Benzofuranoindoline-bearing fungal ripps with Anti-cancer activities

Abstract

Disclosed herein are compounds of the Formula: (I), as well as analogs thereof, wherein the variables are defined herein. Also provided are pharmaceutical compositions thereof. In some aspects, the compounds and compositions provided herein may be used to treat a cancer, such as leukemia. Also provided are methods of administering compounds and compositions provided herein to a patient in need thereof, for example, for the treatment or prevention of diseases or disorders.

Description

DESCRIPTION BENZOFURANOINDOLINE-BEARING FUNGAL RIPPS WITH ANTI-CANCER ACTIVITIES

[0001] This application claims the benefit of priority to United States Provisional Application No. 63 / 664,385 filed June 26, 2024, the entire contents of which are hereby incorporated by reference. FEDERAL FUNDING STATEMENT

[0002] The invention was made with government support under grant No. R35GM138207 awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO A SEQUENCE LISTING

[0003] This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing XML, created on June 24, 2025, is named RICEP0154WO.xml and is 187,960 bytes in size. BACKGROUND OF THE INVENTION I. Field of the Invention

[0004] The present invention relates generally to the field of chemistry, particularly organic chemistry and medicinal chemistry. More particularly, it concerns compounds, compositions, and methods for the treatment and prevention of cancers such as leukemia. II. Description of Related Art

[0005] Peptides have garnered growing interest from the pharmaceutical industry (Dang et al., 2017) and over 80 peptide-based drugs have been developed and introduced to the pharmaceutical market, targeting various human diseases (Zhang & Chen, 2022; Muttenthaler et al., 2021). As a vital family of peptide natural products, ribosomally synthesized and post-translationally modified peptides (RiPPs) exhibit a diverse array of chemical structures and potent biological activities (Arnison et al., 2013, Montalbán-López et al., 2021). While predominantly sourced from bacteria, there exist a handful of RiPPs identified from fungal origins. These fungal RiPPs exhibit a wide range of bioactivities, including theanti-cancer activity of α-amanitin, the nematocidal activity of omphalotin A (Mayer et al., 1997; Ramm et al., 2017; Quijano et al., 2019). the antimitotic activity of ustiloxins (Ford et al., 2022) and phomopsin A (Battilani et al., 2011) as well as the phytotoxic activity of victorin C (Kessler et al., 2020). These examples suggest the enormous potential of fungal RiPPs to serve as new drug leads. Basidiomycete-derived RiPPs including cycloamanides and borosins are distinguished by the head-to-tail macrocyclization mode (FIG. 5) (Ramm et al., 2017; Quijano et al., 2019; Van der Velden et al., 2017). As for ascomycete-derived RiPPs, only five groups of RiPPs have been identified (FIG.1). The structures of these fungal RiPPs all feature cyclic ether motifs, which are proposed to be biosynthesized by fungal-specific DUF3328 oxidases (Sogahata et al., 2021; Nagano et al., 2016). Moreover, their intricate biosynthetic pathways involving cascades of tailoring enzymes highlight the remarkable chemical diversity and biosynthetic versatility of RiPPs in nature.

[0006] Unveiling fungal RiPPs holds great importance due to their promising bioactivities, complex chemical structures, and distinct biosynthetic modifications. However, identification of fungal RiPPs has been hindered by the limited knowledge of their biosynthesis and the lack of efficient toolboxes. Nevertheless, with the expansion of fungal genome database and the development of bioinformatic analysis, a plethora of fungal RiPPs biosynthetic gene clusters have been revealed, underscoring the expansive potential of these well-known prolific producers for RiPPs generation (Nagano et al., 2016; Vignolle et al., 2020) and present remarkable opportunities for the exploration and discovery of new fungal RiPPs.SUMMARY

[0007] The present disclosure provides compounds, compositions, and methods for the treatment and prevention of a disease or disorder.

[0008] In some aspects, the present disclosure provides a compound of the formula:wherein: R1 is hydrogen; or alkyl(C≤12), cycloalkyl(C≤12), acyl(C≤12), heterocycloalkyl(C≤12), aryl(C≤12), aralkyl(C≤12), heteroaryl(C≤12), heteroaralkyl(C≤12), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or −C(O)R12, wherein R12is: hydrogen, hydroxy, or amino; or alkyl(C≤8), cycloalkyl(C≤8), alkoxy(C≤12), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), aralkyl(C≤12), heteroaralkyl(C≤12), hetero- cycloalkyl(C≤8), alkoxy(C≤8), −alkanediyl(C≤12)−aryl(C≤12),−alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or R1 is taken together with R3 as defined below; R2, R4, R5, R6, and R9 are each independently hydrogen, hydroxy, amino or mercapto; or alkyl(C≤8), cycloalkyl(C≤8), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), heterocycloalkyl(C≤8), alkoxy(C≤8), cycloalkoxy(C≤8), acyl(C≤8), amido(C≤8), alkenyloxy(C≤8), aryloxy(C≤8), aralkoxy(C≤8), heteroaryloxy(C≤8), acyloxy(C≤8), alkylamino(C≤8), cycloalkylamino(C≤8), dialkylamino(C≤8), arylamino(C≤8), heteroarylamino(C≤8), alkylsulfonylamino(C≤8), cycloalkylsulfonylamino(C≤8), alkoxyamino(C≤8), hetero- cycloalkylamino(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; R7 and R8 are each independently hydrogen, hydroxy, alkyl(C≤8) or substituted alkyl(C≤8); R3is hydrogen or hydroxy; or alkyl(C≤8), cycloalkyl(C≤8), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), heterocycloalkyl(C≤8), alkoxy(C≤8), cycloalkoxy(C≤8), alkenyloxy(C≤8), aryloxy(C≤8), aralkoxy(C≤8), heteroaryloxy(C≤8), acyloxy(C≤8), alkylamino(C≤8), cyclo- alkylamino(C≤8), dialkylamino(C≤8), arylamino(C≤8), heteroarylamino(C≤8), alkylsulfonylamino(C≤8), cycloalkylsulfonylamino(C≤8), alkoxyamino(C≤8), hetero-cycloalkylamino(C≤8), or a substituted version of any of these groups; or R3 is taken together with R1 and is −C(O)(CR1'R1'')n−, wherein: n is 4 or 5 R1' and R1'' are each independently hydrogen, hydroxy or amino; or alkyl(C≤8), cycloalkyl(C≤8), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), hetero- cycloalkyl(C≤8), alkoxy(C≤8), cycloalkoxy(C≤8), acyl(C≤8), amido(C≤8), alkenyloxy(C≤8), aryloxy(C≤8), aralkoxy(C≤8), heteroaryloxy(C≤8), acyloxy(C≤8), or a substituted version of any of these groups; or −NR2'R2'', wherein R2' and R2'' are each independently hydrogen; or −C(O)R13, wherein R13 is hydrogen, hydroxy, or amino; or alkyl(C≤8), cycloalkyl(C≤8), alkoxy(C≤12), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), aralkyl(C≤12), heteroaralkyl(C≤12), heterocycloalkyl(C≤8), alkoxy(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−amido(C≤18), −alkanediyl(C≤12)−cycloalkyl(C≤12),or a substituted version of any of these groups; R10 is hydrogen, hydroxy or amino; or alkyl(C≤8), cycloalkyl(C≤8), heterocycloalkyl(C≤8), amido(C≤8), alkylamino(C≤8), cycloalkylamino(C≤8), dialkyl- amino(C≤8), arylamino(C≤8), heteroarylamino(C≤8), alkyl- sulfonylamino(C≤8), cycloalkylsulfonylamino(C≤8), alkoxyamino(C≤8), heterocycloalkylamino(C≤8), or a substituted version of any of these groups; or R10 is taken together with R11 as defined below; R11 is hydrogen or hydroxy; or alkyl(C≤8), cycloalkyl(C≤8), heterocycloalkyl(C≤8), alkoxy(C≤8), cycloalkoxy(C≤8), acyl(C≤8), amido(C≤8), alkenyloxy(C≤8), aryloxy(C≤8), aralkoxy(C≤8), heteroaryloxy(C≤8), acyloxy(C≤8), or a substituted version of any of these groups; or R10 is taken together with R11 and is –O(CR3'R3'')m(NH)−, wherein: m is 2, 3 or 4 R3' and R3'' are each independently hydrogen, hydroxy or amino; or alkyl(C≤12), cycloalkyl(C≤12), acyl(C≤12), heterocycloalkyl(C≤12), aryl(C≤12), heteroaryl(C≤12), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or−C(O)R16, wherein R16is: hydrogen, hydroxy, or amino; or alkyl(C≤8), cycloalkyl(C≤8), alkoxy(C≤12), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), aralkyl(C≤12), heteroaralkyl(C≤12), heterocycloalkyl(C≤8), alkoxy(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or a pharmaceutically acceptable salt thereof.

[0009] In some embodiments, the compound is further defined as:wherein: R1is hydrogen; or alkyl(C≤12), cycloalkyl(C≤12), acyl(C≤12), heterocycloalkyl(C≤12), aryl(C≤12), aralkyl(C≤12), heteroaryl(C≤12), heteroaralkyl(C≤12),−alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or −C(O)R12, wherein R12 is: hydrogen, hydroxy, or amino; or alkyl(C≤8), cycloalkyl(C≤8), alkoxy(C≤12), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), aralkyl(C≤12), heteroaralkyl(C≤12), hetero- cycloalkyl(C≤8), alkoxy(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or R1 is taken together with R3 as defined below; R2, R4, R5, R6, and R9 are each independently hydrogen, hydroxy, amino or mercapto; or alkyl(C≤8), cycloalkyl(C≤8), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), heterocycloalkyl(C≤8), alkoxy(C≤8), cycloalkoxy(C≤8), acyl(C≤8), amido(C≤8), alkenyloxy(C≤8), aryloxy(C≤8), aralkoxy(C≤8), heteroaryloxy(C≤8), acyloxy(C≤8), alkylamino(C≤8), cycloalkylamino(C≤8), dialkylamino(C≤8), arylamino(C≤8), heteroarylamino(C≤8), alkylsulfonylamino(C≤8), cycloalkylsulfonylamino(C≤8), alkoxyamino(C≤8), hetero- cycloalkylamino(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups;R3is hydrogen or hydroxy; or alkyl(C≤8), cycloalkyl(C≤8), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), heterocycloalkyl(C≤8), alkoxy(C≤8), cycloalkoxy(C≤8), alkenyloxy(C≤8), aryloxy(C≤8), aralkoxy(C≤8), heteroaryloxy(C≤8), acyloxy(C≤8), alkylamino(C≤8), cyclo- alkylamino(C≤8), dialkylamino(C≤8), arylamino(C≤8), heteroarylamino(C≤8), alkylsulfonylamino(C≤8), cycloalkylsulfonylamino(C≤8), alkoxyamino(C≤8), hetero- cycloalkylamino(C≤8), or a substituted version of any of these groups; or R3is taken together with R1and is −C(O)(CR1'R1'')n−, wherein: n is 4 or 5 R1' and R1'' are each independently hydrogen, hydroxy or amino; or alkyl(C≤8), cycloalkyl(C≤8), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), hetero- cycloalkyl(C≤8), alkoxy(C≤8), cycloalkoxy(C≤8), acyl(C≤8), amido(C≤8), alkenyloxy(C≤8), aryloxy(C≤8), aralkoxy(C≤8), heteroaryloxy(C≤8), acyloxy(C≤8), or a substituted version of any of these groups; or −NR2'R2'', wherein R2' and R2'' are each independently hydrogen; or −C(O)R13, wherein R13is hydrogen, hydroxy, or amino; or alkyl(C≤8), cycloalkyl(C≤8), alkoxy(C≤12), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8),aralkyl(C≤12), heteroaralkyl(C≤12), heterocycloalkyl(C≤8), alkoxy(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−amido(C≤18), −alkanediyl(C≤12)−heterocycloalky l(C≤12), −alkanediyl(C≤12)−cyclo- alkyl(C≤12), or a substituted version of any of these groups; R10 is hydrogen, hydroxy or amino; or alkyl(C≤8), cycloalkyl(C≤8), heterocycloalkyl(C≤8), amido(C≤8), alkylamino(C≤8), cycloalkylamino(C≤8), dialkyl- amino(C≤8), arylamino(C≤8), heteroarylamino(C≤8), alkyl- sulfonylamino(C≤8), cycloalkylsulfonylamino(C≤8), alkoxyamino(C≤8), heterocycloalkylamino(C≤8), or a substituted version of any of these groups; or R10 is taken together with R11 as defined below; R11 is hydrogen or hydroxy; or alkyl(C≤8), cycloalkyl(C≤8), heterocycloalkyl(C≤8), alkoxy(C≤8), cycloalkoxy(C≤8), acyl(C≤8), amido(C≤8), alkenyloxy(C≤8), aryloxy(C≤8), aralkoxy(C≤8), heteroaryloxy(C≤8), acyloxy(C≤8), or a substituted version of any of these groups; or R10 is taken together with R11 and is –O(CR3'R3'')m(NH)−, wherein: m is 2, 3 or 4 R3' and R3'' are each independently hydrogen, hydroxy or amino; oralkyl(C≤12), cycloalkyl(C≤12), acyl(C≤12), heterocycloalkyl(C≤12), aryl(C≤12), heteroaryl(C≤12), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or −C(O)R16, wherein R16is: hydrogen, hydroxy, or amino; or alkyl(C≤8), cycloalkyl(C≤8), alkoxy(C≤12), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), aralkyl(C≤12), heteroaralkyl(C≤12), heterocycloalkyl(C≤8), alkoxy(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or a pharmaceutically acceptable salt thereof.

[0010] In some embodiments, the compound is further defined as:wherein: R2, R4, R5, R6, and R9are each independently hydrogen, hydroxy, amino or mercapto; or alkyl(C≤8), cycloalkyl(C≤8), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), heterocycloalkyl(C≤8), alkoxy(C≤8), cycloalkoxy(C≤8), acyl(C≤8), amido(C≤8), alkenyloxy(C≤8), aryloxy(C≤8), aralkoxy(C≤8), heteroaryloxy(C≤8), acyloxy(C≤8), alkylamino(C≤8), cycloalkylamino(C≤8), dialkylamino(C≤8), arylamino(C≤8), heteroarylamino(C≤8), alkylsulfonylamino(C≤8), cycloalkylsulfonylamino(C≤8), alkoxyamino(C≤8), hetero- cycloalkylamino(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; R10is hydrogen, hydroxy or amino; or alkyl(C≤8), cycloalkyl(C≤8), heterocycloalkyl(C≤8), amido(C≤8), alkylamino(C≤8), cycloalkylamino(C≤8), dialkyl- amino(C≤8), arylamino(C≤8), heteroarylamino(C≤8), alkyl- sulfonylamino(C≤8), cycloalkylsulfonylamino(C≤8), alkoxyamino(C≤8), heterocycloalkylamino(C≤8), or a substituted version of any of these groups; or R10is taken together with R11as defined below; R11is hydrogen or hydroxy; or alkyl(C≤8), cycloalkyl(C≤8), heterocycloalkyl(C≤8), alkoxy(C≤8), cycloalkoxy(C≤8), acyl(C≤8), amido(C≤8), alkenyloxy(C≤8), aryloxy(C≤8), aralkoxy(C≤8), heteroaryloxy(C≤8),acyloxy(C≤8), or a substituted version of any of these groups; or R10 is taken together with R11 and is –O(CR3'R3'')m(NH)−, wherein: m is 2, 3 or 4 R3' and R3'' are each independently hydrogen, hydroxy or amino; or alkyl(C≤12), cycloalkyl(C≤12), acyl(C≤12), heterocycloalkyl(C≤12), aryl(C≤12), heteroaryl(C≤12), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or −C(O)R14, wherein R14 is: hydrogen, hydroxy, or amino; or alkyl(C≤8), cycloalkyl(C≤8), alkoxy(C≤12), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), aralkyl(C≤12), heteroaralkyl(C≤12), heterocycloalkyl(C≤8), alkoxy(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; R15 is hydrogen; or alkyl(C≤12), cycloalkyl(C≤12), acyl(C≤12), heterocycloalkyl(C≤12), aryl(C≤12), heteroaryl(C≤12), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12),−alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or −C(O)R16, wherein R16is: hydrogen, hydroxy, or amino; or alkyl(C≤8), cycloalkyl(C≤8), alkoxy(C≤12), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), aralkyl(C≤12), heteroaralkyl(C≤12), hetero- cycloalkyl(C≤8), alkoxy(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−amido(C≤18), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or a pharmaceutically acceptable salt thereof.

[0011] In some embodiments, the compound is further defined as:wherein:R2, R4, R5, R6, and R9are each independently hydrogen, hydroxy, amino or mercapto; or alkyl(C≤8), cycloalkyl(C≤8), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), heterocycloalkyl(C≤8), alkoxy(C≤8), cycloalkoxy(C≤8), acyl(C≤8), amido(C≤8), alkenyloxy(C≤8), aryloxy(C≤8), aralkoxy(C≤8), heteroaryloxy(C≤8), acyloxy(C≤8), alkylamino(C≤8), cycloalkylamino(C≤8), dialkylamino(C≤8), arylamino(C≤8), heteroarylamino(C≤8), alkylsulfonylamino(C≤8), cycloalkylsulfonylamino(C≤8), alkoxyamino(C≤8), hetero- cycloalkylamino(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; R15is hydrogen; or alkyl(C≤12), cycloalkyl(C≤12), acyl(C≤12), heterocycloalkyl(C≤12), aryl(C≤12), heteroaryl(C≤12), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or −C(O)R14, wherein R14 is: hydrogen, hydroxy, or amino; or alkyl(C≤8), cycloalkyl(C≤8), alkoxy(C≤12), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), aralkyl(C≤12), heteroaralkyl(C≤12), hetero- cycloalkyl(C≤8), alkoxy(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−amido(C≤18), −alkanediyl(C≤12)−heterocycloalkyl(C≤12),−alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or a pharmaceutically acceptable salt thereof.

[0012] In some embodiments, the compound is further defined as:wherein: R9is hydrogen, alkyl(C≤8), or substituted alkyl(C≤8); R15 is hydrogen; or alkyl(C≤12), cycloalkyl(C≤12), acyl(C≤12), heterocycloalkyl(C≤12), aryl(C≤12), heteroaryl(C≤12), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or −C(O)R14, wherein R14is: hydrogen, hydroxy, or amino; or alkyl(C≤8), cycloalkyl(C≤8), alkoxy(C≤12), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), aralkyl(C≤12), heteroaralkyl(C≤12), heterocycloalkyl(C≤8), alkoxy(C≤8), −alkanediyl(C≤12)−aryl(C≤12),−alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−amido(C≤18), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or a pharmaceutically acceptable salt thereof.

[0013] In some embodiments, R9is hydrogen. In other embodiments, R9is methyl. In some embodiments, R15 is −C(O)R14, wherein R14 is: hydrogen, hydroxy, or amino; or alkyl(C≤8), cycloalkyl(C≤8), alkoxy(C≤12), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), aralkyl(C≤12), heteroaralkyl(C≤12), heterocycloalkyl(C≤8), alkoxy(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−amido(C≤18), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups.

[0014] In particular aspects, R14 is −alkanediyl(C≤12)−amido(C≤18). In some embodiments, R2 is hydrogen or hydroxy. In some aspects, R4 is alkyl(C≤8) or substituted alkyl(C≤8). In some embodiments, R5is hydrogen, alkyl(C≤8)or substituted alkyl(C≤8). In some embodiments, R6 is hydrogen, alkyl(C≤8) or substituted alkyl(C≤8). In some embodiments, R10 is taken together with R11 and is –O(CR3'R3'')m(NH)−, wherein: R3' and R3'' are each independently hydrogen, alkyl(C≤12)or −C(O)R16, wherein R16is hydrogen, hydroxy, or amino. In some embodiments, R16 is hydroxy.

[0015] In some embodiments, the compound is further defined as:,,,,,,,

[0016] In some aspects, the present disclosure provides a pharmaceutical composition comprising: (a) a compound disclosed herein; and (b) an excipient.

[0017] In some aspects, the present disclosure provides a method of treating or preventing a disease or disorder in a patient in need thereof comprising administering to the patient a pharmaceutically effective amount of a compound or composition disclosed herein. In some embodiments, the disease or disorder is a cancer, such as leukemia.

[0018] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Note that simply because a particular compound is ascribed to one particular generic formula doesn’t mean that it cannot also belong to another generic formula.BRIEF DESCRIPTION OF THE FIGURES

[0019] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description.

[0020] FIGS. 1A-E show the discovery and engineering of asperigimycins. a.) The candidate cluster of fungal RiPPs from A. flavus and A. oryzae and the MS / MS spectrum details of parent ion m / z 933.411 [M+H]+. Node sizes represent relative precursor ion intensity. b.) Amino acids sequence of the putative precursor peptide ApgA, including a signal peptide (underscored) and nine imperfect repeats. ApgATis indicated in a square bracket. c.) LC-MS analysis of wild-type (WT) and gene deletion mutants including A. flavus WT (i), ΔapgA (ii), and ΔapgA::apgAT(iii). Extracted ion chromatograms (EICs) of 1 (m / z = 1020 [M+H]+), 2 (m / z = 933 [M+H]+), 3 (m / z = 973 [M+H]+), and 4 (m / z = 987 [M+H]+) are shown and labeled, respectively. d.) Chemical structures of asperigimycins A-D (1-4). e.) Engineering the precursor peptide by mutagenesis. Subscript numbers denote the position of amino acids in core peptide for 3. X1, X4, X5, X7, X8, and X9can be replaced by corresponding amino acids indicated.

[0021] FIGS. 2A-C show the characterization of asperigimycin biosynthesis. a.) Putative gene cluster apg from A. flavus. Information for all genes is listed below in the Examples section. b.) Metabolites analysis of A. flavus WT (i) and single-gene knock out mutants including ΔapgYa (ii), ΔapgYb (iii), ΔapgYc (iv), ΔapgYd (v), ΔapgYe (vi), ΔapgYf (vii), ΔapgG (viii). EICs of 1 (m / z = 1020 [M+H]+), 2 (m / z = 933 [M+H]+), 3 (m / z = 973 [M+H]+), 4 (m / z = 987 [M+H]+), 5 (m / z = 1004 [M+H]+), 6 (m / z = 917 [M+H]+), 7 (m / z = 957 [M+H]+), 8 (m / z = 971 [M+H]+), 9 (m / z = 822 [M+H]+), 10 (m / z = 862 [M+H]+), 11 (m / z = 876 [M+H]+), 12 (m / z = 806 [M+H]+), 13 (m / z = 846 [M+H]+), 14 (m / z = 860 [M+H]+), 15 (m / z = 990 [M+H]+), and 16 (m / z = 1004 [M+H]+) are shown. The dashed line indicates the consistency of retention time. c.) Chemical structures of 1-16 and proposed biosynthetic pathway of asperigimycins. pGlu, pyroglutamate.

[0022] FIGS. 3A-F show the N-terminal modification of asperigimycins to enhance their anticancer potency. a. Chemical structures of 1-4. b. Anti-proliferation activity of 1-4, and positive control bufalin against human cancer cell lines (Jurkat, Mino, Molm-14, U937, K562, MCF-7, HeLa, and HepG2). c. LC-MS analysis of ApgG in vitro assays. (i) Standard compound 3 from A. flavus WT, (ii) Boiled ApgGT17+ 0.1 mM 15, (iii) 10 μM ApgGT17+ 0.1mM 15. These aliquots were incubated at 28°C for 30 min. EICs of 3 (m / z = 973 [M+H]+) and 15 (m / z = 990 [M+H]+) are shown, respectively. d. Chemical structures of asperigimycin derivatives (2-L1 to 2-L7) with different N-terminal lipid substitutions at R2of compound 2. e. Cytotoxicity of 2-L1 to 2-L7, cytarabine, and daunorubicin against human cancer cell lines (same as Fig.3b). f. Cytotoxic effects of 2, 2-L6, cytarabine, and daunorubicin on Jurkat, Mino, and Molm-14. All cytotoxicity data in this figure are mean ± s.d.; n = 3 biologically independent samples.

[0023] FIGS. 4A-F show CRISPR screening, which reveals that SLC46A3 serves as the transporter of asperigimycin. a.) The whole workflow of high throughput CRISPR screening with Jurkat and 2-L6. Created in BioRender. Nie, Q. (2025) https: / / BioRender.com / 37bqzdc. b.) Distribution of log2(fold change) (LFC) of model-based analysis of genome-wide CRISPR-Cas9 knockout (MAGeCK) score for each sgRNA target (gene) enriched in 2-L6 treatment versus control cells. Size indicates FDR values, in which FDR denotes the false discovery rate. c.) The FDR and LFC in positive selection of 126 genes with LFC > 1. The top 5 most significant genes were marked. d.) The cell viability of WT, SLC46A3Δ, CCNFΔ, CLASP2Δ, KEAP1Δ, and STMN1Δ Jurkat cells mutants treated by 80 nM 2-L6 for 72 h (n = 3 biologically replicated experiments; mean ± s.d.), indicating depletion of these five genes conferring resistance of Jurkat to 2-L6. e.) Dose-response curves and IC50 values of WT, SLC46A3Δ, CCNFΔ, CLASP2Δ, KEAP1Δ, and STMN1Δ Jurkat cells treated with 2-L6. f.) SLC46A3 mediates asperigimycins uptake. WT and SLC46A3Δ Jurkat cells were incubated with 500 nM 2-L6 and 500 nM 2, respectively, for 12 h and 24 h. Intracellular concentration of compounds was quantified via LC-MS / MS assays and the uptake amount was normalized by cellular protein content. *ND means undetectable. Data are mean ± s.d.; n = 3 biologically independent samples.

[0024] FIG.5 shows the chemical structures of fungal RiPPs from basidiomycetes and ascomycetes.

[0025] FIG.6 shows the biosynthesis of known fungal RiPPs.

[0026] FIG.7 shows the GNPS of metabolites of 12 strains.

[0027] FIG.8 shows the candidate precursor peptides in A. flavus.

[0028] FIGS.9A & B show selected 2D NMR data of Asperigimycin A.

[0029] FIGS.10A & B show the LC-MS of metabolites in ΔapgA::ApgAT mutants.

[0030] FIG.11 shows the homologous gene cluster of apg.

[0031] FIG. 12 shows the HPLC analysis of gene deletion mutants. (i) ΔapgB, (ii) ΔapgC, (iii) ΔapgD, (iv) ΔapgQ, (v) ΔapgE, (vi) ΔapgF, (vii) ΔapgH, (viii) ΔapgI.

[0032] FIG.13 shows the SSN analysis of enzymes with DUF3328 domain.

[0033] FIGS. 14A-E show a.) A comparison of structure of ApgG with human glutaminyl cyclotransferase. b-c) Molecular docking of ApgGTand 15. d.) Relative production ratio of ApgG mutants. e.) Proposed enzymatic mechanisms of ApgG.

[0034] FIG.15 shows the crystal diffraction and refinement data.

[0035] FIG.16 shows the MS / MS spectra of 1-4, 15 and 16.

[0036] FIGS. 17A & B show the PCR verification of apg mutants. Scheme of hygromycin-resistance split-marker approach for gene knockout of genes in apg and verification of mutants by PCR.

[0037] FIG.18 shows the sequencing results of ΔapgA::apgAT mutants.

[0038] FIG.19 shows the antimicrobial activity of 1-4.

[0039] FIGS. 20A & B show the analysis of signal peptide by SignalP 6.0. A. ApgA and B. ApgG.

[0040] FIG.21 shows the SDS-PAGE analysis of ApgGT and its mutants.

[0041] FIG.22 shows the dose-response curves of cell viability assays.

[0042] FIGS. 23A-E show the sequencing results of SLC46A3Δ, CCNFΔ, KEAP1Δ, CLAPS2Δ, and STMN1Δ. It indicated that indels were successfully introduced into the correlated genes.

[0043] FIGS. 24A & B show the cytotoxicity assays of 2-L6toward Jurkat with the treatment of endocytosis inhibitors. Jurkat cells were pre-incubated with 50 μM genistein or 30 μM hydroxy-dynasore for 1 h, followed by treatment with 5 μM 2-L6 at 37°C with 5% CO₂ overnight. a. Cell viability of Jurkat cells with inhibitors and 2-L6 normalized by the group only treated with equivalent DMSO. In control group, 95% and 74% Jurkat cells survived treated with genistein and hydroxy-dynasore. The addition of 2-L6 decreased cell viability to 94%, 56%, and 25% in Jurkat cells pretreated with genistein, hydroxy-dynasore, or DMSO, respectively. Despite the inherent cytotoxicity of endocytosis inhibitors toward Jurkat, they significantly mitigated the cytotoxicity of 2-L6. b. Cell viability was normalized to cells treated with the respective inhibitors. Without inhibitors, 25% of cells survived with 2-L6 treatment. In the present of genistein and hydroxy-dynasore, 98% and 74% of cells survived treated with 2-L6, respectively. Cytotoxicity assays were tested with three biologically independent samples. Data are mean ± s.d.; n = 3 biologically independent samples.

[0044] FIGS. 25A-C show the potential effects of 2-L6on the CCNF-mediated pathway and tubulin polymerization. a. Immunofluorescence microscopy analysis of tubulin polymerization. Jurkat cells were incubated for 12 hours with either DMSO (top panel), 500nM 2 (middle panel) or 500 nM 2-L6 (bottom panel), two times of the experiment were repeated with the same results. b. In vitro tubulin polymerization assay. The fluorescence-based tubulin polymerization assay was performed using the Cytoskeleton kit (BK011P), following the manufacturer’s protocol. Time-response curves for 10 μM 2-L6, 10 μM 2, 10 μM 3, 5 μM paclitaxel (a tubulin polymerization enhancer), and DMSO control are shown. Compounds 2- L6, 2, and 3 inhibited tubulin polymerization in vitro. c. Western blot analysis of E2F1 and RRM2 expression in Jurkat cells treated with increasing concentrations of 2-L6 for 20 h.DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0045] Disclosed herein is a new class of fungal RiPPs, named asperigimycins, produced by Aspergillus flavus through the combination of metabolite MS / MS networking and bioinformatics analysis. Asperigimycins possess an unusual structure of a benzofuranoindoline core structure with three fused macrocycles that form post-translationally, representing a group of natural products that have not been reported previously. Notably, asperigimycins C (3) and D (4) (FIG.2), which possess an N-terminal pyroglutamate moiety, exhibited moderate anti- cancer activity. This activity is notably absent in asperigimycins A (1) and B (2) (FIG.2), both of which contain an N-terminal free amine group. The glutaminyl cyclase ApgG, which is responsible for the pyroglutamate formation was identified and characterized. Furthermore, inspired by the importance of the N-terminal structure for the bioactivities of asperigimycins, the inventors chemically synthesized a series of N-terminal substituted derivatives by coupling with lipid chains of different lengths and topologies. An asperigimycin derivative 2-L6, featuring a C-11 linear lipid chain displays significant improvement in bioactivity. The in vitro IC50value of 2-L6 is less than 100 nM against three leukemia cell lines, which is comparable to that of clinically approved anti-leukemia drugs cytarabine and daunorubicin. Finally, high- throughput CRISPR screening implicated a transporter SLC46A3 participates the uptake of lipidized asperigimycins, which may be responsible for their anticancer efficacy. III. Compounds of the Present Invention

[0046] The compounds of the present invention (also referred to as “compounds of the present disclosure”) are shown, for example in the summary of the invention section, in the examples section, and in the claims. They may be made using the synthetic methods outlined in the Examples section. These methods can be further modified and optimized using the principles and techniques of organic chemistry. Such principles and techniques are taught, for example, in Smith, March’s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, (2013), which is incorporated by reference herein. In addition, the synthetic methods may be further modified and optimized for preparative, pilot- or large-scale production, either batch or continuous, using the principles and techniques of process chemistry as applied by a person skilled in the art. Such principles and techniques are taught, for example, in Anderson, Practical Process Research & Development – A Guide for Organic Chemists (2012), which is incorporated by reference herein.

[0047] All the compounds of the present invention may in some embodiments be used for the prevention and treatment of one or more diseases or disorders discussed herein or otherwise. In some embodiments, one or more of the compounds characterized or exemplified herein as an intermediate, a metabolite, and / or prodrug, may nevertheless also be useful for the prevention and treatment of one or more diseases or disorders. As such unless explicitly stated to the contrary, all the compounds of the present invention are deemed “active compounds” and “therapeutic compounds” that are contemplated for use as active pharmaceutical ingredients (APIs). Actual suitability for human or veterinary use is typically determined using a combination of clinical trial protocols and regulatory procedures, such as those administered by the Food and Drug Administration (FDA). In the United States, the FDA is responsible for protecting the public health by assuring the safety, effectiveness, quality, and security of human and veterinary drugs, vaccines and other biological products, and medical devices.

[0048] In some embodiments, the compounds of the present invention have the advantage that they may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, be more easily absorbed than, more metabolically stable than, more lipophilic than, more hydrophilic than, and / or have a better pharmacokinetic profile (e.g., higher oral bioavailability and / or lower clearance) than, and / or have other useful pharmacological, physical, or chemical properties over, compounds known in the prior art, whether for use in the indications stated herein or otherwise.

[0049] Compounds of the present invention may contain one or more asymmetrically- substituted carbon, nitrogen, sulfur, or phosphorus atom and may be isolated in optically active or racemic form. Thus, all chiral, diastereomeric, racemic form, epimeric form, and all geometric isomeric forms of a chemical formula are intended, unless the specific stereochemistry or isomeric form is specifically indicated. Compounds may occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. In some embodiments, a single diastereomer is obtained. The chiral centers of the compounds of the present invention can have the S or the R configuration. In some embodiments, the present compounds may contain two or more atoms which have a defined stereochemical orientation.

[0050] Chemical formulas used to represent compounds of the present invention will typically only show one of possibly several different tautomers. For example, many types of ketone groups are known to exist in equilibrium with corresponding enol groups. Similarly,many types of imine groups exist in equilibrium with enamine groups. Regardless of which tautomer is depicted for a given compound, and regardless of which one is most prevalent, all tautomers of a given chemical formula are intended.

[0051] In addition, atoms making up the compounds of the present invention are intended to include all isotopic forms of such atoms. Isotopes, as used herein, include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include13C and14C.

[0052] In some embodiments, compounds of the present invention function as prodrugs or can be derivatized to function as prodrugs. Since prodrugs are known to enhance numerous desirable qualities of pharmaceuticals (e.g., solubility, bioavailability, manufacturing, etc.), the compounds employed in some methods of the invention may, if desired, be delivered in prodrug form. Thus, the invention contemplates prodrugs of compounds of the present invention as well as methods of delivering prodrugs. Prodrugs of the compounds employed in the invention may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound. Accordingly, prodrugs include, for example, compounds described herein in which a hydroxy, amino, or carboxy group is bonded to any group that, when the prodrug is administered to a patient, cleaves to form a hydroxy, amino, or carboxylic acid, respectively.

[0053] In some embodiments, compounds of the present invention exist in salt or non- salt form. With regard to the salt form(s), in some embodiments the particular anion or cation forming a part of any salt form of a compound provided herein is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.

[0054] It will be appreciated that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as “solvates.” Where the solvent is water, the complex is known as a “hydrate.” It will also be appreciated that many organic compounds can exist in more than one solid form, including crystalline and amorphous forms. All solid forms of the compounds provided herein, including any solvates thereof are within the scope of the present invention.IV. Pharmaceutical Formulations and Routes of Administration

[0055] In another aspect, for administration to a patient in need of such treatment, pharmaceutical formulations (also referred to as a pharmaceutical preparations, pharmaceutical compositions, pharmaceutical products, medicinal products, medicines, medications, or medicaments) comprise a therapeutically effective amount of a compound disclosed herein formulated with one or more excipients and / or drug carriers appropriate to the indicated route of administration. In some embodiments, the compounds disclosed herein are formulated in a manner amenable for the treatment of human and / or veterinary patients. In some embodiments, formulation comprises admixing or combining one or more of the compounds disclosed herein with one or more of the following excipients: lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia, sodium alginate, polyvinylpyrrolidone, and / or polyvinyl alcohol. In some embodiments, e.g., for oral administration, the pharmaceutical formulation may be tableted or encapsulated. In some embodiments, the compounds may be dissolved or slurried in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and / or various buffers. In some embodiments, the pharmaceutical formulations may be subjected to pharmaceutical operations, such as sterilization, and / or may contain drug carriers and / or excipients such as preservatives, stabilizers, wetting agents, emulsifiers, encapsulating agents such as lipids, dendrimers, polymers, proteins such as albumin, nucleic acids, and buffers.

[0056] Pharmaceutical formulations may be administered by a variety of methods, e.g., orally or by injection (e.g. subcutaneous, intravenous, and intraperitoneal). Depending on the route of administration, the compounds disclosed herein may be coated in a material to protect the compound from the action of acids and other natural conditions which may inactivate the compound. To administer the active compound by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. In some embodiments, the active compound may be administered to a patient in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in- oil-in-water CGF emulsions as well as conventional liposomes.

[0057] The compounds disclosed herein may also be administered parenterally, intraperitoneally, intraspinally, or intracerebrally. Dispersions can be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

[0058] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (such as, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

[0059] The compounds disclosed herein can be administered orally, for example, with an inert diluent or an assimilable edible carrier. The compounds and other ingredients may also be enclosed in a hard or soft-shell gelatin capsule, compressed into tablets, or incorporated directly into the patient’s diet. For oral therapeutic administration, the compounds disclosed herein may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The percentage of the therapeutic compound in the compositions and preparations may, of course, be varied. The amount of the therapeutic compound in such pharmaceutical formulations is such that a suitable dosage will be obtained.

[0060] The therapeutic compound may also be administered topically to the skin, eye, ear, or mucosal membranes. Administration of the therapeutic compound topically may include formulations of the compounds as a topical solution, lotion, cream, ointment, gel, foam, transdermal patch, or tincture. When the therapeutic compound is formulated for topical administration, the compound may be combined with one or more agents that increase the permeability of the compound through the tissue to which it is administered. In other embodiments, it is contemplated that the topical administration is administered to the eye. Such administration may be applied to the surface of the cornea, conjunctiva, or sclera. Withoutwishing to be bound by any theory, it is believed that administration to the surface of the eye allows the therapeutic compound to reach the posterior portion of the eye. Ophthalmic topical administration can be formulated as a solution, suspension, ointment, gel, or emulsion. Finally, topical administration may also include administration to the mucosa membranes such as the inside of the mouth. Such administration can be directly to a particular location within the mucosal membrane such as a tooth, a sore, or an ulcer. Alternatively, if local delivery to the lungs is desired the therapeutic compound may be administered by inhalation in a dry-powder or aerosol formulation.

[0061] In some embodiments, it may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. In some embodiments, the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such a therapeutic compound for the treatment of a selected condition in a patient. In some embodiments, active compounds are administered at a therapeutically effective dosage sufficient to treat a condition associated with a condition in a patient. For example, the efficacy of a compound can be evaluated in an animal model system that may be predictive of efficacy in treating the disease in a human or another animal.

[0062] In some embodiments, the effective dose range for the therapeutic compound can be extrapolated from effective doses determined in animal studies for a variety of different animals. In some embodiments, the human equivalent dose (HED) in mg / kg can be calculated in accordance with the following formula (see, e.g., Reagan-Shaw et al., FASEB J., 22(3):659- 661, 2008, which is incorporated herein by reference): HED (mg / kg) = Animal dose (mg / kg) × (Animal Km / Human Km)

[0063] Use of the Kmfactors in conversion results in HED values based on body surface area (BSA) rather than only on body mass. Kmvalues for humans and various animals are well known. For example, the Kmfor an average 60 kg human (with a BSA of 1.6 m2) is 37, whereas a 20 kg child (BSA 0.8 m2) would have a Kmof 25. Kmfor some relevant animal models are also well known, including: mice Kmof 3 (given a weight of 0.02 kg and BSA of 0.007);hamster Kmof 5 (given a weight of 0.08 kg and BSA of 0.02); rat Kmof 6 (given a weight of 0.15 kg and BSA of 0.025) and monkey Km of 12 (given a weight of 3 kg and BSA of 0.24).

[0064] Precise amounts of the therapeutic composition depend on the judgment of the practitioner and are specific to each individual. Nonetheless, a calculated HED dose provides a general guide. Other factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment and the potency, stability and toxicity of the particular therapeutic formulation.

[0065] The actual dosage amount of a compound of the present disclosure or composition comprising a compound of the present disclosure administered to a patient may be determined by physical and physiological factors such as type of animal treated, age, sex, body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. These factors may be determined by a skilled artisan. The practitioner responsible for administration will typically determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual patient. The dosage may be adjusted by the individual physician in the event of any complication.

[0066] In some embodiments, the therapeutically effective amount typically will vary from about 0.001 mg / kg to about 1000 mg / kg, from about 0.01 mg / kg to about 750 mg / kg, from about 100 mg / kg to about 500 mg / kg, from about 1 mg / kg to about 250 mg / kg, from about 10 mg / kg to about 150 mg / kg in one or more dose administrations daily, for one or several days (depending of course of the mode of administration and the factors discussed above). Other suitable dose ranges include 1 mg to 10,000 mg per day, 100 mg to 10,000 mg per day, 500 mg to 10,000 mg per day, and 500 mg to 1,000 mg per day. In some embodiments, the amount is less than 10,000 mg per day with a range of 750 mg to 9,000 mg per day.

[0067] In some embodiments, the amount of the active compound in the pharmaceutical formulation is from about 2 to about 75 weight percent. In some of these embodiments, the amount if from about 25 to about 60 weight percent.

[0068] Single or multiple doses of the agents are contemplated. Desired time intervals for delivery of multiple doses can be determined by one of ordinary skill in the art employing no more than routine experimentation. As an example, patients may be administered two doses daily at approximately 12-hour intervals. In some embodiments, the agent is administered once a day.

[0069] The agent(s) may be administered on a routine schedule. As used herein a routine schedule refers to a predetermined designated period of time. The routine schedule mayencompass periods of time which are identical, or which differ in length, as long as the schedule is predetermined. For instance, the routine schedule may involve administration twice a day, every day, every two days, every three days, every four days, every five days, every six days, a weekly basis, a monthly basis or any set number of days or weeks there-between. Alternatively, the predetermined routine schedule may involve administration on a twice daily basis for the first week, followed by a daily basis for several months, etc. In other embodiments, the invention provides that the agent(s) may be taken orally and that the timing of which is or is not dependent upon food intake. Thus, for example, the agent can be taken every morning and / or every evening, regardless of when the patient has eaten or will eat. V. Chemical Definitions

[0070] When used in the context of a chemical group: “hydrogen” means −H; “hydroxy” means −OH; “oxo” means =O; “carbonyl” means −C(=O)−; “carboxy” means −C(=O)OH (also written as −COOH or −CO2H); “halo” means independently −F, −Cl, −Br or −I; “amino” means −NH2; “hydroxyamino” means −NHOH; “nitro” means −NO2; imino means =NH; “cyano” means −CN; “isocyanyl” means −N=C=O; “azido” means −N3; in a monovalent context “phosphate” means −OP(O)(OH)2or a deprotonated form thereof; in a divalent context “phosphate” means −OP(O)(OH)O− or a deprotonated form thereof; “mercapto” means −SH; and “thio” means =S; “thiocarbonyl” means −C(=S)−; “sulfonyl” means −S(O)2−; and “sulfinyl” means −S(O)−.

[0071] In the context of chemical formulas, the symbol “−”eans a single bond, “=”means a double bond, and “≡” means triple bond. The symbol “ ” represents an optionalbond, which if present is either single or double. The symbol “ ” represents a single bondor a double bond. Thus, the formulaovers, for example,And it is understood that no one such ring atom forms part of more than one double bond. Furthermore, it is noted that the covalent bond symbol “−”, when connecting one or two stereogenic atoms, does not indicate any preferred stereochemistry. Instead, it covers allstereoisomers as well as mixtures thereof. The symbol “ ”, when drawn perpendicularlyacross a bond (e.g., for methyl) indicates a point of attachment of the group. It is notedthat the point of attachment is typically only identified in this manner for larger groups in orderto assist the reader in unambiguously identifying a point of attachment. The symbol “”means a single bond where the group attached to the thick end of the wedge is “out of the page.”The symbol “ ” means a single bond where the group attached to the thick end of the wedgeis “into the page”. The symbol “ ” means a single bond where the geometry around adouble bond (e.g., either E or Z) is undefined. Both options, as well as combinations thereof are therefore intended. Any undefined valency on an atom of a structure shown in this application implicitly represents a hydrogen atom bonded to that atom. A bold dot on a carbon atom indicates that the hydrogen attached to that carbon is oriented out of the plane of the paper.

[0072] When a variable is depicted as a “floating group” on a ring system, for example, the group “R” in the formula:then the variable may replace any hydrogen atom attached to any of the ring atoms, including a depicted, implied, or expressly defined hydrogen, so long as a stable structure is formed. When a variable is depicted as a “floating group” on a fused ring system, as for example the group “R” in the formula:then the variable may replace any hydrogen attached to any of the ring atoms of either of the fused rings unless specified otherwise. Replaceable hydrogens include depicted hydrogens (e.g., the hydrogen attached to the nitrogen in the formula above), implied hydrogens (e.g., a hydrogen of the formula above that is not shown but understood to be present), expressly defined hydrogens, and optional hydrogens whose presence depends on the identity of a ring atom (e.g., a hydrogen attached to group X, when X equals −CH−), so long as a stable structure is formed. In the example depicted, R may reside on either the 5-membered or the 6-membered ring of the fused ring system. In the formula above, the subscript letter “y” immediately following the R enclosed in parentheses, represents a numeric variable. Unless specified otherwise, this variable can be 0, 1, 2, or any integer greater than 2, only limited by the maximum number of replaceable hydrogen atoms of the ring or ring system.

[0073] For the chemical groups and compound classes, the number of carbon atoms in the group or class is as indicated as follows: “Cn” or “C=n” defines the exact number (n) of carbon atoms in the group / class. “C≤n” defines the maximum number (n) of carbon atoms that can be in the group / class, with the minimum number as small as possible for the group / class in question. For example, it is understood that the minimum number of carbon atoms in the groups “alkyl(C≤8)”, “alkanediyl(C≤8)”, “heteroaryl(C≤8)”, and “acyl(C≤8)” is one, the minimum number of carbon atoms in the groups “alkenyl(C≤8)”, “alkynyl(C≤8)”, and “heterocycloalkyl(C≤8)” is two, the minimum number of carbon atoms in the group “cycloalkyl(C≤8)” is three, and the minimum number of carbon atoms in the groups “aryl(C≤8)” and “arenediyl(C≤8)” is six. “Cn-n′” defines both the minimum (n) and maximum number (n′) of carbon atoms in the group. Thus, “alkyl(C2-10)” designates those alkyl groups having from 2 to 10 carbon atoms. These carbon number indicators may precede or follow the chemical groups or class it modifies and it may or may not be enclosed in parenthesis, without signifying any change in meaning. Thus, the terms “C1-4-alkyl”, “C1-4-alkyl”, “alkyl(C1-4)”, and “alkyl(C≤4)” are all synonymous. Except as noted below, every carbon atom is counted to determine whether the group or compound falls with the specified number of carbon atoms. For example, the group dihexylamino is an example of a dialkylamino(C12)group; however, it is not an example of a dialkylamino(C6)group. Likewise, phenylethyl is an example of an aralkyl(C=8) group. When any of the chemical groups or compound classes defined herein is modified by the term “substituted”, any carbon atom in the moiety replacing the hydrogen atom is not counted. Thus methoxyhexyl, which has a total of seven carbon atoms, is an example of a substituted alkyl(C1-6).Unless specified otherwise, any chemical group or compound class listed in a claim set without a carbon atom limit has a carbon atom limit of less than or equal to twelve.

[0074] The term “saturated” when used to modify a compound or chemical group means the compound or chemical group has no carbon-carbon double and no carbon-carbon triple bonds, except as noted below. When the term is used to modify an atom, it means that the atom is not part of any double or triple bond. In the case of substituted versions of saturated groups, one or more carbon oxygen double bond or a carbon nitrogen double bond may be present in the group replacing the hydrogen atom, as discussed below. And when such a bond is present, then carbon-carbon double bonds that may occur as part of keto-enol tautomerism or imine / enamine tautomerism are not precluded. When the term “saturated” is used to modify a solution of a substance, it means that no more of that substance can dissolve in that solution.

[0075] The term “aliphatic” signifies that the compound or chemical group so modified is an acyclic or cyclic, but non-aromatic compound or group. In aliphatic compounds / groups, the carbon atoms can be joined together in straight chains, branched chains, or non-aromatic rings (alicyclic). Aliphatic compounds / groups can be saturated, that is joined by single carbon- carbon bonds (alkanes / alkyl), or unsaturated, with one or more carbon-carbon double bonds (alkenes / alkenyl) or with one or more carbon-carbon triple bonds (alkynes / alkynyl).

[0076] The term “aromatic” signifies that the compound or chemical group so modified has a planar unsaturated ring of atoms with 4n +2 electrons in a fully conjugated cyclic π system. An aromatic compound or chemical group may be depicted as a single resonance structure; however, depiction of one resonance structure is taken to also refer to any other resonance structure. For example:Aromatic compounds may also be depicted using a circle to represent the delocalized nature of the electrons in the fully conjugated cyclic π system, two non-limiting examples of which are shown below:

[0077] The term “alkyl” refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, and no atoms other than carbon and hydrogen. The groups −CH3 (Me), −CH2CH3 (Et), −CH2CH2CH3 (n-Pr or propyl), −CH(CH3)2(i-Pr,iPr or isopropyl), −CH2CH2CH2CH3(n-Bu), −CH(CH3)CH2CH3(sec-butyl), −CH2CH(CH3)2 (isobutyl), −C(CH3)3 (tert-butyl, t-butyl, t-Bu ortBu), and −CH2C(CH3)3 (neo- pentyl) are non-limiting examples of alkyl groups. The term “alkanediyl” refers to a divalent saturated aliphatic group, with one or two saturated carbon atom(s) as the point(s) of attachment, a linear or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups −CH2− (methylene), −CH2CH2−, −CH2C(CH3)2CH2−, and −CH2CH2CH2− are non-limiting examples of alkanediyl groups. The term “alkylidene” refers to the divalent group =CRR′ in which R and R′ are independently hydrogen or alkyl. Non-limiting examples of alkylidene groups include: =CH2,=CH(CH2CH3), and =C(CH3)2. An “alkane” refers to the class of compounds having the formula H−R, wherein R is alkyl as this term is defined above.

[0078] The term “cycloalkyl” refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, said carbon atom forming part of one or more non- aromatic ring structures, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. If more than one ring is present, the rings may be fused, bridged, or spirocyclic. Non-limiting examples include: −CH(CH2)2(cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl (Cy). As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to a carbon atom of the non-aromatic ring structure. The term “cycloalkanediyl” refers to a divalent saturated aliphatic group with two carbon atoms as points of attachment, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groupa non- limiting example of cycloalkanediyl group. A “cycloalkane” refers to the class of compounds having the formula H−R, wherein R is cycloalkyl as this term is defined above.

[0079] The term “alkenyl” refers to a monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched, acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples include: −CH=CH2(vinyl), −CH=CHCH3, −CH=CHCH2CH3, −CH2CH=CH2 (allyl), −CH2CH=CHCH3, and −CH=CHCH=CH2. The term “alkenediyl” refers to a divalent unsaturated aliphatic group, with two carbon atoms as points of attachment, a linear or branched acyclic structure, at least one nonaromatic carbon- carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. The groups −CH=CH−, −CH=C(CH3)CH2−, −CH=CHCH2−, and −CH2CH=CHCH2− are non-limiting examples of alkenediyl groups. It is noted that while the alkenediyl group is aliphatic, once connected at both ends, this group is not precluded from forming part of an aromatic structure. The terms “alkene” and “olefin” are synonymous and refer to the class of compounds having the formula H−R, wherein R is alkenyl as this term is defined above. Similarly, the terms “terminal alkene” and “α-olefin” are synonymous and refer to an alkene having just one carbon-carbon double bond, wherein that bond is part of a vinyl group at an end of the molecule.

[0080] The term “alkynyl” refers to a monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. As used herein, theterm alkynyl does not preclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups −C≡CH, −C≡CCH3, and −CH2C≡CCH3 are non-limiting examples of alkynyl groups. An “alkyne” refers to the class of compounds having the formula H−R, wherein R is alkynyl.

[0081] The term “aryl” refers to a monovalent unsaturated aromatic group with an aromatic carbon atom as the point of attachment, said carbon atom forming part of a one or more aromatic ring structures, each with six ring atoms that are all carbon, and wherein the group consists of no atoms other than carbon and hydrogen. If more than one ring is present, the rings may be fused or unfused. Unfused rings are connected with a covalent bond. As used herein, the term aryl does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl, −C6H4CH2CH3(ethylphenyl), naphthyl, and a monovalent group derived from biphenyl (e.g., 4-phenylphenyl). The term “arenediyl” refers to a divalent aromatic group with two aromatic carbon atoms as points of attachment, said carbon atoms forming part of one or more six-membered aromatic ring structures, each with six ring atoms that are all carbon, and wherein the divalent group consists of no atoms other than carbon and hydrogen. As used herein, the term arenediyl does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. If more than one ring is present, the rings may be fused or unfused. Unfused rings are connected with a covalent bond. Non-limiting examples of arenediyl groups include:. An “arene” refers to the class of compounds having the formula H−R, wherein R is aryl as that term is defined above. Benzene and toluene are non-limiting examples of arenes.

[0082] The term “aralkyl” refers to the monovalent group −alkanediyl−aryl, in which the terms alkanediyl and aryl are each used in a manner consistent with the definitions provided above. Non-limiting examples are: phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl.

[0083] The term “heteroaryl” refers to a monovalent aromatic group with an aromatic carbon atom or nitrogen atom as the point of attachment, said carbon atom or nitrogen atom forming part of one or more aromatic ring structures, each with three to eight ring atoms, wherein at least one of the ring atoms of the aromatic ring structure(s) is nitrogen, oxygen or sulfur, and wherein the heteroaryl group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. If more than one ring is present, the rings are fused; however, the term heteroaryl does not preclude the presence of one or more alkyl or aryl groups (carbon number limitation permitting) attached to one or more ring atoms. Non-limiting examples of heteroaryl groups include benzoxazolyl, benzimidazolyl, furanyl, imidazolyl (Im), indolyl, indazolyl, isoxazolyl, methylpyridinyl, oxazolyl, oxadiazolyl, phenylpyridinyl, pyridinyl (pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl. The term “N-heteroaryl” refers to a heteroaryl group with a nitrogen atom as the point of attachment. A “heteroarene” refers to the class of compounds having the formula H−R, wherein R is heteroaryl. Pyridine and quinoline are non-limiting examples of heteroarenes.

[0084] The term “heterocycloalkyl” refers to a monovalent non-aromatic group with a carbon atom or nitrogen atom as the point of attachment, said carbon atom or nitrogen atom forming part of one or more non-aromatic ring structures, each with three to eight ring atoms, wherein at least one of the ring atoms of the non-aromatic ring structure(s) is nitrogen, oxygen or sulfur, and wherein the heterocycloalkyl group consists of no atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. If more than one ring is present, the rings may be fused, bridged, or spirocyclic. As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to one or more ring atoms. Also, the term does not preclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, tetrahydropyridinyl, pyranyl, oxiranyl, and oxetanyl. The term “N-heterocycloalkyl” refers to a heterocycloalkyl group with a nitrogen atom as the point of attachment. N-pyrrolidinyl is an example of such a group.

[0085] The term “acyl” refers to the group −C(O)R, in which R is a hydrogen, alkyl, cycloalkyl, or aryl as those terms are defined above. The groups, −CHO, −C(O)CH3(acetyl, Ac), −C(O)CH2CH3, −C(O)CH(CH3)2, −C(O)CH(CH2)2, −C(O)C6H5, and −C(O)C6H4CH3 arenon-limiting examples of acyl groups. A “thioacyl” is defined in an analogous manner, except that the oxygen atom of the group −C(O)R has been replaced with a sulfur atom, −C(S)R. The term “aldehyde” corresponds to an alkyl group, as defined above, attached to a −CHO group.

[0086] The term “alkoxy” refers to the group −OR, in which R is an alkyl, as that term is defined above. Non-limiting examples include: −OCH3 (methoxy), −OCH2CH3 (ethoxy), −OCH2CH2CH3, −OCH(CH3)2(isopropoxy), or −OC(CH3)3(tert-butoxy). The terms “cycloalkoxy”, “alkenyloxy”, “alkynyloxy”, “aryloxy”, “aralkoxy”, “heteroaryloxy”, “heterocycloalkoxy”, and “acyloxy”, when used without the “substituted” modifier, refers to groups, defined as −OR, in which R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl, respectively. The term “alkylthio” and “acylthio” refers to the group −SR, in which R is an alkyl and acyl, respectively. The term “alcohol” corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with a hydroxy group. The term “ether” corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with an alkoxy group.

[0087] The term “alkylamino” refers to the group −NHR, in which R is an alkyl, as that term is defined above. Non-limiting examples include: −NHCH3 and −NHCH2CH3. The term “dialkylamino” refers to the group −NRR′, in which R and R′ can be the same or different alkyl groups. Non-limiting examples of dialkylamino groups include: −N(CH3)2 and −N(CH3)(CH2CH3). The term “amido” (acylamino), when used without the “substituted” modifier, refers to the group −NHR, in which R is acyl, as that term is defined above. A non- limiting example of an amido group is −NHC(O)CH3.

[0088] When a chemical group is used with the “substituted” modifier, one or more hydrogen atom has been replaced, independently at each instance, by −OH, −F, −Cl, −Br, −I, −NH2, −NO2, −CO2H, −CO2CH3, −CO2CH2CH3, −CN, −SH, −OCH3, −OCH2CH3, −C(O)CH3, −NHCH3, −NHCH2CH3, −N(CH3)2, −C(O)NH2, −C(O)NHCH3, −C(O)N(CH3)2, −OC(O)CH3, −NHC(O)CH3, −S(O)2OH, or −S(O)2NH2. For example, the following groups are non-limiting examples of substituted alkyl groups: −CH2OH, −CH2Cl, −CF3, −CH2CN, −CH2C(O)OH, −CH2C(O)OCH3, −CH2C(O)NH2, −CH2C(O)CH3, −CH2OCH3, −CH2OC(O)CH3, −CH2NH2, −CH2N(CH3)2, and −CH2CH2Cl. The term “haloalkyl” is a subset of substituted alkyl, in which the hydrogen atom replacement is limited to halo (i.e. −F, −Cl, −Br, or −I) such that no other atoms aside from carbon, hydrogen and halogen are present. The group, −CH2Cl is a non- limiting example of a haloalkyl. The term “fluoroalkyl” is a subset of substituted alkyl, inwhich the hydrogen atom replacement is limited to fluoro such that no other atoms aside from carbon, hydrogen and fluorine are present. The groups −CH2F, −CF3, and −CH2CF3 are non- limiting examples of fluoroalkyl groups. Non-limiting examples of substituted aralkyls are: (3-chlorophenyl)-methyl, and 2-chloro-2-phenyl-eth-1-yl. The groups, −C(O)CH2CF3, −CO2H (carboxyl), −CO2CH3(methylcarboxyl), −CO2CH2CH3, −C(O)NH2(carbamoyl), and −CON(CH3)2, are non-limiting examples of substituted acyl groups. The groups −NHC(O)OCH3and −NHC(O)NHCH3are non-limiting examples of substituted amido groups.

[0089] The use of the word “a” or “an,” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0090] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects or patients.

[0091] An “active ingredient” (AI) or active pharmaceutical ingredient (API) (also referred to as an active compound, active substance, active agent, pharmaceutical agent, agent, biologically active molecule, or a therapeutic compound) is the ingredient in a pharmaceutical drug that is biologically active.

[0092] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps.

[0093] The term “effective,” as that term is used in the specification and / or claims, means adequate to accomplish a desired, expected, or intended result. “Effective amount,” “Therapeutically effective amount” or “pharmaceutically effective amount” when used in the context of treating a patient or subject with a compound means that amount of the compound which, when administered to the patient or subject, is sufficient to effect such treatment or prevention of the disease as those terms are defined below.

[0094] An “excipient” is a pharmaceutically acceptable substance formulated along with the active ingredient(s) of a medication, pharmaceutical composition, formulation, or drug delivery system. Excipients may be used, for example, to stabilize the composition, to bulk upthe composition (thus often referred to as “bulking agents,” “fillers,” or “diluents” when used for this purpose), or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, or enhancing solubility. Excipients include pharmaceutically acceptable versions of antiadherents, binders, coatings, colors, disintegrants, flavors, glidants, lubricants, preservatives, sorbents, sweeteners, and vehicles. The main excipient that serves as a medium for conveying the active ingredient is usually called the vehicle. Excipients may also be used in the manufacturing process, for example, to aid in the handling of the active substance, such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation or aggregation over the expected shelf life. The suitability of an excipient will typically vary depending on the route of administration, the dosage form, the active ingredient, as well as other factors.

[0095] The term “hydrate” when used as a modifier to a compound means that the compound has less than one (e.g., hemihydrate), one (e.g., monohydrate), or more than one (e.g., dihydrate) water molecules associated with each compound molecule, such as in solid forms of the compound.

[0096] As used herein, the term “IC50” refers to an inhibitory dose which is 50% of the maximum response obtained. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological, biochemical or chemical process (or component of a process, i.e. an enzyme, cell, cell receptor or microorganism) by half.

[0097] An “isomer” of a first compound is a separate compound in which each molecule contains the same constituent atoms as the first compound, but where the configuration of those atoms in three dimensions differs.

[0098] As used herein, the term “patient” or “subject” refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non- limiting examples of human patients are adults, juveniles, infants and fetuses.

[0099] As generally used herein “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and / or bodily fluids ofhuman beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0100] “Pharmaceutically acceptable salts” means salts of compounds disclosed herein which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene- 1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiarybutylacetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like. It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0101] A “pharmaceutically acceptable carrier,” “drug carrier,” or simply “carrier” is a pharmaceutically acceptable substance formulated along with the active ingredient medication that is involved in carrying, delivering and / or transporting a chemical agent. Drug carriers may be used to improve the delivery and the effectiveness of drugs, including for example, controlled-release technology to modulate drug bioavailability,decrease drug metabolism, and / or reduce drug toxicity. Some drug carriers may increase the effectiveness of drug delivery to the specific target sites. Examples of carriers include: liposomes, microspheres (e.g., made of poly(lactic-co-glycolic) acid), albumin microspheres, synthetic polymers, nanofibers, protein-DNA complexes, protein conjugates, erythrocytes, virosomes, and dendrimers.

[0102] A “pharmaceutical drug” (also referred to as a pharmaceutical, pharmaceutical preparation, pharmaceutical composition, pharmaceutical formulation, pharmaceutical product, medicinal product, medicine, medication, medicament, or simply a drug, agent, or preparation) is a composition used to diagnose, cure, treat, or prevent disease, which comprises an active pharmaceutical ingredient (API) (defined above) and optionally contains one or more inactive ingredients, which are also referred to as excipients (defined above).

[0103] “Prevention” or “preventing” includes: (1) inhibiting the onset of a disease in a subject or patient which may be at risk and / or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease, and / or (2) slowing the onset of the pathology or symptomatology of a disease in a subject or patient which may be at risk and / or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease.

[0104] “Prodrug” means a compound that is convertible in vivo metabolically into an active pharmaceutical ingredient of the present invention. The prodrug itself may or may not have activity in its prodrug form. For example, a compound comprising a hydroxy group may be administered as an ester that is converted by hydrolysis in vivo to the hydroxy compound. Non-limiting examples of suitable esters that may be converted in vivo into hydroxy compounds include acetates, citrates, lactates, phosphates, tartrates, malonates, oxalates, salicylates, propionates, succinates, fumarates, maleates, methylene- bis-β-hydroxynaphthoate, gentisates, isethionates, di-p-toluoyltartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, cyclohexylsulfamates, quinates, and esters of amino acids. Similarly, a compound comprising an amine group may be administered as an amide that is converted by hydrolysis in vivo to the amine compound.

[0105] A “stereoisomer” or “optical isomer” is an isomer of a given compound in which the same atoms are bonded to the same other atoms, but where the configuration of those atoms in three dimensions differs. “Enantiomers” are stereoisomers of a given compoundthat are mirror images of each other, like left and right hands. “Diastereomers” are stereoisomers of a given compound that are not enantiomers. Chiral molecules contain a chiral center, also referred to as a stereocenter or stereogenic center, which is any point, though not necessarily an atom, in a molecule bearing groups such that an interchanging of any two groups leads to a stereoisomer. In organic compounds, the chiral center is typically a carbon, phosphorus or sulfur atom, though it is also possible for other atoms to be stereocenters in organic and inorganic compounds. A molecule can have multiple stereocenters, giving it many stereoisomers. In compounds whose stereoisomerism is due to tetrahedral stereogenic centers (e.g., tetrahedral carbon), the total number of hypothetically possible stereoisomers will not exceed 2n, where n is the number of tetrahedral stereocenters. Molecules with symmetry frequently have fewer than the maximum possible number of stereoisomers. A 50:50 mixture of enantiomers is referred to as a racemic mixture. Alternatively, a mixture of enantiomers can be enantiomerically enriched so that one enantiomer is present in an amount greater than 50%. Typically, enantiomers and / or diastereomers can be resolved or separated using techniques known in the art. It is contemplated that that for any stereocenter or axis of chirality for which stereochemistry has not been defined, that stereocenter or axis of chirality can be present in its R form, S form, or as a mixture of the R and S forms, including racemic and non-racemic mixtures. As used herein, the phrase “substantially free from other stereoisomers” means that the composition contains ≤ 15%, more preferably ≤ 10%, even more preferably ≤ 5%, or most preferably ≤ 1% of another stereoisomer(s).

[0106] “Treatment” or “treating” includes (1) inhibiting a disease in a subject or patient experiencing or displaying the pathology or symptomatology of the disease (e.g., arresting further development of the pathology and / or symptomatology), (2) ameliorating a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease (e.g., reversing the pathology and / or symptomatology), and / or (3) effecting any measurable decrease in a disease or symptom thereof in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease.

[0107] The term “unit dose” refers to a formulation of the compound or composition such that the formulation is prepared in a manner sufficient to provide a single therapeutically effective dose of the active ingredient to a patient in a single administration. Such unit dose formulations that may be used include but are not limited to a single tablet, capsule, or other oral formulations, or a single vial with a syringeable liquid or other injectable formulations.

[0108] The above definitions supersede any conflicting definition in any reference that is incorporated by reference herein. The fact that certain terms are defined, however, should not be considered as indicative that any term that is undefined is indefinite. Rather, all terms used are believed to describe the invention in terms such that one of ordinary skill can appreciate the scope and practice the present invention.

[0109] The term “heteroaralkyl” refers to the monovalent group −alkanediyl−heteroaryl, in which the terms alkanediyl and heteroaryl are each used in a manner consistent with the definitions provided above. Non-limiting examples are: pyridinylmethyl and 2-quinolinyl-ethyl.

[0110] The term “heteroarenediyl” refers to a divalent aromatic group, with two aromatic carbon atoms, two aromatic nitrogen atoms, or one aromatic carbon atom and one aromatic nitrogen atom as the two points of attachment, said atoms forming part of one or more aromatic ring structures, each with three to eight ring atoms, wherein at least one of the ring atoms of the aromatic ring structure(s) is nitrogen, oxygen or sulfur, and wherein the divalent group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. If more than one ring is present, the rings are fused; however, the term heteroarenediyl does not preclude the presence of one or more alkyl or aryl groups (carbon number limitation permitting) attached to one or more ring atoms. Non-limiting examples of heteroarenediyl groups include:

[0111] The term “heterocycloalkanediyl” refers to a divalent cyclic group, with two carbon atoms, two nitrogen atoms, or one carbon atom and one nitrogen atom as the two points of attachment, said atoms forming part of one or more ring structure(s) wherein at least one of the ring atoms of the non-aromatic ring structure(s) is nitrogen, oxygen or sulfur, and wherein the divalent group consists of no atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. If more than one ring is present, the rings may be fused, bridged, or spirocyclic. As used herein, the term heterocycloalkanediyl does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to one or more ring atoms. Also, the term does not preclude the presence of one or more double bonds in the ring or ring system,provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkanediyl groups include:

[0112] The terms “alkylsulfonyl” and “alkylsulfinyl” refers to the groups −S(O)2R and −S(O)R, respectively, in which R is an alkyl, as that term is defined above. The terms “cycloalkylsulfonyl”, “alkenylsulfonyl”, “alkynylsulfonyl”, “arylsulfonyl”, “aralkylsulfonyl”, “heteroarylsulfonyl”, and “heterocycloalkylsulfonyl” are defined in an analogous manner.

[0113] The terms “cycloalkylamino”, “alkenylamino”, “alkynylamino”, “arylamino”, “aralkylamino”, “heteroarylamino”, “heterocycloalkylamino”, and “alkoxyamino” when used without the “substituted” modifier, refers to groups, defined as −NHR, in which R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and alkoxy, respectively. A non-limiting example of an arylamino group is −NHC6H5. The terms “dicycloalkylamino”, “dialkenylamino”, “dialkynylamino”, “diarylamino”, “diaralkylamino”, “diheteroarylamino”, “diheterocycloalkylamino”, and “dialkoxyamino”, refers to groups, defined as −NRR′, in which R and R′ are both cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and alkoxy, respectively. Similarly, the term alkyl(cycloalkyl)amino refers to a group defined as −NRR′, in which R is alkyl and R′ is cycloalkyl.

[0114] The term “alkylimino” refers to the divalent group =NR, in which R is an alkyl, as that term is defined above.

[0115] The terms “phosphine” and “phosphane” are used synonymously herein. these terms refer to a compound of the formula PR3, wherein each R is independently hydrogen, alkyl, cycloalkyl, alkenyl, aryl, or aralkyl, as those terms are defined above. Non-limiting examples include PMe3, PPh3, and PCy3 (tricyclohexylphosphine). The terms “trialkylphosphine” and “trialkylphosphane” are also synonymous. Such groups are a subset of phosphine, wherein each R is an alkyl group. The term “diphosphine” refers to a compound of the formula R2−P−L−P−R2, wherein each R is independently hydrogen, alkyl, cycloalkyl, alkenyl, aryl, or aralkyl, and wherein L is alkanediyl, cycloalkanediyl, alkenediyl, or arenediyl.

[0116] The term “phosphine oxide” refers to a compound of the formula O=PR3, wherein each R is independently hydrogen, alkyl, cycloalkyl, alkenyl, aryl, or aralkyl, as those terms are defined above. Non-limiting examples include OPMe3(trimethylphosphine oxide) and PPh3O (triphenylphosphine oxide).

[0117] An “amine protecting group” or “amino protecting group” is well understood in the art. An amine protecting group is a group which modulates the reactivity of the amine group during a reaction which modifies some other portion of the molecule. Amine protecting groups can be found at least in Greene and Wuts, 1999, which is incorporated herein by reference. Some non-limiting examples of amino protecting groups include formyl, acetyl, propionyl, pivaloyl, t–butylacetyl, 2–chloroacetyl, 2–bromoacetyl, trifluoroacetyl, trichloroacetyl, o–nitrophenoxyacetyl, α–chlorobutyryl, benzoyl, 4–chlorobenzoyl, 4– bromobenzoyl, 4–nitrobenzoyl, and the like; sulfonyl groups such as benzenesulfonyl, p– toluenesulfonyl and the like; alkoxy- or aryloxycarbonyl groups (which form urethanes with the protected amine) such as benzyloxycarbonyl (Cbz), p-chlorobenzyloxycarbonyl, p- methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p- bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5- dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4- methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxy- benzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, α,α-dimethyl-3,5- dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl, 2-trimethylsilylethyloxycarbonyl (Teoc), phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl (Fmoc), cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and the like; alkylaminocarbonyl groups (which form ureas with the protect amine) such as ethylaminocarbonyl and the like; aralkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl and the like; and silyl groups such as trimethylsilyl and the like. Additionally, the “amine protecting group” can be a divalent protecting group such that both hydrogen atoms on a primary amine are replaced with a single protecting group. In such a situation the amine protecting group can be phthalimide (phth) or a substituted derivative thereof wherein the term “substituted” is as defined above. In some embodiments, the halogenated phthalimide derivative may be tetrachlorophthalimide (TCphth). When used herein, a “protected amino group”, is a group of the formula PGMANH− or PGDAN− whereinPGMAis a monovalent amine protecting group, which may also be described as a “monovalently protected amino group” and PGDA is a divalent amine protecting group as described above, which may also be described as a “divalently protected amino group”.

[0118] The term “alkylphosphate” refers to the group −OP(O)(OH)(OR), in which R is an alkyl, as that term is defined above. Non-limiting examples of alkylphosphate groups include: −OP(O)(OH)(OMe) and −OP(O)(OH)(OEt). The term “dialkylphosphate” refers to the group −OP(O)(OR)(OR′), in which R and R′ can be the same or different alkyl groups, or R and R′ can be taken together to represent an alkanediyl. Non-limiting examples of dialkylphosphate groups include: −OP(O)(OMe)2, −OP(O)(OEt)(OMe) and −OP(O)(OEt)2.

[0119] The term “epoxide” refers to a class of compounds of the formula:, wherein R1, R2, and R3are each independently hydrogen, alkyl, and R4is hydrogen, alkyl, or aryl.

[0120] As used herein, a “chiral auxiliary” refers to a removable chiral group that is capable of influencing the stereoselectivity of a reaction. Persons of skill in the art are familiar with such compounds, and many are commercially available.

[0121] “Substituent convertible to hydrogen in vivo” means any group that is convertible to a hydrogen atom by enzymological or chemical means including, but not limited to, hydrolysis and hydrogenolysis. Non-limiting examples include hydrolyzable groups, such as acyl groups, groups having an oxycarbonyl group, amino acid residues, peptide residues, o- nitrophenylsulfenyl, trimethylsilyl, tetrahydropyranyl, and diphenylphosphinyl. Non-limiting examples of acyl groups include formyl, acetyl, and trifluoroacetyl. Non-limiting examples of groups having an oxycarbonyl group include ethoxycarbonyl, tert-butoxycarbonyl (−C(O)OC(CH3)3), benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, vinyloxycarbonyl, and β-(p-toluenesulfonyl)ethoxycarbonyl. Suitable amino acid residues include, but are not limited to, residues of Gly (glycine), Ala (alanine), Arg (arginine), Asn (asparagine), Asp (aspartic acid), Cys (cysteine), Glu (glutamic acid), His (histidine), Ile (isoleucine), Leu (leucine), Lys (lysine), Met (methionine), Phe (phenylalanine), Pro (proline), Ser (serine), Thr (threonine), Trp (tryptophan), Tyr (tyrosine), Val (valine), Nva (norvaline), Hse (homoserine), 4-Hyp (4-hydroxyproline), 5-Hyl (5-hydroxylysine), Orn (ornithine) and β-Ala. Examples of suitable amino acid residues also include amino acid residues that are protected with a protecting group. Non-limiting examples of suitable protecting groups include those typically employed in peptide synthesis, including acyl groups (such as formyl and acetyl), arylmethoxycarbonyl groups (such as benzyloxycarbonyl and p-nitrobenzyloxycarbonyl), and tert-butoxycarbonyl groups (−C(O)OC(CH3)3). Suitable peptide residues include peptide residues comprising two to five amino acid residues. The residues of these amino acids or peptides can be present in stereochemical configurations of the D-form, the L-form or mixtures thereof. In addition, the amino acid or peptide residue may have an asymmetric carbon atom. Examples of suitable amino acid residues having an asymmetric carbon atom include residues of Ala, Leu, Phe, Trp, Nva, Val, Met, Ser, Lys, Thr and Tyr. Peptide residues having an asymmetric carbon atom include peptide residues having one or more constituent amino acid residues having an asymmetric carbon atom. Non-limiting examples of suitable amino acid protecting groups include those typically employed in peptide synthesis, including acyl groups (such as formyl and acetyl), arylmethoxycarbonyl groups (such as benzyloxycarbonyl and p- nitrobenzyloxycarbonyl), and tert-butoxycarbonyl groups (−C(O)OC(CH3)3). Other examples of substituents “convertible to hydrogen in vivo” include reductively eliminable hydrogenolyzable groups. Examples of suitable reductively eliminable hydrogenolyzable groups include, but are not limited to, arylsulfonyl groups (such as o-toluenesulfonyl); methyl groups substituted with phenyl or benzyloxy (such as benzyl, trityl and benzyloxymethyl); arylmethoxycarbonyl groups (such as benzyloxycarbonyl and o-methoxy-benzyloxycarbonyl); and haloethoxycarbonyl groups (such as β,β,β-trichloroethoxycarbonyl and β-iodoethoxycarbonyl). VI. Methods of Treatment

[0122] Certain aspects of the present embodiments can be used to prevent or treat a disease or disorder such as cancer. In some embodiments, the cancer may be lung cancer, prostate cancer, stomach cancer, thyroid cancer, breast cancer multiple myeloma, melanoma, colon cancer, or leukemia.

[0123] The term “cancer,” as used herein, may be used to describe a solid tumor, metastatic cancer, or non-metastatic cancer. In certain embodiments, the cancer may originate in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, smallintestine, large intestine, colon, rectum, anus, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus.

[0124] The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget’s disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi’s sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma;osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin’s disease; hodgkin’s; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin’s lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0125] Nonetheless, it is also recognized that the present invention may also be used to treat a non-cancerous disease (e.g., a fungal infection, a bacterial infection, a viral infection, a neurodegenerative disease, and / or a genetic disorder).

[0126] The methods and compositions, including combination therapies, enhance the therapeutic or protective effect, and / or increase the therapeutic effect of another anti-cancer or anti-hyperproliferative therapy. Therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve the desired effect, such as the killing of a cancer cell and / or the inhibition of cellular hyperproliferation. This process may involve contacting the cells with both an antibody or antibody fragment and a second therapy. A tissue, tumor, or cell can be contacted with one or more compositions or pharmacological formulation(s) comprising one or more of the agents (i.e., antibody or antibody fragment or an anti-cancer agent), or by contacting the tissue, tumor, and / or cell with two or more distinct compositions or formulations, wherein one composition provides 1) an antibody or antibody fragment, 2) an anti-cancer agent, or 3) both an antibody or antibody fragment and an anti-cancer agent. Also, it is contemplated that such a combination therapy can be used in conjunction with chemotherapy, radiotherapy, surgical therapy, or immunotherapy.

[0127] The terms “contacted” and “exposed,” when applied to a cell, are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve cell killing, for example, both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.

[0128] An antibody may be administered before, during, after, or in various combinations relative to an anti-cancer treatment. The administrations may be in intervals ranging from concurrently to minutes to days to weeks. In embodiments where the antibody or antibody fragment is provided to a patient separately from an anti-cancer agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two compounds would still be able to exert an advantageously combined effect on the patient. In such instances, it is contemplated that one may provide a patient with the antibody therapy and the anti-cancer therapy within about 12 to 24 or 72 h of each other and, more particularly, within about 6-12 h of each other. In some situations it may be desirable to extend the time period for treatment significantly where several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapse between respective administrations.

[0129] In certain embodiments, a course of treatment will last 1-90 days or more (this such range includes intervening days). It is contemplated that one agent may be given on any day of day 1 to day 90 (this such range includes intervening days) or any combination thereof, and another agent is given on any day of day 1 to day 90 (this such range includes intervening days) or any combination thereof. Within a single day (24-hour period), the patient may be given one or multiple administrations of the agent(s). Moreover, after a course of treatment, it is contemplated that there is a period of time at which no anti-cancer treatment is administered. This time period may last 1-7 days, and / or 1-5 weeks, and / or 1-12 months or more (this such range includes intervening days), depending on the condition of the patient, such as their prognosis, strength, health, etc. It is expected that the treatment cycles would be repeated as necessary.

[0130] Various combinations may be employed. For the example below an antibody therapy is “A” and an anti-cancer therapy is “B”: A / B / A B / A / B B / B / A A / A / B A / B / B B / A / A A / B / B / B B / A / B / B B / B / B / A B / B / A / B A / A / B / B A / B / A / B A / B / B / A B / B / A / A B / A / B / A B / A / A / B A / A / A / B B / A / A / A A / B / A / A A / A / B / A

[0131] Administration of any compound or therapy of the present embodiments to a patient will follow general protocols for the administration of such compounds, taking intoaccount the toxicity, if any, of the agents. Therefore, in some embodiments there is a step of monitoring toxicity that is attributable to combination therapy. A. Chemotherapy

[0132] A wide variety of chemotherapeutic agents may be used in accordance with the present embodiments. The term “chemotherapy” refers to the use of drugs to treat cancer. A “chemotherapeutic agent” is used to connote a compound or composition that is administered in the treatment of cancer. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.

[0133] Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammalI and calicheamicin omegaI1); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, such as mitomycin C,mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; anti-metabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues, such as denopterin, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals, such as mitotane and trilostane; folic acid replenisher, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKpolysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2”- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DFMO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine,plicomycin, gemcitabien, navelbine, farnesyl-protein transferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above. B. Radiotherapy

[0134] Other factors that cause DNA damage and have been used extensively include what are commonly known as γ-rays, X-rays, and / or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated, such as microwaves, proton beam irradiation (U.S. Patents 5,760,395 and 4,870,287), and UV- irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2,000 to 6,000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells. In certain embodiments, an antibody of the present disclosure is used in combination with a radiotherapy, e.g., γ-rays, X-rays, and / or the directed delivery of radioisotopes to tumor cells. C. Immunotherapy

[0135] The skilled artisan will understand that immunotherapies may be used in combination or in conjunction with methods of the embodiments. In the context of cancer treatment, immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is such an example. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0136] In one aspect of immunotherapy, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present embodiments. Common tumor markers include B-cell maturation antigen, CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, GPRC5D, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects. Immune stimulating molecules also exist including: cytokines, such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growth factors, such as FLT3 ligand.

[0137] Examples of immunotherapies currently under investigation or in use are immune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Patents 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapy, e.g., interferons α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapy, e.g., TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward andVillaseca, 1998; U.S. Patents 5,830,880 and 5,846,945); and monoclonal antibodies, e.g., anti- CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Patent 5,824,311). It is contemplated that one or more anti-cancer therapies may be employed with the antibody therapies described herein.

[0138] In some aspects, a combination described herein includes an agent that decreases tumor immunosuppression, such as a chemokine (C-X-C motif) receptor 2 (CXCR2) inhibitor. In some embodiments, the CXCR2 inhibitor is danirixin (CAS Registry Number: 954126-98-8). Danirixin is also known as GSK1325756 or 1-(4-chloro-2-hydroxy-3-piperidin- 3-ylsulfonylphenyl)-3-(3-fluoro-2-methylphenyl)urea. Danirixin is disclosed, e.g., in Miller et al. Eur J Drug Metab Pharmacokinet (2014) 39:173-181; and Miller et al. BMC Pharmacology and Toxicology (2015), 16:18. In some embodiments, the CXCR2 inhibitor is reparixin (CAS Registry Number: 266359-83-5). Reparixin is also known as repertaxin or (2R)-2-[4-(2- methylpropyl)phenyl]-N-methylsulfonylpropanamide. Reparixin is a non-competitive allosteric inhibitor of CXCR1 / 2. Reparixin is disclosed, e.g., in Zarbock et al. British Journal of Pharmacology (2008), 1-8. In some embodiments, the CXCR2 inhibitor is navarixin. Navarixin is also known as MK-7123, SCH527123, PS291822, or 2-hydroxy-N,N-dimethyl- 3-[[2-[[(1R)-1-(5-methylfuran-2-yl)propyl]amino]-3,4-dioxocyclobuten-1- yl]amino]benzamide Navarixin is disclosed, e.g., in Ning et al. Mol Cancer Ther.2012; 11(6):1353-64. In some embodiments, the CXCR2 inhibitor is AZD5069, also known as N- [2-[[(2,3-difluoropheny)methyl]thio]-6-{[(1 R,2S)-2,3-dihydroxy-1-methylpropyl]oxy}-4- pyrimidinyl]-1-azetidinesulfonamide. In some embodiments, the CXCR2 inhibitor is an anti- CXCR2 antibody, such as those disclosed in WO2020 / 028479.

[0139] In some aspects, a combination described herein includes an agent that activates dendritic cells, such as, for example, a TLR agonist. A “TLR agonist” as defined herein is any molecule which activates a toll-like receptor as described in Bauer et al., 2001, Proc. Natl. Acad. Sci. USA 98: 9237-9242. A TLR agonist may be a small molecule, a recombinant protein, an antibody or antibody fragment, a nucleic acid, or a protein. In certain embodiments, the TLR agonist is recombinant, a natural ligand, an immunostimulatory nucleotide sequence, a small molecule, a purified bacterial extract or an inactivated bacteria preparation.

[0140] Several agonists of TLR derived from microbes have been described, such as lipopolysaccharides, peptidoglycans, flagellin and lipoteichoic acid (Aderem et al., 2000, Nature 406:782-787; Akira et al., 2001, Nat. Immunol. 2: 675-680) Some of these ligands can activate different dendritic cell subsets, that express distinct patterns of TLRs(Kadowaki et al., 2001, J. Exp. Med. 194: 863-869). Therefore, a TLR agonist could be any preparation of a microbial agent that possesses TLR agonist properties. Certain types of untranslated DNA have been shown to stimulate immune responses by activating TLRs. In particular, immunostimulatory oligonucleotides containing CpG motifs have been widely disclosed and reported to activate lymphocytes (see, U.S. Patent No. 6,194,388). A “CpG motif” as used herein is defined as an unmethylated cytosine-guanine (CpG) dinucleotide. Immunostimulatory oligonucleotides which contain CpG motifs can also be used as TLR agonists according to the methods of the present invention. The immunostimulatory nucleotide sequence may be stabilized by structure modification such as phosphorothioate modification or may be encapsulated in cationic liposomes to improve in vivo pharmacokinetics and tumor targeting.

[0141] In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints either turn up a signal (e.g., co-stimulatory molecules) or turn down a signal. Immune checkpoints either turn up a signal (e.g., co- stimulatory molecules) or turn down a signal. Immune checkpoint proteins that may be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), CCL5, CD27, CD38, CD8A, CMKLR1, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), CXCL9, CXCR5, glucocorticoid-induced tumour necrosis factor receptor-related protein (GITR), HLA-DRB1, ICOS (also known as CD278), HLA-DQA1, HLA-E, indoleamine 2,3- dioxygenase 1 (IDO1), killer-cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG-3, also known as CD223), Mer tyrosine kinase (MerTK), NKG7, OX40 (also known as CD134), programmed death 1 (PD-1), programmed death-ligand 1 (PD-L1, also known as CD274), PDCD1LG2, PSMB10, STAT1, T cell immunoreceptor with Ig and ITIM domains (TIGIT), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain Ig suppressor of T cell activation (VISTA, also known as C10orf54). In particular, the immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0142] The immune checkpoint inhibitors may be drugs, such as small molecules, recombinant forms of ligand or receptors, or antibodies, such as human antibodies (e.g., International Patent Publication WO2015 / 016718; Pardoll, Nat Rev Cancer, 12(4): 252- 264, 2012; both incorporated herein by reference). Known inhibitors of the immune checkpoint proteins or analogs thereof may be used, in particular chimerized, humanized, or human forms of antibodies may be used. As the skilled person will know, alternative and / or equivalent names may be in use for certain antibodies mentioned in the present disclosure. Such alternativeand / or equivalent names are interchangeable in the context of the present disclosure. For example, it is known that lambrolizumab is also known under the alternative and equivalent names MK-3475 and pembrolizumab.

[0143] In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific aspect, the PD-1 ligand binding partners are PD-L1 and / or PD-L2. In another embodiment, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners. In a specific aspect, PD-L1 binding partners are PD-1 and / or B7-1. In another embodiment, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partners. In a specific aspect, a PD-L2 binding partner is PD-1. The antagonist may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patent Nos.8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art, such as described in U.S. Patent Application Publication Nos.2014 / 0294898, 2014 / 022021, and 2011 / 0008369, all of which are incorporated herein by reference.

[0144] In some embodiments, a PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 binding antagonist is AMP- 224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti- PD-1 antibody described in WO2006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO2009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342.

[0145] Another immune checkpoint protein that can be targeted in the methods provided herein is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has the Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an “off” switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA-4 is similar to the T-cell co-stimulatory protein, CD28, and both molecules bind to CD80 and CD86, also called B7-1 and B7-2 respectively, on antigen-presenting cells. CTLA-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA-4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules.

[0146] In some embodiments, the immune checkpoint inhibitor is an anti- CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Anti- human-CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used. For example, the anti-CTLA-4 antibodies disclosed in US Patent No. 8,119,129; PCT Publn. Nos. WO 01 / 14424, WO 98 / 42752, WO 00 / 37504 (CP675,206, also known as tremelimumab; formerly ticilimumab); U.S. Patent No. 6,207,156; Hurwitz et al. (1998) Proc Natl Acad Sci USA, 95(17): 10067-10071; Camacho et al. (2004) J Clin Oncology, 22(145): Abstract No.2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res, 58:5301-5304 can be used in the methods disclosed herein. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used. For example, a humanized CTLA-4 antibody is described in International Patent Application No. WO2001 / 014424, WO2000 / 037504, and U.S. Patent No.8,017,114; all incorporated herein by reference.

[0147] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX- 010, MDX- 101, and Yervoy®) or antigen binding fragments and variants thereof (see, e.g., WO 01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding with and / or binds to the same epitope on CTLA-4 as the above-mentioned antibodies. In another embodiment, the antibody has an at least about 90% variable region amino acid sequence identity with the above-mentioned antibodies (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab). Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors such as described in U.S. Patent Nos.5844905, 5885796 and International Patent Application Nos. WO1995001994 and WO1998042752; allincorporated herein by reference, and immunoadhesins such as described in U.S. Patent No. 8329867, incorporated herein by reference.

[0148] Another immune checkpoint protein that can be targeted in the methods provided herein is lymphocyte-activation gene 3 (LAG-3), also known as CD223. The complete protein sequence of human LAG-3 has the Genbank accession number NP-002277. LAG-3 is found on the surface of activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells. LAG-3 acts as an “off” switch when bound to MHC class II on the surface of antigen-presenting cells. Inhibition of LAG-3 both activates effector T cells and inhibitor regulatory T cells. In some embodiments, the immune checkpoint inhibitor is an anti- LAG-3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Anti- human-LAG-3 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-LAG-3 antibodies can be used. An exemplary anti-LAG-3 antibody is relatlimab (also known as BMS-986016) or antigen binding fragments and variants thereof (see, e.g., WO 2015 / 116539). Other exemplary anti-LAG-3 antibodies include TSR-033 (see, e.g., WO 2018 / 201096), MK-4280, and REGN3767. MGD013 is an anti-LAG-3 / PD-1 bispecific antibody described in WO 2017 / 019846. FS118 is an anti-LAG-3 / PD-L1 bispecific antibody described in WO 2017 / 220569.

[0149] Another immune checkpoint protein that can be targeted in the methods provided herein is V-domain Ig suppressor of T cell activation (VISTA), also known as C10orf54. The complete protein sequence of human VISTA has the Genbank accession number NP_071436. VISTA is found on white blood cells and inhibits T cell effector function. In some embodiments, the immune checkpoint inhibitor is an anti-VISTA3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Anti-human-VISTA antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-VISTA antibodies can be used. An exemplary anti-VISTA antibody is JNJ-61610588 (also known as onvatilimab) (see, e.g., WO 2015 / 097536, WO 2016 / 207717, WO 2017 / 137830, WO 2017 / 175058). VISTA can also be inhibited with the small molecule CA-170, which selectively targets both PD-L1 and VISTA (see, e.g., WO 2015 / 033299, WO 2015 / 033301).

[0150] Another immune checkpoint protein that can be targeted in the methods provided herein is indoleamine 2,3-dioxygenase (IDO). The complete protein sequence ofhuman IDO has Genbank accession number NP_002155. In some embodiments, the immune checkpoint inhibitor is a small molecule IDO inhibitor. Exemplary small molecules include BMS-986205, epacadostat (INCB24360), and navoximod (GDC-0919).

[0151] Another immune checkpoint protein that can be targeted in the methods provided herein is CD38. The complete protein sequence of human CD38 has Genbank accession number NP_001766. In some embodiments, the immune checkpoint inhibitor is an anti-CD38 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Anti-human-CD38 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CD38 antibodies can be used. An exemplary anti-CD38 antibody is daratumumab (see, e.g., U.S. Patent No.7,829,673).

[0152] Another immune checkpoint protein that can be targeted in the methods provided herein is ICOS, also known as CD278. The complete protein sequence of human ICOS has Genbank accession number NP_036224. In some embodiments, the immune checkpoint inhibitor is an anti-ICOS antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Anti-human-ICOS antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-ICOS antibodies can be used. Exemplary anti- ICOS antibodies include JTX-2011 (see, e.g., WO 2016 / 154177, WO 2018 / 187191) and GSK3359609 (see, e.g., WO 2016 / 059602).

[0153] Another immune checkpoint protein that can be targeted in the methods provided herein is T cell immunoreceptor with Ig and ITIM domains (TIGIT). The complete protein sequence of human TIGIT has Genbank accession number NP_776160. In some embodiments, the immune checkpoint inhibitor is an anti-TIGIT antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Anti-human-TIGIT antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-TIGIT antibodies can be used. An exemplary anti-TIGIT antibody is MK-7684 (see, e.g., WO 2017 / 030823, WO 2016 / 028656).

[0154] Another immune checkpoint protein that can be targeted in the methods provided herein is OX40, also known as CD134. The complete protein sequence of humanOX40 has Genbank accession number NP_003318. In some embodiments, the immune checkpoint inhibitor is an anti-OX40 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Anti-human-OX40 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-OX40 antibodies can be used. An exemplary anti- OX40 antibody is PF-04518600 (see, e.g., WO 2017 / 130076). ATOR-1015 is a bispecific antibody targeting CTLA4 and OX40 (see, e.g., WO 2017 / 182672, WO 2018 / 091740, WO 2018 / 202649, WO 2018 / 002339).

[0155] Another immune checkpoint protein that can be targeted in the methods provided herein is glucocorticoid-induced tumour necrosis factor receptor-related protein (GITR), also known as TNFRSF18 and AITR. The complete protein sequence of human GITR has Genbank accession number NP_004186. In some embodiments, the immune checkpoint inhibitor is an anti-GITR antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Anti-human-GITR antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-GITR antibodies can be used. An exemplary anti-GITR antibody is TRX518 (see, e.g., WO 2006 / 105021).

[0156] In some embodiment, the immune therapy could be adoptive immunotherapy, which involves the transfer of autologous antigen- specific T cells generated ex vivo. The T cells used for adoptive immunotherapy can be generated either by expansion of antigen-specific T cells or redirection of T cells through genetic engineering (Park, Rosenberg et al. 2011). Isolation and transfer of tumor specific T cells has been shown to be successful in treating melanoma. Novel specificities in T cells have been successfully generated through the genetic transfer of transgenic T cell receptors or chimeric antigen receptors (CARs) (Jena, Dotti et al. 2010). CARs are synthetic receptors consisting of a targeting moiety that is associated with one or more signaling domains in a single fusion molecule. In general, the binding moiety of a CAR consists of an antigen-binding domain of a single-chain antibody (scFv), comprising the light and variable fragments of a monoclonal antibody joined by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully. The signaling domains for first generation CARs are derived from the cytoplasmic region of the CD3zeta or the Fc receptor gamma chains. CARs have successfullyallowed T cells to be redirected against antigens expressed at the surface of tumor cells from various malignancies including lymphomas and solid tumors (Jena, Dotti et al.2010).

[0157] In one embodiment, the present application provides for a combination therapy for the treatment of cancer wherein the combination therapy comprises adoptive T cell therapy and a checkpoint inhibitor. In one aspect, the adoptive T cell therapy comprises autologous and / or allogenic T-cells. In another aspect, the autologous and / or allogenic T-cells are targeted against tumor antigens. D. Surgery

[0158] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and / or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs’ surgery).\

[0159] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well. E. Other Agents

[0160] It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiments to improve the anti- hyperproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated toimprove the efficacy of the present embodiments. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with certain aspects of the present embodiments to improve the treatment efficacy. VII. Kits

[0161] In various aspects of the embodiments, a kit is envisioned containing therapeutic agents and / or other therapeutic and delivery agents. In some embodiments, a kit is provided for preparing and / or administering a therapy of the embodiments. The kit may comprise one or more sealed vials containing any of the pharmaceutical compositions of the present embodiments. The kit may include, for example, at least embodiment of the present invention, as well as reagents to prepare, formulate, and / or administer the components of the embodiments or perform one or more steps of the inventive methods. In some embodiments, the kit may also comprise a suitable container, which is a container that will not react with components of the kit, such as an eppendorf tube, an assay plate, a syringe, a bottle, or a tube. The container may be made from sterilizable materials such as plastic or glass.

[0162] The kit may further include an instruction sheet that outlines the procedural steps of the methods set forth herein, and will follow substantially the same procedures as described herein or are known to those of ordinary skill in the art. The instruction information may be in a computer readable media containing machine-readable instructions that, when executed using a computer, cause the display of a real or virtual procedure of delivering a pharmaceutically effective amount of a therapeutic agent. VIII. Examples

[0163] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the example which follows represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1: Discovery of a new class of fungal RiPPs, asperigimycins

[0164] The precursor peptides of fungal RiPPs from ascomycetes usually contain multiple perfect or imperfect repeats interspersed with dibasic residues, notably KR (FIG. 6) (Vogt et al., 2022). These dibasic residues serve as recognition sites for Kexin-like endopeptidases, guiding the cleavage of the precursor peptides to single core peptides (Kessler & Chooi, 2022; Yoshimi et al., 2016). This characteristic facilitates the genome mining of candidate dikaritin precursor peptides. To enhance the efficiency of identifying RiPP compounds within the complex metabolomes of fungal strains, leverage metabolomics and bioinformatic analyses of genomic datasets are leveraged to pinpoint compounds most likely associated with RiPPs.

[0165] Aspergillus species have garnered attention as promising RiPPs producers, owing to the presence of numerous RiPPs gene clusters revealed by bioinformatic analysis (Nagano et al., 2016).12 Aspergillus strains were cultivated, including A. clavatus, A. versicolor, A. sydowii, A. brasiliensis, A. niger, A. tubingensis, A. wentii, A. acidus, A. zonatus, A. aculeatus, A. flavus, and A. oryzae, across various culture media to identify novel fungal RiPPs. Then the metabolites produced by these strains were analyzed using ultra-performance liquid chromatography coupled with electrospray ionization / quadrupole-time-of-flight tandem mass spectrometry (UPLC-ESI-Q-TOF-MS / MS). The acquired MS / MS data were subjected to the GNPS platform (Aron et al., 2020) to generate the molecular networks of untargeted mass spectrometry data and provide fragmentation information of compounds and the complex networks were visualized by Cytoscape. The consolidated GNPS analysis revealed approximately 2500 individual nodes and hundreds of clusters contained within the 12 extracts (FIG. 7). Of particular interest was a molecular cluster associated with A. flavus, containing four main precursor ions with m / z values of 1020 (1), 933 (2), 973 (3), and 987 (4) (FIG.1a). Detailed analysis of the MS spectra of each node revealed distinctive fragments derived from the amino acids Ile and Gly, as exemplified by 2, which contains Ala, two Gly, and Ile fragments (FIG. 1a). Bioinformatic analysis of A. flavus genome identified four genes encoding uncharacterized precursor peptides, characterized by multiple repeats of core peptides and potential Kexin protease cleavage sites (FIG. 1b and FIG. 8). Notably, one of the candidates, XP_041147057.1 (ApgA), encompasses all the identified amino acid fragments (FIG. 8) elucidated by MS / MS spectra (FIG. 1b), distinguishing it from the other three candidates that lack these specific features. The correlation of the amino acid sequence in ApgAand the MS / MS data revealed by GNPS strongly implies that ApgA is associated with the target compounds, possibly representing RiPPs.

[0166] To validate the hypothesis, the split-marker method with pyrG gene was employed as a selection marker to delete apgA (Szewczyk et al., 2006; Catlett et al., 2003). The subsequent absence of the four major peaks (1-4) revealed by LC-MS substantiates that these compounds are fungal RiPPs, with ApgA serving as their precursor peptide (FIG.1c-i, ii). Thus, these four fungal RiPPs were purified, and their NMR data was collected. Comprehensive analysis of the 1D NMR and 2D NMR data of 1 revealed a complete amino acid sequence (Ala1-Leu2-Tyr3-Gly4-Ala5-Trp6-Gly7-Gly8-Ile9-Ser10), which is consistent with the proposed core peptide ALYGAWGGIS in ApgA. Additionally, observed HMBCs from H- 27 to C-24 and C-26, from H-25 to C-23, and from H-22 to C-24 enabled the inventors to establish the benzofuranoindoline core via the connection of Tyr3 and Trp6 residues (FIG.9), while the HMBCs from H-6 to C-9, from H-8 to C-9, C-10, and C-26 indicated the connection between Leu2 and Tyr3 at C-9 (ring A). The connectivity of Trp6 and Ile9 (ring C) was identified based on the downfield chemical shift of C-34 (δC85.3) and the diagnostic ROESY cross-peak between H-32 and H-38. Additionally, the downfield chemical shift of C-12 suggests a hydroxyl substitution at the β-carbon of Tyr3residue. The ribosomal synthesis of compounds 1-4 suggests that the amino acid backbone is in the l-configuration. Collectively, the planar chemical structure of 1 was determined as shown and the compound was named asperigimycin A (Fig. 1d). A detailed comparison of the NMR data of asperigimycin B (2) with those of 1 revealed that their structures are closely related and shared similar amino acid compositions. The primary distinction lies in the absence of a Ser10residue at C-terminal of 2, consistent with the core peptide sequence ALYGAWGGI. Another key difference between 2 and asperigimycin C (3) can be found in the pyroglutamate moiety in the N-terminal of 3 rather than the Ala1residue. As the unique pyroglutamate was reported to be derived from glutaminyl in plant RiPPs (Kersten & Weng, 2018), it was thus suspected that the core peptide for 3 to be QLYGAWGGI. Asperigimycin D (4) is similar to 3, except for the replacement of its Gly8residue by an Ala residue, related with the QLYGAWGAI core region sequence. According to the ROESY spectrum and coupling constants analysis, stereochemistry was proposed as shown (FIG.9 and FIG.1d). The benzofuranoindole core in asperigimycins is a rare structural motif, observed in a few natural products, such as diazonamide A (Lingquist et al., 1991), bipleophylline (Kam et al., 2008), and azonazine (Wu et al., 2010). Moreover, the presence of three additional fused macrocycles surrounding this core further enhances the structural complexity of asperigimycins.A. Precursor peptide engineering

[0167] As RiPP precursor peptides are genetically encoded, it is feasible to modify a specific codon within the gene encoding the core peptide to generate new RiPPs analogues and provide insights into the roles of specific amino acids. In order to assess the potential for generating derivatives of asperigimycin, a series of targeted mutations on the core peptide was conducted. ApgA is predicted to possess a transmembrane domain in the N- terminal leader peptide and nine imperfect repeats separated by KR (FIG. 1b). Given the inherent difficulty of modifying these repetitive sequences, the full-length ApgA to ApgAT was truncated. ApgATretains the leader peptide and the first single-core peptide which is responsible for the production of 3 according to the sequence. The strain with ApgAT produces 3 as the only product, albeit with a much lower titer compared to WT (FIG. 1c-iii). Subsequently, the codon of nine amino acids (located at positions X1-X9) within the core peptide were altered to encode for other amino acids. X1, X4, X5, X7, X8, and X9can be replaced by several similar amino acids to produce the corresponding products that are detectable by LC-MS. The Gln in X1is the most flexible amino acid, which can be substituted with Ser, His, Asn, or Leu, while the Ala in X5, Gly in X4, X7, and X8 can only be replaced by Ser. Besides, Ala in X5can be replaced by Thr and Ile X9can be replaced by Leu and Val (FIG.1e and FIG. 10). However, altering three specific residues (Leu, Tyr, and Trp), which are essential for forming the benzofuranoindoline core structure and closure of ring A, eliminate all derivatives, suggesting the essential nature of these residues within the precursor peptide. Although the detected derivatives cannot be isolated and identified due to the low yield, the observation demonstrated the potential to produce asperigimycin derivatives by mutating the precursor peptide. B. Biosynthesis and characterization of asperigimycins

[0168] The putative apg gene cluster contains 16 genes that encode the precursor peptide (ApgA), six possible DUF3328 oxidases (including ApgYa, ApgYb, ApgYc, ApgYd, ApgYe, and ApgYf), one tyrosinase (ApgQ), three transporters (ApgC, ApgD, and ApgE), one glutaminyl-peptide cyclotransferase (ApgG), and four unknown function enzymes (ApgB, ApgF, ApgH, and ApgI) (Fig.2a). Because DUF3328 oxidases featured by conserved "HXXHC" motifs play a crucial role in the post-translational modification of fungal RiPPs, gene deletion experiments were first conducted to explore the functions of six ApgY enzymes encoded in apg gene cluster. The deletion of each of the six apgY genes completely halted the production of compounds 1-4, underscoring the importance of these enzymes in thebiosynthesis of these compounds (Fig.2b). The deletion of apgYb resulted in the accumulation of four new compounds (5-8), exhibiting 16 Da loss in molecular weight (MW) comparing to compounds 1-4, respectively, suggesting the loss of an oxygen atom. The NMR data of 5-8 confirmed that the hydroxyl group at C-12 was replaced by hydrogen, suggesting that ApgYb is responsible for the hydroxylation of C-12. ΔapgYf mutant produced three compounds 12-14 with m / z 806, 846, and 860 respectively. Their NMR data revealed the absence of the hydroxyl group at C-33 along with the degradation of the IIe residue at the C-terminus, likely due to the lack of cyclization. These results implied that ApgYf can catalyze the hydroxylation of the benzene ring in Trp6. The deletion of apgYc and apgYd both led to the accumulation of three compounds 9-11 whose MWs are 821, 861, and 876, respectively. The additional hydroxyl group at C-33 in 9-11 compared to 12-14 implied that ApgYc and ApgYd are involved in the ether bond formation for the ring closure. Since any stable intermediates were not observed in the metabolites of ΔapgYa and ΔapgYe, these two enzymes are probably involved in the early stage of the pathway. Furthermore, bioinformatic analysis revealed that apgYa and apgYe are highly conserved in homologous gene clusters across different fungal species including Aspergillus, Scedosporium, Pseudocercospora, Glarea, Colletotrichum, and Ophiocordyceps. By analyzing the putative precursor peptides encoded in these gene clusters, it was also found that Leu, Trp, and Tyr are conserved at the 2, 3, and 6 positions of the core peptide (FIG.11). These results indicated ApgYa and ApgYe with these three amino acids play essential roles in the formation of the benzofuranoindoline core and the linkage of Leu2and Trp6by forming the C-8-C-9 bond. The analysis further indicates that this class of fungal RiPPs is prevalent across various fungal species, despite the scaffold not having been previously reported

[0169] Next, the remaining genes in the apg cluster were knocked out to investigate their respective functions. It was found that the deletion of apgB, apgC, apgD, apgQ, apgE, apgF, and apgH genes did not influence the production of the final asperigimycin compounds, implying that these genes may not be involved in the posttranslational- modification of asperigimycins (FIG. 12-i-vii). The deletion of apgG did not influence the production of 1 and 2, but it disrupted the production of 3 and 4 accompanied by the production of two new compounds 15 and 16 with m / z 990 and 1004 (Fig. 2b-viii). ApgG belongs to glutaminyl-peptide cyclotransferases and shares a moderate homology (37.8%) to human glutaminyl cyclase (QC). Human QCs are responsible for the formation of neurotoxic pyroglutamate (pE)-modified β-amyloid peptides, which are associated with human Alzheimer's disease (Huang et al., 2011; Huang et al., 2005). Despite the low yield andinstability of 15 and 16, the LC-HRMS / MS data of 15 and 16 revealed they possess a glutaminyl moiety instead of a N-terminal pyroglutamate in 3 and 4. These results indicate ApgG catalyzes the cyclization of N-terminal glutaminyl to generate the pyroglutamate group of 3 and 4. Moreover, deletion of apgI led to a complete loss of all compounds (FIG.12-viii). ApgI, which is indispensable in the production of asperigimycins, contains the ankyrin repeat domain and bZIP transcription factor domain and may function as a positive regulatory protein.

[0170] Based on the data, the biosynthetic pathway of asperigimycins was proposed: the digested four different core peptides undergo cyclization facilitated by ApgYa and ApgYe, resulting in the formation of intermediates with the benzofuranoindoline scaffold and ring A. While the specific function of ApgYa and ApgYe remains unclear due to the lack of intermediate accumulation in ΔapgYa and ΔapgYe mutants, sequence similarity network (SSN) analysis suggests a close relationship between ApgYe and AprY, which is responsible for C-O bond formation (FIG.13). Therefore, it is plausible that ApgYe likely plays a crucial role in the furan ring formation. The A ring was proposed to be formed through the linkage of Tyr3and Leu2via a C-C bond by ApgYa which may involve C(sctivation, indicating a novel reaction catalyzed by DUF3328 enzymes. Subsequent hydroxylations at C-12 and C-33 were mediated by ApgYb and ApgYf, respectively (Fig.2c). Then, the formation of the ether bond is catalyzed by ApgYc and ApgYd, leading to the production of compounds 1, 2, 15, and 16. Ultimately, the N-terminal glutaminyl of 15 and 16 is cyclized to yield 3 and 4. These findings highlight the functional diversity of DUF3328 enzymes and pave the way for genome mining of fungal RiPPs with specific modifications. Example 2 - Anti-cancer activity of asperigimycins and its improvement by N-terminal modification

[0171] Next, compounds 1-4 were evaluated for their cytotoxicity. Compounds 1 and 2, featuring a free amine at their N-terminus, showed no discernible activity against all tested cell lines. In contrast, compounds 3 and 4, which incorporate a pyroglutamate moiety at their N-terminal, displayed notable cytotoxic effects (FIG.3a and 3b). Both 3 and 4 exhibited high potency against several leukemia cell lines, including Jurkat, Mino, and Molm-14, with half-maximal inhibitory concentration (IC50) values ranging from 0.34 to 2.3 μM, while no obvious cytotoxicity was observed towards cervical cancer cell line HeLa and liver cancer cell line HepG2 (FIG.3e and Table 1). In addition, compound 4 demonstrated modest bioactivity against breast cancer cell line MCF-7 (6.2 μM of IC50).Table 1 IC50 values of asperigimycins against cancer cell lines

[0172] By comparing the chemical structures of asperigimycins and their respective bioactivities, it was hypothesized that the incorporation of the cyclic pyroglutamate ring in compounds 3 and 4 contributes to their anti-cancer bioactivity. Thus, the biochemical characteristics of ApgG, the enzyme responsible for the formation of this bioactivity-linked pyroglutamate in 3 and 4 were investigated. However, the presence of a signal peptide at the N-terminus of ApgG, as predicted by SignalP 6.0, made it challenging to obtain the full-length protein in Escherichia coli. To overcome this limitation, the first 17 amino acids, ApgGT17were truncated, which resulted in the successful production of a soluble enzyme. In the presence of ApgGT17, 15 was completely transformed to 3 in 30 min (FIG.4c). To gain insight into the catalytic mechanism of ApgG, the crystal structure of ApgG was determined and ApgG was found as a dimer within a single unit cell. The overall structure features an open-sandwich topology which shows high similarity with zinc-dependent human glutaminyl cyclase QC (FIG.4d and FIG.14a). Accordingly, the active site architecture of ApgG was demonstrated. The ion observed in its active site was proposed to be a zinc ion which is coordinated by the canonical amino acid residues D137, E185, and H326 of the zinc binding domain (FIG. 4d). As expected, mutation of these three amino acids to alanine all resulted in the complete loss of catalytic activity (FIG. 14d). Based on docking the substrate 15 into the active site of ApgG (FIGS.14b and c), alanine mutations at three residues including E184, D232, and D297 were introduced due to their proximity to the terminal amide of the glutaminyl motif. Among these mutants, E184A and D297A exhibited no catalytic activity, while the catalytic capacity of the D232A mutant decreased by 64%. (FIG. 4d and FIG. 14d). It was proposed that these three amino acids may participate in the formation of hydrogen bonds required for activating the terminal amide. Furthermore, variants F321A and W325A displayed reduced conversion ratesby 48% and 26%, respectively, suggesting that these residues may influence substrate recognition. These results demonstrate that ApgG can catalyze the cyclization of N-terminal glutaminyl to form the pyroglutamate moiety and represents the first instance of a QC enzyme participating in the biosynthesis of fungal RiPPs.

[0173] Inspired by the observation that enhancing the cytotoxicity of asperigimycins can be achieved through the incorporation of pyroglutamate, a group known to enhance the proteolytic stability and hydrophobicity of peptide (Van Coillie et al., 1998; Shih et al., 2014) six variants of 2 (2-L1 to 2-L6) were then chemically synthesized. These variants were designed to introduce different lipid substitutions at the N-terminus with the aim of enhancing stability, hydrophobicity, and permeability to improve their bioactivity.2-L1, which bears a butyramide moiety at the N-terminus, did not exhibit appreciable bioactivity. The incorporation of a biphenyl group (2-L2) resulted in a slight enhancement of its anticancer activity compared to the parent compound. Variants with extended chains (2-L3, 2-L4, 2-L5, and 2-L6) demonstrated notably enhanced bioactivity (FIG.3a and Table 1). In particular, 2- L6, featuring a linear C11-fatty acid chain at its N-terminus, exhibited potent inhibitory effects on three leukemia cell lines (Jurkat, Mino, and Molm-14) with IC50ranging from 40 to 99 nM (FIG.3a, 3e, and Table 1). Meanwhile, 2-L6 exhibited reduced activity against the other two leukemia (K562 and U937) and MCF-7 cell line. Additionally, it exhibited no cytotoxicity against HeLa and HepG2, indicating a potential for selective cytotoxicity. Moreover, 2-L6displayed superior in vitro cytotoxicity compared to cytarabine and demonstrated cytotoxicity comparable to that of daunorubicin, both of which are well-established therapeutics used in clinical leukemia treatment (Table 1). Collectively, the results reveal the importance of N- terminal substitution in improving the cytotoxicity of 2 and indicate that medium length fatty acid conjugation can improve activity (Morstein et al., 2022). It also highlights a promising lead compound (2-L6) for further development as effective drugs for leukemia treatment. Example 3 - CRISPR screening reveals SLC46A3 serves as a transporter for asperigimycins

[0174] Considering the potential elevation in cytotoxicity associated with lipid substitution, a high-throughput CRISPR screening was conducted to uncover the underlying factors driving this enhancement and identify potential targets for asperigimycins. The screening utilized the most potent asperigimycin derivative, 2-L6 and Jurkat, along with the Toronto Knock Out library v3 (TKOv3) (FIG. 4a). Compared to the untreated controls, sgRNAs targeting SLC46A3, CCNF, KEAP1, CLASP2, and STMN1, showed significantenrichment in asperigimycin-treated cells. Among those, SLC46A3, belonging to the solute carrier (SLC) superfamily (Kim et al., 2021; Tomabechi et al., 2023) is potentially involved in the transportation of 2-L6 into the cytoplasm (FIG.4b and 4c). To further validate the role of SLC46A3 in asperigimycin toxicity, CRISPR-Cas9 technology was utilized to generate SLC46A3 knockout Jurkat cell lines (SLC46A3Δ). Upon treating both wild-type (WT) Jurkat and SLC46A3Δ with 80 nM 2-L6 for 72 hours, less than half of the WT cells remained viable, whereas approximately 98% of SLC46A3Δ cells survived (FIG. 4d). Notably, the IC50value of 2-L6 against SLC46A3Δ (2440 nM) was approximately 30-fold higher than that against the WT cells (FIG. 4e). Subsequent cellular uptake assays revealed that 2-L6 exhibited an approximately 8-fold higher concentration in WT cells compared to SLC46A3Δ (FIG.4f). As a comparison, 2 without the lipid chain was undetectable within the cells after 24 hours. These findings suggest that the depletion of SLC46A3 could confer Jurkat cells resistance to asperigimycin cytotoxicity. It also confirms the role of SLC46A3 as a transporter of lipid derivatives of asperigimycins and highlights the contribution of compound penetration by lipid substitution. Additionally, other genes including E3 ubiquitin ligase-encoding genes CCNF (Williams et al., 2016) and KEAP1 (Cuadrado et al., 2019), along with CLASP2 (Luo et al., 2023) and STMN1 (Liu et al., 2021), which play a role in regulating the microtubule filament system, are also associated with asperigimycin resistance revealed by CRISPR Screening results. Depleting these genes demonstrated a moderate increase in resistance of aperigimycins in Jurkat (FIG.4d and 4e), suggesting their potential relevance as drug targets.

[0175] To further investigate this transport process, Jurkat cells were co- incubated with 2-L6 and endocytosis inhibitors (genistein or hydroxy-dynasore) overnight (Gratton et al., 2008). Treatment with endocytosis inhibitors increased cell survival from 25% (no inhibitor control) to 98% (genistein) and 74% (hydroxy-dynasore) (FIGS. 24A and B), demonstrating endocytosis-dependent transport of lipidated asperigimycins to lysosomes is essential for its anticancer activity. It was proposed that lipidated asperigimycin predominantly enters cells via endocytosis and reaches the lysosome (Banushi et al., 2023). Subsequently, the lysosomal transporter SLC46A (Kim et al., 2021; Hamblett et al., 2015), facilitates the compound escape into the cytoplasm where it exerts anticancer activity; Without efficient lysosomal escape, the cyclic peptide likely undergoes degradation, reducing intracellular concentrations. These results further highlight the significance of lipid substitution in improving drug uptake and transport, thereby enhancing their anticancer efficacy. Interestingly, Depmap (Tsherniak et al., 2017) analysis revealed higher expression levels of SLC46A3 in celllines inhibited by asperigimycins (such as Jurkat, Mino, Molm14, K562, and MCF-7) compared to those less affected (such as HepG2 and HeLa). This further implies potential transporter-mediated selective cytotoxicity of asperigimycins.

[0176] Deletion of other positive hits identified from the CRISPR screening experiments, including the E3 ubiquitin ligase-encoding genes CCNF (D’Angiolella et al., 2012) and KEAP1 (Cuadrado et al., 2019) and the microtubule filament regulatory genes CLASP2 (Luo et al., 2023) and STMN1 (Kavallaris et al., 2010), moderately increased asperigimycin resistance in Jurkat cells (FIGS. 4d and e), suggesting their possible roles in pathways underpinning drug sensitivity. Using fluorescent-based tubulin polymerization assays, it was demonstrated that compounds 2, 3, and 2-L6 all inhibit tubulin polymerization in vitro, when no cell penetration is required (FIG.25a). However, immunofluorescence staining revealed that only 2-L6 significantly reduced microtubule polymerization in Jurkat cells compared to cells treated with DMSO or 2 (FIG. 25b). These results confirm the antitubulin activity of asperigimycins and suggest that the enhanced antitumor activity of the modified asperigimycin is likely due to improved intracellular transport rather than intrinsic pharmacological properties. Although no significant alteration in the NRF2 expression level, whose degradation is facilitated by KEAP1 (Cuadrado et al., 2019), was observed, it was found that 2-L6 substantially downregulated RRM2 (D’Angiolella et al., 2012) and E2F1 (Clijsters et al., 2019) proteins in Jurkat cells, known substrates of CCNF, shown in western blot analysis (FIG. 25c). These suggest that asperigimycins engage in the ubiquitination and subsequent proteasomal degradation pathway mediated by CCNF.

[0177] The discovery of fungal RiPPs is challenging, primarily due to limited knowledge of biosynthesis of fungal RiPPs and the lack of effective mining methods. This emphasizes the need to improve our understanding of the biosynthesis and diversity of fungal RiPPs and develop efficient strategies for their discovery and identification. Integrating metabolite MS / MS networking with the recognition of recurring amino acid sequences within precursor peptides offers an effective approach for identifying potential ascomycete-derived RiPPs and establishing the relationship between metabolites and BGCs. Utilizing this strategy, a previously unknown group of fungal RiPPs named asperigimycins was successfully discovered, featuring an unprecedented architecture consisting of benzofuranoindoline multicyclic scaffolds from A. flavus, underscoring the significant potential of fungi in RiPP production. While the chemical synthesis of asperigimycins could be challenging, the benzofuranoindoline core is proposed to be arisen from oxidative C-O and C-C bonds betweenTrp6and Tyr3, facilitated by a DUF3328 oxidase. In contrast, the benzofuranoindoline core in dipeptide azonazine A, a non-ribosomal peptide, was generated via an oxidative cyclization catalyzed by a P450 enzyme (Liu et al., 2023). Furthermore, the emergence of this distinct core in other compounds remains unreported. These findings expand the library of fungal RiPPs, deepen our understanding of fungal RiPPs biosynthesis, and highlight the diverse biosynthetic pathway of fungal peptides.

[0178] Cyclic peptides have significantly contributed to the pharmaceutical industry, with 18 cyclopeptides receiving clinical approval for various applications in the past two decades (Zhang et al., 2022). Despite their pharmaceutical potential, peptides face challenges related to cellular penetration (Dougherty et al., 2019). Hence, the incorporation of noncanonical elements becomes a crucial strategy for optimizing their pharmacokinetic and pharmacodynamic characteristics (Zhang et al., 2022; Wang et al., 2022). Among naturally produced asperigimycins, it has been observed that 3 and 4, containing a pyroglutamate moiety, exhibit greater cytotoxicity than 1 and 2 lacking this functional group. In their biosynthesis, a QC enzyme is verified to be responsible for the formation of this group. Further investigation into the catalytic mechanism of ApgG offered valuable insights for engineering this enzyme as a biocatalyst to introduce the distinct pyroglutamate modifications into peptide natural products. Through chemical synthesis, the incorporation of lipophilic side chains at the N- terminus markedly enhanced the bioactivity of 2, with particular emphasis on the positive impact of the long linear fatty acid chain. Uptake experiments indicate that the lipid chain facilitates the permeability of asperigimycins. Furthermore, research reveals SLC46A3 facilitates the cellular uptaking of 2-L6, emphasizing the crucial role of SLC46A3 in the cytotoxicity of lipid-modified asperigimycins. The absence of SLC46A3 leads to the resistance of Jurkat to asperigimycins, further indicating that SLC46A3 could potentially serve as a biomarker for asperigimycin bearing lipid chains. In summary, findings described herein lay the foundation for the chemical modification of asperigimycins for potential therapeutic applications and highlight the crucial role of peptide modification in shaping their bioactivity.

[0179] Murine KCs (Kup5) were treated with 1ug / ml MET, 1ug / ml SIM, 25ug / ml EGCG or 6.25ug / ml T1 and incubated for 24h. RNA was extracted to measure TNF and IL6 gene expression by quantitative RT-PCR. To assess intracellular killing, Kup5 were infected with USA300 methicillin-resistant SA at an MOI 5, incubated for 1.5h then treated with 100ug / ml gentamicin for 2h to kill extracellular bacteria. Infected cells were treated with25ug / ml EGCG or 6.25ug / ml T1 and incubated for 10h before lysing with 0.1% Triton X. Lysate was streaked on TSA and CFUs were enumerated the next day.

[0180] Compared to no treatment, Kup5s exposed to 1ug / ml MET showed a 1.5X increase in expression of IL-6 but no change in TNF while the opposite was observed with 1ug / ml SIM (4X increase in TNF, no change in IL-6). Interestingly, Kup5s exposed to 25ug / ml EGCG showed no change in either IL-6 or TNF but when exposed to the EGCG analog at 6.25ug / ml (T1), gene expression increased for both IL-6 (1.5x) and TNF (3X) likely due to improved cellular penetration. Treatment of infected Kup5 with 6.25ug / ml of T1 reduced 10h intracellular SA growth by 4-fold compared to EGCG and no treatment (2.3 and 2.2 log CFU increase from baseline respectively). MET and SIM exerted effects on IL-6 or TNF expression while T1 increased expression of both pro-inflammatory mediators which correlated with enhanced intracellular killing of SA in KCs.

[0181] Differential expression of M1 markers was observed with minimal alteration of M2 markers when comparing the addition of different immunomodulatory compounds to vancomycin. Intracellular killing of SA by Kupffer cells does not appear to correlate with M1 / M2 phenotype expression when using markers such as CD38 and TNF-a Egr2, and Arg1. EGCG increased surface marker stimulation (CD38) for M1 phenotype but did not correlate with intracellular killing likely attributable to its poor intracellular penetration. MCC-1 (T1) showed greatest potentiation of vancomycin in intracellular killing suggesting that reducing M2 phenotype expression may be more important than increasing M1 marker expression along with other mechanisms including intrinsic antimicrobial activity. Example 2: Novel synthetic EGCG analogs potentiate intracellular killing of Staphylococcus aureus within liver-resident macrophages

[0182] Three novel (-)-Epigallocatechin gallate (EGCG) analogs with improved lipophilicity and reduced polar surface area predictive of superior membrane penetration were synthesized and the in vitro activity of the EGCG analogs against S. aureus intracellular killing was assessed. Broth microdilution assays were performed following CLSI standardized methodology to determine MICs (minimum inhibitory concentration) for the analogs alone or in combination with cefazolin or oxacillin against GFP USA300 methicillin- resistant S. aureus (GFP SA). The analogs were tested at a concentration range of 1.56- 200ug / ml and cefazolin and oxacillin at 0.5-128ug / ml. MICs were read visually and GFP fluorescence was used to confirm the MIC as EGCG and some analogs have color. MurineKupffer cells (Kup5) were infected with GFP SA at an MOI 5, incubated for 1.5h, then treated with 100ug / ml gentamicin for 2h to kill extracellular bacteria. Sub-MICs of the analogs were added to the cells and incubated for 10h before lysing with 0.1% Triton X. Lysate was streaked on TSA and CFUs were enumerated the next day to assess intracellular killing.

[0183] The MICs for all 3 EGCG analogs were significantly lower than EGCG (12.5 vs 50ug / ml). The addition of EGCG analogs at sub-MIC(6.25ug / ml) concentrations potentiated the activity of cefazolin and oxacillin, reducing their MICs from 64ug / ml to 2ug / ml. Treatment of infected Kup5 with 6.25ug / ml of our lead analog reduced 10h intracellular SA growth by at least 4-fold compared to EGCG and no treatment (2.3 and 2.2 log CFU increase from baseline respectively).

[0184] Chemical modifications to EGCG (FIGS.2 and 6-9) exhibited improved drug-like properties with enhancements of the following: (1) Demonstrated direct antibacterial activity leading to a 4x decrease in MIC value when tested against MRSA; (2) Exhibited synergy with beta-lactam agents and restored activity against MRSA; (3) Improved cell penetrating potential compared to EGCG resulting in enhanced intracellular killing of SA in Kupffer cells. Overall, MCC-1 (T1) displayed a favorable pharmacologic profile (see, for example, FIGS.6-9). Example 3 – Materials and Methods

[0185] Culture media. YES medium (10 g / L yeast extract, 150 g / L sucrose, 0.5 g / L MgSO4·7H2O); Peptone medium (4 g / L glucose, 2 g / L peptone, 0.5 g / L NaCl); PDB medium (4 g / L potato starch, 20 g / L glucose); Maltose medium (10 g / L glucose, 20 g / L maltose, 20 g / L mannitol, 10 g / L monosodium glutamate, 0.5 g / L KH2PO4, 0.3 g / L MgSO4·7H2O, 1 g / L corn steep liquor, and 3 g / L yeast extract, pH 7.0); GMM medium (10 g / L glucose, 50 mL 20 × nitrate salts, 1 mL trace elements, pH 6.5); CD-ST (20 g / L starch, 20 g / L peptone, 50 mL 20 × nitrate salts, 1 mL trace elements); MYPG medium (3 g / L malt extract, 3 g / L yeast extract, 3 g / L peptone, 20 g / L glucose); Rice medium (400 g / L rice); YM medium (20 g / L yeast extract, 20 g / L maltose), PYG (10 g / L peptone, 5 g / L yeast extract, 10 g / L glucose).

[0186] General culturing conditions. All aspergillus strains were cultured on different culture media at 30°C for 7 days. For large-scale fermentation of A. flavus CA14 and the mutants, they were cultured on YES solid medium at 30°C for 7 days.

[0187] Preparation of protoplast of A. flavus CA14. Fresh spores were inoculated into 100 mL liquid PDB medium in 1 L flask and germinated at 30°C and 180 rpm for approximately 12 h. Mycelia were harvested by centrifugation at 3500 rpm for 10 min and washed with 10 mL of Osmotic buffer (1.2 M MgSO4, 10 mM sodium phosphate, pH 5.8). Then the mycelia were transferred to 10 mL of Osmotic buffer containing 30 mg of lysing enzymes from Trichoderma and 20 mg of Yatalase in a 125 mL flask. The flask was shaken at 80 rpm overnight at 30°C. Cells were collected in 50 mL conical centrifuge tubes and overlaid gently by 10 mL of Trapping buffer (0.6 M sorbitol, 0.1 M Tris-HCl, pH 7.0). After centrifugation at 3500 rpm for 15 min at 4°C, the protoplasts were collected at the interface of the two buffers. The protoplasts were then transferred to a sterile 15 mL conical centrifuge Tubes and washed with 10 mL STC buffer (1.2 M sorbitol, 10 mM CaCl2, 10 mM Tris-HCl, pH 7.5). The protoplasts were resuspended in 1 mL STC buffer for transformation (Szewczyk et al., 2006).

[0188] Gene deletion and Transformation of A. flavus CA14. The split-marker approach was used for targeted gene knockout by homologous recombination. The homologous regions (~1.5 kb) were amplified by PCR from the A. flavus genome. The fusion PCR provided the deletion cassettes. These gene fragments were transformed into A. flavus by polyethylene glycol-mediated protoplast transformation following the method described in the reference (Zhao et al., 2023). The DNA fragments were incubated with 50 μL of the protoplasts in the ice water bath for 1 h. Then 1 mL freshly filtered PEG solution (25 % PEG average molecular weight 3350, 0.6 M KCl, 50 mM CaCl2, 10 mM Tris-HCl, pH 7.5) was added and gently mixed. Mix the PEG solution with the protoplast suspension by gently aspirating and ejecting the solution into the tip of the micropipette at least ten times. Incubation at 30°C for 25 min. Plate transformation mixture onto selective plates.

[0189] Metabolite analysis of A. flavus and its mutants. The broth was extracted by 50% (v / v) MeOH aqueous solution plus ultrasonic assistance. A linear gradient program of 5%-90% (v / v) acetonitrile with 0.1% (v / v) Formic acid in H2O of 30 min at a 0.5 mL / min flow rate was used to analyze the metabolite profiles.

[0190] Isolation and characterization. The obtained crude was fraction by a ODS column eluted with MeOH aqueous solution (10%-100%, v / v) to give ten subfractions (Fr.1-Fr.10). Fr.3 was further purified with Sephadex LH-20 chromatography (40-70 μm; GE Healthcare Life Science). The isolates were finally purified by Agilent 1260 Infinity II LCSystem (Agilent Technologies) equipped with a semipreparative column (Ultimate XB-C18, 10 × 250 mm, 5 μm, Welch) using a linear gradient program of 5%-50% (v / v) acetonitrile with 0.1% (v / v) Formic acid in H2O over 30 min at a 4 mL / min flow rate. The obtained compounds were dissolved in deuterated solvents (H2O, D2O, CD3OD, or DMSO-d6) for NMR experiments.

[0191] Expression and purification of ApgG and its mutants from E. coli. The ORF of ApgG was amplified by PCR using the A. flavus cDNA as the template. The recovered DNA fragment was ligated into the vector pET by Gibson assembly. The plasmids were transferred into E. coli BL21 (DE3) for protein expression and purification. E. coli harboring the plasmid was grown overnight in 5 mL of LB with 50 μg / mL ampicillin at 37°C.500 mL of fresh LB with 50 μg / mL ampicillin was inoculated with 5 mL of the overnight culture and incubated at 37°C until the OD600 value reached 0.6-0.8. Next, the cells were induced with 0.1 mM IPTG for 20 h at 16°C. Cells were harvested by centrifugation (3000 g, 15 min). All subsequent procedures were performed at 4°C or on ice. Harvested cells were resuspended in disruption buffer (50 mM Tris-HCl (pH 7.8), 200 mM NaCl). After sonication and centrifugation (17000 g, 60 min, 4°C), the supernatant was subjected to His-tag affinity purification. Tables 2-5 describe the fungal strains, plasmids and primers used, as well as the proteins encoded in apg gene cluster.Table 2. Fungal strainsTable 3. PlasmidsTable 4. PrimersTable 5. Proteins encoded in apg gene cluster.

[0192] In vitro assays of ApgGT17 and its mutants. The enzymatic assays of ApgG were carried out at 28°C in a 50 μL aliquot containing 50 mM Tris-HCl (pH 8.0), 10 μM ApgG, 0.1 mM 15. After 30 min, the assay was quenched by 5 μL MeOH. Samples were analyzed by LC-MS with the following time program: 5%-95% acetonitrile for 15 min, 95% acetonitrile for 5 min, 95%-5% acetonitrile for 5 min, and 5% acetonitrile for 5 min.0.1% of formic acid was added to H2O. The flow rate was 0.5 mL / min.

[0193] Crystallization and structural determination of ApgGT17. Crystal of ApgGT17 were obtained using the hanging-drop vapor-diffusion method against a reservoir solution containing 0.1 M Bis-Tris (pH 6.5), 8% w / v Polyethylene glycol 10,000, and 0.2 M Potassium sodium tartrate tetrahydrate at room temperature for 4 days.

[0194] General cell culturing conditions. The Jurkat, Mino, Molm-14, U937, and K562 cells were cultured in RPMI-1640 medium, supplemented with 10% (v / v) fetal bovine serum and Penicillin-Streptomycin. The HeLa and MCF-7 were cultured in Dulbecco’sModified Eagle Medium and HepG2 was cultured in Eagle's Minimum Essential Medium with the same supplements as above. All cell lines were cultured at 37°C in a humidified 5% CO2 atmosphere.

[0195] Cell viability assays. Plate 4000 cells in 100 μL medium in 96 well plates. For adherent cells, we will let them adapt overnight to attach, for suspension cells, we found there is no need to wait. Prepare drugs to get 7 serial dilutions which is 10-fold of the final desired concentration (10000, 2000, 400, 80, 16, 3.2, 0.64, and 0.128 nM). Add 10 μL to each cell. Incubate cells in drugs for 72 h. The cell viability was assessed according to the protocol of CellTiter-Glo® Kit (Promega, G7572) and observed under microplate readers (Tecan, Infinite 200 PRO).

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Claims

WHAT IS CLAIMED IS:

1. A compound of the formula:wherein: R1is hydrogen; or alkyl(C≤12), cycloalkyl(C≤12), acyl(C≤12), heterocycloalkyl(C≤12), aryl(C≤12), aralkyl(C≤12), heteroaryl(C≤12), heteroaralkyl(C≤12), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or −C(O)R12, wherein R12 is: hydrogen, hydroxy, or amino; or alkyl(C≤8), cycloalkyl(C≤8), alkoxy(C≤12), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), aralkyl(C≤12), heteroaralkyl(C≤12), hetero- cycloalkyl(C≤8), alkoxy(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12),99−alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or R1 is taken together with R3 as defined below; R2, R4, R5, R6, and R9 are each independently hydrogen, hydroxy, amino or mercapto; or alkyl(C≤8), cycloalkyl(C≤8), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), heterocycloalkyl(C≤8), alkoxy(C≤8), cycloalkoxy(C≤8), acyl(C≤8), amido(C≤8), alkenyloxy(C≤8), aryloxy(C≤8), aralkoxy(C≤8), heteroaryloxy(C≤8), acyloxy(C≤8), alkylamino(C≤8), cycloalkylamino(C≤8), dialkylamino(C≤8), arylamino(C≤8), heteroarylamino(C≤8), alkylsulfonylamino(C≤8), cycloalkylsulfonylamino(C≤8), alkoxyamino(C≤8), hetero- cycloalkylamino(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; R7 and R8 are each independently hydrogen, hydroxy, alkyl(C≤8) or substituted alkyl(C≤8); R3 is hydrogen or hydroxy; or alkyl(C≤8), cycloalkyl(C≤8), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), heterocycloalkyl(C≤8), alkoxy(C≤8), cycloalkoxy(C≤8), alkenyloxy(C≤8), aryloxy(C≤8), aralkoxy(C≤8), heteroaryloxy(C≤8), acyloxy(C≤8), alkylamino(C≤8), cyclo- alkylamino(C≤8), dialkylamino(C≤8), arylamino(C≤8), heteroarylamino(C≤8), alkylsulfonylamino(C≤8), cycloalkylsulfonylamino(C≤8), alkoxyamino(C≤8), hetero- cycloalkylamino(C≤8), or a substituted version of any of these groups; orR3is taken together with R1and is −C(O)(CR1'R1'')n−, wherein: n is 4 or 5; R1' and R1'' are each independently hydrogen, hydroxy or amino; or alkyl(C≤8), cycloalkyl(C≤8), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), hetero- cycloalkyl(C≤8), alkoxy(C≤8), cycloalkoxy(C≤8), acyl(C≤8), amido(C≤8), alkenyloxy(C≤8), aryloxy(C≤8), aralkoxy(C≤8), heteroaryloxy(C≤8), acyloxy(C≤8), or a substituted version of any of these groups; or −NR2'R2'', wherein R2' and R2'' are each independently hydrogen; or −C(O)R13, wherein R13 is hydrogen, hydroxy, or amino; or alkyl(C≤8), cycloalkyl(C≤8), alkoxy(C≤12), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), aralkyl(C≤12), heteroaralkyl(C≤12), heterocycloalkyl(C≤8), alkoxy(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−amido(C≤18), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; R10is hydrogen, hydroxy or amino; oralkyl(C≤8), cycloalkyl(C≤8), heterocycloalkyl(C≤8), amido(C≤8), alkylamino(C≤8), cycloalkylamino(C≤8), dialkyl- amino(C≤8), arylamino(C≤8), heteroarylamino(C≤8), alkyl- sulfonylamino(C≤8), cycloalkylsulfonylamino(C≤8), alkoxyamino(C≤8), heterocycloalkylamino(C≤8), or a substituted version of any of these groups; or R10is taken together with R11as defined below; R11is hydrogen or hydroxy; or alkyl(C≤8), cycloalkyl(C≤8), heterocycloalkyl(C≤8), alkoxy(C≤8), cycloalkoxy(C≤8), acyl(C≤8), amido(C≤8), alkenyloxy(C≤8), aryloxy(C≤8), aralkoxy(C≤8), heteroaryloxy(C≤8), acyloxy(C≤8), or a substituted version of any of these groups; or R10is taken together with R11and is –O(CR3'R3'')m(NH)−, wherein: m is 2, 3 or 4 R3' and R3'' are each independently hydrogen, hydroxy or amino; or alkyl(C≤12), cycloalkyl(C≤12), acyl(C≤12), heterocycloalkyl(C≤12), aryl(C≤12), heteroaryl(C≤12), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or −C(O)R16, wherein R16 is: hydrogen, hydroxy, or amino; or alkyl(C≤8), cycloalkyl(C≤8), alkoxy(C≤12), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8),aralkyl(C≤8), heteroaryl(C≤8), aralkyl(C≤12), heteroaralkyl(C≤12), heterocycloalkyl(C≤8), alkoxy(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein the compound is further defined as:wherein: R1is hydrogen; or alkyl(C≤12), cycloalkyl(C≤12), acyl(C≤12), heterocycloalkyl(C≤12), aryl(C≤12), aralkyl(C≤12), heteroaryl(C≤12), heteroaralkyl(C≤12), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or−C(O)R12, wherein R12is: hydrogen, hydroxy, or amino; or alkyl(C≤8), cycloalkyl(C≤8), alkoxy(C≤12), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), aralkyl(C≤12), heteroaralkyl(C≤12), hetero- cycloalkyl(C≤8), alkoxy(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or R1 is taken together with R3 as defined below; R2, R4, R5, R6, and R9 are each independently hydrogen, hydroxy, amino or mercapto; or alkyl(C≤8), cycloalkyl(C≤8), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), heterocycloalkyl(C≤8), alkoxy(C≤8), cycloalkoxy(C≤8), acyl(C≤8), amido(C≤8), alkenyloxy(C≤8), aryloxy(C≤8), aralkoxy(C≤8), heteroaryloxy(C≤8), acyloxy(C≤8), alkylamino(C≤8), cycloalkylamino(C≤8), dialkylamino(C≤8), arylamino(C≤8), heteroarylamino(C≤8), alkylsulfonylamino(C≤8), cycloalkylsulfonylamino(C≤8), alkoxyamino(C≤8), hetero- cycloalkylamino(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; R3is hydrogen or hydroxy; or alkyl(C≤8), cycloalkyl(C≤8), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), heterocycloalkyl(C≤8), alkoxy(C≤8), cycloalkoxy(C≤8), alkenyloxy(C≤8), aryloxy(C≤8), aralkoxy(C≤8),heteroaryloxy(C≤8), acyloxy(C≤8), alkylamino(C≤8), cyclo- alkylamino(C≤8), dialkylamino(C≤8), arylamino(C≤8), heteroarylamino(C≤8), alkylsulfonylamino(C≤8), cycloalkylsulfonylamino(C≤8), alkoxyamino(C≤8), hetero- cycloalkylamino(C≤8), or a substituted version of any of these groups; or R3is taken together with R1and is −C(O)(CR1'R1'')n−, wherein: n is 4 or 5 R1' and R1'' are each independently hydrogen, hydroxy or amino; or alkyl(C≤8), cycloalkyl(C≤8), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), hetero- cycloalkyl(C≤8), alkoxy(C≤8), cycloalkoxy(C≤8), acyl(C≤8), amido(C≤8), alkenyloxy(C≤8), aryloxy(C≤8), aralkoxy(C≤8), heteroaryloxy(C≤8), acyloxy(C≤8), or a substituted version of any of these groups; or −NR2'R2'', wherein R2' and R2'' are each independently hydrogen; or −C(O)R13, wherein R13 is hydrogen, hydroxy, or amino; or alkyl(C≤8), cycloalkyl(C≤8), alkoxy(C≤12), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), aralkyl(C≤12), heteroaralkyl(C≤12), heterocycloalkyl(C≤8), alkoxy(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12),−alkanediyl(C≤12)−amido(C≤18), −alkanediyl(C≤12)−heterocycloalky l(C≤12), −alkanediyl(C≤12)−cyclo- alkyl(C≤12), or a substituted version of any of these groups; R10 is hydrogen, hydroxy or amino; or alkyl(C≤8), cycloalkyl(C≤8), heterocycloalkyl(C≤8), amido(C≤8), alkylamino(C≤8), cycloalkylamino(C≤8), dialkyl- amino(C≤8), arylamino(C≤8), heteroarylamino(C≤8), alkyl- sulfonylamino(C≤8), cycloalkylsulfonylamino(C≤8), alkoxyamino(C≤8), heterocycloalkylamino(C≤8), or a substituted version of any of these groups; or R10 is taken together with R11 as defined below; R11 is hydrogen or hydroxy; or alkyl(C≤8), cycloalkyl(C≤8), heterocycloalkyl(C≤8), alkoxy(C≤8), cycloalkoxy(C≤8), acyl(C≤8), amido(C≤8), alkenyloxy(C≤8), aryloxy(C≤8), aralkoxy(C≤8), heteroaryloxy(C≤8), acyloxy(C≤8), or a substituted version of any of these groups; or R10 is taken together with R11 and is –O(CR3'R3'')m(NH)−, wherein: m is 2, 3 or 4 R3' and R3'' are each independently hydrogen, hydroxy or amino; or alkyl(C≤12), cycloalkyl(C≤12), acyl(C≤12), heterocycloalkyl(C≤12), aryl(C≤12), heteroaryl(C≤12), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12),−alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or −C(O)R16, wherein R16is: hydrogen, hydroxy, or amino; or alkyl(C≤8), cycloalkyl(C≤8), alkoxy(C≤12), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), aralkyl(C≤12), heteroaralkyl(C≤12), heterocycloalkyl(C≤8), alkoxy(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or a pharmaceutically acceptable salt thereof.

3. The compound of either claim 1 or claim 2, wherein the compound is further defined as: Rwherein:R2, R4, R5, R6, and R9are each independently hydrogen, hydroxy, amino or mercapto; or alkyl(C≤8), cycloalkyl(C≤8), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), heterocycloalkyl(C≤8), alkoxy(C≤8), cycloalkoxy(C≤8), acyl(C≤8), amido(C≤8), alkenyloxy(C≤8), aryloxy(C≤8), aralkoxy(C≤8), heteroaryloxy(C≤8), acyloxy(C≤8), alkylamino(C≤8), cycloalkylamino(C≤8), dialkylamino(C≤8), arylamino(C≤8), heteroarylamino(C≤8), alkylsulfonylamino(C≤8), cycloalkylsulfonylamino(C≤8), alkoxyamino(C≤8), hetero- cycloalkylamino(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; R10is hydrogen, hydroxy or amino; or alkyl(C≤8), cycloalkyl(C≤8), heterocycloalkyl(C≤8), amido(C≤8), alkylamino(C≤8), cycloalkylamino(C≤8), dialkyl- amino(C≤8), arylamino(C≤8), heteroarylamino(C≤8), alkyl- sulfonylamino(C≤8), cycloalkylsulfonylamino(C≤8), alkoxyamino(C≤8), heterocycloalkylamino(C≤8), or a substituted version of any of these groups; or R10is taken together with R11as defined below; R11is hydrogen or hydroxy; or alkyl(C≤8), cycloalkyl(C≤8), heterocycloalkyl(C≤8), alkoxy(C≤8), cycloalkoxy(C≤8), acyl(C≤8), amido(C≤8), alkenyloxy(C≤8), aryloxy(C≤8), aralkoxy(C≤8), heteroaryloxy(C≤8), acyloxy(C≤8), or a substituted version of any of these groups; or R10is taken together with R11and is –O(CR3'R3'')m(NH)−, wherein:m is 2, 3 or 4 R3' and R3'' are each independently hydrogen, hydroxy or amino; or alkyl(C≤12), cycloalkyl(C≤12), acyl(C≤12), heterocycloalkyl(C≤12), aryl(C≤12), heteroaryl(C≤12), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or −C(O)R14, wherein R14 is: hydrogen, hydroxy, or amino; or alkyl(C≤8), cycloalkyl(C≤8), alkoxy(C≤12), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), aralkyl(C≤12), heteroaralkyl(C≤12), heterocycloalkyl(C≤8), alkoxy(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; R15is hydrogen; or alkyl(C≤12), cycloalkyl(C≤12), acyl(C≤12), heterocycloalkyl(C≤12), aryl(C≤12), heteroaryl(C≤12), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or−C(O)R16, wherein R16is: hydrogen, hydroxy, or amino; or alkyl(C≤8), cycloalkyl(C≤8), alkoxy(C≤12), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), aralkyl(C≤12), heteroaralkyl(C≤12), hetero- cycloalkyl(C≤8), alkoxy(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−amido(C≤18), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or a pharmaceutically acceptable salt thereof.

4. The compound according to any one of claims 1-3, wherein the compound is further defined as:wherein: R2, R4, R5, R6, and R9 are each independently hydrogen, hydroxy, amino or mercapto; oralkyl(C≤8), cycloalkyl(C≤8), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), heterocycloalkyl(C≤8), alkoxy(C≤8), cycloalkoxy(C≤8), acyl(C≤8), amido(C≤8), alkenyloxy(C≤8), aryloxy(C≤8), aralkoxy(C≤8), heteroaryloxy(C≤8), acyloxy(C≤8), alkylamino(C≤8), cycloalkylamino(C≤8), dialkylamino(C≤8), arylamino(C≤8), heteroarylamino(C≤8), alkylsulfonylamino(C≤8), cycloalkylsulfonylamino(C≤8), alkoxyamino(C≤8), hetero- cycloalkylamino(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; R15 is hydrogen; or alkyl(C≤12), cycloalkyl(C≤12), acyl(C≤12), heterocycloalkyl(C≤12), aryl(C≤12), heteroaryl(C≤12), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or −C(O)R14, wherein R14is: hydrogen, hydroxy, or amino; or alkyl(C≤8), cycloalkyl(C≤8), alkoxy(C≤12), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), aralkyl(C≤12), heteroaralkyl(C≤12), hetero- cycloalkyl(C≤8), alkoxy(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−amido(C≤18), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or a pharmaceutically acceptable salt thereof.

5. The compound according to any one of claims 1-4, wherein the compound is further defined as:wherein: R9 is hydrogen, alkyl(C≤8), or substituted alkyl(C≤8); R15is hydrogen; or alkyl(C≤12), cycloalkyl(C≤12), acyl(C≤12), heterocycloalkyl(C≤12), aryl(C≤12), heteroaryl(C≤12), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or −C(O)R14, wherein R14 is: hydrogen, hydroxy, or amino; or alkyl(C≤8), cycloalkyl(C≤8), alkoxy(C≤12), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), aralkyl(C≤12), heteroaralkyl(C≤12), heterocycloalkyl(C≤8), alkoxy(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−amido(C≤18), −alkanediyl(C≤12)−heterocycloalkyl(C≤12),−alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups; or a pharmaceutically acceptable salt thereof.

6. The compound according to any one of claims 1-5, wherein R9 is hydrogen.

7. The compound according to any one of claims 1-5, wherein R9 is methyl.

8. The compound according to any one of claims 3-7, wherein R15 is −C(O)R14, wherein R14 is: hydrogen, hydroxy, or amino; or alkyl(C≤8), cycloalkyl(C≤8), alkoxy(C≤12), alkenyl(C≤8), alkynyl(C≤8), aryl(C≤8), aralkyl(C≤8), heteroaryl(C≤8), aralkyl(C≤12), heteroaralkyl(C≤12), heterocycloalkyl(C≤8), alkoxy(C≤8), −alkanediyl(C≤12)−aryl(C≤12), −alkanediyl(C≤12)−heteroaryl(C≤12), −alkanediyl(C≤12)−amido(C≤18), −alkanediyl(C≤12)−heterocycloalkyl(C≤12), −alkanediyl(C≤12)−cycloalkyl(C≤12), or a substituted version of any of these groups.

9. The compound of claim 8, wherein R14is −alkanediyl(C≤12)−amido(C≤18).

10. The compound according to any one of claims 1-4 and 6-9, wherein R2is hydrogen or hydroxy.

11. The compound according to any one of claims 1-4 and 6-10, wherein R4 is alkyl(C≤8) or substituted alkyl(C≤8).

12. The compound according to any one of claims 1-4 and 6-11, wherein R5 is hydrogen, alkyl(C≤8)or substituted alkyl(C≤8).

13. The compound according to any one of claims 1-4 and 6-12, wherein R6 is hydrogen, alkyl(C≤8) or substituted alkyl(C≤8).

14. The compound according to any one of claims 1-3, wherein R10is taken together with R11 and is –O(CR3'R3'')m(NH)−, wherein:R3' and R3'' are each independently hydrogen, alkyl(C≤12)or −C(O)R16, wherein R16 is hydrogen, hydroxy, or amino.

15. The compound of claim 14, wherein R16 is hydroxy. The compound of claim 1, wherein the compound is further defined as:,,,,,,,,.

17. A pharmaceutical composition comprising: (a) a compound according to any one of claims 1-16; and (b) an excipient.

18. A method of treating or preventing a disease or disorder in a patient in need thereof comprising administering to the patient a pharmaceutically effective amount of a compound or composition according to any one of claims 1-17.

19. The method of claim 18, wherein the disease or disorder is a cancer.

20. The method of claim 19, wherein the cancer is leukemia.

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