Sting agonists, compositions, and uses thereof

Abstract

The present disclosure provides STING agonists, and compositions, formulations, and methods for treating diseases or disorders (e.g., cancer, inflammatory diseases, autoimmune diseases, and infectious diseases) with the STING agonists or compositions thereof.

Description

STING AGONISTS, COMPOSITIONS, AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No.63 / 594,272, filed on October 30, 2023, which is incorporated herein by reference in its entirety. FIELD

[0002] The present disclosure provides stimulator of interferon (IFN) genes (STING) agonists, compositions, formulations, and methods for inducing or modulating an immune or inflammatory response, and for treating diseases or disorders (e.g., cancer, autoimmune diseases, inflammatory diseases, and infectious diseases) with the STING agonists or compositions thereof. BACKGROUND

[0003] The innate immune agonist STING (Stimulator of Interferon Genes) binds its natural ligand 2'3'-cGAMP (cyclic guanosine-adenosine monophosphate) and then, by way of its signaling pathway, induces the expression of interferons, inflammatory factors, and autophagy genes. Microbial infection, tumor DNA, and self-damaging DNA are three factors that induce the activation of the cGAS(cyclic GMP-AMP synthase)-STING signaling pathway and associate STING with the etiology of cancer, and autoimmune, infectious, and inflammatory diseases. Many natural and synthetic STING agonists have entered clinical development, particularly for cancer treatment, with the first generation demonstrating safety but only modest systemic activity. These STING agonists are mainly divided into two categories, namely, cyclic dinucleotides (CDNs) and non-cyclic dinucleotides (non-CDN). CDNs, as exemplified by ADU-S100 undergoing clinical trial, are cGAMP analogues with remarkable binding affinity. However, CDNs constantly suffer from poor permeability, inferior stability, and synthetic complexity. As a result, these STING agonists are administered via intratumoral injection. In contrast, non-CDN STING agonists show improved pharmacokinetic properties, which makes it possible to develop oral STING agonists. Indeed, MSA-2 is reported as the first oral non-CDN STING agonist with impressive in vivo antitumor efficacy. Nonetheless, most non-CDNs are still limited by weak binding affinity and poor permeability. So far, no STING agonist has been approved for clinical use. Thus, there is a need for non-CDN STING agonists with good efficacy and pharmacokinetic properties.SUMMARY

[0004] In one aspect, disclosed herein is a compound of formula (I):or a pharmaceutically acceptable salt thereof, wherein: R1, R2, R3, and R4 are each independently selected from hydrogen, C1-C6 alkoxy, halo, and C1-C6 alkyl; X1 is selected from S and NH; A is selected from a five- or six-membered cycloalkyl, heterocyclyl, heteroaryl, or aryl, each of which is optionally substituted with 1 or 2 oxo groups; W1 is a bond or O; W2 is selected from C1-C6 alkylene, C3-C6 cycloalkylene, C3-C6 cycloalkylene-C1-C6 alkylene, arylene, and hydroxy-C1-C6-alkylene; and R5 is selected from hydrogen, C1-C80 alkyl, C2-C80 alkenyl, C2-C80 alkynyl, amino, amino-C1-C6-alkyl, di-C1-C6-alkylamino-C1-C6-alkyl, heterocyclyl-C1-C6-alkyl, C1-C80 heteroalkyl, C2-C80 heteroalkenyl, and C2-C80 heteroalkynyl, wherein each of the C1-C80 alkyl, C2-C80 alkenyl, C2-C80 alkynyl, C1-C80 heteroalkyl, C2-C80 heteroalkenyl, and C2-C80 heteroalkynyl is optionally substituted with one or more substituents selected from hydroxy and amino.

[0005] In some embodiments, the compound is a compound of formula (Ia):or a pharmaceutically acceptable salt thereof. In some embodiments, X2 is CH or N, and X3 is CH2 or CO. In some embodiments, X2 is CH. In some embodiments, X2 is N.In some embodiments, X3 is CH2. In some embodiments, X3 is CO.

[0006] In some embodiments, the compound is a compound of formula (Ib):or a pharmaceutically acceptable salt thereof, wherein X4 and X5 are independently selected from N and CH. In some embodiments, X4 is N. In some embodiments, X5 is N. In some embodiments, X5 is CH.

[0007] In some embodiments, the compound is a compound of formula (Ic):or a pharmaceutically acceptable salt thereof. In some embodiments, X6 and X7 are independently selected from CH and N. In some embodiments, X6 is N. In some embodiments, X7 is N.

[0008] In some embodiments, X1 is S. In some embodiments, X1 is NH.

[0009] In some embodiments, W2 is selected from C1-C4 alkylene, C4-C5 cycloalkylene, C4-C5 cycloalkylene-C1-C2 alkylene, phenylene, and hydroxy-C1-C3-alkylene. In some embodiments, wherein W2 is selected from: and

[0010] In some embodiments, R5 is selected from hydrogen, C1-C40 alkyl, C2-C40 alkenyl, C1-C4-alkylamino, di-C1-C6-alkylamino-C1-C6-alkyl, and heterocyclyl-C1-C6-alkyl.

[0011] In some embodiments, R5 is hydrogen or C 5 1-C4 alkyl. In some embodiments, R is hydrogen, methyl, ethyl, or isopropyl. In some embodiments, R5 is hydrogen or methyl.

[0012] In some embodiments, R5 is selected from C12-C40 alkyl and C12-C40 alkenyl. In some embodiments, R5 is selected from C12-C20 alkyl and C12-C20 alkenyl. In some embodiments, R5 is or.

[0013] In some embodiments, R2 and R3 are each independently C1-C6 alkoxy. In some embodiments, R2 and R3 are each methoxy.

[0014] In some embodiments, R1 and R4 are each independently halo or hydrogen. In some embodiments, R1 is halo and R4 is hydrogen.

[0015] In some embodiments, R1 is halo, R2 and R3 are each independently C1-C6 alkoxy, and R4 is hydrogen. In some embodiments, R1 is chloro or bromo, and R2 and R3 are methoxy. In some embodiments, R1 and R4 are hydrogen, and R2 and R3 are methoxy.

[0016] In some embodiments, the compound is selected from:,a d .

[0017] In one aspect, disclosed herein is a compound of formula (II):or a pharmaceutically acceptable salt thereof, wherein: R1a, R1b, R2a, R2b, R3a, R3b, R4a, and R4b are each independently selected from hydrogen, C1-C6 alkoxy, halo, and C1-C6 alkyl; wherein one of R1a, R2a, R3a, and R4a, and one of R1b, R2b, R3b, and R4b represents a point of attachment to Linker; X1a and X1b are each independently selected from NH and S; A’ and A’’ are each independently a five- or six-membered cycloalkyl, heterocyclyl, heteroaryl, or aryl, each of which is optionally substituted with 1 or 2 oxo groups; W1a and W1b are each independently a bond or O; W2a and W2b are each independently selected from C1-C6 alkylene, C3-C6 cycloalkylene, C3-C6 cycloalkylene-C1-C6 alkylene, arylene, and hydroxy-C1-C6-alkylene; R5a and R5b are each independently selected from hydrogen, C1-C80 alkyl, C2-C80 alkenyl, C2-C80 alkynyl, amino, amino-C1-C6-alkyl, di-C1-C6-alkylamino-C1-C6-alkyl, heterocyclyl-C1-C6-alkyl, C1-C80 heteroalkyl, C2-C80 heteroalkenyl, and C2-C80 heteroalkynyl, wherein each of the C1-C80 alkyl, C2-C80 alkenyl, C2-C80 alkynyl, C1-C80 heteroalkyl, C2-C80 heteroalkenyl, and C2-C80 heteroalkynyl is optionally substituted with one or more substituents selected from hydroxy and amino.

[0018] In some embodiments, the compound is a compound of formula (IIa):(IIa) or a pharmaceutically acceptable salt thereof. In some embodiments, X2a and X2b are each independently CH or N, and X3a and X3b are each independently CH2 or CO.

[0019] In some embodiments, X2a and X2b are CH. In some embodiments, X2a and X2b are N.

[0020] In some embodiments, X3a and X3b are CH 3a 3b 2. In some embodiments, X and X are CO.

[0021] In some embodiments, X1a and X1b are S. In some embodiments, X1a and X1b are NH.

[0022] In some embodiments, W2a and W2b are each independently selected from C1-C4 alkylene, C4-C5 cycloalkylene, C4-C5 cycloalkylene-C1-C2 alkylene, phenylene, and hydroxy- C1-C3-alkylene. In some embodiments, W2a and W2b are each independently selected fromand

[0023] In some embodiments, R5a and R5b are each independently selected from hydrogen, C1-C40 alkyl, C2-C40 alkenyl, and C1-C4-alkylamino.

[0024] In some embodiments, R5a and R5b are each independently hydrogen or C1-C4 alkyl. In some embodiments, R5a and R5b are each independently hydrogen, methyl, ethyl, or isopropyl.In some embodiments, R5a and R5b are each independently hydrogen or methyl.

[0025] In some embodiments, R5a and R5b are each independently selected from C12-C40 alkyl and C12-C40 alkenyl.In some embodiments, R5a and R5b are each independently selected from C12-C20 alkyl and C12-C20 alkenyl. In some embodiments, R5a and R5b are each or.

[0026] In some embodiments, R1a and R1b are each independently halo or hydrogen. In some embodiments, R1a and R1b are each hydrogen.

[0027] In some embodiments, R2a and R2b are each the point of attachment to Linker.

[0028] In some embodiments, R3a and R3b are each independently C1-C6 alkoxy. In some embodiments, R3a and R3b are each methoxy.

[0029] In some embodiments, R4a and R4b are each hydrogen.

[0030] In some embodiments, Linker is a group -O-(CH2)m-O-, or a pharmaceutically acceptable salt thereof, wherein m is 1, 2, 3, 4, 5, or 6. In some embodiments, m is 3.

[0031] In some embodiments, the compound is selected from:.

[0032] In another aspect, disclosed herein are compositions. In some embodiments, the compositions comprise the disclosed compounds, or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions further comprise an albumin nanoparticle. In some embodiments, the compositions further comprise a liposome, a PLGA or PLA nanoparticle, a lipid nanoparticle, or a micelle.

[0033] In some embodiments, the composition further comprises at least one additional therapeutic agent. In some embodiments, the at least one additional therapeutic agent comprises an immune modulator, a chemotherapeutic agent, a nucleic acid, a decongestant, a steroid, an analgesic, an antimicrobial agent, or a combination thereof. In some embodiments, the at least one additional therapeutic agent comprises an RNA selected from the group consisting of a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), and mixtures thereof. In some embodiments, the at least one additional therapeutic agent is selected from a chemotherapeutic agent, an IDO inhibitor, a Stat3 inhibitor, a TLR agonist, and a PI3K inhibitor.

[0034] In some embodiments, the composition further comprises one or more cell targeting epitopes. In some embodiments, the one or more cell targeting epitopes are covalently attached or directly conjugated to an albumin. In some embodiments, the cell targeting epitopes comprise an immune cell epitope.

[0035] In some embodiments, the composition further comprises one or more epitopes from a microbiological agent.

[0036] In another aspect, disclosed herein are vaccines comprising a compound disclosed herein, or a pharmaceutically acceptable salt thereof, or a composition as disclosed herein and an antigen or a nucleic acid encoding thereof. In some embodiments, the antigen is a tumor antigen, a self-antigen, or an infectious disease derived antigen. In some embodiments, the nucleic acid is messenger RNA (mRNA).

[0037] In a further aspect, disclosed herein are methods for treating or preventing a disease or disorder in a subject (e.g., a human) comprising administering to the subject an effective amount of a compound disclosed herein, or a pharmaceutically acceptable salt thereof, or a composition or vaccine as disclosed herein. In some embodiments, the disease or disorder comprises cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.

[0038] In some embodiments, the disease or disorder is cancer. In some embodiments, the subject has cancer, has had cancer, is predisposed to cancer, or has a family history of cancer. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer is metastatic cancer. In some embodiments, the method suppresses or eliminates cancer metastasis, decreases tumor growth, prevents tumor recurrences, or any combination thereof.

[0039] In a further aspect, disclosed herein are methods for inducing or modulating an immune or inflammatory response in a subject (e.g., a human) comprising administering to the subject an effective amount of a compound disclosed herein, or a pharmaceutically acceptable salt thereof, or a composition or vaccine as disclosed herein.

[0040] In some embodiments, the method further comprises administering at least one additional therapeutic agent. In some embodiments, the at least one additional therapeutic agent comprises an immune modulator, a chemotherapeutic agent, a nucleic acid, a decongestant, a steroid, an analgesic, an antimicrobial agent, or a combination thereof.

[0041] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description and accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIGS.1A-1F show ZSA-51 and 52 activate both human and mouse STING (hSTING and mSTING, respectively). FIG.1A is a graph of binding affinity of the compounds with hSTING. FIG.1B is a graph of hSTING stimulation activity of the compounds in THP1 cells. The cells were treated with the compounds at indicated Q]\QS\b`ObW]\a T]` ,. V( <?=( +9 Wa O U`O^V ]T W\RcQSR ?<C'q aSQ`SbW]\ W\ H>E+ QSZZa( HVS cells were treated with the compounds at indicated concentrations for 24 h. FIG.1D is a graph of binding affinity of the compounds with mSTING. FIG.1E is a graph of mSTING stimulation activity of the compounds in THP1 cells. The cells were treated with the compounds at indicated concentrations for 24 h. FIG.1F is a graph of the activity of the compounds in THP1 (STING KO) cells. The cells were treated with the compounds at indicated concentrations for 24 h. (t test, **P<0.01)

[0043] FIGS.2A-2I show potent activity for ZSA-53 and ZSA-54 both in vitro and in vivo. FIG.2A is structures of ZSA-53, ZSA-54, ZSA-52NC1 and ZSA-52NC2. FIG.2B is a graph of hSTING stimulation activity of the compounds in THP1 cells. The cells were treated eWbV bVS Q][^]c\Ra Ob W\RWQObSR Q]\QS\b`ObW]\a T]` ,. V( <?=( ,9 Wa O U`O^V ]T W\RcQSR ?<C'q secretion in THP1 cells. The cells were treated with the compounds at indicated concentrations for 24 h. FIG.2D is a graph of mSTING stimulation activity of the compounds in THP1 cells. The cells were treated with the compounds at indicated concentrations for 24 h. FIGS.2E-2G show mice (n = 3) treated with ZSA-51 or ZSA-52 through PO or IV administration and plasma and tissues were collected at indicated time point and analyzed by LC-MS / MS. FIGS.2H-2I is the antitumor efficacy and effect on body weight of the compounds (PO, 60 mg / kg, Q5D) with or without anti-PD1 antibody (IP, Q5D, 0.1 mg / mouse) on MC38-bearing mice (n = 5). (t test, *P<0.05, **P<0.01)

[0044] FIG.3 is a graph of the cellular activity of MSA-2, ZSA-51, ZSA-52D, ZSA-51D and nano ZSA-51D with various concentrations in THP-1 blue ISG cells for 24 h by measuring interferon regulatory factor-inducible SEAP reporter.

[0045] FIGS.4A-4B are particle size distributions of STING agonist ZSA-52N (FIG.4A) and ZSA-51D (FIG. 4B)) loaded human serum albumin nanoparticles by dynamic light scattering (DLS).

[0046] FIGS.5A-5L show the tissue distribution and in vivo antitumor efficacy of ZSA-51 and ZSA-52. FIGS.5A-5C mice (n = 3) were treated with ZSA-51 or ZSA-52 through PO (10 mg / kg) or IV (5 mg / kg) administration and plasma and tissues were collected at indicated time point and analyzed by LC-MS / MS. FIGS.5D-5E show the antitumor efficacy and effecton body weight, respectively, of the compounds (PO, Q5D, 60 mg / kg) with or without anti- PD1 antibody (IP, Q5D, 0.1 mg / mouse) on MC38-bearing C57BL / 6 mice (n = 5). FIGS.5F- 5G show the antitumor efficacy and effect on body weight, respectively, of ZSA-51 (PO, Q4D, 100 mg / kg) with anti-PD1 antibody (IP, Q4D, 0.1 mg / mouse) on MC38-bearing C57BL / 6 mice (n = 5). FIGS.5H-5J show the antitumor efficacy and effect on body weight, respectively, of ZSA-51 (SC, Q4D, 50 mg / kg) with or without anti-PD1 antibody (IP, Q4D, 0.1 mg / mouse) on MC38-bearing C57BL / 6 mice (n = 5) with two tumors planted (the local and distal relative to compound-injected site). FIGS.5K-5L show the antitumor efficacy and effect on body weight, respectively, of ZSA-51 (PO, Q4D) with anti-PD1 antibody (IP, Q4D, 0.1 mg / mouse) on MC38-bearing B6.Cg-Ces1ctm1.1Loc / J mice (Es- / -, n = 5). (t test, *P<0.05, **P<0.01, ***P<0.001)

[0047] FIG.6 is the pharmacokinetics and biodistribution of SH-254 and SH-266 delivery orally or intravenously, as indicated.

[0048] FIG.7 is the STING agonist anticancer efficacy of SH-254 and SH-266 for MC-38 xenograft tumor mice model (top, local; middle, distal; bilateral, lower).

[0049] FIGS.8A-8D show ZSA-51 was effective against pancreatic cancer. FIGS.8A-8B show the antitumor efficacy and effect on body weight, respectively, of the compounds (PO, Q5D) with or without anti-PD1 antibody (IP, Q5D, 0.1 mg / mouse) on KPC-6422-bearing C57BL / 6 mice (n = 5). FIGS.8C-8D show the antitumor efficacy and effect on body weight, respectively, of the compounds (PO, Q4D) with anti-PD1 antibody (IP, Q4D, 0.1 mg / mouse) on KPC-6620-bearing C57BL / 6 mice (n = 5). (t test, **P<0.01, ***P<0.001)

[0050] FIGS.9A-9H show the binding mode investigation of ZSA-52. FIG.9A is images of a docking study of ZSA-52 (blue and yellow) with STING dimer (pink and green, PDB: 6ukz). Hydrogen bond was depicted as red dash line. FIG.9B is overlap of ZSA-52 (blue and yellow) and MSA-2 (cyan). FIG.9C is RMSD of STING protein backbone and ZSA-52. FIG. 9D is total hydrogen number calculation. FIG.9E shows the numbering of atoms involved in hydrogen bond formation. FIGS.9F-9H show the calculation of distance between hydrogen bond donor and acceptor.

[0051] FIG.10 shows human WT Sting binding for ZSA-51D and ZSA-52D.

[0052] FIGS.11A-11G show activation of BMDC in vitro for ZSA-51D and nanoformulated ZSA-51D. FIGS.11A-11D shows the activation analysis of MHC II (FIG. 11A), CD80 (FIG.11B), CD86 (FIG.11C), and CD80 CD86 double positive (FIG.11D) BMDC by flow cytometry after free and nano ZSA-51D treatments for 48 h. FIGS.11E and ++< aV]e Qgb]YW\S aSQ`SbW]\ OaaOga ]T HC<'n #<?=( ++;$ O\R ?<C'q #<?=( ++<$ Pg 8B:9after free and nano ZSA-51D treatments for 48 h. FIG.11G shows the calculated EC 50 of free and nano ZSA-51D on activation of MHC II, CD80, CD86 and CD80 CD86 double positive BMDC.

[0053] FIGS.12A-12G show the polarization of BMDM from M2 to M1 in vitro. FIGS. 12A-12D show the polarization analysis of MHC II (FIG.12A), CD80 (FIG.12B), CD86 (FIG.12C), and CD80 CD86 double positive (FIG.12D) BMDM by flow cytometry after free and nano ZSA-51D treatments for 48 h. FIGS.12E and 12F show cytokine secretion OaaOga ]T HC<'n #<?=( +,;$ O\R?<C'q #<?=( +,<$ Pg 8B:B OTbS` T`SS O\R \O\] LG7' / +: treatments for 48 h. FIG.12G shows the of free and nano ZSA-51D on activation of MHC II, CD80, CD86 and CD80 CD86 double positive BMDM.

[0054] FIGS.13A-13D show in vivo efficacy evaluation on bilateral MC-38 xenograft tumor mice model. FIGS.13A and 13B show near (FIG.13A) and distal (FIG.13B) tumor volume changes of MC-38 xenograft tumor model (n = 5) after different treatments. FIG.13C is a graph of the survival rate of MC-38 xenograft mice after different treatments (n = 5). FIG.13D is a graph of the cured MC-38 xenograft mice (n = 5) rechallenged with MC38 tumor cells at 120 d following the initial tumor inoculation on the opposite flank and tumor growth was assessed to compared with naive control mice (n = 5) given the same tumor challenge.

[0055] FIG.14 is a graph of the in vivo efficacy evaluation on KPC 6620 xenograft tumor mice model. DETAILED DESCRIPTION

[0056] Described herein are stimulator of interferon (IFN) genes (STING) agonists, and compositions and formulations thereof. The disclosed STING agonists are capable of being formulated for oral, intravenous, or subcutaneous administration or in lipid-based and albumin-based nanocarriers to enable delivery with currently available cancer and immune modulatory drugs as combination therapies as well as for use in vaccines and biomolecule delivery (e.g., nucleic acid delivery).

[0057] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting. 1. Definitions

[0058] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms“a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0059] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6- 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0060] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

[0061] As used herein, “treat,” “treating,” and the like means a slowing, stopping, or reversing of progression of a disease or disorder when provided a compound or composition described herein to an appropriate control subject. The term also means a reversing of the progression of such a disease or disorder to a point of eliminating or greatly reducing the symptoms. As such, “treating” means an application or administration of the compositions described herein to a subject, where the subject has a disease or a symptom of a disease, where the purpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease or symptoms of the disease.

[0062] A “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein. Likewise, patient may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., humans and non-humans) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice andguinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment, the mammal is a human.

[0063] As used herein, the terms “providing,” “administering,” and “introducing,” are used interchangeably herein and refer to the placement of the compounds or compositions of the disclosure into a subject by a method or route which results in at least partial localization of the compounds or composition to a desired site. The compounds or compositions can be administered by any appropriate route which results in delivery to a desired location in the subject.

[0064] The term “vaccine,” as used herein, refers to any pharmaceutical composition containing at least one antigenic or immunogenic peptide or other immunogen or at least one nucleic acid encoding at least one antigenic or immunogenic peptide or other immunogen, which can be used to prevent or treat a disease or condition in a subject.

[0065] The term “immunization,” as used herein, refers to a process that increases an organisms' reaction to antigen and therefore improves its ability to resist or overcome infection.

[0066] “Polynucleotide” or “oligonucleotide” or “nucleic acid,” as used herein, means at least two nucleotides covalently linked together. The polynucleotide may be DNA, both genomic and cDNA, RNA, or a hybrid, where the polynucleotide may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. The nucleic acid, whether DNA or RNA may comprise non-natural nucleotides, modified nucleotides, and / or non- nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”). Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods. Polynucleotides may be single- or double- stranded or may contain portions of both double stranded and single stranded sequence. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof.

[0067] A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies. The terms “polypeptide” and “protein,” are used interchangeably herein.

[0068] As used herein, “nucleic acid” or “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and / or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub.1982)). The present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. In some embodiments, a nucleic acid or nucleic acid sequence comprises other kinds of nucleic acid structures such as, for instance, a DNA / RNA helix, peptide nucleic acid (PNA), morpholino nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002)) and U.S. Pat. No.5,034,506), locked nucleic acid (LNA; see Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 97: 5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, J. Am. Chem. Soc., 122: 8595-8602 (2000)), and / or a ribozyme. Hence, the term “nucleic acid” or “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and / or non- nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”); further, the term “nucleic acid sequence” as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or double-stranded, and represent the sense or antisense strand. The terms “nucleic acid,” “polynucleotide,” “nucleotide sequence,” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.

[0069] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Sorrell, Organic Chemistry, 2nd edition, University Science Books, Sausalito, 2006; Smith, March's Advanced Organic Chemistry: Reactions, Mechanism, and Structure, 7th Edition, John Wiley & Sons, Inc., New York, 2013;Larock, Comprehensive Organic Transformations, 3rd Edition, John Wiley & Sons, Inc., New York, 2018; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0070] The term “alkyl,” as used herein, means a straight or branched, saturated hydrocarbon chain. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 4,4- dimethylpentan-2-yl, n-heptyl, n-octyl, n-nonyl, n-decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl and icosyl.

[0071] The term “alkylene,” as used herein, refers to a divalent group derived from a straight or branched, saturated hydrocarbon chain. Representative examples of alkylene include, but are not limited to, -CH2-, -CH2CH2-, -CH(CH3)-, -CH2CH2CH2-, -CH2CH(CH3)-, -CH2CH2CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -CH2CH2CH2CH2CH2-, - CH2CH(CH3)CH2CH2-, -CH(CH3)CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, - CH2CH2CH(CH3)CH2CH2-, -CH2CH(CH3)CH2CH2CH2-, and -CH(CH3)CH2CH2CH2CH2-.

[0072] The term “alkenyl,” as used herein, means a straight or branched hydrocarbon chain containing at least one carbon-carbon double bond. The double bond(s) may be located at any positions with the hydrocarbon chain. Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5- hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl.

[0073] The term “alkynyl,” as used herein, means a straight or branched hydrocarbon chain containing at least one carbon-carbon triple bond. The triple bond(s) may be located at any positions with the hydrocarbon chain. Representative examples of alkynyl include, but are not limited to, ethynyl, propynyl, and butynyl.

[0074] The term “alkoxy,” as used herein, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, and tert-butoxy.

[0075] The term “amino,” as used herein, refers to an -NH2 group. The term “alkylamino,” as used herein, refers to a group -NHR, wherein R is an alkyl group as defined herein. The term “dialkylamino,” as used herein, refers to a group -NR2, wherein each R is independently an alkyl group as defined herein.

[0076] As used herein, the term “aryl” refers to a radical of a monocyclic, bicyclic, or b`WQgQZWQ .\%, O`][ObWQ `W\U agabS[ #S(U(& VOdW\U 0& +*& ]` +. u SZSQb`]\a aVO`SR W\ O QgQZWQ array) having 6-14 ring carbon atoms and zero heteroatoms (“C6-C14 aryl”). In some embodiments, an aryl group has six ring carbon atoms (“C6 aryl,” i.e., phenyl). In some embodiments, an aryl group has ten ring carbon atoms (“C10 aryl,” e.g., naphthyl such as 1- naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (“C14 aryl,” e.g., anthracenyl and phenanthrenyl).

[0077] As used herein, the term “arylene” refers to a divalent aryl radical.

[0078] The term “cycloalkyl,” as used herein, refers to a saturated carbocyclic ring system containing three to ten carbon atoms and zero heteroatoms. The cycloalkyl may be monocyclic, bicyclic, bridged, fused, or spirocyclic. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, adamantyl, bicyclo[2.2.1]heptanyl, bicyclo[3.2.1]octanyl, and bicyclo[5.2.0]nonanyl.

[0079] The term “cycloalkylene,” as used herein, refers to a divalent cycloalkyl group.

[0080] The term “halogen” or “halo,” as used herein, means F, Cl, Br, or I.

[0081] The term “heteroalkyl,” as used herein, means an alkyl group, as defined herein, in which one or more of the carbon atoms (and any associated hydrogen atoms) are each independently replaced with a heteroatom group such as -NH-, -O-, -S-, -S(O)-, -S(O)2-, -O- P(O)(O-)O-, or the like. By way of example, 1, 2, 3, 4, 5, 6, or more carbon atoms may be independently replaced with the same or different heteroatom group. A heteroalkyl group can also include one or more carbonyl moieties (i.e., wherein a carbon atom of the alkyl group is oxidized to a -C(O)- group).

[0082] The term “heteroalkenyl,” as used herein, refers to an alkenyl group, as defined herein, in which one or more of the carbon atoms (and any associated hydrogen atoms) are each independently replaced with a heteroatom group such as -NH-, -O-, -S-, -S(O)-, -S(O)2-, or the like. By way of example, 1, 2, 3, 4, 5, 6, or more carbon atoms may be independently replaced with the same or different heteroatom group. A heteroalkenyl group can also include one or more carbonyl moieties (i.e., wherein a carbon atom of the alkyl group is oxidized to a -C(O)- group).

[0083] The term “heteroalkynyl,” as used herein, refers to an alkynyl group, as defined herein, in which one or more of the carbon atoms (and any associated hydrogen atoms) are each independently replaced with a heteroatom group such as -NH-, -O-, -S-, -S(O)-, -S(O)2-, or the like. By way of example, 1, 2, 3, 4, 5, 6, or more carbon atoms may be independentlyreplaced with the same or different heteroatom group. A heteroalkynyl group can also include one or more carbonyl moieties (i.e., wherein a carbon atom of the alkyl group is oxidized to a -C(O)- group).

[0084] As used herein, the term “heteroaryl” refers to a radical of a 5-10 membered []\]QgQZWQ ]` PWQgQZWQ .\%, O`][ObWQ `W\U agabS[ #S(U(& VOdW\U 0 ]` +* u SZSQb`]\a aVO`SR W\ a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (“5-10 membered heteroaryl”). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused (aryl / heteroaryl) ring system. Bicyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl). Exemplary 5-membered heteroaryl groups containing one heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, without limitation, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, without limitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6- bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.

[0085] As used herein, the term “heterocyclyl” refers to a radical of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“3-10 membered heterocyclyl”). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”), and can be saturated or can be partially unsaturated. Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more cycloalkyl groups wherein the point of attachment is either on the cycloalkyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. A heterocyclyl group may be described as, e.g., a 3-7-membered heterocyclyl, wherein the term “membered” refers to the non-hydrogen ring atoms, i.e., carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon, within the moiety. Exemplary 3-membered heterocyclyl groups containing one heteroatom include, without limitation, azirdinyl, oxiranyl, and thiorenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, without limitation, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, without limitation, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, without limitation, piperidinyl (e.g., 2,2,6,6-tetramethylpiperidinyl), tetrahydropyranyl, dihydropyridinyl, pyridinonyl (e.g., 1-methylpyridin-2-onyl), and thianyl. Exemplary 6- membered heterocyclyl groups containing two heteroatoms include, without limitation, piperazinyl, morpholinyl, pyridazinonyl (2-methylpyridazin-3-onyl), pyrimidinonyl (e.g., 1- methylpyrimidin-2-onyl, 3-methylpyrimidin-4-onyl), dithianyl, dioxanyl. Exemplary 6- membered heterocyclyl groups containing two heteroatoms include, without limitation, triazinanyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include,without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary 5-membered heterocyclyl groups fused to a C6 aryl ring (also referred to herein as a 5,6-bicyclic heterocyclyl ring) include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like. Exemplary 5-membered heterocyclyl groups fused to a heterocyclyl ring (also referred to herein as a 5,5-bicyclic heterocyclyl ring) include, without limitation, octahydropyrrolopyrrolyl (e.g., octahydropyrrolo[3,4-c]pyrrolyl), and the like. Exemplary 6- membered heterocyclyl groups fused to a heterocyclyl ring (also referred to as a 4,6- membered heterocyclyl ring) include, without limitation, diazaspirononanyl (e.g., 2,7- diazaspiro[3.5]nonanyl). Exemplary 6-membered heterocyclyl groups fused to an aryl ring (also referred to herein as a 6,6-bicyclic heterocyclyl ring) include, without limitation, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like. Exemplary 6-membered heterocyclyl groups fused to a cycloalkyl ring (also referred to herein as a 6,7-bicyclic heterocyclyl ring) include, without limitation, azabicyclooctanyl (e.g., (1,5)-8- azabicyclo[3.2.1]octanyl). Exemplary 6-membered heterocyclyl groups fused to a cycloalkyl ring (also referred to herein as a 6,8-bicyclic heterocyclyl ring) include, without limitation, azabicyclononanyl (e.g., 9-azabicyclo[3.3.1]nonanyl).

[0086] The term “hydroxy,” as used herein, refers to an -OH group.

[0087] The term “hydroxyalkyl,” as used herein, refers to an alkyl group, as defined herein, in which at least one hydrogen atom (e.g., one hydrogen atom) is replaced with a hydroxy group.

[0088] As used herein, the term “substituent” refers to a group substituted on an atom of the indicated group.

[0089] When a group or moiety can be substituted, the term “substituted” indicates that one or more (e.g., 1, 2, 3, 4, 5, or 6; in some embodiments 1, 2, or 3; and in other embodiments 1 or 2) hydrogen atoms on the group indicated in the expression using “substituted” can be replaced with a selection of recited indicated groups or with a suitable substituent group known to those of skill in the art (e.g., one or more of the groups recited below), provided that the designated atom’s normal valence is not exceeded. Substituent groups include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, acyl, amino, amido, amidino, aryl, azido, carbamoyl, carboxyl, carboxyl ester, cyano, cycloalkyl, cycloalkenyl, guanidino, halo, haloalkyl, haloalkoxy, heteroalkyl, heteroaryl, heterocyclyl, hydroxy,hydrazino, imino, oxo, nitro, phosphate, phosphonate, sulfonic acid, sulfonamido, thiol, thione, thioxo, or combinations thereof.

[0090] As used herein, in chemical structures the indication:represents a point of attachment of one moiety to another moiety.

[0091] In some instances, the number of carbon atoms in a hydrocarbyl substituent (e.g., alkyl alkenyl) is indicated by the prefix “Cx-Cy”, wherein x is the minimum and y is the maximum number of carbon atoms in the substituent. Thus, for example, “C1-C3 alkyl” refers to an alkyl substituent containing from 1 to 3 carbon atoms.

[0092] For compounds described herein, groups and substituents thereof may be selected in accordance with permitted valence of the atoms and the substituents, such that the selections and substitutions result in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

[0093] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. 2. Compounds

[0094] In one aspect, disclosed is a compound of formula (I):or a pharmaceutically acceptable salt thereof, wherein: R1, R2, R3, and R4 are each independently selected from hydrogen, C1-C6 alkoxy, halo, and C1-C6 alkyl; X1 is selected from S and NH; A is selected from a five- or six-membered cycloalkyl, heterocyclyl, heteroaryl, or aryl, each of which is optionally substituted with 1 or 2 oxo groups; W1 is a bond or O;W2 is selected from C1-C6 alkylene, C3-C6 cycloalkylene, C3-C6 cycloalkylene-C1-C6 alkylene, arylene, and hydroxy-C1-C6-alkylene; and R5 is selected from hydrogen, C1-C80 alkyl, C2-C80 alkenyl, C2-C80 alkynyl, amino, amino-C1-C6-alkyl, di-C1-C6-alkylamino-C1-C6-alkyl, heterocyclyl-C1-C6-alkyl, C1-C80 heteroalkyl, C2-C80 heteroalkenyl, and C2-C80 heteroalkynyl, wherein each of the C1-C80 alkyl, C2-C80 alkenyl, C2-C80 alkynyl, C1-C80 heteroalkyl, C2-C80 heteroalkenyl, and C2-C80 heteroalkynyl is optionally substituted with one or more substituents selected from hydroxy and amino.

[0095] In some embodiments, A is a 5- or 6-membered cycloalkyl. In some embodiments, A is a 5-membered cycloalkyl. In some embodiments, A is a 5- or 6-membered heterocyclyl having 1 or 2 nitrogen atoms. In some embodiments, A is a 5-membered heterocyclyl having 1 nitrogen atom. In some embodiments, A is a 6-membered heterocyclyl having 1 or 2 nitrogen atoms. In some embodiments, A is a 5- or 6-membered heteroaryl having 1 or 2 nitrogen atoms. In some embodiments, A is a 6-membered heteroaryl having 2 nitrogen atoms. In some embodiments, A is substituted with 1 oxo group. In some embodiments, A is substituted with 2 oxo groups.

[0096] In some embodiments, W1 is a bond. In some embodiments, W1 is O.

[0097] In some embodiments, the compound of formula (I) is a compound of formula (Ia):( ) wherein X2 is CH or N, and X3 is CH2 or CO. In some embodiments, X2 is CH. In some embodiments, X2 is N. In some embodiments, X3 is CH2. In some embodiments, X3 is CO. In some embodiments, X2 is CH and X3 is CH2. In some embodiments, X2 is N and X3 is CH2. In some embodiments, X2 is N and X3 is CO.

[0098] In some embodiments, the compound of formula (I) is a compound of formula (Ib):wherein X4 and X5 are independently selected from N and CH. In some embodiments, X4 is N. In some embodiments, X5 is N. In some embodiments, X5 is CH. In some embodiments, X4 and X5 are N. In some embodiments, X4 is N and X5 is CH.

[0099] In some embodiments, the compound of formula (I) is a compound of formula (Ic):wherein X6 and X7 are independently selected from CH and N. In some embodiments, X6 is N. In some embodiments, X7 is N. In some embodiments, X6 and X7 are N.

[0100] In some embodiments, X1 is S. In some embodiments, X1 is NH.

[0101] In some embodiments, W2 is selected from C1-C4 alkylene, C4-C5 cycloalkylene, C4-C5 cycloalkylene-C1-C2 alkylene, phenylene, and hydroxy-C1-C3-alkylene. In some embodiments, W2 is selected from C1-C4 alkylene, C4-C5 cycloalkylene-C1-C2 alkylene, and hydroxy-C1-C3-alkylene. In some embodiments, W2 is selected from: , and. In someembodiments, W2 is.

[0102] In some embodiments, R1, R2, R3, and R4 are each independently selected from hydrogen, C1-C6 alkoxy, and halo. In some embodiments, R1, R2, R3, and R4 are each independently selected from hydrogen, methoxy, chloro, and bromo.

[0103] In some embodiments, R2 and R3 are each independently C1-C6 alkoxy. In some embodiments, R2 and R3 are each methoxy.

[0104] In some embodiments, R1 and R4 are each independently halo or hydrogen. In some embodiments, R1 and R4 are each independently chloro, bromo, or hydrogen. In some embodiments, R1 is halo and R4 is hydrogen. In some embodiments, R1 and R4 are each hydrogen. In some embodiments, R1 is chloro or bromo, and R4 is hydrogen.

[0105] In some embodiments, R1 and R4 are each hydrogen, and R2 and R3 are each independently C1-C6 alkoxy. In some embodiments, R1 and R4 are each hydrogen, and R2 and R3 are each methoxy.

[0106] In some embodiments, R1 is halo, R2 and R3 are each independently C1-C6 alkoxy, and R4 is hydrogen. In some embodiments, R1 is chloro or bromo, R2 and R3 are each methoxy, and R4 is hydrogen.

[0107] In some embodiments, R5 is selected from hydrogen, C1-C80 alkyl, C2-C80 alkenyl, C2-C80 alkynyl, amino, amino-C1-C6-alkyl, di-C1-C6-alkylamino-C1-C6-alkyl, heterocyclyl- C1-C6-alkyl, C1-C80 heteroalkyl, C2-C80 heteroalkenyl, and C2-C80 heteroalkynyl, wherein each of the C1-C80 alkyl, C2-C80 alkenyl, C2-C80 alkynyl, C1-C80 heteroalkyl, C2-C80 heteroalkenyl, and C2-C80 heteroalkynyl is optionally substituted with one or more substituents selected from hydroxy and amino. In some embodiments, R5 is selected from hydrogen, C1-C40 alkyl, C2-C40 alkenyl, C1-C4-alkylamino, di-C1-C6-alkylamino-C1-C6-alkyl, and heterocyclyl-C1-C6-alkyl. In some embodiments, R5 is hydrogen or C1-C4 alkyl. In some embodiments, R5 is hydrogen, methyl, ethyl, isopropyl, or tert-butyl. In some embodiments, R5 is hydrogen, methyl, ethyl, or isopropyl. In some embodiments, R5 is hydrogen or methyl.

[0108] In some embodiments, R5 is selected from C1-C80 alkyl, C2-C80 alkenyl, C2-C80 alkynyl, C1-C80 heteroalkyl, C2-C80 heteroalkenyl, and C2-C80 heteroalkynyl, each of which is optionally substituted with one or more substituents selected from hydroxy and amino. In some embodiments, R5 is selected from C4-C80 alkyl, C4-C80 alkenyl, C4-C80 alkynyl, C4-C80 heteroalkyl, C4-C80 heteroalkenyl, and C4-C80 heteroalkynyl, each of which is optionally substituted with one or more substituents selected from hydroxy and amino. In some embodiments, R5 is selected from C8-C80 alkyl, C8-C80 alkenyl, C8-C80 alkynyl, C8-C80 heteroalkyl, C8-C80 heteroalkenyl, and C8-C80 heteroalkynyl, each of which is optionally substituted with one or more substituents selected from hydroxy and amino. In some embodiments, R5 is selected from C8-C40 alkyl, C8-C40 alkenyl, C8-C40 alkynyl, C8-C40 heteroalkyl, C8-C40 heteroalkenyl, and C8-C40 heteroalkynyl, each of which is optionally substituted with one or more substituents selected from hydroxy and amino.

[0109] For example, in some embodiments, R5 is selected from C8-C80 alkyl and C8-C80 alkenyl. In some embodiments, R5 is selected from C8-C40 alkyl and C8-C40 alkenyl. In some embodiments, R5 is selected from C12-C40 alkyl and C12-C40 alkenyl. In some embodiments, R5 is selected from C12-C20 alkyl and C12-C20 alkenyl. In some such embodiments, R5 is derived from a saturated or unsaturated fatty alcohol. In such embodiments, the O attached to R5 in formula (I) is derived from the oxygen of the alcohol. In some embodiments, R5 is derived from linoleyl alcohol ((9Z,12Z)-octadeca-9,12-dien-1-ol), myristyl alcohol (1- tetradecanol), palmitoleyl alcohol ((Z)-hexadec-9-en-1-ol), oleyl alcohol ((Z)-octadec-9-en-1- ol), elaidyl alcohol (trans-9-octadecenol), cis-vaccenyl alcohol (cis-11-octadecenol), gadoleylalcohol ((Z)-icos-9-en-1-ol), 11-eicosenol, erucyl alcohol (cis-13-docosenol), 15-tetracosen- 1-ol, eicosadienyl alcohol (icosa-11,14-dien-1-ol), linolenyl alcohol ((9Z,12Z,15Z)-9,12,15- ]QbORSQOb`WS\'+']Z$& r'ZW\]ZS\gZ OZQ]V]Z ##0;&3;&+,;$']QbORSQO'0&3&+,'b`WS\'+']Z$& SZS]abSO`gZ alcohol (octadeca-9,11,13-trien-1-ol), icosa-5,8,11-trien-1-ol, eicos-13-en-1-ol, icosa-11,14- 17-trien-1-ol, octadeca-6,9,12,15-tetraen-1-ol, arachidonyl alcohol ((5Z,8Z,11Z,14Z)- icosatetraen-1-ol), 4E,6Z-hexadecadien-1-ol, icosa-5,8,11,14,17-pentaen-1-ol, docosahexaenoyl alcohol (docosa-4,7,10,13,16,19-hexaen-1-ol), docosa-7,10,13,16,19- pentaen-1-ol, tetracosa-6,9,12,15,18,21-hexaen-1-ol, capryl alcohol (1-octanol), pelargonic alcohol (1-nonanol), decyl alcohol (1-decanol), undecyl alcohol (1-undecanol), lauryl alcohol (1-dodecanol), tridecyl alcohol (1-tridecanol), myristyl alcohol (1-tetradecanol), pentadecyl alcohol (1-pentadecanol), cetyl alcohol (1-hexadecanol), palmitoleyl alcohol (cis-9- hexadecen-1-ol), heptadecylalcohol (1-n-heptadecanol), stearyl alcohol (1-octadecanol), oleyl alcohol (1-octadecenol), nonadecyl alcohol (1-nonadecanol), arachidyl alcohol (1-eicosanol), heneicosyl alcohol (1-heneicosanol), behenyl alcohol (1-docosanol), erucyl alcohol (cis-13- docosen-1-ol), 1-tricosanol, lignoceryl alcohol (1-tetracosanol), pentacosylic alcohol (1- pentacosanol), ceryl alcohol (1-hexacosanol), 1-heptacosanol, montanyl alcohol (1- octacosanol), 1-nonacosanol, myricyl alcohol (1-triacontanol), 1-hentriacontanol, 1- dotriacontanol (lacceryl alcohol), 1-tritriacontanol, geddyl alcohol (1-tetratriacontanol), 1- hexatriacontanol, 1-heptatriacontanol, 1-octatriacontanol, nonatriacontan-1-ol, or 1- tetracontanol.

[0110] In some embodiments, R5 is selected from C8-C80 heteroalkyl, C8-C80 heteroalkenyl, and C8-C80 heteroalkynyl. In such embodiments, R5 can be derived from a lipid including one or more heteroatom groups, such as -O-, -NH-, -C(O)-, or the like, or combinations thereof (e.g., -C(O)O- groups).

[0111] For example, in some embodiments, R5 has a formula (A):wherein: n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40; and Ra and Rb are each independently selected from C6-C40 alkyl, C6-C40 alkenyl, C6-C40 heteroalkyl, and C6-C40 heteroalkenyl.

[0112] For example, in some embodiments, n is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, n is 2, 3, or 4. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4.

[0113] In some embodiments, Ra and Rb are each independently selected from C6-C40 alkyl and C6-C40 alkenyl. For example, in some embodiments, Ra and Rb are each independently selected from n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl, henicosyl, docosyl, tricosyl, tetracosyl, pentacosyl, hexacosyl, heptacosyl, octacosyl, nonacosyl, triacontyl, hentriacontyl, dotriacontyl, tritriacontyl, tetratriacontyl, pentatriacontyl, hexatriacontyl, heptatriacontyl, octatriacontyl, nonatriacontyl, tetracontyl, linoleyl ((9Z,12Z)- octadeca-9,12-dien-1-yl), palmitoleyl ((Z)-hexadec-9-en-1-yl), oleyl ((Z)-octadec-9-en-1-yl), elaidyl (trans-9-octadecenyl), cis-vaccenyl (cis-11-octadecenyl), gadoleyl ((Z)-icos-9-en-1- yl), 11-eicosenyl, erucyl (cis-13-docosenyl), 15-tetracosen-1-yl, eicosadienyl (icosa-11,14- RWS\'+'gZ$& ZW\]ZS\gZ ##3L&+,L&+ / L$'3&+,&+ / ']QbORSQOb`WS\'+'gZ$& r'ZW\]ZS\gZ ##0;&3;&+,;$' octadeca-6,9,12-trien-1-yl), eleostearyl (octadeca-9,11,13-trien-1-yl), icosa-5,8,11-trien-1-yl, eicos-13-en-1-yl, icosa-11,14-17-trien-1-yl, octadeca-6,9,12,15-tetraen-1-yl, arachidonyl ((5Z,8Z,11Z,14Z)-icosatetraen-1-yl), 4E,6Z-hexadecadien-1-yl, icosa-5,8,11,14,17-pentaen- 1-yl, docosahexaenoyl (docosa-4,7,10,13,16,19-hexaen-1-yl), docosa-7,10,13,16,19-pentaen- 1-yl, and tetracosa-6,9,12,15,18,21-hexaen-1-yl. In some embodiments, Ra and Rb are each linoleyl.

[0114] In some embodiments, R5 has a formula (B) or (C): wherein:n and p are each independently 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40; Ra1 is selected from C6-C40 alkyl and C6-C40 alkenyl; and Rb is selected from C6-C40 alkyl, C6-C40 alkenyl, C6-C40 heteroalkyl, and C6-C40 heteroalkenyl.

[0115] In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, n is 2, 3, or 4. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4.

[0116] In some embodiments, p is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, p is 3, 4, 5, 6, or 7. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5. In some embodiments, p is 6. In some embodiments, p is 7.

[0117] In some embodiments, Ra1 is C6-C24 alkyl or C6-C24 alkenyl. In some embodiments, Ra1 is selected from C9-C22 alkyl and C9-C22 alkenyl. In some embodiments, Ra1 is selected from straight or branched C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, or C24 alkyl. In some embodiments, Ra1 is selected from straight or branched C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, or C24 alkenyl. In some embodiments, Ra1 is selected from linoleyl, n-nonyl, n-undecyl, henicosan-11-yl, pentadecane-7-yl, and heptadecan-9-yl.

[0118] In some embodiments, Rb is selected from C6-C40 alkyl and C6-C40 alkenyl. For example, in some embodiments, Rb is selected from n-hexyl, n-heptyl, n-octyl, n-nonyl, n- decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl, henicosyl, docosyl, tricosyl, tetracosyl, pentacosyl, hexacosyl, heptacosyl, octacosyl, nonacosyl, triacontyl, hentriacontyl, dotriacontyl, tritriacontyl, tetratriacontyl, pentatriacontyl, hexatriacontyl, heptatriacontyl, octatriacontyl, nonatriacontyl, tetracontyl, linoleyl ((9Z,12Z)-octadeca-9,12-dien-1-yl), palmitoleyl ((Z)-hexadec-9-en-1-yl), oleyl ((Z)- octadec-9-en-1-yl), elaidyl (trans-9-octadecenyl), cis-vaccenyl (cis-11-octadecenyl), gadoleyl ((Z)-icos-9-en-1-yl), 11-eicosenyl, erucyl (cis-13-docosenyl), 15-tetracosen-1-yl, SWQ]aORWS\gZ #WQ]aO'++&+.'RWS\'+'gZ$& ZW\]ZS\gZ ##3L&+,L&+ / L$'3&+,&+ / ']QbORSQOb`WS\'+'gZ$& r' linolenyl ((6E,9E,12E)-octadeca-6,9,12-trien-1-yl), eleostearyl (octadeca-9,11,13-trien-1-yl), icosa-5,8,11-trien-1-yl, eicos-13-en-1-yl, icosa-11,14-17-trien-1-yl, octadeca-6,9,12,15- tetraen-1-yl, arachidonyl ((5Z,8Z,11Z,14Z)-icosatetraen-1-yl), 4E,6Z-hexadecadien-1-yl, icosa-5,8,11,14,17-pentaen-1-yl, docosahexaenoyl (docosa-4,7,10,13,16,19-hexaen-1-yl), docosa-7,10,13,16,19-pentaen-1-yl, and tetracosa-6,9,12,15,18,21-hexaen-1-yl. In some embodiments, Rb is linoleyl.

[0119] In some embodiments, R5 has a formula (D), (E), or (F):wherein:n, p, and q are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40; and Ra1 and Ra2 are each independently selected from C6-C40 alkyl and C6-C40 alkenyl.

[0120] In some embodiments, R5 has formula (D). In some embodiments, R5 has formula (E). In some embodiments, R5 has formula (F). In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, n is 2, 3, or 4. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4.

[0121] In some embodiments, p and q are each independently selected from 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, p and q are each independently selected from 3, 4, 5, 6, and 7. In some embodiments, p and q are each independently selected from 3, 5, and 7. In some embodiments, p and q are each 5. In some embodiments, p and q are each 6. In some embodiments, p and q are each 7.

[0122] In some embodiments, Ra1 and Ra2 are each independently selected from C6-C24 alkyl and C6-C24 alkenyl. In some embodiments, Ra1 and Ra2 are each independently selected from C9-C22 alkyl and C9-C22 alkenyl. In some embodiments, Ra1 and Ra2 are each independently selected from straight or branched C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, or C24 alkyl. In some embodiments, Ra1 and Ra2 are each independently selected from straight or branched C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, or C24 alkenyl. In some embodiments, Ra1 and Ra2 are each independently selected from linoleyl, n-nonyl, n-undecyl, henicosan-11-yl, pentadecane-7-yl, and heptadecan-9-yl.

[0123] In some embodiments, R5 is selected from:

[0124] In some embodiments, R5 is or

[0125] In some embodiments, the compound of formula (I) is selected from:

[0126] In another aspect, disclosed is a compound of formula (II):or a pharmaceutically acceptable salt thereof, wherein:R1a, R1b, R2a, R2b, R3a, R3b, R4a, and R4b are each independently selected from hydrogen, C1-C6 alkoxy, halo, and C1-C6 alkyl; wherein one of R1a, R2a, R3a, and R4a, and one of R1b, R2b, R3b, and R4b represents a point of attachment to Linker; X1a and X1b are each independently selected from NH and S; A’ and A’’ are each independently selected from five- or six-membered cycloalkyl, heteroaryl, and heterocyclyl, each of which is optionally substituted with 1 or 2 oxo groups; W1a and W1b are each independently a bond or O; W2a and W2b are each independently selected from C1-C6 alkylene, C3-C6 cycloalkylene, C3-C6 cycloalkylene-C1-C6 alkylene, arylene, and hydroxy-C1-C6-alkylene; R5a and R5b are each independently selected from hydrogen, C1-C80 alkyl, C2-C80 alkenyl, C2-C80 alkynyl, amino, amino-C1-C6-alkyl, di-C1-C6-alkylamino-C1-C6-alkyl, heterocyclyl-C1-C6-alkyl, C1-C80 heteroalkyl, C2-C80 heteroalkenyl, and C2-C80 heteroalkynyl, wherein each of the C1-C80 alkyl, C2-C80 alkenyl, C2-C80 alkynyl, C1-C80 heteroalkyl, C2-C80 heteroalkenyl, and C2-C80 heteroalkynyl is optionally substituted with one or more substituents selected from hydroxy and amino.

[0127] In some embodiments, W1a and W1b are each a bond. In some embodiments, W1a and W1b are each O.

[0128] In some embodiments, the compound of formula (I) is a compound of formula (Ia):wherein X2a and X2b are each independently CH or N, and X3a and X3b are each independently CH2 or CO. In some embodiments, X2a and X2b are each CH. In some embodiments, X2a and X2b are each N. In some embodiments, X3a and X3b are each CH2. In some embodiments, X3a and X3b are each CO. In some embodiments, X2a and X2b are each CH and X3a and X3b are each CH2. In some embodiments, X2a and X2b are each N, and X3a and X3b are each CH2. In some embodiments, X2a and X2b are each N, and X3a and X3b are each CO.

[0129] In some embodiments, X1a and X1b are S. In some embodiments, X1a and X1b are NH.

[0130] In some embodiments, W2a and W2b are each independently selected from C1-C4 alkylene, C4-C5 cycloalkylene, C4-C5 cycloalkylene-C1-C2 alkylene, phenylene, and hydroxy- C1-C3-alkylene. In some embodiments, W2a and W2b are each independently selected from C1-C4 alkylene, C4-C5 cycloalkylene-C1-C2 alkylene, and hydroxy-C1-C3-alkylene. In some embodiments, W2a and W2b are each independently selected from: , and. In someembodiments, W2a and W2b are each .

[0131] In some embodiments, R1a, R1b, R2a, R2b, R3a, R3b, R4a, and R4b are each independently selected from hydrogen, C1-C6 alkoxy, and halo, wherein one of R1a, R2a, R3a, and R4a, and one of R1b, R2b, R3b, and R4b represents a point of attachment to Linker. In some embodiments, R1a, R1b, R2a, R2b, R3a, R3b, R4a, and R4b are each independently selected from hydrogen, methoxy, chloro, and bromo, wherein one of R1a, R2a, R3a, and R4a, and one of R1b, R2b, R3b, and R4b represents a point of attachment to Linker.

[0132] In some embodiments, R1a and R1b are each independently halo or hydrogen. In some embodiments, R1a and R1b are each hydrogen. In some embodiments, R4a and R4b are each hydrogen.

[0133] In some embodiments, R1a, R1b, R4a, and R4b are each independently halo or hydrogen. In some embodiments, R1a, R1b, R4a, and R4b are each independently chloro, bromo, or hydrogen. In some embodiments, R1a and R1b are each halo and R4a and R4b are each hydrogen. In some embodiments, R1a, R1b, R4a, and R4b are each hydrogen. In some embodiments, R1a and R1b are each chloro or bromo, and R4a and R4b are each hydrogen.

[0134] In some embodiments, R2a, R2b, R3a, and R3b are each independently C1-C6 alkoxy. In some embodiments, R2a, R2b, R3a, and R3b each methoxy. In some embodiments, R2a and R2b are each the point of attachment to Linker. In some embodiments, R3a and R3b are each independently C1-C6 alkoxy. In some embodiments, R3a and R3b are each methoxy.

[0135] In some embodiments, R1a and R1b are each independently halo or hydrogen, R2a and R2b are each the point of attachment to Linker, R3a and R3b are each independently C1-C6 alkoxy, and R4a and R4b are each hydrogen. In some embodiments, R1a and R1b are each independently hydrogen, R2a and R2b are each the point of attachment to Linker, R3a and R3b are each methoxy, and R4a and R4b are each hydrogen.

[0136] The compounds of formula (II) include a linker. In some embodiments, the linker is about 5 Å to 1000 Å in length. In some embodiments, the linker is about 5 Å, 10 Å, 20 Å, 50 Å, 100 Å, 150 Å, 200 Å, 300 Å, 400 Å, 500 Å, 600 Å, 700 Å, 800 Å, 900 Å, or 1000 Å in length, or any suitable range therebetween (e.g., 5-100 Å, 50-500 Å, 150-700 Å, etc.). In some embodiments, the linker comprises about 1-200 atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, or any suitable ranges therebetween (e.g., 2-20, 10-50, etc.)).

[0137] The linker can include one or more groups independently selected from methylene (-CH2-), ether (-O-), amine (-NH-), alkylamine (-NR-, wherein R is an optionally substituted C1-C6 alkyl group), thioether (-S-), disulfide (-S-S-), amide (-C(O)NH-), ester (-C(O)O-), carbamate (-OC(O)NH-), urea (-NHC(O)NH-), and sulfonamide (-S(O)2NH-), and any combination thereof.

[0138] In some embodiments, the linker comprises one or more -(CH2CH2O)- (oxyethylene) groups, e.g., 1-20 -(CH2CH2O)- groups (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 -(CH2CH2O)- groups, or any range therebetween). In some embodiments, the linker comprises a -(CH2CH2O)-, -(CH2CH2O)2-, -(CH2CH2O)3-, - (CH2CH2O)4-, -(CH2CH2O)5-, -(CH2CH2O)6-, -(CH2CH2O)7-, -(CH2CH2O)8-, -(CH2CH2O)9-, or -(CH2CH2O)10- group. In some embodiments, the linker comprises one or more - (CH2CH2CH2O)- (oxypropylene) groups, e.g., 1-20 -(CH2CH2CH2O)- groups (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 -(CH2CH2CH2O)- groups, or any range therebetween). In some embodiments, the linker comprises a -(CH2CH2CH2O)-, - (CH2CH2CH2O)2-, -(CH2CH2CH2O)3-, -(CH2CH2CH2O)4-, -(CH2CH2CH2O)5-, - (CH2CH2CH2O)6-, -(CH2CH2CH2O)7-, -(CH2CH2CH2O)8-, -(CH2CH2CH2O)9-, or - (CH2CH2CH2O)10- group.

[0139] In some embodiments, the linker is a group is a group -O-(CH2)m-O-, wherein m is 1, 2, 3, 4, 5, or 6. In some embodiments, m is 2, 3, or 4. In some embodiments, m is 3.

[0140] In some embodiments, the linker is selected from -OCH2CH2CH2O-, - OCH2CH2CH2-, -CH2CH2CH2-, -OCH2CH2O-, and -OCH2CH2-.

[0141] In some embodiments, the linker comprises one or more alkylene groups (e.g., - (CH2)n-, wherein n is 1-12, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or any suitable range therebetween). In some embodiments, the linker comprises one or more branched alkylene groups.

[0142] In some embodiments, the linker comprises a cleavable (e.g., enzymatically cleavable, chemically cleavable, etc.) moiety.

[0143] In some embodiments, the linker comprises one or more substituents, pendants, side chains, etc., comprising any suitable organic functional groups (e.g., -OH, -NH2, -SH, - CN, =O, =S, halogen (e.g., -F, -Cl, -Br, -I), -COOH, -CONH2, -CH3, etc.).

[0144] The groups R5a and R5b are selected from any of the groups described above for group R5 of formula (I). For example, in some embodiments, R5a and R5b are each independently selected from hydrogen and C1-C4 alkyl. In some embodiments, R5a and R5b are each independently selected from hydrogen, methyl, ethyl, isopropyl, and tert-butyl. In some embodiments, R5a and R5b are each independently selected from C1-C80 alkyl, C2-C80 alkenyl, C2-C80 alkynyl, C1-C80 heteroalkyl, C2-C80 heteroalkenyl, and C2-C80 heteroalkynyl, each of which is optionally substituted with one or more substituents selected from hydroxy and amino, e.g., any of the specific groups described above for R5.

[0145] In some embodiments, the compound of formula (II) is selected from:and, and pharmaceutically acceptable salts thereof.

[0146] The compounds may exist as a stereoisomer wherein asymmetric or chiral centers are present. The stereoisomer is “R” or “S” depending on the configuration of substituents around the chiral carbon atom. The terms “R” and “S” used herein are configurations as defined in IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry, in Pure Appl. Chem., 1976, 45: 13-30. The disclosure contemplates various stereoisomers and mixtures thereof and these are specifically included within the scope of this disclosure. Stereoisomers include enantiomers and diastereomers, and mixtures of enantiomers or diastereomers. Individual stereoisomers of the compounds may be prepared syntheticallyfrom commercially available starting materials, which contain asymmetric or chiral centers or by preparation of racemic mixtures followed by methods of resolution well-known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography and optional liberation of the optically pure product from the auxiliary as described in Furniss, Hannaford, Smith, and Tatchell, “Vogel's Textbook of Practical Organic Chemistry,” 5th edition (1989), Longman Scientific & Technical, Essex CM202JE, England (or more recent versions thereof), or (2) direct separation of the mixture of optical enantiomers on chiral chromatographic columns, or (3) fractional recrystallization methods.

[0147] It should be understood that the compounds may possess tautomeric forms, as well as geometric isomers, and that these also constitute embodiments of the disclosure.

[0148] The present disclosure also includes isotopically-labeled compounds, which is identical to those recited in formula (I), but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes include those for hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as, but not limited to 2H, 3H, 13C, 14C, 15N, 18O, 17O, 31P, 32P , 35S, 18F, and 36Cl, respectively. Substitution with heavier isotopes such as deuterium, for example, 2H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. The compound may incorporate positron-emitting isotopes for medical imaging and positron-emitting tomography (PET) studies for determining the distribution of receptors. Suitable positron- emitting isotopes that can be incorporated in compounds of formula (I) are 11C, 13N, 15O, and 18F. Isotopically-labeled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying examples using appropriate isotopically-labeled reagent in place of non- isotopically-labeled reagent.

[0149] The disclosed compounds may exist as pharmaceutically acceptable salts. The term “pharmaceutically acceptable salt” refers to salts or zwitterions of the compounds which are water or oil-soluble or dispersible, suitable for treatment of disorders without undue toxicity, irritation, and allergic response, commensurate with a reasonable benefit / risk ratio and effective for their intended use. The salts may be prepared during the final isolation and purification of the compounds or separately by reacting an amino group of the compoundswith a suitable acid. For example, a compound may be dissolved in a suitable solvent, such as but not limited to methanol and water and treated with at least one equivalent of an acid, like hydrochloric acid. The resulting salt may precipitate out and be isolated by filtration and dried under reduced pressure. Alternatively, the solvent and excess acid may be removed under reduced pressure to provide a salt. Representative salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, isethionate, fumarate, lactate, maleate, methanesulfonate, naphthylenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, oxalate, maleate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, glutamate, para-toluenesulfonate, undecanoate, hydrochloric, hydrobromic, sulfuric, phosphoric and the like. The amino groups of the compounds may also be quaternized with alkyl chlorides, bromides and iodides such as methyl, ethyl, propyl, isopropyl, butyl, lauryl, myristyl, stearyl and the like.

[0150] Basic addition salts may be prepared during the final isolation and purification of the disclosed compounds by reaction of a carboxyl group with a suitable base such as the hydroxide, carbonate, or bicarbonate of a metal cation such as lithium, sodium, potassium, calcium, magnesium, or aluminum, or an organic primary, secondary, or tertiary amine. Quaternary amine salts can be prepared, such as those derived from methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine O\R C&Cp'RWPS\hgZSbVgZS\SRWO[W\S& SbVgZS\SRWO[W\S& SbVO\]ZO[W\S& RWSbVO\]ZO[W\S& piperidine, piperazine, and the like.

[0151] Compounds may be synthesized according to a variety of methods, including those illustrated in the Examples. Reaction conditions and reaction times for each individual step can vary depending on the particular reactants employed and substituents present in the reactants used. Specific procedures are provided in the Examples section. Reactions can be worked up in the conventional manner, e.g., by eliminating the solvent from the residue and further purified according to methodologies generally known in the art such as, but not limited to, crystallization, distillation, extraction, trituration, and chromatography. Unless otherwise described, the starting materials and reagents are either commercially available or can be prepared by one skilled in the art from commercially available materials using methods described in the chemical literature. Starting materials, if not commerciallyavailable, can be prepared by procedures selected from standard organic chemical techniques, techniques that are analogous to the synthesis of known, structurally similar compounds, or techniques that are analogous to the above described schemes or the procedures described in the synthetic examples section.

[0152] Routine experimentations, including appropriate manipulation of the reaction conditions, reagents and sequence of the synthetic route, protection of any chemical functionality that cannot be compatible with the reaction conditions, and deprotection at a suitable point in the reaction sequence of the method are included in the scope of the disclosure. Suitable protecting groups and the methods for protecting and deprotecting different substituents using such suitable protecting groups are well known to those skilled in the art; examples of which can be found in PGM Wuts and TW Greene, in Greene's book titled Protective Groups in Organic Synthesis (4th ed.), John Wiley & Sons, NY (2006), which is incorporated herein by reference in its entirety. Synthesis of the compounds of the disclosure can be accomplished by methods analogous to those described in the synthetic schemes described hereinabove and in specific examples.

[0153] When an optically active form of a disclosed compound is required, it can be obtained by carrying out one of the procedures described herein using an optically active starting material (prepared, for example, by asymmetric induction of a suitable reaction step), or by resolution of a mixture of the stereoisomers of the compound or intermediates using a standard procedure (such as chromatographic separation, recrystallization, or enzymatic resolution).

[0154] Similarly, when a pure geometric isomer of a compound is required, it can be obtained by carrying out one of the above procedures using a pure geometric isomer as a starting material, or by resolution of a mixture of the geometric isomers of the compound or intermediates using a standard procedure such as chromatographic separation.

[0155] It can be appreciated that the synthetic schemes and specific examples as described are illustrative and are not to be read as limiting the scope of the disclosure as it is defined in the appended claims. All alternatives, modifications, and equivalents of the synthetic methods and specific examples are included within the scope of the claims. 3. Compositions

[0156] The disclosed compounds may be incorporated into compositions that may be suitable for administration to a subject (such as a patient, which may be a human or non- human).a. Pharmaceutical Compositions

[0157] The disclosed compounds may be incorporated into pharmaceutically acceptable compositions. The pharmaceutical compositions may include a “therapeutically effective amount” or a “prophylactically effective amount” of the compound(s). A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the composition may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of a compound of the invention (e.g., a compound of formula (I)) are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0158] The pharmaceutical compositions and formulations may include pharmaceutically acceptable carriers. The term “pharmaceutically acceptable carrier,” as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material, surfactant, cyclodextrins or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as, but not limited to, lactose, glucose and sucrose; starches such as, but not limited to, corn starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as, but not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; surfactants such as, but not limited to, cremophor EL, cremophor RH 60, Solutol HS 15 and polysorbate 80; cyclodextrins such as, but not limited to, alpha-CD, beta-CD, gamma-CD, HP-beta-CD, SBE-beta-CD; glycols; such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non- toxic compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

[0159] The route by which the disclosed compounds are administered and the form of the composition will dictate the type of carrier to be used. The composition may be in a variety of forms, suitable, for example, for systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implants, or parenteral injections) or topical administration (e.g., dermal, pulmonary, nasal, aural, ocular, liposome delivery systems, or iontophoresis).

[0160] Carriers for systemic administration typically include at least one of diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, antioxidants, preservatives, glidants, solvents, suspending agents, wetting agents, surfactants, cyclodextrins combinations thereof, and others. All carriers are optional in the compositions.

[0161] Suitable diluents include sugars such as glucose, lactose, dextrose, and sucrose; diols such as propylene glycol; calcium carbonate; sodium carbonate; sugar alcohols, such as glycerin; mannitol; and sorbitol. The amount of diluent(s) in a systemic or topical composition is typically about 50 to about 90%.

[0162] Suitable lubricants include silica, talc, stearic acid and its magnesium salts and calcium salts, calcium sulfate; and liquid lubricants such as polyethylene glycol and vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma. The amount of lubricant(s) in a systemic or topical composition is typically about 5 to about 10%.

[0163] Suitable binders include polyvinyl pyrrolidone; magnesium aluminum silicate; starches such as corn starch and potato starch; gelatin; tragacanth; and cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, methylcellulose, microcrystalline cellulose, and sodium carboxymethylcellulose. The amount of binder(s) in a systemic composition is typically about 5 to about 50%.

[0164] Suitable disintegrants include agar, alginic acid and the sodium salt thereof, effervescent mixtures, croscarmellose, crospovidone, sodium carboxymethyl starch, sodium starch glycolate, clays, and ion exchange resins. The amount of disintegrant(s) in a systemic or topical composition is typically about 0.1 to about 10%.

[0165] Suitable colorants include a colorant such as an FD&C dye. When used, the amount of colorant in a systemic or topical composition is typically about 0.005 to about 0.1%.

[0166] Suitable flavors include menthol, peppermint, and fruit flavors. The amount of flavor(s), when used, in a systemic or topical composition is typically about 0.1 to about 1.0%.

[0167] Suitable sweeteners include aspartame and saccharin. The amount of sweetener(s) in a systemic or topical composition is typically about 0.001 to about 1%.

[0168] Suitable antioxidants include butylated hydroxyanisole (“BHA”), butylated hydroxytoluene (“BHT”), and vitamin E. The amount of antioxidant(s) in a systemic or topical composition is typically about 0.1 to about 5%.

[0169] Suitable preservatives include benzalkonium chloride, methyl paraben and sodium benzoate. The amount of preservative(s) in a systemic or topical composition is typically about 0.01 to about 5%.

[0170] Suitable glidants include silicon dioxide. The amount of glidant(s) in a systemic or topical composition is typically about 1 to about 5%.

[0171] Suitable solvents include water, isotonic saline, ethyl oleate, glycerine, hydroxylated castor oils, alcohols such as ethanol, dimethyl sulfoxide, N-methyl-2- pyrrolidone, dimethylacetamide and phosphate (or other suitable buffer). The amount of solvent(s) in a systemic or topical composition is typically from about 0 to about 100%.

[0172] Suitable suspending agents include AVICEL RC-591 (from FMC Corporation of Philadelphia, Pa.) and sodium alginate. The amount of suspending agent(s) in a systemic or topical composition is typically about 1 to about 8%.

[0173] Suitable surfactants include lecithin, Polysorbate 80, and sodium lauryl sulfate, and the TWEENS from Atlas Powder Company of Wilmington, Del. Suitable surfactants include those disclosed in the C.T.F.A. Cosmetic Ingredient Handbook, 1992, pp.587-592; Remington's Pharmaceutical Sciences, 15th Ed.1975, pp.335-337; and McCutcheon's Volume 1, Emulsifiers & Detergents, 1994, North American Edition, pp.236-239. The amount of surfactant(s) in the systemic or topical composition is typically about 0.1% to about 5%.

[0174] Suitable cyclodextrins include alpha-CD, beta-CD, gamma-CD, hydroxypropyl PSbORSf #>E'PSbO'9:$& acZT]PcbgZ'SbVS` q'QgQZ]RSfb`W\ #G8;'PSbO'9:$( HVS O[]c\b ]T cyclodextrins in the systemic or topical composition is typically about 0% to about 40%.

[0175] Although the amounts of components in the systemic compositions may vary depending on the type of systemic composition prepared, in general, systemic compositions include 0.01% to 50% of an active compound (e.g., a compound of formula (I)) and 50% to99.99% of one or more carriers. Compositions for parenteral administration typically include 0.1% to 10% of actives and 90% to 99.9% of a carrier including a diluent and a solvent.

[0176] Compositions for oral administration can have various dosage forms. For example, solid forms include tablets, capsules, granules, and bulk powders. These oral dosage forms include a safe and effective amount, usually at least about 5%, and more particularly from about 25% to about 50% of actives. The oral dosage compositions include about 50% to about 95% of carriers, and more particularly, from about 50% to about 75%.

[0177] Tablets can be compressed, tablet triturates, enteric-coated, sugar-coated, film- coated, or multiple-compressed. Tablets typically include an active component, and a carrier comprising ingredients selected from diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, glidants, and combinations thereof. Specific diluents include calcium carbonate, sodium carbonate, mannitol, lactose, and cellulose. Specific binders include starch, gelatin, and sucrose. Specific disintegrants include alginic acid and croscarmellose. Specific lubricants include magnesium stearate, stearic acid, and talc. Specific colorants are the FD&C dyes, which can be added for appearance. Chewable tablets preferably contain sweeteners such as aspartame and saccharin, or flavors such as menthol, peppermint, fruit flavors, or a combination thereof.

[0178] Capsules (including implants, time release and sustained release formulations) typically include an active compound (e.g., a compound of formula (I)), and a carrier including one or more diluents disclosed above in a capsule comprising gelatin. Granules typically comprise a disclosed compound, and preferably glidants such as silicon dioxide to improve flow characteristics. Implants can be of the biodegradable or the non-biodegradable type.

[0179] The selection of ingredients in the carrier for oral compositions depends on secondary considerations like taste, cost, and shelf stability, which are not critical for the purposes of this invention.

[0180] Solid compositions may be coated by conventional methods, typically with pH or time-dependent coatings, such that a disclosed compound is released in the gastrointestinal tract in the vicinity of the desired application, or at various points and times to extend the desired action. The coatings typically include one or more components selected from the group consisting of cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methyl cellulose phthalate, ethyl cellulose, EUDRAGIT® coatings (available from Evonik Industries of Essen, Germany), waxes and shellac.

[0181] Compositions for oral administration can have liquid forms. For example, suitable liquid forms include aqueous solutions, emulsions, suspensions, solutions reconstituted from non-effervescent granules, suspensions reconstituted from non-effervescent granules, effervescent preparations reconstituted from effervescent granules, elixirs, tinctures, syrups, and the like. Liquid orally administered compositions typically include a disclosed compound and a carrier, namely, a carrier selected from diluents, colorants, flavors, sweeteners, preservatives, solvents, suspending agents, and surfactants. Peroral liquid compositions preferably include one or more ingredients selected from colorants, flavors, and sweeteners.

[0182] Other compositions useful for attaining systemic delivery of the subject compounds include sublingual, buccal and nasal dosage forms. Such compositions typically include one or more of soluble filler substances such as diluents including sucrose, sorbitol, and mannitol; and binders such as acacia, microcrystalline cellulose, carboxymethyl cellulose, and hydroxypropyl methylcellulose. Such compositions may further include lubricants, colorants, flavors, sweeteners, antioxidants, and glidants.

[0183] The disclosed compounds can be topically administered. Topical compositions that can be applied locally to the skin may be in any form including solids, solutions, oils, creams, ointments, gels, lotions, shampoos, leave-on and rinse-out hair conditioners, milks, cleansers, moisturizers, sprays, skin patches, and the like. Topical compositions include: a disclosed compound (e.g., a compound of formula (I)), and a carrier. The carrier of the topical composition preferably aids penetration of the compounds into the skin. The carrier may further include one or more optional components.

[0184] The amount of the carrier employed in conjunction with a disclosed compound is sufficient to provide a practical quantity of composition for administration per unit dose of the compound. Techniques and compositions for making dosage forms useful in the methods of this invention are described in the following references: Modern Pharmaceutics, Chapters 9 and 10, Banker & Rhodes, eds. (1979); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms, 2nd Ed., (1976).

[0185] A carrier may include a single ingredient or a combination of two or more ingredients. In the topical compositions, the carrier includes a topical carrier. Suitable topical carriers include one or more ingredients selected from phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols, symmetrical alcohols, aloe vera gel, allantoin, glycerin, vitamin A and E oils, mineral oil, propylene glycol, PPG-2 myristyl propionate, dimethyl isosorbide, castor oil, combinations thereof, and the like. More particularly, carriers for skin applications include propylene glycol, dimethyl isosorbide, andwater, and even more particularly, phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols, and symmetrical alcohols.

[0186] The carrier of a topical composition may further include one or more ingredients selected from emollients, propellants, solvents, humectants, thickeners, powders, fragrances, pigments, and preservatives, all of which are optional.

[0187] Suitable emollients include stearyl alcohol, glyceryl monoricinoleate, glyceryl monostearate, propane-1,2-diol, butane-1,3-diol, mink oil, cetyl alcohol, isopropyl isostearate, stearic acid, isobutyl palmitate, isocetyl stearate, oleyl alcohol, isopropyl laurate, hexyl laurate, decyl oleate, octadecan-2-ol, isocetyl alcohol, cetyl palmitate, di-n-butyl sebacate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, butyl stearate, polyethylene glycol, triethylene glycol, lanolin, sesame oil, coconut oil, arachis oil, castor oil, acetylated lanolin alcohols, petroleum, mineral oil, butyl myristate, isostearic acid, palmitic acid, isopropyl linoleate, lauryl lactate, myristyl lactate, decyl oleate, myristyl myristate, and combinations thereof. Specific emollients for skin include stearyl alcohol and polydimethylsiloxane. The amount of emollient(s) in a skin-based topical composition is typically about 5% to about 95%.

[0188] Suitable propellants include propane, butane, isobutane, dimethyl ether, carbon dioxide, nitrous oxide, and combinations thereof. The amount of propellant(s) in a topical composition is typically about 0% to about 95%.

[0189] Suitable solvents include water, ethyl alcohol, methylene chloride, isopropanol, castor oil, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, dimethylsulfoxide, dimethyl formamide, tetrahydrofuran, and combinations thereof. Specific solvents include ethyl alcohol and homotopic alcohols. The amount of solvent(s) in a topical composition is typically about 0% to about 95%.

[0190] Suitable humectants include glycerin, sorbitol, sodium 2-pyrrolidone-5- carboxylate, soluble collagen, dibutyl phthalate, gelatin, and combinations thereof. Specific humectants include glycerin. The amount of humectant(s) in a topical composition is typically 0% to 95%.

[0191] The amount of thickener(s) in a topical composition is typically about 0% to about 95%.

[0192] Suitable powders include beta-cyclodextrins, hydroxypropyl cyclodextrins, chalk, talc, fullers earth, kaolin, starch, gums, colloidal silicon dioxide, sodium polyacrylate, tetra alkyl ammonium smectites, trialkyl aryl ammonium smectites, chemically-modified magnesium aluminum silicate, organically-modified montmorillonite clay, hydratedaluminum silicate, fumed silica, carboxyvinyl polymer, sodium carboxymethyl cellulose, ethylene glycol monostearate, and combinations thereof. The amount of powder(s) in a topical composition is typically 0% to 95%.

[0193] The amount of fragrance in a topical composition is typically about 0% to about 0.5%, particularly, about 0.001% to about 0.1%.

[0194] Suitable pH adjusting additives include HCl or NaOH in amounts sufficient to adjust the pH of a topical pharmaceutical composition. b. Albumin Compositions

[0195] The disclosure further provides compositions comprising albumin nanoparticles. In some embodiments, a compound as disclosed herein is incorporated into compositions comprising an albumin nanoparticle. The albumin nanoparticles compositions and formulations may also include pharmaceutically acceptable carriers, as described above.

[0196] Albumins include the most abundant plasma proteins in mammals and albumins from a large and diverse number of mammals have been characterized by biochemical methods and / or by sequence information. Any natural, synthetic, or engineered albumin may be used in the context of the nanoparticle compositions described herein. In some embodiments, the albumin is human serum albumin.

[0197] In some embodiments, the albumin nanoparticles further comprise one or more cell targeting epitopes. In some embodiments, the epitopes are covalently attached or directly conjugated to the albumin. In some embodiments, the epitopes are crosslinked to the albumin. In select embodiments, the albumin nanoparticles further comprise one or more immune cell epitopes (e.g., B cell and T cell epitopes). The one or more immune cell antigens may facilitate targeting to lymphatic systems. In select embodiments, the albumin nanoparticles further comprise one or more epitopes from a microbiological agent (e.g., Clostridioides difficile, Bacillus anthracis, clostridium botulinum, Heliobacter pylori, Rotavirus sp., Coronaviridae). c. Lipophilic Formulations

[0198] In some embodiments, the compounds disclosed herein are incorporated into lipophilic compositions comprising a liposome, a lipid nanoparticle, a micelle, or the like. In some embodiments, a disclosed compound is encapsulated in the liposome, the lipid nanoparticle, or the micelle. The formulations may also include pharmaceutically acceptable carriers, as described above.

[0199] In some embodiments, the disclosed compounds are incorporated into lipophilic compositions comprising one or more vesicle forming lipids. Methods of making lipophiliccompositions include, for example, lipid film hydration, optionally coupled with sonication or extrusion, solvent evaporation (e.g., ethanol injection, ether injection, or reverse phase evaporation), solvent-diffusion method, hot homogenization process, detergent removal methods, or combinations thereof. The disclosed compounds can be combined with the lipid(s) before formation of the vesicles (passive loading) or after vesicle formation (active loading). The lipophilic compositions may prolong circulation time in vivo, increase stability of the compound, and prevent degradation in the bloodstream. The lipophilic compositions may increase the distribution of the compounds within the lung, breast, pancreas, and spleen.

[0200] Any naturally occurring or synthetic vesicle forming lipid or combinations thereof can be used. The one or more vesicle forming lipids may be selected from di-aliphatic chain lipids, such as phospholipids; diglycerides; di-aliphatic glycolipids; single lipids such as sphingomyelin or glycosphingolipid; steroidal lipids; hydrophilic polymer derivatized lipids; or mixtures thereof.

[0201] Lipophilic compositions of the disclosure may include one or more cationic and / or ionizable lipids, phospholipids, neutral or non-cationic lipids, polyethylene glycol (PEG)- lipid conjugates, and / or sterols. In some embodiments, the lipid nanoparticle comprises a cationic lipid and / or ionizable lipid, a neutral or non-cationic lipid, and cholesterol.

[0202] Cationic and / or ionizable lipids include, for example, amine-containing lipids that can be readily protonated and may have a positive or partial positive charge at physiological pH due to a pKa value between pH 5 and 8. The polar headgroup of the cationic lipids preferably comprises amine derivatives such as primary, secondary, and / or tertiary amines, quaternary ammonium, various combinations of amines, amidinium salts, or guanidine and / or imidazole groups as well as pyridinium, piperazine and amino acid headgroups such as lysine, arginine, ornithine and / or tryptophan. Cationic lipids include, but are not limited to, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 2,3- di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium bromide (DMRIE), didodecyl(dimethyl)ammonium bromide (DDAB), 1,2-dioleyloxypropyl-3-dimethyl- VgR`]fgSbVgZ O[[]\Wc[ P`][WRS #:DF?;$& -q'MCl#CNCp'RW[SbVgZO[W\]' ethane)carbamoyl]cholesterol (DC-Chol) or dioleyl ether phosphatidylcholine (DOEPC). Ionizable lipids include, but are not limited to, 1,2-dioleyloxy-3-dimethylamino- propane (DODMA).

[0203] In some embodiments, the lipophilic compositions comprise a polyethylene glycol (PEG)-lipid conjugate. A PEG-lipid conjugate may include, but is not limited to, PEG- modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-DMG (1,2- RW[g`Wab]gZ'`OQ'UZgQS`]'-'[SbV]fg^]ZgSbVgZS\S UZgQ]Z$& E;='Q':DB= #F'-'M#t'[SbV]fg poly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristyloxlpropyl-3-amine), PEG-DMA (PEG- dimethacrylate), PEG-DLPE (1,2-didodecanoyl-sn-glycero-3-phosphoethanolamine-PEG), PEG-DMPE (PEG- 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), PEG-DPPC (PEG- dipalmitoyl phosphatidylcholine), PEG-N,N-di(tetradecyl)acetamide, or a PEG-DSPE (1, 2- distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)) lipid. In some embodiments, the lipid nanoparticle comprises PEG-DMG and / or PEG-N,N- di(tetradecyl)acetamide.

[0204] The sterol may comprise cholesterol, fecosterol, ergosterol, campesterol, sitosterol, stigmasterol, brassicasterol, or a sterol ester, such as cholesteryl hemisuccinate, cholesteryl sulfate, or any other derivatives of cholesterol.

[0205] A neutral or non-cationic lipid may include one or more phospholipids. Phospholipids include a phospholipid moiety and one or more fatty acid moieties. A phospholipid moiety may include, but is not limited to, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2- lysophosphatidyl choline, and sphingomyelin. A fatty acid moiety may include, but is not limited to, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0206] Phospholipids suitable for use in the compositions may include, but are not limited to, phosphatidylglycerol (PG) including dimyristoyl phosphatidylglycerol (DMPG) and 1,2- dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG); phosphatidylcholine (PC), including egg yolk phosphatidylcholine, dimyristoyl phosphatidylcholine (DMPC), 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dilinoleoyl-sn-glycero-3- phosphocholine (DLPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di- O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn- glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3- phosphocholine; phosphatidylethanolamine (PE) including 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2- diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine; phosphatidic acid (PA); phosphatidylinositol (PI); phosphatidylserine (PS); and sphingomyelin (SM).

[0207] The positively charged lipid structures described herein may also include other components typically used in the formation of vesicles (e.g., for stabilization). Examples of such other components includes, without being limited thereto, fatty alcohols, fatty acids, and / or any other pharmaceutically acceptable excipients which may affect the surface charge, the membrane fluidity and assist in the incorporation of the lipid into the lipid assembly.

[0208] The lipophilic compositions can also be targeting, e.g., contain one or more targeting moieties or biodistribution modifiers on the surface. A targeting moiety can be any agent that is capable of specifically binding or interacting with a desired target and are generally known in the art, for example ligands such as folic acid, proteins, antibody or antibody fragments, and the like). In some embodiments, the targeting moiety is an immune cell epitope (e.g., B cell and T cell epitopes). In select embodiments, the targeting moiety comprises one or more epitopes from a microbiological agent (e.g., Clostridioides difficile, Bacillus anthracis, clostridium botulinum, Heliobacter pylori, Rotavirus sp., Coronaviridae).

[0209] The lipophilic compositions can have any structure, e.g., structures having an inner space sequestered from the outer medium by one or more lipid bilayers, or any microcapsule that has a semi-permeable membrane with a lipophilic central part where the membrane sequesters an interior. In some embodiments, the lipophilic compositions may comprise unilamellar liposomes, having a single lipid layer. The disclosed compounds may be completely or partially located in the interior space of the liposome or completely or partially within the bilayer membrane of the liposome. In some embodiments, the lipophilic compositions comprise micelles.

[0210] In some embodiments, the disclosed compounds are incorporated into formulations comprising PLA and / or PLGA. PLA or PLGA formulations may be prepared by variousmethods known in the art such as single / double emulsion-solvent evaporation technique, spray drying, spray freeze drying, supercritical fluid drying, and nanoprecipitation. d. Additional Therapeutic Agents

[0211] Any of the above compositions or formulations disclosed herein may further comprise at least one additional therapeutic agent. In some embodiments the at least one additional therapeutic agent comprises an immune modulator, a chemotherapeutic agent, a nucleic acid (e.g., mRNA, aptamers, antisense oligonucleotides, ribozyme nucleic acids, interfering RNAs, antisense and antigene nucleic acids), or a combination thereof.

[0212] Exemplary immune modulators include: indoleamine 2,3-dioxygenase (IDO) inhibitors and analogs thereof, such as, epacadostat, BMS-986205, indoximod, PF-06840003, and analogs thereof; signal transducer and activator of transcription 3 (Stat3) inhibitors and analogs thereof, such as, SM-36 and its analogs; toll-like receptor (TLR) agonists and analogs thereof, such as, imiquimod, resiquimod, selgantolimod, gardiquimod, SM-360320, TMX- 101, TMX-202, TMX-302, TMX-306, GSK2245035, CL097, 852A, AZD-8848, DSP-3025, GS-9620, RO7020531, RO6871765, ANA773, DSP-0509, NJH395, BNT411, TQ-A3334, JNJ-4964, LHC165, CV8102, VTX-1463, VTX-2337, IMO-8400, IMO-3100, IRS-954, and analogs thereof; and statins or other lipid-lowering medications and analogs thereof, such as, atorvastatin, pravastatin, fluvastatin, simvastatin, lovastatin, mevastatin, pitavastatin, rosuvastatin, and analogs thereof.

[0213] In some embodiments, the at least one additional therapeutic agent comprises at least one chemotherapeutic agent. As used herein, the term “chemotherapeutic” or “anti- cancer drug” includes any small molecule or other drug used in cancer treatment or prevention. Chemotherapeutics include, but are not limited to, cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, docetaxel, daunorubicin, bleomycin, vinblastine, dacarbazine, cisplatin, paclitaxel, raloxifene hydrochloride, tamoxifen citrate, abemacicilib, afinitor (Everolimus), alpelisib, anastrozole, pamidronate, anastrozole, exemestane, capecitabine, epirubicin hydrochloride, eribulin mesylate, toremifene, fulvestrant, letrozole, gemcitabine, goserelin, ixabepilone, emtansine, lapatinib, olaparib, megestrol, neratinib, palbociclib, ribociclib, talazoparib, thiotepa, toremifene, methotrexate, and tucatinib.

[0214] In some embodiments, the at least one additional therapeutic agent comprises a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The term “polynucleotide,” in its broadest sense, includes any compound and / or substance that is or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides for use in accordance with the present disclosure include, but are not limited to, one or more ofdeoxyribonucleic acid (DNA), ribonucleic acid (RNA) including messenger mRNA (mRNA), hybrids thereof, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, aptamers, vectors, etc.

[0215] In some embodiments, the at least one additional therapeutic agent is an RNA. RNAs useful in the compositions and methods described herein can be selected from the group consisting of, but are not limited to, shortmers, antagomirs, antisense RNAs , ribozymes, small interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof.

[0216] In certain embodiments, the at least one additional therapeutic agent is an mRNA. An mRNA may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. A polypeptide encoded by an mRNA may be of any size and may have any secondary structure or activity. In some embodiments, a polypeptide encoded by an mRNA may have a therapeutic effect when expressed in a cell.

[0217] In other embodiments, the at least one additional therapeutic agent is an siRNA. An siRNA may be capable of selectively knocking down or down regulating expression of a gene of interest. For example, an siRNA could be selected to silence a gene associated with a particular disease, disorder, or condition upon administration to a subject in need thereof of a nanoparticle composition including the siRNA. An siRNA may comprise a sequence that is complementary to an mRNA sequence that encodes a gene or protein of interest. In some embodiments, the siRNA may be an immunomodulatory siRNA.

[0218] In some embodiments, the at least one additional therapeutic agent is an shRNA or a vector or plasmid encoding the same. An shRNA may be produced inside a target cell upon delivery of an appropriate construct to the nucleus. Constructs and mechanisms relating to shRNA are well known in the relevant arts. e. Vaccines

[0219] The compounds and compositions may also be used for vaccines. The vaccines comprise the compound or compositions disclosed and an antigen or a nucleic acid encoding thereof. Suitable antigens include microbial pathogens, bacteria, viruses, proteins, glycoproteins lipoproteins, peptides, glycopeptides, lipopeptides, toxoids, carbohydrates, and tumor-specific antigens. Mixtures of two or more antigens may be employed.

[0220] The antigen can be derived and / or isolated from essentially any desired source depending on the infectious disease, autoimmune disease, condition, cancer, pathogen, or a disease that is to be treated with a given vaccine composition.

[0221] The vaccines described herein may be capable of providing immunity against one or more conditions related to infectious diseases, including but not limited to, influenza, measles, human papillomavirus (HPV), rabies, meningitis, whooping cough, tetanus, plague, hepatitis, and tuberculosis and can include infectious disease derived antigens and / or epitopes, or nucleic acids encoding thereof.

[0222] The vaccines described herein may also direct an immune response against cancer cells and can include tumor cell derived antigens, epitopes, and / or neoepitopes, or portions thereof, or nucleic acids encoding tumor cell derived antigens, epitopes, and / or neoepitopes. Tumor antigens are surface molecules that are differentially expressed in tumor cells relative to non-tumor tissues. Tumor antigens make tumor cells immunologically distinct from normal cells and provide diagnostic and therapeutic targets for human cancers. Tumor antigens have been characterized either as membrane proteins or as altered carbohydrate molecules of glycoproteins or glycolipids on the cell surface. Cancer cells often have distinctive tumor antigens on their surfaces, such as truncated epidermal growth factor, folate binding protein, epithelial mucins, melanoferrin, carcinoembryonic antigen, prostate-specific membrane antigen, HER2-neu, which are candidates for use in therapeutic cancer vaccines. Because tumor antigens are normal or related to normal components of the body, the immune system often fails to mount an effective immune response against those antigens to destroy the tumor cells. Illustrative cancer types for which this approach can be used include prostate, colon, breast, ovarian, pancreatic, brain, head and neck, melanoma, leukemia, lymphoma, etc.

[0223] In other embodiments, the antigen present in the vaccine composition is not a foreign antigen, but a self-antigen, e.g., the vaccine composition is directed toward an autoimmune disease. Examples of autoimmune diseases include type 1 diabetes, conventional organ specific autoimmunity, neurological disease, rheumatic diseases / connective tissue disease, autoimmune cytopenias, and related autoimmune diseases. Such conventional organ specific autoimmunity may include thyroiditis (Graves+Hashimoto's), gastritis, adrenalitis (Addison's), ovaritis, primary biliary cirrhosis, myasthenia gravis, gonadal failure, hypoparathyroidism, alopecia, malabsorption syndrome, pernicious anemia, hepatitis, anti- receptor antibody diseases and vitiligo. Such neurological diseases may include schizophrenia, Alzheimer's disease, depression, hypopituitarism, diabetes insipidus, sicca syndrome and multiple sclerosis. Such rheumatic diseases / connective tissue diseases mayinclude rheumatoid arthritis, systemic lupus erythematous (SLE) or Lupus, scleroderma, polymyositis, inflammatory bowel disease, dermatomyositis, ulcerative colitis, Crohn's disease, vasculitis, psoriatic arthritis, exfoliative psoriatic dermatitis, pemphigus vulgaris, Sjogren's syndrome. Other autoimmune related diseases may include autoimmune uvoretinitis, glomerulonephritis, post myocardial infarction cardiotomy syndrome, pulmonary hemosiderosis, amyloidosis, sarcoidosis, aphthous stomatitis, and other immune related diseases, as presented herein and known in the related arts.

[0224] In one embodiment, the antigen in a vaccine composition is a peptide, polypeptide, or immunogenic portion thereof. An “immunogenic portion,” as used herein is a portion of a protein that is recognized (e.g., specifically bound) by a B cell and / or T cell surface antigen receptor. Such immunogenic portions generally comprise at least 5 amino acid residues, more preferably at least 10, and still more preferably at least 20 amino acid residues of an antigenic protein or a variant thereof.

[0225] Immunogenic portions of antigen polypeptides may generally be identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3rd ed., 243- 247 (Raven Press, 1993) and references cited therein. Such techniques include screening polypeptides for the ability to react with antigen-specific antibodies, antisera and / or T cell lines or clones. As used herein, antisera and antibodies are “antigen-specific” if they specifically bind to an antigen (e.g., they react with the protein in an ELISA or other immunoassay, and do not react detectably with unrelated proteins). Such antisera and antibodies may be prepared using known techniques. An immunogenic portion of a protein is a portion that reacts with such antisera and / or T cells at a level that is not substantially less than the reactivity of the full length polypeptide (e.g., in an ELISA and / or T cell reactivity assay). Such immunogenic portions may react within such assays at a level that is similar to or greater than the reactivity of the full length polypeptide. Such screens may generally be performed using methods well known to those of ordinary skill in the art, such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. For example, a polypeptide may be immobilized on a solid support and contacted with patient sera to allow binding of antibodies within the sera to the immobilized polypeptide. Unbound sera may then be removed and bound antibodies detected using, for example, 125I-labeled Protein A.

[0226] Peptide and polypeptide antigens may be prepared using any of a variety of well- known techniques. Recombinant polypeptides encoded by DNA sequences may be readily prepared from isolated DNA sequences using any of a variety of expression vectors known tothose of ordinary skill in the art. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast, and higher eukaryotic cells, such as mammalian cells and plant cells. Preferably, the host cells employed are E. coli, yeast, or a mammalian cell line such as COS or CHO.

[0227] Portions and other variants of a protein antigen having less than about 100 amino acids, and generally less than about 50 amino acids, may also be generated by synthetic means, using techniques well known to those of ordinary skill in the art. For example, such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See, Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer / Applied BioSystems Division (Foster City, Calif.), and may be operated according to the manufacturer's instructions.

[0228] In certain embodiments, the nucleic acid encoding the antigen is DNA. Illustrative DNA-based vaccines of this type contain DNA encoding one or more polypeptide antigens, such that the antigen is generated in situ. Alternatively, the vaccine may be an RNA-based vaccine. In certain embodiments, the nucleic acid encoding the antigen is an mRNA. An mRNA may encode any polypeptide antigen of interest, including any naturally or non- naturally occurring or otherwise modified polypeptide. A polypeptide encoded by an mRNA may be of any size and may have any secondary structure or activity. In some embodiments, a polypeptide encoded by the mRNA may stimulate an immune response when expressed in a cell.

[0229] The vaccine compositions of the present disclosure may also contain other compounds, which may be biologically active or inactive. The vaccine or medicament may comprise an adjuvant or immunostimulant, or a polynucleotide encoding an adjuvant or immunostimulant (e.g., an adjuvantive polypeptide). Adjuvants and immunostimulants are compounds or compositions that either directly or indirectly stimulate the immune system’s response to a co-administered antigen. In some embodiments, the vaccines are not adjuvanted or are self-adjuvanting.

[0230] Suitable adjuvants are commercially available as, for example, Glucopyranosyl Lipid Adjuvant (GLA); Pam3CSK4; Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham); mineral salts (for example, aluminum, silica,kaolin, and carbon); aluminum salts such as aluminum hydroxide gel (alum), AlK(SO4)2, AlNa(SO4)2, AlNH4(SO4), and Al(OH)3; salts of calcium (e.g., Ca3(PO4)2), iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polynucleotides (for example, poly IC, poly AU acids, and CpG oligodeoxynucleotides (e.g., Class A or B)); polyphosphazenes; cyanoacrylates; polymerase- (DL-lactide-co- glycoside); bovine serum albumin; diphtheria toxoid; tetanus toxoid; edestin; keyhole-limpet hemocyanin; Pseudomonal Toxin A; choleragenoid; cholera toxin; pertussis toxin; viral proteins; Quil A; aminoalkyl glucosamine phosphate compounds. In addition, adjuvants such as cytokines (e.g., GM-CSF or interleukin-2, -7, or -12), interferons, or tumor necrosis factor, may also be used as adjuvants. Protein and polypeptide adjuvants may be obtained from natural or recombinant sources according to methods well known to those skilled in the art. When obtained from recombinant sources, the adjuvant may comprise a protein fragment comprising at least the immunostimulatory portion of the molecule.

[0231] Other known immunostimulatory macromolecules which can be used include, but are not limited to, polysaccharides, tRNA, non-metabolizable synthetic polymers such as polyvinylamine, polymethacrylic acid, polyvinylpyrrolidone, mixed polycondensates (with relatively high molecular weight) of 4',4-diaminodiphenylmethane-3,3'-dicarboxylic acid and 4-nitro-2- aminobenzoic acid (See, Sela, M., Science 166: 1365-1374 (1969)) or glycolipids, lipids or carbohydrates.

[0232] In some embodiments, the adjuvantive polypeptide comprises immune activator proteins, such as CD70, CD40 ligand, and constitutively active TLR4, or polycationic peptides (e.g., protamine). In some embodiments, the adjuvantive polypeptide is a flagellin polypeptide. Commercially available mRNA encoding adjuvantive polypeptides are available, for example, as TriMix (See Bonehill, A. et al. Mol. Ther.16, 1170–1180 (2008), incorporated herein by reference). In some embodiments, the vaccine may comprise at least two separate polynucleotides, one encoding anti-Müllerian hormone receptor II extracellular domain (AMHR2-ED), as described above, and the other encoding an adjuvantive polypeptide (e.g., a flagellin polypeptide or immune activator protein).

[0233] Vaccine preparation is a well-developed art and general guidance in the preparation and formulation of vaccines is readily available from any of a variety of sources. One such example is New Trends and Developments in Vaccines, edited by Volier et al. University Park Press, Baltimore, Md., U.S.A.1978. Vaccine compositions may generally be used for prophylactic and therapeutic purposes.

[0234] The amount of antigen in each vaccine dose is generally selected as an amount which induces an immunoprotective response without significant adverse side effects in typical vaccines. Such amount will vary depending upon which specific immunogen is employed and how it is presented. Of course, the dosage administered may be dependent upon the age, weight, kind of concurrent treatment, if any, and nature of the antigen administered.

[0235] The immunogenic activity of a given amount of a vaccine composition can be readily determined, for example by monitoring the increase in titer of antibody against the antigen used in the vaccine composition (Dalsgaard, K. Acta Veterinia Scandinavica 69: 1-40 (1978)). Another common method involves injecting CD-l mice intradermally with various amounts of a vaccine composition, later harvesting sera from the mice and testing for anti - immunogen antibody, e.g., by ELISA. These and other similar approaches will be apparent to the skilled artisan. 4. Methods of Use

[0236] The disclosure provides methods for inducing or modulating an immune or inflammatory response. As used herein, the term “modulating” generally refers to the ability to alter, by increasing or decreasing, e.g., directly or indirectly promoting / stimulating / up- regulating or interfering with / inhibiting / down-regulating a specific concentration, level, expression, function or behavior (e.g., of the immune or inflammatory response). In some embodiments, the modulating is an increase and / or decrease of a certain concentration, level, activity, or function relative to a control, or relative to the average level of activity that would generally be expected or relative to a control level of activity.

[0237] Thus, in some embodiments, modulating an immune or inflammatory response refers to the ability of the compounds of the present invention to alter or modulate one or more aspects of the immune or inflammatory response. In some embodiments, the methods polarize macrophages. In some embodiments, the methods induce an interferon response. In some embodiments, the methods active transcription factors (e.g., STAT6, IRF3) of the innate immune response.

[0238] The disclosure further provides methods for treating a disease or disorder comprising administration of a compound or composition as disclosed herein, to a subject in need thereof. In some embodiments, the subject is a human.

[0239] The disease or disorder may comprise cancer, autoimmune diseases, inflammatory diseases, and infectious diseases.

[0240] In some embodiments, the disease or disorder is an inflammatory disease or disorder. Inflammatory diseases are characterized by activation of the immune system in a tissue or an organ to abnormal levels that may lead to abnormal function and / or disease in the tissue or organ. The inflammatory diseases and disorders that may be treated by the methods of the present invention include, but are not limited to, arthritis, rheumatoid arthritis, asthma, inflammatory bowel disease (Crohn's disease or ulcerative colitis), chronic obstructive pulmonary disease (COPD), allergic rhinitis, vasculitis (polyarteritis nodosa, temporal arteritis, Wegener's granulomatosis, Takayasu's arteritis, or Behcet’s syndrome), inflammatory neuropathy, psoriasis, systemic lupus erythematosus (SLE), chronic thyroiditis, Hashimoto's thyroiditis, Addison's disease, polymyalgia rheumatica, Sjogren's syndrome, or Churg-Strauss syndrome.

[0241] In some embodiments, the disease or disorder is an autoimmune disease or disorder. Autoimmune diseases and disorders refer to conditions in a subject characterized by cellular, tissue and / or organ injury caused by an immunologic reaction of the subject to its own cells, tissues and / or organs. Autoimmune diseases and disorders that may be treated by the methods of the present invention include, but are not limited to, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), irritable bowel disease (IBD), IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatics, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, Rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, takayasu arteritis, temporal arteritis / giantcell arteritis, ulcerative colitis, uveitis, vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, and Wegener's granulomatosis.

[0242] Some autoimmune disorders are also associated with an inflammatory condition. Examples of inflammatory disorders which are also autoimmune disorders that can be prevented, treated or managed in accordance with the methods of the invention include, but are not limited to, asthma, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation resulting from chronic viral or bacterial infections. Examples of the types of psoriasis which can be treated in accordance with the compositions and methods of the invention include, but are not limited to, plaque psoriasis, pustular psoriasis, erythrodermic psoriasis, guttate psoriasis and inverse psoriasis.

[0243] In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer comprises a blood cancer or lymphoma. In some embodiments, the cancer is metastatic cancer. In some embodiments, the disclosed compounds, compositions, or methods result in suppression of elimination of metastasis. In some embodiments, the disclosed compounds, compositions, or methods result in decreased tumor growth. In some embodiments, the disclosed compounds, compositions, or methods prevent tumor recurrence.

[0244] The compounds and compositions herein may be useful to treat a wide variety of cancers including carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma. The cancer may be a cancer of the bladder, blood, bone, brain, breast, cervix, colon / rectum, endometrium, head and neck, kidney, liver, lung, lymph nodes, muscle tissue, ovary, pancreas, prostate, skin, spleen, stomach, testicle, thyroid, or uterus.

[0245] In some embodiments, the cancer is invasive and / or metastatic cancer (e.g., stage II cancer, stage III cancer or stage IV cancer). In some embodiments, the cancer is an early stage cancer (e.g., stage 0 cancer, stage I cancer), and / or is not invasive and / or metastatic cancer.

[0246] In some embodiments, the disease or disorder is an infectious disease. Infectious diseases that can be treated or prevented by the methods of the present invention are caused by infectious agents including, but not limited to, viruses, bacteria, fungi, protozoa, helminths, and parasites. The invention is not limited to treating or preventing infectious diseases caused by intracellular or extracellular pathogens. The infectious disease may bederived from: bacteria, such as Mycobacterium tuberculosis, Chlamydia, Francisella tularensis; DNA viruses, such as Herpesviridae (herpes simplex virus-1, Kaposi's sarcoma- associated virus and Epstein-Barr virus), Papillomaviridae (human papilloma virus), Adenovirus and Hepadnaviridae (Hepatitis B virus), or RNA viruses, such as Retroviridae (human immunodeficiency virus) Flaviviridae (Dengue virus, Hepatitis C virus), Orthomyxoviridae (influenza), and Coronaviridae (human coronavirus and SARS coronavirus).

[0247] The compounds and compositions disclosed herein may be administered to a subject by a variety of methods. In any of the uses or methods described herein, administration may be by various routes known to those skilled in the art, including without limitation oral, inhalation, intravenous, intramuscular, topical, subcutaneous, systemic, and / or intraperitoneal administration to a subject in need thereof.

[0248] The amount of the compounds of the present disclosure required for use in the disclosed methods will vary not only with the particular compound selected but also with the route of administration, the nature and / or symptoms of the disease and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician. The determination of effective dosage levels, that is the dosage levels necessary to achieve the desired result, can be accomplished by one skilled in the art using routine methods, for example, human clinical trials, in vivo studies, and in vitro studies. For example, useful dosages can be determined by comparing their in vitro activity, and in vivo activity in animal models.

[0249] Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the modulating effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vivo and / or in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, FIPLC assays or bioassays can be used to determine plasma concentrations. Dosage intervals can also be determined using MEC value. Compositions should be administered using a regimen, which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

[0250] It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels ifthe clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the symptoms to be treated and the route of administration. Further, the dose, and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.

[0251] The compounds and compositions disclosed herein can be evaluated for efficacy and toxicity using known methods. For example, the toxicology of a particular compound or a subset of the compounds sharing certain chemical moieties, or a composition thereof, may be established by determining in vitro toxicity towards a cell line, such as a mammalian, and preferably human, cell line. The results of such studies are often predictive of toxicity in animals, such as mammals, or more specifically, humans. Alternatively, the toxicity of particular compounds in an animal model, such as mice, rats, rabbits, dogs, or monkeys, may be determined using known methods. Efficacy may be established using several recognized methods, such as in vitro methods, animal models, or human clinical trials. When selecting a model to determine efficacy, the skilled artisan can be guided by the state of the art to choose an appropriate model, dose, route of administration and / or regime.

[0252] A wide range of second therapies may be used in conjunction with the compounds of the present disclosure. The second therapy may be administration of an additional therapeutic agent or may be a second therapy not connected to administration of another agent. Such second therapies include, but are not limited to, surgery, immunotherapy, radiotherapy.

[0253] The second therapy may be administered at the same time as the initial therapy, either in the same composition or in a separate composition administered at substantially the same time as the first composition. In some embodiments, the second therapy may precede or follow the treatment of the first therapy by time intervals ranging from hours to months.

[0254] In some embodiments, a therapeutically effective amount of a compound disclosed herein, or compositions thereof, is administered alone or in combination with a therapeutically effective amount of at least one additional therapeutic agent. In some embodiments, effective combination therapy is achieved with a single composition or pharmacological formulation that includes both agents, or with two distinct compositions or formulations, administered at the same time or separated by a time interval, wherein one composition includes a compound of this invention, and the other includes the at least one additional therapeutic agent.

[0255] In some embodiments, the at least one additional therapeutic agent comprises an immune modulator, a chemotherapeutic agent, a nucleic acid (e.g., mRNA, aptamers, antisense oligonucleotides, ribozyme nucleic acids, interfering RNAs, antigene nucleic acids), a decongestant, a steroid, an analgesic, an antimicrobial agent, or a combination thereof.

[0256] Exemplary immune modulators include: indoleamine 2,3-dioxygenase (IDO) inhibitors and analogs thereof, such as, epacadostat, BMS-986205, indoximod, PF-06840003, and analogs thereof; signal transducer and activator of transcription 3 (Stat3) inhibitors and analogs thereof, such as, SM-36 and its analogs; toll-like receptor (TLR) agonists and analogs thereof, such as, imiquimod, resiquimod, selgantolimod, gardiquimod, SM-360320, TMX- 101, TMX-202, TMX-302, TMX-306, GSK2245035, CL097, 852A, AZD-8848, DSP-3025, GS-9620, RO7020531, RO6871765, ANA773, DSP-0509, NJH395, BNT411, TQ-A3334, JNJ-4964, LHC165, CV8102, VTX-1463, VTX-2337, IMO-8400, IMO-3100, IRS-954, and analogs thereof; and statins or other lipid-lowering medications and analogs thereof, such as, atorvastatin, pravastatin, fluvastatin, simvastatin, lovastatin, mevastatin, pitavastatin, rosuvastatin, and analogs thereof.

[0257] In some embodiments, the at least one additional therapeutic agent comprises at least one chemotherapeutic agent. As used herein, the term “chemotherapeutic” or “anti- cancer drug” includes any small molecule or other drug used in cancer treatment or prevention. Chemotherapeutics include, but are not limited to, cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, docetaxel, daunorubicin, bleomycin, vinblastine, dacarbazine, cisplatin, paclitaxel, raloxifene hydrochloride, tamoxifen citrate, abemacicilib, afinitor (Everolimus), alpelisib, anastrozole, pamidronate, anastrozole, exemestane, capecitabine, epirubicin hydrochloride, eribulin mesylate, toremifene, fulvestrant, letrozole, gemcitabine, goserelin, ixabepilone, emtansine, lapatinib, olaparib, megestrol, neratinib, palbociclib, ribociclib, talazoparib, thiotepa, toremifene, methotrexate, and tucatinib. In select embodiments, the chemotherapeutic agent comprises paclitaxel.

[0258] In some embodiments of the methods disclosed herein, the compound or composition can be co-administered with an antimicrobial (e.g., antiviral or antibacterial) agent. In some embodiments, the additional antimicrobial agent is an antiviral agent, including but not limited to, abacavir, acyclovir, adefovir, amantadine, amprenavir, atazanavir, baloxavir marboxil, bictegravir, boceprevir, bulevirtide, cidofovir, cobicistat, daclatasvir, darunavir, delavirdine, didanosine, docosanol, dolutegravir, doravirine, edoxudine, efavirenz, elvitegravir, emtricitabine, enfuvirtide, entecavir, etravirine,famciclovir, fomivirsen, fosamprenavir, foscarnet, ganciclovir, ibacitabine, ibalizumab, idoxuridine, imiquimod, imunovir, indinavir, lamivudine, letermovir, lopinavir, loviride, maraviroc, methisazone, moroxydine, nelfinavir, nevirapine, nexavir, nitazoxanide, oseltamivir, penciclovir, peramivir, penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir, remdesivir, ribavirin, rilpivirine, rilpivirine, rimantadine, rintatolimod, ritonavir, saquinavir, simeprevir, sofosbuvir, stavudine, taribavirin, telaprevir, telbivudine, tenofovir (e.g., tenofovir alafenamide or tenofovir disoproxil), tipranavir, trifluridine, tromantadine, umifenovir, valaciclovir, valganciclovir, vicriviroc, vidarabine, zalcitabine, zanamivir, and zidovudine, and any combination thereof.

[0259] In some embodiments, the additional antimicrobial agent is an antibacterial agent. Exemplary antibacterial agents include sulfonamides, amphenicols, spectinomycin, trimethoprim, glycylcyclines, macrolides (e.g., erythromycin, clarithromycin, azithromycin, roxithromycin), oxazolidinones (e.g., linezolid), tetracyclines (e.g., doxycycline, tetracycline, [W\]QgQZW\S$& q'ZOQbO[a #S(U(& ^S\WQWZZW\& [SbVWQWZZW\& QZ]fOQWZZW\$& QO`PO^S\S[a #S(U(& imipenem, meropenem, aztreonam), aminoglycosides (e.g., gentamicin, tobramycin, amikacin), quinolones and fluoroquinolones (e.g., levofloxacin, ciprofloxacin, moxifloxacin), glycopeptides (e.g., vancomycin), polymyxins (e.g., polymyxin, colistin).

[0260] In some embodiments, the second therapy includes immunotherapy. Immunotherapies include chimeric antigen receptor (CAR) T-cell or T-cell transfer therapies, cytokine therapy, immunomodulators, cancer vaccines, or administration of antibodies (e.g., monoclonal antibodies).

[0261] In some embodiments, the immunotherapy comprises administration of antibodies. The antibodies may target antigens either specifically expressed by tumor cells or antigens shared with normal cells. In some embodiments, the immunotherapy may comprise an antibody targeting, for example, CD20, CD33, CD52, CD30, HER (also referred to as erbB or EGFR), VEGF, CTLA-4 (also referred to as CD152), epithelial cell adhesion molecule (EpCAM, also referred to as CD326), and PD-1 / PD-L1. Suitable antibodies include, but are not limited to, rituximab, blinatumomab, trastuzumab, gemtuzumab, alemtuzumab, ibritumomab, tositumomab, bevacizumab, cetuximab, panitumumab, ofatumumab, ipilimumab, brentuximab, pertuzumab, and the like). In some embodiments, the additional therapeutic agent may comprise anti-PD-1 / PD-L1 antibodies, including, but not limited to, pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, and ipilimumab. The antibodies may also be linked to a chemotherapeutic agent. Thus, in some embodiments, the antibody is an antibody-drug conjugate.

[0262] The immunotherapy (e.g., administration of antibodies) may be administered to a subject by a variety of methods. In any of the uses or methods described herein, administration may be by various routes known to those skilled in the art, including without limitation oral, inhalation, intravenous, intramuscular, topical, subcutaneous, systemic, and / or intraperitoneal administration to a subject in need thereof. The immunotherapy may be administered by parenteral administration (including, but not limited to, subcutaneous, intramuscular, intravenous, intraperitoneal, intracardiac and intraarticular injections). In some embodiments, the immunotherapy may be administered in the same or different manner than the disclosed compounds or compositions. 5. Kits

[0263] In another aspect, the disclosure provides kits comprising at least one disclosed compound or a pharmaceutically acceptable salt thereof, or a composition comprising the compound or a pharmaceutically acceptable salt thereof, and instructions for using the compound or composition.

[0264] The kits can also comprise other agents and / or products co-packaged, co- formulated, and / or co-delivered with other components. For example, a drug manufacturer, a drug reseller, a physician, a compounding shop, or a pharmacist can provide a kit comprising a disclosed compound and / or product and another agent for delivery to a patient.

[0265] The kits can also comprise instructions for using the components of the kit. The instructions are relevant materials or methodologies pertaining to the kit. The materials may include any combination of the following: background information, list of components, brief or detailed protocols for using the compositions, trouble-shooting, references, technical support, and any other related documents. Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.

[0266] It is understood that the disclosed kits can be employed in connection with the disclosed methods. The kit may further contain containers or devices for use with the methods or compositions disclosed herein. The kits optionally may provide additional components such as buffers and disposable single-use equipment (e.g., pipettes, cell culture plates or flasks).

[0267] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Individual member components of the kits may be physically packaged together or separately. 6. Examples

[0268] Abbreviations used in the schemes and examples that follow are: AIBN is azobisisobutyronitrile; BuOK is potassium tert-butoxide; DCM is dichloromethane; DIPEA is N,N-diisopropylethylamine; DMA is dimethylacetamide; DMAP is 4- dimethylaminopyridine; DMF is dimethylformamide; DMSO is dimethyl sulfoxide; EA is ethyl acetate; Et is ethyl; EtOH is ethanol; Et3N is triethylamine; HATU is hexafluorophosphate azabenzotriazole tetramethyl uronium; i-Pr is iso-propyl; Me is methyl; MeOH is methanol; NBS is N-bromosuccinimide; NMO is N-methylmorpholine N-oxide; NXS is N-halogensuccinimide; OAc is acetyloxy, PCC is pyridinium chlorochromate; RT, rt, or r.t. is room temperature; TBAF is yetra-n-butylammonium fluoride; TBDMSCl is tert- butyldimethylsilyl chloride; TEA is triethanolamine; TFA is trifluoroacetic acid; and THF is tetrahydrofuran.

[0269] Unless specified otherwise, all the starting materials, reagents, and solvents are commercially available. All the reactions were monitored by thin-layer chromatography on silica gel plates (GF254) and visualized with UV light (254 and 365 nm). NMR spectra were recorded on Bruker 600 MHz NMR spectrometers. All chemical shifts are reported in parts per million (ppm). The following abbreviations were used to describe peak splitting patterns when appropriate: s (singlet), d (doublet), t (triplet), m (multiplet), dd (doublet of doublets). Coupling constants (J) are expressed in the hertz unit (Hz). Mass spectrum (MS) was obtained by ESI-MS (Skyray instrument, LC-MS 1000). The purity of all the target compounds were determined by HPLC (Shimadzu, method A: Aglient, C18, 2.1 × 50 mm, 5 µm; 5:95 (MeOH:H2O) over 3 min followed by gradient 5:95~75:35 (MeOH:H2O) for 2 min, then keeping 75:35 (MeOH:H2O) for 3 min followed by gradient 75:35~5:95 (MeOH:H2O); flow rate: 1 mL / min. method B: Sunfire, C18, 4.6 × 150 mm, 5 µM). Example 1 Compound Syntheses 1) Monomers Synthesis of 2a~c

[0270] Reagents and conditions: (i) paraformaldehyde, i-Pr2NH.TFA, TFA, 1,4-dioxane, 100 °C, 40 h; (ii) conc. H2SO4, 0°C~rt; (iii) AlCl3, DCM, 0°C~rt Synthesis of 11a~m and 12a~m

[0271] Reagents and conditions: (i) methyl thioglycolate, t-BuOK, DIPEA, DMA, 80°C; (ii) NBS, AIBN, CCl4, 80°C; (iii) NMO, CH3CN, 0°C~rt; (iv) 1) LiOH, EtOH, H2O, 0°C~rt; 2) HCl (aq, 3 M); (v) 1) R2NH2.HC1, TEA, DCM, NaBH3CN; 2) HATU, DIPEA, DCM; (vi) 1) LiOH, EtOH, H2O, rt; 2) HCl (aq, 1 M)Synthesis of 13a~c

[0272] Reagents and conditions: (i) HATU, DIPEA, DMF, rt; (ii) 1) HC1.NH2(CH2)2SO2NH2, K2CO3, NaI, DMF, rt; 2) EtOH, 80°C Synthesis of 15~16

[0273] Reagents and conditions: (i) 1) R2NH2w>9Z& H;7& :9B& CO8>3CN; 2) HATU, DIPEA, DCM; (ii) 1) LiOH, EtOH, H2O, rt; 2) HCl (aq, 1 M) Synthesis of 17a~b and 18a~b

[0274] Reagents and conditions: (i) NXS, CH3CN, 80°C; (ii) 1) LiOH, EtOH, H2O, rt; 2) HCl (aq, 1 M)Synthesis of 22a and 23

[0275] Reagents and conditions (i) HC1·H2N(CH2)2CO2R, HATU, DIPEA, DCM, rt; (ii) NBS, AIBN, CCl4, 80°C; (iii) NMO, CH3CN, 0°C~rt; (iv) PCC, DCM, rt; (v) TFA, DCM, rt Synthesis of 25, 26a~j, 27a~b and 28a~f

[0276] Reagents and conditions: (i) methyl thioglycolate, K2CO3, DIPEA, DMF, rt~80°C; (ii) formamide, formamidine acetate, 150°C; (iii) K2CO3, NaI, DMF, rt or 60°C; (iv) 1) LiOH, THF, H2O, rt; 2) HCl (aq, 1 M); (v) K2CO3, NaI, acetone, 60°C; (vi) EDC HCl, DMAP, rt Synthesis of 31~33

[0277] Reagents and conditions: (i) methyl thioglycolate, K2CO3, DIPEA, DMF, rt~80°C; (ii) formamide, formamidine acetate, 150°C; (iii) K2CO3, NaI, DMF, rt; (iv) 1) LiOH, THF, H2O, rt; 2) HCl (aq, 1 M) General procedure for the synthesis of intermediates 1a~c

[0278] To a glass tube were added MSA-2 (0.102 g, 0.35 mmol), paraformaldehyde (21.0 mg, 0.70 mmol), i-Pr2C>wH<7 #1 / (- [U& *(- / [[]Z$& +&.'RW]fO\S #, [A$ O\R H<7 #,(1 jA& 0.035 mmol). The tube was sealed and heated at 100 °C for 16 h. Then, another portion of paraformaldehyde (21.0 mg, 0.70 mmol) was added. After 24 h, the mixture was cooled to room temperature. And the solvent was removed under vacuum. The residue was isolated using a silica gel chromatography column (DCM:MeOH = 50:1~30:1) to afford 1a.

[0279] 3-(5,6-dimethoxybenzo[b]thiophene-2-carbonyl)but-3-enoic acid (1a) Yellow solid, 62% yield, 1H NMR (500 MHz, DMSO-d6$ o +,(,3 #a& +>$& 1(33 #a& +>$& 1( / 3 #a& +>$& 7.53 (s, 1H), 5.99 (d, J = 2.5 Hz, 2H), 3.85 (s, 3H), 3.81 (s, 3H), 3.44 (s, 2H). MS: calcd for C15H15O5S (M+H), 307.1; found, 307.2.

[0280] 4-(5,6-dimethoxybenzo[b]thiophene-2-carbonyl)pent-4-enoic acid (1b) Yellow solid, 45% yield, 1H NMR (600 MHz, DMSO-d6$ o +,(+- #a& +>$& 1(3. #a& +>$& 1(0+ #a& +>$&7.53 (s, 1H), 5.86 (d, J = 5.3 Hz, 2H), 3.87 (s, 3H), 3.82 (s, 3H), 2.62 (t, J = 7.5 Hz, 2H), 2.44 (t, J = 7.5 Hz, 2H). MS: calcd for C16H17O5S (M+H), 321.1; found, 321.2.

[0281] 5-(5,6-dimethoxybenzo[b]thiophene-2-carbonyl)hex-5-enoic acid (1c) 1c is very difficult to be separated from 4b, so it was used as the mixture in the next step. Yellow solid, 43% yield, MS: calcd for C17H19O5S (M+H), 335.1; found, 335.2. General procedure for the synthesis of intermediates 4a~4b

[0282] 3 (50.0 mg, 0.26 mmol) was dissolved in anhydrous DCM (1 mL). The solution was cooled to 0°C with an ice bath. Glutaric anhydride (44.5 mg, 0.39 mmol) was added portionwise. The mixture was allowed to warm to room temperature and stirred for 13 h. The solvent was removed under reduced pressure, and the residue was isolated using a silica gel chromatography column (DCM:MeOH:EA = 50:1:10) to give 4a.

[0283] 5-(5,6-dimethoxybenzo[b]thiophen-2-yl)-5-oxopentanoic acid (4a) White solid, 46% yield, 1H NMR (500 MHz, DMSO-d6$ o +,(*3 #a& +>$& 2(+. #a& +>$& 1( / 1 #a& +>$& 1(. / (s, 1H), 3.84 (s, 3H), 3.81 (s, 3H), 3.04 (t, J = 7.3 Hz, 2H), 2.30 (t, J = 7.4 Hz, 2H), 1.84 (p, J = 7.3 Hz, 2H). MS: calcd for C15H17O5S (M+H), 309.1; found, 309.2.

[0284] 6-(5,6-dimethoxybenzo[b]thiophen-2-yl)-6-oxohexanoic acid (4b) White solid, 18% yield, 1H NMR (600 MHz, DMSO-d6$ o +,(*, #a& +>$& 2(+2 #a& +>$& 1( / 3 #a& +>$& 1(.0 (s, 1H), 3.86 (s, 3H), 3.83 (s, 3H), 3.02 (t, J = 7.2 Hz, 2H), 2.26 (t, J = 7.3 Hz, 2H), 1.66 (p, J = 7.1 Hz, 2H), 1.57 (p, J = 7.0, 6.6 Hz, 2H). MS: calcd for C16H19O5S (M+H), 323.1; found, 323.1.Synthesis of intermediate 6 and 7

[0285] DIPEA (5.8 mL, 33.1 mmol) and methyl thioglycolate (3.6 mL, 39.7 mmol) were added into the solution of 5 (6.003 g, 33.1 mmol) in DMA (60 mL). The resulting solution was stirred at room temperature for 40 min under nitrogen atmosphere, then t-BuOK (5.570 g, 49.7 mmol) was added. The mixture was heated at 80°C for 4 h under nitrogen atmosphere. Then, the mixture was cooled with an ice bath, and ice water (150 mL) was added. The precipitate was filtered, washed with water, and dried to give intermediate 6. The pH of the filtrate was adjusted to 2 using aqueous HCl (3 M). The precipitate was filtered, washed with water, and dried to give intermediate 7.

[0286] Methyl 5,6-dimethoxy-3-methylbenzo[b]thiophene-2-carboxylate (6) Gray solid, 22% yield, 1H NMR (500 MHz, DMSO-d6$ o 1( / . #R& J = 0.8 Hz, 1H), 7.34 (d, J = 0.9 Hz, 1H), 3.85 (s, 3H), 3.84 (s, 3H), 3.82 (s, 3H), 2.68 (d, J = 0.6 Hz, 3H). MS: calcd for C13H15O4S (M+H), 267.1; found, 267.2.

[0287] 5,6-dimethoxy-3-methylbenzo[b]thiophene-2-carboxylic acid (7) Gray solid, 23% yield, MS: calcd for C12H11O4S (M-H), 251.0; found, 250.3. Synthesis of intermediate 8

[0288] To a flask were added 6 (1.005 g, 3.76 mmol), NBS (0.803 g, 4.51 mmol), AIBN (0.123 g, 0.75 mmol) and CCl4 (30 mL). The mixture was heated at 80°C for 2 h and cooled to room temperature. The solvent was removed, and the residue was isolated using a silica gel chromatography column (DCM:Hexane = 2:1~5:1) to give intermediate 8.

[0289] Methyl 3-(bromomethyl)-5,6-dimethoxybenzo[b]thiophene-2-carboxylate (8) Yellow solid, 90% yield, 1H NMR (500 MHz, DMSO-d6$ o 1(0, #a& +>$& 1( / + #a& +>$& / (-+ #a&2H), 3.87 (s, 3H), 3.87 (s, 3H), 3.85 (s, 3H). MS: calcd for C13H14BrO4S (M+H), 344.0; found, 345.1 (79Br), 347.2 (81Br). Synthesis of intermediate 9

[0290] The suspension of 8 (0.817 g, 2.37 mmol) in CH3CN (15 mL) was stirred with the cooling of an ice bath under nitrogen atmosphere. NMO was added (0.831 g, 7.10 mmol). The mixture was allowed to warm to room temperature. After 13 h, the solvent was removed under reduced pressure. To the residue was added ice water (20 mL). After the ice melted, the precipitate was filtered, washed with water, and dried to afford intermediate 9.

[0291] Methyl 3-formyl-5,6-dimethoxybenzo[b]thiophene-2-carboxylate (9) Yellow solid, 84% yield, 1H NMR (600 MHz, DMSO-d6$ o +*(1- #a& +>$& 2(*. #a& +>$& 1(1- #a& +>$& -(3 / (s, 3H), 3.88 (s, 3H), 3.86 (s, 3H). MS: calcd for C13H12O5SNa (M+H), 303.0; found, 303.0. Synthesis of intermediate 10

[0292] 9 (0.664 g, 2.37 mmol) was suspended in the mixed solvent of EtOH (8 mL) and H2O (4 mL). The suspension was cooled to 0°C with an ice bath, and LiOH (0.568 g, 23.7 mmol) was added. After 0.5 h, the ice bath was removed. The reaction completed after another 0.5 h stirring at room temperature. EtOH was removed under reduced pressure. The pH of the residue was adjusted to 1.5 using aqueous HCl (3 M). The precipitate was filtered, washed with water, and dried to give intermediate 10.

[0293] 3-formyl-5,6-dimethoxybenzo[b]thiophene-2-carboxylic acid (10) Yellow solid, 72% yield, 1H NMR (600 MHz, DMSO-d6$ o +*(12 #R& J = 1.5 Hz, 1H), 8.04 (d, J = 1.5 Hz, 1H), 7.71 (d, J = 1.6 Hz, 1H), 3.87 (d, J = 1.4 Hz, 3H), 3.85 (d, J = 1.4 Hz, 3H). MS: calcd for C12H9O5S (M-H), 265.0; found, 264.7. General procedure for the synthesis of intermediates 19a~b

[0294] To a flask were added 7 (0.200 g, 0.79 mmol), methyl 3-aminopropionate hydrochloride (0.166 g, 1.19 mmol), HATU (0.452 g, 1.19 mmol), DIPEA (346 µL, 1.98 mmol) and anhydrous DCM (3 mL). After being stirred at room temperature for 2 h, the reaction mixture was diluted with DCM (50 mL), washed with water (3 × 20 mL) and saturated brine (20 mL). The organic phase was evaporated under reduced pressure. The residue was dispersed in the mixed solvent of EtOH (4 mL) and H2O (2 mL) through sonication. The resulted slurry was filtered, washed with EtOH:H2O (1:1), and dried to give intermediate 19a.

[0295] Methyl 3-(5,6-dimethoxy-3-methylbenzo[b]thiophene-2-carboxamido)propanoate (19a) Pale yellow solid, 85% yield, 1H NMR (600 MHz, DMSO-d6$ o 2(+, #b& J = 5.6 Hz, 1H), 7.52 (d, J = 1.2 Hz, 1H), 7.28 (d, J = 1.1 Hz, 1H), 3.86 (d, J = 1.1 Hz, 3H), 3.84 (d, J = 1.2 Hz, 3H), 3.62 (d, J = 1.2 Hz, 3H), 3.51 – 3.46 (m, 2H), 2.60 (t, J = 6.9 Hz, 2H), 2.54 (d, J = 1.1 Hz, 3H). MS: calcd for C16H20NO5S (M+H), 338.1; found, 338.2.

[0296] Tert-butyl 3-(5,6-dimethoxy-3-methylbenzo[b]thiophene-2- carboxamido)propanoate (19b) Pale yellow solid, 93% yield, MS: calcd for C19H26NO5S (M+H), 380.2; found, 380.3. General procedure for the synthesis of intermediates 20a~b

[0297] Compounds 20a and 20b was prepared in a manner analogous to 8.

[0298] Methyl 3-(3-(bromomethyl)-5,6-dimethoxybenzo[b]thiophene-2- carboxamido)propanoate (20a) Yellow solid, 68% yield, 1H NMR (600 MHz, CDCl3$ o 1(,2 (s, 1H), 7.24 (s, 1H), 6.74 (t, J = 5.6 Hz, 1H), 5.13 (s, 2H), 4.03 (s, 3H), 3.99 (s, 3H), 3.77 (t,J = 4.7 Hz, 5H), 2.74 – 2.71 (m, 2H). MS: calcd for C16H19BrNO5S (M+H), 416.0; found, 416.1 (79Br), 418.1 (81Br).

[0299] Tert-butyl 3-(3-(bromomethyl)-5,6-dimethoxybenzo[b]thiophene-2- carboxamido)propanoate (20b) Yellow solid, 60% yield, MS: calcd for C19H25BrNO5S (M+H), 458.1; found, 458.1 (79Br), 460.2 (81Br). General procedure for the synthesis of intermediates 21a~b

[0300] Compound 21a was prepared in a manner analogous to 9. This compound is unstable, so it was directly used as mixture in the next step.

[0301] Methyl 3-(1-hydroxy-6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3- c]pyrrol-2-yl)propanoate (21a) Yellow solid, crude yield 52%, MS: calcd for C16H18NO6S (M+H), 352.1; found, 352.2.

[0302] Compound 21b was prepared in a manner analogous to 21a.

[0303] Tert-butyl 3-(1-hydroxy-6,7-dimethoxy-3-oxo-1,3-dihydro-2H- benzo[4,5]thieno[2,3-c]pyrrol-2-yl)propanoate (21b) Yellow solid, crude yield 62%, MS: calcd for C19H24NO6S (M+H), 394.1; found, 394.2. General procedure for the synthesis of target compounds 2a~c

[0304] The flask with 1a (44.4 mg, 0.14 mmol) was cooled to 0°C with an ice bath. Concentrated H2SO4 (0.3 mL), which was pre-cooled with an ice bath, was added. The mixture was stirred at 0°C for 1 h followed by another 2 h stirring at room temperature. Crushed ice was added (approximately 3 mL water). After the ice melted, the precipitate was filtered and washed with water. The filter cake was isolated with a column chromatographycolumn (DCM:MeOH = 50:1:~30:1) to give crude product which was further purified with preparative TLC (DCM:MeOH:EA = 13:1:2.5) to afford 2a.

[0305] 2-(6,7-dimethoxy-3-oxo-2,3-dihydro-1H-benzo[b]cyclopenta[d]thiophen-2- yl)acetic acid (2a). White solid, 12% yield, 1H NMR (500 MHz, DMSO-d6$ o +,(-, #a& +>$& 7.67 (s, 1H), 7.52 (s, 1H), 3.86 (s, 3H), 3.85 (s, 3H), 3.43 (dd, J = 17.5, 6.8 Hz, 1H), 3.28 – 3.24 (m, 1H), 2.89 (dd, J = 17.5, 3.0 Hz, 1H), 2.75 (dd, J = 16.8, 4.2 Hz, 1H), 2.60 (dd, J = 16.8, 8.7 Hz, 1H).13C NMR (150 MHz, DMSO-d6$ o +32( / & +1-( / & +0.(0& + / +( / & +.3(+& 141.5, 137.1, 127.9, 106.3, 105.5, 56.4, 56.3, 47.8, 35.5, 30.6. MS: calcd for C15H15O5S (M+H), 307.1; found, 307.1. HPLC: method A, tR = 5.578 min, 98% purity.

[0306] 3-(6,7-dimethoxy-3-oxo-2,3-dihydro-1H-benzo[b]cyclopenta[d]thiophen-2- yl)propanoic acid (2b) White solid, 13% yield, 1H NMR (500 MHz, DMSO-d6$ o 1(00 #a& 1H), 7.48 (s, 1H), 3.86 (s, 3H), 3.85 (s, 3H), 3.37 (d, J = 6.7 Hz, 1H), 3.03 (dddd, J = 9.2, 7.1, 5.2, 2.6 Hz, 1H), 2.84 (dd, J = 17.7, 2.6 Hz, 1H), 2.37 (dt, J = 8.7, 6.5 Hz, 2H), 2.11 – 2.01 (m, 1H), 1.75 – 1.65 (m, 1H). MS: calcd for C16H17O5S (M+H), 321.1; found, 321.1. HPLC: method A, tR = 5.657 min, 92% purity.

[0307] 4-(6,7-dimethoxy-3-oxo-2,3-dihydro-1H-benzo[b]cyclopenta[d]thiophen-2- yl)butanoic acid (2c) White solid, 15% yield, 1H NMR (500 MHz, DMSO-d6$ o +,(* / #a& 1H), 7.68 (s, 1H), 7.52 (s, 1H), 3.87 (s, 3H), 3.86 (s, 3H), 3.38 (dd, J = 17.6, 6.6 Hz, 1H), 3.02 (dddd, J = 9.3, 6.8, 4.4, 2.5 Hz, 1H), 2.87 (d, J = 2.5 Hz, 1H), 2.27 (td, J = 7.4, 3.4 Hz, 2H), 1.90 – 1.79 (m, 1H), 1.67 – 1.58 (m, 2H), 1.48 (dtd, J = 13.0, 9.5, 5.6 Hz, 1H). MS:calcd for C17H19O5S (M+H), 335.1; found, 335.1. HPLC: method A, tR = 5.707 min, 92% purity. General procedure for the synthesis of target compounds 11a~m

[0308] To a flask were added 10 (44.5 mg, 0.17 mmol), methyl glycinate hydrochloride (42.0 mg, 0.33 mmol), DCM (2 mL) and Et3N (46 µL, 0.33 mmol). Being stirred at room temperature for 12 h, the mixture was cooled to 0°C with an ice bath before the addition of NaBH3CN (15.8 mg, 0.25 mmol). The mixture was allowed to warm to room temperature. After 4 h, DCM (1 mL) was complemented, and the mixture was cooled to 0°C with an ice bath. HATU (76.2 mg, 0.20 mmol) and DIPEA (44 µL, 0.25 mmol) were added. The mixture was allowed to warm to room temperature and stirred for another 2 h. Then, the mixture was diluted with DCM (50 mL), washed with water (3 × 20 mL) and saturated brine (20 mL), and dried with anhydrous Na2SO4. The isolation using a silica gel chromatography column furnished 11a which was crystalized in EA (0.5 mL) to give pure product.

[0309] Methyl 2-(6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2- yl)acetate (11a) Yellow solid, 8% yield, 1H NMR (500 MHz, CDCl3$ o 1(-. #a& +>$& 1(+- #a& 1H), 4.63 (s, 2H), 4.43 (s, 2H), 4.00 (s, 3H), 3.97 (s, 3H), 3.79 (s, 3H).13C NMR (150 MHz, CDCl3$ o +03(2& +0 / (1& +.3(3& +.2(2& +.1(+& +-3(1& +-,(3& +,0(+& +* / (,& +*,(3& / 0(,& / 0(,& 52.4, 49.1, 44.3. MS: calcd for C15H16NO5S (M+H), 322.1; found, 322.1. HPLC: method B, 50% MeOH, tR = 5.945 min, >99% purity.

[0310] Methyl (S)-2-(6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3- c]pyrrol-2-yl)-3-hydroxypropanoate (11b) Yellow solid, 22% yield, 1H NMR (500 MHz, DMSO-d6$ o 1(1* #a& +>$& 1( / 1 #a& +>$& / (-- k / (,2 #[& +>$& .(3* #RR& J = 7.0, 4.0 Hz, 1H), 4.74 (d, J = 2.7 Hz, 2H), 4.01 – 3.97 (m, 1H), 3.94 – 3.90 (m, 1H), 3.86 (s, 3H), 3.84 (s, 3H),3.68 (s, 3H). MS: calcd for C16H18NO6S (M+H), 352.1; found, 352.1. HPLC: method B, 50% MeOH, tR = 5.093 min, 98% purity.

[0311] Methyl (R)-2-(6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3- c]pyrrol-2-yl)-3-hydroxypropanoate (11c) Yellow solid, 21% yield, 1H NMR (500 MHz, DMSO-d6$ o 1(1* #a& +>$& 1( / 1 #a& +>$& / (,3 #b& J = 5.5 Hz, 1H), 4.90 (dd, J = 7.0, 3.9 Hz, 1H), 4.74 (d, J = 2.7 Hz, 2H), 3.99 (ddd, J = 11.5, 7.0, 5.6 Hz, 1H), 3.91 (ddd, J = 11.5, 5.5, 4.0 Hz, 1H), 3.86 (s, 3H), 3.84 (s, 3H), 3.68 (s, 3H). MS: calcd for C16H18NO6S (M+H), 352.1; found, 352.1. HPLC: method B, 50% MeOH, tR = 5.188 min, 97% purity.

[0312] Methyl 3-(6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2- yl)propanoate (11d) Yellow solid, 32% yield, 1H NMR (500 MHz, DMSO-d6$ o 1(02 #a& +>$& 7.49 (s, 1H), 4.60 (s, 2H), 3.85 (s, 3H), 3.84 (s, 3H), 3.75 (t, J = 6.9 Hz, 2H), 3.61 (s, 3H), 2.70 (t, J = 6.9 Hz, 2H). MS: calcd for C15H18NO5S (M+H), 336.1; found, 336.2. HPLC: method B, 50% MeOH, tR = 7.872 min, 95% purity.

[0313] Methyl (S)-3-(6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3- c]pyrrol-2-yl)butanoate (11e) Yellow solid, 19% yield, 1H NMR (500 MHz, DMSO-d6$ o 7.67 (s, 1H), 7.48 (s, 1H), 4.60 – 4.50 (m, 3H), 3.84 (s, 3H), 3.82 (s, 3H), 3.55 (s, 3H), 2.70 (qd, J = 15.2, 7.3 Hz, 2H), 1.28 (d, J = 6.8 Hz, 3H). MS: calcd for C17H20NO5S (M+H), 350.1; found, 350.2. HPLC: method B, 50% MeOH, tR = 9.122 min, 97% purity.

[0314] Methyl (R)-3-(6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3- c]pyrrol-2-yl)butanoate (11f) Yellow solid, 20% yield, 1H NMR (500 MHz, DMSO-d6$ o 7.67 (s, 1H), 7.48 (s, 1H), 4.62 – 4.48 (m, 3H), 3.84 (s, 3H), 3.82 (s, 3H), 2.70 (qd, J = 15.3, 7.3 Hz, 2H), 1.28 (d, J = 6.8 Hz, 3H). MS: calcd for C17H20NO5S (M+H), 350.1; found, 350.2. HPLC: method B, 50% MeOH, tR = 9.171 min, 99% purity.

[0315] Methyl 1-((6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2- yl)methyl)cyclobutane-1-carboxylate (11g) Yellow solid, 23% yield, 1H NMR (500 MHz, DMSO-d6$ o 1(01 #a& +>$& 1( / , #a& +>$& .(.2 #a& ,>$& -(3* #a& ,>$& -(2- #a& ->$& -(2- #a& ->$& 3.65 (s, 3H), 2.34 (ddd, J = 12.2, 9.4, 6.9 Hz, 2H), 2.11 (ddd, J = 11.9, 8.8, 5.5 Hz, 2H), 1.97 (dq, J = 9.8, 7.5 Hz, 1H), 1.88 – 1.79 (m, 1H). MS: calcd for C19H22NO5S (M+H), 376.1; found, 376.1. HPLC: method B, 60% MeOH, tR = 8.044 min, 96% purity.

[0316] Methyl 3-(6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2- yl)-2,2-dimethylpropanoate (11h) Pale yellow solid, 27% yield, 1H NMR (500 MHz, DMSO- d6$ o 1(01 #a& +>$& 1( / , #a& +>$& .( / , #a& ,>$& -(2- #a& ->$& -(2- #a& ->$& -(02 #a& ->$& -(0 / #a& 2H), 1.18 (s, 6H). MS: calcd for C18H22NO5S (M+H), 364.1; found, 364.1. HPLC: method B, 50% MeOH, tR = 9.946 min, >99% purity.

[0317] Methyl (1r,3r)-3-(6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3- c]pyrrol-2-yl)cyclobutane-1-carboxylate (11i) Yellow solid, 20% yield, 1H NMR (500 MHz,DMSO-d6$ o 1(02 #a& +>$& 1(.1 #a& +>$& .(3, k .(2- #[& +>$& .(1- #a& ,>$& -(2. #a& ->$& -(2- (s, 3H), 3.66 (s, 3H), 3.11 – 3.02 (m, 1H), 2.69 – 2.61 (m, 2H), 2.47 – 2.40 (m, 2H). MS: calcd for C18H20NO5S (M+H), 362.1; found, 362.1. HPLC: method B, 60% MeOH, tR = 5.650 min, >99% purity.

[0318] Methyl (1s,3s)-3-(6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3- c]pyrrol-2-yl)cyclobutane-1-carboxylate (11j) Yellow solid, 15% yield, 1H NMR (500 MHz, DMSO-d6$ o 1(01 #a& +>$& 1( / / #a& +>$& .(02 #R& J = 3.9 Hz, 3H), 3.83 (s, 3H), 3.83 (s, 3H), 3.62 (s, 3H), 2.93 (p, J = 9.0 Hz, 1H), 2.45 (t, J = 8.8 Hz, 4H). MS: calcd for C18H20NO5S (M+H), 362.1; found, 362.1. HPLC: method B, 50% MeOH, tR = 7.569 min, >99% purity.

[0319] Methyl 3-(6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2- yl)benzoate (11k) Yellow solid, 21% yield, 1H NMR (500 MHz, DMSO-d6$ o 2(-3 k 2(-2 #[& 1H), 8.17 (ddd, J = 8.3, 2.4, 1.1 Hz, 1H), 7.74– 7.71 (m, 2H), 7.61 (s, 1H), 7.58 (t, J = 8.0 Hz, 1H), 5.19 (s, 2H), 3.88 (s, 3H), 3.86 (s, 3H), 3.86 (s, 3H). MS: calcd for C20H18NO5S (M+H), 384.1; found, 384.2. HPLC: method B, 70% MeOH, tR = 6.781 min, 97% purity.

[0320] Methyl 4-(6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2- yl)butanoate (11l) Yellow solid, 16% yield, 1H NMR (500 MHz, DMSO-d6$ o 1(01 #a& +>$& 7.47 (s, 1H), 4.56 (s, 2H), 3.83 (s, 3H), 3.82 (s, 3H), 3.51 (d, J = 5.7 Hz, 5H), 2.35 (t, J = 7.3 Hz, 2H), 1.86 (p, J = 7.1 Hz, 2H). MS: calcd for C17H20NO5S (M+H), 350.1; found, 350.1. HPLC: method B, 50% MeOH, tR = 9.258 min, 97% purity.

[0321] Methyl 5-(6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2- yl)pentanoate (11m) Yellow solid, 44% yield, 1H NMR (500 MHz, DMSO-d6$ o 1(01 #a& +>$& 7.47 (s, 1H), 4.56 (s, 2H), 3.83 (s, 3H), 3.82 (s, 3H), 3.56 (s, 3H), 3.49 (t, J = 6.7 Hz, 2H), 2.35 (t, J = 7.3 Hz, 2H), 1.61 (tt, J = 9.0, 6.5 Hz, 2H), 1.52 (dq, J = 10.2, 7.2 Hz, 2H). MS: calcd for C18H22NO5S (M+H), 364.1; found, 364.2. HPLC: method B, 60% MeOH, tR = 4.419 min, 98% purity. General procedure for the synthesis of target compounds 12a~m

[0322] To the mixture of 11a (13.5 mg, 0.042 mmol), EtOH (200 µL) and H2O (100 µL) was added LiOH (10.0 mg, 0.42 mmol). The mixture was stirred at room temperature for 0.5 h. The pH was adjusted to 2 using aqueous HCl (1 M). The crystal formed was filtered, washed with water, and dried to give 12a.

[0323] 2-(6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2-yl)acetic acid (12a) Yellow solid, 90% yield, 1H NMR (500 MHz, DMSO-d6$ o +,(30 #a& +>$& 1(1* #a& 1H), 7.52 (s, 1H), 4.64 (s, 2H), 4.27 (s, 2H), 3.86 (s, 3H), 3.84 (s, 3H).13C NMR (150 MHz, DMSO-d6$ o +1+(-& +0 / (-& + / *(*& +.3(*& +.2(1& +-2(0& +-,(+& +,0(.& +*0(.& +*.(2& / 0(-& / 0(,& 49.7, 44.5. MS: calcd for C14H14NO5S (M+H), 308.1; found, 308.2. HPLC: method A, tR = 5.047 min, 95% purity.

[0324] (S)-2-(6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2-yl)- 3-hydroxypropanoic acid (12b) White solid, 82% yield, 1H NMR (500 MHz, DMSO-d6$ o 13.04 (s, 1H), 7.70 (s, 1H), 7.57 (s, 1H), 4.79 (dd, J = 7.3, 3.9 Hz, 1H), 4.74 (d, J = 2.6 Hz, 2H), 3.98 (dd, J = 11.5, 7.4 Hz, 1H), 3.90 (dd, J = 11.5, 3.9 Hz, 1H), 3.86 (s, 3H), 3.84 (s,3H).13C NMR (150 MHz, DMSO-d6$ o +1+(1& +0 / (0& + / *(*& +.3(+& +.3(*& +-2(0& +-,(*& 126.4, 106.3, 104.8, 60.7, 57.2, 56.3, 56.2, 47.6. MS: calcd for C15H16NO6S (M+H), 338.1; found, 338.2. HPLC: method A, tR = 4.858 min, 92% purity.

[0325] (R)-2-(6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2-yl)- 3-hydroxypropanoic acid (12c) White solid, 87% yield, 1H NMR (500 MHz, DMSO-d6$ o 13.12 (s, 1H), 7.70 (s, 1H), 7.57 (s, 1H), 5.25 (s, 1H), 4.79 (dd, J = 7.4, 3.9 Hz, 1H), 4.74 (d, J = 2.5 Hz, 2H), 3.98 (dd, J = 11.5, 7.3 Hz, 1H), 3.90 (dd, J = 11.5, 3.9 Hz, 1H), 3.86 (s, 3H), 3.84 (s, 3H).13C NMR (150 MHz, DMSO-d6$ o +1+(1& +0 / (0& + / *(*& +.3(+& +.3(*& +-2(0& 132.0, 126.4, 106.3, 104.8, 60.7, 57.2, 56.3, 56.2, 47.6. MS: calcd for C15H16NO6S (M+H), 338.1; found, 338.1. HPLC: method A, tR = 4.793 min, 93% purity.

[0326] 3-(6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2- yl)propanoic acid (12d) Yellow solid, 96% yield, 1H NMR (500 MHz, DMSO-d6$ o +,(-. #a& 1H), 7.68 (s, 1H), 7.50 (s, 1H), 4.61 (s, 2H), 3.85 (s, 3H), 3.84 (s, 3H), 3.71 (t, J = 6.9 Hz, 2H), 2.61 (t, J = 6.9 Hz, 2H).13C NMR (150 MHz, DMSO-d6$ o +1-(-& +0.(2& +.3(3& +.3(*& 148.0, 138.4, 132.7, 126.4, 106.4, 104.7, 56.3, 56.2, 49.3, 39.3, 33.7. MS: calcd for C15H16NO5S (M+H), 322.1; found, 322.2. HPLC: method A, tR = 5.350 min, 99% purity.

[0327] (S)-3-(6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2- yl)butanoic acid (12e) 1H NMR (500 MHz, DMSO-d6$ o +,(,2 #a& +>$& 1(01 #a& +>$& 1(.3 #a& 1H), 4.61 – 4.49 (m, 3H), 3.83 (s, 3H), 3.82 (s, 3H), 2.61 (qd, J = 15.3, 7.3 Hz, 2H), 1.27 (d, J = 6.8 Hz, 3H).13C NMR (150 MHz, DMSO-d6$ o +1,(2& +0.(-& +.3(3& +.2(3& +.2(*& +-2(-& 132.9, 126.5, 106.4, 104.6, 56.3, 56.2, 45.6, 45.3, 19.3 (one carbon atom was overlapped withsolvent peak). MS: calcd for C16H18NO5S (M+H), 336.1; found, 336.1. HPLC: method A, tR = 5.480 min, 99% purity.

[0328] (R)-3-(6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2- yl)butanoic acid (12f) Yellow solid, 31% yield, 1H NMR (500 MHz, DMSO-d6$ o +,(,2 #a& 1H), 7.67 (s, 1H), 7.49 (s, 1H), 4.61 – 4.48 (m, 3H), 3.83 (s, 3H), 3.82 (s, 3H), 2.61 (qd, J = 15.3, 7.3 Hz, 2H), 1.27 (d, J = 6.8 Hz, 3H).13C NMR (150 MHz, DMSO-d6$ o +1,(2& +0.(-& 149.9, 148.9, 148.0, 138.3, 132.9, 126.5, 106.4, 104.6, 56.3, 56.2, 45.6, 45.3, 19.2 (one carbon atom was overlapped with solvent peak). MS: calcd for C16H18NO5S (M+H), 336.1; found, 336.1. HPLC: method A, tR = 5.390 min, 99% purity.

[0329] 1-((6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2- yl)methyl)cyclobutane-1-carboxylic acid (12g) Yellow solid, 53% yield, 1H NMR (500 MHz, DMSO-d6$ o +,(. / #a& +>$& 1(01 #a& +>$& 1( / * #a& +>$& .( / , #a& ,>$& -(22 #a& ,>$& -(2- #a& ->$& 3.82 (s, 3H), 2.33 (ddd, J = 12.0, 9.4, 6.7 Hz, 2H), 2.12 – 2.03 (m, 2H), 1.99 – 1.89 (m, 1H), 1.88 – 1.78 (m, 1H).13C NMR (150 MHz, DMSO-d6$ o +11( / & +0 / ( / & +.3(3& +.3(*& +.2(*& 138.5, 132.4, 126.3, 106.3, 104.8, 56.3, 56.2, 50.0, 49.1, 48.1, 28.6 (2C), 15.6. MS: calcd for C18H20NO5S (M+H), 362.1; found, 362.2. HPLC: method A, tR = 5.029 min, 97% purity.

[0330] 3-(6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2-yl)-2,2- dimethylpropanoic acid (12h) Yellow solid, 82% yield, 1H NMR (500 MHz, DMSO-d6$ o 12.48 (s, 1H), 7.67 (s, 1H), 7.48 (s, 1H), 4.57 (s, 2H), 3.83 (s, 3H), 3.82 (s, 3H), 3.64 (s, 2H), 1.15 (s, 6H).13C NMR (150 MHz, DMSO-d6$ o +12(0& +0 / (2& +.3(3& +.3(*& +.2(,& +-2( / &132.4, 126.3, 106.4, 104.7, 56.3, 56.2, 51.7, 50.9, 43.7, 23.9 (2C). MS: calcd for C17H20NO5S (M+H), 350.1; found, 350.2. HPLC: method A, tR = 4.871 min, 93% purity.

[0331] (1r,3r)-3-(6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2- yl)cyclobutane-1-carboxylic acid (12i) Yellow solid, 98% yield, 1H NMR (500 MHz, DMSO- d6$ o +,(-, #a& +>$& 1(02 #a& +>$& 1(.1 #a& +>$& .(3+ k .(2+ #[& +>$& .(1, #a& ,>$& -(2. #a& ->$& 3.83 (s, 3H), 3.00 – 2.91 (m, 1H), 2.66 – 2.56 (m, 2H), 2.42 (ddd, J = 14.3, 9.2, 3.2 Hz, 2H). 13C NMR (150 MHz, DMSO-d6$ o +11(*& +0.(.& +.3(3& +.2(3& +.2(+& +-2( / & +-,(0& +,0(.& 106.4, 104.5, 56.3, 56.2, 45.5, 45.4, 32.3, 31.6 (2C). MS: calcd for C17H18NO5S (M+H), 348.1; found, 348.1. HPLC: method A, tR = 5.609 min, 94% purity.

[0332] (1s,3s)-3-(6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2- yl)cyclobutane-1-carboxylic acid (12j) Yellow solid, 78% yield, 1H NMR (500 MHz, DMSO-d6$ o +,(, / #a& +>$& 1(01 #a& +>$& 1( / 0 #a& +>$& .(01 #R& J = 11.3 Hz, 3H), 3.83 (s, 3H), 3.83 (s, 3H), 2.82 (p, J = 8.9 Hz, 1H), 2.43 (td, J = 9.3, 4.9 Hz, 4H).13C NMR (150 MHz, DMSO-d6$ o +1 / (3& +0.(.& +.3(3& +.2(3& +.2(,& +-2( / & +-,( / & +,0( / & +*0(-& +*.(2& / 0(-& / 0(,& 45.4, 42.6, 32.3 (2C), 30.9. MS: calcd for C17H18NO5S (M+H), 348.1; found, 348.1. HPLC: method A, tR = 5.564 min, 96% purity.

[0333] 3-(6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2- yl)benzoic acid (12k) Yellow solid, 75% yield, 1H NMR (500 MHz, DMSO-d6$ o +-(*2 #a& 1H), 8.35 (t, J = 2.0 Hz, 1H), 8.17 (ddd, J = 8.2, 2.4, 1.0 Hz, 1H), 7.74 (s, 1H), 7.70 (dt, J = 7.6, 1.2 Hz, 1H), 7.62 (s, 1H), 7.55 (t, J = 7.9 Hz, 1H), 5.20 (s, 2H), 3.86 (s, 3H), 3.85 (s,3H). MS: calcd for C19H16NO5S (M+H), 370.1; found, 370.1. HPLC: method A, tR = 5.870 min, 91% purity.

[0334] 4-(6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2- yl)butanoic acid (12l) Yellow solid, 54% yield, 1H NMR (500 MHz, DMSO-d6$ o +,(*0 #a& 1H), 7.67 (s, 1H), 7.48 (s, 1H), 4.57 (s, 2H), 3.83 (s, 3H), 3.82 (s, 3H), 3.51 (t, J = 6.9 Hz, 2H), 2.25 (t, J = 7.2 Hz, 2H), 1.83 (p, J = 7.1 Hz, 2H).13C NMR (150 MHz, DMSO-d6$ o 174.5, 165.0, 149.8, 148.9, 147.9, 138.4, 132.8, 126.5, 106.4, 104.7, 56.3, 56.2, 48.8, 42.4, 31.4, 24.1. MS: calcd for C16H18NO5S (M+H), 336.1; found, 336.1. HPLC: method A, tR = 4.712 min, 97% purity.

[0335] 5-(6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2- yl)pentanoic acid (12m) Yellow solid, 75% yield, 1H NMR (600 MHz, DMSO-d6$ o +,(*, #a& 1H), 7.69 (s, 1H), 7.49 (s, 1H), 4.58 (s, 2H), 3.85 (s, 3H), 3.84 (s, 3H), 3.51 (t, J = 6.9 Hz, 2H), 2.27 (t, J = 7.3 Hz, 2H), 1.66 – 1.60 (m, 2H), 1.54 – 1.48 (m, 2H).13C NMR (150 MHz, DMSO-d6$ o +1.(2& +0.(3& +.3(2& +.2(3& +.1(2& +-2(.& +-,(3& +,0( / & +*0(.& +*.(0& / 0(-& / 0(,& 48.7, 42.4, 33.7, 28.1, 22.3. MS: calcd for C17H20NO5S (M+H), 350.1; found, 350.2. HPLC: method A, tR = 5.623 min, 93% purity. General procedure for the synthesis of target compounds 13a~c

[0336] To a flask were added 12d (10.0 mg, 0.031 mmol), HATU (23.6 mg, 0.062 mmol), NH4Cl (8.3 mg, 0.155 mmol), DMF (0.5 mL) and DIPEA (16 µL, 0.093 mmol). The mixture was stirred at room temperature for 3 h. The solvent was removed under vacuum. The residue was isolated on a silica gel chromatography column (DCM:MeOH = 50:1~20:1). The crude product was triturated in the mixed solvent of EtOH (300 µL) and H2O (300 µL). Then the precipitate was filtered, washed with EtOH:H2O (1:1), and dried to give 13a.

[0337] 3-(6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2- yl)propenamide (13a) Yellow solid, 40% yield, 1H NMR (500 MHz, DMSO-d6$ o 1(00 #a& 1H), 7.49 (s, 1H), 7.41 (s, 1H), 6.85 (s, 1H), 4.58 (s, 2H), 3.83 (s, 3H), 3.82 (s, 3H), 3.69 (t, J = 6.7 Hz, 2H), 2.41 (t, J = 6.7 Hz, 2H).13C NMR (150 MHz, DMSO-d6$ o +1,(3& +0.(2& 149.8, 149.0, 148.0, 138.3, 132.8, 126.4, 106.4, 104.6, 56.3, 56.2, 49.5, 35.0 (one carbon atom was overlapped with solvent peak). MS: calcd for C15H17N2O4S (M+H), 321.1; found, 321.2. HPLC: method B, 45% MeOH, tR = 3.143 min, 97% purity.

[0338] 3-(6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2-yl)-N- methoxypropanamide (13b) Yellow solid, 60% yield, 1H NMR (500 MHz, DMSO-d6$ o 11.09 (s, 1H), 7.67 (s, 1H), 7.50 (s, 1H), 4.57 (s, 2H), 3.83 (s, 3H), 3.82 (s, 3H), 3.70 (t, J = 6.7 Hz, 2H), 3.52 (s, 3H), 2.31 (t, J = 6.7 Hz, 2H). 13C NMR (150 MHz, DMSO-d6$ o +01(1& 164.8, 149.9, 149.0, 148.0, 138.4, 132.7, 126.4, 106.4, 104.7, 63.6, 56.3, 56.2, 49.5, 32.5 (one carbon atom was overlapped with solvent peak). MS: calcd for C16H19N2O5S (M+H), 351.1; found, 351.2. HPLC: method B, 50% MeOH, tR = 4.324 min, 92% purity.

[0339] 2-(3-((isopropylamino)oxy)-3-oxopropyl)-6,7-dimethoxy-1,2-dihydro-3H- benzo[4,5]thieno[2,3-c]pyrrol-3-one (13c) Yellow solid, 50% yield, 1H NMR (500 MHz, DMSO-d6$ o 1(01 #a& +>$& 1(0, #R& J = 5.6 Hz, 1H), 7.47 (s, 1H), 4.60 (s, 2H), 3.83 (s, 3H), 3.83 (s, 3H), 3.75 (t, J = 6.8 Hz, 2H), 2.72 (t, J = 6.8 Hz, 2H), 0.93 (d, J = 6.3 Hz, 6H).13C NMR (150 MHz, DMSO-d6$ o +1+(0& +0.(3& +.3(3& +.3(*& +.2(+& +-2(.& +-,(0& +,0(.& +*0(.& 104.6, 56.3, 56.2, 51.5, 49.3, 39.3, 32.2, 20.0 (2C). MS: calcd for C18H23N2O5S (M+H), 379.1; found, 379.1. HPLC: method B, 50% MeOH, tR = 10.120 min, 94% purity.Synthesis of target compound 13d

[0340] To a flask were added 8 (64.6 mg, 0.19 mmol), 3-aminopropylsulphonamide hydrochloride (60.1 mg, 0.38 mmol), K2CO3 (51.6 mg, 0.38 mmol), NaI (catalytic amount) and DMF (3 mL). The mixture was stirred at room temperature for 12 h. The solvent was removed under vacuum. The residue was dispersed in EtOH (2 mL) and heated at 80°C for 3 h. The solvent was removed under reduced pressure. The residue was isolated on a silica gel chromatography column (DCM:MeOH = 50:1~30:1) to furnish crude product which was triturated in the mixed solvent of EtOH (200 µL) and H2O (200 µL). The slurry was filtered, washed with EtOH:H2O (1:1), and dried to afford 13d.

[0341] 2-(6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2- yl)ethane-1-sulfonamide (13d) Yellow solid, 24% yield, 1H NMR (500 MHz, DMSO-d6$ o 7.68 (s, 1H), 7.47 (s, 1H), 6.98 (s, 2H), 4.66 (s, 2H), 3.89 (dd, J = 8.1, 6.2 Hz, 2H), 3.84 (s, 3H), 3.83 (s, 3H), 3.37 – 3.32 (m, 2H).13C NMR (150 MHz, DMSO-d6$ o +0 / (*& + / *(*& 149.0, 148.3, 138.5, 132.3, 126.4, 106.4, 104.6, 56.3, 56.2, 53.2, 49.4, 38.6. MS: calcd for C14H17N2O5S2 (M+H), 357.1; found, 357.2. HPLC: method B, 45% MeOH, tR = 4.933 min, 99% purity.

[0342] Methyl 3-(6,7-dimethoxy-3-oxo-3,4-dihydropyrrolo[3,4-b]indol-2(1H)- yl)propanoate (15) The synthesis of 15 is the same as 11a. Yellow solid, 43% yield, 1H NMR (500 MHz, DMSO-d6$ o ++( / + #a& +>$& 1(+, #a& +>$& 0(22 #a& +>$& .(-1 #a& ,>$& -(12 #a& ->$& 3.75 (s, 3H), 3.70 (t, J = 7.0 Hz, 2H), 3.59 (s, 3H), 2.66 (t, J = 7.0 Hz, 2H). MS: calcd for C16H19N2O5 (M+H), 319.1; found, 319.1. HPLC: method B, 50% MeOH, tR = 5.743 min, 96% purity.

[0343] 3-(6,7-dimethoxy-3-oxo-3,4-dihydropyrrolo[3,4-b]indol-2(1H)-yl)propanoic acid (16) The synthesis of 16 is the same as 12a. Yellow solid, 71% yield, 1H NMR (500 MHz, DMSO-d6$ o +,(-* #a& +>$& ++( / + #a& +>$& 1(+, #a& +>$& 0(22 #a& +>$& .(-2 #a& ,>$& -(11 #a& 3H), 3.75 (s, 3H), 3.67 (t, J = 7.0 Hz, 2H), 2.57 (t, J = 7.0 Hz, 2H).13C NMR (150 MHz, DMSO-d6$ o +1-(.& +0,(0& +.2(1& +. / (0& +-0( / & +--(*& +, / (3& ++.(1& +*,(*& 30(,& / 0(,& / / (3& 46.6, 39.3, 34.0. MS: calcd for C15H17N2O5 (M+H), 305.1; found, 305.1. HPLC: method A, tR = 5.257 min, 94% purity. General procedure for the synthesis of target compounds 17a~b

[0344] To a flask were added 11d (20.0 mg, 0.06 mmol), NBS (16.0 mg, 0.09 mmol) and CH3CN (0.3 mL). The mixture was heated at 80°C for 1 h. The mixture was evaporated under reduced pressure and isolated on a silica gel chromatography column (hexane:EA = 1:1) to give product which was crystalized with EtOH (0.3 mL) to afford 17a.

[0345] Methyl 3-(8-bromo-6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3- c]pyrrol-2-yl)propanoate (17a) Yellow solid, 81% yield, 1H NMR (500 MHz, DMSO-d6$ o 7.87 (s, 1H), 4.80 (s, 2H), 3.91 (s, 3H), 3.78 (s, 3H), 3.74 (t, J = 7.0 Hz, 2H), 3.59 (s, 3H), 2.71 (t, J = 7.0 Hz, 2H). MS: calcd for C16H17BrNO5S (M+H), 414.0; found, 414.0 (79Br), 416.1 (81Br). HPLC: method B, 60% MeOH, tR = 11.178 min, 94% purity.

[0346] Methyl 3-(8-chloro-6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3- c]pyrrol-2-yl)propanoate (17b) Yellow solid, 45% yield, 1H NMR (500 MHz, DMSO-d6$ o 7.84 (s, 1H), 4.78 (s, 2H), 3.91 (s, 3H), 3.80 (s, 3H), 3.74 (t, J = 7.0 Hz, 2H), 3.59 (s, 3H),2.71 (t, J = 7.0 Hz, 2H). MS: calcd for C16H17ClNO5S (M+H), 370.1; found, 370.2 (35Cl), 372.3 (37Cl). HPLC: method B, 60% MeOH, tR = 10.918 min, 95% purity.

[0347] 3-(8-bromo-6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol- 2-yl)propanoic acid (18a) The synthesis of 18a is the same as 12a. Yellow solid, 75% yield, 1H NMR (500 MHz, DMSO-d6$ o +,(-. #a& +>$& 1(22 #a& +>$& .(2* #a& ,>$& -(3+ #a& ->$& -(12 (s, 3H), 3.72 (t, J = 7.0 Hz, 2H), 2.62 (t, J = 7.0 Hz, 2H).13C NMR (150 MHz, DMSO-d6$ o 173.4, 164.1, 153.2, 147.4, 145.8, 141.9, 134.6, 127.0, 111.0, 107.4, 60.9, 57.0, 50.8, 39.4, 33.6. MS: calcd for C15H15BrNO5S (M+H), 400.0; found, 400.1 (79Br), 402.2 (81Br). HPLC: method A, tR = 5.810 min, 98% purity.

[0348] 3-(8-chloro-6,7-dimethoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2- yl)propanoic acid (18b) The synthesis of 18b is the same as 12a. Yellow solid, 50% yield, 1H NMR (500 MHz, DMSO-d6$ o +,(-0 #a& +>$& 1(20 #a& +>$& .(2* #a& ,>$& -(3. #a& ->$& -(2- #a& 3H), 3.74 (t, J = 7.0 Hz, 2H), 2.64 (t, J = 7.0 Hz, 2H).13C NMR (150 MHz, DMSO-d6$ o 173.4, 164.0, 153.5, 146.5, 144.7, 141.7, 134.4, 125.4, 121.5, 106.8, 61.1, 57.0, 50.5, 39.3, 33.5. MS: calcd for C15H15ClNO5S (M+H), 356.0; found, 356.1 (35Cl), 358.2 (37Cl). General procedure for the synthesis of target compounds 22a-b

[0349] To a flask were added 21a (crude, 0.203 g, 0.58 mmol), DCM (3 mL) and PCC (0.250 g, 1.16 mmol). The mixture was stirred at room temperature for 16 h, and evaporated under reduced pressure. The residue was isolated using flash silica gel chromatography column (hexane:EA = 3:1) to give product which was triturated in the mixed solvent of EA (2 mL) and hexane (1 mL). The precipitate was filtered, washed with EA:hexane (1:1), and dried to give 22a.

[0350] Methyl 3-(6,7-dimethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3- c]pyrrol-2-yl)propanoate (22a) Yellow solid, 26% yield, 1H NMR (500 MHz, DMSO-d6$ o 7.88 (s, 1H), 7.43 (s, 1H), 3.90 (s, 3H), 3.89 (s, 3H), 3.79 (t, J = 7.1 Hz, 2H), 3.60 (s, 3H), 2.69 (t, J = 7.1 Hz, 2H).139 CBF #+ / * B>h& :BGD$ o +1+(0& +0.(-& +0-( / & + / *(3& + / *( / & 141.0, 138.9, 137.7, 124.4, 106.5, 103.4, 56.4, 56.2, 52.0, 34.3, 33.0. MS: calcd for C16H16NO6S (M+H), 350.1; found, 350.2. HPLC: method B, 70% MeOH, tR = 5.181 min, 96% purity.

[0351] Tert-butyl 3-(6,7-dimethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3- c]pyrrol-2-yl)propanoate (22b) Yellow solid, 67% yield, 1H NMR (500 MHz, DMSO-d6$ o 7.87 (s, 1H), 7.42 (s, 1H), 3.89 (s, 3H), 3.88 (s, 3H), 3.74 (t, J = 7.0 Hz, 2H), 2.57 (t, J = 7.0 Hz, 2H), 1.33 (s, 9H). MS: calcd for C19H22NO6S (M+H), 392.1; found, 392.2. Synthesis of target compound 23

[0352] To the solution of 22b (0.102 g, 0.26 mmol) in DCM (2 mL) was added TFA (0.5 mL). The resulting solution was stirred at room temperature for 3 h. The solvent was removed. The residue was triturated in EA (1 mL), filtered and dried to furnish 23.

[0353] 3-(6,7-dimethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2- yl)propanoic acid (23) Yellow solid, 76% yield, 1H NMR (500 MHz, DMSO-d6$ o +,(-0 #a& 1H), 7.86 (s, 1H), 7.42 (s, 1H), 3.89 (s, 3H), 3.88 (s, 3H), 3.75 (t, J = 7.3 Hz, 2H), 2.60 (t, J = 7.3 Hz, 2H).139 CBF #+ / * B>h& :BGD$ o +1,(0& +0.(.& +0-(0& + / *(3& + / *( / & +.+(*& +-2(3&137.7, 124.4, 106.5, 103.5, 56.5, 56.2, 34.4, 33.2. MS: calcd for C15H14NO6S (M+H), 336.1; found, 336.1. HPLC: method A, tR = 5.787 min, 98% purity.

[0354] 3-(6,7-dimethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2- yl)propenamide (ZSA-52NC1) The synthesis of ZSA-52NC1 is the same as 13a. Yellow solid, 56% yield, 1H NMR (600 MHz, DMSO-d6$ o 1(21 #a& +>$& 1(., #a& ,>$& 0(2 / #a& +>$& 3.89 (s, 3H), 3.89 (s, 3H), 3.73 (t, J = 7.3 Hz, 2H), 2.42 (t, J = 7.3 Hz, 2H). 13C NMR (150 B>h& :BGD$ o +1,(+& +0.(.& +0-(0& + / *(2& + / *( / & +.*(3& +-3(*& +-1(2& +,.( / & +*0( / & +*-( / & 56.5, 56.2, 35.1, 34.4. MS: calcd for C15H14N2O5S (M+H), 335.1; found, 335.2. HPLC: method B, 50% MeOH, tR = 6.998 min, 95% purity.

[0355] 3-(6,7-dimethoxy-1,3-dioxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2-yl)- N-methylpropanamide (ZSA-52NC2) The synthesis of ZSA-52NC1 is the same as 13a. Yellow solid, 51% yield, 1H NMR (600 MHz, DMSO-d6$ o 1(3+ k 1(22 #[& +>$& 1(21 #a& +>$& 7.43 (s, 1H), 3.89 (s, 3H), 3.89 (s, 3H), 3.73 (t, J = 7.3 Hz, 2H), 2.53 (d, J = 4.6 Hz, 3H), 2.42 (t, J = 7.3 Hz, 2H).13C NMR (150 MHz, DMSO-d6$ o +1*(.& +0.(.& +0-(0& + / *(2& + / *( / & 140.9, 139.0, 137.8, 124.5, 106.5, 103.5, 56.5, 56.2, 35.2, 34.6, 25.9. MS: calcd for C16H17N2O5S (M+H), 349.1; found, 349.1. HPLC: method B, 50% MeOH, tR = 8.337 min, 92% purity.

[0356] 2-morpholinoethyl 3-(6,7-dimethoxy-1,3-dioxo-1,3-dihydro-2H- benzo[4,5]thieno[2,3-c]pyrrol-2-yl)propanoate (ZSA-53) The synthesis of ZSA-53 is the same as 13a. Yellow solid, 35% yield, 1H NMR (600 MHz, CDCl3$ o 1( / 0 #a& +>$& 1(-. #a& 1H), 4.25 (t, J = 5.9 Hz, 2H), 4.02 (s, 3H), 4.02 (s, 3H), 3.98 (t, J = 7.1 Hz, 2H), 3.72 – 3.67(m, 4H), 2.78 (t, J = 7.1 Hz, 2H), 2.63 (t, J = 5.9 Hz, 2H), 2.49 (s, 4H).13C NMR (150 MHz, CDCl3$ o +1*(2& +0.(-& +0-( / & + / *(2& + / *(-& +.+(,& +-2(1& +-1(3& +,.(3& +*.(.& +*-(3& 00(3 (2C), 61.9, 57.0, 56.3, 56.3, 53.8 (2C), 34.1, 33.3. MS: calcd for C21H25N2O7S (M+H), 449.1; found, 449.1. HPLC: method B, 65% MeOH, tR = 5.650 min, >99% purity.

[0357] 2-(dimethylamino)ethyl 3-(6,7-dimethoxy-1,3-dioxo-1,3-dihydro-2H- benzo[4,5]thieno[2,3-c]pyrrol-2-yl)propanoate (ZSA-54) The synthesis of ZSA-54 is the same as 13a. Yellow solid, 53% yield, 1H NMR (600 MHz, CDCl3$ o 1( / 1 #a& +>$& 1(-. #a& 1H), 4.22 (t, J = 5.8 Hz, 2H), 4.02 (s, 3H), 4.02 (s, 3H), 3.98 (t, J = 7.1 Hz, 2H), 2.79 (t, J = 7.1 Hz, 2H), 2.59 (t, J = 5.8 Hz, 2H), 2.29 (s, 6H).13C NMR (150 MHz, CDCl3$ o +1+(*& 164.3, 163.5, 150.8, 150.3, 141.2, 138.8, 137.9, 124.9, 104.4, 103.9, 62.5, 57.7, 56.3, 56.3, 45.7 (2C), 34.1, 33.3. MS: calcd for C19H23N2O6S (M+H), 407.1; found, 407.1. HPLC: method B, 60% MeOH, tR = 5.193 min, 98% purity. General procedure for the synthesis of intermediates 24 and 30

[0358] A mixture of 23 (1.495 g, 8.25 mmol), DIPEA (1440 µL, 8.25 mmol) and methyl thioglycolate (740 µL, 8.25 mmol) in anhydrous N,N-dimethylformamide (5 mL) was stirred for 30 min at room temperature. K2CO3 (1.138 g, 8.25 mmol) was added, and the resulting mixture was stirred at 80°C for 16 h. Ice water was added. The precipitate was filtered, washed with water, and dried to give 24.

[0359] Methyl 3-amino-5,6-dimethoxybenzo[b]thiophene-2-carboxylate (24) Gray solid, 92% yield, MS: calcd for C12H14NO4S (M+H), 268.1; found, 268.2.

[0360] Methyl 3-amino-benzo[b]thiophene-2-carboxylate (30) White solid, 85% yield, MS: calcd for C10H10NO2S (M+H), 208.0; found, 208.2. General procedure for the synthesis of intermediates 25 and 31

[0361] A mixture of 24 (0.970 g, 3.62 mmol) in formamide (5 mL) was heated to 150°C. Formamidine acetate (0.376 g, 3.62 mmol) was added, and the mixture was heated at 150 °C for 45 min. The addition of formamidine acetate (0.376 g, 3.62 mmol) was repeated every 45 min for 6 h. Ice water (15 mL) was added. The precipitate was filtered, washed with water, and dried to give 25.

[0362] 7,8-dimethoxybenzo[4,5]thieno[3,2-d]pyrimidin-4(3H)-one (25) Tanned solid, 90% yield, MS: calcd for C12H11N2O3S (M+H), 263.0; found, 263.2.

[0363] Benzo[4,5]thieno[3,2-d]pyrimidin-4(3H)-one (31) Tanned solid, 86% yield, MS: calcd for C10H7N2OS (M+H), 203.0; found, 203.2. General procedure for the synthesis of intermediates 26a~j, 27a~b and 32

[0364] To a flask were added 25 (0.115 g, 0.44 mmol), K2CO3 (0.121 g, 0.88 mmol), catalytic amount of NaI, DMF (1 mL) and ethyl 2-bromoacetate (100 µL, 0.88 mmol). The mixture was stirred at room temperature overnight. The solvent was removed. The residue was isolated using flash column chromatography (hexanes:EA = 2:1~1:1) to afford 26a. For 26d~j, the reaction was carried out at 60 °C.

[0365] Ethyl 2-(7,8-dimethoxy-4-oxobenzo[4,5]thieno[3,2-d]pyrimidin-3(4H)-yl)acetate (26a) Yellow solid, 75% yield, 1H NMR (499 MHz, CDCl3$ o 2(-- #RRR& J = 7.9, 1.4, 0.8 Hz,1H), 8.16 (s, 1H), 7.94 (dt, J = 8.2, 0.9 Hz, 1H), 7.62 (ddd, J = 8.1, 7.1, 1.4 Hz, 1H), 7.56 (ddd, J = 8.1, 7.1, 1.1 Hz, 1H), 4.83 (s, 2H), 4.30 (q, J = 7.1 Hz, 2H), 1.32 (t, J = 7.1 Hz, 3H). MS: calcd for C16H17N2O5S (M+H), 349.1; found, 349.2.

[0366] Methyl 2-(7,8-dimethoxy-4-oxobenzo[4,5]thieno[3,2-d]pyrimidin-3(4H)-yl)acetate (26b) Yellow solid, 60% yield, 1H NMR (499 MHz, CDCl3$ o 2(+, #a& +>$& 1(02 #a& +>$& 1(-- (s, 1H), 4.83 (s, 2H), 4.04 (s, 3H), 4.03 (s, 3H), 3.84 (s, 3H). MS: calcd for C15H15N2O5S (M+H), 335.1; found, 335.3.

[0367] Isopropyl 3-(7,8-dimethoxy-4-oxobenzo[4,5]thieno[3,2-d]pyrimidin-3(4H)-yl)-2- oxopropanoate (26c) Yellow solid, 72% yield, 1H NMR (499 MHz, CDCl3$ o 2(+, #a& +>$& 7.68 (s, 1H), 7.33 (s, 1H), 5.14 (hept, J = 6.3 Hz, 1H), 4.78 (s, 2H), 4.04 (s, 3H), 4.02 (s, 3H), 1.31 (s, 3H), 1.30 (s, 3H). MS: calcd for C17H19N2O5S (M+H), 363.1; found, 363.3.

[0368] Ethyl 2-(7,8-dimethoxy-4-oxobenzo[4,5]thieno[3,2-d]pyrimidin-3(4H)- yl)propanoate (26d) Yellow solid, 45% yield, 1H NMR (499 MHz, CDCl3$ o 2(,- #a& +>$& 7.69 (s, 1H), 7.33 (s, 1H), 5.62 (q, J = 7.4 Hz, 1H), 4.27 (qd, J = 7.1, 1.0 Hz, 2H), 4.04 (s, 3H), 4.02 (s, 3H), 1.81 (d, J = 7.4 Hz, 3H), 1.30 (t, J = 7.1 Hz, 3H). MS: calcd for C17H19N2O5S (M+H), 363.1; found, 363.3.

[0369] Ethyl 2-(7,8-dimethoxy-4-oxobenzo[4,5]thieno[3,2-d ]pyrimidin-3(4H)- yl)propanoate (26e). Yellow solid, 51% yield, *H NMR (499 MHz, CDCh) 8 8.20 (s, 1H), 7.66 (s, 1H), 7.30 (s, 1H), 5.59 (q, J= 7.4 Hz, 1H), 4.01 (d, J= 6.7 Hz, 7H), 3.79 (s, 3H), 1.80 (d, J = 7.4 Hz, 3H). MS: calcd for C16H17N2O5S (M+H), 349.1; found, 349.3.

[0370] Methyl 3-(7,8-dimethoxy-4-oxobenzo[4,5]thieno[3,2-d ]pyrimidin-3(4H)- yl)propanoate (26f) Yellow solid, 91% yield, H NMR (499 MHz, CDCh) 5 8.36 (s, 1H), 7.67 (s, 1H), 7.32 (s, 1H), 4.37 (t, J= 6.0 Hz, 2H), 4.03 (s, 3H), 4.02 (s, 3H), 3.70 (s, 3H), 2.96 (t, J= 6.0 Hz, 2H), 1.58 (s, 3H). MS: calcd for C16H17N2O5S (M+H), 349.1; found, 349.3.

[0371] Methyl (S)-3-(7,8-dimethoxy-4-oxobenzo[4,5]thieno[3,2-d ]pyrimidin-3(4H )-yl)-2- methylpropanoate (26g) Yellow solid, 55% yield, *H NMR (499 MHz, CDCh) 5 8.24 (s, 1H), 7.65 (s, 1H), 7.31 (s, 1H), 4.26 (dd, J= 13.4, 4.9 Hz, 1H), 4.12 (dd, J= 13.5, 9.3 Hz, 1H), 4.01 (d, J= 3.8 Hz, 6H), 3.66 (s, 3H), 3.23 (ddd, 9.3, 7.2, 4.8 Hz, 1H), 1.32 (d, J= 7.3 Hz, 3H). 13C NMR (126 MHz, CDCh) 5 174.79, 157.66, 152.67, 151.81, 148.96, 148.57, 135.26, 127.39, 121.95, 104.22, 103.84, 56.27, 56.21, 52.13, 49.71, 38.52, 15.43. MS: calcd for C17H19N2O5S (M+H), 363.1; found, 363.3.

[0372] Methyl 3-(7,8-dimethoxy-4-oxobenzo[4,5]thieno[3,2-d]pyrimidin-3(4H)- yl)butanoate. Yellow solid, 49% yield, 1 H NMR (499 MHz, CDCh) 5 8.22 (s, 1H), 7.65 (s, 1H), 7.31 (s, 1H), 5.09 (q, J - 6.9 Hz, 1H), 4.01 (d, J= 5.1 Hz, 6H), 3.65 (s, 3H), 3.23 (dd, J = 16.8, 7.6 Hz, 1H), 2.94 - 2.85 (m, 1H), 1.70 (d, J= 7.0 Hz, 3H). 13C NMR (126 MHz,CDC13) δ 170.91, 157.57, 151.76, 148.94, 146.83, 135.24, 127.34, 104.25, 103.83, 56.26, 56.21, 51.95, 38.67, 19.30. MS: calcd for C17H19N2O5S (M+H), 363.1; found, 363.3.

[0373] Methyl 4-(7,8-dimethoxy-4-oxobenzo[4,5]thieno[3,2-d]pyrimidin-3(4H)- yl)butanoate. Yellow solid, 82% yield, 1H NMR (499 MHz, CDC13) δ 8.79 (s, 1H), 7.84 (s, 1H), 7.32 (s, 1H), 4.65 (s, 2H), 4.03 (d, J = 6.8 Hz, 6H), 3.71 (s, 3H), 2.57 (t, J = 7.3 Hz, 2H), 2.28 – 2.20 (m, 2H).13C NMR (126 MHz, CDC13) δ 173.37, 163.89, 158.37, 154.21, 148.76, 134.45, 126.68, 104.26, 66.05, 56.31, 56.28, 51.74, 30.65, 24.26. MS: calcd for C17H19N2O5S (M+H), 363.1; found, 363.3.

[0374] Methyl 5-(7,8-dimethoxy-4-oxobenzo[4,5]thieno[3,2-d]pyrimidin-3(4H)- yl)pentanoate (26j). Yellow solid, 77% yield, 1H NMR (499 MHz, CDC13) δ 2(13 #a& +>$& 7.84 (s, 1H), 7.31 (s, 1H), 4.61 (s, 2H), 4.03 (d, J = 7.6 Hz, 6H), 3.68 (s, 3H), 2.44 (t, J = 7.3 Hz, 2H), 1.94 (dd, J = 8.5, 6.2 Hz, 2H), 1.86 (dd, J = 8.6, 7.1 Hz, 2H). MS: calcd for C18H21N2O5S (M+H), 377.1; found, 377.3.

[0375] Methyl 4-((7,8-dimethoxybenzo[4,5]thieno[3,2-d]pyrimidin-4-yl)oxy)butanoate (27a). Yellow solid, 32% yield, 1H NMR (499 MHz, CDC13) δ 8.17 (s, 1H), 7.65 (s, 1H), 7.31 (s, 1H), 4.17 (t, J = 7.1 Hz, 2H), 4.01 (d, J = 5.9 Hz, 6H), 3.68 (s, 3H), 2.44 (t, J = 7.0 Hz, 2H), 2.18 (p, J = 7.1 Hz, 2H).13C NMR (126 MHz, CDC13) δ 172.98, 157.58, 152.60, 151.78, 148.96, 147.81, 135.23, 127.37, 122.21, 104.24, 103.80, 56.27, 56.22, 51.84, 46.02, 30.54, 24.66. MS: calcd for C17H19N2O5S (M+H) 3631; found 3633.

[0376] Methyl 5-((7,8-dimethoxybenzo[4,5]thieno[3,2-d]pyrimidin-4-yl)oxy)pentanoate (27b). Yellow solid, 39% yield, 1H NMR (499 MHz, CDCl3$ o 2(+- #a& +>$& 1(0, #a& +>$& 7.29 (s, 1H), 4.09 (t, J = 7.3 Hz, 2H), 4.00 (d, J = 5.6 Hz, 6H), 3.65 (s, 3H), 2.38 (t, J = 7.3 Hz, 2H), 1.88 (ddd, J = 9.1, 6.2, 1.8 Hz, 2H), 1.76 – 1.70 (m, 3H). MS: calcd for C18H21N2O5S (M+H), 377.1; found, 377.3.

[0377] Ethyl 2-(4-oxobenzo[4,5]thieno[3,2-d]pyrimidin-3(4H)-yl)acetate (32) Yellow solid, 68% yield, 1H NMR (499 MHz, CDCl3$ o 2(-- #RRR& J = 7.9, 1.4, 0.8 Hz, 1H), 8.16 (s, 1H), 7.94 (dt, J = 8.2, 0.9 Hz, 1H), 7.62 (ddd, J = 8.1, 7.1, 1.4 Hz, 1H), 7.56 (ddd, J = 8.1, 7.1, 1.1 Hz, 1H), 4.83 (s, 2H), 4.30 (q, J = 7.1 Hz, 2H), 1.32 (t, J = 7.1 Hz, 3H). MS: calcd for C14H13N2O3S (M+H), 289.1; found, 289.3. General procedure for the synthesis of intermediates 28a~f and 33

[0378] To the mixture of 26a (20.5 mg, 0.059 mmol), THF (200 µL) and H2O (100 µL) was added LiOH (14.2 mg, 0.59 mmol). The mixture was stirred at room temperature for 0.5 h. The pH was adjusted to 2 using aqueous HCl (1 M). The precipitate formed was filtered, washed with water, and dried to give 28a.

[0379] 2-(7,8-dimethoxy-4-oxobenzo[4,5]thieno[3,2-d]pyrimidin-3(4H)-yl)acetic acid (28a) Yellow solid, 79% yield, 1> CBF #.33 B>h& R[a]$ o 2( / / #a& +>$& 1(1, #a& +>$& 1(0+ (s, 1H), 4.81 (s, 2H), 3.89 (d, J = 3.7 Hz, 6H). MS: calcd for C14H13N2O5S (M+H), 321.1; found, 321.3.

[0380] 2-(7,8-dimethoxy-4-oxobenzo[4,5]thieno[3,2-d]pyrimidin-3(4H)-yl)propanoic acid (28b) Yellow solid, 81% yield, MS: calcd for C15H15N2O5S (M+H), 335.1; found, 335.3.

[0381] 3-(7,8-dimethoxy-4-oxobenzo[4,5]thieno[3,2-d]pyrimidin-3(4H)-yl)propanoic acid (28c) Yellow solid, 75% yield, 1H NMR (499 MHz, cd3]R$ o 2(.2 #a& +>$& 1(1* #a& +>$& 1(.- (s, 1H), 4.35 (t, J = 6.2 Hz, 2H), 3.97 (d, J = 0.9 Hz, 6H), 2.90 (t, J = 6.2 Hz, 2H). MS: calcd for C15H15N2O5S (M+H), 335.1; found, 335.3.

[0382] (S)-3-(7,8-dimethoxy-4-oxobenzo[4,5]thieno[3,2-d]pyrimidin-3(4H)-yl)-2- methylpropanoic acid (28d) Yellow solid, 73% yield, 1> CBF # / 33 B>h& :BGD$ o +,( / 0 (s, 1H), 8.53 (d, J = 1.5 Hz, 1H), 7.72 (d, J = 3.0 Hz, 1H), 7.61 (d, J = 2.5 Hz, 1H), 4.23 (dd, J = 13.5, 8.2 Hz, 1H), 4.12 (dd, J = 13.5, 6.7 Hz, 1H), 3.90 (d, J = 2.9 Hz, 6H), 3.00 (dt, J = 7.9, 6.8 Hz, 1H), 1.14 (d, J = 7.2 Hz, 3H).139 CBF #+,0 B>h& R[a]$ o +1 / ( / 2& + / 1(.0& 152.74, 152.03, 150.57, 149.22, 134.55, 127.27, 120.91, 105.83, 104.19, 56.46, 56.18, 49.12, 38.51, 15.07. MS: calcd for C16H17N2O5S (M+H), 349.1; found, 349.3.

[0383] 3-(7,8-dimethoxy-4-oxobenzo[4,5]thieno[3,2-d]pyrimidin-3(4H)-yl)butanoic acid (28e) Yellow solid, 65% yield, MS: calcd for C16H17N2O5S (M+H), 349.1; found, 349.3.

[0384] 4-(7,8-dimethoxy-4-oxobenzo[4,5]thieno[3,2-d]pyrimidin-3(4H)-yl)butanoic acid (28f) Yellow solid, 90% yield, 1> CBF #.33 B>h& R[a]$ o +,(+- #a& +>$& 2( / , #a& +>$& 1(03 (s, 1H), 7.58 (s, 1H), 4.07 (t, J = 7.0 Hz, 2H), 3.88 (s, 3H), 3.87 (s, 3H), 2.29 (t, J = 7.4 Hz, 2H), 1.95 (t, J = 7.1 Hz, 2H).139 CBF #+,0 B>h& R[a]$ o +1.(+2& + / 1(.*& + / ,(1-& + / +(3,& 150.09, 149.15, 134.44, 127.30, 121.08, 105.80, 104.14, 56.42, 56.14, 45.96, 31.18, 24.76. MS: calcd for C16H17N2O5S (M+H), 349.1; found, 349.3.

[0385] 2-(4-oxobenzo[4,5]thieno[3,2-d]pyrimidin-3(4H)-yl)acetic acid (33) Yellow solid, 79% yield, 1> CBF #.33 B>h& R[a]$ o 2(0, #a& +>$& 2(,1 #Rb& J = 7.8, 1.0 Hz, 1H), 8.18 (d, J = 8.1 Hz, 1H), 7.69 (ddd, J = 8.3, 7.1, 1.3 Hz, 1H), 7.62 (ddd, J = 8.0, 7.0, 1.0 Hz, 1H), 4.84 (s, 2H). MS: calcd for C12H9N2O3S (M+H), 261.0; found, 261.2. Synthesis of Compounds 29a and 29b

[0386] (9Z,12Z)-Octadeca-9,12-dien-1-yl (S)-3-(7,8-dimethoxy-4- oxobenzo[4,5]thieno[3,2-d]pyrimidin-3(4H)-yl)-2-methylpropanoate (29a).T Yellow solid, 80% yield, 1H NMR (599 MHz, CDCl3$ o 2(,0 #a& +>$& 1(02 #R& J = 7.9 Hz, 1H), 7.33 (d, J = 3.5 Hz, 1H), 5.52 – 5.29 (m, 2H), 4.29 (dd, J = 13.4, 4.9 Hz, 1H), 4.20 – 3.95 (m, 7H), 3.25 (d, J = 7.3 Hz, 1H), 2.78 (t, J = 6.7 Hz, 1H), 2.13 – 1.96 (m, 2H), 1.79 – 1.51 (m, 5H), 1.49 – 1.02 (m, 15H), 0.90 (t, J = 6.8 Hz, 3H). MS: calcd for C34H49N2O5S (M+H), 597.3; found, 597.2.

[0387] Octadecyl (S)-3-(7,8-dimethoxy-4-oxobenzo[4,5]thieno[3,2-d]pyrimidin-3(4H)- yl)-2-methylpropanoate (29b) Yellow solid, 81% yield, 1H NMR (599 MHz, CDCl3$ o 2(,0 (s, 1H), 7.68 (d, J = 7.9 Hz, 1H), 7.33 (d, J = 3.5 Hz, 1H), 5.52 – 5.29 (m, 2H), 4.29 (dd, J = 13.4, 4.9 Hz, 1H), 4.20 – 3.95 (m, 7H), 3.25 (d, J = 7.3 Hz, 1H), 2.78 (t, J = 6.7 Hz, 1H), 2.13 – 1.96 (m, 2H), 1.79 – 1.51 (m, 5H), 1.49 – 1.02 (m, 15H), 0.90 (t, J = 6.8 Hz, 3H). MS: calcd for C34H53N2O5S (M+H), 601.4; found, 601.3. Synthesis of target compound 34 (ZSA-52N)

[0388] To the mixture of 23 (30.0 mg, 0.089 mmol), HATU (50.8 mg, 0.134 mmol), DCM (1 mL) and DIPEA (31 µL, 0.178 mmol) were added linoleyl alcohol (57 µL, 0.178 mmol) and catalytic amount of DMAP. The resulting mixture was stirred at room temperature for 5 h. The solvent was evaporated, and the residue was isolated on a silica gel column (hexanes:EA = 5:1) to afford 34.(9Z,12Z)-octadeca-9,12-dien-1-yl 3-(6,7-dimethoxy-1,3-dioxo-1,3-dihydro-2H- benzo[4,5]thieno[2,3-c]pyrrol-2-yl)propanoate (34) Yellow solid, 48% yield, MS: calcd for C33H45NO6SNa (M+H), 606.3; found, 606.4. Synthesis of Compound 39

[0389] Reagents and conditions: i) NaNO2, KI, HCl (aq, 6 M), H2O, 60~0~60°C; ii) a) LiOH (aq, 4 M), EtOH, THF, rt, 5 h; b) HCl (aq, 1 M); iii) tert-butyl 3-aminopropionate hydrochloride, HATU, DIPEA, DCM, rt, 2 h; iv) ethynyltrimethylsilane, Pd(PPh3)2Cl2, CuI, Et3N, DMF, rt, 24 h; v) TBAF, THF, 45°C, 1.5 h; vi) TFA, DCM, rt, 2 h

[0390] Experimental procedure for the synthesis of intermediate 34. 24 (2.90 mmol, 0.774 g) was suspended in HCl (6 M, 5 mL) and heated at 60°C for 20 min. After the mixture was cooled in an ice bath for 5 min, NaNO2 (4.35 mmol, 0.300 g) was added portionwise within 20 min. The suspension was stirred for 1 h before it was added to the solution of KI (5.80 mmol, 0.963 g) in water (3 mL). Being heated at 60°C for 0.5 h, the resulted mixture was diluted with water (30 mL), extracted with DCM (3 × 30 mL). The organic phase was washed with saturated brine, dried with anhydrous Na2SO4 and evaporated under reduced pressure. The residue was isolated using flash chromatography (hexanes:DCM = 2:1~1:3) to give 34 (methyl 3-iodo-5,6-dimethoxybenzo[b]thiophene-2-carboxylate). Pale yellow solid, 63% yield, 1H NMR (600 MHz, CDCl3$ o 1(-. #a& +>$& 1(,. #a& +>$& .(* / #a& ->$& .(*, #a& ->$& -(32 #a& ->$( MS: calcd for C12H12IO4S (M+H), 379.0; found, 379.1.

[0391] Experimental procedure for the synthesis of intermediate 35. A flask was charged with 34 (1.02 mmol, 0.385 g), NaOH (aq, 4 M, 2.6 mL), EtOH (2.6 mL) and THF (2.6 mL). After the mixture was stirred at room temperature for 16 h, organic solvent was removed under airflow. The pH of the residue was adjusted to 2.0 using HCl (1 M). The precipitate was filtered, washed with water and dried at 60°C overnight to afford 35 (3-iodo-5,6- dimethoxybenzo[b]thiophene-2-carboxylic acid). White solid, 89% yield, 1H NMR (600 MHz, DMSO-d6$ o +-( / , #a& +>$& 1(0 / #a& +>$& 1(,. #a& +>$& -(3* #a& ->$& -(22 #a& ->$(

[0392] Experimental procedure for the synthesis of intermediate 36. To a flask were added 35 (0.33 mmol, 120.0 mg), tert-butyl 3-aminopropionate hydrochloride (0.40 mmol, 72.1 mg), HTAU (0.50 mmol, 0.188 g), DMF (1 mL) and DIPEA (0.99 mmol, 173 µL). The mixture was stirred at room temperature for 2 h. The solvent was removed under reduced pressure. The residue was isolated using flash chromatography (heaxanes:EA = 5:1~1:1) to give 36 (tert-butyl 3-(3-iodo-5,6-dimethoxybenzo[b]thiophene-2-carboxamido)propanoate). White solid, 51% yield, 1H NMR (600 MHz, CDCl3$ o 1(.0 k 1(., #[& +>$& 1(, / #a& +>$& 1(,, (s, 1H), 4.04 (s, 3H), 4.00 (s, 3H), 3.78 (p, J = 6.3 Hz, 2H), 2.65 – 2.61 (m, 2H), 1.50 (s, 9H). MS: calcd for C18H23INO5S (M+H), 492.0; found, 492.1.

[0393] Experimental procedure for the synthesis of intermediate 37. 36 (0.20 mmol, 100.1 mg), DMF (1.0 mL) and Et3N (0.80 mmol, 110 µL) were added into a flask and stirred under N2 atmosphere followed by the addition of Pd(PPh3)2Cl2 (0.007 mmol, 4.9 mg) and CuI(0.016 mmol, 3.1 mg). The solution was stirred at room temperature under N2 atmosphere for 1 h before the addition of ethynyltrimethylsilane (0.60 mmol, 0.08 mL). The resulted solution was stirred at room temperature for another 23 h. The solvent was removed under vacuum. The residue was isolated using flash chromatography (hexanes:DCM = 5:1~2:1) to give 37 (tert- butyl 3-(5,6-dimethoxy-3-((trimethylsilyl)ethynyl)benzo[b]thiophene-2-carboxamido) propanoate). Yellow solid, 52% yield, 1H NMR (600 MHz, DMSO-d6$ o 2(+0 #b& J = 5.8 Hz, 1H), 7.71 (s, 1H), 7.26 (s, 1H), 3.94 (s, 3H), 3.93 (s, 3H), 3.61 (q, J = 6.3 Hz, 2H), 2.60 (t, J = 6.4 Hz, 2H), 1.47 (s, 9H), 0.44 (s, 9H). MS: calcd for C23H32NO5SSi (M+H), 462.2; found, 462.2.

[0394] Experimental procedure for the synthesis of intermediate 38. 37 (0.061 mmol, 28.2 mg) was dissolved in THF (0.5 mL) and heated at 45°C. TBAF (1 M in THF, 0.182 mmol, 180 µL) was added. The solution was stirred at room temperature for 1.5 h. The solvent was removed, and the residue was isolated using flash chromatography (hexanes:DCM = 5:1~1:1) to give 38 (tert-butyl 3-(6,7-dimethoxy-1-oxobenzo[4,5]thieno[2,3-c]pyridin-2(1H)- yl)propanoate). Yellow solid, 25% yield, 1H NMR (600 MHz, CDCl3$ o 1(.3 #R& J = 7.1 Hz, 1H), 7.40 (s, 1H), 7.37 (s, 1H), 6.84 (d, J = 7.1 Hz, 1H), 4.34 (t, J = 6.4 Hz, 2H), 4.03 (s, 3H), 4.03 (s, 3H), 2.85 (t, J = 6.4 Hz, 2H), 1.44 (s, 9H).

[0395] Experimental procedure for the synthesis of 39. A flask was charged with 38 (0.013 mmol, 5.1 mg), DCM (300 µL) and TFA (150 µL). The solution was stirred at room temperature for 2 h. The solvent was removed under airflow. The residue was triturated in water (0.5 mL), filtered and dried to give 39 (3-(6,7-dimethoxy-1-oxobenzo[4,5]thieno[2,3- c]pyridin-2(1H)-yl)propanoic acid). Yellow solid, 45% yield, MS: calcd for C16H16NO5S (M+H), 334.1; found, 334.2. 2) Dimers

[0396] Synthesis scheme of ZSA-51DSynthesis of intermediate 2

[0397] 1 (2.003 g, 6.23 mmol) was dissolved in anhydrous THF (20 mL). The solution was cooled to 0°C before MeMgBr (4 mL, 3 M in diethyl ether) was added dropwise. The mixture was allowed to warm to room temperature and stirred overnight. Saturated NH4Cl (aq, 5 mL) was added dropwise with the cooling of an ice bath to quench the reaction followed by the addition of water (20 mL). The water phase was extracted by EA (3×20 mL). The organic phase was washed with saturated brine, dried with anhydrous Na2SO4, and evaporated under reduced pressure to give yellow oil which was used directly in the next step.

[0398] 1-(5-(benzyloxy)-2-bromo-4-methoxyphenyl)ethan-1-ol (2) Yellow oil, 80% yield, MS: calcd for C16H18BrO3 (M+H), 337.0; found, 337.3 (79Br), 339.2 (81Br). Synthesis of intermediate 3 (1-(5-(benzyloxy)-2-bromo-4-methoxyphenyl)ethan-1-one)

[0399] PCC (2.430 g, 11.28 mmol) was added to the solution of 2 (1.902 g, 5.64 mmol) in DCM (15 mL). The mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure. The residue was isolated using flash chromatography (hexanes:EA = 10:1~5:1) to give desired product (3). Yellow solid, 76% yield, MS: calcd for C16H16BrO3 (M+H), 335.0; found, 335.3 (79Br), 337.3 (81Br). Synthesis of intermediate 4 (5-(benzyloxy)-6-methoxy-3-methylbenzo[b]thiophene-2- carboxylic acid)

[0400] To a flask were added 3 (1.790 g, 5.34 mmol), DMA (10 mL), DIPEA (5.34, 930 µL) and methyl thioglycolate (955 µL, 10.68 mmol). The mixture was stirred at room temperature under nitrogen atmosphere for 40 min. Then, t-BuOK (8.01, 0.898 g) was added. The resulting mixture was heated at 80°C under nitrogen atmosphere for 14 h. The solvent was removed under vacuum. The residue was dispersed into water (15 mL), extracted by DCM (3×15 mL). The solid formed in the process of extraction was filtered. Then, the pH of the water phase was adjusted to 2 using HCl (aq, 3 M). The precipitate was filtered and washed with water. This filter cake was combined with the previous filter cake and dried to give gray solid.23% yield, MS: calcd for C18H17O4S (M+H), 329.1; found, 329.2. Synthesis of intermediates 5a~5b (Methyl 3-(5-(benzyloxy)-6-methoxy-3- methylbenzo[b]thiophene-2-carboxamido)propanoate (5a); Tert-butyl 3-(5-(benzyloxy)-6- methoxy-3-methylbenzo[b]thiophene-2-carboxamido)propanoate (5b))

[0401] To a flask were added 4 (0.105 g, 0.32 mmol), methyl 3-aminopropionate hydrochloride (58.1 mg, 0.42 mmol), HATU (0.182 g, 0.48 mmol), DCM (2 mL) and DIPEA (160 µL, 0.93 mmol). The mixture was stirred at room temperature for 2 h. Then mixture was diluted with DCM (50 mL), washed with water (3×15 mL), and evaporated under reduced pressure. The residue was triturated in the mixed solvent of EtOH (1 mL) and H2O (1 mL). The precipitate was filtered, washed with EtOH:H2O (1:1), and dried to give 5a. Yellow solid, 55% yield, MS: calcd for C22H24NO5S (M+H), 414.1; found, 414.2.

[0402] Intermediate 5b was prepared in the same manner as 5a, using 3-aminopropionate hydrochloride instead of methyl 3-aminopropionate hydrochloride. Yellow solid, 72% yield, MS: calcd for C25H30NO5S (M+H), 456.2; found, 456.2. Synthesis of intermediates 6a and 6b (Methyl 3-(5-hydroxy-6-methoxy-3- methylbenzo[b]thiophene-2-carboxamido)propanoate (6a); Tert-butyl 3-(5-hydroxy-6- methoxy-3-methylbenzo[b]thiophene-2-carboxamido)propanoate (6b)

[0403] To a flask were added 5a (0.402 g, 0.97 mmol), Pd / C (5%, 80.1 mg) and EtOH (5 mL). The mixture was heated at 70°C under hydrogen gas atmosphere for 16 h before it was filtered through a pad of celite and washed with DCM:MeOH. The filtrate was evaporated under reduced pressure to give 6a without further purification. Gray solid, 95% yield, MS: calcd for C15H18NO5S (M+H), 324.1; found, 324.2.

[0404] Intermediate 6b was prepared in the same manner as 6a, using intermediate 5b as the starting material. Gray solid, 92% yield, MS: calcd for C18H24NO5S (M+H), 366.1; found, 366.2. Synthesis of intermediates 7a~7b (Methyl 3-(5-((tert-butyldimethylsilyl)oxy)-6-methoxy-3- methylbenzo[b]thiophene-2-carboxamido)propanoate (7a); Tert-butyl 3-(5-((tert- butyldimethylsilyl)oxy)-6-methoxy-3-methylbenzo[b]thiophene-2-carboxamido)propanoate (7b))

[0405] To the solution of 6a (0.244 g, 0.71 mmol) in DCM (4 mL) were added TBDMSCl (0.213 g, 1.42 mmol), imidazole (0.145 g, 2.13 mmol) and DMAP (8.7 mg, 0.071 mmol). After 0.5 h stirring at room temperature, the solvent was removed. The residue was isolated using flash chromatography (hexanes:EA = 5:1~3:1) to give 7a. Yellow oil, 86% yield, MS: calcd for C21H32NO5SSi (M+H), 438.2; found, 438.4.

[0406] Intermediate 7b was prepared in the same manner as 7a, using intermediate 6b as the starting material. Yellow oil, 81% yield, MS: calcd for C24H38NO5SSi (M+H), 480.2; found, 480.4. Synthesis of intermediates 8a~8b (Methyl 3-(3-(bromomethyl)-5-((tert- butyldimethylsilyl)oxy)-6-methoxybenzo[b]thiophene-2-carboxamido)propanoate (8a); Tert- butyl 3-(3-(bromomethyl)-5-((tert-butyldimethylsilyl)oxy)-6-methoxybenzo[b]thiophene-2- carboxamido)propanoate (8b)).

[0407] To a flask were added 7a (0.190 g, 0.43 mmol), NBS (0.116 g, 0.65 mmol), AIBN (14.1 mg, 0.086 mmol) and CCl4 (4 mL). The mixture was heated at 80°C for 2 h. The solvent was removed. The residue was isolated using flash chromatography (hexanes:EA = 3:1~2:1) to afford 8a. Yellow oil, 73% yield, MS: calcd for C21H31BrNO5SSi (M+H), 516.1; found, 516.3 (79Br), 518.3 (81Br).

[0408] Intermediate 8b was prepared in the same manner as 8a, using intermediate 7b as the starting material. Yellow oil, 61% yield, MS: calcd for C24H37BrNO5SSi (M+H), 558.1; found, 558.3 (79Br), 560.3 (81Br). Synthesis of intermediates 9a~9b (Methyl 3-(7-((tert-butyldimethylsilyl)oxy)-1-hydroxy-6- methoxy-3-oxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2-yl)propanoate (9a); Tert- butyl 3-(7-((tert-butyldimethylsilyl)oxy)-1-hydroxy-6-methoxy-3-oxo-1,3-dihydro-2H- benzo[4,5]thieno[2,3-c]pyrrol-2-yl)propanoate (9b))

[0409] To the solution of 8a (0.164 g, 0.32 mmol) in CH3CN (2 mL) was added NMO (0.112 g, 0.96 mmol) with the cooling of an ice bath. After 10 min, the ice bath was removed, and the mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure. The residue was isolated using flash chromatography (hexanes:EA = 2:1~1:1) to furnish crude 9a. This product is not stable. Yellow oil, 81% crude yield, MS: calcd for C21H30NO6SSi (M+H), 452.1; found, 452.3

[0410] Intermediate 9b was prepared in the same manner as 9a, using intermediate 8b as the starting material. This product is not stable. Yellow oil, 61% crude yield, MS: calcd for C24H36NO6SSi (M+H), 494.2; found, 494.3. Synthesis of intermediates 10a~10b (Methyl 3-(7-((tert-butyldimethylsilyl)oxy)-6-methoxy- 1,3-dioxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrol-2-yl)propanoate (10a); Tert-butyl 3- (7-((tert-butyldimethylsilyl)oxy)-6-methoxy-1,3-dioxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3- c]pyrrol-2-yl)propanoate (10b))

[0411] To the solution of 9a (0.120 g, 0.26 mmol) in DCM (2 mL) was added PCC (0.112 g, 0.52 mmol). The mixture was stirred at room temperature for 16 h. The solvent was removed. The residue was isolated using flash chromatography (hexanes:EA = 5:1) to give 10a. Yellow solid, 62% yield, MS: calcd for C21H28NO6SSi (M+H), 450.1; found, 450.3.

[0412] Intermediate 10b was prepared in the same manner as 10a, using intermediate 9b as the starting material. Yellow solid, 81% yield, MS: calcd for C24H34NO6SSi (M+H), 492.2; found, 492.3 Synthesis of intermediates 11a~11b

[0413] TBAF (0.25 mL, 1 M in THF) was added into the solution of 10a (75.0 mg, 0.17 mmol) in THF (2 mL). The solution was stirred at room temperature for 0.5 h. The solvent was removed under reduced pressure. The residue was isolated using flash chromatography (hexanes:EA = 2:1~1:1~DCM:EA = 8:1) to afford 11a. Yellow solid, 64% yield, MS: calcd for C15H14NO6S (M+H), 336.1; found, 336.3.

[0414] Intermediate 11b was prepared in the same manner as 11a, using intermediate 10b as the starting material. Yellow solid, 63% yield, MS: calcd for C18H20NO6S (M+H), 378.1; found, 378.3. Synthesis of target compound ZSA-51D and Compound 13b (Dimethyl 3,3'-((propane- 1,3-diylbis(oxy))bis(6-methoxy-1,3-dioxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrole- 7,2-diyl))dipropionate (ZSA-51D); Ditert-butyl 3,3'-((propane-1,3-diylbis(oxy))bis(6- methoxy-1,3-dioxo-1,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrole-7,2-diyl))dipropionate (13b))

[0415] To a flask were added 11a (20.7 mg, 0.06 mmol), K2CO3 (16.6 mg, 0.12 mmol), catalytic amount of NaI, CH3CN (1 mL) and 1,3-dibromopropane (18 µL, 0.18 mmol). The mixture was heated at 80°C for 3 h. The solvent was removed under high vacuum to give crude 12a. MS: calcd for C18H19BrNO6S (M+H), 456.1; found, 456.3 (79Br), 458.3 (81Br).

[0416] To the residue above were added 11a (14.0 mg, 0.04 mmol), K2CO3 (16.6 mg, 0.12 mmol), catalytic amount of NaI and CH3CN (1 mL). The resulted mixture was heated at 80 °C for 4 h. The solvent was removed. The residue was isolated on a silica gel chromatography column (hexanes:EA = 2:1~1:1) to give product which was triturated in the mixed solvent of hexanes (0.5 mL) and EA (0.5 mL). The slurry was filtered, washed with hexanes:EA (1:1), and dried to furnish ZSA-51D. Yellow solid, 9% overall yield, MS: calcd for C33H31N2O12S2 (M+H), 711.1; found, 711.3.

[0417] Compound 13b was prepared in the same manner as ZSA-51D, using intermediate 11b as the starting material. Yellow solid, 24% overall yield, MS: calcd for C39H43N2O12S2 (M+H), 795.2; found, 795.3.Synthesis of target compound ZSA-52D (3,3'-((propane-l,3-diylbis(oxy))bis(6-methoxy- l,3-dioxo-l,3-dihydro-2H-benzo[4,5]thieno[2,3-c]pyrrole-7,2-diyl))dipropionic acid)

[0418] To a flask were added 13b (10.7 mg, 0.013 mmol), TFA (200 μL) and DCM (0.5 mL). The mixture was stirred at room temperature for 3 h. The solvent was removed under airflow. The residue was triturated in EA (200 μL), filtered and dried to give ZSA-52D.Yellow solid, 83% yield, MS: calcd for C31H27N2O12S2 (M+H), 683.1; found, 683.2.Example 2 Activity for STING

[0419] ISG (interferon-s timulated gene) reporter assay. STING activation activity of the compounds indicated below was tested using a THP-1 ISG reporter cell assay. THPl-Blue- ISG Cells, which were specifically designed for monitoring the interferon signaling pathway, were obtained from InvivoGen. The assay was developed for the monitoring of Human IRF activation by determining the activity of secreted embryonic alkaline phosphatase (SEAP) reporter construct, which can be readily assessed with QUANTI-Blue™, a SEAP detection reagent. Briefly, 100 μL of different concentrations of the compounds were incubated with 100 μL of THPl-Blue-ISG cell suspension (106 cells / mL) for 24 h at 37 °C. The SEAP levels were measured using Synergy 2 microplate reader (Biotek) at 620 nm in absorbance model.

[0420] THP 1 -Dual mouse STING (mSTING) cells and THP 1 -Dual (STING KO) cells were purchased from InvivoGen. THP 1 -Dual (mSTING) cells express the wild-type mSTING while THP 1 -Dual (STING KO) cells were generated from THP1 cells by stable knockout of the STING gene. Briefly, 100 μL of different concentrations of the compounds were incubated with 100 μL of THP 1 -Dual (mSTING) cells or THP 1 -Dual (STING KO) cells (106 cells / mL) for 24 h at 37 °C. The IRF responses of the two cell lines were detected using Quanti-Luc reagent Synergy 2 microplate reader (Biotek) at 620 nm in luminescence model.

[0421] Concentration measurement oflFN-fi. THP1 Cell Line (Monocyte) was purchased from ATCC. The concentrations of Human IFN-β in supernatants of cell medium were measure by ELISA using Quantikine ELISA kit (DIFNBO) purchased from R&D systems (Minneapolis, MN, United States).

[0422] STING-binding assay. Human STING WT binding kit (64BDSTGPEG) was obtained from Perkin Elmer. The assays were conducted according to manufacturer’s protocols in white opaque 386-well microplates. Briefly, 5 μL different concentrations of compounds were mixed with 5 μL human STING WT protein 6His-tagged protein, and 10 μL of premixed STING WT ligand d2 reagent and 6 His Tb antibody working solutions. The mixture was incubated at room temperature for 3hrs. The HTRF signal of the reaction mixture was read on a Synergy 2 microplate reader (Biotek). Mouse STING WT binding kit (64BDSTGMPEG) was obtained from Perkin Elmer. The assay protocol was the same as Human STING WT binding kit.

[0423] The cellular STING activation activity was evaluated on human monocytic THP1 cells (THPl-Blue ISG) harboring an IRF-inducible secreted embryonic alkaline phosphatase (SEAP) reporter of interferon-stimulated gene (ISG). The data was expressed as fold change of compound-induced reported activity relative to DMSO-treated reporter activity. The binding affinity was determined using cGAMP displacement assay. First, the cyclopentanone-containing compounds (2a~c, Table 1) were inspected. 2a, with one-atom linker, showed moderate cellular activity and binding affinity, although it was weaker than MSA-2. This result, however, indicated that fusing a ring to thiophene was tolerable. The activity disappeared when the linker was lengthened to two (2b) or three carbon atoms (2c), maybe due to the loss of an interaction between the carboxyl group and Arg238.

[0424] Subsequently, the CH group on the cyclopentanone moiety (2a~c, Table 1) was changed to nitrogen (lla~m, 12a~m, Table 2). The CH group of 2a was replaced with nitrogen to produce ester Ila. Surprisingly, Ila retained nearly the same cellular activity as 2a. However, the corresponding acid 12a showed the loss of the cellular activity, and both Ila and 12a exhibited very weak binding affinity within tested concentration (IC50>50 μM, Table 2). Attaching a hydroxylmethyl group (llb~c, 9 folds at 5 μM, Table 2) to the linker of Ila (4 folds at 5 μM, Table 2) slightly improved the cellular activity. Next, the linker was extended to three carbon atoms to afford ester lid and acid 12d. lid demonstrated STING activation activity on the cellular level but lost binding affinity. In contrast, 12d showed high binding affinity (IC50= 6 μM, Table 2) which was the same as MSA-2 (IC50 = 6 μM, Table 2), while the cellular activity was only observed at 50 μM. This result indicated that the acid12d was active form with low permeability, while the ester lid served as a prodrug that increased the permeability of the molecule and can be hydrolyzed in cells to release the active acid 12d.

[0425] Based on lid and 12d, the substituents on the a-carbon or (3-carbon were explored relative to carboxyl or methyl carboxylate group of the linker. The methyl group on the (3-carbon eliminated the binding affinity regardless of its configuration (11e~f, 12e~f, Table 2) implying that the modification on (β-carbon is not practical. As a result, the substituent on the a-carbon was varied. The bulky a-cyclobutyl group largely decreased the binding affinity (11g and 12g, Table 2). A dimethyl group was attached to the position (llh and 12h, Table 2). However, both the cellular activity and binding affinity was undetectable, indicating the dimethyl group likely dramatically reduced the binding with STING. As shown from 11j~k and 12j~k, the cyclobutyl or phenyl linker turned out to be inferior. Moreover, further linker extension dramatically decreased the cellular activity and binding affinity (11II~m, 121~m).

[0426] Then, the carboxyl group was replaced with amide (13a), A-methoxyamide (13b), (O-acyl-A-isopropylhydroxylamine (13c) or sulfonamide (13d). Only 13c displayed STING activation in cells as it can be cleaved to release active acid 12d, while 13a, 13b and 13d, which were difficult to be hydrolyzed in cells, did not show any binding affinity and cellular activity. Thus, the linear three-carbon chain (12d) was confirmed to be the optimal linker.

[0427] Next, the tricyclic scaffold was investigated (Table 3). Changing sulfur (12d) to nitrogen (15-16, Table 3) significantly diminished the binding affinity. Attaching a bromine atom (17a and 18a) to phenyl ring eliminated the cellular activity, while the smaller chlorine atom (17b and 18b) retained some cellular activity. The newly established pyrrolidone ring was modified by replacing the CH2with carbonyl group expecting to build new interactions with STING. 23 (ZSA-52) demonstrated both good binding affinity (IC50= 5 μM, Table 2 and Figure 3A) and cellular activity (13 folds at 5 μM). Meanwhile, the corresponding ester 22a induced strong response in the cells (15 folds at 5 μM). Therefore, ZSA-51 and 52 were further evaluated below.Table 1. Activity of 2a~c for STING®"All the data were collected for at least two individual experiments.Table 2. Activity of lla~m and 12a~m for human STING (hSTING)""All the data were collected for at least two individual experiments. ND: not determined.Table 3. Activity of 15~16, 17a~b, 18a~b, 22a and 23 for STING"15-16, 17a~b, 18a~b, 22a, 23"All the data were collected for at least two individual experiments. ND: not determined.

[0428] ZSA-51 and ZSA-52 activate both hSTING and mSTING. The binding affinity of ZSA-51 and ZSA-52 was shown in detail in FIG. 1 A. ZSA-52 displayed slightly better binding affinity (IC50 = 5 μM) than MSA-2 (IC50 = 6 μM), while ester ZSA-51 was almost non-responsive. Importantly, as seen from FIG. IB, ester ZSA-51 (EC50 = 0.1 μM) showed nanomolar STING activation activity on the cellular level, which was far more potent than MSA-2 (EC50 = 3.2 μM) and ZSA-52 (EC50 = 2.9 μM). As the induced production of IFN-β is crucial for antitumor immunity, their ability to accelerate the secretion of IFN-β was further tested. As a result, ZSA-51 and -52 were able to effectively increase the secretion of IFN-β , which surpassed MSA-2 at the concentration of 5 μM (FIG. 1C). DMXAA was reported to distinguish between hSTING and mSTING, so the activity of ZSA-51 and 52 on mSTING was investigated. ZSA-52 was able to bind with mSTING with an IC50 of 1 μM, while MSA-2 (IC50 = 2 μM) and DMXAA (IC50 = 5 μM) were less effective (FIG. ID). In the cells harboring mSTING, both ZSA-51 and -52 exhibited the activity of mSTING activation with EC50 values of 9 and 14 μM, respectively (FIG. IE). To confirm the target specificity, the compounds were tested on STING knock out (KO) THP1 cells. As expected, all the compounds were non-responsive which suggested that the immune-stimulation function of ZSA-51 and -52 was attributed to STING (FIG. IF).

[0429] The activity of ZSA-51 was further investigated after modifying the ester group. A tertiary amine moiety was attached, resulting in ZSA-53 and ZSA-54 which demonstrated improved solubility (FIG. 2A). Subsequently, the in vitro STING stimulation activity of ZSA-53 and ZSA-54 was assessed. In THP1 cells harboring hSTING, ZSA-53 and ZSA-54 significantly activated STING with EC50 values of 0.6 and 1.4 μM, respectively (FIG. 2B). Furthermore, a high concentration of IFN-β was produced by THP1 cells in the presence of ZSA-53 or ZSA-54 which largely surpassed MSA-2 (FIG. 2C). Additionally, ZSA-53 and ZSA-54 were effective for mSTING with EC50 values of 10 and 12 μM, respectively, in THP1 cells harboring mSTING (FIG. 2D).Table 4. Activity of 26a~j, 27a~b, 28a~f and 31~33 for STING®All the data were collected for at least two individual experiments. ND: not determined.

[0430] ZSA-5 ID and nano-ZSA-5 ID are significantly more potent than its monomer ZSA-51 and MSA-2 (Pan et al., Science 369, 935 (2020)) (FIG. 3).

[0431] The below table shows the calculated EC 50 of MSA-2, ZSA-51, ZSA-52D, ZSA- 5 ID and nano ZSA-5 ID in THP-1 blue ISG cells.

[0432] The below table shows data for Compound 39.Compound ECso in THPl-Blue-ISG Binding affinity with h-cells (μM) STING (ICso, μM) 25 4.7Example 3Albumin F ormulations

[0433] Albumin nanoformulations of ZSA-52N were prepared. ZSA-52N was dissolved in 1 mL of chloroform (organic phase) and then added dropwise into human serum albumin (66kDa) dissolved in 20 mL Milli Q water (water phase) to generate a milky emulsion using a rotor-stator homogenizer. A crude ZSA-52N emulsion was obtained after running 6 cycles of low pressure (20000 psi) on a high-pressure homogenizer (Nano DeBEE). The organic solvent was removed by rotary evaporation at 25°C. After being filtered with a 0.22 pm strainer, the resulting nanoformulation was lyophilized to obtain dry powder for long-term storage at -20 °C. FIG. 4A shows the particle size distribution for the obtained nanoformulations

[0434] ZSA-5 ID (10 mg) was dissolved in 1 ml of chloroform and then added into 100 mg of human or mouse serum albumin dissolved in 20 ml of Milli-Q water to generate a milky emulsion using a rotor-stator homogenizer. The nanosuspension was obtained after running five cycles at 25,000 psi on a high-pressure homogenizer (Nano DeBEE) at 4°C. The organic solvent was removed using a rotary evaporator at 30°C under reduced pressure. After filtering (0.45 μm), the ZSA-5 ID human serum albumin NPs was lyophilized and stored at -20°C.

[0435] The ZSA-5 ID albumin NPs is stable and size around 140 nm with good PDI less than 0.2 (FIG. 4B).Example 4 Pharmacokinetics, Biodistribution, and Antitumor Efficacy

[0436] Plasma and liver microsome stability

[0437] Plasma stability. The tested compounds were prepared as 100 μM stock solution. 396 μL of plasma (Innovative Research) was added to a microcentrifuge tube pre-warmed at37 °C for 5 min. 4 μL of the tested compound was added to the tube. The mixture was incubated at 37°C for 5, 15, 30, 60, 120, and 240 min. Then, an aliquot of the reaction solution (40 μL) was mixed with 160 μL of clofazimine (100 ng / mL) and centrifuged at 3500 rpm for 10 minutes to precipitate protein. The supernatant was used for LC-MS / MS analysis.

[0438] Liver microsome stability. The tested compounds were prepared as 10 μM stock solution. The microsomes (XEno TECH) were prepared in phosphate buffer (100 mM) containing MgCh (3 mM) and NADPH (1 μM). The solution was prewarmed at 37°C for 5 min. Then, 40 μL of the tested compound was added to the solution. The resulting mixture was incubated at 37°C for 5, 10, 15, 30, 45, and 60 min. Then, an aliquot of the reaction solution (40 μL) was mixed with 160 μL of clofazimine (100 ng / mL) and centrifuged at 3500 rpm for 10 minutes to precipitate protein. The supernatant was used for LC-MS / MS analysis.

[0439] In vivo PK study. The tested compounds were orally (10 mg / kg) or intravenously (5 mg / kg) administered to three mice with DMSO / PEG-400 / PBS (2 / 3 / 5, v / v / v) as formulation at Img / mL. Tissue or blood samples were collected at the indicated time point. Blood samples were collected using heparinized calibrated pipettes and centrifuged at 15000 rpm for 10 min. Subsequently, plasma was collected from the supernatant. The plasma was frozen at -80°C for later analysis. Tissues were taken out and frozen at -80°C immediately for later preparation and analysis.

[0440] LC-MS / MS was performed using a HPLC System, Prominence ULLC (Shimadzu). Separation was achieved on a Waters XBridge C18 column (2.1 mm x 50 mm,3.5 pm). The autosampler and column oven temperatures were 4 and 30 °C, respectively, and the sample injection volume was 5 μL. The mobile phase consisted of formic acid 0.10% (spontaneous pH) in water as solvent A and formic acid 0.10% (spontaneous pH) in acetonitrile as solvent B at a flow rate of 400 μL / min. The gradient was as follows: 0-0.70 min (99% A and 1% B), 1.0-3.5 min (1% A and 99% B), 3.6 min (99% A and 1% B), and5.6 min (99% A and 1% B). The gradient step was linear. Mass spectrometry was performed using a Triple Quad 5500 (Sciex) mass spectrometer. The detection of analytes was achieved by electrospray ionization (ESI) in the positive mode with an appropriate MRM transition. Other mass spectrometer settings were as follows: capillary voltage and cone voltage were optimized for each compound; the desolation temperature was 550 °C. The LC- MS / MS instrument was controlled by Analyst software (Sciex).

[0441] In vivo antitumor efficacy. All animal experimental procedures were performed according to the protocols approved by the unit for laboratory animal medicine (ULAM) and the institution animal care and use committee (IACUC) University of Michigan. Eight-week-old female C57BL / 6 mice were obtained from Charles River (Wilmington, MA, USA) and eight-week-old female B6.Cg-Ceslctml.lLoc / J mice (Es~ ) were obtained from the Jackson laboratory (Bar Harbor, ME, USA). 5 x 105MC38 cells (murine colon adenocarcinoma cells), KPC-6422 or KPC-6620 cells (murine pancreatic cell line) in 100 μL FBS-free DMEM medium were injected into right lower flank or bilateral sides of the mice. The mice were assigned to treatment arms of 5 mice each when the mean tumors volume reached approximate 50 mm3among all treatment groups. Tumor and body weight measurements were performed using calipers and weigh scale respectively. Tumor volume was calculated with the formula V = (length x width2) / 2. Mice were euthanized when tumor diameter is greater than approached 20 mm, weight loss exceeds 20%, or tumors ulcerate.

[0442] Graph and statistical analysis. All the statistical graphs were generated by GraphPad Prism 9. Statistical test was performed through t tests (*P < 0.05, **P < 0.01, ***P < 0.001).

[0443] To preliminarily evaluate the clearance of the compounds in vivo, plasma and liver microsome stability were determined. Procaine and verapamil were used as control. The stability was reflected by the change of compound concentration after incubation with plasma or microsome. ZSA-51, the ester bond of which can be easily hydrolyzed to release active ZSA-52, was remarkably unstable in both plasma and liver microsome (Table 5). In contrast, ZSA-52 showed much better stability, which laid the foundation for its in vivo efficacy (Table 5).

[0444] Tissue exposure is crucial for a drug to exert in vivo efficacy and lower toxicity. Hence, the tissue distribution of ZSA-51 and ZSA-52 was investigated. As expected, ZSA-51 was not detected in plasma and tissue because of fast hydrolysis of the ester bond. Only ZSA-52 was detected after administration of ZSA-51. As shown in FIG. 5 A, peak plasma concentration of ZSA-52 was reached in plasma after oral (PO) or intravenous (IV) administration. The bioavailability of ZSA-51 and ZSA-52 reached 49% and 51%, respectively. After 7 h, the concentration was still as high as -900 ng / mL (2.7 μM) which is nearly the same as the EC50 of ZSA-52 (2.9 μM, FIG. IB). The tissue concentration was determined in liver, spleen, lymph node, fat pad and pancreas. First, the tissue concentration was investigated after 7 h treatment with ZSA-51 or ZSA-52. As displayed in FIG. 5B, ZSA-52 was able to be detected in all the tissues. In particular, ZSA-52 was confirmed to infiltrate into lymph node and spleen which are pivotal immune response-related tissues. Moreover, compared to ZSA-52 administered, ZSA-51 afforded higher concentration of ZSA-52 in spleen (IV), lymph node (PO and IV), fat pad (IV) and pancreas (PO). Then, in considerationof high tissue concentration reached by ZSA-51, the tissue distribution of ZSA-51 was studied at earlier times (2 h and 4 h, FIG. 5C). Short time treatment led to high tissue concentration especially in lymph node (-1000 ng / g after 2 h treatment (IV)).

[0445] The in vivo pharmacokinetics and biodistribution of SH-254 and SH-266 were tested on Balb / c mice after oral dosing (P.O. lOmg / kg) and intravenous dosing (I.V. 5mg / kg) of SH-254 (mice SH-254) or SH-266 (mice SH-266) (FIG. 6).

[0446] The in vivo antitumor efficacy of ZSA-51 and ZSA-52 was also evaluated. As depicted in FIG. 5D, ZSA-51 alone effectively inhibited the tumor growth in MC38-bearing mice with 42% tumor growth inhibition (TGI), while ZSA-52 was less responsive.Noticeably, ZSA-51 plus anti-PDl antibody (anti-PDl) dramatically suppressed the tumor growth with 81 % TGI. Minimum body weight change of the mice revealed low toxicity of the compounds (FIG. 5E). High dose of ZSA-51 (100 mg / kg) plus anti-PDl reached 85% TGI without causing significant weight loss indicating ZSA-51 was well tolerated (FIG. 5F- 5G). Subsequently, the in vivo activity of ZSA-51 was further investigated through subcutaneous (SC) injection (FIGS. 5H-5J). To better demonstrate the systemic immune- simulating effect of ZSA-51, two tumors (the local and distal relative to the compoundinjecting site) were planted. Interestingly, both local and distal tumors are dramatically responsive to ZSA-51 plus anti-PDl with local tumor almost completely suppressed.

[0447] Carboxylesterase (CES) was reported to be abundant in mouse plasma but absent in human plasma. Mice lacking exon 5 of the Ceslc gen e (Esl'1') were employed to mimic human. The tumor growth was nearly terminated, which further suggested ZSA-51 would be suitable for human use (FIG. 5K-5L).

[0448] The antitumor efficacy of SH-254 and SH-266 was tested in an MC-38 xenograft tumor mice model (FIG. 7).Table 5 In vitro lasma and liver microsome stabilit of ZSA-51 and ZSA-52 (Z1 / 2 min)a

[0449] Pancreatic cancer is tricky and believed to be insensitive to immunotherapy because of the immunosuppressive microenvironment. Therefore, response to the immune-stimulating agents was explored in a pancreatic cancer model. ZSA-51 and ZSA-52 were tested in pancreatic cancer cell KPC-6422 -bearing mice. As seen in FIG. 8A, ZSA-51 (53 mg / kg, PO) and ZSA-52 (80 mg / kg, PO) significantly suppressed the tumor growth with (TGI = 39% and 68%, respectively) or without anti-PDl (TGI = 33% and 43%, respectively). Moreover, no significant body loss was observed implying low toxicity (FIG. 8B). To further confirm the efficacy against pancreatic cancer, KPC-6620 bearing mice were employed. Both SC and PO administrations of ZSA-51 dramatically suppressed the tumor growth with TGI of 75% and 65%, respectively, without obvious toxicity (FIGS. 8C-8D). These results demonstrated that ZSA-51 was a potent systemic STING-stimulating agent with superior PK properties for the treatment of cancer.

[0450] The PK properties of ZSA-53 and ZSA-54 were also investigated. Their short halflife time in both plasma and liver microsome suggested that the ester bonds of ZSA-53 and ZSA-54 can be easily cleaved (Table 5). As shown in FIG. 2E, ZSA-53 and ZSA-54 generated considerably higher plasma concentration of ZSA-52 with oral bioavailability of 78% and 84%, respectively. Importantly, after 7 h, oral administration of ZSA-53 and ZSA- 54 afforded a plasma concentration of ZSA-52 as high as 2527 ng / mL (7.7 μM) and 3486 ng / mL (10.4 μM), respectively, which were larger than EC50 of ZSA-52 (2.9 μM). As displayed in FIGS. 2F-2G, ZSA-53 and ZSA-54 were able to penetrate spleen and lymph node which are critical immunity-related tissues. Interestingly, ZSA-54 showed priority for lymph node, especially after 4 h oral administration, which was beneficial for its potency and low toxicity (FIG. 2G).

[0451] To validate that the in vivo efficacy relied on STING, the acid group of ZSA-52 was converted to amide group to obtain ZSA-52NC1 and ZSA-52NC2 as negative control (FIG. 2A). ZSA-52NC2 was nonresponsive to STING at concentrations as high as 100 μM (FIG. 2B). Therefore, ZSA-52NC2 was selected as negative control for in vivo study. As can be seen in FIGS. 2H-2I, ZSA-53 and ZSA-54 dramatically inhibited tumor growth in MC38- bearing mice, especially when combined with anti-PD 1 without causing weight loss, which was nearly the same as ZSA-51. Negative control ZSA-52NC2 did not show any in vivo efficacy suggesting that the in vivo antitumor efficacy of ZSA-53 and ZSA-54 relied on STING. These results suggested that ZSA-53 and ZSA-54 were potent STING-stimulating agents for the treatment of cancer, which were comparable to ZSA-51.Example 5Structure-Function

[0452] Molecular docking and MD simulation. Docking studies were performed employing Autodock4. The STING dimer protein (PDB: 6ukz) was processed using Autodocktools. The water molecules were removed. Kollman charges and hydrogen atoms were added. The conformation with lowest binding energy was selected and processed in Pymol. The MD simulation was performed in GROMACS for the docking results of ZSA-52. The potential energy was minimized to -8.6 kJ / mol. The pressure, temperature and density were maintained at 1 atm, 300 K and 1 kg / m3, respectively. Production dynamics simulations were conducted for 10 ns. The data were exported and analyzed in GraphPad Prism 9.

[0453] The binding mode of ZSA-52 was investigated employing Autodock4. ZSA-52 was docked into STING dimer (PDB: 6ukz). Two conformations (yellow and blue, FIG. 9A were extracted with the yellow one showing the lowest binding energy. These two conformations formed a dimer occupying cGAMP-binding pocket of STING dimer, which was consistent with reported binding modes of other STING agonists. In terms of yellow conformation, the carboxyl group formed critical hydrogen bonds with Arg238 of STING monomer B. The two carbonyl groups on the scaffold made hydrogen bond interactions with Thr263 and Arg238 of monomer A. Hydrogen bonds were also observed between the methoxy groups and Seri 62 of monomer A. The binding mode was very similar to that of MSA-2 as further suggested by the overlapping with MSA-2 (FIG. 9B, cyan).To further validate this binding mode, 10 ns molecular dynamic (MD) simulations employing GROMACS were performed. From the trajectory snapshots, the root mean square deviation (RMSD) of backbone Ca atoms of STING and those of the heavy atoms of ZSA- 52 were plotted. As shown in FIG. 9C, RMSD of STING protein and ZSA-52 were below 0.15 and 0.25 A, respectively, suggesting they form a stable ligand-protein complex. To confirm the hydrogen bond network depicted from docking (FIG. 9A), the hydrogen bond number was calculated. As depicted in FIG. 9D, almost four hydrogen bonds stably formed in the whole simulation process. To further validate the existence of those hydrogen bonds the distance of six hydrogen bonds indicated in FIG. 9E were calculated (FIGS. 9F-9H). The distances of four hydrogen bonds were shorter than 0.35 nm (3.5 A), which was consistent with total hydrogen bonds calculated in FIG. 9D. These results indicated that ZSA-52 stably bound to STING dimer with at least four hydrogen bonds formed.

Claims

CLAIMS1. A compound of formula (I):or a pharmaceutically acceptable salt thereof, wherein:R1, R2, R3, and R4are each independently selected from hydrogen, C1-C6alkoxy, halo, and C1-C6alkyl;X1is selected from S and NH;A is selected from a five- or six-membered cycloalkyl, heterocyclyl, heteroaryl, or aryl, each of which is optionally substituted with 1 or 2 oxo groups;W1is a bond or O;W2is selected from C1-C6alkylene, C3-C6 cycloalkylene, C3-C6 cycloalkylene-C1-C6alkylene, arylene, and hydroxy-C1-C6-alkylene; andR5is selected from hydrogen, C1-C80alkyl, C2-C80alkenyl, C2-C80alkynyl, amino, amino-C1-C6-alkyl, di-C1-C6-alkylamino-C1-C6-alkyl, heterocyclyl-C1-C6-alkyl, Ci-Cso heteroalkyl, C2-C80heteroalkenyl, and C2-C80heteroalkynyl, wherein each of the Ci-Cso alkyl, C2-C80alkenyl, C2-C80alkynyl, Ci-Cso heteroalkyl, C2-C80heteroalkenyl, and C2-C80heteroalkynyl is optionally substituted with one or more substituents selected from hydroxy and amino.

2. The compound of claim 1, wherein the compound is a compound of formula (la):or a pharmaceutically acceptable salt thereof, wherein X2is CH or N, and X3is CH2 or CO.

3. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein X2is CH.

4. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein X2is N.

5. The compound of any of claims 2-4, or a pharmaceutically acceptable salt thereof, wherein X3is CH2.

6. The compound of any of claims 2-4, or a pharmaceutically acceptable salt thereof, wherein X3is CO.

7. The compound of claim 1, wherein the compound is a compound of formula (lb):or a pharmaceutically acceptable salt thereof, wherein X4and X5are independently selected from N and CH.

8. The compound of claim 7, or a pharmaceutically acceptable salt thereof, wherein X4is N.

9. The compound of claim 7 or 8, or a pharmaceutically acceptable salt thereof, wherein X5is N.

10. The compound of claim 7 or 8, or a pharmaceutically acceptable salt thereof, wherein X5is CH.

11. The compound of claim 1, wherein the compound is a compound of formula (Ic):or a pharmaceutically acceptable salt thereof, wherein X6and X7are independently selected from CH and N.

12. The compound of claim 11, or a pharmaceutically acceptable salt thereof, wherein X6is N.

13. The compound of claim 11 or 12, or a pharmaceutically acceptable salt thereof, wherein X7is N.

14. The compound of any of claims 1-13, or a pharmaceutically acceptable salt thereof, wherein X1is S.

15. The compound of any of claims 1-13, or a pharmaceutically acceptable salt thereof, wherein X1is NH.

16. The compound of any of claims 1-13, or a pharmaceutically acceptable salt thereof, wherein W2is selected from C1-C4alkylene, C4-C5cycloalkylene, C4-C5cycloalkylene-C1-C2alkylene, phenylene, and hydroxy-C1-C3-alkylene.

17. The compound of any of claims 1-16, or a pharmaceutically acceptable salt thereof, wherein W2is selected from:

18. The compound of any of claims 1-17, or a pharmaceutically acceptable salt thereof, wherein R5is selected from hydrogen, C1-C40alkyl, C2-C40alkenyl, C1-C4-alkylamino, di-C1- C6-alkylamino-C1-C6-alkyl, and heterocyclyl-C1-C6-alkyl.

19. The compound of any of claims 1-18, or a pharmaceutically acceptable salt thereof, wherein R5is hydrogen or C1-C4alkyl.

20. The compound of any of claims 1-19, or a pharmaceutically acceptable salt thereof, wherein R5is hydrogen, methyl, ethyl, or isopropyl.

21. The compound of any of claims 1 -20, or a pharmaceutically acceptable salt thereof, wherein R5is hydrogen or methyl.

22. The compound of any of claims 1-21, or a pharmaceutically acceptable salt thereof, wherein R5is selected from C12-C40alkyl and C12-C40alkenyl.

23. The compound of any of claims 1-22, or a pharmaceutically acceptable salt thereof, wherein R5is selected from C12-C20alkyl and C12-C20alkenyl.

24. The compound of any of claims 1-23, or a pharmaceutically acceptable salt thereof,25. The compound of any of claims 1-23, or a pharmaceutically acceptable salt thereof, wherein R2and R3are each independently C1-C6alkoxy.

26. The compound of claim 25, or a pharmaceutically acceptable salt thereof, wherein R2and R3are each methoxy.

27. The compound of any of claims 1-26, or a pharmaceutically acceptable salt thereof, wherein R1and R4are each independently halo or hydrogen.

28. The compound of claim 27, or a pharmaceutically acceptable salt thereof, wherein R1is halo and R4is hydrogen.

29. The compound of any of claims 1-28, or a pharmaceutically acceptable salt thereof, wherein R1is halo, R2and R3are each independently C1-C6alkoxy, and R4is hydrogen.

30. The compound of claim 29, or a pharmaceutically acceptable salt thereof, wherein R1is chloro or bromo, and R2and R3are methoxy.

31. The compound of any of claims 1 -27, or a pharmaceutically acceptable salt thereof, wherein R1and R4are hydrogen, and R2and R3are methoxy.

32. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of:or a pharmaceutically acceptable salt thereof, wherein:R1a, R1b, R2a, R2b, R3a, R3b, R4a, and R4bare each independently selected from hydrogen, C1-C6alkoxy, halo, and C1-C6alkyl; wherein one of R1a, R2a, R3a, and R4a, and one of R1b, R2b, R3b, and R4brepresents a point of attachment to Linker;X1aand X1bare each independently selected from NH and S;A’ and A” are each independently a five- or six-membered cycloalkyl, heterocyclyl, heteroaryl, or aryl, each of which is optionally substituted with 1 or 2 oxo groups;W1aand W1bare each independently a bond or O;W2aand W2bare each independently selected from C1-C6alkylene, C3-C6cycloalkylene, C3-C6cycloalkylene-C1-C6alkylene, arylene, and hydroxy-C1-C6-alkylene;R5aand R5bare each independently selected from hydrogen, C1-C80alkyl, C2-C80alkenyl, C2-C80alkynyl, amino, amino-C1-C6-alkyl, di-C1-C6-alkylamino-C1-C6-alkyl, heterocyclyl-C1-C6-alkyl, Ci-Cso heteroalkyl, C2-C80heteroalkenyl, and C2-C80heteroalkynyl, wherein each of the Ci-Cso alkyl, C2-C80alkenyl, C2-C80alkynyl, Ci-Cso heteroalkyl, C2-C80heteroalkenyl, and C2-C80heteroalkynyl is optionally substituted with one or more substituents selected from hydroxy and amino.

34. The compound of claim 33, wherein the compound is a compound of formula (Ila):or a pharmaceutically acceptable salt thereof, wherein X2aand X2bare each independently CH or N, and X3aand X3bare each independently CH2 or CO.

35. The compound of claim 34, or a pharmaceutically acceptable salt thereof, wherein X2aand X2bare CH.

36. The compound of claim 34, or a pharmaceutically acceptable salt thereof, wherein X2aand X2bare N.

37. The compound of any of claims 34-35, or a pharmaceutically acceptable salt thereof, wherein X3aand X3bare CH2.

38. The compound of any of claims 34-36, or a pharmaceutically acceptable salt thereof, wherein X3aand X3bare CO.

39. The compound of any of claims 33-38, or a pharmaceutically acceptable salt thereof, wherein X1aand X1bare S.

40. The compound of any of claims 33-38, or a pharmaceutically acceptable salt thereof, wherein X1aand X1bare NH.

41. The compound of any of claims 33-40, or a pharmaceutically acceptable salt thereof, wherein W2aand W2bare each independently selected from C1-C4alkylene, C4-C5cycloalkylene, C4-C5cycloalkylene-C1-C2alkylene, phenylene, and hydroxy-C1-C3-alkylene.

42. The compound of any of claims 33-41, or a pharmaceutically acceptable salt thereof, wherein W2aand W2bare each independently selected from43. The compound of any of claims 33-42, or a pharmaceutically acceptable salt thereof, wherein R5aand R5bare each independently selected from hydrogen, C1-C40alkyl, C2-C40alkenyl, and C1-C4-alkylamino.

44. The compound of any of claims 33-43, or a pharmaceutically acceptable salt thereof, wherein R5aand R5bare each independently hydrogen or C1-C4alkyl.

45. The compound of claim 44, or a pharmaceutically acceptable salt thereof, wherein R5aand R5bare each independently hydrogen, methyl, ethyl, or isopropyl.

46. The compound of claim 45, or a pharmaceutically acceptable salt thereof, wherein R5aand R5bare each independently hydrogen or methyl.

47. The compound of any of claims 33-42, or a pharmaceutically acceptable salt thereof, wherein R5aand R5bare each independently selected from C12-C40alkyl and C12-C40alkenyl.

48. The compound of claim 47, or a pharmaceutically acceptable salt thereof, wherein R5aand R5bare each independently selected from C12-C20alkyl and C12-C20alkenyl.

49. The compound of claim 48, or a pharmaceutically acceptable salt thereof, wherein R5a50. The compound of any of claims 33-49, or a pharmaceutically acceptable salt thereof, wherein R1aand R1bare each independently halo or hydrogen.

51. The compound of claim 50, or a pharmaceutically acceptable salt thereof, wherein R1aand R1bare each hydrogen.

52. The compound of any of claims 33-51, or a pharmaceutically acceptable salt thereof, wherein R2aand R2bare each the point of attachment to Linker.

53. The compound of any of claims 33-52, or a pharmaceutically acceptable salt thereof, wherein R3aand R3bare each independently C1-C6alkoxy.

54. The compound of claim 53, or a pharmaceutically acceptable salt thereof, wherein R3aand R3bare each methoxy.

55. The compound of any of claims 33-54, or a pharmaceutically acceptable salt thereof, wherein R4aand R4bare each hydrogen.

56. The compound of any of claims 33-55, or a pharmaceutically acceptable salt thereof, wherein Linker is a group -0-(CH2)m-0-, or a pharmaceutically acceptable salt thereof, wherein m is 1, 2, 3, 4, 5, or 6.

57. The compound of claim 56, or a pharmaceutically acceptable salt thereof, wherein m is 3.

58. The compound of claim 33, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

59. A pharmaceutical composition comprising an effective amount of a compound of any one of claims 1-58, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

60. The pharmaceutical composition of claim 59, wherein the composition comprises albumin nanoparticles, liposomes, micelles, or lipid nanoparticles.

61. The pharmaceutical composition of claim 59 or 60, wherein the composition further comprises at least one additional therapeutic agent.

62. The pharmaceutical composition of claim 61, wherein the at least one additional therapeutic agent comprises an immune modulator, a chemotherapeutic agent, a nucleic acid, a decongestant, a steroid, an analgesic, an antimicrobial agent, or a combination thereof.

63. The pharmaceutical composition of claim 61 or 62, wherein the at least one additional therapeutic agent comprises an RNA selected from the group consisting of a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicersubstrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), and mixtures thereof.

64. The pharmaceutical composition of claim 61 or 62, wherein the at least one additional therapeutic agent is selected from a chemotherapeutic agent, an IDO inhibitor, a Stat3 inhibitor, a TLR agonist, and a PI3K inhibitor.

65. The pharmaceutical composition of any of claims 59-64, further comprising one or more cell targeting epitopes.

66. The pharmaceutical composition of claim 65, wherein the one or more cell targeting epitopes are covalently attached or directly conjugated to an albumin.

67. The pharmaceutical composition of claim 65 or 66, wherein the cell targeting epitopes comprise an immune cell epitope.

68. The pharmaceutical composition of claim 65 or 66, further comprising one or more epitopes from a microbiological agent.

69. A vaccine comprising an effective amount of: a compound of any one of claims 1-58, or a pharmaceutically acceptable salt thereof, or a composition of any of one of claims 59-68; and an antigen or a nucleic acid encoding thereof.

70. The vaccine of claim 69, the antigen is a tumor antigen, a self-antigen, or an infectious disease derived antigen.

71. The vaccine of claim 69 or 70, wherein the nucleic acid is messenger RNA (mRNA).

72. A method of treating or preventing a disease or disorder comprising administering an effective amount of a compound of any one of claims 1-58, or a pharmaceutically acceptable salt thereof, a composition of any of claims 59-68, or a vaccine of any of claims 69-71, to a subject in need thereof.

73. The method of claim 72, wherein the disease or disorder comprises cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.

74. The method of claim 72 or 73, wherein the disease or disorder is cancer.

75. The method of claim 74, wherein the subject has cancer, has had cancer, is predisposed to cancer, or has a family history of cancer.

76. The method of any of claims 73-75, wherein the cancer comprises a solid tumor.

77. The method of any of claims 73-76, wherein the cancer is metastatic cancer.

78. The method of any of claims 73-77, wherein the method suppresses or eliminates cancer metastasis, decreases tumor growth, prevents tumor recurrences, or any combination thereof.

79. The method of any of claims 73-78, wherein the administering comprises an initial administration and at least one subsequent administration.

80. A method of inducing or modulating an immune or inflammatory response in a subject comprising administering the composition of a compound of any one of claims 1-58, or a pharmaceutically acceptable salt thereof, a composition of any of claims 59-68, or the vaccine of any of claims 69-71, to a subject in need thereof.

81. The method of any of claims 73-80, wherein the subject is human.

82. The method of any of claims 73-81, further comprising administering at least one additional therapeutic agent.

83. The method of claim 82, wherein the at least one additional therapeutic agent comprises an immune modulator, a chemotherapeutic agent, a nucleic acid, a decongestant, a steroid, an analgesic, an antimicrobial agent, or a combination thereof.

84. Use of a compound of any one of claims 1-58, or a pharmaceutically acceptable salt thereof, or a composition of any of claims 59-6967 in the manufacture of a medicament for the treatment or prevention of a disease or disorder.

85. The use of claim 84, wherein the disease or disorder comprises cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.

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