WO2023039170A1 - Selective targeting of cd38 activity as an immunostimulatory and antitumor strategy - Google Patents

Abstract

The present invention relates compounds having the general structure of formulas I and II, and other compounds having similar activity, including any pharmaceutically acceptable salts thereof. The invention includes the use of such compounds for the treatment of diseases, for example, for the treatment of cancer.

Description

SELECTIVE TARGETING OF CD38 ACTIVITY AS AN IMMUNOSTIMULATORY AND ANTITUMOR STRATEGY

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority of U.S. Provisional Application No. 63/320,876, filed March 17, 2022 and U.S. Provisional Application No. 63/242,321, filed September 9, 2021, the entire contents of each of which are hereby incorporated by reference herein.

FIELD OF THE INVENTION

The invention relates generally to chemical compounds and use thereof in treatment of diseases associated with altered CD38 enzymatic activity and/or altered NAD⁺/Ca²⁺ signaling.

All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes and to the same extent as if each individual publication, patent, patent application or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present invention.

BACKGROUND OF THE INVENTION

Considerable effort has been focused on the development of new methods to stimulate the human immune system in the tumor microenvironment (TME) as an antitumor strategy. Monoclonal immune checkpoint inhibitors (ICIs) targeting immunosuppressive complexes such as PD-1 :PD- L1 and CTLA4:CD80/CD81 promote an unhindered adaptive immune response. As a result, 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.

The ectoenzyme known as cluster of differentiation 38 (CD38) 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). Recent literature suggests that 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).

SUMMARY OF THE INVENTION

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.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph showing that in using two distinct catalytic reactions, CD38 produces ADP- ribose (ADPR) from both NAD⁺ and cADPR.

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. In each structure 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

It is to be understood that the descriptions of the present invention have been simplified to illustrate elements that are relevant for a clear understanding of the present invention, while eliminating, for the purpose of clarity, many other elements found in typical pharmaceutical compositions. Those of ordinary skill in the art will recognize that other elements and/or steps are desirable and/or required in implementing the present invention. However, because such elements and steps are well known in the art, and because they do not facilitate a better understanding of the present invention, a discussion of such elements and steps is not provided herein. The disclosure herein is directed to all such variations and modifications to such elements and methods known to those skilled in the art. Furthermore, the embodiments identified and illustrated herein are for exemplary purposes only, and are not meant to be exclusive or limited in their description of the present invention.

Technical and scientific terms used herein have the meaning commonly understood by one of skill in the art to which the present invention pertains, unless otherwise defined. Reference is made herein to various methodologies and materials known to those of skill in the art. Standard reference works setting forth the general principles of pharmacology include Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10^th Ed., McGraw Hill Companies Inc., New York (2001). Any suitable materials and/or methods known to those of skill can be utilized in carrying out the present invention. However, preferred materials and methods are described. Materials, reagents and the like to which reference are made in the following description and examples are obtainable from commercial sources, unless otherwise noted.

A compound according to the invention is inherently intended to comprise all stereochemically isomeric forms thereof. The term "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. Unless otherwise mentioned or indicated, the chemical designation of compounds denotes the mixture of all possible stereochemically isomeric forms. In particular, 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 (entgegen) 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.

Stereochemically isomeric forms of the compounds disclosed herein are obviously intended to be embraced within the scope of this invention. Additionally, stereochemically pure compounds are also within the scope of this invention.

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.

When a specific stereoisomeric form is indicated, this means that said form is substantially free, i.e. associated with less than 50%, preferably less than 20%, more preferably less than 10%, even more preferably less than 5%, further preferably less than 2% and most preferably less than 1% of the other isomer(s). Thus, when a compound of formula (I) is for instance specified as (R,S), this means that the compound is substantially free of the (S,R) isomer.

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. Preferably if a specific stereoisomer is desired, said compound will be synthesized by stereospecific methods of preparation. These methods will advantageously employ enantiomerically pure starting materials.

Definitions

The articles “a” and “an” are used in this disclosure to refer to one or more than one (i.e., to at least one) of the grammatical object of the article.

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.

The term “carrier”, as used in this disclosure, 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.

The term “treating,” with regard to a subject, 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. The term “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" or "administering to the (human) patient" 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. As used herein, "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.

As used herein, a "unit dose", "unit doses" and "unit dosage form(s)" mean a single drug administration entity/entities.

As used herein, "effective" or “therapeutically effective” when referring to an amount of a substance, for example an antagonist, refers to the quantity of the substance that is sufficient to yield a desired therapeutic response. In certain embodiments, 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. Thus, the combination of the two active compounds may be administered:

• as a combination that is part of the same medicament formulation, the two active compounds are then administered simultaneously, or

• as a combination of two units, each with one of the active substances giving rise to the possibility of simultaneous, sequential or separate administration.

As used 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.

As used herein, "add-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.

Any known CD38 antagonist may be utilized in the practice of the invention, a broad variety of which are known and disclosed in the art. The CD38 antagonist preferably neutralizes biological function after binding. The CD38 antagonist is preferably a human CD38 antagonist. Optionally, 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. Optionally, 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. Preferably a fragment is pegylated or encapsulated (e.g. for stability). The CD38 antagonist may also be a camelid antibody. As used herein, CD38 antagonists include but are not limited to CD38 receptor inhibitors.

Any known PD-1 antagonist may be utilized in the practice of the invention, a broad variety of which are known and disclosed in the art. The PD-1 antagonist preferably neutralizes biological function after binding. The PD-1 antagonist is preferably a human PD-1 antagonist. Optionally, 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. Optionally, 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. Preferably a fragment is pegylated or encapsulated (e.g. for stability). The PD-1 antagonist may also be a camelid antibody. As used herein, 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.

As used herein, the term "alkyl", alone or in combination with other groups, 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.

The term "lower alkyl", alone or in combination with other groups, 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 chemical structures provided herein are to be understood from the perspective of a person of ordinary skill in the art. It should be understood that when a structure has two or more possible configurations, both configurations are disclosed. For example, to the extent that A in the embodiments below can be connected in two different configurations, each configuration is independently disclosed.

Dosage and Administration:

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). The term "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.

The term “excipient” as used herein 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. The phrase “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, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-l -carboxylic acid, glucoheptonic acid, 3 -phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.

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. In powders, the carrier generally is a finely divided solid which is a mixture with the finely divided 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.

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. Examples of oily or nonaqueous carriers, diluents, solvents or vehicles 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. Alternatively, 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. The aerosol may conveniently also contain a surfactant such as lecithin. The dose of drug may be controlled by a metered valve. Alternatively 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, for example, 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.

As discussed above, 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. For oral administration, 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. Thus, for administration to a 70 kg person, 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. In such form, 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. Also, 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. Currently available 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. Thus, there is a critical, unmet medical need for smallmolecule CI agents aimed at new or traditional CI drug targets. 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. In addition, since 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.

As discussed above, 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. Other studies have shown that 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).

In tumor cells such as multiple myeloma (MM), CD38-targeted antibodies such as daratumumab and isatuximab bind to CD38 and promote cancer cell death by stimulating a robust antitumor immune response. These observations demonstrate the importance of CD38 in the activated immune response towards cancer and as a target for the discovery of novel cancer immunotherapies. Although ICIs are achieving unprecedented success in a percentage of cases, these therapies are expensive, and high rates of resistance limit their efficacy. Furthermore, 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. These limitations in CD38 targeted ICIs demonstrate that there is an urgent and unmet medical need for small molecule agents that boost the immune response to cancer.

As discussed herein, 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. However, other CD38 expressing cancers (e.g., acute myeloid leukemia, chronic lymphocytic leukemia, prostate cancer, pancreatic cancer, lung cancer, hepatocellular cancer, and triplenegative breast cancer) are also expected to be treatable with the herein described agents. Moreover, combination therapy with the disclosed compounds and candidate CD38 inhibitors and approved agents (e.g., bortezomib, carfilzomib, lenalidomide, panobinostat, dexamethasone, and selinexor) or approved antibody therapies (daratumumab, isatuximab) is contemplated.

As outlined above, 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. As such, the CD38 specific antibody therapies daratumumab and isatuximab have been used effectively against MM by binding CD38 and promoting tumor cell death through ADCC. Ironically, 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. The development of resistance to antibody therapies like daratumumab, coupled with their high cost and inconvenient route of administration, make it clear that there is a continuing medical need for small molecule inhibitors that can be used alone or in combination with antibody therapy for the treatment of cancer. To date, a handful of studies have been published describing small molecule inhibitors of CD38 for use in treating metabolic disorders, but none of these agents were designed to engage cancer targets. In addition, some existing small molecule CD38 inhibitors have been shown to produce unacceptable toxicities. This application describes newly discovered novel small molecule CD38 inhibitors. These inhibitors could potentially be used alone or in combination with anti-CD38 monoclonal antibodies (MAbs) and other agents for the treatment of MM and other tumor types, including solid tumors.

There are very few compounds that are known to inhibit the ectoenzyme CD38, and the hydrolase/cyclase selectivity profiles of known CD38 inhibitors remains undefined. As we have shown below, existing non-selective compounds such as 78c are less effective stimulators of immune function, and as such as less suitable for immunotherapy or for use as chemical tools. Further, no small molecule CD38 inhibitors have been designed to engage antitumor targets. The compounds disclosed herein selectively target the hydrolase or cyclase activity of CD38. The compounds herein have a significant effect on the release of IFNg and other cytokines, and lead to significant T-cell or NK cell activation. The compounds disclosed herein have favorable safety, efficacy and selectivity profiles.

EXAMPLES

The following examples further describe and demonstrate particular embodiments within the scope of the present invention. Techniques and formulations generally are found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.). The disclosure is further illustrated by the following examples, which are not to be construed as limiting this disclosure in scope or spirit to the specific procedures herein described. It is to be understood that the examples are provided to illustrate certain embodiments and that no limitation to the scope of the disclosure is intended thereby. It is to be further understood that resort may be had to various other embodiments, modifications, and equivalents thereof which may suggest themselves to those skilled in the art without departing from the spirit of the present disclosure and/or scope of the appended claims.

Example 1

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). Because 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).

Ironically, resistance to CD38-targeted MABs is mediated by CD38 itself. Importantly, 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). In vitro and in vivo studies demonstrate that CD38 inhibits CD8+ T-cell function via adenosine (ADO) receptor signalling. In addition, resistance to PD-1/PD-L1 blocking antibodies is mediated through upregulation of CD38 and subsequent production of ADO (Chen 2018). 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). Taken together, these data indicate that CD38 or ADO receptor blockade are effective strategies to overcome this resistance (Quarona 2013).

For multiple reasons, 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.

Role of CD38 in cellular energy metabolism

As an ectoenzyme, 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

(cADPR)(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. For example, it has been demonstrated that 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).

Role of CD38 in ADO generation Adenosine is considered a crucial mediator of the immune response. 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). While the 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). In naive CD4+ T cells, A2AAR stimulation down regulates IL-4 and IFN-g production (Antonioli 2019). Regulatory T cells (Tregs) 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). Likewise, stimulation of A2AAR on natural killer (NK) cells suppresses their tumor cell cytotoxicity and their production of IFN-g, tumor necrosis factor (TNFa), and granulocyte/macrophage colony- stimulating factor (GM-CSF), which are critical for effective antibody-dependent cell-mediated cytotoxicity (ADCC) (Lokshin 2006, Raskovalova 2005). Additionally, 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). Collectively these transformations cause a shift from an ATP-driven proinflammatory environment to an anti-inflammatory milieu induced by ADO-mediated down regulation of immune function (Gabrilovich 2014, Graeff 2006). The coupled immunosuppressive and pro-angiogenic effects of ADO signaling in the TME indicate that targeting ADO generation could be a useful therapeutic strategy, particularly in the context of improving ADCC treatment for cancer using immune checkpoint inhibitors such as daratumumab.

CD38 in cancer

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. In 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. 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). In addition, 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. Currently, all clinically available CD38- targeted immunotherapies are MAbs that have the same mechanism of action: binding to CD38 and facilitating ADCC in tumor cells. As such, patients experience a conserved panel of adverse events independent of the prescribed immunotherapy. In addition, 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. Thus, there is a need for novel small molecules for immunomodulation through specific inhibition of CD38. These agents could also become an important new class of antitumor agent for use in patients.

Although antibody-based immune checkpoint inhibitors are achieving unprecedented success in a percentage of cases, these therapies are expensive, and high rates of resistance limit their efficacy (Koyama 2016, Sharma 2017, Tumeh 2014). Importantly, CD38 up regulation appears to be one of the most important factors in mediating resistance to checkpoint blockade in MM and other cancers (Koyama 2016, Tumeh 2014, Chen 2018). In addition, resistance to PD-l/PD- L1 blocking antibodies is mediated through up regulation of CD38 and subsequent production of ADO (Chen 2018). To address this issue, antibodies targeting CD38 have emerged as promising immunotherapies. Two antibody therapies are available that target CD38; 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).

Over the past several decades, numerous small molecule 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. Studies involving 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). Herein details a medicinal chemistry approach to screen for activity- selective small molecule inhibitors of CD38 and to optimize their activity through structural modification. These compounds will be useful as chemical probes to better understand the mechanism by which CD38 produces resistance. Moreover, selected compounds are evaluated for antitumor efficacy in murine models of MM and neuroblastoma.

Results and discussion

The observations above led to the initiation of a screen for small molecule inhibitors of CD38 that are selective for either the cyclase or hydrolase activity of CD38 and to optimize their immunostimulatory activity through structural modification. In order to identify novel non- NAD⁺ mimetic scaffolds as CD38 inhibitors, a model consensus structure of CD38 was constructed via in silico analysis of published substrate/inhibitor co-crystals of CD38 (PDB 2165, 2O3U, 4F45, 4XJT and 2O3T) using the Molecular Operating Environment software (Chemical Computing Group, Montreal, CA) (Fig. 3) (Graeff 2006, Graeff 2009, Liu 2007, Liu 2005, Liu 2006). After preparing each crystal structure at pH 7.4 and 310 K (see below for a further description), structures were superimposed to generate a model of the CD38 active site. The model was then used to confirm the importance of GLU226 for catalysis via its ability to H-bond with 2' and 3' -OH of the nicotinamide-ribose. Furthermore, SERI 93 in the model structure of CD38 was observed proximal to the Cl' of the nicotinamide-ribose and has been implicated in the reaction mechanism for NAD⁺ degradation. The orientation of nicotinamide was mediated through aromatic interactions with TRP189. In both the original X-ray structure 4XJT and the generated model structure, phosphate groups were found stabilizing the complex and active site that were not involved with catalysis. However, despite ligand diversity across substrates and inhibitors, a consistent active site conformation was observed for all ligands screened virtually in the model structure of CD38. This geometric orientation of the active site and the cyclic nature of cADPR suggested the evaluation of heterocycles and macrocycles. Additionally, to mimic substrate interactions with TRP189, SER193, GLU146 and GLU226, nicotinamide and purine bio-isosteres were investigated. Most notably, an emphasis was placed on non-biological bioisosteres with structures not mimicking NAD⁺, since NAD⁺ analogues would have a greater potential for off-target effects at other NAD⁺ binding sites. 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.

Initial screening was performed at a concentration of 50 pM of each compound in technical and experimental triplicates. Hydrolase or cyclase activities were monitored independently by using N6-ethenonicotinamide adenine dinucleotide (ε-NAD⁺) or nicotinamide guanine dinucleotide (NGD⁺), respectively, as substrate, and monitoring fluorescence at 410 nm (Fig. 4)( Camacho- Perreira 2016). The flavanoid apigenin, which has been shown to be an inhibitor of the hydrolase activity of CD38, and quercitin, which has been shown to be an inhibitor of the cyclase activity of CD38 (Escande 2013), were used as positive controls for the hydrolase and cyclase reaction, respectively. It should be noted that in our experiments, neither compound showed significant selectivity for either activity, and both were significantly less potent than previously reported. Of the 24 selected compounds, 16 produced significant inhibition of CD38 (Table 1) and were then assayed for selectivity between the hydrolase and cyclase activities. Apigenin, a known non-selective CD38 inhibitor, was used as a positive control. Based on these data, compounds 1 (Fig. 5, panel A) and 12 (Fig. 5 panel B) were highly selective for their respective targets and were chosen for further study. 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. The 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. By contrast, compound 12 was relatively non-toxic up to 50 pM, which is 2.5-fold higher that its IC50 value.

The 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. To determine the effect of CD38 inhibition on stimulation of the immune response in vitro, 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 (Fig. 13) demonstrate that 1 is highly effective at promoting NK cell-mediated tumor toxicity (12% reduction after 90 min). Importantly, 1 is significantly more effective than 78c (IC50 7 nM), despite a significantly higher IC50.

To further demonstrate the effect of 1 and 12 on inhibition of CD38 and to monitor the biological efficacy of our hit molecules, increases in NADH⁺ in activated PBMCs were monitored at 30 and 60 minutes after addition of the inhibitor. The known CD38 inhibitor 78c (Haffner 2015) was used as a control. Compound 78c had a modest effect on NADH⁺ levels (Fig. 8), while 1 and 12 produced a 40.1 and 82.0% increase at 0.5 h, respectively. In both cases, elevated NADH⁺ levels remained relatively stable at 1 h.

The effects of inhibitors 78c, 1 and 12 on IFNy levels was next examined after 48 hours and 12 days, as shown in Fig. 9. PBMCs were incubated with the appropriate inhibitor at 10 pM for the indicated time, and IFNy levels were measured by ELISA. At 48 hours, 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. At 12 days, 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. In this model, the 2-thioxothiazolidin-4-one ring nitrogen appears to form hydrogen bonds with LYS 129 and ASP 155. In addition, 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. This arrangement is consistent with the observed uncompetitive kinetics of inhibition since optimal binding depends on the presence of the substrate NAD⁺. As the structure-activity relationships for binding of 12 and its analogues are further elucidated, the in silico model may be refined accordingly.

Library generation

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).

Scheme 1

Four of the compounds shown in Table 2 exhibited a lower IC50 against CD38 cyclase activity than 12 (compounds 20, 22, 23 and 32). The ability of these compounds to increase IFNy content in activated PBMCs from 3 separate donors was determined at 48 hours, as described above, and the results of these studies appear in Fig. 11. Compounds 12, 23 and 32 were more effective than 78c at increasing IFNy, and the percent increase caused by compounds 22, 23 and 32 was inversely proportional to their observed IC50 values. Among compounds 12, 20, 22, 23 and 32, compound 12 exhibited the greatest effect on IFNy levels at 1.0 pM, despite having the highest IC50. Because CD38 carries out its enzyme function both inside and outside the cell (Lee 2019), this finding may be due to an enhanced ability of 12 to penetrate into PBMCs.

Additionally, the following compound was found to have 65% hydrolase activity remaining at 25

Conclusions

A number of small molecule 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. The parent molecule in this series, 12, produced an 82% increase in NAD⁺ content in naive PBMCs after 30 minutes, and a 212% increase in IFNy levels after 48 hours that persisted for 12 days. Importantly, 12 was significantly more potent than the known CD38 inhibitor 78c with regard to promoting increases in IFNy. Compound 78c was evaluated as an inhibitor of CD38 hydrolase, but not specifically as an inhibitor of CD38 cyclase. Since 12 and its analogues are cyclase- selective, the data suggests that the cyclase plays a greater role in activation of T-cell function than was previously thought.

With regard to structure-activity relationships (SAR), 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. In addition, because these compounds have IC50 values in the micromolar range, the possibility of off-target effects exists. A number of cyclase-selective inhibitors of CD38 have been described that will be of use in determining the role that the hydrolase and cyclase activities of CD38 play in metabolism, activation of immune cells and in the development of resistance to immune checkpoint inhibitors. It is not currently known whether the CD38 hydrolase or the CD38 cyclase activity makes the greatest contribution to down regulation of the immune response, or which activity contributes to the development of resistance to CD38 immune checkpoint inhibitors. The data suggests that the CD38 cyclase plays a significant role on down regulation of the immune response and the development of resistance. 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.

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. Decreases in 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.

Methods and supplemental information

Possible synthetic routes for compounds 1 and 12 are shown in Schemes 1 and 2.

Scheme 2

Compound 1 was synthesized in 3 steps, as shown in Scheme 2. Compound 12 was synthesized in a single step using a modified Claisen-Schmidt reaction (Scheme 1). Each synthesis was designed to allow incorporation of chemical diversity to facilitate hit-to-lead optimization.

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.

Synthesis All reagents and dry solvents were purchased from Aldrich Chemical Co. (Milwaukee, WI), Sigma Chemical Co. (St. Louis, MO), VWR (Radnor, PA) or Fisher Scientific (Chicago, IL) and were used without further purification except as noted below. Triethylamine was distilled from potassium hydroxide and stored in a nitrogen atmosphere. Dry methanol, ethyl acetate, tetrahydrofiiran, dimethyl formamide and hexane were either purchased (VWR) or prepared using a Glass Contour Solvent Purification System (Pure Process Technology, LLC, Nashua, NH). 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).

All 1H- and 13C-NMR spectra were recorded on a Bruker Avance 600 MHz spectrometer, and all chemical shifts are reported as d values referenced to TMS or DSS. Splitting patterns are indicated as follows: s, singlet; d, doublet; t, triplet; m, multiplet; br, broad peak. In all cases, 1H- NMR, 13C-NMR and MS spectra were consistent with assigned structures, and 13C peak assignments appear on the spectrum. Mass spectra were recorded by LC/MS on a Waters UPLC/MS system with a model QDa mass spectrometer detector. Prior to biological testing procedures, all compounds were determined to be >95% pure by UPLC chromatography (9: 1 H2O: acetonitrile, +0.1% formic acid to 1 :9 H2O/ acetonitrile +0.1% formic acid over 8 minutes) using a Waters Acquity H-series ultrahigh-performance liquid chromatograph fitted with a Cl 8 reversed-phase column (Acquity UPLC BEH C18 1.7 M, 2.1 X 100 mm). Compounds 20-46 were synthesized according to the general procedure described below.

Synthesis of (Z)-5-((5-(4-methoxyphenyl)isoxazol-3-yl)methylene)-2-thioxothiazolidin-4-one (20): A 0.05 g portion of 2-thioxothiazolidin-4-one 18 (0.43 mmol), 0.087 g (0.43 mmol) of 5- (4-methoxyphenyl)isoxazole-3-carbaldehyde 47 and 0.66 mg (0.86 mmol) of ammonium acetate were added to a microwave vial along with 2 mL of glacial acetic acid, a stirring bar was added and the vial was sealed. 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. Residual water was removed from the solid by lyophilization to afford (Z)-5-((5-(4-methoxyphenyl)isoxazol-3- yl)methylene)-2-thioxothiazolidin-4-one 20 (104 mg, 76.1%) as a yellowish solid. ^XH NMR (600 MHz, D6-DMSO/TMS): d 7.84 (d, J = 8.7 Hz, 2H, H-7, H-l l), 7.49 (s, 1H, H-21), 7.19 (s, 1H, H4), 7.13-7.09 (m, 2H, H-10, H-8), 3.83 ppm (s, 3H, H-13). ¹³C NMR (600 MHz, D6- DMSO/TMS) d 55.48 (CH3-O); 100.39 (N-C-CH); 114.85 (O-C-CH); 116.38 (O-C-CH); 127.60 (CH-C); 158.24 (N-C); 161.30 (O-C); 168.68 (CO); 170.05 (CS). UPLC retention time: 7.23 min. MS: calculated, 319.3650, found, 318.9710.

Synthesis of (Z)-5-(4-(methylsulfonyl)benzylidene)-2-thioxothiazolidin-4-one (21): Compound 21 was synthesized from 2-thioxothiazolidin-4-one and 4-(methylsulfonyl)benzaldehyde exactly as described for compound 20 and isolated as a dark yellow solid in 71.2% yield. ’H NMR (600 MHz, D6-DMSO/TMS) d 7.84 (d, J = 8.7 Hz, 2H, H-7, H-l l), 7.49 (s, 1H, H-21), 7.19 (s, 1H, H-4), 7.13- 7.09 (m, 2H, H-10, H-8), 3.83 ppm (s, 3H, H-13). ¹³C NMR (600 MHz, D6- DMSO/TMS) d 43.80 (CH3); 128.18 (C-CH); 129.61 (C-CH); 129.66 (C-CH); 138.18 (C-CH); 142.02 (C-CH); 195.84 (CS). UPLC retention time: 5.21 min. MS: calculated, 298.3770, found, 298.0614.

Synthesis of (Z)-2-thioxo-5-(4-(trifluoromethoxy)benzylidene)thiazolidin-4-one (22): Compound 22 was synthesized from 2-thioxothiazolidin-4-one and 4-(trifhioromethoxy)benzaldehyde exactly as described for compound 20 and isolated as a dark orange-yellow solid in 70.4% yield. ^1H NMR (600 MHz, D6-DMSO/TMS) 6 7.75-7.67 (m, 3H, H-7, H-18, H-9), 7.52 ppm (d, J = 8.0 Hz, 2H, H10, H-17). ¹³C NMR (600 MHz, D6-DMSO/TMS) 6 122.07 (C-CH); 127.16 (C-CH); 130.32 (C-CH); 132.63 (C-CH); 132.90 (C-CH); 149.76 (C-CH); 169.71 (CO); 195.89 (CS). UPLC retention time 7.56 min. MS: calculated, 304.2892, found, 304.0641.

Synthesis of (Z)-2-thioxo-5-(4-(trifluoromethyl)benzylidene)thiazolidin-4-one (23): Compound 23 was synthesized from 2-thioxothiazolidin-4-one and 4-(trifluoromethyl)benzaldehyde exactly as described for compound 20 and isolated as a yellow solid in 79.8% yield. ’H NMR (600 MHz, D6-DMSO/TMS) 6 7.90-7.79 (m, 4H, H-10, H-16, H-17, H-9), 7.71 ppm (s, 1H, H-7). UPLC retention time 7.43 min. MS calculated, 288.0568, found, 288.0568.

Synthesis of (Z)-5-((4' -methoxy- [ 1, 1 '-biphenyl]-4-yl)methylene)-3-methyl-2-thioxothiazolidin-4- one (24): Compound 24 was synthesized from 3-methyl-2-thioxothiazolidin-4-one and 4'- methoxy-[l,l'-biphenyl]-4-carbaldehyde exactly as described for compound 20 and isolated as a yellow solid in 70.6% yield. ^1H NMR (600 MHz, D6-DMSO/TMS) 6 7.85-7.82 (m, 3H), 7.74- 7.69 (m, 4H), 7.05 (d, J = 8.7 Hz, 2H), 3.81 (s, 3H, H-23), 3.41 ppm (s, 3H, H-8). ¹³C NMR (600 MHz, D6-DMSO/TMS) 6 55.74 (CH3); 115.08 (C-CH); 127.40 (C-CH); 128.55 (C-CH); 131.87 (C-CH); 132.97 (C-CH). UPLC retention time 8.817 min. MS calculated, 342.05442, found, 342.0713.

Synthesis of (Z)-5-(quinolin-6-ylmethylene)-2-thioxothiazolidin-4-one (25): Compound 25 was synthesized from 2-thioxothiazolidin-4-one and quinoline-6-carbaldehyde exactly as described for compound 20 and isolated as a light yellow solid in 50.7% yield. ’H NMR (600 MHz, D6- DMSO/TMS) 6 8.97 (dd, J = 0.9, 4.1 Hz, 1H), 8.52 (d, J = 8.2 Hz, 1H), 8.22 (s, 1H), 8.11 (d, J = 8.7 Hz, 1H), 7.93 (dd, J = 1.8, 8.7 Hz, 1H), 7.80 (s, 1H), 7.61 ppm (dd, J = 4.2, 8.3 Hz, 1H, H- 11). ¹³C NMR (600 MHz, D6-DMSO/TMS) 6 123.00 (C-CH); 128.42 (C-CH); 130.52 (C-CH); 130.71 (C-CH); 130.89 (C-CH); 131.51 (C-CH); 137.41 (C-CH); 148.21 (CO); 152.79 (C-CH); 196.41 (CS). UPLC retention time 1.55 min. MS calculated, 273.00780, found, 272.9719.

Synthesis of (Z)-5-(dibenzo[b,d]furan-2-ylmethylene)-2-thioxothiazolidin-4-one (26): Compound

26 was synthesized from 2-thioxothiazolidin-4-one and dibenzo[Z>, ]furan-2-carbaldehyde exactly as described for compound 20 and isolated as an off-white solid in 71.1% yield. ’H NMR (600 MHz, D6-DMSO/TMS) 6 8.31 (s, 1H, H-8), 8.20 (d, J = 7.7 Hz, 1H), 7.83 (d, J = 8.6 Hz, 1H), 7.77 (s, 1H), 7.72 (d, J = 8.5 Hz, 2H), 7.57 (t, J = 7.7 Hz, 1H), 7.44 ppm (t, J = 7.4 Hz, 1H). ¹³C NMR (600 MHz, D6-DMSO/TMS) 6 112.36 (C-CH); 113.34(C-CH); 122.18 (C-CH);

123.18 (C-CH); 123.90 (C-CH); 124.20 (C-CH); 125.24 (C-CH); 128.82 (C-CH); 129.06 (C- CH); 130.66 (C-CH); 132.32 (C-CH); 156.81 (CO). UPLC retention time 7.94 min. MS calculated, 311.00747, found, 312.0881.

Synthesis of (Z)-5-(4-phenoxybenzylidene)-2-thioxothiazolidin-4-one (27): Compound 27 was synthesized from 2-thioxothiazolidin-4-one and 4-phenoxybenzaldehyde exactly as described for compound 20 and isolated as a yellow solid in 55.9% yield. ’H NMR (600 MHz, ) D6- DMSO/TMS 6 7.62-7.59 (m, 3H), 7.47-7.42 (m, 2H), 7.25-7.07 ppm (m, 5H). ¹³C NMR (600 MHz, D6-DMSO/TMS) 6 117.96 (C-CH); 120.19 (C-CH); 124.25 (C-CH); 125.14 (C-CH); 128.12 (C-CH); 130.17 (C-CH); 131.66 (C-CH); 133.23 (C-CH); 155.54 (C-CH); 159.66 (C- CH); 169.84 (CO); 195.96 (CS). UPLC retention time 7.73 min. MS calculated, 314.02312, found, 314.0204.

Synthesis of (Z)-5-((2, 3-dihydrobenzo[b ][ 1, 4 ]dioxin-6-yl)methylene)-2-thioxothiazolidin-4-one (28): Compound 28 was synthesized from 2-thioxothiazolidin-4-one and 2,3- dihydrobenzo[Z>][l,4]dioxine-6-carbaldehyde exactly as described for compound 20 and isolated as a dark orange solid in 63.8% yield. ’H NMR (600 MHz, D6-DMSO/TMS) 6 7.52 (s, 1H, H- 15), 7.09-7.06 (m, 2H), 7.00 (d, J = 8.5 Hz, 1H), 4.30 ppm (dd, J = 4.5, 17.6 Hz, 4H, H-13, H- 14). ¹³C NMR (600 MHz, D6-DMSO/TMS) 6 64.46 (CH2); 65.01 (CH2); 118.65 (C-CH); 118.67 (C-CH); 119.60 (C-CH); 119.62 (C-CH); 124.84 (C-CH); 124.86 (C-CH); 126.73 (C- CH); 132.22 (C-CH); 132.24 (CO); 144.29 (CS). UPLC retention time 5.83 min. MS calculated, 280.00238, found, 279.9747.

Synthesis of (Z)-5-((4' -methyl- [ 1 , l'-biphenyl]-3-yl)methylene)-2-thioxothiazolidin-4-one (29): Compound 29 was synthesized from 2-thioxothiazolidin-4-one and 4'-methyl-[l,l'-biphenyl]-3- carbaldehyde exactly as described for compound 20 and isolated as a dark red-orange solid in 64.3% yield. ^XH NMR (600 MHz, D6-DMSO/TMS) 6 7.80 (s, 1H, H-8), 7.74-7.55 (m, 5H), 7.50 (d, J = 7.7 Hz, 1H), 7.28 (d, J = 7.7 Hz, 2H), 2.35-2.31 ppm (m, 3H, H-21). ¹³C NMR (600 MHz, D6-DMSO/TMS) 6 21.15 (CH3); 126.43 (C-CH); 127.07 (C-CH); 129.09 (C-CH); 129.13 (C-CH); 130.08 (C-CH); 130.17 (C-CH); 130.52 (C-CH); 132.10 (C-CH); 134.14 (C-CH);

136.62 (C-CH); 137.93 (C-CH); 141.59 (C-CH); 169.90 (CO); 196.13 (CS). UPLC retention time 8.34 min. MS calculated, 312.04386, found, 312.0044.

Synthesis of (Z)-5-( 6-chloro-[ 1, 3 ]dioxolo[ 4, 5-g] quinolin- 7-yl)methylene)-2-thioxothiazolidin-4- one (30): Compound 30 was synthesized from 2-thioxothiazolidin-4-one and 6-chloro- [l,3]dioxolo[4,5-g]quinoline-7-carbaldehyde exactly as described for compound 20 and isolated as a dark orange solid in 40.2% yield. ’H NMR (600 MHz, D6-DMSO/TMS) 6 8.12 (s, 1H), 7.65 (s, 1H), 7.58 (s, 1H), 7.28 (s, 1H), 6.25 ppm (s, 2H, H-18). ¹³C NMR (600 MHz, D6- DMSO/TMS) 6 60.73 (CH2); 72.71 (C-CH); 103.41 (C-CH); 104.05 (C-CH); 104.68 (C-CH);

123.63 (C-CH); 124.49 (C-CH); 125.25 (C-CH); 137.04 (C-CH); 145.98 (C-CH); 148.00 (C- CH); 149.25 (C-CH); 153.55 (CO). UPLC retention time 6.48 min. MS calculated, 350.95866, found, 350.9773.

Synthesis of (Z)-5-((9H-fluoren-3-yl)methylene)-2-thioxothiazolidin-4-one (31): Compound 31 was synthesized from 2-thioxothiazolidin-4-one and 9Z7-fluorene-3-carbaldehyde exactly as described for compound 20 and isolated as an orange solid in 72.4% yield. ’H NMR (600 MHz, D6-DMSO/TMS) 6 8.06-8.03 (m, 1H), 7.97 (s, 1H, H-8), 7.78-7.68 (m, 2H), 7.62 (s, 2H), 7.44- 7.36 (m, 2H), 4.00 ppm (s, 2H, H-9). ¹³C NMR (600 MHz, D6-DMSO/TMS) 6 36.89 (CH2); 121.33 (C-CH); 121.41 (C-CH); 125.82 (C-CH); 127.41 (C-CH); 127.56 (C-CH); 128.53 (C- CH); 130.57 (C-CH);131.85 (C-CH); 132.71 (C-CH); 144.21 (C-CH); 144.62 (C-CH); 170.02 (CO). UPLC retention time 8.10 min. MS calculated, 310.02821, found, 309.9631.

Synthesis of (Z)-2-thioxo-5-((4'-(trifhwromethyl)-[lf '-biphenyl] -4-yl)methylene)thiazolidin-4- one (32). Compound 32 was synthesized from 2-thioxothiazolidin-4-one and 4'- (trifhioromethyl)-[l,r-biphenyl]-4-carbaldehyde exactly as described for compound 20 and isolated as an orange solid in 66.5% yield. ^1H NMR (600 MHz, D6-DMSO/TMS) 6 7.99-7.69 ppm (m, 9H). ¹³C NMR (600 MHz, D6-DMSO/TMS) 6 125.95 (CF3, C-CH); 127.60 (C-CH); 127.79 (C-CH); 127.99 (C-CH)); 130.90 (C-CH); 131.23 (C-CH); 132.12 (C-CH); 140.34 (C- CH); 142.81 (C-CH); 169.36 (CO). UPLC retention time 8.51 min. MS: calculated, 364.3882, found, 364.0916.

Synthesis of (Z)-5-((4' -methyl- [ 1 , r-biphenyl]-4-yl)methylene)-2-thioxothiazolidin-4-one (33): Compound 33 was synthesized from 2-thioxothiazolidin-4-one and 4'-methyl-[l,l'-biphenyl]-4- carbaldehyde exactly as described for compound 20 and isolated as a yellow solid in 77.6% yield. ^1H NMR (600 MHz, D6-DMSO/TMS) 6 7.85-7.81 (m, 2H), 7.68-7.62 (m, 5H), 7.30 (d, J = 7.4 Hz, 2H), 2.34 ppm (s, 3H, H-16). ¹³C NMR (600 MHz, D6-DMSO/TMS) 6 20.74 (CH3); 125.04 (C-CH); 126.67 (C-CH); 127.23(C-CH)); 129.75 (C-CH, C-CH); 131.22 (C-CH); 137.94(C-CH); 142.08 (C-CH); 169.39(CO); 195.54 (CS). UPLC retention time 8.50 min. MS calculated, 310.4170, found, 310.0669.

Synthesis of (Z)-5-(4-(pyridin-2-yl)benzylidene)-2-thioxothiazolidin-4-one (34): Compound 34 was synthesized from 2-thioxothiazolidin-4-one and 4-(pyridin-2-yl)benzaldehyde exactly as described for compound 20 and isolated as a yellow solid in 68.8% yield. ’H NMR (600 MHz, D6-DMSO/TMS) 6 8.69 (d, J = 4.6 Hz, 1H), 8.23 (d, J = 8.2 Hz, 2H), 8.02 (d, J = 8.0 Hz, 1H), 7.90 (t, J = 7.6 Hz, 1H), 7.70-7.65 (m, 3H), 7.41-7.36 ppm (m, 1H). ¹³C NMR (600 MHz, D6- DMSO/TMS) 6 121.30 (C-CH); 123.82 (C-CH); 127.81 (C-CH); 131.39 (C-CH); 131.43 (C- CH); 133.92 (C-CH); 137.98 (C- CH); 140.78 (C-CH); 155.11 (CO). UPLC retention time 5.56 min. MS calculated, 299.02345, found, 298.9749.

Synthesis of (Z)-2-thioxo-5-((5-(3-(trifluoromethyl)phenyl)furan-2-yl)methylene)thiazolidin-4- one (35): Compound 35 was synthesized from 2-thioxothiazolidin-4-one and 5-(3- (trifluoromethyl)phenyl)furan-2-carbaldehyde exactly as described for compound 20 and isolated as a yellow solid in 69.0% yield. ^1H NMR (600 MHz, D6-DMSO/TMS) 6 8.08 (s, 1H, H-7), 8.03 (d, J = 7.7 Hz, 1H), 7.77-7.70 (m, 2H), 7.47-7.45 (m, 2H), 7.27 ppm (d, J = 3.7 Hz, 1H). ¹³C NMR (600 MHz, D6-DMSO/TMS) 6 111.98 (C-CH); 117.46 (C-CH); 121.07 (C-CH); 122.56 (C-CH); 123.35 (C-CH); 125.73 (C-CH); 128.25 (C-CH); 130.03 (C-CH); 130.47 (CF3); 130.68 (CF3); 131.03 (C-CH); 150.12 (C-CH); 156.13 (C-CH); 169.37(CO); 196.52 (CS). UPLC retention time 7.69 min. MS calculated, 355.99485, found, 356.0168. Synthesis of (Z)-5-((6-methoxynaphthalen-2-yl)methylene)-2-thioxothiazolidin-4-one (36): Compound 36 was synthesized from 2-thioxothiazolidin-4-one and 6-methoxy-2-naphthaldehyde exactly as described for compound 20 and isolated as an orange solid in 68.4% yield. ’H NMR (600 MHz, D6-DMSO/TMS) b 8.04 (s, 1H), 7.91 (dd, J = 8.8, 29.0 Hz, 2H), 7.68 (s, 1H, H-8), 7.56 (dd, J = 1.3, 8.6 Hz, 1H), 7.34 (d, J = 1.8 Hz, 1H), 7.22 (dd, J = 2.3, 9.0 Hz, 1H), 3.89 ppm (s, 3H, H-20). 13C NMR (600 MHz, D6-DMSO/TMS) b 55.93 (CH3); 106.687 (C-CH); 120.26 (C-CH); 127.39 (C-CH)); 128.41 (C-CH); 128.70 (); 131.07 (C-CH); 132.04 (C-CH); 132.49 (C- CH); 135.69 (C-CH); 159.66 (CO). UPLC retention time 7.20 min. MS calculated, 302.02312, found, 301.9300.

Synthesis of (Z)-5-((4' -methyl- [ 1 , r-biphenyl]-4-yl)methylene)2-thioxothiazolidin-4-one (37): Compound 37 was synthesized from 2-thioxothiazolidin-4-one and 4'-methyl-[l,l'-biphenyl]-4- carbaldehyde exactly as described for compound 20 and isolated as an off-white solid in 50.3% yield. ^1H NMR (600 MHz, D6-DMSO/TMS) b 11.10-11.07 (m, 1H, H-5), 7.84-7.82 (m, 3H, H- 12, H-8, H-6), 7.70 (d, J = 8.2 Hz, 2H, H-l l, H-9), 7.66-7.62 (m, 2H, H-18, H- 14), 7.33-7.29 (m, 2H, H-17, H-15), 2.37 ppm (s, 3H, H-19). ¹³C NMR (600 MHz, D6-DMSO/TMS) b 21.18 (CH3); 123.61 (C-CH); 127.08 (C-CH); 127.54 (C-CH); 130.15 (C-CH); 131.15 (C-CH); 131.85 (C-CH); 132.24 (C-CH); 136.40 (C-CH);138.21 (C-CH); 142.19 (C-CH); 167.82 (CO); 168.26 (CO). UPLC retention time 7.61 min. MS, calculated, 294.3560, found, 294.1762.

Synthesis of (Z)-5-(4-(benzyloxy)benzylidene)-2-thioxothiazolidin-4-one (38): Compound 38 was synthesized from 2-thioxothiazolidin-4-one and 4-(benzyloxy)benzaldehyde exactly as described for compound 20 and isolated as a yellow-orange solid in 16.8% yield. ’H NMR (600 MHz, D6- DMSO/TMS) b 12.18 (s, 1H, H-5), 7.61-7.58 (m, 3H), 7.50 (d, J = 7.4 Hz, 2H), 7.40 (t, J = 7.6 Hz, 2H), 7.34 (t, J = 7.3 Hz, 1H), 7.23-7.20 (m, 2H), 5.20 ppm (s, 2H, H-16). ¹³C NMR (600 MHz, D6-DMSO/TMS) b 70.06 (CH2); 116.36 (C-CH); 122.89 (C-CH); 126.17 (C-CH); 128.27 (C-CH); 128.50 (C-CH); 128.96 (C-CH); 132.27 (C-CH); 133.13 (C-CH); 136.92 (C-CH); 160.91 (C-CH); 169.88 (CO); 195.95 (CS). UPLC retention time 8.49 min. MS calculated, 328.03877, found, 328.0041. Synthesis of (Z)-2-thioxo-5-(3,4,5-trimethoxybenzylidene)thiazolidin-4-one (39): Compound 39 was synthesized from 2-thioxothiazolidin-4-one and 3,4,5-trimethoxybenzaldehyde exactly as described for compound 20 and isolated as a dark red-orange solid in 8.3% yield. ’H NMR (600 MHz, D6-DMSO/TMS) 6 12.21 (d, J = 20.6 Hz, 1H, H-17), 7.57 (s, 1H, H-13), 6.90 (s, 2H, H-2, H6), 3.92 (s, 6H, H-12, H-10), 3.81 ppm (s, 3H, H-l l). ¹³C NMR (600 MHz, D6-DMSO/TMS) 6 56.56 (O-CH3); 60.72 (O-CH3); 108.51 (C-CH); 128.93 (C-CH); 132.51 (C-CH); 153.78 (CO). UPLC retention time 5.74 min. MS calculated, 312.02860, found, 311.9912.

Synthesis of (Z)-5-([l,r-biphenyl]-4-ylmethylene)-2-thioxothiazolidin-4-one (40): Compound 40 was synthesized from 2-thioxothiazolidin-4-one and [l,l'-biphenyl]-4-carbaldehyde exactly as described for compound 20 and isolated as a dark orange solid in 58.9% yield. ’H NMR (600 MHz, D6-DMSO/TMS) 6 12.31-12.20 (m, 1H, H-4), 7.88 (d, J = 8.2 Hz, 2H, H-14, H-18), 7.74 (dd, J = 7.9, 18.0 Hz, 4H, H-l l, H-9, H-8, H-12), 7.68 (s, 1H, H-6), 7.50 (dd, J = 6.9, 8.2 Hz, 2H, H-17, H-15), 7.42 ppm (t, J = 7.3 Hz, 1H, H-16). ¹³C NMR (600 MHz, D6-DMSO/TMS) 6 125.71 (C-CH); 127.22 (C-CH); 127.93 (C-CH); 128.81 (C-CH); 129.59 (C-CH); 131.62 (C- CH); 132.34 (C-CH); 139.11 (C-CH); 142.54 (C-CH); 169.93 (CO); 196.00 (CS). UPLC retention time 7.85 min. MS calculated, 298.3900, found, 297.9619.

Synthesis of (Z)-2-thioxo-5-(2-(trifluoromethyl)benzylidene)thiazolidin-4-one (41): Compound 41 was synthesized from 2-thioxothiazolidin-4-one and 2-(trifhioromethyl)benzaldehyde exactly as described for compound 20 and isolated as a an off-white solid in 60.7% yield. ’H NMR (600 MHz, D6-DMSO/TMS) 6 12.41 (s, 1H, H-15), 7.92-7.81 (m, 3H, H-5, H-3, H-2), 7.76-7.69 ppm (m, 2H, H-6, H-4). ¹³C NMR (600 MHz, D6-DMSO/TMS) 6 123.39 (C-CH); 125.21 (C- CH); 125.90 (C-CH); 127.02 (C-CH); 127.29 (C-CH); 127.33 (C-CH); 127.37(C-CH); 128.03 (CF3); 128.23 (CF3); 128.42 (CF3); 128.62 (CF3); 129.77 (C-CH); 131.04 (C-CH); 131.35 (C- CH); 131.67 (C-CH); 133.96 (C-CH), 169.27 (CO); 196.06 (CS). UPLC retention time 6.68 min. MS calculated, 287.98429, found, 288.0781.

Synthesis of (Z)-2-thioxo-5-(2-(trifluoromethoxy)benzylidene)thiazolidin-4-one (42): Compound 42 was synthesized from 3-methyl-2-thioxothiazolidin-4-one and 4'-methoxy-[l,l'-biphenyl]-4- carbaldehyde exactly as described for compound 20 and isolated as a an off-white solid in 70.6% yield. *HNMR (600 MHz, D6-DMSO/TMS) 6 12.40 (s, 1H, H-14), 7.76 (s, 1H, H-6), 7.70-7.59 (m, 3H, H-10, H-l 1, H-12), 7.53 ppm (d, J = 8.3 Hz, 1H, H-9). ¹³C NMR (600 MHz, D6- DMSO/TMS) 6 122.29 (C-CH); 123.06 (C-CH); 126.62 (C-CH); 128.96 (C-CH); 129.83 (C- CH); 129.88 (C-CH); 132.94 (C-CH); 147.50 (C-CH); 169.56 (CO); 195.80 (CS). UPLC retention time 6.93 min. MS calculated, 304.2892, measured 304.0848.

Synthesis of (Z)-3-methyl-5-((4'-methyl-[ 1 , 1 '-biphenyl] -4-yl)methylene)-2-thioxothiazolidin-4- one (43): Compound 43 was synthesized from 4'-methyl-[l,l'-biphenyl]-4-carbaldehyde exactly as described for compound 24 and isolated as a yellow solid in 30.1% yield. 1H NMR (600 MHz, D6-acetone): 6 7.88- 7.85 (m, 2H, H-l l, H-15), 7.80 (s, 1H, H-9), 7.72 (d, J = 8.2 Hz, 2H, H-14, H-12), 7.66-7.64 (m, 2H, H21, H-17), 7.32 (d, J = 8.0 Hz, 2H, H-20, H-18), 3.49 (s, 3H, H-8), 2.38 ppm (s, 3H, H-22). ¹³C NMR (600 MHz, D6-DMSO/TMS) 6 21.20 (CH3); 31.68 (CH3); 127.15 (C-CH); 127.43 (C-CH); 130.21 (C-CH); 131.84 (C-CH); 132.22 (C-CH); 132.87 (C-CH); 138.42 (C-CH). UPLC retention time 9.49min. MS calculated, 326.4440, measured 326.0348.

Synthesis of (Z)-2-(5-((4'-methyl-[ 1, 1 '-biphenyl]-4-yl)methylene)-4-oxo-2-thioxothiazolidin-3- yl)acetic acid (44): Compound 44 was synthesized from 4'-methyl-[l,l'-biphenyl]-4- carbaldehyde exactly as described from compound 24 and isolated as a yellow solid. 1H NMR (600 MHz, D6-Acetone): 6 7.89-7.85 (m, 3H, H-l l, H-15, H-9), 7.75 (d, J = 8.2 Hz, 2H, H-12, H-14), 7.67-7.65 (m, 2H, H-17, H-21), 7.32 (d, J = 7.8 Hz, 2H, H-18, H-20), 4.85 (s, 2H, H-8), 2.38 ppm (s, 3H, H-22). ¹³C NMR (600 MHz, D6-DMSO/TMS) 6 47.05 (CH3); 127.14 (C-CH); 127.71 (C-CH); 130.18 (C-CH); 131.85 (C-CH); 132.16 (C-CH); 133.16 (C-CH); 136.26 (C- CH); 138.39 (C-CH);142.72 (C-CH); 167.09 (CO); 167.59 (COOH); 193.50 (CS). UPLC retention time 8.20 min. MS calculated, 368.4530, measured 368.2621.

Synthesis of (Z)-5-((4' -methoxy- [ 1, 1 '-biphenyl]-4-yl)methylene)-2-thioxothiazolidin-4-one (45): Compound 45 was synthesized from 4'-methoxy-[l,l'-biphenyl]-4-carbaldehyde exactly as described from compound 24 and isolated as an orange solid. 1H NMR (600 MHz, D6-DMSO): 6 7.81 (d, J = 8.2 Hz, 2H), 7.73-7.63 (m, 5H), 7.05 (d, J = 8.6 Hz, 2H), 3.80 ppm (s, 3H, H-22). ¹³C NMR (600 MHz, D6-DMSO/TMS) 6 55.72 (O-CH3); 115.05 (C-CH); 127.30 (C-CH); 128.49 (C-CH); 131.18 (C-CH); 131.47 (C-CH); 131.60 (C-CH); 131.88 (C-CH); 142.10 (C- CH); 160.09 (CO). UPLC retention time 7.69min. MS calculated, 328.03877, measured 328.0452.

Synthesis of (Z)-5-((4' -(trifluoromethyl) -[1,1 '-biphenyl] -4-yl)methylene)-2-thioxothiazolidin-4- one (46): Compound 46 was synthesized from 4'-(trifhioromethyl)-[l,T-biphenyl]-4- carbaldehyde exactly as described from compound 24 and isolated as an off white solid. 1H NMR (600 MHz, D6-DMSO): 6 7.94 (dd, J = 8.3, 28.4 Hz, 4H), 7.85-7.82 (m, 3H), 7.74-7.71 ppm (m, 2H). ¹³C NMR (600 MHz, D6-DMSO/TMS) > 124.57 (C-CH); 126.33 (C-CH); 128.07 (C-CH); 128.26 (C-CH); 131.17 (C-CH); 131.45 (C-CH); 133.55 (C-CH); 140.46 (C-CH); 143.31 (C-CH); 167.76 (CO); 168.17 (CO). UPLC retention time 7.69 min. MS calculated, 349.03843, measured 342.0713.

Enzyme Assay

CD38 cyclase activity. Compounds were screened for the ability to inhibit the cyclase activity of recombinant human CD38 in a fluorometric assay. In brief, 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. After 15 mins incubation on an orbital shaker, 50 pL of a 50 pM solution of NGD⁺ (2x) was added to each well, initiating the reaction. The final concentration of CD38 and NGD⁺ in the reaction are, 10 nM and 25 pM respectively. With an excitation at 300nm and 410nm emission, the reaction was monitored for 10 mins. Initial rates (velocities) were calculated by determining the slope over the first 5 mins. Mean fluorescence for the vehicle treated control was used to normalize each treatment group, and values were expressed as percent activity of CD38. Each assay was conducted in at least technical duplicates and experimental triplicates and compared to the literature described CD38 cyclase inhibitor quercetin (Q). CD38 hydrolase activity. The 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.

Dose dependence and kinetic mechanism of inhibition. Selected hit compounds were assayed for dose dependent inhibition of CD38 cyclase using the general workflow described above at concentrations between 0 and 100 pM. Serial dilutions of each compound were adjusted to establish an eight-point concentration curve. Likewise, Michalis-Menton enzyme kinetics were assessed by varying substrate (8-point curve) and compound concentrations. All enzyme reactions were kept at a standard ratio of CD38/compound/substrate (4x/4x/2x). Data from each experiment was collected in technical and experimental triplicates.

Cell Culture

Primary culture. All PBMC and T cells were cultured in accordance with the Stem Cell Technology T-cell Expansion Protocol. In brief, for expansion of T cells in PBMC cultures, an initial culture was seeded at 1 X 10⁶ 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.

Cytokine Analysis (Interferon- k). 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. 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.

Example 2

Bispecific T cell engager induced cytotoxicity assay

Bispecific T cell engagers (BiTEs) 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. A 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). Notably, it was also observed that 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. The activity of novel CD38 inhibitor scaffolds using this assay system are evaluated and are further adapt the assay to tailor it to MM models. Additionally the B cell maturation antigen (BCMA) is targeted, which is highly expressed by MM cells, using a commercial BCMAxCD3 bispecific T cell engager antibody (BPS Biosciences). 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 cell assay

To confirm the observed cytotoxic effects are due to the inhibition of CD38 and not due to a random off-target effect, 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), 1x10⁶ 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). The fluorescence is then measured (Ex/Em = 300/410 nm) in kinetic mode for 30-60 min at 37 °C. Using a standard curve, the sample’s CD38 activity is determined at nmol/min/mg (U/mg).

Drug metabolism and pharmacokinetic (DMPK) studies

To further prioritize compounds, drug metabolism and pharmacokinetic (DMPK) studies are performed.

The kinetic aqueous solubility assay is a turbidimetric method proposed by Lipinski (Lipinski 2001). In this assay, compounds to be studied are dissolved in dimethylsulfoxide (DMSO) and added to a defined volume of buffer in small aliquots until precipitation occurs (OD 600-820 nm). This rapid assay provides an understanding of the aqueous solubility of compounds and assists in the prioritization of compounds to move forward in the pipeline. Desirable metrics for solubility will be > 60 pg/ml.

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.

The 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.

Pharmacokinetic (PK) 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). For this study, 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. Oral bioavailability (F) of compounds is calculated using the equation [%F= 100 x (AUC_pox Di_v)/(AUCiv x D_po)] where D is the dose administered. 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.

Alternative methods. Although the LDH assay from Promega is an efficient way to determine cytotoxicity in suspension and adherent cells (Archer 2010), LDH assays from other companies (Roche or Sigma) 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

2018). To better determine IC50 values, 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

2019). Additional drug combinations may be tested in the isobologram studies, e.g., inhibitors of dual-specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2), a protein that phosphorylates and activates the proteasome (Banerjee 2018, Banergee 2019, Guo 2016) and may induce synergism with CD38 inhibitors, bortezomib or carfilzomib DYRK2 inhibitors (curcumin, LDN192960, harmine).

Example 3

Evaluate the anti-tumor effects of selected compounds in vivo using four preclinical murine cancer models for MM and NB,

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. For NB, a patient-derived xenograft (PDX) model88 and the well-established transgenic TH-MYCN mouse model95 is used.

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. MTD and LD are determined using both female and male NOD-SCID IL2Rgamma^null(NSG) mice (Jackson Laboratory). Selected compounds are tested in a pilot single-dose toxicity study to compare tolerability of intravenous (/. v. ) versus intraperitoneal (/./?.) routes of administration, as we previously described. If the compound is soluble, the carrier is PBS. If the compound is insoluble, the carrier is 5% EtOH (or DMSO) in PBS or Cremophor. Mice are assigned to four groups including a control group (no drug): 0, 25, 50, 100 mg/kg/dose (n=4 per group). If the lowest dose is lethal, concentration is decreased or vice versa.

Mouse model # 1 : MM xenotransplant model. NOD-SCID IL2Rgamma^null(NSG) mice are inoculated with MM cell suspensions (MM.1 S BzR, 1x10⁶ 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.

Experiments are replicated using male and female mice. Drug treatments begins 14 days after the injection of cell suspensions. Treatments with vehicle, positive control bortezomib (0.6 mg/kg, i.p., days 1 and 3 of each 7-day cycle) and 3 selected compounds are administered i.p. (daily) based on each compound’s MTD, for up to 30 days, using the formula (MTD X 1.5)/4 according to published protocol (Bachmann). Compounds with suitable oral bioavailability as determined in PK studies are administered by daily oral gavage. Animal survival is an endpoint and toxicity/tolerability is evaluated qualitatively by body condition scoring and quantitatively by monitoring animal body weight. For the study endpoint where 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. Comprehensive toxicology profiling is also conducted following blood collection using Vetscan profiling kits (Abaxis Global Diagnostics). Complete blood count (CBC) with differential is also be carried out. Biostatistics. For survival studies in NSG mice measuring time to death, 11 mice per group were recommended. In preliminary studies, it was observed that >95% (N=60) of untreated mice have died of MM between days 40 and 50. Based on this experience a conservative assumption was made that if 20% of the control group survived for 6 weeks, and 70% of the treatment group were surviving, the log-rank test would have a power of 80% with 11 mice per group. Statistical tests were level a=0.05. Power analysis was also conducted for the proposed animal experiments that will quantify numbers of MM cells in the bone marrow aspirates of mice. Assuming that 20% of animals will have minimal tumor take, data were simulated with N=8 samples per group, resulting in power of 81% when the true mean difference between groups was approximately 2.9 standard deviations. All statistical tests were level a=0.05. Kaplan-Meier survival analysis and non-parametric Wilcoxin rank-sum tests will be used for survival and cellular analysis studies, respectively.

Mouse model #2: MM Immunocompetent Vk*MYC transgenic model. Vk*MYC immunocompetent transgenic mice are provided. In Vk*MYC mice, 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. These 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. 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. 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). In this study, twoMYCN-amplified Stage IV PDX (COG-N-623 and COG-N-421) are used. Each PDX is implanted by injecting 15 x 10⁶ cells subcutaneously in female or male athymic nu/nu mice. Tumor volume is measured twice weekly using the formula V (mm³) = length x width²/2. Animals with 100 mm³ tumors are randomly divided into four groups (n=10/group). 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 mm³.

Mouse model #4: NB transgenic TH-MYCN mouse model. In this experiment, 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. Hemizygous TH-MYCN mice are in a 129xl/svJ background which are a well- characterized, immunocompetent NB mouse model (Weiss 1997). TH-MYCN mice are bred and their pups are weaned at 21 days post birth and genotyped. Hemizygous and homozygous TH-MYCN mice are randomly divided into groups (minimum N=15/group for both hemi- and homozygous animals). CD38 inhibitors are chosen and each are assigned to its own group and the last group is used as a control group. Mice are monitored daily and weighed twice weekly and imaged once weekly by high-frequency ultrasound to detect for signs of tumor formation. Treatment begins once tumors are detectable by ultrasound. Drug dose and route of administration (i.p. or z.v.) is selected based on outcomes of toxicity studies. Mice are euthanized using a Euthanex Prodigy carbon dioxide delivery system if animals display signs of altered gait or 15% weight loss or no later than 180 days post initiation of treatment. Tumors are split into approximate halves. One half is formalin fixed for subsequent immunohistochemistry (IHC) staining. The other half is snap-frozen for Western blot analysis. A minimum of 132 TH-MYCN mice are needed. N=15 minimum/group x 4 groups x 2 (for both hemi- and homozygous mice) + 10% attrition rate for study loss.

Correlative biomarker response studies. 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. Specifically, 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.

Alternatives. We have experience with a variety of in vivo animal cancer models including MM xenograft models. If the NSG xenotransplant model or the transgenic Vk*MYC model should fail other well-established MM models may be used (Rossi 2018, Lwin 2016). For example, transplantable Vk*MYC-derived MM cell lines Vkl2598 and Vkl2653 that grow in immunocompetent C57BL/6 mice may be used. These have been used to evaluate the efficacy of anti-MM drugs (Chesi 2012). If the i.p. route of administration of a compound is not effective, the compound may be administered via i.v. injections (2x/week) using the same carriers. Compounds may also be tested in solid tumor murine models against CD38-positive cancers (e.g. hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), melanoma, pancreatic ductal adenocarcinoma (PDAC), glioma or breast cancer) (Wo 2019). In addition, pharmacodynamic (PD) 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.

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The invention is further described in the following numbered paragraphs:

1) A compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein

X is S or O; is a single bond or a double bond;

Ri is H, alkyl, lower alkyl, or CH2COOH;

A is

R2 is H, CH3, CF3, OCH3, OCF3, SO2CH3, or a phenyl group optionally substituted with at least one of CH₃, CF₃, OCF3, OCH₃, SO2CH3, or

or R2 is

2) The compound of paragraph 1, wherein Ri is H, methyl or CH2COOH.

3) A compound of paragraph 1 or 2, which has the structure of formula la, lb, or Ic:

or a pharmaceutically acceptable salt thereof.

4) A compound of formula II:

II or a pharmaceutically acceptable salt thereof, wherein R3 is lower alkyl, or OCH3; R₄ is lower alkyl; R₅ is lower alkyl, or OCH₃; R₆ is NHR₁₂ , lower alkyl or phenyl; R7 is R₁₁

R₈ is H, lower alkyl, R9 , R10 and R11 are each independently lower alkyl, and R12 is alkyl or lower alkyl. 5) The compound of paragraph 4, wherein the compound is a compound of formula IIa, IIb, or IIc, or a pharmaceutically acceptable salt thereof:

R₁₂

7) The compound of any one of paragraphs 4-6, wherein R3 is methyl; R₄ is ethyl; R₅ is methyl; R6 is methyl or phenyl; R₁₁ is methyl; Z is C; R9 is methyl; and/or R10 is methyl. 8) A compound which is:

or a pharmaceutically acceptable salt thereof.

9) The compound of any one of paragraphs 1-8 which is an CD38 inhibitor.

10) The compound any one of paragraphs 1-9 which is an inhibitor the cyclase activity of CD38.

11) The compound of any one of paragraphs 1-10 which is an inhibitor of the cyclase activity of CD38.

12) 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.

13) 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.

14) The method of paragraph 13, further comprising increasing the activity of T cells and/or natural killer cells within the subject.

15) The method of paragraph 13 or 14, further comprising (a) preventing reduced levels of extracellular NAD+, (b) increasing NADH+ levels, (c) inhibiting the NADase function of CD38, or (d) promoting NK cell-mediated tumor toxicitiy, in the subject.

16) The method of any one of paragraphs 13-15, wherein 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.

17) The method of any one of paragraphs 13-16, wherein the cancer comprises a tumor cell which highly expresses CD38.

18) The method of any one of paragraphs 13-17, wherein the cancer is drug resistant multiple myeloma.

19) The method according to any one of paragraphs 13-18, wherein the subject is a human patient.

20) The method according to any one of paragraphs 13-19, wherein the administration is oral, intravenous or intraperitoneal.

21) The method of any one of paragraphs 13-20, further comprising administering a pharmaceutically acceptable carrier with the compound.

22) The method of any one of paragraphs 13-21, in combination with a second antagonist therapy which is a second cluster of differentiation 38 (CD38) antagonist therapy, or a PD1 or PD-Ll antagonist therapy.

23) The method of paragraph 22, wherein 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.

24) The method of paragraph 23, wherein 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.

25) The pharmaceutical combination according to paragraph 23, wherein the second antagonist is a monoclonal antibody (mAb).

26) The method of paragraph 25, wherein the monoclonal antibody is human or humanized. 27) The method of paragraph 23, wherein the second antagonist is a CDS 8 antagonist which is daratumumab, isatuximab, bortezomib, carfilzomib, lenalidomide, panobinostat, dexamethasone, or selinexor.

28) The method of any one of paragraphs 23-27, wherein the second antagonist is a CDS 8 antagonist and binds to CDS 8 receptor on a surface of a tumor cell.

29) The method of any one of paragraphs 23-28, wherein the second antagonist is a PD-1 antagonist which is nivolumab, pembrolizumab, avelumab, durvalumab, cemiplimab, or atezolizumab.

30) The method of any one of paragraphs 23-29, wherein the second antagonist is a CTLA-4 antagonist which is ipilimumab or tremelimumab.

31) The method of any one of paragraphs 23-30, wherein the administration of the compound precedes the administration of the second antagonist.

32) The method according to any one of paragraphs 23-30, wherein the administration of the second antagonist precedes the administration of the compound.

33) The method according to any one of paragraphs 23-30, wherein the second antagonist is administered adjunctively to the compound.

34) The method according to any one of paragraphs 23-30, wherein the compound is administered adjunctively to the second antagonist.

35) The method according to any one of paragraphs 23-30, wherein the subject is receiving second antagonist therapy prior to initiating the compound therapy.

36) The method according to any one of paragraphs 22-30, wherein the subject is receiving the compound therapy prior to initiating second antagonist therapy.

37) The method according to any one of claims 22-36, wherein in the subject is receiving a first therapy for at least 8 weeks, at least 10 weeks, at least 24 weeks, at least 28 weeks, at least 48 weeks or at least 52 weeks prior to initiating a second therapy.

38) The method according to any one of paragraphs 22-37, wherein the periodic administration of the second antagonist and/or the compound continues for at least 3 days, for at least 30 days, for at least 42 days, for at least 8 weeks, for at least 12 weeks, for at least 24 weeks or for at least 6 months. 39) The method according to any one of paragraphs 23-38, wherein each of the amount of second antagonist when taken alone, and the amount of the compound when taken alone is effective to treat the subject.

40) The method according to any one of paragraphs 23-38, wherein either the amount of second antagonist when taken alone, the amount of the compound when taken alone, or each such amount when taken alone is not effective to treat the subject.

41) The method according to any one of paragraphs 23-38, wherein either the amount of second antagonist when taken alone, the amount of compound when taken alone, or each such amount when taken alone is less effective to treat the subject.

42) The method according to any one of paragraphs 22-41, wherein the patient previously received second antagonist therapy and ceased receiving second antagonist therapy prior to the combination therapy.

43) The method according to any one of paragraphs 22-42, wherein the patient previously failed to respond to the second antagonist therapy or the second antagonist failed to treat the subject.

44) 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.

45) 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.

46) The pharmaceutical composition according to paragraph 44 or 45, comprising essentially an amount of an second antagonist and an amount of the compound.

47) The pharmaceutical composition according to any one of paragraphs 44-46, for use in treating a subject afflicted with cancer, wherein the amount of the second antagonist and an amount of the compound are to be administered simultaneously, contemporaneously or concomitantly. 48) 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.

49) 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.

50) The compound of any one of paragraphs 1-11 for use as an add-on therapy or in combination with second antagonist in treating a subject afflicted with cancer, wherein the second antagonist is a CD38 antagonist, or a PD1 or PD-L1 antagonist.

51) Use of an amount of second antagonist and an amount of an compound of any one of paragraphs 1-11 in the preparation of a combination for treating a subject afflicted with cancer wherein the second antagonist and the compound are prepared to be administered simultaneously, contemporaneously or concomitantly, wherein the second antagonist is a CD38 antagonist, or a PDl or PD-L1 antagonist.

52) A combination of second antagonist and an compound of any one of paragraphs 1-11 for use in the manufacture of a medicament, wherein the second antagonist is a CD38 antagonist, or a PD1 or PD-L1 antagonist.

53) The combination according to paragraph 52, wherein the medicament is for the treatment, prevention, or alleviation of a symptom of cancer.

54) 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. 55) The method of paragraph 54, wherein the disease is a metabolic disease, diabetes, obesity, dyslipidemia, nonalcoholic fatty liver, an infectious disease or COVID-19.

* * *

It is to be understood that the invention is not limited to the particular embodiments of the invention described above, as variations of the particular embodiments may be made and still fall within the scope of the appended claims.

Claims

CLAIMS:

1) A compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein

X is S or O; is a single bond or a double bond;

Ri is H, alkyl, methyl, CH2COOH;

- 82 -

R2 is H, CH3, CF3, OCH3, OCF3, SO2CH3, or a phenyl group optionally substituted with at least

2) The compound of claim 1, wherein Ri is H, methyl or CH2COOH;

3) A compound of claim 1, which has the structure of formula la, lb, or Ic:

- 83 -

or a pharmaceutically acceptable salt thereof.

4) A compound of formula II:

II or a pharmaceutically acceptable salt thereof, wherein

- 84 - Z is CRs , NRs, S, or O;

R3 is lower alkyl, or OCH3;

R4 is lower alkyl;

Rs is lower alkyl, or OCH3;

Rs is NHR12 , lower alkyl or phenyl;

R7 is

Rs is H, lower alkyl,

R9 , Rio and Rn are each independently lower alkyl, and

R12 is alkyl.

5) The compound of claim 4, wherein the compound is a compound of formula Ila, lib, or lie:

- 85 -

or a pharmaceutically acceptable salt thereof.

6) The compound of claim 4 having the structure:

- 86 -

or a pharmaceutically acceptable salt thereof.

7) The compound of claim 4, wherein

R3 is methyl;

R4 is ethyl;

Rs is methyl;

Rs is methyl or phenyl;

R11 is methyl;

Z is C;

R9 is methyl; and/or Rio is methyl.

8) A compound which is:

46

- 93 -

47 or a pharmaceutically acceptable salt thereof.

9) The compound of claim 1 which is an CD38 inhibitor.

10) The compound of claim 1 which is an inhibitor the cyclase activity of CD38.

11) The compound of claim 1 which is an inhibitor of the cyclase activity of CD38.

12) A pharmaceutical composition, comprising a therapeutically effective amount of a compound according to claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

13) 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 claim 1, or a pharmaceutically acceptable salt thereof, thereby treating the subject.

14) The method of claim 13, further comprising increasing the activity of T cells and/or natural killer cells within the subject.

15) The method of claim 13, further comprising (a) preventing reduced levels of extracellular NAD⁺, (b) increasing NADH⁺ levels, (c) inhibiting the NADase function of CD38, or (d) promoting NK cell-mediated tumor toxicitiy, in the subject.

16) The method of claim 13, wherein 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, triplenegative breast cancer, follicular lymphoma, or mantle cell lymphoma.

- 94 - ) The method of claim 13, wherein the cancer comprises a tumor cell which highly expresses CD38. ) The method of claim 13, wherein the cancer is drug resistant multiple myeloma. ) The method according to claim 13, wherein the subject is a human patient. ) The method according to claim 13, wherein the administration is oral, intravenous or intraperitoneal. ) The method of claim 13, further comprising administering a pharmaceutically acceptable carrier with the compound. ) The method of claim 13, in combination with a second antagonist therapy which is a second cluster of differentiation 38 (CD38) antagonist therapy, or a PD1 or PD-L1 antagonist therapy. ) The method of claim 22, wherein 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 method of claim 23, wherein 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 pharmaceutical combination according to claim 23, wherein the second antagonist is a monoclonal antibody (mAb). ) The method of claim 25, wherein the monoclonal antibody is human or humanized. ) The method of claim 23, wherein the second antagonist is a CD38 antagonist which is daratumumab, isatuximab, bortezomib, carfilzomib, lenalidomide, panobinostat, dexamethasone, or selinexor. ) The method of claim 23, wherein the second antagonist is a CD38 antagonist and binds to CD38 receptor on a surface of a tumor cell.

- 95 - ) The method of claim 23, wherein the second antagonist is a PD-1 antagonist which is nivolumab, pembrolizumab, avelumab, durvalumab, cemiplimab, or atezolizumab. ) The method of claim 23, wherein the second antagonist is a CTLA-4 antagonist which is ipilimumab or tremelimumab. ) The method of claim 23, wherein the administration of the compound precedes the administration of the second antagonist. ) The method according to claim 23, wherein the administration of the second antagonist precedes the administration of the compound. ) The method according to claim 23, wherein the second antagonist is administered adjunctively to the compound. ) The method according to claim 23, wherein the compound is administered adjunctively to the second antagonist. ) The method according to claim 23, wherein the subject is receiving second antagonist therapy prior to initiating the compound therapy. ) The method according to claim 22, wherein the subject is receiving the compound therapy prior to initiating second antagonist therapy. ) The method according to claim 23, wherein periodic administration of the second antagonist and/or the compound continues for at least 3 days, for at least 30 days, for at least 42 days, for at least 8 weeks, for at least 12 weeks, for at least 24 weeks or for at least 6 months. ) The method according to claim 23, wherein each of the amount of second antagonist when taken alone, and the amount of the compound when taken alone is effective to treat the subject. ) The method according to claim 23, wherein either the amount of second antagonist when taken alone, the amount of the compound when taken alone, or each such amount when taken alone is not effective to treat the subject. ) The method according to claim 23, wherein either the amount of second antagonist when taken alone, the amount of compound when taken alone, or each such amount when taken alone is less effective to treat the subject.

- 96 - ) The method according to claim 22, wherein the patient previously received second antagonist therapy and ceased receiving second antagonist therapy prior to the combination therapy. ) The method according to claim 22, wherein the patient previously failed to respond to the second antagonist therapy or the second antagonist failed to treat the subject. ) A kit for treating a patient suffering from cancer, comprising a therapeutically effective amount of the compound of claim 1, 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 claim 1, wherein the second antagonist is a CD38 antagonist or a PD1 or PD-L1 antagonist. ) The pharmaceutical composition according to claim 43, comprising essentially an amount of an second antagonist and an amount of the compound. ) The pharmaceutical composition according to claim 43, for use in treating a subject afflicted with cancer, wherein the amount of the second antagonist and an amount of the compound are to be administered simultaneously, contemporaneously or concomitantly.) 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 claim 1 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 claim 1 in treating a subject afflicted with cancer, wherein the second antagonist is a CD38 antagonist, or a PDl or PD-L1 antagonist.

- 97 - ) The compound of claim 1 for use as an add-on therapy or in combination with second antagonist in treating a subject afflicted with cancer, wherein the second antagonist is a CD38 antagonist, or a PDl or PD-L1 antagonist. ) Use of an amount of second antagonist and an amount of an compound of claim 1 in the preparation of a combination for treating a subject afflicted with cancer wherein the second antagonist and the compound are prepared to be administered simultaneously, contemporaneously or concomitantly, wherein the second antagonist is a CD38 antagonist, or a PD1 or PD-L1 antagonist. ) A combination of second antagonist and an compound of claim 1 for use in the manufacture of a medicament, wherein the second antagonist is a CD38 antagonist, or a PD1 or PD-Ll antagonist. ) The combination according to paragraph 51, wherein the medicament is for the treatment, prevention, or alleviation of a symptom of cancer. ) 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 claim 1, 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 method of claim 53, wherein the disease is a metabolic disease, diabetes, obesity, dyslipidemia, nonalcoholic fatty liver, an infectious disease or COVID-19.

- 98 -