Patent Application
20220265691
Selectins are a class of cell adhesion molecules that have well-characterized roles in leukocyte homing. One of these, E-selectin (endothelial selectin), is expressed by endothelial cells at sites of inflammation or injury. Recent investigations have suggested that cancer cells are immunostimulatory and interact with selectins to extravasate and metastasize.
The most common types of cancer include prostate, breast, lung, colorectal, melanoma, bladder, non-Hodgkins lymphoma, kidney, thyroid, leukemias, endometrial and pancreatic cancers based on estimated incidence data.
The cancer with the highest expected incidence is prostate cancer. The highest mortality rate is for patients who have lung cancer. Despite enormous investment of financial and human resources, cancers such as colorectal cancer remain one of the major causes of death. Colorectal cancer is the second leading cause of cancer-related deaths in the United States of cancers that affect both men and women. Over the last several years, more then 50,000 patients with colorectal cancer have died every year.
The four hematological cancers that are most common are acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML) and acute myelogenous leukemia (AML). Leukemias and other cancers of the blood, bone marrow, and lymphatic system affect 10 times more adults than children. However, leukemia is one of the most common childhood cancers and 75% of childhood leukemias are ALL.
AML is a cancer of myeloid stem cells, characterized by the rapid growth of abnormal cells that build up in the bone marrow and blood and interfere with normal blood cells. Symptoms may include fatigue, shortness of breath, easy bruising and bleeding, and increased risk of infection. It is an acute form of leukemia, which can progress rapidly and is typically fatal within weeks or months if left untreated. AML is the most common leukemia in adults. Approximately 47,000 new cases are diagnosed every year and approximately 23,500 people die every year from leukemia. The 5-year survival rate for AML is 27.4%. It accounts for roughly 1.8% of cancer deaths in the United States.
The underlying mechanism of AML is believed to involve uncontrolled expansion of immature myeloid cells in the bone marrow, which results in a drop in counts of red blood cells, platelets, and normal white blood cells. Diagnosis is generally based on bone marrow aspiration and specific blood tests. AML has several subtypes for which treatments and outcomes may vary.
First-line treatment of AML consists primarily of chemotherapy with an anthracycline/cytarabine combination and is divided into two phases: induction and post-remission (or consolidation) therapy. The goal of induction therapy is to achieve a complete remission by reducing the number of leukemic cells to an undetectable level; the goal of consolidation therapy is to eliminate any residual undetectable disease and achieve a cure. The specific genetic mutations present within the cancer cells may guide therapy, as well as determine how long that person is likely to survive.
Despite advances in our understanding of the pathogenesis of AML, the short- and long-term outcomes for AML patients have remained unchanged over three decades (Roboz et al., (2012) Curr. Opin. Oncol., 24: 711-719). The median age at diagnosis is 66 years with cure rates of less than 10% and median survival of less than 1 year (Burnett et al., (2010), J. Clin. Oncol., 28: 586-595). Although 70-80% of patients younger than 60 years achieve complete remission, most eventually relapse, and overall survival is only 40-50% at 5 years (Fernandez et al., (2009) N. Engl. J. Med., 361: 1249-1259; Mandelli et al., (2009) J. Clin. Oncol., 27: 5397-5403; Ravandi et al., (2006) Clin. Can. Res., 12(2): 340-344). Relapse is thought to occur due to leukemic stem cells that escape initial induction therapy and drive reoccurrence of AML (Dean et al., (2005) Nat. Rev. Cancer, 5(4): 275-294; Guan et al., (2003) Blood, 101(8): 3142; and Konopleva et al., (2002) Br. J. Haematol. 118(2): 521-534). Chemoresistance, the ability of cancer cells to evade or to cope in the presence of therapeutics, is also a key challenge for therapeutic success.
Selectins are a group of structurally similar cell surface receptors important for mediating leukocyte binding to endothelial cells. These proteins are type 1 membrane proteins and are composed of an amino terminal lectin domain, an epidermal growth factor (EGF)-like domain, a variable number of complement receptor related repeats, a hydrophobic domain spanning region and a cytoplasmic domain. The binding interactions appear to be mediated by contact of the lectin domain of the selectins and various carbohydrate ligands.
There are three known selectins: E-selectin, P-selectin, and L-selectin. E-selectin is a transmembrane adhesion protein expressed on the surface of activated endothelial cells, which line the interior wall of capillaries. E-selectin binds to the carbohydrate sialyl-Lewisx (sLex), which is presented as a glycoprotein or glycolipid on the surface of certain leukocytes (monocytes and neutrophils) and helps these cells adhere to capillary walls in areas where surrounding tissue is infected or damaged; and E-selectin also binds to sialyl-Lewisa (sLea), which is expressed on many tumor cells. P-selectin is expressed on inflamed endothelium and platelets, and also recognizes sLex and sLea, but also contains a second site that interacts with sulfated tyrosine. The expression of E-selectin and P-selectin is generally increased when the tissue adjacent to a capillary is infected or damaged. L-selectin is expressed on leukocytes. Selectin-mediated intercellular adhesion is an example of a selectin-mediated function.
With few exceptions, E-selectin is not normally expressed in the vasculature but must be stimulated to be synthesized and expressed by inflammatory mediators. One of those exceptions is the microvasculature of the bone marrow (BM) where E-selectin is constitutively expressed.
In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the disclosed embodiments may be practiced without these details. In other instances, well-known structures have not been shown or described in detail to avoid unnecessarily obscuring descriptions of the embodiments. These and other embodiments will become apparent upon reference to the following detailed description and attached drawings.
In order to better understand the disclosure, certain exemplary embodiments are discussed herein. In addition, certain terms are discussed to aid in the understanding.
Disclosed herein are methods of screening cancer patients for treatment, and upon screening the patients, treating a subset of them meeting certain criteria with an E-selectin inhibitor for purposes of treating the cancer and lengthening overall survival.
According to one embodiment, a method of screening a cancer patient may include obtaining or having obtained a biological sample from the cancer patient.
The biological sample may be any sample that is taken from a cancer patient. Examples include, but are not limited to, blood, plasma, saliva, pleural fluid, sweat, ascitic fluid, bile, urine, serum, pancreatic juice, stool, cervical smear samples, tumor biopsies, or any other sample that contains nucleic acids such as DNA and RNA.
In these embodiments, the method of screening the cancer patient may include performing or having performed an assay on the biological sample obtained from the cancer patient to determine the gene expression level of one or more E-selectin ligand-forming genes in the sample.
In these embodiments, performing the assay may further comprise measuring the number of mRNA transcripts or the amount of protein expressed.
The assay may be any assay that allows determination of a gene expression level, including but not limited to Sanger sequencing, high throughput sequencing, quantitative polymerase chain reaction, reverse transcriptase qPCR, RNA sequencing, microarray analysis, Northern blots, RNA-seq, high coverage mRNA sequencing, flow analysis, flow cytometry, immunohistology, immunostaining, immunohistochemistry, affinity purification, mass spectrometry, Western blotting, enzyme-linked immunoadsorbent assay, and multidimensional flow cytometry.
In some embodiments, the assay may use reagents chosen from a HECA-452-FITC monoclonal antibody, an E-selectin/hIg chimera, and chimera/PE.
In some embodiments, if the biological sample has an increased gene expression level for one or more particular genes relative to the expression level for that particular gene in a cancer-free subject, a newly diagnosed cancer subject, or a subject diagnosed with the same cancer as the patient, the method of screening the cancer patient may include selecting the patient for treatment comprising one or more E-selectin inhibitors. In some embodiments, the gene is an E-selectin ligand-forming gene.
In some embodiments, if at least 10%, at least 15%, at least 20%, or at least 25% of the blast cells in the biological sample express one or more particular genes, the method of screening the cancer patient may include selecting the patient for treatment comprising one or more E-selectin inhibitors. In some embodiments, the gene is an E-selectin ligand-forming gene.
In another embodiment, a method of treating a cancer patient may include obtaining or having obtained a biological sample from the cancer patient.
The biological sample may be any sample that is taken from a cancer patient. Examples include, but are not limited to, blood, plasma, saliva, pleural fluid, sweat, ascitic fluid, bile, urine, serum, pancreatic juice, stool, cervical smear samples, tumor biopsies, or any other sample that contains nucleic acids such as DNA and RNA.
In these embodiments, the method of treating the cancer patient may include performing or having performed an assay on the biological sample obtained from the cancer patient to determine the gene expression level of one or more E-selectin ligand-forming genes in the sample.
In these embodiments, performing the assay may further comprise measuring the number of mRNA transcripts or the amount of protein expressed.
The assay may be any assay that allows determination of a gene expression level, including but not limited to Sanger sequencing, high throughput sequencing, quantitative polymerase chain reaction, reverse transcriptase qPCR, RNA sequencing, microarray analysis, Northern blots, RNA-seq, high coverage mRNA sequencing, flow analysis, flow cytometry, immunohistology, immunostaining, immunohistochemistry, affinity purification, mass spectrometry, Western blotting, enzyme-linked immunoadsorbent assay, and multidimensional flow cytometry.
In some embodiments, the assay may use reagents chosen from a HECA-452-FITC monoclonal antibody, an E-selectin/hIg chimera, and chimera/PE.
In some embodiments, if the biological sample has an increased gene expression level for one or more particular genes relative to the expression level for that particular gene in a non-cancer subject, a newly diagnosed cancer subject, or a subject diagnosed with the same cancer as the patient, the method of screening the cancer patient may include selecting the patient for treatment comprising one or more E-selectin inhibitors. In some embodiments, the gene is an E-selectin ligand-forming gene.
In some embodiments, if at least 10%, at least 15%, at least 20%, or at least 25% of the blast cells in the biological sample express one or more particular genes, the method of screening the cancer patient may include selecting the patient for treatment comprising one or more E-selectin inhibitors. In some embodiments, the gene is ane E-selectin ligand-forming gene.
In these embodiments, the method of treating a cancer patient may include administering a therapeutically effective amount of a composition comprising one or more E-selectin inhibitors.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All references cited herein are incorporated by reference in their entireties. To the extent terms or discussion in references conflict with this disclosure, the latter shall control.
As used herein, the singular forms of a word also include the plural form of the word, unless the context clearly dictates otherwise; as examples, the terms “a,” “an,” and “the” are understood to be singular or plural. By way of example, “an element” means one or more element. The term “or” shall mean “and/or” unless the specific context indicates otherwise.
“About” can be understood as within +/−10%, e.g., +/−10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. When used in reference to a percentage value, “about” can be understood as within ±1% (e.g., “about 5%” can be understood as within 4%-6%). All ranges used herein encompass the endpoints.
As used herein, the terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof from occurring in the first place and/or can be therapeutic in terms of a partial or complete cure for a disease and/or adverse effects attributable to the disease. As an example, the term “treatment” and the like, as used herein, encompasses any treatment of cancers such as AML or any of its subtypes and related hematologic cancers in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject, e.g., a subject identified as predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) delaying onset or progression of the disease, e.g., as compared to the anticipated onset or progression of the disease in the absence of treatment: (c) inhibiting the disease, i.e., arresting its development; and/or (d) relieving the disease, i.e., causing regression of the disease. In some embodiments, “treating” refers to administering e.g., subcutaneously, an effective dose, or effective multiple doses of a composition e.g., a composition comprising an inhibitor, e.g., an E-selectin inhibitor, as disclosed herein to an animal (including a human being) suspected of suffering or already suffering from AML or another related cancer. It can also refer to reducing, eliminating, or at least partially arresting, as well as to exerting any beneficial effect, on one or more symptoms of the disease and/or associated with the disease and/or its complications.
As used herein, the terms “blasts” and “blast cells” are used interchangeably to refer to undifferentiated, precursor blood stem cells. As used herein, the term “blast count” refers to the number of blast cells in a sample.
The terms “acute myeloid leukemia,” “acute myelogenous leukemia,” “acute myeloblastic leukemia,” “acute granulocytic leukemia,” and “acute nonlymphocytic leukemia,” and “AML” are used interchangeably and as used herein, refer to a cancer of the bone marrow characterized by abnormal proliferation of myeloid stem cells. AML as used herein, refers to any or all known subtypes of the disease, including but not limited to subtypes classified by the World Health Organization (WHO) 2016 classification of AML, e.g., AML with myelodysplasia-related changes or myeloid sarcoma, and the French-American-British (FAB) classification system, e.g., M0 (acute myeloblastic leukemia, minimally differentiated) or M1 (acute myeloblastic leukemia, without maturation). Falini et al., (2010) Discov. Med., 10(53): 281-92; Lee et al., (1987) Blood, 70(5): 1400-1406.
The term “E-selectin ligand” as used herein, refers to a carbohydrate structure that contains the epitope shared by sialyl Lea and sialyl Lex. Carbohydrates are secondary gene products synthesized by enzymes known as glycosyltransferases which are the primary gene products coded for by DNA. Each glycosyltransferase adds a specific monosaccharide in a specific stereochemical linkage to a specific donor carbohydrate chain.
The terms “E-selectin antagonist” and “E-selectin inhibitor” are used interchangeably herein. E-selectin inhibitors are known in the art. Some E-selectin inhibitors are specific for E-selectin only. Other E-selectin inhibitors have the ability to inhibit not only E-selectin but additionally P-selectin or L-selectin or both P-selectin and L-selectin. In some embodiments, an E-selectin inhibitor inhibits E-selectin, P-selectin, and L-selectin.
In some embodiments, an E-selectin inhibitor is a specific glycomimetic antagonist of E-selectin. Examples of E-selectin inhibitors (specific for E-selectin or otherwise) are disclosed in U.S. Pat. No. 9,109,002, the disclosure of which is expressly incorporated by reference in its entirety.
In some embodiments, the E-selectin antagonists suitable for the disclosed compounds and methods include pan-selectin antagonists.
Non-limiting examples of suitable E-selectin antagonists include small molecules, such as nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, glycomimetics, lipids and other organic (carbon containing) or inorganic molecules. Suitably, the selectin antagonist is selected from antigen-binding molecules that are immuno-interactive with a selectin, peptides that bind to the selectin and that block cell-cell adhesion, and carbohydrate or peptide mimetics of selectin ligands. In some embodiments, the E-selectin antagonist reduces the expression of a selectin gene or the level or functional activity of an expression product of that gene. For example, the E-selectin antagonist may antagonize the function of the selectin, including reducing or abrogating the activity of at least one of its ligand-binding sites.
In some embodiments, the E-selectin antagonist inhibits an activity of E-selectin or inhibits the binding of E-selectin to one or more E-selectin ligands (which in turn may inhibit a biological activity of E-selectin).
E-selectin antagonists include the glycomimetic compounds described herein. E-selectin antagonists also include antibodies, polypeptides, peptides, peptidomimetics, and aptamers which bind at or near the binding site on E-selectin to inhibit E-selectin interaction with sialyl Lea (sLea) or sialyl Lex (sLex).
Further disclosure regarding E-selectin antagonists suitable for the disclosed methods and compounds may be found in U.S. Pat. No. 9,254,322, issued Feb. 9, 2016, and U.S. Pat. No. 9,486,497, issued Nov. 8, 2016, which are both hereby incorporated by reference in their entireties. In some embodiments, the selectin antagonist is chosen from E-selectin antagonists disclosed in U.S. Pat. No. 9,109,002, issued Aug. 18, 2015, which is hereby incorporated by reference in its entirety. In some embodiments, the E-selectin antagonist is chosen from heterobifunctional antagonists disclosed in U.S. Pat. No. 8,410,066, issued Apr. 2, 2013, and US Publication No. US2017/0305951, published Oct. 26, 2017, which are both hereby incorporated by reference in their entireties. Further disclosure regarding E-selectin antagonists suitable for the disclosed methods and compounds may be found in PCT Publication Nos. WO2018/068010, published Apr. 12, 2018, WO2019/133878, published Jul. 4, 2019, and WO2020/139962, published Jul. 2, 2020, which are hereby incorporated by reference in their entireties.
The term “at least one” refers to one or more, such as one, two, etc. For example, the term “at least one C1-4 alkyl group” refers to one or more C1-4 alkyl groups, such as one C1-4 alkyl group, two C1-4 alkyl groups, etc.
The term “pharmaceutically acceptable salts” includes both acid and base addition salts. Non-limiting examples of pharmaceutically acceptable acid addition salts include chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, methane sulfonates, formates, tartrates, maleates, citrates, benzoates, salicylates, and ascorbates. Non-limiting examples of pharmaceutically acceptable base addition salts include sodium, potassium, lithium, ammonium (substituted and unsubstituted), calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Pharmaceutically acceptable salts may, for example, be obtained using standard procedures well known in the field of pharmaceuticals.
The term “prodrug” includes compounds that may be converted, for example, under physiological conditions or by solvolysis, to a biologically active compound described herein. Thus, the term “prodrug” includes metabolic precursors of compounds described herein that are pharmaceutically acceptable. A discussion of prodrugs can be found, for example, in Higuchi, T., et al., “Pro-drugs as Novel Delivery Systems,” A.C.S. Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987. The term “prodrug” also includes covalently bonded carriers that release the active compound(s) as described herein in vivo when such prodrug is administered to a subject. Non-limiting examples of prodrugs include ester and amide derivatives of hydroxy, carboxy, mercapto and amino functional groups in the compounds described herein.
This application contemplates all the isomers of the compounds disclosed herein. “Isomer” as used herein includes optical isomers (such as stereoisomers, e.g., enantiomers and diastereoisomers), geometric isomers (such as Z (zusammen) or E (entgegen) isomers), and tautomers. The present disclosure includes within its scope all the possible geometric isomers, e.g., Z and E isomers (cis and trans isomers), of the compounds as well as all the possible optical isomers, e.g. diastereomers and enantiomers, of the compounds. Furthermore, the present disclosure includes in its scope both the individual isomers and any mixtures thereof, e.g. racemic mixtures. The individual isomers may be obtained using the corresponding isomeric forms of the starting material or they may be separated after the preparation of the end compound according to conventional separation methods. For the separation of optical isomers, e.g., enantiomers, from the mixture thereof conventional resolution methods, e.g. fractional crystallization, may be used.
The present disclosure includes within its scope all possible tautomers. Furthermore, the present disclosure includes in its scope both the individual tautomers and any mixtures thereof. Each compound disclosed herein includes within its scope all possible tautomeric forms. Furthermore, each compound disclosed herein includes within its scope both the individual tautomeric forms and any mixtures thereof. With respect to the methods, uses and compositions of the present application, reference to a compound or compounds is intended to encompass that compound in each of its possible isomeric forms and mixtures thereof. Where a compound of the present application is depicted in one tautomeric form, that depicted structure is intended to encompass all other tautomeric forms.
E-selectin antagonists, such as the compound of Formula I, which interrupt leukemic cell homing to the vascular niche and increase susceptibility to cytotoxic therapies, can be potent adjuncts to therapeutics.
The pre-screening of patients amenable to treatment with an E-selectin inhibitor such as the compound of Formula I is also contemplated, e.g., according to the methods of identifying cancers disclosed herein, as well as the administration of treatment to patients identified according to criteria disclosed herein. In some embodiments, one or more diagnostic assays may be used to pre-screen cancer patients amenable to treatment with an E-selectin inhibitor. In some embodiments, the cancer patients amenable to treatment with an E-selectin inhibitor have leukemia. In some embodiments the cancer patients amenable to treatment with an E-selectin inhibitor have AML. In some embodiments, the AML patients may have one or more genetic mutations to the FLT3 gene. In some embodiments, the one or more diagnostic assays may be used to identify FLT3 patients expressing E-selectin ligand on their AML cells.
Pre-screening of patients who are likely to benefit from the treatments disclosed herein are also contemplated. Without being bound by theory, patients who express high amounts of E-selectin ligands on blast cells are chemo-resistant (relapsed/refractory) by a mechanism involving E-selectin, and therefore treatment with E-selectin antagonists shows greater efficacy. Accordingly, expression levels of genes involved in the synthesis or degradation of E-selectin ligands may be useful in pre-screening patients who may be more likely to benefit from treatment with E-selectin antagonists, e.g., the compound of Formula I. The disclosure herein is based on the surprising discovery that while AML patients with the highest expression of genes involved in synthesis or degradation of E-selectin ligands, e.g., ST3GAL4 and FUT7 genes, have poorer outcomes and shorter overall survival, relapsed/refractory patients expressing higher levels of these genes have better outcomes when treated with a combination of chemotherapy and the compositions disclosed herein.
Methods to measure gene expression levels are known to persons of skill in the art. Gene expression may be measured by the number of mRNA transcripts or the amount of protein expressed. Exemplary methods to measure the amount of mRNA include but are not limited to Sanger sequencing, high throughput sequencing, quantitative polymerase chain reaction (qPCR), reverse transcriptase qPCR (RT-qPCR), RNA sequencing, microarray analysis, and Northern blots. In some embodiments, gene expression level is measured by RNA-seq. In some embodiments, gene expression level is measured by high coverage mRNA sequencing.
In some embodiments, gene expression level is measured by the amount of mRNA. In some embodiments, the method comprises measuring the amount of mRNA encoding one or more of the following genes in a patient sample: FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9, ST3GAL1, ST3GAL2, ST3GAL3, ST3GAL4, ST3GAL5, ST3GAL6, NEU1, NEU2, NEU3, NEU4, FUCA1, and/or FUCA2.
Gene expression may also be measured by the amount of protein in a patient sample. Exemplary methods to measure the amount of protein include but are not limited to immunostaining, immunohistochemistry, affinity purification, mass spectrometry, Western blotting, and enzyme-linked immunoadsorbent assay (ELISA).
In some embodiments, gene expression level is measured by the amount of protein in a patient sample. In some embodiments, the method comprises measuring the amount of one or more of the following proteins in a patient sample: FUT3 protein, FUT4 protein, FUT5 protein, FUT7 protein, FUT8 protein, FUT9 protein, ST3GAL1 protein, ST3GAL2 protein, ST3GAL3 protein, ST3GAL4 protein, ST3GAL5 protein, ST3GAL6 protein, NEU1 protein, NEU2 protein, NEU3 protein, NEU4 protein, FUCA1 protein, and/or FUCA2 protein.
In some embodiments, high coverage single strand mRNA sequencing may be performed on clinical samples from pediatric AML patients (0 to 30 years old). In some embodiments, the data from this analysis may then be screened for expression of the 24 different genes listed in
In some embodiments, the one or more diagnostic assays may comprise assays to detect expression of E-selectin ligand on the surface of FLT3 AML cells, and may include flow analysis, flow cytometry, or immunohistology using the appropriate reagents. In some embodiments, the reagents for immunohistology may include a HECA-452-FITC monoclonal antibody, or similar reagents. In other embodiments, the reagents for immunohistology may include an E-selectin/hIg chimera/PE, or similar reagents.
In some embodiments, the expression level of a gene involved in the synthesis of sialic acids is measured. In some embodiments, the sialic acid is an α2-3 sialic acid. In some embodiments, the expression level of a gene involved in the degradation of sialic acids is measured. In some embodiments, the expression level of a gene involved in the synthesis of fucose linkages in E-selectin ligands is measured. In some embodiments, the expression level of a gene involved in the degradation of fucose linkages in E-selectin ligands is measured. In some embodiments, the expression level of a gene that encodes a glycotransferase in a patient is measured. In some embodiments, the expression level of a gene that encodes a glycosidase in a patient is measured. In some embodiments, 24 different genes (i.e., those shown in
In some embodiments, the method comprises measuring the expression level(s) of one or more of the following genes in a patient sample: FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9, ST3GAL1, ST3GAL2, ST3GAL3, ST3GAL4, ST3GAL5, ST3GAL6, NEU1, NEU2, NEU3, NEU4, FUCA1, and/or FUCA2.
In some embodiments, one or more diagnostic assays may be used to identify cancer patients likely to benefit from treatment with an E-selectin inhibitor. In some embodiments, the cancer patients likely to benefit from treatment with an E-selectin inhibitor have leukemia. In some embodiments the cancer patients likely to benefit from treatment with an E-selectin inhibitor have AML. In some embodiments the cancer patients likely to benefit from treatment with an E-selectin inhibitor have ALL. In some embodiments the cancer patients likely to benefit from treatment with an E-selectin inhibitor have CLL. In some embodiments the cancer patients likely to benefit from treatment with an E-selectin inhibitor have CML. In some embodiments the cancer patients likely to benefit from treatment with an E-selectin inhibitor have non-Hodgkins lymphoma. In some embodiments the cancer patients likely to benefit from treatment with an E-selectin inhibitor have Hodgkins lymphoma. In some embodiments the cancer patients likely to benefit from treatment with an E-selectin inhibitor have multiple myeloma. In some embodiments the cancer patients likely to benefit from treatment with an E-selectin inhibitor have colorectal cancer. In some embodiments the cancer patients likely to benefit from treatment with an E-selectin inhibitor have liver cancer. In some embodiments the cancer patients likely to benefit from treatment with an E-selectin inhibitor have gastric cancer. In some embodiments the cancer patients likely to benefit from treatment with an E-selectin inhibitor have lung cancer. In some embodiments the cancer patients likely to benefit from treatment with an E-selectin inhibitor have brain cancer. In some embodiments the cancer patients likely to benefit from treatment with an E-selectin inhibitor have kidney cancer. In some embodiments the cancer patients likely to benefit from treatment with an E-selectin inhibitor have bladder cancer. In some embodiments the cancer patients likely to benefit from treatment with an E-selectin inhibitor have thyroid cancer. In some embodiments the cancer patients likely to benefit from treatment with an E-selectin inhibitor have prostrate cancer. In some embodiments the cancer patients likely to benefit from treatment with an E-selectin inhibitor have ovarian cancer. In some embodiments the cancer patients likely to benefit from treatment with an E-selectin inhibitor have cervical cancer. In some embodiments the cancer patients likely to benefit from treatment with an E-selectin inhibitor have uterine cancer. In some embodiments the cancer patients likely to benefit from treatment with an E-selectin inhibitor have endometrial cancer. In some embodiments the cancer patients likely to benefit from treatment with an E-selectin inhibitor have melanoma. In some embodiments the cancer patients likely to benefit from treatment with an E-selectin inhibitor have breast cancer. In some embodiments the cancer patients likely to benefit from treatment with an E-selectin inhibitor have pancreatic cancer. In some embodiments, the one or more diagnostic assays comprises quantitative PCR (polymerase chain reaction).
In some aspects, a method of treating a patient suffering from cancer comprises: (a) determining the gene expression level of one or more genes in the patient or a sample from the patient; (b) comparing the gene expression level from (a) to a control sample from a cancer-free subject, a newly diagnosed cancer subject, or a subject diagnosed with the same cancer as the patient, and when the gene expression level exceeds that in the control sample; then (c) administering one or more doses of a pharmaceutical composition comprising an E-selectin inhibitor to the patient. In some embodiments, the one or more genes is chosen from ST3GAL4, FUT5, and FUT7. In some embodiments, the E-selectin inhibitor is administered in combination with an anti-cancer agent. In some embodiments, gene expression level is determined by high coverage single-strand mRNA sequencing. In some embodiments, the sample from the patient is peripheral blood.
In some aspects, a method of treating a cancer patient comprises: (a) obtaining or having obtained a biological sample comprising blast cells from the cancer patient; (b) performing or having performed an assay on the biological sample to determine the gene expression level of one or more E-selectin ligand-forming genes in the sample; and (c) if the blast cells in the sample have an increased gene expression level of the one or more E-selectin ligand-forming genes relative to a control sample from a non-cancer subject, a newly-diagnosed cancer subject, or a subject having the same cancer as the patient, then administering a therapeutically effective amount of a composition comprising one or more E-selectin inhibitors.
In some embodiments, the control sample is from a person diagnosed with the same cancer as that of the patient. In some embodiments, the control sample is the distribution of gene expression levels of ST3GAL4 in a population of people diagnosed with the same cancer as that of the patient. In some embodiments, the threshold is the 90th percentile, 85th percentile, 80th percentile, 75th percentile, 70th percentile, 65th percentile, 60th percentile, 55th percentile, or 50th percentile level of expression of ST3GAL4 in a population of people diagnosed with the same cancer as that of the patient.
In some embodiments, the control sample is from a person diagnosed with the same cancer as that of the patient. In some embodiments, the control sample is the distribution of gene expression levels of FUT5 in a population of people diagnosed with the same cancer as that of the patient. In some embodiments, the threshold is the 90th percentile, 85th percentile, 80th percentile, 75th percentile, 70th percentile, 65th percentile, 60th percentile, 55th percentile, or 50th percentile level of expression of FUT5 in a population of people diagnosed with the same cancer as that of the patient.
In some embodiments, the control sample is from a person diagnosed with the same cancer as that of the patient. In some embodiments, the control sample is the distribution of gene expression levels of FUT7 in a population of people diagnosed with the same cancer as that of the patient. In some embodiments, the threshold is the 90th percentile, 85th percentile, 80th percentile, 75th percentile, 70th percentile, 65th percentile, 60th percentile, 55th percentile, or 50th percentile level of expression of FUT7 in a population of people diagnosed with the same cancer as that of the patient.
In some aspects, a method of treating a patient suffering from cancer comprises: (a) determining the gene expression level of one or more genes in the patient or a sample from the patient; and (b) administering one or more doses of a pharmaceutical composition comprising an E-selectin inhibitor to the patient if at least 10% of the blast cells in the patient or a sample from the patient express the one or more genes. In some embodiments, the one or more genes are chosen from ST3GAL4, FUT5, and FUT7. In some embodiments, the E-selectin inhibitor is administered in combination with an anti-cancer agent. In some embodiments, gene expression level is determined by high coverage single-strand mRNA sequencing. In some embodiments, the sample from the patient is peripheral blood.
In some aspects, a method of treating a cancer patient comprises: (a) obtaining or having obtained a biological sample comprising blast cells from the cancer patient; (b) performing or having performed an assay on the biological sample to determine the gene expression level of one or more E-selectin ligand-forming genes in the sample; and (c) if at least 10% of the blast cells in the sample express the one or more E-selectin ligand-forming genes, then administering a therapeutically effective amount of a composition comprising one or more E-selectin inhibitors.
In some embodiments, one or more doses of a pharmaceutical composition comprising an E-selectin inhibitor, e.g., the compound of Formula I, is administered in combination with an anti-cancer agent to a patient who has been pre-screened by the criteria as disclosed herein as having, e.g., increased expression of ST3GAL4, FUT5, or FUT7.
In some aspects, a method of selecting a patient to treat for cancer comprises: (a) determining the gene expression level of one or more genes in the patient or a sample from the patient; (b) selecting the patient for treatment when the patient or sample from the patient has an increased gene expression level relative to a control sample; and (c) treating the patient by administering one or more doses of a pharmaceutical composition comprising an E-selectin inhibitor. In some embodiments, the one or more genes are chosen from ST3GAL4, FUT5, and FUT7. In some embodiments, the E-selectin inhibitor is administered in combination with an anti-cancer agent. In some embodiments, gene expression level is determined by high coverage single-strand mRNA sequencing. In some embodiments, the sample from the patient is peripheral blood.
In some aspects, a method of screening a cancer patient for treatment comprises: (a) obtaining or having obtained a biological sample comprising blast cells from the cancer patient; (b) performing or having performed an assay on the biological sample to determine the gene expression level of one or more E-selectin ligand-forming genes in the sample; and (cxi) if the blast cells in the sample have an increased expression level of the one or more E-selectin ligand-forming genes relative to a control sample from a non-cancer subject, a newly-diagnosed cancer subject, or a subject having the same cancer as the patient, or (c)(ii) if at least 10% of the blast cells in the sample express the one or more E-selectin ligand-forming genes, then (d) selecting the patient for treatment comprising one or more E-selectin inhibitors.
In some embodiments, the control sample is from a patient suffering from AML. In some embodiments, the control sample is the distribution of gene expression levels of ST3GAL4 in a population of patients suffering from AML. In some embodiments, the threshold is the 90th percentile, 85th percentile, 80th percentile, 75th percentile, 70th percentile, 65th percentile, 60th percentile, 55th percentile, or 50th percentile level of expression of ST3GAL4 in a population of AML patients. In some embodiments, the control sample is the distribution of gene expression levels of FUT5 in a population of patients suffering from AML. In some embodiments, the threshold is the 90th percentile, 85th percentile, 80th percentile, 75th percentile, 70th percentile, 65th percentile, 60th percentile, 55th percentile, or 50th percentile level of expression of FUT5 in a population of AML patients. In some embodiments, the control sample is the distribution of gene expression levels of FUT7 in a population of patients suffering from AML. In some embodiments, the threshold is the 90th percentile, 85th percentile, 80th percentile, 75th percentile, 70th percentile, 65th percentile, 60th percentile, 55th percentile, or 50th percentile level of expression of FUT7 in a population of AML patients.
In some embodiments, the treated patient has expression of ST3GAL4 greater than that of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of patients with relapsed/refractory AML. In some embodiments, the treated patient has expression of FUT5 greater than that of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of patients with relapsed/refractory AML. In some embodiments, the treated patient has expression of FUT7 greater than that of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of patients with relapsed/refractory AML. In some embodiments, the treated patient has expression of ST3GAL4 and FUT5 greater than that of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of patients with relapsed/refractory AML. In some embodiments, the treated patient has expression of ST3GAL4 and FUT7 greater than that of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of patients with relapsed/refractory AML. In some embodiments, the treated patient has expression of FUT5 and FUT7 greater than that of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of patients with relapsed/refractory AML. In some embodiments, the treated patient has expression of ST3GAL4, FUT5, and FUT7 greater than that of 55%, 600%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of patients with relapsed/refractory AML.
In some aspects, a method of selecting a patient to treat for cancer comprises: (a) determining the gene expression level of one or more genes in the patient or a sample from the patient; (b) selecting the patient for treatment when at least 10% of the blast cells from the patient or sample from the patient expresses the one or more genes; and (c) treating the patient by administering one or more doses of a pharmaceutical composition comprising an E-selectin inhibitor. In some embodiments, the one or more genes are chosen from ST3GAL4, FUT5, and FUT7. In some embodiments, the E-selectin inhibitor is administered in combination with an anti-cancer agent. In some embodiments, gene expression level is determined by high coverage single-strand mRNA sequencing. In some embodiments, the sample from the patient is peripheral blood.
In some embodiments, a method of treating FLT3 AML patients with antagonists of E-selectin is disclosed, the method comprising administering to a FLT3 AML patient an effective amount of at least one E-selectin antagonist and/or a pharmaceutical composition comprising at least one E-selectin antagonist. In some embodiments, the at least one E-selectin antagonist is the compound of Formula I.
In some embodiments, the method further comprises administering at least one additional therapeutic agent. In some embodiments, the at least one additional therapeutic agent is chosen from chemotherapy agents and kinases inhibitors targeting FLT3.
Methods of treating AML comprising administering to a subject in need thereof an effective amount of the compound of Formula I and compositions comprising the same have been reported. See, e.g., PCT/US2019/020574. The compound of Formula I was rationally designed based on the bioactive conformation of sialyl Lea/x in the binding site of E-selectin and is a potent and specific glycomimetic antagonist of E-selectin.
Contemplated herein are compositions for treating cancer patients in need thereof, comprising E-selectin inhibitors. E-selectin is a transmembrane adhesion protein expressed on the surface of endothelial cells lining the blood vessel. E-selectin recognizes and binds to sialylated carbohydrates, e.g., members of the Lewis X and Lewis A families found on monocytes, granulocytes, and T-lymphocytes. When expressed, it causes cells which express E-selectin ligands on their surface to adhere.
As discussed in detail herein, the disease or disorder to be treated is a cancer and related metastasis and includes cancers that comprise solid tumors and cancers that comprise liquid tumors. E-selectin plays a central role in the progression of cancer. The invasive properties of cancer cells depend, at least in part, on the capability of cancer cells to breach the endothelial barrier. Cancer cells, for example, colon cancer cells, may express E-selectin ligands that are capable of binding to endothelial cells that express E-selectin on their cell surface. Without wishing to be limited to any theory, binding of cancer cells to the endothelial cells can contribute to extravasation of the cancer cells.
Cancers that may be prevented from metastasizing include cancers that comprise solid tumors and those that comprise liquid tumors (e.g., hematological malignancies). Examples of solid tumors that may be treated with the agents described herein include colorectal cancer, liver cancer, gastric cancer, lung cancer, brain cancer, kidney cancer, bladder cancer, thyroid cancer, prostrate cancer, ovarian cancer, cervical cancer, uterine cancer, endometrial cancer, melanoma, breast cancer and pancreatic cancer. Liquid tumors occur in the blood, bone marrow, and lymph nodes and include leukemia (e.g., AML, ALL, CLL, and CML), lymphoma (e.g., non-Hodgkins lymphoma and Hodgkins lymphoma) and myeloma (e.g., multiple myeloma). Reports have described that liquid tumors such as multiple myeloma follow a similar invasion-metastasis cascade as observed with solid tumors and that E-selectin ligands are present on liquid tumor cells, such as myeloma cells. Others have observed that ligands of E-selectin may be important for extravascular infiltration of leukemia cells. Liquid tumor cells may also adhere to bone marrow, which may further lead to sequestration and quiescence of the tumor cells to chemotherapy, which phenomenon is referred to as adhesion mediated drug resistance. Studies have also indicated that bone marrow contains anatomic regions that comprise specialized endothelium, which expresses the E-selectin. Accordingly, an E-selectin antagonist, such as those described herein, may be useful for inhibiting metastasis of cancers that comprise either a solid or liquid tumor by inhibiting binding of an E-selectin ligand to E-selectin.
Methods of treating cancer are known to a skilled artisan, and may include, but are not limited to chemotherapy, radiation therapy, chemotherapy with stem cell transplant, other drugs such as arsenic trioxide and all-trans retinoic acid, and targeted therapy (e.g. a monoclonal antibody).
Contemplated herein are methods of treating cancer patients in need thereof, comprising administering a therapeutically effective amount of a composition comprising an E-selectin inhibitor, e.g., the compound of Formula I. The composition disclosed herein may be administered by parenteral, topical, intradermal, intravenous, oral, subcutaneous, intraperitoneal, intranasal or intramuscular means for prophylactic and/or therapeutic treatment.
Methods of treating cancer comprising administering to a subject in need thereof an effective amount of a compound of Formula I and compositions comprising the same have been reported. See, e.g., PCT/US2019/020574, the disclosure of which is expressly incorporated by reference in its entirety. The compound of Formula I was rationally designed based on the bioactive conformation of sialyl Lea/x in the binding site of E-selectin and is a potent and specific glycomimetic antagonist of E-selectin.
In some embodiments, the composition is delivered by subcutaneous delivery. In some embodiments, the composition is delivered by subcutaneous delivery to the upper arm. In some embodiments, the composition is delivered by subcutaneous delivery to the abdomen. In some embodiments, the composition is delivered by subcutaneous delivery to the thigh. In some embodiments, the composition is delivered by subcutaneous delivery to the upper back. In some embodiments the composition is delivered by subcutaneous delivery to the buttock.
In some embodiments, the composition is delivered by intravenous infusion.
In some embodiments, the composition is delivered in combination with one or more anti-cancer agents. In some embodiments, the composition is delivered in combination with chemotherapy. Chemotherapy may comprise one or more chemotherapeutic agent(s). For example, chemotherapy agents, radiotherapy agents, inhibitors of phosphoinoditide-3 kinase (PI3K), and inhibitors of VEGF may be used in combination with an agent described herein. Examples of inhibitors of PI3K include the compound named Exelixis as “XL499”. Examples of VEGF inhibitors include the compound “cabo” (previously known as XL184). Many other chemotherapeutics are small organic molecules. As understood by a person skilled in the art, chemotherapy may also refer to a combination of two or more chemotherapeutic molecules that are administered coordinately and which may be referred to as combination chemotherapy. Numerous chemotherapeutic drugs are used in the oncology art and include, for example, alkylating agents, antimetabolites, anthracyclines, plant alkaloids and topoisomerase inhibitors. Examples of therapeutic agents administered for chemotherapy are well known to the skilled artisan. In some embodiments, the composition is delivered in combination with induction chemotherapy. In some embodiments, the composition is delivered in combination with mitoxantrone. In some embodiments, the composition is delivered in combination with etoposide. In some embodiments, the composition is delivered in combination with cytarabine. In some embodiments, the composition is delivered together with at least one of mitoxantrone, etoposide, and cytarabine. In some embodiments, the composition is delivered in combination with consolidation chemotherapy. In some embodiments, the composition is delivered in combination with daunomycin. In some embodiments, the composition is delivered in combination with idarubicin. In some embodiments, the composition is delivered in combination with MEC (mitoxantrone, etoposide, cytarabine) chemotherapy. In some embodiments, the composition is delivered in combination with 7+3 (cytarabine for 7 days then daunorubicin, idarubicin, or mitoxantrone for 3 days) chemotherapy.
In some embodiments, the anti-cancer agents are anti-leukemic agents. Examples of anti-leukemic agents are well-known to the skilled artisan, and include but are not limited to cyclophosphamide, methotrexate, and etoposide. In some embodiments, the composition is delivered in combination with 6-mercaptopurine. In some embodiments, the composition is delivered in combination with 6-thioguanine. In some embodiments, the composition is delivered in combination with aminopterin. In some embodiments, the composition is delivered in combination with arsenic trioxide. In some embodiments, the composition is delivered in combination with asparaginase. In some embodiments, the composition is delivered in combination with cladribine. In some embodiments, the composition is delivered in combination with clofarabine. In some embodiments, the composition is delivered in combination with cyclophosphamide. In some embodiments, the composition is delivered in combination with cytosine arabinoside. In some embodiments, the composition is delivered in combination with dasatinib. In some embodiments, the composition is delivered in combination with decitabine. In some embodiments, the composition is delivered in combination with dexamethasone. In some embodiments, the composition is delivered in combination with fludarabine. In some embodiments, the composition is delivered in combination with gemtuzumab ozogamicin. In some embodiments, the composition is delivered in combination with imatinib mesylate. In some embodiments, the composition is delivered in combination with interferon-α. In some embodiments, the composition is delivered in combination with interleukin-2. In some embodiments, the composition is delivered in combination with melphalan. In some embodiments, the composition is delivered in combination with methotrexate. In some embodiments, the composition is delivered in combination with nelarabine. In some embodiments, the composition is delivered in combination with nilotinib. In some embodiments, the composition is delivered in combination with oblimersen. In some embodiments, the composition is delivered in combination with pegaspargase. In some embodiments, the composition is delivered in combination with pentostatin. In some embodiments, the composition is delivered in combination with ponatinib. In some embodiments, the composition is delivered in combination with prednisone. In some embodiments, the composition is delivered in combination with rituximab. In some embodiments, the composition is delivered in combination with tretinoin. In some embodiments, the composition is delivered in combination with vincristine.
In some embodiments, the anti-cancer agent may be radiation. In some embodiments, the composition may be delivered in combination with external beam radiation.
In various embodiments, the composition is administered over one or more doses, with one or more intervals between doses. In some embodiments, the composition is administered over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 doses. In some embodiments, the composition is administered at 6-hour, 12-hour, 18-hour, 24-hour, 48-hour, 72-hour, or 96-hour intervals. In some embodiments, the composition is administered at one interval, and then administered at a different interval, e.g., 1 dose 24 hours before chemotherapy, then twice-daily doses throughout chemotherapy. In some embodiments, the composition is administered at 1 dose 24 hours before chemotherapy, then twice-daily doses throughout chemotherapy up till 48 hours post-chemotherapy.
In some embodiments, the methods and materials disclosed herein are indicated for and can be used in the treatment of AML, e.g., by subcutaneous or intravenous administration to a patient showing the symptoms of the disease. In some embodiments, the methods and materials disclosed herein are indicated for and can be used in the treatment of ALL. In some embodiments, the methods and materials disclosed herein are indicated for and can be used in the treatment of CLL. In some embodiments, the methods and materials disclosed herein are indicated for and can be used in the treatment of CML. In some embodiments, the methods and materials disclosed herein are indicated for and can be used in the treatment of non-Hodgkins lymphoma. In some embodiments, the methods and materials disclosed herein are indicated for and can be used in the treatment of Hodgkins lymphoma. In some embodiments, the methods and materials disclosed herein are indicated for and can be used in the treatment of multiple myeloma. In some embodiments, the methods and materials disclosed herein are indicated for and can be used in the treatment of colorectal cancer. In some embodiments, the methods and materials disclosed herein are indicated for and can be used in the treatment of liver cancer. In some embodiments, the methods and materials disclosed herein are indicated for and can be used in the treatment of gastric cancer. In some embodiments, the methods and materials disclosed herein are indicated for and can be used in the treatment of lung cancer. In some embodiments, the methods and materials disclosed herein are indicated for and can be used in the treatment of brain cancer. In some embodiments, the methods and materials disclosed herein are indicated for and can be used in the treatment of kidney cancer. In some embodiments, the methods and materials disclosed herein are indicated for and can be used in the treatment of bladder cancer. In some embodiments, the methods and materials disclosed herein are indicated for and can be used in the treatment of thyroid cancer. In some embodiments, the methods and materials disclosed herein are indicated for and can be used in the treatment of prostrate cancer. In some embodiments, the methods and materials disclosed herein are indicated for and can be used in the treatment of ovarian cancer. In some embodiments, the methods and materials disclosed herein are indicated for and can be used in the treatment of In some embodiments, the methods and materials disclosed herein are indicated for and can be used in the treatment of cervical cancer. In some embodiments, the methods and materials disclosed herein are indicated for and can be used in the treatment of uterine cancer. In some embodiments, the methods and materials disclosed herein are indicated for and can be used in the treatment of endometrial cancer. In some embodiments, the methods and materials disclosed herein are indicated for and can be used in the treatment of melanoma. In some embodiments, the methods and materials disclosed herein are indicated for and can be used in the treatment of breast cancer. In some embodiments, the methods and materials disclosed herein are indicated for and can be used in the treatment of pancreatic cancer.
In some embodiments, an effective dose is a dose that partially or fully alleviates (i.e., eliminates or reduces) at least one symptom associated with the disorder/disease state being treated, that slows, delays, or prevents onset or progression to a disorder/disease state, that slows, delays, or prevents progression of a disorder/disease state, that diminishes the extent of disease, that reverse one or more symptom, that results in remission (partial or total) of disease, and/or that prolongs survival. Examples of disease states contemplated for treatment are set out herein. In some embodiments, the patient currently has cancer, was once treated for cancer and is in remission, or is at risk of relapsing after treatment for the cancer.
In some embodiments, a pharmaceutical composition as disclosed herein is administered, e.g., subcutaneously or intravenously, to a patient in need of treatment for AML. In some embodiments, the patient has been diagnosed with AML as per the World Health Organization (WHO) criteria. Arber D A et al., “The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia.” Blood (2016) 127(20):2391-2405. In some embodiments, the patients are ≥18 years of age with relapsed or refractory AML after ≤2 prior induction regiments, at least one containing anthracyclines. In some embodiments, the patient is ≥60 years of age with newly diagnosed AML. In some embodiments, the patient has an absolute blast count 9ABC) of ≤40,000/mm. In some embodiments, the patient is medically eligible to receive MEC chemotherapy. In some embodiments, the patient is medically eligible to receive 7+3 cytarabine/idarubicin chemotherapy. In some embodiments, the patient has an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2. In some embodiments, the patient has hemodynamically stable and adequate organ function. In some embodiments, the patient does not have acute promyelocytic leukemia. In some embodiments, the patient does not have acute leukemia of ambiguous lineage. In some embodiments, the patient does not have active signs or symptoms of CNS involvement by malignancy. In some embodiments, the patient has no prior G-CSF, GM-CSF or plerixafor within 14 days of treatment with the pharmaceutical composition disclosed herein. In some embodiments, the patient has no known history or evidence of active hepatitis A, B, or C or HIV. In some embodiments, the patient does not have uncontrolled acute life-threatening bacterial, viral, or fungal infection. In some embodiments, the patient does not have active graft versus host disease (GVHD)≥Grade 2 or extensive chronic GVHD requiring immunosuppressive therapy. In some embodiments, the patient does not have hematopoietic stem cell transplantation ≤4 months prior to the treatments disclosed herein. In some embodiments, the patient does not have clinically significant cardiovascular disease.
In some embodiments, the E-selectin inhibitor is chosen from the compound of Formula I, prodrugs of the compound of Formula I, and pharmaceutically acceptable salts of any of the foregoing. In some embodiments, the E-selectin inhibitor is the compound of Formula I. In some embodiments, the E-selectin inhibitor is chosen from pharmaceutically acceptable salts of the compound of Formula I. In some embodiments, the pharmaceutically acceptable salt is a sodium salt.
In some embodiments, the E-selectin antagonist is chosen from compounds of Formula Ix:
prodrgus of Formula Ix, and pharmaceutically acceptable salts of any of the foregoing, wherein:
In some embodiments, the E-selectin antagonist is chosen from compounds of Formula Ix, wherein the non-glycomimetic moiety comprises polyethylene glycol.
In some embodiments, the E-selectin antagonist is chosen from compounds of Formula Ix, wherein the linker is —C(═O)NH(CH2)1-4NHC(═O)— and the non-glycomimetic moiety comprises polyethylene glycol.
In some embodiments, the E-selectin inhibitor is chosen from the compound of Formula Ix, prodrugs of compounds of Formula Ix and pharmaceutically acceptable salts of any of the foregoing. In some embodiments, the E-selectin inhibitor is the compound of Formula Ix. In some embodiments, the E-selectin inhibitor is chosen from pharmaceutically acceptable salts of the compound of Formula Ix.
In some embodiments, the E-selectin antagonist is chosen from compounds of Formula Ia:
and pharmaceutically acceptable salts thereof, wherein n is chosen from integers ranging from 1 to 100. In some embodiments, n is chosen from 4, 8, 12, 16, 20, 24, and 28. In some embodiments n is 12.
In some embodiments, the E-selectin antagonist is a heterobifunctional antagonist chosen from compounds of Formula II:
prodrugs of compounds of Formula II, and pharmaceutically acceptable salts of any of the foregoing, wherein:
In some embodiments, the E-selectin antagonist is a heterobifunctional antagonist chosen from compounds of Formula IIa:
and pharmaceutically acceptable salts thereof.
In some embodiments, the linker groups of Formula Ix and/or Formula II are independently chosen from groups comprising spacer groups, such spacer groups as, for example, —(CH2)p— and —O(CH2)p—, wherein p is chosen from integers ranging from 1 to 30. In some embodiments, p is chosen from integers ranging from 1 to 20.
Other non-limiting examples of spacer groups include carbonyl groups and carbonyl-containing groups such as, for example, amide groups. A non-limiting example of a spacer group is
In some embodiments, the linker groups of Formula Ix and/or Formula II are independently chosen from
Other linker groups, such as, for example, polyethylene glycols (PEGs) and —C(═O)—NH—(CH2)p—C(═O)—NH—, wherein p is chosen from integers ranging from 1 to 30, or wherein p is chosen from integers ranging from 1 to 20, will be familiar to those of ordinary skill in the art and/or those in possession of the present disclosure.
In some embodiments, at least one linker group of Formula Ix and/or Formula II is
In some embodiments, at least one linker group of Formula Ix and/or Formula II is
In some embodiments, at least one linker group of Formula Ix and/or Formula II is chosen from —C(═O)NH(CH2)2NH—, —CH2NHCH2—, and —C(═O)NHCH2—. In some embodiments, at least one linker group is —C(═O)NH(CH2)2NH—.
In some embodiments, the E-selectin antagonist is chosen from Compound B:
and pharmaceutically acceptable salts thereof.
In some embodiments, the E-selectin antagonist is chosen from compounds of Formula III:
prodrugs of compounds of Formula III, and pharmaceutically acceptable salts of any of the foregoing, wherein:
In some embodiments, the E-selectin antagonist is chosen from compounds of Formula IV:
prodrugs of compounds of Formula IV, and pharmaceutically acceptable salts of any of the foregoing, wherein:
In some embodiments, the E-selectin antagonist of Formula III or Formula IV is chosen from compounds of the following Formula IIIa/IVa (see definitions of L and m for Formula III or IV above):
In some embodiments, the E-selectin antagonist of Formula III or Formula IV is chosen from compounds of the following Formula IIIb/IVb (see definitions of L and m for Formula III or IV above):
In some embodiments, the E-selectin antagonist is Compound C:
In some embodiments, the E-selectin antagonist is a heterobifunctional inhibitor of E-selectin and Galectin-3, chosen from compounds of Formula V:
prodrugs of compounds of Formula V, and pharmaceutically acceptable salts of any of the foregoing, wherein:
In some embodiments, the E-selectin antagonist is chosen from compounds having the following Formulae:
In some embodiments, the E-selectin antagonist is chosen from compounds having the following Formulae:
In some embodiments, the E-selectin antagonist is Compound D:
In some embodiments, the E-selectin antagonist is chosen from compounds of Formula VI:
prodrugs of compounds of Formula VI, and pharmaceutically acceptable salts of any of the foregoing, wherein:
In some embodiments of Formula VI, M is chosen from
In some embodiments of Formula VI, M is chosen from
In some embodiments of Formula VI, linker groups may be chosen from groups comprising spacer groups, such spacer groups as, for example, —(CH2)— and —O(CH2)—, wherein t is chosen from integers ranging from 1 to 20. Other non-limiting examples of spacer groups include carbonyl groups and carbonyl-containing groups such as, for example, amide groups. A non-limiting example of a spacer group is
In some embodiments of Formula VI, the linker group is chosen from
In some embodiments of Formula VI, the linker group is chosen from polyethylene glycols (PEGs), —C(═O)NH(CH2)vO—, —C(═O)NH(CH2)vNHC(═O)—, —C(═O)NHC(═O)(CH2)NH—, and —C(═O)NH(CH2)vC(═O)NH— groups, wherein v is chosen from integers ranging from 2 to 20. In some embodiments, v is chosen from integers ranging from 2 to 4. In some embodiments, v is 2. In some embodiments, v is 3. In some embodiments, v is 4.
In some embodiments of Formula VI, the linker group is
In some embodiments of Formula VI, the linker group is
In some embodiments of Formula VI, the linker group is
In some embodiments of Formula VI, the linker group is
In some embodiments of Formula VI, the linker group is
In some embodiments of Formula VI, the linker group is
In some embodiments of Formula VI, the linker group is
In some embodiments of Formula VI, the linker group is
In some embodiments of Formula VI, the linker group is
In some embodiments, the E-selectin antagonist is a multimeric inhibitor of E-selectin, Galectin-3, and/or CXCR4, chosen from compounds of Formula VII:
prodrugs of compounds of Formula VII, and pharmaceutically acceptable salts of any of the foregoing, wherein:
In some embodiments of Formula VII, at least one linker group is chosen from groups comprising spacer groups, such spacer groups as, for example, —(CH2)z— and —O(CH2)z—, wherein z is chosen from integers ranging from 1 to 250. Other non-limiting examples of spacer groups include carbonyl groups and carbonyl-containing groups such as, for example, amide groups. A non-limiting example of a spacer group is
In some embodiments of Formula VII, at least one linker group is chosen from
Other linker groups for certain embodiments of Formula VII, such as, for example, polyethylene glycols (PEGs) and —C(═O)—NH—(CH2)z—C(═O)—NH—, wherein z is chosen from integers ranging from 1 to 250, will be familiar to those of ordinary skill in the art and/or those in possession of the present disclosure.
In some embodiments of Formula VII, at least one linker group is
In some embodiments of Formula VII, at least one linker group is
In some embodiments of Formula VII, at least one linker group is chosen from —C(═O)NH(CH2)2NH—, —CH2NHCH2—, and —C(═O)NHCH2—. In some embodiments of Formula VII, at least one linker group is —C(═O)NH(CH2)2NH—.
In some embodiments of Formula VII, L is chosen from dendrimers. In some embodiments of Formula VII, L is chosen from polyamidoamine (“PAMAM”) dendrimers. In some embodiments of Formula VII, L is chosen from PAMAM dendrimers comprising succinamic. In some embodiments of Formula VII, L is PAMAM GO generating a tetramer. In some embodiments of Formula VII, L is PAMAM G1 generating an octamer. In some embodiments of Formula VII, L is PAMAM G2 generating a 16-mer. In some embodiments of Formula VII, L is PAMAM G3 generating a 32-mer. In some embodiments of Formula VII, L is PAMAM G4 generating a 64-mer. In some embodiments, L is PAMAM G5 generating a 128-mer.
In some embodiments of Formula VII, m is 2 and L is chosen from
In some embodiments of Formula VII, L is chosen from
In some embodiments of Formula VII, L is chosen from
In some embodiments of Formula VII, L is
In some embodiments of Formula VII, L is chosen from
In some embodiments of Formula VII, L is chosen from
In some embodiments of Formula VII, L is chosen from
In some embodiments of Formula VII, L is
In some embodiments of Formula VII, L is chosen from
In some embodiments of Formula VII, L is
In some embodiments of Formula VII, L is
In some embodiments of Formula VII, L is
In some embodiments of Formula VII, L is chosen from
In some embodiments of Formula VII, L is
In some embodiments of Formula VII, L is chosen from
wherein each y, which may be identical or different, is independently chosen from integers ranging from 0 to 250.
In some embodiments of Formula VII, L is chosen from
wherein each y, which may be identical or different, is independently chosen from integers ranging from 0 to 250.
In some embodiments of Formula VII, L is chosen from
In some embodiments, at least one compound is chosen from compounds of Formula VII, wherein each R1 is identical, each R2 is identical, each R3 is identical, each R4 is identical, each R5 is identical, and each X is identical. In some embodiments, at least one compound is chosen from compounds of Formula VII, wherein said compound is symmetrical.
Provided are pharmaceutical compositions comprising at least one compound chosen from compounds of Formula Ix, Ia, II, IIa, III, IV, IIIa/IVa, IIIb/IVb, V, VI, and VII, and pharmaceutically acceptable salts of any of the foregoing. Also provided are pharmaceutical compositions comprising at least one compound chosen from the compound of Formula I, compound B, compound C, and compound D, and pharmaceutically acceptable salts of any of the foregoing. These compounds and compositions may be used in the methods described herein.
Compound 3: A mixture of compound 1 (preparation described in WO 2007/028050) and compound 2 (preparation described in WO 2013/096926) (1.7 eq) is azeotroped 3 times from toluene. The mixture is dissolved in DCM under argon and cooled on an ice bath. To this solution is added boron trifluoride etherate (1.5 eq). The reaction mixture is stirred 12 hours at room temperature. The reaction is quenched by the addition of triethylamine (2 eq). The reaction mixture is transferred to a separatory funnel and washed 1 time with half saturated sodium bicarbonate solution and 1 time with water. The organic phase is dried over sodium sulfate, filtered, and concentrated. The residue is purified by flash chromatography to afford compound 3.
Compound 4: Compound 3 is dissolved in methanol at room temperature. A solution of sodium methoxide in methanol (0.1 eq) is added and the reaction mixture stirred overnight at room temperature. The reaction mixture is quenched by the addition of acetic acid. The reaction mixture is diluted with ethyl acetate, transferred to a separatory funnel and washed 2 times with water. The organic phase is dried over magnesium sulfate, filtered and concentrated. The residue is separated by flash chromatography to afford compound 4.
Compound 5: To a solution of compound 4 in dichloromethane cooled on an ice bath is added DABCO (1.5 eq) followed by monomethoxytrityl chloride (1.2 eq). The reaction mixture is stirred overnight allowing to warm to room temperature. The reaction mixture is transferred to a separatory funnel and washed 2 times with water. The organic phase is concentrated and the residue is purified by flash chromatography to afford compound 5.
Compound 7: To a solution of compound 5 in methanol is added dibutyltin oxide (1.1 eq). The reaction mixture is refluxed for 3 hours then concentrated. The residue is suspended in DME. To this suspension is added compound 6 (preparation described in Thoma et. al. J. Med. Chem., 1999, 42, 4909) (1.5 eq) followed by cesium fluoride (1.2 eq). The reaction mixture is stirred at room temperature overnight. The reaction mixture is diluted with ethyl acetate, transferred to a separatory funnel, and washed with water. The organic phase is dried over sodium sulfate, filtered and concentrated. The residue is purified by flash chromatography to afford compound 7.
Compound 8: To a degassed solution of compound 7 in anhydrous DCM at 0° C. is added Pd(PPh3)4 (0.1 eq), Bu3SnH (1.1 eq) and N-trifluoroacetyl glycine anhydride (2.0 eq) (preparation described in Chemische Berichte (1955), 88(1), 26). The resulting solution is stirred for 12 hrs allowing the temperature to increase to room temperature. The reaction mixture is diluted with DCM, transferred to a separatory funnel, and washed with water. The organic phase is dried over Na2SO4, then filtered and concentrated. The residue is purified by flash chromatography to afford compound 8.
Compound 9: To a stirred solution of compound 8 in DCM/MeOH (25/1) at room temperature is added orotic acid chloride (5 eq) and triphenylphosphine (5 eq). The reaction mixture is stirred 24 hours. The solvent is removed and the residue is separated by column chromatography to afford compound 9.
Compound 10: Compound 9 is dissolved in methanol and degassed. To this solution is added Pd(OH)2/*C. The reaction mixture is vigorously stirred under a hydrogen atmosphere for 12 hours. The reaction mixture is filtered through a Celite pad. The filtrate is concentrated under reduced pressure to give compound 10.
Compound 11: Compound 10 is dissolved in methanol at room temperature. A solution of sodium methoxide in methanol (1.1 eq) is added and the reaction mixture stirred overnight at room temperature. The reaction mixture is quenched by the addition of acetic acid. The reaction mixture is concentrated. The residue is separated by C-18 reverse phase chromatography to afford compound 11.
Compound 12: Compound 12 can be prepared in an analogous fashion to
Compound 13: Compound 10 is dissolved in DMF and cooled on an ice bath. Diisopropylethylamine (1.5 eq) is added followed by HATU (1.1 eq). The reaction mixture is stirred 15 minutes on the ice bath then azetidine (2 eq) is added. The ice bath is removed and the reaction mixture is stirred overnight at room temperature. The solvent is removed under reduced pressure and the residue is separated by flash chromatography to afford compound 13.
Compound 14: Compound 13 is dissolved in methanol at room temperature. A solution of sodium methoxide in methanol (0.3 eq) is added and the reaction mixture stirred overnight at room temperature. The reaction mixture is quenched by the addition of acetic acid. The reaction mixture is concentrated. The residue is separated by C-18 reverse phase chromatography to afford compound 14.
Compound 15: Compound 15 can be prepared in an analogous fashion to
Compound 16: Compound 16 can be prepared in an analogous fashion to
Compound 17: Compound 17 can be prepared in an analogous fashion to
Compound 18: Compound 18 can be prepared in an analogous fashion to
Compound 21: Compound 19 can be prepared in an analogous fashion to
Compound 21: A solution of compound 20 (0.4 eq) in DMSO is added to a solution of compound 11 (1 eq) and DIPEA (10 eq) in anhydrous DMSO at room temperature. The resulting solution is stirred overnight. The solution is dialyzed against distilled water for 3 days with dialysis tube MWCO 1000 while distilled water is changed every 12 hours. The solution in the tube is lyophilized to give compound 21.
Compound 22: A solution of compound 21 in ethylenediamine is stirred overnight at 70° C. The reaction mixture is concentrated under reduced pressure and the residue is purified by reverse phase chromatography to give compound 22.
Compound 23: Compound 23 can be prepared in an analogous fashion to
Compound 24: Compound 24 can be prepared in an analogous fashion to
Compound 25: Compound 25 can be prepared in an analogous fashion to
Compound 26: Compound 26 can be prepared in an analogous fashion to
Compound 27: Compound 27 can be prepared in an analogous fashion to
Compound 28: Compound 28 can be prepared in an analogous fashion to
Compound 29: Compound 29 can be prepared in an analogous fashion to
Compound 30: Compound 30 can be prepared in an analogous fashion to
Compound 31: Compound 31 can be prepared in an analogous fashion to
Compound 32: Compound 32 can be prepared in an analogous fashion to
Compound 33: Compound 33 can be prepared in an analogous fashion to
Compound 34: Compound 34 can be prepared in an analogous fashion to
Compound 36: To a solution of compound 12 in MeOH at room temperature is added compound 35 followed by cesium acetate (2.5 eq). The reaction mixture is stirred at room temperature until completion. The solvent is removed under reduced pressure. The product is purified by reverse phase chromatography to give compound 36.
Compound 37: Compound 36 is dissolved in ethylenediamine and the reaction mixture is stirred overnight at 70° C. The reaction mixture is concentrated under reduced pressure and the residue is purified by reverse phase chromatography to give compound 37.
Compound 38: Compound 38 can be prepared in an analogous fashion to
Compound 39: Compound 39 can be prepared in an analogous fashion to
Compound 40: Compound 40 can be prepared in an analogous fashion to
Compound 41: To a stirred solution of compound 7 in DCM/MeOH (25/1) at room temperature is added orotic acid chloride (5 eq) and triphenylphosphine (5 eq). The reaction mixture is stirred 24 hours. The solvent is removed and the residue is separated by column chromatography to afford compound 41.
Compound 42: To a degassed solution of compound 41 in anhydrous DCM at 0° C. is added Pd(PPh3)4 (0.1 eq), Bu3SnH (1.1 eq) and azidoacetic anhydride (2.0 eq). The ice bath is removed and the solution is stirred for 12 hrs under a N2 atmosphere at room temperature. The reaction mixture is diluted with DCM, washed with water, dried over Na2SO4, then concentrated. The crude product is purified by column chromatography to give compound 42.
Compound 44: A solution of bispropagyl PEG-5 (compound 43) and compound 42 (2.4 eq) in MeOH is degassed at room temperature. A solution of CuSO4/THPTA in distilled water (0.04 M) (0.2 eq) and sodium ascorbate (0.2 eq) are added successively and the resulting solution is stirred 12 hrs at 70° C. The solution is cooled to room temperature and concentrated under reduced pressure. The crude product is purified by chromatography to give compound 44.
Compound 45: Compound 44 is dissolved in MeOH/i-PrOH (2/1) and hydrogenated in the presence of Pd(OH)2 (20 wt %) at 1 atm of H2 gas pressure for 24 hrs at room temperature. The solution is filtered through a Celite pad. The filtrate is concentrated to give compound 45.
Compound 46: Compound 45 is dissolved in ethylenediamine and stirred for 12 hrs at 70° C. The reaction mixture is concentrated under reduced pressure. The crude product is purified by C-18 column chromatography followed by lyophilization to give a compound 46.
Compound 47: Compound 47 can be prepared in an analogous fashion to
Compound 48: Compound 48 can be prepared in an analogous fashion to
Compound 49: Compound 49 can be prepared in an analogous fashion to
Compound 50: Compound 50 can be prepared in an analogous fashion to
Compound 51: Compound 51 can be prepared in an analogous fashion to
Compound 52: Compound 52 can be prepared in an analogous fashion to
Compound 53: Compound 53 can be prepared in an analogous fashion to
Compound 54: Compound 54 can be prepared in an analogous fashion to
Compound 55: Compound 54 is dissolved in DMF and cooled on an ice bath. Diisopropylethylamine (2.5 eq) is added followed by HATU (2.2 eq). The reaction mixture is stirred 15 minutes on the ice bath then azetidine (10 eq) is added. The ice bath is removed and the reaction mixture is stirred overnight at room temperature. The solvent is removed under reduced pressure and the residue is separated by flash chromatography to afford compound 55.
Compound 56: Compound 55 is dissolved in ethylenediamine and stirred for 12 hrs at 70° C. The reaction mixture is concentrated under reduced pressure. The crude product is purified by C-18 column chromatography followed by lyophilization to give a compound 56.
Compound 57: Compound 57 can be prepared in an analogous fashion to
Compound 58: Compound 58 can be prepared in an analogous fashion to
Compound 59: Compound 59 can be prepared in an analogous fashion to
Compound 60: To a stirred solution of compound 1 in DCM/MeOH (25/1) at room temperature is added orotic acid chloride (5 eq) and triphenylphosphine (5 eq). The reaction mixture is stirred 24 hours. The solvent is removed and the residue is separated by column chromatography to afford compound 60.
Compound 62: Compound 61 is dissolved in acetonitrile at room temperature. Benzaldehyde dimethylacetal (1.1 eq) is added followed by camphorsulfonic acid (0.2 eq). The reaction mixture is stirred until completion. Triethylamine is added. The solvent is removed and the residue separated by flash chromatography to afford compound 62.
Compound 63: Compound 62 is dissolved in pyridine at room temperature. Dimethylaminopyridine (0.01 eq) is added followed by chloroacetyl chloride (2 eq). The reaction mixture is stirred until completion. The solvent is removed under educed pressure. The residue is dissolved in ethyl acetate, transferred to a separatory funnel and washed two times with 0.1N HCl and two times with water. The organic phase is dried over sodium sulfate, filtered, and concentrated. The residue is separated by column chromatograph to afford compound 63.
Compound 64: Activated powdered 4 Å molecular sieves are added to a solution of compound 60 and compound 63 (2 eq) in dry DCM under argon. The mixture is stirred for 2 hours at room temperature. Solid DMTST (1.5 eq) is added in 4 portions over 1.5 hours. The reaction mixture is stirred overnight at room temperature. The reaction mixture is filtered through Celite, transferred to a separatory funnel and washed two times with half saturated sodium bicarbonate and two times with water. The organic phase is dried over sodium sulfate, filtered and concentrated. The residue is separated by flash chromatography to afford compound 64.
Compound 65: Compound 64 is dissolved in DMF. Sodium azide (1.5 eq) is added and the reaction mixture is stirred at 50° C. until completion. The reaction mixture is cooled to room temperature, diluted with ethyl acetate and transferred to a separatory funnel. The organic phase is washed 4 times with water then dried over sodium sulfate and concentrated. The residue is separated by column chromatography to afford compound 65.
Compound 66: A solution of bispropagyl PEG-5 (compound 43) and compound 65 (2.4 eq) in MeOH is degassed at room temperature. A solution of CuSO4/THPTA in distilled water (0.04 M) (0.2 eq) and sodium ascorbate (0.2 eq) are added successively and the resulting solution is stirred 12 hrs at 50° C. The solution is concentrated under reduced pressure. The crude product is purified by chromatography to give a compound 66.
Compound 67: To a solution of compound 66 in dioxane/water (4/1) is added Pd(OH)2/C. The reaction mixture is stirred vigorously overnight under a hydrogen atmosphere. The reaction mixture is filtered through Celite and concentrated. The residue is purified by C-19 reverse phase column chromatography to afford compound 67.
Compound 68: Compound 67 is dissolved in ethylenediamine and stirred for 12 hrs at 70° C. The reaction mixture is concentrated under reduced pressure. The crude product is purified by C-18 column chromatography followed by lyophilization to afford compound 68.
Compound 69: Compound 69 can be prepared in an analogous fashion to
Compound 70: Compound 70 can be prepared in an analogous fashion to
Compound 71: Compound 71 can be prepared in an analogous fashion to
Compound 72: Compound 67 is dissolved in DMF and cooled on an ice bath. Diisopropylethylamine (2.5 eq) is added followed by HATU (2.2 eq). The reaction mixture is stirred 15 minutes on the ice bath then azetidine (10 eq) is added. The ice bath is removed and the reaction mixture is stirred overnight at room temperature. The solvent is removed under reduced pressure and the residue is separated by flash chromatography to afford compound 72.
Compound 73: Compound 72 is dissolved in ethylenediamine and stirred for 12 hrs at 70° C. The reaction mixture is concentrated under reduced pressure. The crude product is purified by C-18 column chromatography followed by lyophilization to afford compound 73.
Compound 75: To a degassed solution of compound 74 (synthesis described in WO 2013/096926) (0.5 g, 0.36 mmole) in anhydrous DCM (10 mL) at 0° C. was added Pd(PPh3)4 (42 mg, 36.3 μmole, 0.1 eq), Bu3SnH (110 μL, 0.4 μmole, 1.1 eq) and azidoacetic anhydride (0.14 g, 0.73 mmole, 2.0 eq). The resulting solution was stirred for 12 hrs under N2 atmosphere while temperature was gradually increased to room temperature. After the reaction was completed, the solution was diluted with DCM (20 mL), washed with distilled water, dried over Na2SO4, then concentrated. The crude product was purified by combi-flash (EtOAc/Hex, Hex only—3/2, v/v) to give compound 75 (0.33 g, 67%). MS: Calculated (C81H95N4O16, 1376.6), ES—Positive (1400.4, M+Na)).
Compound 76: A solution of bispropargyl PEG-5 (compound 43, 27 mg, 0.1 mmole) and compound 75 (0.33 g, 0.24 mmole, 2.4 eq) in a mixed solution (MeOH/1,4 dioxane, 2/1, v/v, 12 mL) was degassed at room temperature. A solution of CuSO4/THPTA in distilled water (0.04 M) (0.5 mL, 20 μmole, 0.2 eq) and sodium ascorbate (4.0 mg, 20 μmole, 0.2 eq) were added successively and the resulting solution was stirred 12 hrs at 70° C. The solution was cooled to room temperature and concentrated under reduced pressure. The crude product was purified by combi-flash (EtOAc/MeOH, EtOAc only—4/1, v/v) to give a compound 76 as a white foam (0.23 g, 70%).
Compound 77: A solution of compound 76 (0.23 g, 0.76 μmole) in solution of MeOH/i-PrOH (2/1, v/v, 12 mL) was hydrogenated in the presence of Pd(OH)2 (0.2 g) and 1 atm of H2 gas pressure for 24 hrs at room temperature. The solution was filtered through a Celite pad and the cake was washed with MeOH. The combined filtrate was concentrated under reduced pressure. The crude product was washed with hexane and dried under high vacuum to give compound 77 as a white solid (0.14 g, quantitative). MS: Calculated (C80H130N8O35, 1762.8), ES—positive (1785.4, M+Na), ES—Negative (1761.5, M-1, 879.8).
Compound 78: Compound 77 (60 mg, 34.0 μmole) was dissolved in ethylenediamine (3 mL) and the homogeneous solution was stirred for 12 hrs at 70° C. The reaction mixture was concentrated under reduced pressure and the residue was dialyzed against distilled water with MWCO 500 dialysis tube. The crude product was further purified by C-18 column chromatography with water/MeOH (9/1-1/9, v/v) followed by lyophilization to give a compound 78 as a white solid (39 mg, 63%).
1H NMR (400 MHz, Deuterium Oxide) δ 8.00 (s, 2H), 5.26-5.14 (two d, J=16.0 Hz, 4H), 4.52 (d, J=4.0 Hz, 2H), 4.84 (dd, J=8.0 Hz, J=4.0 Hz, 2H), 4.66 (s, 4H), 4.54 (broad d, J=12 Hz, 2H), 3.97 (broad t, 2H), 3.91-3.78 (m, 6H), 3.77-3.58 (m, 28H), 3.57-3.46 (m, 4H), 3.42 (t, J=8.0 Hz, 6H), 3.24 (t, J=12.0 Hz, 2H), 3.02 (t, J=6.0 Hz, 4H), 2.67 (s, 2H), 2.32 (broad t, J=12 Hz, 2H), 2.22-2.06 (m, 2H), 1.96-1.74 (m, 4H), 1.73-1.39 (m, 18H), 1.38-1.21 (m, 6H), 1.20-0.99 (m, J=8.0 Hz, 14H), 0.98-0.73 (m, J=8.0 Hz, 10H).
Compound 79: Compound 79 can be prepared in an analogous fashion to
Compound 80: Compound 80 can be prepared in an analogous fashion to
Compound 81: Compound 81 can be prepared in an analogous fashion to
Compound 82: Compound 82 can be prepared in an analogous fashion to
Compound 83: Compound 83 can be prepared in an analogous fashion to
Compound 84: Compound 84 can be prepared in an analogous fashion to
Compound 85: Compound 85 can be prepared in an analogous fashion to
Compound 86: Compound 77 is dissolved in DMF and cooled on an ice bath. Diisopropylethylamine (2.5 eq) is added followed by HATU (2.2 eq). The reaction mixture is stirred 15 minutes on the ice bath then azetidine (10 eq) is added. The ice bath is removed and the reaction mixture is stirred overnight at room temperature. The solvent is removed under reduced pressure and the residue is separated by flash chromatography to afford compound 86.
Compound 87: Compound 86 is dissolved in ethylenediamine stirred for 12 hrs at 70° C. The reaction mixture was concentrated under reduced pressure. The residue was purified by C-18 column chromatography followed by lyophilization to give a compound 87.
Compound 88: Compound 88 can be prepared in an analogous fashion to
Compound 89: Compound 89 can be prepared in an analogous fashion to
Compound 90: Compound 90 can be prepared in an analogous fashion to
Compound 91: Compound 91 can be prepared in an analogous fashion to
Compound 92: Compound 92 can be prepared in an analogous fashion to
Compound 93: Compound 93 can be prepared in an analogous fashion to
Compound 95: Compound 22 and compound 94 (5 eq) preparation described in WO/2016089872) is co-evaporated 3 times from methanol and stored under vacuum for 1 hour. The mixture is dissolved in methanol under an argon atmosphere and stirred for 1 hour at room temperature. Sodium triacetoxyborohydride (15 eq) is added and the reaction mixture is stirred overnight at room temperature. The solvent is removed and the residue is separated by C-18 reverse phase chromatography.
The purified material is dissolved in methanol at room temperature. The pH is adjusted to 12 with 1N NaOH. The reaction mixture is stirred at room temperature until completion. The pH is adjusted to 9. The solvent is removed under vacuum and the residue is separated by C-18 reverse phase chromatography to afford compound 95.
Compound 96: Compound 96 can be prepared in an analogous fashion to
Compound 97: Compound 97 can be prepared in an analogous fashion to
Compound 98: Compound 98 can be prepared in an analogous fashion to
Compound 99: Compound 99 can be prepared in an analogous fashion to
Compound 100: Compound 100 can be prepared in an analogous fashion to
Compound 101: Compound 101 can be prepared in an analogous fashion to
Compound 102: Compound 102 can be prepared in an analogous fashion to
Compound 103: Compound 103 can be prepared in an analogous fashion to
Compound 104: Compound 104 can be prepared in an analogous fashion to
Compound 105: Compound 105 can be prepared in an analogous fashion to
Compound 106: Compound 106 can be prepared in an analogous fashion to
Compound 107: Compound 107 can be prepared in an analogous fashion to
Compound 108: Compound 108 can be prepared in an analogous fashion to
Compound 109: Compound 109 can be prepared in an analogous fashion to
Compound 110: Compound 110 can be prepared in an analogous fashion to
Compound 111: Compound 111 can be prepared in an analogous fashion to
Compound 112: Compound 112 can be prepared in an analogous fashion to
Compound 113: Compound 113 can be prepared in an analogous fashion to
Compound 114: Compound 114 can be prepared in an analogous fashion to
Compound 115: Compound 115 can be prepared in an analogous fashion to
Compound 116: Compound 116 can be prepared in an analogous fashion to
Compound 117: Compound 117 can be prepared in an analogous fashion to
Compound 118: Compound 118 can be prepared in an analogous fashion to
Compound 119: Compound 119 can be prepared in an analogous fashion to
Compound 120: Compound 120 can be prepared in an analogous fashion to
Compound 121: Compound 121 can be prepared in an analogous fashion to
Compound 122: Compound 122 can be prepared in an analogous fashion to
Compound 123: Compound 123 can be prepared in an analogous fashion to
Compound 124: Compound 124 can be prepared in an analogous fashion to
Compound 125: Compound 125 can be prepared in an analogous fashion to
Compound 126: Compound 126 can be prepared in an analogous fashion to
Compound 127: Compound 127 can be prepared in an analogous fashion to
Compound 128: Compound 128 can be prepared in an analogous fashion to
Compound 129: Compound 129 can be prepared in an analogous fashion to
Compound 130: Compound 130 can be prepared in an analogous fashion to
Compound 131: Compound 131 can be prepared in an analogous fashion to
Compound 132: Compound 132 can be prepared in an analogous fashion to
Compound 133: Compound 133 can be prepared in an analogous fashion to
Compound 134: Compound 134 can be prepared in an analogous fashion to
Compound 135: Compound 135 can be prepared in an analogous fashion to
Compound 136: Compound 136 can be prepared in an analogous fashion to
Compound 137: Compound 137 can be prepared in an analogous fashion to
Compound 138: Compound 138 can be prepared in an analogous fashion to
Compound 139: Compound 139 can be prepared in an analogous fashion to
Compound 140: Compound 140 can be prepared in an analogous fashion to
Compound 141: Compound 141 can be prepared in an analogous fashion to
Compound 142: Compound 142 can be prepared in an analogous fashion to
Compound 143: Compound 143 can be prepared in an analogous fashion to
Compound 144: Compound 144 can be prepared in an analogous fashion to
Compound 315: To a solution of compound 314 (1 gm, 3.89 mol) (preparation described in WO 2007/028050) and benzyl trichloroacetaimidate (1.1 ml, 5.83 mmol) in anhydrous dichloromethane (10 ml) was added trimethylsilyl trifluoromethanesulfonate (70 uL, 0.4 mmol). The mixture was stirred at ambient temperature for 12 h. After this period the reaction was diluted with dichloromethane, washed with saturated NaHCO3, dried over MgSO4 and concentrated. The residue was purified by column chromatography to give compound 315 (0.8 gm, 60%).
Compound 316: To a solution of compound 315 (800 mg, 2.3 mmol) in anhydrous methanol (1 ml) and anhydrous methyl acetate (5 ml) was added 0.5M sodium methoxide solution in methanol (9.2 ml). The mixture was stirred at 40° C. for 4 h. The reaction was quenched with acetic acid and concentrated. The residue was purified by column chromatography to afford compound 316 as mixture of epimers at the methyl ester with 75% equatorial and 25% axial epimer (242 mg, 35%).
1H NMR (400 MHz, Chloroform-d) δ 7.48-7.32 (m, 6H), 4.97 (d, J=11.1 Hz, 1H), 4.72 (dd, J=11.1, 5.7 Hz, 1H), 3.77-3.65 (m, 6H), 3.22-3.15 (m, 1H), 2.92-2.82 (m, 1H), 2.39 (dddd, J=15.7, 10.6, 5.1, 2.7 Hz, 2H), 1.60 (dtd, J=13.9, 11.2, 5.4 Hz, 3H). MS: Calculated for C15H19N3O4=305.3, Found ES—positive m/z=306.1 (M+Na+).
Compound 318: A solution of compound 317 (5 gm, 11.8 mmol) (preparation described in WO 2009/139719) in anhydrous methanol (20 ml) was treated with 0.5 M solution of sodium methoxide in methanol (5 ml) for 3 h. Solvent was removed in vacuo and the residue was co-evaporated with toluene (20 ml) three times. The residue was dissolved in pyridine (20 ml) followed by addition of benzoyl chloride (4.1 ml, 35.4 mmol) over 10 minutes. The reaction mixture was stirred at ambient temperature under an atmosphere of argon for 22 h. The reaction mixture was concentrated to dryness, dissolved in dichloromethane, washed with cold 1N hydrochloric acid and cold water, dried over MgSO4, filtered, and concentrated. The residue was purified by column chromatography to give compound 318. MS: Calculated for C33H27N3O7S=609.2, Found ES—positive m/z=610.2 (M+Na+).
Compound 319: A mixture of compound 318 (2.4 gm, 3.93 mmol), diphenyl sulfoxide (1.5 gm, 7.3 mmol) and 2,6-di-tert-butyl pyridine (1.8 gm, 7.8 mmol) was dissolved in anhydrous dichloromethane (10 ml) at room temperature. The reaction mixture was cooled to −60° C. Triflic anhydride (0.62 ml, 3.67 mmol) was added dropwise and the mixture was stirred for 15 minutes at the same temperature. A solution of compound 316 (0.8 gm, 2.6 mmol) in anhydrous dichloromethane (10 ml) was added dropwise to the reaction mixture. The mixture was allowed to warm to 0° C. over 2 h. The reaction mixture was diluted with dichloromethane, transferred to a separatory funnel and washed with saturated sodium bicarbonate solution followed by brine. The organic phase was dried over MgSO4, filtered, and concentrated. The residue was separated by column chromatography to afford compound 319 as a white solid (1.2 gm, 57%). MS: Calculated for C42H40N6O11=804.3, Found ES—positive m/z=805.3 (M+Na+).
Compound 320: To a solution of compound 319 (1.2 gm 2.067 mmol) and 2-fluorophenyl acetylene (1.2 ml, 10.3 mmol) in methanol (30 ml) was added a stock solution of copper sulfate and tris(3-hydroxypropyltriazolylmethyl) amine in water (2.58 ml). The reaction was initiated by addition of an aqueous solution of sodium ascorbate (0.9 gm, 4.5 mmol) and the mixture was stirred at ambient temperature for 16 hours. The mixture was co-evaporated with dry silica gel and purified by column chromatography to afford compound 320 as a white solid (1.2 gm, 77%).
Stock solution of Copper Sulfate/THPTA—(100 mg of copper sulfate pentahydrate and 200 mg of tris(3-hydroxypropyltriazolylmethyl)amine were dissolved in 10 ml of water).
1H NMR (400 MHz, Chloroform-d) δ 8.07-8.00 (m, 2H), 7.96 (ddd, J=9.8, 8.2, 1.3 Hz, 4H), 7.79 (d, J=5.4 Hz, 2H), 7.65-7.53 (m, 5H), 7.43 (ddt, J=22.4, 10.7, 5.0 Hz, 7H), 7.25-7.01 (m, 9H), 6.92 (td, J=7.6, 7.1, 2.2 Hz, 1H), 6.13-6.02 (m, 2H), 5.58 (dd, J=11.6, 3.2 Hz, 1H), 5.15 (d, J=7.5 Hz, 1H), 4.98 (d, J=10.3 Hz, 1H), 4.68 (dd, J=11.2, 5.7 Hz, 1H), 4.52 (dq, J=22.1, 6.6, 5.6 Hz, 2H), 4.35 (dd, J=11.1, 7.6 Hz, 1H), 4.28-4.18 (m, 1H), 4.11 (d, J=10.3 Hz, 1H), 3.87 (t, J=9.1 Hz, 1H), 3.71 (s, 3H), 2.95 (s, 1H), 2.62-2.43 (m, 3H), 1.55 (dt, J=12.7, 6.1 Hz, 1H). MS: Calculated for C58H50N6O11=1044.4, Found ES—positive m/z=1045.5 (M+Na+).
Compound 145: To a solution of compound 320 (1.2 gm, 1.1 mmol) in iso-propanol (40 ml) was added Na-metal (80 mg, 3.4 mmol) at ambient temperature and the mixture was stirred for 12 hours at 50° C. 10% aqueous sodium hydroxide (2 ml) was added to the reaction mixture and stirring continued for another 6 hours at 50° C. The reaction mixture was cooled to room temperature and neutralized with 50% aqueous hydrochloric acid. To the mixture was added 10% Pd(OH)2 on carbon (0.6 gm) and the reaction mixture was stirred under an atmosphere of hydrogen for 12 hours. The reaction mixture was filtered through a Celite pad and concentrated. The residue was separated by HPLC to give compound 145 as a white solid (0.5 gm, 70%). HPLC Conditions—Waters preparative HPLC system was used with ELSD & PDA detectors. Kinetex XB-C18, 100 A, 5 uM, 250×21.2 mm column (from Phenomenex) was used with 0.2% formic acid in water as solvent A and acetonitrile as solvent B at a flow rate of 20 mL/min.
1H NMR (400 MHz, DMSO-d6) δ 8.77 (s, 1H), 8.68 (s, 1H), 7.77-7.60 (m, 5H), 7.49 (tdd, J=8.3, 6.1, 2.6 Hz, 3H), 7.15 (tt, J=8.6, 3.2 Hz, 3H), 4.83 (dd, J=10.9, 3.1 Hz, 1H), 4.63 (d, J=7.5 Hz, 1H), 4.53-4.41 (m, 1H), 4.10 (dd, J=10.9, 7.5 Hz, 1H), 3.92 (d, J=3.2 Hz, 1H), 3.74 (h, J=6.0, 5.6 Hz, 3H), 3.65-3.24 (m, 5H), 2.37 (d, J=13.4 Hz, 1H), 2.24-2.04 (m, 2H), 1.93 (q, J=12.5 Hz, 1H), 1.46 (t, J=12.1 Hz, 1H). MS: Calculated for C29H30F2N6O8=628.2, Found ES—positive m % z=629.2 (M+Na+).
Compound 146: To a solution of compound 145 (3 eq) in anhydrous DMF was added HATU (3.3 eq) and DIPEA (5 eq). The mixture was stirred at ambient temperature for 15 minutes followed by addition of compound 22 (1 eq). The mixture was stirred at ambient temperature for 12 h. The solvent was removed in vacuo and the residue was purified by HPLC to afford compound 146.
Compound 147: Compound 147 can be prepared in an analogous fashion to
Compound 148: Compound 148 can be prepared in an analogous fashion to
Compound 149: Compound 149 can be prepared in an analogous fashion to
Compound 150: Compound 150 can be prepared in an analogous fashion to
Compound 151: Compound 151 can be prepared in an analogous fashion to
Compound 152: Compound 152 can be prepared in an analogous fashion to
Compound 153: Compound 153 can be prepared in an analogous fashion to
Compound 154: Compound 154 can be prepared in an analogous fashion to
Compound 155: Compound 155 can be prepared in an analogous fashion to
Compound 156: Compound 156 can be prepared in an analogous fashion to
Compound 157: Compound 157 can be prepared in an analogous fashion to
Compound 158: Compound 158 can be prepared in an analogous fashion to
Compound 159: Compound 159 can be prepared in an analogous fashion to
Compound 160: Compound 160 can be prepared in an analogous fashion to
Compound 161: Compound 161 can be prepared in an analogous fashion to
Compound 162: Compound 162 can be prepared in an analogous fashion to
Compound 163: Compound 163 can be prepared in an analogous fashion to
Compound 164: Compound 164 can be prepared in an analogous fashion to
Compound 165: Compound 165 can be prepared in an analogous fashion to
Compound 166: Compound 166 can be prepared in an analogous fashion to
Compound 167: Compound 167 can be prepared in an analogous fashion to
Compound 168: Compound 168 can be prepared in an analogous fashion to
Compound 169: Compound 169 can be prepared in an analogous fashion to
Compound 170: Compound 170 can be prepared in an analogous fashion to
Compound 171: Compound 171 can be prepared in an analogous fashion to
Compound 172: Compound 172 can be prepared in an analogous fashion to
Compound 173: Compound 173 can be prepared in an analogous fashion to
Compound 174: Compound 174 can be prepared in an analogous fashion to
Compound 175: Compound 175 can be prepared in an analogous fashion to
Compound 176: Compound 176 can be prepared in an analogous fashion to
Compound 177: Compound 177 can be prepared in an analogous fashion to
Compound 178: Compound 178 can be prepared in an analogous fashion to
Compound 179: Compound 179 can be prepared in an analogous fashion to
Compound 180: Compound 180 can be prepared in an analogous fashion to
Compound 181: Compound 181 can be prepared in an analogous fashion to
Compound 182: Compound 182 can be prepared in an analogous fashion to
Compound 183: Compound 183 can be prepared in an analogous fashion to
Compound 184: Compound 184 can be prepared in an analogous fashion to
Compound 185: Compound 185 can be prepared in an analogous fashion to
Compound 186: Compound 186 can be prepared in an analogous fashion to
Compound 187: Compound 187 can be prepared in an analogous fashion to
Compound 188: Compound 188 can be prepared in an analogous fashion to
Compound 189: Compound 189 can be prepared in an analogous fashion to
Compound 190: Compound 190 can be prepared in an analogous fashion to
Compound 191: Compound 191 can be prepared in an analogous fashion to
Compound 192: Compound 192 can be prepared in an analogous fashion to
Compound 193: Compound 193 can be prepared in an analogous fashion to
Compound 194: Compound 194 can be prepared in an analogous fashion to
Compound 195: Compound 195 can be prepared in an analogous fashion to
Compound 197: To a solution of compound 22 (1 eq) in anhydrous DMSO was acetic acid NHS ester (compound 196) (5 eq). The mixture was stirred at ambient temperature for 12 hours. The solvent was removed in vacuo and the residue was purified by HPLC to afford compound 197.
Compound 198: Compound 198 can be prepared in an analogous fashion to
Compound 199: Compound 199 can be prepared in an analogous fashion to
Compound 200: Compound 200 can be prepared in an analogous fashion to
Compound 201: Compound 201 can be prepared in an analogous fashion to
Compound 202: Compound 202 can be prepared in an analogous fashion to
Compound 203: Compound 203 can be prepared in an analogous fashion to
Compound 205: A solution of compound 204 (synthesis described in Mead, G. et. al., Bioconj. Chem., 2015, 25, 1444-1452) (0.25 g, 0.53 mmole) and propiolic acid (0.33 mL, 5.30 mmole, 10 eq) in distilled water (1.5 mL) was degassed. A solution of CuSO4/THPTA in distilled water (0.04 M) (1.3 mL, 53 μmole, 0.1 eq) and sodium ascorbate (21 mg, 0.11 mmole, 0.2 eq) were added successively and the resulting solution was stirred 3 hrs at room temperature. The reaction mixture was concentrated under reduced pressure and partially purified by C-18 column chromatography (water/MeOH, water only—5/5, v/v). The resulting material was further purified by C-18 column chromatography eluting with water to afford compound 205 (0.16 g, 0.34 mmole, 64%). MS: (Calculated for C8H103N3Na3O14S3, 537.34), ES—Negative (513.5, M-Na-1).
Compound 206: To a solution of compound 205 (7.5 mg, 14 μmole), DIPEA (2.4 μL, 14 μmole) and a catalytic amount of DMAP in DMF/DMSO (3/1, v/v, 0.15 mL) at 0° C. was added EDCI (1.6 mg, 8.22 μmole). The solution was stirred for 20 min. This solution was slowly added to a solution of compound 78 (5.0 mg, 2.7 μmole) in DMF/DMSO (3/1, v/v, 0.2 mL) cooled at 0° C. The resulting solution was stirred 12 hrs allowing the reaction temperature to increase to room temperature. The reaction mixture was purified directly by HPLC. The product portions were collected, concentrated under reduced pressure, then lyophilized to give compound 206 as a white solid (0.4 mg, 1.15 μmole, 1.1%). MS: Calculated (C98H154N18Na6O59S6, 2856.7), ES—Negative (907.7, M/3; 881.0, M-1SO3/3; 854.1 M-2SO3/3; 685.8 M+1Na/4; 680.5 M/4); Fraction of RT=10.65 min, 1399.4, M+7Na-1SO3/2; 959.3 M+7Na/3; M+7Na-1SO3/3; 724.8, M+8Na/4; 549. M+1Na/5; 460.9 M+2Na/6; 401. M+4Na/7).
Compound 207: Compound 207 can be prepared in an analogous fashion to
Compound 208: Compound 208 can be prepared in an analogous fashion to
Compound 209: Compound 209 can be prepared in an analogous fashion to
Compound 210: Compound 210 can be prepared in an analogous fashion to
Compound 211: Compound 211 can be prepared in an analogous fashion to
Compound 213: Prepared according to Bioorg. Med. Chem. Lett. 1995, 5, 2321-2324 starting with D-threonolactone.
Compound 214: Compound 213 (500 mg, 1 mmol) was dissolved in 9 mL acetonitrile. Potassium hydroxide (1 mL of a 2M solution) was added and the reaction mixture was stirred at 50° C. for 12 hours. The reaction mixture was partitioned between dichloromethane and water. The phases were separated and the aqueous phase was extracted 3 times with dichloromethane. The aqueous phase was acidified with 1N HCl until pH˜1 and extracted 3 times with dichloromethane. The combined dichloromethane extracts from after acidification of the aqueous phase were concentrated in vacuo to give compound 214 as a yellow oil (406 mg). LCMS (C-18; 5-95 H2O/MeCN) UV (peak at 4.973 mi), positive mode: m/z=407 [M+H]+; negative mode: m/z=405 [M−H]− C25H26O5 (406).
Compound 215: Prepared in an analogous fashion to compound 214 using L-erythronolactone as the starting material. LCMS (C-18; 5-95 H2O/MeCN): ELSD (5.08 min), UV (peak at 4.958 min), positive mode: m/z=407 [M+H]+; negative mode: m/z=405 [M−H]− C25H26O5 (406).
Compound 216: Prepared in an analogous fashion to compound 214 using L-threonolactone as the starting material. LCMS (C-18; 5-95 H2O/MeCN): ELSD (5.08 min), UV (peak at 4.958 min), positive mode: m-z=407 [M+H]+; negative mode: m-z=405 [M−H]− C25H26O5 (406).
Compound 217: Prepared in an analogous fashion to compound 214 using D-erythronolactone as the starting material. LCMS (C-18; 5-95 H2O/MeCN): ELSD (5.08 min), UV (peak at 4.958 min), positive mode: m-z=407 [M+H]+; negative mode: m/z=405 [M−H]− C25H26O5 (406).
Compound 218: To a solution of compound 214 (3 eq) in anhydrous DMF was added HATU (3.3 eq) and DIPEA (5 eq). The mixture was stirred at ambient temperature for 15 minutes followed by addition of compound 78 (1 eq). The mixture was stirred at ambient temperature for 12 h. The solvent was removed in vacuo and the residue was purified by HPLC to afford compound 218.
Compound 219: Compound 218 is dissolved in methanol and degassed. To this solution is added Pd(OH)2/C. The reaction mixture is vigorously stirred under a hydrogen atmosphere for 12 hours. The reaction mixture is filtered through a Celite pad. The filtrate is concentrated under reduced pressure to give compound 219.
Compound 220: A solution of the sulfur trioxide pyridine complex (100 eq) and compound 219 (1 eq) in pyridine was stirred at 67° C. for 1 h. The reaction mixture was concentrated under vacuum. The resulting solid was dissolved in water and cooled to 0° C. A 1N solution of NaOH was then added slowly until pH-10 and the latter was freeze dried. The resulting residue was purified by Gel Permeation (water as eluent). The collected fractions were lyophilised to give compound 220.
Compound 221: Compound 221 can be prepared in an analogous fashion to
Compound 222: Compound 222 can be prepared in an analogous fashion to
Compound 223: Compound 223 can be prepared in an analogous fashion to
Compound 224: To a solution of compound 78 in anhydrous DMSO was added a drop of DIPEA and the solution was stirred at room temperature until a homogeneous solution was obtained. A solution of succinic anhydride (2.2 eq) in anhydrous DMSO was added and the resulting solution was stirred at room temperature overnight. The solution was lyophilized to dryness and the crude product was purified by HPLC to give compound 224.
Compound 225: Compound 225 can be prepared in an analogous fashion to
Compound 226: Compound 226 can be prepared in an analogous fashion to
Compound 227: Compound 227 can be prepared in an analogous fashion to
Compound 228: Compound 228 can be prepared in an analogous fashion to
Compound 229: Compound 229 can be prepared in an analogous fashion to
Compound 231: A mixture of compound 230 (preparation described in Schwizer, et. al., Chem. Eur. J., 2012, 18, 1342) and compound 2 (preparation described in WO 2013/096926) (1.7 eq) is azeotroped 3 times from toluene. The mixture is dissolved in DCM under argon and cooled on an ice bath. To this solution is added boron trifluoride etherate (1.5 eq). The reaction mixture is stirred 12 hours at room temperature. The reaction is quenched by the addition of triethylamine (2 eq). The reaction mixture is transferred to a separatory funnel and washed 1 time with half saturated sodium bicarbonate solution and 1 time with water. The organic phase is dried over sodium sulfate, filtered, and concentrated. The residue is purified by flash chromatography to afford compound 231.
Compound 232: Compound 231 is dissolved in methanol at room temperature. A solution of sodium methoxide in methanol (0.1 eq) is added and the reaction mixture stirred overnight at room temperature. The reaction mixture is quenched by the addition of acetic acid. The reaction mixture is diluted with ethyl acetate, transferred to a separatory funnel and washed 2 times with water. The organic phase is dried over magnesium sulfate, filtered and concentrated. The residue is separated by flash chromatography to afford compound 232.
Compound 233: To a solution of compound 232 in dichloromethane cooled on an ice bath is added DABCO (1.5 eq) followed by monomethoxytrityl chloride (1.2 eq). The reaction mixture is stirred overnight allowing to warm to room temperature. The reaction mixture is concentrated and the residue is purified by flash chromatography to afford compound 233.
Compound 234: To a solution of compound 233 in methanol is added dibutyltin oxide (1.1 eq). The reaction mixture is refluxed for 3 hours then concentrated. The residue is suspended in DME. To this suspension is added compound 6 (preparation described in Thoma el. al. J. Med. Chem., 1999, 42, 4909) (1.5 eq) followed by cesium fluoride (1.2 eq). The reaction mixture is stirred at room temperature overnight. The reaction mixture is diluted with ethyl acetate, transferred to a separatory funnel, and washed with water. The organic phase is dried over sodium sulfate, filtered and concentrated. The residue is purified by flash chromatography to afford compound 234.
Compound 235: To a degassed solution of compound 234 in anhydrous DCM at 0° C. is added Pd(PPh3)4 (0.1 eq), Bu3SnH (1.1 eq) and N-trifluoroacetyl glycine anhydride (2.0 eq) (preparation described in Chemische Berichte (1955), 88(1), 26). The resulting solution is stirred for 12 hrs allowing the temperature to increase to room temperature. The reaction mixture is diluted with DCM, transferred to a separatory funnel, and washed with water. The organic phase is dried over Na2SO4, then filtered and concentrated. The residue is purified by flash chromatography to afford compound 235.
Compound 236: Compound 235 is dissolved in methanol and degassed. To this solution is added Pd(OH)2/C. The reaction mixture is vigorously stirred under a hydrogen atmosphere for 12 hours. The reaction mixture is filtered through a Celite pad. The filtrate is concentrated under reduced pressure to give compound 236.
Compound 237: Compound 236 is dissolved in methanol at room temperature. A solution of sodium methoxide in methanol (1.1 eq) is added and the reaction mixture stirred overnight at room temperature. The reaction mixture is quenched by the addition of acetic acid. The reaction mixture is concentrated. The residue is separated by C-18 reverse phase chromatography to afford compound 237.
Compound 238: Compound 238 can be prepared in an analogous fashion to
Compound 239: Compound 239 can be prepared in an analogous fashion to
Compound 240: Compound 236 is dissolved in DMF and cooled on an ice bath. Diisopropylethylamine (1.5 eq) is added followed by HATU (1.1 eq). The reaction mixture is stirred 15 minutes on the ice bath then azetidine (2 eq) is added. The ice bath is removed and the reaction mixture is stirred overnight at room temperature. The solvent is removed under reduced pressure and the residue is separated by flash chromatography to afford compound 240.
Compound 241: Compound 240 is dissolved in methanol at room temperature. A solution of sodium methoxide in methanol (0.3 eq) is added and the reaction mixture stirred overnight at room temperature. The reaction mixture is quenched by the addition of acetic acid. The reaction mixture is concentrated. The residue is separated by C-18 reverse phase chromatography to afford compound 241.
Compound 242: Compound 242 can be prepared in an analogous fashion to
Compound 243: Compound 243 can be prepared in an analogous fashion to
Compound 244: Compound 244 can be prepared in an analogous fashion to
Compound 245: A solution of compound 20 (0.4 eq) in DMSO is added to a solution of compound 237 (1 eq) and DIPEA (10 eq) in anhydrous DMSO at room temperature. The resulting solution is stirred overnight. The reaction mixture is separated by reverse phase chromatography and the product lyophilized to give compound 245.
Compound 246: Compound 246 can be prepared in an analogous fashion to
Compound 247: Compound 247 can be prepared in an analogous fashion to
Compound 248: Compound 248 can be prepared in an analogous fashion to
Compound 249: Compound 249 can be prepared in an analogous fashion to
Compound 250: Compound 250 can be prepared in an analogous fashion to
Compound 251: Compound 251 can be prepared in an analogous fashion to
Compound 252: Compound 252 can be prepared in an analogous fashion to
Compound 253: Compound 253 can be prepared in an analogous fashion to
Compound 254: Compound 254 can be prepared in an analogous fashion to
Compound 255: Compound 255 can be prepared in an analogous fashion to
Compound 256: Compound 256 can be prepared in an analogous fashion to
Compound 257: To a solution of compound 238 in MeOH at room temperature is added compound 35 followed by cesium acetate (2.5 eq). The reaction mixture is stirred at room temperature until completion. The solvent is removed under reduced pressure. The product is purified by reverse phase chromatography to give compound 257.
Compound 258: Compound 258 can be prepared in an analogous fashion to
Compound 259: Compound 259 can be prepared in an analogous fashion to
Compound 260: To a degassed solution of compound 234 in anhydrous DCM at 0° C. is added Pd(PPh3)4 (0.1 eq), Bu3SnH (1.1 eq) and azidoacetic anhydride (2.0 eq). The ice bath is removed and the solution is stirred for 12 hrs under a N2 atmosphere at room temperature. The reaction mixture is diluted with DCM, washed with water, dried over Na2SO4, then concentrated. The crude product is purified by column chromatography to give compound 260.
Compound 261: A solution of bis-propagyl PEG-5 (compound 43) and compound 260 (2.4 eq) in MeOH is degassed at room temperature. A solution of CuSO4/THPTA in distilled water (0.04 M) (0.2 eq) and sodium ascorbate (0.2 eq) are added successively and the resulting solution is stirred 12 hrs at 70° C. The solution is cooled to room temperature and concentrated under reduced pressure. The crude product is purified by chromatography to give compound 261.
Compound 262: Compound 261 is dissolved in MeOH and hydrogenated in the presence of Pd(OH)2 (20 wt %) at 1 atm of H2 gas pressure for 24 hrs at room temperature. The solution is filtered through a Celite pad. The filtrate is concentrated to give compound 262.
Compound 263: Compound 262 is dissolved in DMF and cooled on an ice bath. Diisopropylethylamine (2.5 eq) is added followed by HATU (2.2 eq). The reaction mixture is stirred 15 minutes on the ice bath then azetidine (10 eq) is added. The ice bath is removed and the reaction mixture is stirred overnight at room temperature. The solvent is removed under reduced pressure and the residue is separated by reverse phase chromatography to afford compound 263.
Compound 264: Compound 264 can be prepared in an analogous fashion to
Compound 265: Compound 265 can be prepared in an analogous fashion to
Compound 266: Compound 266 can be prepared in an analogous fashion to
Compound 267: Compound 267 can be prepared in an analogous fashion to
Compound 268: Compound 268 can be prepared in an analogous fashion to
Compound 269: Compound 269 can be prepared in an analogous fashion to
Compound 270: Compound 270 can be prepared in an analogous fashion to
Compound 271: Compound 271 can be prepared in an analogous fashion to
Compound 272: Activated powdered 4 Å molecular sieves are added to a solution of compound 230 and compound 63 (2 eq) in dry DCM under argon. The mixture is stirred for 2 hours at room temperature. Solid DMTST (1.5 eq) is added in 4 portions over 1.5 hours. The reaction mixture is stirred overnight at room temperature. The reaction mixture is filtered through Celite, transferred to a separatory funnel and washed two times with half saturated sodium bicarbonate and two times with water. The organic phase is dried over sodium sulfate, filtered and concentrated. The residue is separated by flash chromatography to afford compound 272.
Compound 273: Compound 272 is dissolved in DMF. Sodium azide (1.5 eq) is added and the reaction mixture is stirred at 50° C. until completion. The reaction mixture is cooled to room temperature, diluted with ethyl acetate and transferred to a separatory funnel. The organic phase is washed 4 times with water then dried over sodium sulfate and concentrated. The residue is separated by column chromatography to afford compound 273.
Compound 274: A solution of bispropagyl PEG-5 (compound 43) and compound 273 (2.4 eq) in MeOH is degassed at room temperature. A solution of CuSO4/THPTA in distilled water (0.04 M) (0.2 eq) and sodium ascorbate (0.2 eq) are added successively and the resulting solution is stirred 12 hrs at 50° C. The solution is concentrated under reduced pressure. The crude product is purified by chromatography to give a compound 274.
Compound 275: To a solution of compound 274 in dioxane/water (4/1) is added Pd(OH)2/C. The reaction mixture is stirred vigorously overnight under a hydrogen atmosphere. The reaction mixture is filtered through Celite and concentrated. The residue is purified by C-18 reverse phase column chromatography to afford compound 275.
Compound 276: Compound 275 is dissolved in DMF and cooled on an ice bath. Diisopropylethylamine (2.5 eq) is added followed by HATU (2.2 eq). The reaction mixture is stirred 15 minutes on the ice bath then azetidine (10 eq) is added. The ice bath is removed and the reaction mixture is stirred overnight at room temperature. The solvent is removed under reduced pressure and the residue is separated by reverse phase chromatography to afford compound 276.
Compound 277: Compound 277 can be prepared in an analogous fashion to
Compound 278: Compound 278 can be prepared in an analogous fashion to
Compound 279: Compound 279 can be prepared in an analogous fashion to
Compound 280: Compound 280 can be prepared in an analogous fashion to
Compound 281: Compound 281 can be prepared in an analogous fashion to
Compound 282: Compound 282 can be prepared in an analogous fashion to
Compound 284: A mixture of compound 283 (preparation described in WO 2007/028050) and compound 2 (preparation described in WO 2013/096926) (1.7 eq) is azeotroped 3 times from toluene. The mixture is dissolved in DCM under argon and cooled on an ice bath. To this solution is added boron trifluoride etherate (1.5 eq). The reaction mixture is stirred 12 hours at room temperature. The reaction is quenched by the addition of triethylamine (2 eq). The reaction mixture is transferred to a separatory funnel and washed 1 time with half saturated sodium bicarbonate solution and 1 time with water. The organic phase is dried over sodium sulfate, filtered, and concentrated. The residue is purified by flash chromatography to afford compound 284.
Compound 285: Compound 284 is dissolved in methanol at room temperature. A solution of sodium methoxide in methanol (0.1 eq) is added and the reaction mixture stirred overnight at room temperature. The reaction mixture is quenched by the addition of acetic acid. The reaction mixture is diluted with ethyl acetate, transferred to a separatory funnel and washed 2 times with water. The organic phase is dried over magnesium sulfate, filtered and concentrated. The residue is separated by flash chromatography to afford compound 285.
Compound 286: To a solution of compound 285 in dichloromethane cooled on an ice bath is added DABCO (1.5 eq) followed by monomethyoxytrityl chloride (1.2 eq). The reaction mixture is stirred overnight allowing to warm to room temperature. The reaction mixture is transferred to a separatory funnel and washed 2 times with water. The organic phase is concentrated and the residue is purified by flash chromatography to afford compound 286.
Compound 287: To a solution of compound 286 in methanol is added dibutyltin oxide (1.1 eq). The reaction mixture is refluxed for 3 hours then concentrated. The residue is suspended in DME. To this suspension is added compound 6 (preparation described in Thoma et. al. J. Med Chem., 1999, 42, 4909) (1.5 eq) followed by cesium fluoride (1.2 eq). The reaction mixture is stirred at room temperature overnight. The reaction mixture is diluted with ethyl acetate, transferred to a separatory funnel, and washed with water. The organic phase is dried over sodium sulfate, filtered and concentrated. The residue is purified by flash chromatography to afford compound 287.
Compound 288: To a degassed solution of compound 287 in anhydrous DCM at 0° C. is added Pd(PPh3)4 (0.1 eq), Bu3SnH (1.1 eq) and N-trifluoroacetyl glycine anhydride (2.0 eq) (preparation described in Chemische Berichte (1955), 88(1), 26). The resulting solution is stirred for 12 hrs allowing the temperature to increase to room temperature. The reaction mixture is diluted with DCM, transferred to a separatory funnel, and washed with water. The organic phase is dried over Na2SO4, then filtered and concentrated. The residue is purified by flash chromatography to afford compound 288.
Compound 289: To a stirred solution of compound 288 in DCM/MeOH (25/1) at room temperature is added orotic acid chloride (5 eq) and triphenylphosphine (5 eq). The reaction mixture is stirred 24 hours. The solvent is removed and the residue is separated by column chromatography to afford compound 289.
Compound 290: Compound 289 is dissolved in methanol and degassed. To this solution is added Pd(OH)2/C. The reaction mixture is vigorously stirred under a hydrogen atmosphere for 12 hours. The reaction mixture is filtered through a Celite pad. The filtrate is concentrated under reduced pressure to give compound 290.
Compound 291: Compound 290 is dissolved in methanol at room temperature. A solution of sodium methoxide in methanol (1.1 eq) is added and the reaction mixture stirred overnight at room temperature. The reaction mixture is quenched by the addition of acetic acid. The reaction mixture is concentrated. The residue is separated by C-18 reverse phase chromatography to afford compound 291.
Compound 292: Compound 292 can be prepared in an analogous fashion to
Compound 293: Compound 293 can be prepared in an analogous fashion to
Compound 294: A solution of compound 291 (0.4 eq) in DMSO is added to a solution of compound 20 (1 eq) and DIPEA (10 eq) in anhydrous DMSO at room temperature. The resulting solution is stirred overnight. The reaction mixture is separated by reverse phase chromatography and the product lyophilized to give compound 294.
Compound 295: Compound 294 is dissolved in DMF and cooled on an ice bath. Diisopropylethylamine (2.5 eq) is added followed by HATU (2.2 eq). The reaction mixture is stirred 15 minutes on the ice bath then azetidine (10 eq) is added. The ice bath is removed and the reaction mixture is stirred overnight at room temperature. The solvent is removed under reduced pressure and the residue is separated by reverse phase chromatography to afford compound 295.
Compound 296: Compound 296 can be prepared in an analogous fashion to
Compound 297: Compound 297 can be prepared in an analogous fashion to
Compound 298: Compound 298 can be prepared in an analogous fashion to
Compound 299: Compound 299 can be prepared in an analogous fashion to
Compound 300: Compound 300 can be prepared in an analogous fashion to
Compound 301: Compound 301 can be prepared in an analogous fashion to
Compound 302: Compound 302 can be prepared in an analogous fashion to
Compound 303: To a stirred solution of compound 287 in DCM/MeOH (25/1) at room temperature is added orotic acid chloride (5 eq) and triphenylphosphine (5 eq). The reaction mixture is stirred 24 hours. The solvent is removed and the residue is separated by column chromatography to afford compound 303.
Compound 304: To a degassed solution of compound 303 in anhydrous DCM at 0° C. is added Pd(PPh3)4 (0.1 eq), Bu3SnH (1.1 eq) and azidoacetic anhydride (2.0 eq). The ice bath is removed and the solution is stirred for 12 hrs under a N2 atmosphere at room temperature. The reaction mixture is diluted with DCM, washed with water, dried over Na2SO4, then concentrated. The crude product is purified by column chromatography to give compound 304.
Compound 305: A solution of bispropagyl PEG-5 (compound 43) and compound 304 (2.4 eq) in MeOH is degassed at room temperature. A solution of CuSO4/THPTA in distilled water (0.04 M) (0.2 eq) and sodium ascorbate (0.2 eq) are added successively and the resulting solution is stirred 12 hrs at 50° C. The solution is cooled to room temperature and concentrated under reduced pressure. The crude product is purified by chromatography to give compound 305.
Compound 306: Compound 305 is dissolved in MeOH and hydrogenated in the presence of Pd(OH)2 (20 wt %) at 1 atm of H2 gas pressure for 24 hrs at room temperature. The solution is filtered through a Celite pad. The filtrate is concentrated to give compound 306.
Compound 307: Compound 306 is dissolved in DMF and cooled on an ice bath. Diisopropylethylamine (2.5 eq) is added followed by HATU (2.2 eq). The reaction mixture is stirred 15 minutes on the ice bath then azetidine (10 eq) is added. The ice bath is removed and the reaction mixture is stirred overnight at room temperature. The solvent is removed under reduced pressure and the residue is separated by reverse phase chromatography to afford compound 307.
Compound 308: Compound 308 can be prepared in an analogous fashion to
Compound 309: Compound 309 can be prepared in an analogous fashion to
Compound 310: Compound 310 can be prepared in an analogous fashion to
Compound 311: Compound 311 can be prepared in an analogous fashion to
Compound 312: Compound 312 can be prepared in an analogous fashion to
Compound 313: Compound 313 can be prepared in an analogous fashion to
Compound 321: Compound 317 (1.1 g, 2.60 mmoles) was dissolved in methanol (25 mL) at room temperature. Sodium methoxide (0.1 mL, 25% sol. in MeOH) was added and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture neutralized by the addition of Amberlyst acidic resin, filtered and concentrated to give crude 321, which was used for the next step without further purification. LCMS (ESI): m/z calculated for C12H15N3O4S: 297.3, found 298.1 (M+1); 320.1 (M+Na).
Compound 322: Crude compound 321 (2.60 mmoles), 3,4,5-trifluorophenyl-1-acetylene (2.5 equiv), THPTA (0.11 equiv), and copper (II) sulfate (0.1) were dissolved in methanol (15 mL) at room temperature. Sodium ascorbate (2.4 equiv) dissolved in water was added and the reaction mixture was stirred overnight at room temperature. The resultant precipitate was collected by filtration, washed with hexanes and water, and dried to give compound 322 as a pale yellow solid (1.2 g, 100% yield for 2 steps). LCMS (ESI): m/z calculated for C20H18F3N3O4S: 453.1, found 454.2 (M+1); 476.2 (M+Na).
Compound 323: Compound 322 (1.2 g, 2.65 mmoles) was dissolved in DMF (15 mL) and cooled on an ice bath. Sodium hydride (60% oil dispersion, 477 mg, 11.93 mmoles) was added and the mixture stirred for 30 minutes. Benzyl bromide (1.42 mL, 11.93 mmoles) was added and the reaction was warmed to room temperature and stirred overnight. The reaction mixture was quenched by the addition of aqueous saturated ammonium chloride solution, transferred to a separatory funnel and extracted 3 times with ether. The combined organic phases were dried over magnesium sulfate, filtered, and concentrated. The residue was purified by flash chromatography to afford compound 323 (1.8 g, 94% yield). LCMS (ESI): m/z calculated for C41H36F3N3O4S: 723.2, found 724.3 (M+1); 746.3 (M+Na).
Compound 324: Compound 323 (1.8 g, 2.49 mmol) was dissolved in acetone (20 mL) and water (2 mL) and cooled on an ice bath. Trichloroisocyanuric acid (637 mg, 2.74 mmoles) was added and the reaction mixture stirred on the ice bath for 3 h. The acetone was removed in vacuo and the residue was diluted with DCM, transferred to a separatory funnel, and washed with saturated aqueous NaHCO3. The organic phase was concentrated and the residue was purified by flash chromatography to afford compound 324 (1.5 g, 95%). LCMS (ESI): m/z calculated for C35H32F3N3O5: 631.2, found 632.2 (M+1); 654.2 (M+Na).
Compound 325: Compound 324 (1.0 g, 1.58 mmoles) was dissolved in DCM (20 mL) and cooled on an ice bath. Dess-Martin periodinane (1.0 g, 2.37 mmoles) was added and mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture quenched by the addition of aqueous saturated NaHCO3, transferred to a separatory funnel, and extracted 2 times with DCM. The combined organic phases were dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography to afford compound 325 (520 mg, 52% yield). LCMS (ESI): m/z calculated for C35H30F3N3O5: 629.2, found 652.2 (M+Na); 662.2 (M+MeOH+1); 684.2 (M+MeOH+Na).
Compound 326: Methyl bromoacetate (253 mg, 1.65 mmoles) dissolved in 0.5 mL of THF was added dropwise to a solution of lithium bis(trimethylsilyl)amide (1.0 M in THF, 1.65 mL, 1.65 mmoles) cooled at −78 C. The reaction mixture was stirred for 30 minutes at −78 C. Compound 325 (260 mg, 0.41 mmoles) dissolved in THF (2.0 mL) was then added. The reaction mixture was stirred at −78 C for 30 minutes. The reaction was quenched by the addition of aqueous saturated NH4Cl and warmed to rt. The reaction mixture was transferred to a separatory funnel and extracted 3 times with ethyl acetate. The combined organic phases were dried over sodium sulfate, filtered and concentrated. The residue was separated by flash chromatography to afford compound 326 (183 mg, 64% yield).
1H NMR (400 MHz, Chloroform-d) δ 7.38-7.22 (m, 9H), 7.15-7.11 (m, 3H), 7.09 (dd, J=8.4, 6.6 Hz, 1H), 7.06-7.00 (m, 2H), 6.98-6.93 (m, 2H), 5.11 (dd, J=11.3, 3.2 Hz, 1H), 4.60 (d, J=11.8 Hz, 1H), 4.57-4.49 (m, 2H), 4.49-4.42 (m, 2H), 4.35 (d, J=11.8 Hz, 1H), 4.14 (d, J=3.2 Hz, 1H), 4.05 (s, 1H), 4.02 (d, J=7.0 Hz, 1H), 3.84 (d, J=11.0 Hz, 1H), 3.81 (s, 3H), 3.70 (dd, J=9.5, 7.7 Hz, 1H), 3.62 (dd, J=9.4, 6.0 Hz, 1H). LCMS (ESI): m/z calculated for C38H34F3N3O7: 701.2, found 702.3 (M+1); 724.3 (M+Na).
Compound 327: Compound 326 (5.0 g, 7.13 mmol) was azeotroped with toluene two times under reduced pressure, and then dried under high vacuum for 2 hours. It was then dissolved in anhydrous CH2Cl2 (125 mL) and cooled on an ice bath while stirring under an atmosphere of argon. Tributyltin hydride (15.1 mL, 56.1 mmol) was added dropwise and the solution was allowed to stir for 25 minutes on the ice bath. Trimethylsilyl triflate (2.1 mL, 11.6 mmol) dissolved in 20 mL of anhydrous CH2Cl2 was then added dropwise over the course of 5 minutes. The reaction was slowly warmed to ambient temperature and stirred for 16 hours. The reaction mixture was then diluted with CH2C12 (50 mL), transferred to a separatory funnel, and washed with saturated aqueous NaHCO3 (50 mL). The aqueous phase was separated and extracted with CH2Cl2 (50 mL×2). The combined organic phases were washed with saturated aqueous NaHCO3 (50 mL), dried over Na2SO4, filtered, and concentrated. The residue was purified by flash chromatography (hexanes to 40% EtOAc in hexanes, gradient) to afford compound 327 (2.65 g, 48%).
1H-NMR (400 MHz, CDCl3): δ 7.65 (s, 1H), 7.36-7.22 (m, 8H), 7.16-7.06 (m, 7H), 6.96-6.90 (m, 2H), 5.03 (dd, J=10.7, 3.2 Hz, 1H), 4.72 (d, J=2.3 Hz, 1H), 4.51 (dt, J=22.6, 11.4 Hz, 3H), 4.41 (d, J=10.9 Hz, 1H), 4.32 (dd, J=10.7, 9.2 Hz, 1H), 4.07 (d, J=3.1 Hz, 1H), 3.94 (d, J=10.9 Hz, 1H), 3.92-3.84 (m, 3H), 3.78-3.71 (m, 4H), 3.65 (dd, J=9.1, 5.5 Hz, 1H), 0.24 (s, 9H). LCMS (ESI): m/z (M+Na) calculated for C41H44F3N3O7SiNa: 798.87, found 798.2.
Compound 328: To a solution of compound 327 (2.65 g, 3.4 mmol) in anhydrous MeOH (40 mL) was added Pd(OH)2 (0.27 g, 200% by wt). The mixture was cooled on an ice bath and stirred for 30 minutes. Triethylsilane (22 mL, 137 mmol) was added dropwise. The solution was allowed to slowly warm to ambient temperature and stirred for 16 hours. The reaction mixture was filtered through a bed of Celite and concentrated. The residue was purified by flash chromatography (hexanes to 100% EtOAc, gradient) to afford compound 328 (1.09 g, 73%).
1H-NMR (400 MHz, CD3OD): δ 8.57 (s, 1H), 7.77-7.53 (m, 2H), 4.91-4.82 (m, 1H), 4.66-4.59 (m, 1H), 4.55 (dd, J=10.8, 9.4 Hz, 1H), 4.13 (d, J=2.8 Hz, 1H), 3.86 (dd, J=9.4, 2.1 Hz, 1H), 3.81 (s, 3H), 3.77-3.74 (m, 1H), 3.71-3.68 (m, 2H). LCMS (ESI): m/z (M+Na) calculated for C17H18F3N3O7Na: 456.33, found 456.0.
Compound 329: Compound 328 (1.09 g, 2.5 mmol) and CSA (0.115 g, 0.49 mmol) were suspended in anhydrous MeCN (80 mL) under an argon atmosphere. Benzaldehyde dimethyl acetal (0.45 mL, 2.99 mmol) was added dropwise. The reaction mixture was allowed to stir for 16 hours at ambient temperature, during which time it became a homogenous solution. The reaction mixture was then neutralized with a few drops of Et3N, and concentrated. The residue was purified via flash chromatography (CH2Cl2 to 10% MeOH in CH2C12, gradient) to afford compound 329 (978 mg, 75%).
1H NMR (400 MHz, DMSO-d6): δ 8.84 (s, 1H), 7.95-7.73 (m, 2H), 7.33 (qdt, J=8.4, 5.6, 2.7 Hz, 5H), 5.51 (t, J=3.8 Hz, 2H), 5.47 (d, J=6.8 Hz, 1H), 5.14 (dd, J=10.8, 3.6 Hz, 1H), 4.54 (dd, J=6.7, 2.2 Hz, 1H), 4.47 (ddd, J=10.8, 9.3, 7.5 Hz, 1H), 4.40 (d, J=4.0 Hz, 1H), 4.09-3.99 (m, 2H), 3.85 (dd, J=9.3, 2.2 Hz, 1H), 3.81-3.76 (m, 1H), 3.71 (s, 3H). LCMS (ESI): m/z (M+Na) calculated for C24H22F3N3O7Na: 544.43, found 544.1.
Compound 330: Compound 329 (25.2 mg, 0.048 mmol) was azeotroped with toluene 2 times under reduced pressure, dried under high vacuum for 2 hours, then dissolved in anhydrous DMF (2 mL) and cooled on an ice bath. Benzyl bromide (6 uL, 0.05 mmol) dissolved in 0.5 mL of anhydrous DMF was added and the reaction and was stirred under an atmosphere of argon for 30 minutes at 0° C. Sodium hydride (2 mg, 0.05 mmol, 60%) was added and the reaction was allowed to gradually warm to ambient temperature while stirring for 16 hours. The reaction mixture was diluted with EtOAc (20 mL), transferred to a separatory funnel, and washed with H2O (10 mL). The aqueous phase was separated and extracted with EtOAc (10 mL×3). The combined organic phases were washed with H2O (10 mL×3), dried over Na2SO4, filtered, and concentrated. The residue was purified via preparative TLC (5% MeOH in CH2Cl2) to afford compound 330 (6.3 mg, 21%). LCMS (ESI): m/z (M+Na) calculated for C31H28F3N3O7Na: 634.55, found 634.1.
Compound 331: Compound 330 (6.3 mg, 0.01 mmol) was dissolved in anhydrous MeOH (1 mL) containing CSA (0.26 mg, 0.001 mmol). The reaction mixture was heated to 76° C. in a screw-cap scintillation vial while stirring. After 2 hours, an additional 0.13 mg of CSA in 0.5 mL of MeOH was added. The reaction mixture was stirred at 76° C. for 16 hours. The reaction mixture concentrated under reduced pressure. The residue was purified via preparative TLC (10° % MeOH in CH2Cl2) to afford compound 331 (4.2 mg, 80%).
1H NMR (400 MHz, DMSO-d6) δ 8.80 (s, 1H), 7.94-7.86 (m, 2H), 7.48-7.42 (m, 2H), 7.38 (t, J=7.4 Hz, 2H), 7.36-7.28 (m, 1H), 5.46 (d, J=7.7 Hz, 1H), 5.28 (d, 1=6.0 Hz, 1H), 4.85 (dd, =10.7, 2.9 Hz, 1H), 4.67 (d, J=11.0 Hz, 1H), 4.62-4.58 (m, 1H), 4.54 (d, J=11.1 Hz, 1H), 4.44 (d, J=2.5 Hz, 1H), 4.36 (q, J=9.5 Hz, 1H), 3.95-3.90 (m, 1H), 3.78 (dd, J=9.3, 2.5 Hz, 1H), 3.71 (s, 3H), 3.61-3.54 (m, 1H), 3.52-3.43 (m, 1H), 3.43-3.38 (m, 1H). LCMS (ESI): m/z (M+Na) calculated for C24H24F3N3O7Na: 546.45, found 546.0.
Compound 332: To a solution of compound 331 (3.5 mg, 0.007 mmoles) in methanol (0.5 mL) was added 1.0 M NaOH solution (0.1 mL). The reaction mixture was stirred overnight at room temperature then neutralized with acidic resin, filtered and concentrated. The residue was purified by reverse phase chromatography using a C-8 matrix to afford 3.0 mg compound 332 (90%).
1H NMR (400 MHz, Deuterium Oxide) δ 8.39 (s, 1H), 8.37 (s, 2H), 7.54-7.45 (m, 1H), 7.43 (d, J=7.4 Hz, 2H), 7.35 (dt, J=14.3, 7.2 Hz, 3H), 4.86 (dd, J=11.0, 2.9 Hz, 1H), 4.76 (d, J=11.0 Hz, 1H), 4.40-4.30 (m, 2H), 4.16 (d, J=1.9 Hz, 1H), 4.04 (d, J=3.0 Hz, 1H), 3.81 (d, J=9.6 Hz, 1H), 3.73 (d, J=3.9 Hz, 0H), 3.67 (d, J=7.6 Hz, 1H), 3.56 (dd, J=11.7, 3.9 Hz, 1H). LCMS (ESI): m/z+Na calculated for C23H22F3N3O7: 509.1, found 508.2 (M−H).
Compound 333: Compound 333 can be prepared in an analogous fashion to
Compound 334: Compound 334 can be prepared in an analogous fashion to
Compound 335: Compound 335 can be prepared in an analogous fashion to
Compound 336: Compound 336 can be prepared in an analogous fashion to
Compound 337: Compound 337 can be prepared in an analogous fashion to
Compound 338: Compound 338 can be prepared in an analogous fashion to
Compound 339: Compound 339 can be prepared in an analogous fashion to
Compound 340: Compound 340 can be prepared in an analogous fashion to
Compound 341: Compound 341 can be prepared in an analogous fashion to
Compound 342: Compound 342 can be prepared in an analogous fashion to
Compound 343: Compound 342 can be prepared in an analogous fashion to
The inhibition assay to screen and characterize antagonists of E-selectin is a competitive binding assay, from which IC50 values may be determined. E-selectin/Ig chimera are immobilized in 96 well microtiter plates by incubation at 37° C. for 2 hours. To reduce nonspecific binding, bovine serum albumin is added to each well and incubated at room temperature for 2 hours. The plate is washed and serial dilutions of the test compounds are added to the wells in the presence of conjugates of biotinylated, sLea polyacrylamide with streptavidin/horseradish peroxidase and incubated for 2 hours at room temperature.
To determine the amount of sLea bound to immobilized E-selectin after washing, the peroxidase substrate, 3,3′,5,5′ tetramethylbenzidine (TMB) is added. After 3 minutes, the enzyme reaction is stopped by the addition of H3PO4, and the absorbance of light at a wavelength of 450 nm is determined. The concentration of test compound required to inhibit binding by 50% is determined.
Galectin-3 antagonists can be evaluated for their ability to inhibit binding of galectin-3 to a Galβ1-3GlcNAc carbohydrate structure. The detailed protocol is as follows. A 1 ug/mL suspension of a Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ-PAA-biotin polymer (Glycotech, catalog number 01-096) is prepared. A 100 uL aliquot of the polymer is added to the wells of a 96-well streptavidin-coated plate (R&D Systems, catalog number CP004). A 100 uL aliquot of 1×Tris Buffered Saline (TBS, Sigma, catalog number T5912—10×) is added to control wells. The polymer is allowed to bind to the streptavidin-coated wells for 1.5 hours at room temperature. The contents of the wells are discarded and 200 uL of 1×TBS containing 1% bovine serum albumin (BSA) is added to each well as a blocking reagent and the plate is kept at room temperature for 30 minutes. The wells are washed three times with 1×TBS containing 0.1% BSA. A serial dilution of test compounds is prepared in a separate V-bottom plate (Corning, catalog number 3897). A 75 uL aliquot of the highest concentration of the compound to be tested is added to the first well in a column of the V-bottom plate then 15 ul is serially transferred into 60 uL 1×TBS through the remaining wells in the column to generate a 1 to 5 serial dilution. A 60 uL aliquot of 2 ug/mL galectin-3 (IBL, catalog number IBATGP0414) is added to each well in the V-bottom plate. A 100 uL aliquot of the galectin-3/test compound mixture is transferred from the V-bottom plate into the assay plate containing the Galβ1-3GlcNAc polymer. Four sets of control wells in the assay plate are prepared in duplicate containing 1) both Galβ1-3GlcNAc polymer and galectin-3, 2) neither the polymer nor galectin-3, 3) galectin-3 only, no polymer, or 4) polymer only, no galectin-3. The plate is gently rocked for 1.5 hours at room temperature. The wells are washed four times with TBS/0.1% BSA. A 100 uL aliquot of anti-galectin-3 antibody conjugated to horse radish peroxidase (R&D Systems, from DGAL30 kit) is added to each well and the plate is kept at room temperature for 1 hour. The wells are washed four times with TBS/0.1% BSA. A 100 uL aliquot of TMB substrate solution is added to each well. The TMB substrate solution is prepared by making a 1:1 mixture of TMB Peroxidase Substrate (KPL, catalog number 5120-0048) and Peroxidase Substrate Solution B (KPL, catalog number 5120-0037). The plate is kept at room temperature for 10 to 20 minutes. The color development is stopped by adding 100 uL 10% phosphoric acid (RICCA Chemical Co., catalog number 5850-16). The absorbance at 450 nm (A450) is measured using a FlexStation 3 plate reader (Molecular Devices). Plots of A450 versus test compound concentration and IC50 determinations are made using GraphPad Prism 6.
The CXCR4-cAMP assay measures the ability of a glycomimetic CXCR4 antagonist to inhibit the binding of CXCL12 (SDF-1α) to CHO cells that have been genetically engineered to express CXCR4 on the cell surface. Assay kits may be purchased from DiscoveRx (95-0081E2CP2M; cAMP Hunter eXpress CXCR4 CHO-K1). The Gi-coupled receptor antagonist response protocol described in the kit instruction manual can be followed. GPCRs, such as CXCR4, are typically coupled to one of the 3 G-proteins: Gs, Gi, or Gq. In the CHO cells supplied with the kit, CXCR4 is coupled to Gi. After activation of CXCR4 by ligand binding (CXCL12), Gi dissociates from the CXCR4 complex, becomes activated, and binds to adenylyl cyclase, thus inactivating it, resulting in decreased levels of intracellular cAMP. Intracellular cAMP is usually low, so the decrease of the low level of cAMP by a Gi-coupled receptor will be difficult to detect. Forskolin is added to the CHO cells to directly activate adenylyl cyclase (bypassing all GPCRs), thus raising the level of cAMP in the cell, so that a Gi response can be more easily observed. CXCL12 interaction with CXCR4 decreases the intracellular level of cAMP and inhibition of CXCL12 interaction with CXCR4 by a CXCR4 antagonist increases the intracellular cAMP level, which is measured by luminescence.
Acute myelogenous leukemia (AML) cells may express the carbohydrate structures that contain the E-selectin ligand. When these AML cells circulate through the BM microvasculature, they will adhere to E-selectin, which in turn activates the NfkB pathway, causing chemoresistance. See
The result is that AML patients undergoing chemotherapy treatment will have those AML cells expressing high levels of the E-selectin ligands adhered to E-selectin in the microvasculature of these protective microdomains in the BM. These bound AML cells are chemoresistant and will be a source of surviving AML cells during relapse. This mechanism predicts that AML cells from relapsed patients should express higher levels of the E-selectin ligands. Indeed, mice engrafted with murine AML cells from the MLL-AF9 cell line showed higher expression of E-selectin on the surface of bone marrow endothelial cells than control animals (
In order to treat AML patients effectively, there is a need for understanding the mechanisms of leukemic cell chemotherapy evasion. Drugs, e.g., E-selectin inhibitors, that can be used alone, or in combination with chemotherapy, to treat relapsed/refractory AML are also desired. It may also be useful to identify patient subpopulations that are more or less likely to build chemoresistance, and protein or gene biomarkers, e.g., those involved in E-selectin ligand biosynthesis or metabolism, that may serve as effective biomarkers for identifying such patient subpopulations.
E-selectin antagonists, such as the compound of Formula I, which interrupt leukemic cell homing to the vascular niche and increase susceptibility to cytotoxic therapies, can be potent adjuncts to therapeutics.
Recent data demonstrated a correlation between leukemic cell surface levels of E-selectin ligands and response to the compound of Formula I, linking E-selecting ligand expression to E-selectin antagonist response (DeAngelo et al. 2018).
Multiple genes involved in the glycan synthesis of E-selectin ligands are highly expressed in pediatric AML. These genes provide novel therapeutic targets for overcoming drug resistance induced by the tumor microenvironment and lend support for the use of E-selectin ligand glycosylation genes as predictive biomarkers.
24 different genes (
High coverage single strand mRNA sequencing may be performed on clinical samples from pediatric AML patients (0 to 30 years old). The data from this analysis may then be screened for expression of the 24 different genes listed in
We questioned whether transcriptome profiling of E-selectin ligand forming glycosylation genes can be used to identify elevated E-selectin ligand expression in patients with cancers such as acute myeloid leukemia (AML), and subsequently which patients might benefit from and respond to E-selectin antagonists.
RNA-seq data from patients treated in COG AAML1031 (N=1,074) was available for evaluation. We examined transcriptome expression of 24 genes that code for enzymes involved in glycosylation of E-selectin ligands. All analyses were performed in R (v 3.5.2). Cox proportional hazards models were generated using the survival package (v 2.44-1.1). Multidimensional flow cytometry (MDF) was used to detect cell surface E-selectin ligand expression by two techniques: direct binding of an E-sel/hIg, PE labeled chimera, and the anti-sLex antibody HECA-452.
Seven of the 24 genes examined had minimal expression (mean <1 TPM) and were excluded from further analysis. The remaining 17 were variably expressed (
ST3GAL4 and FUT7 were targeted for further evaluation, as they directly synthesize sLex (
To verify surface protein expression of the two genes, leukemic specimens from SFhigh patients (N=10) and SFlow patients (N=10) underwent cell surface expression evaluation of glycosylated E-selectin ligands using two MDF assays. SFlow patients had low or undetectable levels of cell surface E-selectin ligands by both assays, whereas SFhigh patients had significantly higher expression of E-selectin ligands (p<0.001,
Expression levels of ST3GAL4 and FUT7, are associated with poor outcome. Additionally, high expression of these genes is detectable at the transcript level and associated with cell surface E-selectin ligand expression (Leonti et al. 2019).
Transcriptome profiling of E-selectin ligand-forming glycosylation genes was extended with an emphasis on ST3GAL4 and FUT7 in different cancers and in adult AML. Initially, expression levels of ST3GAL4 and FUT7 in 10,258 samples covering 33 cancer types from the TCGA PanCanAtlas were investigated. ST3GAL4 and FUT7 were consistently expressed in most of the cancers evaluated. The cancer types that expressed ST3GAL4 most highly were melanoma (uvual and skin), kidney chromphobe adrenocortical carcinoma and bladder urothelial carcinoma, while FUT7 was expressed most highly by AML, diffuse large B cell lymphoma, thymoma, testicular germ cell tumors, and head and neck squamous cell carcinoma.
Of particular interest was the identification of adult AML for the highest expression of FUT7 with high expression levels of ST3GAL4 (mean log 2 gene expression=8.1 and 9.4, respectively). Augmented expression of FUT7 was also observed in an analysis of 39 AML cell lines among the 1,457 cell lines comprising the Cancer Cell Line Encyclopedia RNAseq data set. The prognostic significance of FUT7 and ST3GAL4 in adult AML was further assessed using the TCGA-LAML RNAseq dataset for differential expression and associations with overall survival (OS).
The observed expression may then be correlated with the clinical outcome of overall survival (OS).
Treatment of AML relapsed/refractory patients with the compound of Formula I and chemotherapy were evaluated for response. Those patients with higher percentages of AML blasts expressing E-selectin ligands either in the BM (
The association was also observed to contribute to better overall survival (OS). As shown in
The data set of the present disclosure included 151 RNAseq profiles of bone marrow samples from adult patients with AML, and within this data set the status of the FMS-like tyrosine kinase 3 (FLT3) proto-oncogene was considered.
Mutational alterations of FLT3 are associated with higher risk of relapse and shorter OS compared with wild-type FLT3. ST3GAL4 and FUT7 were both identified as being upregulated (fold-change=1.73 and 1.40, respectively) in the mutated FLT3 subset (n=46) as compared to wild type FLT-3 (p=0.000033 and 0.046, respectively). Notably in the FLT3-ITD mutated subset expression of FUT7 was significantly associated with a poor prognosis and decreased OS (Hazard Ratio=0.223, p=0.015).
Mutations in FLT3 tyrosine kinase are detected in about 1/3 of patients newly diagnosed for acute myelogenous leukemia (AML). About 3/4 of these mutations are internal tandem duplications (FLT3-ITD) and the rest (1/4) have missense mutations within the tyrosine kinase domain activation loop (TKD) (See Thiede C. et al., Blood 99: 4326-4335 (2002), which is incorporated by reference in its entirety). Both mutations cause constitutive kinase activation and are associated with aggressive proliferative disease and poor survival (See Yamamoto Y. et al., Blood 97: 2434-2439 (2001), which is incorporated by reference in its entirety). In particular, the FLT3-ITD mutation is a strong risk factor for relapse after treatment (See Schnittger S. et al., Blood 100: 59-66 (2002), which is incorporated by reference in its entirety).
Patients expressing various subtypes of AML blast cells (M2, M3, M4, and M5) are known to contain high levels of TNFα circulating in their peripheral blood (See Volk A. et al., J. Exp. Med. 211: 1093-1108 (2014), which is incorporated by reference in its entirety;
Cytokines TNFα, IL-1, IL-6, IL-10 and endostatin were measured in AML patients, and only TNFα levels correlated with poor survival. (See Tsimberidou A. M. et al., Cancer, 113: 1605-1613 (2008), which is incorporated by reference in its entirety). Of these cytokines, TNFα is well known to stimulate expression of E-selectin. Previous data show that a high serum TNFα level is an adverse prognostic factor for overall survival and event-free survival in patients with untreated AML or high-risk MDS. In contrast, low TNFα levels (<10 pg/ml) were associated with higher rates of complete remission (P=0.003), survival (P=0.0003), and event-free survival (EFS) (P=0.0009). High expression of TNFα is associated with poor survival and also poor event free survival (See Tsimberidou A. M. et al., Cancer, 113: 1605-1613 (2008),
The hallmark of the AML cells containing mutations in the FLT3 gene is the constitutive kinase activation of these cancer cells. These highly activated cells are expected to produce higher levels of cytokines. A previous group examined relationships among cytokines, adhesion molecules and AML status. They showed that the FLT3-ITD mutation in AML patients was significantly associated with the expression of E-selectin. (See Kupsa T. et al., Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub., 160: 94-99 (2016), which is incorporated by reference in its entirety). The correlation of higher E-selectin expression in patients containing the FLT3-ITD mutation in their AML cells is strongly significant (P=0.0010) (See id.,
Analysis of a public database of AML patients, which is known as TCGA (The Cancer Genome Atlas) from NCI containing 151 paired data with Overall Survival was performed and expression of the gene (FUT7) which codes for the fucosyltransferase that adds the terminal fucose required for binding activity of the E-selectin ligand, sialyl Lex was correlated. This synthetic pathway is shown in
As discussed supra, FUT7 gene expression correlates to expression of the E-selectin ligand (sialyl Lex) on the surface of AML cells in patients. As shown in
Correlation of poor survival with expression of the E-selectin ligand as determined by FUT7 expression in FLT3-ITD patients is statistically significant (P=0.015). This suggests that the binding of AML cells to E-selectin drives the poor survival observed with AML patients containing the FLT3 mutations.
Collectively, these studies extend the prognostic importance of the E-selectin ligand glycosylation genes, ST3GAL4 and FUT7, to adult AML, where these genes may be useful as predictive biomarkers. In addition, these studies suggest potential additional tumor types beyond AML where treatment protocols with E-selectin inhibitors may have therapeutic benefits.
As shown in
The gene products of the ST3GAL4 and FUT7 genes, sialyltransferase ST3GAL4 (see Mondal N. et al., Blood 125: 687-696 (2015)) and fucosyltransferase FUT7 (see Maly P. et al., Cell 86: 643-653 (1996)), respectively, are known to add terminal sugars to synthesize the E-selectin ligand, sialyl Lex, as shown in
The database of gene expression from AML patients was therefore screened for the expression of both ST3GAL4 and FUT7 and correlated with OS. As can be seen in
These data support the observation that cancer patients that express high levels of the E-selectin ligand (sialyl Lea/x) on their tumors have a poorer outcome, as was reviewed in a meta-analysis of over ten years of publications on the role of sialyl Lex in cancer. In this review, the authors conclude that “our meta-analysis showed that a high level of sialyl Lex expression was significantly associated with lymphatic invasion, venous invasion, deep invasion, lymph node metastasis, distant metastasis, tumor stage, tumor recurrence, and OS in cancer.” Liang J. et al., Oncotargets and Therapy 9: 3113-3125 (2016).
Interestingly, those relapsed/refractory AML patients expressing high levels of sialyl Lex on their blasts show the greatest therapeutic response when treated with the compound of Formula I. This clinical observation supports the action of the compound of Formula I in inhibiting the binding of tumor sialyl Lex to E-selectin and preventing or breaking the E-selectin mediated chemoresistance as well.
Expression of E-selectin ligand on AML blasts: AML blasts from relapsed patients were isolated. The expression of E-selectin ligand was measured by immunofluorescence. As shown in
Phase I/II trial of Formula I in combination with chemotherapy for AML: A specific glycomimetic antagonist of E-selectin (Formula I) was rationally designed based on the bioactive conformation of sialyl Lea/x in the binding site of E-selectin. Treatment of AML relapsed/refractory patients with the compound of Formula I and chemotherapy were evaluated for response. In a Phase I trial, 19 patients were treated with the compound of Formula I twice a day at 5 mg/kg (n=6), 10 mg/kg (n=7), and 20 mg/kg doses (n=6), in combination with induction MEC (mitoxantrone, epotoside, and cytarabine) chemotherapy. In a Phase II trial, the treatment regimen comprised one 10 mg/kg dose of the compound of Formula I 24 hours prior to chemotherapy, then twice daily 10 mg/kg doses of the compound of Formula I throughout either MEC (mitoxantrone, etoposide, and cytarabine) or 7+3 (cytarabine for 7 days followed by 3 days of daunorubicin, idarubicin or mitoxantrone) chemotherapy up till 48 hours post-chemotherapy. AML blasts from the patients' bone marrow and peripheral blood were isolated, and the expression of E-selectin ligand on the blasts was measured using immunofluorescence. Patient response to treatment was also assessed.
For patients with relapsed or refractory AML, the response rate (CR/CRi) was 41% and this was higher than expected given the high-risk cytogenetic and other disease features. After a single course of induction treatment with the compound of Formula I, a higher CR/CRi rate (47%) was seen compared to historical controls of similar populations treated with MEC. The durability of response was sufficient to allow patients to proceed to stem cell transplant (n=9).
Interestingly, those patients with higher percentages of AML blasts expressing E-selectin ligands either in the BM (
Similarly,
The association was also observed to contribute to better overall survival (OS). As shown in
Biomarkers for clinical outcome and overall survival: High coverage single strand mRNA sequencing was performed on clinical samples from 1111 pediatric AML patients (0 to 30 years old) from the COG AAML1031 trial. The data from this analysis was screened for expression of the 24 different genes listed in
The difference in survival probability is starker when the highest-expressing quartiles of ST3GAL4 and FUT7 patients are compared to all other patients. As shown in
Clinical and RNAseq expression data for 10,258 samples covering 33 cancer types from the PanCanAtlas of The Cancer Genome Atlas (TCGA) were accessed via the NIH Genomics Data Commons (GDC) (
The number of samples from each tumor type varied, ranging from 45 samples which were available for cholangiocarcinoma (CHOL) to 1,188 for breast invasive carcinoma (BRCA), with a median of 198 samples/tumor type.
Expression data was log 2 transformed. (
The E-selectin ligand glycosylation genes, FUT7 and ST3GAL4 are consistently expressed in the majority of cancer subtypes. The top five cancer types, based in mean expression:
The E-selectin ligan glycosylation genes, FUT7 and ST3GAL4 are also consistently expressed in tumor cell lines comprising the Cancer Cell Line Encyclopedia database (
The TCGA-LAML RNAseq dataset was characterized for expression of FUT7 and ST3GAL4. (
Survival analysis was performed with the Cox proportional model, associating the expression levels of FUT7 and ST3GAL4 with overall survival (OS) (
These studies extend the prognostic importance of the E-selectin ligand glycosylation genes FUT7 and ST3GAL4 to adult AML. AML patients harboring the FLT3 ITD mutation with high expressions of FUT7 and ST3GAL4 experience poor survival compared to patients with low expression of FUT7 and ST3GAL4. These studies suggest additional tumor types beyond AML where treatment protocols with an E-selectin antagonist of Formula I may have therapeutic benefits.
This application claims priority to U.S. Provisional Patent Application No. 62/873,634, filed Jul. 12, 2019; U.S. Provisional Patent Application No. 62/881,312, filed Jul. 31, 2019; U.S. Provisional Patent Application No. 62/898,530, filed Sep. 10, 2019; U.S. Provisional Patent Application No. 62/914,812, filed Oct. 14, 2019; U.S. Provisional Patent Application No. 62/944,343, filed Dec. 5, 2019; and U.S. Provisional Patent Application No. 63/032,683, filed May 31, 2020; the disclosures of all of which are incorporated herein by reference in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/041740 | 7/12/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62873634 | Jul 2019 | US | |
| 62881312 | Jul 2019 | US | |
| 62898530 | Sep 2019 | US | |
| 62914812 | Oct 2019 | US | |
| 62944343 | Dec 2019 | US | |
| 63032683 | May 2020 | US |
