting or preventing development of symptoms characteristic of cancer; (f) inhibiting worsening of symptoms characteristic of cancer; (g) limiting or preventing recurrence of symptoms characteristic of cancer in subjects that were previously symptomatic; (i) increasing subject survival time; and (j) limiting or reducing morbidity of therapy by enhancing current therapies, permitting decreased dose of current standard of care therapies.
  • the methods of the can be used to treat any suitable tumor type.
  • the methods are used to treat any tumor type that over-expresses QSOX1.
  • Expression of QSOX1 can be assessed by any suitable method, including but not limited to immunohistochemistry of suitable tissue sample, polymerase chain reaction, or detection of QSOX1 peptides in suitable tissue.
  • techniques that can be used in the analysis include mass spectrometry (MS), two dimensional gel electrophoresis, Western blotting, immunofluorescence, ELISAs, antigen capture assays (including dipstick antigen capture assays) and mass spec immunoassay (MSIA).
  • Non-limiting tumor types that can be treated using the methods of the include pancreatic, kidney, breast cancer (Luminal A, TNBC, Ductal), prostate cancer, squamous cell tumors, squamous cell carcinoma (e.g., squamous cell lung cancer or squamous cell head and neck cancer), neuroendocrine tumors, urothelial cancer, vulvar cancer, mesothelioma, liver cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, lung cancer, small cell lung cancer, non-small cell lung cancer, cutaneous or intraocular malignant melanoma, renal cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, non-Hodgkin's lympho
  • the tumor is a pancreatic tumor, such as a pancreatic adenocarcinoma or a neuroendocrine tumor.
  • the tumor comprises a pancreatic adenocarcinoma.
  • cancer refers to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • cancer examples include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include, but are not limited to, breast cancer (Luminal A, TNBC, Ductal), prostate cancer, squamous cell tumors, squamous cell carcinoma (e.g., squamous cell lung cancer or squamous cell head and neck cancer), neuroendocrine tumors, urothelial cancer, vulvar cancer, mesothelioma, liver cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, lung cancer, small cell lung cancer, non-small cell lung cancer, cutaneous or intraocular malignant melanoma, renal cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium
  • the inhibitors for QSOX1 as disclosed herein can be administered via any suitable technique or formulation, including but not limited to lipid, virus, polymer, or any other physical, chemical or biological agent, but are generally administered as part of a pharmaceutical composition together with a pharmaceutically acceptable carrier, diluent, or excipient.
  • a pharmaceutically acceptable carrier e.g., a pharmaceutically acceptable styrene, a pharmaceutically acceptable styrene, or a pharmaceutically acceptable i.e., contain amounts of non-pharmaceutically acceptable components lower than permitted by U.S. regulatory requirements at the time of filing this application.
  • the composition if the compound is dissolved or suspended in water, the composition further optionally comprises an additional pharmaceutically acceptable carrier, diluent, or excipient.
  • the pharmaceutical compositions described herein are solid pharmaceutical compositions (e.g., tablet, capsules, etc.).
  • compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be via physical injection with a needle to, for example, a tumor in the subject; topical (including ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), ocular, oral or parenteral.
  • routes for example, a tumor in the subject
  • topical including ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery
  • pulmonary e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal
  • Methods for ocular delivery can include topical administration (eye drops), subconjunctival, periocular or intravitreal injection or introduction by balloon catheter or ophthalmic inserts surgically placed in the conjunctival sac.
  • Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.
  • Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • the inhibitor described herein can also be formulated in combination with one or more additional active ingredients as desired.
  • methods for limiting tumor metastasis comprising contacting a subject having a tumor with an amount effective of an inhibitor of QSOX1 activity, or pharmaceutically acceptable salt thereof, to limit metastasis of the tumor in the subject.
  • this application features a method of treating (e.g., curing, suppressing, ameliorating, delaying or preventing the onset of, or preventing recurrence or relapse of) or preventing a QSOX1 -associated disorder in which QXOS1 activity is detrimental, in a subject.
  • the method includes: administering to the subject an SBI- 183-analog, or any QSOxl-biding compound that modulates (e.g., inhibits or activates) QSOX1 activity, in an amount sufficient to treat or prevent the QSOXl-associated disorder.
  • the QSOX1 antagonist e.g., the SBI-183-analog or QSOXl-binding agent, can be administered to the subject, alone or in combination with other therapeutic modalities as described herein.
  • a disorder in which QSOX1 activity is detrimental is intended to include diseases and other disorders in which the presence of QSOX1 in a subject suffering from the disorder has been shown to be or is suspected of being either responsible for the pathophysiology of the disorder or a factor that contributes to a worsening of the disorder. Accordingly, a disorder in which QSOX1 activity is detrimental is a disorder in which reduction of QSOX1 activity is expected to alleviate the symptoms and/or progression of the disorder.
  • Such disorders may be evidenced, for example, by an increase in the concentration of QSOX1 in a biological cell, fluid or tissue of a subject suffering from the disorder (e.g., an increase in the concentration of QSOX1 in a tumor, serum, plasma, synovial fluid, etc. of the subject), which can be detected, for example, using an anti- QSOX1 antibody well-known in the art.
  • SBI-183 has been shown to inhibit the enzymatic activity of QSOX1 and suppress tumor cell invasion in vitro and metastasis of MDA-MB-231 breast tumor cells in vivo.
  • SPX-009 an analog of SBI-183 with increased potency, has also been shown to suppress invasion and metastasis in vitro and in vivo.
  • SPX-009 Two dimensional (2D) invasion assays and 3D tumor spheroid assays were employed to measure the effect of the analogs on invasion.
  • one potent analog SPX-009; also referred to herein as SPX-1009
  • SPX-1009 was evaluated for the ability to suppress tumor growth and metastasis in mice bearing MDA-MB-231-luc breast cancer orthotopic xenografts.
  • Fig. IB shows inhibition of invasion of shQSOXl (sh742)-transduced breast cancer cell line (BT549) in a modified Boyden chamber invasion assay. Inhibition of invasion could be rescued by addition of recombinant human QSOX1 (rhQSOXl, bar 4 in Fig. IB), but not with an alanine mutant (dead) rhQSOXl enzyme (Fig. IB, bar 5).
  • RNAse A protein 8 thiols
  • H202 is produced by QSOX1 as a result of thiol oxidation of RNAse A. H202 generation is monitored using a commercial fluorescence detection kit. SBI-183 was chosen from the 20 active compounds because it inhibited QSOX1 at lower concentrations than other compounds in the primary enzymatic screen.
  • MST Microscale thermophoresis
  • mice were treated with ⁇ 14mg/kg per day by oral gavage. As shown in on one embodiment depicted in Fig. 5A, tumors grew 86% more slowly in SBI-183-treated mice than vehicle- treated controls.
  • immunodeficient mice bearing triple negative breast cancer cells (MDA-MB-231-Luciferase expressing cells) were treated daily by oral gavage with SBI-183 (and other text SBI- 183- analogs) for 28 days with lOOmg/kg dosage.
  • SBI-183 More than eighty (80) structural analogs for SBI-183 were tested thus far in the cell-free enzymatic HVA assay (Fig. 2); 53 of these were commercially available and at least 27 have been newly synthesized.
  • the hit compound SBI-183 is a rather simple structure comprised of two main fragments (a) phenylpyrrolidine and (b) methoxybenzamide linked by an amide bond.
  • the objective of this preliminary study was to determine how alterations in one or both parts of the molecule affect relative activity.
  • Table 1A-F which shows the inhibitory activity of a total of 89 SBI-183-analogs.
  • Table 1A- F shows both active and inactive structures tested in fluorimetric homovanillic acid (HVA) assay at luM inhibitory concentration with dithiothreitol (DTT as enzyme substrate).
  • HVA activity is ranked from 0 to 100, with 100 meaning complete inhibition of QSOX1 activity; and 0 meaning that no activity observed.
  • the HVA values are based on 0- 100% scale, where 0 corresponds to no inhibition of QSOX1 (i.e. equivalent to vehicle) while 100 corresponds to 100% inhibition at 20 micromolar.
  • an “active” SBI-183-analog in the context of its inhibitory activity in afluorimetric homovanillic acid (HVA) assay at luM inhibitory concentration with dithiothreitol (DTT as enzyme substrate) refers to an SBI-183-analog having and HVA value selected from at least 10, 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, and 95.
  • the SBI-183-analogs have an HVA value of at least 25.
  • the SBI-183-analogs have an HVA value of at least 50.
  • the SBI-183-analogs have an HVA value of at least 60.
  • the SBI-183-analogs have an HVA value of at least 70. In another embodiment, the SBI-183-analogs have an HVA value of at least 75. In another embodiment, the SBI-183-analogs have an HVA value of at least 80. In another embodiment, the SBI-183-analogs have an HVA value of at least 85. In another embodiment, the SBI-183-analogs have an HVA value of at least 90. In another embodiment, the SBI- 183-analogs have an HVA value of at least 95.
  • SPX-009 inhibits migration of tumor cells growing as 3D spheroids in Matrigel:
  • SBI- 183-analog SPX-009 a modification of SBI-183 with two extra methoxy groups in para and meta positions (Table 1), about lOx more potently inhibits both tumor types compared to the parent SBI-183 compound.
  • SPX-009 blunts the formation of MDA-MB-231 tumor cell tendrils spreading radially from the spheroid compared to vehicle at 3 days.
  • SPX-009 also suppresses RCJ-41T2 tumor invasion through Matrigel (RCJ-41T2 is a primary tumor cell line derived from a patient at Mayo Clinic) (Fig. 6B). This result is similar to shRNA knockdown of QSOX1 in 786-0 kidney cancer cells.
  • the benzamide group is modified with R1,R2,R3 groups in a systematic manner - for example, in one embodiment, two methoxy groups are kept initially in place while replacing the third, etc.
  • substitutions of pyrrolidine group are introduced at R4.
  • smaller rings and pyrrolidine derivatives are employed.
  • the central linkage is modified.
  • alkyls and alkenes of various sizes C,U,Z
  • heterocycles A,B are introduced in lieu of phenyls into the structure.
  • the amide bond is formed by the condensation of substituted benzoic acids with amine building blocks.
  • the syntheses is done in parallel using ‘catch and release’ solid phase chemistry techniques. Specifically, the carboxylic acid is reacted with resin-bound N-Benzyl-N'-cyclohexylcarbodiimide. The resin-bound activated acid is reacted with sub-stoichiometric amounts of corresponding amine to release the nearly pure amide coupling product. The solvent is removed in vacuo and the residue purified by column chromatography on CombiFlash automated chromatography system.
  • the enzymatic inhibition assay was performed in duplicate as reported by Thorpe et al. and as performed in Fig 2. Briefly, QSOX1 enzyme was preincubated for 10 min with a compound dissolved at decreasing concentrations in 10% DMSO. The reaction mix was added to the assay buffer composed of HRP, homovanillic acid (HVA) in lxPBS with ImM EDTA. The enzyme reaction, initiated by the addition of dithiothreitol (DTT) serving as a substrate for QSOX1, generates hydrogen peroxide which is detected by fluorescence at 420nm.
  • DTT dithiothreitol
  • Percent inhibition relative to SPX-009 set forth as the HVA value for each compound in Table 1A-F, was used to prioritize new leads for advancement to the orthogonal assay validation below.
  • Compounds having at least sub- micromolar affinity and corresponding increases in potency with respect to inhibiting QSOX1, relative to SBI-183 and/or SPX-009, in the enzymatic assay as well as in tumor cells are particularly useful in the methods of inhibiting QSOX1 and treating cancer provided herein.
  • Analogs that showed activity in the enzymatic inhibition assay were identified using an oxygen consumption assay.
  • the distinct advantage of this assay compared to the screening assay is that it does not require addition of horseradish peroxidase (HRP) and a fluorogenic substrate.
  • Controls for this assay are the addition of the chemical analog alone in the oxygen sensor to ensure the compound itself does not consume oxygen.
  • SBI-183-analogs have been found to inhibit migration of tumor cells growing as 3D spheroids in Matrigel.
  • HVA cell-free enzyme assay
  • active compounds were advanced to tumor cell assays. The results for an exemplary subset of these active compounds are depicted in Figures 8-18.
  • a 3D tumor cell spheroid assay cells are allowed to form spheroids in media for 2 days, then placed into Matrigel. Inhibitory compounds are then added to the spheroids in Matrigel. Images were taken on day 3 and spheroid area calculated using Image J.
  • Analog SPX-1009 (also referred to herein as SPX- 009) which contains 3 methoxy groups in para, ortho and meta positions on the benzamide ring more potently inhibits both tumor types compared to the parent SBI-183 compound.
  • SPX-009 blunts the formation of MDA-MB-231 tumor cell tendrils spreading radially from the spheroid compared to vehicle at 3 days.
  • SPX-1009 also suppresses RCJ-41T2 tumor invasion through Matrigel (RCJ-41T2 is a primary tumor cell line derived from a patient at Mayo Clinic).
  • SPX-9230 is more potent than SPX-009 (aka SPX-1009), in either one or more of these cell line biological assays, in amount selected from the group consisting of at least: 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold, or more.
  • inhibitors of QSOX1 have a minor effect on tumor cell viability, but a major effect on tumor cell invasion in vitro and metastasis in vivo. Accordingly, in particular embodiments, combination therapy with one or more additional therapeutic agents is contemplated herein, e.g., combining a QSOX1 inhibitor with a cytotoxic agent is contemplated herein to provide a means to inhibit metastasis of cells that are resistant to cytotoxic agents.
  • Exemplary therapeutic agents contemplated herein for combination with SBI-183- analogs include formulation with, and/or co-administered with, one or more additional therapeutic agents, e.g., one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents (e.g., systemic anti-inflammatory agents), anti-fibrotic agents, metabolic inhibitors, checkpoint inhibitors, enzyme inhibitors, and/or cytotoxic or cytostatic agents, mitotic inhibitors, antitumor antibiotics, immunomodulating agents, vectors for gene therapy, alkylating agents, antiangiogenic agents, antimetabolites, boron-containing agents, chemoprotective agents, hormones, antihormone agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, tyrosine kinase inhibitors, radiosensitizers, or the like.
  • additional therapeutic agents e.g., one or more cytokine

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Abstract

Provided herein are compounds and methods for targeting QSOX1 and ameliorating QSOX1-mediated diseases.

Description

COMPOUNDS AND METHODS TARGETING QSOX1
Field
[0001] This disclosure relates to the field of anti-neoplastic compounds and methods, and more particularly to such compounds and methods that modulate QSOX1.
Background
[0002] Cancer (and other hyperproliferative diseases) is characterized by uncontrolled cell proliferation. The desmoplasia (fibrosis) that forms around many solid tumors is a highly complex mixture of stellate cells, cancer associated fibroblasts, myofibroblasts, macrophages, and even endothelial cells and adipocytes. Each of these cell types secretes ECM proteins that contribute to the complex fibrotic barrier around the tumor. Components of this fibrotic barrier include fibronectin, laminin, decorin, tenascin, periostin, multiple types of collagen and various stroma and tumor-derived enzymes.
[0003] Among the enzymes in the ECM is Quiescin Sulfhydryl Oxidase 1 (QSOX1), an over-expressed, but understudied sulfhydryl oxidase. An association of QSOX1 expression with poor outcome has been found in several cancers including pancreatic ductal adenocarcinoma (PDAC) and luminal B breast cancer. QSOX1 is a FAD-dependent sulfhydryl oxidase that is over-produced by many types of tumor cells and is undetectable by antibody staining in adjacent non-malignant tissues.
[0004] QSOX1 catalyzes disulfide bond formation during protein folding. It is the only known enzyme that both generates and shuttles disulfides in proteins. Of note, protein disulfide isomerase (PD I) requires QSOX1 for protein folding but QSOX1 does not require PDI. QSOX1 shuttles disulfides using its CxxC motifs, ultimately using FAD to transfer electrons to 02, producing H202 as a byproduct. Without QSOX1 to form disulfide bonds, cysteines have free thiols which can indiscriminately cross link as they are secreted from the cell. This leads to improper disulfide cross-linking and incorrect folding of proteins, aggregate formation and a disorganized, disrupted ECM.
[0005] Researchers have explored cancer treatment approaches that inhibit various proteins involved in uncontrolled cell growth. What remains needed, however, are additional and improved inhibitors of regulatory proteins. SUMMARY
[0006] Quiescin sulfhydryl oxidase 1 (QSOX1) is an enzyme that is over-expressed in many tumor types. Two transcript variants encoding two different isoforms of QSOX1 have been found. Recombinant QSOX1 has been expressed and used to screen a library of chemical compounds to identify any compounds that inhibit the enzymatic activity of QSOX1.
[0007] Provided herein are compounds having a structure according to formula I:
Figure imgf000004_0001
wherein,
Ri-Rio are each independently selected from: -OMe, -OCH2OH, -OCfhOme. -OCH2OCH3, - OCH2F, -SMe, -SEt, -SCH2OH, -C(0)CH=CH2, -C(0)NH2, -Me, -Et, -CH2CH=CH2, - CH2COOH, -CHO, -CH2CHO, -CH2F, -CH2CH2F, -CH2CH2Br, -NHMe, -CH2NH2, - NHCH2CH2OH, -CH2C(0)CH2CH3, F, Cl, Br, I;
AI-6 are each independently selected from C ORN;
X and Y are each independently zero or central expansion with NH, -S-, -CH2-, -CH2CH2-, - CH2CH2CH2-, -CH=CH-;
Z is independently selected from C OR S;
W is independently selected from O, O2 or NH; and wherein Rs can be further independently selected from
Figure imgf000005_0001
[0008] In on embodiment the compound is set forth in Table 1A-F. In another embodiment, the compounds correspond to structures 1-9 and 64-89 in Table 1A-F. In a particular embodiment, the compound comprises the structure corresponding to SPX-9230.
[0009] Also provided herein are compositions comprising the compounds, or an analogs thereof; and a pharmaceutically acceptable carrier. In particular embodiments, said composition is a stereoisomer, prodrug form, or pharmaceutically acceptable salt of an SBI- 183 -analog compound, or an analogue thereof.
[0010] Also provided herein is a method for neoplastic cell growth inhibition, comprising the step of contacting said neoplastic cell with an effective amount of an SBI-183-analog compound or an analogue thereof or a pharmaceutically acceptable salt thereof. Also provided herein is an SBI-183-analog compound, or an analogue thereof or a pharmaceutically acceptable salt thereof for use in the treatment of a tumor or a cancer.
[0011] Also provided herein, is a method for treating cancer, comprising administering to a subject having a tumor an amount effective of an SBI- 183 -analog compound, or an analogue thereof or a pharmaceutically acceptable salt thereof. In particular embodiments, the cancer is selected from the group consisting of: carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include, but are not limited to, breast cancer (Luminal A, TNBC, Ductal), prostate cancer, squamous cell tumors, squamous cell carcinoma (e.g., squamous cell lung cancer or squamous cell head and neck cancer), neuroendocrine tumors, urothelial cancer, vulvar cancer, mesothelioma, liver cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, lung cancer, small cell lung cancer, non-small cell lung cancer, cutaneous or intraocular malignant melanoma, renal cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, environmentally induced cancers including those induced by asbestos, hematologic malignancies including, for example, multiple myeloma, B-cell lymphoma, Hodgkin lymphoma/primary mediastinal B-cell lymphoma, non-Hodgkin's lymphomas, acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), myeloproliferative disorders (MPD), chronic lymphoid leukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fimgoides, anaplastic large cell lymphoma, T-cell lymphoma, and precursor T-lymphoblastic lymphoma, PVNS, acute myeloid leukemia, adrenocortico carcinoma, ladder urothelial carcinoma, cervical squamous cell carcinoma, endocervical adenocarcinoma, diffuse large B cell lymphoma, glioblastoma multiforme, chronic lymphocytic leukemia, brain lower grade glioma, head and neck squamous cell carcinoma, hepatocellular carcinoma, lung adenocarcinoma, large squamous cell carcinoma, cutaneous melanoma, ovarial serous cystadenocarcinoma, gastric cancer, soft tissue sarcoma, testicular germ cell cancer, thymoma, thyroid carcinoma, uterine corpus endometrial carcinoma, uterine carcinosarcoma, kidney renal clear cell carcinoma, and kidney renal papillary cell carcinoma, and any combinations of said cancers. In another embodiment, the cancer is a cancer of the breast. In another embodiment, the cancer is a cancer of the pancreas. In another embodiment, the cancer is a cancer of the kidney. In another embodiment, the cancer is myeloma.
[0012] Accordingly, in particular aspects, this disclosure relates to analogs of a compound corresponding to, 3-methoxy-N-[4-(l- pyrrolidinyl)phenyl]benzamide (SBI-183- analogs for short):
Figure imgf000007_0001
[0013] In an additional aspect, this disclosure relates to stereoisomers, prodrugs and pharmaceutically acceptable salts of SBI-183-analogs.
[0014] In another aspect, this disclosure relates to the use of SBI-183-analogs and related compounds to modulate QSOX1, e.g., either inhibit (antagonize) or activate (agonize) QSOX1.
[0015] In a further aspect, this disclosure relates to the use of SBI-183-analogs and related compounds to inhibit growth and invasive properties of neoplastic cells.
[0016] In still another aspect, this disclosure relates to the use of SBI-183-analogs and related compounds to inhibit neoplastic cells that over-express the QSOX1 enzyme, such as in, for example, myeloma and pancreatic, renal, and breast cancer.
[0017] An SB-183-analog compound of the present disclosure or a pharmaceutically acceptable salt thereof, can be administered as such to a human patient or can be administered in pharmaceutical compositions in which the foregoing materials are mixed with suitable carriers or excipient(s). Techniques for formulation and administration of drugs may be found, for example, in REMINGTON'S PHARMACOLOGICAL SCIENCES, Mack Publishing Co., Easton, PA, latest edition.
[0018] These and other aspects will be apparent upon reference to the following detailed description and figures. To that end, any patent and other documents cited herein are hereby incorporated by reference in their entirety. BRIEF DESCRIPTION OF DRAWINGS
[0019] Figure 1A. Suppression of xenograft growth in mice of human pancreatic tumor cell line MIAPaCa2 transduced with shQSOXl constructs sh742 and sh528. Average tumor weight is shown in the y-axis.
[0020] Figure IB. Sh742 -transduced BT549 tumor cells did not invade through Matrigel- coated trans-well inserts (bar 3) compared to controls (bars 1&2). Invasion was rescued by exogenous addition of functional rhQSOXl (bar 4), but not mutated inactive rhQSOXl (bar 5). Number of cells invading through Matrigel is shown in the Y-axis.
[0021] Figure 1C . Diagram of 16 hour 2-D invasion assay.
[0022] Figure 2. An in vitro assay to screen for QSOX1 inhibitors; Fluorogenic assay (also can be HTS). Enzymatically active recombinant QSOX1 is incubated with individual compounds in individual wells of a 384 well plate. If compounds inhibit the activity of the enzyme, no H202 will be made by the enzyme because it can't fold RNAse A (surrogate substrate). Therefore, no fluorescence is observed. If compounds do not inhibit QSOX1, the enzyme with re-fold RNAse A, produce H202 and the fluorescent indicator will fluoresce.
[0023] Figure 3. Fluorogenic QSOX1 Activity Assay. Data were recorded at time = 15 minutes (steady state) after addition of substrate.
[0024] Figure 4. MST titrations of recombinant QSOX1 with SBI-183. Red and green curves represent two independent titrations of 50nM Dylight® 650-labeled QSOX1 with increasing amounts of SBI-183 (0.0076 to 250uM). Data fitting yielded Kd= 20±7 uM.
[0025] Figure 5A. Treatment of human tumor xenograft-bearing mice with SBI-183 suppresses tumor growth. Treatment was started on day 7 after tumor implantation. Experiment was terminated on day 41. Tumor volume, shown on the Y -axis was calculated using length and width measurements.
[0026] Figure 5B. Treatment of mice bearing MDA-MB-231 -Luc tumors with lOOmg/kg SBI-183, daily resulted in decreased lung metastasis in treated mice. Mice were injected with luciferin at the end of the experiment followed by ISIS 2000 imaging of lung mets and calculation of mean lung radiance. [0027] Figure 6A. 3D Spheroid Invasion Assay. Graphs are shown on the left as area of MDA-MB-231 as in Matrigel at 0, 1 and 3 days. Replicates of 6 spheroids for each concentration of compound was performed. Different color lines show 2-fold serial dilutions of SPX-009 compound compared to parent compound SBI-183 at 1250nM.Photographs on the right show invasive growth of spheroids on Day 3 in either 0.01% DMSO vehicle.
[0028] Figure 6B. 3D Spheroid Invasion Assay. Graphs are shown on the left as area of RCJ-41T2 spheroids as in Matrigel at 0, 1 and 3 days. Replicates of 6 spheroids for each concentration of compound was performed. Different color lines show 2-fold serial dilutions of SPX-009 compound compared to parent compound SBI-183 at 1250nM. Photographs on the right show invasive growth of spheroids on Day 3 in either 0.01% DMSO vehicle.
[0029] Figure 7. SBI-183-analog structure scaffold; sample structures; and representative examples of structural elements to be used for SAR optimization. The exact nature of these elements will be adjusted based on the outcome of each round of screening.
[0030] Figure 8. Shows results from a 3D tumor cell spheroid assay in MDA-MB-231 cells for SPX-52091.
[0031] Figure 9. Shows results from a 3D tumor cell spheroid assay in MDA-MB-231 cells for SPX-52092.
[0032] Figure 10. Shows results from a 3D tumor cell spheroid assay in MIA PaCa2 cells for SPX-52091.
[0033] Figure 11. Shows results from a 3D tumor cell spheroid assay in MDA-MB-231 cells for SPX-52092.
[0034] Figure 12. Shows results from a 3D tumor cell spheroid assay in RCJ-41T2 cells for SPX-52091.
[0035] Figure 13. Shows results from a 3D tumor cell spheroid assay in RCJ-41T2 cells for SPX-52092.
[0036] Figure 14. Shows results from a 3D tumor cell spheroid assay in MDA-MB-231 cells for SPX-5229.1,-5229.2, and -5229.3. [0037] Figure 15. Shows results from a 3D tumor cell spheroid assay in MIA PaCa2 cells for SPX-5229.1,-5229.2, and -5229.3.
[0038] Figure 16. Shows results from a 3D tumor cell spheroid assay in MIA PaCa2 cells for comparing SPX-5226 and SPX-5226R. SPX-5226 is chromatography purified, whereas SPX-5226R is the same structure that was also recrystallized.
[0039] Figure 17. Shows results from a 3D tumor cell spheroid assay in RCJ-41T2 cells for SPX-5229.1,-5229.2, and -5229.3.
[0040] Figure 18. Shows results from a 3D tumor cell spheroid assay in RCJ-41T2 cells for comparing SPX-5226 and SPX-5226R.
DETAILED DESCRIPTION
[0041] As used herein, "QSOX1" is Quiescin Sulfhydryl Oxidase 1, also called QSCN6. The protein accession number for the long variant of QSOX1 on the NCBI database is NP — 002817, and the accession number for the short form is NP — 001004128. As used herein, "QSOX1" refers to both the long and short variants of QSOX1.
[0042] In screening for inhibitors, recombinant QSOX1 was produced and tested in fluorogenic assays to make sure it was active. The recombinant enzyme then was sent to the Sanford Burnham Institute for use in the same/similar assay to screen libraries for chemical compounds that inhibit the enzymatic activity of QSOX1.
[0043] In one embodiment, a compound derived from a chemical library in the Sanford Burnham collection of compounds, 3-methoxy-N-[4-(l-pyrrolidinyl)phenyl]benzamide was discovered to be an inhibitor (designated SBI-0143183 or SBI-183 for short) of QSOX1.
[0044] A peptide was identified in plasma from pancreatic cancer patients that mapped back to the QSOX1 parent protein. QSOX1 was shown to be over-expressed in many human and murine tumor types. A high throughput screening assay (HTS) was employed to screen a library of chemical compounds (50,000 compounds). The assay detects H202 produced by QSOX1 when it is enzymatically active (Figure 2). Inhibition of the enzymatic activity of QSOX1 (with a chemical compound) suppresses H202 production, decreasing fluorescent or bioluminescent signal in the assay. [0045] A secondary screening of 20 lead compounds identified as promising hits in HTS. The secondary screen consisted of incubating pancreatic (MIAPaCa2) and kidney (786-0) tumor cell lines with 20, 10, and 5 uM SBI-183 (0143183) for 48 hours followed by MTT assay (measure of mitochondrial respiration).
[0046] It has been found that SBI-183 suppressed invasion of 786-0 cells through Matrigel (in vitro surrogate for a basement membrane) in a dose-dependent manner (see US2019/0008829A1; which is incorporated herein by reference in its entirety for all purposes). SBI-183 inhibits deposition of laminin-oc4 into the extracellular matrix at 15 uM. It has been found that SBI-183 suppresses invasion through Matrigel of MIAPaCa2 pancreatic tumor cells and highly invasive MCF-IOA breast cancer cell lines containing mutant p53. SBI-183 suppresses the growth of UOK 117 renal cell carcinoma xenografts implanted into nude mice compared to vehicle-treated and untreated mice.
[0047] The QSOX1 enzyme is over-expressed in every histological type of tumor tested so far including blood cancers. Thus, in another embodiment, SBI-183 slows the growth and invasion of pancreatic, renal and breast cancer cells. In further embodiments, SBI-183 has been tested in-vivo in a murine model of cancer. SBI-183 suppresses the growth of UOK 1 17 renal cell carcinoma xenografts in nude mice. Moreover, MTT assay results from exposure of myeloma cell lines to SBI-183 shows decreased viability at higher does. Taken together, the results disclosed herein indicate that the use of SBI-183 and related compounds inhibit neoplastic cells that over-express the QSOX1 enzyme, such as in, for example, myeloma and pancreatic, renal, and breast cancer.
[0048] In other embodiments, methods for neoplastic cell inhibition, tumor inhibition, and cancer treatment involving contacting a cell, a tumor, or a subject having cancer with an effective amount of SBI-183 or a pharmaceutically acceptable salt thereof. The subject can be any mammal, preferably a human. As used herein, the phrase "an amount effective" refers to the amount of inhibitor that provides a suitable inhibition and/or treatment effect.
[0049] As used herein, "treating tumors" means accomplishing one or more of the following: (a) reducing tumor mass; (b) slowing the increase in tumor mass; (c) reducing tumor metastases; (d) slowing the incidence of tumor metastases; (e) limiting or preventing development of symptoms characteristic of cancer; (f) inhibiting worsening of symptoms characteristic of cancer; (g) limiting or preventing recurrence of symptoms characteristic of cancer in subjects that were previously symptomatic; (i) increasing subject survival time; and (j) limiting or reducing morbidity of therapy by enhancing current therapies, permitting decreased dose of current standard of care therapies.
[0050] The methods of the can be used to treat any suitable tumor type. In one preferred embodiment, the methods are used to treat any tumor type that over-expresses QSOX1. Expression of QSOX1 can be assessed by any suitable method, including but not limited to immunohistochemistry of suitable tissue sample, polymerase chain reaction, or detection of QSOX1 peptides in suitable tissue.
[0051] In various non-limiting embodiments, techniques that can be used in the analysis include mass spectrometry (MS), two dimensional gel electrophoresis, Western blotting, immunofluorescence, ELISAs, antigen capture assays (including dipstick antigen capture assays) and mass spec immunoassay (MSIA).
[0052] Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et ah, 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, Calif.), "Guide to Protein Purification" in Methods in Enzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, Calif), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R. I . Freshney. 1987. Liss, Inc. New York, N.Y.), Gene Transfer and Expression Protocols, pp. 109-128, ed. E . J . Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, Tex.).
[0053] All embodiments of any aspect of the can be used in combination, unless the context clearly dictates otherwise.
[0054] Non-limiting tumor types that can be treated using the methods of the include pancreatic, kidney, breast cancer (Luminal A, TNBC, Ductal), prostate cancer, squamous cell tumors, squamous cell carcinoma (e.g., squamous cell lung cancer or squamous cell head and neck cancer), neuroendocrine tumors, urothelial cancer, vulvar cancer, mesothelioma, liver cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, lung cancer, small cell lung cancer, non-small cell lung cancer, cutaneous or intraocular malignant melanoma, renal cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, environmentally induced cancers including those induced by asbestos, hematologic malignancies including, for example, multiple myeloma, B-cell lymphoma, Hodgkin lymphoma/primary mediastinal B- cell lymphoma, non-Hodgkin's lymphomas, acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), myeloproliferative disorders (MPD), chronic lymphoid leukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B -lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fimgoides, anaplastic large cell lymphoma, T-cell lymphoma, and precursor T-lymphoblastic lymphoma, PVNS, acute myeloid leukemia, adrenocortico carcinoma, ladder urothelial carcinoma, cervical squamous cell carcinoma, endocervical adenocarcinoma, diffuse large B cell lymphoma, glioblastoma multiforme, chronic lymphocytic leukemia, brain lower grade glioma, head and neck squamous cell carcinoma, hepatocellular carcinoma, lung adenocarcinoma, large squamous cell carcinoma, cutaneous melanoma, ovarial serous cystadenocarcinoma, gastric cancer, soft tissue sarcoma, testicular germ cell cancer, thymoma, thyroid carcinoma, uterine corpus endometrial carcinoma, uterine carcinosarcoma, kidney renal clear cell carcinoma, and kidney renal papillary cell carcinoma, and any combinations of said cancers. In a preferred embodiment, the tumor is a pancreatic tumor, such as a pancreatic adenocarcinoma or a neuroendocrine tumor. In a further preferred embodiment, the tumor comprises a pancreatic adenocarcinoma.
[0055] Accordingly, provided herein are methods of treating (e.g., curing, suppressing, ameliorating, delaying or preventing the onset of, or preventing recurrence or relapse of) or preventing cancer in a subject in need thereof, comprising administering to the subject an SBI- 183-analog, SPX-013, SPX-014, and/or the like, in an amount sufficient to treat or prevent the QSOX1 -associated cancer. [0056] As used herein, the term “cancer,” refers to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include, but are not limited to, breast cancer (Luminal A, TNBC, Ductal), prostate cancer, squamous cell tumors, squamous cell carcinoma (e.g., squamous cell lung cancer or squamous cell head and neck cancer), neuroendocrine tumors, urothelial cancer, vulvar cancer, mesothelioma, liver cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, lung cancer, small cell lung cancer, non-small cell lung cancer, cutaneous or intraocular malignant melanoma, renal cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, environmentally induced cancers including those induced by asbestos, hematologic malignancies including, for example, multiple myeloma, B-cell lymphoma, Hodgkin lymphoma/primary mediastinal B-cell lymphoma, non- Hodgkin's lymphomas, acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), myeloproliferative disorders (MPD), chronic lymphoid leukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B -lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fimgoides, anaplastic large cell lymphoma, T-cell lymphoma, and precursor T- lymphoblastic lymphoma, PVNS, acute myeloid leukemia, adrenocortico carcinoma, ladder urothelial carcinoma, cervical squamous cell carcinoma, endocervical adenocarcinoma, diffuse large B cell lymphoma, glioblastoma multiforme, chronic lymphocytic leukemia, brain lower grade glioma, head and neck squamous cell carcinoma, hepatocellular carcinoma, lung adenocarcinoma, large squamous cell carcinoma, cutaneous melanoma, ovarial serous cystadenocarcinoma, gastric cancer, soft tissue sarcoma, testicular germ cell cancer, thymoma, thyroid carcinoma, uterine corpus endometrial carcinoma, uterine carcinosarcoma, kidney renal clear cell carcinoma, and kidney renal papillary cell carcinoma, and any combinations of said cancers. The present is also applicable to treatment of metastatic cancers.
[0057] The inhibitors for QSOX1 as disclosed herein can be administered via any suitable technique or formulation, including but not limited to lipid, virus, polymer, or any other physical, chemical or biological agent, but are generally administered as part of a pharmaceutical composition together with a pharmaceutically acceptable carrier, diluent, or excipient. Such compositions are substantially free of non-pharmaceutically acceptable components, i.e., contain amounts of non-pharmaceutically acceptable components lower than permitted by U.S. regulatory requirements at the time of filing this application. In some embodiments of this aspect, if the compound is dissolved or suspended in water, the composition further optionally comprises an additional pharmaceutically acceptable carrier, diluent, or excipient. In other embodiments, the pharmaceutical compositions described herein are solid pharmaceutical compositions (e.g., tablet, capsules, etc.).
[0058] These compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be via physical injection with a needle to, for example, a tumor in the subject; topical (including ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), ocular, oral or parenteral. Methods for ocular delivery can include topical administration (eye drops), subconjunctival, periocular or intravitreal injection or introduction by balloon catheter or ophthalmic inserts surgically placed in the conjunctival sac. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
[0059] The inhibitor described herein can also be formulated in combination with one or more additional active ingredients as desired. [0060] In another embodiment, methods are provided for limiting tumor metastasis, comprising contacting a subject having a tumor with an amount effective of an inhibitor of QSOX1 activity, or pharmaceutically acceptable salt thereof, to limit metastasis of the tumor in the subject.
[0061] Several analogues of SBI-183 have been tested (e.g., SBI- 183-analogs) and several have also been found effective limiting tumor metastasis as described herein. It should be noted that SPX-013 and SPX-014 are not analogues, but rather new compounds. U.S. Patent Publication 2016/0166553, which is incorporated herein by reference in its entirety discloses compounds that are contemplated herein to bind to QSOX1 and inhibit QSOX1 activity. These SBI- 183 -analogs and related compounds are contemplated herein to be particularly useful for ameliorating or preventing a Q SOX 1 -associated disorder.
[0062] Thus in another embodiment, this application features a method of treating (e.g., curing, suppressing, ameliorating, delaying or preventing the onset of, or preventing recurrence or relapse of) or preventing a QSOX1 -associated disorder in which QXOS1 activity is detrimental, in a subject. The method includes: administering to the subject an SBI- 183-analog, or any QSOxl-biding compound that modulates (e.g., inhibits or activates) QSOX1 activity, in an amount sufficient to treat or prevent the QSOXl-associated disorder. The QSOX1 antagonist, e.g., the SBI-183-analog or QSOXl-binding agent, can be administered to the subject, alone or in combination with other therapeutic modalities as described herein.
[0063] As used herein, the phrase “a disorder in which QSOX1 activity is detrimental” is intended to include diseases and other disorders in which the presence of QSOX1 in a subject suffering from the disorder has been shown to be or is suspected of being either responsible for the pathophysiology of the disorder or a factor that contributes to a worsening of the disorder. Accordingly, a disorder in which QSOX1 activity is detrimental is a disorder in which reduction of QSOX1 activity is expected to alleviate the symptoms and/or progression of the disorder. Such disorders may be evidenced, for example, by an increase in the concentration of QSOX1 in a biological cell, fluid or tissue of a subject suffering from the disorder (e.g., an increase in the concentration of QSOX1 in a tumor, serum, plasma, synovial fluid, etc. of the subject), which can be detected, for example, using an anti- QSOX1 antibody well-known in the art. [0064] As noted SBI-183 has been shown to inhibit the enzymatic activity of QSOX1 and suppress tumor cell invasion in vitro and metastasis of MDA-MB-231 breast tumor cells in vivo. SPX-009, an analog of SBI-183 with increased potency, has also been shown to suppress invasion and metastasis in vitro and in vivo.
EXAMPLES
[0065] Methods Summary: Commercially available and new synthesized analogs for SBI-183 (e.g., SBI-183 -analogs) were screened in a cell-free fluorescent screening assay for the ability to inhibit QSOX1. SBI- 183-analog compounds that inhibited QSOX1 in a concentration-dependent manner were then tested for the ability to inhibit growth and invasion of triple negative breast cancer (TNBC) cells (MDA-MB-231), sarcomatoid kidney cancer cells (RCJ-41T2) and pancreatic adenocarcinoma cells (MIAPaCa2). MTT assays were used to measure the effect of the compounds on tumor growth. Two dimensional (2D) invasion assays and 3D tumor spheroid assays were employed to measure the effect of the analogs on invasion. In one embodiment, one potent analog (SPX-009; also referred to herein as SPX-1009) was evaluated for the ability to suppress tumor growth and metastasis in mice bearing MDA-MB-231-luc breast cancer orthotopic xenografts.
[0066] Results Summary: Numerous SBI-183-analogs were found to inhibit the enzymatic activity of QSOX1 at levels similar to the parent SBI-183 compound. However in one embodiment, one of those compounds, referred to herein as SPX-009, suppressed invasion of breast, kidney and pancreas tumor cells in 2D and 3D invasion assays at > 10-fold lower concentrations than the parent compound (SBI-183). In a non-metastatic xenograft of kidney cancer, SBI-183 suppressed primary kidney tumor growth by 51%, but in a metastatic xenograft breast cancer model (MDA-MB-231-luc TNBC), SBI-183 reduced lung metastasis by 76% compared to control. Human-mouse xenograft studies have also been conducted with the more potent analog (SPX-009).
[0067] Upon discovery that QSOX1 was over-expressed in pancreatic cancer as well as other tumor types, the expression of QSOX1 in tumor cell lines was genetically silenced to determine if a phenotype appeared in the knockdown. It was found that tumor cells did not adhere very strongly to substrate and lost the ability to invade, likely through multiple mechanisms, one of which includes post-translational folding of MMP-2 and -9. Additionally, tumors transduced with short hairpin (sh) QSOX1 demonstrated poor growth in human tumor xenografts in immunodeficient mouse models. Growth of shQSOXl (sh528 or sh742)-transduced MIAPaCa2 pancreatic tumor cells in nude mice was suppressed compared to untreated tumor and a shScramble as shown in Fig. 1A. Fig. IB shows inhibition of invasion of shQSOXl (sh742)-transduced breast cancer cell line (BT549) in a modified Boyden chamber invasion assay. Inhibition of invasion could be rescued by addition of recombinant human QSOX1 (rhQSOXl, bar 4 in Fig. IB), but not with an alanine mutant (dead) rhQSOXl enzyme (Fig. IB, bar 5).
Identification of Small Molecule Inhibitors for QSOX1 in vitro (enzymatic assay).
[0068] Twenty QSOX1 inhibitors were identified with IC50’s ranging from 2-100 uM using a high throughput screening assay. Fig. 2 demonstrates the screening strategy to detect inhibition of QSOX1 -mediated oxidation of reduced RNAse A protein (8 thiols) as a model substrate. H202 is produced by QSOX1 as a result of thiol oxidation of RNAse A. H202 generation is monitored using a commercial fluorescence detection kit. SBI-183 was chosen from the 20 active compounds because it inhibited QSOX1 at lower concentrations than other compounds in the primary enzymatic screen. We confirmed the ability of SBI-183 to inhibit the enzymatic activity of QSOX1 in a concentration-dependent manner as shown in Fig. 3. The control of SBI-183 + horse radish peroxidase (HRP) was included to be sure the compound did not inhibit HRP (first bar in Fig. 3).
Microscale thermophoresis (MST) experiment demonstrates binding of SBI-183 to QSOX1.
[0069] Two independent MST experiments (Fig. 4) indicate that SBI-183 binds to QSOX1 at a Kd of ~20 ±7uM. Taken together, Figs 3 and 4 suggest that SBI-183 binds and inhibits the enzymatic activity of QSOX1.
In vivo studies:
[0070] In a human-xenograft mouse model, 786-0 renal cell carcinoma cells were implanted into the hind flanks of immunodeficient mice. Seven days after implantation, mice were treated with ~14mg/kg per day by oral gavage. As shown in on one embodiment depicted in Fig. 5A, tumors grew 86% more slowly in SBI-183-treated mice than vehicle- treated controls. [0071] Since it is beleived that QSOX1 facilitates tumor invasion and metastasis, immunodeficient mice bearing triple negative breast cancer cells (MDA-MB-231-Luciferase expressing cells) were treated daily by oral gavage with SBI-183 (and other text SBI- 183- analogs) for 28 days with lOOmg/kg dosage. Although for SBI-183 there was no change in primary tumor size, lung metastases were reduced in treated vs control mice (Fig. 5B). In a related experiment, we determined that the maximal tolerated dose was >200mg/kg SBI-183 and was limited by the solubility of the compound.
Preliminary SAR studies:
[0072] More than eighty (80) structural analogs for SBI-183 were tested thus far in the cell-free enzymatic HVA assay (Fig. 2); 53 of these were commercially available and at least 27 have been newly synthesized. The hit compound SBI-183 is a rather simple structure comprised of two main fragments (a) phenylpyrrolidine and (b) methoxybenzamide linked by an amide bond. The objective of this preliminary study was to determine how alterations in one or both parts of the molecule affect relative activity. The data is partly summarized in Table 1A-F, which shows the inhibitory activity of a total of 89 SBI-183-analogs. Table 1A- F shows both active and inactive structures tested in fluorimetric homovanillic acid (HVA) assay at luM inhibitory concentration with dithiothreitol (DTT as enzyme substrate). HVA activity is ranked from 0 to 100, with 100 meaning complete inhibition of QSOX1 activity; and 0 meaning that no activity observed. In other words, the HVA values are based on 0- 100% scale, where 0 corresponds to no inhibition of QSOX1 (i.e. equivalent to vehicle) while 100 corresponds to 100% inhibition at 20 micromolar.
[0073] As used herein, an “active” SBI-183-analog in the context of its inhibitory activity in afluorimetric homovanillic acid (HVA) assay at luM inhibitory concentration with dithiothreitol (DTT as enzyme substrate) ; refers to an SBI-183-analog having and HVA value selected from at least 10, 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, and 95. In another embodiment, the SBI-183-analogs have an HVA value of at least 25. In another embodiment, the SBI-183-analogs have an HVA value of at least 50. In another embodiment, the SBI-183-analogs have an HVA value of at least 60. In another embodiment, the SBI-183-analogs have an HVA value of at least 70. In another embodiment, the SBI-183-analogs have an HVA value of at least 75. In another embodiment, the SBI-183-analogs have an HVA value of at least 80. In another embodiment, the SBI-183-analogs have an HVA value of at least 85. In another embodiment, the SBI-183-analogs have an HVA value of at least 90. In another embodiment, the SBI- 183-analogs have an HVA value of at least 95.
(a) Phenylpyrrolidine:
[0074] The most drastic change in activity occurred when we modified the phenylpyrrolidine part of the molecule. Almost universally, those compounds became inactive. Replacing the pyrrolidine group with pyrrolidone invariably resulted in complete loss of activity (e.g. SPX-034, 036, 038, 046, 051, 052). Converting the pyrrolidine ring into conjugated pyrrole (SPX-5180) or benzo[b] pyrrole (HC-4233) or imidazole abolished activity. Bridging pyrrolidine function with a methylene group also resulted in inactive compound SPX-5211 as well as introducing six-member ring piperazine groups (AN-1498, QC-2306, AN-3618) also almost completely vanquished the activity. Piperidine and morpholine groups, on the other hand, reduced but did not eliminate activity. Noteworthy is that installation of trifluoromethyl group next to pyrrolidine ring (QA-3179) resulted in activity loss as well. At the same time, substitution with smaller fluorine atom at the same position resulted in a highly active compound AM-2172. AM-2172 is used for FAXS NMR studies to understand compound binding dynamics, obtain Kd values and for screening other stronger binding analogs (vide infra). Moving the pyrrolidine group from a para- to meta- (SPX-010, SPX-51261) and to ortho-positions progressively lowered but did not abolish activity. These results indicate that the pyrrolidine ring directly attached to phenyl ring is an important prerequisite for QSOX1 binding and inhibition. This is evidenced by the active structures in Table 1A-F that have been tested so far where each active compound maintained same phenylpyrrolidine structural element. One explanation may be that the delocalization of amine lone electron pair is responsible for the activity loss, although this does not explain effects of piperazine rings. b) Methoxybenzamide:
[0075] As can be seen in Table 1A-F, the SBI-183-analogs’ inhibitory activity remained fairly intact when the benzamide moiety was altered in numerous ways. Most notable modulators of activity were alkoxy substituents. Increasing the bulkiness of an alkoxy substituent progressively reduced inhibitory activity. Thus, replacing the methoxy group (SBI-183) with isopropyloxy, butyloxy and benzyloxy reduced activity by 20-50%. The positioning and number of methoxy groups seemed to modulate the apparent activity, with trimethoxy modification being the most potent. In fact, the trimethoxy analog of SBI-183 (SPX-009) was advanced to tumor cell assays and pK studies (vide infra). Despite having similar activity to SBI-183 in enzymatic assay, SPX-009 proved to be ten-fold more potent in tumor inhibition assay, possibly because of more favorable LogP, polarization and/or number ofH-bonds.
SPX-009 inhibits migration of tumor cells growing as 3D spheroids in Matrigel:
[0076] In a 3D tumor cell spheroid assay, cells were allowed to form spheroids in media for 2 days, then placed into Matrigel. Inhibitory test compounds (e.g., SBI-183, SPX-009, - 52091, -52092, -5229.1, -5229.2, -5229.3, -6003, and the like) were then added to the spheroids in Matrigel. Images were taken on day 3 and spheroid area calculated using Image J. SBI- 183-analog SPX-009, a modification of SBI-183 with two extra methoxy groups in para and meta positions (Table 1), about lOx more potently inhibits both tumor types compared to the parent SBI-183 compound. In Fig. 6A, SPX-009 blunts the formation of MDA-MB-231 tumor cell tendrils spreading radially from the spheroid compared to vehicle at 3 days. SPX-009 also suppresses RCJ-41T2 tumor invasion through Matrigel (RCJ-41T2 is a primary tumor cell line derived from a patient at Mayo Clinic) (Fig. 6B). This result is similar to shRNA knockdown of QSOX1 in 786-0 kidney cancer cells.
[0077] Over 80 commercially available and in-house synthesized SBI- 183-analogs were screened for more potent inhibitory activity that SBI-183. One of them, SPX-009 was nearly 10-fold more potent than the parent compound in cellular assays. In the following example section, we describe NMR-guided SAR studies to identify potent biologically active QSOX1 inhibitors with favorable ADME predictions using QikProp software (Shroedinger).
Synthesis and screening structural analogs of SPX-009 for the ability to inhibit QSOX1 enzyme.
[0078] Based on preliminary SAR data (e.g. Table 1A-F), SAR studies were continued by systematically synthesizing compound evolutionary libraries around the lead SPX-009 as a scaffold to guide us through initial lead optimization efforts with the goal of a double digit nM potency, while attempting to maintain favorable ADME properties. Fig. 7 shows a non- exhaustive list of functional groups contemplated in the SBI-183-analogs for increasing polarity, hydrogen bonding and, to a reasonable extent, lipophilicity in an iterative manner. [0079] Substitutions at benzamide, phenylpyrrolidine and central parts of the molecule are contemplated herein. In a first embodiment, while keeping the phenylpyrrolidine group intact, the benzamide group is modified with R1,R2,R3 groups in a systematic manner - for example, in one embodiment, two methoxy groups are kept initially in place while replacing the third, etc. In a second embodiment, while keeping benzamide group intact, substitutions of pyrrolidine group are introduced at R4. In further particular embodiments, smaller rings and pyrrolidine derivatives are employed. In yet a further embodiment, the central linkage is modified. For example, in one embodiment the amide linkage can be expanded with sulfonamide (U=S, W=02), alkyls and alkenes of various sizes (C,U,Z) to explore effects of H-bonding, polarity, hydrophobicity, lengthening, and planarity at the central linkage. In another embodiment, heterocycles (A,B) are introduced in lieu of phenyls into the structure. Once the NMR analysis is complete, SAR findings are applied to fine tune new lead candidate.
Chemical synthesis:
[0080] In one embodiment, the amide bond is formed by the condensation of substituted benzoic acids with amine building blocks. The syntheses is done in parallel using ‘catch and release’ solid phase chemistry techniques. Specifically, the carboxylic acid is reacted with resin-bound N-Benzyl-N'-cyclohexylcarbodiimide. The resin-bound activated acid is reacted with sub-stoichiometric amounts of corresponding amine to release the nearly pure amide coupling product. The solvent is removed in vacuo and the residue purified by column chromatography on CombiFlash automated chromatography system.
Assays:
[0081] In the initial screen, the enzymatic inhibition assay was performed in duplicate as reported by Thorpe et al. and as performed in Fig 2. Briefly, QSOX1 enzyme was preincubated for 10 min with a compound dissolved at decreasing concentrations in 10% DMSO. The reaction mix was added to the assay buffer composed of HRP, homovanillic acid (HVA) in lxPBS with ImM EDTA. The enzyme reaction, initiated by the addition of dithiothreitol (DTT) serving as a substrate for QSOX1, generates hydrogen peroxide which is detected by fluorescence at 420nm. Percent inhibition relative to SPX-009, set forth as the HVA value for each compound in Table 1A-F, was used to prioritize new leads for advancement to the orthogonal assay validation below. Compounds having at least sub- micromolar affinity and corresponding increases in potency with respect to inhibiting QSOX1, relative to SBI-183 and/or SPX-009, in the enzymatic assay as well as in tumor cells are particularly useful in the methods of inhibiting QSOX1 and treating cancer provided herein.
Characterize the biological activity of potent SBI-183-analogs in tumor cell invasion assays.
[0082] Analogs that showed activity in the enzymatic inhibition assay were identified using an oxygen consumption assay. The distinct advantage of this assay compared to the screening assay is that it does not require addition of horseradish peroxidase (HRP) and a fluorogenic substrate. Controls for this assay are the addition of the chemical analog alone in the oxygen sensor to ensure the compound itself does not consume oxygen.
Tumor Invasion assays:
[0083] Analogs that showed increased potency in the cell-free enzyme assays were advanced to tumor cell invasion assays. Because SBI-183 suppresses tumor cell invasion through Matrigel in a modified Boyden chamber assay (see Figure 1C for how the assay works) and in the 3D tumor spheroid migration assay (see Figure 5) similar to a genetic knockdown, different tumor types that over-express QSOX1 were used to evaluate more potent analogs identified in cell -free screening , which tumor type assays include: the triple negative breast cancer (TNBC) cell line (MDA-MB-231 (triple negative breast), a human lung epithelial tumor cell line (A549 (lung), a pancreatic tumor cell line (MiaPaCa2 (pancreas) and RCJ-41T2 a (primary sarcomatoid kidney cancer)). Since effective therapies are lacking for both TNBC and sarcomatoid kidney cancer, compounds that affect the growth, migration and invasion of these tumor types are contemplated herein to be particularly useful in treating various cancers. The other advantage to using these cell lines is that they grow orthotopically in immunodeficient mice and produce metastases which is how the most potent SBI-183- analogs will be evaluated.
[0084] SBI-183-analogs have been found to inhibit migration of tumor cells growing as 3D spheroids in Matrigel. After screening 89 analogs in cell-free enzyme assay (HVA), active compounds were advanced to tumor cell assays. The results for an exemplary subset of these active compounds are depicted in Figures 8-18. In a 3D tumor cell spheroid assay, cells are allowed to form spheroids in media for 2 days, then placed into Matrigel. Inhibitory compounds are then added to the spheroids in Matrigel. Images were taken on day 3 and spheroid area calculated using Image J. Analog SPX-1009 (also referred to herein as SPX- 009) which contains 3 methoxy groups in para, ortho and meta positions on the benzamide ring more potently inhibits both tumor types compared to the parent SBI-183 compound. SPX-009 blunts the formation of MDA-MB-231 tumor cell tendrils spreading radially from the spheroid compared to vehicle at 3 days. SPX-1009 also suppresses RCJ-41T2 tumor invasion through Matrigel (RCJ-41T2 is a primary tumor cell line derived from a patient at Mayo Clinic).
[0085] Six of the 89 compounds were screened with several cancer cell lines (MDA = breast, RCJ = kidney, MIA-PaCa2 = pancreatic). It was found that Sulfonamide analogs SPX-5209-1,2,3 were less active than SBI-183. Bulky substituents (ethoxy, e.g., SPX-5226) in analogs of SPX-1009 (methoxy) result in reduced activity. The most biologically potent compound was found to be SPX-1009. Additional medicinal chemistry is contemplated herein in accordance with the present to improve the solubility of lead analogs. Upon completion of thee assays, analogs of SPX-009 that are > 10-fold more potent than the parent compound are identified.
[0086] For example, SPX-9230, and the other SBI- 183-analog claimed and/or set forth herein in Table 1A-F, are screened with several cancer cell lines (MDA = breast, RCJ = kidney, MIA-PaCa2 = pancreatic), and the like. In one embodiment, it is contemplated herein that SPX-9230 is more potent than SPX-009 (aka SPX-1009), in either one or more of these cell line biological assays, in amount selected from the group consisting of at least: 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold, or more.
[0087] In particular embodiments, it has been found that inhibitors of QSOX1 have a minor effect on tumor cell viability, but a major effect on tumor cell invasion in vitro and metastasis in vivo. Accordingly, in particular embodiments, combination therapy with one or more additional therapeutic agents is contemplated herein, e.g., combining a QSOX1 inhibitor with a cytotoxic agent is contemplated herein to provide a means to inhibit metastasis of cells that are resistant to cytotoxic agents.
[0088] Exemplary therapeutic agents contemplated herein for combination with SBI-183- analogs, include formulation with, and/or co-administered with, one or more additional therapeutic agents, e.g., one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents (e.g., systemic anti-inflammatory agents), anti-fibrotic agents, metabolic inhibitors, checkpoint inhibitors, enzyme inhibitors, and/or cytotoxic or cytostatic agents, mitotic inhibitors, antitumor antibiotics, immunomodulating agents, vectors for gene therapy, alkylating agents, antiangiogenic agents, antimetabolites, boron-containing agents, chemoprotective agents, hormones, antihormone agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, tyrosine kinase inhibitors, radiosensitizers, or the like.
[0089] The claims are not intended to be limited to the materials, methods, embodiments and examples described herein.
Figure imgf000026_0001
Table 1-A
Figure imgf000027_0001
Table 1-B
Figure imgf000028_0001
Table 1-C
Figure imgf000029_0001
Table 1-D
Figure imgf000030_0001
Table 1-E
Figure imgf000031_0001
Table 1-F
Figure imgf000032_0001

Claims

CLAIMS What is claimed is
1. A compound having a structure according to formula I:
Figure imgf000033_0001
wherein,
R1-R10 are each independently selected from: -OMe, -OCH2OH, -OQrhOme, -OCH2OCH3, - OCH2F, -SMe, -SEt, -SCH2OH, -C(0)CH=CH2, -C(0)NH2, -Me, -Et, -CH2CH=CH2, - CH2COOH, -CHO, -CH2CHO, -CH2F, -CH2CH2F, -CH2CH2Br, -NHMe, -CH2NH2, -
NHCH2CH2OH, -CH2C(0)CH2CH3, F, Cl, Br, I;
Ai-6 are each independently selected from C ORN;
X and Y are each independently zero or central expansion with NH, -S-, -CH2-, -CH2CH2-, - CH2CH2CH2-, -CH=CH-;
Z is independently selected from C OR S;
W is independently selected from O, O2 or NH; and wherein Rs can be further independently selected from:
Figure imgf000033_0002
2. The compound of claim 1, wherein the compound is set forth in Table 1A-F.
3. The compound of claim 1, wherein the compounds correspond to structures 1- 9 and 64-89 in Table 1A-F.
4. A compound comprising the structure corresponding to SPX-9230.
5. A composition comprising the compound of claims 1-4, or an analogs thereof; and a pharmaceutically acceptable carrier.
6. The composition of claim 5, wherein said composition is a stereoisomer, prodrug form, or pharmaceutically acceptable salt of a compound according to claims
1-4, or an analogue thereof.
7. A method for neoplastic cell growth inhibition, comprising the step of contacting said neoplastic cell with an effective amount of a compound according to claims 1-4 or an analogue thereof or a pharmaceutically acceptable salt thereof.
8. A compound according to claims 1-4, or an analogue thereof or a pharmaceutically acceptable salt thereof for use in the treatment of a tumor or a cancer.
9. A method for treating cancer, comprising administering to a subject having a tumor an amount effective of a compound according to claims 1-4, or an analogue thereof or a pharmaceutically acceptable salt thereof.
10. The method of claim 9, wherein said cancer is selected from the group consisting of: carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include, but are not limited to, breast cancer (Luminal A, TNBC, Ductal), prostate cancer, squamous cell tumors, squamous cell carcinoma (e.g., squamous cell lung cancer or squamous cell head and neck cancer), neuroendocrine tumors, urothelial cancer, vulvar cancer, mesothelioma, liver cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, lung cancer, small cell lung cancer, non-small cell lung cancer, cutaneous or intraocular malignant melanoma, renal cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, environmentally induced cancers including those induced by asbestos, hematologic malignancies including, for example, multiple myeloma, B-cell lymphoma, Hodgkin lymphoma/primary mediastinal B-cell lymphoma, non- Hodgkin's lymphomas, acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), myeloproliferative disorders (MPD), chronic lymphoid leukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B -lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fimgoides, anaplastic large cell lymphoma, T-cell lymphoma, and precursor T- lymphoblastic lymphoma, PVNS, acute myeloid leukemia, adrenocortico carcinoma, ladder urothelial carcinoma, cervical squamous cell carcinoma, endocervical adenocarcinoma, diffuse large B cell lymphoma, glioblastoma multiforme, chronic lymphocytic leukemia, brain lower grade glioma, head and neck squamous cell carcinoma, hepatocellular carcinoma, lung adenocarcinoma, large squamous cell carcinoma, cutaneous melanoma, ovarial serous cystadenocarcinoma, gastric cancer, soft tissue sarcoma, testicular germ cell cancer, thymoma, thyroid carcinoma, uterine corpus endometrial carcinoma, uterine carcinosarcoma, kidney renal clear cell carcinoma, and kidney renal papillary cell carcinoma, and any combinations of said cancers.
11. The method of claim 9, wherein said cancer is a cancer of the breast.
12. The method of claim 9, wherein said cancer is a cancer of the pancreas.
13. The method of claim 9, wherein said cancer is a cancer of the kidney.
14. The method of claim 9, wherein said cancer is myeloma.
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