Patent Application
20240383910
Bruton's Tyrosine Kinase (BTK) is a Tec-family tyrosine kinase present in all blood cells except for T cells and natural killer cells. Overexpression of BTK is associated with various B cell malignancies, including mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). BTK is a clinically proven target to attenuate B cell receptor (BCR) signaling and induce cell death in these cancer cells. Several BTK kinase inhibitors have been approved to treat B cell malignancies. Not only serving as a kinase, BTK can also enhance antigen receptor-induced calcium influx in a kinase-independent manner. Therefore, developing BTK degraders to abolish both kinase-dependent and -independent functions attracts significant interests in academia and industry.
Described herein are compounds of the following formula:
or a pharmaceutically acceptable salt or prodrug thereof. Also described herein are compositions comprising a compound as described above and a pharmaceutically acceptable carrier, along with kits comprising the compounds and compositions described herein.
Also described herein are methods of treating or preventing a BTK-related disease in a subject, comprising administering to the subject an effective amount of a compound or composition as described herein. Optionally, the BTK-related disease is cancer (e.g., bladder cancer, blood cancer, a bone marrow cancer, brain cancer, breast cancer, bronchus cancer, colorectal cancer, cervical cancer, chondrosarcoma, endometrial cancer, gastrointestinal cancer, gastric cancer, genitourinary cancer, head and neck cancer, hepatic cancer, hepatocellular carcinoma, leukemia, liver cancer, lung cancer, lymphoma, melanoma of the skin, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, skin cancer, testicular cancer, thyroid cancer, or uterine cancer). Optionally, the BTK-related disease is a neurodegenerative disorder or an inflammatory disease. The methods described herein can further include administering a second compound, biomolecule, or composition. Optionally, the second compound, biomolecule, or composition comprises a chemotherapeutic agent.
Further described herein are methods of inducing BTK degradation in a cell, comprising contacting a cell with an effective amount of a compound as described herein. The contacting can be performed in vitro or in vivo.
The details of one or more embodiments are set forth in the drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.
Described herein are novel small molecule proteolysis-targeting chimeras (PROTACs) and methods for their use as Bruton's tyrosine kinase (BTK) degraders. A proteolysis targeting chimera (PROTAC) is a heterobifunctional molecule that can bind both a targeted protein and an E3 ubiquitin ligase to facilitate the formation of a ternary complex, leading to ubiquitination and ultimate degradation of the target protein. Using the PROTAC approach, described herein are orally available BTK degraders. The reversible covalent BTK PROTACs described herein are highly potent and selective. The compounds described herein are capable of selectivity inducing degradation of IKZF1 and IKZF3. In addition, lower molecular weight compounds are described herein along with their uses as potent triple degraders of BTK and lymphoid transcription factors Ikaros (IKZF1) and Aiolos (IKZF3). The small molecule PROTACs described herein are useful in treating and/or preventing cancer, neurodegenerative disorders, inflammatory diseases, metabolic disorders, and other BTK-related diseases.
In some cases, the PROTACs described herein are compounds as shown below:
An additional compound described herein, optionally for use in the methods described herein includes the following:
The compounds described herein also include isotopic substitutions (e.g., a deuterium or tritium variant) of the compounds. In particular, one or more hydrogen atoms can be substituted by a hydrogen isotope (e.g., a deuterium or a tritium). For example, a methoxy group (—OCH3) can be substituted with one or more isotopic groups to form, for example, —OCDH2, —OCD2H, or —OCD3.
The compounds described herein can be prepared in a variety of ways. The compounds can be synthesized using various synthetic methods. At least some of these methods are known in the art of synthetic organic chemistry. The compounds described herein can be prepared from readily available starting materials. Optimum reaction conditions can vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.
Variations on the compounds described herein include the addition, subtraction, or movement of the various constituents as described for each compound. Similarly, when one or more chiral centers are present in a molecule, all possible chiral variants are included.
Additionally, compound synthesis can involve the protection and deprotection of various chemical groups. The use of protection and deprotection, and the selection of appropriate protecting groups can be determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Wuts, Greene's Protective Groups in Organic Synthesis, 5th. Ed., Wiley & Sons, 2014, which is incorporated herein by reference in its entirety.
Reactions to produce the compounds described herein can be carried out in solvents, which can be selected by one of skill in the art of organic synthesis. Solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products under the conditions at which the reactions are carried out, i.e., temperature and pressure.
Reactions can be carried out in one solvent or a mixture of more than one solvent. Product or intermediate formation can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1H or 13C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography such as high-performance liquid chromatography (HPLC) or thin layer chromatography.
Exemplary methods for synthesizing compounds as described herein are provided in Example 1 below.
The compounds described herein or derivatives thereof can be provided in a pharmaceutical composition. Depending on the intended mode of administration, the pharmaceutical composition can be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, or suspensions, preferably in unit dosage form suitable for single administration of a precise dosage. The compositions will include a therapeutically effective amount of the compound described herein or derivatives thereof in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, or diluents. By pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, which can be administered to an individual along with the selected compound without causing unacceptable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it is contained.
As used herein, the term carrier encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington: The Science and Practice of Pharmacy, 22d Edition, Loyd et al. eds., Pharmaceutical Press and Philadelphia College of Pharmacy at University of the Sciences (2012). Examples of physiologically acceptable carriers include buffers, such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, such as TWEEN® (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICS™ (BASF; Florham Park, NJ).
Compositions containing the compound described herein or derivatives thereof suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
These compositions may also contain adjuvants, such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be promoted by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents, for example, sugars, sodium chloride, and the like may also be included. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
Solid dosage forms for oral administration of the compounds described herein or derivatives thereof include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compounds described herein or derivatives thereof is admixed with at least one inert customary excipient (or carrier), such as sodium citrate or dicalcium phosphate, or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (e) solution retarders, as for example, paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol, and glycerol monostearate, (h) adsorbents, as for example, kaolin and bentonite, and (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents.
Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethyleneglycols, and the like.
Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and others known in the art. They may contain opacifying agents and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.
Liquid dosage forms for oral administration of the compounds described herein or derivatives thereof include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols, and fatty acid esters of sorbitan, or mixtures of these substances, and the like.
Besides such inert diluents, the composition can also include additional agents, such as wetting, emulsifying, suspending, sweetening, flavoring, or perfuming agents.
Suspensions, in addition to the active compounds, may contain additional agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
Compositions of the compounds described herein or derivatives thereof for rectal administrations are optionally suppositories, which can be prepared by mixing the compounds with suitable non-irritating excipients or carriers, such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and, therefore, melt in the rectum or vaginal cavity and release the active component.
Dosage forms for topical administration of the compounds described herein or derivatives thereof include ointments, powders, sprays, inhalants, and skin patches. The compounds described herein or derivatives thereof are admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants as may be required. Ophthalmic formulations, ointments, powders, and solutions are also contemplated as being within the scope of the compositions.
Optionally, the compounds described herein can be contained in a drug depot. A drug depot comprises a physical structure to facilitate implantation and retention in a desired site (e.g., a synovial joint, a disc space, a spinal canal, abdominal area, a tissue of the patient, etc.). The drug depot can provide an optimal concentration gradient of the compound at a distance of up to about 0.1 cm to about 5 cm from the implant site. A depot, as used herein, includes but is not limited to capsules, microspheres, microparticles, microcapsules, microfibers particles, nanospheres, nanoparticles, coating, matrices, wafers, pills, pellets, emulsions, liposomes, micelles, gels, antibody-compound conjugates, protein-compound conjugates, or other pharmaceutical delivery compositions. Suitable materials for the depot include pharmaceutically acceptable biodegradable materials that are preferably FDA approved or GRAS materials. These materials can be polymeric or non-polymeric, as well as synthetic or naturally occurring, or a combination thereof. The depot can optionally include a drug pump.
The compositions can include one or more of the compounds described herein and a pharmaceutically acceptable carrier. As used herein, the term pharmaceutically acceptable salt refers to those salts of the compound described herein or derivatives thereof that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds described herein. The term salts refers to the relatively non-toxic, inorganic and organic acid addition salts of the compounds described herein. These salts can be prepared in situ during the isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate, lactobionate, methane sulphonate, and laurylsulphonate salts, and the like. These may include cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. (See S. M. Barge et al., J. Pharm. Sci. (1977) 66, 1, which is incorporated herein by reference in its entirety, at least, for compositions taught therein.)
Administration of the compounds and compositions described herein or pharmaceutically acceptable salts thereof can be carried out using therapeutically effective amounts of the compounds and compositions described herein or pharmaceutically acceptable salts thereof as described herein for periods of time effective to treat a disorder. The effective amount of the compounds and compositions described herein or pharmaceutically acceptable salts thereof as described herein may be determined by one of ordinary skill in the art and includes exemplary dosage amounts for a mammal of from about 0.0001 to about 200 mg/kg of body weight of active compound per day, which may be administered in a single dose or in the form of individual divided doses, such as from 1 to 4 times per day. Alternatively, the dosage amount can be from about 0.01 to about 150 mg/kg of body weight of active compound per day, about 0.1 to 100 mg/kg of body weight of active compound per day, about 0.5 to about 75 mg/kg of body weight of active compound per day, about 0.5 to about 50 mg/kg of body weight of active compound per day, about 0.01 to about 50 mg/kg of body weight of active compound per day, about 0.05 to about 25 mg/kg of body weight of active compound per day, about 0.1 to about 25 mg/kg of body weight of active compound per day, about 0.5 to about 25 mg/kg of body weight of active compound per day, about 1 to about 20 mg/kg of body weight of active compound per day, about 1 to about 10 mg/kg of body weight of active compound per day, about 20 mg/kg of body weight of active compound per day, about 10 mg/kg of body weight of active compound per day, about 5 mg/kg of body weight of active compound per day, about 2.5 mg/kg of body weight of active compound per day, about 1.0 mg/kg of body weight of active compound per day, or about 0.5 mg/kg of body weight of active compound per day, or any range derivable therein. Optionally, the dosage amounts are from about 0.01 mg/kg to about 10 mg/kg of body weight of active compound per day. Optionally, the dosage amount is from about 0.01 mg/kg to about 5 mg/kg. Optionally, the dosage amount is from about 0.01 mg/kg to about 2.5 mg/kg.
Those of skill in the art will understand that the specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors, including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the species, age, body weight, general health, sex and diet of the subject, the mode and time of administration, rate of excretion, drug combination, and severity of the particular condition.
The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each subject's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems. Further, depending on the route of administration, one of skill in the art would know how to determine doses that result in a plasma concentration for a desired level of response in the cells, tissues and/or organs of a subject.
Provided herein are methods to treat, prevent, or ameliorate a BTK-related disease in a subject. The methods include administering to a subject an effective amount of one or more of the compounds or compositions described herein, or a pharmaceutically acceptable salt or prodrug thereof. Effective amount, when used to describe an amount of compound in a method, refers to the amount of a compound that achieves the desired pharmacological effect or other biological effect. The effective amount can be, for example, the concentrations of compounds at which BTK is degraded in vitro, as provided herein. Also contemplated is a method that includes administering to the subject an amount of one or more compounds described herein such that an in vivo concentration at a target cell in the subject corresponding to the concentration administered in vitro is achieved.
The compounds and compositions described herein or pharmaceutically acceptable salts thereof are useful for treating BTK-related diseases in humans, including, without limitation, pediatric and geriatric populations, and in animals, e.g., veterinary applications.
In some embodiments, the BTK-related disease is cancer. Optionally, the cancer is a poor prognosis cancer. The term poor prognosis, as used herein, refers to a prospect of recovery from a disease, infection, or medical condition that is associated with a diminished likelihood of a positive outcome. In relation to a disease such as cancer, a poor prognosis may be associated with a reduced patient survival rate, reduced patient survival time, higher likelihood of metastatic progression of said cancer cells, and/or higher likelihood of chemoresistance of said cancer cells. Optionally, a poor prognosis cancer can be a cancer associated with a patient survival rate of 50% or less. Optionally, a poor prognosis cancer can be a cancer associated with a patient survival time of five years or less after diagnosis. In some embodiments, the cancer is an invasive cancer.
Optionally, the cancer is a cancer that has an increased expression of BTK as compared to non-cancerous cells of the same cell type. Optionally, the cancer is bladder cancer, brain cancer, breast cancer (e.g., triple negative breast cancer), bronchus cancer, colorectal cancer (e.g., colon cancer, rectal cancer), cervical cancer, chondrosarcoma, endometrial cancer, gastrointestinal cancer, gastric cancer, genitourinary cancer, glioblastoma, head and neck cancer, hepatic cancer, hepatocellular carcinoma, leukemia, liver cancer, lung cancer, lymphoma, melanoma of the skin, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, skin cancer, testicular cancer, thyroid cancer, or uterine cancer. Optionally, the cancer is a cancer that affects one or more of the following sites: oral cavity and pharynx (e.g., tongue, mouth, pharynx, or other oral cavity); digestive system (e.g., esophagus, stomach, small intestine, colon, rectum, anus, anal canal, anorectum, liver and intrahepatic bile duct, gallbladder and other biliary, pancreas, or other digestive organs); respiratory system (e.g., larynx, lung and bronchus, or other respiratory organs); bones and joints; soft tissue (e.g., heart); skin (e.g., melanoma of the skin or other nonepithelial skin); breast; genital system (e.g., uterine cervix, uterine corpus, ovary, vulva, vagina and other female genital areas, prostate, testis, penis and other male genital areas); urinary system (e.g., urinary bladder, kidney and renal pelvis, and ureter and other urinary organs); eye and orbit; brain and other nervous system; endocrine system (e.g., thyroid and other endocrine); lymphoma (e.g., Hodgkin lymphoma and non-Hodgkin lymphoma); myeloma; or leukemia (e.g., acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, or other leukemia). Optionally, the cancer is a drug resistant cancer, such as an ibrutinib-resistant cancer.
In some embodiments, the BTK-related disease is a metabolic disorder (e.g., obesity, diabetes, and genetic disorders). Optionally, the BTK-related disease is a neurodegenerative disorder. Optionally, the neurodegenerative disorder is Parkinson's disease. Optionally, the neurodegenerative disorder is Alexander disease, Alper's disease, Alzheimer disease, amyotrophic lateral sclerosis, ataxia telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjogren-Batten disease), Canavan disease, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease, Huntington's disease, Kennedy's disease, Krabbe disease, Lewy body dementia, Machado-Joseph disease, Spinocerebellar ataxia type 3, multiple sclerosis, multiple system atrophy, Pelizaeus-Merzbacher disease, Pick's disease, Primary lateral sclerosis, Refsum's disease, Sandhoff disease, Schilder's disease, Spielmeyer-Vogt-Sjogren-Batten disease (also known as Batten disease), Spinocerebellar ataxia (multiple types with varying characteristics), Spinal muscular atrophy, Steele-Richardson-Olszewski disease, Tay-Sachs, Transmissible spongiform encephalopathies (TSE), or Tabes dorsalis.
Optionally, the BTK-related disease is an inflammatory disease. Generally, inflammatory disorders include, but are not limited to, respiratory or pulmonary disorders (including asthma, COPD, chronic bronchitis and cystic fibrosis); cardiovascular related disorders (including atherosclerosis, post-angioplasty, restenosis, coronary artery diseases and angina); inflammatory diseases of the joints (including rheumatoid and osteoarthritis); skin disorders (including dermatitis, eczematous dermatitis and psoriasis); post transplantation late and chronic solid organ rejection; multiple sclerosis; autoimmune conditions (including systemic lupus erythematosus, dermatomyositis, polymyositis, Sjogren's syndrome, polymyalgia rheumatica, temporal arteritis, Behcet's disease, Guillain Barre, Wegener's granulomatosus, polyarteritis nodosa); inflammatory neuropathies (including inflammatory polyneuropathies); vasculitis (including Churg-Strauss syndrome, Takayasu's arteritis); inflammatory disorders of adipose tissue; and proliferative disorders (including Kaposi's sarcoma and other proliferative disorders of smooth muscle cells).
Optionally, the BTK-related disease is ischemia, a gastrointestinal disorder, a viral infection (e.g., human immunodeficiency virus (HIV), including HIV type 1 (HIV-1) and HIV type 2 (HIV-2)), a bacterial infection, a central nervous system disorder, a spinal cord injury, or peripheral neuropathy.
The methods of treating or preventing a BTK-related disease (e.g., cancer) in a subject can further comprise administering to the subject one or more additional agents. The one or more additional agents and the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof can be administered in any order, including concomitant, simultaneous, or sequential administration. Sequential administration can be administration in a temporally spaced order of up to several days apart. The methods can also include more than a single administration of the one or more additional agents and/or the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof. The administration of the one or more additional agents and the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof can be by the same or different routes and concurrently or sequentially.
Additional therapeutic agents include, but are not limited to, chemotherapeutic agents, anti-depressants, anxiolytics, antibodies, antivirals, steroidal and non-steroidal anti-inflammatories, conventional immunotherapeutic agents, cytokines, chemokines, and/or growth factors. The additional therapeutic agents can be biomolecules.
A chemotherapeutic agent is a compound or composition effective in inhibiting or arresting the growth of an abnormally growing cell. Thus, such an agent may be used therapeutically to treat cancer as well as other diseases marked by abnormal cell growth. Illustrative examples of chemotherapeutic compounds include, but are not limited to, bexarotene, gefitinib, erlotinib, gemcitabine, paclitaxel, docetaxel, topotecan, irinotecan, temozolomide, carmustine, vinorelbine, capecitabine, leucovorin, oxaliplatin, bevacizumab, cetuximab, panitumumab, bortezomib, oblimersen, hexamethylmelamine, ifosfamide, CPT-11, deflunomide, cycloheximide, dicarbazine, asparaginase, mitotant, vinblastine sulfate, carboplatin, colchicine, etoposide, melphalan, 6-mercaptopurine, teniposide, vinblastine, antibiotic derivatives (e.g. anthracyclines such as doxorubicin, liposomal doxorubicin, and diethylstilbestrol doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil (FU), 5-FU, methotrexate, floxuridine, interferon alpha-2B, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cisplatin, vincristine and vincristine sulfate); hormones (e.g., medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and testolactone);
nitrogen mustard derivatives (e.g., mephalen, chlorambucil, mechlorethamine (nitrogen mustard) and thiotepa); and steroids (e.g., bethamethasone sodium phosphate).
Therapeutic agents further include, but are not limited to, levadopa, a dopamine agonist, an anticholinergic agent, a monoamine oxidase inhibitor, a COMT inhibitor, amantadine, rivastigmine, an NMDA antagonist, a cholinesterase inhibitor, riluzole, an anti-psychotic agent, an antidepressant, and tetrabenazine.
Any of the aforementioned therapeutic agents can be used in any combination with the compositions described herein. Combinations are administered either concomitantly (e.g., as an admixture), separately but simultaneously (e.g., via separate intravenous lines into the same subject), or sequentially (e.g., one of the compounds or agents is given first followed by the second). Thus, the term combination is used to refer to concomitant, simultaneous, or sequential administration of two or more agents.
Optionally, a compound or therapeutic agent as described herein may be administered in combination with a radiation therapy, an immunotherapy, a gene therapy, or a surgery.
The methods and compounds as described herein are useful for both prophylactic and therapeutic treatment. For prophylactic use, a therapeutically effective amount of the compounds and compositions or pharmaceutically acceptable salts thereof as described herein are administered to a subject prior to onset (e.g., before obvious signs of a BTK-related disease), during early onset (e.g., upon initial signs and symptoms of a BTK-related disease), or after the development of a BTK-related disease. Prophylactic administration can occur for several days to years prior to the manifestation of symptoms of a BTK-related disease. Therapeutic treatment involves administering to a subject a therapeutically effective amount of the compounds and compositions or pharmaceutically acceptable salts thereof as described herein after a BTK-related disease is diagnosed.
The compounds described herein are also useful in modulating BTK in a cell. Optionally, the compounds and compositions described herein are useful for inducing BTK degradation in a cell. The methods for inducing BTK degradation in a cell includes contacting a cell with an effective amount of one or more of the compounds as described herein. Optionally, the contacting is performed in vivo. Optionally, the contacting is performed in vitro.
The methods herein for prophylactic and therapeutic treatment optionally comprise selecting a subject with or at risk of developing a BTK-related disease. A skilled artisan can make such a determination using, for example, a variety of prognostic and diagnostic methods, including, for example, a personal or family history of the disease or condition, clinical tests (e.g., imaging, biopsy, genetic tests), and the like. Optionally, the methods herein can be used for preventing relapse of cancer in a subject in remission (e.g., a subject that previously had cancer).
Also provided herein are kits for treating or preventing a BTK-related disease (e.g., cancer, a neurodegenerative disorder, an inflammatory diseases, and/or a metabolic disorder) in a subject. A kit can include any of the compounds or compositions described herein. A kit can further include one or more additional agents, such as one or more chemotherapeutic agents. A kit can include an oral formulation of any of the compounds or compositions described herein. A kit can include an intravenous formulation of any of the compounds or compositions described herein. A kit can additionally include directions for use of the kit (e.g., instructions for treating a subject), a container, a means for administering the compounds or compositions (e.g., a syringe), and/or a carrier.
As used herein the terms treatment, treat, or treating refer to a method of reducing one or more symptoms of a disease or condition. Thus in the disclosed method, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of one or more symptoms of the disease or condition. For example, a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms or signs (e.g., size of the tumor or rate of tumor growth) of the disease in a subject as compared to a control. As used herein, control refers to the untreated condition (e.g., the tumor cells not treated with the compounds and compositions described herein). Thus the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.
As used herein, the terms prevent, preventing, and prevention of a disease or disorder refer to an action, for example, administration of a composition or therapeutic agent, that occurs before or at about the same time a subject begins to show one or more symptoms of the disease or disorder, which inhibits or delays onset or severity of one or more symptoms of the disease or disorder.
As used herein, references to decreasing, reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater as compared to a control level. Such terms can include, but do not necessarily include, complete elimination.
As used herein, subject means both mammals and non-mammals. Mammals include, for example, humans; non-human primates, e.g., apes and monkeys; cattle; horses; sheep; rats; mice; pigs; and goats. Non-mammals include, for example, fish and birds.
Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application.
The examples below are intended to further illustrate certain aspects of the methods and compositions described herein, and are not intended to limit the scope of the claims.
Materials: All chemicals were purchased from Sigma-Aldrich, Combi-blocks or Alfa Aesar, unless otherwise specified. All solvents and reagents were used as obtained without further purification.
Instrumentation: 1H NMR and 13C NMR spectra were on a Varian (Palo Alto, CA) 400-MR spectrometer. Chemical shifts (8) are reported in ppm, and coupling constants (J) are in Hertz (Hz). The following abbreviations were used to explain the multiplicities: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, br=broad. Flash chromatography was performed on a Teledyne ISCO CombiFlash Rf 200. ESI mass spectrometry was measured on an Agilent Mass Spectrometer.
To a 250 mL of Schlenk tube equipped with a magnetic stir bar were added compound 1 (3 g, 21.6 mmol), 2 (4 g, 21.6 mmol) and DIPEA (6.5 g, 50 mmol) in MeOH (300 mL). The mixture was stirred under air at room temperature overnight. Upon the completion of the reaction, the resulting solid was filtered and dried over under a reduced pressure to obtain the title compound 3 (3.7 g, 60%). 1H NMR (400 MHZ, DMSO-d6) δ 8.63 (d, J=2.3 Hz, 1H), 8.34 (t, J=2.3 Hz, 1H), 8.23 (dd, J=8.0, 2.0 Hz, 1H), 7.96-7.87 (m, 1H), 7.82 (t, J=8.2 Hz, 1H), 7.29 (d, J=2.2 Hz, 1H).
To a 25 mL of Schlenk tube equipped with a magnetic stir bar were added compound 3 (1.5 g, 5 mmol), 4 (1.4 g, 5 mmol), K2CO3 (1.4 g, 10 mmol), Pd2(dba)3 (456 mg, 10 mol %) and X-phos (457 mg, 20 mol %). Then dioxane (20 mL) was added under N2. The Schlenk tube was screw capped and heated to 100° C. for 12 hours. Then the reaction mixture was cooled to room temperature, and sat. NH4Cl aq. was poured into the reaction mixture and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, and filtered. The filtrate was concentrated in vacuo and the residue was purified by flash column chromatography to afford 5 (1.9 g, 73%) as a yellow solid. MS (EI): m/z 533.2 [M+H]+.
To a flask was added compound 5 (1.06 g, 2 mmol) and Pd/C (110 mg, 10%) in MeOH (50 mL). The mixture was stirred under 1 atm H2 at room temperature overnight. LC-MS showed compound 5 converted into compound 6 completely. Then the reaction mixture was filtered, and the filtrate was concentrated in vacuo. The mixture was extracted with EtOAc and washed with brine, dried over Na2SO4, and filtered. The filtrate was concentrated in vacuo and the residue was purified by flash column chromatography to afford 6 (1.9 g, 90%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.12 (s, 1H), 8.26 (d, J=2.2 Hz, 1H), 7.48 (d, J=8.7 Hz, 2H), 7.10 (t, J=8.0 Hz, 1H), 6.92 (d, J=2.2 Hz, 1H), 6.78 (d, J=9.1 Hz, 2H), 6.51 (dd, J=7.9, 2.1 Hz, 1H), 6.45 (t, J=2.2 Hz, 1H), 6.41 (dd, J=7.9, 2.3 Hz, 1H), 5.32 (s, 2H), 3.49-3.39 (m, 4H), 2.95 (t, J=5.1 Hz, 4H), 1.42 (s, 9H). MS (EI): m/z 503.2 [M+H].
To compound 6 (500 mg, 1 mmol) was added compound (E)-2-cyano-4,4-dimethylpent-2-enoyl chloride (257 mg, 1.5 mol) and DIPEA (387 mg, 3 mmol) in DCM (20 mL). The mixture was stirred at room temperature for 30 minutes. LC-MS showed compound 6 converted into compound 7 completely. Then the reaction mixture was concentrated in vacuo to give compound 7. MS (EI): m/z 638.2 [M+H]+.
In a 25 mL flask was added 7 (32 mg, 0.05 mmol) in TFA/DCM (5 mL, 1/1). The mixture was stirred for 30 minutes at room temperature. Then the solvent was removed in vacuo to give the deprotected intermediate, which was used for next step without further purification. To the above intermediate was added compound PS-6 (33 mg, 0.1 mol), HATU (38 mg, 0.1 mmol) and DIPEA (32 mg, 0.25 mmol) in DMF (2 mL). The mixture was stirred at room temperature for 30 minutes. Then the reaction mixture was concentrated in vacuo and the residue was purified by PrepHPLC with a reverse phase C18 column to afford the product as a yellow solid PS-RC-1 (13 mg, 30%). 1H NMR (400 MHZ, DMSO-d6) δ 11.09 (s, 1H), 10.44 (s, 1H), 9.18 (s, 1H), 8.30 (d, J=2.2 Hz, 1H), 7.71-7.55 (m, 3H), 7.53-7.45 (m, 2H), 7.42 (d, J=8.4 Hz, 2H), 7.16-7.03 (m, 4H), 6.95 (d, J=2.2 Hz, 1H), 6.76 (d, J=8.5 Hz, 2H), 5.08 (dd, J=12.9, 5.5 Hz, 1H), 4.24 (d, J=4.5 Hz, 2H), 3.71-3.54 (m, 4H), 3.12-2.97 (m, 4H), 2.89 (td, J=17.6, 15.6, 5.4 Hz, 1H), 2.67-2.52 (m, 2H), 2.04 (d, J=12.1 Hz, 1H), 1.27 (s, 9H). 13C NMR (100 MHZ, DMSO-d6) δ 173.3, 170.5, 169.3, 167.8, 167.1, 166.8, 161.2, 156.7, 156.6, 153.4, 153.1, 152.6, 145.9, 145.8, 139.9, 136.6, 134.1, 132.5, 130.4, 129.0, 120.0, 1187, 118.2, 116.9, 115.5, 114.4, 111.3, 110.0, 109.6, 107.4, 50.0, 49.7, 49.0, 44.2, 44.1, 41.9, 34.9, 31.4, 29.0, 22.6. HRMS (m/z): [M+H]+ calcd. for C45H43N10O8, 851.3265; found: 851.3267.
(E)-2-cyano-4,4-dimethyl-N-(3-((2-((4-(4-((2-(1-methyl-2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)glycyl)piperazin-1-yl)phenyl)amino)furo[3,2-d]pyrimidin-4-yl)oxy)phenyl)pent-2-enamide (PS-RC-1-Me)
The general procedure outlined above for synthesizing PS-RC-1 was modified and used to prepare PS-RC-1-Me.
1H NMR (400 MHZ, DMSO-d6) δ 10.43 (s, 1H), 9.18 (s, 1H), 8.30 (d, J=2.2 Hz, 1H), 7.67-7.53 (m, 3H), 7.52-7.45 (m, 2H), 7.42 (d, J=8.6 Hz, 2H), 7.17-7.01 (m, 4H), 6.95 (d, J=2.2 Hz, 1H), 6.77 (d, J=8.3 Hz, 2H), 5.14 (dd, J=13.0, 5.4 Hz, 1H), 4.25 (d, J=4.5 Hz, 2H), 3.65 (d, J=10.2 Hz, 4H), 3.07 (s, 2H), 3.03 (s, 3H), 3.00-2.90 (m, 2H), 2.82-2.72 (m, 1H), 2.62-2.50 (m, 2H), 2.06 (d, J=10.3 Hz, 1H), 1.27 (s, 9H). 13C NMR (100 MHZ, DMSO-d6) δ 172.3, 170.3, 169.2, 167.8, 167.0, 166.8, 164.9, 161.2, 156.7, 156.6, 153.4, 153.1, 152.8, 152.6, 145.9, 145.7, 139.9, 136.7, 134.1, 132.4, 130.4, 129.0, 120.0, 118.7, 118.2, 116.9, 115.5, 114.4, 111.3, 110.0, 107.4, 50.0, 49.7, 49.6, 44.1, 41.9, 34.9, 31.6, 29.3, 29.0, 27.01, 23.6. HRMS (m/z): [M+H]+ calcd. for C46H45N10O8, 865.3422; found: 865.3412.
(E)-2-cyano-N-(3-((2-((4-(4-(3-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)acetamido)ethoxy)propanoyl)piperazin-1-yl)phenyl)amino)furo[3,2-d]pyrimidin-4-yl)oxy)phenyl)-4,4-dimethylpent-2-enamide. (PS-RC-2)
The general procedure outlined above for synthesizing PS-RC-1 was modified and used to prepare PS-RC-2.
1H NMR (400 MHZ, DMSO-d6) δ 9.13 (d, J=3.9 Hz, 1H), 8.27 (d, J=2.2 Hz, 1H), 8.14 (t, J=5.6 Hz, 1H), 7.64-7.51 (m, 3H), 7.50-7.39 (m, 2H), 7.37 (d, J=8.3 Hz, 2H), 7.06 (dd, J=11.9, 7.1 Hz, 2H), 6.92 (t, J=4.1 Hz, 2H), 6.83 (d, J=8.5 Hz, 1H), 6.70 (d, J=8.5 Hz, 2H), 5.04 (dd, J=12.8, 5.4 Hz, 1H), 3.91 (d, J=5.6 Hz, 2H), 3.60 (t, J=6.4 Hz, 2H), 3.54 (s, 4H), 3.39 (t, J=5.8 Hz, 2H), 3.23 (d, J=5.7 Hz, 2H), 2.99-2.88 (m, 4H), 2.88-2.79 (m, 1H), 2.62-2.50 (m, 4H), 1.99 (d, J=11.9 Hz, 1H), 1.24 (s, 5H), 1.01-0.89 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 173.3, 170.5, 169.2, 169.1, 169.0, 167.8, 161.3, 156.7, 156.6, 153.4, 153.1, 152.8, 146.3, 145.9, 139.9, 136.7, 134.0, 132.5, 130.6, 129.0, 120.0, 117.9, 116.8, 111.4, 110.3, 107.4, 69.1, 66.9, 50.3, 49.8, 49.0, 45.6, 45.3, 43.2, 33.2, 31.4, 29.3, 29.1, 23.6, 22.6. MS (EI): m/z 966.3 [M+H]+.
(E)-2-cyano-N-(3-((2-((4-(4-(3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)acetamido)ethoxy)ethoxy)propanoyl)piperazin-1-yl)phenyl)amino)furo[3,2-d]pyrimidin-4-yl)oxy)phenyl)-4,4-dimethylpent-2-enamide (PS-RC-3)
The general procedure outlined above for synthesizing PS-RC-1 was modified and used to prepare PS-RC-3.
1H NMR (400 MHZ, DMSO-d6) δ 9.15 (d, J=3.8 Hz, 1H), 8.30 (t, J=1.8 Hz, 1H), 8.17 (t, J=5.7 Hz, 1H), 7.59 (td, J=14.6, 13.7, 8.3 Hz, 3H), 7.53-7.42 (m, 2H), 7.40 (d, J=8.4 Hz, 2H), 7.08 (dd, J=14.4, 7.5 Hz, 2H), 6.94 (d, J=1.9 Hz, 2H), 6.85 (d, J=8.4 Hz, 1H), 6.72 (d, J=8.5 Hz, 2H), 5.06 (dd, J=12.9, 5.4 Hz, 1H), 3.93 (d, J=5.5 Hz, 2H), 3.63 (t, J=6.7 Hz, 2H), 3.56 (d, J=5.2 Hz, 4H), 3.48 (d, J=3.5 Hz, 4H), 3.40 (d, J=5.8 Hz, 2H), 3.25 (d, J=6.0 Hz, 2H), 3.02-2.90 (m, 4H), 2.88-2.81 (m, 1H), 2.65-2.51 (m, 4H), 2.01 (d, J=11.8 Hz, 1H), 1.26 (d, J=1.3 Hz, 5H), 1.04-0.90 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 173.3, 170.5, 169.2, 169.1, 169.0, 167.8, 164.9, 156.7, 156.6, 153.4, 153.1, 152.8, 152.7, 152.6, 146.2, 145.9, 139.9, 136.6, 134.0, 132.5, 130.6, 130.2, 129.0, 120.0, 119.9, 117.9, 116.8, 111.4, 110.3, 107.4, 70.0, 69.4, 67.2, 50.3, 49.7, 49.0, 45.6, 45.4, 43.2, 39.1, 33.2, 31.4, 29.3, 29.1, 23.6, 22.6. MS (EI): m/z 1010.4 [M+H]+.
(E)-2-cyano-N-(3-((2-((4-(4-(1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-2-oxo-6,9,12-trioxa-3-azapentadecan-15-oyl)piperazin-1-yl)phenyl)amino)furo[3,2-d]pyrimidin-4-yl)oxy)phenyl)-4,4-dimethylpent-2-enamide (PS-RC-4)
The general procedure outlined above for synthesizing PS-RC-1 was modified and used to prepare PS-RC-4.
1H NMR (400 MHZ, DMSO-d6) δ 9.16 (s, 1H), 8.30 (d, J=2.2 Hz, 1H), 8.16 (t, J=5.7 Hz, 1H), 7.65-7.52 (m, 3H), 7.47 (d, J=7.3 Hz, 2H), 7.40 (d, J=8.7 Hz, 2H), 7.13-7.03 (m, 2H), 6.94 (t, J=3.5 Hz, 2H), 6.85 (d, J=8.6 Hz, 1H), 6.73 (d, J=8.4 Hz, 2H), 5.07 (dd, J=12.9, 5.4 Hz, 1H), 3.93 (d, J=5.6 Hz, 2H), 3.63 (t, J=6.6 Hz, 2H), 3.56 (d, J=5.5 Hz, 4H), 3.48 (d, J=6.1 Hz, 8H), 3.41 (t, J=5.7 Hz, 2H), 3.24 (t, J=5.9 Hz, 2H), 2.96 (d, J=23.0 Hz, 4H), 2.90-2.81 (m, 1H), 2.64-2.52 (m, 4H), 2.02 (d, J=11.5 Hz, 1H), 1.27 (s, 5H), 0.98 (d, J=27.6 Hz, 4H). 13C NMR (100 MHz, DMSO-d6) δ 173.3, 170.5, 169.2, 169.1, 169.0, 167.8, 153.4, 153.1, 152.8, 152.6, 146.3, 145.9, 136.6, 134.0, 132.5, 120.0, 119.9, 117.9, 116.9, 116.8, 111.4, 110.3, 107.4, 70.21, 70.16, 70.1, 70.0, 69.4, 67.2, 50.3, 49.7, 49.0, 45.6, 45.4, 43.2, 39.1, 33.2, 31.4, 29.3, 23.6, 22.6. MS (EI): m/z 1054.4 [M+H]+.
The general procedure outlined above for synthesizing PS-RC-1 was modified and used to prepare PS-IRC-1.
1H NMR (400 MHZ, DMSO-d6) δ 11.11 (s, 1H), 10.37 (s, 1H), 9.17 (s, 1H), 8.30 (d, J=2.2 Hz, 1H), 7.73-7.59 (m, 3H), 7.51-7.34 (m, 3H), 7.17-7.03 (m, 4H), 6.95 (d, J=2.2 Hz, 1H), 6.75 (d, J=8.7 Hz, 2H), 6.44 (dd, J=17.0, 10.1 Hz, 1H), 6.27 (dd, J=17.0, 2.1 Hz, 1H), 5.78 (dd, J=10.0, 2.1 Hz, 1H), 5.08 (dd, J=12.9, 5.4 Hz, 1H), 4.25 (d, J=4.5 Hz, 2H), 3.65 (dd, J=11.4, 5.9 Hz, 4H), 3.11-2.96 (m, 4H), 2.89 (td, J=17.0, 15.3, 5.4 Hz, 1H), 2.65-2.51 (m, 2H), 2.12-2.00 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 173.3, 170.5, 169.2, 167.8, 166.8, 163.8, 156.7, 156.6, 153.4, 153.1, 152.7, 145.9, 145.8, 140.8, 136.6, 134.1, 132.5, 132.1, 130.4, 129.0, 127.8, 120.0, 118.7, 117.2, 117.0, 116.9, 113.3, 111.3, 110.0, 107.4, 50.0, 49.7, 49.0, 44.1, 41.9, 31.4, 22.6. HRMS (m/z): [M+H]+ calcd. for C40H36N9O8, 770.2687; found: 770.2690.
The general procedure outlined above for synthesizing PS-RC-1 was modified and used to prepare PS-RNC-1.
1H NMR (400 MHZ, DMSO-d6) δ 11.11 (s, 1H), 10.08 (s, 1H), 9.15 (s, 1H), 8.30 (d, J=2.2 Hz, 1H), 7.69-7.57 (m, 2H), 7.55 (d, J=8.2 Hz, 1H), 7.41 (t, J=8.2 Hz, 3H), 7.19-7.06 (m, 3H), 7.04-6.99 (m, 1H), 6.95 (d, J=2.1 Hz, 1H), 6.77 (d, J=8.7 Hz, 2H), 5.08 (dd, J=12.9, 5.4 Hz, 1H), 4.25 (d, J=4.5 Hz, 2H), 3.65 (d, J=10.9 Hz, 4H), 3.05 (dd, J=25.9, 6.1 Hz, 4H), 2.89 (ddd, J=17.3, 13.9, 5.5 Hz, 1H), 2.66-2.52 (m, 2H), 2.33 (q, J=7.5 Hz, 2H), 2.12-1.96 (m, 1H), 1.07 (t, J=7.5 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 173.3, 172.7, 170.5, 169.2, 167.8, 166.8, 156.7, 156.5, 153.4, 153.0, 152.8, 145.9, 145.8, 141.2, 136.6, 134.2, 132.5, 130.3, 129.0, 120.0, 118.7, 116.9, 116.7, 116.6, 112.8, 111.3, 110.0, 107.4, 50.03, 49.7, 49.0, 44.2, 44.1, 41.9, 31.4, 30.0, 22.6, 10.0. HRMS (m/z): [M+H]+ calcd. for C40H38N9O8, 772.2843; found: 772.2831.
To a 25 mL of Schlenk tube equipped with a magnetic stir bar were added compound 9 (49 mg, 0.2 mmol), 10 (55 mg, 0.2 mmol), K2CO3 (55 mg, 0.4 mmol), Pd2(dba)3 (18 mg, 10 mol %) and X-phos (19 mg, 20 mol %). Then dioxane (5 mL) was added under N2. The Schlenk tube was screw capped and heated to 100° C. for 12 hours. Then the reaction mixture was cooled to room temperature, and sat. NH4Cl aq. was poured into the reaction mixture and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, and filtered. The filtrate was concentrated in vacuo and the residue was purified by flash column chromatography to afford 11 (63 mg, 65%) as a white solid. 1H NMR (400 MHZ, DMSO-d6) δ 9.38-9.28 (m, 1H), 8.29 (dd, J=5.5, 1.9 Hz, 1H), 7.54-7.45 (m, 2H), 7.40-7.27 (m, 3H), 7.27-7.19 (m, 2H), 6.72 (d, J=8.5 Hz, 2H), 6.33 (dd, J=5.6, 1.9 Hz, 1H), 3.43 (d, J=5.4 Hz, 4H), 2.94 (t, J=5.2 Hz, 4H), 1.41 (d, J=2.0 Hz, 9H). MS (EI): m/z 488.2 [M+H]+.
In a 25 mL flask was added 11 (27 mg, 0.05 mmol) in TFA/DCM (5 mL, 1/1). The mixture was stirred for 30 min at room temperature. Then the solvent was removed in vacuo to give the deprotected intermediate, which was used for next step without further purification. To the above intermediate was added compound PS-6 (33 mg, 0.1 mol), HATU (38 mg, 0.1 mmol) and DIPEA (32 mg, 0.25 mmol) in DMF (2 mL). The mixture was stirred at room temperature for 30 minutes. Then the reaction mixture was concentrated in vacuo and the residue was purified by PrepHPLC with a reverse phase C18 column to afford the product as a yellow solid PS-1 (14 mg, 40%). 1H NMR (400 MHZ, DMSO-d6) δ 11.12 (s, 1H), 9.12 (s, 1H), 8.28 (d, J=2.2 Hz, 1H), 7.61 (t, J=7.8 Hz, 1H), 7.55-7.47 (m, 2H), 7.42 (d, J=8.5 Hz, 2H), 7.36 (d, J=7.6 Hz, 3H), 7.15-7.05 (m, 3H), 6.94 (d, J=2.2 Hz, 1H), 6.76 (d, J=8.8 Hz, 2H), 5.08 (dd, J=12.9, 5.4 Hz, 1H), 4.24 (d, J=4.6 Hz, 2H), 3.65 (dt, J=12.2, 4.9 Hz, 4H), 3.04 (dt, J=25.5, 5.1 Hz, 4H), 2.89 (ddd, J=17.2, 14.0, 5.5 Hz, 1H), 2.65-2.52 (m, 2H), 2.10-1.98 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 173.3, 170.5, 169.3, 167.8f, 166.8, 156.7, 156.4, 153.5, 152.9, 152.5, 145.9, 145.8, 136.6, 134.2, 132.5, 130.2, 129.1, 126.1, 122.4, 120.0, 118.7, 117.0, 111.3, 110.0, 107.4, 50.0, 49.8, 49.0, 44.2, 44.1, 42.0, 31.4, 22.6. HRMS (m/z): [M+H]+ calcd. for C37H33N8O7, 701.2472; found: 701.2459.
To a 25 mL of Schlenk tube equipped with a magnetic stir bar were added compound 12 (41 mg, 0.2 mmol), 10 (55 mg, 0.2 mmol), K2CO3 (55 mg, 0.4 mmol), Pd2(dba)3 (18 mg, 10 mol %) and X-phos (19 mg, 20 mol %). Then dioxane (5 mL) was added under N2. The Schlenk tube was screw capped and heated to 100° C. for 12 hours. Then the reaction mixture was cooled to room temperature, and sat. NH4Cl aq. was poured into the reaction mixture and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, and filtered. The filtrate was concentrated in vacuo and the residue was purified by flash column chromatography to afford 13 (67 mg, 75%) as a white solid. 1H NMR (400 MHZ, DMSO-d6) δ 9.11 (s, 1H), 8.28 (t, J=1.5 Hz, 1H), 7.54-7.48 (m, 2H), 7.44-7.31 (m, 5H), 6.94 (t, J=1.5 Hz, 1H), 6.72 (d, J=8.7 Hz, 2H), 3.44 (t, J=5.1 Hz, 4H), 2.94 (t, J=5.1 Hz, 4H), 1.42 (s, 9H). MS (EI): m/z 448.2 [M+H]+.
In a 25 mL flask was added 13 (22 mg, 0.05 mmol) in TFA/DCM (5 mL, 1/1). The mixture was stirred for 30 min at room temperature. Then the solvent was removed in vacuo to give the deprotected intermediate, which was used for next step without further purification. To the above intermediate was added compound PS-6 (33 mg, 0.1 mol), HATU (38 mg, 0.1 mmol) and DIPEA (32 mg, 0.25 mmol) in DMF (2 mL). The mixture was stirred at room temperature for 30 min. Then the reaction mixture was concentrated in vacuo and the residue was purified by PrepHPLC with a reverse phase C18 column to afford the product as a yellow solid PS-2 (11 mg, 34%). 1H NMR (400 MHZ, DMSO-d6) δ 11.11 (s, 1H), 9.37 (s, 1H), 8.29 (dd, J=5.6, 1.6 Hz, 1H), 7.62 (dd, J=8.5, 7.0 Hz, 1H), 7.52-7.44 (m, 2H), 7.34 (dt, J=14.8, 7.9 Hz, 3H), 7.26-7.20 (m, 2H), 7.16-7.03 (m, 3H), 6.76 (d, J=8.5 Hz, 2H), 6.34 (dd, J=5.6, 1.6 Hz, 1H), 5.08 (dd, J=12.7, 5.2 Hz, 1H), 4.25 (d, J=4.2 Hz, 2H), 3.63 (d, J=9.7 Hz, 4H), 3.12-2.99 (m, 4H), 2.93-2.79 (m, 1H), 2.65-2.52 (m, 2H), 2.09-1.96 (m, 1H). 13C NMR (100 MHZ, DMSO-d6) δ 173.3, 170.5, 169.8, 169.2, 167.8, 166.8, 160.3, 160.2, 152.9, 145.9, 136.6, 133.4, 132.5, 130.2, 125.9, 122.4, 120.5, 118.7, 116.9, 111.3, 110.0, 49.9, 49.7, 49.0, 44.1, 41.9, 31.4, 22.6. HRMS (m/z): [M+H]+ calcd. for C35H33N8O6, 661.2523; found: 661.2514.
To a 25 mL of Schlenk tube equipped with a magnetic stir bar were added compound 14 (23 mg, 0.2 mmol), 10 (55 mg, 0.2 mmol), K2CO3 (55 mg, 0.4 mmol), Pd2(dba)3 (18 mg, 10 mol %) and X-phos (19 mg, 20 mol %). Then dioxane (5 mL) was added under N2. The Schlenk tube was screw capped and heated to 100° C. for 12 hours. Then the reaction mixture was cooled to room temperature, and sat. NH4Cl aq. was poured into the reaction mixture and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, and filtered. The filtrate was concentrated in vacuo and the residue was purified by flash column chromatography to afford 15 (50 mg, 70%) as a white solid. 1H NMR (400 MHZ, DMSO-d6) δ 9.35 (s, 1H), 8.40 (d, J=4.7 Hz, 2H), 7.57 (d, J=8.9 Hz, 2H), 6.90 (d, J=8.9 Hz, 2H), 6.74 (t, J=4.8 Hz, 1H), 3.45 (t, J=5.0 Hz, 4H), 3.00 (t, J=5.1 Hz, 4H), 1.42 (s, 9H). MS (EI): m/z 356.2 [M+H]+.
In a 25 mL flask was added 15 (18 mg, 0.05 mmol) in TFA/DCM (5 mL, 1/1). The mixture was stirred for 30 min at room temperature. Then the solvent was removed in vacuo to give the deprotected intermediate, which was used for next step without further purification. To the above intermediate was added compound PS-6 (33 mg, 0.1 mol), HATU (38 mg, 0.1 mmol) and DIPEA (32 mg, 0.25 mmol) in DMF (2 mL). The mixture was stirred at room temperature for 30 min. Then the reaction mixture was concentrated in vacuo and the residue was purified by PrepHPLC with a reverse phase C18 column to afford the product as a yellow solid PS-3 (13 mg, 45%). 1H NMR (400 MHZ, DMSO-d6) δ 11.11 (s, 1H), 9.37 (s, 1H), 8.41 (d, J=4.8 Hz, 2H), 7.65-7.52 (m, 3H), 7.15-7.03 (m, 3H), 6.94 (d, J=9.0 Hz, 2H), 6.75 (t, J=4.8 Hz, 1H), 5.07 (dd, J=12.9, 5.4 Hz, 1H), 4.26 (d, J=4.5 Hz, 2H), 3.67 (d, J=10.6 Hz, 4H), 3.10 (d, J=26.3 Hz, 4H), 2.95-2.80 (m, 1H), 2.67-2.55 (m, 2H), 2.09-1.97 (m, 1H). 13C NMR (100 MHZ, DMSO-d6) δ 173.3, 170.5, 169.3, 167.8, 166.9, 160.6, 158.4, 146.3, 145.9, 136.6, 133.7, 132.5, 120.7, 118.7, 117.1, 112.1, 111.3, 110.0, 49.9, 49.6, 49.0, 44.1, 41.9, 31.4, 22.6. HRMS (m/z): [M+H]+ calcd. for C29H29N8O5, 569.2261; found: 569.2249.
To a 100 mL flask was added compound 16 (18 mg, 0.1 mmol), PS-6 (50 mg, 0.15 mmol), HATU (57 mg, 0.15 mmol) and DIPEA (65 mg, 0.5 mmol) in DMF (2 mL). The mixture was stirred at room temperature for 30 min. Then the reaction mixture was concentrated in vacuo and the residue was purified by PrepHPLC with a reverse phase C18 column to afford the product as a yellow solid PS-4 (24 mg, 50%). 1H NMR (400 MHZ, DMSO-d6) δ 11.11 (s, 1H), 7.95 (s, 2H), 7.61 (t, J=7.8 Hz, 1H), 7.19-7.00 (m, 3H), 6.79-6.67 (m, 1.5H), 6.57-6.40 (m, 1.5H), 5.07 (dd, J=12.8, 5.4 Hz, 1H), 4.23 (d, J=4.6 Hz, 2H), 3.62 (dt, J=12.2, 4.9 Hz, 4H), 2.99-2.89 (m, 4H), 2.85 (d, J=5.7 Hz, 1H), 2.64-2.52 (m, 2H), 2.04 (m, 1H). 13C NMR (100 MHZ, DMSO-d6) δ 173.3, 170.5, 167.9, 166.8, 162.8, 145.9, 143.3, 142.4, 136.6, 132.5, 119.2, 118.7, 115.1, 111.3, 110.0, 51.3, 51.0, 49.0, 44.4, 44.1, 42.2, 31.2, 22.6. HRMS (m/z): [M+H]+ calcd. for C25H27N6O5, 491.2043; found: 491.2042.
To a 100 mL flask was added compound 17 (10 mg, 0.1 mmol), PS-6 (50 mg, 0.15 mmol), HATU (57 mg, 0.15 mmol) and DIPEA (65 mg, 0.5 mmol) in DMF (2 mL). The mixture was stirred at room temperature for 30 min. Then the reaction mixture was concentrated in vacuo and the residue was purified by PrepHPLC with a reverse phase C18 column to afford the product as a yellow solid PS-5 (27 mg, 65%). 1H NMR (400 MHZ, DMSO-d6) δ 11.11 (s, 1H), 7.60 (dd, J=8.5, 7.1 Hz, 1H), 7.12-7.04 (m, 3H), 5.07 (dd, J=12.9, 5.3 Hz, 1H), 4.18 (d, J=4.5 Hz, 2H), 3.49 (dt, J=10.2, 4.9 Hz, 4H), 2.89 (ddd, J=17.6, 13.9, 5.5 Hz, 1H), 2.64-2.51 (m, 2H), 2.33 (dt, J=25.4, 5.0 Hz, 4H), 2.21 (s, 3H), 2.07-1.99 (m, 1H). 13C NMR (100 MHZ, DMSO-d6) δ 173.3, 170.5, 169.2, 167.8, 166.8, 145.9, 136.6, 132.5, 118.7, 111.2, 110.0, 54.9, 54.6, 49.0, 46.0, 44.1, 44.0, 41.9, 31.4, 22.6 HRMS (m/z): [M+H]+ calcd. for C20H24N5O5, 414.1777; found: 414.1768.
To a flask was added compound 18 (6.5 mg, 0.01 mmol) and Pd/C (1 mg) in MeOH (2 mL). The mixture was stirred under 1 atm H2 at room temperature overnight. LC-MS showed compound 18 converted into compound 19 completely. Then the reaction mixture was filtered, and the filtrate was concentrated in vacuo to provide compound 19 (5.5 mg, 90%) without further purification.
In a 25 mL flask was added 19 (5 mg, 0.05 mmol) in TFA/DCM (5 mL, 1/1). The mixture was stirred for 30 min at room temperature. Then the solvent was removed in vacuo to give the deprotected intermediate, which was used for next step without further purification. To the above intermediate was added BODIPY-FL (2 mg, 0.005 mol), and DIPEA (3.2 mg, 0.025 mmol) in DMF (2 mL). The mixture was stirred at room temperature for 30 min. Then the reaction mixture was concentrated in vacuo and the residue was purified by PrepHPLC with a reverse phase C18 column to afford the product as a dark blue solid 20 (2 mg, 50%). MS (EI): m/z 816.4.
HEK-293T17 cells were engineered to stably express nLuc-fused to the C-terminal of BTK or IKZF1 or IKZF3 via lentivectors. Briefly, BTK/IKZF1/IKZF3 expression constructs (DNASU: Cat. No. HsCD00514411, HsCD00512327, and HsCD00513042) were Gateway cloned into pLenti6.2-ccdB-Nanoluc vector (Addgene: Cat. No. 87078), which were co-transfected into HEK-293T cells with lentiviral envelope protein construct pCMV-VSV-G (Addgene, Cat. No. 8454) and lentiviral packaging plasmid (Addgene, Cat. No. 8454) for viral production. Transfections were carried out using calcium phosphate-mediated transfection method (Promega, Cat. No. E1200). Virus was harvested at 48 hours and 72 hours post transfection and transfection was performed in the presence of 8 μg/mL of polybrene. HEK-293T17 cells were treated with the concentrated viral particles. Following lentiviral transduction, stable cell lines were selected by 7.5 μg/mL of blasticidin. Successful establishment of stable cell lines were confirmed with Nano-Glo Luciferase Assay (Promega, Cat. No. N1110).
HEK-293T17, Mino, Jeko-1, Ramos, and A20 were obtained from the American Type Culture Collection (ATCC, Cat. No. CRL-11268, CRL-3000, CRL3006, CRL1596, and TIB-208). MOLM14 and Rec-R cells were obtained from non-commercial sources.
All cell lines were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Cat. No. MT10040CV), except engineered HEK-293T17-BTK/IKZF1/IKZF3-nLuc cell lines, which were cultured in DMEM medium (Thermo Fisher Scientific, Cat. No. MT10013CV). All media contained 10% fetal bovine serum (GE Healthcare, Cat. No. SH30071.03) plus 1% Pen/Strep (Thermo Fisher Scientific, Cat. No. 15140163). All cells were grown in a humidified incubator at 37° C. with 5% CO2.
Mino cells were seeded into the wells of six-well plates at the density of 5×105 cells per mL in 2 mL of complete RPMI-1640 culture medium. After overnight adaptation, cells were treated with serially diluted compounds (from 1,000-1.6 nM, 5-fold dilution) for 24 hours. After treatment, whole cell lysates for immunoblotting were prepared by pelleting Mino cells at 4° C. and 200×g for 5 minutes. The resulting cell pellets were washed once with ice-cold PBS and lysed in 1×RIPA lysis buffer (Alfa Aesar, Cat. No. J62524) supplemented with protease and phosphatase inhibitor cocktail (Thermo Fisher, Cat. No. 78430). Lysates were centrifuged at 15,000×g for 10 min at 4° C. and protein concentrations were assessed using BCA assay (Pierce, Cat. No. 23225). Same amounts of protein (30 μg) for each sample were loaded onto sodium dodecyl sulfate-polyacrylamide gel, separated by electrophoresis (Bio-Rad) at 120 V for 1.5 h and transferred to PVDF membrane using a Transblot Turbo system (Bio-Rad). After blocking for 1 h at room temperature in 1% BSA-TBST, the membranes were immunoblotted with the specified primary antibodies at the dilution of 1:1000 in TBST (Cell Signaling Technology: anti-BTK Cat. No. 8547, anti-IKZF1 Cat. No. 14859, anti-IKZF3 Cat. No. 15103, anti-β-actin Cat. No. 4570, Proteintech: anti-GSPT1, Cat. No. 10763-1-AP) overnight at 4° C. and the HRP-conjugated secondary antibodies (Cell Signaling, Cat. No. 7074, 1:1000 in TBST) for 1 h at room temperature. Imaging was performed using the ECL Prime chemiluminescent Western blot detection reagents (Kindle Biosciences, Cat. No. R1100) by visualization of the blots with an Imager (Kindle Biosciences, Cat. No. D1001). All Western blots were subsequently processed and quantified with Imager software ImageJ and protein level was normalized to the B-actin loading controls.
For endpoint assays, engineered HEK-293T17 cells were plated in white opaque 96-well plate (Thermo Fisher) at a density of 4,000 cells per well in either 100 μL of Opti-MEM (4 h treatment) or 100 μL of DMEM whole medium (24 h treatment). The nanoluc activity was determined using the furimazine substrate (Promega, Cat. No. N1110). Briefly, after 4 h incubation of indicated compounds, cells were directly lysed by addition of 100 μL of nanoluciferase detection solution, containing lysis buffer and furimazine (1/50 dilution). For the 24-hour treatment group, the old medium was replaced with 100 μL fresh opti-MEM, followed by the addition of equal volume of lysis buffer plus furimazine (1/50 dilution). The endpoint luminescence was measured with a microplate reader (BioTek Synergy H1).
For protein degradation kinetics assays, HEK-293T17 cells stably expressing nLuc fusion proteins were plated in white opaque 96-well plates (Thermo Fisher) at a density of 4,000 cells per well in 100 μL of Opti-MEM. Cells were allowed to attach overnight and treated with 40 nM or 200 nM of indicated compounds plus endurazine (1:2000, Promega, Cat. No. N2570) and extracellular nanoluc inhibitor (1:2000, Promega, Cat. No. N2162), after which the real-time luminescence was measured with a microplate reader (BioTek Synergy H1) immediately every 5 min for 24 hours. The kinetics data were fitted in the “one phase decay” model in Prism 9 to calculate the half-life, defined as time to reach 50% of maximal protein degradation.
Mino cells were plated in 96-well plates (Corning, Cat. No. 3598) at a density of 6,000 cells per well in 100 μL of medium. Cells were treated the next day with indicated compounds in a five-fold dilution series (from 1000 to 0.64 nM), followed by 72 h incubation at 37° C. with 5% CO2. Cell viabilities were measured using the Alarma Blue assay by adding pre-warmed Resazuerin sodium (Sigma, Cat. No. 199303) solution (1 mg mL−1 in PBS) in an amount equal to 10% of the volume in the well. Four hours after incubation, fluorescence signals were measured with a BioTek Synergy H1 microplate reader at excitation/emission 544/590 nm from top with a gain of 60. The EC50 values were calculated using GraphPad Prism 9.3 (GraphPad Software, La Jolla, CA) with the nonlinear fitting model of “[Inhibitor] vs. response—Variable slope (four parameters)”. The IC50 values, defined as compound concentrations that reduce cell viabilities by 50%, were extrapolated based on the nonlinear fitting model and reported in figure panels.
Time-resolved fluorescence resonance energy transfer (TR-FRET) assay was carried out to evaluate the binding of indicated compounds and BTK by competition with a BODIPY-FL labeled BTK tracer (See
Two million of Mino cells were treated with DMSO or 200 nM of PS-2 for 4 hours in biological triplicates and cells were harvested by centrifugation (200×g, 4° C., 5 min). Lysis buffer (100 mM Triethylammonium bicarbonate (TEAB, Thermo Fisher Scientific, Cat. No. 90114), 2% SDS) was added to the cell pellets and homogenized with a microtip sonicator (Branson) to achieve a cell lysate with a protein concentration of ˜2 mg mL−1. Protein concentration was determined using BCA assay (Pierce, Cat. No. 23225) and normalized to 1 mg mL−1. One hundred μg of protein for each sample were reduced and alkylated with 10 mM Tris(2-carboxyethyl) phosphine hydrochloride (TCEP, Sigma, Cat. No. C4706) and 17 mM iodoacetamide (IAA, Sigma, Cat. No. 16125-5G), followed by digestion with trypsin (1:20, enzyme: protein, Thermo Fisher Scientific, Cat. No. 90058) for 16 h shaking at 37° C. Tandem mass tag (TMT) reagents (Thermo Fisher Scientific, Cat. No. A44520) were dissolved in anhydrous acetonitrile (ACN, Sigma, Cat. No. 271004), 0.08 mg of label reagent was used for 10 μg of protein digest. The 16-plex labeling reactions were performed for 1 hour at room temperature and the reaction was quenched by the addition of 5% hydroxylamine solution for 15 min at room temperature. The sample channels were combined at a 1:1:1:1:1:1 ratio, desalted using peptide desalting columns (Thermo Fisher Cat. No. 89852). After drying down in a speed vacuum overnight, the combined sample was resuspended in 1% formic acid and subjected to Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA).
A series of reversible covalent BTK PROTACs with different linkers were synthesized (
The potencies of PS-RC-1, PS-RC-2, PS-RC-3 and PS-RC-4 in inhibiting cell growth were assessed with poseltinib as a positive control (
To measure the binding affinities between PROTACs and BTK, a time-resolved fluorescence resonance energy transfer (TR-FRET) competition binding assay was developed. A BTK tracer was synthesized by conjugating an ibrutinib-based reversible noncovalent binder with the BODIPY-FL dye (
CC-885 and CC-220, two commercially available derivatives of lenalidomide, potently degrade the cell cycle regulator and translation termination factor GSPT1 as a neo-substrate for Cereblon. GSPT1 is known as a common off-target for phthalimide-based PROTACs. Additionally, depending on the linker position on thalidomide, the CRBN engaged PROTACs can also degrade IKZF1 and IKZF3. To test these three off-target degradations, Mino cells were treated with PS-RC-1 at different concentrations for 24 hours, followed by Western blotting. PS-RC-1 does not degrade GSPT1 (
Not to be bound by theory, it is possible that the reversible covalent analog of poseltinib (PS-RC-Ctrl) may bind other off-targets, contributing to the observed cell growth inhibition effect of PS-RC-1 in Mino cells. It was found that PS-RC-Ctrl has no appreciable effect on cell growth in Mino cells. To test whether the phenotype of PS-RC-1 in Mino cells is due to its bivalent structure-based protein degradation or a monovalent molecular glue, Mino cells were pretreated with a large excess of PS-RC-Ctrl (2 and 10 μM) for 2 h to prevent ternary complex formation with PS-RC-1, followed by PS-RC-1 treatment for 24 h. An appreciable IC50 shift was not observed for PS-RC-1 in Mino cells upon PS-RC-Ctrl pre-treatment, suggesting that the reversible covalent poseltinib moiety in PS-RC-1 does not significantly contribute to the observed toxicity in Mino cells (
As PS-RC-1 degrades IKZF1/3 instead of BTK, it was determined whether the molecular size could be reduced by removing the BTK binding moiety. A series of PS-RC-1 analogs, PS-1, PS-2, PS-3, PS-4, PS-5 and PS-6 (
IMiDs are commonly used to treat multiple myeloma (MM). PS-1 was further tested in MM.1S cells, an MM cell line. As shown in
Next, tandem mass tagging (TMT)-based quantitative proteomic profiling was performed to evaluate the degradation specificity of PS-2. PS-2 was chosen for the proteomics study because it is the most potent to induce degradation of IKZF1 and IKZF3 in Mino cells. To avoid the potential secondary effects induced by PS-2 treatment, Mino cells were treated with 200 nM of PS-2 for 4 h. Quantitative proteomics employing TMT chemical labeling coupled with LC/MS/MS enabled the detection of >2,500 unique proteins without fractionation (
Compared with PS-1, the BTK binding moiety of PS-2 lacks the furan ring fused to pyrimidine, which may be flexible enough to allow formation of a more stable ternary complex with BTK and CRBN in cells. In contrast, PS-3, whose warhead is further reduced compared with PS-2, loses its ability to function as either a BTK degrader or a potent IKZF1/3 molecular glue. To confirm this observation, BTK degradation induced by PS-1, PS-2, and PS-3 was tested in both a human B lymphocyte cell line Ramos and mouse BALB/c B cell lymphoma line A20, with DD-03-171 from the Gray group as a positive control (
The degradation kinetics of IKZF1 and IKZF3 were further examined using the HEK-293 cells stably expressing IKZF1-nLuc or IKZF3-nLuc. PS-1 and PS-2 induce the fastest degradation of IKZF1 and IKZF3 with degradation half-lives of ˜2.8 and ˜0.8 h, respectively (
To further illustrate the BTK degradation ability of PS-2, BTK-Hibit knock-in Ramos cells were treated with PS-2 for 24 hours, using Nurix and DD-03171 as controls (
A BTK ligand competition assay was also conducted to shed light on the mechanism of PS-2. Ramos cells were incubated overnight, and then the indicated compounds were added and incubated for 24 hours, followed by Western blotting for BTK degradation (
To quantify the binding ability of PS-2 to BTK, a TRFRET assay for BTK was performed (
PD data results are shown in
To verify the metabolic stability of PS-2, PS-2 was co-incubated with mouse liver S9 enzymes and LC-MS was used to test the remaining amount of PS-2 compound. DD-03-171 was used as a control (
The compounds and methods of the appended claims are not limited in scope by the specific compounds and methods described herein, which are intended as illustrations of a few aspects of the claims and any compounds and methods that are functionally equivalent are within the scope of this disclosure. Various modifications of the compounds and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compounds, methods, and aspects of these compounds and methods are specifically described, other compounds and methods are intended to fall within the scope of the appended claims. Thus, a combination of steps, elements, components, or constituents can be explicitly mentioned herein; however, all other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.
This application claims priority to U.S. Provisional Application No. 63/502,209, filed May 15, 2023, which is incorporated herein by reference in its entirety.
This invention was made with government support under Grant Nos. R01-GM115622 and R01-CA250503, awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63502209 | May 2023 | US |
