Patent Application
20250170120
This application contains a Sequence Listing that has been submitted in XML format via Patent Center and is hereby incorporated by reference in its entirety. The XML file, created Jan. 30, 2023, is named UTSD4040_106546-748634_Sequence_Listing.xml and is 9 kilobytes in size.
The present disclosure is generally directed to compositions and methods for modulating STING. Also provided are methods of use thereof.
Stimulator of interferon genes (STING) is an essential adaptor protein in innate immunity against DNA viruses or bacteria. STING is a transmembrane (TM) dimeric protein located in the endoplasmic reticulon (ER) or the Golgi apparatus. STING is activated by binding of its cytoplasmic ligand-binding domain (LBD) to cyclic dinucleotides, produced by either the cytosolic DNA-sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS) or invading bacteria. Cyclic dinucleotides induce the formation of high-order oligomers of STING, which is essential for triggering the downstream signaling pathways. cGAMP induces a conformational change in the STING LBD, which promotes its oligomerization. However, the cGAMP-induced STING oligomers appeared weak and have not been resolved to high resolution, hampering the understanding of the activation mechanism.
The present disclosure is based, at least in part, on the surprising discovery that a small molecular agonist, compound 53 (C53), promotes human STING oligomerization and activation through a mechanism orthogonal to that of cGAMP. Accordingly, C53 and related compounds may be useful to modulate STING-mediated immunity for vaccines or cancer immune-therapies. In various exemplary embodiments described herein, high-resolution cryo-EM structures and mutational analysis reveal that cGAMP and C53 can synergistically induce the oligomerization and activation of STING, thereby allowing for modulation of STING without use of cGAMP.
In some aspects, the present disclosure provides a method of allosterically inhibiting activity of a stimulator of interferon genes protein (STING), the method comprising contacting a compound of Formula (I), Formula (II) or Formula III to STING:
or a salt thereof,
In some aspects, the method of allosterically inhibiting activity of STING is an in vitro method. In other aspects, the method of allosterically inhibiting activity of STING is an in vivo method.
In further aspects, the present disclosure also provides for a method of treating a condition caused or related to disrupted STING signaling in a subject in need thereof, the method comprising administering a STING antagonist to the subject, wherein the STING antagonist is a compound of Formula (I), Formula (II) or Formula III:
or a salt thereof,
In some aspects, the condition caused or related to disrupted STING signaling in a subject can comprise inflammation, allergies, an autoimmune condition, an infectious disease, a neurodegenerative disease, a liver disease, a cancer, and/or a renal disease. The disclosed method further comprises administering the STING antagonist in a pharmaceutical composition comprising at least one carrier or excipient. In some aspects, the subject in need thereof is a mammal. In some aspects, the subject in need thereof is human.
In any of the methods provided herein, L1 may be absent or
Further, in any of the methods provided herein, R1 may be selected from the group consisting of
For example, in some aspects, R1 may be selected from the group consisting of
In another non-limiting example, R1 may be selected from the group consisting of
In various methods provided herein, R2 may be selected from the group consisting of
—CN, H, and —CF3. For example, in certain aspects, R2 can be selected from the group consisting of
In further aspects, R3 can be hydrogen.
In any of the methods provided herein, the compound and/or STING antagonist may be selected from the group consisting of:
and any salt thereof.
For example, the compound or STING antagonist may be selected from the group consisting of:
and any salt thereof.
In still further aspects, the compound and/or STING antagonist can be selected from the group consisting of:
In various aspects, the compound or STING antagonist may be selected from the group consisting of:
and any salt thereof.
In some aspects, the compound or STING antagonist may be selected from the group consisting of:
and any salt thereof.
Further aspects of the disclosure provides for a compound of Formula I, Formula II or Formula III:
or a salt thereof,
R2 is selected from the group consisting of H, CF3, F, CN,
and R3 is selected from the group consisting of H, CN or CF3; with the provisos that (i) at least one of R2 and R3 is not hydrogen and (ii) R1 is not
when L1 is
In various aspects, L1 may be absent,
For example, L1 can be absent or
In some aspects, R1 may be selected from the group consisting of
For example, in various embodiments, R1 may be selected from the group consisting of
For example, R1 may be selected from the group consisting of
In various aspects, R2 may be selected from the group consisting of
—CN, H, and —CF3. For example, in various aspects, R2 may be selected from the group consisting of
In any of the foregoing or related aspects R3 may be hydrogen.
In some aspects the compound provided herein is selected from the group consisting of:
and any salt thereof.
In further aspects, the present disclosure further provides for a pharmaceutical composition comprising any one of the compounds (i.e., STING antagonists) provided herein and at least one carrier or excipient.
Embodiments of the present inventive concept are illustrated by way of example in which like reference numerals indicate similar elements and in which:
The drawing figures do not limit the present inventive concept to the specific embodiments disclosed and described herein. The drawings are not necessarily to scale, emphasis instead being placed on clearly illustrating principles of certain embodiments of the present inventive concept.
The following detailed description references the accompanying drawings that illustrate various embodiments of the present inventive concept. The drawings and description are intended to describe aspects and embodiments of the present inventive concept in sufficient detail to enable those skilled in the art to practice the present inventive concept. Other components can be utilized and changes can be made without departing from the scope of the present inventive concept. The following description is, therefore, not to be taken in a limiting sense. The scope of the present inventive concept is defined only by the appended claims, along with the full scope of equivalents to which such claims are entitled.
The present disclosure is based, at least in part, on an allosteric strategy to modulate STING activities, including small molecule STING antagonist and agonist compounds that bind to the corresponding allosteric site, methods of preparation of the compounds, pharmaceutical compositions comprising the compounds, and their use in medical therapy. In particular, the present disclosure provides STING modulator compounds, which find utility as inhibitors or activators of STING. An advantage of the compounds provided herein is that a broad range of pharmacological activities is possible, ranging from antagonizing STING activity induced by cyclic dinucleotide cGAMP to acting alone or synergizing with cGAMP to activate STING. In addition, the disclosure provides methods of using the antagonist compounds described herein for the treatment of inflammatory, allergic, autoimmune, and infectious diseases, and agonist compounds to treat cancer. The antagonist compounds can also be used for the treatment of senescence- or age-related diseases, such as neurodegenerative diseases, cardiovascular diseases, liver and renal diseases, and premature aging.
The phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. For example, the use of a singular term, such as, “a” is not intended as limiting of the number of items. Also, the use of relational terms such as, but not limited to, “top,” “bottom,” “left,” “right,” “upper,” “lower,” “down,” “up,” and “side,” are used in the description for clarity in specific reference to the figures and are not intended to limit the scope of the present inventive concept or the appended claims.
Further, as the present inventive concept is susceptible to embodiments of many different forms, it is intended that the present disclosure be considered as an example of the principles of the present inventive concept and not intended to limit the present inventive concept to the specific embodiments shown and described. Any one of the features of the present inventive concept may be used separately or in combination with any other feature. References to the terms “embodiment,” “embodiments,” and/or the like in the description mean that the feature and/or features being referred to are included in, at least, one aspect of the description. Separate references to the terms “embodiment,” “embodiments,” and/or the like in the description do not necessarily refer to the same embodiment and are also not mutually exclusive unless so stated and/or except as will be readily apparent to those skilled in the art from the description. For example, a feature, structure, process, step, action, or the like described in one embodiment may also be included in other embodiments, but is not necessarily included. Thus, the present inventive concept may include a variety of combinations and/or integrations of the embodiments described herein. Additionally, all aspects of the present disclosure, as described herein, are not essential for its practice. Likewise, other systems, methods, features, and advantages of the present inventive concept will be, or become, apparent to one with skill in the art upon examination of the figures and the description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present inventive concept, and be encompassed by the claims.
Any term of degree such as, but not limited to, “substantially” as used in the description and the appended claims, should be understood to include an exact, or a similar, but not exact configuration. For example, “a substantially planar surface” means having an exact planar surface or a similar, but not exact planar surface. Similarly, the terms “about” or “approximately,” as used in the description and the appended claims, should be understood to include the recited values or a value that is three times greater or one third of the recited values. For example, about 3 mm includes all values from 1 mm to 9 mm, and approximately 50 degrees includes all value from 16.6 degrees to 150 degrees. For example, they can refer to less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to 1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%.
The terms “comprising,” “including” and “having” are used interchangeably in this disclosure. The terms “comprising,” “including” and “having” mean to include, but not necessarily be limited to the things so described.
Lastly, the terms “or” and “and/or,” as used herein, are to be interpreted as inclusive or meaning any one or any combination. Therefore, “A, B or C” or “A, B and/or C” mean any of the following: “A,” “B” or “C”; “A and B”; “A and C”; “B and C”; “A, B and C.” An exception to this definition will occur only when a combination of elements, functions, steps or acts are in some way inherently mutually exclusive.
Various aspects of the present disclosure are directed to STING allosteric modulators. As demonstrated herein, high-resolution cryo-EM structure of human STING in complex with both cGAMP and compounds of the present disclosure define a novel binding site in the transmembrane domain (TMD) of STING, paving the way for developing more modulators that target this site. Compounds that target the TMD site might be better STING modulators than cGAMP mimetics for therapeutic purposes because they are more hydrophobic and therefore more permeable to the cell membrane. In addition, the structural analyses clarify the direct role of the TMD in the oligomerization and activation of STING. The coupling between the induced opening of the TMD pocket by an illustrative agonist (C53) and the high-order oligomerization of human STING suggests that this conformational change might be an integral part of its activation mechanism in vivo. Surprisingly, it has been found that minor changes in a structure of a compound can effectively interfere with this conformational change, while still binding at the same site—turning a potent agonist into an antagonist in an unpredictable and surprising way.
In view of these discoveries, the present disclosure is directed to particular methods of allosterically inhibiting activity of a STING protein by contacting the STING protein with a STING antagonist described herein. In certain aspects, the STING antagonist may be a compound of Formula (I), Formula (II), or Formula (III):
or a salt thereof,
In various aspects, L1 may be absent
For example, in some aspects L1 may be absent. In other aspects, L1 may be
In other aspects, L1 may be
In further aspects of the present disclosure, R1 can be selected from the group consisting of
For example, R1 may be selected from the group consisting of
As another example, R1 may be selected from the group consisting of
In still further aspects of the present disclosure, R2 may be selected from the group consisting of
—CN, H, and —CF3. For example, in some aspects, R2 is selected from the group consisting of
In certain aspects, R3 can be hydrogen, CN or CF3. In certain aspects, R3 is hydrogen.
In accord with the above, the allosteric inhibitors of the present disclosure may be selected from the group consisting of
and any salt thereof.
For example, in certain aspects, the allosteric inhibitor of the STING protein (i.e., a STING antagonist) may be selected from the group consisting of
For example, in certain aspects, the compounds presented herein may be selected from the group consisting of
and any salt thereof.
In various aspects, a STING antagonist provided herein may be selected from the group consisting of:
and any salt thereof.
In still further aspects, a STING antagonist may be selected from the group consisting of:
and any salt thereof.
In various aspects, the compounds provided herein may be active inhibitors of STING signaling. For example, in some aspects, the compounds may show >50% (i.e., >50%, greater than 60%, greater than 70%, greater than 80% or greater than 90%) inhibition at 10 μM upon cGAMP (100 μM) or MSA-2 (10 μM) stimulation in THP-1 cells. In other aspects, the compounds may show less than 50% inhibition at 10 μM upon cGAMP (100 μM) or MSA-2 (10 μM) stimulation in THP-1 cells. In still other aspects, the compounds may show greater than 50% inhibition at 32 μM under basal conditions (no external stimulation) in THP-1 cells. In other aspects, the compounds may show less than 50% inhibition at 32 μM under basal conditions (no external stimulation) in THP-1 cells.
Methods of measuring STING activity and by extension, the activity of the compounds discussed herein, are known in the art. As an example, the STING pathway activity may be evaluated using reporter cell lines where a reporter gene (i.e., luciferase, GFP) is expressed under direct or indirect control of interferon response elements. For example, in an exemplary embodiment, the STING pathway can be measured using THP1-Dual™ cells (InvivoGen) which express the Lucia gene (a secreted luciferase reporter gene) under the control of an ISG54 minimal promoter in conjunction with five IFN-stimulated response elements. The STING pathway, when activated, stimulates interferon release which, in this model, will increase luciferase expression in a detectable manner. When inhibited, interferon release will be decreased, reducing luciferase expression in a detectable manner. The compounds can optionally be tested in the presence of external stimulation (i.e., using cGAMP, a natural agonist of STING, or MSA-2).
As described in more detail below, the inhibitor compounds provided herein may be used to decrease interferon production and thereby treat or ameliorate various conditions. Without being bound by theory, it is considered that the hydrophobic nature of these and related compounds may make these compounds more suitable as therapeutic agents since they can more easily partition across the plasma membrane and reach the target protein on the endoplasmic reticulum.
In further aspects of the present disclosure, pharmaceutical compositions are provided. The pharmaceutical compositions may comprise any of the agonist or antagonist compounds described herein and a pharmaceutically suitable carrier or excipient.
The composition may comprise at least one excipient. Suitable excipients include pharmaceutically acceptable excipients, such as diluents, binders, fillers, buffering agents, pH modifying agents, disintegrants, dispersants, preservatives, lubricants, taste-masking agents, flavoring agents, coloring agents, or combinations thereof. The amount and types of excipients utilized to form pharmaceutical compositions may be selected according to known principles of pharmaceutical science.
In one embodiment, the excipient may be a diluent. The diluent may be compressible (i.e., plastically deformable) or abrasively brittle. Non-limiting examples of suitable compressible diluents include microcrystalline cellulose (MCC), cellulose derivatives, cellulose powder, cellulose esters (i.e., acetate and butyrate mixed esters), ethyl cellulose, methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, corn starch, phosphated corn starch, pregelatinized corn starch, rice starch, potato starch, tapioca starch, starch-lactose, starch-calcium carbonate, sodium starch glycolate, glucose, fructose, lactose, lactose monohydrate, sucrose, xylose, lactitol, mannitol, maltitol, sorbitol, xylitol, maltodextrin, and trehalose. Non-limiting examples of suitable abrasively brittle diluents include dibasic calcium phosphate (anhydrous or dihydrate), calcium phosphate tribasic, calcium carbonate, and magnesium carbonate.
In another embodiment, the excipient may be a binder. Suitable binders include, but are not limited to, starches, pregelatinized starches, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C12-C18 fatty acid alcohol, polyethylene glycol, polyols, or saccharides.
In another embodiment, the excipient may be a filler. Suitable fillers include, but are not limited to, carbohydrates, inorganic compounds, and polyvinylpyrrolidone. By way of non-limiting example, the filler may be calcium sulfate, both di- and tri-basic, starch, calcium carbonate, magnesium carbonate, microcrystalline cellulose, dibasic calcium phosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc, modified starches, lactose, sucrose, mannitol, or sorbitol.
In still another embodiment, the excipient may be a buffering agent. Representative examples of suitable buffering agents include, but are not limited to, phosphates, carbonates, citrates, tris buffers, and buffered saline salts (e.g., Tris buffered saline or phosphate buffered saline).
In various embodiments, the excipient may be a pH modifier. By way of non-limiting example, the pH modifying agent may be sodium carbonate, sodium bicarbonate, sodium citrate, citric acid, or phosphoric acid.
In a further embodiment, the excipient may be a disintegrant. The disintegrant may be non-effervescent or effervescent. Suitable examples of non-effervescent disintegrants include, but are not limited to, starches such as corn starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, micro-crystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar, locust bean, karaya, pecitin, and tragacanth. Non-limiting examples of suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid and sodium bicarbonate in combination with tartaric acid.
In yet another embodiment, the excipient may be a dispersant or dispersing enhancing agent. Suitable dispersants may include, but are not limited to, starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose.
In another alternate embodiment, the excipient may be a preservative. Non-limiting examples of suitable preservatives include antioxidants, such as BHA, BHT, vitamin A, vitamin C, vitamin E, or retinyl palmitate, citric acid, sodium citrate; chelators such as EDTA or EGTA; and antimicrobials, such as parabens, chlorobutanol, or phenol.
The compositions disclosed herein may be formulated into various dosage forms and administered by a number of different means that will deliver a therapeutically effective amount of the active ingredient. The excipients included in the compositions comprising the STING agonist or antagonists described herein, acids thereof, and/or salts thereof may be based on the form of administering such compositions. Such compositions may be administered orally, parenterally, or topically in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. Topical administration may also involve the use of transdermal administration such as transdermal patches or iontophoresis devices. The term, “parenteral,” as used herein includes subcutaneous, intravenous, intramuscular, or intrasternal injection, or infusion techniques. Formulation of drugs is discussed in, for example, Gennaro, A. R., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (18th ed, 1995), and Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Dekker Inc., New York, N.Y. (1980).
In various aspects, methods of using the STING agonists and/or antagonists described herein are provided. In various aspects, the STING modulators may be used to treat various diseases related to hyper or hypo active native immunity (e.g., interferon production).
As described above, the present disclosure describes a novel allosteric binding site in the transmembrane domain (TMD) of the STING protein. Accordingly, in various aspects, a method is provided for modulating (i.e., antagonizing or activating) STING by targeting the allosteric binding site in the transmembrane domain (TMD).
In various aspects, the methods comprise activating the STING protein. This may occur, for example, by applying a compound which targets the allosteric binding site in the TMD and facilitates the oligomerization and/or phosphorylation of the STING complex—thereby triggering downstream signaling pathways that trigger interferon release and activation of innate immunity. In various aspects, the methods may comprise applying a STING agonist, described herein, to a cell. In various aspects the STING agonist may comprise
In additional aspects, the method may comprise antagonizing the STING protein. This may be done by applying a compound that also targets the allosteric binding site, but which blocks oligomerization and/or blocks binding of a native agonist (e.g., cGAMP) to its binding site. In various aspects, the STING antagonist may be a compound of Formula I, Formula II or Formula III as defined above.
In various aspects, the methods of allosterically modulating the STING protein may comprise contacting a cell with any of the compounds described herein. In various aspects, the cell may be in vitro or in vivo. Accordingly, in various aspects a method of treating a subject is provided, the method comprising administering a therapeutically effective amount of any of the STING antagonists or agonists provided herein to the subject. In some aspects, the subject may be suffering from an inflammatory, allergic, autoimmune, and/or infectious diseases, atherosclerosis, arthritis (e.g., osteoarthritis or rheumatoid arthritis), an inflammatory bowel disease (e.g., ulcerative colitis or Crohn's disease). In some aspects, the subject may have senescence- or age-related diseases, such as neurodegenerative diseases, cardiovascular diseases, liver and renal diseases, or be afflicted with premature aging. In some aspects, the subject may have cancer. In some aspects, the method may comprise administering a therapeutically effective amount of a STING antagonist to a subject with an inflammatory, allergic, autoimmune, and/or infectious disease. In some aspects, the method may comprise administering a therapeutically effective amount of a STING antagonist to a subject with senescence- or age-related diseases, such as neurodegenerative diseases, cardiovascular diseases, liver and renal diseases, or premature aging. In some aspects, the method may comprise administering a therapeutically effective amount of a STING agonist to a subject with cancer.
Dosage amounts of the disclosed compounds can be in the range of from about 0.0001 mg/kg/day, 0.001 mg/kg/day or 0.01 mg/kg/day to about 100 mg/kg/day. Dosage may be adjusted based on the stage and severity of cancer and subject characteristics. In some aspects, the subject is administered at least about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg/kg (or any range derivable therein) of the STING modulator, sufficient to effect treatment of the disease. The effective amount to be administered depends upon a number of factors including, for example, the age and weight of the subject (e.g., a mammal such as a human), the precise condition requiring treatment and its severity, the route of administration, and will ultimately be at the discretion of the attendant physician or veterinarian. It will be understood, however, that the specific dose level for any particular subject will depend on a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex and diet of the subject being treated; the time and route of administration; the rate of excretion; other drugs which have previously been administered; and the severity of the particular condition undergoing therapy, as is well understood by those skilled in the art.
A dose may be administered on an as needed basis or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 hours (or any range derivable therein) or 1, 2, 3, 4, 5, 6, 7, 8, 9, or times per day (or any range derivable therein). A dose may be first administered before or after signs of disease are exhibited or felt by a subject or after a clinician evaluates the subject for an infection. In some aspects, the subject is administered a first dose of a regimen 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours (or any range derivable therein) or 1, 2, 3, 4, or 5 days after the subject experiences or exhibits signs or symptoms of a disease (or any range derivable therein). The subject may be treated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days (or any range derivable therein) or until symptoms of the disease have disappeared or been reduced or after 6, 12, 18, or 24 hours or 1, 2, 3, 4, or 5 days after symptoms of a disease have disappeared or been reduced.
In some aspects, the disclosure provides methods for modulating an immune response in a subject having a disease or disorder associated with altered STING function. These methods can include the step of administering to the subject an amount of a pharmaceutical composition including a disclosed STING modulator compound and a pharmaceutically acceptable carrier, wherein amount the pharmaceutical composition is effective to ameliorate the altered STING function in the subject.
Also described herein are methods of treating cancer in a subject having a cancer or tumor with altered STING function and/or infiltrated with inflammatory immune cells. These methods comprise administering to the subject in need thereof, an amount of a pharmaceutical composition including a disclosed STING modulator and a pharmaceutically acceptable carrier, wherein amount the pharmaceutical composition is effective for treating cancer. Treating cancer can comprise inhibition of the proliferation, growth, and/or spread of cancer or tumor cells, inhibition of cancer progression and/or metastases, inhibition of an increase in tumor volume, a reduction in tumor volume, a reduction in tumor growth, an eradication of a tumor and/or cancer cell, or any combinations thereof. The method can also result in a prolonging survival of a subject or improving the quality of the life for the subject. Further, the method can include reducing the number of inflammatory immune cells infiltrating the cancer or tumor (e.g., by at least 20, 30, 40, 50, 60, 70, 80, or 90%, or until reduction of inflammatory cell infiltration is detectably reduced by histology or scanning).
Methods disclosed herein may be used in combination with additional cancer therapy. In some aspects, the distinct cancer therapy comprises surgery, radiotherapy, chemotherapy, toxin therapy, immunotherapy, cryotherapy or gene therapy. In some aspects, the cancer is a chemotherapy-resistant or radio-resistant cancer. Combination therapy may be achieved by use of a single pharmaceutical composition that includes both agents, or by administering two distinct compositions at the same time, wherein one composition includes the STING modulator and the other includes the second agent(s).
As such, one aspect of the disclosure encompasses treatment of any STING related cancer or neoplasm. As it will be recognized by individuals skilled in the art, cancer as used throughout the instant disclosure may be one or more neoplasm or cancer. The neoplasm may be malignant or benign, the cancer may be primary or metastatic; the neoplasm or cancer may be early stage or late stage. Non-limiting examples of neoplasms or cancers that may be treated include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytomas (childhood cerebellar or cerebral), basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brainstem glioma, brain tumors (cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic gliomas), breast cancer, bronchial adenomas/carcinoids, Burkitt lymphoma, carcinoid tumors (childhood, gastrointestinal), carcinoma of unknown primary, central nervous system lymphoma (primary), cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma in the Ewing family of tumors, extracranial germ cell tumor (childhood), extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancers (intraocular melanoma, retinoblastoma), gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumors (childhood extracranial, extragonadal, ovarian), gestational trophoblastic tumor, gliomas (adult, childhood brain stem, childhood cerebral astrocytoma, childhood visual pathway and hypothalamic), gastric carcinoid, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma (childhood), intraocular melanoma, islet cell carcinoma, Kaposi sarcoma, kidney cancer (renal cell cancer), laryngeal cancer, leukemias (acute lymphoblastic, acute myeloid, chronic lymphocytic, chronic myelogenous, hairy cell), lip and oral cavity cancer, liver cancer (primary), lung cancers (non-small cell, small cell), lymphomas (AIDS-related, Burkitt, cutaneous T-cell, Hodgkin, non-Hodgkin, primary central nervous system), macroglobulinemia (Waldenstrom), malignant fibrous histiocytoma of bone/osteosarcoma, medulloblastoma (childhood), melanoma, intraocular melanoma, Merkel cell carcinoma, mesotheliomas (adult malignant, childhood), metastatic squamous neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome (childhood), multiple myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases, myelogenous leukemia (chronic), myeloid leukemias (adult acute, childhood acute), multiple myeloma, myeloproliferative disorders (chronic), nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma/malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer (surface epithelial-stromal tumor), ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, pancreatic cancer (islet cell), paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and supratentorial primitive neuroectodermal tumors (childhood), pituitary adenoma, plasma cell neoplasia, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter transitional cell cancer, retinoblastoma, rhabdomyosarcoma (childhood), salivary gland cancer, sarcoma (Ewing family of tumors, Kaposi, soft tissue, uterine), Sezary syndrome, skin cancers (nonmelanoma, melanoma), skin carcinoma (Merkel cell), small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer with occult primary (metastatic), stomach cancer, supratentorial primitive neuroectodermal tumor (childhood), T-Cell lymphoma (cutaneous), testicular cancer, throat cancer, thymoma (childhood), thymoma and thymic carcinoma, thyroid cancer, thyroid cancer (childhood), transitional cell cancer of the renal pelvis and ureter, trophoblastic tumor (gestational), unknown primary site (adult, childhood), ureter and renal pelvis transitional cell cancer, urethral cancer, uterine cancer (endometrial), uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma (childhood), vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor (childhood).
In some aspects, administration of STING modulator disclosed herein, prevents (neuroprotective) or treats neurological disorders. These methods comprise administering to the subject in need thereof, an amount of a pharmaceutical composition including a disclosed STING modulator and a pharmaceutically acceptable carrier, wherein amount the pharmaceutical composition is effective for treating the neurological disorder. In such cases, the administration of disclosed STING modulators can improve cognitive function, suppress neuronal apoptosis, suppress amyloidosis of cranial nerves, lower a total count of immune microglia in brain, reduce inflammation in brain, and/or prevent the progression of neurological disorder. Neurological disorder refers to any disorder of the nervous system and/or visual system. “Neurological disorders” include disorders that involve the central nervous system (brain, brainstem and cerebellum), the peripheral nervous system (including cranial nerves), and the autonomic nervous system (parts of which are located in both central and peripheral nervous system). Neurodegenerative diseases, include, for example, Alzheimer's Disease, stroke, multiple sclerosis etc.
Several neurological disorders, symptoms, signs and syndromes that can be treated using compositions and methods according to the present invention include: acquired epileptiform aphasia; acute disseminated encephalomyelitis; adrenoleukodystrophy; age-related macular degeneration; agenesis of the corpus callosum; agnosia; Aicardi syndrome; Alexander disease; Alpers' disease; alternating hemiplegia; Alzheimer's disease; Vascular dementia; amyotrophic lateral sclerosis; anencephaly; Angelman syndrome; angiomatosis; anoxia; aphasia; apraxia; arachnoid cysts; arachnoiditis; Anronl-Chiari malformation; arteriovenous malformation; Asperger syndrome; ataxia telegiectasia; attention deficit hyperactivity disorder; autism; autonomic dysfunction; back pain; Batten disease; Behcet's disease; Bell's palsy; benign essential blepharospasm; benign focal; amyotrophy; benign intracranial hypertension; Binswanger's disease; blepharospasm; Bloch Sulzberger syndrome; brachial plexus injury; brain abscess; brain injury; brain tumors (including glioblastoma multiforme); spinal tumor; Brown-Sequard syndrome; Canavan disease; carpal tunnel syndrome; causalgia; central pain syndrome; central pontine myelinolysis; cephalic disorder; cerebral aneurysm; cerebral arteriosclerosis; cerebral atrophy; cerebral gigantism; cerebral palsy; Charcot-Marie-Tooth disease; chemotherapy-induced neuropathy and neuropathic pain; Chiari malformation; chorea; chronic inflammatory demyelinating polyneuropathy; chronic pain; chronic regional pain syndrome; Coffin Lowry syndrome; coma, including persistent vegetative state; congenital facial diplegia; corticobasal degeneration; cranial arteritis; craniosynostosis; Creutzfeldt-Jakob disease; cumulative trauma disorders; Cushing's syndrome; cytomegalic inclusion body disease; cytomegalovirus infection; dancing eyes-dancing feet syndrome; Dandy-Walker syndrome; Dawson disease; De Morsier's syndrome; Dejerine-Klumke palsy; dementia; dermatomyositis; diabetic neuropathy; diffuse sclerosis; dysautonomia; dysgraphia; dyslexia; dystonias; early infantile epileptic encephalopathy; empty sella syndrome; encephalitis; encephaloceles; encephalotrigeminal angiomatosis; epilepsy; Erb's palsy; essential tremor; Fabry's disease; Fahr's syndrome; fainting; familial spastic paralysis; febrile seizures; Fisher syndrome; Friedreich's ataxia; fronto-temporal dementia and other “tauopathies”; Gaucher's disease; Gerstmann's syndrome; giant cell arteritis; giant cell inclusion disease; globoid cell leukodystrophy; Guillain-Barre syndrome; HTLV-1-associated myelopathy; Hallervorden-Spatz disease; head injury; headache; hemifacial spasm; hereditary spastic paraplegia; heredopathia atactica polyneuritiformis; herpes zoster oticus; herpes zoster; Hirayama syndrome; HIV-associated dementia and neuropathy (also neurological manifestations of AIDS); holoprosencephaly; Huntington's disease and other polyglutamine repeat diseases; hydranencephaly; hydrocephalus; hypercortisolism; hypoxia; immune-mediated encephalomyelitis; inclusion body myositis; incontinentia pigmenti; infantile phytanic acid storage disease; infantile refsum disease; infantile spasms; inflammatory myopathy; intracranial cyst; intracranial hypertension; Joubert syndrome; Kearns-Sayre syndrome; Kennedy disease Kinsbourne syndrome; Klippel Feil syndrome; Krabbe disease; Kugelberg-Welander disease; kuru; Lafora disease; Lambert-Eaton myasthenic syndrome; Landau-Kleffner syndrome; lateral medullary (Wallenberg) syndrome; learning disabilities; Leigh's disease; Lennox-Gustaut syndrome; Lesch-Nyhan syndrome; leukodystrophy; Lewy body dementia; Lissencephaly; locked-in syndrome; Lou Gehrig's disease (i.e., motor neuron disease or amyotrophic lateral sclerosis); lumbar disc disease; Lyme disease—neurological sequelae; Machado-Joseph disease; macrencephaly; megalencephaly; Melkersson-Rosenthal syndrome; Menieres disease; meningitis; Menkes disease; metachromatic leukodystrophy; microcephaly; migraine; Miller Fisher syndrome; mini-strokes; mitochondrial myopathies; Mobius syndrome; monomelic amyotrophy; motor neuron disease; Moyamoya disease; mucopolysaccharidoses; milti-infarct dementia; multifocal motor neuropathy; multiple sclerosis and other demyelinating disorders; multiple system atrophy with postural hypotension; p muscular dystrophy; myasthenia gravis; myelinoclastic diffuse sclerosis; myoclonic encephalopathy of infants; myoclonus; myopathy; myotonia congenital; narcolepsy; neurofibromatosis; neuroleptic malignant syndrome; neurological manifestations of AIDS; neurological sequelae of lupus; neuromyotonia; neuronal ceroid lipofuscinosis; neuronal migration disorders; Niemann-Pick disease; O'Sullivan-McLeod syndrome; occipital neuralgia; occult spinal dysraphism sequence; Ohtahara syndrome; olivopontocerebellar atrophy; opsoclonus myoclonus; optic neuritis; orthostatic hypotension; overuse syndrome; paresthesia; Parkinson's disease; paramyotonia congenital; paraneoplastic diseases; paroxysmal attacks; Parry Romberg syndrome; Pelizaeus-Merzbacher disease; periodic paralyses; peripheral neuropathy; painful neuropathy and neuropathic pain; persistent vegetative state; pervasive developmental disorders; photic sneeze reflex; phytanic acid storage disease; Pick's disease; pinched nerve; pituitary tumors; polymyositis; porencephaly; post-polio syndrome; postherpetic neuralgia; postinfectious encephalomyelitis; postural hypotension; Prader-Willi syndrome; primary lateral sclerosis; prion diseases; progressive hemifacial atrophy; progressive multifocal leukoencephalopathy; progressive sclerosing poliodystrophy; progressive supranuclear palsy; pseudotumor cerebri; Ramsay-Hunt syndrome (types I and II); Rasmussen's encephalitis; reflex sympathetic dystrophy syndrome; Refsum disease; repetitive motion disorders; repetitive stress injuries; restless legs syndrome; retrovirus-associated myelopathy; Rett syndrome; Reye's syndrome; Saint Vitus dance; Sandhoff disease; Schilder's disease; schizencephaly; septo-optic dysplasia; shaken baby syndrome; shingles; Shy-Drager syndrome; Sjögren's syndrome; sleep apnea; Soto's syndrome; spasticity; spina bifida; spinal cord injury; spinal cord tumors; spinal muscular atrophy; Stiff-Person syndrome; stroke; Sturge-Weber syndrome; subacute sclerosing panencephalitis; subcortical arteriosclerotic encephalopathy; Sydenham chorea; syncope; syringomyelia; tardive dyskinesia; Tay-Sachs disease; temporal arteritis; tethered spinal cord syndrome; Thomsen disease; thoracic outlet syndrome; Tic Douloureux; Todd's paralysis; Tourette syndrome; transient ischemic attack; transmissible spongiform encephalopathies; transverse myelitis; traumatic brain injury; tremor; trigeminal neuralgia; tropical spastic paraparesis; tuberous sclerosis; vascular dementia (multi-infarct dementia); vasculitis including temporal arteritis; Von Hippel-Lindau disease; Wallenberg's syndrome; Werdnig-Hoffman disease; West syndrome; whiplash; Williams syndrome; Wildon's disease; and Zellweger syndrome.
In another aspect, disclosed herein are methods of administration of STING modulators to prevent or treat subjects suffering from an autoimmune disease. Examples of autoimmune diseases comprise: rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel diseases (IBDs) comprising Crohn disease (CD), ulcerative colitis (UC) psoriasis, diabetes and as agents to suppress transplant rejection. The method comprises administering to a subject, an amount of a disclosed STING modulator, or a pharmaceutically acceptable salt thereof, for curing, reversing, alleviating, palliative and/or prophylactic treatment of the autoimmune disease or one or more symptoms associated with the disease. Examples of other autoimmune diseases which may be treated using the compositions of the present invention include, but are not limited to, alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, autoimmune juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, lupus, myasthenia gravis, some forms of myocarditis, multiple sclerosis, pemphigus/pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma/systemic sclerosis, Sjögren's syndrome, systemic lupus erythematosus, some forms of thyroiditis, some forms of uveitis, vitiligo, and granulomatosis with polyangiitis (Wegener's).
In a further aspect provided herein is a method of treating inflammation, and/or allergy comprising administering to a subject in need thereof a therapeutically effective amount of a disclosed STING modulator or a pharmaceutically acceptable salt thereof. Inflammatory and/or allergic conditions which may be treated with the disclosed STING modulators include, for example, asthma, appendicitis, dermatitis, dermatomyositis, endocarditis, fibrositis, gingivitis, glossitis, hepatitis, hidradenitis suppurativa, iritis, laryngitis, mastitis, myocarditis, nephritis, otitis, pancreatitis, parotitis, percarditis, peritonoitis, pharyngitis, pleuritis, pneumonitis, prostatistis, pyelonephritis, and stomatisi, transplant rejection (involving organs such as kidney, liver, heart, lung, pancreas (e.g., islet cells), bone marrow, cornea, small bowel, skin allografts, skin homografts, and heart valve xengrafts, sewrum sickness, and graft vs host disease), acute pancreatitis, chronic pancreatitis, acute respiratory distress syndrome. Sexary's syndrome, congenital adrenal hyperplasis, nonsuppurative thyroiditis, hypercalcemia associated with cancer, pemphigus, bullous dermatitis herpetiformis, severe erythema multiforme, exfoliative dermatitis, seborrheic dermatitis, seasonal or perennial allergic rhinitis, bronchial asthma, contact dermatitis, astopic dermatitis, drug hypersensistivity reactions, allergic conjunctivitis, keratitis, herpes zoster ophthalmicus, iritis and oiridocyclitis, chorioretinitis, optic neuritis, symptomatic sarcoidosis, fulminating or disseminated pulmonary tuberculosis chemotherapy, idiopathic thrombocytopenic purpura in adults, secondary thrombocytopenia in adults, acquired (autroimmine) haemolytic anemia, leukaemia and lymphomas in adults, acute leukaemia of childhood, regional enteritis, autoimmune vasculitis, multiple sclerosis, chronic obstructive pulmonary disease, solid organ transplant rejection, sepsis. The STING modulators may additionally be used to treat T-cell mediated hypersensitivity diseases having an inflammatory component. Such conditions include contact hypersensitivity, contact dermatitis (including that due to poison ivy), uticaria, skin allergies, respiratory allergies (hayfever, allergic rhinitis) and gluten-sensitive enteropathy (Celliac disease).
In some aspects this disclosure provides a method for delaying onset or progression of senescence or an age-related disease or condition in a subject comprising administering to the subject a STING modulator provided herein, or a pharmaceutically acceptable salt thereof. In some aspects the method delays the onset of senescence or an age-related disease or condition. In some aspects the method delays the progression of senescence or an age-related disease or condition. In some aspects the age-related disease or condition is selected from atherosclerosis, cardiovascular disease, cancer, arthritis, dementia, cataract, osteoporosis, diabetes, hypertension, age-related fat loss, vertebral disc degeneration, age-related muscular atrophy and kidney disease.
In some aspects, provided herein, is a method of treating infections using disclosed STING modulators. The method comprises administering an amount of a STING modulator to a subject in need thereof, to reduce or inhibit one or more symptoms associated with infection. Infections can be viral infections or bacterial infections. In some aspects, the method inhibits viral or bacterial replication in a subject. In other aspects, the disclosed STING modulators reduce the viral or bacterial load in the subject. In some aspects, STING modulators disclosed herein, can be used as a vaccine adjuvant, to enhance the potency of the vaccine. In some aspects, the infection is a hepatitis B viral (HBV) infection. In some aspects, the infection is a Alphaviral infection, such as the ones caused by West Nile Virus (WNV), Vaccinia Virus (VACV), and Chikungunya virus (CHIKV), Venezuelan Equine Encephalitis Virus (VEEV), Eastern equine encephalitis (EEE). Administration of the STING modulators can elicit the desired activity or biological response in the subject, such as, can decrease the sign or symptom by, for example by at least 20%, at least 40%, at least 50%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, as compared to how the sign or symptom would have progressed in the absence of the composition.
Typical subjects include animals (e.g., mammals, birds, amphibians, reptiles, etc.). In various embodiments, the subject is a mammal. Any suitable mammal can be treated by a method or composition described herein. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In certain embodiments a mammal is a human. In certain embodiments a mammal is a non-rodent mammal (e.g., human, pig, goat, sheep, horse, dog, or the like). In certain embodiments a non-rodent mammal is a human. A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female. In certain embodiments a mammal can be an animal disease model. In an aspect, a subject is a human. In some aspects, a subject may be suffering from, and/or susceptible to a disease, disorder, and/or condition associated with altered STING activity (e.g., cancer, neurodegenerative diseases, cardiovascular diseases etc.).
Having described several embodiments, it will be recognized by those skilled in the art that various modifications, alternative constructions, and equivalents may be used without departing from the spirit of the present inventive concept. Additionally, a number of well-known processes and elements have not been described in order to avoid unnecessarily obscuring the present inventive concept. Accordingly, this description should not be taken as limiting the scope of the present inventive concept.
Those skilled in the art will appreciate that the presently disclosed embodiments teach by way of example and not by limitation. Therefore, the matter contained in this description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense. The following claims are intended to cover all generic and specific features described herein, as well as all statements of the scope of the method and assemblies, which, as a matter of language, might be said to fall there between.
The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.
Innate immunity provides the first-line defense against infection. A major innate immunity pathway for detecting invading viruses or bacteria is mediated by cyclic GMP-AMP (cGAMP) synthase (cGAS). cGAS serves as a DNA sensor by directly binding to pathogen DNA in the cytosol and uses GTP and ATP as substrates to produce the second messenger cGAMP. cGAMP binds and activates the downstream adaptor protein STING (Stimulator of interferon genes), which initiates several downstream signaling pathways, including the type-I interferon pathway, the NFκB pathway and autophagy, to eliminate the pathogen. cGAS/STING signaling can be activated by DNA from tumor cells and launch antitumor immunity by promoting the production of type I interferons, cell senescence and the adaptive immunity against tumor cells. Targeting cGAS-STING dependent signaling has shown promise in clinical application on anti-tumor treatment. In the past few years, a surge effort has been made in the development of STING agonists as novel anti-cancer therapeutics.
STING is a transmembrane protein containing four TM helices that forms the TM domain (TMD), which is followed by a cytoplasmic ligand-binding domain (LBD) that binds cGAMP. In addition, STING has a C-terminal tail that contains the PXPLRXD (SEQ ID NO: 1 wherein X is any residue) motif that recruits the TANK-binding kinase 1 (TBK1). Binding of TBK1 to this motif promotes the phosphorylation of Ser366 in the STING tail, which subsequently recruits and promotes the phosphorylation of the transcription factor interferon regulatory factor 3 (IRF3), ultimately leading to the expression of type I interferons. Structural approaches have been used to elucidate the mechanisms that control these activation steps of STING. STING exists as a constitutive domain-swapped dimer that is stabilized by interactions contributed by both the TMD and LBD. cGAMP binds the cleft at the center of the butterfly-shaped LBD dimer, inducing concerted conformational changes in the LBD that include inward rotation of the two wings and closure of the binding pocket. These conformational changes were coupled to a 180°-rotation of the LBD relative to the TMD, as well as a downward tilt of the LBDα2-LBDα3 loop, which mediated the formation of the side-by-side high-order oligomer of STING that is essential for the recruitment of TBK1 and the subsequent phosphorylation events. STING oligomerization and activation were also coupled to cGAMP-induced translocation from the ER to the Golgi apparatus, for which the mechanism is not well understood.
Despite the high affinity of STING for cGAMP, STING oligomers induced by cGAMP alone appeared weak in solution, which contradicts the cell-based imaging results that cGAMP induced robust puncta formation of STING on ER or Golgi membrane. In addition, previous cryo-EM structures of the chicken STING tetramer at low resolution (6.5 Å) show inter-dimer interactions in the LBD, but the role of the TMD in the oligomerization remains unclear. These observations together suggested that the stability of the high-order oligomer of STING may involve additional factors interacting with the TMD. In search for such factors, the interaction of a STING activator compound 53 (C53) with human STING was examined. A cryo-EM structure of human STING bound with both cGAMP and C53, was solved, revealing a novel agonist binding pocket in the TMD of STING. These structural and functional analyses further showed that the concurrent bindings of cGAMP and C53 to LBD and TMD of STING, respectively, promoted the formation of higher-order STING oligomers, and thereby efficiently boost the activation of STING.
Protein expression and purification. The expression constructs and expression and purification procedures used for human STING are similar to those described previously (e.g., Shang, G. et al., Cryo-EM structures of STING reveal its mechanism of activation by cyclic GMP-AMP. Nature 567, 389-393, (2019), incorporated herein by reference in its entirety). The coding sequence for human STING residues 1-343, excluding the C-terminal tail that is not a part of the folded structure of the protein, fused to a cleavage site for the human rhinovirus 3C protease and T6SS immunity protein 3 (Tsi3) from Pseudomonas aeruginosa at the C terminus in tandem were inserted into the pEZT-BM vector. Mutations were introduced by PCR-based mutagenesis. The plasmids were transfected using polyethylenimine (PEI) into HEK293F cells cultured in suspension in FreeStyle293 Expression medium (Gibco, Cat #12338-018), with 1000 μg DNA and 4 ml PEI at 1 mg/ml for 1 L cells. These and other cells used were assumed to be authenticated by the commercial sources, and therefore were not authenticated in the study. DAPI (4′,6-diamidino-2-phenylindole) staining and the e-Myco Mycoplasma PCR Detection Kit (Bulldog Bio) were used to ensure cells not contaminated by mycoplasma. Cells were harvested 72 hours after transfection, re-suspended in buffer A (containing 20 mM HEPES pH 7.5, 150 mM NaCl, 5 mM CaCl2, 1% DNase I, 0.2 mM AEBSF and 0.5 mM TECP) and disrupted by French press. The lysates were centrifuged for 10 min at 5000 g to remove debris. Membrane fraction was pelleted by centrifugation at 100,000 g for 1 hour. Proteins in lipid membrane were extracted by 1% n-Dodecyl-B-D-Maltoside (DDM) and 0.2% cholesteryl hemisuccinate tris salt (CHS) in buffer A. The sample was subjected to another round of centrifugation to remove insoluble fraction. The affinity purification step of human STING was based on the high-affinity interaction between the C-terminal Tsi3 tag and the T6SS effector protein Tse38. Detergent solubilized STING was captured by Tse3-conjugated Sepharose 4B resin (GE Healthcare) equilibrated in buffer B (20 mM HEPES pH 7.5, 150 mM NaCl, 5 mM CaCl2, 20 mM imidazole, 0.5 mM TECP, 0.03% DDM and 0.006% CHS). Unbound proteins were removed by extensive wash with buffer B. STING was eluted by cleavage from the Tsi3 tag with the 3C protease on resin at 4° C. for 12 hours. The eluted protein was further purified on a Superdex S200 increase 10/300 column (GE healthcare) in buffer C (25 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM TECP, 0.03% DDM and 0.006% CHS). Peak fractions were pooled, concentrated and kept at −80° C. before use.
Synthesis of C53. All solvents for synthesis (N,N′-dimethylformamide (DMF), tetrahydrofuran (THF) and methylene chloride (DCM)) were obtained by passing commercially available pre-dried, oxygen-free formulations through activated alumina columns. All reagents were purchased at high commercial quality (Sigma-Aldrich, Oakwood and AK Scientific) and used without further purification, unless otherwise stated. Reactions were monitored by thin-layer chromatography (TLC) carried out on 0.25 mm E. Merck silica gel plates (60F-254) and visualized under UV light and/or by appropriate staining method (an ethanolic solution of phosphomolybdic acid or cerium sulphate). Flash column chromatography was performed using E. Merck silica gel (60, particle size 0.04-0.063 mm). NMR spectra were recorded on a Bruker Ascend 400 and calibrated using residual not perdeuterated solvent (DMSO-d6: δH=2.50 ppm, δC=39.52 ppm) or using an external reference for 19F NMR [δF=0 (CCl3F) ppm] at 298 K. All 13C NMR spectra were broadband 1H decoupled. The chemical shifts of the peaks of the major rotamer were reported and coupling constants were given in Hz only for the major rotamer. LC-MS was performed on Agilent 1260 Infinity II Single Quadrupole with an Agilent Eclipse XDB-C18 5 μm 4.6×150 mm column. Buffer A was 0.1% CF3CO2H in H2O, and buffer B was 0.1% CF3CO2H in MeCN. Analytical gradient (0.0-7.0 min, buffer B from 10% to 60%; 7.0-10.0 min, buffer B from 60% to 100%; 10.0-15.0 min, buffer B maintains 100%) was performed with flow of 0.8 mL/min. For details of the chemical synthesis and characterization, as described below.
Native gel analyses of STING oligomerization. Purified human STING wild type or mutants in buffer C at 50 μM were incubated with DMSO as control, cGAMP at 100 μM, C53 at 100 μM or both at 4° C. for 2 hrs. Samples (15 μg protein) were mixed with the Native gel loading buffer (Invitrogen, Cat #BN20032) and resolved using 3-12% gradient native gel (Invitrogen, Cat #BN1003BOX).
Cryo-EM data collection and image processing. Purified wild type human STING at 50 μM were incubated with cGAMP at 100 μM and C53 at 100 μM for formation of the protein/ligands complex. The complex was purified using a Superose 6 10/300 gel filtration column (GE healthcare) in buffer C. Peak fractions were collected and concentrated to 2.9 mg/ml. Additional cGAMP (100 μM) and C53 (100 μM) were added to ensure saturation of the protein by the ligands. The sample was applied to a glow-discharged Quantifoil R1.2/1.3 300-mesh gold holey carbon grid (Quantifoil, Micro Tools GmbH, Germany), blotted under 100% humidity at 4° C. and plunged into liquid ethane using a Mark IV Vitrobot (FEI).
Micrographs were collected on a Titan Krios microscope (FEI) with a K3 Summit direct electron detector (Gatan) in the super-resolution counting mode operated at 300 kV. The slit width of the GIF-Quantum energy filter was set to 20 eV. The nominal magnification was 81,000× and the pixel size of 1.08 Å. Micrographs were dose-fractioned into 36 frames with a total exposure time of 7.2 s at the dose rate of 1.6 e−/Å2/frame in the correlated double sampling (CDS) mode. Movie frames were motion-corrected and dose-weighted using the Motioncorr2 program (version 1.2). GCTF 1.06 was used for CTF correction. Two sets of Particles were picked by using Topaz 0.2 and template-based picking in RELION 3.1, respectively. The two sets were combined with duplicates removed. The rest of the image processing was done in RELION (
Model building, refinement and analyses. Model building was initiated by docking the structure of the human STING dimer in the apo state (PDB ID: 6NT5) into the cryo-EM density, followed by manual adjustments in Coot 0.94 (Emsley, P. et al., Features and development of Coot. Acta crystallographica 66, 486-501, (2010), incorporated herein by reference in its entirety). The high quality of the density allows most of the residue sidechains to be clearly identified. C53 and cGAMP were manually fit into the density in Coot. cGAMP is an asymmetric molecule with a 3′-5′ and 2′-5′ phosphodiester bond linking the AMP and GMP moieties, which could bind to the symmetric STING dimer in two alternative orientations. The subtle asymmetric of cGAMP however do not usually cause obvious asymmetric in the STING LBD. As a result, either way of fitting cGAMP into its binding pocket was equally valid. One orientation was arbitrarily chosen to fit cGAMP into the density (
Statistics of the refined model are summarized in
Sequence alignment was rendered with ESPript 3 (Robert, X. & Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res 42, W320-324, doi:10.1093/nar/gku316 (2014), incorporated herein by reference in its entirety). Two-dimensional interaction diagram between C53 and STING was generated with LigPLot+2.2 (Laskowski, R. A. & Swindells, M. B. LigPlot+: multiple ligand-protein interaction diagrams for drug discovery. J Chem Inf Model 51, 2778-2786, doi:10.1021/ci200227u (2011), incorporated herein by reference in its entirety).
Western blot analyses of phosphorylation of STING, TBK1 and IRF3. The coding sequence for full-length human STING fused with a C-terminal FLAG-tag was cloned into the pcDNA3.1A(+) vector (Invitrogen). Mutations were introduced by PCR-based mutagenesis. Plasmids were transfected with 500 ng into HEK293T cells in 6-well plates and cultured for additional 24 hours. Stimulation of cells with cGAMP and C53 was carried out by using digitonin-mediated permeabilization that allows cGAMP to penetrate the cell membrane and enter the cell. cGAMP (1 μM), C53 (10 μM) or both in a buffer containing 50 mM HEPES pH7.5, 100 mM KCl, 3 mM MgCl2, 0.1 mM DTT, 85 mM sucrose, 0.2% BSA, 1 mM ATP, 0.1 mM GTP and 10 μg/ml digitonin were used to treat cells for 1 hour. Cells were lysed in RIPA buffer and lysates were subjected to western blot analyses. Human STING protein was detected by anti-FLAG primary antibody (Bimake, Cat #A5712; 3000× dilution) and anti-Mouse IgG HRP-linked secondary antibody (Cell Signaling Technology, Cat #7076S; 3000× dilution). Phosphorylated STING was detected by Rabbit anti-phospho-STING (S366) antibody (Cell Signaling Technology, Cat #19781S; 1000× dilution) and anti-Rabbit IgG HRP-linked secondary antibody (Cell Signaling Technology, Cat #7074S; 3000× dilution). TBK1 was detected by Mouse anti-TBK1/NAK antibody (Cell Signaling Technology, Cat #51872S; 1000× dilution). Phosphorylated TBK1 was detected by Rabbit anti-phospho-TBK1/NAK antibody (Cell Signaling Technology, Cat #5483S; 1000× dilution). IRF3 was detected by Mouse anti-IRF3 antibody (Abcam, Cat #ab50772; 100× dilution) and phosphorylated IRF was detected by Rabbit anti-phospho-IRF3 (S386) (Abcam, Cat #ab76493; 1000× dilution).
Fluorescence microscopy of STING oligomerization in cells. The coding sequence for full-length human STING fused with a C-terminal GFP-tag was cloned into the pmEGFP-N1 vector (Addgene). Mutations were introduced by PRC-based mutagenesis. Plasmids were transfected into Hela cells cultured on glass coverslips in 6-well plates and cultured for additional 24 hours. Stimulation of cells with cGAMP and C53 was performed in the same manner as described above. One hour after stimulation, cells were washed in PBS and fixed in 4% paraformaldehyde. STING-GFP was imaged by using a DeltaVision fluorescence microscope with a 40× objective. Nuclei were stained with DAPI. The experiment for each STING constructs was repeated three times. The percentage of cells containing STING puncta were obtained by counting number of GFP-positive cells with or without STING puncta respectively in images taken from random fields of coverslips. The number of cells counted for each STING construct in each repeat ranged from 60-452. The data was plotted with GraphPad Prism 9.
The atomic coordinates and the cryo-EM map will be deposited into the RCSB and EMD databases and released to the public upon. Additionally, see
To stabilize the cGAMP-induced active oligomer of human STING for mechanistic analyses, STING agonists were identified that are chemically distinct from cGAMP and less likely act as cGAMP mimetics. To this end, a series of studies were performed on small mostly hydrophobic benzothiazinone-like compounds that activate human STING selectively and potently but lack the negative charge of cGAMP. One of these agonists—C53, induces STING-mediated secretion of interferons with an EC50 of 185 nM. A native gel assay was used to assess the effect of C53 on oligomerization of purified human STING in detergent solution (
To understand how C53 activates STING, single-particle cryo-EM was used to determine the structure of human STING oligomer with both C53 and cGAMP bound (
Previous structural analyses of chicken STING in both the apo- and cGAMP-bound states have shown that cGAMP induces a 180°-rotation of the LBD with respect to the TMD, converting the two connectors linking the LBD and TMD in the STING dimer from the crossover to parallel configuration. The human STING dimer bound to both cGAMP and C53 here exhibited the parallel configuration of the connector, in contrast to the crossover configuration in the apo-state solved previously (
The cryo-EM structure of human STING tetramer showed that two STING dimers bound with both C53 and cGAMP assemble in a side-by-side fashion similar to the chicken STING tetramer (
Strikingly, the high-resolution cryo-EM map showed a strong density peak at the luminal side of the TMD of each STING dimer, which was assigned to C53 unequivocally based on clear asymmetric “C”-shape and local chemical environment (
The 2-Cl-6-F-phenyl ring at one end of C53 is juxtaposed closely with the trifluoro-phenyl ring at the other end, resulting in the overall C-shape (
The mouth of the binding pocket facing the ER/Golgi lumen was sealed by the TM3-TM4 loop from protomer A of the STING dimer (
To validate this binding mode of C53, mutations were introduced to residues in the binding pocket (H50A, S53L, Y106A, and M120L) and the TM3-TM4 loop (V113Q, G114Q and P115Q) of STING. Native-gel results showed that, compared to STING wild type (STING-WT), these mutants showed no or greatly reduced high-order oligomerization upon stimulation by C53 and cGAMP (
Structural comparison of STING without or with C53 bound revealed that TMs that surround C53 in the STING dimer, especially TM2 and TM4, undergo substantial sideway expansion upon C53 binding (
The open-ended nature of the STING tetramer allowed assembly of larger oligomers by adding additional dimeric units to both sides, enabling STING to serve as a signaling platform for efficient recruitment and activation of TBK1 and IRF3. The induction of the TMD-TMD interaction therefore likely underlies the activating effect of C53 on STING. To test this model, mutations were introduced to the C53-induced TMD-TMD interface, including L26A, L30A, L44A and Y104A. Compared to STING-WT, these mutants showed substantially reduced oligomerization in solution in the presence of both C53 and cGAMP (
In this work, high-resolution cryo-EM structure of human STING in complex with both cGAMP and C53 defined a novel agonist binding site in the TMD of STING, paving the way for developing more agonists that target this site. Compounds that target the TMD site might be better STING agonists than cGAMP mimetics for therapeutic purposes because they are more hydrophobic and therefore more permeable to the cell membrane. In addition, structural analyses clarified the direct role of the TMD in the oligomerization and activation of STING. The coupling between the induced opening of the TMD pocket by C53 and the high-order oligomerization of human STING suggested that this conformational change might be an integral part of its activation mechanism in vivo. This notion raises the question how this conformational change is triggered in the cell. It is possible that cGAMP-induced 180°-rotation of the LBD may allosterically induce the open conformation of the TMD on the ER/Golgi membrane in the cell, which could not be recapitulated with purified protein in vitro. Alternatively, a cellular endogenous ligand may bind to the TMD pocket and act as a secondary activator to facilitate cGAMP-induced STING activation.
Compound UT009 was synthesized according to the scheme shown in
To a solution of Compound 1 (300 mg, 1.37 mmol, 1 eq) in DMF (3 mL) was added NaH (82.10 mg, 2.05 mmol, 60% purity, 1.5 eq) and stirred at 0° C. for 0.5 hour. Then to the mixture was added compound 1A (422.05 mg, 1.64 mmol, 1.2 eq), the mixture was stirred at 25° C. for 0.5 hour. The reaction mixture was diluted with water (5 mL) and extracted with EtOAc (5 mL×3). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue which was purified by flash silica gel chromatography (ISCO®; 4 g Sepa Flash® Silica Flash Column, Eluent of 0˜20% Ethyl acetate/Petroleum ether gradient @40 mL/min) to give Compound 2 (400 mg, 1.01 mmol, 73.94% yield) as a white solid.
LCMS: MS (ESI) Retention time: 0.577 min, [M+1]+=396.3
1H NMR (400 MHz, DMSO-d6) δ=7.63 (dd, J=7.6, 0.8 Hz, 1H), 7.45 (d, J=8.0 Hz, 1H), 7.34-7.26 (m, 1H), 7.19 (s, 1H), 7.16-7.13 (m, 1H), 7.12-7.05 (m, 1H), 5.10 (s, 2H), 3.73 (s, 3H), 1.30 (s, 6H).
To a solution of Compound 2 (400 mg, 1.01 mmol, 1 eq) in THE (1 mL) and MeOH (1 mL) was added LiOH (121.15 mg, 5.06 mmol, 5 eq) in H2O (1 mL). The mixture was stirred at 20° C. for 1 hour. The reaction mixture was concentrated under the reduced pressure to obtain Compound 3 (530 g, crude) as a white solid and the mixture was used directly for the next step.
LCMS: MS (ESI) Retention time: 0.468 min, [M+1]+=382.0
To a solution of Compound 3 (100 mg, 262.25 umol, 1 eq) and Compound 3A (42.25 mg, 262.25 umol, 1 eq) in DMF (2 mL) was added HATU (149.57 mg, 393.37 umol, 1.5 eq) and DIEA (101.68 mg, 786.74 umol, 137.04 μL, 3 eq). The mixture was stirred at 20° C. for 12 hour. The reaction mixture was concentrated under the reduced pressure to give the crude product which was purified by prep-HPLC (column: Phenomenex Luna C18 100*30 mm*5 um; mobile phase: [water (FA)-ACN]; B %: 47%-87%, 8 min) and lyophilized to obtain UT009 (6.86 mg, 12.74 μmol, 4.86% yield, 97.4% purity) as a yellow solid.
LCMS: LCMS: MS (ESI) Retention time: 0.528 min, [M+1]+=525.1
HPLC: Retention time: 3.684 min, Purity=98.11%, 220 nm
1H NMR (400 MHz, DMSO-d6) δ=8.73 (t, J=5.2 Hz, 1H), 7.72-7.64 (m, 1H), 7.64-7.56 (m, 1H), 7.55-7.47 (m, 2H), 7.45-7.40 (m, 1H), 7.26 (d, J=0.8 Hz, 1H), 7.17 (t, J=8.8 Hz, 2H), 5.10 (s, 2H), 4.42 (d, J=5.0 Hz, 2H), 1.27 (s, 6H)
Compound UT019 was synthesized according to the scheme illustrated in
To a solution of Compound 7 (3 g, 18.68 mmol, 1 eq) in DCM (30 mL) was added SOCl2 (4.45 g, 37.37 mmol, 2.71 mL, 2 eq), Then was added DMF (136.56 mg, 1.87 mmol, 143.75 uL, 0.1 eq). The mixture was stirred at 0° C. for 1 h. The reaction mixture was concentrated under the reduced pressure to obtain Compound 1A (4.49 g, crude) as a colorless liquid.
1H NMR (400 MHz, DMSO-d6): δ=7.51-7.44 (m, 1H), 7.43-7.37 (m, 1H), 7.37-7.27 (m, 1H), 5.75 (s, 1H), 4.82 (s, 2H)
To a solution of Compound 1 (2 g, 9.12 mmol, 1 eq) and Compound 1A (1.63 g, 9.12 mmol, 1.17 mL, 1 eq) in ACN (30 mL) was added K2CO3 (7.56 g, 54.74 mmol, 6 eq). The mixture was stirred at 80° C. for 2 h. The reaction mixture was filtered and the filtrate was concentrated under the reduced pressure to give the product. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0˜13% Ethyl acetate/Petroleum ether gradient @80 mL/min) and concentrated under the reduced pressure to obtain Compound 2 (3.60 g, crude) as an off-white solid.
LCMS: MS (ESI) Retention time: 0.653 min, [M+1]+=361.9 1H NMR (400 MHz, DMSO-d6) (EC2637-135-P1A): δ=7.65 (dd, J=1.2, 7.6 Hz, 1H), 7.51 (d, J=8.0 Hz, 1H), 7.45-7.34 (m, 3H), 7.30-7.21 (m, 1H), 5.08 (s, 2H), 3.81 (s, 3H), 1.30 (s, 6H)
To a solution of Compound 2 (3.27 g, 9.04 mmol, 1 eq) in THF (15 mL) was added LiOH·H2O (568.92 mg, 13.56 mmol, 1.5 eq) in H2O (15 mL). The mixture was stirred at 60° C. for 2 h. Acidify the reaction mixture by adding, with shaking, 10 mL of HCl until pH around 3, and then extracted with EtOAc (20 ml×3). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated to obtain Compound 3 (3.10 g, 8.92 mmol, 98.69% yield) as a yellow solid.
LCMS: MS (ESI) Retention time: 0.563 min, [M+1]+=348.0
1H NMR (400 MHz, DMSO-d6): δ=13.26-12.47 (m, 1H), 7.64 (dd, J=1.2, 7.6 Hz, 1H), 7.50-7.33 (m, 4H), 7.25 (ddd, J=1.2, 8.0, 10.0 Hz, 1H), 5.08 (s, 2H), 1.30 (s, 6H)
To a solution of Compound 3 (500 mg, 1.44 mmol, 1 eq) and TEA (174.58 mg, 1.73 mmol, 240.14 uL, 1.2 eq) in THF (10 mL) was cooled to −10° C. and Compound 3A (216.00 mg, 1.58 mmol, 207.69 uL, 1.1 eq) was added. After 20 min was added NaBH4 (435.15 mg, 11.50 mmol, 8 eq) at 0° C. The mixture was stirred at 0° C. for 30 min. The reaction was quenched by the addition of 10% citric acid (50 mL). The mixture was extracted with ethyl acetate (20 mL×3). The combined organic extracts were washed with brine (20 mL×3), dried, filtered and concentrated. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, Eluent of 0˜23% Ethyl acetate/Petroleum ether gradient @50 mL/min) and concentrated to obtain Compound 4 (337 mg, 1.01 mmol, 70.22% yield) as a white solid.
LCMS: MS (ESI) Retention time: 0.436 min, [M+1]+=333.9
1H NMR (400 MHz, DMSO-d6): δ=7.49-7.33 (m, 2H), 7.33-7.16 (m, 2H), 7.07-6.73 (m, 2H), 5.10 (t, J=5.6 Hz, 1H), 5.02 (s, 1H), 4.37 (d, J=5.2 Hz, 2H), 1.26 (s, 6H)
To a solution of Compound 4 (180 mg, 539.27 umol, 1 eq) in toluene (2 mL) was added DBU (106.73 mg, 701.05 umol, 105.67 uL, 1.3 eq) and DPPA (178.09 mg, 647.13 umol, 140.23 uL, 1.2 eq). The mixture was stirred at 25° C. for 12 h. The mixture was extracted with ethyl acetate (10 mL×3). The combined organic extracts were washed with brine (10 mL×3), dried, filtered and concentrated. The residue was purified by prep-TLC (SiO2, Petroleum ether:Ethyl acetate=5:1, Rf=0.2) and eluted with acetonitrile (5 mL) to obtain Compound 5 (160 mg, 436.94 μmol, 81.02% yield, 97.982% purity) as a white solid.
LCMS: MS (ESI) Retention time: 0.536 min, [M+1]+=360.0
1H NMR (400 MHz, DMSO-d6): δ=7.48-7.34 (m, 3H), 7.28-7.20 (m, 1H), 6.99 (d, J=7.2 Hz, 1H), 6.80 (s, 1H), 5.04 (s, 2H), 4.36 (s, 2H), 1.28 (s, 6H)
To a solution of Compound 5 (140 mg, 390.19 μmol, 1 eq) in THE (2 mL) was added PtV/C (10.19 mg, 39.02 umol, 0.1 eq). The mixture was stirred at 25° C. for 12 h under H2 (15 psi) atmosphere. The mixture was filtered and concentrated under reduced pressure to give Compound 6 (129 mg, 387.62 μmol, 99.34% yield) as an off-white solid.
LCMS: MS (ESI) Retention time: 0.342 min, [M+1]+=315.9.
A mixture of Et3N (45.61 mg, 450.72 umol, 62.73 uL, 3 eq) in CH2Cl2 (1 mL) was Compound 6 (50 mg, 150.24 umol, 1 eq), and was degassed and purged with N2 for 3 times, then was added Compound 6A (34.64 mg, 150.24 umol, 1 eq) in CH2Cl2 (1 mL) and then the mixture was stirred at 0° C. for 2 h under N2 atmosphere. The mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100*30 mm*5 um; mobile phase: [water (FA)-ACN]; B %: 43%-73%, 8 min) and lyophilized to obtain UT019 (25.97 mg, 48.10 μmol, 32.01% yield, 97.591% purity) as a white solid.
LCMS: MS (ESI) Retention time: 0.511 min, [M+1]+=527.2.
HPLC: Retention time: 0.511 min, Purity=97.59%, 220 nm
1H NMR (400 MHz, DMSO-d6) δ=8.78 (s, 1H), 7.45-7.34 (m, 2H), 7.29-7.21 (m, 1H), 7.19-7.11 (m, 3H), 6.86 (d, J=6.8 Hz, 1H), 6.63 (s, 1H), 4.96 (s, 2H), 4.06 (s, 2H), 1.21 (s, 6H)
Synthesis of UT073 occurred according to the scheme shown in
To a solution of compound 1 (5.24 g, 33.05 mmol, 1 eq) and compound 1A (4.3 g, 33.05 mmol, 1 eq) in DMF (50 mL) was added K2CO3 (6.85 g, 49.58 mmol, 1.5 eq). The mixture was stirred at 120° C. for 12 h. The reaction mixture was partitioned between EtOAc (100 mL×3) and water (100 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give Compound 2 (3.5 g, crude) as a blackish brown solid.
To a solution of Compound 2 (3.5 g, 13.03 mmol, 1 eq) in MeOH (40 mL) was added NaBH4 (739.35 mg, 19.54 mmol, 1.5 eq) at 0° C. The mixture was stirred at 0° C. for 0.5 h. The reaction was quenched by MeOH (100 mL). The mixture was filtered and concentrated under the reduced pressure to obtain Compound 3 (3.46 g, crude) as a yellow oil.
To a solution of Compound 3 (500 mg, 2.39 mmol, 1 eq) in THE (10 mL) was added Pd (127.15 mg, 119.48 μmol, 10% purity, 0.05 eq) under N2 atmosphere. The suspension was degassed and purged with H2 for 3 times. The mixture was stirred under H2 (15 Psi) at 25° C. for 2 h. The residue was filtered and the filtrate was concentrated in vacuum to obtain Compound 4 (400 mg, crude) as a yellow oil.
To a solution of Compound 6 (1.0 g, 4.97 mmol, 1 eq) in THE (20 mL) was added bromocopper;methylspLfanylmethane (102.21 mg, 497.15 μmol, 0.1 eq) and t-BuOK (2.79 g, 24.86 mmol, 5.0 eq) at −78° C. After addition, mixture was added CH3I (1.45 g, 10.19 mmol, 634.47 μL, 2.05 eq) in THE (20 mL). And the mixture was stirred at 25° C. for 2 h. The mixture was poured into 5% of citric acid (100 mL) and extracted with EtOAc (100×3). The combined organic phase was washed with brine (100 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 200*40 mm*10 um; mobile phase: [water (FA)-ACN]; B %: 35%-65%, 10 min) to obtain Compound 5 (427 mg, 1.86 mmol, 37.47% yield) as a white solid.
1H NMR (400 MHz, CDCl3) δ=8.66 (s, 1H), 7.37-7.32 (m, 1H), 7.32-7.28 (m, 1H), 7.19 (s, 1H), 1.44 (s, 6H)
A mixture of Compound 4 (100 mg, 345.90 μmol, 1 eq), Compound 5 (79.28 mg, 345.90 μmol, 1 eq), K2CO3 (95.61 mg, 691.79 μmol, 2.0 eq) in MeCN (4 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 60° C. for 2 h under N2 atmosphere. The mixture was filtered and concentrated in vacuum to give a residue.
The residue was purified by prep-HPLC (column: Phenomenex luna C18 150*25 mm*10 um; mobile phase: [water (FA)-ACN]; B %: 70%-100%, 9 min) to give Compound UT073 (34.63 mg, 70.43 μmol, 20.36% yield, 98% purity) was obtained as a off-white solid.
LCMS: MS (ESI) Retention time: 0.625 min, [M+1]+=482.0
HPLC: Retention time: 4.835 min, Purity=98.07%, 220 nm
1H NMR (400 MHz, DMSO-d6) δ=7.51 (d, J=7.6 Hz, 1H), 7.44-7.37 (m, 2H), 7.34 (dd, J=0.8, 7.6 Hz, 1H), 7.23 (s, 1H), 7.04 (dd, J=2.0, 7.2 Hz, 1H), 6.96 (tt, J=2.4, 9.6 Hz, 1H), 6.46 (dd, J=2.2, 8.4 Hz, 2H), 5.12 (s, 2H), 1.20 (s, 6H)
Compound UT122 was synthesized according to
To a solution of Compound 1 (100 mg, 254.94 umol, 1 eq) in DMF (1.5 mL) was added HATU (145.40 mg, 382.41 umol, 1.5 eq) at 0° C., and the mixture was stirred at 0° C. for 30 min. After was added Compound 1A (37.27 mg, 254.94 umol, 1 eq) and DIEA (98.85 mg, 764.83 umol, 133.22 uL, 3 eq), and the mixture was stirred at 25° C. for 30 min. The residue was purified by Prep-HPLC (41%-71% MeCN in water (FA), 9 min), to obtain UT122 (10.04 mg, 19.24 μmol, 7.55% yield, 99.74% purity) as a white solid.
LCMS: MS (ESI) Retention time: 0.613 min, [M+1]+=521.8
HPLC: Retention time: 3.651 min, Purity=99.74%, 220 nm
1H NMR (400 MHz, DMSO-d6)=10.92 (s, 1H), 8.88 (t, J=5.6 Hz, 1H), 7.64 (dd, J=7.6, 1.2 Hz, 1H), 7.53-7.39 (m, 3H), 7.38-7.28 (m, 1H), 7.05-6.98 (m, 1H), 6.97-6.90 (m, 1H), 6.27 (d, J=0.8 Hz, 1H), 4.97 (s, 2H), 4.60 (d, J=5.6 Hz, 2H), 3.75 (s, 3H), 2.06 (s, 3H), 1.34 (s, 6H).
Compounds UT009, UT019, UT073 and UT122 were tested for their ability to interfere with cGAMP induced STING signaling.
Two compounds were also tested for their ability to induce STING signaling (e.g., act as agonists or activators). Agonist activity was measured by measuring interferon levels in the presence of increasing concentrations of each compound. Dose response curves for each of the two compounds (C1 and C2) are shown in
Compound UT017 was synthesized according to
To a solution of Compound 1 (1.6 g, 7.30 mmol, 1 eq) and Compound 1A (3.26 g, 14.60 mmol, 2 eq) in ACN (16 mL) was added K2CO3 (2.02 g, 14.60 mmol, 2 eq). The mixture was stirred at 60° C. for 1 h. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0˜35% Ethyl acetate/Petroleum ether gradient @80 mL/min) to obtain Compound 2 (800 mg, 2.15 mmol, 29.49% yield, 97.316% purity) as a white solid.
LCMS: [M+H]+=362.0
1H NMR (400 MHz, DMSO-d6) δ=7.66 (d, J=7.6 Hz, 1H), 7.51 (d, J=7.6 Hz, 1H), 7.41-7.35 (m, 3H), 7.25 (t, J=8.0 Hz, 1H), 5.08 (s, 2H), 3.81 (s, 3H), 1.30 (s, 6H).
To a solution of Compound 2 (800 mg, 2.21 mmol, 1 eq) in THF (1.6 mL) and MeOH (1.6 mL) was added LiOH·H2O (371.16 mg, 8.84 mmol, 4 eq) in H2O (0.8 mL). The mixture was stirred at 20° C. for 1 h. Acidify the reaction mixture by adding, with shaking, 6 mL of HCL until pH around 3, and then extracted with EtOAc (20 ml×3). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated to give a residue to obtain Compound 3 (654 mg, 1.88 mmol, 85.05% yield) as an off-white solid.
LCMS: [M+H]+=348.0
To a solution of Compound 3 ((400 mg, 1.15 mmol, 1 eq) and TEA (139.66 mg, 1.38 mmol, 192.11 uL, 1.2 eq) in THE (20 mL) was cooled to −10° C. and Compound 3A (172.80 mg, 1.27 mmol, 166.15 uL, 1.1 eq) was added. After 20 min was added NaBH4 (130.54 mg, 3.45 mmol, 3 eq) at 0° C., and the mixture was stirred at 0° C. for 30 min. Acidify the reaction mixture by adding, with shaking, 10 mL of HCl until pH around 7. The solvent was evaporated and the residue triturated under water. The product was filtered and dried under vacuum to obtain Compound 4 (376 mg, crude) as a yellow oil.
LCMS: [M+H]+=334.1
1H NMR (400 MHz, DMSO-d6) δ=7.48-7.32 (m, 2H), 7.30-7.18 (m, 2H), 6.97-6.90 (m, 1H), 6.79 (s, 1H), 5.17-5.07 (m, 1H), 5.03 (s, 2H), 4.37 (d, J=5.6 Hz, 2H), 1.26 (s, 6H).
To a solution of Compound 4 (260 mg, 778.95 umol, 1 eq) in toluene (3 mL) was added DBU (154.16 mg, 1.01 mmol, 152.63 uL, 1.3 eq) and DPPA (257.24 mg, 934.74 umol, 202.55 uL, 1.2 eq). The mixture was stirred at 25° C. for 12 h. The reaction mixture was filtered and the filtrate was concentrated under the reduced pressure to give Compound 5 (470 mg, crude) was obtained as a yellow oil.
LCMS: [M+H]+=359.1
To a solution of Compound 5 (400 mg, 1.11 mmol, 1 eq) in MeOH (5 mL) was added PtV/C (29.10 mg, 111.48 umol, 0.1 eq) and the mixture was stirred at 25° C. for 3 h under H2 (2.25 mg, 1.11 mmol) atmosphere (15 PSI). The reaction mixture was filtered and the filtrate was concentrated under the reduced pressure to obtain compound 6 (340 mg, crude) as a yellow solid.
LCMS: [M+H]+=359.1
To a solution of Compound 6 (160 mg, 480.77 umol, 1 eq) and Et3N (48.65 mg, 480.77 umol, 66.92 uL, 1 eq) in ACN (1 mL) was added Compound 6A (93.53 mg, 480.77 umol, 1 eq) at 0° C. The mixture was stirred at 0° C. for 0.5 h. The reaction mixture was filtered and the filtrate was concentrated under the reduced pressure to give the product. The residue was purified by Prep-HPLC (44%-74% MeCN in water (FA), 9 min), to obtain UT017 (19.68 mg, 40.09 μmol, 8.34% yield, 100% purity) as a white solid.
LCMS: [M+H]+=491.2
1H NMR (400 MHz, DMSO-d6) δ=9.15 (t, J=6.0 Hz, 1H), 7.51-7.25 (m, 5H), 7.24-7.13 (m, 1H), 6.97 (d, J=7.6 Hz, 1H), 6.78 (s, 1H), 5.01 (s, 2H), 4.36 (d, J=6.0 Hz, 2H), 1.27 (s, 6H).
Compound UT018 was synthesized according to
To a solution of Compound 1 (2 g, 9.43 mmol, 1 eq) in THF (20 mL) was colded to −78° C., and was added LDA (2 M, 9.43 mL, 2 eq) dropwise there into followed by stirring for 10 min. After adding LDA (2 M, 4.72 mL, 1 eq), the reaction mixture was brought to 25° C. and stirred for 1 h. Then the reaction solution was cooled to −78° C. again and Mel (1.34 g, 9.43 mmol, 587.18 uL, 1 eq) was added dropwise there into followed by stirring for 10 min. After adding Mel (1.34 g, 9.43 mmol, 587.18 uL, 1 eq), the reaction mixture was brought to 25° C. and stirred for 1 h. The reaction mixture was diluted with NH4Cl and extracted with EtOAc (30 mL×2), the combined organic layers dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by reversed-phase HPLC (44% ACN in water (0.1% FA), 33 min), to obtain Compound 2 (870 mg, 3.62 mmol, 38.42% yield) as a white solid.
LCMS: [M+H]+=239.7
1H NMR (400 MHz, CHLOROFORM-d6) δ=9.00 (s, 1H), 7.23-7.15 (m, 1H), 7.13 (d, J=1.6 Hz, 1H), 7.06 (d, J=8.0 Hz, 1H), 1.40 (s, 6H).
To a solution of Compound 2 (870 mg, 3.62 mmol, 1 eq) in THF (10 mL) was added NaH (173.91 mg, 4.35 mmol, 60% purity, 1.2 eq) at 0° C. for 15 min, and then was added Compound 2A (1.21 g, 5.44 mmol, 1.5 eq). The mixture was stirred at 0° C. for 45 min. The residue was diluted with H2O (20 mL) and extracted with EtOAc (20 mL×3). The combined organic layers were washed with brine (20 mL×3), the combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, Eluent of 0˜15% Ethyl acetate/Petroleum ether gradient @80 L/min), to obtain Compound 3 (1.19 g, 3.11 mmol, 85.82% yield) as an off-white solid.
LCMS: [M+H]+=383.9
1H NMR (400 MHz, CHLOROFORM-d6) δ=7.24-7.11 (m, 2H), 7.06 (dd, J=8.0, 1.2 Hz, 1H), 7.01-6.88 (m, 2H), 6.83 (d, J=1.2 Hz, 1H), 5.01 (s, 2H), 1.30 (s, 6H).
A mixture of Compound 3 (990 mg, 2.59 mmol, 1 eq), Compound 3A (642.67 mg, 5.17 mmol, 606.30 uL, 2 eq), Pd2(dba)3 (47.38 mg, 51.74 umol, 0.02 eq), Xantphos (59.88 mg, 103.49 umol, 0.04 eq) and DIEA (668.75 mg, 5.17 mmol, 901.28 uL, 2 eq) in toluene (10 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 100° C. for 12 h. The reaction mixture was concentrated under the reduced pressure, and then the mixture was quenched by addition water (50 mL), and extracted with EtOAc (50 mL×3). The combined organic phase was washed with brine (50 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by flash silica gel chromatography (ISCO®; 12 SepaFlash® Silica Flash Column, Eluent of 0˜20% thylacetate/Petroleum ether gradient @80 mL/min), to obtain Compound 4 (800 mg, 1.88 mmol, 72.60% yield) as a yellow oil.
LCMS: [M+H]+=426.1
1H NMR (400 MHz, CHLOROFORM-d) δ=7.22-7.10 (m, 7H), 6.97 (d, J=7.6 Hz, 1H), 6.94-6.80 (m, 2H), 6.67 (s, 1H), 5.01 (s, 2H), 3.94 (s, 2H), 1.30 (d, J=0.8 Hz, 6H).
To a solution of Compound 4 (100 mg, 234.77 umol, 1 eq), HOAc (0.15 mL), H2O (0.1 mL) and 1,3-dichloro-5,5-dimethyl-imidazolidine-2,4-dione (92.51 mg, 469.54 umol, 2 eq) in ACN (4 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 0° C. for 30 min under N2 atmosphere. The reaction mixture was quenched by addition water (10 mL), and extracted with DCM (10 mL×3). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain Compound 5 (95 mg, crude) as a yellow oil.
LCMS: [M+H]+=402.0
To a solution of Compound 5 (95 mg, 236.16 umol, 1 eq) in DCM (2 mL) was added TEA (71.69 mg, 708.49 umol, 98.61 uL, 3 eq) and Compound 5A (57.08 mg, 354.24 umol, 1.5 eq). The mixture was stirred at 0° C. for 1 h. The residue was purified by Prep-HPLC (47%-77% ACN in water (FA), 9 min), to obtain UT018 (51.14 mg, 94.85 umol, 40.16% yield, 97.73% purity) as a white solid.
LCMS: [M+H]+=527.2
1H NMR (400 MHz, DMSO-d6) δ=7.52 (dd, J=7.6, 1.2 Hz, 1H), 7.38-7.29 (m, 2H), 7.29-7.20 (m, 2H), 7.17-7.06 (m, 1H), 6.50 (t, J=8.0 Hz, 2H), 5.19 (s, 2H), 5.16 (t, J=6.8 Hz, 1H), 4.25 (d, J=6.8 Hz, 2H), 1.47 (s, 6H).
Compound UT65 was synthesized according to
To a solution of Compound 1 (3 g, 18.74 mmol, 1 eq) in DCE (45 mL) was added Compound 1A (2.27 g, 18.74 mmol, 1 eq). To the mixture was added Ti(i-PrO)4 (8.52 g, 29.98 mmol, 8.85 mL, 1.6 eq) gradually at 0° C. The reaction mixture was stirred at 80° C. for 3 h. The stirred mixture was cooled down to 0° C. To the vigorously stirred cold solution was added Celite (30 g) then added water (15 mL). The slurry is vigorously stirred at 20° C. for 30 min. The mixture was filtered and the Celite cake was washed with DCM (100 mL). The filtrate was concentrated. The residue was dissolved with EtOAc (100 mL), washed with brine (100 mL), dried, filtered and concentrated under the reduced pressure to obtain Compound 2 (4.4 g, 16.71 mmol, 89.18% yield) as a yellow oil.
LCMS: [M+H]+=207.9
1H NMR (400 MHz, CHLOROFORM-d) δ=8.74 (s, 1H), 6.78 (t, J=8.4 Hz, 2H), 1.27 (s, 9H).
To a solution of Compound 2 (1 g, 3.80 mmol, 1 eq) in DCM (15 mL) was added MeMgBr (3 M, 3.04 mL, 2.4 eq) dropwise at −20° C. The reaction was stirred at −20° C. for 1 h then 20° C. for 2 h. The reaction was quenched by the addition of aq. NH4Cl (30 mL). The aqueous phase was extracted with DCM (30 mL). The combined organic extracts were washed with brine (30 mL), dried, filtered and concentrated. The residue was purified by flash silica gel chromatography (ISCO®; 4 g SepaFlash® Silica Flash Column, Eluent of 0˜25% Ethylacetate/Petroleum ether gradient @80 mL/min) and concentrated under the reduced pressure to obtain Compound 3A (190 mg, 680.22 μmol, 17.91% yield) as a yellow oil and Compound 3B (400 mg, 1.43 mmol, 37.65% yield) as a yellow oil.
LCMS: [M+H]+=280.1
1H NMR (400 MHz, CHLOROFORM-d) δ=6.65 (t, J=8.8 Hz, 2H), 4.88-4.74 (m, 1H), 3.78 (d, J=9.6 Hz, 1H), 1.57 (d, J=7.2 Hz, 3H), 1.21 (s, 9H).
1H NMR (400 MHz, CHLOROFORM-d) δ=6.65 (t, J=8.8 Hz, 2H), 4.93 (m, 1H), 3.63 (d, J=6.8 Hz, 1H), 1.65 (d, J=6.8 Hz, 3H), 1.15 (s, 9H).
To a solution of Compound 3A (190 mg, 680.22 umol, 1 eq) in dioxane (1.5 mL) was added HCl/dioxane (4 M, 170.06 uL, 1 eq). The mixture was stirred at 20° C. for 1 hr. The reaction mixture was concentrated under the reduced pressure to obtain Compound 4 (400 mg, crude, HCl) as an off-white solid.
LCMS: [M+H]+=176.1
1H NMR (400 MHz, DMSO-d6) δ=8.57 (s, 3H), 7.32 (t, J=9.2 Hz, 2H), 5.75 (s, 1H), 4.61 (s, 1H), 1.60-1.55 (m, 3H).
To a solution of Compound 4 (50.00 mg, 236.28 umol, 1 eq, HCl) and Compound 4A (123.26 mg, 354.42 umol, 1.5 eq) in DMF (1 mL) was added HATU (134.76 mg, 354.42 umol, 1.5 eq) and DIEA (152.69 mg, 1.18 mmol, 205.78 uL, 5 eq). The mixture was stirred at 20° C. for 12 hr. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 150*25 mm*10 um; mobile phase: [water (FA)-ACN]; B %: 53%-83%, 10 min) and lyophilized to obtain UT065 (48.09 mg, 90.76 μmol, 38.41% yield, 95.293% purity) as a white solid.
LCMS: [M+H]+=505.0
1H NMR (400 MHz, CHLOROFORM-d) δ=7.45 (d, J=7.6 Hz, 1H), 7.24 (dd, J=1.6, 5.2 Hz, 3H), 7.16 (s, 1H), 7.05-6.98 (m, 1H), 6.68 (t, J=8.4 Hz, 2H), 6.55 (d, J=8.4 Hz, 1H), 5.75-5.66 (m, 1H), 5.16 (d, J=3.2 Hz, 2H), 1.59 (s, 3H), 1.41 (s, 6H).
Compound UT066 was synthesized according to
To a solution of Compound 1 (190 mg, 680.22 umol, 1 eq) in dioxane (1.5 mL) was added HCl/dioxane (4 M, 170.06 μL, 1 eq). The mixture was stirred at 20° C. for 1 hr. The reaction mixture was concentrated under the reduced pressure to give Compound 2 (160 mg, crude, HCl) as an off-white oil.
LCMS: [M+H]+=176.1
1H NMR (400 MHz, DMSO-d6) δ=8.47 (s, 2H), 7.37-7.32 (m, 1H), 4.69-4.56 (m, 1H), 4.68-4.56 (m, 1H), 1.56 (d, J=7.6 Hz, 3H).
To a solution of Compound 2 (50.00 mg, 236.28 umol, 1 eq, HCl) and Compound 2A (123.26 mg, 354.42 umol, 1.5 eq) in DMF (1 mL) was added HATU (134.76 mg, 354.42 umol, 1.5 eq) and DIEA (152.69 mg, 1.18 mmol, 205.78 uL, 5 eq). The mixture was stirred at 20° C. for 12 hr. The residue was purified by prep-TLC (SiO2, Petroleum ether:Ethyl acetate=3:1, Rf=0.4) and eluted with acetonitrile (5 mL) to give UT066 (39.25 mg, 76.43 μmol, 32.35% yield, 98.316% purity) was obtained as an off-white solid.
LCMS: [M+H]+=505.2
1H NMR (400 MHz, DMSO-d6) δ=8.72 (d, J=6.4 Hz, 1H), 7.55 (dd, J=1.2, 7.6 Hz, 1H), 7.45-7.33 (m, 3H), 7.26-7.20 (m, 2H), 7.12 (t, J=8.8 Hz, 2H), 5.27 (t, J=6.8 Hz, 1H), 5.10-5.00 (m, 2H), 1.52 (d, J=7.2 Hz, 3H), 1.28 (d, J=2.4 Hz, 6H).
Compound UT071 was synthesized according to
A mixture of Compound 1 (100 mg, 351.27 μmol, 1 eq), SOCl2 (83.58 mg, 702.53 μmol, 50.96 uL, 2.0 eq) in DCM (3 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 0° C. for 1 h under N2 atmosphere. The mixture was concentrated in vacuum to obtain Compound 2 (100 mg, crude) as a white solid.
A mixture of Compound 2 (100 mg, 329.89 μmol, 1 eq), Compound 6 (75.61 mg, 329.89 μmol, 1 eq), K2CO3 (91.19 mg, 659.78 μmol, 2.0 eq) in MeCN (3 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 60° C. for 2 h under N2 atmosphere. The mixture was filtered and concentrated in vacuum to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100*30 mm*5 um; mobile phase: [water (FA)-ACN]; B %: 65%-95%, 8 min) to obtain Compound UT071A (18.97 mg, 38.05 μmol, 11.53% yield, 99.46% purity) as a off-white solid.
LCMS: [M+H]+=496.1
1H NMR (400 MHz, DMSO-d6) δ=7.53 (d, J=7.6 Hz, 1H), 7.39-7.28 (m, 2H), 7.22-7.01 (m, 6H), 5.18 (s, 2H), 5.10 (s, 2H), 1.25 (s, 6H).
Compound UT072 was synthesized according to
To a solution of Compound 1 (5 g, 31.94 mmol, 1 eq) and Compound 1A (6.61 g, 31.94 mmol, 4.13 mL, 1 eq) in ACN (30 mL) was added K2CO3 (5.30 g, 38.32 mmol, 1.2 eq). The mixture was stirred at 80° C. for 1 h. The reaction mixture was filtered and the filtrate was concentrated under the reduced pressure to give the product. The residue was purified by column chromatography (SiO2, Petroleum ether/Dichloromethane=1/1 to 1/1) was concentrated under the reduced pressure to yield Compound 2 (6.79 g, 24.02 mmol, 75.22% yield) as a white solid.
LCMS: [M+H]+=283.0
1H NMR (400 MHz, DMSO-d6) δ=10.45 (s, 1H), 7.59 (t, J=8.4 Hz, 1H), 7.30-7.14 (m, 5H), 5.30 (s, 2H).
To a solution of Compound 2 (6.7 g, 23.70 mmol, 1 eq) in MeOH (70 mL) was added NaBH4 (896.73 mg, 23.70 mmol, 1 eq) at 0° C. The mixture was stirred at 0° C. for 0.5 hr. The reaction was quenched by the addition of water (200 mL). The mixture was extracted with ethyl acetate (200 mL×2). The combined organic extracts were washed with brine (200 mL), dried, filtered and concentrated under the reduced pressure to yield Compound 3 (6.58 g, 23.12 mmol, 97.54% yield) as a white solid.
LCMS: [M+H]+=267.0
1H NMR (400 MHz, DMSO-d6) δ=7.32-7.12 (m, 4H), 7.08-6.97 (m, 2H), 5.20 (s, 2H), 4.92 (t, J=5.2 Hz, 1H), 4.66 (d, J=5.2 Hz, 2H).
To a solution of TMAD (2.28 g, 13.23 mmol, 2.5 eq) in THE (10 mL) was added PBu3 (3.47 g, 13.23 mmol, 2.5 eq) at 0° C. and the mixture was stirred at 0° C. for 30 min, then Compound 3A (800 mg, 5.29 mmol, 1.08 mL, 1 eq) and Compound 3 (1.51 g, 5.29 mmol, 1 eq) in THE (10 mL) was added and the mixture was stirred at 0° C. for 1.5 h. The reaction was quenched by the addition of water (30 mL). The mixture was extracted with ethyl acetate (30 mL×2). The combined organic extracts were washed with brine (30 mL), dried, filtered and concentrated. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0˜10% Ethyl acetate/Petroleum ether gradient @80 mL/min) and concentrated under the reduced pressure to yield Compound 4 (689 mg, crude) as an off-white solid.
LCMS: [M+H]+=418.1
To a solution of Compound 4 (300 mg, 718.03 umol, 1 eq) in THE (5 mL) was added NaH (34.46 mg, 861.58 umol, 60% purity, 1.20 eq) and Mel (203.83 mg, 1.44 mmol, 89.40 uL, 2 eq) at 0° C. The mixture was stirred at 25° C. for 2 h. The reaction mixture was diluted with NH4Cl and extracted with EtOAc (10 mL×2), the combined organic layers dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex luna C18 150*25 mm*10 um; mobile phase: [water (FA)-ACN]; B %: 56%-86%, 10.5 min) and lyophilized to yield UT072 (14.71 mg, 32.78 μmol, 4.57% yield, 99.366% purity) as an off-white solid.
LCMS: [M+H]+=446.1
1H NMR (400 MHz, DMSO-d6) δ=7.31-7.31 (m, 1H), 7.37-7.26 (m, 1H), 7.20-7.03 (m, 5H), 6.72 (ddd, J=2.4, 8.0, 10.0 Hz, 1H), 6.56 (dd, J=2.4, 10.0 Hz, 1H), 5.18 (s, 2H), 5.03 (s, 2H), 1.19 (s, 6H).
Compound UT074 was synthesized according to
To a solution of TMAD (2.28 g, 13.23 mmol, 2.5 eq) in THE (8 mL) was added tributylphosphane (2.68 g, 13.23 mmol, 3.26 mL, 2.5 eq) at 0° C. and the mixture was stirred at 0° C. for 30 min, then Compound 1 (800 mg, 5.29 mmol, 1.08 mL, 1 eq) and Compound 1A (1.43 g, 5.29 mmol, 1 eq) in THE (8 mL) was added and the mixture was stirred at 0° C. for another 90 min. The mixture was quenched by H2O (20 mL) and extracted with EtOAc (30 mL), the organic layer was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=20/1 to 10/1) to yield Compound 2 (320 mg, 792.51 μmol, 14.97% yield) as a yellow solid.
LCMS: [M+H]+=404.0
1H NMR (400 MHz, DMSO-d6) δ=7.46-7.44 (m, 2H), 7.43-7.41 (m, 1H), 7.20-7.12 (m, 1H), 7.06 (t, J=1.2 Hz, 1H), 6.72-6.70 (m, 2H), 6.39-6.36 (m, 2H), 5.03 (s, 2H), 3.23 (s, 2H).
To a solution of Compound 2 (300.00 mg, 742.98 umol, 1 eq) in THE (5 mL) was added NaH (59.43 mg, 1.49 mmol, 60% purity, 2 eq) and Mel (210.91 mg, 1.49 mmol, 92.51 uL, 2 eq) at 0° C. The mixture was stirred at 25° C. for 2 h. The reaction mixture was diluted with NH4Cl and extracted with EtOAc (10 mL×2), the combined organic layers dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (62%-92% MeCN in water (FA), 9 min), to yield UT074 (135.81 mg, 312.43 μmol, 42.05% yield, 99.34% purity) as a yellow solid.
LCMS: [M+H]+=432.1
1H NMR (400 MHz, DMSO-d6) δ=7.45-7.37 (m, 2H), 7.30 (dd, J=8.4, 5.6 Hz, 1H), 7.05 (dd, J=6.4, 2.8 Hz, 1H), 6.97 m, 1H), 6.84-6.66 (m, 2H), 6.53 (dd, J=8.4, 2.0 Hz, 2H), 5.04 (s, 2H), 1.15 (s, 6H).
Compound UT114 was synthesized according to
To a solution of Compound 1 (5 g, 30.84 mmol, 1 eq) in DCM (50 mL) was added SOCl2 (7.34 g, 61.69 mmol, 4.47 mL, 2 eq) at 0° C. The mixture was stirred at 0° C. for 1 h. The reaction mixture was concentrated under the reduced pressure to give the product. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0˜10% Ethylacetate/Petroleum ether gradient @80 mL/min) and was concentrated under the reduced pressure to give Compound 2 (3.7 g, 20.49 mmol, 66.44% yield) as a colorless liquid.
1H NMR (400 MHz, DMSO-d6) δ=7.35-7.17 (m, 2H), 4.74 (s, 2H).
To a solution of methyl Compound 2A (2 g, 9.12 mmol, 1 eq) and Compound 2 (1.65 g, 9.12 mmol, 1 eq) in ACN (20 mL) was added K2CO3 (7.56 g, 54.74 mmol, 6 eq). The mixture was stirred at 65° C. for 2 h. The reaction mixture was filtered and the filtrate was concentrated under the reduced pressure to give the product. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0˜20% Ethylacetate/Petroleum ether gradient @80 mL/min) and was concentrated under the reduced pressure to obtain Compound 3 (1.7 g, 4.68 mmol, 51.29% yield) as a white solid.
1H NMR (400 MHz, DMSO-d6) δ=7.68 (dd, J=1.6, 8.0 Hz, 1H), 7.52 (d, J=7.6 Hz, 1H), 7.46 (d, J=1.2 Hz, 1H), 7.22 (t, J=9.2 Hz, 2H), 4.98 (s, 2H), 3.83 (s, 3H), 1.29 (s, 6H).
To a solution of Compound 3 (1.7 g, 4.68 mmol, 1 eq) in THE (10 mL) was added LiOH (294.52 mg, 7.02 mmol, 1.5 eq) in H2O (10 mL). The mixture was stirred at 20° C. for 1 h. Acidify the reaction mixture by adding, with shaking, 10 mL of HCl until pH around 3, and then extracted with EtOAc (20 mL×3). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated to obtain Compound 4 (1.61 g, 4.60 mmol, 98.33% yield) as a white solid.
LCMS: [M+H]+=350.0
1H NMR (400 MHz, DMSO-d6) δ=14.08-11.88 (m, 1H), 7.67 (dd, J=1.2, 7.6 Hz, 1H), 7.52-7.42 (m, 2H), 7.22 (t, J=8.8 Hz, 2H), 4.98 (s, 2H), 1.29 (s, 6H).
To a solution of Compound 4 (200 mg, 572.57 umol, 1 eq) in DMF (2 mL) was added HATU (326.56 mg, 858.85 umol, 1.5 eq) at 0° C. The mixture was stirred at this temperature for 30 min. Then was added Compound 4A (84.27 mg, 572.57 umol, 1 eq) and DIEA (222.00 mg, 1.72 mmol, 299.19 uL, 3 eq) at 0° C. The mixture was stirred at 25° C. for 1.5 h. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 100*30 mm*5 um; mobile phase: [water (FA)-ACN]; B %: 47%-77%, 8 min) and lyophilized to yield UT114 (123.55 mg, 253.34 μmol, 44.25% yield, 98.107% purity) as an off-white solid.
LCMS: [M+H]+=479.0
1H NMR (400 MHz, DMSO-d6) δ=9.04 (t, J=5.6 Hz, 1H), 7.64-7.54 (m, 2H), 7.50 (d, J=8.0 Hz, 1H), 7.45 (d, J=7.6 Hz, 1H), 7.41 (s, 1H), 7.36-7.02 (m, 4H), 6.71 (s, 1H), 4.94 (s, 2H), 4.60 (d, J=5.6 Hz, 2H), 1.26 (s, 6H).
Compound UT115 was synthesized according to
To a solution of t-BuOK (4.06 g, 36.22 mmol, 2.56 eq) in THE (30 mL) was added bromocopper;methylsulfanylmethane (290.86 mg, 1.41 mmol, 0.1 eq) and Compound 1 (3 g, 14.15 mmol, 1 eq) at 0° C., then was added Mel (2.41 g, 16.98 mmol, 1.06 mL, 1.2 eq) at 8° C. for 1 h. The mixture was stirred at 25° C. for 1 h. Acidify the reaction mixture by adding, with shaking, 50 mL of citric acid until pH around 8, and then extracted with EtOAc (50 mL×3). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated to give a residue. The water-course was quenched by addition aq. NaClO (50 mL) and discarded. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0˜20% Ethylacetate/Petroleum ether gradient @50 mL/min) to obtain Compound 2 (1.17 g, 4.87 mmol, 34.44% yield) as a pink solid.
LCMS: [M+H]+=239.7
1H NMR (400 MHz, DMSO-d6) δ=10.45 (s, 1H), 7.25 (d, J=7.6 Hz, 1H), 7.14 (dd, J=8.0, 2.0 Hz, 1H), 6.98 (d, J=1.6 Hz, 1H), 1.23 (s, 6H).
To a solution of Compound 2 (1 g, 4.16 mmol, 1 eq) and Compound 2A (1.12 g, 5.41 mmol, 1.3 eq) in ACN (15 mL) was added K2CO3 (2.88 g, 20.82 mmol, 5 eq). The mixture was stirred at 60° C. for 2 h. The reaction mixture was filtered and the filtrate was concentrated under the reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0˜12% Ethylacetate/Petroleum ether gradient @60 mL/min) to yield Compound 3 (1.45 g, 3.96 mmol, 95.07% yield) as a red solid.
LCMS: [M+H]+=365.9
1H NMR (400 MHz, DMSO-d6) δ=7.42 (m, 1H), 7.32 (d, J=7.6 Hz, 1H), 7.21 (dd, J=7.6, 1.2 Hz, 1H), 7.17-7.00 (m, 3H), 4.98 (s, 2H), 1.25 (s, 6H).
To a solution of Compound 4 (4 g, 24.99 mmol, 1 eq) in MeOH (20 mL) was added 1-(isocyanomethylsulfonyl)-4-methyl-benzene (4.88 g, 24.99 mmol, 1 eq) and K2CO3 (5.18 g, 37.48 mmol, 1.5 eq). The mixture was stirred at 80° C. for 12 h. The residue was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=10/1=5:1) to obtain Compound 3A (2.5 g, 12.55 mmol, 50.25% yield) as a white solid.
1H NMR (400 MHz, CHLOROFORM-d) δ=8.03 (s, 1H), 7.49 (s, 1H), 7.03-6.62 (m, 2H).
A mixture of Compound 3 (183.90 mg, 502.19 umol, 1 eq) and Compound 3A (100 mg, 502.19 umol, 1 eq) Pd(OAC)2 (11.27 mg, 50.22 μmol, 0.1 eq), Cs2CO3 (327.24 mg, 1.00 mmol, 2 eq) and tris-o-tolylphosphane (30.57 mg, 100.44 umol, 0.2 eq) in DMF (2 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 80° C. for 17 h under N2 atmosphere. The reaction mixture was filtered and the filtrate was concentrated under the reduced pressure to give a residue. The residue was purified by prep-HPLC (62%-92% MeCN in water (FA), 9 min), to obtain UT115 (92.65 mg, 190.95 mol, 38.02% yield, 99.84% purity) as an off-white solid.
LCMS: [M+H]+=485.1
1H NMR (400 MHz, DMSO-d6) δ=7.77-7.67 (m, 2H), 7.56 (d, J=7.6 Hz, 1H), 7.54-7.45 (m, 3H), 7.45-7.37 (m, 1H), 7.13 (t, J=8.0 Hz, 2H), 5.06 (s, 2H), 1.33 (s, 6H).
Compound UT117 was synthesized according to
To a solution of Compound 1 (49.08 mg, 352.74 umol, 1 eq) and Compound 1A (180 mg, 440.92 umol, 1.25 eq) in EtOAc (3 mL) was added AgOTf (113.29 mg, 440.92 umol, 1.25 eq). The mixture was stirred at 70° C. for 12 h under dark. The mixture was cooled to 20° C. and diluted with ethyl acetate (3 mL) and was added brine (3 mL), the mixture was stirred at 25° C. for 4 h. The mixture was filtered and the filtrate was stratified with a separating funnel. The organic layer was washed with water (4 mL) and 5% NaHCO3 (4 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure and purified by prep-HPLC (column: Phenomenex luna C18 150*25 mm*10 um; mobile phase: [water (FA)-ACN]; B %: 66%-96%, 9 min) and lyophilized to obtain UT117 (8.76 mg, 19.24 μmol, 5.45% yield, 98.467% purity) as a white solid.
LCMS: [M+H]+=449.0
1H NMR (400 MHz, DMSO-d6) δ=8.66 (s, 1H), 7.89 (d, J=7.6 Hz, 1H), 7.78 (td, J=9.2, 2.4 Hz, 1H), 7.65 (dt, J=8.0, 5.8 Hz, 1H), 7.57-7.50 (m, 1H), 7.48-7.35 (m, 4H), 7.19-7.04 (m, 2H), 5.04 (s, 2H), 1.30 (s, 6H).
Compound UT118 was synthesized according to
A mixture of Compound 1 (400 mg, 1.09 mmol, 1 eq), Compound 1A (128.74 mg, 1.31 mmol, 181.58 uL, 1.2 eq), Pd(dppf)Cl2 (79.92 mg, 109.23 umol, 0.1 eq), TEA (221.06 mg, 2.18 mmol, 304.07 uL, 2 eq) and CuI (10.40 mg, 54.62 umol, 0.05 eq) in ACN (2 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 60° C. for 2 h under N2 atmosphere. The reaction mixture was filtered and the filtrate was concentrated under the reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 4 g SepaFlash® Silica Flash Column, Eluent of 0˜20% Ethylacetate/Petroleum ether gradient @50 mL/min) to obtain Compound 2 (310 mg, 654.75 μmol, 14.99% yield, 81% purity) as a black brown oil.
LCMS: [M+H]+=384.5
To a solution of Compound 3 (310 mg, 654.75 umol, 81% purity, 1 eq) in MeOH (3 mL) was added K2CO3 (271.47 mg, 1.96 mmol, 3 eq). The mixture was stirred at 60° C. for 2 h. The reaction mixture was filtered and the filtrate was concentrated under the reduced pressure to give a residue. The residue was purified by prep-TLC (SiO2, Petroleum ether:Ethyl acetate=3:1, Rf=0.3) and eluted with ethyl acetate (20 mL) to obtain Compound 3 (190 mg, 610.30 μmol, 93.21% yield) as a white solid.
LCMS: [M+H]+=312.3
1H NMR (400 MHz, CHLOROFORM-d) δ=7.27-7.22 (m, 1H), 7.21-7.16 (m, 1H), 7.16-7.12 (m, 1H), 6.98-6.83 (m, 3H), 5.02 (s, 2H), 3.04 (s, 1H), 1.39 (s, 6H).
To a solution of Compound 4 (300 mg, 2.46 mmol, 1 eq) in MeOH (5 mL) was added NaN3 (300 mg, 4.61 mmol, 1.88 eq) and Cu(OAc)2 (44.69 mg, 246.04 μmol, 0.1 eq). The reaction was stirred at 20° C. for 12 h. The reaction mixture was diluted with ethyl acetate (20 mL) and water (20 mL). The layers were separated and the aqueous layer was extracted with ethyl acetate (10 mL). The combined organic layers were washed with brine (20 mL), dried, filtered and concentrated till half amount of solvent remained. EtOH (20 mL) was added and the organic solution was concentrated half amount of solvent again. The solvent replacement was repeat for 3 times to obtain Compound 3A (167 mg, 1.40 mmol, 56.98% yield) as a yellow liquid.
GCMS: [M+H]+=119.1
To a solution of Compound 3 (57.40 mg, 481.81 umol, 1.5 eq) in DCM (0.5 mL), t-BuOH (0.5 mL) and H2O (0.5 mL) was added SODIUM ASCORBATE (15.27 mg, 77.09 umol, 0.24 eq), Compound 3A (100 mg, 321.21 umol, 1 eq) and CuSO4 (4.10 mg, 25.70 umol, 3.94 uL, 0.08 eq). The mixture was stirred at 50° C. for 4 h. The reaction mixture was filtered and the filtrate was concentrated under the reduced pressure to give a residue. The residue was purified by prep-HPLC (52%-82% MeCN in water (FA), 9 min), to obtain UT118 (94.79 mg, 219.53 μmol, 68.34% yield, 99.69% purity) as a white solid.
LCMS: [M+H]+=431.1
1H NMR (400 MHz, DMSO-d6) δ=9.22 (s, 1H), 8.01-7.89 (m, 2H), 7.64 (t, J=7.6 Hz, 2H), 7.59-7.46 (m, 4H), 7.45-7.35 (m, 1H), 7.12 (t, J=8.0 Hz, 2H), 5.05 (s, 2H), 2.07 (s, 1H), 1.31 (s, 6H).
Compound UT120 was synthesized according to
To a solution of Compound 1 (200 mg, 706.04 umol, 1 eq) in DCM (1 mL) was added TFA (1.54 g, 13.51 mmol, 1 mL, 19.13 eq). The mixture was stirred at 0° C. for 1 h. The reaction mixture was added NaHCO3 until pH=8, and then extracted with EtOAc (10 ml×3). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated to obtain Compound 2 (232 mg, crude) as a yellow oil.
LCMS: [M+H]+=167.0
To a solution of Compound 2A (200 mg, 572.57 umol, 1 eq) in DMF (2 mL) was added HATU (326.56 mg, 858.85 umol, 1.5 eq) at 0° C. The mixture was stirred at this temperature for 30 min. Compound 2 was then added (104.87 mg, 572.57 umol, 1 eq) and DIEA (222.00 mg, 1.72 mmol, 299.19 uL, 3 eq) at 0° C. The mixture was stirred at 25° C. for 11.5 h. The residue was purified by prep-HPLC (column: Phenomenex luna C18 150*25 mm*10 um; mobile phase: [water (HCl)-ACN]; B %: 51%-81%, 10 min) and lyophilized to obtain UT120 (199.55 mg, 386.55 μmol, 67.51% yield, 99.653% purity) as an off-white solid.
LCMS: [M+H]+=515.0
1H NMR (400 MHz, DMSO-d6) δ=9.07 (t, J=5.2 Hz, 1H), 7.80 (dd, J=6.4, 10.0 Hz, 1H), 7.72-7.55 (m, 2H), 7.54-7.37 (m, 2H), 7.36-7.12 (m, 2H), 6.90-6.65 (m, 1H), 4.96 (s, 2H), 4.61 (d, J=5.2 Hz, 2H), 1.28 (s, 6H).
Compound UT121 was synthesized according to
To a solution of Compound 1 (103.71 mg, 264.40 umol, 1 eq) in DMF (1.5 mL) was added HATU (150.80 mg, 396.59 umol, 1.5 eq) at 0° C., and the mixture was stirred at 0° C. for 30 min. After was added Compound 1A (43.16 mg, 264.40 umol, 1 eq) and DIEA (102.51 mg, 793.19 umol, 138.16 uL, 3 eq), and the mixture was stirred at 25° C. for 30 min. The residue was purified by Prep-HPLC (46%-76% MeCN in water (FA), 9 min, and 37%-67% MeCN in water (NH3H2O), 8 min), to obtain UT121 (9.33 mg, 17.72 μmol, 6.95% yield, 99.21% purity) was obtained as a white solid.
LCMS: [M+H]+=539.0
1H NMR (400 MHz, DMSO-d6) δ=9.15 (t, J=6.0 Hz, 1H), 7.89 (d, J=8.0 Hz, 1H), 7.77 (d, J=7.6 Hz, 1H), 7.60 (dd, J=7.6, 1.2 Hz, 1H), 7.49 (d, J=7.6 Hz, 1H), 7.45 (s, 1H), 7.40-7.22 (m, 3H), 4.97 (s, 2H), 4.72 (d, J=5.6 Hz, 2H), 3.75 (s, 3H), 2.06 (s, 3H), 1.33 (s, 6H).
Compound UT126 was synthesized according to
A mixture of Compound 1 (400 mg, 686.90 umol, 1 eq), Pd(dppf)Cl2 (50.26 mg, 68.69 umol, 0.1 eq) and TEA (139.01 mg, 1.37 mmol, 191.21 uL, 2 eq) in MeOH (4 mL) was degassed and purged with CO for 3 times, and then the mixture was stirred under 15 psi at 80° C. for 12 h under CO atmosphere. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was diluted with H2O (3 mL) and extracted with EtOAc (4 mL×3), dried over Na2SO4, filtered and concentrated under reduced pressure and purified by flash silica gel chromatography (ISCO®; 4 g SepaFlash® Silica Flash Column, Eluent of 0˜20% Ethyl acetate/Petroleum ether gradient @50 mL/min) to obtain Compound 2 (220 mg, 427.63 μmol, 62.25% yield) as a white solid.
1H NMR (400 MHz, CHLOROFORM-d6) δ=7.58 (d, J=7.6 Hz, 1H), 7.46 (dd, J=7.6, 1.6 Hz, 1H), 7.34 (dt, J=8.0, 5.2 Hz, 1H), 7.28 (s, 1H), 7.24-7.11 (m, 2H), 6.70 (t, J=8.2 Hz, 2H), 6.38 (t, J=5.2 Hz, 1H), 5.32 (s, 2H), 4.66 (d, J=5.6 Hz, 2H), 3.94 (s, 3H), 1.37 (s, 6H).
To a solution of Compound 2 (220 mg, 427.63 μmol, 1 eq) in THE (2 mL) and H2O (1 mL) was added LiOH (35.89 mg, 855.25 umol, 2 eq). The mixture was stirred at 25° C. for 1 h. The residue was purified by flash silica gel chromatography (ISCO®; 4 g SepaFlash® Silica Flash Column, Eluent of 0˜45% Ethyl acetate/Petroleum ether gradient @80 mL/min) to obtain Compound 3 (200 mg, 398.86 μmol, 93.27% yield, 99.802% purity) as a white solid.
LCMS: [M+H]+=500.9
1H NMR (400 MHz, DMSO-d6) δ=12.13 (s, 1H), 8.72 (t, J=4.8 Hz, 1H), 7.63 (d, J=7.6 Hz, 1H), 7.49 (dd, J=8.0, 1.2 Hz, 1H), 7.46-7.30 (m, 4H), 7.17 (t, J=8.8 Hz, 2H), 5.29 (s, 2H), 4.43 (d, J=54.8 Hz, 2H), 1.24 (s, 6H).
To a solution of Compound 3 (180 mg, 359.68 umol, 1 eq) in DMF (4 mL) was added NH4Cl (28.86 mg, 539.52 umol, 1.5 eq), DIEA (139.46 mg, 1.08 mmol, 187.95 uL, 3 eq) and HATU (205.14 mg, 539.52 umol, 1.5 eq). The mixture was stirred at 25° C. for 1 h. The residue was purified by prep-HPLC (column: Phenomenex luna C18 150*25 mm*10 um; mobile phase: [water (FA)-ACN]; B %: 32%-62%, 9 min) and lyophilized to obtain UT126 (40.16 mg, 79.98 μmol, 22.24% yield, 99.469% purity) as a white solid.
LCMS: [M+H]+=500.3
1H NMR (400 MHz, DMSO-d6) δ=8.64 (t, J=5.2 Hz, 1H), 8.04 (s, 1H), 7.65 (s, 1H), 7.52-7.44 (m, 2H), 7.43-7.33 (m, 2H), 7.26 (dd, J=7.6, 0.8 Hz, 1H), 7.24-7.12 (m, 3H), 5.10 (s, 2H), 4.44 (d, J=5.2 Hz, 2H), 1.26 (s, 6H).
Compound C1 was synthesized according to
To a solution of Compound 1 (578.0 mg, 3.11 mmol, 1.0 eq) in DCM (6 mL) was added (Boc)2O (1.1 mL, 4.66 mmol, 1.5 eq) at 25° C., followed by Et3N (0.87 mL, 6.21 mmol, 2.0 eq). The reaction mixture was stirred at the same temperature for 1 hour. The reaction mixture was concentrated under the reduced pressure to give the crude product, which was purified by flash column chromatography (SiliaFlash® Irregular Silica Gel, P60 40-63 μm, 60 Å, 10%˜20% Ethyl acetate/Petroleum ether gradient, manually) to obtain Compound 2 (675.0 mg, 2.36 mmol, 75.84% yield) as a light-yellow oil.
LCMS: [M+Na]+=308.1 1H NMR (400 MHz, CDCl3) δ=7.54 (dd, J=8.0, 1.3 Hz, 1H), 7.42-7.34 (m, 1H), 7.29 (td, J=7.5, 1.3 Hz, 1H), 7.14 (td, J=7.6, 1.8 Hz, 1H), 5.01 (s, 1H), 4.39 (d, J=6.3 Hz, 2H), 1.45 (s, 9H).
To a stirred solution of Compound 2 (550.0 mg, 1.92 mmol, 1.0 eq) in 1,4-dioxane (20 mL) was added pinacol vinylboronate (342.8 mg, 2.11 mmol, 1.1 eq) at 25° C. Then, Cs2CO3 (1.252 g, 3.84 mmol, 2.0 eq) and PPh3 (100.8 mg, 0.38 mmol, 0.2 eq) were added to the reaction mixture, followed by Pd(OAc)2 (21.6 mg, 0.10 mmol, 5 mol %). The reaction mixture was degassed with nitrogen five times and heated to 75° C. After 12 hours, the resulting mixture was allowed to cool to 25° C. and filtered through a pad of Celite. The filtrate was washed with H2O (20 mL) and extracted with EtOAc (2×20 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by flash column chromatography (SiliaFlash® Irregular Silica Gel, P60 40-63 μm, 60 Å, 10%˜20% Ethyl acetate/Petroleum ether gradient, manually) to obtain Compound 3 (443.3 mg, 1.90 mmol, 98.96% yield) as a light-yellow oil.
LCMS: [M+Na]+=256.2
1H NMR (400 MHz, CDCl3) δ=7.50-7.41 (m, 1H), 7.34-7.27 (m, 0.4H), 7.26-7.15 (m, 2.5H), 7.11-7.03 (m, 0.5H), 6.95-6.84 (m, 0.6H), 5.67 (dd, J=17.3, 1.4 Hz, 0.6H), 5.35 (dd, J=11.0, 1.4 Hz, 0.6H), 5.01-4.86 (m, 0.4H), 4.68-4.51 (m, 0.4H), 4.37-4.21 (m, 2H), 1.41-1.36 (m, 9H).
To a stirred solution of Compound 4 (109.9 mg, 0.50 mmol, 1.0 eq) in DMF (2 mL) was added NaH (20.0 mg, 0.50 mmol, 1.0 eq, 60% suspension in mineral oil) at 0° C. The resulting mixture was stirred for 15 min at the same temperature before addition of Compound 4A (142.6 mg, 0.50 mmol, 1.0 eq). The reaction mixture was slowly warmed to 25° C. and stirred for 1 hour. The reaction was monitored by TLC. After completion, NH4Cl (1 mL) was added to the mixture, followed by EtOAc (2 mL). The organic phase was separated, washed with H2O (2×1 mL) and brine (2 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by flash column chromatography (SiliaFlash® Irregular Silica Gel, P60 40-63 μm, 60 Å, 25%˜50% Ethyl acetate/Petroleum ether gradient, manually) to obtain Compound 5 (188.1 mg, 0.44 mmol, 88.77% yield) as a light-yellow solid.
LCMS: [M+H]+=422.1
1H NMR (400 MHz, CDCl3) δ=7.72 (dd, J=7.7, 1.4 Hz, 1H), 7.57 (dd, J=8.1, 1.2 Hz, 1H), 7.41 (d, J=1.4 Hz, 1H), 7.39 (dd, J=8.3, 1.4 Hz, 1H), 7.24 (d, J=7.7 Hz, 1H), 7.13 (t, J=8.0 Hz, 1H), 5.27 (s, 2H), 3.87 (s, 3H), 1.42 (s, 6H).
To a stirred solution of Compound 5 (30.2 mg, 0.071 mmol, 1.0 eq) in DMA (1 mL) was added Compound 3 (25.0 mg, 0.107 mmol, 1.5 eq) at 25° C. Then, Et3N (20.0 μL, 0.142 mmol, 2.0 eq) and Tri-o-tolylphosphine (1.3 mg, 0.004 mmol, 6 mol %) were added to the reaction mixture, followed by Pd(OAc)2 (0.5 mg, 0.002 mmol, 3 mol %). The reaction mixture was degassed with nitrogen five times and heated to 140° C. After 40 hours, the resulting mixture was allowed to cool to 25° C. and filtered through a pad of Celite. The filtrate was washed with H2O (20 mL) and extracted with EtOAc (2×20 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by flash column chromatography (SiliaFlash® Irregular Silica Gel, P60 40-63 μm, 60 Å, 20%˜50% Ethyl acetate/Petroleum ether gradient, manually) to yield Compound 6 (18.2 mg, 0.032 mmol, 44.29% yield) as a white solid.
LCMS: [M+Na]+=597.3
To a stirred solution of Compound 6 (18.2 mg, 0.032 mmol, 1.0 eq) in ethyl acetate (2 mL) was added Pd/C (1.8 mg, 10 wt %) at 25° C. The reaction mixture was degassed three times with a H2 balloon before heating to 40° C. After 4 hours, the resulting mixture was allowed to cool to 25° C. and filtered through a pad of Celite. The filtrate was concentrated under reduced pressure to obtain Compound 7 (14.4 mg, 0.025 mmol, 78.62% yield) as a white solid.
LCMS: [M+Na]+=599.3
1H NMR (400 MHz, CDCl3) δ=7.70 (dd, J=7.7, 1.4 Hz, 1H), 7.37 (d, J=1.4 Hz, 1H), 7.32 (dd, J=7.6, 1.7 Hz, 1H), 7.29-7.24 (m, 1H), 7.23-7.14 (m, 5H), 7.08 (dt, J=5.1, 3.5 Hz, 1H), 5.06 (s, 2H), 4.93 (s, 1H), 4.27 (d, J=5.6 Hz, 2H), 3.85 (s, 3H), 3.07-3.00 (m, 2H), 2.92 (dd, J=9.1, 6.0 Hz, 2H), 1.41 (s, 9H), 1.32 (s, 6H).
To a stirred solution of Compound 7 (14.4 mg, 0.025 mmol, 1.0 eq) in a mixture of THE (0.2 mL) and H2O (0.1 mL) was added LiOH·H2O (4.2 mg, 0.100 mmol, 4.0 eq) at 25° C. The resulting mixture was stirred at 40° C. for 20 hours. The reaction was monitored by TLC and LC-MS. After completion, H2O (0.5 mL) was added to the mixture, followed by EtOAc (0.5 mL). The aqueous phase was separated and acidified with 1N HCl to pH=5. The aqueous mixture was extracted with EtOAc (2×0.5 mL). The combined organic layers were washed with brine (0.5 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure to yield Compound 8 (12.6 mg, 0.022 mmol, 89.68% yield) as a white solid, which was used for the next step without any further purification.
LCMS: [M+Na]+=585.3
To a stirred solution of Compound 8 (12.6 mg, 0.022 mmol, 1.0 eq) in DCM (1 mL) was added TFA (0.2 mL). The resulting mixture was stirred at 25° C. for 2 hours and concentrated under reduced pressure to obtain Compound 9 (12.3 mg, 0.021 mmol, 95.00% yield) as a white solid, which was used for the next step without any further purification.
LCMS: [M+H]+=463.2
To a stirred solution of Compound 9 (12.3 mg, 0.021 mmol, 1.0 eq) in DCM (0.5 mL) was added Et3N (8.9 μL, 0.064 mmol, 3.0 eq) and HATU (9.7 mg, 0.026 mmol, 1.2 eq) at 0° C. The reaction mixture was allowed to warm to 25° C. and stirred a further 1 hour. The reaction was monitored by TLC and LC-MS. After completion, H2O (0.5 mL) was added to the mixture. The organic phase was separated, washed with H2O (2×0.5 mL) and brine (0.5 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by prep-HPLC (C18 column) to obtain C1 (2.1 mg, 0.0047 mmol, 22.14% yield) as a white solid.
LCMS: [M+H]+=445.2
1H NMR (600 MHz, CD3CN) δ=7.61 (dd, J=21.1, 7.8 Hz, 2H), 7.33 (tt, J=24.8, 7.4 Hz, 5H), 7.21 (q, J=7.5 Hz, 2H), 7.08-6.96 (m, 1H), 5.29 (d, J=15.3 Hz, 1H), 4.70-4.55 (m, 2H), 4.48 (s, 1H), 4.19 (d, J=13.6 Hz, 1H), 3.63 (t, J=11.7 Hz, 2H), 3.54 (q, J=12.6, 11.1 Hz, 1H), 3.01 (t, J=11.8 Hz, 1H), 1.37-1.26 (m, 6H).
Compound C2 was synthesized according to
To a solution of Compound 8 (457.9 mg, 2.23 mmol, 1.0 eq) in chloroform (2 mL) was added NBS (436.3 mg, 2.45 mmol, 1.1 eq) at 25° C., followed by AlBN (73.2 mg, 0.45 mmol, 0.2 eq). The reaction mixture was refluxed for 5 hours. After cooling to 25° C., the reaction mixture was concentrated under the reduced pressure to give the crude product, which was purified by flash column chromatography (SiliaFlash® Irregular Silica Gel, P60 40-63 μm, 60 Å, 0%˜10% Ethyl acetate/Petroleum ether gradient, manually) to obtain Compound 1A (560.2 mg, 1.97 mmol, 88.40% yield) as a yellow solid.
LCMS: [M+H]+=282.3
1H NMR (400 MHz, CDCl3) δ=7.59 (d, J=2.4 Hz, 1H), 7.38 (dd, J=8.5, 2.4 Hz, 1H), 7.26 (d, J=8.5 Hz, 1H), 4.52 (s, 2H).
To a stirred solution of Compound 1 (70.0 mg, 0.32 mmol, 1.0 eq) in DMF (2 mL) was added NaH (12.8 mg, 0.32 mmol, 1.0 eq, 60% suspension in mineral oil) at 0° C. The resulting mixture was stirred for 15 min at the same temperature before addition of Compound 1A (90.8 mg, 0.32 mmol, 1.0 eq). The reaction mixture was slowly warmed to 25° C. and stirred for 1 hour. The reaction was monitored by TLC. After completion, NH4Cl (1 mL) was added to the mixture, followed by EtOAc (2 mL). The organic phase was separated, washed with H2O (2×1 mL) and brine (2 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by flash column chromatography (SiliaFlash® Irregular Silica Gel, P60 40-63 μm, 60 Å, 25%˜50% Ethyl acetate/Petroleum ether gradient, manually) to yield Compound 2 (104.9 mg, 0.25 mmol, 77.73% yield) as a light-yellow solid.
LCMS: [M+H]+=422.1
1H NMR (400 MHz, CDCl3) δ=7.82 (dd, J=7.7, 1.4 Hz, 1H), 7.36 (d, J=1.5 Hz, 1H), 7.34 (d, J=2.5 Hz, 1H), 7.31 (d, J=5.5 Hz, 1H), 7.29-7.26 (m, 1H), 7.09 (d, J=2.3 Hz, 1H), 5.01 (s, 2H), 3.89 (s, 3H), 1.48 (s, 6H).
To a stirred solution of Compound 2 (35.6 mg, 0.084 mmol, 1.0 eq) in 1,4-dioxane (1 mL) was added pinacol vinylboronate (15.0 mg, 0.093 mmol, 1.1 eq) at 25° C. Then, Cs2CO3 (54.7 mg, 0.17 mmol, 2.0 eq) and PPh3 (4.4 mg, 0.017 mmol, 0.2 eq) were added to the reaction mixture, followed by Pd(OAc)2 (1.0 mg, 0.0042 mmol, 5 mol %). The reaction mixture was degassed with nitrogen five times and heated to 75° C. After 12 hours, the resulting mixture was allowed to cool to 25° C. and filtered through a pad of Celite. The filtrate was washed with H2O (1 mL) and extracted with EtOAc (2×1 mL). The combined organic layers were washed with brine (1 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by flash column chromatography (SiliaFlash® Irregular Silica Gel, P60 40-63 μm, 60 Å, 10%˜20% Ethyl acetate/Petroleum ether gradient, manually) to yield Compound 3 (27.4 mg, 0.074 mmol, 87.96% yield) as a light-yellow solid.
LCMS: [M+H]+=370.2
1H NMR (400 MHz, CDCl3) δ=7.79 (dd, J=7.7, 1.4 Hz, 1H), 7.40 (d, J=1.4 Hz, 1H), 7.36 (d, J=8.2 Hz, 1H), 7.31 (d, J=7.7 Hz, 1H), 7.27-7.23 (m, 1H), 7.03 (d, J=2.1 Hz, 1H), 6.53 (dd, J=17.6, 10.9 Hz, 1H), 5.56 (dd, J=17.6, 0.7 Hz, 1H), 5.18 (dd, J=10.8, 0.6 Hz, 1H), 5.05 (s, 2H), 3.87 (s, 3H), 1.48 (s, 6H).
To a stirred solution of Compound 3 (21.2 mg, 0.057 mmol, 1.0 eq) in DCM (6 mL) was added Compound 3A (33.4 mg, 0.14 mmol, 2.5 eq) at 25° C., followed by Grubbs-II catalyst (2.4 mg, 0.0029 mmol, 5 mol %). The reaction mixture was refluxed for 24 hours. After cooling to 25° C., the reaction mixture was filtered through a pad of Celite. The filtrate was concentrated under the reduced pressure to give the crude product, which was purified by flash column chromatography (SiliaFlash® Irregular Silica Gel, P60 40-63 μm, 60 Å, 10%˜50% Ethyl acetate/Petroleum ether gradient, manually) to yield Compound 4 (15.1 mg, 0.026 mmol, 45.81% yield) as a yellow solid.
LCMS: [M+Na]+=597.3
1H NMR (400 MHz, CDCl3) δ=7.80 (dd, J=7.7, 1.4 Hz, 1H), 7.52 (t, J=8.6 Hz, 2H), 7.44-7.37 (m, 2H), 7.31 (d, J=7.7 Hz, 1H), 7.29-7.21 (m, 4H), 7.08 (d, J=2.2 Hz, 1H), 6.80 (d, J=16.0 Hz, 1H), 5.07 (s, 2H), 4.67 (s, 1H), 4.39 (d, J=5.8 Hz, 2H), 3.87 (s, 3H), 1.50 (s, 6H), 1.38 (s, 9H).
To a stirred solution of Compound 4 (15.1 mg, 0.026 mmol, 1.0 eq) in ethyl acetate (2 mL) was added Pd/C (1.5 mg, 10 wt %) at 25° C. The reaction mixture was degassed three times with a H2 balloon before heating to 40° C. After 4 hours, the resulting mixture was allowed to cool to 25° C. and filtered through a pad of Celite. The filtrate was concentrated under reduced pressure to yield Compound 5 (12.9 mg, 0.022 mmol, 85.13% yield) as a white solid.
LCMS: [M+Na]+=599.3
To a stirred solution of Compound 5 (12.9 mg, 0.022 mmol, 1.0 eq) in a mixture of THE (0.2 mL) and H2O (0.1 mL) was added LiOH·H2O (3.8 mg, 0.089 mmol, 4.0 eq) at 25° C. The resulting mixture was stirred at 40° C. for 25 hours. The reaction was monitored by TLC and LC-MS. After completion, H2O (0.5 mL) was added to the mixture, followed by EtOAc (0.5 mL). The aqueous phase was separated and acidified with 1N HCl to pH=5. The aqueous mixture was extracted with EtOAc (2×0.5 mL). The combined organic layers were washed with brine (0.5 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure to yield Compound 6 (11.8 mg, 0.021 mmol, 93.75% yield) as a white solid, which was used for the next step without any further purification.
LCMS: [M+Na]+=585.3
To a stirred solution of Compound 6 (11.8 mg, 0.021 mmol, 1.0 eq) in DCM (1 mL) was added TFA (0.2 mL). The resulting mixture was stirred at 25° C. for 2 hours and concentrated under reduced pressure to obtain Compound 7 (11.0 mg, 0.019 mmol, 90.97% yield) as a white solid, which was used for the next step without any further purification.
LCMS: [M+H]+=463.2
To a stirred solution of Compound 7 (11.0 mg, 0.019 mmol, 1.0 eq) in DCM (0.5 mL) was added Et3N (8.0 μL, 0.057 mmol, 3.0 eq) and HATU (8.7 mg, 0.023 mmol, 1.2 eq) at 0° C. The reaction mixture was allowed to warm to 25° C. and stirred a further 1 hour. The reaction was monitored by TLC and LC-MS. After completion, H2O (0.5 mL) was added to the mixture. The organic phase was separated, washed with H2O (2×0.5 mL) and brine (0.5 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by prep-HPLC (C18 column) to obtain C2 (4.6 mg, 0.010 mmol, 54.23% yield) as a white solid.
LCMS: [M+H]+=445.2
1H NMR (400 MHz, CDCl3) δ=7.67 (d, J=7.8 Hz, 1H), 7.63 (d, J=2.2 Hz, 1H), 7.37-7.46 (m, 2H), 7.24-7.14 (m, 3H), 6.99 (d, J=8.1 Hz, 1H), 6.75 (dd, J=8.2, 2.1 Hz, 1H), 6.69 (d, J=1.5 Hz, 1H), 5.02 (s, 2H), 4.90 (s, 1H), 4.41 (d, J=4.0 Hz, 2H), 3.34-3.28 (m, 2H), 3.26-3.18 (m, 2H), 1.39 (s, 6H).
Compounds C53 and UT009 were tested for their ability to interfere STING signaling. As shown in
Compounds UT014 and UT122 were tested for its ability to regulate STING signaling. Activities of the compounds were analyzed by measuring interferon levels in the presence of increasing concentration of the compounds in THP-1 cells. UT014 has a O moiety in the R2 group, while UT122 has a NH moiety at the same position in R2 group. UT014 was found to exhibit agonistic activity, by enhancing the induction of interferon levels in a dose dependent manner (
High-resolution cryo-EM structure of human STING oligomer in complex with the agonist C53 or the antagonist compound UT009 were analyzed to understand the conformation and position of the target binding site of C53 and UT009. As shown in
Compounds UT017, UT018, UT019 and UT0122 were tested for their ability to interfere with STING signaling. The structures of each of these compounds is shown in
Further, compounds UT141, UT142, UT157, UT153, UT156 and UT158 were tested for their ability to interfere with STING signaling. Structures of each of these compounds are shown in
The results from the above described examples showed that substituting moieties across the groups of the disclosed formulas may result in surprisingly different effects on the activity of the STING protein. Conversion of a STING agonist to a STING antagonist can be made with as little as a single substitution to the moiety in a structural group. Additionally, even though STING agonists and antagonists have opposite modulatory activities, the STING antagonist was found to bind STING in a similar conformation and manner, as an agonist. The STING modulators described herein therefore provide a range of compounds that are useful in a wide range of therapeutic applications, as STING agonists or antagonists.
To a solution of Compound 1 (30 mg, 0.128 mmol, 1 eq) in DMF (0.5 mL) was added NaH (6.1 mg, 0.154 mmol, 60% purity, 1.2 eq) and stirred at 0° C. for 15 min. Then Compound 1A (18.5 uL, 0.14 mmol, 1.1 eq) was added to the mixture. The mixture was stirred at 25° C. for 2 h. The reaction mixture was diluted with water (1 mL) and extracted with EtOAc (1 mL×3). The combined organic layers were washed with brine (1 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue which was purified by flash silica gel chromatography to give Compound 2 (39 mg, 0.103 mmol, 80% yield). LCMS: [M+H]+=379.1 1H NMR (400 MHz, CDCl3) δ 7.66-7.61 (m, 2H), 7.06 (d, J=8.1 Hz, 1H), 6.63 (t, J=8.4 Hz, 2H), 5.79 (dd, J=15.9, 1.4 Hz, 1H), 4.80 (d, J=15.8 Hz, 1H), 4.38 (q, J=6.6 Hz, 1H), 3.90 (s, 3H), 3.09 (s, 3H), 1.30 (d, J=6.6 Hz, 3H). 19F NMR (376 MHz, CDCl3) δ −108.49 (s, 1F), −111.32 (d, J=6.5 Hz, 2F).
To a solution of Compound 2 (10 mg, 0.026 mmol, 1 eq) in THE (1 mL) and MeOH (0.5 mL) was added LiOH·H2O (3.3 mg, 0.079 mmol, 3 eq) in H2O (0.5 mL). The mixture was stirred at 25° C. for 12 h. The reaction mixture was concentrated under the reduced pressure to give a residue which was purified by flash silica gel chromatography to give Compound 3 (9.5 mg, 0.026 mmol, 99% yield). LCMS: [M+H]+=365.1 1H NMR (600 MHz, CDCl3) δ 7.75-7.67 (m, 2H), 7.11 (d, J=7.8 Hz, 1H), 6.65 (t, J=8.3 Hz, 2H), 5.82 (dd, J=15.8, 1.5 Hz, 1H), 4.82 (d, J=15.8 Hz, 1H), 4.41 (q, J=6.6 Hz, 1H), 3.11 (s, 3H), 1.32 (d, J=6.6 Hz, 3H). 19F NMR (565 MHz, CDCl3) δ −108.39 (p, J=7.5 Hz, 1F), −111.24 (t, J=7.4 Hz, 2F).
To a solution of Compound 3 (13.9 mg, 0.038 mmol, 1 eq) in DCM (1 mL) was added Et3N (16 uL, 0.11 mmol, 3 eq) and HBTU (18.8 mg, 0.05 mmol, 1.3 eq). The mixture was stirred at 25° C. for 5 min, then Compound 3A (6.7 mg, 0.046 mmol, 1.2 eq) was added. The mixture was stirred at 25° C. for 2 h. The reaction mixture was concentrated under the reduced pressure to give a residue which was purified by flash silica gel chromatography to give UT156 (18.2 mg, 0.037 mmol, 97% yield). LCMS: [M+H]+=493.2
1H NMR (400 MHz, CD3CN) δ 9.36 (s, 1H), 7.53-7.43 (m, 2H), 7.38 (td, J=7.9, 1.3 Hz, 2H), 7.17 (d, J=7.8 Hz, 1H), 7.10 (ddd, J=8.2, 7.1, 1.2 Hz, 1H), 7.02 (ddd, J=8.0, 7.1, 1.1 Hz, 1H), 6.76 (t, J=8.8 Hz, 2H), 6.36 (dd, J=2.2, 0.9 Hz, 1H), 5.74-5.51 (m, 1H), 4.85 (d, J=16.0 Hz, 1H), 4.71-4.58 (m, 2H), 4.48 (q, J=6.6 Hz, 1H), 2.99 (s, 3H), 1.24 (d, J=6.6 Hz, 3H). 19F NMR (376 MHz, CD3CN) δ −110.65 (t, J=6.5 Hz, 1F), −112.53 (d, J=6.3 Hz, 2F).
To a solution of Compound 1 (31 mg, 0.14 mmol, 1 eq) in DMF (0.5 mL) was added NaH (6.7 mg, 0.17 mmol, 60% purity, 1.2 eq) and stirred at 0° C. for 15 min. Then Compound 1A (20.3 uL, 0.15 mmol, 1.1 eq) was added to the mixture. The mixture was stirred at 25° C. for 2 h. The reaction mixture was diluted with water (1 mL) and extracted with EtOAc (1 mL×3). The combined organic layers were washed with brine (1 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue which was purified by flash silica gel chromatography to give Compound 2 (51 mg, 0.14 mmol, 99% yield). LCMS: [M+H]+=364.1. 1H NMR (400 MHz, CDCl3) δ 7.78 (dd, J=7.7, 1.4 Hz, 1H), 7.49 (d, J=1.4 Hz, 1H), 7.28 (s, 1H), 6.70 (dd, J=8.7, 7.8 Hz, 2H), 5.02 (s, 2H), 3.92 (s, 3H), 1.41 (s, 6H). 19F NMR (376 MHz, CDCl3) δ −107.17 (t, J=6.7 Hz, 1F), −110.52 (d, J=6.9 Hz, 2F).
To a solution of Compound 2 (51 mg, 0.14 mmol, 1 eq) in THE (1 mL) and MeOH (0.5 mL) was added LiOH·H2O (17.7 mg, 0.42 mmol, 3 eq) in H2O (0.5 mL). The mixture was stirred at 25° C. for 12 h. The reaction mixture was concentrated under the reduced pressure to give a residue which was purified by flash silica gel chromatography to give Compound 3 (48 mg, 0.14 mmol, 98% yield). LCMS: [M+H]+=350.1. 1H NMR (400 MHz, CDCl3) δ 7.86 (dt, J=7.7, 1.2 Hz, 1H), 7.54 (d, J=1.4 Hz, 1H), 7.31 (d, J=7.7 Hz, 1H), 6.70 (t, J=8.2 Hz, 2H), 5.03 (s, 2H), 1.42 (s, 6H). 19F NMR (376 MHz, cdcl3) δ −107.03 (p, J=7.6 Hz, 1F), −110.50 (t, J=7.4 Hz, 2F).
To a solution of Compound 3 (6.8 mg, 0.020 mmol, 1 eq) in DCM (1 mL) was added Et3N (8.1 uL, 0.058 mmol, 3 eq) and HATU (9.6 mg, 0.025 mmol, 1.3 eq). The mixture was stirred at 25° C. for 5 min, then Compound 3A (3.4 mg, 0.023 mmol, 1.2 eq) was added. The mixture was stirred at 25° C. for 2 h. The reaction mixture was concentrated under the reduced pressure to give a residue which was purified by flash silica gel chromatography to give UT141 (9.0 mg, 0.019 mmol, 97% yield). LCMS: [M+H]+=478.1. 1H NMR (400 MHz, DMSO) δ 10.92 (s, 1H), 8.93 (t, J=5.7 Hz, 1H), 7.63 (dd, J=7.7, 1.5 Hz, 1H), 7.51-7.41 (m, 3H), 7.33 (dq, J=8.1, 0.9 Hz, 1H), 7.26-7.16 (m, 2H), 7.02 (ddd, J=8.2, 7.0, 1.3 Hz, 1H), 6.94 (ddd, J=8.0, 7.0, 1.1 Hz, 1H), 6.26 (dd, J=2.2, 1.0 Hz, 1H), 4.96 (s, 2H), 4.60 (d, J=5.6 Hz, 2H), 1.29 (s, 6H). 19F NMR (376 MHz, DMSO) δ −109.62 (t, J=6.8 Hz, 1F), −112.24 (d, J=6.8 Hz, 2F).
To a solution of Compound 1 (22.1 mg, 0.10 mmol, 1 eq) in DMF (0.5 mL) was added NaH (4.8 mg, 0.12 mmol, 60% purity, 1.2 eq) and stirred at 0° C. for 15 min. Then Compound 1A (28.5 mg, 0.11 mmol, 1.1 eq) was added to the mixture. The mixture was stirred at 25° C. for 2 h. The reaction mixture was diluted with water (1 mL) and extracted with EtOAc (1 mL×3). The combined organic layers were washed with brine (1 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue which was purified by flash silica gel chromatography to give Compound 2 (37.9 mg, 0.095 mmol, 95% yield). LCMS: [M+H]+=396.1 1H NMR (400 MHz, CDCl3) δ 7.76 (dd, J=7.7, 1.4 Hz, 1H), 7.59 (d, J=7.9 Hz, 1H), 7.47-7.39 (m, 1H), 7.33 (d, J=1.4 Hz, 1H), 7.30-7.27 (m, 1H), 7.22 (ddd, J=10.0, 8.4, 1.2 Hz, 1H), 5.24 (s, 2H), 3.87 (s, 3H), 1.44 (s, 6H). 19F NMR (376 MHz, CDCl3) δ −58.62 (s, 3F), −112.65 (s, 1F).
To a solution of Compound 2 (31.3 mg, 0.079 mmol, 1 eq) in THE (1 mL) and MeOH (0.5 mL) was added LiOH·H2O (10.0 mg, 0.24 mmol, 3 eq) in H2O (0.5 mL). The mixture was stirred at 25° C. for 12 h. The reaction mixture was concentrated under the reduced pressure to give a residue which was purified by flash silica gel chromatography to give Compound 3 (22.8 mg, 0.060 mmol, 76% yield). LCMS: [M+H]+=382.1. 1H NMR (400 MHz, CDCl3) δ 7.82 (dd, J=7.7, 1.4 Hz, 1H), 7.58 (d, J=7.9 Hz, 1H), 7.42 (td, J=8.1, 5.1 Hz, 1H), 7.34 (d, J=1.4 Hz, 1H), 7.30 (d, J=7.7 Hz, 1H), 7.24-7.17 (m, 1H), 5.23 (s, 2H), 1.44 (s, 6H). 19F NMR (376 MHz, CDCl3) δ −58.64 (s, 3F), −112.71 (s, 1F).
To a solution of Compound 3 (20.6 mg, 0.054 mmol, 1 eq) in DCM (1 mL) was added Et3N (22.6 uL, 0.16 mmol, 3 eq) and HATU (26.7 mg, 0.070 mmol, 1.3 eq). The mixture was stirred at 25° C. for 5 min, then Compound 3A (9.5 mg, 0.065 mmol, 1.2 eq) was added. The mixture was stirred at 25° C. for 2 h. The reaction mixture was concentrated under the reduced pressure to give a residue which was purified by flash silica gel chromatography to give UT142 (20.1 mg, 0.039 mmol, 73% yield). LCMS: [M+H]+=510.2. 1H NMR (400 MHz, CD3CN) δ 9.34 (s, 1H), 7.61 (d, J=7.9 Hz, 1H), 7.54-7.41 (m, 3H), 7.37-7.27 (m, 3H), 7.22 (d, J=1.4 Hz, 1H), 7.09 (ddd, J=8.2, 7.0, 1.3 Hz, 1H), 7.01 (ddd, J=8.0, 7.1, 1.1 Hz, 1H), 6.33 (dd, J=2.1, 1.0 Hz, 1H), 5.15 (s, 2H), 4.61 (dd, J=5.8, 0.8 Hz, 2H), 1.33 (s, 6H). 19F NMR (376 MHz, CD3CN) δ −59.10 (s, 3F), −114.00 (s, 1F).
To a solution of Compound 1 (18.3 mg, 0.078 mmol, 1 eq) in DMF (0.5 mL) was added NaH (3.7 mg, 0.094 mmol, 60% purity, 1.2 eq) and stirred at 0° C. for 15 min. Then Compound 1A (22.1 mg, 0.086 mmol, 1.1 eq) was added to the mixture. The mixture was stirred at 25° C. for 2 h. The reaction mixture was diluted with water (1 mL) and extracted with EtOAc (1 mL×3). The combined organic layers were washed with brine (1 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue which was purified by flash silica gel chromatography to give Compound 2 (30.9 mg, 0.075 mmol, 96% yield). LCMS: [M+H]+=411.1 1H NMR (400 MHz, CDCl3) δ 7.63 (dd, J=7.8, 1.5 Hz, 1H), 7.53 (d, J=7.9 Hz, 1H), 7.50 (d, J=1.5 Hz, 1H), 7.33 (td, J=8.1, 5.1 Hz, 1H), 7.18-7.10 (m, 1H), 7.09 (d, J=7.8 Hz, 1H), 5.79 (d, J=17.0 Hz, 1H), 5.18 (d, J=17.0 Hz, 1H), 4.48 (q, J=6.6 Hz, 1H), 3.83 (s, 3H), 3.10 (s, 3H), 1.41 (d, J=6.6 Hz, 3H). 19F NMR (376 MHz, CDCl3) δ −59.15 (s, 3F), −114.29 (s, 1F).
To a solution of Compound 2 (30.9 mg, 0.075 mmol, 1 eq) in THE (1 mL) and MeOH (0.5 mL) was added LiOH·H2O (9.5 mg, 0.226 mmol, 3 eq) in H2O (0.5 mL). The mixture was stirred at 25° C. for 12 h. The reaction mixture was concentrated under the reduced pressure to give a residue which was purified by flash silica gel chromatography to give Compound 3 (27.7 mg, 0.070 mmol, 93% yield). LCMS: [M+H]+=397.1 1H NMR (400 MHz, CDCl3) δ 7.69 (dd, J=7.8, 1.4 Hz, 1H), 7.57-7.51 (m, 2H), 7.33 (td, J=8.1, 4.9 Hz, 1H), 7.19-7.09 (m, 2H), 5.77 (d, J=16.9 Hz, 1H), 5.21 (d, J=17.0 Hz, 1H), 4.49 (q, J=6.6 Hz, 1H), 3.11 (s, 3H), 1.42 (d, J=6.7 Hz, 3H). 19F NMR (376 MHz, CDCl3) δ −59.22 (s, 3F), −114.43 (s, 1F).
To a solution of Compound 3 (13.8 mg, 0.035 mmol, 1 eq) in DCM (1 mL) was added Et3N (14.6 uL, 0.10 mmol, 3 eq) and HBTU (17.2 mg, 0.045 mmol, 1.3 eq). The mixture was stirred at 25° C. for 5 min, then Compound 3A (5.1 uL, 0.042 mmol, 1.2 eq) was added. The mixture was stirred at 25° C. for 2 h. The reaction mixture was concentrated under the reduced pressure to give a residue which was purified by flash silica gel chromatography to give UT153 (16.8 mg, 0.031 mmol, 89% yield). LCMS: [M+H]+=540.2 1H NMR (400 MHz, CDCl3) δ 7.47 (d, J=7.9 Hz, 1H), 7.39 (dd, J=7.7, 1.5 Hz, 1H), 7.32 (td, J=8.1, 5.0 Hz, 1H), 7.17-7.03 (m, 3H), 6.77-6.62 (m, 2H), 6.15 (t, J=5.7 Hz, 1H), 5.78 (d, J=17.1 Hz, 1H), 5.12 (d, J=17.0 Hz, 1H), 4.60 (d, J=5.6 Hz, 2H), 4.45 (q, J=6.6 Hz, 1H), 3.09 (s, 3H), 1.38 (d, J=6.6 Hz, 3H). 19F NMR (376 MHz, CDCl3) δ −59.13 (s, 3F), −107.72 (t, J=6.3 Hz, 1F), −111.75 (d, J=6.4 Hz, 2F), −113.99 (s, 1F).
To a solution of Compound 4 (213.5 mg, 1.04 mmol, 1 eq) in DCM (4 mL) was added PBr3 (98 uL, 1.04 mmol, 1 eq) dropwise at 25° C. The reaction mixture was stirred at 25° C. for 2 h. The reaction mixture was diluted with water (1 mL) and extracted with DCM (1 mL×3). The combined organic layers were washed with brine (1 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue which was purified by flash silica gel chromatography to give Compound 1A (234.4 mg, 0.87 mmol, 84% yield). LCMS: [M+H]+=267.0. 1H NMR (400 MHz, CDCl3) δ 4.44 (s, 2H), 3.86 (s, 3H), 2.21 (s, 3H).
To a solution of Compound 1 (26.7 mg, 0.114 mmol, 1 eq) in DMF (1 mL) was added NaH (5.5 mg, 0.137 mmol, 60% purity, 1.2 eq) and stirred at 0° C. for 15 min. Then Compound 1A (33.6 mg, 0.125 mmol, 1.1 eq) was added to the mixture. The mixture was stirred at 25° C. for 2 h. The reaction mixture was diluted with water (1 mL) and extracted with EtOAc (1 mL×3). The combined organic layers were washed with brine (1 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue which was purified by flash silica gel chromatography to give Compound 2 (44.9 mg, 0.107 mmol, 94% yield). LCMS: [M+H]+=421.1. 1H NMR (400 MHz, CDCl3) δ 7.69 (td, J=4.0, 1.4 Hz, 2H), 7.13-7.06 (m, 1H), 5.72 (d, J=16.3 Hz, 1H), 4.78 (d, J=16.3 Hz, 1H), 4.46 (q, J=6.6 Hz, 1H), 3.90 (s, 3H), 3.83 (s, 3H), 3.11 (s, 3H), 2.20 (s, 3H), 1.37 (d, J=6.6 Hz, 3H).
To a solution of Compound 2 (40.0 mg, 0.095 mmol, 1 eq) in THE (1 mL) and MeOH (0.5 mL) was added LiOH·H2O (11.2 mg, 0.285 mmol, 3 eq) in H2O (0.5 mL). The mixture was stirred at 25° C. for 12 h. The reaction mixture was concentrated under the reduced pressure to give a residue which was purified by flash silica gel chromatography to give Compound 3 (38.3 mg, 0.094 mmol, 99% yield). LCMS: [M+H]+=407.1. 1H NMR (400 MHz, CDCl3) δ 7.73 (td, J=4.0, 1.4 Hz, 2H), 7.13-7.08 (m, 1H), 5.69 (d, J=16.4 Hz, 1H), 4.79 (d, J=16.3 Hz, 1H), 4.46 (q, J=6.7 Hz, 1H), 3.86 (s, 3H), 3.10 (s, 3H), 2.18 (s, 3H), 1.38 (d, J=6.6 Hz, 3H).
To a solution of Compound 3 (8.4 mg, 0.021 mmol, 1 eq) in DCM (1 mL) was added Et3N (8.6 uL, 0.062 mmol, 3 eq) and HBTU (10.2 mg, 0.027 mmol, 1.3 eq). The mixture was stirred at 25° C. for 5 min, then Compound 3A (3.6 mg, 0.025 mmol, 1.2 eq) was added. The mixture was stirred at 25° C. for 2 h. The reaction mixture was concentrated under the reduced pressure to give a residue which was purified by flash silica gel chromatography to give UT151 (11.6 mg, 0.022 mmol, 99% yield). LCMS: [M+H]+=535.2. 1H NMR (400 MHz, CD3CN) δ 9.38 (s, 1H), 7.50 (dq, J=7.9, 0.9 Hz, 1H), 7.47-7.40 (m, 2H), 7.37 (dq, J=8.1, 0.9 Hz, 1H), 7.19 (dt, J=7.6, 0.6 Hz, 1H), 7.10 (ddd, J=8.2, 7.1, 1.2 Hz, 1H), 7.01 (ddd, J=8.0, 7.1, 1.1 Hz, 1H), 6.36 (dq, J=1.6, 0.8 Hz, 1H), 5.52 (d, J=16.4 Hz, 1H), 4.78 (d, J=16.4 Hz, 1H), 4.64 (dt, J=5.9, 1.0 Hz, 2H), 4.54 (q, J=6.6 Hz, 1H), 3.78 (s, 3H), 3.00 (s, 3H), 2.03 (s, 3H), 1.32 (d, J=6.6 Hz, 3H).
To a solution of Compound 4 (228.0 mg, 1.36 mmol, 1 eq) in THE (1.5 mL) was added LiAlH4 (51.5 mg, 1.36 mmol, 1 eq) at 0° C. The reaction mixture was warmed to 25° C. and stirred for 2 h. H2O (0.05 mL) was added, followed by 15% NaOH (0.05 mL). Additional H2O (0.15 mL) was added. The mixture was extracted with EtOAc (1 mL×3). The combined organic layers were washed with brine (1 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give crude Compound 5 which was used directly for the next step. LCMS: [M+H]+=141.2
To a solution of crude Compound 5 (1.36 mmol, 1 eq) in DCM (5 mL) was added PBr3 (128 uL, 1.36 mmol, 1 eq) dropwise at 25° C. The reaction mixture was stirred at 25° C. for 2 h. The reaction mixture was diluted with water (1 mL) and extracted with DCM (1 mL×3). The combined organic layers were washed with brine (1 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue which was purified by flash silica gel chromatography to give Compound 1A (165.5 mg, 0.81 mmol, 60% yield for 2 steps). LCMS: [M+H]+=203.1. 1H NMR (400 MHz, CDCl3) δ 4.41 (s, 2H), 3.79 (s, 3H), 2.15 (s, 3H), 1.96 (s, 3H).
To a solution of Compound 1 (23.7 mg, 0.11 mmol, 1 eq) in DMF (1 mL) was added NaH (5.2 mg, 0.13 mmol, 60% purity, 1.2 eq) and stirred at 0° C. for 15 min. Then Compound 1A (24.1 mg, 0.12 mmol, 1.1 eq) was added to the mixture. The mixture was stirred at 25° C. for 2 h. The reaction mixture was diluted with water (1 mL) and extracted with EtOAc (1 mL×3). The combined organic layers were washed with brine (1 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue which was purified by flash silica gel chromatography to give Compound 2 (29.5 mg, 0.086 mmol, 80% yield). LCMS: [M+H]+=342.2. 1H NMR (400 MHz, CDCl3) δ 7.79 (dd, J=7.7, 1.4 Hz, 1H), 7.43 (d, J=1.4 Hz, 1H), 7.28 (d, J=1.1 Hz, 1H), 4.90 (s, 2H), 3.91 (s, 3H), 3.74 (s, 3H), 2.20 (d, J=2.2 Hz, 6H), 1.43 (s, 6H).
To a solution of Compound 2 (29.4 mg, 0.086 mmol, 1 eq) in THE (1 mL) and MeOH (0.5 mL) was added LiOH·H2O (10.8 mg, 0.258 mmol, 3 eq) in H2O (0.5 mL). The mixture was stirred at 25° C. for 12 h. The reaction mixture was concentrated under the reduced pressure to give a residue which was purified by flash silica gel chromatography to give Compound 3 (27.9 mg, 0.085 mmol, 99% yield). LCMS: [M+H]+=328.2. 1H NMR (400 MHz, CDCl3) δ 7.86 (dd, J=7.7, 1.3 Hz, 1H), 7.44 (d, J=1.4 Hz, 1H), 7.30 (d, J=7.7 Hz, 1H), 4.91 (s, 2H), 3.80 (s, 3H), 2.21 (s, 3H), 2.17 (s, 3H), 1.43 (s, 6H).
To a solution of Compound 3 (6.4 mg, 0.0195 mmol, 1 eq) in DCM (1 mL) was added Et3N (8.2 uL, 0.0586 mmol, 3 eq) and HATU (9.7 mg, 0.0254 mmol, 1.3 eq). The mixture was stirred at 25° C. for 5 min, then Compound 3A (3.4 mg, 0.0235 mmol, 1.2 eq) was added. The mixture was stirred at 25° C. for 2 h. The reaction mixture was concentrated under the reduced pressure to give a residue which was purified by flash silica gel chromatography to give UT157 (8.4 mg, 0.0184 mmol, 94% yield). LCMS: [M+H]+=456.2. 1H NMR (400 MHz, CD3CN) δ 9.39 (s, 1H), 7.48 (ddd, J=13.9, 7.8, 1.3 Hz, 2H), 7.39-7.32 (m, 2H), 7.26 (d, J=1.5 Hz, 1H), 7.09 (ddd, J=8.2, 7.1, 1.2 Hz, 1H), 7.01 (ddd, J=8.1, 7.0, 1.1 Hz, 1H), 6.36 (dt, J=2.2, 0.9 Hz, 1H), 4.86 (s, 2H), 4.64 (dd, J=5.8, 0.8 Hz, 2H), 3.64 (s, 3H), 2.03 (s, 3H), 2.01 (s, 3H), 1.35 (s, 6H).
Compound UT158 was synthesized according to the following reaction scheme.
To a solution of Compound 1 (13.9 mg, 0.035 mmol, 1 eq) in DCM (1 mL) was added Et3N (14.7 uL, 0.11 mmol, 3 eq) and HBTU (17.3 mg, 0.046 mmol, 1.3 eq). The mixture was stirred at 25° C. for 5 min, then Compound 1A (6.2 mg, 0.042 mmol, 1.2 eq) was added. The mixture was stirred at 25° C. for 2 h. The reaction mixture was concentrated under the reduced pressure to give a residue which was purified by flash silica gel chromatography to give UT158 (13.3 mg, 0.025 mmol, 72% yield). LCMS: [M+H]+=525.2 1H NMR (400 MHz, CD3CN) δ 9.36 (s, 1H), 7.55 (d, J=7.9 Hz, 1H), 7.51-7.30 (m, 5H), 7.27-7.15 (m, 2H), 7.08 (ddd, J=8.2, 7.1, 1.3 Hz, 1H), 7.00 (ddd, J=8.0, 7.0, 1.1 Hz, 1H), 6.32 (dd, J=2.1, 1.0 Hz, 1H), 5.53 (d, J=16.9 Hz, 1H), 5.19 (d, J=16.9 Hz, 1H), 4.59 (ddd, J=5.8, 1.6, 0.8 Hz, 2H), 4.53 (q, J=6.6 Hz, 1H), 2.97 (s, 3H), 1.33 (d, J=6.6 Hz, 3H). 19F NMR (376 MHz, CD3CN) δ −59.53 (s, 3F), −115.97 (s, 1F).
To a solution of Compound 1 (30.0 mg, 0.128 mmol, 1 eq) in DMF (1 mL) was added NaH (6.1 mg, 0.154 mmol, 60% purity, 1.2 eq) and stirred at 0° C. for 15 min. Then Compound 1A (28.6 mg, 0.141 mmol, 1.1 eq) was added to the mixture. The mixture was stirred at 25° C. for 2 h. The reaction mixture was diluted with water (1 mL) and extracted with EtOAc (1 mL×3). The combined organic layers were washed with brine (1 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue which was purified by flash silica gel chromatography to give Compound 2 (43.3 mg, 0.122 mmol, 95% yield). LCMS: [M+H]+=357.2. 1H NMR (400 MHz, CDCl3) δ 7.66 (dd, J=7.8, 1.5 Hz, 1H), 7.53 (d, J=1.5 Hz, 1H), 7.08 (d, J=7.8 Hz, 1H), 5.69 (d, J=16.3 Hz, 1H), 4.66 (d, J=16.3 Hz, 1H), 4.45 (q, J=6.6 Hz, 1H), 3.89 (s, 3H), 3.80 (s, 3H), 3.10 (s, 3H), 2.15 (s, 3H), 2.11 (s, 3H), 1.35 (d, J=6.6 Hz, 3H).
To a solution of Compound 2 (30.7 mg, 0.086 mmol, 1 eq) in THE (1 mL) and MeOH (0.5 mL) was added LiOH·H2O (10.8 mg, 0.259 mmol, 3 eq) in H2O (0.5 mL). The mixture was stirred at 25° C. for 12 h. The reaction mixture was concentrated under the reduced pressure to give a residue which was purified by flash silica gel chromatography to give Compound 3 (29.2 mg, 0.085 mmol, 99% yield). LCMS: [M+H]+=343.2
To a solution of Compound 3 (8.1 mg, 0.024 mmol, 1 eq) in DCM (1 mL) was added Et3N (9.9 uL, 0.071 mmol, 3 eq) and HBTU (11.7 mg, 0.031 mmol, 1.3 eq). The mixture was stirred at 25° C. for 5 min, then Compound 3A (4.2 mg, 0.028 mmol, 1.2 eq) was added. The mixture was stirred at 25° C. for 2 h. The reaction mixture was concentrated under the reduced pressure to give a residue which was purified by flash silica gel chromatography to give UT160 (9.3 mg, 0.020 mmol, 83% yield). LCMS: [M+H]+=471.2. 1H NMR (400 MHz, CD3CN) δ 9.43 (s, 1H), 7.54 (s, 1H), 7.50 (dq, J=7.7, 0.9 Hz, 1H), 7.41-7.32 (m, 3H), 7.16 (dt, J=7.4, 0.7 Hz, 1H), 7.09 (ddd, J=8.2, 7.1, 1.2 Hz, 1H), 7.01 (ddd, J=8.1, 7.1, 1.1 Hz, 1H), 6.35 (dq, J=1.7, 0.8 Hz, 1H), 5.46 (d, J=16.4 Hz, 1H), 4.74 (d, J=16.4 Hz, 1H), 4.64 (ddd, J=5.8, 2.6, 0.8 Hz, 2H), 4.53 (q, J=6.6 Hz, 1H), 3.68 (s, 3H), 3.01 (s, 3H), 1.97 (s, 3H), 1.90 (s, 3H), 1.28 (d, J=6.6 Hz, 3H).
To a solution of Compound 2 (53.5 mg, 0.176 mmol, 1 eq) in DCM (1 mL) was PBr3 (16.5 uL, 0.176 mmol, 1 eq). The mixture was stirred at 25° C. for 2 h. The reaction mixture was diluted with water (1 mL) and extracted with DCM (1 mL×3). The combined organic layers were washed with brine (1 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue which was purified by flash silica gel chromatography to give Compound 1A (25.2 mg, 0.069 mmol, 39% yield). 1H NMR (400 MHz, CDCl3) δ 7.50 (dd, J=8.0, 1.3 Hz, 1H), 7.46-7.39 (m, 1H), 7.13 (dd, J=8.2, 1.3 Hz, 1H), 6.68-6.55 (m, 3H), 4.68 (s, 2H). 19F NMR (376 MHz, CDCl3) δ −59.48 (s, 3F), −107.58 (s, 2F).
To a solution of Compound 1 (2.6 mg, 0.011 mmol, 1 eq) in DMF (0.5 mL) was added NaH (0.5 mg, 0.014 mmol, 60% purity, 1.2 eq) and stirred at 0° C. for 15 min. Then Compound 1A (4.5 mg, 0.012 mmol, 1.1 eq) was added to the mixture. The mixture was stirred at 25° C. for 2 h. The reaction mixture was diluted with water (1 mL) and extracted with EtOAc (1 mL×3). The combined organic layers were washed with brine (1 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue which was purified by flash silica gel chromatography to give UT149 (5.7 mg, 0.011 mmol, 97% yield). LCMS: [M+H]+=516.1. 1H NMR (400 MHz, CDCl3) δ 7.66-7.58 (m, 1H), 7.50-7.39 (m, 1H), 7.29-7.25 (m, 1H), 7.20 (d, J=7.7 Hz, 1H), 7.09 (dd, J=8.3, 1.2 Hz, 1H), 6.91-6.84 (m, 1H), 6.49 (tt, J=8.8, 2.3 Hz, 1H), 6.23-6.14 (m, 2H), 5.19 (s, 2H), 1.23 (s, 6H). 19F NMR (376 MHz, CDCl3) δ −58.03 (s, 3F), −62.60 (s, 3F), −107.49 (s, 2F).
Compound UT202 was synthesized according to the reaction scheme depicted below.
To a solution of Compound 1 (2.6 mg, 0.011 mmol, 1 eq) in DMF (0.5 mL) was added NaH (0.5 mg, 0.014 mmol, 60% purity, 1.2 eq) and stirred at 0° C. for 15 min. Then Compound 1A (4.5 mg, 0.012 mmol, 1.1 eq) was added to the mixture. The mixture was stirred at 25° C. for 2 h. The reaction mixture was diluted with water (1 mL) and extracted with EtOAc (1 mL×3). The combined organic layers were washed with brine (1 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue which was purified by flash silica gel chromatography to give UT202 (4.2 mg, 0.008 mmol, 72% yield). LCMS: [M+H]+=516.1. 1H NMR (400 MHz, CD3CN) δ 7.71-7.66 (m, 1H), 7.56-7.49 (m, 2H), 7.45 (ddd, J=8.4, 2.1, 1.0 Hz, 1H), 7.20 (d, J=8.3 Hz, 1H), 6.82 (d, J=8.3 Hz, 1H), 6.62 (tt, J=9.3, 2.3 Hz, 1H), 6.30-6.16 (m, 2H), 5.15 (s, 2H), 1.16 (s, 6H). 19F NMR (376 MHz, CD3CN) δ −58.42 (s, 3F), −61.93 (s, 3F), −109.95 (s, 2F).
Compound UT200 was synthesized according to the reaction scheme depicted below.
To a solution of Compound 1 (2.2 mg, 0.012 mmol, 1 eq) in DMF (0.5 mL) was added NaH (0.6 mg, 0.014 mmol, 60% purity, 1.2 eq) and stirred at 0° C. for 15 min. Then Compound 1A (4.8 mg, 0.013 mmol, 1.1 eq) was added to the mixture. The mixture was stirred at 25° C. for 2 h. The reaction mixture was diluted with water (1 mL) and extracted with EtOAc (1 mL×3). The combined organic layers were washed with brine (1 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue which was purified by flash silica gel chromatography to give UT200 (5.2 mg, 0.011 mmol, 93% yield). LCMS: [M+H]+=473.1. 1H NMR (400 MHz, CDCl3) δ 7.64 (dd, J=7.9, 1.2 Hz, 1H), 7.48 (t, J=8.1 Hz, 1H), 7.32 (dd, J=7.6, 1.4 Hz, 1H), 7.20 (d, J=7.6 Hz, 1H), 7.10 (d, J=8.2 Hz, 1H), 6.81 (d, J=1.3 Hz, 1H), 6.51 (tt, J=8.9, 2.3 Hz, 1H), 6.24-6.16 (m, 2H), 5.18 (s, 2H), 1.22 (s, 6H). 19F NMR (376 MHz, CDCl3) δ −57.94 (s, 3F), −107.18 (s, 2F).
Compound UT201 was synthesized according to the reaction scheme depicted below.
To a solution of Compound 1 (2.4 mg, 0.013 mmol, 1 eq) in DMF (0.5 mL) was added NaH (0.6 mg, 0.015 mmol, 60% purity, 1.2 eq) and stirred at 0° C. for 15 min. Then Compound 1A (5.1 mg, 0.014 mmol, 1.1 eq) was added to the mixture. The mixture was stirred at 25° C. for 2 h. The reaction mixture was diluted with water (1 mL) and extracted with EtOAc (1 mL×3). The combined organic layers were washed with brine (1 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue which was purified by flash silica gel chromatography to give UT201 (5.6 mg, 0.012 mmol, 92% yield). LCMS: [M+H]+=473.1. 1H NMR (400 MHz, CDCl3) δ 7.62 (dd, J=8.0, 1.2 Hz, 1H), 7.51-7.39 (m, 2H), 7.37 (d, J=1.6 Hz, 1H), 7.08 (dd, J=8.3, 1.2 Hz, 1H), 6.66 (d, J=8.2 Hz, 1H), 6.52 (tt, J=8.8, 2.2 Hz, 1H), 6.23-6.15 (m, 2H), 5.20 (s, 2H), 1.23 (s, 6H). 19F NMR (376 MHz, CDCl3) δ −57.89 (s, 3F), −107.09 (s, 2F).
Compound UT203 was synthesized according to the reaction scheme depicted below.
To a solution of Compound 1 (2.2 mg, 0.011 mmol, 1 eq) in DMF (0.5 mL) was added NaH (0.5 mg, 0.013 mmol, 60% purity, 1.2 eq) and stirred at 0° C. for 15 min. Then Compound 1A (4.5 mg, 0.012 mmol, 1.1 eq) was added to the mixture. The mixture was stirred at 25° C. for 2 h. The reaction mixture was diluted with water (1 mL) and extracted with EtOAc (1 mL×3). The combined organic layers were washed with brine (1 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue which was purified by flash silica gel chromatography to give UT203 (4.5 mg, 0.009 mmol, 85% yield). LCMS: [M+H]+=492.2. 1H NMR (400 MHz, CD3CN) δ 7.67 (d, J=7.9 Hz, 1H), 7.52 (t, J=8.1 Hz, 1H), 7.20 (d, J=8.3 Hz, 1H), 6.77 (s, 1H), 6.64 (tt, J=9.3, 2.3 Hz, 1H), 6.33-6.25 (m, 3H), 5.85 (s, 2H), 5.06 (s, 2H), 1.09 (s, 6H). 19F NMR (376 MHz, CD3CN) δ −58.39 (s, 3F), −110.01 (s, 2F).
The STING pathway activity was evaluated in THP1-Dual™ cells (Invivogen). 1.5×105 THP1-Dual™ cells were seeded in 96-well plates and treated with the test compound followed by cGAMP (100 uM) or MSA-2 (10 uM, Science 2020, 369, eaba6098) 2 hours later. After incubating at 37° C. for 16 hours, the luciferase activity was measured by QUANTI-Luc™ (Invivogen).
Table 1 depicts compounds analyzed and their relative activity (see below for Activity codes).
This application claims priority to U.S. Provisional Application No. 63/305,060, filed Jan. 31, 2022, which is incorporated by reference herein in its entirety.
This invention was made with government support under Grant Nos. CA226419 and GM130289 awarded by the National Institutes of Health. The government has certain rights in this invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2023/061685 | 1/31/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63305060 | Jan 2022 | US |
