Methods and compositions for treatment of fibrosis

Information

  • Patent Grant
  • 10112933
  • Patent Number
    10,112,933
  • Date Filed
    Friday, July 18, 2014
    10 years ago
  • Date Issued
    Tuesday, October 30, 2018
    6 years ago
Abstract
Embodiments of the invention include methods of treating, preventing, and/or reducing the risk of fibrosis in an individual in need thereof. In some embodiments, particular small molecules are employed for treatment, prevention, and/or reduction of the risk of fibrosis. In at least particular cases, the small molecules are inhibitors of STAT3.
Description
TECHNICAL FIELD

The present invention generally concerns at least the fields of cell biology, molecular biology, and medicine.


BACKGROUND OF THE INVENTION

Fibrosis is a pathological process involving the accumulation of excessive extra-cellular matrix in tissues, leading to tissue damage and organ dysfunction, which can progress to organ failure and death. In systemic sclerosis, an idiopathic fibrosis disease, the trigger is postulated to be an autoimmune response that leads to tissue injury, production of growth factors, pro-inflammatory and pro-fibrotic cytokines, and accumulation of myofibroblasts. Two potential sources of myofibroblasts are the differentiation of local fibroblasts and the process of epithelial-to-mesenchymal transition (EMT). IL-6 is a proinflammatory and pro-fibrotic cytokine increasingly recognized as an important mediator of fibrosis that may contribute to the accumulation of myofibroblasts. After engaging its receptor, IL-6 signals through the STAT3. To be fully active, STAT3 becomes phosphorylated on Y705; levels of pY705-STAT3 are measured as an indicator of the level of activated STAT3.


The present disclosure satisfies a need in the art to provide novel compounds and methods for treating and/or preventing fibrosis in individuals.


SUMMARY OF THE INVENTION

Embodiments of the invention include methods and compositions for the treatment, prevention, or reduction in the risk of fibrosis. In specific embodiments, the fibrosis is not pulmonary fibrosis or myelofibrosis.


Embodiments of the invention include methods and/or compositions for the treatment of fibrosis in an individual known to have the fibrosis, suspected of having fibrosis, or at risk for having fibrosis. Patients at risk of fibrosis may be those that have autoimmune diseases such as schleroderma or systemic schlerosis, those exposed to certain drugs including chemotherapy, those exposed to environmental or other toxins or allergens, those who have suffered an ischemia/reperfusion injury such as myocardial infarction or hypotension, and/or those with idiopathic pulmonary fibrosis, idiopathic liver fibrosis, hepatitis induced by alcohol, toxins, drugs or infections, liver cirrhosis, primary biliary cirrhosis, viral infections involving the heart, liver, or lung, and idiopathic retroperiotoneal fibrosis. The compositions include small molecules and functional derivatives as described herein. In some embodiments, the individual is receiving an additional therapy for fibrosis. An individual in need thereof is an individual that has at least one symptom of fibrosis or is susceptible to or at risk of having fibrosis.


In embodiments of the invention, an individual is given more than one dose of one or more compositions described herein or functional derivatives thereof. The dosing regimen may include doses separated in time by hours, days, months or years. Delivery of the composition of the invention may occur by any suitable route, including systemic or local, although in specific embodiments, the delivery route is oral, intravenous, topical, subcutaneous, intraarterial, intraperitoneal, buccal, and so forth.


In some embodiments of the invention, the methods and/or compositions of the invention are useful for treating and/or preventing and/or reducing the risk or severity of fibrosis, and in specific cases such treatment occurs by inhibiting Stat3 and/or Stat1 activity, although in some aspects the composition(s) does not inhibit Stat3 or Stat1 or both. In certain embodiments, the compositions inhibit Stat3 but fail to inhibit Stat1. In some embodiments, compounds of the invention interact with the Stat3 SH2 domain, competitively inhibit recombinant Stat3 binding to its immobilized pY-peptide ligand, and/or inhibit IL-6-mediated tyrosine phosphorylation of Stat3, for example. In particular embodiments, the compositions of the invention fulfills the criteria of interaction analysis (CIA): 1) global minimum energy score≤−30; 2) formation of a salt-bridge and/or H-bond network within the pY-residue binding site of Stat3; and/or 3) formation of a H-bond with or blocking access to the amide hydrogen of E638 of Stat3, for example. In some embodiments, the composition(s) interacts with a hydrophobic binding pocket with the Stat3 SH2 domain.


In a specific embodiment of the invention, there is a method of treating, preventing, and/or reducing the risk of fibrosis in an individual comprising delivering to the individual a therapeutically effective amount of a compound selected from the group consisting of N-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide, N-(3,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(4,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(5,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(6,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(7,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(8,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, 4-Bromo-N-(1,6′-dihydroxy-[2,2′]binaphthalenyl-4-yl)-benzenesulfonamide, 4-Bromo-N-[4-hydroxy-3-(1H-[1,2,4]triazol-3-ylsulfanyl)-naphthalen-1-yl]-benzenesulfonamide; a functionally active derivative thereof; and a mixture thereof.


In a specific embodiment of the invention, there is a method of treating, preventing, and/or reducing the risk of fibrosis in an individual comprising delivering to the individual a therapeutically effective amount of a compound selected from the group consisting of 4-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-oxo-1-propen-1-yl]benzoic acid; 4{5-[(3-ethyl-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-2-furyl}benzoic acid; 4-[({3-[(carboxymethyl)thio]-4-hydroxy-1-naphthyl}amino) sulfonyl]benzoic acid; 3-({2-chloro-4-[(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)methyl]-6-ethoxyphenoxy}methyl)benzoic acid; methyl 4-({[3-(2-methyoxy-2-oxoethyl)-4,8-dimethyl-2-oxo-2H-chromen-7-yl]oxy}methyl)benzoate; 4-chloro-3-{5-[(1,3-diethyl-4,6-dioxo-2-thioxotetrahydro-5(2H)-pyrimidinylidene)methyl]-2-furyl}benzoic acid; a functionally active derivative thereof; and a mixture thereof. In a specific embodiment, any of the compounds disclosed herein are suitable to treat and/or prevent fibrosis, for example.


In another embodiment, the inhibitor comprises the general formula:




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wherein R1 and R2 may be the same or different and are selected from the group consisting of hydrogen, carbon, sulfur, nitrogen, oxygen, flourine, chlorine, bromine, iodine, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne-based derivative, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives, heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid, and benzoic acid-based derivatives.


In another embodiment of the invention, the composition comprises the general formula:




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wherein R1, and R3 may be the same or different and are selected from the group consisting of hydrogen, carbon, nitrogen, sulfur, oxygen, flouring, chlorine, bromine, iodine, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne-based derivative, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives, heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid, and benzoic acid-based derivatives; and R2 and R4 may be the same or different and are selected from the group consisting of hydrogen, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne-based derivative, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives, heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid, and benzoic acid-based derivatives.


In another embodiment of the invention, the composition comprises the general formula:




embedded image


wherein R1, R2, and R3 may be the same or different and are selected from the group consisting of hydrogen, carbon, nitrogen, sulfur, oxygen, fluorine, chlorine, bromine, iodine, carboxyl, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne-based derivative, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives, heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid, and benzoic acid-based derivatives.


In specific embodiments, the individual does not have cancer and/or is not suspected of having cancer nor has been diagnosed with cancer.


Mammals may be treated with the methods and/or compositions of the invention, including humans, dogs, cats, horses, cows, pigs, sheep, and goats, for example.


In other embodiments of the invention, there are methods of treating fibrosis in an individual wherein the composition(s) is an inhibitor of any members of the STAT protein family, including STAT1, STAT2, STAT3, STAT4, STAT5 (STAT5A and STAT5B), or STATE, for example.


Embodiments of the invention include methods for preventing or treating a fibrotic disease or complications thereof in a subject comprising providing to the subject an effective amount of a composition as described herein. The fibrosis may occur externally on the individual, such as on the skin, and/or the fibrosis may occur internally in the individual.


In some embodiments of the invention, the composition(s) are employed to treat or prevent scarring, such as of a wound or surgical incision, for example.


In one embodiment, there is a method of treating, preventing, or reducing the risk or severity of fibrosis in an individual that has fibrosis or that is at risk of having or susceptible to having fibrosis, comprising the step of providing to the individual an effective amount of one or more compositions of Table 6-11, FIG. 15, FIG. 16, or a functional derivative thereof. In specific embodiments, the fibrosis is not pulmonary fibrosis or myelofibrosis. The fibrosis may be of the lung, skin, heart, intestine, pancreas, joint, liver, or retroperionteum. The fibrosis may be of the skin, heart, intestine, pancreas, joint, liver, or retroperionteum. In some cases, the individual is provided the composition in multiple doses, such as multiple doses are separated by hours, days, or weeks. In specific embodiments, the individual is provided with an additional therapy for the fibrosis. In certain aspects, the composition is selected from the group consisting of N-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide, N-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide, N-(3,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(4,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(5,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(6,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(7,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(8,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, 4-Bromo-N-(1,6′-dihydroxy-[2,2′]binaphthalenyl-4-yl)-benzene sulfonamide, 4-Bromo-N-[4-hydroxy-3-(1H-[1,2,4]triazol-3-ylsulfanyl)-naphthalen-1-yl]-benzenesulfonamide, a functionally active derivative thereof, and a mixture thereof. The composition may inhibit Stat3, Stat1, or both, in specific embodiments. In particular aspects, a method further comprises the step of diagnosing the fibrosis.


Compositions may be provided intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, injection, infusion, continuous infusion, localized perfusion, via a catheter, via a lavage, in lipid compositions, in liposome compositions, or as an aerosol. The composition may be provided systemically or locally. In certain cases, the individual does not have cancer. In certain cases, the individual is not suspected of having cancer. In specific embodiments, the fibrosis is scleroderma.


The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present invention.





DESCRIPTION OF THE DRAWINGS


FIG. 1 demonstrates inhibition of Stat3 binding to immobilized phosphopeptide ligand by compounds. Binding of recombinant Stat3 (500 nM) to a BiaCore sensor chip coated with a phosphododecapeptide based on the amino acid sequence surrounding Y1068 within the EGFR was measured in real time by SPR (Response Units) in the absence (0 μM) or presence of increasing concentrations (0.1 to 1,000 μM) of Cpd3 (panel A), Cpd30 (panel B), Cpd188 (panel C), Cpd3-2 (panel D), Cpd3-7 (panel E) and Cpd30-12 (panel F). Data shown are representative of 2 or more experiments. The equilibrium binding levels obtained in the absence or presence of compounds were normalized (response obtained in the presence of compound÷the response obtained in the absence of compound×100), plotted against the log concentration (nM) of the compounds (panel G). The experimental points fit to a competitive binding curve that uses a four-parameter logistic equation (see exemplary methods for details). These curves were used to calculate IC50 (Table 1).



FIG. 2 demonstrates inhibition of IL-6-mediated activation of Stat3 by compounds. HepG2 cells were pretreated with DMSO alone or DMSO containing Cpd3 (panel A), Cpd188 (panel B), Cpd30 (panel C), Cpd3-2 (panel D), Cpd3-7 (panel E) or Cpd30-12 (panel F) at the indicated concentration for 60 min. Cells were then stimulated with IL-6 (30 ng/ml) for 30 min. Protein extracts of cells were separated by SDS-PAGE, blotted and developed serially with antibodies to pStat3, total Stat3 and β-actin. Blots were stripped between each antibody probing. The bands intensities of immunoblot were quantified by densitometry. The value of each pStat3 band's intensity was divided by each corresponding value of total Stat3 band intensity and the results normalized to the DMSO-treated control value and plotted as a function of the log compound concentration. The best-fit curves were generated based on 4 Parameter Logistic Model/Dose Response One Site/XLfit 4.2, IDBS. Each panel is representative of 3 or more experiments.



FIG. 3 provides exemplary chemical formulas and names of compounds. The chemical formulas and names are indicated for Cpd3 (panel A), Cpd30 (panel B), Cpd188 (panel C), Cpd3-2 (panel D), Cpd3-7 (panel E) and Cpd30-12 (panel F).



FIG. 4 shows effect of compounds on Stat1 activation. HepG2 cells were pretreated with DMSO alone or DMSO containing each of the compounds at a concentration of 300 μM for 60 min. Cells were then stimulated with IFN-γ (30 ng/ml) for 30 min. Protein extracts of cells were separated by SDS-PAGE and immunoblotted serially with antibodies to pStat1, total Stat1 and β-actin. Blots were stripped between each immunoblotting. The results shown are representative of 2 or more experiments.



FIGS. 5A-5C provides comparisons of the Stat3 and Stat1 SH2 domain sequences, 3-D structures and van der Waals energies of compound binding. Sequence alignment of Stat3 and Stat1 SH2 domains is shown in panel A. The residues that bind the pY residue are highlighted in and pointed to by a solid arrow, the residue (E638) that binds to the +3 residue highlighted and pointed to by a dotted arrow and LoopβC-βD and LoopαB-αC, which comprise the hydrophobic binding site consisting, are highlighted and pointed to by dot-dashed and dashed arrows, respectively. Panel B shows an overlay of a tube-and-fog van der Waals surface model of the Stat3 SH2 domain and a tube-and-fog van der Waals surface model of the Stat1 SH2. The residues of the Stat3 SH2 domain represents LoopβC-βD are highlighted and shown by dotted circles and the residues represent LoopαB-αC are highlighted and shown by a dotted-dashed circle; the corresponding loop residues within the Stat1 SH2 domain are shown in a light fog surrounding the circles. This overlay is shown bound by Cpd3-7 as it would bind to the Stat3 SH2 domain. The van der Waals energy of each compound bound to the Stat1 SH2 domain or the Stat3 SH2 domain was calculated, normalized to the value for Stat1 and depicted in panel C.



FIGS. 6A-6F shows a computer model of each compound bound by the Stat3 SH2 domain. The results of computer docking to the Stat3 SH2 domain is shown for Cpd3 (panel A), Cpd30 (panel B), Cpd188 (panel C), Cpd3-2 (panel D), Cpd3-7 (panel E) and Cpd30-12 (panel F). The image on the left of each panel shows the compound binding to a spacefilling model of the Stat3 SH2 domain. The pY-residue binding site is represented by dashed circle, the +3 residue binding site is represented by a solid circle, loop LoopβC-βD is represented by dotted circle and loop LoopαB-αC is represented by dot-dashed circle. Residues R609 and K591 critical for binding pY are shown within a dashed circle, residue E638 that binds the +3 residue shown within a solid circle and the hydrophobic binding site consisting of LoopβC-βD and LoopαB-αC is shown within a dash-dot and dotted circle, respectively. The image on the right side of each panel is a closer view of this interaction with hydrogen bonds indicated by dotted lines. In FIG. 6A the negatively charged benzoic acid moiety of Cpd3 has electrostatic interactions with the positively-charge pYresidue binding site consisting mainly of the guanidinium cation group of R609 and the basic ammonium group of K591. The benzoic acid group also forms a hydrogen-bond network consisting of double H-bonds between the carboxylic oxygen and the ammonium hydrogen of R609 and the amide hydrogen of E612. H-bond formation also occurs between the benzoic acid carbonyl oxygen and the side chain hydroxyl hydrogen of Serine 611. Within the +3 residue-binding site, the oxygen atom of 1,4-benzodioxin forms a hydrogen bond with the amide hydrogen of E638. In addition, the 2,3-dihydro-1,4-benzodioxin of Cpd3 interacts with the loops forming the hydrophobic binding site. In FIG. 6B the carboxylic terminus of the benzoic acid moiety of Cpd30, which is negatively charged under physiological conditions, forms a salt bridge with the guanidinium group of R609 within the pYresidue binding site. Within the +3 residue-binding site, the oxygen of the thiazolidin group forms a H-bond with the peptide backbone amide hydrogen of E638. In addition, the thiazolidin moiety plunges into the hydrophobic binding site. In FIG. 6C there is an electrostatic interaction between the (carboxymethyl)thio moiety of Cpd188 carrying a negative charge and the pY-residue binding site consisting of R609 and K591 carrying positive charge under physiological conditions. There are H-bonds between the hydroxyloxygen of the (carboxymethyl) thio group of Cpd188 and the guanidinium hydrogen of R609, between the hydroxyl-oxygen of the (carboxymethyl)thio group and the backbone amide hydrogen of E612, and between the carboxyl-oxygen of the (carboxymethyl)thio group of Cpd188 and the hydroxyl-hydrogen of 5611. Within the +3 residue-binding site, there is a H-bond between the hydroxyl-oxygen of benzoic acid group of Cpd188 and the amide-hydrogen of E638. In addition, the benzoic acid group extends and interacts with the hydrophobic binding site. In FIG. 6D the benzoic acid group of Cpd3-2 has significant electrostatic interactions with the pY-residue binding site pocket, mainly contributed by R609 and K591, and forms two H bonds; the carboxylic oxygen of the benzoic acid group binds the guanidinium hydrogen of R609, and the carbonyl oxygen of the benzoic acid group binds to the carbonyl hydrogen of S611. Within the +3 residue-binding site, oxygen within the 1,3-dihydro-2H-inden-2-ylidene group forms an H bond to the backbone amide-hydrogen of E638. In addition, the 1,3-dihydro-2H-inden-2-ylidene group plunges into the hydrophobic binding site. In FIG. 6E H-bonds are formed between the carbonyl-oxygen of the methyl 4-benzoate moiety of Cpd 3-7 and the side chain guanidinium of R609 and between the methoxy-oxygen and the hydrogen of the ammonium terminus of K591. The (2-methoxy-2-oxoethyl)-4,8-dimethyl-2-oxo-2H-chromen group of Cpd3-7 blocks access to the amide hydrogen of E638 within the +3 residue-binding site. In addition, this group plunges into the hydrophobic binding site. In FIG. 6F there are electrostatic interactions between the benzoic acid derivative group of Cpd30-12 and R609 and 591 within the pY-residue binding site. Also, H-bonds are formed between the hydroxyl-oxygen of Cpd30-12 and the guanidinium-hydrogen of R609, between the carboxyl-oxygen of Cpd30-12 and the hydroxyl-hydrogen of S611 and between the furyl group of Cpd30-12 and the hydrogen of ammonium of K591. The 1,3-diethyl-4,6-dioxo-2-thioxotetrahydro-5(2H)-pyrimidinylidene groups blocks access to the +3 residue binding site; however, it extends into the groove between the pY-residue binding site and LoopβC-βD, while sparing the hydrophobic binding site.



FIGS. 7A-7B shows inhibition of cytoplasmic-to-nuclear translocation of Stat3 assessed by confocal and high-throughput fluorescence microscopy. In panel A, MEF/GFP-Stat3 cells grown on coverslips were pretreated with DMSO that either contained (row four) or did not contain (row three) Cpd3 (300 μM) for 60 min before being stimulated without (row one) or with IL-6 (200 ng/ml) and IL-6sR (250 ng/ml) for 30 minutes (rows two, three and four). Coverslips were examined by confocal fluorescent microscopy using filters to detect GFP (column one), DAPI (column two) or both (merge; column three). In panel B, MEF-GFP-Stat3 cells were grown in 96-well plates with optical glass bottoms and pretreated with the indicated compound at the indicated concentrations in quadruplicate for 1 hour then stimulated with IL-6 (200 ng/ml) and IL-6sR (250 ng/ml) for 30 minutes. Cells were fixed and the plates were examined by high-throughput microscopy to determine the fluorescence intensity in the nucleus (FLIN) and the % ΔFLINMax was calculated as described in Example 1. Data shown are mean±SD and are representative of 2 or more studies. Best-fit curves were generated based on 4 Parameter Logistic Model/Dose Response One Site/XLfit 4.2, IDBS and were used to calculate IC50 (Table 1).



FIG. 8 demonstrates inhibition of Stat3 DNA binding by compounds. Electrophoretic mobility shift assays were performed using whole-cell extracts prepared from HepG2 cells without and with stimulation with IL-6 (30 ng/ml) for 30 min. Protein (20 μg) was incubated with radiolabeled duplex oligonucleotide (hSIE) and DMSO without or with the indicated compounds (300 uM) for 60 minutes at 37° C. then separated by PAGE. The gel was dried and autoradiographed; the portion of the gel corresponding to the Stat3-bound hSIE band is shown. Data shown are representative of 2 studies.



FIG. 9 shows Cpd3, Cpd30 and Cpd188 and the hydrophobicity or hydrophilicity of the surface of the molecule. The dashed arrows point to hydrophilic surfaces, and the solid arrows point to hydrophobic surfaces.



FIG. 10 illustrates exemplary compound 3 (Cpd3). The top-left picture of FIG. 11 shows Cpd3 docked into Stat3 and the interaction between Cpd3 and the surface of the protein and derivatives of Cpd3 that can fit into the surface of the protein. Stars represent atoms and chemical groups that can be replaced with other atoms or chemical groups to create one or more functional derivatives. The hydrophobic/hydrophilic surfaces of Cpd3 are also demonstrated on the top-right picture. The dashed arrows point to hydrophilic surfaces, and the solid arrows point to hydrophobic surfaces. R1 and R2 could be identical or different and may comprise hydrogen, carbon, sulfur, nitrogen, oxygen, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne-based derivative, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives, heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid, or benzoic acid-based derivatives.



FIG. 11 illustrates exemplary compound 30 (Cpd30). The top-left picture of FIG. 12 shows Cpd30 docked into Stat3 and the interaction between Cpd30 and the surface of the protein, and derivatives of Cpd30 that fit into the surface of the protein. Stars represent atoms and chemical groups that can be replaced with other atoms or chemical groups to create one or more functional derivatives. The hydrophobic/hydrophilic surfaces of Cpd30 are also demonstrated on the top-right picture. The dashed arrows point to hydrophilic surfaces, and the solid arrows point to hydrophobic surfaces. 2-D structure of Cpd30 shown on the bottom picture, R1, R2, R3 and R4 could identical or different and may comprise be hydrogen, carbon, sulfur, nitrogen, oxygen, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne-based derivative, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives, heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid, or benzoic acid-based derivatives.



FIG. 12 illustrates exemplary compound 188 (Cpd188). The top picture of FIG. 12 shows Cpd188 docked into Stat3 SH2 domain and the interaction between Cpd188 and the surface of the protein, and derivatives of Cpd188 that fit into the surface of the protein. Stars represent atoms and chemical groups that can be replaced with other atoms or chemical groups to create one or more functional derivative. The hydrophobic/hydrophilic surfaces of Cpd188 are also demonstrated on the left picture on the bottom. The dashed arrows point to hydrophilic surfaces, and the solid arrows point to hydrophobic surfaces. Shown on the right bottom picture, R1 and R2 could be identical or different and may comprise hydrogen, carbon, sulfur, nitrogen, oxygen, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne-based derivative, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives, heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid, or benzoic acid-based derivatives.



FIG. 13 illustrates schematic diagrams of Stat1 and Stat3.



FIG. 14 demonstrates that SPR IC50 of 2nd generation Stat3 chemical probes is inversely correlated with 3-D pharmacophore score.



FIGS. 15A-15C shows SPR IC50 and AML apoptosis EC50 of parent Cpd188 and two 2nd generation 188-like Stat3 chemical probes.



FIG. 16 provides an illustration of structure-activity relationships of 38 Cpd188-like, 2nd generation Stat3 probes.



FIG. 17 shows an exemplary modification scheme for 3rd generation Stat3 probe development using Cpd188-15 as a scaffold.



FIG. 18 provides illustration of the electrostatic surface of Stat3 SH2 domain (positive area in blue, neutral in white and negative in red in a color figure) and 20 docking poses of 5 (R=CH2PO32−), showing strong interactions between phosphonate groups (in purple and red) and K591/R609.



FIG. 19 shows increased STAT-3 activation in a skin fibrosis model.



FIG. 20 illustrates generation of an exemplary subcutaneous Bleomycin model for skin fibrosis and exemplary STAT3 inhibitory studies.



FIG. 21 demonstrates that STAT-3 inhibition is associated with diminished fibrosis (Hematoxylin and eosin stain).



FIG. 22 shows quantification that STAT-3 inhibition leads to diminished fibrosis (dermal thickness).



FIG. 23 illustrates that STAT-3 inhibition is associated with diminished fibrosis, using Masson's trichome stain as an indicator.



FIG. 24 demonstrates that STAT-3 inhibition is associated with diminished fibrosis, based on alpha smooth muscle actin staining.



FIGS. 25A-25C shows the effect of C188-9 on dermal fibroblast production of type I collagen (Col1a1), smooth muscle actin (aSMA) and the transcription factor Snail.



FIG. 26 demonstrates quantification of the antifibrotic effects of C188-9 in a bleomycin skin fibrosis model. RT-PCR was used to assess fibrotic gene expression that was increased with bleomycin but blunted by C188-9 treatment.





DETAILED DESCRIPTION OF THE INVENTION

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.


In some embodiments, there is a method of treating, preventing, and/or reducing the risk of fibrosis in an individual, comprising delivering to the individual one or more particular compounds. The fibrosis may be of any kind, although in some cases the fibrosis is not pulmonary fibrosis or myelofibrosis. In some embodiments, the compound(s) is a STAT3 inhibitor. In certain embodiments the compound(s) is not a STAT3 inhibitor. In particular cases, the compound(s) is a STAT1 inhibitor, but in particular cases it is not a STAT1 inhibitor. In certain aspects, there are some compounds that are both STAT3 and STAT1 inhibitors or is neither a STAT3 or STAT1 inhibitor.


In certain embodiments of the invention, there is a compound for use in the prevention, treatment, and/or reduction in risk for fibrosis, wherein the compound is selected from the group consisting of N-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide, N-(3,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(4,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(5,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(6,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(7,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(8,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, 4-Bromo-N-(1,6′-dihydroxy-[2,2′]binaphthalenyl-4-yl)-benzenesulfonamide, 4-Bromo-N-[4-hydroxy-3-(1H-[1,2,4]triazol-3-yl sulfanyl)-naphthalen-1-yl]-benzenesulfonamide; a functionally active derivative and a mixture thereof.


In certain embodiments of the invention, there is a compound for use in the prevention, treatment, and/or reduction in risk for fibrosis, wherein the compound is selected from the group consisting of 4-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-oxo-1-propen-1-yl]benzoic acid; 4{5-[(3-ethyl-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-2-furyl}benzoic acid; 4-[({3-[(carboxymethyl)thio]-4-hydroxy-1-naphthyl}amino)sulfonyl]benzoic acid; 3-({2-chloro-4-[(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)methyl]-6-ethoxyphenoxy}methyl)benzoic acid; methyl 4-({[3-(2-methyoxy-2-oxoethyl)-4,8-dimethyl-2-oxo-2H-chromen-7-yl]oxy}methyl)benzoate; 4-chloro-3-{5-[(1,3-diethyl-4,6-dioxo-2-thioxotetrahydro-5(2H)-pyrimidinylidene)methyl]-2-furyl}benzoic acid; a functionally active derivative and a mixture thereof. In a specific embodiment of the invention, the composition is a Stat3 inhibitor but does not inhibit Stat1.


In a specific embodiment of the invention, the composition is delivered in vivo in a mammal. In another embodiment the mammal is a human. In another specific embodiment the human is known to have fibrosis, is suspected of having fibrosis, or is at risk for developing fibrosis. In another embodiment, the human is known to have fibrosis and is receiving an additional therapy for the fibrosis and/or an underlying condition that is related to the fibrosis. Composition(s) of the disclosure treat, prevent, and/or reduce the risk or severity of fibrosis, in particular embodiments.


I. Definitions

As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one. As used herein “another” may mean at least a second or more. Still further, the terms “having”, “including”, “containing” and “comprising” are interchangeable and one of skill in the art is cognizant that these terms are open ended terms. Some embodiments of the invention may consist of or consist essentially of one or more elements, method steps, and/or methods of the invention. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.


The term “inhibitor” as used herein refers to one or more molecules that interfere at least in part with the activity of Stat3 to perform one or more activities, including the ability of Stat3 to bind to a molecule and/or the ability to be phosphorylated.


The phrase “therapeutically effective amount” as used herein means that amount of a compound, material, or composition comprising a compound of the present invention that is effective for producing some desired therapeutic effect, e.g., treating (i.e., preventing and/or ameliorating) cancer in a subject, or inhibiting protein-protein interactions mediated by an SH2 domain in a subject, at a reasonable benefit/risk ratio applicable to any medical treatment. In one embodiment, the therapeutically effective amount is enough to reduce or eliminate at least one symptom. One of skill in the art recognizes that an amount may be considered therapeutically effective even if the cancer is not totally eradicated but improved partially. For example, the spread of the cancer may be halted or reduced, a side effect from the cancer may be partially reduced or completed eliminated, life span of the subject may be increased, the subject may experience less pain, and so forth.


The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.


The phrase “at risk for having fibrosis” is used herein to refer to individuals that have a chance to have fibrosis because of past, present, or future factors.


As used herein, “binding affinity” refers to the strength of an interaction between two entities, such as a protein-protein interaction. Binding affinity is sometimes referred to as the Ka, or association constant, which describes the likelihood of the two separate entities to be in the bound state. Generally, the association constant is determined by a variety of methods in which two separate entities are mixed together, the unbound portion is separated from the bound portion, and concentrations of unbound and bound are measured. One of skill in the art realizes that there are a variety of methods for measuring association constants. For example, the unbound and bound portions may be separated from one another through adsorption, precipitation, gel filtration, dialysis, or centrifugation, for example. The measurement of the concentrations of bound and unbound portions may be accomplished, for example, by measuring radioactivity or fluorescence, for example. Ka also can be inferred indirectly through determination of the Ki or inhibitory constant. Determination of the Ki can be made several ways for example by measuring the Ka of STAT3 binding to its phosphopeptide ligand within the EGFR at position Y1068 and by measuring the concentration of a molecule that reduces binding of STAT3 by 50%. In certain embodiments of the invention, the binding affinity of a Stat3 inhibitor for the SH2 domain of Stat3 is similar to or greater than the affinity of the compounds listed herein.


The term “domain” as used herein refers to a subsection of a polypeptide that possesses a unique structural and/or functional characteristic; typically, this characteristic is similar across diverse polypeptides. The subsection typically comprises contiguous amino acids, although it may also comprise amino acids that act in concert or that are in close proximity due to folding or other configurations. An example of a protein domain is the Src homology 2 (SH2) domain of Stat3. The term “SH2 domain” is art-recognized, and, as used herein, refers to a protein domain involved in protein-protein interactions, such as a domain within the Src tyrosine kinase that regulates kinase activity. The invention contemplates modulation of activity, such as activity dependent upon protein-protein interactions, mediated by SH2 domains of proteins (e.g., tyrosine kinases such as Src) or proteins involved with transmission of a tyrosine kinase signal in organisms including mammals, such as humans.


As used herein, a “mammal” is an appropriate subject for the method of the present invention. A mammal may be any member of the higher vertebrate class Mammalia, including humans; characterized by live birth, body hair, and mammary glands in the female that secrete milk for feeding the young. Additionally, mammals are characterized by their ability to maintain a constant body temperature despite changing climatic conditions. Examples of mammals are humans, cats, dogs, cows, mice, rats, and chimpanzees. Mammals may be referred to as “patients” or “subjects” or “individuals”.


II. General Embodiments

General embodiments include one or more compositions for the treatment and/or prevention of fibrosis and methods of use. The fibrosis may have an unknown cause or it may be associated with an underlying condition. The underlying condition may be a catabolic condition. The underlying condition may be autoimmune, chemical intoxication, a viral infection, and so forth.


In some cases an individual is suspected of having fibrosis; such suspicion may be because the individual has changes from normal in the characteristics and function of the skin, lung, heart, liver, and kidneys. In certain aspects, such suspicion may be because the individual has tight skin, shortness of breath with exertion, cough, yellowing of the sclera or skin and/or reduced urine production. In some cases, an individual may have at least one symptom of fibrosis but may have other symptoms as well.


In certain cases, an individual is at risk of having fibrosis. In such cases, the individual has a medical condition that can be associated with fibrosis and has not had enough progression of the medical condition to manifest fibrosis or has not yet had a detectable symptom of fibrosis.


In some embodiments, the individual is known to have an underlying condition that often has fibrosis as at least one symptom, and that individual may or may have not shown other symptoms or signs of having fibrosis. In cases wherein an individual has an underlying condition that often has fibrosis as at least one symptom, the individual may be provided with an effective amount of one or more compositions of the invention prior to and/or after the appearance of fibrosis. When the individual is provided one of more compositions prior to the appearance of fibrosis, the onset of fibrosis may be delayed or completely inhibited and/or the severity of the fibrosis may be reduced, compared to the condition of the individual without having received the composition(s), for example.


In particular embodiments, an individual has been diagnosed with an underlying condition known to have fibrosis as at least one symptom, and methods of the invention may include steps of diagnosing of the fibrosis and/or the underlying condition of the individual. An individual may be tested for fibrosis by standard means in the art.


Fibrosis can occur in response to or as part of autoimmune diseases such as systemic sclerosis, systemic lupus erythematosus or mixed connective tissue disease, for example. Fibrosis of the liver can occur in response to chronic liver disease from viral infections, parasitic infections, and toxins (such as alcohol), for example.


Embodiments concern STAT3 inhibitors as they related to biological mechanisms associated with fibrosis. To evaluate if STAT3 contributes to the development of tissue fibrosis in the lung and skin and whether it does so in part through the modulation of EMT, fibrotic tissue was examined from systemic sclerosis patients, idiopathic pulmonary fibrosis patients, and mouse models of lung and skin fibrosis. Tissues were assessed by immunohistology using a monoclonal antibody to the pY705 epitope within STAT3. The findings demonstrated that levels of pY705-STAT3 were increased in fibrotic tissue from systemic sclerosis patients, idiopathic pulmonary fibrosis patients, and mouse models of lung and skin fibrosis.


It was further determined that STAT3 signaling contributed to the development of fibrosis by using an example of a small molecule STAT-3 inhibitor. N-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide (herein also referred to as C188-9) was administered to mice in both the intraperitoneal (IP) bleomycin mouse model of lung fibrosis and the subcutaneous (SC) bleomycin mouse model of skin fibrosis. C-188-9 administration decreased fibrotic endpoints (collagen deposition by Sircol), expression of alpha-smooth muscle actin (SMA), and improved arterial oxygen saturation) in the IP bleomycin pulmonary fibrosis model. C-188-9 also decreased the development of dermal fibrosis in the SC bleomycin model as assessed by decreased dermal thickness, a reduction of alpha-SMA accumulation, and decreased collagen deposition.


The role of STAT3 in EMT and myofibroblast differentiation was determined by using C188-9 in tissue culture experiments with alveolar epithelial cells (AEC; MLE-12 and primary AEC) and murine lung fibroblasts. In vitro studies showed that TGF-beta or IL-6 trans-signaling (IL-6/sIL-6R-alpha) were able to induce EMT in primary AEC and MLE-12 cell line, as well as differentiation of fibroblasts into myofibroblast. C188-9 prevented TGF-beta and IL-6/sIL-6R-alpha induced EMT of AEC assessed by Col1a, alpha-SMA, Twist, and Snail mRNA levels and reduced myofibroblast differentiation as assessed by Col1a and alpha-SMA mRNA levels. Thus, the findings demonstrated that STAT3 contributes to the development of tissue fibrosis in the skin and the lung and plays a role in the development of myofibroblasts in vitro. Furthermore, the data indicate that STAT3 is a useful a therapeutic target in the treatment of dermal and pulmonary fibrosis, as well as fibrosis of any other tissue.


III. Fibrosis

Embodiments of the invention concern methods of treatment and/or prevention of any kind of fibrosis. Fibrosis includes the formation of excess fibrous connective tissue in a reparative or reactive process, such as in an organ or tissue. The establishment of fibrosis can be a reactive, benign, or pathological state. In injury cases, the fibrosis may be referred to as scarring. In particular embodiments, the fibrosis is related to overproduction of the protein collagen. The fibrosis may be related to excessive accumulation of extracellular matrix (ECM) proteins. The fibrosis may be related to accumulation of myofibroblasts.


In physiological embodiments, the fibrosis encompasses deposition of connective tissue that can severely impact the structure and/or function of the affected organ and/or tissue. In certain aspects, fibrosis is the pathological state of excess deposition of fibrous tissue and/or the state of undesirable connective tissue deposition in healing that is not necessarily pathological.


Although fibrosis can occur in many tissues and organs within the body, such as the result of inflammation or damage, for example, in some cases the fibrosis is not pulmonary fibrosis or myelofibrosis.


The fibrotic tissue may be present in the heart, lung, skin, intestine, joint, and/or liver, and so forth, in some cases, although in certain aspects the fibrotic tissue is not in the lung.


Specific types of fibrosis that may be treated and/or prevented in the present invention include at least liver cirrhosis, endomyocardial fibrosis, myocardial infarction, atrial fibrosis, mediastinal fibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, Crohn's Disease, Keloid, scleroderma/systemic sclerosis, arthrofibrosis, adhesive capsulitis, fibrosis following exposure to certain drugs such as chemotherapy, fibrosis following exposure to environmental or other toxins or allergens, fibrosis occurring after an ischemia/reperfusion injury such as myocardial infarction or hypotension, fibrosis occurring after radiation, fibrosis following hepatitis induced by alcohol, toxins, drugs or infections, primary biliary cirrhosis, fibrosis following viral infections involving the heart, liver, or lung, and/or idiopathic retroperiotoneal fibrosis.


In embodiments of the invention, the fibrosis is skin fibrosis (that may also be referred to as dermal fibrosis), which may or may not be part of scleroderma. Diagnosis of skin fibrosis may include examination by a medical care provider and may include a biopsy. The skilled artisan recognizes that fibrosis may be evaluated, in certain settings (including research settings) in one or more of a variety of ways, including at least tissue staining (such as Hematoxylin and eosin (H& E) stain, Masson's trichrome stain (extracellular matrix (ECM, collagen)), or Sircol (for collagen); immunohistochemistry; western blot; and/or nucleic acid analysis, for example. Exemplary means of nucleic acid analysis includes expression analysis, such as using PCR (such as QRT-PCR); examples of genes for PCR analysis include at least (alpha smooth muscle actin) α-SMA and collagen, type 1, alpha 1 (Col1a).


In particular embodiments and in certain settings (such as research), one can analyze the fibrosis or suspicion of fibrosis by histology using H&E and Masson's trichrome and one can quantify certain measures by dermal thickness.


In particular embodiments and in certain settings (such as clinical), one can analyze the fibrosis or suspicion thereof. The analysis can include physical exam. For example, scleroderma patients have tight and fibrotic skin that starts in their fingers and creeps proximally (in some patients it totally encases them—diffuse systemic sclerosis/scleroderma; while in others it remains distal to the elbows—limited systemic sclerosis/scleroderma). The diagnosis can be confirmed by histology/skin biopsy.


In some embodiments, the skin fibrosis may be part of sclerodactyl), scleroderma, Amyloidosis, Dupytren's contracture, Peyronie's disease, Polymyositis, Carcinoid tumour, or Graft versus host disease, for example. Causes of skin fibrosis may be of any kind, such as unknown, Ainhum, Amyloidosis, Atrophoderma of Pasini and Pierini, Carcinoid tumours and carcinoid syndrome, Dupuytren's contracture, Eosinophilic fasciitis, Graft versus host disease, Hutchinson-Gilford progeria syndrome, Lichen myxedematosus, Mixed connective tissue disease, Morphoea, Nephrogenic systemic fibrosis, Peyronie's disease, Polymyositis, Porphyria cutanea tarda type 1 (sporadic), Scleredema adultorum, or systemic sclerosis.


An individual may be at risk for skin fibrosis because of family history or exposure to events or conditions that leaves them prone to fibrosis, such as radiation therapy or surgery.


Another type of fibrosis that may be treated, prevented, or have the risk reduced is cystic fibrosis (CF). CF includes scarring and cyst formation within the pancreas and can also affect the lungs, liver, and intestine. Symptoms include breathing difficulty, frequent lung infection, sinus infections, poor growth, infertility, accumulation of thick, sticky mucus, and salty skin. An individual may be at risk for CF if there is a family history, for example. Management of CF symptoms include intravenous, inhaled, and oral antibiotics; transplants; medications and/or mechanical techniques to dislodge and expectorate sputum, and so forth. Composition(s) of the invention may be utilized in conjuction with such management regimen(s).


Fibrosis of the liver can occur in reponse to chornic liver disease from viral infections, parasitic infections, and toxins (such as alcohol).


Renal fibrosis can occur from chronic kidney diseases, hypertension, diabetes, and autoimmune diseases, for example.


IV. Compositions

Embodiments of the invention encompass compositions that are useful for treating, preventing, and/or reducing the risk of fibrosis. Specific compositions are disclosed herein, but one of skill in the art recognizes that functional derivatives of such compositions are also encompassed by the invention. The term “derivative” as used herein is a compound that is formed from a similar compound or a compound that can be considered to arise from another compound, if one atom is replaced with another atom or group of atoms. Derivative can also refer to compounds that at least theoretically can be formed from the precursor compound.


In particular embodiments, compositions and functionally active derivatives as described herein are utilized in treatment and/or prevention of fibrosis. Specific but nonlimiting examples of different R groups for the compositions are provided in Tables 1, 2, and 3.


The term “functionally active derivative” or “functional derivative” is a derivative as previously defined that retains the function of the compound from which it is derived. In one embodiment of the invention, a derivative of N-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide, N-(3,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(4,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(5,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(6,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(7,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(8,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, 4-Bromo-N-(1,6′-dihydroxy-[2,2′]binaphthalenyl-4-yl)-benzene sulfonamide, 4-Bromo-N-[4-hydroxy-3-(1H-[1,2,4]triazol-3-yl sulfanyl)-naphthalen-1-yl]-benzene sulfonamide, 4-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-oxo-1-propen-1-yl]benzoic acid, 4{5-[(3-ethyl-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-2-furyl}benzoic acid, 4-[({3-[(carboxymethyl)thio]-4-hydroxy-1-naphthyl}amino)sulfonyl]benzoic acid, 3-({2-chloro-4-[1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)methyl]-6-ethoxyphenoxy}methyl)benzoic acid, methyl 4-({[3-(2-methyoxy-2-oxoethyl)-4,8-dimethyl-2-oxo-2H-chromen-7-yl]oxy}methyl)benzoate, or 4-chloro-3-{5-[(1,3-diethyl-4,6-dioxo-2-thioxotetrahydro-5(2H)-pyrimidinylidene)methyl]-2-furyl}benzoic acid retains Stat3 inhibitory activity. In another embodiment of the invention, a derivative of 4-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-oxo-1-propen-1-yl]benzoic acid, 4{5-[(3-ethyl-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-2-furyl}benzoic acid, 4-[({3-[(carboxymethyl)thio]-4-hydroxy-1-naphthyl}amino)sulfonyl]benzoic acid, 3-({2-chloro-4-[(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)methyl]-6-ethoxyphenoxy}methyl)benzoic acid, methyl 4-({[3-(2-methyoxy-2-oxoethyl)-4,8-dimethyl-2-oxo-2H-chromen-7-yl]oxy}methyl)benzoate, or 4-chloro-3-{5-[(1,3-diethyl-4,6-dioxo-2-thioxotetrahydro-5(2H)-pyrimidinylidene)methyl]-2-furyl}benzoic acid retains Stat3 inhibitory activity and, in specific embodiments, also retains non-inhibition of Stat1, although in some cases it may also inhibit Stat1.


In a specific embodiment of the invention, there is a method of treating and/or preventing fibrosis in an individual comprising delivering to the individual a compound selected from the group consisting of N-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide, N-(3,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(4,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(5,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(6,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(7,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(8,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, 4-Bromo-N-(1,6′-dihydroxy-[2,2′]binaphthalenyl-4-yl)-benzenesulfonamide, 4-Bromo-N-[4-hydroxy-3-(1H-[1,2,4]triazol-3-ylsulfanyl)-naphthalen-1-yl]-benzenesulfonamide; 4-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-oxo-1-propen-1-yl]benzoic acid 4{5-[(3-ethyl-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-2-furyl}benzoic acid; 4-[({3-[(carboxymethyl)thio]-4-hydroxy-1-naphthyl}amino)sulfonyl]benzoic acid; 3-({2-chloro-4-[(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)methyl]-6-ethoxyphenoxy}methyl)benzoic acid; methyl 4-({[3-(2-methyoxy-2-oxoethyl)-4,8-dimethyl-2-oxo-2H-chromen-7-yl]oxy}methyl)benzoate; 4-chloro-3-{5-[(1,3-diethyl-4,6-dioxo-2-thioxotetrahydro-5(2H)-pyrimidinylidene)methyl]-2-furyl}benzoic acid; and a mixture thereof.


In another embodiment, the composition comprises the general formula:




embedded image


wherein R1 and R2 may be the same or different and are selected from the group consisting of hydrogen, carbon, sulfur, nitrogen, oxygen, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne-based derivative, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives, heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid, and benzoic acid-based derivatives.


In another embodiment of the invention, the composition comprises the general formula:




embedded image


wherein R1, and R3 may be the same or different and are selected from the group consisting of hydrogen, carbon, nitrogen, sulfur, oxygen, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne-based derivative, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives, heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid, and benzoic acid-based derivatives, and R2 and R4 may be the same or different and are selected from the group consisting of hydrogen, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne-based derivative, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives, heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid, and benzoic acid-based derivatives.


In another embodiment of the invention, the composition comprises the general formula:




embedded image


wherein R1, R2, and R3 may be the same or different and are selected from the group consisting of hydrogen, carboxyl, alkanes, cyclic alkanes, alkane-based derivatives, alkenes, cyclic alkenes, alkene-based derivatives, alkynes, alkyne-based derivative, ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives, carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters and ester-based derivatives, amines, amino-based derivatives, amides, amide-based derivatives, monocyclic or polycyclic arene, heteroarenes, arene-based derivatives, heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid, and benzoic acid-based derivatives.


An exemplary and illustrative list of alkanes, cyclic alkanes, and alkane-based derivates are described herein. Non-limiting examples of ketones, ketone-based derivatives, aldehydes, aldehyde-based derivatives; carboxylic acids, carboxylic acid-based derivatives, ethers, ether-based derivatives, esters, ester-based derivatives, amines, amino-based derivatives, amides, and amide-based derivatives are listed herein. Exemplary monocyclic or polycyclic arene, heteroarenes, arene-based or heteroarene-based derivatives, phenols, phenol-based derivatives, benzoic acid and benzoic acid-based derivatives are described herein.












TABLE 1







Chemical names
Formulas









Methyl
CH3



Ethyl
C2H5



Vinyl (ethenyl)
C2H3



Ethynyl
C2H



Cyclopropyl
C3H5



Cyclobutyl
C4H7



Cyclopentyl
C5H9



Cyclohexyl
C6H11




















TABLE 2







Chemical names
Chemical formulas









Acetonyl
C3H5O



Methanal (formaldehyde)
CH2O



Paraldehyde
C6H12O3



Ethanoic acid
CH3COOH



Diethyl ether
C4H10O



Trimethylamine
C3H9N



Acetamide
C2H5NO



Ethanol
C2H5OH



Methanol
CH3OH




















TABLE 3







Chemical names
Chemical formulas









Benzol
C6H6



Phenol
C6H6O



Benzoic acid
C7H6O2



Aniline
C6H7N



Toluene
C7H8



Pyridazine
C4H4N2



Pyrimidine
C4H4N2



Pyrazine
C4H4N2



Biphenyl
C12H10










The compositions of the present invention and any functionally active derivatives thereof may be obtained by any suitable means. In specific embodiments, the derivatives of the invention are provided commercially, although in alternate embodiments the derivatives are synthesized. The chemical synthesis of the derivatives may employ well known techniques from readily available starting materials. Such synthetic transformations may include, but are not limited to protection, de-protection, oxidation, reduction, metal catalyzed C—C cross coupling, Heck coupling or Suzuki coupling steps (see for example, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structures, 5th Edition John Wiley and Sons by Michael B. Smith and Jerry March, incorporated here in full by reference).


V. Embodiments for Targeting Stat3

STAT proteins, of which there are seven (1, 2, 3, 4, 5A, 5B and 6), transmit peptide hormone signals from the cell surface to the nucleus. Detailed structural information of STAT proteins currently is limited to Stat1 and Stat3. Stat1 was the first STAT to be discovered (Fu et al., 1992) and is required for signaling by the Type I and II IFNs (Meraz et al., 1996; Wiederkehr-Adam et al., 2003; Durbin et al., 1996; Haan et al., 1999). Studies in Stat1-deficient mice (Meraz et al., 1996; Durbin et al., 1996; Ryan et al., 1998) support an essential role for Stat1 in innate immunity, notably against viral pathogens. In addition, Stat1 is a potent inhibitor of growth and promoter of apoptosis (Bromberg and Darnell, 2000). Also, because tumors from carcinogen-treated wild-type animals grow more rapidly when transplanted into the Stat1-deficient animals than they do in a wild-type host, Stat1 contributes to tumor surveillance (Kaplan et al., 1998).


Stat3 was originally termed acute-phase response factor (APRF) because it was first identified as a transcription factor that bound to IL-6-response elements within the enhancer-promoter region of various acute-phase protein genes (Akira, 1997). In addition to receptors for the IL-6 cytokine family, other signaling pathways are linked to Stat3 activation include receptors for other type I and type II cytokine receptors, receptor tyrosine kinases, G-protein-coupled receptors and Src kinases (Schindler and Darnell, 1995; Turkson et al., 1998). Targeted disruption of the mouse Stat3 gene leads to embryonic lethality at 6.5 to 7.5 days (Takeda et al., 1997) indicating that Stat3 is essential for early embryonic development possibly gastrulation or visceral endoderm function (Akira, 2000). Tissue-specific deletion of Stat3 using Cre-lox technology has revealed decreased mammary epithelial cell apoptosis resulting in delayed breast involution during weaning (Chapman et al., 1999). Recent findings indicate that switching of the predominant STAT protein activated by a given receptor can occur when a STAT downstream of that receptor is genetically deleted (Costa-Pereira et al., 2002; Qing and Stark, 2004). These findings suggest the possibility that the effect of Stat3 deletion in breast tissue may be mediated indirectly by increased activation of other STAT proteins, especially Stat5.


Stat1 and Stat3 Isoforms. Two isoforms of Stat1 and Stat3 have been identified—α(p91 and p92, respectively) and β (p84 and p83, respectively) (Schindler et al., 1992; Schaefer et al., 1995; Caldenhoven et al., 1996; Chakraborty et al., 1996)—that arise due to alternative mRNA splicing (FIG. 13). In contrast to Stat1β (712 aa), in which the C-terminal transactivation is simply deleted, the 55 amino acid residues of Stat3α are replaced in Stat3 β by 7 unique amino acid residues at its C-terminus. Unlike Stat1β, Stat3β is not simply a dominant-negative of Stat3α (Maritano et al., 2004) and regulates gene targets in a manner distinct from Stat3β (Maritano et al., 2004; Yoo et al., 2002). Stat3α has been demonstrated to contribute to transformation in cell models and many human cancers including breast cancer. Stat3α was shown to be constitutively activated in fibroblasts transformed by oncoproteins such as v-Src (Yu et al., 1995; Garcia and Jove, 1998) and to be essential for v-Src-mediated transformation (Turkson et al., 1998; Costa-Pereira et al., 2002). In contrast to Stat3α, Stat3β antagonized v-Src transformation mediated through Stat3α (Turkson et al., 1998). Overexpression of a constitutively active form of Stat3α in immortalized rat or mouse fibroblasts induced their transformation and conferred the ability to form tumors in nude mice (Bromberg et al., 1999). Stat3 has been shown to be constitutively activated in a variety of hematological and solid tumors including breast cancer (Dong et al., 2003; Redell and Tweardy, 2003) as a result of either autocrine growth factor production or dysregulation of protein tyrosine kinases. In virtually all cases, the isoform demonstrating increased activity is Stat3α.


Targeting Stat3α While Sparing Stat1.


Given its multiple contributory roles to oncogenesis, Stat3 has recently gained attention as a potential target for cancer therapy (Bromberg, 2002; Turkson, 2004). While several methods of Stat3 inhibition have been employed successfully and have established proof-of-principle that targeting Stat3 is potentially beneficial in a variety of tumor systems including breast cancer in which Stat3 is constitutively activated (Epling-Burnette et al., 2001; Yoshikawa et al., 2001; Li and Shaw, 2002; Catlett-Falcone et al., 1999; Mora et al., 2002; Grandis et al., 2000; Leong et al., 2003; Jing et al., 2003; Jing et al., 2004; Turkson et al., 2001; Ren et al., 2003; Shao et al., 2003; Turkson et al., 2004; Uddin et al., 2005); all have potential limitations for translation to clinical use for cancer therapy related to issues regarding delivery, specificity or toxicity.


Specific strategies that target Stat3 by identifying inhibitors of Stat3 recruitment and/or dimerization have been pursued by several groups (Turkson et al., 2001; Ren et al., 2003; Shao et al., 2003; Uddin et al., 2005; Song et al., 2005; Schust et al., 2006). As outlined below, this strategy has the potential to achieve specificity based on the observation that the preferred pY peptide motif of each STAT protein is distinct. When coupled to a small molecule approach, this strategy has the potential to overcome issues of delivery and toxicity.


Targeting Stat3α while Sparing Stat3β.


Some of the distinct biochemical features of Stat3β vs. Stat3α, notably constitutive activation and a 10-to-20 fold increased DNA binding affinity, have been attributed to the absence of the C-terminal transactivation domain (TAD) resulting in increased Stat3β dimer stability (Park et al., 1996; Park et al., 2000). Increased dimer stability likely results from higher binding affinity of the SH2 domain to pY peptide motifs when in the context of Stat3β compared to Stat3α because of reduced steric hindrance conferred by removal of the TAD. These differential biochemical features between Stat3α and Stat3β are exploited to develop a chemical compound that selectively targets Stat3 α, in some embodiments. This selectivity enhances the anti-tumor effect of such compounds, in certain cases, because they would spare Stat3β, which functions to antagonize the oncogenic functions of Stat3α.


In certain embodiments of the invention, specific therapies targeting Stat3 signaling are useful for treatment of fibrosis.


VI. Combination Therapy

It is an aspect of this invention that a composition as disclosed herein is used in combination with another agent or therapy method, such as another fibrosis treatment. In embodiments wherein the fibrosis is skin fibrosis, the additional therapy may be phototherapy, such as UVA1 phototherapy, ciprofloxacin, bosentan, methotrexate, E4 peptide (synthetic version of a peptide building block obtained from the natural protein endostatin; Yamaguchi et al., 2012), P144, a compound that is a known inhibitor of TGF-β (U.S. Pat. No. 7,582,609) and so forth.


The composition(s) (which may or may not be a Stat3 inhibitor) may precede or follow the other agent treatment by intervals ranging from minutes to weeks, for example. In embodiments where the other agent and the composition of the invention are applied separately to an individual with fibrosis, such as upon delivery to an individual suspected of having fibrosis, known to have fibrosis, or at risk for having fibrosis, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and composition of the invention would still be able to exert an advantageously combined effect on the individual.


For example, in such instances, it is contemplated that one may contact the cell, tissue or organism with one, two, three, four or more modalities substantially simultaneously (i.e., within less than about a minute) with the composition of the invention. In other aspects, one or more agents may be administered within about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, to about 48 hours or more prior to and/or after administering the composition of the invention. In certain other embodiments, an agent may be administered within of from about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20, to about 21 days prior to and/or after administering the composition of the invention, for example. In some situations, it may be desirable to extend the time period for treatment significantly, such as where several weeks (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7 or about 8 weeks or more) lapse between the respective administrations. In some situations, it may be desirable to extend the time period for treatment significantly, such as where several months (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7 or about 8 weeks or more) lapse between the respective administrations.


Various combinations may be employed, the composition of the invention is “A” and the secondary agent, which can be any other cancer therapeutic agent, is “B”:



















A/B/A
B/A/B
B/B/A
A/A/B
A/B/B
B/A/A


A/B/B/B
B/A/B/B
B/B/B/A
B/B/A/B
A/A/B/B
A/B/A/B


A/B/B/A
B/B/A/A
B/A/B/A
B/A/A/B
A/A/A/B
B/A/A/A


A/B/A/A
A/A/B/A









Administration of the therapeutic compositions of the present invention to a patient will follow general protocols for the administration of drugs, taking into account the toxicity. It is expected that the treatment cycles would be repeated as necessary.


In some cases, the combination therapy is an antimicrobial agents. Any antimicrobial agent(s) may be used, such as one or more of methyl propyl and chlorocresol, for example.


VII. Pharmaceutical Compositions

Pharmaceutical compositions of the present invention comprise an effective amount of a composition as disclosed herein dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases “pharmaceutical” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one Stat3 inhibitor of the invention, and in some cases an additional active ingredient, will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.


As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.


The composition(s) may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it needs to be sterile for such routes of administration such as injection. The composition(s) can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in lipid compositions (e.g., liposomes), as an aerosol, or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).


The actual dosage amount of a composition of the present invention administered to an individual can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, and the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.


In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of a composition. In other embodiments, the active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from about 0.1 mg/kg/body weight, 0.5 mg/kg/body weight, 1 mg/kg/body weight, about 5 mg/kg/body weight, about 10 mg/kg/body weight, about 20 mg/kg/body weight, about 30 mg/kg/body weight, about 40 mg/kg/body weight, about 50 mg/kg/body weight, about 75 mg/kg/body weight, about 100 mg/kg/body weight, about 200 mg/kg/body weight, about 350 mg/kg/body weight, about 500 mg/kg/body weight, about 750 mg/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 10 mg/kg/body weight to about 100 mg/kg/body weight, etc., can be administered, based on the numbers described above. In certain embodiments of the invention, various dosing mechanisms are contemplated. For example, the composition may be given one or more times a day, one or more times a week, or one or more times a month, and so forth.


In any case, the composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including, but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.


The composition may be formulated in a free base, neutral or salt form. Pharmaceutically acceptable salts include the salts formed with the free carboxyl groups derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.


In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising, but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example, liquid polyol or lipids; by the use of surfactants such as, for example, hydroxypropylcellulose; or combinations thereof such methods. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.


Sterile injectable solutions are prepared by incorporating the instant invention in the required amount of the appropriate solvent with various amounts of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.


The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.


In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.


VIII. Kits of the Invention

Any of the compositions described herein may be comprised in a kit, and they are housed in a suitable container. The kits will thus comprise, in suitable container means, one or more compositions and, in some cases, an additional agent of the present invention. In some cases, there are one or more agents other than the composition of the disclosure that are included in the kit, such as one or more other agents for the treatment of fibrosis and/or one or more agents for the treatment of an underlying condition associated with fibrosis. In particular embodiments, there is an apparatus or any kind of means for the diagnosing of fibrosis.


The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the composition, additional agent, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.


Compositions may also be formulated into a syringeable composition. In which case, the container means may itself be a syringe, pipette, and/or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, and/or even applied to and/or mixed with the other components of the kit. However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.


EXAMPLES

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.


Example 1
Exemplary Materials and Methods

Virtual Ligand Screening.


The inventors isolated the three-dimensional structure of the Stat3 SH2 domain from the core fragment structure of phosphorylated Stat3 homodimers bound to DNA (Becker et al., 1998) deposited in the RCSB Protein Data Bank (PDB) databank (PDB code 1BG1) and converted it to be an Internal Coordinate Mechanics (ICM)-compatible system by adding hydrogen atoms, modifying unusual amino acids, making charge adjustments and performing additional cleanup steps. In addition, the inventors retrieved the coordinates of the Stat1 SH2 domain from the PDB databank (PDB code 1BF5) for use in computational selectivity analysis (Chen et al., 1998). Commercial chemical databases (Chembridge, Asinex, ChemDiv, Enamine, Keyorganics and Life Chemicals) were chosen as sources of compounds for screening in silico. Selection was of the amide hydrogen of E638 within the site that binds the +3 residue (Q, C or T) within the pY-peptide ligand (Shao et al., 2006) as the central point of the binding pocket, which consisted of a cube with dimensions 16.0×16.9×13.7 angstrom. In addition to the +3 binding site, this cube contained the pY residue binding site consisting mainly of R609 and K591 (Shao et al., 2006) and a hydrophobic binding site consisting of LoopβC-βD and LoopαB-αC. Sequence alignment and overlay of the Stat3 and Stat1 structures revealed substantial differences in sequence of these loops; lack of their superimposition indicated that this region might serve as a selectivity filter (Cohen et al., 2005). A flexible docking calculation (Totrov and Abagyan 1997) was performed in order to determine the global minimum energy score and thereby predict the optimum conformation of the compound within the pocket. A compound was selected for purchase and biochemical testing based on fulfilling the criteria of interaction analysis (CIA): 1) global minimum energy score 30, 2) formation of a salt-bridge and/or H-bond network within the pY-residue binding site and 3) formation of a H-bond with or blocking access to the amide hydrogen of E638. Most, but not all, compounds also interacted with the hydrophobic binding site.


Stat3 SH2/pY-peptide binding assay. Stat3 binding assays were performed at 25° C. with a BIAcore 3000 biosensor using 20 mM Tris buffer pH 8 containing 2 mM mercaptoethanol and 5% DMSO as the running buffer (Kim et al., 2005). Phosphorylated and control non-phosphorylated biotinylated EGFR derived dodecapeptides based on the sequence surrounding Y1068 (Shao et al., 2004) were immobilized on a streptavidin coated sensor chip (BIAcore inc., Picataway N.J.). The binding of Stat3 was conducted in 20 mM Tris buffer pH 8 containing 2 mM β-mercaptoethanol at a flow rate of 10 uL/min for 1-2 minute. Aliquots of Stat3 at 500 nM were premixed with compound to achieve a final concentration of 1-1,000 uM and incubated at 4° C. prior to being injected onto the sensor chip. The chip was regenerated by injecting 10 uL of 100 mM glycine at pH 1.5 after each sample injection. A control (Stat3 with DMSO but without compound) was run at the beginning and the end of each cycle (40 sample injections) to ensure that the integrity of the sensor chip was maintained throughout the cycle run. The average of the two controls was normalized to 100% and used to evaluate the effect of each compound on Stat3 binding. Responses were normalized by dividing the value at 2 min by the response obtained in the absence of compounds at 2 min and multiplying by 100. IC50 values were determined by plotting % maximum response as a function of log concentration of compound and fitting the experimental points to a competitive binding model using a four parameter logistic equation: R=Rhigh−(Rhigh−Rlow)/(1+conc/A1)^A2, where R=percent response at inhibitor concentration, Rhigh=percent response with no compound, Rlow=percent response at highest compound concentration, A2=fitting parameter (slope) and A1=IC50 (BIAevaluation Software version 4.1).


Immunoblot assay. The human hepatocellular carcinoma cell line (HepG2) was grown in 6-well plates under standard conditions. Cells were pretreated with compounds (0, 1, 3, 10, 30, 100 and 300 uM) for 1 hour then stimulated under optimal conditions with either interferon gamma (IFN-γ; 30 ng/ml for 30 min) to activate Stat1 or interleukin-6 (IL-6; 30 ng/ml for 30 min) to activate Stat3 (30-31). Cultures were then harvested and proteins extracted using high-salt buffer, as described (Shao et al., 2006). Briefly, extracts were mixed with 2× sodium dodecyl sulfate (SDS) sample buffer (125 mmol/L Tris-HCL pH 6.8; 4% SDS; 20% glycerol; 10% 2-mercaptoethanol) at a 1:1 ratio and heated for 5 minutes at 100° C. Proteins (20 μg) were separated by 7.5% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Waltham, Mass.) and immunoblotted. Prestained molecular weight markers (Biorad, Hercules, Calif.) were included in each gel. Membranes were probed serially with antibody against Stat1 pY701 or Stat3 pY705 followed by antibody against Stat1 or Stat3 (Transduction labs, Lexington, Ky.) then antibody against β-actin (Abcam, Cambridge, Mass.). Membranes were stripped between antibody probing using Restore™ Western Blot Stripping Buffer (Thermo Fisher Scientific Inc., Waltham, Mass.) per the manufacturer's instructions. Horseradish peroxidase-conjugated goat-anti-mouse IgG was used as the secondary antibody (Invitrogen Carlsbad, Calif.) and the membranes were developed with enhanced chemiluminescence (ECL) detection system (Amersham Life Sciences Inc.; Arlington Heights, Ill.).


Similarity screen. Three compounds identified in the initial virtual ligand screening (VLS)—Cpd3, Cpd30 and Cpd188—inhibited Stat3 SH2/pY-peptide binding and IL-6-mediated Stat3 phosphorylation and were chosen as reference molecules for similarity screening. A fingerprint similarity query for each reference compound was submitted to Molcart/ICM (Max Distance, 0.4). Similarity between each reference molecule and each database molecule was computed and the similarity results were ranked in decreasing order of ICM similarity score (Eckert and Bajorath 2007). The databases searched included ChemBridge, LifeChemicals, Enamine, ChemDiv, Asinex, AcbBlocks, KeyOrganics and PubChem for a total of 2.47 million compounds. All compounds identified were docked into the binding pocket of Stat3 SH2 domain in silico. Compounds that fulfilled CIA criteria were purchased and tested as described for compounds identified in the primary screen.


Electrophoretic Mobility Shift Assay (EMSA): EMSA was performed using the hSIE radiolabeled duplex oligonucleotide as a probe as described (Tweardy et al., 1995). Briefly, high salt extracts were prepared from HepG2 cells incubated without or with IL-6 (30 ng/ml) for 30 minutes. Protein concentration was determined by Bradford Assay and 20 ug of extract was incubated with compound (300 uM) for 60 minutes at 37° C. Bound and unbound hSIE probe was separated by polyacrylamide gel electrophoresis (4.5%). Gels were dried and autoradiographed.


Molecular modeling. All 3-D configurations of the Stat3 SH2 domain complexed with compounds were determined by global energy optimization that involves multiple steps: 1) location of organic molecules were adjusted as a whole in 2 Å amplitude by pseudo-Brownian random translations and rotations around the molecular center of gravity, 2) the internal variables of organic molecules were randomly changed. 3) coupled groups within the Stat3 SH2 domain side-chain torsion angles were sampled with biased probability shaking while the remaining variables of the protein were fixed, 4) local energy minimizations were performed using the Empirical Conformation Energy Program for Peptides type-3 (ECEPP3) in a vacuum (Nemethy et al., 1992) with distance-dependent dielectric constant ε=4r, surface-based solvent energy and entropic contributions from the protein side chains evaluated added and 5) conformations of the complex, which were determined by Metropolis criteria, were selected for the next conformation-scanning circle. The initial 3-dimensional configuration of the Stat1 SH2 domain in a complex with each compound was predicted and generated by superimposing, within the computational model, the 3-dimensional features of the Stat1 SH2 onto the 3-dimensional configuration of the Stat3 SH2 domain in a complex with each compound. The final computational model of Stat1 SH2 in a complex with each compound was determined by local minimization using Internal Coordinate Force Field (ICFF)-based molecular mechanics (Totrov and Abagyan 1997). The inventors computed the van der Waals energy of the complex of Stat1 or 3-SH2 bound with each compound using Lennard-Jones potential with ECEPP/3 force field (Nemethy et al., 1992).


Confocal and high-throughput fluorescence microscopy. Confocal and highthroughput fluorescence microscopy (HTFM) of MEF/GFP-Stat3α cells were performed as described (Huang et al., 2007). Briefly, for confocal fluorescence microscopy, cells were grown in 6-well plates containing a cover slip. For HTFM, cells were seeded into 96-well CC3 plates at a density of 5,000 cells/well using an automated plating system. Cells were cultured under standard conditions until 85-90% confluent. Cells were pretreated with compound for 1 hour at 37° C. then stimulated with IL-6 (200 ng/ml) and IL-6sR (250 ng/ml) for 30 minutes. Cells were fixed with 4% formaldehyde in PEM Buffer (80 mM Potassium PIPES, pH 6.8, 5 mM EGTA pH 7.0, 2 mM MgCl2) for 30 minutes at 4° C., quenched in 1 mg/ml of NaBH4 (Sigma) in PEM buffer and counterstained for 1 min in 4,6-diamidino-2-phenylindole (DAPI; Sigma; 1 mg/ml) in PEM buffer. Cover slips were examined by confocal fluorescent microscopy. Plates were analyzed by automated HTFM using the Cell Lab IC Image Cytometer (IC100) platform and CytoshopVersion 2.1 analysis software (Beckman Coulter). Nuclear translocation is quantified by using the fraction localized in the nucleus (FLIN) measurement (Sharp et al., 2006).


Breast cancer cell line apoptosis assay. Human breast carcinoma cell lines MDA-MB-468, MDA-MB-231, MBA-MD-435 and MCF7 were kindly provided by Dr. Powel H. Brown (Breast Cancer Center, Baylor College of Medicine). Breast cancer cell line, MDA-MB-453 was kindly provided by Dr. Shou Jiang (Breast Cancer Center, Baylor College of Medicine). All cell lines were grown in DMEM medium supplemented with 10% fetal bovine serum (FBS), 25,000 units penicillin G, 25,000 ug streptomycin, and 131.4 mg L-Glutamine and cultured in the incubator under the condition of 95% air, 5% CO2 at 37° C. (Garcia et al., 2001). Cells were seeded at 2,500 cells/cm2 into 12-well plates. At 80% confluency, cells were washed with PBS and supplemented with fresh medium containing compound or the topoisomerase I-inhibitor, camptothecin, at 0, 0.1, 03, 1, 3, 10, 30, 100, 300 μM. At 24 hours, treatment was terminated by removing the medium from each well. Cells were lysed with cell lysis buffer (600 for 30 minutes at 25° C.). Cell lysate (200 μl) was centrifuged at 200×g for 10 minutes and 20 of each supernatant was assayed for nucleosomes using the Cell Death Detection ELISA (Roche Applied Science) as described by the manufacturer. The percent maximum nucleosome level was calculated by dividing the nucleosome level by the maximum nucleosome level achieved in the assay and multiplying by 100. This value was plotted as a function of the log compound concentration and the best-fitting curve generated using 4-Parameter Logistic Model/Dose Response/XLfit 4.2, IDBS software.


Example 2
Identification by VLS of Compounds that Blocked Stat3 Binding to its Phosphopeptide Ligand and Inhibited IL-6-Mediated Phosphorylation of Stat3

The VLS protocol was used to evaluate a total of 920,000 drug-like compounds. Of these, 142 compounds fulfilled CIA criteria. These compounds were purchased and tested for their ability to block Stat3 binding to its phosphopeptide ligand in a surface plasmon resonance (SPR)-based binding assay and to inhibit IL-6-mediated phosphorylation of Stat3. SPR competition experiments showed that of the 142 compounds tested, 3 compounds—Cpd3, Cpd30 and Cpd188—were able to directly compete with pY-peptide for binding to Stat3 with IC50 values of 447, 30, and 20 μM, respectively (FIGS. 1 and 3; Table 4).









TABLE 4







IC50 values (μM) of 6 active compounds













Assay
Cpd3
Cpd30
Cpd188
Cpd3-2
Cpd3-7
Cpd30-12
















SPR

4471

30
20
256
137
114


pStat3
 91
18
73
144
63
60


HTM
131
77
39
150
20
>300






1Data presented are the mean or mean ± SD;



ND = not determined.






In addition, each compound inhibited IL-6-mediated phosphorylation of Stat3 with IC50 values of 91, 18 and 73 μM respectively (FIG. 2; Table 4).


Similarity screening with Cpd3, Cpd30 and Cpd188 identified 4,302 additional compounds. VLS screening was performed with each of these compounds, which identified 41 compounds that fulfilled CIA criteria; these were purchased and tested. SPR competition experiments showed that of these 41 compounds, 3 compounds—Cpd3-2, Cpd3-7 and Cpd30-12—were able to directly compete with pY-peptide for binding to Stat3 with IC50 values of 256, 137 and 114 μM, respectively (FIGS. 1 and 3; Table 4). In addition, each compound inhibited IL-6-mediated phosphorylation of Stat3 with IC50 values of 144, 63 and 60 μM, respectively (FIG. 2; Table 4).


Example 3
Compound-Mediated Inhibition of Ligand-Stimulated Phosphorylation of Stat3 is Specific for Stat3 Vs. Stat1

While Stat3 contributes to oncogenesis, in part, through inhibition of apoptosis, Stat1 is anti-oncogenic; it mediates the apoptotic effects of interferons and contributes to tumor surveillance (Kaplan et al., 1998; Ramana et al., 2000). Consequently, compounds that target Stat3 while sparing Stat1, leaving its anti-oncogenic functions unopposed, may result in a synergistic anti-tumor effect. To assess the selectivity of the compounds for Stat3 vs. Stat1, HepG2 cells were incubated with Cpd3, Cpd30, Cpd188, Cpd3-2, Cpd3-7, and Cpd30-12 (300 μM) for 1 hour at 37° C. before IFN-γ stimulation (FIG. 4). Only treatment with Cpd30-12 blocked Stat1 phosphorylation while each of the other five compounds—Cpd3, Cpd30, Cpd188, Cpd3-2 and Cpd3-7—did not. Thus, five of the six exemplary compounds identified were selective and inhibited ligand-stimulated phosphorylation of Stat3 but not Stat1.


Example 4
Sequence Analysis and Molecular Modeling of the Interaction of Each Compound with the Stat3 Vs. Stat1 Sh2 Domain

To understand at the molecular level the basis for the selectivity of Cpds 3, 30, 188, 3-2 and 3-7 and the absence of selectivity in the case of Cpd 30-12, the amino acid sequence and available structures of the Stat1 and Stat3 SH2 domain were compared and also it was examined how each compound interacted with both. Sequence alignment revealed identity in the residues within Stat3 and Stat1 corresponding to the binding site for the pYresidue and the +3 residue (FIG. 5A). In addition, overlay of the Stat3 and Stat1 SH2 structures revealed that the loops that contained these binding sites were superimposed (FIG. 5B). In contrast, sequence alignment revealed substantial differences in the sequence of the regions of the SH2 domain corresponding to the loops forming the hydrophobic binding site (FIG. 5A). In addition, review of the overlay of Stat3 and Stat1 SH2 domains revealed that, in contrast to the close apposition of the two loops of Stat3 that form the hydrophobic binding site, the corresponding two loops of Stat1 are not closely apposed to form a pocket (FIG. 5B).


Review of computational models of Cpd3, Cpd30, Cpd188, Cpd3-2 and Cpd3-7 in a complex with the Stat3 SH2 domain revealed that each has significant interactions with the Stat3 SH2 domain binding pocket at all three binding sites, the pY-residue binding site, the +3 residue binding site and the hydrophobic binding site (FIGS. 6A, B, C, D, and E). In contrast, Cpd30-12 interacts with the pY-residue binding site and blocks access to the +3 residue-binding site but does not interact with or block access to the hydrophobic binding site (FIG. 6F). In addition, van der Waals energies of the 5 selective compounds were much more favorable for their interaction with the loops of Stat3 forming the hydrophobic binding site than with corresponding loops of Stat1 (FIG. 5C). Thus, computer modeling indicated that activity of compounds against Stat3 derives from their ability to interact with the binding sites for the pY and the +3 residues within the binding pocket, while selectivity for Stat3 vs. Stat1 derives from the ability of compounds to interact with the hydrophobic binding site within the Stat3 SH2 binding pocket, which served as a selectivity filter.


Example 5
Inhibition of Nuclear Translocation of Phosphorylated Stat3 by CPD3, CPD30, CPD188, CPD3-2 and CPD3-7 Assessed by HTFM

Following its phosphorylation on Y705, Stat3 undergoes a change in conformation from head-to-head dimerization mediated through its N-terminal oligomerization domain to tail-to-tail dimerization mediated by reciprocal SH2/pY705-peptide ligand interactions. This conformational change is followed by nuclear accumulation. Compounds that targeted SH2/pY-peptide ligand interactions of Stat3 would be expected to inhibit nuclear accumulation of Stat3. To determine if this was the case with the compounds herein, a nuclear translocation assay (FIG. 7) was employed using murine embryonic fibroblast (MEF) cells that are deficient in endogenous Stat3 but constitutively express GFP-tagged Stat3α at endogenous levels, MEF/GFP-Stat3 α (Huang et al., 2007). Preincubation of MEF/GFP-Stat3 cc cells with Cpd3, Cpd30, Cpd188, Cpd3-2 and Cpd3-7, but not Cpd30-12, blocked ligand-mediated nuclear translocation of GFP-Stat3 cc with IC50 values of 131, 77, 39, 150 and 20 μM (FIG. 7 and Table 4).


Example 6
Destabilization of Stat3-DNA Complexes by CPD3 and CPD3-7

Once in the nucleus, Stat3 dimers bind to specific DNA elements to activate and, in some instances, repress gene transcription. Tyrosine-phosphorylated dodecapeptides based on motifs within receptors that recruit Stat3 have previously been shown to destabilize Stat3 (Chakraborty et al., 1999; Shao et al., 2003). Compounds that bind to the phosphopeptide-binding site of Stat3 might be expected to do the same. To determine if this was the case for any of the identified compounds, extracts of IL-6-stimulated HepG2 cells were incubated in binding reactions containing radiolabeled hSIE (FIG. 8) and each of the five selective compounds (300 μM). Incubation with Cpd3 or Cpd3-7 reduced the amount of hSIE shifted by half or greater. The other compounds did not have a detectable effect on the Stat3:hSIE band intensity. Thus, 2 of the 5 selective compounds destabilized Stat3:hSIE complexes.


Example 7
Exemplary Approach for Stat3 Inhibitors for Cancer Stem Cells

In the field of Stat3 probe development the inventors have focused on small molecule Stat3 probes (Xu et al., 2009), and several features of the small molecule program are useful, including: 1) a clearly defined mode of action of these probes: they target the Stat3 Src-homology (SH) 2 domain that is involved in 2 steps in the Stat3 activation pathway; 2) their specificity of action; and 3) the potential for using lead probes identified so far to identify probes with 2-to-3 logs greater activity based on recent and exemplary SAR analysis and medicinal chemistry considerations outlined below.


In specific embodiments, compound affinity is improved upon gaining a log greater affinity upon moving from 1St generation to 2nd generation probes using 3-D pharmacophore analysis. In addition, selectivity is improved through modeling embodiments, in particular through identification of a distinct hydrophobic binding domain in the phosphopeptide binding pocket of Stat3 SH2 vs. the Stat1 SH2 (Xu et al., 2009).


Identification of 1st Generation Stat3 Chemical Probes.


To develop chemical probes that selectively target Stat3, the inventors virtually screened 920,000 small drug-like compounds by docking each into the peptide-binding pocket of the Stat3 SH2 domain, which consists of three sites—the pY-residue binding site, the +3 residue-binding site and a hydrophobic binding site, which served as a selectivity filter (Xu et al., 2009). Three compounds (Cpd3, Cpd30 and Cpd188) satisfied criteria of interaction analysis, competitively inhibited recombinant Stat3 binding to its immobilized pY-peptide ligand and inhibited IL-6-mediated tyrosine phosphorylation of Stat3. These compounds were used in a similarity screen of 2.47 million compounds, which identified 3 more compounds (Cpd3-2, Cpd3-7 and Cpd30-12) with similar activities. Examinations of the 6 active compounds for the ability to inhibit IFN-γ-mediated Stat1 phosphorylation revealed that all but Cpd30-12 were selective for Stat3. Molecular modeling of the SH2 domains of Stat3 and Stat1 bound to compound revealed that compound interaction with the hydrophobic binding site was the basis for selectivity. All 5 selective compounds inhibited nuclear-tocytoplasmic translocation of Stat3, while 3 of 5 compounds (Cpd3, Cpd30 and Cpd188) induced apoptosis preferentially of exemplary breast cancer cell lines with constitutive Stat3 activation.


Identification of 2nd Generation Stat3 Chemical Probes.


The similarity screening described above did not yield any hits using Cpd188, the most active of the 3 lead compounds, as the query compound. Consequently, the inventors repeated 2-D similarity screening using the scaffold of Cpd188 as the query structure and the Life Chemicals library, which yielded 207 hits. 3-D pharmacophore analysis was performed on these 207 compounds using Ligand Scout and the top 39 scoring compounds were purchased and tested for inhibition of Stat3 binding to its phosphopeptide ligand by SPR. All but six of these 39 compounds have measurable SPR IC50s, with 19 having IC50 values equal to or less than the parent compound and 2 (Cpd188-9 and Cpd188-15) having IC50 values one log lower. Examination of these 19 compounds has revealed a statistically significant correlation between 3-D pharmacophore scores and SPR IC50s and as well as 3-D pharmacophore score and IC50s for inhibition of ligand-mediated cytoplasmic-to-nuclear translocation. In addition, both Cpd188-9 and Cpd188-15 exhibited a log greater activity in inducing human leukemic cell line apoptosis than the parent Cpd188 (FIG. 15). In addition, Cpd188-38 exhibited a 2 logs greater activity than parent Cpd188 in inhibiting cytoplasmic-to-nuclear translocation in HTFM assay, while Cpd188-15 exhibited a 1 log greater activity than parent Cpd188 in decreasing MSFE (Table 5). Furthermore, several of the second-generation 188-like compounds represent a substantial improvement over Cpd188 from a medicinal chemistry, metabolism and bioavailability standpoint. In particular, Cpd188-9 lacked both carboxyl groups, which in particular cases improves cell permeability and/or the thioether group, which is subject to oxidation. R2=0.2 P=0.013 (μM)









TABLE 5







Summary of Certain 2nd Generation 188-like Compounds




embedded image







*mean ± SD


**Xu et al PLoS ONE


***SUM159PT and HS578T cells plated (6 wells per test) without or with compound at 1, 10 or 100 μM, incubated 3 d; spheres counted on day 3.






Structure-Activity Relationship (SAR) Analysis of 2nd Generation Stat3 Probes.


All of the 39 second generation compounds described above, plus Cpd188 itself, are derivatives of N-naphth-1-yl benzenesulfamide. Upon careful analysis of their structure-activity relationships (SAR), the inventors found that most of these Cpd188-like compounds (38 out of 40: the rest of 2 are weak and will be described below in EXP ID) can be divided into three structural groups in a general trend of decreased activity, as shown in FIG. 16. Five compounds in Group III are actually the parents of compounds in Groups I and II. Addition of a variety of groups (the —R group highlighted in red in the general structure of Group I in FIG. 16), such as a triazole-3-yl-mercapto (188-15) or a chloro (188-10) group, to the 3-position of the naphthylamine ring led to the Group I compounds, which are the most potent series of Stat3 probes. In a specific embodiment, this is the most important contributor to the inhibitory activity: a total of eight 3-substituents are found in Group I compounds, which invariably enhance the activity by several orders of magnitude.


Most Stat3 probes in Group II contain a 5-membered ring that combines the 3-R and 4-OR2 groups, such as a furan (188-11). However, the compounds in this group are, in average, ˜5× less active than the Group I compounds, which indicates that in certain aspects the H atom of the 4-hydroxy group (highlighted in blue in the general structure of Group I in FIG. 16) is important, e.g., involved in a favorable H-bond with the protein. Lacking the ability to form the H-bond attributes to the weaker activities of Group II probes, in particular cases. These considerations underlie a medicinal chemistry approach outlined below.


Example 8
Medicinal Chemistry for Synthesis of 3RD Generation 188-Like Sulfamide STAT3 Probes

The crystal structure of Stat3 shows that the SH2 domain has a large, widely dispersed and generally shallow binding area with several valleys and hills that recognize the pY-peptide ligand (FIG. 18). Structure-based molecular modeling (docking) was useful in identifying the contribution of the hydrophobic binding surface of the Stat3 SH2 domain as a selectivity filter (Xu et al., 2009). However, different docking programs gave distinct binding poses for the same probe over the binding surface with similar predicted binding affinities. The inventors therefore in particular embodiments, based on initial SAR results outlined above, use traditional medicinal chemistry to further carry out an exemplary comprehensive structure activity relationship study, to optimize the activity as well as the selectivity of this novel class of sulfamide probes of Stat3. Compound 188-15 serves as a scaffold for making the new generation compounds, as shown schematically (FIG. 16).


In addition, chemistry for making these compounds is straightforward with a good yield, involving the reaction of a sulfonyl chloride with an aniline/amine, which can be either obtained commercially or synthesized readily.


For the proposed modifications described below, one can consult FIG. 17. EXP IA. Modification 1. Since almost all of the 2nd generation probes contain a phenylsulfonyl group, the first step towards activity optimization focuses on synthesizing a series of compounds that have a larger (e.g., bicyclic or tricyclic) or an alkyl sulfonyl group. The general synthetic route is shown as follows:


There are about 4,300 commercially available sulfonyl chlorides, among which 25, such as those shown above, are selected to make probes. Aniline 2, which is the amine component of compound 188-10 (FIG. 16), one the most active probes, is readily made in a simple two step reaction from nitro compound 1. One can first make 25 (for example) compounds and test their activities in an in vitro rapid throughput SPR and in vivo HTFM assays. Based on the outcomes of structure-activity relationship study, more compounds can be designed and synthesized and tested in an iterative manner until optimization of this modification.


EXP IB. Modification 2. Next, one can modify the 3-substituent of the naphthylamine ring, based on either the structure of compound 188-15, for example. Prior SAR studies demonstrated this substituent is useful to the activity of this class of probes, in certain embodiments. However, a total of 8 groups at this position with a huge difference in size, from a single atom Cl to a large, bicyclic benzothiazole-2-ylmercapto group, showed similar activities. This feature indicates that in certain embodiments modifications at this position should be more focused on other properties, such as electrostatic interactions with the protein, as exemplified below. In addition, many of these groups are thioethers, which may be subjected to oxidation/degradation in vivo and lead to an unfavorable pharmacokinetic profile, in particular aspects. The central —S— atom is changed to a more metabolically stable isosteres, such as —CH2—, —NH—, and —O—, in certain cases. In certain aspects one can synthesize the following compounds to optimize the 3-substituent:


The synthesis is also started from 1, in certain cases. Regio-selective halogenation and formylation at the 3-position gives rise to two compounds, i.e., bromo- or iodo-compound 3 and aldehyde 4, which are versatile, common starting compounds for introducing a wide range of substituents at this position (e.g., those listed above).


Moreover, the crystal structure of Stat3 SH2 domain also provides strong evidence that more compounds with different electrostatic properties are useful for characterization. The electrostatic molecular surface of the protein shows two distinct features, as shown in FIG. 18. The first one is the negatively charged Glu638 surface stands out in the center. Next, of particular interest is a positively charged area, composed of Arg609 and Lys591 located in the edge of the domain, which is actually the pY (phosphorylated tyrosine) binding site of the receptor. The inventors also found that introducing a negatively charged group targeting the pY binding site leads to particularly active probes, in certain embodiments. For example, the docking study of the 3-phosphomethyl compound 5 (R═CH2PO32−) showed all of the phosphonate groups of the 20 docking poses are tightly clustered together and located in the pY binding site, indicating strong electrostatic and H-bond interactions with the residues Arg609 and Lys591 (FIG. 18).


EXP IC. Modifications 3 and 4. Collectively, Modifications 3 and 4 test the effects of changing the substituents at the 4, 5, and 6-positions. The —OH at 4-position may be superior to —OR, in certain aspects. One can test whether the H atom in —OH is responsible for a better activity by synthesizing compounds 6 (acylated or alkylated 5), as schematically shown below. In addition, dehydroxy compounds 7 may also be made, starting from 3-bromonaphthyl-1-amine.


Regarding the general synthetic methods for modifying positions 5 and 6, one can first synthesize about a dozen of these compounds in this category and if very active compounds emerge, one can make more compounds to optimize the activity for these two positions.


EXP ID. Modification 5. The only two compounds not included in the SAR analysis (due to a different 4-substituent) are shown here, as well as their inhibitory activities against Stat3:


Despite the weak activity, masking the polar H of the sulfamide for the second compound is favorable, in certain aspects, which provides an easy route to making more potent probes. One can therefore use the following method to make a series of N-acyl or N-alkyl sulfamides 5:


Example 9
Identification of Stat3-Selective Chemical Probes from Sulfamide Compounds Synthesized in Example 11

Each novel sulfamide compound is tested for the ability to inhibit Stat3 binding to its phosphopeptide ligand by SPR and the ability to block IL-6-stimulated cytoplasmic-to-nuclear translocation in the HTFM assay. Probes with activity in these assays equivalent to or greater than the most active 2nd generation compounds are tested for inhibition of IL-6-stimulated Stat3 phosphorylation and lack of ability to inhibit IFN-γ-stimulated Stat1 phosphorylation as outlined below.


EXP IIA. Stat3/pY-peptide SPR binding inhibition assay. Stat3 pY-peptide binding assays is performed at 25° C. using a BIAcore 3000 biosensor as described (Xu et al., 2009). Briefly, phosphorylated and control nonphosphorylated biotinylated EGFR derived dodecapeptides based on the sequence surrounding Y1068 are immobilized on a streptavidin coated sensor chip (BIAcore Inc., Piscataway N.J.). The binding of Stat3 is performed in 20 mM Tris buffer pH 8 containing 2 mM β-mercaptoethanol at a flow rate of 10 uL/min for 1-2 minute. Aliquots of Stat3 at 500 nM are premixed with compound to achieve a final concentration of 1-1,000 uM and incubated at 4° C. prior to being injected onto the sensor chip. The chip is regenerated by injecting 10 uL of 100 mM glycine at pH 1.5 after each sample injection. A control (Stat3 with DMSO but without compound) is run at the beginning and the end of each cycle (40 sample injections) to ensure that the integrity of the sensor chip is maintained throughout the cycle run. The average of the two controls is normalized to 100% and used to evaluate the effect of each compound on Stat3 binding. Responses are normalized by dividing the value at 2 min by the response obtained in the absence of compounds at 2 min and multiplying by 100. IC50 values are determined by plotting % maximum response as a function of log concentration of compound and fitting the experimental points to a competitive binding model using a four parameter logistic equation: R=Rhigh−(Rhigh−Rlow)/(1+conc/A1)A2, where R=percent response at inhibitor concentration, Rhigh=percent response with no compound, Rlow=percent response at highest compound concentration, A2=fitting parameter (slope) and A1=IC50 (BIAevaluation Software version 4.1).


EXP IIB. High throughput fluorescence microscopy (HTFM), cytoplasm-to-nucleus translocation inhibition assays. HTFM of MEF/GFP-Stat3α cells is performed to assess the ability of probes to inhibit GFP-Stat3 cytoplasmic-to-nuclear translocation, as described (Xu et al., 2009), using the robotic system available as part of the John S. Dunn Gulf Coast Consortium for Chemical Genomics at the University of Texas-Houston School of Medicine. Briefly, cells are seeded into 96-well CC3 plates at a density of 5,000 cells/well and cultured under standard conditions until 85-90% confluent. Cells are pre-treated with compound for 1 hour at 37° C. then stimulated with IL-6 (100 ng/ml) and IL-6sR (150 ng/ml) for 30 minutes. Cells are fixed with 4% formaldehyde in PEM Buffer (80 mM Potassium PIPES, pH 6.8, 5 mM EGTA pH 7.0, 2 mM MgCl2) for 30 minutes at 4° C., quenched in 1 mg/ml of NaBH4 (Sigma) in PEM buffer and counterstained for 1 min in 4,6-diamidino-2-phenylindole (DAPI; Sigma; 1 mg/ml) in PEM buffer. Plates are analyzed by automated HTFM using the Cell Lab IC Image Cytometer (IC100) platform and CytoshopVersion 2.1 analysis software (Beckman Coulter).


Nuclear translocation is quantified by using the fraction localized in the nucleus (FLIN) measurement. FLIN values are normalized by subtracting the FLIN for unstimulated cells then dividing this difference by the maximum difference (delta, A) in FLIN (FLIN in cells stimulated with IL-6/sIL-6R in the absence of compound minus FLIN of unstimulated cells). This ratio is multiplied by 100 to obtain the percentage of maximum difference in FLIN and is plotted as a function of the log compound concentration. The best-fitting curve and IC50 value are determined using 4-Parameter LogisticModel/Dose Response/XLfit 4.2, IDBS software.


EXP IIC. Ligand-mediated pStat3 and pStat1 inhibition assays. Newly synthesized Stat3 probes with activity equivalent to or greater than parent compound 188 in the SPR and HTFM assays will be tested for the ability to selectively inhibit ligand-mediated phosphorylation of Stat3 as described (Xu et al., 2009). Briefly, human hepatocellular carcinoma cells (HepG2) are grown in 6-well plates and pretreated with compounds (0, 0.1, 0.3, 1, 3, 10, 30, 100 μM) for 1 hour then stimulated under optimal conditions with either interleukin-6 (IL-6; 30 ng/ml for 30 min) to activate Stat3 or interferon gamma (IFN-γ; 30 ng/ml for 30 min) to activate Stat1. Cells are harvested and proteins extracted using high-salt buffer, mixed with 2× sodium dodecyl sulfate (SDS) sample buffer (125 mmol/L Tris-HCL pH 6.8; 4% SDS; 20% glycerol; 10%2-mercaptoethanol) at a 1:1 ratio then heated for 5 minutes at 100° C. Proteins (20 μg) are separated by 7.5% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Waltham, Mass.) and immunoblotted. Membranes are probed serially with antibody against Stat1 pY701 or Stat3 pY705 followed by antibody against Stat1 or Stat3 (Transduction labs, Lexington, Ky.) then antibody against β-actin (Abcam, Cambridge, Mass.). Membranes are stripped between antibody probings using Restore™ Western Blot Stripping Buffer (Thermo Fisher Scientific Inc., Waltham, Mass.) per the manufacturer's instructions. Horseradish peroxidase-conjugated goat-anti-mouse IgG is used as the secondary antibody (Invitrogen Carlsbad, Calif.) and the membranes are developed with enhanced chemiluminescence (ECL) detection system (Amersham Life Sciences Inc.; Arlington Heights, Ill.). Band intensities are quantified by densitometry. The value of each pStat3 band is divided by its corresponding total Stat3 band intensity; the results are normalized to the DMSO-treated control value. This value was plotted as a function of the log compound concentration. The best-fitting curve is determined using 4-Parameter Logistic Model/Dose Response/XLfit 4.2, IDBS software and was used to calculate the IC50 value.


EXP IID. Molecular modeling of probe-Stat3 interactions. The results of modeling of the binding of the first generation probe to the Stat3 vs. Stat1 SH2 domains suggested that the basis for experimental selectivity of probes for Stat3 vs. Stat1 rested on the ability of the probes to have greater interaction with the hydrophobic binding site within the pY-peptide binding pocket of Stat3 compared to Stat1. Thus, the hydrophobic binding site served as a selectivity filter. To test if this remains the case for newly synthesized 3rd generation probes, one can use 2 complementary docking programs GLIDE (Schrödinger) and ICM (MolSoft) to determine the lowest energy docking configuration of each probe within the pY-peptide binding domain of Stat3 and Stat1 SH2 domain. One can review the computational models of each probe in a complex with the Stat3 vs. Stat1 SH2 domain and, in particular, compare the van der Waals energies and determine if they are equivalent for their interaction with the Stat3 SH2 domain vs. the Stat1 SH2 domain. It was this calculation that determined the selectivity of 1st generation probes for Stat3 vs. Stat1. In particular, van der Waals energy calculations implicated residues that form the hydrophobic binding site (W623, Q635, V637, Y640 and Y657) as critical for this selectivity.


In specific embodiments of the invention, there is identification of probes with one log or greater activity than 2nd generation probes in SPR, HTFM and pStat3 assays. Furthermore, in certain aspects some of the most active 3rd generation probes that emerge from this analysis are selective for Stat3 vs. Stat1 based on their greater interaction with the hydrophobic binding site within the Stat3 vs. Stat1 SH2 pY-peptide binding pocket.


Example 10
Exemplary Compositions of the Disclosure

Exemplary composition(s) of the disclosure are provided in Tables 6-11 below.













TABLE 6





IDNUMBER
Structure
Formula structure
MW
LogP



















F1566-0306


embedded image


C22H17NO3S2
407.5137
5.846





F1566-0318


embedded image


C23H19NO3S2
421.5408
6.144





F1566-0330


embedded image


C22H16ClNO3S2
441.9587
6.438





F1566-0342


embedded image


C22H16BrNO3S2
486.4097
6.644





F1566-0366


embedded image


C24H21NO3S2
435.5679
6.477





F1566-0414


embedded image


C24H21NO3S2
435.5679
6.477





F1566-0438


embedded image


C24H21NO3S2
435.5679
6.619





F1566-0450


embedded image


C23H19NO4S2
437.5402
5.802





F1566-0462


embedded image


C24H21NO4S2
451.5673
6.143





F1566-0486


embedded image


C26H25NO3S2
463.6221
7.345





F1566-0510


embedded image


C26H19NO3S2
457.5742
7.105





F1566-0546


embedded image


C22H16N2O5S2
452.5112
5.818





F1566-0558


embedded image


C23H18N2O5S2
466.5383
6.114





F1566-0618


embedded image


C20H15NO3S3
413.5395
5.359





F1566-1606


embedded image


C25H18N2O3S2
458.5618
6.046





F1566-1818


embedded image


C18H17NO3S2
359.4691
4.705





F1566-1832


embedded image


C19H19NO3S2
373.4962
5.147





F1566-1846


embedded image


C20H21NO3S2
387.5233
5.589





F1566-1860


embedded image


C17H15NO3S2
345.442
4.192





F5749-0371


embedded image


C22H16N2O5S2
452.5112
5.781





F5749-0372


embedded image


C22H23NO3S2
413.5615
6.171





F5749-0373


embedded image


C25H23NO4S2
465.5944
6.468





F5749-0374


embedded image


C23H18ClNO4S2
471.9852
6.429





F5749-0375


embedded image


C24H21NO3S2
435.5679
6.438





F5749-0376


embedded image


C24H19NO5S2
465.5507
5.787





F5749-0377


embedded image


C24H20N2O4S2
464.566
5.137





F5749-0378


embedded image


C24H21NO5S2
467.5667
5.54474





F5749-0379


embedded image


C24H19NO5S2
465.5507
5.441





F5749-0380


embedded image


C21H16N2O3S2
408.5013
4.613





F5749-0381


embedded image


C18H18N2O3S2
374.4838
3.74





F5749-0382


embedded image


C24H21NO3S2
435.5679
6.477





F5749-0383


embedded image


C22H16N2O5S2
452.5112
5.779





F5749-0384


embedded image


C23H19NO3S2
421.5408
5.98





F5749-0385


embedded image


C20H14ClNO3S3
447.9845
6.649





F5749-0386


embedded image


C22H15F2NO3S2
443.4946
6.187





F5749-0387


embedded image


C21H19N3O3S2
425.5319
4.956





F5749-0388


embedded image


C21H18N2O4S2
426.5166
4.99





F5749-0389


embedded image


C23H22N2O5S2
470.5702
3.633





F5749-0390


embedded image


C23H18FNO4S2
455.5306
5.99





F5749-0391


embedded image


C24H21NO4S2
451.5673
6.135





F5749-0392


embedded image


C26H20N2O3S2
472.5889
6.305





F5749-0393


embedded image


C22H19NO3S3
441.5936
6.497





F5749-0394


embedded image


C21H17NO3S3
427.5665
6.022





F5749-0395


embedded image


C24H19NO3S2
433.5519
6.204





F5749-0396


embedded image


C22H16FNO3S2
425.5041
5.997





F5749-0397


embedded image


C23H19NO4S2
437.5402
5.839





F5749-0398


embedded image


C22H16FNO3S2
425.5041
6.036





F5749-0399


embedded image


C22H15ClFNO3S2
459.9492
6.626





F5749-0400


embedded image


C23H16F3NO4S2
491.5115
7.24476





F5749-0401


embedded image


C23H18ClNO3S2
455.9858
6.771





F5749-0402


embedded image


C24H19NO4S2
449.5513
5.736





F5749-0403


embedded image


C24H19NO4S2
449.5513
5.699





F5749-0404


embedded image


C23H18ClNO3S2
455.9858
6.732





F5749-0405


embedded image


C23H19NO4S2
437.5402
5.8





F5749-0406


embedded image


C24H21NO4S2
451.5673
6.141





F5749-0407


embedded image


C22H15F2NO3S2
443.4946
6.148





F5749-0408


embedded image


C19H19NO3S2
373.4962
5.339





F5749-0409


embedded image


C23H16F3NO3S2
475.5121
6.81776





F5749-0410


embedded image


C23H16F3NO3S2
475.5121
6.78076





F5749-0411


embedded image


C22H16ClNO3S2
441.9587
6.475





F5749-0412


embedded image


C23H17Cl2NO3S2
490.4308
7.398





F5749-0413


embedded image


C22H15F2NO3S2
443.4946
6.187





F5749-0414


embedded image


C25H23NO3S2
449.595
7.061





F5749-0415


embedded image


C26H23NO3S2
461.6061
6.933





F5749-0416


embedded image


C26H20N2O5S2
504.5877
4.973





F5749-0417


embedded image


C27H22N2O5S2
518.6148
5.415





F5749-0418


embedded image


C23H20N2O4S3
484.6189
5.149





F5749-0419


embedded image


C20H15N3O5S2
441.4877
2.891





F5749-0420


embedded image


C25H20N2O4S2
476.5772
5.042





F5749-0421


embedded image


C24H18N2O4S2
462.5501
4.954





F5749-0422


embedded image


C22H19N3O5S2
469.5418
2.955





F5749-0423


embedded image


C26H22N2O4S2
490.6042
5.277





F5749-0424


embedded image


C23H18FNO3S2
439.5312
6.133





F5749-0425


embedded image


C23H18FNO3S2
439.5312
6.17





F5749-0426


embedded image


C25H23NO4S2
465.5944
6.206





F5749-0427


embedded image


C28H25N3O3S2
515.6578
6.125





F5749-0428


embedded image


C19H15N3O3S2
397.4777
3.986





F5749-0429


embedded image


C27H23N3O3S2
501.6307
5.991





F5749-0430


embedded image


C29H23NO5S2
529.6384
7.16174





F5749-0431


embedded image


C28H20ClNO4S2
534.0569
8.046





F5749-0432


embedded image


C29H23NO4S2
513.639
7.754





F5749-0433


embedded image


C23H15ClF3NO3S2
509.9571
7.40776





F5749-0434


embedded image


C28H21NO4S2
499.6119
7.456





F5749-0435


embedded image


C22H16BrNO3S2
486.4097
6.642





F5749-0436


embedded image


C22H16BrNO3S2
486.4097
6.681





F5749-0437


embedded image


C22H15BrFNO3S2
504.4002
6.832





F5749-0438


embedded image


C23H15BrF3NO3S2
554.4081
7.61376





F5749-0439


embedded image


C22H16ClNO3S2
441.9587
6.436





F5749-0440


embedded image


C22H17NO5S3
471.5765
5.046





F5749-0441


embedded image


C23H16F3NO4S2
491.5115
7.24276




















TABLE 7





IDNUMBER
Structure
Formula structure
MW
LogP







F0808-0081


embedded image


C28H23NO4S
469.5638
7.101





F0808-0084


embedded image


C28H23NO5S
485.5632
6.767





F0808-0085


embedded image


C26H18BrNO4S
520.4057
7.268





F0808-0086


embedded image


C28H23NO4S
469.5638
7.243





F0808-0089


embedded image


C30H21NO4S
491.5702
7.729





F0808-0091


embedded image


C26H18FNO4S
459.5001
6.623





F0808-0092


embedded image


C28H23NO4S
469.5638
7.101





F0808-0094


embedded image


C26H18ClNO4S
475.9547
7.062





F1269-0222


embedded image


C24H17NO4S2
447.5354
5.983





F1269-2003


embedded image


C27H20N2O6S
500.5343
6.738





F1566-1138


embedded image


C29H20N2O4S
492.5578
6.67





F5749-0001


embedded image


C21H17NO4S
379.4379
4.816





F5749-0002


embedded image


C26H18N2O6S
486.5072
6.405





F5749-0003


embedded image


C26H25NO4S
447.5575
6.795





F5749-0004


embedded image


C29H25NO5S
499.5903
7.092





F5749-0005


embedded image


C27H20ClNO5S
505.9812
7.053





F5749-0006


embedded image


C28H23NO4S
469.5638
7.062





F5749-0007


embedded image


C28H21NO6S
499.5467
6.411





F5749-0008


embedded image


C28H22N2O5S
498.5619
5.761





F5749-0009


embedded image


C28H23NO6S
501.5626
6.16874





F5749-0010


embedded image


C28H21NO6S
499.5467
6.065





F5749-0011


embedded image


C25H18N2O4S
442.4972
5.237





F5749-0012


embedded image


C22H19NO4S
393.465
5.329





F5749-0013


embedded image


C28H23NO6S
501.5626
6.417





F5749-0014


embedded image


C22H20N2O4S
408.4797
4.364





F5749-0015


embedded image


C28H23NO4S
469.5638
7.101





F5749-0016


embedded image


C26H18N2O6S
486.5072
6.403





F5749-0017


embedded image


C23H21NO4S
407.4921
5.771





F5749-0018


embedded image


C27H21NO4S
455.5367
6.604





F5749-0019


embedded image


C24H23NO4S
421.5192
6.213





F5749-0020


embedded image


C24H16ClNO4S2
481.9804
7.273





F5749-0021


embedded image


C26H17F2NO4S
477.4905
6.811





F5749-0022


embedded image


C25H21N3O4S
459.5278
5.58





F5749-0023


embedded image


C25H20N2O5S
460.5126
5.614





F5749-0024


embedded image


C27H24N2O6S
504.5661
4.257





F5749-0025


embedded image


C27H20FNO5S
489.5266
6.614





F5749-0026


embedded image


C28H23NO5S
485.5632
6.759





F5749-0027


embedded image


C30H22N2O4S
506.5848
6.929





F5749-0028


embedded image


C26H21NO4S2
475.5896
7.121





F5749-0029


embedded image


C25H19NO4S2
461.5625
6.646





F5749-0030


embedded image


C28H21NO4S
467.5479
6.828





F5749-0031


embedded image


C26H18FNO4S
459.5001
6.621





F5749-0032


embedded image


C27H21NO5S
471.5361
6.463





F5749-0033


embedded image


C26H18FNO4S
459.5001
6.66





F5749-0034


embedded image


C26H17ClFNO4S
493.9451
7.25





F5749-0035


embedded image


C27H18F3NO5S
525.5074
7.86876





F5749-0036


embedded image


C27H20ClNO4S
489.9818
7.395





F5749-0037


embedded image


C28H21NO5S
483.5473
6.36





F5749-0038


embedded image


C28H21NO5S
483.5473
6.323





F5749-0039


embedded image


C27H20ClNO4S
489.9818
7.356





F5749-0040


embedded image


C27H21NO5S
471.5361
6.424





F5749-0041


embedded image


C28H23NO5S
485.5632
6.765





F5749-0042


embedded image


C26H17F2NO4S
477.4905
6.772





F5749-0043


embedded image


C23H21NO4S
407.4921
5.963





F5749-0044


embedded image


C27H18F3NO4S
509.508
7.44176





.F5749-0045


embedded image


C27H18F3NO4S
509.508
7.40476





F5749-0046


embedded image


C26H18ClNO4S
475.9547
7.099





F5749-0047


embedded image


C27H19Cl2NO4S
524.4268
8.022





F5749-0048


embedded image


C26H17F2NO4S
477.4905
6.811





F5749-0049


embedded image


C29H25NO4S
483.5909
7.685





F5749-0050


embedded image


C30H25NO4S
495.6021
7.557





F5749-0051


embedded image


C30H22N2O6S
538.5836
5.597





F5749-0052


embedded image


C31H24N2O6S
552.6107
6.039





F5749-0053


embedded image


C27H22N2O5S2
518.6148
5.773





F5749-0054


embedded image


C24H17N3O6S
475.4836
3.515





F5749-0055


embedded image


C29H22N2O5S
510.5731
5.666





F5749-0056


embedded image


C28H20N2O5S
496.546
5.578





F5749-0057


embedded image


C26H21N3O6S
503.5378
3.579





F5749-0058


embedded image


C30H24N2O5S
524.6002
5.901





F5749-0059


embedded image


C27H20FNO4S
473.5272
6.757





F5749-0060


embedded image


C27H20FNO4S
473.5272
6.794





F5749-0061


embedded image


C29H25NO5S
499.5903
6.83





F5749-0062


embedded image


C32H27N3O4S
549.6537
6.749





F5749-0063


embedded image


C23H17N3O4S
431.4736
4.61





F5749-0064


embedded image


C31H25N3O4S
535.6266
6.615





F5749-0065


embedded image


C33H25NO6S
563.6343
7.78574





F5749-0066


embedded image


C32H22ClNO5S
568.0528
8.67





F5749-0067


embedded image


C33H25NO5S
547.6349
8.378





F5749-0068


embedded image


C27H17ClF3NO4S
543.953
8.03176





F5749-0069


embedded image


C32H23NO5S
533.6078
8.08





F5749-0070


embedded image


C26H18BrNO4S
520.4057
7.266





F5749-0071


embedded image


C26H18BrNO4S
520.4057
7.305





F5749-0072


embedded image


C26H17BrFNO4S
538.3961
7.456





F5749-0073


embedded image


C27H17BrF3NO4S
588.404
8.23776





F5749-0074


embedded image


C26H18ClNO4S
475.9547
7.06





F5749-0075


embedded image


C26H19NO6S2
505.5724
5.67





F5749-0076


embedded image


C27H18F3NO5S
525.5074
7.86676




















TABLE 8





IDNUMBER
Structure
Formula structure
MW
Log P







F1566-0329


embedded image


C26H20N2O352
472.5889
6.344





F1566-0341


embedded image


C25H17ClN2O3S2
493.0068
6.638





F1566-0353


embedded image


C25H17BrN2O3S2
537.4578
6.844





F1566-0377


embedded image


C27H22N2O3S2
486.616
6.677





F1566-0425


embedded image


C27H22N2O3S2
486.616
6.677





F1566-0449


embedded image


C27H22N2O3S2
486.616
6.819





F1566-0473


embedded image


C27H22N2O4S2
502.6154
6.343





F1566-0497


embedded image


C29H26N2O3S2
514.6702
7.545





F1566-0521


embedded image


C29H20N2O3S2
508.6224
7.305





F1566-0557


embedded image


C25H17N3O5S2
503.5593
6.018





F1566-0569


embedded image


C26H19N3O5S2
517.5864
6.314





F1566-0617


embedded image


C27H22N2O5S2
518.6148
5.993





F1566-0629


embedded image


C23H16N2O3S3
464.5876
5.559





F1566-1608


embedded image


C28H19N3O3S2
509.6099
6.246





F1566-1821


embedded image


C21H18N2O3S2
410.5172
4.905





F1566-1835


embedded image


C22H20N2O3S2
424.5443
5.347





F1566-1849


embedded image


C23H22N2O3S2
438.5714
5.789





F1566-1863


embedded image


C20H16N2O3S2
396.4901
4.392





F5749-0077


embedded image


C25H17N3O5S2
503.5593
5.981





F5749-0078


embedded image


C25H24N2O3S2
464.6096
6.371





F5749-0079


embedded image


C28H24N2O4S2
516.6425
6.668





F5749-0080


embedded image


C26H19ClN2O4S2
523.0333
6.629





F5749-0081


embedded image


C27H22N2O3S2
486.616
6.638





F5749-0082


embedded image


C27H20N2O5S2
516.5989
5.987





F5749-0083


embedded image


C27H21N3O4S2
515.6141
5.337





F5749-0084


embedded image


C27H22N2O5S2
518.6148
5.74474





F5749-0085


embedded image


C27H20N2O5S2
516.5989
5.641





F5749-0086


embedded image


C24H17N3O3S2
459.5494
4.813





F5749-0087


embedded image


C21H19N3O3S2
425.5319
3.94





F5749-0088


embedded image


C27H22N2O3S2
486.616
6.677





F5749-0089


embedded image


C25H17N3O5S2
503.5593
5.979





F5749-0090


embedded image


C26H20N2O3S2
472.5889
6.18





F5749-0091


embedded image


C23H15ClN2O3S3
499.0326
6.849





F5749-0092


embedded image


C25H16F2N2O3S2
494.5427
6.387





F5749-0093


embedded image


C24H20N4O3S2
476.58
5.156





F5749-0094


embedded image


C24H19N3O4S2
477.5647
5.19





F5749-0095


embedded image


C26H23N3O5S2
521.6183
3.833





F5749-0096


embedded image


C26H19FN2O4S2
506.5787
6.19





F5749-0097


embedded image


C27H22N2O4S2
502.6154
6.335





F5749-0098


embedded image


C29H21N3O3S2
523.637
6.505





F5749-0099


embedded image


C25H20N2O3S3
492.6418
6.697





F5749-0100


embedded image


C24H18N2O3S3
478.6147
6.222





F5749-0101


embedded image


C27H20N2O3S2
484.6001
6.404





F5749-0102


embedded image


C25H17FN2O3S2
476.5522
6.197





F5749-0103


embedded image


C26H20N2O4S2
488.5883
6.039





F5749-0104


embedded image


C25H17FN2O3S2
476.5522
6.236





F5749-0105


embedded image


C25H16ClFN2O3S2
510.9973
6.826





F5749-0106


embedded image


C26H17F3N2O4S2
542.5596
7.44476





F5749-0107


embedded image


C26H19ClN2O3S2
507.0339
6.971





F5749-0108


embedded image


C27H20N2O4S2
500.5995
5.936





F5749-0109


embedded image


C27H20N2O4S2
500.5995
5.899





F5749-0110


embedded image


C26H19ClN2O3S2
507.0339
6.932





F5749-0111


embedded image


C26H20N2O4S2
488.5883
6





F5749-0112


embedded image


C27H22N2O4S2
502.6154
6.341





F5749-0113


embedded image


C25H16F2N2O3S2
494.5427
6.348





F5749-0114


embedded image


C22H20N2O3S2
424.5443
5.539





F5749-0115


embedded image


C26H17F3N2O3S2
526.5602
7.01776





F5749-0116


embedded image


C26H17F3N2O3S2
526.5602
6.98076





F5749-0117


embedded image


C25H17ClN2O3S2
493.0068
6.675





F5749-0118


embedded image


C26H18Cl2N2O3S2
541.479
7.598





F5749-0119


embedded image


C25H16F2N2O3S2
494.5427
6.387





F5749-0120


embedded image


C28H24N2O3S2
500.6431
7.261





F5749-0121


embedded image


C29H24N2O3S2
512.6542
7.133





F5749-0122


embedded image


C29H21N3O5S2
555.6358
5.173





F5749 0123


embedded image


C30H23N3O5S2
569.6629
5.615





F5749-0124


embedded image


C26H21N3O4S3
535.667
5.349





F5749-0125


embedded image


C23H16N4O5S2
492.5358
3.091





F5749-0126


embedded image


C28H21N3O4S2
527.6253
5.242





F5749-0127


embedded image


C27H19N3O4S2
513.5982
5.154





F5749-0128


embedded image


C25H20N4O5S2
520.59
3.155





F5749-0129


embedded image


C29H23N3O4S2
541.6524
5.477





F5749-0130


embedded image


C26H19FN2O3S2
490.5793
6.333





F5749-0131


embedded image


C26H19FN2O3S2
490.5793
6.37





F5749-0132


embedded image


C28H24N2O4S2
516.6425
6.406





F5749-0133


embedded image


C31H26N4O3S2
566.7059
6.325





F5749-0134


embedded image


C22H16N4O3S2
448.5258
4.186





F5749-0135


embedded image


C30H24N4O3S2
552.6788
6.191





F5749-0136


embedded image


C32H24N2O5S2
580.6865
7.36174





F5749-0137


embedded image


C31H21ClN2O4S2
585.105
8.246





F5749-0138


embedded image


C32H24N2O4S2
564.6871
7.954





F5749-0139


embedded image


C26H16ClF3N2O3S2
561.0052
7.60776





F5749-0140


embedded image


C31H22N2O4S2
550.66
7.656





F5749-0141


embedded image


C25H17BrN2O3S2
537.4578
6.842





F5749-0142


embedded image


C25H17BrN2O3S2
537.4578
6.881





F5749-0143


embedded image


C25H16BrFN2O3S2
555.4483
7.032





F5749-0144


embedded image


C26H16BrF3N2O3S2
605.4562
7.81376





F5749-0145


embedded image


C25H17ClN2O3S2
493.0068
6.636





F5749-0146


embedded image


C25H18N2O5S3
522.6246
5.246





F5749-0147


embedded image


C26H17F3N2O4S2
542.5596
7.44276




















TABLE 9





IDNUMBER
Structure
Formula structure
MW
LogP



















F1565-0253


embedded image


C18H14N4O3S2
398.4653
3.698





F1566-0328


embedded image


C19H16N4O3S2
412.4924
3.996





F1566-0340


embedded image


C18H13ClN4O3S2
432.9103
4.29





F1566-0520


embedded image


C22H16N4O3S2
448.5258
4.957





F1566-0556


embedded image


C18H13N5O5S2
443.4628
3.67





F1566-0568


embedded image


C19H15N5O5S2
457.4899
3.966





F1566-0616


embedded image


C20H18N4O5S2
458.5183
3.645





F1566-0628


embedded image


C16H12N4O3S3
404.491
3.211





F5749-0148


embedded image


C13H12N4O3S2
336.3936
2.044





F5749-0149


embedded image


C18H13N5O5S2
443.4628
3.633





F5749-0150


embedded image


C18H20N4O3S2
404.5131
4.023





F5749-0151


embedded image


C21H20N4O4S2
456.546
4.32





F5749-0152


embedded image


C19H15ClN4O4S2
462.9368
4.281





F5749-0153


embedded image


C20H18N4O3S2
426.5195
4.29





F5749-0154


embedded image


C20H16N4O5S2
456.5023
3.639





F5749-0155


embedded image


C20H17N5O4S2
455.5176
2.989





F5749-0156


embedded image


C20H18N4O5S2
458.5183
3.39674





F5749-0157


embedded image


C20H16N4O5S2
456.5023
3.293





F5749-0158


embedded image


C17H13N5O3S2
399.4529
2.465





F5749-0159


embedded image


C14H14N4O3S2
350.4207
2.557





F5749-0160


embedded image


C14H15N5O3S2
365.4354
1.592





F5749-0161


embedded image


C20H18N4O3S2
426.5195
4.329





F5749-0162


embedded image


C18H13N5O5S2
443.4628
3.631





F5749-0163


embedded image


C15H16N4O3S2
364.4478
2.999





F5749-0164


embedded image


C19H16N4O3S2
412.4924
3.832





F5749-0165


embedded image


C16H18N4O3S2
378.4749
3.441





F5749-0166


embedded image


C16H11ClN4O3S3
438.9361
4.501





F5749-0167


embedded image


C18H12F2N4O3S2
434.4461
4.039





F5749-0168


embedded image


C17H16N6O3S2
416.4835
2.808





F5749-0169


embedded image


C17H15N5O4S2
417.4682
2.842





F5749-0170


embedded image


C19H19N5O5S2
461.5218
1.485





F5749-0171


embedded image


C19H15FN4O4S2
446.4822
3.842





F5749-0172


embedded image


C20H18N4O4S2
442.5189
3.987





F5749-0173


embedded image


C22H17N5O3S2
463.5405
4.157





F5749-0174


embedded image


C21H15N5O3S2
449.5134
3.898





F5749-0175


embedded image


C18H16N4O3S3
432.5452
4.349





F5749-0176


embedded image


C17H14N4O3S3
418.5181
3.874





F5749-0177


embedded image


C20H16N4O3S2
424.5035
4.056





F5749-0178


embedded image


C18H13FN4O3S2
416.4557
3.849





F5749-0179


embedded image


C19H16N4O4S2
428.4918
3.691





F5749-0180


embedded image


C18H13FN4O3S2
416.4557
3.888





F5749-0181


embedded image


C18H12ClFN4O3S2
450.9007
4.478





F5749-0182


embedded image


C19H13F3N4O4S2
482.4631
5.09676





F5749-0183


embedded image


C19H15ClN4O3S2
446.9374
4.623





F5749-0184


embedded image


C20H16N4O4S2
440.5029
3.588





F5749-0185


embedded image


C20H16N4O4S2
440.5029
3.551





F5749-0186


embedded image


C19H15ClN4O3S2
446.9374
4.584





F5749-0187


embedded image


C19H16N4O4S2
428.4918
3.652





F5749-0188


embedded image


C20H18N4O4S2
442.5189
3.993





F5749-0189


embedded image


C18H12F2N4O3S2
434.4461
4





F5749-0190


embedded image


C15H16N4O3S2
364.4478
3.191





F5749-0191


embedded image


C19H13F3N4O3S2
466.4637
4.66976





F5749-0192


embedded image


C19H13F3N4O3S2
466.4637
4.63276





F5749-0193


embedded image


C18H13ClN4O3S2
432.9103
4.327





F5749-0194


embedded image


C19H14Cl2N4O3S2
481.3824
5.25





F5749-0195


embedded image


C18H12F2N4O3S2
434.4461
4.039





F5749-0196


embedded image


C21H20N4O3S2
440.5466
4.913





F5749-0197


embedded image


C22H20N4O3S2
452.5577
4.785





F5749-0198


embedded image


C22H17N5O5S2
495.5393
2.825





F5749-0199


embedded image


C23H19N5O5S2
509.5664
3.267





F5749-0200


embedded image


C19H17N5O4S3
475.5704
3.001





F5749-0201


embedded image


C16H12N6O5S2
432.4392
0.743





F5749-0202


embedded image


C21H17N5O4S2
467.5287
2.894





F5749-0203


embedded image


C20H15N5O4S2
453.5017
2.806





F5749-0204


embedded image


C18H16N6O5S2
460.4934
0.807





F5749-0205


embedded image


C22H19N5O4S2
481.5558
3.129





F5749-0206


embedded image


C19H15FN4O3S2
430.4828
3.985





F5749-0207


embedded image


C19H15FN4O3S2
430.4828
4.022





F5749-0208


embedded image


C21H20N4O4S2
456.546
4.058





F5749-0209


embedded image


C24H22N6O3S2
506.6093
3.977





F5749-0210


embedded image


C15H12N6O3S2
388.4293
1.838





F5749-0211


embedded image


C23H20N6O3S2
492.5823
3.843





F5749-0212


embedded image


C25H20N4O5S2
520.59
5.01374





F5749-0213


embedded image


C24H17ClN4O4S2
525.0085
5.898





F5749-0214


embedded image


C25H20N4O4S2
504.5906
5.606





F5749-0215


embedded image


C19H12ClF3N4O3S2
500.9087
5.25976





F5749-0216


embedded image


C24H18N4O4S2
490.5635
5.308





F5749-0217


embedded image


C18H13BrN4O3S2
477.3613
4.494





F5749-0218


embedded image


C18H13BrN4O3S2
477.3613
4.533





F5749-0219


embedded image


C18H12BrFN4O3S2
495.3517
4.684





F5749-0220


embedded image


C19H12BrF3N4O3S2
545.3597
5.46576





F5749-0221


embedded image


C18H13ClN4O3S2
432.9103
4.288





F5749-0222


embedded image


C18H14N4O5S3
462.5281
2.898





F5749-0223


embedded image


C19H13F3N4O4S2
482.4631
5.09476




















TABLE 10





IDNUMBER
Structure
Formula structure
MW
LogP



















F0808-0128


embedded image


C25H20N2O3S3
492.6418
6.892





F0808-0132


embedded image


C23H16N2O3S3
464.5876
6.261





F0808-0133


embedded image


C23H15ClN2O3S3
499.0326
6.853





F0808-0134


embedded image


C24H18N2O3S3
478.6147
6.559





F0808-0136


embedded image


C25H20N2O3S3
492.6418
7.034





F0808-0137


embedded image


C23H15BrN2O3S3
543.4836
7.059





F1269-0225


embedded image


C21H14N2O3S4
470.6133
5.774





F1269-1420


embedded image


C24H18N2O4S3
494.6141
6.217





F1566-1144


embedded image


C26H17N3O3S3
515.6357
6.461





F1566-1584


embedded image


C24H17N3O5S3
523.6122
6.529





F1566-1596


embedded image


C25H20N2O5S3
524.6406
6.208





F1566-1816


embedded image


C19H16N2O3S3
416.543
5.12





F1566-1830


embedded image


C20H18N2O3S3
430.5701
5.562





F1566-1844


embedded image


C21H20N2O3S3
444.5972
6.004





F1566-1858


embedded image


C18H14N2O3S3
402.5159
4.607





F5749-0224


embedded image


C23H15N3O5S3
509.5851
6.196





F5749-0225


embedded image


C23H22N2O3S3
470.6354
6.586





F5749-0226


embedded image


C26H22N2O4S3
522.6682
6.883





F5749-0227


embedded image


C24H17ClN2O4S3
529.0591
6.844





F5749-0228


embedded image


C25H20N2O3S3
492.6418
6.853





F5749-0229


embedded image


C25H18N2O5S3
522.6246
6.202





F5749-0230


embedded image


C25H19N3O4S3
521.6399
5.552





F5749-0231


embedded image


C25H20N2O5S3
524.6406
5.95974





F5749-0232


embedded image


C25H18N2O5S3
522.6246
5.856





F5749-0233


embedded image


C22H15N3O3S3
465.5752
5.028





F5749-0234


embedded image


C19H17N3O3S3
431.5576
4.155





F5749-0235


embedded image


C25H20N2O3S3
492.6418
6.892





F5749-0236


embedded image


C23H15N3O5S3
509.5851
6.194





F5749-0237


embedded image


C24H18N2O3S3
478.6147
6.395





F5749-0238


embedded image


C21H13ClN2O3S4
505.0584
7.064





F5749-0239


embedded image


C23H14F2N2O3S3
500.5684
6.602





F5749-0240


embedded image


C22H18N4O3S3
482.6058
5.371





F5749-0241


embedded image


C22H17N3O4S3
483.5905
5.405





F5749-0242


embedded image


C24H21N3O5S3
527.6441
4.048





F5749-0243


embedded image


C24H17FN2O4S3
512.6045
6.405





F5749-0244


embedded image


C25H20N2O4S3
508.6412
6.55





F5749-0245


embedded image


C27H19N3O3S3
529.6628
6.72





F5749-0246


embedded image


C23H18N2O3S4
498.6675
6.912





F5749-0247


embedded image


C22H16N2O3S4
484.6404
6.437





F5749-0248


embedded image


C25H18N2O3S3
490.6258
6.619





F5749-0249


embedded image


C23H15FN2O3S3
482.578
6.412





F5749-0250


embedded image


C24H18N2O4S3
494.6141
6.254





F5749-0251


embedded image


C23H15FN2O3S3
482.578
6.451





F5749-0252


embedded image


C23H14ClFN2O3S3
517.023
7.041





F5749-0253


embedded image


C24H15F3N2O4S3
548.5854
7.65976





F5749-0254


embedded image


C24H17ClN2O3S3
513.0597
7.186





F5749-0255


embedded image


C25H18N2O4S3
506.6252
6.151





F5749-0256


embedded image


C25H18N2O4S3
506.6252
6.114





F5749-0257


embedded image


C24H17ClN2O3S3
513.0597
7.147





F5749-0258


embedded image


C24H18N2O4S3
494.6141
6.215





F5749-0259


embedded image


C25H20N2O4S3
508.6412
6.556





F5749-0260


embedded image


C23H14F2N2O3S3
500.5684
6.563





F5749-0261


embedded image


C20H18N2O3S3
430.5701
5.754





F5749-0262


embedded image


C24H15F3N2O3S3
532.586
7.23276





F5749-0263


embedded image


C24H15F3N2O3S3
532.586
7.19576





F5749-0264


embedded image


C23H15ClN2O3S3
499.0326
6.89





F5749-0265


embedded image


C24H16Cl2N2O3S3
547.5047
7.813





F5749-0266


embedded image


C23H14F2N2O3S3
500.5684
6.602





F5749-0267


embedded image


C26H22N2O3S3
506.6688
7.476





F5749-0268


embedded image


C27H22N2O3S3
518.68
7.348





F5749-0269


embedded image


C27H19N3O5S3
561.6616
5.388





F5749-0270


embedded image


C28H21N3O5S3
575.6887
5.83





F5749-0271


embedded image


C24H19N3O4S4
541.6927
5.564





F5749-0272


embedded image


C21H14N4O5S3
498.5615
3.306





F5749-0273


embedded image


C26H19N3O4S3
533.651
5.457





F5749-0274


embedded image


C25H17N3O4S3
519.6239
5.369





F5749-0275


embedded image


C23H18N4O5S3
526.6157
3.37





F5749-0276


embedded image


C27H21N3O4S3
547.6781
5.692





F5749-0277


embedded image


C24H17FN2O3S3
496.6051
6.548





F5749-0278


embedded image


C24H17FN2O3S3
496.6051
6.585





F5749-0279


embedded image


C26H22N2O4S3
522.6682
6.621





F5749-0280


embedded image


C29H24N4O3S3
572.7316
6.54





F5749-0281


embedded image


C20H14N4O3S3
454.5516
4.401





F5749-0282


embedded image


C28H22N4O3S3
558.7045
6.406





F5749-0283


embedded image


C30H22N2O5S3
586.7122
7.57674





F5749-0284


embedded image


C29H19ClN2O4S3
591.1308
8.461





F5749-0285


embedded image


C30H22N2O4S3
570.7128
8.169





F5749-0286


embedded image


C24H14ClF3N2O3S3
567.031
7.82276





F5749-0287


embedded image


C29H20N2O4S3
556.6858
7.871





F5749-0288


embedded image


C23H15BrN2O3S3
543.4836
7.057





F5749-0289


embedded image


C23H15BrN2O3S3
543.4836
7.096





F5749-0290


embedded image


C23H14BrFN2O3S3
561.474
7.247





F5749-0291


embedded image


C24H14BrF3N2O3S3
611.482
8.02876





F5749-0292


embedded image


C23H15ClN2O3S3
499.0326
6.851





F5749-0293


embedded image


C23H16N2O5S4
528.6504
5.461





F5749-0294


embedded image


C24H15F3N2O4S3
548.5854
7.65776




















TABLE 11





IDNUMBER
Structure
Formula structure
MW
LogP



















F0433-0038


embedded image


C16H12ClNO3S
333.7959
4.192





F0433-0041


embedded image


C17H14ClNO3S
347.823
4.49





F0433-0044


embedded image


C16H11Cl2NO3S
368.241
4.784





F0433-0047


embedded image


C17H14ClNO4S
363.8224
4.148





F0433-0050


embedded image


C20H14ClNO3S
383.8565
5.451





F0808-1895


embedded image


C18H16ClNO3S
361.8501
4.823





F0808-1902


embedded image


C16H11BrClNO3S
412.692
4.99





F0808-1909


embedded image


C16H11ClN2O5S
378.7935
4.164





F0808-1913


embedded image


C18H16ClNO3S
361.8501
4.823





F0808-1914


embedded image


C20H20ClNO3S
389.9043
5.691





F1269-0272


embedded image


C14H10ClNO3S2
339.8217
3.705





F1269-1995


embedded image


C17H13ClN2O5S
392.8206
4.46





F1566-1223


embedded image


C19H13ClN2O3S
384.8441
4.392





F5749-0295


embedded image


C11H10ClNO3S
271.7243
2.538





F5749-0296


embedded image


C16H11ClN2O5S
378.7935
4.127





F5749-0297


embedded image


C16H18ClNO3S
339.8438
4.517





F5749-0298


embedded image


C19H18ClNO4S
391.8766
4.814





F5749-0299


embedded image


C17H13Cl2NO4S
398.2675
4.775





F5749-0300


embedded image


C18H16ClNO3S
361.8501
4.784





F5749-0301


embedded image


C18H14ClNO5S
391.833
4.133





F5749-0302


embedded image


C18H15ClN2O4S
390.8483
3.483





F5749-0303


embedded image


C18H16ClNO5S
393.8489
3.89074





F5749-0304


embedded image


C18H14ClNO5S
391.833
3.787





F5749-0305


embedded image


C15H11ClN2O3S
334.7835
2.959





F5749-0306


embedded image


C12H12ClNO3S
285.7513
3.051





F5749-0307


embedded image


C18H16ClNO5S
393.8489
4.139





F5749-0308


embedded image


C12H13ClN2O3S
300.766
2.086





F5749-0309


embedded image


C18H16ClNO3S
361.8501
4.823





F5749-0310


embedded image


C16H11ClN2O5S
378.7935
4.125





F5749-0311


embedded image


C13H14ClNO3S
299.7784
3.493





F5749-0312


embedded image


C17H14ClNO3S
347.823
4.326





F5749-0313


embedded image


C14H16ClNO3S
313.8055
3.935





F5749-0314


embedded image


C14H9Cl2NO3S2
374.2667
4.995





F5749-0315


embedded image


C16H10ClF2NO3S
369.7768
4.533





F5749-0316


embedded image


C15H14ClN3O3S
351.8141
3.302





F5749-0317


embedded image


C15H13ClN2O4S
352.7989
3.336





F5749-0318


embedded image


C17H17ClN2O5S
396.8524
1.979





F5749-0319


embedded image


C17H13ClFNO4S
381.8129
4.336





F5749-0320


embedded image


C18H16ClNO4S
377.8495
4.481





F5749-0321


embedded image


C20H15ClN2O3S
398.8712
4.651





F5749-0322


embedded image


C16H14ClNO3S2
367.8759
4.843





F5749-0323


embedded image


C15H12ClNO3S2
353.8488
4.368





F5749-0324


embedded image


C18H14ClNO3S
359.8342
4.55





F5749-0325


embedded image


C16H11ClFNO3S
351.7864
4.343





F5749-0326


embedded image


C17H14ClNO4S
363.8224
4.185





F5749-0327


embedded image


C16H11ClFNO3S
351.7864
4.382





F5749-0328


embedded image


C16H10Cl2FNO3S
386.2314
4.972





F5749-0329


embedded image


C17H11ClF3NO4S
417.7937
5.59076





F5749-0330


embedded image


C17H13Cl2NO3S
382.2681
5.117





F5749-0331


embedded image


C18H14ClNO4S
375.8336
4.082





F5749-0332


embedded image


C18H14ClNO4S
375.8336
4.045





F5749-0333


embedded image


C17H13Cl2NO3S
382.2681
5.078





F5749-0334


embedded image


C17H14ClNO4S
363.8224
4.146





F5749-0335


embedded image


C18H16ClNO4S
377.8495
4.487





F5749-0336


embedded image


C16H10ClF2NO3S
369.7768
4.494





F5749-0337


embedded image


C13H14ClNO3S
299.7784
3.685





F5749-0338


embedded image


C17H11ClF3NO3S
401.7943
5.16376





F5749-0339


embedded image


C17H11ClF3NO3S
401.7943
5.12676





F5749-0340


embedded image


C16H11Cl2NO3S
368.241
4.821





F5749-0341


embedded image


C17H12Cl3NO3S
416.7131
5.744





F5749-0342


embedded image


C16H10ClF2NO3S
369.7768
4.533





F5749-0343


embedded image


C19H18ClNO3S
375.8772
5.407





F5749-0344


embedded image


C20H18ClNO3S
387.8884
5.279





F5749-0345


embedded image


C20H15ClN2O5S
430.87
3.319





F5749-0346


embedded image


C21H17ClN2O5S
444.897
3.761





F5749-0347


embedded image


C17H15ClN2O4S2
410.9011
3.495





F5749-0348


embedded image


C14H10ClN3O5S
367.7699
1.237





F5749-0349


embedded image


C19H15ClN2O4S
402.8594
3.388





F5749-0350


embedded image


C18H13ClN2O4S
388.8323
3.3





F5749-0351


embedded image


C16H14ClN3O5S
395.8241
1.301





F5749-0352


embedded image


C20H17ClN2O4S
416.8865
3.623





F5749-0353


embedded image


C17H13ClFNO3S
365.8135
4.479





F5749-0354


embedded image


C17H13ClFNO3S
365.8135
4.516





F5749-0355


embedded image


C19H18ClNO4S
391.8766
4.552





F5749-0356


embedded image


C22H20ClN3O3S
441.94
4.471





F5749-0357


embedded image


C13H10ClN3O3S
323.76
2.332





F5749-0358


embedded image


C21H18ClN3O3S
427.9129
4.337





F5749-0359


embedded image


C23H18ClNO5S
455.9206
5.50774





F5749-0360


embedded image


C22H15Cl2NO4S
460.3392
6.392





F5749-0361


embedded image


C23H18ClNO4S
439.9212
6.1





F5749-0362


embedded image


C17H10Cl2F3NO3S
436.2394
5.75376





F5749-0363


embedded image


C22H16ClNO4S
425.8941
5.802





F5749-0364


embedded image


C16H11BrClNO3S
412.692
4.988





F5749-0365


embedded image


C16H11BrClNO3S
412.692
5.027





F5749-0366


embedded image


C16H10BrClFNO3S
430.6824
5.178





F5749-0367


embedded image


C17H10BrClF3NO3S
480.6904
5.95976





F5749-0368


embedded image


C16H11Cl2NO3S
368.241
4.782





F5749-0369


embedded image


C16H12ClNO5S2
397.8587
3.392





F5749-0370


embedded image


C17H11ClF3NO4S
417.7937
5.58876









Example 11
Stat3 Inhibitor as a Treatment for Dermal Fibrosis

Skin biopsies from the subcutaneous bleomycin skin model in wild type mice and human subjects (healthy control vs. scleroderma) are illustrated in FIG. 19. Scleroderma is an autoimmune disease that manifests as fibrosis in the skin and internal organs. Both bleomycin injected skin with fibrosis and scleroderma skin demonstrate increased staining for phosphorylated (active) Stat3, indicating activation of Stat3 signaling in these fibrotic tissues.



FIG. 20 illustrates an exemplary study design using a subcutaneous (SQ) bleomycin model for skin fibrosis. In a basic model, bleomycin is administered to wild type mice (C57/Bl6) via subcutaneous injection 5 days a week for a total of 4 weeks. PBS is used as a control for the bleomycin. Fibrosis is assessed in the biopsied skin on day 28. Fibrosis is assessed using histology (hematoxylin and eosin stain), where the dermal thickness is quantified as the average linear distance from the dermis-epidermis junction to the subQ muscle layer. Masson's trichrome staining is also performed to stain collagen blue. Immunohistochemistry is performed for alpha-smooth muscle actin, the marker of the myofibroblasts (cell that makes collagen during the process of fibrosis). Future endpoints are assessed including collagen content. To illustrate that STAT3 inhibitor is a useful therapy at least for dermal fibrosis, an example of a STAT3 inhibitor (C188-9) was given via the intraperitoneal route daily, starting in week 3 of the bleomycin model. DMSO was used as a control for the STAT3 inhibitor.


As shown in FIG. 21, wild-type mice given Bleomycin (BLM, middle panel) have increased dermal thickness that is decreased when given the STAT3 inhibitor (right panel). Images are representative images from 1 mouse per group total of 5 mice per group in the study.



FIG. 22 provides quantitation of the images in FIG. 22, and all 5 in each group are averaged. There was no difference in dermal thickness between PBS-injected mice with treatment with DMSO or STAT3 inhibitor. However, bleomycin increases the dermal thickness that is reduced in the Bleo/STAT3 inhibitor group.


Wild type mice given Bleomycin (BLM, middle panel) have increased collagen content (blue in Masson's trichrome stain) that is decreased when given the STAT3 inhibitor (right panel) (FIG. 23). Images are representative images from 1 mouse per group total of 5 mice per group in the study.


In FIG. 24, wild type mice given Bleomycin (BLM, middle panel) have increased alpha Smooth muscle actin staining (bright pink) that is decreased when given the STAT3 inhibitor (right panel). Images are representative images from 1 mouse per group total of 5 mice per group in the experiment. The aSMA stains myofibroblasts; therefore, STAT3 inhibitor decreases fibrosis and the accumulation of myofibroblasts in the subcutaneous bleomycin model.



FIG. 25 shows the effect of Cpd188-9 on dermal fibroblast production of type I collagen (Col1a1), smooth muscle actin (aSMA) and the transcription factor Snail. Col1a1, aSMA and Snail are important genes involved in the development of fibrosis of the skin and other organs. During fibrosis, TGFbeta and/or IL-6 can increase the expression of these genes in dermal fibroblasts and contribute to the development of tissue fibrosis. As see in FIG. 25, Cpd188-9 blocks the increased expression of Col1a1, aSMA and Snail mRNA induced by TGF-beta or IL6 (plus the IL6 receptor) in mouse dermal fibroblasts. These data demonstrate that Cpd188-9 is antifibrotic in vitro and confirm and extend the in vivo findings in the dermal fibrosis model.



FIG. 26 provides quantitative real time PCR assessment of fibrotic gene expression in the skin of wild type mice in the subcutaneous bleomycin model treated with control (DMSO) or Cpd188-9 (exemplary Stat3 inhibitor). As expected, 28 days of subcutaneous bleomycin injection increased the dermal expression of smooth muscle actin (Acta2) and type I collagen (Col1a1 and Col1a2) transcripts. These increases were prevented with intraperitoneal treatment of mice with Cpd188-9 from days 14-28 of the 4 week model. N=5 mice per group.


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All patents and publications cited herein are hereby incorporated by reference in their entirety herein. Full citations for the references cited herein are provided in the following list.


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Claims
  • 1. A method of treating an individual that has fibrosis, comprising the step of providing to the individual an effective amount of one or more compositions selected from the group consisting of N-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide, N-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide, N-(3,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(4,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(5,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(6,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(7,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(8,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, 4-Bromo-N-(1,6′-dihydroxy-[2,2′]binaphthalenyl-4-yl)-benzenesulfonamide, 4-Bromo-N-[4-hydroxy-3-(1H-[1,2,4]triazol-3-ylsulfanyl)-naphthalen-1-yl]-benzenesulfonamide, and a functional derivative thereof, wherein the fibrosis is not pulmonary fibrosis or myelofibrosis.
  • 2. The method of claim 1, wherein the fibrosis is of the skin, heart, intestine, pancreas, joint, liver, or retroperionteum.
  • 3. The method of claim 1, wherein the individual is provided the composition in multiple doses.
  • 4. The method of claim 3, wherein the multiple doses are separated by hours, days, or weeks.
  • 5. The method of claim 1, wherein the individual is provided with an additional therapy for the fibrosis.
  • 6. The method of claim 1, wherein the composition is selected from the group consisting of N-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide, N-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide, N-(3,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(4,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(5,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(6,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(7,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(8,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, 4-Bromo-N-(1,6′-dihydroxy-[2,2′]binaphthalenyl-4-yl)-benzenesulfonamide, 4-Bromo-N-[4-hydroxy-3-(1H-[1,2,4]triazol-3-ylsulfanyl)-naphthalen-1-yl]-benzenesulfonamide, a functionally active derivative thereof, and a mixture thereof.
  • 7. The method of claim 1, wherein the composition inhibits Stat3, Stat1, or both.
  • 8. The method of claim 1, further comprising the step of diagnosing the fibrosis.
  • 9. The method of claim 1, wherein the composition is provided intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, injection, infusion, continuous infusion, localized perfusion, via a catheter, via a lavage, in lipid compositions, in liposome compositions, or as an aerosol.
  • 10. The method of claim 1, wherein the composition is provided by injection.
  • 11. The method of claim 1, wherein the composition is provided topically.
  • 12. The method of claim 1, wherein the composition is provided subcutaneously.
  • 13. The method of claim 1, wherein the composition is provided systemically.
  • 14. The method of claim 1, wherein the composition is provided locally.
  • 15. The method of claim 1, wherein the individual does not have cancer.
  • 16. The method of claim 1, wherein the individual is not suspected of having cancer.
  • 17. The method of claim 1, wherein the fibrosis is scleroderma.
  • 18. The method of claim 1, wherein the composition comprises N-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide.
  • 19. A method of treating an individual, comprising the step of providing to the individual an effective amount of one or more compositions selected from the group consisting of N-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide, N-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide, N-(3,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(4,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(5,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(6,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(7,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(8,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, 4-Bromo-N-(1,6′-dihydroxy-[2,2′]binaphthalenyl-4-yl)-benzenesulfonamide, 4-Bromo-N-[4-hydroxy-3-(1H-[1,2,4]triazol-3-ylsulfanyl)-naphthalen-1-yl]-benzenesulfonamide, and a functional derivative thereof, wherein the fibrosis is not pulmonary fibrosis or myelofibrosis, and wherein the fibrosis is of the heart, pancreas, joint, liver, or retroperionteum.
  • 20. The method of claim 19, wherein the composition is selected from the group consisting of N-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide, N-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide, N-(3,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(4,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(5,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(6,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(7,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, N-(8,1′-Dihydroxy-[1,2′]binaphthalenyl-4′-yl)-4-methoxy-benzenesulfonamide, 4-Bromo-N-(1,6′-dihydroxy-[2,2′]binaphthalenyl-4-yl)-benzenesulfonamide, 4-Bromo-N-[4-hydroxy-3-(1H-[1,2,4]triazol-3-ylsulfanyl)-naphthalen-1-yl]-benzenesulfonamide, a functionally active derivative thereof, and a mixture thereof.
  • 21. The method of claim 19, wherein the composition is provided intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, injection, infusion, continuous infusion, localized perfusion, via a catheter, via a lavage, in lipid compositions, in liposome compositions, or as an aerosol.
  • 22. The method of claim 19, wherein the fibrosis is scleroderma.
  • 23. The method of claim 19, wherein the composition comprises N-(1′,2-dihydroxy-1,2′-binaphthalen-4′-yl)-4-methoxybenzenesulfonamide.
Parent Case Info

This application claims priority to U.S. Provisional Patent Application Ser. No. 61/847,744, filed Jul. 18, 2013, which is incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under P50 CA058183, K08 HL085018-01A2, P50 CA097007, R21CA149783, and R41CA153658 awarded by National Institutes of Health. The United States Government has certain rights in the invention.

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Related Publications (1)
Number Date Country
20150051233 A1 Feb 2015 US
Provisional Applications (1)
Number Date Country
61847744 Jul 2013 US