Polyfluorinated thalidomide analogs and uses thereof

Abstract

Polyfluorinated thalidomide analogs have a structure according to formula I, wherein R is aliphatic. The compounds may be used to inhibit cancer cell proliferation and / or to treat subjects with cancer or an inflammatory process. In some aspects, the cancer is multiple myeloma. In certain aspects, the compounds are used to inhibit proliferation of drug-resistant multiple myeloma cells and / or to treat subjects with drug-resistant multiple myeloma.

Description

POLYFLUORINATED THALIDOMIDE ANALOGS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of the earlier filing date of U.S. Provisional Application No. 63 / 518,823, filed August 10, 2023, which is incorporated by reference in its entirety herein.ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under project number Z01# ZIA BC 010547 awarded by the National Institutes of Health, National Cancer Institute. The Government has certain rights in the invention.FIELD

[0003] Polyfluorinated thalidomide analogs and uses thereof are disclosed.BACKGROUND

[0004] Thalidomide and its analogs have demonstrated preclinical and clinical activity in numerous malignancies involving immunomodulatory, angiogenic, and inflammatory pathways. The development of novel thalidomide analogs with antiangiogenic, anti-inflammatory, and anti-cancer properties is extremely relevant in the field of hematological malignancies, particularly multiple myeloma. Two analogs of thalidomide, lenalidomide and pomalidomide, are currently FDA- approved for use in multiple myeloma and share a common protein target, cereblon (CRBN), a substrate recruiter element of the E3 cullin 4-RING ubiquitin ligase complex. Patients eventually become resistant to these thalidomide analogs, and resistance acquisition is strongly associated with a downregulation of CRBN. A need exists for compounds to treat multiple myeloma, particularly lenalidomide- and pomalidomide -resistant multiple myeloma.SUMMARY

[0005] Polyfluorinated thalidomide analogs and uses thereof are disclosed. In some aspects, a pharmaceutical composition includes a compound according to formulawherein R is aliphatic, and a pharmaceutically acceptable carrier. The pharmaceutical composition is formulated for parenteral or oral administration. In some implementations, R is unsubstituted Ci- Cio straight alkyl, branched alkyl, or cycloalkyl. Exemplary compounds according to formula Iinclude

[0006] Aspects of a method for inhibiting proliferation of cancer cells include contacting the cancer cells with an effective amount of a compound as disclosed herein. Contacting the cancer cells may include administering the effective amount of the compound to a subject having cancer. In some implementations, the cancer cells are multiple myeloma (MM) cells. Aspects of a method for treating cancer or an inflammatory process include administering to a subject having cancer or an inflammatory process a therapeutically effective amount of a compound disclosed herein or a therapeutically effective amount of a pharmaceutical composition comprising the compound. In some aspects, the subject has MM.

[0007] In any of the foregoing or following aspects, the MM cells may be drug resistant and / or the subject may have drug-resistant MM. Resistance may be to one or more immunomodulatory drugs, such as one or more of thalidomide, lenalidomide, and pomalidomide. Some aspects of the disclosed compounds inhibit the proliferation of drug-resistant MM cells and / or drug-resistant MM with an IC50 of from 0.2 pM to 10 pM. In any of the foregoing implementations, the compound may not increase degradation of cereblon substrates IKZF1, IKZF3, or IKZF1 and IKZF3.

[0008] The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIGS. 1 A- IE are bar graphs showing effects of several thalidomide analogs on multiple myeloma cell proliferation in vitro. FIG. 1 A shows effects on RPMI-8226 cells after 72 hours of incubation with the analogs at 10 pM concentration compared to a control of 0.5% DMSO.FIG. IB shows effects on JJN3 cells after 24 hours of incubation with the analogs at 10 pM concentration compared to a control of 0.5% DMSO. FIG. 1C shows dose-dependent (0.1 pM - 20 pM) effects of four analogs on RPMI-8226 cells after 72 hours of incubation. FIG. ID shows dosedependent (0.1 pM - 20 pM) effects of four analogs on JJN3 cells after 24 hours of incubation.FIG. IE shows dose-dependent (0.1 pM - 10 pM) effects of four analogs on MOLP-8 cells after 72 hours of incubation. The data are representative of at least three independent experiments with at least three replicates per experiment. (Control = 0.5% DMSO, LEN = 10 pM lenalidomide, POM = 1 pM pomalidomide.)

[0010] FIGS. 2A-2D are bar graphs showing that treatment with several thalidomide analogs inhibits multiple myeloma cell proliferation in vitro. FIG. 2A - 72-hour treatment of MM1 / R10R cells with four lead analogs at a range of concentrations (0.1 pM - 10 pM) shows dose-dependent response, with Gul215 exhibiting the most potent inhibition (***p<0.0001). FIG. 2B - 72-hour treatment of MM1.S cells with four lead analogs at a range of concentrations (0.1 pM - 10 pM) shows dose-dependent response, with Gul210 exhibiting the most potent inhibition (***p<0.0001). FIG. 2C - 72-hour treatment of U266 / R10R cells with four lead analogs at a range of concentrations (0.05 pM - 10 pM) shows dose-dependent response, with Gul210 exhibiting the most potent inhibition (***p<0.0001). FIG. 2D - 72-hour treatment of U266 cells with four lead analogs at a range of concentrations (0.05 pM - 10 pM) shows dose-dependent response, with Gul215 exhibiting the most potent inhibition (***p<0.0001). These data are representative of at least three independent experiments with at least three replicates per experiment. (Control = 0.5% DMSO, LEN = 10 pM lenalidomide.)

[0011] FIGS. 3A-3D are bar graphs and representative images showing that treatment with several thalidomide analogs reduces spheroid growth in vitro. FIG. 3 A - 72-hour treatment of MOLP-8 spheroids at a range of concentrations (0.1 - 10 M) produces a dose-dependent response, with Gul213 exhibiting the most potent inhibition of spheroid growth. FIG. 3B - 72-hour treatment of RPMI-8226 spheroids with the lead compounds at a range of concentrations (0.1 - 10 pM) also produces a dose-dependent response, with Gul 215 exhibiting the most potent inhibition of spheroid growth. **** p < 0.0001, *** p < 0.001. FIGS. 3C and 3D are representative images of the spheroids from FIGS. 3A and 3B, respectively. These data represent at least three independent experiments performed in triplicates. (Control = 0.5% DMSO, LEN = 10 pM lenalidomide.)

[0012] FIG. 4 is a bar graph showing results of in vitro testing was conducted to assess thalidomide analogs’ effects on the inflammatory response in THP-1 cells, using a TNF-a ELISA. Vehicle control was 0.5% DMSO, while thalidomide (200 pM) was used as a comparator. Results shown are representative of at least three independent experiments with at least three replicates per experiment.

[0013] FIGS. 5A and 5B show results of in vitro screening in an endothelial tube formation angiogenesis assay. Vehicle control was 0.5% DMSO, CPS49 (30 pM) was used as a positive control, and Thalidomide (100 pM) was used as a comparator. FIG. 5 A is a graph representing the mean area of lattice formation relative to vehicle control. Treatment of HUVEC cells with four lead analogs (Gul210, Gul213, Gul214, and Gul215) at a range of concentrations (1 pM - 10 pM) showed a dose-dependent inhibition of tube formation (***p<0.0001, **p<0.05). FIG. 5B is representative images of the tube formation assay. Results shown are representative of at least threeindependent experiments with at least three replicates per experiment. (Control = 0.5% DMSO, CPS-39 = 30 M CPS-49, THAL = 100 pM thalidomide.)

[0014] FIGS. 6 A and 6B show results of ex vivo testing of thalidomide analogs in the human saphenous vein model of angiogenesis. Vehicle control was 0.5% DMSO, TNP-470 (50 pM) was used as a positive control, and thalidomide (100 pM) was included as a comparator. FIG. 6A - Graph represents the mean area of micro vessel outgrowth relative to vehicle control. 14-day incubation of human saphenous vein rings with Gul 213 and Gul215 at l0 pM and 25 pM demonstrates dose-dependent response, with Gul215 exhibiting the most potent angiogenesis inhibition (***p<0.0001). FIG. 6B - Representative images of human saphenous vein rings treated with vehicle control, 50 pM TNP-470, 100 pM Thalidomide, or the indicated thalidomide analog at 10 pM for 14 days. Results shown are representative of at least two independent experiments with at least two rings per experiment. (Control = 0.5% DMSO, TNP-470 = 50 pM TNP-470, THAL = 100 pM thalidomide.)

[0015] FIGS. 7 A and 7B show that expression of Ikaros and Aiolos proteins remain unaffected after 24-hour treatment of both MM1.S (FIG. 7A) and MOLP-8 cells (FIG. 7B) with Gul215 at 0.1 pM, 0.5 pM, and 1 pM. Results shown are representative of at least three independent experiments.DETAILED DESCRIPTION

[0016] Polyfluorinated thalidomide analogs are disclosed. The compounds may be used to treat cancers and inflammatory processes. In some aspects, the cancer is multiple myeloma (MM). In certain aspects, the compounds are used to treat drug-resistant MM.

[0017] MM is a hematological malignancy that makes up nearly 2% of all cancers and accounts for more than 12,500 deaths annually (Siegel et al., CA Cancer J. Clin. 2023, 73(1) : 17-48). Characterized by the accumulation of monoclonal plasma cells in the bone marrow which overproduce a monoclonal immunoglobulin known as M-protein, MM results in complications such as hypercalcemia, renal insufficiency, anemia, bone deterioration, and immunosuppression (van de Donk et al., Lancet 2021, 397(10272):410-27; Davis et al. , Cancers (Basel) 2021 , 13(7). Current standard therapies include a variety of treatment classes, with proteasome inhibitors, CD38-targeting antibodies, and immunomodulatory (IMiD) drugs representing current standard of care (van de Donk et al., Lancet 2021, 397(10272):410-27; Pinto et al., Cancers (Basel) 2020, 12(2)). The IMiDs lenalidomide, a second-generation thalidomide analog, and pomalidomide, a third-generation analog of thalidomide, are among these approved agents. However, most patients with MM relapse multiple times, with increasingly shorter remissions, and eventually develop disease refractory to all available therapies van de Donk et al., Lancet 2021, 397(10272):410-27;Kumar et al., Leukemia 2012, 26(1): 149-57). In order to improve clinical outcomes for these patients with relapsed-refractory multiple myeloma (RRMM), novel treatment modalities are required. Aspects of the disclosed polyfluorinated thalidomide compounds may fill this need.I. Definitions and Abbreviations

[0018] The following explanations of terms and abbreviations are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein, “comprising” means “including” and the singular forms “a” or “an” or “the” include plural references unless the context clearly dictates otherwise. The term “or” refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise.

[0019] Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features of the disclosure are apparent from the following detailed description and the claims.

[0020] The disclosure of numerical ranges should be understood as referring to each discrete point within the range, inclusive of endpoints, unless otherwise noted. Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, percentages, temperatures, times, and so forth, as used in the specification or claims are to be understood as being modified by the term “about.” Accordingly, unless otherwise implicitly or explicitly indicated, or unless the context is properly understood by a person of ordinary skill in the art to have a more definitive construction, the numerical parameters set forth are approximations that may depend on the desired properties sought and / or limits of detection under standard test conditions / methods as known to those of ordinary skill in the art. When directly and explicitly distinguishing embodiments from discussed prior art, the embodiment numbers are not approximates unless the word “about” is recited.

[0021] Although there are alternatives for various components, parameters, operating conditions, etc. set forth herein, that does not mean that those alternatives are necessarily equivalent and / or perform equally well. Nor does it mean that the alternatives are listed in a preferred order unless stated otherwise.

[0022] Definitions of common terms in chemistry may be found in Richard J. Lewis, Sr. (ed.), Hawley ’s Condensed Chemical Dictionary, published by John Wiley & Sons, Inc., 2016 (ISBN978-1-118-13515-0). The presently disclosed compounds also include all isotopes of atoms present in the compounds, which can include, but are not limited to, deuterium, tritium,18F,14C, etc. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341); and other similar references.

[0023] In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:

[0024] Aliphatic: A substantially hydrocarbon-based compound, or a radical thereof (e.g., Hn, for a hexane radical), including alkanes, alkenes, alkynes, including cyclic versions thereof (also referred to as cycloaliphatic), and further including straight- and branched-chain arrangements, and all stereo and position isomers as well. Unless expressly stated otherwise, an aliphatic group contains from one to twenty-five carbon atoms; for example, from one to fifteen, from one to ten, from one to six, or from one to four carbon atoms. Unless expressly referred to as an “unsubstituted aliphatic,” an aliphatic group can either be unsubstituted or substituted. An aliphatic group can be substituted with one or more substituents (up to two substituents for each methylene carbon in an aliphatic chain, or up to one substituent for each carbon of a -C=C- double bond in an aliphatic chain, or up to one substituent for a carbon of a terminal methine group). Exemplary substituents include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, acyl, aldehyde, amide, amino, aminoalkyl, aryl, arylalkyl, carboxyl, cyano, cycloalkyl, dialkylamino, halo, haloaliphatic, heteroaliphatic, heteroaryl, heterocycloaliphatic, hydroxyl, oxo, sulfonamide, sulfhydryl, thioalkoxy, or other functionality. A substituted aliphatic group must include at least one sp3-hybridized carbon or two sp2-hybridized carbons bonded with a double bond or at least two sp-hybridized carbons bonded with a triple bond.

[0025] Alkyl: A hydrocarbon group having a saturated carbon chain. The chain may be cyclic, branched or unbranched and includes at least one sp3-hybridized carbon atom. Cyclic groups may be referred to as cycloalkyl. Examples, without limitation, of alkyl groups include methyl, ethyl, propyl, and isopropyl (2-propyl). Unless otherwise specified, an alkyl group may be substituted or unsubstituted.

[0026] CRBN: cereblon

[0027] DMSO: dimethyl sulfoxide

[0028] Effective amount or therapeutically effective amount: An amount sufficient to provide a beneficial, or therapeutic, effect to a subject or a given percentage of subjects.

[0029] HSV : human saphenous vein

[0030] HUVECs: human umbilical vein endothelial cells

[0031] IMiDs: immunomodulatory agents

[0032] LPS: lipopolysaccharide

[0033] MM: multiple myeloma

[0034] Pharmaceutically acceptable carrier: The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21stEdition (2005), describes compositions and formulations suitable for pharmaceutical delivery of one or more thalidomide analogs as disclosed herein.

[0035] In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. In some examples, the pharmaceutically acceptable carrier may be sterile to be suitable for administration to a subject (for example, by parenteral, intramuscular, or subcutaneous injection). In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0036] Pharmaceutical composition: A composition that includes an amount (for example, a unit dosage) of one or more of the disclosed compounds together with one or more non-toxic pharmaceutically acceptable additives, including carriers, diluents, and / or adjuvants, and optionally other biologically active ingredients. Such pharmaceutical compositions can be prepared by standard pharmaceutical formulation techniques such as those disclosed in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA (19th Edition).

[0037] Subject: An animal (human or non-human) subjected to a treatment, observation or experiment. Includes both human and veterinary subjects, including human and non-human mammals, such as rats, mice, cats, dogs, pigs, horses, cows, and non-human primates.

[0038] TNF-a: tumor necrosis factor-alpha

[0039] Treat(ing) or treatment: As used herein, these terms refer to ameliorating a disease or condition of interest in a patient or subject, particularly a human having the disease or condition of interest, and includes by way of example, and without limitation:

[0040] (i) inhibiting the disease or condition, for example, arresting or slowing its development;

[0041] (ii) relieving the disease or condition, for example, causing regression of the disease or condition or a symptom thereof; or

[0042] (iii) stabilizing the disease or condition.

[0043] As used herein, the terms “disease” and “condition” can be used interchangeably or can be different in that the particular malady or condition may not have a known causative agent (so that etiology has not yet been determined) and it is therefore not yet recognized as a disease but only as an undesirable condition or syndrome, where a more or less specific set of symptoms have been identified by clinicians.II. Compounds and Pharmaceutical Compositions

[0044] Polyfluorinated thalidomide analogs are disclosed. In some aspects, the compounds have a formula according to formulawherein R is aliphatic. In some implementations, R is unsubstituted aliphatic, such as unsubstituted alkyl. In certain aspects, R is unsubstituted C1-C10 straight alkyl, branched alkyl, or cycloalkyl. In some implementations, R is C2-C4 straight or branched alkyl or cyclohexyl. Exemplary compounds according to formula I

[0045] A pharmaceutical composition includes a compound according to formula I and a pharmaceutically acceptable carrier. The disclosed compounds can be further combined with excipients, and optionally sustained-release matrices, such as biodegradable polymers. The composition may comprise a unit dosage form of the composition, and may further comprise instructions for administering the composition to a subject.

[0046] The disclosed pharmaceutical compositions can be in the form of tablets, capsules, powders, granules, lozenges, liquid or gel preparations, such as oral or sterile parenteral solutions or suspensions, and other forms known in the art. Pharmaceutical compositions can be administeredsystemically in any manner appropriate to the treatment of a given condition, including orally, parenterally, rectally, nasally, buccally, topically, by inhalation spray, or via an implanted reservoir. The term "parenterally" as used herein includes, but is not limited to subcutaneous, intravenous, intramuscular, intrastemal, intrasynovial, intrathecal, intrahepatic, intralesional, and intracranial administration, for example, by injection or infusion. In some aspects, the pharmaceutical composition is formulated for parenteral or oral administration.

[0047] Pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffers (such as phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, poly acrylates, waxes, polyethylene-polyoxypropylene -block polymers, polyethylene glycol, and wool fat.

[0048] Tablets and capsules for oral administration can be in a form suitable for unit dose presentation and can contain conventional pharmaceutically acceptable excipients. Examples of these include binding agents such as syrup, acacia, gelatin, sorbitol, tragacanth, and polyvinylpyrrolidone; fillers such as lactose, sugar, corn starch, calcium phosphate, sorbitol, or glycine; tableting lubricants, such as magnesium stearate, talc, polyethylene glycol, or silica; disintegrants, such as potato starch; and dispersing or wetting agents, such as sodium lauryl sulfate. Oral liquid preparations can be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or can be presented as a dry product for reconstitution with water or other suitable vehicle before use.

[0049] The pharmaceutical compositions can also be administered parenterally in a sterile aqueous or oleaginous medium. The composition can be dissolved or suspended in a non-toxic parenterally-acceptable diluent or solvent, e.g., as a solution in 1,3 -butanediol. Commonly used vehicles and solvents include water, physiological saline, Hank's solution, Ringer's solution, and sterile, fixed oils, including synthetic mono- or di-glycerides, etc. For topical application, the drug may be made up into a solution, suspension, cream, lotion, or ointment in a suitable aqueous or non-aqueous vehicle. Additives may also be included, for example, buffers such as sodium metabisulfite or disodium edetate; preservatives such as bactericidal and fungicidal agents, including phenyl mercuric acetate or nitrate, benzalkonium chloride or chlorhexidine, and thickening agents, such as hypromellose.III. Methods of Use

[0050] The disclosed compounds may be used for treating cancers or inflammatory process. In some aspects, the disclosed compounds are used for inhibiting proliferation of cancer cells. In some aspects, a method of inhibiting proliferation of cancer cells includes contacting the cells with an effective amount of a compound as disclosed herein. The cells may be contacted in vitro, in vivo, or ex vivo. In certain aspects, contacting the cell with the effective amount of the compound comprises administering the effective amount of the compound to a subject having cancer. In some implementations, the cancer is multiple myeloma. In certain implementations, the MM cells are drug resistant.

[0051] A method for treating a cancer or an inflammatory process includes administering to a subject having cancer or an inflammatory process a therapeutically effective amount of a compound as disclosed herein, or a therapeutically effective amount of a pharmaceutical composition comprising the compound. In some aspects, the cancer is MM and treating the MM comprises inhibiting proliferation of MM cells. In some implementations, the subject has drugresistant MM as discussed below.100521 Some MM cells are drug resistant, resulting in drug-resistant MM. In particular, the cells may be resistant to one or immunomodulatory drugs, such as thalidomide, lenalidomide, and / or pomalidomide. The drug resistance may be intrinsic or may be acquired following exposure to, or treatment with, one or more immunomodulatory drugs. Some of the disclosed compounds inhibit drug-resistant MM cell proliferation with an IC50 of from 0.2 pM to 10 pM. In some aspects, the compound has an IC50 from 0.5 pM to 5 pM, 0.5 pM to 3pM, or 0.5 pM to 2 pM.

[0053] In any of the foregoing or following aspects, the compound according to formula I may beany combination thereof. In certain aspects, the compound i

[0054] In any of the foregoing aspects, contacting the MM cells with the effective amount of the compound, or administering the therapeutically effective amount of the compound to the subject, may further inhibit angiogenesis, TNF-a expression, or angiogenesis and TNF-a expression.Additionally, the compounds may reduce MM cell viability.

[0055] The primary target of thalidomide and its analogs is cereblon (CRBN), the substrate recognition element of the E3 cullin 4-RING ubiquitin ligase complex (Ito et al., Science 2010, 327(5971):1345-50).) The interaction of IMiDs with CRBN triggers ubiquitination and subsequent proteasomal degradation of neo-substrates Ikaros (IKZF1) and Aiolos (IKZF3) (Kronke et al., Science 2014, 343(6168) :301 -5 ; Lu et al., Science 2014, 343(6168):305-9), leading to downregulation of transcription factors interferon regulatory 4 (IRF4) and c-MYC. Since these transcription factors play a role in promoting the survival and proliferation of the monoclonal MM plasma cells, this IMiD-CRBN interaction explains, in part, the anti-myeloma effects of these compounds (Davis et al., Cancers (Basel) 2021, 13(7)). Down-regulation of CRBN has been postulated to be one of the mechanisms of acquired resistance to IMiDs (Zhu et al., Blood 2011, 118(18):4771-9). Genetic alterations in CRBN may also contribute to acquired IMiD resistance, as genomic aberrations have been found to increase incrementally as MM patients progress from newly-diagnosed to lenalidomide-resistant and finally to pomalidomide-resistant (Gooding et al. , Blood 2021, 137(2):232-7). Advantageously, some of the disclosed compounds fail to promote degradation of CRBN substrates, such as Ikaros (IKZF1) and Aiolos (IKZF3), indicating the compounds act through a CRBN -independent mechanism. Thus, these compounds may be useful for treating drug-resistant MM, or other cancers and inflammatory processes.

[0056] Illustrative inflammatory process that may be treated with the compounds disclosed herein include Sarcopenia, traumatic brain injury, spinal cord injury, Alzheimer’s disease, Crohn’s disease, rheumatoid arthritis, immune arthritis, degenerative arthritis, celiac disease, glomerulonephritis, lupus nephritis, prostatitis, inflammatory bowel disease, pelvic inflammatory disease, graft-versus-host disease, interstitial cystitis, autoimmune thyroiditis, Graves’ disease; autoimmune pancreatitis, Sjogren’s syndrome, myocarditis, autoimmune hepatitis, primary biliary cirrhosis, autoimmune angioedema, bullous pemphigoid, discoid lupus erythematosus, erythema nodosum leprosum, sarcoidosis, pemphigus vulgaris psoriasis, POEMS syndrome, polymyositis, human immune deficiency virus / acquired immune deficiency syndrome, vasculitis, stroke, Parkinson’s disease, amyotrophic lateral sclerosis, human immunodeficiency virus dementia, Huntington’s disease, multiple sclerosis, cerebral amyloid angiopathy, a tauopathy, peripheral neuropathy, macular degeneration, hearing loss, cochlear injury, epilepsy, a non-epileptic seizure disorder, and major depressive disorder.IV. Representative Aspects

[0057] Certain representative aspects are exemplified in the following numbered clauses.

[0058] 1. A pharmaceutical composition comprising: a compound according to formula I,wherein R is aliphatic; and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is formulated for parenteral or oral administration.

[0059] 2. The pharmaceutical composition of clause 1, wherein R is unsubstituted Ci-Cio straight alkyl, branched alkyl, or cycloalkyl.

[0060] 3. The pharmaceutical composition of clause 1, wherein the compound isany combination thereof.

[0061] 4. A method of inhibiting proliferation of cancer cells, comprising contacting the cancer cells with an effective amount of a compound according to formulawherein R is aliphatic.

[0062] 5. The method of clause 4, wherein R is unsubstituted C2-C10 straight alkyl, branched alkyl, or cycloalkyl.

[0063] 6. The method of clause 4, wherein the compoundr any combination thereof.

[0064] 7. The method of any one of clauses 4-6, wherein contacting the cancer cells with the effective amount of the compound comprises administering the effective amount of the compound to a subject having cancer.

[0065] 8. The method of any one of clauses 4-7, wherein the cancer is multiple myeloma and the method comprises contacting the multiple myeloma cells with the effective amount of the compound.

[0066] 9. The method of clause 8, wherein the multiple myeloma cells are drug resistant.

[0067] 10. The method of clause 9, wherein the multiple myeloma cells are resistant to one or more immunomodulatory drugs.

[0068] 11. The method of clause 9 or clause 10, wherein the multiple myeloma cells are resistant to one or more of thalidomide, lenalidomide, and pomalidomide.

[0069] 12. The method of any one of clauses 8-11, wherein the compound inhibits multiple myeloma cell proliferation with an IC50 of from 0.2 pM to 10 pM.

[0070] 13. The method of any one of clauses 4-12, wherein contacting the multiple myeloma cells with the effective amount of the compound does not increase degradation of cereblon substrate IKZF1, IKZF3, or IKZF1 and IKZF3.

[0071] 14. The method of any one of clauses 4-13, wherein the compound is

[0072] 15. A method of treating a cancer or inflammatory process, comprising administering to a subject having cancer or an inflammatory process a therapeutically effective amount of a compound according to formulawherein R is aliphatic, or a therapeutically effective amount of a pharmaceutical composition comprising the compound.

[0073] 16. The method of clause 15, wherein R is unsubstituted C1-C10 straight alkyl, branched alkyl, or cycloalkyl.

[0075] 18. The method of any one of clauses 15-17, wherein the cancer is multiple myeloma.

[0076] 19. The method of clause 18, wherein treating the multiple myeloma comprises inhibiting proliferation of multiple myeloma cells.

[0077] 20. The method of clause 18 or clause 19, wherein the subject has drug-resistant multiple myeloma.

[0078] 21. The method of clause 20, wherein the drug-resistant multiple myeloma is resistant to one or more immunomodulatory drugs.

[0079] 22. The method of clause 20 or clause 21 , wherein the drug-resistant multiple myeloma is resistant to one or more of thalidomide, lenalidomide, and pomalidomide.

[0080] 23. The method of clause 22, wherein the compound inhibits the proliferation of drugresistant multiple myeloma cells with an ICso of from 0.2 pM to 10 pM.

[0081] 24. The method of any one of clauses 15-23, wherein the compound does not increase degradation of cereblon substrate IKZF1, IKZF3, or IKZF1 and IKZF3.

[0082] 25. The method of any one of clauses 14-23, wherein the compound isV. Examples

[0083] Materials and Methods

[0084] Thalidomide analogs: Analogs were synthesized to a chemical purity of > 99.5% (as assessed by LC / MS,]H NMR, and13C NMR) at the University of Bonn, Germany (Steinebach el al., ChemMedChem. October 8, 2018, 13(19):2080-2089). The analogs are shown in Table 1.

[0085] Cell lines and reagents: Dimethyl sulfoxide (DMSO) was purchased from Sigma Aldrich (St. Louis, MO). Human umbilical vein endothelial cells (HUVEC; RRID:CVCL_2959) were purchased from Lonza (Walkersville, MD), cultured in EGM-plus media (Lonza), seeded to collagen-I coated flasks (Corning Life Sciences, Tewksbury, MA), and used before passage 12. Human leukemic monocytic cell line THP-1 (RRID:CVCL_0006) and human myeloma cell lines RPMI-8226 (RRID:CVCL_0014) and MM1.S (RRID:CVCL_8792) were purchased from American Type Culture Collection (ATCC, Manassas, VA) and grown in RPML1640 (Life Technologies, Carlsbad, CA) with 10% FBS and 1% P / S. The human myeloma cell line JJN3 (RRID:CVCL_2078) was purchased from DSMZ (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and grown in 40% DMEM and 40% IMDM (Life Technologies, Carlsbad, CA) with 20% FBS. The pomalidomide -resistant human myeloma cell line MOLP-8 (RRID:CVCL_2124) was also purchased from DSMZ and grown in RPMI-1640 with 15% FBS and 1% P / S. The lenalidomide-resistant MM1.S (MM1 / R10R) and U266 (U266 / R10R) cell lines, and their respective wild-type counterparts (MM1.S, RR1D:CVCL_8792; U266, RR1D:CVCL_O566), were kindly provided by Dr. Robert Orlowski (The University of Texas MD Anderson Cancer Center, Houston, TX) (Bjorkland et al., J Biol Chem. 2011, 286(13): 11009-20). The MM1.S and MM1 / R10R cell lines were grown in RPMI- 1640 with 10% FBS and 1% P / S, and the U266 and U266 / R10R cell lines were grown in RPMI- 1640 with 15% FBS and 1% P / S. All cell lines were cultured in 5% CO2 and 95% air at 37°C and underwent cell authentication and mycoplasma testing through ATCC.

[0086] Cell proliferation assay: Compounds were tested for their ability to inhibit the proliferation of various cell lines in vitro using a CCK-8 assay. RPML8226 (10,000 cells / well), JJN3 (20,000 cells / well), MOLP-8 (20,000 cells / well), MM1.S (R10R & WT) (20,000 cells / well), and U266 (R10R & WT) (20,000 cells / well) cells were plated in 96- well plates and allowed to incubate overnight at 37°C in 5% CO2. An equal amount of treatment media containing vehicle (0.5% DMSO) or test compounds at a range of concentrations was then plated on top of the seeded cells for 24, 48, or 72 hours of incubation. Cell proliferation was evaluated by adding 10 pL of CCK-8 reagent (Dojindo Laboratories, Rockville, MD), incubating for 2-4 hours, and measuring the absorbance at 450 nm on a microplate reader (SpectraMax® iD3, Molecular Devices, San Jose, CA).

[0087] Spheroid assay: Three-dimensional (3D) spheroids were cultured as previously described (Selby etal., SIAS Discov. 2017, 22(5):473-83). Briefly, MOLP-8 and RPMI-8226 cells were seeded at 1250 and 800 cells / well, respectively, in 50 pL volumes in 96-well ultra-low attachmentplates and allowed to incubate for 30 minutes at 37°C in 5% CO2, 95% air. The seeded cells were then treated with equal volumes of media containing vehicle or test compounds at a range of concentrations. Following 72 hours of incubation, cell viability within the spheroids was evaluated using the CellTiter-Glo® 3D Cell Viability Assay (Promega, Madison, WI). 100 pL of roomtemperature CellTiter-Glo® 3D reagent was added to each well, and plates were placed on a shaker for 5 minutes and then allowed to sit at room temperature for 25 additional minutes. Subsequently, luminescence was measured on a microplate reader (SpectraMax® iD3) with an integration time of 1000 ms.

[0088] THP-1 inflammatory assay: THP-1 macrophages, when stimulated with lipopolysaccharide (LPS) bacteria, secrete the inflammatory cytokine tumor necrosis factor-alpha (TNF-a) (Takashiba et al., Infect Immun. 1999, 67(1 1):5573-8). THP-1 cells were plated in 24-well plates at 45,000 cells / well and allowed to attach overnight at 37°C in 5% CO2. THP-1 cells were then pretreated for 1 hour with either vehicle (0.5% DMSO) or test compound and subsequently challenged with LPS (1 ng / mL). After three hours, the cells were spun at 12,000 rpm for 7 minutes and the supernatant was collected. The levels of TNF-a were measured by ELISA using human TNF-a ELISA kit (cat. no. KHC3011; Thermo Fisher) according to manufacturer’s instructions.

[0089] Endothelial cell tube formation assay (lattice assay): An in vitro angiogenesis assay kit was purchased from EMD Millipore (Darmstadt, Germany). An amount of 50 pL / well of ECMatrix™ laminin substrate (Sigma- Aldrich, Inc., St. Louis MO) was plated to a 96- well plate and left to set for at least 30 minutes. To harvest cells, HUVECs were detached using TrypLE™ Express enzyme (Thermo Fisher), spun at 1300 rpm, and resuspended in EGM-plus media. Cells were plated atop the gel (35,000 cells / well) and treated with the vehicle control (0.5% DMSO), the positive control (30 pM CPS49), or test compounds at a range of concentrations. The wells were imaged after 18 hours of incubation. Tubule formation was quantified using ImageJ (version 1.54j).

[0090] Human saphenous vein (HSV) angiogenesis assay: The antiangiogenic effects of the test compounds were evaluated in the human saphenous vein angiogenesis model. Through an institution-approved protocol between the National Cancer Institute (Bethesda, MD) and the INOVA Heart and Vascular Institute (Fairfax, VA), specimens of human saphenous vein were harvested during surgery. Subsequently, 24-well tissue culture plates were covered with 350 pL of Matrigel® matrix (Coming Life Sciences) and allowed to set for one hour at 37°C. Following excision of fibroadipose tissue, the veins were cut into 2-mm cross sections, placed on the Matrigel® matrix-coated wells, and layered with additional Matrigel® matrix (450 pL). These were then allowed to set for one hour, after which the cross-sectional rings were covered with EGM-I1 endothelial cell growth media (Lonza) and incubated under 5% CO2 / 95% air at 37°C overnight.EGM-II consists of endothelial cell basal medium-2 (EBM-II) and endothelial cell growth medium- 2 SingleQuots™ supplements and growth factors (Lonza Bioscience, Walkersville, MD). The next day, the rings were treated with media containing vehicle control (0.5% DMSO), a known angiogenesis inhibitor (TNP-470) as the positive control, or test compounds at a range of concentrations. Rings were incubated for a total of 14 days, being re-treated every 2 to 3 days, with all growth factors removed from the medium starting on day 7. The rings were then imaged on day 15 using an EVOS scope. This was independently replicated two times using specimens from 2-3 different donors. The area of angiogenic sprouting, reported in square pixels, was quantified using Adobe Photoshop. Data were presented as percent growth based on the vehicle control, which was normalized to 100% growth.

[0091] Western blot: MM1.S and MOLP-8 cells were plated in 6-well plates at 1.5 x 106cells / well and allowed to incubate overnight at 37°C in 5% CO2, 95% air. The cells were treated with vehicle with or without drugs for 24 hours. Cells were collected from the 6-well plates, washed with ice- cold dPBS (Dulbecco’s phosphate-buffered saline), centrifuged at 1300 rpm, then lysed with RIPA lysis buffer (Sigma Aldrich) and complete protease inhibitor (Catalog no. 25955-11, Nacalai USA, San Diego, CA). Samples were left on ice for 10 minutes, briefly vortexed, and returned to the ice for another 10 minutes. Lysed cells were then centrifuged at 13,000 rpm at 4°C for 10 minutes. The protein-containing supernatant was carefully removed, and the amount of protein in the supernatant was quantified using the Pierce bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Waltham, MA). Samples were run on 4-20% SDS-PAGE Mini-PROTEAN TGX™ Precast Gels (Bio-Rad Laboratories Inc., Hercules, CA) and separated by electrophoresis at 80 V for 90 minutes. Gels were transferred to 0.2 pm nitrocellulose membranes using the Mini Trans-Blot Turbo semidry transfer system (Bio-Rad). Nitrocellulose membranes were blocked with 5% nonfat dry milk in 0.1% TBST (Tris-buffered saline with Tween® 20 surfactant (Millipore Sigma, St. Louis, MO)) for 60 minutes and probed overnight at 4°C with antibodies against Aiolos (CST catalog no. 15103;1 :1000; Cell Signaling Technology, Danvers, MA), Ikaros (CST catalog no. 5443; 1:100; Cell Signaling Technology, Danvers, MA), and GAPDH (SC-47724; 1:2000; Santa Cruz Biotechnology). Membranes were washed 3 times with 0.1% TBST and incubated in Goat AntiMouse IRDye 680RD (1: 10000, LI-COR 925-68070; LI-COR, Lincoln, NE) and Goat Anti-Rabbit IRDye 800CW (1 :10000, Licor 925-32211) for one hour at room temperature. Blots were imaged on LI-COR Odyssey Fc (LI-COR).

[0092] Statistics All results are presented as mean ± SEM. For the TNF-oc ELISA, treatment groups were compared using Mann-Whitney U test, followed by the Hochberg method for adjusting p- values for multiple comparisons. All other data were compared utilizing a one-wayanalysis of variance (ANOVA) and employing the Satterthwaite method to adjust the degrees of freedom of each pairwise difference to account for the varying standard deviations across concentrations.Example 1Effects on Intrinsically-IMiD-resistant Multiple Myeloma Cells

[0093] A library of 26 thalidomide analogs (Table 1) was screened for anti-cancer activity in MM cell lines. Two MM cell lines, RPMI-8226 and JJN3, have been shown to carry intrinsic resistance to lenalidomide (Thakurta et al., Leukemia 2014 28(5): 1129-31). Based on initial testing, all 26 analogs were evaluated at a screening concentration of 10 pM for their ability to inhibit the growth of these two intrinsically IMiD-resistant MM cell lines. In the presence or absence of analogs (10 pM concentration), RPMI-8226 and JJN3 cells were incubated for 72 and 24 hours, respectively, to allow for at least one doubling time to pass for each respective cell line, after which a CCK-8 cell proliferation assay was conducted to determine cytotoxicity. Four compounds (Gul210, Gul213, Gul214, and Gul215) exhibited the most potent activity in both RPMI-8226 and JJN3 cells, with each analog inhibiting growth by >90% and >50%, respectively (FIGS 1A and IB).

[0094] The four lead analogs - Gul 210, Gul 213, Gul 214, and Gul215 - were further evaluated to determine the effects of each compound on cell proliferation at a range of concentrations (0.10 pM to 20 pM) in both RPMI-8226 and JJN3 cells. All four analogs inhibited cell proliferation in a dose-dependent manner in both MM-resistant cell lines (FIGS. 1C and ID). In RPMI-8226 cells, Gul210 and Gul214 demonstrated ICso values of 2.5 pM and 3.0 pM, respectively, while Gul213 and Gul 215 more potently inhibited growth of cells, with ICsos of 2.4 pM and 1.6 pM, respectively (FIG. 1C; ***p<0.0001). In JJN3 cells, Gul215 was the most potent of the four analogs, inhibiting cell proliferation with a demonstrated ICso value of 3.4 pM [vs. Gul213 (5.3 pM), Gul210 and Gul214 (ICso values >10 pM)] (FIG. ID; ***p<0.0001; the 5 pM dose represents two independent replicates).

[0095] The four lead analogs were also tested in the MOLP-8 MM cell line, which carries intrinsic resistance to lenalidomide as well as some intrinsic resistance to pomalidomide (Bosseler et al., Int J Mol Sci. 2018, 19(6): 1551). In this cell line, compounds were tested at 0.1 pM, 1 pM, 5 pM, and 10 pM. As in the other intrinsically IMiD-resistant cells lines, each of the analogs inhibited cell proliferation in a dose-dependent manner. Gul 214 demonstrated the most potent inhibition of cell proliferation in this cell line, exhibiting an IC50 of 1.00 pM, followed by Gul210, Gul215, and Gu 1213, with ICsos of 1.16, 1.23, and 1.91, respectively (FIG. IE).Example 2Effects on Acquired-IMiD-resistant MM Cells

[0096] The effects of the four lead compounds (Gul 210, Gul 213, Gul 214, and Gul 215) in two acquired-lenalidomide-resistant MM cell lines (MM1 / R10R, U266 / R10R) and their respective wild-type counterparts (MM1.S, U266) at a concentration range of 50 nM - 10 pM. The resistant cell lines were developed by continuous exposure of cells to increasing concentrations of lenalidomide. The four lead analogs each inhibited cell proliferation in a dose-dependent manner in all cell lines (FIGS. 2A-2D). ). In MM1 / R10R cells, Gul210, Gul213, and Gul214 all showed similar IC50 values of 5.6 pM, 4.9 pM, and 5.0 pM, respectively, while Gul215 inhibited proliferation most potently, with an IC50 of 2.7 pM (FIG. 2A). In the parental MM1.S cell line, Gul210 produced the greatest growth inhibition, yielding an IC50 value of 0.45 pM (FIG. 2B). The other three analogs were similarly potent, with IC50S of 0.60 pM for Gul213, 0.52 pM for Gul214, and 0.78 pM for Gul215 (FIG. 2B). In U266 / R10R cells, Gul213, Gul214, and Gul215 inhibited proliferation with respective IC50 values of 2.67 pM, 3.10 pM, and 3.09 pM, while Gul210, the most potent lead analog in this cell line, demonstrated inhibition with an IC50 value of 1.71 pM (FIG. 2C). In the wild-type counterpart (U266), Gul215 exhibited the most potent inhibition of cell proliferation, with an IC50 of 2.23 pM (FIG. 2D). Gul210, Gul213, and Gul214 each exhibited slightly less potent inhibitions, with IC50 values of 2.34 pM, 3.33 pM, and 3.50 pM, respectively (FIG. 2D).Example 3Effects on 3D Myeloma Spheroid Growth

[0097] Following evaluation of the polyfluorinated thalidomide analogs in cell proliferation assays, the compounds’ anti-cancer activity was evaluated in three-dimensional spheroid models in the two resistant MM cell lines, MOLP-8 and RPMI-8226. Following 72-hour treatment of the MOLP-8 spheroids with the four leads at varying concentrations [0.1 pM-10 pM], cell viability within the spheroids was reduced in a dose-dependent manner. Gul213 produced the most potent inhibition of spheroid growth, with an IC50 of 0.85 pM, followed closely by Gul210 (0.89 pM) > Gul214 (1.06 pM) > Gul215 (1.12 pM) (FIGS. 3A, 3C). After 72-hour treatment of the RPMI-8226 spheroids, Gul215 produced the greatest inhibition of spheroid growth, with an IC50 of 0.99 pM. Gu 1210, Gul213, and Gul214 exhibited IC50S of 1.73 pM, 1.97 pM, and 2.30 pM, respectively in this spheroid model (FIGS. 3B, 3D).Example 4In Vitro Inflammatory Response

[0098] The compounds’ abilities to inhibit the expression of the inflammatory cytokine TNF-a from THP-1 cells, induced by challenging with lipopolysaccharides (LPS) from E. coli were evaluated. Thalidomide was included as a comparator. After pretreating with vehicle (0.5% DMSO) or compound for 1 hour, THP-1 cells were challenged with LPS (1 ng / mL) for 3 hours and the extent of TNF-a expression was determined by ELISA. For this assay, screening concentrations of 5 pM and 10 pM were set based on initial assays conducted to determine the potency of these compounds. Out of all 26 compounds tested at the screening concentrations, four of the analogs (Gul210, Gul213, Gul214, and Gul215) demonstrated increased potencies and were selected for further evaluation. Each compound was tested at concentrations of 250 nM, 500 nM, and 750 nM, and all four analogs inhibited TNF-a expression in a dose-dependent fashion. Of the four compounds tested at these doses, Gul215 exhibited the greatest potency in this assay (ICso=161 nM, ***p<0.0001). Gul210, Gul213, and Gul214 demonstrated IC50 values of 338 nM, 301 nM, and 253 nM, respectively (FIG. 4).Example 5Effects on Endothelial Tube Formation

[0099] The compounds’ antiangiogenic properties were assessed with the endothelial tube formation (lattice) assay, a frequently-used in vitro angiogenesis assay (Kruger et al., Semin Oncol. 2001, 28(6):570-6). CPS49, a thalidomide analog with potent antiangiogenic activity (Therapontos et al., PNAS USA 2009, 106(21) : 8573-8), served as a positive control, while thalidomide was included as a comparator. The ability of human umbilical vein endothelial cells (HUVECs) to form tubules was not markedly inhibited by thalidomide at 100 pM (FIG. 5 A), consistent with previously published data (ELAarag et al., Int Immunopharmacol. 2014, 21(2):283-92). Also consistent with previous data (Beedie et al., Mol Cancer Ther. 2015, 14(10):2228-37; Therapontos et al., PNAS USA 2009, 106(21 ):8573-8), CPS49 at 30 pM significantly reduced tube formation by >80% (FIG. 5A). HUVECs were treated with the four lead compounds (Gul210, Gul213, Gul214, and Gul215; 1 pM-10 pM) to determine the effect of these compounds in this assay. All four analogs demonstrated >50% inhibition of tube formation at 5 pM (Gul214 ~ Gul215 ~ Gul213 > Gul210) (FIG. 5A). Representative images of tube formation are shown in FIG. 5B.Example 6 Antiangiogenic Effects in ex vivo Human Saphenous Vein Model

[0100] The antiangiogenic properties of the four lead compounds (Gul210, Gul213, Gul214, and Gul215) were assessed in an ex vivo human saphenous vein model of angiogenesis. TNP-470, a known angiogenesis inhibitor, served as a positive control, while thalidomide was included as a comparator. The analogs were tested at 10 pM and 25 pM, dosed repeatedly over a two-week period. On day 15, the extent of microvessel outgrowth in each test group was assessed. At 50 pM, TNP-470 inhibited microvessel outgrowth by 98.90% (FIG. 6A). Outgrowth was inhibited by 19.92% by thalidomide at 100 pM (FIG. 6A). All four lead compounds demonstrated dosedependent inhibition of microvessel outgrowth with GU1213 and GU1215 exhibiting the most potent activity (FIG. 6A; Gul210 and Gul214 data not shown). At 10 pM, Gul215 produced the most potent inhibition, reducing microvessel outgrowth by 83.22%. Gul213 was the next most potent analog, inhibiting outgrowth by 79.72%. Gul214 and Gul210 inhibited outgrowth by 45.78% and 29.65% at 10 pM (data not shown). At 25 pM, Gul213 and Gul215 reduced microvessel outgrowth by >98% (FIG. 6A), and Gul210 and Gul214 by 87.80% and 91.57%, respectively (data not shown). Representative images of microvessel outgrowth are shown in FIG. 6B.Example 7 Effects on Expression of Proteins Downstream of Cereblon (CRBN)

[0101] The effect of the primary lead compound (Gul215) on proteins downstream of CRBN was assessed. CRBN is the known molecular target of lenalidomide and pomalidomide (Ito et al. , Science 2010, 327(5971):1345-50). IMiD-induced conformational changes in CRBN promote its binding and subsequent degradation of two protein substrates, Ikaros (IKZF1) and Aiolos (IKZF3). After 24-hour treatment of MM1.S cells with lenalidomide or pomalidomide, both IKZF1 and IKZF3 exhibited decreased expression in Western blot analysis (FIG. 7A), consistent with previously reported data (Hsueh et al., ACS Pharmacol Transl Sci. 2021, 4(2):980-1000). In contrast, neither IKZF1 nor IKZF3 demonstrated decreased expression following 24-hour treatment with Gul215 at concentrations of 0. 1 pM, 0.5 pM, or 1 pM (FIG. 7A). Likewise, 24-hour treatment of MOLP-8 cells with lenalidomide or pomalidomide reduced IKZF1 / 3 expression, but treatment with Gul215 did not affect the expression of either substrate at 0.1, 0.5, or 1 pM (FIG. 7B). Treatment with Gul215 at higher doses was cytotoxic (data not shown).

[0102] Discussion

[0103] Polyfluorinated thalidomide analogs were screened to determine their antiangiogenic, antiinflammatory, and anti-cancer activity in lenalidomide- and pomalidomide-refractory MM cells. Four lead thalidomide analogs (Gul210, Gul213, Gul214, Gul215) exhibiting varying degrees of anti-inflammatory, antiangiogenic, and anti-cancer properties were identified.

[0104] The ability of each lead compound to inhibit the proliferation of a number of MM cell lines, particularly those that carry intrinsic or acquired resistance to iMIDs (e.g., lenalidomide, pomalidomide), was assessed. All compounds reduced the extent of cell proliferation in each cell line tested, with Gul215 as the lead candidate in inhibiting cell proliferation in most MM cell lines. In three-dimensional spheroid models, all compounds reduced tumor spheroid growth in a dosedependent manner, with Gul215 exhibiting potent activity in RPMI-8226 spheroids and Gul213 exhibiting the greatest potency in the MOLP-8 spheroid model.

[0105] The ability of the lead compounds to inhibit the expression of TNF-oc using a TNF-oc ELISA was evaluated, with Gul215 exhibiting the most potent anti-inflammatory effects. The antiinflammatory properties of the lead compounds compared favorably to structurally similar thalidomide analogs; each compound demonstrated more significant inhibition of TNF-a expression at lower concentrations (500 nM) than those tested previously at 25 pM (Gutschow et al., Bioorg. Med. Chem. 2001, 9: 1059-1065).

[0106] The four lead compounds also were evaluated in the well-established human umbilical vein endothelial cell tube formation assay, which confirmed that Gul 210, Gul 213, Gul 214, and Gul215 are potent angiogenesis inhibitors. Gul215 and Gul213 produced the most marked inhibition, reducing microvessel outgrowth by over 80% at 10 p M (FIG. 6A).

[0107] The precise mechanisms of action of thalidomide and its analogs exerting antiangiogenic effects are not completely understood. Initial studies identified cereblon (CRBN) as the protein target of thalidomide and its close analogs lenalidomide and pomalidomide (Ito et al. , Science 2010, 327(5971): 1345-50; Chamberlain et al., Nat Struct Mol Biol. 2014, 21(9):803-9; Fischer et al., Nature 2014, 512(7512):49-53). However, loss of CRBN alone does not eliminate thalidomide’s antiangiogenic activity (Beedie et al., FASEB J. 2020, 34(9): 11395-404). In subsequent studies using, for example, in silico CRBN-docking experiments, it was found that enhanced cereblon binding does not correlate with antiangiogenic effects of thalidomide analogs (Peach et al., Molecules 2020, 25(23); Heim et al. , Biochem Biophys Res Cornmun. 2021, 534:67- 72). A recent study using FRET pairing to investigate cereblon-binding abilities of a variety of 5- and 6-membered rings found that at least one carbonyl group is required for binding (Boichenko et al., ACS Omega 2018, 3(9) : 11163-71 ). In this study, Gul215, which lacks a carbonyl group on itscyclohexane ring or any other moiety to be engaged in polar or hydrogen-bond interactions (Table 1), nevertheless demonstrated potent antiangiogenic properties in both angiogenesis assays. Taken together, these data suggest that IMiD-induced antiangiogenic effects may be exerted through a non-CRBN protein target.

[0108] Acquired resistance to lenalidomide is associated with a significant decrease or absence of CRBN protein expression compared to isogenic lenalidomide-sensitive cell lines (Dimopoulos et al., Mol Oncol 2018, 12(2): 180-95; Lopez-Girona et al., Leukemia 2012, 26(1 l):2326-35; Zhu et al., Blood Cancer J. 2019, 9(2): 19). Conversely, baseline CRBN expression levels are not correlated with IMiD sensitivity, as intrinsic-resistant MM cell lines have baseline CRBN expression levels comparable to their IMiD-sensitive counterparts lines (Dimopoulos et al. , Mol Oncol 2018, 12(2): 180-95). Additionally, treatment of MM1.S and MOLP-8 cells with Gul215 had no effect on IKZF1 and IKZF3, the downstream proteins of CRBN that exhibit decreased expression when treated with lenalidomide and pomalidomide (FIG. 2E). Therefore, the data suggest that the lead compounds may be exerting their anti-myeloma effects through a non-CRBN target and a mechanism that is yet to be understood. Given its efficacy in all assays conducted in this study, particularly against IMiD-resistant MM cell lines, Gul215 warrants further investigation as a potential drug candidate for lenalidomide- and pomalidomide-resistant multiple myeloma.

[0109] In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the disclosure and should not be taken as limiting the scope of the disclosure. Rather, the scope of the disclosure is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

4239-110465-02We claim:

1. A pharmaceutical composition comprising: a compound according to formulawherein R is aliphatic; and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is formulated for parenteral or oral administration.

2. The pharmaceutical composition of claim 1 , wherein R is unsubstituted C1-C10 straight alkyl, branched alkyl, or cycloalkyl.

3. The pharmaceutical composition of claim 1 , wherein the compound isany combination thereof.

4. A method of inhibiting proliferation of cancer cells, comprising contacting the cancer cells with an effective amount of a compound according to formulawherein R is aliphatic.

5. The method of claim 4, wherein R is unsubstituted C2-C10 straight alkyl, branched alkyl, or cycloalkyl.

6. The method of claim 4, wherein the compound isor any combination thereof.

7. The method of any one of claims 4-6, wherein contacting the cancer cells with the effective amount of the compound comprises administering the effective amount of the compound to a subject having cancer.

8. The method of any one of claims 4-7, wherein the cancer is multiple myeloma and the method comprises contacting the multiple myeloma cells with the effective amount of the compound.

9. The method of claim 8, wherein the multiple myeloma cells are drug resistant.

10. The method of claim 9, wherein the multiple myeloma cells are resistant to one or more immunomodulatory drugs.

11. The method of claim 9 or claim 10, wherein the multiple myeloma cells are resistant to one or more of thalidomide, lenalidomide, and pomalidomide.

12. The method of any one of claims 8-11, wherein the compound inhibits multiple myeloma cell proliferation with an ICso of from 0.2 pM to 10 pM.

13. The method of any one of claims 4-12, wherein contacting the multiple myeloma cells with the effective amount of the compound does not increase degradation of cereblon substrate IKZF1, IKZF3, or IKZF1 and IKZF3.

14. The method of any one of claims 4-13, wherein the compound is15. A method of treating a cancer or inflammatory process, comprising administering to a subject having cancer or an inflammatory process a therapeutically effective amount of a compound according to formulawherein R is aliphatic, or a therapeutically effective amount of a pharmaceutical composition comprising the compound.

16. The method of claim 15, wherein R is unsubstituted Ci-Cio straight alkyl, branched alkyl, or cycloalkyl.

17. The method of claim 15, wherein the compound isor any combination thereof.

18. The method of any one of claims 15-17, wherein the cancer is multiple myeloma.

19. The method of claim 18, wherein treating the multiple myeloma comprises inhibiting proliferation of multiple myeloma cells.

20. The method of claim 18 or claim 19, wherein the subject has drug-resistant multiple myeloma.

21. The method of claim 20, wherein the drug -resistant multiple myeloma is resistant to one or more immunomodulatory drugs.

22. The method of claim 20 or claim 21, wherein the drug -resistant multiple myeloma is resistant to one or more of thalidomide, lenalidomide, and pomalidomide.

23. The method of claim 22, wherein the compound inhibits the proliferation of drugresistant multiple myeloma cells with an IC50 of from 0.2 pM to 10 pM.

24. The method of any one of claims 15-23, wherein the compound does not increase degradation of cereblon substrate IKZF1, IKZF3, or IKZF1 and IKZF3.

25. The method of any one of claims 15-24, wherein the compound isRECTIFIED SHEET (RULE 91) ISA / EP

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