Novel therapies for erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP)

Abstract

Novel compositions and methods of using the same for the treatment of erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP) are disclosed. Methods and compositions related to the treatment of erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP) are disclosed. Therapeutic agents capable of inhibiting ABCG2 are also described. For example, therapeutic agents defined by Formula I are described herein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 850,061, filed May 20, 2019, which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Grant No. R01 DK090305 awarded by the National Institute of Diabetes and Digestive and Kidney Diseases. The government has certain rights in this invention. [Background technology]

[0003] Porphyrias are a group of metabolic disorders of the heme biosynthetic pathway. Erythropoietic protoporphyria (EPP) is the third most common type of porphyria and the most common childhood porphyria. EPP is characterized by the loss of Fe 2+ EPP is caused by loss-of-function mutations in ferrochelatase (FECH), the final enzyme in the heme biosynthetic pathway that combines α-aminolevulinic acid with protoporphyrin IX (PPIX) to form heme. Due to FECH deficiency, PPIX accumulates significantly in patients with EPP, primarily in red blood cells (RBCs), plasma, and the liver. In addition to EPP, PPIX accumulation also occurs in X-linked protoporphyria (XLP), another type of porphyria caused by gain-of-function mutations in δ-aminolevulinic acid synthase 2 (ALAS2), a rate-limiting enzyme in the heme biosynthetic pathway. Furthermore, many clinically used drugs and environmental toxins, including rifampicin (RIF), isoniazid (INH), diethoxycarbonyl-1,4-dihydrocollidine (DDC), and griseofulvin (GSF), can lead to PPIX accumulation in the liver by inducing ALAS and / or inhibiting FECH. Novel treatments for EPP and XLP are needed. Summary of the Invention [Means for solving the problem]

[0004] Methods and compositions related to the treatment of erythropoietic protoporphyria and X-linked protoporphyria are disclosed.

[0005] Also described are therapeutic agents capable of inhibiting ABCG2, for example, therapeutic agents defined by Formula I:

[0006] [ka] (In the formula, A is,

[0007] [ka] selected from the group consisting of: n is an integer from 0 to 6; X, if present, is selected from the group consisting of CH2, O and S; R 1 H, halo, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl, CN, NO2, OR a , S.R. a , C(O)R b , C(O)NR c R d , C(O)OR a ,OC(O)R b , OC(O)NR c R d , N.R. c R d , N.R. c OR d , N.R. c C(O)R b , N.R. c C(O)OR a , N.R.c C(O)NR c R d , C(=NR e )R b , C(=NR e )NR c R d , N.R. c C(=NR e )NR c R d , N.R. c S(O)R b , N.R. c S(O)2R b , N.R. c S(O)NR c R d , S(O)R b , S(O)NR c R d , S(O)2R b , and S(O)NR c R d wherein C is selected from the group consisting of 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A optionally substituted with a group; R 2 and R 3 H, halo, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl, CN, NO2, OR a , and S.R. a wherein C is independently selected from the group consisting of 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A optionally substituted with a group; R 4 and R 5 is H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 independently selected from the group consisting of cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl; 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A optionally substituted with a group; R 6 and R 7 is H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 each independently selected from the group consisting of cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl; 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A optionally substituted with a group or R 6 and R 7 together with the N atoms to which they are attached, form one, two, or three independently selected R Agroups, each forming an optionally substituted 4- to 9-membered heterocycloalkyl group or a 5- to 6-membered heteroaryl group; R 8 and R 9 is H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 independently selected from the group consisting of cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl; 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A optionally substituted with a group or R 8 and R 9 together with the atoms to which they are attached, form one, two, or three independently selected R A groups, each forming an optionally substituted 4- to 9-membered heterocycloalkyl group or a 5- to 6-membered heteroaryl group; R 10 is C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl, 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A optionally substituted with a group; Each R a, R b , R c , and R d is H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 independently selected from cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl; C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A each optionally substituted with a group; Each R e are H, CN, and C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkylthio, C 1-6 Alkylsulfonyl, C 1-6 Alkyl carbonyl, C 1-6 Alkylaminosulfonyl, Carbamyl, C 1-6 Alkylcarbamyl, di(C 1-6 Alkyl)carbamyl, aminosulfonyl, C 1-6 Alkylaminosulfonyl, and di(C 1-6 alkyl)aminosulfonyl; and Each R A OH, NO2, CN, halo, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, Cyano-C 1-3 Alkyl, HO-C 1-3 Alkyl, Amino, C 1-6 Alkylamino, di(C 1-6 Alkyl)amino, thio, C 1-6 Alkylthio, C1-6 Alkylsulfinyl, C 1-6 Alkyl sulfonyl, carbamyl, C 1-6 Alkylcarbamyl, di(C 1-6 Alkyl) carbamyl, carboxy, C 1-6 Alkyl carbonyl, C 1-6 Alkoxycarbonyl, C 1-6 Alkylcarbonylamino, C 1-6 Alkyl sulfonyl amino, amino sulfonyl, C 1-6 Alkylaminosulfonyl, di(C 1-6 Alkyl)aminosulfonyl, aminosulfonylamino, C 1-6 Alkylaminosulfonylamino, di(C 1-6 Alkyl)aminosulfonylamino, aminocarbonylamino, C 1-6 Alkylaminocarbonylamino, and di(C 1-6 alkyl)aminocarbonylamino; or a pharmaceutically acceptable salt, ester, or N-oxide thereof provided herein.

[0008] Also disclosed herein are pharmaceutical compositions comprising the therapeutic agents of any of the above aspects, further comprising rifampicin (RIF), isoniazid (INH), diethoxycarbonyl-1,4-dihydrocollidine (DDC), or griseofulvin (GSF).

[0009] In one embodiment, disclosed herein is a method of treating, preventing, reducing, or inhibiting erythropoietic protoporphyria (EPP) or X-linked protoporphyria (XLP) in a subject, the method comprising administering to the subject a therapeutic agent that inhibits ABCG2 activity, including but not limited to any therapeutic agent that inhibits ABCG2 of any of the foregoing embodiments.

[0010] Also disclosed herein is a method for reducing PPIX efflux from red blood cells or hepatocytes in a subject, the method comprising administering to the subject a therapeutic agent that inhibits ABCG2 activity.

[0011] Also disclosed herein, in one aspect, is a method of treating, preventing, reducing, or inhibiting purpura, erythema, edema, or burning sensation in the skin of a subject with EPP, the method comprising administering to the subject a therapeutic agent that inhibits ABCG2 activity.

[0012] In one aspect, disclosed herein is a method of treating, preventing, reducing, or inhibiting hepatotoxicity in the skin of a subject (e.g., having EPP), the method comprising administering to the subject a therapeutic agent that inhibits ABCG2 activity.

[0013] In one embodiment, disclosed herein is a method of treating, preventing, reducing, or inhibiting EPP, XLP, PPIX excretion from red blood cells or hepatocytes, skin purpura, erythema, edema, or burning sensation of any preceding embodiment, wherein the therapeutic agent is an antibody, peptide, protein, RNAi, small molecule, or a targeted nucleic acid integration system (e.g., a clustered regularly interspaced short palindromic repeat (CRISPR) / CRISPR-associated 9 (Cas9) integration system) comprising a guide RNA targeting the ABCG2 gene, or an RNAi targeting the ABCG2 gene), such as, for example, an anti-ABCG2 antibody.

[0014] In one aspect, disclosed herein is a method of treating, preventing, reducing, or inhibiting EPP, XLP, PPIX excretion from red blood cells or hepatocytes, skin purpura, erythema, edema, or burning sensation of any of the preceding aspects, wherein the agent comprises a tissue-specific targeting moiety, or an expression vector.

[0015] In one embodiment, disclosed herein are cells comprising an ABCG2 knockout. Also disclosed herein are transgenic animals comprising the cells of any of the above embodiments.

[0016] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and, together with the specification, explain the compositions and methods of the present disclosure. [Brief explanation of the drawings]

[0017] [Figure 1] Figures 1A, 1B, 1C, 1D, 1E, and 1F show the role of ABCG2 in ERP-related phototoxicity. Figure 1A shows the genotyping results for WT, Abcg2-null, Fech-mut, and Fech-mut / Abcg2-null mice. Fech-mut / Abcg2-null mice are deficient in both Fech and Abcg2. Figure 1B shows the macroscopic findings of mice after light exposure. The hair on the backs of mice was shaved and exposed to UV light (395–410 nm) for 30 minutes daily for 5 days. Figures 1C, 1D, 1E, and 1F show histological sections of mouse skin after UV light exposure and H&E staining. Bars equal 40 μm. [Figure 2] Figures 2A, 2B, and 2C show oxidative stress and inflammation in the skin of WT, Abcg2-null, Fech-mut, and Fech-mut / Abcg2-null mice after exposure to UV light. The back skin of the mice was shaved and exposed to UV light (395–410 nm) for 30 minutes daily for 5 days. Figure 2A shows glutathione (GSH) levels in the skin. Figures 2B and 2C show the mRNA expression of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the skin. All data are expressed as mean ± SEM (n = 4 per group). *P < 0.05, ***P < 0.001 by one-way analysis of variance (ANOVA). [Figure 3]Figures 3A, 3B, 3C, and 3D show that ABCG2 regulates PPIX distribution in red blood cells (RBCs), plasma, and skin in an EPP mouse model. Figure 3A shows PPIX levels in RBCs. Figure 3B shows PPIX levels in serum. Figure 3C shows PPIX levels in mouse skin after exposure to UV light. PPIX was analyzed by UPLC-QTOFMS. All data are presented as mean ± SEM (n = 4 per group). *P < 0.05, ***P < 0.001 (analysis by one-way ANOVA). Figure 3D shows an overview demonstrating that ABCG2 deficiency reduces PPIX distribution to the skin and suppresses PPIX-mediated phototoxicity in EPP. [Figure 4-1] Figures 4A, 4B, 4C, 4D, 4E, 4F, 4G, 4H, and 4I show that EPP-associated hepatotoxicity is dependent on ABCG2. WT, Abcg2-null, Fech-mut, and Fech-mut / Abcg2-null mice were maintained under identical conditions and sacrificed at similar ages. Figures 4A, 4B, and 4C show serum activities of alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP). [Figure 4-2] Figures 4A, 4B, 4C, 4D, 4E, 4F, 4G, 4H, and 4I show that EPP-associated hepatotoxicity is dependent on ABCG2. WT, Abcg2-null, Fech-mut, and Fech-mut / Abcg2-null mice were maintained under identical conditions and sacrificed at similar ages. Figure 4D shows serum total bilirubin. Figure 4E shows PPIX in the liver analyzed by UPLC-QTOFMS. Data are presented as mean ± SEM (n = 4 per group). ***P < 0.001 (analysis by one-way ANOVA). Figures 4F, 4G, 4H, and 4I show representative H&E-stained liver sections. Arrows indicate bile plugs. Bars equal 10 μm. PV, portal vein; BD, bile duct. [Figure 5]Figures 5A, 5B, 5C, 5D, 5E, and 5F show that ABCG2 deficiency abolishes DDC-induced PPIX accumulation and hepatotoxicity. WT and Abcg2-null mice were treated with the porphyrin-generating agent DDC for 14 days. Figures 5A, 5B, and 5C show serum activities of ALT, AST, and ALP. Figure 5D shows PPIX in the liver analyzed by UPLC-QTOFMS. All data are expressed as mean ± SEM (n = 3-4 per group). **P < 0.01, ***P < 0.001 by one-way analysis of variance (ANOVA). Figures 5E and 5F show histological analysis of the liver by H&E staining. Arrows indicate bile plugs. Bars equal 10 μm. PV, portal vein; BD, bile duct. [Figure 6] Figures 6A, 6B, 6C, 6D, 6E, and 6F show that ABCG2 deficiency abolishes griseofulvin (GSF)-induced PPIX accumulation and hepatotoxicity. WT and Abcg2-null mice were treated with the porphyrin-generating agent GSF for 14 days. Figures 6A, 6B, and 6C show serum activity of ALT, AST, and ALP. Figure 6D shows PPIX in the liver. All data are expressed as mean ± SEM (n = 3-4 per group). ***P < 0.001 by one-way analysis of variance (ANOVA). Figures 6E and 6F show histological analysis of the liver by H&E staining. Arrows indicate bile plugs. Bars equal 10 μm. PV, portal vein; BD, bile duct. [Figure 7]Figures 7A, 7B, 7C, 7D, 7E, and 7F show PPIX accumulation and hepatotoxicity in hPXR and hPXR / Abcg2-null mice treated with rifampicin (RIF) and isoniazid (INH). Figure 7A shows the genotyping results of hPXR and hPXR / Abcg2-null mice. Figures 7B and 7C show serum ALT and ALP activities. Figure 7D shows PPIX in the liver analyzed by UPLC-QTOFMS. All data are expressed as mean ± SEM (n = 3-4 per group). *P < 0.05, **P < 0.001 by one-way analysis of variance (ANOVA). Figures 7E and 7F show histological analysis of the liver by H&E staining. Arrows indicate bile plugs. PV, portal vein; BD, bile duct. [Figure 8-1] Figures 8A, 8B, 8C, 8D, 8E, 8F, 8G, 8H, and 8I show that ABCG2 deficiency regulates the distribution, metabolism, and excretion of PPIX. Figures 8A, 8B, 8C, 8D, 8E, and 8F show metabolomic analysis in WT and Abcg2-null mice treated with deuterium-labeled aminolevulinic acid (D2-ALA), a precursor of PPIX. Liver and bile samples were collected 1 hour after D2-ALA treatment. Figure 8A shows a score plot of the liver samples generated by principal component analysis (PCA). Each point represents a mouse sample. [Figure 8-2] Figures 8A, 8B, 8C, 8D, 8E, 8F, 8G, 8H, and 8I show that ABCG2 deficiency modulates the distribution, metabolism, and excretion of PPIX. Figures 8A, 8B, 8C, 8D, 8E, and 8F show metabolomic analysis in WT and Abcg2-null mice treated with deuterium-labeled aminolevulinic acid (D2-ALA), a precursor of PPIX. Liver and bile samples were collected 1 hour after D2-ALA treatment. Figures 8B and 8C show S-plots of the liver and bile metabolome generated by orthogonal partial least squares discriminant analysis (OPLS-DA). Each point represents a metabolite. All metabolites were analyzed by UPLC-QTOFMS. [Figure 8-3]Figures 8A, 8B, 8C, 8D, 8E, 8F, 8G, 8H, and 8I show that ABCG2 deficiency modulates the distribution, metabolism, and excretion of PPIX. Figures 8A, 8B, 8C, 8D, 8E, and 8F show metabolomic analysis in WT and Abcg2-null mice treated with deuterium-labeled aminolevulinic acid (D2-ALA), a precursor of PPIX. Liver and bile samples were collected 1 hour after D2-ALA treatment. Figure 8D shows D16-PPIX in bile. Figures 8E and 8F show D16-protoporphyrin-1-O-acyl-glucuronide (D16-PPIX-glu) in liver and bile. All data are expressed as mean ± SEM. Data for WT mice are set as 100% or 1. *P<0.05, **P<0.001 by two-tailed Student's t-test. Figure 8G shows the structures of PPIX and its conjugated metabolites with glucuronic acid, xylose, and glucose. [Figure 8-4] Figures 8A, 8B, 8C, 8D, 8E, 8F, 8G, 8H, and 8I show that ABCG2 deficiency regulates the distribution, metabolism, and excretion of PPIX. Figures 8A, 8B, 8C, 8D, 8E, and 8F show metabolomic analysis in WT and Abcg2-null mice treated with deuterium-labeled aminolevulinic acid (D2-ALA), a precursor of PPIX. Liver and bile samples were collected 1 hour after D2-ALA treatment. Figure 8H shows the proportions of PPIX and its conjugated metabolites in the bile of Fech-mut and Fech-mut / Abcg2-null mice. Figure 8I shows an overview demonstrating that ABCG2 deficiency ameliorates EPP-associated liver injury by regulating the distribution, metabolism, and excretion of PPIX. [Figure 9-1] Figures 9A, 9B, 9C, and 9D show the identification of protoporphyrin-1-O-acyl-glucuronide (PPIX-glu). Figure 9A shows the extraction chromatogram of D16-PPIX-glu in the liver of Abcg2-null mice treated with D2-ALA. [Figure 9-2]Figures 9A, 9B, 9C, and 9D show the identification of protoporphyrin-1-O-acyl-glucuronide (PPIX-glu). Figure 9B shows the MS / MS of D16-PPIX-glu. [Figure 9-3] Figures 9A, 9B, 9C, and 9D show the identification of protoporphyrin-1-O-acyl-glucuronide (PPIX-glu). Figure 9C shows a schematic illustrating the synthetic route of PPIX-glu. [Figure 9-4] Figures 9A, 9B, 9C, and 9D show the identification of protoporphyrin-1-O-acyl-glucuronide (PPIX-glu). Figure 9D shows the MS / MS of the synthesized PPIX-glu. [Figure 10] Figures 10A and 10B show an overview of the role of ABCG2 in the pathophysiology of EPP. ABCG2 is expressed in red blood cells (RBCs) and hepatocytes. Figure 10A shows an overview showing that ABCG2 drives phototoxicity and hepatotoxicity in EPP by (1) increasing PPIX distribution to the skin and enhancing photosensitivity; (2) increasing PPIX delivery to the hepatobiliary system and causing bile duct obstruction and cholestatic liver injury; and (3) ABCG2-dependent bile duct obstruction further increases PPIX accumulation in the body, which then enhances both phototoxicity and hepatotoxicity. FIG. 10B shows an overview demonstrating that ABCG2 deficiency in EPP abolishes phototoxicity and hepatotoxicity by (1) reducing PPIX distribution to the skin and decreasing photosensitivity, (2) reducing PPIX delivery to the hepatobiliary system and alleviating PPIX-mediated bile duct obstruction, (3) PPIX retained in hepatocytes may be further metabolized to conjugated products and more easily excreted, and (4) preventing PPIX-mediated bile duct obstruction reduces PPIX accumulation in the body and alleviates both phototoxicity and hepatotoxicity. [Figure 11] FIG. 11 shows the pharmacokinetic analysis of KO143, K2, K31, and K34 in mice. [Figure 12]Figures 12A and 12B show the effectiveness of K31 against EPP-related phototoxicity. The Fech-mut mouse model was used as an EPP model. Figure 12A shows the appearance of the skin in EPP mice before light exposure. Figure 12B shows the appearance of the skin in EPP mice that were pretreated with or without K31 and then exposed to light. [Figure 13] Figure 13 shows phototoxicity in Fech-mut mice after withdrawal of K31. Mice were pretreated with K31 and then exposed to light on days 1 to 4. From day 5 onwards, mice were exposed to light only, without K31 treatment. [Figure 14] Figure 14 shows the effect of K31 on PPIX efflux from RBCs in Fech-mut mice. Results are expressed as mean ± SEM (n = 3). **P < 0.01, ***P < 0.001, ****P < 0.0001. DETAILED DESCRIPTION OF THE INVENTION

[0018] Before the present compounds, compositions, articles, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to particular synthetic methods, or to particular recombinant biotechnology methods, unless otherwise specified, or to particular reagents, unless otherwise specified, which, as such, can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0019] definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

[0020] Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it is understood that the particular value forms another embodiment. It is further understood that the endpoints of each range are meaningful both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are several values ​​disclosed herein, and that each value is also disclosed herein as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that when a value "less than or equal to" a value is disclosed, then "greater than or equal to" that value and possible ranges between values ​​are also disclosed, as appropriately understood by one of ordinary skill in the art. For example, if the value "10" is disclosed, then "less than or equal to 10" and "greater than or equal to 10" are also disclosed. It is also understood that throughout this application, data are provided in several different formats, and that this data also represents endpoints and starting points, and ranges for any combination of the data points. For example, if a specific data point "10" and a specific data point 15 are disclosed, it is understood that not only 10 to 15, but also greater than 10 to 15, greater than or equal to 10 to 15, less than 10 to 15, less than or equal to 10 to 15, and equal to 10 to 15 are also considered disclosed. It is also understood that each unit between two specified units is disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0021] In this specification and the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings.

[0022] "Optional" or "Optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances in which the event or circumstance occurs and instances in which it does not occur.

[0023] "Primers" are a subset of probes that can support some type of enzymatic manipulation and can hybridize to a target nucleic acid so that the enzymatic manipulation can occur. Primers can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art that do not interfere with the enzymatic manipulation.

[0024] A "probe" is a molecule that can interact with a target nucleic acid, typically in a sequence-specific manner, for example, by hybridization. Nucleic acid hybridization is well understood in the art and is discussed herein. Typically, a probe can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art.

[0025] A DNA sequence that "encodes" a particular RNA is a DNA nucleic acid sequence that is transcribed into RNA. A DNA polynucleotide can encode an RNA (mRNA) that is translated into a protein (thus both DNA and mRNA encode proteins), or a DNA polynucleotide can encode an RNA that is not translated into a protein (e.g., tRNA, rRNA, microRNA (miRNA), "non-coding" RNA (ncRNA), guide RNA, etc.).

[0026] A "protein coding sequence," or a sequence encoding a specific protein or polypeptide, is a nucleic acid sequence that is transcribed into mRNA (in the case of DNA) and translated into a polypeptide (in the case of mRNA) in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5'-terminus (N-terminus) and a translation stop nonsense codon at the 3'-terminus (C-terminus). Coding sequences can include, but are not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic nucleic acids. A transcription termination sequence is typically located 3' to the coding sequence.

[0027] The terms "naturally occurring" or "unmodified" or "wild-type," as used herein as applied to a nucleic acid, polypeptide, cell, or organism, refer to a nucleic acid, polypeptide, cell, or organism that is found in nature. For example, a polypeptide or polynucleotide sequence that can be isolated from a natural source and is present in an organism (including a virus) that has not been intentionally modified by man in the laboratory is wild-type (and naturally occurring).

[0028] "Administration" to a subject includes any route of introducing or delivering a therapeutic agent to a subject. Administration can be by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-articular, parenteral, intraarteriolar, intradermal, intracerebroventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injection or infusion), and the like. "Concurrent administration," "administration in combination," "simultaneous administration," or "administered simultaneously," as used herein, means that compounds are administered at the same time or essentially consecutively and immediately following each other. In the latter case, it means that the two compounds are administered at times sufficiently close that the observed results are indistinguishable from those obtained when the compounds are administered at the same time. "Systemic administration" refers to the introduction or delivery of a therapeutic agent to a subject via a route that introduces or delivers the therapeutic agent to a wide area of ​​the subject's body (e.g., more than 50% of the body), for example, through an entry into the circulatory or lymphatic system. In contrast, "local administration" refers to the introduction or delivery of a therapeutic agent to a subject via a route that introduces or delivers the therapeutic agent to an area immediately adjacent to the area of ​​administration or the point of administration, but does not introduce a therapeutically significant amount of the therapeutic agent into the whole body. For example, a locally administered agent is readily detectable locally near the point of administration, but is undetectable or detectable in only small amounts in distant parts of the subject's body. Administration includes self-administration and administration by another.

[0029] An "effective amount" of a therapeutic agent refers to a sufficient amount of the therapeutic agent to produce the desired effect. The amount of an "effective" agent will vary from subject to subject, depending on many factors, such as the subject's age and general condition, the specific agent, etc. Therefore, it is not always possible to specify a quantified "effective amount." However, an appropriate "effective amount" for any subject can be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless otherwise specifically stated, an "effective amount" of a therapeutic agent can refer to an amount that encompasses both a therapeutically effective amount and a prophylactically effective amount. The "effective amount" of a therapeutic agent required to achieve a therapeutic effect may vary depending on factors such as the subject's age, sex, and weight. The administration regimen can be adjusted to produce the optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0030] A "pharmaceutically acceptable" ingredient may refer to an ingredient that is not biologically undesirable or otherwise undesirable. That is, the ingredient can be incorporated into the pharmaceutical formulations of the present invention as described herein and administered to a subject without causing significant undesirable biological effects or interacting in a harmful manner with any of the other ingredients of the formulation in which the ingredient is contained. When used in relation to human administration, the term generally means that the ingredient has met the necessary standards for toxicological testing and manufacturing testing, or that the ingredient is included in the "Inactive Ingredients Guide" prepared by the U.S. Food and Drug Administration.

[0031] A "pharmaceutically acceptable carrier" (sometimes referred to as a "carrier") refers to a carrier or excipient useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic, and includes carriers that are acceptable for pharmaceutical or therapeutic use in animals and / or humans. The term "carrier" or "pharmaceutically acceptable carrier" can include, but is not limited to, phosphate buffered saline, water, emulsions (such as oil / water or water / oil emulsions), and / or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material known in the art for use in pharmaceutical formulations and as further described herein.

[0032] "Pharmacologically active" (or simply "active"), when used in "pharmacologically active" derivatives or analogs, can refer to derivatives or analogs (e.g., salts, esters, amides, conjugates, metabolites, isomers, fragments, etc.) that have the same type of pharmacological activity as the parent compound to approximately the same extent.

[0033] A "therapeutic agent" refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., non-immunogenic cancer). The term also encompasses pharmaceutically acceptable, pharmacologically active derivatives of the beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term "therapeutic agent" is used, or when a particular agent is specifically identified, it should be understood that the term includes the therapeutic agent itself, as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, and the like.

[0034] A "therapeutically effective amount" or "therapeutically effective dose" of a composition (e.g., a composition comprising a therapeutic agent) refers to an amount effective to achieve a desired therapeutic result. In some embodiments, the desired therapeutic result is control of type 1 diabetes. In some embodiments, the desired therapeutic result is control of obesity. The therapeutically effective amount of a given therapeutic agent typically varies with factors such as the type and severity of the disorder or disease being treated, as well as the age, sex, and weight of the subject. The term may also refer to the amount of therapeutic agent or the rate of delivery of the therapeutic agent (e.g., amount over time) effective to promote a desired therapeutic effect, such as pain relief. The precise desired therapeutic effect will vary depending on the condition being treated, the subject's tolerance, the therapeutic agent and / or pharmaceutical formulation being administered (e.g., potency of the therapeutic agent, concentration of the agent in the formulation, etc.), and various other factors understood by those skilled in the art. In some examples, the desired biological or medical response is obtained after administering multiple doses of the composition to the subject over a period of days, weeks, or years.

[0035] The term "n-membered," where n is an integer, typically describes the number of ring-forming atoms in a moiety where n is the number of ring-forming atoms. For example, piperidinyl is an example of a 6-membered heterocycloalkyl ring, pyrazolyl is an example of a 5-membered heteroaryl ring, pyridyl is an example of a 6-membered heteroaryl ring, and 1,2,3,4-tetrahydro-naphthalene is an example of a 10-membered cycloalkyl group.

[0036] As used herein, the phrase "optionally substituted" means unsubstituted or substituted. As used herein, the term "substituted" means that a hydrogen atom has been removed and replaced with a substituent. It should be understood that substitution at a given atom is limited by valence.

[0037] Throughout the definition, the term "C n-m " indicates a range inclusive of the endpoints, and n and m are integers indicating the number of carbons. Examples include C 1-4 , C 1-6 etc. are included.

[0038] As used herein, the term "C" when used alone or in combination with other terms means n-m "Alkyl" refers to a saturated hydrocarbon group having n to m carbons, which may be straight-chained or branched. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; higher homologs such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, and the like. In some embodiments, alkyl groups contain 1 to 6 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms.

[0039] As used herein, "C n-m "Alkenyl" refers to an alkyl group having one or more carbon-carbon double bonds and having n to m carbons. Exemplary alkenyl groups include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl, and the like. In some embodiments, the alkenyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

[0040] As used herein, "C n-m "Alkynyl" refers to an alkyl group having one or more carbon-carbon triple bonds and having n to m carbons. Exemplary alkynyl groups include, but are not limited to, ethynyl, propyn-1-yl, propyn-2-yl, and the like. In some embodiments, the alkynyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

[0041] As used herein, the term "C" when used alone or in combination with other terms means n-m"Alkylene" refers to a divalent alkyl linking group having n to m carbons. Examples of alkylene groups include, but are not limited to, ethane-1,2-diyl, propane-1,3-diyl, propane-1,2-diyl, butane-1,4-diyl, butane-1,3-diyl, butane-1,2-diyl, 2-methyl-propane-1,3-diyl, and the like. In some embodiments, the alkylene moiety contains 2 to 6, 2 to 4, 2 to 3, 1 to 6, 1 to 4, or 1 to 2 carbon atoms.

[0042] As used herein, the term "C" when used alone or in combination with other terms means n-m "Alkoxy" refers to a group of the formula -O-alkyl, where the alkyl group has n to m carbons. Exemplary alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), tert-butoxy, and the like. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0043] As used herein, the term "C n-m "Alkylamino" refers to a group of formula -NH(alkyl), where the alkyl group has n to m carbons. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0044] As used herein, the term "C n-m "Alkoxycarbonyl" refers to a group of formula -C(O)O-alkyl, where the alkyl group has n to m carbons. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0045] As used herein, the term "C n-m "Alkylcarbonyl" refers to a group of formula -C(O)-alkyl, where the alkyl group has n to m carbons. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0046] As used herein, the term "C n-m "Alkylcarbonylamino" refers to a group of formula -NHC(O)-alkyl, where the alkyl group has n to m carbons. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0047] As used herein, the term "C n-m "Alkylsulfonylamino" refers to a group of formula -NHS(O)2-alkyl, where the alkyl group has n to m carbons. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0048] As used herein, the term "aminosulfonyl" refers to a group of formula -S(O)2NH2.

[0049] As used herein, the term "C n-m "Alkylaminosulfonyl" refers to a group of formula -S(O)NH(alkyl), where the alkyl group has n to m carbons. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0050] As used herein, the term "di(C n-m "(Alkyl)aminosulfonyl" refers to a group of formula -S(O)N(alkyl), where each alkyl group independently has n to m carbon atoms. In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0051] As used herein, the term "aminosulfonylamino" refers to a group of formula -NHS(O)2NH2.

[0052] As used herein, the term "C n-m "Alkylaminosulfonylamino" refers to a group of formula -NHS(O)NH(alkyl), where the alkyl group has n to m carbons. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0053] As used herein, the term "di(C n-m "NHS(O)2N(alkyl)aminosulfonylamino" refers to a group of formula -NHS(O)2N(alkyl)2, where each alkyl group independently has n to m carbon atoms. In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0054] As used herein, the term "aminocarbonylamino," used alone or in combination with other terms, refers to a group of formula -NHC(O)NH2.

[0055] As used herein, the term "C n-m "Alkylaminocarbonylamino" refers to a group of formula -NHC(O)NH(alkyl), where the alkyl group has n to m carbons. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0056] As used herein, the term "di(C n-m "NHC(O)N(alkyl)aminocarbonylamino" refers to a group of formula -NHC(O)N(alkyl), where each alkyl group independently has n to m carbon atoms. In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0057] As used herein, the term "C n-m "Alkylcarbamyl" refers to a group of formula -C(O)-NH(alkyl), where the alkyl group has n to m carbons. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0058] As used herein, the term "thio" refers to a group of formula -SH.

[0059] As used herein, the term "C n-m"Alkylsulfinyl" refers to a group of the formula -S(O)-alkyl, where the alkyl group has n to m carbons. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0060] As used herein, the term "C n-m "Alkylsulfonyl" refers to a group of formula -S(O)2-alkyl, where the alkyl group has n to m carbons. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0061] As used herein, the term "amino" refers to a group of formula -NH2.

[0062] As used herein, the term "aryl," used alone or in combination with other terms, refers to an aromatic hydrocarbon group which may be monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings). n-m "Aryl" refers to an aryl group having n to m ring carbon atoms. Aryl groups include, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and the like. In some embodiments, an aryl group has from 6 to about 20 carbon atoms, from 6 to about 15 carbon atoms, or from 6 to about 10 carbon atoms. In some embodiments, an aryl group is substituted or unsubstituted phenyl.

[0063] As used herein, the term "carbamyl" refers to a group of formula -C(O)NH2.

[0064] As used herein, the term "carbonyl," employed alone or in combination with other terms, refers to a -C(=O)- group, which may also be written as C(O).

[0065] As used herein, the term "di(C n-m"-N(alkyl)amino" refers to a group of formula -N(alkyl)2, where the two alkyl groups each independently have n to m carbon atoms. In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0066] As used herein, the term "di(C n-m "-C(O)N(alkyl)carbamyl" refers to a group of formula -C(O)N(alkyl)2, where the two alkyl groups each independently have n to m carbon atoms. In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0067] As used herein, the term "halo" refers to F, Cl, Br, or I. In some embodiments, halo is F, Cl, or Br. In some embodiments, halo is F or Cl.

[0068] As used herein, "C n-m "Haloalkoxy" refers to a group of the formula -O-haloalkyl having n to m carbon atoms. An exemplary haloalkoxy group is OCF. In some embodiments, the haloalkoxy group is exclusively fluorinated. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0069] As used herein, the term "C" when used alone or in combination with other terms means n-m "Haloalkyl" refers to an alkyl group having 1 halogen atom to 2s+1 halogen atoms, which may be the same or different, where "s" is the number of carbon atoms in the alkyl group, wherein the alkyl group has n to m carbon atoms. In some embodiments, the haloalkyl group is exclusively fluorinated. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0070] As used herein, "cycloalkyl" refers to a non-aromatic cyclic hydrocarbon, including cyclized alkyl and / or alkenyl groups. Cycloalkyl groups can include monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings) groups and spirocycles. Cycloalkyl groups can have 3, 4, 5, 6, 7, 8, 9, or 10 ring-forming carbons (C 3-10 ) The ring-forming carbon atoms of a cycloalkyl group can be optionally substituted with oxo or sulfido (e.g., C(O) or C(S)). Cycloalkyl groups also include cycloalkylidenes. Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcarnyl, and the like. In some embodiments, a cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentyl, or adamantyl. In some embodiments, a cycloalkyl has 6 to 10 ring-forming carbon atoms. In some embodiments, a cycloalkyl is adamantyl. Also included within the definition of cycloalkyl are moieties having one or more aromatic rings fused to (i.e., sharing a bond with) the cycloalkyl ring, e.g., benzo- or thienyl derivatives of cyclopentane, cyclohexane, and the like. Cycloalkyl groups containing fused aromatic rings may be attached through any ring-forming atom, including ring-forming atoms of the fused aromatic ring.

[0071] As used herein, "heteroaryl" refers to a monocyclic or polycyclic aromatic heterocycle having at least one heteroatom ring member selected from sulfur, oxygen, and nitrogen. In some embodiments, the heteroaryl ring has 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, any ring-forming N in the heteroaryl moiety may be an N-oxide. In some embodiments, the heteroaryl has 5 to 10 ring atoms and 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, the heteroaryl has 5 to 6 ring atoms and 1 or 2 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, the heteroaryl is a 5- or 6-membered heteroaryl ring. A 5-membered heteroaryl ring is a heteroaryl containing a ring having 5 ring atoms, in which one or more (e.g., 1, 2, or 3) ring atoms are independently selected from N, O, and S. Exemplary 5-membered heteroaryls are thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl. A 6-membered heteroaryl ring is a heteroaryl containing a ring having 6 ring atoms, where one or more (e.g., 1, 2, or 3) ring atoms are independently selected from N, O, and S. Exemplary 6-membered heteroaryls are pyridyl, pyrazinyl, pyrimidinyl, triazinyl, and pyridazinyl.

[0072] As used herein, "heterocycloalkyl" refers to a non-aromatic monocyclic or polycyclic heterocycle having one or more ring-forming heteroatoms selected from O, N, or S. Included in heterocycloalkyl are monocyclic 4-, 5-, 6-, and 7-membered heterocycloalkyl groups. Heterocycloalkyl groups can also include spirocycles. Exemplary heterocycloalkyl groups include pyrrolidin-2-one, 1,3-isoxazolidin-2-one, pyranyl, tetrahydrofuran, oxetanyl, azetidinyl, morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, azepanyl, benzazapene, and the like. The ring-forming carbon atoms and heteroatoms of a heterocycloalkyl group may be optionally substituted with oxo or sulfido (e.g., C(O), S(O), C(S), or S(O)). A heterocycloalkyl group can be bonded through a ring-forming carbon atom or ring-forming heteroatom. In some embodiments, a heterocycloalkyl group contains 0 to 3 double bonds. In some embodiments, a heterocycloalkyl group contains 0 to 2 double bonds. Also included within the definition of heterocycloalkyl are moieties having one or more aromatic rings fused (i.e., sharing a bond) to a cycloalkyl ring, e.g., benzo- or thienyl derivatives such as piperidine, morpholine, azepine, and the like. Heterocycloalkyl groups containing fused aromatic rings can be bonded through any ring-forming atom, including a ring-forming atom of the fused aromatic ring. In some embodiments, a heterocycloalkyl contains 1 or 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and has 4 to 10, 4 to 7, or 4 to 6 ring atoms with one or more oxidized ring members.

[0073] In certain places, definitions or embodiments refer to specific rings (e.g., azetidine rings, pyridine rings, etc.). Unless otherwise specified, these rings can be attached to any ring member, provided that the valence of the atom is not exceeded. For example, an azetidine ring can be attached at any position on the ring, while a pyridin-3-yl ring is attached at the 3-position.

[0074] The term "compound," as used herein, is intended to include all stereoisomers, geometric isomers, tautomers, and isotopes of the depicted structures. Compounds identified herein by name or structure as one particular tautomeric form are intended to include other tautomeric forms unless otherwise specified.

[0075] The compounds provided herein also include tautomeric forms. Tautomeric forms result from the exchange of a single bond with an adjacent double bond, accompanied by the simultaneous migration of a proton. Tautomeric forms include prototropic tautomers, which are isomeric protonation states with the same empirical formula and total charge. Exemplary prototropic tautomers include ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, and cyclic forms in which protons can occupy two or more positions in a heterocyclic ring system, such as 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms may be in equilibrium, or may be stereochemically fixed to one form by appropriate substitution.

[0076] In some embodiments, the compounds described herein may contain one or more asymmetric centers and thus may occur as racemates and racemic mixtures, enantiomer-enriched mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures (e.g., including (R) and (S) enantiomers, diastereomers, (D) isomers, (L) isomers, (+) (dextrorotatory) forms, (-) (levorotatory) forms, racemic mixtures thereof, and mixtures thereof). Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomeric forms of these compounds, and mixtures thereof, are expressly included in this description. The compounds described herein may also contain or further contain bonds (e.g., carbon-carbon bonds, carbon-nitrogen bonds such as amide bonds) in which bond rotation is restricted about that particular bond (e.g., restrictions resulting from the presence of a ring or double bond). Thus, all cis / trans and E / Z isomers and rotational isomers are expressly included in this description. Unless otherwise stated or indicated, the chemical designation of a compound encompasses the mixture of all possible stereochemically isomeric forms of that compound.

[0077] Optical isomers can be obtained in pure form by standard procedures known to those skilled in the art, including, but not limited to, diastereomeric salt formation, kinetic resolution, and asymmetric synthesis. See, for example, Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, SH, et al., Tetrahedron 33:2725 (1977); Eliel, EL Stereochemistry of Carbon Compounds (McGrawHill, NY, 1962); Wilen, SH Tables of Resolving Agents and Optical Resolutions p. 268 (EL Eliel, Ed., University of Notre Dame Press, Notre Dame, IN 1972). Each of these is incorporated herein by reference in its entirety. It is also understood that the compounds described herein include all possible regioisomers, and mixtures thereof, which can be obtained in pure form by standard separation procedures known to those skilled in the art, including, but not limited to, column chromatography, thin layer chromatography, and high performance liquid chromatography.

[0078] Unless specifically defined, the compounds provided herein may include all isotopes of atoms occurring in intermediates or final compounds. Isotopes include atoms with the same atomic number but different mass numbers. Unless otherwise specified, atoms may be isotopes or radioisotopes (e.g., deuterium, [ 11 C], [ 18 When an atom is represented as "D" or "deuterium," the atom is understood to include the isotope or radioisotope in an amount at least greater than the natural abundance of the isotope or radioisotope. For example, when an atom is represented as "D" or "deuterium," the position is understood to have deuterium in an abundance at least 3000 times greater than the natural abundance of deuterium, which is 0.015% (i.e., at least 45% incorporation of deuterium).

[0079] All compounds, and pharmaceutically acceptable salts thereof, may be found together with other substances such as water and solvents (eg, hydrates and solvates), or may be isolated.

[0080] In some embodiments, preparation of compounds may involve, for example, the addition of acids or bases to catalyze a desired reaction or to affect the formation of salt forms, such as acid addition salts.

[0081] Exemplary acids may be inorganic or organic and may include, but are not limited to, strong and weak acids. Some exemplary acids include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, p-toluenesulfonic acid, 4-nitrobenzoic acid, methanesulfonic acid, benzenesulfonic acid, trifluoroacetic acid, and nitric acid. Some weak acids include, but are not limited to, acetic acid, propionic acid, butanoic acid, benzoic acid, tartaric acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, and decanoic acid.

[0082] Exemplary bases include lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, and sodium bicarbonate. Some exemplary strong bases include, but are not limited to, hydroxides, alkoxides, metal amides, metal hydroxides, metal dialkylamides, and arylamines (alkoxides include the lithium, sodium, and potassium salts of methyl, ethyl, and t-butyl oxide; metal amides include sodium amide, potassium amide, and lithium amide; metal hydroxides include sodium hydroxide, potassium hydroxide, and lithium hydroxide; and metal dialkylamides include the lithium, sodium, and potassium salts of methyl, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl, trimethylsilyl, and cyclohexyl substituted amides).

[0083] The present application also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds in which the parent compound is modified by converting an existing acid or base moiety into its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the present application include conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present application can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or free base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, alcohol (e.g., methanol, ethanol, isopropanol, or butanol), or acetonitrile (MeCN) are preferred. Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977). Conventional methods for preparing salt forms are described, for example, in Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCR, 2002.

[0084] In some embodiments, the compounds provided herein or salts thereof are substantially isolated. "Substantially isolated" means that the compounds are at least partially or substantially separated from the environment in which they were formed or detected. Partial separation can include, for example, compositions enriched with the compounds provided herein. Substantial separation can include compositions containing at least about 50% by weight, at least about 60% by weight, at least about 70% by weight, at least about 80% by weight, at least about 90% by weight, at least about 95% by weight, at least about 97% by weight, or at least about 99% by weight of the compounds provided herein or salts thereof. Methods for isolating compounds and their salts are routine in the art.

[0085] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

[0086] composition Disclosed herein are not only the compositions themselves used in the methods disclosed herein, but also the components used to prepare the disclosed compositions. These and other materials are disclosed herein. When combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that each is specifically contemplated and described herein, even if specific reference to various individual and collective combinations and permutations of each of these compounds is not expressly disclosed. For example, if a particular compound is disclosed and discussed, and several modifications that can be made at several positions in the compound are discussed, any and all combinations and permutations of the compound and possible modifications are specifically contemplated unless specifically indicated to the contrary. Thus, if classes of molecules A, B, and C are disclosed, as well as classes of molecules D, E, and F, and an example of a combined molecule, AD, each is individually and collectively contemplated, meaning that combinations AE, AF, BD, BE, BF, CD, CE, and CF are considered to be disclosed, even if each is not individually listed. Similarly, any subset or combination of these is also disclosed. Thus, for example, the subgroups of AE, BF, and CE would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, to the extent that there are various additional steps that may be performed, it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

[0087] Accumulation of protoporphyrin IX (PPIX) in the skin leads to phototoxicity in patients with erythropoietic protoporphyria (EPP). PPIX is an electron-rich tetrapyrrole. When EPP patients are exposed to light, PPIX becomes excited and releases its energy to oxygen, which generates free radicals and can lead to skin damage. Symptoms of the skin reaction include purpura, erythema, edema, and a burning sensation. Therefore, EPP patients must avoid light by reducing outdoor activities and / or using protective clothing, significantly reducing their social and work activities and overall quality of life. Current therapies for phototoxicity in EPP patients focus on reducing light penetration into the skin and / or managing skin lesions resulting from photoexcited PPIX. Beta-carotene has been used in EPP patients due to its antioxidant properties and its ability to increase skin pigmentation and reduce light penetration into the skin, but its effectiveness has been modest. Afamelanotide reduces skin symptoms in patients with EPP by increasing melanin synthesis and decreasing light penetration into the skin. Despite these treatment options, no therapy currently addresses the underlying cause of phototoxicity in EPP: accumulation of PPIX in the skin. Similarly, accumulation of PPIX has been shown to be an underlying cause of phototoxicity in X-linked protoporphyria (XLP).

[0088] The composition can include a small molecule ABCG2 inhibitor. ABCG2 inhibitors are known and described in the art (see, for example, Ricci et al. J Develop Drugs, 2015, 4: 138, which is attached as an appendix to this application and is incorporated herein by reference). Exemplary ABCG2 inhibitors are also described in U.S. Patent Application Publication No. 2017 / 0224837 to Chang et al., which is incorporated herein by reference in its entirety.

[0089] Examples of ABCG2 inhibitors include, for example, 1,4-dihydropyridines; artesunate; AST1306; bifendate-chalcone hybrids; botryllamide; cadmium; calcium channel blockers (e.g., nicardipine, nitrendipine, nimodipine, dipyridamole); camptothecin analogs (e.g., ST1481, CHO793076); cannabinoids; CCT129202; chalcone; curcumin; cyclosporins (e.g., e.g., cyclosporine A); dihydropyridines and pyridines; dimethoxyaurone; dofequidar fumarate; EGFR inhibitors; flavones and benzoflavones; flavonoids; bergamottin; 6',7'-dihydroxybergamottin; tangeretin; nobiletin; hesperidin; hesperetin; quercetin; kaempferol; fumitremorgin C; fumitremorgin C analogs (e.g., KO143); gefitinib; GF120918; BNP1350; GW58 3340; GW2974; HM30181 and its derivatives; cathelicidins (e.g., human cathelicidin); imatinib mesylate; MBL-II-141; ML753286; lapatinib; LY294002; MBLI-87; methoxystilbenes; mithramycin A; quercetin derivatives; naphthopyrones; nilotinib; novobiocin; NP-1250; olomoucine II and purvalanol A; organochlorines and pyrethroids; OSI-930; phytoestrogens / flavonoids; piperazinobenes These include zopyranone (Pipernzinobenzopyranone); phenalkyaminobenzopyranone; ponatinib; PZ-39; quinazolines; quizartinib; sildenafil; sorafenib; substituted chromones; sunitinib; tandutinib; tariquidar; terpenoids; CI1033; toremifene; XR9577; WK-X-34; WK-X-50; WK-X-84; YHO-13177; and YHO-13351.

[0090] In some embodiments, the ABCG2 inhibitor may include fumitremorgin C, KO143, GF120918, YHO-13351, curcumin, CID44640177, CID1434724, CID46245505, CCT129202, artesunate, ST1481, dihydropyridine, dofequidar fumarate, gefitinib, imatinib mesylate, lapatinib, WK-X-34, YHO-13177, MBL-II-141, ML753286, or any combination thereof.

[0091] In some embodiments, the ABCG2 inhibitor may include fumitremorgin C, KO143, GF120918, YHO-13351, curcumin, CID44640177, CID1434724, CID46245505, CCT129202, artesunate, ST1481, dihydropyridine, dofequidar fumarate, gefitinib, imatinib mesylate, lapatinib, WK-X-34, YHO-13177, or any combination thereof.

[0092] The structures of some of these ABCG2 inhibitors are shown below.

[0093] [ka]

[0094] [ka]

[0095] [ka]

[0096] In some embodiments, the ABCG2 inhibitor has the following formula I

[0097] [ka] (In the formula, A is,

[0098] [ka] selected from the group consisting of: n is an integer from 0 to 6; X, if present, is selected from the group consisting of CH, O and S; R 1 H, halo, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl, CN, NO2, OR a , S.R. a , C(O)R b , C(O)NR c R d , C(O)OR a ,OC(O)R b , OC(O)NR c R d , N.R. c R d , N.R. c OR d , N.R. c C(O)R b , N.R. c C(O)OR a , N.R. c C(O)NR c R d , C(=NR e )R b , C(=NR e )NR c R d , N.R. c C(=NR e )NR c R d , N.R. c S(O)R b , N.R. c S(O)2R b , N.R. c S(O)NR c R d , S(O)R b , S(O)NR cR d , S(O)2R b , and S(O)NR c R d wherein C is selected from the group consisting of 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A optionally substituted with a group; R 2 and R 3 H, halo, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl, CN, NO2, OR a , and S.R. a wherein C is independently selected from the group consisting of 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A optionally substituted with a group; R 4 and R 5 is H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 independently selected from the group consisting of cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl; 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A optionally substituted with a group; R 6 and R 7 is H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 each independently selected from the group consisting of cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl; 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A optionally substituted with a group or R 6 and R 7 together with the N atoms to which they are attached, form one, two, or three independently selected R A groups, each forming an optionally substituted 4- to 9-membered heterocycloalkyl group or a 5- to 6-membered heteroaryl group; R 8 and R 9 is H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 independently selected from the group consisting of cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl; 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A optionally substituted with a group or R 8 and R 9 together with the atoms to which they are attached, form one, two, or three independently selected R A groups, each forming an optionally substituted 4- to 9-membered heterocycloalkyl group or a 5- to 6-membered heteroaryl group; R 10 is C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl, 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A optionally substituted with a group; Each R a , R b , R c , and R d is H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 independently selected from cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl; 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A each optionally substituted with a group; Each R e are H, CN, and C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkylthio, C 1-6 Alkylsulfonyl, C 1-6 Alkyl carbonyl, C 1-6 Alkylaminosulfonyl, Carbamyl, C 1-6 Alkylcarbamyl, di(C 1-6 Alkyl)carbamyl, aminosulfonyl, C 1-6 Alkylaminosulfonyl, and di(C 1-6 alkyl)aminosulfonyl; and Each R A OH, NO2, CN, halo, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, Cyano-C 1-3 Alkyl, HO-C 1-3 Alkyl, Amino, C 1-6 Alkylamino, di(C 1-6 Alkyl)amino, thio, C 1-6 Alkylthio, C 1-6 Alkylsulfinyl, C 1-6 Alkyl sulfonyl, carbamyl, C 1-6 Alkylcarbamyl, di(C 1-6 Alkyl) carbamyl, carboxy, C 1-6 Alkyl carbonyl, C 1-6 Alkoxycarbonyl, C 1-6 Alkylcarbonylamino, C 1-6 Alkyl sulfonyl amino, amino sulfonyl, C 1-6 Alkylaminosulfonyl, di(C 1-6 Alkyl)aminosulfonyl, aminosulfonylamino, C1-6 Alkylaminosulfonylamino, di(C 1-6 Alkyl)aminosulfonylamino, aminocarbonylamino, C 1-6 Alkylaminocarbonylamino, and di(C 1-6 alkyl)aminocarbonylamino; or a pharmaceutically acceptable salt, ester, or N-oxide thereof.

[0099] In some embodiments, R 4 and R 5 may both be hydrogen.

[0100] In some embodiments, R 2 and R 3 H and C 1-6 alkyl, wherein C 1-6 Alkyl is one, two, three, or four independently selected R A Optionally substituted with a group.

[0101] In some embodiments, R 2 may be an isobutyl group.

[0102] In some embodiments, R 3 may be hydrogen. In other embodiments, R 3 may be methyl.

[0103] In some embodiments, R 1 is H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, and OR a may be selected from the group consisting of: 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, and C 1-4 Haloalkyl is one, two, three, or four independently selected R Aoptionally substituted with a group; and R a If present, H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 haloalkyl; 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, and C 1-4 Haloalkyl is one, two, three, or four independently selected R A Each is optionally substituted with a group.

[0104] In certain embodiments, R 1 may be hydrogen. In other embodiments, R 1 is hydroxy, C 1-4 Alkyl, and C 1-4 alkoxy, wherein C 1-4 Alkyl and C 1-4 Alkoxy is one, two, three, or four independently selected R A For example, in certain embodiments, R 1 may be a methoxy group.

[0105] In some embodiments, X, if present, is selected from the group consisting of O and S.

[0106] The composition comprises a small molecule compound defined by formula IA:

[0107] [ka] (In the formula, n is an integer from 0 to 6; R 6 and R 7 is H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10each independently selected from the group consisting of cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl; 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A optionally substituted with a group or R 6 and R 7 together with the N atoms to which they are attached, form one, two, or three independently selected R A forming a 4- to 9-membered heterocycloalkyl group or a 5- to 6-membered heteroaryl group, each optionally substituted with a group; and Each R A OH, NO2, CN, halo, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, Cyano-C 1-3 Alkyl, HO-C 1-3 Alkyl, Amino, C 1-6 Alkylamino, di(C 1-6 Alkyl)amino, thio, C 1-6 Alkylthio, C 1-6 Alkylsulfinyl, C 1-6 Alkyl sulfonyl, carbamyl, C 1-6 Alkylcarbamyl, di(C 1-6 Alkyl) carbamyl, carboxy, C 1-6 Alkyl carbonyl, C 1-6 Alkoxycarbonyl, C 1-6 Alkylcarbonylamino, C 1-6 Alkyl sulfonyl amino, amino sulfonyl, C 1-6 Alkylaminosulfonyl, di(C 1-6 Alkyl)aminosulfonyl, aminosulfonylamino, C 1-6Alkylaminosulfonylamino, di(C 1-6 Alkyl)aminosulfonylamino, aminocarbonylamino, C 1-6 Alkylaminocarbonylamino, and di(C 1-6 alkyl)aminocarbonylamino; or a pharmaceutically acceptable salt, ester, or N-oxide thereof.

[0108] In some of these embodiments, n may be an integer from 1 to 4.

[0109] In some of these embodiments, R 6 and R 7 is H, C 1-6 Alkyl, C 1-4 Haloalkyl, C 3-10 each independently selected from the group consisting of cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl; 1-6 Alkyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A In other embodiments, R 6 and R 7 together with the N atom to which they are attached, can be one, two, or three independently selected R A The groups form an optionally substituted 4- to 9-membered heterocycloalkyl group or a 5- to 6-membered heteroaryl group, respectively.

[0110] The composition comprises a small molecule compound defined by formula IB:

[0111] [ka] (In the formula, n is an integer from 0 to 6; R 8 and R9 is H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 independently selected from the group consisting of cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl; 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A optionally substituted with a group or R 8 and R 9 together with the atom to which they are attached form a 4- to 9-membered heterocycloalkyl group or a 5- to 6-membered heteroaryl group, each optionally substituted with 1, 2, or 3 independently selected R A groups; and Each R A OH, NO2, CN, halo, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, Cyano-C 1-3 Alkyl, HO-C 1-3 Alkyl, Amino, C 1-6 Alkylamino, di(C 1-6 Alkyl)amino, thio, C 1-6 Alkylthio, C 1-6 Alkylsulfinyl, C 1-6 Alkyl sulfonyl, carbamyl, C 1-6 Alkylcarbamyl, di(C 1-6 Alkyl) carbamyl, carboxy, C 1-6 Alkyl carbonyl, C 1-6 Alkoxycarbonyl, C 1-6 Alkylcarbonylamino, C 1-6Alkyl sulfonyl amino, amino sulfonyl, C 1-6 Alkylaminosulfonyl, di(C 1-6 Alkyl)aminosulfonyl, aminosulfonylamino, C 1-6 Alkylaminosulfonylamino, di(C 1-6 Alkyl)aminosulfonylamino, aminocarbonylamino, C 1-6 Alkylaminocarbonylamino, and di(C 1-6 alkyl)aminocarbonylamino; or a pharmaceutically acceptable salt, ester, or N-oxide thereof.

[0112] In some of these embodiments, n may be an integer from 1 to 4.

[0113] In some embodiments, R 9 is hydrogen.

[0114] In some embodiments, R 8 is H, C 1-6 Alkyl, C 1-4 Haloalkyl, C 3-10 cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl, wherein said C 1-6 Alkyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A In other embodiments, R 8 and R 9 together with the atoms to which they are attached, form one, two, or three independently selected R A The groups form an optionally substituted 4- to 9-membered heterocycloalkyl group or a 5- to 6-membered heteroaryl group, respectively.

[0115] The composition comprises a small molecule compound defined by the formula IC:

[0116] [ka] (In the formula, n is an integer from 0 to 6; R 6 and R 7 is H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 each independently selected from the group consisting of cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl; 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A optionally substituted with a group or R 6 and R 7 together with the N atoms to which they are attached, form one, two, or three independently selected R A forming a 4- to 9-membered heterocycloalkyl group or a 5- to 6-membered heteroaryl group, each optionally substituted with a group; and Each R A OH, NO2, CN, halo, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, Cyano-C 1-3 Alkyl, HO-C 1-3 Alkyl, Amino, C 1-6 Alkylamino, di(C 1-6 Alkyl)amino, thio, C 1-6 Alkylthio, C 1-6 Alkylsulfinyl, C 1-6Alkyl sulfonyl, carbamyl, C 1-6 Alkylcarbamyl, di(C 1-6 Alkyl) carbamyl, carboxy, C 1-6 Alkyl carbonyl, C 1-6 Alkoxycarbonyl, C 1-6 Alkylcarbonylamino, C 1-6 Alkyl sulfonyl amino, amino sulfonyl, C 1-6 Alkylaminosulfonyl, di(C 1-6 Alkyl)aminosulfonyl, aminosulfonylamino, C 1-6 Alkylaminosulfonylamino, di(C 1-6 Alkyl)aminosulfonylamino, aminocarbonylamino, C 1-6 Alkylaminocarbonylamino, and di(C 1-6 alkyl)aminocarbonylamino; or a pharmaceutically acceptable salt, ester, or N-oxide thereof.

[0117] In some of these embodiments, n may be an integer from 1 to 4.

[0118] In some of these embodiments, R 6 and R 7 is H, C 1-6 Alkyl, C 1-4 Haloalkyl, C 3-10 each independently selected from the group consisting of cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl; 1-6 Alkyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A In other embodiments, R 6 and R 7 together with the N atom to which they are attached, can be one, two, or three independently selected R AThe groups form an optionally substituted 4- to 9-membered heterocycloalkyl group or a 5- to 6-membered heteroaryl group, respectively.

[0119] In some embodiments, the ABCG2 inhibitor has the following formula ID

[0120] [ka] (In the formula, n is an integer from 0 to 6; X is selected from the group consisting of CH2, O and S; R 10 is C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl, 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A optionally substituted with a group; and Each R A OH, NO2, CN, halo, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, Cyano-C 1-3 Alkyl, HO-C 1-3 Alkyl, Amino, C 1-6 Alkylamino, di(C 1-6 Alkyl)amino, thio, C 1-6 Alkylthio, C 1-6 Alkylsulfinyl, C 1-6Alkyl sulfonyl, carbamyl, C 1-6 Alkylcarbamyl, di(C 1-6 Alkyl) carbamyl, carboxy, C 1-6 Alkyl carbonyl, C 1-6 Alkoxycarbonyl, C 1-6 Alkylcarbonylamino, C 1-6 Alkyl sulfonyl amino, amino sulfonyl, C 1-6 Alkylaminosulfonyl, di(C 1-6 Alkyl)aminosulfonyl, aminosulfonylamino, C 1-6 Alkylaminosulfonylamino, di(C 1-6 Alkyl)aminosulfonylamino, aminocarbonylamino, C 1-6 Alkylaminocarbonylamino, and di(C 1-6 alkyl)aminocarbonylamino; or a pharmaceutically acceptable salt, ester, or N-oxide thereof.

[0121] In some embodiments, X, if present, is selected from the group consisting of O and S. In some embodiments, X is O. In some embodiments, X is S.

[0122] In some embodiments, n is an integer from 1 to 4 (eg, n is 3).

[0123] The ABCG2 inhibitors described above (e.g., ABCG2 inhibitors described by Formula I, Formula IA, Formula IB, Formula IC, and / or Formula ID) can also be used in conjunction with other therapeutic methods involving the inhibition of ABCG2. For example, these ABCG2 inhibitors can be used to enhance chemotherapy treatment of tumor cells, reduce resistance of cancer cells to chemotherapeutic agents, reduce ABCG2 transporter activity in cancer cells, or a combination thereof. Such methods are described, for example, in U.S. Pat. Nos. 9,937,217; 9,314,448; and 9,056,111, each of which is incorporated herein by reference in its entirety. Thus, the ABCG2 inhibitors described above (e.g., ABCG2 inhibitors described by Formula I, Formula IA, Formula IB, Formula IC, and / or Formula ID) can be used, for example, in the methods described in U.S. Pat. Nos. 9,937,217; 9,314,448; and 9,056,111.

[0124] Pharmaceutical Carriers / Drug Product Delivery As described above, the composition can also be administered in vivo in a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means that the material is not biologically undesirable or otherwise undesirable. That is, the material can be administered to a subject together with a nucleic acid or vector without causing any undesirable biological effects or interacting in a harmful manner with any of the other components of the pharmaceutical composition in which the component is contained. Of course, the carrier will be selected to minimize any degradation of the active ingredient and minimize any adverse side effects in the subject, as is known to those skilled in the art.

[0125] The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically, etc., including topical administration or administration by inhalation. As used herein, "topical intranasal administration" refers to delivery of a composition to the nose and nasal cavity through one or both nostrils and may include delivery by a spray or droplet mechanism, or by aerosolization of the nucleic acid or vector. Administration of a composition by inhalation may be through the nose or mouth by delivery by a spray or droplet mechanism. Delivery may also be directly to any area of ​​the respiratory system (e.g., lungs) by intubation. The exact amount of composition required will vary from subject to subject, depending on the species, age, weight, and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration, etc. Therefore, it is not possible to specify an exact amount for each composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation, given the teachings herein.

[0126] When used, parenteral administration of composition is generally characterized by injection.Injection can be prepared in any conventional form, either as liquid solution or suspension, as solid suitable for the solution of suspension in liquid before injection, or as emulsion.The more recent approach of parenteral administration is accompanied by the use of slow-release or sustained-release system to maintain a constant dosage.For example, see U.S. Patent No. 3,610,795, which is incorporated herein by reference.

[0127] The materials may be in solution, suspension (e.g., incorporated into microparticles, liposomes, or cells), and may be targeted to specific cell types via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target proteins specific to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, KD, Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol. 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody-conjugated liposomes (including lipid-mediated drugs targeting colon cancer), receptor-mediated targeting of DNA through cell-specific ligands, lymphocyte-directed tumor targeting, and highly specific therapeutic retroviral targeting of mouse glial cells in vivo. The following references are examples of the use of this technology to target proteins specific to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general, receptors are involved in either constitutive or ligand-induced endocytic pathways.These receptor clusters in clathrin-coated pits enter cells via clathrin-coated vesicles, pass through acidified endosomes where the receptors are sorted, and then recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. Internalization pathways perform various functions, including nutrient absorption, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, ligand dissociation and degradation, and control of receptor levels. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, ligand type, ligand valency, and ligand concentration. The molecular and cellular mechanisms of receptor-mediated endocytosis have been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

[0128] Pharmaceutically acceptable carrier The compositions containing the antibodies can be used therapeutically in combination with a pharmaceutically acceptable carrier.

[0129] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic. Examples of pharmaceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5. Additional carriers include sustained-release preparations, such as semipermeable matrices of solid hydrophobic polymers containing the antibody, where the matrices are in the form of shaped articles, e.g., films, liposomes, or microparticles. It will be apparent to those skilled in the art that certain carriers will be more preferable depending, for example, on the route of administration and concentration of the administered composition.

[0130] Pharmaceutical carriers are known to those skilled in the art.These will be the standard carriers for administering drugs to humans, most typically, including solutions such as sterile water, physiological saline, and buffer solutions at physiological pH.Compositions can also be administered intramuscularly or subcutaneously.Other compounds will be administered according to the standard procedures used by those skilled in the art.

[0131] In addition to the molecule of choice, pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surfactants, etc. Pharmaceutical compositions may also include one or more active ingredients such as antibacterial agents, anti-inflammatory agents, anesthetics, etc.

[0132] The pharmaceutical compositions can be administered in several ways, depending on whether local or systemic treatment is desired and the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, or intranasal), oral, by inhalation, or parenteral, such as by intravenous drip, subcutaneous, intraperitoneal, or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

[0133] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as, for example, antibacterial agents, antioxidants, chelating agents, and inert gases and the like.

[0134] Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0135] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable.

[0136] Some of the compositions may be administered as pharmaceutically acceptable acid or base addition salts formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, tri-alkyl-, and aryl-amines and substituted ethanolamines.

[0137] therapeutic use Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill of the art. The dosage range for administering the compositions is large enough to produce the desired effect on the symptoms of the disorder. The dosage should not be so large as to cause adverse side effects, such as undesirable cross-reactions, anaphylactic reactions, etc. Generally, dosages vary depending on the patient's age, condition, sex, and extent of disease, the route of administration, or whether other drugs are included in the regimen, and can be determined by one of ordinary skill in the art. Dosages can be adjusted by the individual physician if any counterindications occur. Dosages can vary and can be administered in one or multiple doses daily for one or several days. Guidance can be found in the literature regarding appropriate dosages for a given class of pharmaceutical products. For example, guidance in selecting an appropriate dose of an antibody can be found in literature on the therapeutic use of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ, (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. Typical daily dosages of antibodies used alone can range from about 1 μg / kg to up to 100 mg / kg body weight or more per day, depending on the factors mentioned above.

[0138] Methods for treating erythropoietic protoporphyria (EPP) or X-linked protoporphyria (XLP) It is understood, and contemplated herein, that PPIX excretion and the resulting accumulation of PPIX (i.e., the problem that causes phototoxicity) depend on the activity of ABCG2. Therefore, the disclosed ABCG2 inhibitors can prevent PPIX excretion, thereby treating EPP and XLP and any symptoms associated therewith. Thus, in one aspect, disclosed herein is a method for treating, preventing, reducing, or inhibiting erythropoietic protoporphyria (EPP) or X-linked protoporphyria (XLP) in a subject, comprising administering to the subject any of the therapeutic agents disclosed herein that inhibit ABCG2 activity.

[0139] As used herein, "treat," "treating," "treatment," and grammatical variations thereof as used herein include administration of a composition intended or intended to partially or completely prevent, delay, cure, heal, alleviate, relieve, alter, treat, ameliorate, improve, stabilize, alleviate, and / or reduce the intensity or frequency of one or more diseases or conditions, symptoms of a disease or condition, or the underlying causes of a disease or condition. Treatment according to the present invention may be applied preventively, prophylactically, palliatively, or therapeutically. Prophylactic treatment is administered to a subject pre-symptomatically (e.g., before overt signs of EPP or XLP), early in the course of the disease (e.g., at the first signs and symptoms of EPP or XLP), or after the established development of cancer. Prophylactic administration may occur days to years before the onset of symptoms of infection.

[0140] "Reduction" can refer to any change in gene expression, protein production, symptom, disease, composition, condition, or activity that results in a smaller amount. A substance is also understood to reduce the genetic output of a gene if the genetic output of the gene product containing the substance is lower compared to the output of the gene product without the substance. Similarly, for example, a reduction can be a change in a symptom of a disorder such that the symptom is less than previously observed. A reduction can be any individual, median, or average reduction in a condition, symptom, activity, or composition by a statistically significant amount. Thus, the reduction can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% reduction, as long as it is statistically significant.

[0141] "Inhibit," "inhibiting," and "inhibition" refer to reducing an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete elimination of the activity, response, condition, or disease. It can also include, for example, a 10% reduction in the activity, response, condition, or disease compared to native or control levels. Thus, a reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction compared to native or control levels.

[0142] The terms "prevent," "preventing," "prevention," and grammatical variations thereof, as used herein, refer to a method of partially or completely delaying or eliminating the onset or recurrence of a disease and / or one or more of its attendant symptoms, or preventing a subject from acquiring or re-acquiring a disease, or reducing a subject's risk of acquiring or re-acquiring a disease or one or more of its attendant symptoms.

[0143] It is understood and contemplated herein that treating, inhibiting, preventing, or reducing EPP or XLP does not necessarily involve a curative outcome, but may include alleviating symptoms of EPP or XLP, including, but not limited to, cutaneous phototoxicity, purpura, erythema, edema, or burning. Thus, in one aspect, disclosed herein is a method of treating, preventing, reducing, or inhibiting cutaneous phototoxicity, purpura, erythema, edema, or burning in a subject, comprising administering to the subject any of the therapeutic agents disclosed herein that inhibit ABCG2 activity.

[0144] In addition to the skin, the liver is another target organ of PPIX toxicity in EPP, and its severity is affected by polymorphisms in genes controlling porphyrin homeostasis. The liver is involved in the excretion of PPIX from the body via the hepatobiliary system. Due to the highly hydrophobic nature of PPIX, excess PPIX in bile accumulates, causing bile duct obstruction and cholestatic liver injury. This is a vicious cycle, as PPIX-mediated bile duct obstruction can lead to further PPIX accumulation in the body. In clinical settings, several pharmacological approaches have been attempted to manage EPP-associated liver injury, but none have produced satisfactory results. While liver transplantation is effective, it fails to restore FECH function in the bone marrow and prevent PPIX accumulation in the liver, making recurrence of EPP-associated liver injury common. It is understood and contemplated herein that the ABCG2 inhibitor therapeutic agents disclosed herein can be used to manage EPP-associated liver injury. Accordingly, in one aspect, disclosed herein is a method of reducing PPIX efflux from hepatocytes in a subject, the method comprising administering to the subject a therapeutic agent that inhibits ABCG2 activity.

[0145] In EPP, PPIX is primarily produced in the bone marrow and delivered to other organs (including the skin and liver) via the circulatory system by RBCs and plasma. PPIX efflux from RBCs into plasma is dependent on the transporter ABCG2, and exposure to light promotes PPIX efflux. These data led to the hypothesis that inhibition of ABCG2 reduces PPIX deposition in the skin and alleviates phototoxicity in EPP. Additionally, retention of PPIX in hepatocytes and Kupffer cells, rather than the biliary system, attenuates EPP-associated hepatotoxicity. ABCG2 is expressed in hepatocytes and is responsible for PPIX efflux from hepatocytes into the biliary system. Therefore, we hypothesize that inhibition of ABCG2 reduces the amount of PPIX in the biliary system and alleviates PPIX-mediated bile duct obstruction and cholestatic liver injury. Thus, in one aspect, disclosed herein is a method for reducing PPIX efflux from red blood cells in a subject, comprising administering to the subject a therapeutic agent that inhibits ABCG2 activity.

[0146] In one embodiment, therapeutic agents used in the methods of the present disclosure may include, but are not limited to, any of the small molecules disclosed herein, as well as GF120918, MBL-II-141, ML753286, and antibodies, oligonucleotides, small interfering RNA, RNAi, peptides, proteins, and / or targeted nucleic acid integration systems. Reducing or inhibiting the expression of a gene or the protein it encodes can have unexpected consequences, especially when induced gene expression or activity is inhibited off-target. In one embodiment, ABCG2 inhibitor therapeutic agents can be engineered to express in specific tissues (e.g., under the control of a tissue-specific promoter), or to express under inducible conditions (e.g., a cre-lox, flp, or tet inducible promoter system activated by administration of a trigger compound such as tetracycline, or a construct that expresses Cre and removes the floxed cassette is transfected into the ABCG2 knockout locus), or to target specific tissues (e.g., a tissue-specific bispecific antibody or bivalent construct). In one embodiment, the therapeutic agent can be a targeted nucleic acid integration system (e.g., a CRISPR / Cas9 system in which a guide RNA (gRNA) targets the ABCG2 gene).

[0147] antibody As noted above, the therapeutic methods of the present disclosure may involve the administration of anti-ABCG2 antibodies (including immunotoxins, variants, or fragments thereof).

[0148] Antibody Overview The term "antibody" is used broadly herein and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, the term "antibody" also includes fragments or polymers of these immunoglobulin molecules, as well as human or humanized versions of immunoglobulin molecules or fragments thereof, including bivalent single-chain antibodies, diabodies, triabodies, and tetrabodies, so long as they are selected for their ability to interact with ABCG2 to inhibit ABCG2 from enabling PPIX excretion. Antibodies can be tested for their desired activity using the in vitro assays described herein or similar methods, and then their in vivo therapeutic and / or prophylactic activity is tested according to known clinical trial methods. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes) (e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2). Those skilled in the art will recognize the equivalent classes to mice. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

[0149] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for naturally occurring mutations that may be present in a small subset of antibody molecules. Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided that they exhibit the desired antagonistic activity.

[0150] The monoclonal antibodies of the present disclosure can be produced using any method for producing monoclonal antibodies. For example, the monoclonal antibodies of the present disclosure can be prepared using a hybridoma method, such as that described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, typically, a mouse or other suitable host animal is immunized with an immunizing agent to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0151] Monoclonal antibodies can also be produced by recombinant DNA methods. DNA encoding the monoclonal antibodies of the present disclosure can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies). Libraries of antibodies or active antibody fragments can also be generated and sequenced using phage display technology, for example, as described in U.S. Patent No. 5,804,440 to Burton et al. and U.S. Patent No. 6,096,441 to Barbas et al.

[0152] In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof (particularly Fab fragments) can be achieved using routine techniques known in the art. For example, digestion can be carried out using papain. Examples of papain digestion are described in WO 94 / 29348, published December 22, 1994, and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen-binding fragments, called Fab fragments (each with a single antigen-binding site), and a residual Fc fragment. Pepsin treatment yields a fragment with two antigen-binding sites that is still capable of cross-linking antigen.

[0153] In one embodiment, an ABCG2-binding antibody may include an antibody fragment. As used herein, the term "antibody or fragment thereof" encompasses fragments (e.g., F(ab')2, Fab', Fab, Fv, scFv, and the like) including chimeric and hybrid antibodies with dual or multiple antigen or epitope specificities, as well as hybrid fragments. Thus, antibody fragments that retain the ability to bind to a specific antigen are provided. For example, antibody fragments that retain ABCG2-binding activity are included within the meaning of the term "antibody or fragment thereof." Such antibodies and fragments may be produced by techniques known in the art and screened for specificity and activity according to the methods described in the Examples and in General Methods for Generating and Screening Antibodies for Specificity and Activity (see Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, (1988)).

[0154] Also included within the meaning of "antibody or fragment thereof" is a conjugate of an antibody fragment with an antigen-binding protein (single-chain antibody). A conjugated antibody or fragment refers to an antibody or fragment that is operably linked to or otherwise physically or functionally associated with an effector moiety or tag (e.g., a toxic substance, a radioactive substance, a fluorescent substance, a liposome, or an enzyme, among others, as described, for example, in U.S. Pat. No. 4,704,692, the contents of which are incorporated herein by reference).

[0155] Fragments may also contain insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues, whether or not linked to other sequences, provided that the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the unmodified antibody or antibody fragment. These modifications may confer additional properties (e.g., removing / adding amino acids capable of disulfide bonding, increasing biolongevity, altering secretion characteristics, etc.). In any case, the antibody or antibody fragment must retain biological properties, such as specific binding to its conjugated antigen. Functional or active regions of an antibody or antibody fragment may be identified by mutagenesis of specific regions of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to those skilled in the art and may include site-directed mutagenesis of nucleic acids encoding the antibody or antibody fragment. (Zoller, MJ Curr. Opin. Biotechnol. 3:348-354, 1992).

[0156] As used herein, the term "antibody" or "antibodies" can also refer to human antibodies and / or humanized antibodies. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans and, therefore, may provoke an undesired immune response when administered to humans. Thus, the use of human or humanized antibodies in the present methods reduces the likelihood that antibodies administered to humans will provoke an undesired immune response.

[0157] Human antibodies The human antibodies of the present disclosure can be prepared using any technique. The human antibodies of the present disclosure can also be obtained from transgenic animals. For example, transgenic mutant mice that can produce a full repertoire of human antibodies in response to immunization have been described (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993)). Specifically, the homozygous deletion of the antibody heavy chain joining region (J(H)) gene in these chimeric and germline mutant mice completely inhibits endogenous antibody production, and successful transfer of a human germline antibody gene array into such germline mutant mice results in the production of human antibodies upon antigen challenge. Antibodies with the desired activity are selected using the Env-CD4-co-receptor complexes described herein.

[0158] humanized antibodies Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Thus, humanized forms of non-human antibodies (or fragments thereof) are chimeric antibodies or chimeric antibody chains (or fragments thereof, such as sFv, Fv, Fab, Fab', F(ab')2, or other antigen-binding portions of antibodies) that contain portions of the antigen-binding site from the non-human (donor) antibody incorporated into the framework of a human (recipient) antibody.

[0159] To generate a humanized antibody, residues from one or more complementarity-determining regions (CDRs) of a recipient (human) antibody molecule are replaced with residues from one or more CDRs of a donor (non-human) antibody molecule that have been found to have the desired antigen-binding properties (e.g., a particular level of specificity and affinity for the target antigen). In some cases, Fv framework (FR) residues of the human antibody are replaced with corresponding non-human residues. Humanized antibodies may also contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, humanized antibodies have one or more amino acid residues introduced from a non-human source. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. A humanized antibody generally comprises at least a portion of an antibody constant region (Fc), typically that of a human antibody (Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-327 (1988); and Presta, Curr. Opin. Struct. Biol., 2:593-596 (1992)).

[0160] Methods for humanizing non-human antibodies are known in the art. For example, humanized antibodies can be produced according to the method of Winter and coworkers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)) by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Methods that can be used to produce humanized antibodies are also described in U.S. Pat. No. 4,816,567 (Cabilly et al.), U.S. Pat. No. 5,565,332 (Hoogenboom et al.), U.S. Pat. No. 5,721,367 (Kay et al.), U.S. Pat. No. 5,837,243 (Deo et al.), U.S. Pat. No. 5,939,598 (Kucherlapati et al.), U.S. Pat. No. 6,130,364 (Jakobovits et al.), and U.S. Pat. No. 6,180,377 (Morgan et al.).

[0161] Antibody administration Antibodies can be administered as disclosed herein. Nucleic acid approaches for antibody delivery also exist. Anti-ABCG2 antibodies and antibody fragments can also be administered to patients or subjects as nucleic acid preparations (e.g., DNA or RNA) encoding the antibodies or antibody fragments, so that the patient's or subject's own cells take up the nucleic acid and produce and secrete the encoded antibodies or antibody fragments. Nucleic acid delivery can be carried out, for example, by any means disclosed herein.

[0162] Transgenic Cells and Animals We developed an EPP mouse model with a loss-of-function mutation in FECH (Fech-mut). To test our hypothesis, we generated an EPP mouse model lacking ABCG2 (Fech-mut / Abcg2-null). We found that ABCG2 deficiency abolished both phototoxicity and hepatotoxicity in EPP mice. We also found that Abcg2-null mice failed to develop PPIX accumulation and hepatotoxicity when challenged with liver-specific porphyrin-producing chemicals. Metabolomic analysis revealed that ABCG2 deficiency prevented PPIX-mediated phototoxicity and hepatotoxicity by modulating the distribution, metabolism, and excretion of PPIX.

[0163] In various embodiments, provided herein are genetically modified cells and non-human animals (e.g., rodents, such as mice or rats) that comprise in their genomes (e.g., in their germline genomes) a nucleic acid sequence encoding a null mutation in the ABCG2 gene. In some aspects, the modified cells can further comprise a mutation in the FECH gene.

[0164] The term "cell" includes any cell suitable for expressing a recombinant nucleic acid sequence, including prokaryotic and eukaryotic cells (unicellular or multicellular), bacterial cells (e.g., strains of E. coli, Bacillus species, Streptomyces species, etc.), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusions (e.g., hybridomas or quadromas). In some embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the cells are eukaryotic and selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60 (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, Sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived from the aforementioned cells. In some embodiments, the cells contain, e.g., are retinal cells (e.g., PER.C6.™ cells) that contain, e.g., express, one or more viral genes. In some embodiments, the cells are ES cells.

[0165] In one aspect, the transgenic cells may contain a selection cassette that confers inducible and / or tissue-specific expression. A selection cassette is a nucleotide sequence inserted into a targeting construct to facilitate selection of cells (e.g., bacterial cells, ES cells) that have integrated the desired construct. Many suitable selection cassettes are known in the art (e.g., Neo, Hyg, Pur, CM, SPEC, etc.). Additionally, the selection cassette may be flanked by recombination sites that allow deletion of the selection cassette upon treatment with a recombinase. Commonly used recombination sites are loxP and Frt, recognized by Cre and Flp enzymes, respectively, although others are known in the art. The selection cassette may be located anywhere in the construct outside the coding region. In one embodiment, the selection cassette is inserted upstream of a human ABCG2 null insertion sequence.

[0166] The selection cassette used in this method can be removed by methods known to those skilled in the art. For example, ES cells carrying an ABCG2 knockout locus can be transfected with a construct that expresses Cre to remove the floxed cassette. The selection cassette can optionally be removed by breeding with mice that express Cre recombinase. Optionally, the selection cassette is retained in the mice. [Example]

[0167] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices, and / or methods claimed herein may be made and evaluated, and are intended to be purely illustrative and are not intended to limit the present disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is °C or is ambient temperature, and pressure is at or near atmospheric.

[0168] Example 1: "Gene deletion of the transporter ABCG2 protects against phototoxicity and hepatotoxicity in erythropoietic protoporphyria (EPP)" result "ABCG2 deficiency protects against EPP-associated phototoxicity" Fech-mut / Abcg2-null mice have a loss-of-function mutation in FECH and are deficient in ABCG2 (Figure 1A). When Fech-mut / Abcg2-null and Fech-mut mice were exposed to light with the excitation wavelength of PPIX, Fech-mut mice developed severe skin lesions, whereas this phenotype was absent in Fech-mut / Abcg2-null mice (Figures 1B, 1C, 1D, 1E, and 1F). In addition, oxidative stress and inflammation were observed in the skin of Fech-mut mice after light exposure, but not in Fech-mut / Abcg2-null mice (Figures 2A, 2B, and 2C). These data indicate that ABCG2 is a major mediator of EPP-related phototoxicity. Next, we analyzed PPIX levels in RBCs, serum, and skin of the EPP mouse model. We found that ABCG2 deficiency significantly increased PPIX levels in RBCs but reduced PPIX levels in serum and skin (Figures 3A, 3B, and 3C), indicating that ABCG2 dysfunction blocks PPIX excretion from RBCs into plasma, thus reducing PPIX distribution to the skin and attenuating PPIX-mediated phototoxicity (Figure 3D).

[0169] "ABCG2 deficiency protects against EPP-associated hepatotoxicity" As expected, liver injury occurred in Fech-mut mice but was abolished in Fech-mut / Abcg2-null mice (Figure 4). Compared with Fech-mut mice, serum biomarkers of liver injury were significantly reduced in Fech-mut / Abcg2-null mice (Figure 4A, 4B, 4C, and 4D). Decreased serum alkaline phosphatase (ALP) activity in Fech-mut / Abcg2-null mice (Figure 4C) indicates attenuation of cholestatic liver injury. Indeed, PPIX levels in the livers of Fech-mut / Abcg2-null mice were significantly reduced (Figure 4E), and PPIX-mediated bile duct obstruction and bile thrombus were not observed in the livers of Fech-mut / Abcg2-null mice (Figure 4F, 4G, 4H, and 4I). Liver fibrosis is a key step in the progression of EPP-associated liver injury. Liver fibrosis was observed in Fech-mut mice but not in Fech-mut / Abcg2-null mice (measured by total fibrotic area and mRNA expression). The data showed that Fech-mut mice had more than 15-fold higher expression levels of collagen 1a1 and 1a2 than Fech-mut / Abcg2-null mice. In addition, compared with Fech-mut mice, which had an 8-fold increase in fibrotic area, Fech-mut / Abcg2-null mice had background levels of fibrosis. These data indicate that ABCG2 plays an essential role in the development of EPP-associated liver injury.

[0170] "ABCG2 deficiency protects against chemical-induced PPIX accumulation and hepatotoxicity" To further determine the role of ABCG2 in PPIX-mediated liver injury, wild-type (WT) and Abcg2-null mice were challenged with DDC or GSF, two model chemicals that induce hepatic PPIX accumulation and hepatotoxicity. DDC and GSF induced liver injury, PPIX accumulation, and bile thrombus formation in WT mice, but not in Abcg2-null mice (Figures 5 and 6). In addition to DDC and GSF, previous studies have shown that cotreatment with RIF and INH induces hepatic PPIX accumulation and hepatotoxicity via the human pregnane X receptor (hPXR)-mediated pathway. To determine the role of ABCG2 in RIF- and INH-induced PPIX accumulation and hepatotoxicity, we generated an ABCG2-deficient hPXR mouse model (hPXR / Abcg2-null) (Figure 7A). In hPXR mice cotreated with RIF and INH, hepatotoxicity was observed along with PPIX accumulation and bile thrombus formation, whereas these phenotypes were abolished in hPXR / Abcg2-null mice (Figures 7B, 7C, 7D, 7E, and 7F). These data further confirmed that PPIX-mediated liver injury is dependent on ABCG2.

[0171] "ABCG2 deficiency regulates the distribution, metabolism, and excretion of PPIX" To understand the mechanism by which ABCG2 deficiency abolishes PPIX accumulation and hepatotoxicity, we performed metabolomic analysis in liver and bile samples from WT and Abcg2-null mice treated with deuterium-labeled aminolevulinic acid (D2-ALA), a precursor of PPIX (Figures 8A, 8B, and 8C). 16 We identified D2-PPIX as a downstream metabolite of D2-ALA. Consistent with the idea of ​​PPIX as an ABCG2 substrate, D2-PPIX was transported into bile. 16 Excretion of PPIX was significantly reduced in Abcg2-null mice compared with WT mice (Fig. 8D), indicating that ABCG2 deficiency directly reduces PPIX levels in the biliary system, thereby preventing PPIX-mediated biliary obstruction. Interestingly, a small amount of D was detected in the bile of Abcg2-null mice. 16PPIX was detected in the liver of ABCG2-deficient mice (Fig. 8D), suggesting that transporters other than ABCG2 may be involved in PPIX efflux, albeit to a lesser extent. We observed compensatory changes in efflux transporters (e.g., Mdr1, Mdr2, Bsep, Mrp2, Abcg5, and Abcg8) in the livers of ABCG2-deficient mice.

[0172] In metabolomic analysis, D 16 D16-protoporphyrin-1-O-acyl-glucuronide (D16-protoporphyrin-1-O-acyl-glucuronide), a conjugated metabolite of PPIX 16 -PPIX-glu) was also discovered. 16 The structure of PPIX-glu was verified by comparison with a synthetic chemical standard of PPIX-glu (Figure 9). Interestingly, D in the liver and bile of Abcg2-null mice was significantly reduced. 16 The PPIX-glu level was significantly increased compared with that of WT mice (Figures 8E and 8F). PPIX-glu was also identified in the bile of Fech-mut / Abcg2-null mice (Figure 8G). In addition to PPIX-glu, two known conjugated metabolites of PPIX, protoporphyrin-1-O-acyl-β-glucoside and protoporphyrin-1-O-acyl-β-xyloside, were identified in the bile of Fech-mut / Abcg2-null mice (Figure 8G). Overall, conjugated metabolites of PPIX were significantly increased in the bile of Fech-mut / Abcg2-null mice compared with Fech-mut mice (Figure 8H). These conjugated metabolites are considered to be detoxified metabolites because they are more hydrophilic than the parent compound and are easily excreted. Therefore, these data indicate that ABCG2 deficiency in hepatocytes increases the conjugation pathway of PPIX, promoting its excretion under EPP conditions (Figure 8I). Furthermore, ABCG2 deficiency in RBCs reduces plasma PPIX levels (Figure 3B), which in turn reduces PPIX uptake by hepatocytes and reduces PPIX levels in the biliary tree, thereby alleviating PPIX-mediated biliary obstruction (Figure 8I).

[0173] Consideration Phototoxicity is the most common symptom in patients with EPP. This study demonstrated that phototoxicity in EPP is dependent on ABCG2. Compared with Fech-mut mice, PPIX levels were significantly elevated in RBCs but decreased in serum and skin in Fech-mut / Abcg2-null mice. Consistently, the phototoxicity observed in Fech-mut mice was abolished in Fech-mut / Abcg2-null mice. These data indicate that ABCG2 in RBCs promotes phototoxicity in EPP by increasing PPIX distribution to the skin (Figure 10A). In addition, ABCG2-dependent delivery of PPIX to the biliary system causes bile duct obstruction, which further increases PPIX accumulation in the body and subsequently enhances phototoxicity (Figure 10A). The data suggest that ABCG2 deficiency reduces PPIX accumulation in the skin and prevents phototoxicity, suggesting that ABCG2 inhibition may be used as a novel strategy for managing EPP-associated phototoxicity (Figure 10B).

[0174] In EPP, PPIX in the liver is primarily derived from the bone marrow via the circulation, followed by a low level of de novo synthesis in hepatocytes. PPIX accumulation in the liver leads to potentially life-threatening liver injury due to liver failure. Here, we demonstrate that EPP-related hepatotoxicity is dependent on ABCG2, leading to high levels of PPIX accumulation in the biliary system, causing bile duct obstruction and cholestatic liver injury (Figure 10A). Specifically, we demonstrate that ABCG2 deficiency abolishes EPP-related hepatotoxicity by reducing PPIX delivery to the hepatobiliary system and alleviating PPIX-mediated bile duct obstruction (Figure 10B). Additionally, ABCG2 deficiency results in the retention of PPIX in hepatocytes, which can be metabolized to conjugated products for enhanced excretion. Overall, ABCG2 deficiency interrupts the vicious cycle of PPIX accumulation in EPP patients, reducing PPIX accumulation throughout the body and attenuating liver toxicity (Figure 10B). These data indicate that ABCG2 is a target for the management of EPP-associated liver toxicity.

[0175] The research disclosed herein changes the current research paradigm regarding the role of ABCG2 in porphyrin homeostasis and toxicity. Previous reports have shown that Abcg2-null mice are sensitive to the exogenous phototoxin pheophorbide A (PPA), a PPIX analog and ABCG2 substrate. This phototoxin model is distinct from EPP, because oral treatment with PPA bypasses ABCG2 in RBCs and delivers PPA directly to the skin. However, in EPP, PPIX distribution to the skin is dependent on ABCG2 in RBCs, and ABCG2 deficiency reduces PPIX distribution to the skin. Additionally, ABCG2 deficiency in the intestine increases PPA bioavailability by preventing PPA reflux into the intestinal lumen. Furthermore, PPA cannot be further metabolized via the conjugation pathway because a methyl group already occupies one of the conjugation positions in PPA. Furthermore, ABCG2 deficiency in hepatocytes blocks the excretion of PPA through the biliary system, allowing it to return to the circulation and accumulate more in the skin, increasing phototoxicity.

[0176] Some relevant questions regarding ABCG2 dysfunction in EPP are: is PPIX accumulation in RBCs safe? What is the fate of high levels of PPIX in RBCs? We analyzed mean corpuscular hemoglobin (MCH) and total hemoglobin (tHb) in the blood of an EPP mouse model. Reduced MCH and tHb were observed in Fech-mut mice, but not in Fech-mut / Abcg2-null mice. Additionally, Fech-mut / Abcg2-null mice appear healthy, with normal reproductive patterns compared with Fech-mut mice, which are difficult to breed. Spleen enlargement has been observed in EPP patients. Interestingly, ABCG2 deficiency attenuates EPP-associated splenomegaly, yet PPIX levels in the spleens of Fech-mut / Abcg2-null mice were higher than those of Fech-mut mice. These data indicate that PPIX accumulation in RBCs is safe in EPP, where ABCG2 is deficient. RBCs have a lifespan of approximately 120 days, after which they are recycled by macrophages in the spleen and liver. Thus, the high levels of PPIX in RBCs from Fech-mut / Abcg2-null mice ultimately end up in splenic and liver macrophages. This is protective, as retention of PPIX in resident liver macrophages, Kupffer cells, attenuates EPP-associated hepatotoxicity.

[0177] In summary, this study demonstrates that the transporter ABCG2 is a key mediator in the pathophysiology of EPP, indicating that ABCG2 is a therapeutic target for EPP. These findings in EPP may also be applicable to the management of porphyrin-generating agent / chemical toxicity and another type of porphyria, XLP, because they all share a similar biochemical basis for PPIX accumulation as EPP.

[0178] material and method Animal development, characterization, and maintenance Fech-mut / Abcg2-null mice were generated by crossing Fech-mut mice with Abcg2-null mice. Abcg2-null mice were originally generated in Dr. Schinkel's group and obtained from Taconic Biosciences, Inc. (Hudson, NY). Fech-mut mice were purchased from Jackson Laboratory (Bar Harbor, ME), which originally developed them based on a loss-of-function mutation in FECH. Fech-mut / Abcg2-null mice were verified by PCR genotyping of Fech mutation and mouse Abcg2. hPXR / Abcg2-null mice were generated by crossing hPXR mice with Abcg2-null mice. hPXR mice were originally generated by bacterial artificial chromosome (BAC) recombination. hPXR / Abcg2-null mice were verified by PCR genotyping of human PXR, mouse Pxr, and mouse Abcg2. All mice (2-4 months old, male) were housed under a standard 12-hour light / dark cycle and handled in accordance with a research protocol approved by the Institutional Animal Care and Use Committee.

[0179] Animal studies to determine the role of ABCG2 in EPP-associated phototoxicity The role of ABCG2 in EPP-related phototoxicity was determined using wild-type, Abcg2-null, Fech-mut, and Fech-mut / Abcg2-null mice. Briefly, the back skin of mice was shaved and exposed to UV light (395–410 nm) for 30 minutes daily for 5 days. On day 6, all mice were sacrificed. The back skin was harvested for histological analysis. Skin samples were also used to measure PPIX.

[0180] Animal studies to determine the role of ABCG2 in EPP-associated hepatotoxicity WT, Abcg2-null, Fech-mut, and Fech-mut / Abcg2-null mice were housed under identical conditions and sacrificed at similar ages. Liver and blood samples were collected to assess liver injury. Liver, bile, spleen, and blood samples were used to analyze PPIX and its metabolites.

[0181] Animal studies to determine the role of ABCG2 in chemically induced PPIX accumulation and hepatotoxicity WT and Abcg2-null mice were treated with DDC (0.1% in diet) or GSF (2.5% in diet) for 2 weeks. In addition, hPXR and hPXR / Abcg2-null mice were treated with RIF (100 mg / kg diet) and INH (400 mg / L drinking water) for 4 weeks. After treatment, blood and liver samples were collected to assess liver injury and analyze PPIX.

[0182] Animal studies to determine the role of ABCG2 in regulating the distribution, metabolism, and excretion of PPIX WT and Abcg2-null mice were treated with D2-ALA (50 mg / kg, i.p.), a stable isotope-labeled precursor of PPIX. One hour after D2-ALA treatment, liver and bile samples were collected for metabolomic analysis. Briefly, liver and bile samples were analyzed by ultra-performance liquid chromatography coupled to a quadrupole time-of-flight mass spectrometer (UPLC-QTOFMS, Waters Corp, Milford, MA). Mass-centered and integrated mass chromatographic data were processed using MarkerLynx software (Waters Corp, Milford, MA) to generate a multivariate data matrix. These data were then exported to SIMCA-P+ software (Umetrics, Kinnelon, NJ) for multivariate data analysis. Principal component analysis (PCA) and orthogonal projection to latent structure-discriminant analysis (OPLS-DA) were performed to represent the major latent variables in the data matrix. Variables that significantly contributed to the discrimination between groups were subjected to structure identification.

[0183] statistics Data are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism 7.0. One-way analysis of variance (ANOVA) with Tukey's post-hoc test was used to compare differences between multiple groups. Two-tailed Student's t-test was used to compare differences between two groups. A P value <0.05 was considered statistically significant.

[0184] Chemicals and Reagents Protoporphyrin IX (PPIX), 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), griseofulvin (GSF), rifampicin (RIF), isoniazid (INH), N-methylmorpholine, and glucuronic acid were purchased from Sigma-Aldrich (St. Louis, MO). Deuterium-labeled aminolevulinic acid (D2-ALA) was purchased from CDN Isotopes (Pointe-Claire, Quebec, Canada). 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) was purchased from Oakwood Products, Inc. (West Columbia, SC). All solvents used for metabolite analysis were of the highest commercially available grade.

[0185] Sample preparation for metabolite analysis PPIX and / or its metabolites were analyzed in RBCs, serum, liver, skin, spleen, and bile. Briefly, 3 × 10 PPIX samples were collected using 100 μl of 80% methanol. 7PPIX was extracted from RBCs. The mixture was sonicated for 10 seconds and centrifuged at 15,000 g for 10 minutes. 30 μl of serum sample was added to 70 μl of methanol, then vortexed and centrifuged at 15,000 g for 10 minutes. Liver and spleen samples were homogenized in water (100 mg of tissue in 500 μl of water), and then 200 μl of acetonitrile:methanol (1:1, v / v) was added to 100 μl of each homogenate, followed by vortexing and centrifugation at 15,000 g for 10 minutes. Skin tissue was ground to powder under liquid nitrogen and then digested in lysis buffer (0.1 M Tris.HCl (pH = 8), 5 mM EDTA, 0.2% SDS, 0.2 M NaCl, and 0.2 mg / ml proteinase K) for 18 hours at 54 °C (100 mg tissue powder in 500 μl buffer). To 100 μl of each skin mixture, 200 μl of acetonitrile:methanol (1:1, v / v) was added, followed by vortexing and centrifugation at 15,000 g for 10 minutes. To 80 μl of 50% aqueous acetonitrile, 2 μl of bile sample was added, followed by vortexing for 30 seconds and centrifugation at 15,000 g for 10 minutes. Each supernatant was transferred to an autosampler vial, and 1.0 μl was injected into a UPLC-QTOFMS for metabolite analysis.

[0186] UPLC-QTOFMS analysis An Acquity BEH C18 column (2.1 × 100 mm, 1.7 μm, Waters, Milford, MA) was used for metabolite separation. The mobile phase flow rate was 0.5 ml / min, and a gradient ranging from 5% to 95% acetonitrile / water containing 2 mM NH4HCO3 and 0.05% aqueous ammonia was used. The column temperature was maintained at 50 °C. The QTOFMS was operated in positive mode with electrospray ionization. The source and desolvation temperatures were set at 150 °C and 500 °C, respectively. Argon was applied as the collision gas. Nitrogen was applied as the cone and desolvation gas. The capillary and cone voltages were set at 0.8 kV and 40 V, respectively. MS data (50–1,000 Da) were acquired in center-of-mass format. Metabolite structures were elucidated using tandem mass fragmentation scans with collision energy ramps from 20 to 60 eV.

[0187] Synthesis of protoporphyrin-1-O-acyl-glucuronide (PPIX-glu) Based on the MS / MS data, we proposed PPIX-glu as a novel metabolite of PPIX. To confirm this structure, PPIX-glu was synthesized. Briefly, PPIX was first activated using HATU in the presence of N-methylmorpholine at room temperature for 14 hours, and then reacted with D-glucuronic acid to form PPIX-glu.

[0188] clinical chemistry Liver injury was assessed by analyzing serum alanine aminotransaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) (Pointe Scientific, Canton, MI), and total bilirubin (Sigma-Aldrich, St. Louis, MO). These liver injury biomarkers were analyzed according to standard procedures provided by the manufacturer. Hematologic toxicity was assessed by analyzing mean corpuscular hemoglobin (MCH) (HESKA HemaTrue; Loveland, CO) and total hemoglobin (tHb) (AVOXimeter 4000; Edison, NJ).

[0189] Histological analysis Liver and skin tissues were first fixed overnight in 4% formaldehyde phosphate solution, then dehydrated and embedded in paraffin. 4 μm sections were cut and stained. For hematoxylin and eosin (H&E) staining, tissue sections were stained with hematoxylin solution for 5 minutes, washed with tap water for 1 minute, then fractionated in 1% acetic acid solution for 1 minute, followed by staining with eosin solution for 1 minute. For Sirius Red staining, tissue sections were stained with 0.1% picrosirius red solution for 1 hour, then washed twice with 1% acetic acid solution.

[0190] Fluorescence analysis of PPIX Frozen liver tissue was mounted in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA) and cut into 10 μm sections. The liver sections were then analyzed under a fluorescence microscope (BZ-X710; Keyence Corporation, Osaka, Japan). PPIX was visualized in red. Nuclei were stained with DAPI and visualized in blue.

[0191] Quantitative PCR (qPCR) analysis Total mRNA was extracted from liver and skin tissues using TRIzol Reagent (Invitrogen, Carlsbad, CA). Complementary DNA (cDNA) was then generated from 1 μg of total RNA using the SuperScript II Reverse Transcriptase kit and random oligonucleotides (Invitrogen). qPCR analysis was performed using 25 ng of cDNA and 150 nM of each primer (TNF-α forward primer (SEQ ID NO: 1), TNF-α reverse primer (SEQ ID NO: 2), IL-1β forward primer (SEQ ID NO: 3), IL-1β reverse primer (SEQ ID NO: 4), collagen 1a1 forward primer (SEQ ID NO: 5), collagen 1a1 reverse primer (SEQ ID NO: 6), collagen 1a2 forward primer (SEQ ID NO: 7), collagen 1a2 reverse primer (SEQ ID NO: 8), Mdr1 forward primer (SEQ ID NO: 9), and Mdr1 reverse primer (SEQ ID NO: 10). (SEQ ID NO: 10), Mdr2 forward primer (SEQ ID NO: 11), Mdr2 reverse primer (SEQ ID NO: 12), Bsep forward primer (SEQ ID NO: 13), Bsep reverse primer (SEQ ID NO: 14), Mrp2 forward primer (SEQ ID NO: 15), Mrp2 reverse primer (SEQ ID NO: 16), Abcg5 forward primer (SEQ ID NO: 17), Abcg5 reverse primer (SEQ ID NO: 18), Abcg8 forward primer (SEQ ID NO: 19), and Abcg8 reverse primer (SEQ ID NO: 20)), and 5 μL of SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). qPCR plates were read on an ABI-Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA) and quantified using the comparative CT method.

[0192] Example 2: "Development and Use of ABCG2 Inhibitors for the Treatment of Erythropoietic Protoporphyria (EPP)" Although several structures with diverse scaffolds have been published as ABCG2 inhibitors, only a few of these function effectively in vivo. KO143, a structural analog of fumitremorgin C, is a potent and selective ABCG2 inhibitor. However, KO143 has a poor oral pharmacokinetic profile. Due to rapid hydrolysis of the tert-butyl ester group in the KO143 structure by carboxylesterase 1, KO143 was found to be highly unstable (Scheme 1). In this study, KO143 analogs were developed with a focus on improving metabolic stability. The inhibitory activity and cytotoxicity of this novel ABCG2 inhibitor were also determined. Additionally, the efficacy of this novel ABCG2 inhibitor was evaluated in an EPP mouse model.

[0193] result Table 1 shows the novel ABCG2 inhibitors developed based on amide I and their inhibitory activity, cytotoxicity, and metabolic stability. 50 , half-maximal inhibitory concentration; CC 50 , 50% cytotoxic concentration.

[0194] [Table 1]

[0195] Table 2 shows the novel ABCG2 inhibitors developed based on amide II and their inhibitory activity, cytotoxicity, and metabolic stability. 50 , half-maximal inhibitory concentration; CC 50 , 50% cytotoxic concentration.

[0196] [Table 2]

[0197] Table 3 shows the novel ABCG2 inhibitors developed based on amide III and their inhibitory activity, cytotoxicity, and metabolic stability. 50 , half-maximal inhibitory concentration; CC 50 , 50% cytotoxic concentration.

[0198] [Table 3]

[0199] Table 4 shows the novel O-ether-based ABCG2 inhibitors developed and their inhibitory activity, cytotoxicity, and metabolic stability. 50 , half-maximal inhibitory concentration; CC 50 , 50% cytotoxic concentration.

[0200] [Table 4]

[0201] Pharmacokinetics Based on their inhibitory activity, cytotoxicity, and metabolic stability, K2, K31, and K34 were selected for pharmacokinetic studies in mice. All three KO143 analogs, especially K31, showed favorable pharmacokinetic profiles compared with KO143 (Figure 11).

[0202] Efficacy of K31 against phototoxicity in the EPP mouse model K31 has high ABCG2 inhibitory activity, low cytotoxicity, high metabolic stability, and an ideal pharmacokinetic profile (Table 2 and Figure 11). Therefore, we further investigated the effect of K31 on phototoxicity in the EPP mouse model. We found that treatment with K31 completely protected EPP mice from phototoxicity (Figure 12). A withdrawal study of K31 in Fech-mut mice also showed phototoxicity immediately after withdrawal of K31 (Figure 13).

[0203] Effect of ABCG2 inhibitors on PPPIX efflux from RBCs. The effect of K31 on PPIX efflux from RBCs was evaluated. As shown in Figure 14, K31 significantly inhibited PPIX efflux from RBCs.

[0204] Summary and Discussion In summary, this study developed a novel ABCG2 inhibitor with better metabolic stability, and we also demonstrated herein that ABCG2 inhibitors can be used to treat EPP-associated phototoxicity, the most common symptom in EPP patients.

[0205] In addition to EPP treatment, newly developed ABCG2 inhibitors can be used to prevent multidrug resistance (MDR) in cancer therapy. Many clinically used anticancer drugs are substrates of ABCG2, but ABCG2 is overexpressed in cancer cells and excretes anticancer drugs, causing MDR and chemotherapy failure. Therefore, ABCG2 is a target for overcoming MDR in cancer therapy. Newly developed ABCG2 inhibitors can be tested for preventing MDR in cancer therapy.

[0206] method Design of ABCG2 inhibitors To develop stable ABCG2 inhibitors, we herein replaced the tert-butyl ester group with various carboxylesterase-resistant groups, including amides and ethers, based on this ester moiety. The structures of three series of amides (I, II, III) are shown in Scheme 1.

[0207] [ka]

[0208] chemical synthesis Twenty-five target compounds (Tables 1, 2, 3, and 4) were synthesized. The synthetic strategies used to prepare these compounds are detailed in Schemes 2-4 below.

[0209] [ka] Scheme 2. Synthesis of glutamate-linked Ko143 analogs. Reagents and conditions: (a) R6R7NH, EDCI, HOBt, CHCl2, rt, overnight; (b) Pd / C, MeOH, rt, 3–5 h; (c) Fmoc N-hydroxysuccinimide ester, NaHCO3, 1,4-dioxane, rt, overnight; (d) compound 4, SOCl2, DMF, CHCl2, 0 °C–rt, 2 h, then EtN, rt, overnight; or 2-chloro-1,3-dimethylimidazolinium hexafluorophosphate (CIP), DIEA N-methylpyrrolidone, rt, 5 days; (e) piperidine, THF, rt, overnight.

[0210] [ka] Scheme 3. Synthesis of aspartic acid-linked Ko143 analogs. Reagents and conditions: (a) compound 5, CIP, DIEA N-methylpyrrolidone, rt, 5 days; (b) piperidine, THF, rt, overnight; (c) Pd / C, MeOH, rt, 3 h; (d) R6R7NH, EDCI, HOBt, CHCl2, rt, overnight.

[0211] [ka] Scheme 3. Synthesis of lysine-linked Ko143 analogs. (a) Compound 5, CIP, DIEA N-methylpyrrolidone, rt, 5 days; (b) piperidine, THF, rt, overnight; (c) Pd / C, HCl, MeOH, rt, 4 h; (d) RCOCl, EtN, CHCl, 0 °C–rt, 4 h; or RCOOH, EDCI, HOBt, CHCl, rt, overnight.

[0212] The synthesis of exemplary compounds is detailed below. (S)-Benzyl 2-(((benzyloxy)carbonyl)amino)-5-(tert-butylamino)-5-oxopentanoate (2a)

[0213] [ka] (S)-5-(benzyloxy)-4-(((benzyloxy)carbonyl)amino)-5-oxopentanoic acid (1) was purchased from Chem-Impex (Wood Dale, IL). To a solution of compound 1 (8 g, 21.5 mmol) in CHCl was added EDCI (7 g, 36 mmol), HOBt (4.9 g, 36.5 mmol), and tert-butylamine (3.37 mL, 32.3 mmol). The mixture was stirred overnight at room temperature and then quenched with saturated aqueous NaHCO and extracted with CHCl. ​​The combined organic phases were washed with brine, dried over MgSO, and concentrated. The residue was purified by silica gel chromatography (PE / EtOAc = 2:1) to give compound 2a (8.9 g, 97%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 7.34 (m, 10H), 5.66 (d, J = 8.0 Hz, 1H), 5.44 (brs, 1H), 5.23 - 5.12 (m, 2H), 5.10 (s, 2H), 4.37 (m, 1H), 2.24 - 2.06 (m, 3H), 1.99 (m, 1H), 1.29 (s, 9H).HRMS(ESI):m / z(M+Na) + C 24 H 30 Calculated value of N2O5Na: 449.2052, measured value: 449.2062.

[0214] (S)-2-Amino-5-(tert-butylamino)-5-oxopentanoic acid (3a)

[0215] [ka] To a solution of compound 2a (8.9 g, 20.9 mmol) in MeOH was added 10% Pd / C (900 mg), and the suspension was hydrogenated at room temperature for 5 hours until the starting material disappeared. The mixture was then filtered, and the filtrate was concentrated to give compound 3a (2.09 g, 49%) as a white solid. HRMS (ESI): m / z (M+H) + C9H 19 Calculated N2O3 value: 203.1396, Measured value: 203.1392.

[0216] (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butylamino)-5-oxopentanoic acid (4a)

[0217] [ka] Acid 3a (7.16 g, 35.4 mmol) was dissolved in aqueous NaHCO (8.92 g, 106.2 mmol, 100 mL HO) and a solution of Fmoc N-hydroxysuccinimide ester (11.94 g, 35.4 mmol) in 1,4-dioxane (100 mL) was slowly added. The reaction mixture was stirred overnight at room temperature. The resulting mixture was then concentrated to remove most of the organic solvent, and the aqueous residue was adjusted to pH = 1 with concentrated HCl. The mixture was extracted with EtOAc, and the combined organic phases were washed with brine, dried over MgSO, and concentrated. The residue was purified by silica gel chromatography (CHCl / MeOH = 10:1) to give compound 4a (13 g, 87%) as a white solid. 1 H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 7.5 Hz, 2H), 7.59 (m, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.31 (t, J = 7.2 Hz, 2H), 6.06 (d, 1H), 5.76 (brs, 1H), 4.43 - 4.27 (m, 3H), 4.21 (t, J = 6.9 Hz, 1H), 2.49 (m, 1H), 2.37 (m, 1H), 2.23 - 2.11 (m, 1H), 2.06 (m, 1H), 1.35 (s, 9H).HRMS(ESI):m / z(MH) - C 24 H 27 Calculated value of N2O5: 423.1920, measured value: 423.1922.

[0218] N-(tert-butyl)-3-((3S,6S,12aS)-6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)propanamide (K2)

[0219] [ka] To a solution of compound 4a (8 g, 19 mmol) in dry CHCl was added dry DMF (0.3 mL, 3.8 mmol) followed by SOCl (13.8 mL, 190 mmol) under nitrogen at 0°C. The mixture was stirred at room temperature for 2 hours until all starting material was consumed. The mixture was concentrated, and the residue was washed three times with CHCl to evaporate most of the remaining SOCl. The crude acyl chloride was dissolved in dry CHCl, and this solution was added dropwise to a mixture of compound 5 (1.8 g, 5.7 mmol, see Li Y, Hayman E, Plesescu M, et al. Synthesis of potent BCRP inhibitor-Ko143. Tetrahedron Letters 2008;49:1480-1483) and EtN (2.35 mL, 17 mmol) in CHCl at 0°C. The mixture was then warmed to room temperature and stirred overnight. The reaction was quenched with saturated aqueous NaHCO3 and extracted with CHCl2. The combined organic phases were washed with brine, dried over MgSO4, and concentrated. The residue was purified by silica gel chromatography (PE / EtOAc = 2:1) to give 3.0 g of crude (1S,3S)-methyl 2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butylamino)-5-oxopentanoyl)-1-isobutyl-7-methoxy-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate (6a). HRMS (ESI): m / z (M+H) + C 42 H 51 Calculated value of N4O7: 723.3758, Measured value: 723.3754.

[0220] The crude compound 6a was dissolved in THF (60 mL), and then piperidine (3 mL) was added. The mixture was stirred at room temperature overnight. The resulting mixture was concentrated, and the crude material was dissolved in EtOAc. The organic phase was washed with brine, dried over MgSO4, and concentrated. The residue was purified by silica gel chromatography (PE / EtOAc = 1:1), followed by recrystallization in PE / EtOAc / CHCl2 to give K2 (820 mg, 31%) as a pale yellow solid. 1 H NMR (400 MHz, CDCl3) δ 7.92 (s, 1H), 7.62 (s, 1H), 7.44 (d, J = 8.6 Hz, 1H), 6.89 (d, J = 2.1 Hz, 1H), 6.83 (dd, J = 8.6, 2.2 Hz, 1H), 5.45 (m, 2H), 4.05 - 3.95 (m, 2H), 3.85 (s, 3H), 3.52 (dd, J = 15.8, 5.0 Hz, 1H), 3.04 (dd, J = 15.8, 11.7 Hz, 1H), 2.42 - 2.34 (m, 3H), 2.30 - 2.20 (m, 1H), 1.73 (m, 1H), 1.59 - 1.48 (m, 2H), 1.36 (s, 9H), 1.06 (d, J = 6.5 Hz, 3H), 0.83 (d, J = 6.4 Hz, 3H).HRMS(ESI):m / z(M+H) + C 26 H 37 Calculated N4O4 value: 469.2815, Measured value: 469.2818.

[0221] (S)-Benzyl 2-(((benzyloxy)carbonyl)amino)-5-(cyclopropylamino)-5-oxopentanoate (2b)

[0222] [ka] Compound 2b (2.0 g, 92%) was prepared as a white solid from cyclopropylamine (0.75 mL, 10.76 mmol) following a procedure similar to that described for the preparation of compound 2a. 1 H NMR (400 MHz, CDCl3) δ 7.33 (m, 10H), 5.86 (brs, 1H), 5.62 (m, 1H), 5.22 - 5.11 (m, 2H), 5.10 (s, 2H), 4.37 (m, 1H), 2.66 (m, 1H), 2.27 - 2.08 (m, 3H), 1.97 (m, 1H), 0.77 - 0.66 (m, 2H), 0.46 (m, 2H).HRMS(ESI):m / z(M+Na) + C 23 H 26 Calculated value of N2O5Na: 433.1739, Measured value: 433.1741.

[0223] (S)-2-Amino-5-(cyclopropylamino)-5-oxopentanoic acid (3b)

[0224] [ka] To a solution of compound 2b (2 g, 4.9 mmol) in THF / MeOH, 10% Pd / C (200 mg) was added, and the suspension was hydrogenated at room temperature for 3 hours until the starting material disappeared. The mixture was then dissolved in water, filtered, and the filtrate was concentrated and lyophilized to give crude compound 3b (1.2 g). 1 H NMR (400 MHz, DMSO) δ 3.11 (m, 1H), 2.60 (m, 1H), 2.19 (t, J = 7.5 Hz, 2H), 1.86 (m, 2H), 0.58 (m, 2H), 0.37 (m, 2H).HRMS(ESI):m / z(MH) - C8H 13 Calculated N2O3 value: 185.0926, Measured value: 185.0942.

[0225] (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-(cyclopropylamino)-5-oxopentanoic acid (4b)

[0226] [ka] Compound 4b (1.2 g, 60% yield over two steps) was prepared as a white solid from compound 3b (1.2 g) following a procedure similar to that described for the preparation of compound 4a. 1 H NMR (400 MHz, DMSO) δ 7.89 (m, 3H), 7.73 (d, J = 7.4 Hz, 2H), 7.60 (d, J = 8.0 Hz, 1H), 7.42 (t, J = 7.3 Hz, 2H), 7.33 (t, J = 7.4 Hz, 2H), 4.33 - 4.14 (m, 3H), 3.92 (m, 1H), 2.58 (m, 1H), 2.12 (t, J = 7.6 Hz, 2H), 1.97 (m, 1H), 1.76 (m, 1H), 0.62 - 0.50 (m, 2H), 0.41 - 0.31 (m, 2H).HRMS(ESI):m / z(M−H) - C 23 H 23 Calculated value of N2O5: 407.1607, measured value: 407.1541.

[0227] N-Cyclopropyl-3-((3S,6S,12aS)-6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)propanamide (K12)

[0228] [ka] To a solution of compound 5 (158 mg, 0.5 mmol) in N-methylpyrrolidone, compound 4b (306 mg, 0.75 mmol), DIEA (131 μL, 0.75 mmol), and CIP (140 mg, 0.5 mmol) were added. The reaction mixture was stirred at room temperature for 24 hours. After 24 hours, the second and third portions of compound 4b, DIEA, and CIP were added, respectively, and the mixture was stirred for another 24 hours. After 4 days, the reaction was monitored by TLC until almost no starting material remained. The resulting mixture was then diluted with water and extracted with EtOAc. The organic phase was washed with brine, dried over MgSO4, and concentrated. The residue was purified by silica gel chromatography (PE / EtOAc=1:1) to give (1S,3S)-methyl 2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(cyclopropylamino)-5-oxopentanoyl)-1-isobutyl-7-methoxy-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate (6b, 130 mg crude) as a pale yellow solid. HRMS (ESI): m / z (M+H) + C 25 H 32 Calculated N4O4 value: 707.3445, Measured value: 707.3452.

[0229] 120 mg (0.17 mmol) of compound 6b was dissolved in THF, and piperidine (0.3 mL) was added. The mixture was stirred at room temperature overnight. The resulting mixture was concentrated, and the crude material was dissolved in EtOAc. The organic phase was washed with brine, dried over MgSO4, and concentrated. The residue was purified by silica gel chromatography (PE / EtOAc = 2:1) to give K12 (40 mg, 52%) as a white solid. 1H NMR (400 MHz, CDCl3, mixture of atropisomers approximately 4:1) δ7.90 (s, 0.8H), 7.85 (s, 0.2H), 7.60 (s, 0.2H), 7.51 (s, 0.8H), 7.44 (d, J = 8.6 Hz, 1H), 6.89 (d, J = 1.8 Hz, 1H), 6.83 (dd, J = 8.7, 2.2 Hz, 1H), 5.89 (brs, 0.2H), 5.80 (brs, 0.8H), 5.44 (m, 1H), 4.02 (m, 2H), 3.85 (s, 3H), 3.52 (dd, J = 15.8, 4.9 Hz, 1H), 3.04 (dd, J = 15.7, 11.6 Hz, 1H), 2.78 - 2.68 (m, 1H), 2.40 (m, 2H), 2.30 (m, 1H), 1.71 (m, 1H), 1.54 (m, 2H), 1.06 (m, 3H), 0.87 - 0.75 (m, 5H), 0.60 (m, 0.4H), 0.52 (m, 1.6H).HRMS(ESI):m / z(M+H) + C 25 H 32 Calculated N4O4 value: 453.2502, Measured value: 453.2508.

[0230] (S)-Benzyl 2-(((benzyloxy)carbonyl)amino)-5-(cyclohexylamino)-5-oxopentanoate (2c)

[0231] [ka] Compound 2c (2.14 g, 87%) was prepared as a white solid from cyclohexylamine (1.22 mL, 10.76 mmol) following a procedure similar to that described for the preparation of compound 2a. 1H NMR (400 MHz, CDCl3) δ 7.44 - 7.32 (m, 10H), 5.71 (d, J = 7.8 Hz, 1H), 5.58 (d, J = 7.0 Hz, 1H), 5.25 - 5.14 (m, 2H), 5.13 (s, 2H), 4.41 (m, 1H), 3.81 - 3.68 (m, 1H), 2.28 - 2.13 (m, 3H), 2.07 - 1.97 (m, 1H), 1.89 (d, J = 12.1 Hz, 2H), 1.76 - 1.62 (m, 3H), 1.44 - 1.27 (m, 2H), 1.23 - 1.01 (m, 3H).HRMS(ESI):m / z(M+Na) + C 26 H 32 Calculated value of N2O5Na: 475.2209, Measured value: 475.2202.

[0232] (S)-2-Amino-5-(cyclohexylamino)-5-oxopentanoic acid (3c)

[0233] [ka] Compound 3c (1.06 g, 98%) was prepared as a white solid from compound 2c (2.14 g, 4.7 mmol) following a procedure similar to that described for the preparation of compound 3b. 1 H NMR (400 MHz, D2O) δ 3.64 (t, J = 6.1 Hz, 1H), 3.49 (m, 1H), 2.35 - 2.21 (m, 2H), 2.02 (m, 2H), 1.72 (m, 2H), 1.68 - 1.57 (m, 2H), 1.51 (m, 1H), 1.31 - 1.01 (m, 5H).HRMS(ESI):m / z(MH) - C 11 H 19 Calculated N2O3 value: 227.1396, Measured value: 227.1402.

[0234] (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-(cyclohexylamino)-5-oxopentanoic acid (4c)

[0235] [ka] Compound 4c (2.09 g, 93%) was prepared as a white solid from compound 3c (1.05 g, 4.6 mmol) following a procedure similar to that described for the preparation of compound 4a. 1 H NMR (400 MHz, DMSO) δ 12.57 (brs, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.73 (d, J = 7.4 Hz, 2H), 7.66 (t, J = 8.1 Hz, 2H), 7.42 (dt, J = 7.5, 3.8 Hz, 2H), 7.33 (t, J = 7.5 Hz, 2H), 4.31 - 4.15 (m, 3H), 3.94 (m, 1H), 3.50 (d, J = 7.4 Hz, 1H), 2.15 (t, J = 7.6 Hz, 2H), 2.02 - 1.92 (m, 1H), 1.73 (m, 5H), 1.53 (m, 1H), 1.24 (m, 2H), 1.10 (m, 3H).HRMS(ESI):m / z(MH) - C 26 H 29 Calculated value of N2O5: 449.2076, measured value: 449.2077.

[0236] N-Cyclohexyl-3-((3S,6S,12aS)-6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)propanamide (K14)

[0237] [ka] Following a procedure similar to that described for the preparation of compound K12, K14 (73 mg, 30%) was prepared from compound 4c (1.01 g, 2.25 mmol) as a light gray solid. 1 H NMR (400 MHz, CDCl3) δ 8.45 (brs, 1H), 7.83 (brs, 1H), 7.41 (d, J = 8.7 Hz, 1H), 6.89 (d, J = 2.1 Hz, 1H), 6.81 (dd, J = 8.6, 2.0 Hz, 1H), 5.88 (brs, 1H), 5.52 - 5.43 (m, 1H), 4.06 - 3.93 (m, 2H), 3.84 (s, 3H), 3.83 - 3.72 (m, 1H), 3.51 (dd, J = 15.8, 4.9 Hz, 1H), 3.04 (dd, J = 15.7, 11.7 Hz, 1H), 2.51 - 2.24 (m, 4H), 1.97 - 1.87 (m, 2H), 1.77 - 1.66 (m, 3H), 1.58 (m, 3H), 1.36 (m, 2H), 1.23 - 1.09 (m, 3H), 1.03 (d, J = 6.2 Hz, 3H), 0.81 (d, J = 6.3 Hz, 3H).HRMS(ESI):m / z(M+H) + C 28 H 39 Calculated N4O4 value: 495.2971, Measured value: 495.2969.

[0238] 3-((3S,6S,12aS)-6-Isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)-N,N-dimethylpropanamide (K18)

[0239] [ka] Following a procedure similar to that described for the preparation of compound K12, K18 was prepared as a white solid. 1H NMR (400 MHz, CDCl3) δ 8.02 (s, 1H), 7.82 (s, 1H), 7.43 (d, J = 8.5 Hz, 1H), 6.88 (d, J = 2.0 Hz, 1H), 6.82 (dd, J = 8.6, 2.0 Hz, 1H), 5.45 (m, 1H), 4.00 (m, 2H), 3.84 (s, 3H), 3.50 (dd, J = 15.9, 5.0 Hz, 1H), 3.08 - 3.00 (m, 1H), 3.00 (s, 3H), 2.98 (s, 3H), 2.56 (m, 3H), 2.34 (m, 1H), 1.77 - 1.68 (m, 1H), 1.60 - 1.49 (m, 2H), 1.06 (d, J = 6.4 Hz, 3H), 0.83 (d, J = 6.3 Hz, 3H).HRMS(ESI):m / z(M+H) + C 24 H 33 Calculated N4O4 value: 441.2502, Measured value: 441.2499.

[0240] (S)-Benzyl 2-(((benzyloxy)carbonyl)amino)-5-oxo-5-(phenylamino)pentanoate (2d)

[0241] [ka] Compound 2d (2.2 g, 93%) was prepared as a white solid from aniline (0.98 mL, 10.76 mmol) following a procedure similar to that described for the preparation of compound 2a. 1 H NMR (400 MHz, CDCl3) δ 7.94 (brs, 1H), 7.53 (d, J = 7.9 Hz, 2H), 7.39 - 7.28 (m, 12H), 7.10 (t, J = 7.4 Hz, 1H), 5.63 (d, J = 7.6 Hz, 1H), 5.22 - 5.13 (m, 2H), 5.09 (m, 2H), 4.47 (m, 1H), 2.37 (m, 3H), 2.03 (m, 1H).HRMS(ESI):m / z(M+Na)+ C 26 H 26 Calculated value of N2O5Na: 469.1739, Measured value: 469.1734.

[0242] (S)-2-Amino-5-oxo-5-(phenylamino)pentanoic acid (3d)

[0243] [ka] Compound 3d (1.08 g, 99%) was prepared as a white solid from compound 2d (2.2 g, 5.0 mmol) following a procedure similar to that described for the preparation of compound 3b. 1 H NMR (400 MHz, DMSO) δ 7.56 (d, J = 7.7 Hz, 2H), 7.26 (t, J = 7.9 Hz, 2H), 7.00 (t, J = 7.4 Hz, 1H), 3.15 (m, 1H), 2.35 (s, 2H), 1.82 (m, 2H).HRMS(ESI):m / z(MH) - C 11 H 13 Calculated N2O3 value: 221.0926, Measured value: 221.0936.

[0244] (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-oxo-5-(phenylamino)pentanoic acid (4d)

[0245] [ka] Compound 4d (1.88 g, 86%) was prepared as a pale yellow solid from compound 3d (1.08 g, 4.9 mmol) following a procedure similar to that described for the preparation of compound 4a. 1H NMR (400 MHz, DMSO) δ 10.01 (s, 1H), 7.90 (d, J = 7.4 Hz, 2H), 7.73 (d, J = 7.5 Hz, 2H), 7.59 (d, J = 7.9 Hz, 2H), 7.42 (t, J = 7.3 Hz, 2H), 7.31 (m, 4H), 7.02 (t, J = 7.4 Hz, 1H), 4.25 (m, 3H), 3.96 (m, 1H), 2.42 (t, J = 7.6 Hz, 2H), 2.15 - 2.04 (m, 1H), 1.89 (m, 1H).HRMS(ESI):m / z(MH) - C 26 H 23 Calculated value of N2O5: 443.1607, measured value: 443.1604.

[0246] 3-((3S,6S,12aS)-6-Isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)-N-phenylpropanamide (K19)

[0247] [ka] Following a procedure similar to that described for the preparation of K12, K19 (45 mg, 18%) was prepared from compound 4d (0.99 g, 2.25 mmol) as a pale yellow solid. 1H NMR (400 MHz, CDCl3) δ 9.55 (s, 1H), 7.52 (d, J = 8.1 Hz, 2H), 7.38 (d, J = 8.6 Hz, 1H), 7.29 (d, J = 8.2 Hz, 2H), 7.12 - 7.03 (m, 1H), 6.87 (d, J = 2.1 Hz, 1H), 6.76 (dd, J = 8.5, 2.0 Hz, 1H), 5.46 - 5.36 (m, 1H), 3.99 (m, 2H), 3.82 (s, 3H), 3.47 (dd, J = 15.6, 4.7 Hz, 1H), 3.39 (m, 1H), 3.05 - 2.92 (dd, J = 15.6, 11.7 Hz, 1H), 2.52 (m, 2H), 2.39 - 2.26 (m, 2H), 1.65 (m, 1H), 1.60 - 1.46 (m, 2H), 1.01 (d, J = 6.1 Hz, 3H), 0.77 (d, J = 6.1 Hz, 3H).HRMS(ESI):m / z(M+H) + C 28 H 33 Calculated N4O4 value: 489.2502, Measured value: 489.2481.

[0248] (S)-Benzyl 2-(((benzyloxy)carbonyl)amino)-5-oxo-5-(pyrrolidin-1-yl)pentanoate (2e)

[0249] [ka] Compound 2e (2.3 g, 100%) was prepared as a colorless oil from pyrrolidine (0.88 mL, 10.76 mmol) following a procedure similar to that described for the preparation of compound 2a. 1H NMR (400 MHz, CDCl3) δ 7.40 - 7.28 (m, 10H), 6.04 (d, J = 7.4 Hz, 1H), 5.22 - 5.03 (m, 4H), 4.39 (m, 1H), 3.41 (t, J = 6.7 Hz, 2H), 3.22 (t, J = 6.6 Hz, 2H), 2.36 - 2.17 (m, 3H), 2.15 - 2.02 (m, 1H), 1.93 - 1.75 (m, 4H).HRMS(ESI):m / z(M+H) + C 24 H 29 Calculated value of N2O5: 425.2076, Measured value: 425.2076.

[0250] (S)-2-Amino-5-oxo-5-(pyrrolidin-1-yl)pentanoic acid (3e)

[0251] [ka] Compound 3e (1.07 g, 98%) was prepared as a white solid from compound 2e (2.3 g, 5.4 mmol) following a procedure similar to that described for the preparation of compound 3a. 1 H NMR (400 MHz, MeOD) δ 3.61 (t, J = 5.8 Hz, 1H), 3.53 - 3.44 (m, 2H), 3.42 (t, J = 6.9 Hz, 2H), 2.61 - 2.54 (m, 2H), 2.16 - 2.09 (m, 2H), 2.02 - 1.93 (m, 2H), 1.92 - 1.83 (m, 2H).HRMS(ESI):m / z(MH) - C9H 15 Calculated N2O3: 199.1083, Measured: 199.1049.

[0252] (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-oxo-5-(pyrrolidin-1-yl)pentanoic acid (4e)

[0253] [ka] Compound 4e (1.76 g, 78%) was prepared as a white solid from compound 3e (1.07 g, 5.34 mmol) following a procedure similar to that described for the preparation of compound 4a. 1 H NMR (400 MHz, DMSO) δ 7.90 (d, J = 7.5 Hz, 2H), 7.72 (dd, J = 7.1, 4.8 Hz, 2H), 7.66 (d, J = 7.8 Hz, 1H), 7.42 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 4.32 - 4.16 (m, 3H), 3.98 (m, 1H), 3.42 - 3.29 (m, 4H), 2.40 - 2.21 (m, 2H), 2.00 (m, 1H), 1.88 - 1.69 (m, 5H).HRMS(ESI):m / z(MH) - C 24 H 25 Calculated value of N2O5: 421.1763, measured value: 421.1749.

[0254] (3S,6S,12aS)-6-Isobutyl-9-methoxy-3-(3-oxo-3-(pyrrolidin-1-yl)propyl)-2,3,12,12a-tetrahydropyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4(6H,7H)-dione (K20)

[0255] [ka] Following a procedure similar to that described for the preparation of K12, K20 (92 mg, 40%) was prepared as a white solid from compound 4e (0.95 g, 2.25 mmol). 1H NMR (400 MHz, CDCl3) δ 8.08 (s, 2H), 7.42 (d, J = 8.6 Hz, 1H), 6.88 (d, J = 2.1 Hz, 1H), 6.82 (dd, J = 8.6, 2.2 Hz, 1H), 5.45 (m, 1H), 4.00 (m, 2H), 3.84 (s, 3H), 3.54 - 3.44 (m, 3H), 3.38 (m, 2H), 3.04 (dd, J = 15.8, 11.7 Hz, 1H), 2.61 - 2.43 (m, 3H), 2.40 - 2.27 (m, 1H), 2.02 - 1.92 (m, 2H), 1.91 - 1.81 (m, 2H), 1.77 - 1.68 (m, 1H), 1.60 - 1.47 (m, 2H), 1.06 (d, J = 6.4 Hz, 3H), 0.82 (d, J = 6.3 Hz, 3H).HRMS(ESI):m / z(M+H) + C 26 H 35 Calculated N4O4 value: 467.2658, Measured value: 467.2629.

[0256] N,N-Diethyl-3-((3S,6S,12aS)-6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)propanamide (K21)

[0257] [ka] Following a procedure similar to that described for the preparation of K12, K21 was prepared as a pale yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.00 (s, 1H), 7.87 (s, 1H), 7.43 (d, J = 8.6 Hz, 1H), 6.88 (d, J = 1.8 Hz, 1H), 6.82 (dd, J = 8.6, 2.0 Hz, 1H), 5.46 (m, 1H), 3.99 (m, 2H), 3.84 (s, 3H), 3.50 (dd, J = 15.8, 4.9 Hz, 1H), 3.40 (m, 2H), 3.31 (m, 2H), 3.04 (dd, J = 15.7, 11.7 Hz, 1H), 2.66 - 2.44 (m, 3H), 2.38 - 2.28 (m, 1H), 1.72 (m, 1H), 1.60 - 1.48 (m, 2H), 1.15 (m, 6H), 1.06 (d, J = 6.3 Hz, 3H), 0.82 (d, J = 6.3 Hz, 3H).HRMS(ESI):m / z(M+H) + C 26 H 37 Calculated N4O4 value: 469.2815, Measured value: 469.2802.

[0258] (S)-Benzyl 2-(((benzyloxy)carbonyl)amino)-5-oxo-5-(piperidin-1-yl)pentanoate (2f)

[0259] [ka] Compound 2f (2.2 g, 93%) was prepared as a colorless oil from piperidine (0.98 mL, 10.76 mmol) following a procedure similar to that described for the preparation of compound 2a. 1H NMR (400 MHz, CDCl3) δ 7.39 - 7.28 (m, 10H), 5.85 (d, J = 7.6 Hz, 1H), 5.22 - 5.13 (m, 2H), 5.13 - 5.04 (m, 2H), 4.40 (m, 1H), 3.53 - 3.45 (m, 2H), 3.27 - 3.18 (m, 2H), 2.40 - 2.16 (m, 3H), 2.11 - 2.04 (m, 1H), 1.60 (m, 2H), 1.49 (m, 4H).HRMS(ESI):m / z(M+H) + C 25 H 31 Calculated value of N2O5: 439.2233, measured value: 439.2239.

[0260] (S)-2-Amino-5-oxo-5-(piperidin-1-yl)pentanoic acid (3f)

[0261] [ka] Compound 3f (1.02 g, 95%) was prepared as a white solid from compound 2f (2.2 g, 5.0 mmol) following a procedure similar to that described for the preparation of compound 3a. 1 H NMR (400 MHz, DMSO) δ 3.46 - 3.36 (m, 5H), 3.21 - 3.13 (m, 2H), 2.39 (m, 2H), 1.94 - 1.75 (m, 2H), 1.57 (m, 2H), 1.48 (m, 2H), 1.40 (m, 2H).HRMS(ESI):m / z(MH) - C 10 H 17 Calculated N2O3 value: 213.1239, measured value: 213.1227.

[0262] (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-oxo-5-(piperidin-1-yl)pentanoic acid (4f)

[0263] [ka] Compound 4f (1.82 g, 88%) was prepared as a white solid from compound 3f (1.02 g, 4.76 mmol) following a procedure similar to that described for the preparation of compound 4a. 1 H NMR (400 MHz, DMSO) δ 7.90 (d, J = 7.4 Hz, 2H), 7.72 (m, 2H), 7.64 (m, 1H), 7.47 - 7.36 (t, J = 7.2 Hz, 2H), 7.33 (t, J = 7.2 Hz, 2H), 4.33 - 4.14 (m, 3H), 4.01 (m, 1H), 3.41 (m, 4H), 2.36 (m, 2H), 1.97 (m, 1H), 1.81 (m, 1H), 1.55 (m, 2H), 1.42 (m, 4H).HRMS(ESI):m / z(MH) - C 25 H 27 Calculated value of N2O5: 435.1920, measured value: 435.1913.

[0264] (3S,6S,12aS)-6-Isobutyl-9-methoxy-3-(3-oxo-3-(piperidin-1-yl)propyl)-2,3,12,12a-tetrahydropyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4(6H,7H)-dione (K24)

[0265] [ka] Following a procedure similar to that described for the preparation of K12, K24 (42 mg, 18%) was prepared as a white solid from compound 4f (0.97 g, 2.25 mmol). 1H NMR (400 MHz, CDCl3) δ 7.97 (s, 1H), 7.79 (s, 1H), 7.43 (d, J = 8.6 Hz, 1H), 6.88 (d, J = 2.1 Hz, 1H), 6.82 (dd, J = 8.6, 2.2 Hz, 1H), 5.45 (dd, J = 9.2, 4.2 Hz, 1H), 4.03 - 3.95 (m, 2H), 3.84 (s, 3H), 3.64 - 3.54 (m, 2H), 3.51 (dd, J = 15.9, 5.0 Hz, 1H), 3.43 - 3.37 (m, 2H), 3.04 (dd, J = 15.8, 11.7 Hz, 1H), 2.64 - 2.45 (m, 3H), 2.31 (m, 1H), 1.73 (m, 1H), 1.67 - 1.60 (m, 2H), 1.60 - 1.48 (m, 6H), 1.06 (d, J = 6.5 Hz, 3H), 0.83 (d, J = 6.4 Hz, 3H).HRMS(ESI):m / z(M+H) + C 27 H 37 Calculated N4O4 value: 481.2815, Measured value: 481.2817.

[0266] (S)-Benzyl 2-(((benzyloxy)carbonyl)amino)-5-morpholino-5-oxopentanoate (2g)

[0267] [ka] Compound 2g (2 g, 84%) was prepared as a pale yellow solid from morpholine (0.94 mL, 10.76 mmol) following a procedure similar to that described for the preparation of compound 2a. 1H NMR (400 MHz, CDCl3) δ 7.39 - 7.29 (m, 10H), 5.71 (d, J = 7.6 Hz, 1H), 5.24 - 5.04 (m, 4H), 4.46 - 4.37 (m, 1H), 3.64 - 3.59 (m, 2H), 3.56 (m, 4H), 3.27 (m, 2H), 2.39 - 2.18 (m, 3H), 2.04 (m, 1H).HRMS(ESI):m / z(M+H) + C 24 H 29 Calculated value of N2O6: 441.2026, measured value: 441.2032.

[0268] (S)-2-Amino-5-morpholino-5-oxopentanoic acid (3g)

[0269] [ka] Compound 3g (0.98 g, 99%) was prepared as a white solid from compound 2g (2 g, 4.54 mmol) following a procedure similar to that described for the preparation of compound 3a. 1 H NMR (400 MHz, DMSO) δ 3.77 - 3.17 (m, 10H), 2.45 (m, 1H), 2.01 - 1.76 (m, 2H).HRMS(ESI):m / z(MH) - C9H 15 Calculated N2O4 value: 215.1032, Measured value: 215.1031.

[0270] (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-morpholino-5-oxopentanoic acid (4g)

[0271] [ka] Compound 4g (1.57 g, 79%) was prepared as a white solid from compound 3g (0.98 g, 4.53 mmol) following a procedure similar to that described for the preparation of compound 4a. 1H NMR (400 MHz, DMSO) δ 12.59 (brs, 1H), 7.97 - 7.87 (m, 2H), 7.73 (m, 2H), 7.66 (m, 1H), 7.49 - 7.39 (m, 2H), 7.34 (m, 2H), 5.77 (m, 1H), 4.27 (m, 3H), 4.01 (m, 1H), 3.48 - 3.30 (m, 7H), 2.55 (m, 1H), 2.39 (m, 2H), 2.00 (m, 1H), 1.89 - 1.73 (m, 1H).HRMS(ESI):m / z(MH) - C 24 H 25 Calculated value of N2O6: 437.1713, measured value: 437.1725.

[0272] (3S,6S,12aS)-6-Isobutyl-9-methoxy-3-(3-morpholino-3-oxopropyl)-2,3,12,12a-tetrahydropyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4(6H,7H)-dione (K22)

[0273] [ka] Following a procedure similar to that described for the preparation of K12, K22 (48 mg, 20%) was prepared as a white solid from compound 4g (0.99 g, 2.25 mmol). 1H NMR (400 MHz, CDCl3) δ 8.00 (s, 1H), 7.62 (s, 1H), 7.43 (d, J = 8.6 Hz, 1H), 6.88 (d, J = 1.9 Hz, 1H), 6.83 (dd, J = 8.6, 2.1 Hz, 1H), 5.44 (dd, J = 9.3, 4.1 Hz, 1H), 4.01 (m, 2H), 3.84 (s, 3H), 3.73 - 3.61 (m, 6H), 3.55 - 3.43 (m, 3H), 3.04 (dd, J = 15.7, 11.8 Hz, 1H), 2.67 - 2.43 (m, 3H), 2.34 (m, 1H), 1.78 - 1.72 (m, 1H), 1.60 - 1.48 (m, 2H), 1.06 (d, J = 6.4 Hz, 3H), 0.82 (d, J = 6.3 Hz, 3H).HRMS(ESI):m / z(M+H) + C 26 H 35 Calculated value of N4O5: 483.2607, Measured value: 483.2609.

[0274] N-Cyclohexyl-3-((3S,6S,12aS)-6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)-N-methylpropanamide (K26)

[0275] [ka] Following a procedure similar to that described for the preparation of K12, K26 was prepared as a white solid. 1H NMR (400 MHz, CDCl3, mixture of atropisomers approximately 1:1) δ7.98 (s, 1H), 7.88 (s, 1H), 7.43 (d, J = 8.6 Hz, 1H), 6.88 (d, J = 2.1 Hz, 1H), 6.82 (dd, J = 8.6, 2.2 Hz, 1H), 5.46 (dd, J = 9.4, 4.6 Hz, 1H), 4.50 - 4.40 (m, 0.5H), 4.04 - 3.94 (m, 2H), 3.84 (s, 3H), 3.61 - 3.54 (m, 0.5H), 3.50 (dd, J = 15.9, 5.0 Hz, 1H), 3.04 (dd, J = 15.8, 11.7 Hz, 1H), 2.84 (s, 1.5H) 2.83 (s, 1.5H), 2.70 - 2.44 (m, 3H), 2.39 - 2.26 (m, 1H), 1.90 - 1.23 (m, 12H), 1.15 (m, 1H), 1.06 (d, J = 6.4 Hz, 3H), 0.83 (d, J = 6.2 Hz, 3H).HRMS(ESI):m / z(M+H) + C 29 H 41 Calculated N4O4 value: 509.3128, Measured value: 509.3125.

[0276] 3-((3S,6S,12aS)-6-Isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)-N-isopropylpropanamide (K34)

[0277] [ka] Following a procedure similar to that described for the preparation of K12, K34 was prepared as a pale yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.17 (s, 1H), 7.76 (s, 1H), 7.42 (d, J = 8.6 Hz, 1H), 6.89 (d, J = 2.0 Hz, 1H), 6.82 (dd, J = 8.6, 2.2 Hz, 1H), 5.66 (d, J = 7.9 Hz, 1H), 5.47 (dd, J = 9.2, 4.1 Hz, 1H), 4.13 - 4.07 (m, 1H), 4.06 - 3.96 (m, 2H), 3.85 (s, 3H), 3.51 (dd, J = 15.8, 5.0 Hz, 1H), 3.04 (dd, J = 15.6, 11.7 Hz, 1H), 2.48 - 2.23 (m, 4H), 1.74 - 1.65 (m, 1H), 1.61 - 1.47 (m, 2H), 1.17 (d, J = 6.6 Hz, 6H), 1.04 (d, J = 6.5 Hz, 3H), 0.82 (d, J = 6.4 Hz, 3H).HRMS(ESI):m / z(M+H) + C 25 H 35 Calculated N4O4 value: 455.2658, Measured value: 455.2663.

[0278] Benzyl 2-((3S,6S,12aS)-6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)acetate (K16)

[0279] [ka] Following a procedure similar to that described for the preparation of K12, K16 (1.1 g, 37%) was prepared as a white solid from Fmoc-Asp(OBzl)-OH (10.8 g, 27 mmol). 1H NMR (400 MHz, CDCl3) δ 7.91 (s, 1H), 7.47 - 7.30 (m, 6H), 6.89 (d, J = 2.0 Hz, 1H), 6.83 (dd, J = 8.8, 2.0 Hz, 1H), 6.78 (s, 1H), 5.42 (dd, J = 8.9, 3.3 Hz, 1H), 5.25 - 5.14 (m, 2H), 4.41 - 4.32 (dd, J = 8.7, 3.8 Hz, 1H), 4.06 (dd, J = 11.6, 4.5 Hz, 1H), 3.85 (s, 3H), 3.55 (dd, J = 15.9, 4.6 Hz, 1H), 3.36 (dd, J = 17.2, 3.9 Hz, 1H), 2.99 (dd, J = 15.6, 11.7 Hz, 1H), 2.78 (dd, J = 17.3, 9.3 Hz, 1H), 1.75 (m, 1H), 1.54 (m, 2H), 1.04 (d, J = 6.2 Hz, 3H), 0.81 (d, J = 6.1 Hz, 3H).HRMS(ESI):m / z(M+H) + C 28 H 32 Calculated value of N3O5: 490.2342, Measured value: 490.2345.

[0280] 2-((3S,6S,12aS)-6-Isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)acetic acid (K17)

[0281] [ka] Following a procedure similar to that described for the preparation of compound 3a, K17 (750 mg, 96%) was prepared as a light gray solid from K16 (950 mg, 4.33 mmol). 1H NMR (400 MHz, MeOD) δ 10.46 (brs, 1H), 7.40 (d, J = 8.7 Hz, 1H), 6.92 (s, 1H), 6.74 (dd, J = 8.9, 2.0 Hz, 1H), 5.44 (m, 1H), 4.42 (m, 1H), 4.23 (m, 1H), 3.83 (s, 3H), 3.66 (brs, 1H), 3.48 (dd, J = 15.2, 4.5 Hz, 1H), 3.09 - 2.80 (m, 4H), 1.75 (m, 1H), 1.61 (m, 2H), 1.01 (d, J = 6.1 Hz, 3H), 0.83 (d, J = 6.3 Hz, 3H).HRMS(ESI):m / z(M+H) + C 21 H 26 Calculated value of N3O5: 400.1872, Measured value: 400.1877.

[0282] N-(tert-butyl)-2-((3S,6S,12aS)-6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)acetamide (K3)

[0283] [ka] To a solution of K17 (105 mg, 0.26 mmol) in CHCl was added EDCI (100 mg, 0.52 mmol), HOBt (70 mg, 0.52 mmol), and tert-butylamine (55 μL, 0.52 mmol). The mixture was stirred at room temperature overnight. The resulting mixture was then quenched with saturated aqueous NaHCO and extracted with CHCl. ​​The combined organic phases were washed with brine, dried over MgSO, and concentrated. The residue was purified by silica gel chromatography (CHCl / MeOH=40:1) to give K3 (48 mg, 40%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.94 (s, 1H), 7.43 (d, J = 8.6 Hz, 1H), 7.10 (s, 1H), 6.88 (d, J = 2.0 Hz, 1H), 6.83 (dd, J = 8.8, 2.0 Hz, 1H), 5.82 (s, 1H), 5.48 - 5.39 (m, 1H), 4.27 (dd, J = 9.4, 4.9 Hz, 1H), 4.04 (dd, J = 11.2, 4.2 Hz, 1H), 3.85 (s, 3H), 3.55 (dd, J = 15.8, 4.7 Hz, 1H), 2.97 (m, 2H), 2.63 (dd, J = 15.0, 8.6 Hz, 1H), 1.72 (m, 1H), 1.61 - 1.47 (m, 2H), 1.35 (s, 9H), 1.04 (d, J = 6.3 Hz, 3H), 0.82 (d, J = 6.2 Hz, 3H).HRMS(ESI):m / z(M+H) + C 25 H 35 Calculated N4O4 value: 455.2658, Measured value: 455.2657.

[0284] 2-((3S,6S,12aS)-6-Isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)-N-isopropylacetamide (K31)

[0285] [ka] Following a procedure similar to that described for the preparation of K3, K31 (50 mg, 44%) was prepared as a white solid from isopropylamine hydrochloride (50 mg, 0.52 mmol). 1H NMR (400 MHz, CDCl3) δ 7.92 (s, 1H), 7.43 (d, J = 8.6 Hz, 1H), 7.11 (s, 1H), 6.89 (d, J = 2.0 Hz, 1H), 6.83 (dd, J = 8.6, 2.0 Hz, 1H), 5.85 (d, J = 7.6 Hz, 1H), 5.42 (dd, J = 9.2, 4.0 Hz, 1H), 4.28 (dd, J = 8.5, 3.8 Hz, 1H), 4.12 - 4.01 (m, 2H), 3.85 (s, 3H), 3.55 (dd, J = 15.8, 4.9 Hz, 1H), 2.99 (m, 2H), 2.66 (dd, J = 15.1, 8.5 Hz, 1H), 1.72 (m, 1H), 1.60 - 1.48 (m, 2H), 1.17 (m, 6H), 1.05 (d, J = 6.4 Hz, 3H), 0.81 (d, J = 6.4 Hz, 3H).HRMS(ESI):m / z(M+H) + C 24 H 33 Calculated N4O4 value: 441.2502, Measured value: 441.2507.

[0286] N-Cyclohexyl-2-((3S,6S,12aS)-6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)acetamide (K23)

[0287] [ka] Following a procedure similar to that described for the preparation of K3, K23 (55 mg, 44%) was prepared as a light gray solid from cyclohexylamine (60 μL, 0.52 mmol). 1H NMR (400 MHz, CDCl3) δ 8.04 (s, 1H), 7.42 (d, J = 8.6 Hz, 1H), 7.19 (s, 1H), 6.88 (d, J = 2.1 Hz, 1H), 6.82 (dd, J = 8.6, 2.2 Hz, 1H), 6.03 (d, J = 8.2 Hz, 1H), 5.42 (dd, J = 9.2, 4.0 Hz, 1H), 4.28 (dd, J = 8.0, 4.0 Hz, 1H), 4.03 (dd, J = 11.7, 4.7 Hz, 1H), 3.84 (s, 3H), 3.81 - 3.69 (m, 1H), 3.54 (dd, J = 15.8, 4.8 Hz, 1H), 3.05 - 2.94 (m, 2H), 2.68 (dd, J = 15.0, 8.2 Hz, 1H), 1.90 (m, 2H), 1.78 - 1.65 (m, 3H), 1.65 - 1.48 (m, HRMS(ESI):m / z(M+H) + C 27 H 37 Calculated N4O4 value: 481.2815, Measured value: 481.2814.

[0288] (3S,6S,12aS)-6-Isobutyl-9-methoxy-3-(2-oxo-2-(piperidin-1-yl)ethyl)-2,3,12,12a-tetrahydropyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4(6H,7H)-dione (K25)

[0289] [ka] Following a procedure similar to that described for the preparation of K3, K25 (62 mg, 51%) was prepared as a white solid from piperidine (48 μL, 0.52 mmol). 1H NMR (400 MHz, CDCl3 / d4-MeOD) δ 8.87 (s, 1H), 7.40 (d, J = 8.5 Hz, 1H), 6.86 (d, J = 1.1 Hz, 1H), 6.78 (dd, J = 8.3, 1.5 Hz, 1H), 5.38 (dd, J = 9.9, 4.1 Hz, 1H), 4.36 (d, J = 9.7 Hz, 1H), 4.03 (dd, J = 12.3, 4.8 Hz, 1H), 3.82 (s, 3H), 3.54 (m, 3H), 3.41 (m, 3H), 2.99 - 2.87 (dd, J = 11.7, 15.52 Hz, 1H), 2.56 (dd, J = 17.1, 10.6 Hz, 1H), 1.75 - 1.45 (m, 10H), 1.01 (d, J = 6.1 Hz, 3H), 0.78 (d, J = 6.3 Hz, 3H).HRMS(ESI):m / z(M+H) + C 26 H 35 Calculated value of N4O4: 467.2658, Measured value: 467.2670.

[0290] N-Benzyl-2-((3S,6S,12aS)-6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)acetamide (K33)

[0291] [ka] Following a procedure similar to that described for the preparation of K3, K33 (68 mg, 53%) was prepared as a white solid from benzylamine (57 μL, 0.52 mmol). 1H NMR (400 MHz, CDCl3) δ 7.94 (s, 1H), 7.45 (d, J = 8.6 Hz, 1H), 7.39 - 7.33 (m, 2H), 7.32 - 7.26 (m, 3H), 7.17 (s, 1H), 6.91 (d, J = 1.9 Hz, 1H), 6.86 (dd, J = 8.7, 2.2 Hz, 1H), 6.45 (m, 1H), 5.46 - 5.37 (m, 1H), 4.47 (qd, J = 14.8, 5.9 Hz, 2H), 4.34 (dd, J = 8.0, 3.5 Hz, 1H), 4.05 (dd, J = 11.7, 4.2 Hz, 1H), 3.87 (s, 3H), 3.56 (dd, J = 15.8, 4.7 Hz, 1H), 3.06 (m, 2H), 2.75 (dd, J = 15.1, 8.2 Hz, 1H), 1.65 (m, 1H), 1.52 (m, 2H), 1.04 (d, J = 6.3 Hz, 3H), 0.81 (d, J = 6.3 Hz, 3H).HRMS(ESI):m / z(M+H) + C 28 H 33 Calculated N4O4 value: 489.2502, Measured value: 489.2509.

[0292] Benzyl (4-((3S,6S,12aS)-6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)butyl)carbamate (K8)

[0293] [ka] K8 was prepared as a white solid from Fmoc-Lys(Z)-OH following a procedure similar to that described for the preparation of K12. 1H NMR (400 MHz, CDCl3) δ 7.92 (s, 1H), 7.44 (d, J = 8.6 Hz, 1H), 7.32 (m, 5H), 6.89 (d, J = 1.9 Hz, 1H), 6.83 (dd, J = 8.5, 2.0 Hz, 1H), 6.68 (brs, 1H), 5.44 (m, 1H), 5.17 - 5.07 (m, 2H), 5.02 (m, 1H), 3.98 (m, 2H), 3.84 (s, 3H), 3.53 (dd, J = 15.7, 4.7 Hz, 1H), 3.41 - 3.12 (m, 2H), 3.01 (dd, J = 15.6, 11.7 Hz, 1H), 2.09 (m, 1H), 1.97 (m, 1H), 1.73 (m, 1H), 1.57 (m, 6H), 1.04 (d, J = 6.4 Hz, 3H), 0.82 (d, J = 6.2 Hz, 3H).HRMS(ESI):m / z(M+H) + C 31 H 39 Calculated value of N4O5: 547.2920, measured value: 547.2919.

[0294] (3S,6S,12aS)-3-(4-aminobutyl)-6-isobutyl-9-methoxy-2,3,12,12a-tetrahydropyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4(6H,7H)-dione (K9)

[0295] [ka] To a solution of K8 (1.06 g) in MeOH, 10% Pd / C (100 mg) and concentrated HCl (1 mL) were added. The mixture was hydrogenated at room temperature for 4 hours until the starting material disappeared. The suspension was then filtered, and the filtrate was concentrated. The residue was crude K9 hydrochloride as a light green solid. 1H NMR (400 MHz, DMSO) δ 10.95 (s, 1H), 8.33 (s, 1H), 8.00 (brs, 3H), 7.40 (d, J = 8.6 Hz, 1H), 6.86 (d, J = 2.1 Hz, 1H), 6.66 (dd, J = 8.6, 2.2 Hz, 1H), 5.33 (dd, J = 7.9, 4.5 Hz, 1H), 4.13 (dd, J = 11.5, 4.8 Hz, 1H), 4.02 (m, 1H), 3.75 (s, 3H), 3.30 (dd, J = 15.6, 4.7 Hz, 1H), 2.77 (m, 3H), 1.92 - 1.79 (m, 1H), 1.75 (m, 1H), 1.57 (m, 4H), 1.50 - 1.35 (m, 3H), 0.90 (d, J = 6.4 Hz, 3H), 0.75 (d, J = 6.5 Hz, 3H).HRMS(ESI):m / z(M+H) + C 23 H 33 Calculated N4O3 value: 413.2553, measured value: 413.2543.

[0296] N-(4-((3S,6S,12aS)-6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)butyl)pivalamide (K10)

[0297] [ka] N-(4-((3S,6S,12aS)-6-isobutyl-9-methoxy-1,4-dioxo-2-pivaloyl-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)butyl)pivalamide (K11)

[0298] [ka] To a solution of K9 (90 mg, 0.22 mmol) and EtN (89 μL, 0.64 mmol) in dichloromethane was added trimethylacetyl chloride (40 μL, 0.32 mmol) at 0 °C. The resulting mixture was stirred at room temperature for 4 h and then quenched with water. The organic phase was washed with brine, dried over MgSO, and concentrated. The residue was purified by silica gel chromatography (CHCl / MeOH = 30:1) to give K10 (28 mg, 26%) and K11 (26 mg, 18%) as a white solid.

[0299] K10: 1 H NMR (400 MHz, CDCl3) δ 8.14 (brs, 1H), 7.43 (d, J = 8.6 Hz, 1H), 6.89 (d, J = 2.0 Hz, 1H), 6.82 (dd, J = 8.6, 2.0 Hz, 1H), 6.75 (brs, 1H), 5.89 (m, 1H), 5.45 (dd, J = 9.1, 3.9 Hz, 1H), 4.06 - 3.94 (m, 2H), 3.84 (s, 3H), 3.52 (dd, J = 15.8, 4.8 Hz, 1H), 3.40 (m, 1H), 3.25 (m, 1H), 3.01 (dd, J = 15.5, 11.9 Hz, 1H), 2.13 (m, 1H), 2.01 (m, 1H), 1.73 (m, 1H), 1.64 - 1.49 (m, 4H), 1.49 - 1.35 (m, 2H), 1.23 (s, 9H), 1.03 (d, J = 6.4 Hz, 3H), 0.81 (d, J = 6.3 Hz, 3H).HRMS(ESI):m / z(M+H) + C 28 H 41 Calculated N4O4 value: 497.3128, Measured value: 497.3101.

[0300] K11: 1H NMR (400 MHz, CDCl3) δ 7.93 (s, 1H), 7.41 (d, J = 8.7 Hz, 1H), 6.88 (d, J = 2.0 Hz, 1H), 6.83 (dd, J = 8.4, 2.0 Hz, 1H), 5.84 (m, 1H), 5.34 (m, 1H), 4.42 (m, 1H), 4.06 (dd, J = 11.3, 4.2 Hz, 1H), 3.85 (s, 3H), 3.62 (dd, J = 15.4, 4.0 Hz, 1H), 3.26 (m, 2H), 2.95 (dd, J = 15.3, 11.6 Hz, 1H), 2.04 (m, 1H), 1.95 - 1.68 (m, 3H), 1.66 - 1.41 (m, 5H), 1.34 (s, 9H), 1.20 (s, 9H), 0.98 (d, J = 6.5 Hz, 3H), 0.86 (d, J = 6.5 Hz, 3H).HRMS(ESI):m / z(M+H) + C 33 H 49 Calculated value of N4O5: 581.3703, measured value: 581.3714.

[0301] N-(4-((3S,6S,12aS)-6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)butyl)cyclohexanecarboxamide (K28)

[0302] [ka] N-(4-((3S,6S,12aS)-2-(cyclohexanecarbonyl)-6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)butyl)cyclohexanecarboxamide (K27)

[0303] [ka] Following a procedure similar to that described for the preparation of K10, K27 and K28 were prepared as white solids from cyclohexanecarbonyl chloride.

[0304] K28: 1 H NMR (400 MHz, CDCl3) δ 8.15 (s, 1H), 7.43 (d, J = 8.6 Hz, 1H), 6.89 (d, J = 2.0 Hz, 1H), 6.82 (dd, J = 8.6, 2.2 Hz, 1H), 6.72 (s, 1H), 5.73 (t, J = 6.0 Hz, 1H), 5.46 (dd, J = 9.1, 4.1 Hz, 1H), 4.03 (dd, J = 11.5, 4.7 Hz, 1H), 3.97 (t, J = 4.8 Hz, 1H), 3.84 (s, 3H), 3.52 (dd, J = 15.8, 4.8 Hz, 1H), 3.38 (m, 1H), 3.26 (m, 1H), 3.01 (dd, J = 15.7, 11.7 Hz, 1H), 2.18 - 2.05 (m, 2H), 1.98 (m, 1H), 1.87 (d, J = 13.4 Hz, 2H), 1.78 (t, J = 9.2 Hz, 2H), 1.72 - 1.63 (m, 2H), 1.62 - 1.52 (m, 4H), 1.46 (dd, J = 14.0, 9.0 Hz, 4H), 1.33 - 1.18 (m, 3H), 1.03 (d, J = 6.5 Hz, 3H), 0.82 (d, J = 6.4 Hz, 3H).HRMS(ESI):m / z(M+H) + C 30 H 43 Calculated N4O4 value: 523.3284, Measured value: 523.3279.

[0305] K27: 1H NMR (400 MHz, CDCl3) δ 7.94 (s, 1H), 7.38 (d, J = 8.6 Hz, 1H), 6.88 (d, J = 2.0 Hz, 1H), 6.82 (dd, J = 8.6, 2.2 Hz, 1H), 5.55 (m, 1H), 5.04 (t, J = 6.0 Hz, 1H), 4.83 (t, J = 6.0 Hz, 1H), 4.29 (dd, J = 11.6, 3.9 Hz, 1H), 3.85 (s, 3H), 3.56 (dd, J = 15.4, 3.6 Hz, 1H), 3.29 (m, 1H), 3.20 (m, 2H), 2.97 (dd, J = 14.5, 12.6 Hz, 1H), 2.30 - 2.21 (m, 1H), 2.12 - 1.97 (m, 2H), 1.90 - 1.60 (m, 10H), 1.58 - 1.46 (m, 3H), 1.47 - 1.34 (m, 7H), 1.22 (m, 7H), 0.94 (d, J = 6.6 Hz, 3H), 0.90 (d, J = 6.6 Hz, 3H).HRMS(ESI):m / z(M+H) + C 37 H 53 Calculated value of N4O5: 633.4016, measured value: 633.4008.

[0306] N-(4-((3S,6S,12aS)-6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)butyl)benzamide (K29)

[0307] [ka] K29 was prepared as a white solid from benzoyl chloride following a procedure similar to that described for the preparation of K10. 1H NMR (400 MHz, CDCl3) δ 8.16 (s, 1H), 7.86 - 7.79 (m, 2H), 7.48 (m, 1H), 7.45 - 7.38 (m, 3H), 6.90 (s, 1H), 6.88 (d, J = 2.1 Hz, 1H), 6.81 (dd, J = 8.6, 2.2 Hz, 1H), 6.57 (t, J = 6.0 Hz, 1H), 5.45 (dd, J = 9.1, 4.1 Hz, 1H), 4.01 (m, 2H), 3.83 (s, 3H), 3.60 (m, 1H), 3.55 - 3.45 (m, 2H), 2.99 (dd, J = 15.9, 12.0 Hz, 1H), 2.18 (m, 1H), 2.08 - 1.96 (m, 1H), 1.76 - 1.66 (m, 3H), 1.62 - 1.47 (m, 4H), 1.01 (d, J = 6.4 Hz, 3H), 0.78 (d, J = 6.3 Hz, 3H).HRMS(ESI):m / z(M+H) + C 30 H 37 Calculated N4O4 value: 517.2815, Measured value: 517.2822.

[0308] N-(4-((3S,6S,12aS)-6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)butyl)cyclopentanecarboxamide (K30)

[0309] [ka] Following a procedure similar to that described for the preparation of K10, K30 was prepared as a white solid. 1H NMR (400 MHz, CDCl3) δ 8.09 (s, 1H), 7.43 (d, J = 8.6 Hz, 1H), 6.89 (d, J = 2.1 Hz, 1H), 6.82 (dd, J = 8.6, 2.2 Hz, 1H), 6.71 (s, 1H), 5.77 (t, J = 6.0 Hz, 1H), 5.45 (dd, J = 9.1, 4.1 Hz, 1H), 4.03 (dd, J = 11.7, 4.8 Hz, 1H), 3.97 (t, J = 4.8 Hz, 1H), 3.85 (s, 3H), 3.52 (dd, J = 15.7, 4.8 Hz, 1H), 3.41 (m, 1H), 3.26 (m, 1H), 3.01 (dd, J = 15.7, 11.7 Hz, 1H), 2.53 (m, 1H), 2.13 (m, 1H), 2.05 - 1.94 (m, 1H), 1.92 - 1.70 (m, 7H), 1.64 - 1.51 (m, 6H), 1.50 - 1.40 (m, 2H), 1.04 (d, J = 6.5 Hz, 3H), 0.82 (d, J = 6.4 Hz, 3H).HRMS(ESI):m / z(M+H) + C 29 H 41 Calculated N4O4 value: 509.3128, Measured value: 509.3136.

[0310] N-(4-((3S,6S,12aS)-6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)butyl)isobutyramide (K32)

[0311] [ka] Following a procedure similar to that described for the preparation of K10, K32 was prepared as a white solid. 1H NMR (400 MHz, CDCl3) δ 8.11 (s, 1H), 7.43 (d, J = 8.6 Hz, 1H), 6.89 (d, J = 2.0 Hz, 1H), 6.82 (dd, J = 8.6, 2.0 Hz, 1H), 6.70 (s, 1H), 5.78 (t, J = 5.7 Hz, 1H), 5.46 (dd, J = 9.0, 4.0 Hz, 1H), 4.07 - 3.94 (m, 2H), 3.84 (s, 3H), 3.52 (dd, J = 15.8, 4.8 Hz, 1H), 3.42 (m, 1H), 3.25 (m, 1H), 3.01 (dd, J = 15.6, 11.7 Hz, 1H), 2.44 - 2.31 (m, 1H), 2.17 - 2.06 (m, 1H), 2.00 (m, 1H), 1.77 - 1.70 (m, 1H), 1.63 - 1.40 (m, 6H), 1.19 (m, 6H), 1.03 (d, J = 6.4 Hz, 3H), 0.82 (d, J = 6.4 Hz, 3H).HRMS(ESI):m / z(M+H) + C 27 H 39 Calculated N4O4 value: 483.2971, Measured value: 483.2964.

[0312] (3S,6S,12aS)-3-(3-(tert-butoxy)propyl)-6-isobutyl-9-methoxy-2,3,12,12a-tetrahydropyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4(6H,7H)-dione (K7)

[0313] [ka] Following a procedure similar to that described for the preparation of K12, K7 was prepared as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.98 (s, 1H), 7.72 (s, 1H), 7.44 (d, J = 8.6 Hz, 1H), 6.88 (d, J = 2.0 Hz, 1H), 6.83 (dd, J = 8.6, 2.1 Hz, 1H), 5.45 (dd, J = 9.3, 4.1 Hz, 1H), 4.01 (dd, J = 11.7, 4.9 Hz, 1H), 3.89 - 3.81 (m, 4H), 3.54 - 3.36 (m, 3H), 3.04 (dd, J = 15.8, 11.6 Hz, 1H), 2.38 (m, 1H), 1.88 (m, HRMS(ESI):m / z(M+H) + C 26 H 38 Calculated value of N3O4: 456.2862, Measured value: 456.2852

[0314] ABCG2 inhibition assay A protoporphyrin IX (PPIX)-based ABCG2 inhibition assay was developed using the A549 cancer cell line. For each KO143 analog, the half-maximal inhibitory concentration (IC 50 ) values ​​were measured. KO143 was used as a positive control for the ABCG2 inhibition assay.

[0315] Cytotoxicity assay Cytotoxicity was determined using the MTT assay, and 50% cytotoxic concentration (CC50) values ​​were calculated for each KO143 analog.

[0316] Metabolic stability assay The metabolic stability of the synthesized KO143 analogs was determined by incubating them with human liver microsomes (HLMs). After incubation (0–60 min), the remaining parent compound was analyzed by UPLC-QTOFMS.

[0317] Pharmacokinetic studies in mice. Pharmacokinetic analysis was performed on KO143 analogs with high ABCG2 inhibitory activity, low cytotoxicity, and high metabolic stability. Briefly, WT mice (6-8 weeks old, male) were treated with selected KO143 analogs (50 mg / kg) by oral gavage. Blood samples were collected at 0, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours post-treatment. Serum concentrations of target compounds were analyzed by UPLC-QTOFMS.

[0318] Efficacy of ABCG2 inhibitors against EPP-associated phototoxicity in vivo This study was carried out on KO143 analogs with high ABCG2 inhibitory activity, low cytotoxicity, and high metabolic stability. Using the Fech-mut mouse model as an EPP model, we determined the efficacy of ABCG2 inhibitors against EPP-associated phototoxicity, the most common symptom in EPP patients (6-8). Briefly, Fech-mut mice with hair removed from their backs were pretreated with selected KO143 analogs for 30 minutes and then exposed to UV light (395-410 nm) for 30 minutes. After 5 days of treatment, all mice were sacrificed and skin damage was evaluated.

[0319] To further verify the protective effect of ABCG2 inhibitors against PPIX-mediated phototoxicity, a withdrawal study was performed using a KO143 analog. Briefly, Fech-mut mice were pretreated with a KO143 analog (100 mg / kg, po). 30 minutes after treatment, the mice were exposed to UV light for 30 minutes. The same treatment (drug + light exposure) was repeated once daily for 4 days. From the 5th day, drug treatment was stopped, but light exposure was continued for another 4 days. The macroscopic findings of the mouse skin were recorded daily. On the 9th day, all mice were sacrificed and the back skin was collected to evaluate phototoxicity.

[0320] Effect of ABCG2 inhibitors on PPIX efflux from RBCs RBCs were collected from Fech-mut mice and treated with each of the selected KO143 analogs (10 μM PPIX in RBCs and PPIX in the culture medium were incubated with PPIX, respectively. The mixture was extracted and analyzed by UPLC-QTOFMS. Examples of the invention of this application include the following. [1] A method for treating, preventing, reducing, or inhibiting erythropoietic protoporphyria (EPP) in a subject, comprising administering to the subject a therapeutic agent that inhibits ABCG2 activity. [2] A method of treating, preventing, reducing, or inhibiting X-linked protoporphyria (XLP) in a subject, comprising administering to the subject a therapeutic agent that inhibits ABCG2 activity. [3] A method for reducing PPIX excretion from red blood cells or hepatocytes in a subject, the method comprising administering to the subject a therapeutic agent that inhibits ABCG2 activity. [4] A method for treating, preventing, reducing, or inhibiting purpura, erythema, edema, or burning sensation in the skin of a subject with EPP, comprising administering to the subject a therapeutic agent that inhibits ABCG2 activity. [5] A method for treating, preventing, reducing, or inhibiting hepatotoxicity in a subject, comprising administering to the subject a therapeutic agent that inhibits ABCG2 activity. [6] The method according to any one of [1] to [5] above, wherein the therapeutic agent is an antibody, a peptide, a protein, an RNAi, a small molecule, or a targeted nucleic acid incorporation system. [7] The method according to [6] above, wherein the therapeutic agent is an anti-ABCG2 antibody. [8] The method according to [6] above, wherein the target nucleic acid integration system is a CRISPR (clustered regularly interspaced short palindromic repeat) / CRISPR-associated 9 (Cas9) integration system containing a guide RNA targeting the ABCG2 gene. [9] The method according to any one of [6] to [8] above, wherein the therapeutic agent comprises a tissue-specific targeting moiety or an expression vector.

[10] The method according to [6] above, wherein the therapeutic agent comprises a small molecule.

[11] The small molecule is a compound defined by Formula I: [ka] (In the formula, A is, [ka] selected from the group consisting of: n is an integer from 0 to 6; X, if present, is selected from the group consisting of O and S; R 1 H, halo, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl, CN, NO 2 , OR a , S.R. a , C(O)R b , C(O)NR c R d , C(O)OR a ,OC(O)Rb , OC(O)NR c R d , N.R. c R d , N.R. c OR d , N.R. c C(O)R b , N.R. c C(O)OR a , N.R. c C(O)NR c R d , C(=NR e )R b , C(=NR e )NR c R d , N.R. c C(=NR e )NR c R d , N.R. c S(O)R b , N.R. c S(O) 2 R b , N.R. c S(O) 2 NR c R d , S(O)R b , S(O)NR c R d , S(O) 2 R b , and S(O) 2 NR c R d wherein C is selected from the group consisting of 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A optionally substituted with a group; R 2 and R 3 H, halo, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl, CN, NO 2 , OR a , and S.R. a wherein C is independently selected from the group consisting of 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A optionally substituted with a group; R 4 and R 5 is H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C(O)R b 、C 3-10 independently selected from the group consisting of cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl; 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A optionally substituted with a group; R 6 and R 7 is H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 each independently selected from the group consisting of cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl; 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A optionally substituted with a group or R 6 and R 7 together with the N atoms to which they are attached, form one, two, or three independently selected R A groups, each forming an optionally substituted 4- to 9-membered heterocycloalkyl group or a 5- to 6-membered heteroaryl group; R 8 and R 9 is H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 independently selected from the group consisting of cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl; 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A optionally substituted with a group or R 8 and R 9 together with the atoms to which they are attached, form one, two, or three independently selected R A R 10 is C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C(O)R b 、C3-10 cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl, 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A optionally substituted with a group; Each R a 、R b 、R c , and R d is H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 independently selected from cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl; 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are each selected from 1, 2, 3, or 4 independently selected R A each optionally substituted with a group; Each R e are H, CN, and C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkylthio, C 1-6 Alkylsulfonyl, C 1-6 Alkyl carbonyl, C 1-6 Alkylaminosulfonyl, Carbamyl, C 1-6 Alkylcarbamyl, di(C 1-6 Alkyl)carbamyl, aminosulfonyl, C 1-6 Alkylaminosulfonyl, and di(C 1-6 alkyl)aminosulfonyl; and Each R A OH NO 2 ,CN,Haro,C 1-6 Alkyl, C2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, Cyano-C 1-3 Alkyl, HO-C 1-3 Alkyl, Amino, C 1-6 Alkylamino, di(C 1-6 Alkyl)amino, thio, C 1-6 Alkylthio, C 1-6 Alkylsulfinyl, C 1-6 Alkyl sulfonyl, carbamyl, C 1-6 Alkylcarbamyl, di(C 1-6 Alkyl) carbamyl, carboxy, C 1-6 Alkyl carbonyl, C 1-6 Alkoxycarbonyl, C 1-6 Alkylcarbonylamino, C 1-6 Alkyl sulfonyl amino, amino sulfonyl, C 1-6 Alkylaminosulfonyl, di(C 1-6 Alkyl)aminosulfonyl, aminosulfonylamino, C 1-6 Alkylaminosulfonylamino, di(C 1-6 Alkyl)aminosulfonylamino, aminocarbonylamino, C 1-6 Alkylaminocarbonylamino, and di(C 1-6 alkyl)aminocarbonylamino; or a pharmaceutically acceptable salt, ester, or N-oxide thereof.

[12] R 4 and R 5 The method according to

[11] above, wherein both are hydrogen.

[13] R 2 and R 3 is H and C 1-6 alkyl, wherein C 1-6 R is 1, 2, 3, or 4 independently selected alkyl A The method according to any one of the above

[11] to

[12] , wherein the aryl group is optionally substituted with a group.

[14] R 2 The method according to any one of the above

[11] to

[13] , wherein is isobutyl.

[15] R 3 The method according to any one of the above

[11] to

[14] , wherein is hydrogen.

[16] R 3 The method according to any one of the above

[11] to

[15] , wherein is methyl.

[17] R 1 H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 Haloalkyl, and OR a selected from the group consisting of: Said C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, and C 1-4 haloalkyl is one, two, three, or four independently selected R A optionally substituted with a group; and R a If exists, H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-4 haloalkyl; 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, and C 1-4 haloalkyl is one, two, three, or four independently selected R A The method according to any one of the above

[11] to

[16] , wherein each of the groups is optionally substituted with a group.

[18] R 1 The method according to

[17] above, wherein is hydrogen.

[19] R 1 is hydroxy, C 1-4 Alkyl, and C 1-4 alkoxy, wherein C 1-4 Alkyl and C 1-4 Alkoxy is selected from 1, 2, 3, or 4 independently selected R A The method according to

[17] above, wherein each of the groups is optionally substituted.

[20] R 1 The method according to

[19] above, wherein is methoxy.

[21] The small molecule is a compound defined by the following formula IA:

change

[22] The small molecule is a compound defined by the following formula IB:

change

[23] The small molecule is a compound defined by the following formula IC:

change

[24] The small molecule is a compound defined by the following formula ID:

change

[25] The method according to

[24] above, wherein X is selected from the group consisting of O and S.

[26] The method according to

[25] above, wherein X is O.

[27] The method according to any one of

[10] to

[26] above, wherein n is an integer of 1 to 4.

[28] The method of

[10] above, wherein the small molecule comprises a compound selected from the group consisting of fumitremorgin C, KO143, GF120918, YHO-13351, curcumin, CID44640177, CID1434724, CID46245505, CCT129202, artesunate, ST1481, dihydropyridine, dofequidar fumarate, gefitinib, imatinib mesylate, lapatinib, WK-X-34, YHO-13177, MBL-II-141, ML753286, and combinations thereof.

[29] cells containing ABCG2 knockout.

[30] A non-human animal comprising the cells described in

[29] above.

[31] A compound defined by the following formula IA:

change

[32] The compound according to

[31] above, wherein n is an integer of 1 to 4.

[33] The compound according to

[32] above, wherein n is 1 or 2.

[34] R 6 and R 7 But H, C 1-6 Alkyl, C 1-4 Haloalkyl, C 3-10 each independently selected from the group consisting of cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl; 1-6 Alkyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are selected from 1, 2, 3, or 4 independently selected R A The compound according to any one of the above

[31] to

[33] , optionally substituted with a group.

[35] R6 and R 7 together with the N atom to which they are attached, form one, two, or three independently selected R A The compound according to any one of the above

[31] to

[33] , wherein the compound is a 4- to 9-membered heterocycloalkyl group or a 5- to 6-membered heteroaryl group, each of which is optionally substituted with a group.

[36] A compound defined by the following formula IB:

change

[37] The compound according to

[36] above, wherein n is an integer of 1 to 4.

[38] The compound according to

[37] above, wherein n is 4.

[39] R 9 The compound according to any one of the above

[36] to

[38] , wherein is hydrogen.

[40] R 8 But H, C 1-6 Alkyl, C 1-4 Haloalkyl, C 3-10 cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl, 1-6 Alkyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are selected from 1, 2, 3, or 4 independently selected R A The compound according to any one of the above

[36] to

[39] , optionally substituted with a group.

[41] R 8 and R 9 together with the atoms to which they are attached to form one, two, or three independently selected R A The compound according to any one of the above

[36] to

[40] , wherein the compound is a 4- to 9-membered heterocycloalkyl group or a 5- to 6-membered heteroaryl group, each of which is optionally substituted with a group.

[42] A compound defined by the following formula IC

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[43] The compound according to

[42] above, wherein n is an integer of 1 to 4.

[44] The compound according to

[43] above, wherein n is 4.

[45] R 6 and R 7 But H, C 1-6 Alkyl, C 1-4 Haloalkyl, C 3-10 each independently selected from the group consisting of cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl; 1-6 Alkyl, C 1-4 Haloalkyl, C 3-10 Cycloalkyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, and 4- to 10-membered heterocycloalkyl are selected from 1, 2, 3, or 4 independently selected R A The compound according to any one of the above

[42] to

[44] , optionally substituted with a group.

[46] R 6 and R 7 together with the N atom to which they are attached, form one, two, or three independently selected R A The compound according to any one of the above

[42] to

[44] , wherein the compound forms a 4- to 9-membered heterocycloalkyl group or a 5- to 6-membered heteroaryl group, each of which is optionally substituted with a group.

[47] A compound defined by the following formula ID:

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[48] ​​The compound according to

[47] above, wherein X is selected from the group consisting of O and S.

[49] The compound according to

[48] above, wherein X is O.

[50] The compound according to any one of the above

[47] to

[49] , wherein n is an integer of 1 to 4.

[51] The compound according to

[50] above, wherein n is 3.

[52] A method for treating multidrug resistance in a tumor or cancer by inhibiting ABCG2 transporter activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the compound described in any one of

[31] to

[51] above.

[53] A method for enhancing chemotherapy treatment of a tumor or cancer using a chemotherapeutic agent, or a method for improving the bioavailability of an ABCG2 substrate drug in a mammal, comprising administering to the mammal an effective amount of the chemotherapeutic agent together with an effective amount of the compound described in any one of

[31] to

[51] above, thereby inhibiting ABCG2 protein.

[54] A method for reducing resistance of a tumor or cancer to a chemotherapeutic agent by inhibiting ABCG2 in a mammal, the method comprising administering to the mammal an effective amount of a compound described in any one of

[31] to

[51] above in combination with the administration of the chemotherapeutic agent.

[55] The method according to any one of

[52] to

[54] above, wherein the tumor or cancer is selected from solid tumors, malignant melanoma, non-small cell lung cancer, colon tumors, prostate tumors, brain tumors, lymphomas, breast tumors, ovarian tumors, lung tumors, and gastric tumors.

[56] The method according to any one of

[53] to

[55] above, wherein the chemotherapeutic agent is selected from the group consisting of mitoxantrone, topotecan, camptothecin, camptothecin derivative SN-38, irinotecan, flavopiridol, gefitinib, rhodamine, daunomycin, imatinib, doxorubicin, colchicine, vinblastine, paclitaxel, cisplatin, adriamycin, danofloxacin mesylate, docetaxel, and combinations thereof.

[0321] References

[0322] Table 5-1

[0323] Table 5-2

[0324] Table 5-3

[0325] Table 5-4

[0326] Arrange

[0327]

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[0328]

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Claims

1. below: 【Chemistry 1】 or a pharmaceutically acceptable salt, ester or N-oxide thereof.

2. below: 【Chemistry 2】 or a pharmaceutically acceptable salt, ester or N-oxide thereof.

3. A pharmaceutical composition comprising a compound of claim 1 or 2, or a pharmaceutically acceptable salt, ester or N-oxide thereof, and a pharmaceutically acceptable carrier.

4. 4. The pharmaceutical composition of claim 3 for use in treating, preventing, reducing or inhibiting erythropoietic protoporphyria (EPP) or X-linked protoporphyria (XLP) in a subject.

5. 4. The pharmaceutical composition of claim 3 for use in treating, preventing, reducing or inhibiting purpura, erythema, edema or burning sensation in the skin of a subject with EPP or XLP.

6. 4. The pharmaceutical composition of claim 3 for use in treating, preventing, reducing or inhibiting hepatotoxicity in a subject.

7. 4. The pharmaceutical composition of claim 3 for use in reducing PPIX efflux from red blood cells or hepatocytes in a subject.

8. 4. The pharmaceutical composition of claim 3 for use in treating multidrug resistance in a tumor or cancer in a subject.

9. 4. The pharmaceutical composition of claim 3 for use in reducing the resistance of a tumor or cancer to a chemotherapeutic agent.

10. 4. The pharmaceutical composition of claim 3 for use in enhancing chemotherapy treatment of tumors or cancers using chemotherapeutic agents.

11. The pharmaceutical composition of claim 3 for use in improving the bioavailability of an ABCG2 substrate drug in a mammal.

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