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WO2023039170A1 - Ciblage sélectif de l'activité de cd38 en tant que stratégie immunostimulatrice et antitumorale - Google Patents

Ciblage sélectif de l'activité de cd38 en tant que stratégie immunostimulatrice et antitumorale Download PDF

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WO2023039170A1
WO2023039170A1 PCT/US2022/043065 US2022043065W WO2023039170A1 WO 2023039170 A1 WO2023039170 A1 WO 2023039170A1 US 2022043065 W US2022043065 W US 2022043065W WO 2023039170 A1 WO2023039170 A1 WO 2023039170A1
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antagonist
compound
cancer
subject
amount
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Patrick M. Woster
Yuri Peterson
Thomas Benton
Catherine Mills
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MUSC Foundation for Research and Development
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MUSC Foundation for Research and Development
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    • A61P35/00—Antineoplastic agents
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/12—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D309/34—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D309/36—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with oxygen atoms directly attached to ring carbon atoms
    • C07D309/38—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with oxygen atoms directly attached to ring carbon atoms one oxygen atom in position 2 or 4, e.g. pyrones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
    • C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
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    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/34—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 3 only
    • C07D311/36—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 3 only not hydrogenated in the hetero ring, e.g. isoflavones
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    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
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    • C07D—HETEROCYCLIC COMPOUNDS
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    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/10—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems
    • C07D491/056—Ortho-condensed systems with two or more oxygen atoms as ring hetero atoms in the oxygen-containing ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems

Definitions

  • the invention relates generally to chemical compounds and use thereof in treatment of diseases associated with altered CD38 enzymatic activity and/or altered NAD + /Ca 2+ signaling.
  • TEE tumor microenvironment
  • ICIs Monoclonal immune checkpoint inhibitors
  • PD-1 :PD- L1 and CTLA4:CD80/CD81 promote an unhindered adaptive immune response.
  • immune cells that infiltrate the tumor microenvironment are able to elicit cytotoxic and inflammatory effects, resulting in a rapid reduction in tumor burden.
  • the ectoenzyme CD38 serves as a recognition glycoprotein that binds to CD31 on the surface of T-cells, causing them to produce a variety of cytokines, but it is also highly expressed on the cell surface of multiple tumor cell types.
  • CD38 cluster of differentiation 38
  • the ectoenzyme known as cluster of differentiation 38 primarily exists as a 34 kDa transmembrane glycoprotein containing a small N-terminal cytosolic tail, a single pass transmembrane domain, and a large C-terminal extracellular domain (Lee 2006).
  • CD38 is expressed on the surface of mature immune cells, and as adaptive immune cells mature and undergo phenotypic reprogramming, cell surface markers are expressed as part of phenotypic remodeling (Fagerberg 2014, Shubinsky 1997). In both B and T cells, CD38 expression is indicative of cellular activation and serves as a receptor for lymphocyte transmigration (Shubinsky 1997).
  • CD38 expression is highly contextual in that it depends on cellular location and the pathophysiological context in which it is embedded (Chini 2018). Within cells and extracellularly NAD + is converted to ADPR and/or cADPR by CD38. These second messengers go on to regulate Ca2 + signaling within cells and function extracellularly on receptors and channels.
  • Groups have targeted CD38 as an NAD boosting therapy in the context of aging, mitochondrial dysfunction, obesity, and diabetes (Chini 2018, Chini 2009, Dong 2011, Escade 2013, Becherer 2015). Notably, some of these inhibitors increased the levels of NAD + in vitro and in vivo, but were reportedly toxic in animal models or possessed undesirable pharmacokinetic profiles. In addition, none of these agents were evaluated for antitumor or immunostimulatory effects (Chini 2018, Dong 2011, Becherer 2015, Haffner 2015).
  • the present invention is directed to compounds having the general structure of formulas I and II, and other compounds having similar activity.
  • the invention also provides a method for the treatment of a cancer, comprising the step of administering to a subject in need thereof a therapeutically effective amount of a compound, or a pharmaceutically acceptable salt thereof, thereby treating the subject, optionally in in combination with a second antagonist therapy which is a second cluster of differentiation 38 (CD38) directed therapy, or a PD1 or PD-L1 directed therapy.
  • a second antagonist therapy which is a second cluster of differentiation 38 (CD38) directed therapy, or a PD1 or PD-L1 directed therapy.
  • Figure 1 is a graph showing that in using two distinct catalytic reactions, CD38 produces ADP- ribose (ADPR) from both NAD + and cADPR.
  • ADPR ADP- ribose
  • Figure 2 shows CD38- and CD39-mediated adenosinergic pathways.
  • Figure 3 shows superimposition of PDB codes 2165, 203 S, 2O3U, and 4F45 displaying a consensus catalytic site and substrate orientation (NAD + , NGD, cADPr (magenta), and NAADP).
  • the nicotinamide ribose retains a conserved binding pose with substrates: NAD + , NGD + , and NAADP displayed. Purine location is solvent exposed and tends to lack a consensus binding position.
  • TRP189, SERI 93, and GLU226 are responsible for forming critical contracts and mediate catalysis of each substrate. For clarity only the heavy atoms are shown, dotted lines represent hydrogen bonds, dashed line represent TI- it interactions.
  • Figure 4 shows reactions comprising the CD38 hydrolase (top) and cyclase (bottom) assays, and structures of the standard inhibitors apigenin and quercitin.
  • Figure 5 shows a comparison of CD38 hydrolase- and cyclase-selective inhibitors at 50 pM.
  • Panel A top 7 hydrolase-selective inhibitors
  • panel B top 7 cyclase-selective inhibitors
  • panel C IC50 determination for compound 1 against CD38 hydrolase
  • panel D IC50 determination for compound 12 against CD38 cyclase. All data points are the result of at least 3 determinations + SEM.
  • Figure 6 shows enzyme inhibition kinetics for the hydrolase and cyclase activities of CD38 by compounds 1 and 12.
  • Panel A inhibition of CD38 hydrolase by compound 1;
  • panel B inhibition of CD38 cyclase by compound 12. All data points are the result of 3 determinations ⁇ SEM.
  • Figure 7 shows cytotoxicity of 78c, 1 and 12 in naive PBMC cells at concentrations between 0.01 and 100 pM. All data points are the average of nine determinations (3 donors each run in triplicate in separate experiments) ⁇ SEM.
  • Figure 8 shows an increase in cellular NADH + levels in activated PBMCs following treatment at 1 pM concentrations of 78c, 1 and 12. All data points are the average of 3 determinations ⁇ SEM.
  • Figure 9 shows effect of 78c, 1 and 12 at 10 pM on IFNy levels. Panel A: 48 hours; panel B: 12 days.
  • Figure 10 shows an in silico representation of compound 12 bound to the CD38 active site. Amino acids involved in binding and 12. The darker structure in the middle of the figure is NAD + .
  • Figure 11 shows 48 hour increase in IFNy in the presence of 1.0 pM compounds 78c, 12, 20, 22, 23 and 24 in activated PBMCs from three separate donors. Each data point is the average of 3 determinations. These averages were then replotted as % increase in IFNy ⁇ SEM.
  • Figure 12 shows the cytotoxicity of 1, 12 and 78c in the NCI-H929 human multiple myeloma cell line.
  • Panel A Proportion of CD38-expressing cells by flow cytometry;
  • Panel B Cell viability dose-response curves for 1, 12 and 78c.
  • Figure 13 shows the effect on the response of natural killer (NK) cells of compounds 78c and 1 against SH-SY5Y neuroblastoma cells transfected with GFP.
  • NK cells are stained. Cells were treated with 50 ng/mL of recombinant chl4.18 IL-2 and 1 uM of each analogue for 90 minutes.
  • Panel A DMSO
  • Panel B 78c
  • Panel C compound 1
  • Panel D graphical representation of the remaining GFP fluorescence
  • Panel E neuroblastoma cells (upper right corner) being destroyed by NK cells (center and lower right corner). Images were produced on a Biotek Cytation 5 cell imager. Data points are the average of 3 determinations from different plates + SEM.
  • Figure 14 shows the effect of CD38 inhibitors 1 and 78C in a co-culture of human PBMCs with CD 19+ REH human acute leukemia cells. Each data point represents the average of 3 determinations + SEM. DETAILED DESCRIPTION OF THE INVENTION
  • a compound according to the invention is inherently intended to comprise all stereochemically isomeric forms thereof.
  • stereochemically isomeric forms as used hereinbefore or hereinafter defines all the possible stereoisomeric forms which the compounds of the formulas disclose herein and their N-oxides, pharmaceutically acceptable salts or physiologically functional derivatives may possess.
  • the chemical designation of compounds denotes the mixture of all possible stereochemically isomeric forms.
  • stereogenic centers may have the R- or S-configuration; substituents on bivalent cyclic (partially) saturated radicals may have either the cis- or trans-configuration.
  • Compounds encompassing double bonds can have an E (ent ought) or Z (zusammen)-stereochemistry at said double bond.
  • the terms cis, trans, R, S, E and Z are well known to a person skilled in the art.
  • Compounds of the present invention can have one or more asymmetric carbon atoms and can exist in the form of optically pure enantiomers, mixtures of enantiomers such as, for example, racemates, optically pure diastereoisomers, mixtures of diastereoisomers, diastereoisomeric racemates or mixtures of diastereoisomeric racemates.
  • the optically active forms can be obtained for example by resolution of the racemates, by asymmetric synthesis or asymmetric chromatography (chromatography with a chiral adsorbents or eluant). The invention embraces all of these forms.
  • the compounds disclosed herein may be synthesized in the form of mixtures, in particular racemic mixtures, of enantiomers which can be separated from one another following art-known resolution procedures.
  • the racemic compounds of the compounds disclosed herein may be converted into the corresponding diastereomeric salt forms by reaction with a suitable chiral acid. Said diastereomeric salt forms are subsequently separated, for example, by selective or fractional crystallization and the enantiomers are liberated therefrom by alkali.
  • An alternative manner of separating the enantiomeric forms of the compounds disclosed herein involves liquid chromatography using a chiral stationary phase.
  • Said pure stereochemically isomeric forms may also be derived from the corresponding pure stereochemically isomeric forms of the appropriate starting materials, provided that the reaction occurs stereospecifically.
  • said compound will be synthesized by stereospecific methods of preparation. These methods will advantageously employ enantiomerically pure starting materials.
  • a “subject” is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or nonhuman primate, such as a monkey, chimpanzee, baboon or rhesus monkey, and the terms “patient” and “subject” are used interchangeably herein.
  • carrier encompasses carriers, excipients, and diluents and means a material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a pharmaceutical agent from one organ, or portion of the body, to another organ, or portion of the body.
  • treating encompasses, e.g., inducing inhibition, regression, or stasis of a disease or disorder; or curing, improving, or at least partially ameliorating the disorder; or alleviating, lessening, suppressing, inhibiting, reducing the severity of, eliminating or substantially eliminating, or ameliorating a symptom of the disease or disorder.
  • “Inhibition" of disease progression or disease complication in a subject means preventing or reducing the disease progression and/or disease complication in the subject.
  • a "symptom” associated with cancer includes any clinical or laboratory manifestation associated with cancer and is not limited to what the subject can feel or observe.
  • disorder is used in this disclosure to mean, and is used interchangeably with, the terms disease, condition, or illness, unless otherwise indicated.
  • administering to the subject means the giving of, dispensing of, or application of medicines, drugs, or remedies to a subject/patient to relieve, cure, or reduce the symptoms associated with a condition, e.g., a pathological condition.
  • the administration can be periodic administration.
  • periodic administration means repeated/recurrent administration separated by a period of time. The period of time between administrations is preferably consistent from time to time. Periodic administration can include administration, e.g., once daily, twice daily, three times daily, four times daily, weekly, twice weekly, three times weekly, four times a week and so on, etc.
  • unit dose means a single drug administration entity/entities.
  • an effective or “therapeutically effective” when referring to an amount of a substance refers to the quantity of the substance that is sufficient to yield a desired therapeutic response.
  • an effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • a therapeutically effective amount of an antagonist or inhibitor of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibodies to elicit a desired response in the individual.
  • a therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the antibody or antibodies are outweighed by the therapeutically beneficial effects.
  • the combination of the invention may be formulated for its simultaneous, separate or sequential administration, with at least a pharmaceutically acceptable carrier, additive, adjuvant or vehicle as described herein.
  • a pharmaceutically acceptable carrier, additive, adjuvant or vehicle as described herein.
  • “combination” means an assemblage of reagents for use in therapy either by simultaneous or contemporaneous administration.
  • Simultaneous administration refers to administration of an admixture (whether a true mixture, a suspension, an emulsion or other physical combination) of two or more components.
  • Contemporaneous administration, or concomitant administration refers to the separate administration of two or more components at the same time, or at times sufficiently close together that a synergistic activity relative to the activity of either component alone is observed or in close enough temporal proximately to allow the individual therapeutic effects of each component to overlap.
  • additive-on or “add-on therapy” means an assemblage of reagents for use in therapy, wherein the subject receiving the therapy begins a first treatment regimen of one or more reagents prior to beginning a second treatment regimen of one or more different reagents in addition to the first treatment regimen, so that not all of the reagents used in the therapy are started at the same time. For example, adding one antagonist therapy (including therapy with the compounds disclosed herein) to a patient already receiving a different antagonist therapy.
  • the CD38 antagonist preferably neutralizes biological function after binding.
  • the CD38 antagonist is preferably a human CD38 antagonist.
  • the CD38 antagonist may be an antibody, such as a monoclonal antibody or fragment thereof; a chimeric monoclonal antibody (such as a human-murine chimeric monoclonal antibody); a fully human monoclonal antibody; a recombinant human monoclonal antibody; a humanized antibody fragment; a soluble CD38 antagonist, including small molecule CD38 blocking agents.
  • the CD38 antagonist is a functional fragment or fusion protein comprising a functional fragment of a monoclonal antibody, such as a Fab, F(ab')2, Fv and preferably Fab.
  • a fragment is pegylated or encapsulated (e.g. for stability).
  • the CD38 antagonist may also be a camelid antibody.
  • CD38 antagonists include but are not limited to CD38 receptor inhibitors.
  • the PD-1 antagonist preferably neutralizes biological function after binding.
  • the PD-1 antagonist is preferably a human PD-1 antagonist.
  • the PD-1 antagonist may be an antibody, such as a monoclonal antibody or fragment thereof; a chimeric monoclonal antibody (such as a human-murine chimeric monoclonal antibody); a fully human monoclonal antibody; a recombinant human monoclonal antibody; a humanized antibody fragment; a soluble PD-1 antagonist, including small molecule PD-1 blocking agents.
  • the PD-1 antagonist is a functional fragment or fusion protein comprising a functional fragment of a monoclonal antibody, such as a Fab, F(ab')2, Fv and preferably Fab.
  • a fragment is pegylated or encapsulated (e.g. for stability).
  • the PD-1 antagonist may also be a camelid antibody.
  • PD-1 antagonists include but are not limited to PD-1 receptor inhibitors.
  • the PD-1 antagonist may be selected, for example, from one or a combination of nivolumab, pembrolizumab, avelumab, durvalumab, cemiplimab, or atezolizumab, or a functional fragment thereof.
  • alkyl refers to a branched or straight-chain monovalent saturated aliphatic hydrocarbon radical of one to twenty carbon atoms, in one embodiment one to sixteen carbon atoms, in another embodiment one to ten carbon atoms.
  • lower alkyl refers to a branched or straight-chain alkyl radical of one to nine carbon atoms, in one embodiment one to six carbon atoms, in another embodiment one to four carbon atoms, in a further embodiment four to six carbon atoms.
  • This term is further exemplified by radicals such as methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, n-pentyl, 3 -methylbutyl, n- hexyl, 2-ethylbutyl and the like.
  • the compounds of the present invention may be formulated in a wide variety of oral administration dosage forms and carriers.
  • Oral administration can be in the form of tablets, coated tablets, dragees, hard and soft gelatin capsules, solutions, emulsions, syrups, or suspensions.
  • Compounds of the present invention are efficacious when administered by other routes of administration including continuous (intravenous drip) topical parenteral, intramuscular, intravenous, subcutaneous, transdermal (which may include a penetration enhancement agent), buccal, nasal, inhalation and suppository administration, among other routes of administration.
  • the preferred manner of administration is generally oral using a convenient daily dosing regimen which can be adjusted according to the degree of affliction and the patient's response to the active ingredient.
  • a compound or compounds of the present invention, as well as their pharmaceutically useable salts, together with one or more conventional excipients, carriers, or diluents, may be placed into the form of pharmaceutical compositions and unit dosages.
  • the pharmaceutical compositions and unit dosage forms may be comprised of conventional ingredients in conventional proportions, with or without additional active compounds or principles, and the unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed.
  • the pharmaceutical compositions may be employed as solids, such as tablets or filled capsules, semisolids, powders, or liquids such as solutions, suspensions, emulsions, elixirs, or filled capsules for oral use; or in the form of sterile injectable solutions for parenteral use.
  • a typical preparation will contain from about 5% to about 95% active compound or compounds (w/w).
  • preparation or “dosage form” is intended to include both solid and liquid formulations of the active compound and one skilled in the art will appreciate that an active ingredient can exist in different preparations depending on the target organ or tissue and on the desired dose and pharmacokinetic parameters.
  • excipient refers to a compound that is useful in preparing a pharmaceutical composition, generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipients that are acceptable for veterinary use as well as human pharmaceutical use.
  • the compounds of this invention can be administered alone but will generally be administered in admixture with one or more suitable pharmaceutical excipients, diluents or carriers selected with regard to the intended route of administration and standard pharmaceutical practice.
  • “Pharmaceutically acceptable” means that which is usefill in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary as well as human pharmaceutical use.
  • a "pharmaceutically acceptable salt” form of an active ingredient may also initially confer a desirable pharmacokinetic property on the active ingredient which were absent in the non-salt form, and may even positively affect the pharmacodynamics of the active ingredient with respect to its therapeutic activity in the body.
  • pharmaceutically acceptable salt of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound.
  • Such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxy ethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid,
  • Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.
  • a solid carrier may be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
  • the carrier In powders, the carrier generally is a finely divided solid which is a mixture with the finely divided active component.
  • the active component In tablets, the active component generally is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired.
  • Suitable carriers include but are not limited to magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.
  • Solid form preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
  • Liquid formulations also are suitable for oral administration include liquid formulation including emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions. These include solid form preparations which are intended to be converted to liquid form preparations shortly before use. Emulsions may be prepared in solutions, for example, in aqueous propylene glycol solutions or may contain emulsifying agents such as lecithin, sorbitan monooleate, or acacia. Aqueous solutions can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing, and thickening agents. Aqueous suspensions can be prepared by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.
  • viscous material such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.
  • the compounds of the present invention may be formulated for parenteral administration (e.g., by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative.
  • the compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol.
  • oily or nonaqueous carriers, diluents, solvents or vehicles examples include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate), and may contain formulatory agents such as preserving, wetting, emulsifying or suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water.
  • the compounds of the present invention may be formulated for topical administration to the epidermis as ointments, creams or lotions, or as a transdermal patch.
  • Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents.
  • Lotions may be formulated with an aqueous or oily base and will in general also containing one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents.
  • Formulations suitable for topical administration in the mouth include lozenges comprising active agents in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
  • the compounds of the present invention may be formulated for nasal administration.
  • the solutions or suspensions are applied directly to the nasal cavity by conventional means, for example, with a dropper, pipette or spray.
  • the formulations may be provided in a single or multidose form. In the latter case of a dropper or pipette, this may be achieved by the patient administering an appropriate, predetermined volume of the solution or suspension. In the case of a spray, this may be achieved for example by means of a metering atomizing spray pump.
  • the compounds of the present invention may be formulated for aerosol administration, particularly to the respiratory tract and including intranasal administration.
  • the compound will generally have a small particle size for example of the order of five (5) microns or less. Such a particle size may be obtained by means known in the art, for example by micronization.
  • the active ingredient is provided in a pressurized pack with a suitable propellant such as a chlorofluorocarbon (CFC), for example, dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, or carbon dioxide or other suitable gas.
  • CFC chlorofluorocarbon
  • the aerosol may conveniently also contain a surfactant such as lecithin.
  • the dose of drug may be controlled by a metered valve.
  • the active ingredients may be provided in a form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidine (PVP).
  • the powder carrier will form a gel in the nasal cavity.
  • the powder composition may be presented in unit dose form for example in capsules or cartridges of e.g., gelatin or blister packs from which the powder may be administered by means of an inhaler.
  • suitable formulations along with pharmaceutical carriers, diluents and excipients are described in Remington: The Science and Practice of Pharmacy 1995, edited by E. W. Martin, Mack Publishing Company, 19th edition, Easton, Pennsylvania.
  • a skilled formulation scientist may modify the formulations within the teachings of the specification to provide numerous formulations for a particular route of administration without rendering the compositions of the present invention unstable or compromising their therapeutic activity.
  • the modification of the present compounds to render them more soluble in water or other vehicle may be easily accomplished by minor modifications (salt formulation, esterification, etc.), which are well within the ordinary skill in the art. It is also well within the ordinary skill of the art to modify the route of administration and dosage regimen of a particular compound in order to manage the pharmacokinetics of the present compounds for maximum beneficial effect in patients.
  • the term "therapeutically effective amount” as used herein means an amount required to reduce symptoms of the disease in an individual.
  • the dose will be adjusted to the individual requirements in each particular case. That dosage can vary within wide limits depending upon numerous factors such as the severity of the disease to be treated, the age and general health condition of the patient, other medicaments with which the patient is being treated, the route and form of administration and the preferences and experience of the medical practitioner involved.
  • a daily dosage of between about 0.01 and about 1000 mg/kg body weight per day should be appropriate in monotherapy and/or in combination therapy.
  • a preferred daily dosage is between about 0.1 and about 500 mg/kg body weight, more preferred 0.1 and about 100 mg/kg body weight, and most preferred 1.0 and about 15 mg/kg body weight per day.
  • the dosage range in one embodiment would be about 70 mg to .7 g per day.
  • the daily dosage can be administered as a single dosage or in divided dosages, typically between 1 and 5 dosages per day. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect for the individual patient is reached.
  • One of ordinary skill in treating diseases described herein will be able, without undue experimentation and in reliance on personal knowledge, experience and the disclosures of this application, to ascertain a therapeutically effective amount of the compounds of the present invention for a given disease and patient.
  • the pharmaceutical preparations are preferably in unit dosage forms.
  • the preparation is subdivided into unit doses containing appropriate quantities of the active component.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules.
  • the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
  • Compounds of the present invention can be prepared beginning with commercially available starting materials and utilizing general synthetic techniques and procedures known to those skilled in the art.
  • Chemicals may be purchased from companies such as for example SigmaAldrich, Argonaut Technologies, VWR and Lancaster. Chromatography supplies and equipment may be purchased from such companies as for example AnaLogix, Inc, Burlington, Wis.; Biotage AB, Charlottesville, Va.; Analytical Sales and Services, Inc., Pompton Plains, N.J.; Teledyne Isco, Lincoln, Nebr.; VWR International, Bridgeport, N.J.; and Waters Corporation, Milford, MA. Biotage, ISCO and Analogix columns are pre-packed silica gel columns used in standard chromatography.
  • Antitumor immunotherapies work by priming immune cells to eradicate cancer. As immunotherapies have continued to gain traction in the clinic, great effort has been afforded to the validation of new immune-targets for drug discovery. In the last decade, checkpoint immunotherapies (Cis) have revolutionized the field of immuno-oncology and shaped current thinking about cancer mechanisms and the treatment of advanced malignancies. Many of the 11 Cis on the market today stimulate a subset of immune cells by preventing the ligation of immunosuppressive receptors. As such, immune cells that infiltrate the tumor microenvironment are able to elicit cytotoxic and inflammatory effects, resulting in a rapid reduction in tumor burden.
  • CI therapies are all antibody preparations targeted to specific checkpoint proteins, and as such they are expensive, must be administered by periodic injection, and have a high rate of development of resistance. Patients who respond to antibody-based Cis experience increased survival rates, but the number of non-responding patients, or patients who develop resistance, is untenably high.
  • Agents of this type would offer significant advantages with respect to cost, route of administration and developent of resistance. These agents can be used in place of expensive and difficult to administer antibody therapies as antitumor therapies for use in a variety of tumor types.
  • resistance to antitumor antibody therapies is mediated through CD38 up regulation, these small molecule agents could be used in combination with existing agents such as daratumumab, isatuximab, and PD1/PD-L1 inhibitors.
  • CD38 primarily exists as a 34 kDa transmembrane glycoprotein. Prominent protein expression of CD38 is found in male reproductive tissues and on the surface of mature immune cells, with the highest expression levels on the antibody-producing plasma cells (Fagerberg 2014). A number of studies have indicated that transmembrane expression of CD38 in the immune compartment indicates that it possesses activities beyond its catalytic function. For example, as adaptive immune cells mature and undergo phenotypic reprogramming, cell surface markers are expressed as part of phenotypic remodeling. In both B and T cells, CD38 expression is indicative of cellular activation (ligation of B- and T cell receptors) and serves as an activation marker.
  • CD38 may act as a receptor for CD31 (platelet endothelial cell adhesion molecule) and hyaluronic acid, suggesting that CD38 plays a role in lymphocyte transmigration (Shubinsky 1997). Recent literature suggests that CD38 expression is highly contextual and depends on cellular location and the pathophysiological context in which it is embedded (Chini 2018).
  • CD38-targeted antibodies such as daratumumab and isatuximab bind to CD38 and promote cancer cell death by stimulating a robust antitumor immune response.
  • ICIs are achieving unprecedented success in a percentage of cases, these therapies are expensive, and high rates of resistance limit their efficacy.
  • CD38 has 2 enzymatic activities, a hydrolase that promotes the formation of adenosine (ADO) and an NADase that depletes NAD+. Ironically, both of these activities promote immunosuppression.
  • novel CD38 inhibitors for use in immunotherapy have been identified. Specifically, compounds have been identified that are selective for either the hydrolase (compound 1) or the cyclase (compound 12) activity of CD38. These compounds have been shown to promote the activation of T cells in vitro. Potent and effective CD38 inhibitors have been identified for use alone or in combination with existing CD38 antibody therapies. Selective small molecule inhibitors of the hydrolase or cyclase activity of CD38 can serve as chemical probes to determine the mechanism by which CD38 promotes cancer progression, and could become novel and effective immunotherapies for the treatment of cancer. Although a MM model system is described herein because it represents a tumor line that highly expresses CD38.
  • CD38 expressing cancers e.g., acute myeloid leukemia, chronic lymphocytic leukemia, prostate cancer, pancreatic cancer, lung cancer, hepatocellular cancer, and triplenegative breast cancer
  • CD38 inhibitors and approved agents e.g., bortezomib, carfilzomib, lenalidomide, panobinostat, dexamethasone, and selinexor
  • approved antibody therapies daratumumab, isatuximab
  • CD38 is a major factor causing immunosuppression in the tumor microenvironment by reducing extracellular NAD + , and by production of ADO.
  • CD38 also acts as a surface recognition protein for T-cell binding, resulting in the release of cytokines.
  • the CD38 specific antibody therapies daratumumab and isatuximab have been used effectively against MM by binding CD38 and promoting tumor cell death through ADCC.
  • over expression of CD38 on tumor cells appears to be one of the most important factors in mediating resistance to antibody-based immune modulatory therapies.
  • CD38 is a target for cancer immunotherapy, since it is highly expressed on the surface of a variety of tumor cells, most notably multiple myeloma (MM).
  • the monoclonal antibodies (MABs) daratumumab and isatuximab specifically bind to CD38 on the surface of these tumor cells and promote tumor cell death by Fc-dependent immune effector mechanisms including complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and apoptosis upon secondary cross-linking(Overdijk 2016, van de Donk 2018 Blood, van de Donk 2016).
  • CDC complement-dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • CD38 is also highly expressed on the surface of regulatory T-cells (Tregs), regulatory B-cells and myeloid suppressor cells, these MABs also reduce the number of immune suppressors, resulting in an increase in cytotoxic T-cells (Krejcik 2016, van de Donk 2018 Immunol Lettl).
  • CD38 up regulation appears to be one of the most important factors in mediating resistance to checkpoint blockade in MM and other cancers (Chen 2018, Koyama 2016, Tumeh 2014).
  • ADO adenosine
  • Extracellular ADO binds to ADO receptors on immune cells, including T cells, natural killer (NK) cells, neutrophils, macrophages and dendritic cells, preventing their activation (Goh 2019, Passarelli 2019).
  • ADO produced by CD38 also promotes myeloid-derived suppressor cell (MDSC) expansion, macrophage M2 polarization, and CD4+ Treg generation, all of which support tumor cell progression (Kennedy 2020).
  • CD38 is considered a promising target for small molecule inhibitors.
  • Highly proliferating cells, such as T cells and many tumor cell lines, are highly dependent on nicotinamide adenine dinucleotide (NAD + ), suggesting that their activity is likely to be further enhanced by inhibition of the NADase function of CD38.
  • NAD + nicotinamide adenine dinucleotide
  • CD38 plays a critical role in the homeostatic regulation of cellular energetics (Chini 2018 and Chini 2009). By metabolizing the cofactor NAD + , CD38 removes an essential electron acceptor, thus limiting the energetic capacity of a cell. CD38 is known for its glycohydrolytic activity, though in some circumstances it uses two reactions that may be sequentially coupled (Fig. 1). In the first reaction, CD38 functions as a cyclase, removing nicotinamide from NAD + and producing cyclic adenosine diphosphate-ribose
  • CD38 (Chini 2018, Egea 2012). CD38 also mediates an energetically more favorable hydrolase reaction that produces adenosine diphosphate-ribose (ADPR) from both NAD + and cADPR (Lee 2006, Chini 2018).
  • CD38 over expression in immune cells and tumor cells within the tumor microenvironment (TME) causes a reduction in NAD + levels, leading to a down regulation of the immune response against tumor cells.
  • CD38 plays an extremely labile role in cellular stimulation, immunogenicity and stem-like memory (Shubinsky 1997). In particular, rapid induction of effector function proteins in response to pathological distress requires quick bursts of energy. Oftentimes this process is dependent on NAD + for transient reduction and cellular electron flow.
  • CD38-knockout CD4+ T cells more effectively control tumor growth when compared to wild type CD4+ T cells, express twice the level of interferon-gamma (IFNy), and up regulate Sirtl,23,24 a key NAD-dependent regulator of effector function within T cells (Fernandez 2018).
  • IFNy interferon-gamma
  • Adenosine is considered a crucial mediator of the immune response.
  • Fig. 2 There are two adenosinergic pathways (Fig. 2) associated with exogenous adenosine (ADO) generation.
  • the better-known pathway involves the nucleoside triphosphate diphosphohydrolase known as cluster of differentiation 39 (CD39).
  • CD39 performs two sequential hydrolysis reactions: ADO triphosphate (ATP) is converted to ADO diphosphate (ADP) followed by the conversion of ADP to ADO monophosphate (AMP). Finally, the hydrolysis of AMP to ADO is mediated by the ectoenzyme cluster of differentiation 73 (CD73) (Yegutkin 2002).
  • CD39 pathway has been regarded as the predominant source of exogenous ADO, there is skepticism regarding the complete functionality of this pathway in vivo.
  • the optimal pH for CD39-mediated hydrolysis of ATP and ADP is 8.0-8.5, suggesting that ADO production via CD38 ADO may predominate in the acidic TME (Leal 2005, Milosevic 2012, Gordon 1986).
  • a lesser known adenosinergic pathway is mediated by CD38, which hydrolyzes NAD + to ADPR.
  • ADPR is in turn converted to ADO by the ectoenzymes cluster of differentiation 203a (CD203a) and CD73 (Fig. 2) (Horenstein 2013).
  • Adenosine receptors are known to be expressed in various immune cells, where they mediate the regulation of immune and inflammatory responses (Pasquini 2021).
  • Extracellular ADO which is prominent in the TME, stimulates the ADO receptor, A2AR, on the surface of immune effector cells, including T cells, natural killer cells, neutrophils, macrophages and dendritic cells, preventing their activation (Blay 1997, Gabrilovich 2014).
  • ADO produced by CD38 also promotes myeloid-derived suppressor cell (MDSC) expansion, macrophage M2 polarization, and CD4 + T regulatory cell generation, all of which support tumor cell progression (Kennedy 2020).
  • MDSC myeloid-derived suppressor cell
  • A2AAR stimulation down regulates IL-4 and IFN-g production (Antonioli 2019).
  • Regulatory T cells also produce adenosine, which stimulates A2AARS and reduces proinflammatory cytokine release via nuclear factor kB (NFkB) activation, leading to additional immunosuppression with a self-reinforcing loop (Ohta 2021, Romio 2011).
  • A2AAR Activated killer cells
  • NK natural killer cells
  • IFN-g tumor necrosis factor
  • TNFa tumor necrosis factor
  • GM-CSF granulocyte/macrophage colony- stimulating factor
  • ADO is implicated in promoting angiogenesis in endothelial cells and cultured tumor cells under hypoxic conditions via modulation of hypoxia-inducible-factor- 1 (HIF-la) (Auchampach 2007, Kazemi 2018, Maugeri 2019).
  • HIF-la hypoxia-inducible-factor- 1
  • CD38 The role of CD38 as an immunomodulator in cancer has been recently reviewed (Li 2020, Wo 2019).
  • the ectoenzyme CD38 serves both as an NADase that reduces the cellular availability of NAD + , and is also involved in the generation of ADO.
  • tumor cells that highly express CD38 such as multiple myeloma (MM), neuroblastoma (NB) and acute lymphocytic leukemia (ALL), both of these pathways lead to immunosuppression.
  • MM multiple myeloma
  • NB neuroblastoma
  • ALL acute lymphocytic leukemia
  • CD38 activity results in lower levels of extracellular NAD + , which supports Warburg metabolism and enhances the production of building blocks for cancer proliferation via anaerobic glycolysis (Yaku 2018).
  • T cell dysfunctionality in tumors is a dynamic process, wherein the CD38-NAD + -Sirtl axis acts as a key determinant of the therapeutic efficacy of anti-tumor T cells (Chatterjee 2018, Chatterjee 2019). While MM is regarded as a prime target for CD38 immunotherapy, it is also a prognostic factor for neuroblastoma, acute myeloid leukemia, chronic lymphocytic leukemia, prostate cancer, pancreatic cancer, acute B lymphoblastic leukemia, lung cancer, hepatocellular cancer and triple-negative breast cancer (Li 2020), suggesting that CD38 inhibition is a viable strategy for a variety of tumor types. Highly proliferating cells, such as T cells and many tumor cell lines, are dependent on NAD + , suggesting that their activity is likely to be further enhanced by inhibition of the NADase function of CD38. Target CD38 may be used as an NAD boosting therapy.
  • CD38 as a therapeutic target Immunotherapy is on the frontier of cancer treatment, but to date there are no FDA approved small molecule agents for cancer immunotherapy.
  • CD38- targeted immunotherapies are MAbs that have the same mechanism of action: binding to CD38 and facilitating ADCC in tumor cells.
  • patients experience a conserved panel of adverse events independent of the prescribed immunotherapy.
  • the administration of these antibody therapies by IV infusion is not optimal, and the cost of a course of therapy with these agents is untenably high.
  • novel small molecules for immunomodulation through specific inhibition of CD38 could also become an important new class of antitumor agent for use in patients.
  • daratumumab has been approved for use in diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma and MM (Lokhorst 2015), while isatuximab has been approved for refractory MM (Martin 2019). Resistance has already been noted in the case of daratumumab (Saltarella 2020), and is expected to appear for isatuximab, although the resistance mechanism for CD38-targeted antibodies appears to be complex (Franssen 2020).
  • NAD + mimetics have been developed and tested for CD38 inhibition in the context of aging, mitochondria dysfunction, obesity, and diabetes (Chini 2019, Chini 2009, Don 2011, Escande 2013, Becherer 2015). Notably, some of these inhibitors were successful in enzymatic studies, and in some cases promoted increases in the levels of NAD + in vivo, but none were evaluated for antitumor or immunostimulatory effects (Chini 2018, Dong 2011, Becherer 2015, Haffner 2015). Many of the CD38 inhibitors described to date are reportedly toxic in animal models or possess undesirable pharmacokinetic profiles including poor oral bioavailability.
  • CD38 knockout cell lines and mice underscore the suitability and importance of CD38 as a drug target (Covarrubias 2020, Guerreiro 2020, Gurney 2020, Higashida 2011, Bu 2018, Ogiya 2020).
  • selected compounds are evaluated for antitumor efficacy in murine models of MM and neuroblastoma.
  • a curated compound library was then constructed consisting of compounds meeting the above criteria. A total of 100 compounds were selected for the initial in silico screen that included hemi-peptidomimetic macrocycles, as well as mono-, bi-, and tricyclic heterocycles. An active site model is then used to perform virtual screening of the curated library using standard docking experiments (Molecular Operating Environment, Montreal, CA). The 24 compounds with the most favorable docking scores were then selected and obtained for physical screening.
  • Compound 1 exhibited an IC50 value of 4.0 pM against CD38 hydrolase (Fig. 5, panel C) and >100 pM against CD38 cyclase, while 12 had an IC50 of 20.8 pM against CD38 cyclase (Fig. 5 panel D) and >100 pM against CD38 hydrolase.
  • Compound 12 was selected for structural optimization primarily due to its selectivity for the CD38 cyclase activity and because it was the most synthetically feasible for library construction..
  • the kinetic mechanism of inhibition for the hydrolase and cyclase activities of CD38 were determined using a standard Michaelis-Menten approach, as shown in Fig. 6. The results suggest that both 1 and 12 produce a mixed/uncompetitive inhibition of the hydrolase and cyclase activities, respectively.
  • the known inhibitor 78c (Becherer 2015, Haffner 2015) is a potent inhibitor of the hydrolase (IC50 76 nM), but has very little activity against the cyclase. These data are consistent with the reported IC50 value for 17, which exhibited an IC50 of 7 nM against CD38 hydrolase via mixed kinetics (Haffner 2015). The difference in the IC50 value obtained in our experiment is likely due to differences in assay conditions.
  • cytotoxicity of 1 was monitored in activated peripheral blood mononuclear cells (PBMCs) over a concentration range of 1 nM to 100 pM, as shown in Fig. 7.
  • Compound 1 began to exhibit cytotoxic effects in PBMC cells at 1.0 pM, which is lower than its IC50 value.
  • compound 12 was relatively non-toxic up to 50 pM, which is 2.5-fold higher that its IC50 value.
  • cytotoxicity of 1 and 12 against MM in vitro was determined using the NCI-H929 human MM line (Fig. 12). These cells exhibit significant expression of CD38 (Panel A), as shown by flow cytometry. Cells were incubated for 48 h over a 0.01 - 100 mM range and viability was determined using the CellTiter-Glo assay (Panel B). Compounds 1 and 12 exhibited IC50 values of 3.37 and 28.6 mM, respectively, compared to a 4.16 mM for 78c.
  • a coculture experiment was conducted to compare the ability of CD38 inhibitors 78c and 1 to promote immune cell-based cytotoxicity in vitro.
  • Compounds were added at 1 rnM to a culture containing a GD2-targeted immunokine (antibody clone chl4.18) conjugated to recombinant IL- 2 (50 ng/mL), SH-SY5Y NB (30,000 cells/well) and NK cells (30,000 cells/well).
  • the results demonstrate that 1 is highly effective at promoting NK cell-mediated tumor toxicity (12% reduction after 90 min).
  • 1 is significantly more effective than 78c (IC50 7 nM), despite a significantly higher IC50.
  • IFNy levels were measured by ELISA.
  • compounds 1 and 12 produced 160.1 and 212.0% increases in IFNy, respectively, compared to an 82.9% increase in IFNy following treatment with 78c.
  • IFNy levels were 21.1, 108.1 and 145.9% for 78c, 1 and 12, respectively, slightly lower than IFNy levels observed after 48 hour incubation.
  • the binding mode of 12 in the CD38 active site was simulated starting with X-ray structure 4XJT, as shown in Fig. 10.
  • the bound inhibitor 4-[(2,6-dimethylbenzyl)amino]-2- methylquinoline-8-carboxamide (Becherer 2015) and the covalent bond between ADPR and CD38 were removed, and compound 12 was docked in the active site as described above.
  • the 2-thioxothiazolidin-4-one ring nitrogen appears to form hydrogen bonds with LYS 129 and ASP 155.
  • the thiazolidine ring nitrogen and an adjacent carbonyl coordinate a water molecule in the active site. In this orientation, there appears to be a pi stacking arrangement between 12 and ADPR that stabilizes binding.
  • a library of analogues of 12 was produced similar synthesis shown in Scheme 2 below.
  • Compound 12 may be synthesized in a single microwave step using a modified Knoevenagel condensation reaction between 2-thioxothiazolidin-4-one 18 and 5-(3- (trifhioromethyl)phenyl)furan-2-carbaldehyde 19 (Scheme 1) predominantly as the Z-isomer (Kandeel 2009, Russell 2009, Song 1999). This synthetic approach was then employed to introduce chemical diversity during structural optimization, resulting in compounds 20- 46 (Table 2).
  • CD38 inhibitors of CD38 have been reported, but to date none have been evaluated for stimulatory effects in immune cells. Indeed, we are the first to use small molecules to target the enzymatic activity of CD38 as an antitumor strategy.
  • a series of CD38 inhibitors have been identified based on a Z-5-ethylidinethiazolidine-2, 4-dione scaffold that are potent inhibitors of CD38, and that in some cases show a marked selectivity for inhibition of cyclase activity. The most potent of these compounds, 32, exhibits an IC50 value of 3.15 pM in the CD38 cyclase assay. It has been demonstrated that these compounds inhibit CD38 by a mixed/uncompetitive mechanism.
  • SAR structure-activity relationships
  • the most active analogues (12, 20, 22, 23, 30, 32) generally possess large phenyl or biphenyl substituents that are in conjugation with the Z-double bond moiety, and except for 20 are substituted with a halogencontaining substituent, indicating that binding to the cyclase catalytic site depends on TI- interactions with nearby residues on the protein.
  • these compounds have IC50 values in the micromolar range, the possibility of off-target effects exists.
  • CD38 inhibitors that are structurally related to 1 are being used as a chemical probe for the hydrolase activity of CD38. With selective hydrolase and cyclase inhibitors as chemical tools, a determination as to what extent selective inhibition of the hydrolase and cyclase activities of CD38 contribute to the activation of immune cells, and which activity plays the greatest role in development of resistance will be possible.
  • CD38 The critical role of CD38, which is expressed on B, NK and T-cells as well as macrophages, has been well studied as a target for cancer immunotherapy (Gao 2021, Morandi 2018, Morandi 2021). As mentioned above, CD38 metabolism has been shown to be a major factor in the development of resistance to MABs. As such, the CD38 inhibitors described herein may be of great value as adjuncts to existing therapy with checkpoint inhibitors such as daratumumab and isatuximab. However, CD38 inhibition could also be exploited in the treatment of other diseases resulting from depletion of NAD + and/or over production of ADO.
  • NAD + levels are associated with various metabolic diseases (diabetes, obesity, dyslipidemia and nonalcoholic fatty liver) (Li 2021, Okabe 2019) and in aging (Zapata 2021, Massudi 2012, Zhu 2015). It also plays a major role in the immune response to infectious disease (Groth 2021) including CO VID- 19 (Habeichi 2021). ADO is a potent immunosuppressant, and plays a similar role in the development and progression of disease (Habeichi 2021). Taken together, these observations indicate an increasing medical need for small molecule inhibitors of CD38 for use in diseases where dysregulated NAD + and ADO metabolism are a factor.
  • a modified PDB was prepared from PDB 4XJT (human CD38 complexed with inhibitor 2 [4- [(2,6-dimethylbenzyl)amino]-2-methylquinoline-8-carboxamide]).
  • PDB 4XJT was imported into the AMBER10 program, and prepared by first breaking the bond between ADPr phosphate and Gln226 (mutated from Glu226) followed by substitution of a hydroxyl on the 1 ’ position of ADPr phosphate. The Gln226 was repacked as Glu226. The 2’ phosphate was removed from ADPr phosphate rendering ADPr. The complex was protonated at 310 K and pH 7.4 and was minimized.
  • a compound (ligand) database of the 100 compounds populating the curated database outlined in the main manuscript was created, and structures were protonated at pH 7.4 and minimized.
  • the 2-methylquinoline-8-carboxamide inhibitor in PDB 4XJT was removed from the complex and dummy atoms were used to map the active site.
  • Compounds were docked into the active site using a triangle matcher and 50 poses were scored using London dG. Placements were further refined using an induced fit method and the top 5 poses were scored with GBVI/WSA dG.
  • the docking campaign was designed such that ADPr and CD38 active site moieties would interact with docking compounds — simulating uncompetitive binding.
  • Protein Ligand Interaction Fingerprints PLIFs were used to assess and annotate predictive binding poses along with observations from cocrystal structures described herein.
  • Microwave synthetic procedures were conducted on an Initiator 8 microwave synthesizer (Biotage, Charlotte, NC).
  • Preparative scale chromatographic procedures were carried out using a Biotage Selekt chromatography system (Biotage, Charlotte, NC) fitted with silica gel 60 cartridges (230-440 mesh).
  • Thin layer chromatography was conducted on Merck precoated silica gel 60 F-254.
  • Apigenin, quercetin and compound 17 were purchased from Selleckchem (Houston, TX), and compounds 1-16 were purchased from Chembridge (San Diego, CA).
  • the sealed vial was microwave irradiated for 10 minutes at 180 °C, during which time the reaction mixture changed from a colorless solution to a vibrant orangeyellow suspension.
  • the reaction mixture was allowed to cool to room temperature, the vial was opened and completion of the reaction was verified by TLC (1 : 1 EtOAc/hexane).
  • the reaction product was precipitated by adding 5 mL of water, the mixture was centrifuged (10,000 X G, 10°C, 5 minutes) and the liquid was decanted. The resulting solid was then washed with an additional 5 mL of water, re-centrifuged and the liquid was decanted.
  • CD38 cyclase activity Compounds were screened for the ability to inhibit the cyclase activity of recombinant human CD38 in a fluorometric assay.
  • recombinant CD38 was diluted to a working concentration of 40 nM (4x) in assay diluent (PBS, 0.002% Tween-20, pH7.4) and 25 pL were pipetted into a black 96 well plate. Screening compounds were diluted using assay diluent to 4x the desired screening concentration and 25 pL of this mixture was added to each wells. DMSO was screened as a vehicle control.
  • hydrolase screening assay was conducted using the same procedure as the cyclase screen, substituting 20pM s-NAD + working solution for NGD + , resulting in a final concentration of lOpM. Initial rates were calculated for the first 2.5mins.
  • PBMC and T cells were cultured in accordance with the Stem Cell Technology T-cell Expansion Protocol.
  • an initial culture was seeded at 1 X 10 6 cells/mL in ImmunoCult XF expansion media with 3-10 ng/mL of IL-2 (complete media).
  • the culture was diluted 4- to 8-fold every 2 to 3 days with complete media.
  • T cell activation PBMCs were cultured as above with the addition of 20 pL/mL of anti- CD3/CD38 tetramer on day 0. Activated T cell cultures were maintained for 14 days on average. For re-stimulation challenges, 20 pL/mL of anti-CD3/CD28 was added to activated T cells (day 9-12).
  • PBMC toxicity Cytotoxicity of CD38 hit inhibitors were assessed against T cell activated PBMCs. After a 48 hr treatment, cell viability was assessed using CellTiter-Glo (Promega), with all experiments performed according to the manufacturer’s directions. Viability was assessed in three separate donors. Experiments were conducted in triplicates and mean values normalized to vehicle treated controls.
  • T cells were activated and cultured as described above for 12 days. Secreted cytokines were measured in the supernatants from T cells by ELISA (Biolegend). For acute T cell activity and screening of CD38 inhibitors, supernatants were analyzed 48 hrs post-activation. Supernatants were also collected on day 14, 48 hrs after restimulation of T cell cultures to assess effects of CD38 inhibitors in a re-challenge/expansion model.
  • Initial interferon- X screens were conducted with a single donor in triplicate and results recorded as percent difference from vehicle treated control. Screening of novel CD38 inhibitors was conducted with three donors and presented as mean percent difference from vehicle treated control.
  • Cellular NADH levels were measured using the NAD-Glo kit (Promega) — with all experiments performed according to the manufacturer’s directions. In short, NADH was measured in resting PBMCs, and PBMCs 30 and 60 mins after activation.
  • Bispecific T cell engagers such as blinatumomab, an FDA-approved treatment for B cell Acute lymphoblastic leukemia (ALL), are a new class of cancer immunotherapy that harness the anti-tumor potential of T cells.
  • Blinatumomab simultaneously engages CD 19 on leukemic B cells and CD3 on T cells, creating an immunological synapse between cancer cells and T cells that closely resembles the synapse formed between T cells and antigen presenting cells (APCs) (Offner 2006, Mack 1995). Formation of the synapse stimulates T cell activation via the T cell receptor (TCR) complex and directs a potent anti-cancer T cell response.
  • TCR T cell receptor
  • T cell dependent cellular cytotoxicity (TDCC) assay was established by co-culturing CD 19+ Raji and REH B cell ALL cell lines with primary PBMCs, which are composed of -70% naive T cells.
  • the addition of blinatumomab to these co-cultures induced significant T cell activation, as determined by IFNy release, and resulted in T cell dependent killing of ALL cancer cells (Fig. 14).
  • the CD38 inhibitor 1 enhanced T cell activation induced by low-dose blinatumomab, giving support to our hypothesis that inhibition of CD38 catalytic activity using small molecule inhibitors will induce an immune related response and enhance the activity of T cell-based therapies given the immunosuppresive function of CD38.
  • BCMA B cell maturation antigen
  • BPS Biosciences BCMAxCD3 bispecific T cell engager antibody
  • Multiple BCMA targeted immunotherapies including bispecific T cell engagers, chimeric antigen receptors (CARs), and antibody drug conjugates are currently in clinical studies for the treatment of MM. Thus, these studies have high translational relevance.
  • T cell activation will be measured by ELISA for IFNy and TNFa and by multicolor flow cytometry for CD69-CD44-CD4-CD8 expression.
  • B cell ALL and MM cell specific death are measured using the LDH release assay described above and by flow cytometric analysis of the apoptosis marker cleaved caspase-3 in GFP-labeled cancer cells.
  • CD38 activity is determined with a fluorometric assay kit according to the manufacturer’s instructions (BioVision; K2042). MM or NB cells are incubated with selected compounds (at IC50, as determined above). At the end of treatment (48 hrs), 1x10 6 cells are collected in 200 pl ice-cold CD38 lysis buffer on ice and centrifuged at 10,000 x g for 10 min. Supernatants (50 pl) are added to a well of a 96-well white plate (flat bottom) including blank controls (buffer only) and positive control (included in kit).
  • DMPK drug metabolism and pharmacokinetic
  • the kinetic aqueous solubility assay is a turbidimetric method proposed by Lipinski (Lipinski 2001).
  • DMSO dimethylsulfoxide
  • OD 600-820 nm precipitation occurs
  • the plasma/blood stability assay (Di 2005) is used to measure protein binding and degradation of compounds in plasma and blood, since compounds which rapidly degrade in plasma generally show poor in vivo efficacy (except pro-drugs).
  • Compounds (10 pM) are incubated for 0, 15, 30, 60, 120 min with plasma from mice, rats, dogs, monkeys, and humans and the free fractions are separated by ultracentrifugation. A positive compound that undergoes degradation in plasma is used as control.
  • microsomal stability assay assesses the intrinsic clearance of new compounds (3 pM) by incubation with mouse liver microsomes (0.5 mg/ml) at 30 min. The disappearance of analogs in samples is analyzed using LC-MS/MS. Both initial and overall disappearance rates and half-lives (T1/2) are calculated. Desirable metrics are TI/2 > 30 min.
  • PK Pharmacokinetic in vivo studies are performed with both male and female C57BL/6 mice (Jackson Laboratory) as previously performed for DFMO in mouse plasma and brain tissue (Schultz 2021).
  • selected compounds are administered to mice by intravenous (i.v.) injection and oral gavage (p.o.) and multiple blood samples collected.
  • Mice are anesthetized with 240 mg/kg Avertin by i.p. injection at 10 min, 30 min, 1 hr, 2 hrs, 4 hrs, and 6 hrs post treatment. Blood is drawn for serum collection by intracardial puncture and collected from 2 mice/group/ time point. A refrigerated centrifuge (3,500 rpm for 10 min) is used to separate whole blood from plasma.
  • Plasma samples are transferred directly to cryotubes and stored at -80°C until LC analysis.
  • N 24 C57BL/6 mice/compound.
  • Mouse plasma samples are analyzed.
  • PK parameters are estimated using standard noncompartmental methods.
  • Area under the curve (AUC) for plasma and concentrations is calculated using the trapezoidal rule.
  • the AUC from the last measured time point to infinity (AUCO-inf) is estimated by dividing the last measured concentration by the elimination rate constant. Apparent plasma clearance is calculated by dividing the dose given by AUCO-inf.
  • Metrics for advancement is T1/2 > 1 hr.
  • Compounds with %F>35 is generally considered suitable for administration by oral gavage. Results'. It is contemplated that CD38 inhibitor candidates are identified with high potency, superior PK characteristics, and favorable therapeutic index.
  • LDH assays from Promega are an efficient way to determine cytotoxicity in suspension and adherent cells (Archer 2010)
  • LDH assays from other companies are also available. Further assay optimization may be required, and if cytotoxicity is too low or too high after 48 hrs, drug exposure time is increased or decreased to 72 hrs or 24 hrs, respectively.
  • a number of other cell culture assays may be used to test viability/metabolic rate (MTS), proliferation (BrdU), and apoptosis (cleaved caspase-3, Annexin V) as well as flow cytometry to measure cell cycle arrest and apoptosis (Schultz 2018, Uhl
  • the drug range for each compound may be determined empirically by first testing each drug at 10 pM and then adjusting the range based on the % inhibition received at this initial concentration/exp eriment.
  • Other MM and NB cell lines are available for testing (MM: MM1.S, MM1.S BzR, MM1.R, U266, U266 BzR, ARD; NB: IMR- 32, CHP-134, , SK-N-BE, LAN1, Kelly) and many others representing heterogeneity and different cytogenetic subclasses (Schultz 2018, Pierce 2018, Pierce 2020, Nolan 2020, Mooney
  • the anti-tumor efficacy of compounds are validated in vivo, using two preclinical murine models that resemble the human MM pathology and two reliable NB mouse models. Following drug tolerance studies for each compound, the NSG xenotransplant model and the immunocompetent Vk*MYC transgenic model are used to determine the potential clinical utility of these drug candidates in mice that develop MM.
  • NB a patient-derived xenograft (PDX) model88 and the well-established transgenic TH-MYCN mouse model95 is used.
  • MTD and LD Drug tolerance studies in vivo.
  • the goal of this study is to determine the maximum tolerated dose (MTD) and lethal dose (LD) of selected CD38 inhibitors in vivo.
  • Mouse model # 1 MM xenotransplant model.
  • NOD-SCID IL2Rgamma null (NSG) mice are inoculated with MM cell suspensions (MM.1 S BzR, 1x10 6 cells) systemically via the lateral tail vein. It has previously been shown that this gives rise to an aggressive and reliable model of MM (Robinson 2019). To date, >95% of all untreated or control treated mice have died with detectable levels of MM in their bone marrow between days 40-50 post injection (Robinson 2019). Study Endpoints. This model offers multiple study endpoints including animal survival and quantification of MM cells that are harvested from mouse bone marrow aspirates and detected using the plasma cell specific marker CD138+. Treatment Groups and Dosing.
  • MM cells will be quantified in the bone marrow aspirates of mice, randomly selected mice are sacrificed on day 35. Animals are euthanized and bone marrow aspirates from individual mice are harvested from the femurs of hind limbs. Cells will then be stained with fluorescent-conjugated antibodies specific for human CD138 or human HLA-ABC cell surface antigens as previously published (Robinson 2019). The percentage of cells that are positive for the indicated markers are then be quantified by flow cytometry at the Van Andel Research Institute (VARI) flow cytometry core facility (fee-for- service, core agreement attached) to determine the extent of MM tumor burden.
  • VARI Van Andel Research Institute
  • Vk*MYC immunocompetent transgenic mice are provided.
  • the range of bone marrow PC infiltration encompasses true MGl .'S disease up to overt MM.
  • These mice have on average lower hemoglobin levels, reduced BMD and MM like kidney damage, thus resembling the human disease (Rossi 2018).
  • the expression of the MYC oncogene is driven by regulatory elements of the IgK light chain gene, which are active in cells of the B lineage and significantly increase during plasma cell differentiation.
  • mice develop a slowly progressing monoclonal expansion of plasma cells within the bone marrow, and this phenotype closely resembles the human MM pathology (Chesi 2008). In addition to offering a pathologically relevant model system in animals with an intact immune system, this model was also shown to faithfully predict single agent clinical MM activity (Chesi 2012), with an updated positive clinical predictive value of 73% and negative predictive value of 92%.
  • Vk*MYC Endpoints Vk*MYC mice produce high levels of serum immunoglobulins, resulting in an M spike that is readily detectable by serum protein electrophoresis (SPEP) starting at 20 weeks of age.
  • SPEP serum protein electrophoresis
  • Serum M spike quantification is a biomarker of tumor burden in these mice and has been used previously to evaluate therapeutic responses to FDA approved and experimental agents (Chesi 2016, Schmidt 2013).
  • SPEP M spike quantification is an endpoint in adult Vk*MYC transgenic experiments.
  • Vk*MYC Treatment Groups and Dosing Treatments with vehicle, positive control bortezomib (0.25 mg/kg, i.p., days 1 and 3 of each 7-day cycle) and selected compounds will be administered i.p. (daily) at the dose determined by MTD experiments in C57BL/6 mice.
  • Mouse model #3 NB patient-derived xenograft (PDX) model.
  • PDX patient-derived xenograft
  • a total of 5 PDX models are provided which have been expanded in mice and show that PDX grow well and can be used for drug studies, as we previously performed with PDX COG-N-623 (Schultz).
  • twoMYCN-amplified Stage IV PDX COG-N-623 and COG-N-421) are used.
  • CD38 inhibitors chosen. Each one of these CD38 inhibitors is assigned to its own group and the last group is the control group. Drug dose is determined based on MTD outcomes.
  • Female or male athymic nu/nu mice are used with two or more treatment groups x2 routes of administration x 2 PDX xlO mice/group, plus 10% attrition rate for study loss. Mice will be euthanized with a Euthenex Prodigy CO2 delivery system when tumor volumes reach 2500 mm 3 .
  • Mouse model #4 NB transgenic TH-MYCN mouse model.
  • hemizygous and homozygous transgenic TH-MYCN mice are used. These mice spontaneously develop tumors that are histologically similar to those arising in Stage IV, MYCN -amplified NB, including syntenic gain and loss of chromosomes (Weiss 1997).
  • TH-MYCN mice express the human MYCN gene under the control of the rat tyrosine hydroxylase promoter (TH-MYCN), as previously described for the ODC inhibitor DFMO.
  • TH-MYCN rat tyrosine hydroxylase promoter
  • Drug dose and route of administration is selected based on outcomes of toxicity studies.
  • IHC immunohistochemistry
  • the collected bone marrow samples are analyzed for CD138 by flow cytometry and immunohistochemical (IHC) methods.
  • Femurs from hind limbs of NSG mice are harvested at sacrifice (day 35) and half of the tumor material fixed in 10% neutral buffered formalin and paraffin embedded for histologic studies.
  • Ki-67 (proliferation), caspase 3/8 (apoptosis), p27/p-Rb (cell cycle), and CD138 are detected in collected bone marrow samples by IHC.
  • Formalin-fixed paraffin embedded bone marrow samples are decalcified and analysis of immune cell components in the tumor microenvironment are assessed to correlate the effects of CD38 inhibition on anti-myeloma immune cell activity.
  • T cell activity in MM lesions is evaluated and quantified using an IHC multiplexing approach with an optimized panel of T cell markers including CD8, Tbet/CD4, and FoxP3 for cytotoxic T-cells, helper T- cells, and T-regs, respectively.
  • T cell markers including CD8, Tbet/CD4, and FoxP3 for cytotoxic T-cells, helper T- cells, and T-regs, respectively.
  • CD38-positive cancers e.g. hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), melanoma, pancreatic ductal adenocarcinoma (PDAC), glioma or breast cancer
  • HCC hepatocellular carcinoma
  • NSCLC non-small cell lung cancer
  • PDAC pancreatic ductal adenocarcinoma
  • glioma or breast cancer glioma or breast cancer
  • PD pharmacodynamic studies are performed to test whether the selected inhibitors have reached the target site, by measuring CD38 enzyme activity in excised tumors using the fluorometric CD38 activity assay for biological samples (K2042, BioVision).
  • TH-MYCN transgenic mice are frequently used to study drug efficacy against mouse NB in an immunocompetent environment and will be an excellent model to assess selected CD38 inhibitor analogs developed in this study.
  • Saltarella I. et al., Cells 2020, 9 (1).
  • X is S or O; is a single bond or a double bond;
  • Ri is H, alkyl, lower alkyl, or CH2COOH
  • R2 is H, CH3, CF3, OCH3, OCF3, SO2CH3, or a phenyl group optionally substituted with at least one of CH 3 , CF 3 , OCF3, OCH 3 , SO2CH3, or or R2 is
  • R3 is lower alkyl, or OCH3;
  • R 4 is lower alkyl;
  • R 5 is lower alkyl, or OCH 3 ;
  • R 6 is NHR 12 , lower alkyl or phenyl;
  • R7 is R 11
  • R 8 is H, lower alkyl, R9 , R10 and R11 are each independently lower alkyl, and
  • R12 is alkyl or lower alkyl.
  • a pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of paragraphs 1-11, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • a method for the treatment of a cancer comprising the step of administering to a subject in need thereof a therapeutically effective amount of a compound according to any one of paragraphs 1-11, or a pharmaceutically acceptable salt thereof, thereby treating the subject.
  • the cancer is multiple myeloma (MM), acute lymphocytic leukemia, neuroblastoma (NB), neuroblastoma, acute myeloid leukemia, chronic lymphocytic leukemia, prostate cancer, pancreatic cancer, lung cancer, acute B lymphoblastic leukemia, diffuse large B cell lymphoma, hepatocellular cancer, triple-negative breast cancer, follicular lymphoma, or mantle cell lymphoma.
  • MM multiple myeloma
  • NB neuroblastoma
  • neuroblastoma acute myeloid leukemia
  • chronic lymphocytic leukemia chronic lymphocytic leukemia
  • pancreatic cancer lung cancer
  • acute B lymphoblastic leukemia diffuse large B cell lymphoma
  • hepatocellular cancer triple-negative breast cancer
  • follicular lymphoma or mantle cell lymphoma.
  • the second antagonist therapy comprises administering a therapeutically effective amount of a second antagonist other than the compound, wherein the second antagonist is a CD38 antagonist, or a PD1 or PD-L1 antagonist.
  • the second antagonist is: a. an antibody, or antigen binding fragment of an antibody, that specifically binds to, and inhibits activation of, an second receptor, or b. a soluble form of an second receptor that specifically binds to a second ligand and inhibits the second ligand from binding to the second receptor, wherein the second receptor is a CD38 receptor or a PD1 receptor and the second ligand is a CD38 ligand, or a PD1 ligand.
  • the second antagonist is a PD-1 antagonist which is nivolumab, pembrolizumab, avelumab, durvalumab, cemiplimab, or atezolizumab.
  • a kit for treating a patient suffering from cancer comprising a therapeutically effective amount of the compound of any one of paragraphs 1-11, a therapeutically effective amount of a second antagonist other than the compound, and an insert comprising instructions for use of the kit, wherein, wherein the second antagonist is a CD38 antagonist, or a PD1 or PD-L1 antagonist.
  • a pharmaceutical composition comprising an amount of an second antagonist and an amount of the compound of any one of paragraphs 1-11, wherein the second antagonist is a CD38 antagonist or a PD1 or PD-L1 antagonist.
  • a therapeutic package for dispensing to, or for use in dispensing to, a subject afflicted with cancer which comprises: a) one or more unit doses, each such unit dose comprising: i) amount of a second antagonist and ii) an amount of a compound of any one of paragraphs 1-11 wherein the respective amounts of said second antagonist and said compound in said unit dose are effective, upon concomitant administration to said subject, to treat the subject, and b) a finished pharmaceutical container therefor, said container containing said unit dose or unit doses, said container further containing or comprising labeling directing the use of said package in the treatment of said subject, wherein the second antagonist is a CD38 antagonist, or a PD1 or PD-L1 antagonist.
  • a second antagonist for use as an add-on therapy or in combination with a compound of any one of paragraphs 1-11 in treating a subject afflicted with cancer wherein the second antagonist is a CD38 antagonist, or a PD1 or PD-L1 antagonist.
  • a method for the treatment of a disease comprising the step of administering to a subject in need thereof a therapeutically effective amount of a compound according to any one of paragraphs 1-11, or a pharmaceutically acceptable salt thereof, thereby treating the subject, wherein the disease is a disease resulting from depletion of NAD + or a disease resulting in over production of ADO.
  • the disease is a metabolic disease, diabetes, obesity, dyslipidemia, nonalcoholic fatty liver, an infectious disease or COVID-19.

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Abstract

La présente invention concerne des composés ayant la structure générale des formules I et II, et d'autres composés ayant une activité similaire, y compris tous les sels pharmaceutiquement acceptables de ceux-ci. L'invention comprend l'utilisation de tels composés pour le traitement de maladies, par exemple, pour le traitement du cancer.
PCT/US2022/043065 2021-09-09 2022-09-09 Ciblage sélectif de l'activité de cd38 en tant que stratégie immunostimulatrice et antitumorale Ceased WO2023039170A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080108677A1 (en) * 2004-08-30 2008-05-08 Karyon-Ctt Ltd Thioxothiazolidinone Compounds For Use As Pharmaceuticals
US20130090339A1 (en) * 2010-06-17 2013-04-11 The Uab Research Foundation Compounds useful as antiviral agents, compositions, and methods of use
US20150051185A1 (en) * 2008-07-02 2015-02-19 Astrazeneca Ab Chemical Compounds 251
US20200317657A1 (en) * 2015-06-12 2020-10-08 Gb006, Inc. Solid forms of(z)-4-(5-((3-benzyl-4-oxo-2-thioxothiazolidin-5-ylidene)methyl)furan-2-yl)benzoic acid

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080108677A1 (en) * 2004-08-30 2008-05-08 Karyon-Ctt Ltd Thioxothiazolidinone Compounds For Use As Pharmaceuticals
US20150051185A1 (en) * 2008-07-02 2015-02-19 Astrazeneca Ab Chemical Compounds 251
US20130090339A1 (en) * 2010-06-17 2013-04-11 The Uab Research Foundation Compounds useful as antiviral agents, compositions, and methods of use
US20200317657A1 (en) * 2015-06-12 2020-10-08 Gb006, Inc. Solid forms of(z)-4-(5-((3-benzyl-4-oxo-2-thioxothiazolidin-5-ylidene)methyl)furan-2-yl)benzoic acid

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Title
DATABASE Pubchem 25 October 2006 (2006-10-25), ANONYMOUS : "5-(Furan-2-ylmethyl)-2-sulfanylidene-1,3-thiazolidin-4-one", XP093048030, Database accession no. CID 10176726 *

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