NZ780825B2 - Lanthionine c-like protein 2 ligands, cells prepared therewith, and therapies using same - Google Patents

Abstract

Provided are compounds that target the lanthionine synthetase C-like protein 2 pathway. The compounds can be used to treat a number of conditions, including autoimmune diseases, inflammatory diseases, chronic inflammatory diseases, diabetes, and infectious diseases, such as lupus, rheumatoid arthritis, type 1 diabetes, inflammatory bowel disease, viral diseases, and nonalcoholic steatohepatitis. The compounds can also be used to generate cells, such as immune cells, for treating the conditions.

Description

LANTHIONINE C-LIKE PROTEIN 2 LIGAND S, CELLS PREPARED THEREWITH, AND THERAPIES USING SAME FIELD OF THE INVENTION The present invention relates to the ?eld of l treatments for diseases and disorders. More speci?cally, the present invention relates to classes of biologically active compounds that treat and prevent in?ammatory and immune mediated diseases such as in?ammatory bowel disease, type 1 diabetes, lupus, Sjogren’s syndrome, rheumatoid arthritis, psoriasis, and multiple sclerosis, as well as chronic in?ammatory diseases and disorders such as insulin resistance, impaired glucose tolerance, prediabetes, type 2 diabetes, and obesity-related ation, among .

BACKGROUND Lanthionine C-like protein 2 (LANCL2) (also called "lanthionine synthetase C-like n 2" or "lanthionine synthetase ent C-like protein 2") is a membrane receptor expressed in immune and epithelial cells of various mucosa, including respiratory and GI, as well as reproductive and neural s that can be controlled by direct binding of ligands. Activation of the LANCL2 pathway has proven bene?cial in multiple autoimmune, in?ammatory and metabolic disorders ranging from glycemic control and 2O insulin sensitivity in diabetes to promotion of survival and regulatory effects in viral and bacterial infectious disease to ssion of in?ammation in in?ammatory bowel disease.

Autoimmune disorders such as in?ammatory bowel disease (IBD), systemic lupus erythematosus, rheumatoid tis, type 1 es, psoriasis, and multiple sclerosis, are rising in incidence throughout the world. ile, other chronic diseases of metabolic origin, including pre-diabetes, type 2 diabetes and lic syndrome, are estimated to af?ict nearly half of the adult population in the United States. Across these disorders, current treatments have mild ef?cacy with the potential for severe side effects such as heart attack and stroke (TZDs for type 2 diabetes) or sed rates of cancer and infection (biologics for in?ammatory bowel diseases and other autoimmune diseases) and t much increase in overall quality of life. Type 1 diabetes has no ed medications other than life-long insulin therapy. Other diseases such as multiple sclerosis and systemic lupus matosus have very limited options to slow the progression of disease towards debilitating physical conditions that require organ transplants and complete living care. The LANCL2 pathway offers an innovative solution to these diseases normalizing metabolism, restoring immunological tolerance, and ssing the in?ammation contributing to a ed prognosis in many of these diseases. ic acid ("ABA") is one of the natural compounds found that binds to LANCL2. There is an enormous number of compounds described in the ?eld of tic organic chemistry. Various compounds are ed by the following references: WOl997/036866 to Diana et al., et al., et al., WO 2008/079277 to Adams et al., JP 2008/056615 to Urasoe et al., WO 66898 to Stoessel et al., US 2013/0142825 to Bassaganya-Riera et al., and US. Pat.

No. 7,741,367 to Bassaganya—Riera et al. Some of the compounds described in these references are known to activate the LANCL2 pathway and others are not.

Previous LANCL2 targeting compounds, such as BT-ll, are highly restricted to the gastrointestinal tract with limited systemic eXposure. While this localization can be advantageous for the treatment of gastrointestinal disorders and infections, further classes of LANCL2 based therapeutics are needed for the treatment of systemic diseases and non- GI disorders with varying pharrnacokinetic properties and potency. BT-ll and its therapeutic uses are described in US Patent 9,556,146 to Bassaganya-Riera et al., US Patent 9,839,635 to Bassaganya-Riera et al., US Patent 10,028,950 to Bassaganya—Riera et al.‘ US Patent 10,201,538 to Bassaganya—Riera et al., US Patent 10,493,072 to Bassaganya-Riera et al., US Patent 10,682,349 to Bassaganya-Riera et al., US 2019/0160100 A1 to Bassaganya-Riera et al., Bissel et a1, 2016 (Bissel P, Boes K, Hinckley l, er BS, Magnintissel G, Werro SR, Ehrich M, Carbo A, Philipson C, illas R, Philipson N Gandour RD, Bassagaiiya~Riera l, Exploratory Studies Willi BT—ll: A Proposed Orally Active 'i‘lierapeutic for Crohn‘s Disease. In? J l. 2016 Sep;35(5):521—9), and Carbo et a1. 2016 (Garbo A, i' RD, Horitecillas R, Philipson N Ui‘en A, Bassaganya—Riera 1. An N,N—Bis(benzimidazolylpicolinoyl)piperazine (BT— 11): A Novel Lanthionine Synthetase (CT—Like 2—Based Therapeutic for Inflammatory Bowel Disease. J?fed Chem. 2016 Nov 23;59(22):101 13—10126); Leber et al. 2018 (Leber A, Honteci?as R, imRodriguez V, Bassaganya—Riera 1. Activation of LANCL2 by 1391211 Ameliorates 1131) by Supporting Regulatory '1‘ Cell ity 'l‘hrough immunometabolic Mechanisms. m Bowel Dis. 2018 Aug 16;24(9):l978—199l); Leber at al, 2019 ,im J beicof. (Leber A, Honiecillas R, Zoccoli—Rodi‘iguez V, Ehricli M, Davis 3, Chauhan 1,13assaganya-Riera 3'. N’onclinical Toxicology and ’170Xicolrinetic Profile of an Oral i‘11'iioi'iirie Synlhetase C~Li1 Tbxicoi. 2019 Mar/Apr;38(2196—109), Leber et a}. 2019 Jimmmmi. {Leber A, l—lontecillas R, Zoccoli—Rodriguez V, Chauhan I, Bassaganya—Riera .l'. Qral Treatment with BTmll Ameliorates lnfl amn'iatory Bowel e by Enhancing Regulatory T Cell Responses in the Gut. J Mammal. 20l9 Apr l;202(7):2095-2104), and Leber et al, 2020 {Leber A, Honteeillas R, ZoocolimRodriguez V, Colombel IF, Chauhan 3, Ehrieh M, Farinola N, Bassaganya-Riera 34 The Safety, 'i‘olerability, and Pharmacokinetics Profile of BT—ll, an Oral, stricted Lanthionine Synthetase C~Like 2 Agonist igational New Drug for inflammatory Bowel Disease: A Randomized, Double—Blind, Placebo—Controlled Phase I al Trial. Jig/filmy" Bowel Dis. 3029 Mar 4;26{4):643—652).

The invention provides compounds that have been developed by novel medicinal chemistry approaches, and ed using in silica, in vilro, and in vivo techniques, to balance an ability to selectively bind to the LANCL2 protein with systemic bioavailability and potency. These compounds can affect a bene?cial response in various disease conditions, including but not limited to, autoimmune, chronic in?ammatory, atory, metabolic, and infectious diseases.

SUMTVIARY OF THE ION The invention provides compounds of formula Z-Y-Q-Y' or pharmaceutically acceptable salts or esters thereof, n: Z is: a r- Q.’ 13:? a ‘\\ y - A2 ’5 x A0 "A1 .

Y is: é?j?a or ix? E Aiéi’l Q is piperazine-1,4-diyl, 2,5-diazabicyclo[2,2,l]heptane-2,5-diyl, 2,5- diazabicyclo[2,2,2]octane-2,5-diyl; 1,4-diazepane-1,4-diyl, benzene-1,4-diamine-N1,N4- diyl, ethane-1 ,2-diamine-N1,N2-diyl, N1,N2-dialkylethane-1,2-diamine-N1,N2-diyl, propane-1,3 -diamine-N1,N3-diyl, N1,N3-dialkylpropane-1,3 -diamine-N1,N3-diyl, 1,4- oanthracene-9,10-dione-l,4-diyl; sub stituted piperazine- l ,4-diyl, or sub stituted C6 arene-1,4-diamine-N1,N4-diyl, Y' is: 14 ?g:\ A ‘ 17 \A15 "’R \ x! or or i 2 --- between A15 and A16 is a bond or absent; A1; A2; A3; A4; A6, A8, A9; A10; A11; A12; A13; A17; A18, A19; A20; A21; and A22 are each independently C(R3) or N.

A5; A7; and A14 are each independently N(R3); C(R3)2; O; or S; A15 and A16 are each independently N or C(R3) when --- is a bond; and are each independently N(R3); C(R3)2; O; or S when --- is absent; R1 is optionally substituted alkylene optionally containing one or two heteroatom(s); optionally tuted alkenylene optionally containing one or two heteroatom(s); optionally tuted alkynylene optionally containing one or two heteroatom(s); an oxygen atom; a sulfur atom; or N(R4); R2 is an electron pair; a hydrogen atom; optionally substituted alkyl; optionally substituted alkenyl; optionally substituted alkynyl; ally substituted cycloalkyl; ally substituted cycloalkenyl; optionally substituted aryl; ally tuted heteroaryl; or an optionally substituted non-aromatic heterocyclic group; R3 in each instance is independently a hydrogen atom; a halogen atom; optionally substituted alkyl; optionally substituted alkenyl; optionally substituted alkynyl; optionally substituted cycloalkyl; optionally substituted cycloalkenyl; hydroxy; carboxy; optionally substituted alkyloxy; optionally substituted alkenyloxy; ally substituted alkynyloxy; optionally substituted cycloalkyloxy; optionally substituted cycloalkenyloxy; mercapto; optionally substituted hio; optionally substituted alkenylthio; optionally substituted alkynylthio; optionally substituted alkylsul?nyl; optionally substituted alkylsulfonyl; optionally substituted alkylsulfonyloxy; optionally substituted cycloalkylthio; optionally tuted cycloalkylsul?nyl; optionally tuted cycloalkylsulfonyl; optionally substituted cycloalkylsulfonyloxy; optionally substituted cycloalkenylthio; optionally substituted cycloalkenylsul?nyl; optionally tuted cycloalkenylsulfonyl; optionally substituted cycloalkenylsulfonyloxy; optionally substituted amino; acyl; optionally substituted alkyloxycarbonyl; optionally substituted alkenyloxycarbonyl; optionally substituted loxycarbonyl; optionally tuted aryloxycarbonyl; optionally substituted carbamoyl; ally substituted oyl; cyano; nitro; optionally substituted aryl; optionally substituted aryloxy; optionally substituted arylthio; optionally substituted arylsul?nyl, optionally substituted arylsulfonyl, optionally substituted arylsulfonyloxy, optionally substituted heteroaryl, optionally substituted heteroaryloxy, optionally tuted heteroarylthio, optionally substituted heteroarylsul?nyl, optionally substituted heteroarylsulfonyl, optionally substituted heteroarylsulfonyloxy, or an optionally substituted non-aromatic heterocyclic group; and R4 is a hydrogen atom, optionally substituted alkyl, ally tuted alkenyl, optionally substituted alkynyl, optionally tuted cycloalkyl, acyl, optionally substituted alkyloxy, optionally substituted aryl, optionally substituted heteroaryl, or an optionally substituted non-aromatic heterocyclic group.

In some versions, A1 is C(R3). In some versions, A1 is N. In some versions, A2 is C(R3). In some versions, A2 is N. In some versions, A3 is C(R3). In some ns, A3 is N.

In some versions, A4 is C(R3). In some versions, A4 is N. In some versions, A5 is N(R3). In some ns, A5 is O. In some versions, A6 is C(R3). In some versions, A6 is N. In some versions, A1, A2, and A3 are C(R3), A5 is N(R3), and A6 is N. In some versions, A1, A2, A3, and A4 are C(R3), A5 is N(R3), and A6 is N.

In some ns, Y is: A? /? -$\9>/\?‘3\ In some versions, A7 is N(R3). In some versions, A7 is N(R3), wherein the R3 in the N(R3) is hydrogen or halogen. In some versions, A7 is O. In some versions, A8 is C(R3). In some versions, A8 is N. In some versions, A9 is C(R3). In some versions, A9 is N.

In some versions, Y is: O\ 3/ A10 \T11 £22 A In some versions, A10 is C(R3). In some versions, A10 is N. In some versions, A11 is C(R3).

In some versions, A11 is N. In some versions, A12 is C(R3). In some versions, A12 is N. In some versions, A13 is C(R3). In some ns, A13 is N. In some ns, A", A", and A13 are C(R3). In some versions, A10 is N, and A", A", and A13 are C(R3).

In some versions, Q is piperazine-l,4-diyl or tuted piperazine-l,4-diyl. In some versions, Q is piperazine-l,4-diyl. In some versions, Q is substituted piperazine-l,4- diyl. In some versions, the substituted piperazine-l,4-diyl is: %~ ~% \._ In some versions one or both of R5 and R5, are independently alkyl optionally substituted at one to three position(s) with a substituent independently selected from a halogen atom and alkyloxy, oxo, cycloalkyl, alkenyl, alkynyl, hydroxy, alkyloxy optionally substituted at one to three position(s) with a substituent independently ed from a halogen atom and phenyl optionally substituted with one to three substituent(s) ndently selected from a halogen atom, alkyl, alkyloxy, cyano, and nitro, aryloxy optionally substituted at one to three positions with a tuent independently ed from a halogen atom, alkyl, alkyloxy, cyano, and nitro, mercapto, alkylthio, a halogen atom, nitro, cyano, carboxy, alkyloxycarbonyl, acyl, alkylsulfonyl, optionally substituted amino, optionally substituted oyl, aryl optionally substituted with one to three substituent(s) independently selected from a halogen atom, alkyl, alkyloxy, cyano, and nitro, heteroaryl optionally substituted at one to three position(s) with a substituent independently selected from a halogen atom and alkyl, and non-aromatic heterocyclic group optionally substituted at one to three position(s) with a substituent independently selected from a halogen atom and alkyl. In some ns, one or both of R5 and R5, are independently oxo, hydroxy, or ally substituted alkyloxy. In some versions, one or both of R5 and R5, are oxo. In some ns, R5 and R5, are each oxo.

In some versions, Y' is: \§R1 \ 15 \ If 17 1’16 In some versions, A14 is N(R3). In some versions, A14 is N(R3), wherein the R3 in the N(R3) is hydrogen or halogen. In some versions, A14 is O. In some versions, --- is a bond, and A15 is C(R3). In some versions, --- is a bond, and A15 is N. In some versions, --- is a bond, and A16 is C(R3). In some ns, --- is a bond, and A16 is N. In some versions, --- is absent, and A15 is C(R3)2. In some versions, --- is absent, and A15 is N(R3). In some versions, --- is absent, and A16 is C(R3)2. In some versions, --- is absent, and A16 is - C(H)(Cl-C3 alkyl)-. In some versions, --- is absent, and A16 is N(R3). In some versions, A17 is C(R3). In some versions, A17 is N.

In some versions, Y' is: §R1, A‘S *xA‘Q A22 {LEG \ :4": In some versions, A18 is C(R3). In some versions, A18 is N. In some ns, A19 is C(R3).

In some versions, A19 is N. In some versions, A20 is C(R3). In some versions, A20 is N. In some versions, A21 is C(R3). In some versions, A21 is N. In some versions, A22 is C(R3). In some versions, A22 is N. In some versions, A18, A20, and A22 are C(R3). In some ns, A18, A19, A20, A21, and A22 are C(R3). In some versions, A19 is N. In some versions, A21 is N. In some versions, A19 and A21 are both N. In some versions, A18, A20 and A22 are C(R3), and one or both of A19 and A21 is N. In some versions, A18, A20, and A22 are C(R3), and both of A19 and A21 are N. In some versions, A20 is C(R3), and the R3 on the C(R3) of A20 is any substituent described herein for R3 except hydrogen. In some versions, A20 is C(R3), and the R3 on the C(R3) of A20 is any substituent described herein for R3 except en and halogen. In some versions, A20 is C(R3), and the R3 on the C(R3) of A20 is cyano. In some versions, A18, A19, A21, A22 are each CH, A20 is C(R3), and the R3 on the C(R3) of A20 is cyano.

In some ns, Y' is: In some versions, R2 is an electron pair. In some versions, R2 is a hydrogen atom.

In some versions, R1 is optionally tuted alkylene optionally ning one or two heteroatom(s). In some ns, R1 is optionally substituted alkylene. In some versions, R1 is optionally substituted C1, C2, or C3 alkylene. In some versions, R1 is unsubstituted C1, C2, or C3 alkylene. In some versions, R1 is tituted Cl alkylene.

In some versions, R1 is C1, C2, or C3 alkylene substituted with one or more alkyl groups.

In some versions, R1 is Cl alkylene substituted with one or two alkyl groups. In some versions, R1 is Cl alkylene substituted with two alkyl groups.

In some versions, Z-Y-Q-Y' is: A3XA\\:[ASll2 f A x" E \A1 A ll2 f A f" {’3 \A1 A 4 18 '19 A 5 A:A AT \ A \A2-3 A2 /- f: \22 4-1 \At A' AwA 4 ‘i _ 14 A a 7 Q R A 15 2 / \EQ’Aa / \LlAlB "Km?" A6 O In some versions, Z-Y-Q-Y' is: A 5 l3! TA2 f ' "0*11 A\,A1§ A?g xiA :H??\22 /21 Q is piperazine-l,4-diyl or substituted piperazine-l,4-diyl, A1, A2, A3, A4, A", A12, A13, A18, A20 and A22 are C(R3), A5 is N(R3), A6 and A10 are N, and R1 is 7 optionally substituted alkylene.

In some ns, R3 in each instance, unless otherwise de?ned, is independently a en atom, a halogen atom, Cl-C6 alkyl, hydroxy, methoxy, carboxyl, acetyl, tri?uoromethyl, amino, cycloalkyl, a non-aromatic heterocyclic group, aryl, or heteroaryl.

In some versions the compound is selected from: 23;": .9 O\ x \ o --------4 ; \N———\ \ ("m-w m \\ 1" H /, \ -----x \ ----- ! ****** ’ H I \ /—————\\ //'\\\ 1/»N\ /N:< ‘--\< M ’7****** \\\ // {k [l/M H ,; l LM o w w /> l \ f J & o 0 ~—9 \\/’ N \;....../.

/ \ /\,—\\ ’ \ \\ \?' N - or a salt or ester of any of the foregoing.

Any version described above or elsewhere herein can be combined with any one or more other tually exclusive versions described above or elsewhere herein.

The invention provides methods of treating a condition in an animal with any one or more of the compounds described herein. The methods may comprise administering an effective amount of one or more of the compounds described herein to the animal. The effective amount is an amount effective to treat the condition in the animal. The condition can comprise any one or more of the ions described herein. In some versions, the condition comprises an autoimmune disease. In some versions, the condition comprises an in?ammatory disease. In some versions, the condition comprises a chronic in?ammatory disease. In some versions, the condition comprises in?ammatory bowel disease, such as ulcerative colitis and/or Crohn’s disease. In some versions, the condition comprises diabetes. In some versions, the condition comprises an infectious disease. In some versions, the condition ses lupus, such as systemic lupus erythematosus. In some versions, the condition comprises Sjogren’s syndrome. In some versions, the condition comprises rheumatoid arthritis. In some versions, the condition comprises type 1 diabetes.

In some versions, the condition ses a viral disease such as in?uenza, Zika virus infection, and coronavirus infection. In some versions, the condition comprises oholic steatohepatitis.

In some versions, the condition comprises a roliferative disorder, an inborn error of metabolism, a chronic immunometabolic disease, organ transplant rejection, and/or chronic pain. In some versions, the hyperproliferative disorder comprises cancer. In some versions, the cancer comprises a cancer of the gastrointestinal tract. In some versions, the cancer of the gastrointestinal tract comprises colorectal cancer. In some versions, the hyperproliferative disorder comprises al atous sis. In some versions, the inborn error of metabolism comprises a glycogen storage disease. In some ns, the en storage e comprises en disease. In some versions, the chronic immunometabolic disease comprises cardiovascular disease. In some versions, the cardiovascular disease comprises sclerosis. In some versions, the chronic immunometabolic disease comprises hypertension. In some versions, the autoimmune disease comprises a cancer-immunotherapy-induced autoimmune disease. In some versions, the cancer-immunotherapy-induced autoimmune disease comprises a cancer immunotherapy-induced rheumatic disease. In some versions, the atory er comprises acute colonic diverticulitis. In some versions, the in?ammatory disorder comprises radiation-induced in?ammation of the gastrointestinal tract. In some versions, the radiation-induced in?ammation of the gastrointestinal tract comprises at least 3O one of radiation proctitis, ion enteritis, and radiation proctosigmoiditis. In some versions, the chronic pain comprises fibromyalgia.

The invention also es methods of generating a prepared cell from a precursor cell with the compounds described herein. The methods may comprise contacting the precursor cell, in vilro, with one or more of the compounds described herein to te the prepared cell. The precursor cell can be contacted with the compound in an amount and for a time effective to induce a compound-dependent difference in the prepared cells with respect to the precursor cells. In some versions, the contacting comprises contacting the precursor cells with the compound and an agent comprising one or more of all-trans- retinoic acid, TGF-B, phorbol myristate acetate, ionomycin, rapamycin, and IL-2. In some versions, the precursor cells se immune cells. In some versions, the precursor cells comprise white blood cells. In some versions, the precursor cells se cells selected from the group consisting of peripheral blood mononuclear cells and lamina propria mononuclear cells. In some versions, the sor cells se T cells. In some versions, the precursor cells comprise na'1've CD4+ T cells. In some versions, the prepared cells comprise Treg cells. In some ns, the prepared cell is differentiated from the precursor cell. In some versions, the nd-dependent difference comprises a difference in gene expression in the prepared cells with respect to the precursor cells. In some versions, the compound-dependent difference comprises at least one of an increase in expression of IL-10 or an ortholog thereof, an se in expression of FOXP3 or an ortholog thereof, a decrease in expression of TNFOL or an ortholog f, a decrease in expression of IFNy or an ortholog thereof, a decrease in expression of Tbet or an ortholog thereof, an increase in expression ofLag3 or an ortholog thereof, an se in expression of Socs2 or an ortholog thereof, an increase in sion of Irf7 or an ortholog thereof, an increase in expression of P2rx7 or an ortholog thereof, an increase in expression of Capn3 or an ortholog thereof, an increase in expression of Isz2 or an og thereof, an increase in expression of Slal5a or an ortholog thereof, an increase in expression of Plen or an ortholog f, an increase in expression of Foxo] or an ortholog thereof, an increase in expression of Phlpp] or an ortholog thereof, an increase in phosphorylation of STATSa or an ortholog thereof, an increase in FOXOl phosphorylation or an ortholog thereof, and an increase in te kinase activity.

The invention also es isolated cells generated by contacting a precursor cell, in vilro, with one or more of the compounds described herein to generate a prepared cell.

The invention also provides methods of treating a condition in an animal with a prepared cell as described herein. The methods comprise administering the prepared cell to 3O the animal in an amount suf?cient to treat the condition. The condition can comprise any condition described above or ere herein.

The objects and advantages of the invention will appear more fully from the following detailed description of the preferred ment of the invention made in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE GS FIGS. 1A-1C. Computational prediction of binding of ed compounds to LANCL2 in kcal/mol.

Pharrnacokinetics of BT-63 after oral dosing with 10 and 40 mg/kg. logical validation of BT-63 activity in CD4+ splenocytes.

Percentages of IFNy+ and ILlO+ FOXP3+ CD4+ T cells were measured by ?ow , TNFOH‘, cytometry after in vitro treatment of cells with BT-63 at concentrations of 0.1, 1 and 10 uM in wild type and LANCL2 de?cient cells. Statistical signi?cance (p < 0.05) is marked by asterisks.

FIGS. 4A and 4B. Activity of compounds in CD4+ splenocytes. Percentages of IFNy+ and FOXP3+ CD4+ T cells were measured by ?ow cytometry after in vitro treatment of cells with BT-63 and BT-62 at concentrations of 0.1, 1 and 10 micromolar (FIG 4A). Percentages of IFNy+ and TNF+ CD4+ T cells were measured by ?ow cytometry after in vitro ent of cells with BTA, BTB and BTC at a trations of 0.1 micromolar (). Statistical signi?cance (p < 0.05) is marked by asterisks.

Ef?cacy of BT-63 in a NOD model of T1D. Weekly blood glucose and percent hyperglycemic monitoring of NOD mice treated with vehicle or 10 or 20 mg/kg BT-63 daily.

Immune effects of BT-63 in vivo in a NOD model of type 1 diabetes (TlD). Ratio of splenic ILlO producing cells to IFNy producing cells, c BCL6+ ILZl+ CD4+ T helper cells and CD4+ PD1+ T cells at 21 weeks of age with oral treatment of vehicle or 10 or 20 mg/kg BT-63 daily.

Plasma biomarker evaluation of BT-63 in a NOD model of T1D. Plasma concentrations of C-peptide, HbAlc, MCP-l, and TNF at 21 weeks of age with oral treatment of vehicle or 10 or 20 mg/kg BT-63 daily. y of BT-63 in a TLR7/9 induced model of SLE. Plasma antibodies to double-stranded DNA, urine n levels, spleen CD8+ IFNy+ and IL6+ CD45+ cells after two weeks of oral treatment with vehicle or 20 mg/kg BT-63.

Ef?cacy of BT-63 in an MDRla-/— model of IBD. Colonic lamina propria Th1, Th17, CD25+ FOXP3+ CD4+ Tregs, and neutrophils after four weeks of oral treatment with vehicle or 20 mg/kg BT-63.

. Reduction of ic in?ammation in Mdrla-/- mice treated with BT-63.

Splenic Th1 and CD25+ Tregs after four weeks of oral treatment with vehicle or 20 mg/kg BT-63.

. Ef?cacy of BT-63 in a mouse model of in?uenza A virus infection. al and disease activity index over 12 days post-infection with in?uenza A (H1N1) with daily oral ent with vehicle or 20 mg/kg BT-63.

. Modulation of lung immune responses to in?uenza A infection by BT-63.

Lung lLlO+ CD8+ T cells, alveolar macrophages, CD4+ TNF+ T cells and neutrophils at day 12 post-infection with daily oral treatment with e or 20 mg/kg BT-63.

. Ef?cacy of BT-63 in an NZB/W F1 model of SLE. Body weight changes from baseline over 12 weeks of vehicle or 20 mg/kg BT-63 treatment. Urine protein score, plasma anti-dsDNA antibodies and plasma IFN—d concentration after 12 weeks of oral treatment with vehicle or BT-63 (20 mg/kg).

. Immunological responses to BT-63 in an NZB/W F1 model. Splenic CD19+ IgD+ Ing" follicular B cells, CD138hi CD191° MHCII10 plasma cells, CXCR3-- plasma cells, IL6+ CD11b+ CD11c+ myeloid cells, CD4+ CD25+ FOXP3+ Tregs, CD4-- ILZl+ BCL6+ follicular helper T cells after 12 weeks of oral ent with vehicle or BT-63 (20 mg/kg).

. Additional exemplary compounds of the invention (BTA, BT B, BTC) that bind to LANCL2 and that can be used in any method described herein.

The conversion of the piperazine in BT-63 to piperazine-2,6-dione, as present in BT A, or the addition of the gem-dimethyl or nitrile groups to BT-63, as present in BTB 2O or -C, respectively, serve to improve the systemic half-life of the target compound through the improvement of the resistance of the methylene group in BT-63 to enzymatic metabolism.

FIGS. 16A and 16B. In vivo validation of BTB y in a CCl4 model of nonalcoholic hepatitis. Liver weight (A) and ?brosis score (B) after 4 weeks of bi-weekly carbon tetrachloride ions in vehicle and BTB (20 mg/kg) treated mice. Statistical signi?cance (P < 0.05) is marked by asterisks.

FIGS. 17A-17C. In vivo validation of -B y in a collagen induced model of arthritis. Percentages of ILl7+ (A), IFNy+ CD4+ T cells (B), and TNF+ CD11b+ CD11c+ d cells (C) in the spleens of collagen induced arthritis mice after 4 weeks of daily oral treatment with vehicle or BTB (20 mg/kg).

Statistical signi?cance (P < 0.05) is marked by asterisks.

FIGS. 18A-18C. In vivo validation of BTB ef?cacy in an NZB/W F1 model of SLE. Percentages of ILl7+ (A), IL21+ (B), and CD25+ FOXP3+ CD4+ T cells (C) in the spleens ofNZB/W F1 mice after 12 weeks of daily oral treatment with vehicle or BTB (20 mg/kg). Statistical signi?cance (P < 0.05) is marked by asterisks.

DETAILED DESCRIPTION OF THE INVENTION Unless otherwise stated, the following de?nitions are used throughout the present application: Analysis of Variance (ANOVA): Arithmetic process for partitioning the overall variation in data sets into speci?c components based on sources of variation. It has been used to determine whether numerical differences between treatment groups are statistically signi?cant.

Conjugated diene: A molecule containing two double bonds separated by a single bond.

Enantiomer: Optical ; chemical classi?cation of molecules based on their ability to rotate the plain of polarization clockwise (+) or anti-clockwise (—).

Substantially pure: Having a purity of at least 90% by weight, preferably at least 95% by weight such as at least 98%, 99% or about 100% by weight.

IBD: In?ammatory bowel disease (IBD) involves chronic in?ammation of all or part of your digestive tract. IBD primarily includes ulcerative colitis and Crohn's disease.

Both usually involve severe diarrhea, pain, fatigue and weight loss. IBD can be tating and mes leads to life-threatening complications. tive colitis (UC): UC is an IBD that causes long-lasting ation and sores (ulcers) in the innermost lining of your large intestine (colon) and rectum.

Crohn's Disease: s disease is an IBD that cause ation of the lining of your digestive tract. In Crohn's disease, in?ammation often spreads deep into affected tissues.

The in?ammation can involve different areas of the digestive tract—the large intestine, small intestine or both. lL-lO: eukin-10 (IL-10), also known as human cytokine synthesis inhibitory factor (CSIF), is an anti-in?ammatory cytokine. In , lL-lO is encoded by the lLlO gene.

FOXP3: FOXP3 ead box P3) also known as scur?n is a protein involved in immune system responses. A member of the FOX protein family, FOXP3 appears to function as a master regulator (transcription ) in the development and function of regulatory T cells.

TNF-alpha: Tumor is factor (TNF, cacheXin, or cachectin, and formerly known as tumor necrosis factor alpha or TNFOL) is cytokine involved in systemic in?ammation and is a member of a group of cytokines that stimulate the acute phase reaction.

MCPl: Monocyte chemoattractant protein-l. An older term for a CC cytokine which is critical for development of sclerotic lesions, found in elial cells, macrophages and in vascular smooth muscle cells of patients undergoing coronary artery bypass procedures. The of?cially preferred term is now chemokine (C-C motif) ligand 2.

Interferon gamma: eron gamma is a pro-in?ammatory dimerized e cytokine that is the only member of the type 11 class of interferons.

Leukocytic in?ltration: Leukocyte in?ltration refers to the process of moving or ating of the leukocytes into the injured tissue to begin the repair process.

Type 1 diabetes: An mune disease terized as a chronic condition in which the pancreas produces little to no insulin as a result of immunological destruction of insulin-producing beta cells within pancreatic islets. The insulin de?ciency leads to chronic hyperglycemia that can cause organ damage, ned an and reduced quality of life. The disease is also referred to as le diabetes or insulin-dependent diabetes.

Systemic lupus erythematosus: An autoimmune disease in which the immune system reacts to nuclear antigens and forms immune complexes that can aggregate or cause damage to multiple organ systems ing skin, joints, kidneys, brain, the heart 2O and cardiovascular systems and other organs.

The term "halogen atom" refers to a ?uorine atom, a chlorine atom, a e atom and an iodine atom. A ?uorine atom, a chlorine atom, and a bromine atom are preferred.

The term "hetero atom" refers to an oxygen atom, a sulfur atom, and a nitrogen atom.

The term "alkyl" includes a monovalent straight or branched hydrocarbon group having one to eight carbon atom(s). Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, yl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neo-pentyl, n-hexyl, isohexyl, n-heptyl, n-octyl, and the like. C1-C6 alkyl is preferred. Cl-C4 alkyl or Cl-C3 alkyl is 3O further preferred. When a number of carbons is speci?ed, it means "alkyl" having the carbon number within the range.

The term "alkenyl" includes a monovalent straight or branched hydrocarbon group having two to eight carbon atoms and one or more double bond(s). Examples e vinyl, allyl, l-propenyl, 2-butenyl, 2-pentenyl, 2-hexenyl, 2-heptenyl, 2-octenyl, and the like. C2-C6 alkenyl is preferred. C2-C4 alkenyl is further preferred.

The term "alkynyl" includes a monovalent straight or branched hydrocarbon group having two to eight carbon atoms and one or more triple bond(s). Examples e ethynyl, l-propynyl, 2-propynyl, 2-butynyl, ynyl, 2-hexynyl, 2-heptynyl, nyl, and the like. C2-C6 alkynyl is preferred. C2-C4 alkynyl is further preferred.

The term "cycloalkyl" includes a cycloalkyl having three to eight carbon atoms.

Examples e cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like. C3-C6 cycloalkyl is preferred.

The term "cycloalkenyl" includes a cycloalkenyl having three to eight carbon atoms. Examples include cyclopropenyl, utenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cycloocentyl, and the like. C3-C6 cycloalkenyl is preferred.

The term oxy" includes a group wherein an oxygen atom is substituted with one "alkyl" as described herein. Examples include methyloxy, ethyloxy, n-propyloxy, isopropyloxy, n-butyloxy, isobutyloxy, sec-butyloxy, tert-butyloxy, n-pentyloxy, isopentyloxy, 2-pentyloxy, 3-pentyloxy, n-hexyloxy, isohexyloxy, 2-hexyloxy, 3- hexyloxy, n-heptyloxy, n-octyloxy, and the like. Cl-C6 alkyloxy is preferred. Cl-C4 alkyloxy or Cl-C3 alkyloxy is further preferred. When a number of carbons is speci?ed, it means "alkyloxy" having the carbon number within the range.

The term "alkenyloxy" includes a group wherein an oxygen atom is substituted with one "alkenyl" as described herein. Examples e xy, allyloxy, l- propenyloxy, 2-butenyloxy, 2-pentenyloxy, 2-hexenyloxy, 2-heptenyloxy, 2-octenyloxy, and the like. C2-C6 alkenyloxy is preferred. Moreover, C2-C4 alkenyloxy is r preferred. When a number of carbons is speci?ed, it means "alkenyloxy" having the carbon number within the range.

The term "alkynyloxy" includes a group wherein an oxygen atom is tuted with one "alkynyl" as described herein. Examples include ethynyloxy, l-propynyloxy, 2- propynyloxy, 2-butynyloxy, 2-pentynyloxy, 2-hexynyloxy, 2-heptynyloxy, 2-octynyloxy, and the like. C2-C6 alkynyloxy is preferred. C2-C4 alkynyloxy is further preferred. When a number of carbons is speci?ed, it means "alkynyloxy" having the carbon number within the range.

The term "cycloalkyloxy" es a group wherein an oxygen atom is tuted with one "cycloalkyl" as described herein. Examples include cyclopropyloxy, utyloxy, cyclopentyloxy, cyclohexyloxy, cycloheptyloxy, and cyclooctyloxy. C3- C6 cycloalkyloxy is preferred. When a number of carbons is speci?ed, it means "cycloalkyloxy" having the carbon number within the range.

The term "cycloalkenyloxy" es a group wherein an oxygen atom is substituted with one "cycloalkenyl" as bed herein. Examples include cyclopropenyloxy, cyclobutenyloxy, cyclopentenyloxy, cyclohexenyloxy, cycloheptenyloxy, and cyclooctenyloxy. C3-C6 cycloalkenyloxy is preferred. When a number of carbons is speci?ed, it means "cycloalkenyloxy" having the carbon number within the range.

The term "alkylthio" es a group wherein a sulfur atom is substituted with one "alkyl" as described herein. Examples include methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, isobutylthio, sec-butylthio, tert-butylthio, n-pentylthio, isopentylthio, 2-pentylthio, 3-pentylthio, n-hexylthio, isohexylthio, 2-hexylthio, 3- hio, n-heptylthio, n-octylthio, and the like. Cl-C6 Alkylthio is preferred. Cl-C4 alkylthio is further preferred. When a number of carbons is speci?ed, it means "alkylthio" haVing the carbon number within the range.

The term "alkenylthio" includes a group wherein a sulfur atom is substituted with one "alkenyl" as described herein. Examples include Vinylthio, allylthio, l-propenylthio, 2-butenylthio, 2-pentenylthio, 2-hexenylthio, 2-heptenylthio, 2-octenylthio, and the like.

C2-C6 lthio is preferred. C2-C4 alkylthio is further preferred. When a number of s is speci?ed, it means "alkenylthio" haVing the carbon number within the range.

The term "alkynylthio" includes a group wherein a sulfur atom is substituted with one "alkynyl" as described herein. Examples include ethynylthio, ynylthio, 2- propynylthio, 2-butynylthio, 2-pentynylthio, 2-hexynylthio, 2-heptynylthio, 2-octynylthio, and the like. C2-C6 alkynylthio is preferred. C2-C4 lthio is further preferred. When a number of carbons is speci?ed, it means "alkynylthio" haVing the carbon number within the range.

The term "alkylsulfmyl" includes a group wherein sulf1nyl is substituted with one "alkyl" as described herein. es include methylsul?nyl, ul?nyl, n- propylsulf1nyl, pylsulf1nyl, n-butylsulf1nyl, isobutylsulf1nyl, sec-butylsulf1nyl, tert- butylsulflnyl, n-pentylsulf1nyl, isopentylsulf1nyl, 2-pentylsulf1nyl, 3-pentylsulf1nyl, n- hexylsulf1nyl, isohexylsulf1nyl, 2-hexylsulf1nyl, 3-hexylsulf1nyl, n-heptylsulf1nyl, n- 3O octylsulflnyl, and the like. Cl-C6 alkylsulf1nyl is preferred. Cl-C4 ulf1nyl is further preferred.

The term "alkylsulfonyl" es a group n sulfonyl is substituted with one "alkyl" as described herein. Examples include methylsulfonyl, ethylsulfonyl, n- propylsulfonyl, isopropylsulfonyl, n-butylsulfonyl, isobutylsulfonyl, sec-butylsulfonyl, tert-butylsulfonyl, n-pentylsulfonyl, isopentylsulfonyl, ylsulfonyl, 3-pentylsulfonyl, n-hexylsulfonyl, isohexylsulfonyl, 2-hexylsulfonyl, 3-hexylsulfonyl, n-heptylsulfonyl, n- octylsulfonyl, and the like. C1-C6 alkylsulfonyl is preferred. Cl-C4 alkylsulfonyl is further preferred.

The term sulfonyloxy" includes a group n an oxygen atom is substituted with one "alkylsulfonyl" as described herein. Examples include methylsulfonyloxy, ethylsulfonyloxy, n-propylsulfonyloxy, isopropylsulfonyloxy, n- butylsulfonyloxy, isobutylsulfonyloxy, sec-butylsulfonyloxy, tert-butylsulfonyloxy, n- pentylsulfonyloxy, isopentylsulfonyloxy, 2-pentylsulfonyloxy, 3-pentylsulfonyloxy, n- hexylsulfonyloxy, isohexylsulfonyloxy, 2-hexylsulfonyloxy, 3-hexylsulfonyloxy, n- heptylsulfonyloxy, n-octylsulfonyloxy, and the like. Cl-C6 alkylsulfonyl is preferred. C1- C4 alkylsulfonyl is further preferred.

The term "cycloalkylthio" includes a group wherein a sulfur atom is substituted with one "cycloalkyl" as described herein. Examples e cyclopropylthio, utylthio, cyclopentylthio, cyclohexylthio, cycloheptylthio, cyclooctylthio, and the like. C3-C6 cycloalkylthio is preferred. When a number of s is specified, it means "cycloalkylthio" haVing the carbon number within the range.

The term "cycloalkylsulfinyl" includes a group in which sulf1nyl is substituted with one "cycloalkyl" as described . Examples include cyclopropylsulf1nyl, cyclobutylsulf1nyl, cyclopentylsulf1nyl, cyclohexylsul?nyl, eptylsulf1nyl, and cyclooctylsulf1nyl are exempli?ed. Preferably C3-C6 cycloalkylsulfmyl.

The term "cycloalkylsulfonyl" includes a group in which sulfonyl is substituted with one "cycloalkyl" as described herein. Examples include cyclopropylsulfonyl, cyclobutylsulfonyl, entylsulfonyl, cyclohexylsulfonyl, cycloheptylsulfonyl, and cyclooctylsulfonyl. C3-C6 cycloalkylsulfonyl is preferred.

The term "cycloalkylsulfonyloxy" includes a group in which an oxygen atom is substituted with one alkylsulfonyl" as described herein. Examples include ropylsulfonyloxy, cyclobutylsulfonyloxy, cyclopentyl sulfonyloxy, cyclohexyl sulfonyloxy, cycloheptylsulfonyloxy, and cyclooctylsulfonyloxy. C3-C6 cycloalkylsulfonyloxy is preferred.

The term "cycloalkenylthio" includes a group in which a sulfur atom is tuted with one "cycloalkenyl" as described herein. Examples include cyclopropenylthio, cyclobutenylthio, cyclopentenylthio, cyclohexenylthio, cycloheptenylthio, and cyclooctenylthio. C3-C6 cycloalkenylthio is preferred. When a number of s is speci?ed, it means "cycloalkenylthio" haVing the carbon number within the range.

The term "cycloalkenylsul?nyl" includes a group in which sulf1nyl is substituted with one "cycloalkenyl" as described herein. Examples include ropenylsulf1nyl, cyclobutenylsulf1nyl, cyclopentenylsulfinyl, cyclohexenylsul?nyl, eptenylsulf1nyl, and cyclooctenylsulf1nyl. C3-C6 cycloalkenylsul?nyl is preferred.

The term "cycloalkenylsulfonyl" includes a group in which sulfonyl is substituted with one "cycloalkenyl" as described herein. Examples include cyclopropenylsulfonyl, cyclobutenylsulfonyl, cyclopentenylsulfonyl, cyclohexenylsulfonyl, cycloheptenylsulfonyl, and cyclooctenylsulfonyl. Preferably C3-C6 cycloalkenylsulfonyl is preferred.

The term "cycloalkenylsulfonyloxy" es a group in which an oxygen atom is substituted with one "cycloalkenylsulfonyl" described as bed herein. Examples include cyclopropenylsulfonyloxy, cyclobutenylsulfonyloxy, entenylsulfonyloxy, cyclohexenylsulfonyloxy, cycloheptenylsulfonyloxy, and ctenylsulfonyloxy. C3-C6 cycloalkenylsulfonyloxy is preferred.

The term "alkyloxycarbonyl" includes a group in which carbonyl is substituted with one "alkyloxy" as bed herein. Examples e methyloxycarbonyl, ethyloxycarbonyl, n-propyloxycarbonyl, isopropyloxycarbonyl, n-butyloxycarbonyl, tert- butyloxycarbonyl, and n-pentyloxycarbonyl. C1-C6 or Cl-C4 alkyloxycarbonyl is preferred. Cl-C2 alkyloxycarbonyl is further preferred. 2O The term "alkenyloxycarbonyl" includes a group in which carbonyl is substituted with one "alkenyloxy" as described herein. Examples include Vinyloxycarbonyl, xycarbonyl, l-propenyloxycarbonyl, 2-butenyloxycarbonyl, and 2- yloxyarbonyl. C2-C6 or C2-C4 alkyloxycarbonyl is preferred.

The term "alkynyloxycarbonyl" includes a group in which carbonyl is substituted with one "alkynyloxy" as described herein. Examples include ethynyloxycarbonyl, l- propynyloxycarbonyl, 2-propynyloxycarbonyl, 2-butynyloxyarbonyl, and 2- pentynyloxycarbonyl. C2-C6 or C2-C4 alkynyloxycarbonyl is red.

The term "acyl" includes alkylcarbonyl n the part of alkyl is "alkyl" as described herein, alkenylcarbonyl wherein the part of alkenyl is "alkenyl" as described 3O herein, alkynylcarbonyl wherein the part of alkynyl is "alkynyl" as described herein, cycloalkylcarbonyl wherein the part of cycloalkyl is "cycloalkyl" as described herein, arylcarbonyl wherein the part of aryl is "aryl" as described herein, heteroarylcarbonyl wherein the part of heteroaryl is oaryl" as described herein, and non-aromatic heterocycliccarbonyl n the part of non-aromatic cyclic group is "non- aromatic heterocyclic group" as described herein. "Alkyl," "alkenyl," "alkynyl," "cycloalkyl," "aryl," "heteroaryl," and (Lnon-aromatic heterocyclic group" may be substituted respectively with substituent groups i?ed in nally substituted alkyl," "optionally substituted alkenyl," nally substituted alkynyl," "optionally substituted cycloalkyl," "optionally substituted aryl," "optionally substituted heteroaryl," and (L optionally substituted non-aromatic heterocyclic group" as described herein.

Examples of the acyl group include acetyl, propionyl, butyroyl, cyclohexylcarbonyl, benzoyl, pyridinecarbonyl, and the like.

The term "optionally substituted amino" es an amino group which may be substituted with one or two group(s) of "alkyl" as described , "alkenyl" as described herein, yl" as described herein, "cycloalkyl" as described herein, "cycloalkynyl" as described herein, "aryl" as described herein, "heteroaryl" as described herein, "acyl" as described herein, "alkyloxycarbonyl" as described herein, "alkenyloxycarbonyl" as bed herein, "alkynyloxycarbonyl" as described herein, "alkyl sulfonyl," "alkenylsulfonyl," "alkynylsulfonyl," L ‘arylsulfonyl," and/or "heteroarylsulfonyl" as described herein. Examples of the ally substituted amino group include amino, methylamino, ylamino, ethylamino, diethylamino, ethylmethylamino, benzylamino, acetylamino, lamino, methyloxycarbonylamino, and methanesulfonylamino.

Amino, methylamino, dimethylamino, ethylmethylamino, diethylamino, acetylamino, and methanesulfonylamino are preferred.

The term "optionally substituted carbamoyl" includes an aminocarbonyl group wherein the part of optionally substituted amino is "optionally substituted amino" as described herein. Examples of the optionally substituted carbamoyl group includes carbamoyl, N—methylcarbamoyl, methylcarbamoyl, N—ethyl-N-methylcarbamoyl, N,N—diethylcarbamoyl, N—phenylcarbamoyl, N—benzylcarbamoyl, N—acetylcarbamoyl, and ylsulfonylcarbamoyl etc. Carbamoyl, N—methylcarbamoyl, N,N— dimethylcarbamoyl, and N—methylsulfonylcarbamoyl etc. are preferred.

The term "optionally substituted sulfamoyl" includes an aminosulfonyl group wherein the part of optionally tuted amino is "optionally substituted amino" as described herein. Examples of the optionally tuted sulfamoyl group e 3O sulfamoyl, N—methylsulfamoyl, N,N—dimethylsulfamoyl, N—ethyl-N-methyl sulfamoyl, N,N—diethylsulfamoyl, N—phenylsulfamoyl, N—benzylsulfamoyl, N—acetylsulfamoyl, and N—methylsulfonylsulfamoyl etc. Sulfamoyl, N—methylsulfamoyl, N,N—dimethylsulfamoyl, and ylsulfonylsulfamoyl etc. are preferred.

The term "alkylene" means a straight or branched alkylene group haVing one to eight carbon atom(s). Examples include methylene, ne, l-methylethylene, trimethylene, l-methyltrimethylene, pentamethylene, thylene, and the like. C1-C4 or Cl-3 alkylenes are preferred. Cl-C2 alkylene is further preferred.

The term "aryl" includes an aromatic monocyclic or aromatic fused cyclic hydrocarbons. It may be fused with "cycloalkyl" as bed herein, "cycloalkenyl" as described herein or "non-aromatic heterocyclic group" as described herein at any possible position. Both of monocyclic ring and fused ring may be tuted at any position.

Examples include phenyl, l-naphthyl, 2-naphthyl, anthryl, tetrahydronaphthyl, l,3- benzodioxolyl, l,4-benzodioxanyl etc. Phenyl, thyl, and 2-naphthyl are preferred.

Phenyl is further preferred.

The term romatic heterocyclic group" es a 5- to 7-membered non- aromatic heterocyclic ring ning one or more of heteroatom(s) selected independently from oxygen, sulfur, and nitrogen atoms or a multicyclic ring formed by fusing the two or more rings thereof. Examples include pyrrolidinyl (e.g., l-pyrrolidinyl, 2-pyrrolidinyl), pyrrolinyl (e.g., 3-pyrrolinyl), imidazolidinyl (e.g., 2-imidazolidinyl), imidazolinyl (e.g., imidazolinyl), pyrazolidinyl (e.g., l-pyrazolidinyl, zolidinyl), pyrazolinyl (e.g., pyrazolinyl), piperidyl (e.g., piperidino, 2-piperidyl), piperazinyl (e.g., l-piperazinyl), indolinyl (e.g., l-indolinyl), isoindolinyl (e.g., isoindolinyl), morpholinyl (e.g., morpholino, 3-morpholinyl) etc.

The term "heteroaryl" includes a 5- to 6-membered aromatic ring containing one or more of heteroatom(s) selected independently from oxygen, sulfur, and nitrogen atoms. It may be fused with "cycloalkyl" as described herein, "aryl" as described herein, "non- aromatic cyclic group" as bed herein, or other heteroaryl at any le position. The heteroaryl group may be substituted at any on whenever it is a clic ring or a fused ring. Examples include pyrrolyl (e.g., l-pyrrolyl, 2-pyrrolyl, 3- pyrrolyl), furyl (e.g., 2-furyl, 3-furyl), thienyl (e.g., 2-thienyl, 3-thienyl), imidazolyl (e.g., 2-imidazolyl, 4-imidazolyl), pyrazolyl (e.g., l-pyrazolyl, 3-pyrazolyl), isothiazolyl (e.g., 3-isothiazolyl), isoxazolyl (e.g., 3-isoxazolyl), oxazolyl (e.g., olyl), thiazolyl (e.g., 2-thiazolyl), pyridyl (e.g., 2-pyridyl, 3-pyridyl, 4-pyridyl), pyrazinyl (e.g., zinyl), pyrimidinyl (e.g., 2-pyrimidinyl, 4-pyrimidinyl), zinyl (e.g., 3-pyridazinyl), 3O tetrazolyl (e.g., lH-tetrazolyl), oxadiazolyl (e.g., l,3,4-oxadiazolyl), thiadiazolyl (e.g., l,3,4-thiadiazolyl), indolidinyl (e.g., 2-indolidinyl, 6-indolidinyl), isoindolynyl (e.g., 2- isoindolynyl), indolyl (e.g., l-indolyl, lyl, 3-indolyl), indazolyl (e.g., 3-indazolyl), purinyl (e.g., 8-purinyl), quinolidinyl (e.g., olidinyl), isoquinolyl (e.g., 3- isoquinolyl), quinolyl (e.g., 2-quinolyl, 5-quinolyl), phtharazinyl (e.g., l-phtharazinyl), naphthylidinyl (e.g., 2-naphthylidinyl), quinolanyl (e.g., 2-quinolanyl), quinazolinyl (e.g., 2-quinazolinyl), inyl (e.g., 3-cinnolinyl), pteridinyl (e.g., 2-pteridinyl), carbazolyl (e.g., 2-carbazolyl, 4-carbazolyl), phenanthridinyl (e.g., 2-phenanthridinyl, 3- phenanthridinyl), acridinyl (e.g., l-acridinyl, 2-acridinyl), dibenzofuranyl (e.g., l- dibenzofuranyl, 2-dibenzofuranyl), midazolyl (e.g., 2-benzoimidazolyl), benzoisoxazolyl (e.g., 3-benzoisoxazolyl), benzooxazolyl (e.g., 2-benzooxazolyl), benzooxadiazolyl (e.g., 4-benzooxadiazolyl), benzoisothiazolyl (e.g., 3-benzoisothiazolyl), hiazolyl (e.g., 2-benzothiazolyl), benzofuryl (e.g., 3-benzofuryl), benzothienyl (e.g., 2-benzothienyl), dibenzothienyl (e.g., 2-dibenzothienyl), and benzodioxolyl (e.g., 1,3- benzodioxolyl), etc.

The term "aryloxy" includes a group in which an oxygen atom is substituted with one "aryl" as described . Examples include phenyloxy and naphthyloxy, etc.

The term hio" includes a group in which a sulfur atom is substituted with one "aryl" as described herein. Examples include phenylthio and naphthylthio, etc.

The term "arylsul?nyl" includes a group in which sul?nyl is tuted with one "aryl" as described herein. Examples include phenylsul?nyl and naphthylsul?nyl, etc.

The term "arylsulfonyl" includes a group in which sulfonyl is substituted with one "aryl" as described herein. es e phenylsulfonyl and naphthylsulfoinyl, etc. es of "arylsulfonyloxy" include phenylsulfonyloxy and naphthylsulfonyloxy, etc. 2O The term "aryloxycarbonyl" includes a group in which carbonyl is substituted with one xy" as described herein. Examples include phenyloxycarbonyl, l- naphthyloxycarbonyl and 2-naphthyloxycarbonyl, etc.

The term "heteroaryloxy" es a group in which an oxygen atom is substituted with one "heteroaryl" as described herein. Examples e pyrrolyloxy, furyloxy, loxy, imidazolyloxy, pyrazolyloxy, isothiazolyloxy, isoxazolyloxy, oxazolyloxy, thiazolyloxy, pyridyloxy, pyrazinyloxy, pyrimidinyloxy, pyridazinyloxy, tetrazolyloxy, oxadiazolyloxy, thiadiazolyloxy, indolidinyloxy, isoindolynyloxy, indolyloxy, indazolyloxy, purinyloxy, quinolidinyloxy, nolyloxy, quinolyloxy, phtharazinyloxy, naphthylidinyloxy, quinolanyloxy, quinazolinyloxy, cinnolinyloxy, pteridinyloxy, 3O carbazolyloxy, phenanthridinyloxy, acridinyloxy, dibenzofuranyloxy, benzoimidazolyloxy, benzoisoxazolyloxy, benzooxazolyloxy, benzooxadiazolyloxy, benzoisothiazolyloxy, benzothiazolyloxy, benzofuryloxy, benzothienyloxy, dibenzothienyloxy, and benzodioxolyloxy are exempli?ed. Preferably furyloxy, thienyloxy, imidazolyloxy, pyrazolyloxy, azolyloxy, isoxazolyloxy, oxazolyloxy, thiazolyloxy, pyridyloxy, pyrazinyloxy, pyrimidinyloxy, and pyridazinyloxy, etc.

The term "heteroarylthio" includes a group in which a sulfur atom is substituted with one "heteroaryl" as described herein. Examples include pyrrolylthio, furylthio, thienylthio, imidazolylthio, pyrazolylthio, isothiazolylthio, isoxazolylthio, oxazolylthio, thiazolylthio, lthio, pyrazinylthio, pyrimidinylthio, pyridazinylthio, tetrazolylthio, oxadiazolylthio, azolylthio, indolidinylthio, isoindolynylthio, indolylthio, indazolylthio, purinylthio, idinylthio, isoquinolylthio, quinolylthio, phtharazinylthio, naphthylidinylthio, quinolanylthio, quinazolinylthio, cinnolinylthio, pteridinylthio, carbazolylthio, phenanthridinylthio, acridinylthio, dibenzofuranylthio, benzoimidazolylthio, benzoisoxazolylthio, benzooxazolylthio, benzooxadiazolylthio, benzoisothiazolylthio, benzothiazolylthio, benzofurylthio, benzothienylthio, dibenzothienylthio, and benzodioxolylthio etc. are exempli?ed. Preferably furylthio, thienylthio, imidazolylthio, pyrazolylthio, isothiazolylthio, isoxazolylthio, oxazolylthio, thiazolylthio, pyridylthio, pyrazinylthio, pyrimidinylthio, and pyridazinylthio, etc.

The term oarylsulfinyl" includes a group in which yl is substituted with one "heteroaryl" as described herein. Examples include pyrrolylsulfmyl, furylsul?nyl, lsulf1nyl, imidazolylsulflnyl, pyrazolylsulflnyl, isothiazolylsulflnyl, isoxazolylsulfmyl, oxazolylsulflnyl, thiazolylsulflnyl, pyridylsulflnyl, pyrazinylsulflnyl, pyrimidinylsulflnyl, pyridazinylsulflnyl, tetrazolylsulflnyl, oxadiazolylsulflnyl, azolylsulflnyl, indolidinylsulf1nyl, isoindolylsulf1nyl, indolylsulf1nyl, indazolylsulflnyl, purinylsulf1nyl, quinolidinylsulflnyl, isoquinolylsulflnyl, quinolylsulfinyl, phtharazinylsulflnyl, naphthylidinylsulflnyl, quinolanylsulflnyl, olinylsulflnyl, cinnolinylsulf1nyl, pteridinylsulf1nyl, carbazolylsulflnyl, phenanthridinylsulf1nyl, acridinylsulf1nyl, dibenzofuranylsulflnyl, midazolylsulflnyl, benzoisoxazolylsulflnyl, xazolylsulflnyl, benzooxadiazolylsulfinyl, benzoisothiazolylsulfmyl, benzothiazolylsulfmyl, benzofurylsulflnyl, benzothienylsulfinyl, dibenzothienylsulflnyl, and benzodioxolylsulflnyl etc. are exempli?ed. Preferably furylsulf1nyl, thienylsulf1nyl, imidazolylsulflnyl, lylsulflnyl, isothiazolylsulflnyl, isoxazolylsulflnyl, oxazolylsulflnyl, thiazolylsulfinyl, pyridylsulflnyl, pyrazinylsulflnyl, pyrimidinylsulflnyl, 3O and pyridazinylsulflnyl, etc.

The term "heteroarylsulfonyl" includes a group in which sulfonyl is substituted with one "heteroaryl" as described herein. Examples e pyrrolylsulfonyl, furylsulfonyl, thienylsulfonyl, imidazolylsulfonyl, pyrazolylsulfonyl, isothiazolylsulfonyl, isoxazolylsulfonyl, oxazolylsulfonyl, thiazolylsulfonyl, pyridylsulfonyl, pyrazinylsulfonyl, pyrimidinylsulfonyl, pyridazinylsulfonyl, olylsulfonyl, oxadiazolylsulfonyl, thiadiazolylsulfonyl, indolizinylsulfonyl, olylsulfonyl, lsulfonyl, indazolylsulfonyl, purinylsulfonyl, quinolidinylsulfonyl, isoquinolylsulfonyl, quinolylsulfonyl, phtharazinylsulfonyl, naphthilidinylsulfonyl, quinolanylsulfonyl, quinazolinylsulfonyl, cinnolinyl yl, pteridinyl sulfonyl, carbazolylsulfonyl, phenanthridinylsulfonyl, acridinylsulfonyl, dibenzofuranylsulfonyl, benzoimidazolylsulfonyl, benzoisoxazolylsulfonyl, benzooxazolylsulfonyl, benzooxadiazolylsulfonyl, benzoisothiazolylsulfonyl, benzothiazolylsulfonyl, benzofurylsulfonyl, benzothienylsulfonyl, dibenzothienylsulfonyl, and benzodioxolylsulfonyl, etc. Furylsulfonyl, thienylsulfonyl, imidazolylsulfonyl, pyrazolylsulfonyl, isothiazolylsulfonyl, isoxazolylsulfonyl, ylsulfonyl, thiazolylsulfonyl, pyridylsulfonyl, pyrazinylsulfonyl, pyrimidinylsulfonyl, and pyridazinylsulfonyl are red.

The term "heteroarylsulfonyloxy" includes a group in which an oxygen atom is substituted with one "heteroarylsulfonyl" as described herein. Examples e pyrrolylsulfonyloxy, furylsulfonyloxy, thienylsulfonyloxy, imidazolylsulfonyloxy, pyrazolylsulfonyloxy, isothiazolylsulfonyloxy, isoxazolylsulfonyloxy, oxazolylsulfonyloxy, thiazolylsulfonyloxy, pyridylsulfonyloxy, pyrazinylsulfonyloxy, pyrimidinylsulfonyloxy, pyridazinylsulfonyloxy, tetrazolylsulfonyloxy, oxadiazolylsulfonyloxy, thiadiazolylsulfonyloxy, indolizinylsulfonyloxy, isoindolylsulfonyloxy, indolylsulfonyloxy, indazolylsulfonyloxy, purinylsulfonyloxy, quinolidinylsulfonyloxy, isoquinolylsulfonyloxy, quinolylsulfonyloxy, phtharazinylsulfonyloxy, naphthilidinylsulfonyloxy, quinolanyl sulfonyloxy, quinazolinylsulfonyloxy, cinnolinylsulfonyloxy, pteridinylsulfonyloxy, carbazolylsulfonyloxy, thridinylsulfonyloxy, acridinylsulfonyloxy, dibenzofuranylsulfonyloxy, benzoimidazolylsulfonyloxy, benzoisoxazolylsulfonyloxy, benzooxazolylsulfonyloxy, benzooxadiazolylsulfonyloxy, benzoisothiazolylsulfonyloxy, benzothiazolylsulfonyloxy, benzofuryl sulfonyloxy, benzothienylsulfonyloxy, dibenzothienylsulfonyloxy, and benzodioxolylsulfonyloxy etc. are exempli?ed. ably, ulfonyloxy, thienylsulfonyloxy, imidazolylsulfonyloxy, 3O pyrazolylsulfonyloxy, isothiazolylsulfonyloxy, isoxazolylsulfonyloxy, oxazolylsulfonyloxy, lylsulfonyloxy, pyridylsulfonyloxy, pyrazinylsulfonyloxy, pyrimidinylsulfonyloxy, and pyridazinylsulfonyloxy, etc.

The term "aromatic carbocyclic ring" includes an ic monocyclic or aromatic fused carbocyclic ring. Examples include a e ring, a naphthalene ring, and an anthracene ring. A benzene ring is preferred.

The term "aromatic cyclic ring" includes an aromatic monocyclic or aromatic fused heterocyclic ring. Examples include a pyrrole ring, a furan ring, a thiophen ring, a pyrazole ring, an imidazole ring, an isothiazole ring, an isoxazole ring, an oxazole ring, a thiazole ring, a pyrazine ring, a pyrimidine ring, a pyridazine ring, a tetrazole ring, an oxadiazole ring, a thiadiazole ring, an indolizine ring, an isoindole ring, an indole ring, an indazole ring, a purine ring, a quinolidine ring, an isoquinoline ring, a quinoline ring, a phtharazine ring, a naphthyridine ring, a quinolane ring, a quinazoline ring, a cinnoline ring, a pteridine ring, a carbazole ring, a phenanthridine ring, an acridine ring, a dibenzofuran ring, a benzoimidazole ring, a benzoisoxazole ring, a xazole ring, a xadiazole ring, a sothiazole ring, a benzothiazole ring, a benzofuran ring, a benzothiophen ring, a dibenzothiophen ring, and a benzodixolane ring. A pyridine ring, a furan ring, and a thiophen ring are preferred.

The term "Cl-C6 alkylene" includes a straight or branched ne group having one to six carbon ). Examples include —CH2—, —CH(CH3)—, —C(CH3)2—, —CH2CH2—, —CH(CH3)CH2—, —C(CH3)2CH2—, —CH2CH2CH2—, —CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2— and 7 —CH2CH2CH2CH2CH2CH2—.

Preferred are —CH2—, —CH2CH2—, —CH2CH2CH2—, and —CH2CH2CH2CH2—.

The term "alkylene optionally containing one or two heteroatom(s)" of "optionally substituted alkylene optionally containing one or two heteroatom(s)" includes a straight or branched alkylene group haVing one to six carbon atoms, optionally containing one or two heteroatom(s) which may be substituted with "alkyl" as described herein. Examples include —CH2—, —CH(CH3)—, —C(CH3)2—, —CH2CH2—, —CH2CH2CH2—, —CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2—, CHzO , OCHz , CH2CHzO—, —OCH2CH2 , Cst , SCHz , —CH2Cst—, —SCH2CH2—, —CH2CH20CH2CH2—, —OCH2CHzO—, —, —NHCH2—, —N(CH3)CH2—, —N+(CH3)2CH2—, —NHCH2CH2CH2—, and —N(CH3)CH2CH2CH2—, etc. Preferred are —CH2—, 2—, 2CH2—, —CH2CH2CH2CH2—, —OCH2CHzO—, —OCHzO—, and —N(CH3)CH2CH2CH2—.

The term "alkenylene optionally ning one or two heteroatom(s)" of "optionally tuted alkenylene ally containing one or two heteroatom(s)" includes a straight or ed alkenylene group haVing two to six carbon atoms, optionally containing one or two heteroatom(s) which may be substituted with "alkyl" as described herein. Examples include —CH=CHCH=CH—, O—, H—, —CH=CHS—, —SCH=CH—, —CH=CHNH—, —NHCH=CH—, —CH=CH—CH=N—, and —N=CH—CH=CH—. Preferred are, —CH=CHCH=CH—, —CH=CHCH=N—, and —N=CHCH=CH—.

The term ylene optionally containing one or two heteroatom(s)" includes a ht or branched lene group haVing two to six carbon atoms, optionally containing one or two atom(s) which may be substituted with "alkyl" as described herein. Examples include C=CCH2 , CH2C=CCH2 , CH2C=CCHzO—, —OCH2CECH—, —CH2CECCH2S—, —SCH2CECH—, —CH2CECCH2NH—, —NHCH2CECH—, —CH2CECCH2N(CH3)—, and —N(CH3)CH2CECH—.

Especially, CH2C=CCH2 ,and OCHzCECH— are preferred.

The term "3- to ered nitrogen-containing non-aromatic heterocyclic ring" includes a ring of any of the formulas described as such in US. Patent 8,143,285, which is incorporated herein by reference in its entirety.

The term "3- to 8-nitrogen-containing ic heterocyclic ring" includes a 3- to 8-membered aromatic heterocyclic ring containing one or more of nitrogen atom(s), and further optionally an oxygen atom and/or sulfur atom in the ring. Examples include pyrrolyl (e.g., l-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl), imidazolyl (e.g., 2-imidazolyl, 4- imidazolyl), pyrazolyl (e.g., zolyl, 3-pyrazolyl), isothiazolyl (e.g., 3-isothiazolyl), isoxazolyl (e.g., 3-isoxazolyl), oxazolyl (e.g., 2-oxazolyl), thiazolyl (e.g., 2-thiazolyl), pyridyl (e.g., 2-pyridyl, 3-pyridyl, 4-pyridyl), pyrazinyl (e.g., 2-pyrazinyl), pyrimidinyl 2O (e.g., 2-pyrimidinyl, midinyl), pyridazinyl (e.g., 3-pyridazinyl), tetrazolyl (e.g., lH- tetrazolyl), oxadiazolyl (e.g., l,3,4-oxadiazolyl), and thiadiazolyl (e.g., l,3,4-thiadiazolyl).

The term "4- to 8-membered nitrogen-containing heterocyclic ring containing one or two nitrogen atom(s)" means a ring of any of the formulas described as such in US.

Patent 8,143,285, which is orated herein by reference in its entirety.

"Optionally substituted" is used interchangeably herein with "substituted or unsubstituted." In the present specif1cation, examples of substituents in "optionally substituted alkyl," "optionally substituted alkyloxy, 77 (L optionally substituted hio, 77 (4 optionally substituted alkylsulflnyl," "optionally substituted alkylsulfonyl," "optionally substituted 3O alkylsulfonyloxy," and "the optionally tuted xycarbonyl" include cycloalkyl, alkylene optionally containing one or two heteroatom(s), hydroxy, oxo, xy optionally substituted with a substituent group A at one to three position(s), mercapto, alkylthio, a halogen atom, nitro, cyano, carboxy, alkyloxycarbonyl, optionally substituted amino, optionally tuted oyl, acyl, aryl (e.g., phenyl) optionally substituted with a substituent group B at one to three position(s), heteroaryl (e.g., pyridyl, furyl, thienyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl) optionally substituted with a substituent group C at one to three position(s), an optionally substituted non-aromatic cyclic ring group (e.g., morpholinyl, pyrrolidinyl, piperazinyl) which may be substituted with a substituent group C at one to three position(s), aryloxy (e.g., phenyloxy) optionally substituted with a substituent group B at one to three position(s), alkylsulfonyl, and the like. The above-referenced nally tuted" moieties can be substituted with one to three of the above-referenced substituent(s) at any possible position.

In the present speci?cation, examples of tuents in nally substituted alkenyl," "optionally substituted alkynyl," "optionally substituted alkenyloxy, 77 (L optionally substituted loxy,77 (L optionally substituted alkenylthio, 77 (L optionally substituted lthio," "optionally substituted loxycarbonyl," "optionally substituted alkynyloxycarbonyl," "optionally substituted cycloalkyl," "optionally substituted cycloalkenyl," nally substituted cycloalkyloxy, "optionally substituted cycloalkenyloxy," "optionally substituted lkylthio," "optionally substituted cycloalkenylthio,77 (L optionally substituted cycloalkylsul?nyl," "optionally substituted cycloalkenylsul?nyl," "optionally substituted cycloalkylsulfonyl," "optionally substituted cycloalkenylsulfonyl," "optionally substituted cycloalkylsulfonyloxy," "optionally substituted cycloalkenylsulfonyloxy," nally substituted alkenyloxycarbonyl," "optionally substituted alkylene,77 (L optionally substituted Cl-C6 alkylene,77 (L optionally tuted alkylene optionally containing 77 (L one or two heteroatom(s), optionally substituted alkenylene, 77 (L optionally substituted alkenylene optionally containing one or two atom(s), 77 (4 optionally substituted alkynylene," and "optionally substituted alkynylene optionally containing one or two atom(s)" include alkyl (such as dialkyl) optionally substituted with a substituent group D at one to three position(s), cycloalkyl, y, oxo, alkyloxy optionally substituted with a substituent group A at one to three position(s), mercapto, alkylthio, a halogen atom, nitro, cyano, carboxy, alkyloxycarbonyl, optionally substituted amino, optionally substituted carbamoyl, acyl acyloxy, aryl (e.g., phenyl) optionally substituted with a substituent group B at one to three position(s), heteroaryl (e.g., pyridyl, furyl, thienyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl) 3O optionally substituted with a substituent group C at one to three position(s), non-aromatic heterocyclic group (e.g., morpholinyl, idinyl, zinyl) optionally tuted with a substituent group C at one to three position(s), aryloxy (e.g., phenyloxy) optionally substituted with a substituent group C at one to three position(s), alkylsulfonyl, and the like. The above-referenced "optionally substituted" moieties can be substituted with one or more of the above-referenced substituent(s) at any possible position.

In the present speci?cation, examples of substituents in "optionally substituted aryl," "optionally substituted phenoxy,77 (L optionally substituted y,77 (L ally tuted phenylthio," "optionally substituted arylthio," "optionally substituted arylsul?nyl," "optionally tuted arylsulfonyl," "optionally substituted arylsulfonyloxy," "optionally substituted heteroaryl," "optionally substituted heteroaryloxy," "optionally substituted heteroarylthio," "optionally substituted heteroarylsul?nyl," "optionally substituted heteroarylsulfonyl," "optionally substituted heteroarylsulfonyloxy," "optionally substituted non-aromatic heterocyclic " "optionally substituted piperazine-l,4-diyl," "substituted piperazine-l,4-diyl," "optionally substituted C6 arene-l,4-diamine-N1,N4-diyl," and substituted C6 arene-l,4-diamine- N1,N4-diyl," include alkyl optionally substituted with a substituent group D at one to three position(s), oxo, cycloalkyl, alkenyl, alkynyl, hydroxy, alkyloxy optionally substituted with a substituent group A at one to three position(s), aryloxy (e.g., phenoxy) optionally substituted with a substituent group B at one to three position(s), mercapto, alkylthio, a halogen atom, nitro, cyano, carboxy, alkyloxycarbonyl, acyl, alkylsulfonyl, optionally substituted amino, optionally substituted carbamoyl, aryl (e.g., phenyl) optionally substituted with a substituent group B at one to three position(s), heteroaryl (e.g., pyridyl, furyl, l, imidazolyl, oxazolyl, lyl, pyrazolyl) optionally substituted with a substituent group C at one to three position(s), non-aromatic cyclic group (e.g., 2O morpholinyl, pyrrolidinyl, zinyl) ally substituted with a substituent group C at one to three position(s), and the like. The above-referenced nally substituted" moieties can be substituted with one or more of the referenced substituent(s) at any possible position.

Substituent group A is comprised of a halogen atom and phenyl optionally substituted with one to three substituent(s) selected from the Substituent group B.

Substituent group B is comprised of a halogen atom, alkyl, alkyloxy, cyano, and nitro.

Substituent group C is comprised of a halogen atom and alkyl.

Substituent group D is comprised of a halogen atom and alkyloxy. 3O In compounds in which R2 is an electron pair, the oxygen is negatively charged and may form a salt with a cation.

In some nds, the R3 on at least 1, at least 2, at least 3 of A1, A2, A3, A4, and A5 is a hydrogen atom or Cl-C6 alkyl.

In the course of the methods of the present invention, a therapeutically effective amount of compounds of the invention can be administered to an , including mammals and humans, in many ways. While in the preferred embodiment, the compounds of the invention are administered orally, parenterally, or topically, other forms of administration such as through medical compounds or aerosols are also contemplated.

For oral administration, the effective amount of compounds may be administered in, for example, a solid, semi-solid, liquid, or gas state. Speci?c examples include tablet, capsule, powder, granule, solution, suspension, syrup, and elixir agents. r, the compounds are not limited to these forms.

To formulate the compounds of the invention into tablets, capsules, powders, granules, solutions, or suspensions, the compound is preferably mixed with a , a disintegrating agent and/or a lubricant. If ary, the resultant composition may be mixed with a diluent, a buffer, an in?ltrating agent, a preservative and/or a ?avor, using known methods. Examples of the binder include crystalline cellulose, cellulose derivatives, arch, extrins, and gelatin. Examples of the disintegrating agent include cornstarch, potato starch, and sodium carboxymethylcellulose. Examples of the lubricant include talc and magnesium stearate. Further, additives, which have been conventionally used, such as lactose and mannitol, may also be used.

For parenteral administration, the nds of the present invention may be administered rectally or by ion. For rectal administration, a suppository may be used.

The suppository may be ed by mixing the compounds of the present invention with a pharmaceutically suitable excipient that melts at body ature but remains solid at room temperature. Examples include but are not limited to cacao , carbon wax, and polyethylene glycol. The resulting ition may be molded into any desired form using methods known to the ?eld.

For administration by injection, the compounds of the t ion may be injected hypodermically, intracutaneously, intravenously, or intramuscularly. Medicinal drugs for such injection may be ed by dissolving, suspending or emulsifying the compounds of the invention into an aqueous or non-aqueous solvent such as ble oil, glyceride of synthetic resin acid, ester of higher fatty acid, or propylene glycol by a known method. If desired, additives such as a solubilizing agent, an osmoregulating agent, an emulsi?er, a izer, or a preservative, which has been conventionally used may also be added. While not required, it is preferred that the composition be sterile or sterilized.

To formulate the compounds of the invention into suspensions, syrups, or elixirs, a pharmaceutically suitable solvent may be used. Included among these is the non-limiting example of water.

For topical administration, l formulations can be in a form of gel, cream, lotion, liquid, emulsion, ointment, spray, solution, suspension, and patches. Inactive ingredients in the topical formulations for example include, but not limited to, lauryl e (emollient/permeation enhancer), diethylene glycol monoethylether ient/permeation enhancer), DMSO (solubility enhancer), silicone elastomer (rheology/texture modi?er), caprylic/capric triglyceride, (emollient), octisalate, (emollient/UV ?lter), ne ?uid (emollient/diluent), squalene (emollient), sun?ower oil (emollient), and silicone dioxide (thickening agent).

The compounds of the invention may also be used er with an additional compound having other pharmaceutically suitable activity to prepare a medicinal drug. A drug, either containing a compound of the ion as a stand-alone compound or as part of a composition, may be used in the treatment of subjects in need thereof.

The compounds of the invention may also be administered in the form of an l or nt prepared by charging the compounds in the form of a liquid or ?ne powder, together with a gaseous or liquid spraying agent and, if necessary, a known auxiliary agent such as an ng agent, into a non-pressurized ner such as an aerosol container or a nebulizer. A pressurized gas of for 7 example, ro?uoromethane, propane or nitrogen may be used as the spraying agent.

The compounds of the invention may be administered to an animal, including mammals and humans, in need f as a pharmaceutical composition, such as tablets, capsules, solutions, or emulsions. Administration of other forms of the compounds described in this invention, including but not limited to esters thereof, pharmaceutically suitable salts thereof, metabolites thereof, structurally d compounds thereof, analogs thereof, and combinations thereof, in a single dose or a le dose, are also plated by the present invention.

The compounds of the invention may also be stered to an animal in need thereof as a nutritional additive, either as a food or nutraceutical supplement.

The terms "preventing,77 (Ltreating," or "ameliorating" and similar terms used herein, include prophylaxis and full or partial treatment. The terms may also include 3O reducing symptoms, ameliorating symptoms, reducing the severity of symptoms, ng the incidence of the disease, or any other change in the condition of the patient, which improves the therapeutic outcome.

The compounds described in this invention are preferably used and/or administered in the form of a composition. le compositions are, preferably, a pharmaceutical composition, a foodstuff, or a food supplement. These compositions provide a convenient form in which to deliver the nds. Compositions of the invention may comprise an antioxidant in an amount effective to increase the ity of the compounds with respect to oxidation or solubility.

The amount of compound that is administered in the method of the invention or that is for administration in the use of the invention is any suitable amount. It is preferably from 1 ng/kg body weight to 20 g/kg body , more preferably in the range of l ug/kg body weight to 1 g/kg body weight, such as 1 mg/kg body weight to 100 mg/kg body weight of compound per day. le compositions can be ated accordingly. Those of skill in the art of dosing of biologically active agents will be able to develop particular dosing regimens for various subjects based on known and well tood parameters.

A preferred composition according to the invention is a pharmaceutical composition, such as in the form of tablets, pills, capsules, s, multiparticulates (including granules, beads, pellets and micro-encapsulated particles), powders, elixirs, syrups, suspensions, and solutions. Pharmaceutical compositions will typically comprise a pharmaceutically acceptable diluent or carrier. Pharmaceutical compositions are preferably adapted for administration parenterally or orally. Orally administrable compositions may be in solid or liquid form and may take the form of tablets, powders, suspensions, and , among other things. Optionally, the compositions comprise one or more ?avoring and/or coloring agents. In general, therapeutic and nutritional compositions may comprise any substance that does not significantly interfere with the action of the compounds on the subject. ceutically acceptable rs suitable for use in such compositions are well known in the art of pharmacy. The compositions of the invention may contain 0.01-99% by weight of the compounds of the invention. The compositions of the ion are generally prepared in unit dosage form. Preferably the unit dosage of compounds described in the present invention is from 0.1 mg to 2000 mg, more preferably from 50 mg to 1000 mg. The excipients used in the preparation of these compositions are the excipients known in the art.

Further examples of product forms for the composition are food supplements, such as in the form of a soft gel or a hard capsule comprising an encapsulating material selected from the group consisting of gelatin, starch, modi?ed starch, starch derivatives such as glucose, sucrose, lactose, and se. The encapsulating material may optionally contain cross-linking or rizing agents, stabilizers, antioxidants, light absorbing agents for protecting sensitive fills, preservatives, and the like. Preferably the unit dosage of compounds described in the present invention is from 0.1 mg to 2000 mg, more preferably from 50 mg to 1000 mg.

In general, the term carrier may be used throughout this application to represent a composition with which the compounds described may be mixed, be it a pharmaceutical carrier, foodstuff, nutritional ment, or dietary aid. The materials described above may be considered carriers for the purposes of the invention. In certain embodiments of the invention, the carrier has little to no biological activity on the compounds of the invention.

Dose: The methods of the present invention can comprise administering a therapeutically effective amount of nd to an animal in need thereof. The effective amount of compound depends on the form of the compound administered, the duration of the administration, the route of administration (e.g., oral or parenteral), the age of the animal, and the condition of the animal, ing mammals and humans.

For instance, an amount of a nd effective to treat or prevent type 1 diabetes, lupus, ulcerative colitis, s disease, gastrointestinal in?ammation, or any other condition described herein in an animal can range from 1 ng/kg/day to 20 g/kg/day.

A preferred effective amount of compound is 50 day to 5 g/kg/day, with a more preferred dose being 1 to 100 mg/kg/day. The effective amount of compound is most effective in treating or preventing type 1 diabetes, lupus, ulcerative colitis, Crohn's e, gastrointestinal in?ammation, or any other condition described herein of an animal when administered to an animal for periods ranging from about 1 to 1000 days, with a preferred period of 7 to 300 days, and a most preferred period of 30 to 90 days, whereby most ive is de?ned as an identi?cation of the induction of bene?cial responses. The effective amount of compound may be continued beyond these periods for maintenance of ial responses in chronic diseases.

An amount of compound most effective in preventing over-activation of the immune system can range from 1 ng/kg/day to 20 g/kg/day, with a preferred dose of l to 100 mg/kg/day.

When the effective amount of the compound of the present ion is administered in a nutritional, therapeutic, medical, or veterinary composition, the preferred dose ranges from about 0.01 to 2.0% wt/wt to the food or nutraceutical product.

In certain other embodiments, the t invention es for use of the compounds described herein in the treatment and tion of type 1 diabetes, lupus, IBD and GI tract in?ammation.

In addition, in general, the present invention relates to inhibition or activation of in?ammation ically, wherein the relevant components include the pancreas, spleen, lung, heart, central nervous system, joints, liver, kidneys, or in the GI tract, n the relevant ents include the esophagus, stomach, small intestine, cecum, large intestine, and rectum. The effect results from the re of compound to various cells types in the body that induces a biological effect. The cells may include those from GI tract tissues, immune cells (i.e. macrophages, monocytes, lymphocytes), pancreatic islet cells, endothelial cells, neurons, or epithelial cells. In certain embodiments, the invention provides for treating ts with a compound of the invention, for example as a y supplement, to reduce or prevent in?ammation related to type 1 diabetes, lupus or in?ammatory bowel e, either Crohn's disease or ulcerative colitis.

When practiced, the methods of the invention can be by way of administering the compounds to a subject via any acceptable stration route using any acceptable form, as is described above, and ng the body of the subject to distribute the compounds to the target cell h natural processes. As is described above, administering can likewise be by direct injection to a site (e.g., organ, tissue) containing a target cell (i.e., a cell to be treated).

Furthermore, administering can follow any number of regimens. It thus can comprise a single dose or dosing of experimental compound, or multiple doses or dosings over a period of time. Accordingly, treatment can comprise repeating the administering step one or more times until a desired result is achieved. In certain embodiments, treating can continue for extended periods of time, such as weeks, months, or years. Dosing regimens can preferably entail administration of compound between 6 times daily to once per week, with a more preferred regimen between three times daily to once daily. Those of skill in the art are fully capable of easily developing suitable dosing regimens for individuals based on known parameters in the art. The dosage s for compounds of the invention may be used in the methods of these embodiments of the invention. For the treatment of type 1 diabetes, lupus or IBD, it is preferred that the compounds be stered at amounts of about 10 ng/day to 10 g/day. 3O The amount to be administered will vary depending on the subject, stage of disease or disorder, age of the subject, general health of the subject, and various other parameters known and routinely taken into consideration by those of skill in the medical arts. As a l matter, a suf?cient amount of compound will be administered in order to make a able change in the amount of in?ammation in the pancreas, GI tract or systemically.

Reduction of in?ammation may be related to amount of pain enced by the subject, insulin, anti-nuclear antigen antibodies, TNFOL or C-reactive protein levels in the blood, the percent of regulatory s in the blood or concentration of calprotectin in feces. Suitable amounts are sed herein, and additional suitable amounts can be identi?ed by those of skill in the art t undue or excessive experimentation, based on the amounts sed herein.

It should be evident that the present invention provides -binding compound therapy for use in ting cells, such as in treating cells of a subject. The above discussion focuses on the use of the compounds of the present invention as part of a composition for use in what could generally be ered a pharmaceutical or medical setting.

The compounds described in this invention for the treatment of autoimmune disease including type 1 diabetes, systemic lupus erythematosus, IBD, Sjogren’s syndrome, and other conditions described herein may be formulated as a pharmaceutical, nutritional composition, functional food composition, or dietary aid, as are described in greater detail above.

As an alternative or in addition to the methods of treating conditions by administering the compounds directly, the conditions can be treated with prepared cells generated from precursor cells with the compounds.

The term rsor cell" is used herein to refer generally to any cell that serves as a ng cell that is treated to generate a prepared cell. The cell may be a cell upstream in a differentiation lineage leading to the ed cell, such as a stem cell, a progenitor cell, or a "precursor cell" (as the term is used in the art to refer to an ediate between a stem cell and a differentiated cell) with, e. g., totipotent, multipotent or unipotent properties, but does not necessarily have to be so. Accordingly, in some versions, generating the ed cell from the precursor cell involves differentiating the precursor cell into the prepared cell. In other versions, ting the prepared cell from the precursor cell merely involves inducing changes such as gene expression changes.

The prepared cells can be generated from precursor cells by contacting the precursor cells in vitro with one or more of the compounds of the invention to thereby 3O generate the prepared cells. The terms "in vitro" and "ex vivo" are used interchangeably herein in contrast to "in vivo" and refer to a state of being outside of a living sm.

The precursor and/or prepared cells of the invention may comprise immune cells.

Exemplary immune cells include granulocytes, mast cells, monocytes, macrophages, neutrophils, dendritic cells, natural killer cells, T cells, and B cells, among others.

Exemplary granulocytes include basophils, eosinophils, and neutrophils.

The precursor and/or prepared cells of the invention may comprise white blood cells (leukocytes). Exemplary white blood cells e neutrophils, eosinophils (acidophiles), basophils, lymphocytes, and monocytes.

The precursor and/or prepared cells of the invention may comprise peripheral blood mononuclear cells (PBMCs) or lamina propria mononuclear cells (LPMCs).

Exemplary PBMCs and LPMCs include lymphocytes (T cells, B cells, NK cells) and monocytes.

The sor and/or prepared cells of the invention may comprise T cells. T cells are divided into two broad categories: CD8+ T cells or CD4+ T cells, based on which protein is present on the cell’s surface. T cells carry out multiple functions, including killing infected cells and activating or ting other immune cells. CD8+ T cells also are called cytotoxic T cells or cytotoxic lymphocytes (CTLs). They are crucial for recognizing and removing virus-infected cells and cancer cells. The major CD4+ T-cell subsets are na'1've CD4+ T cells, THl cells, TH2 cells, THl7 cells, and Treg cells, with "TH" referring to "T helper cell." Na'1've CD4+ T cells are T cells that are not differentiated into any of THl cells, TH2 cells, THl7 cells, and Treg cells. Regulatory T cells (Tregs) monitor and inhibit the activity of other T cells. They prevent adverse immune activation and in tolerance, or the prevention of immune responses against the body’s own cells and antigens. In some ns, the precursor cells comprise na'1've CD4+ T cells, and the prepared cells comprise Treg cells.

Generating the prepared cells from the precursor cells may comprise contacting an amount of one or more compounds of the invention for a time effective to induce a compound-dependent difference in the prepared cells with respect to the precursor cells.

As used herein, "compound-dependent difference" refers to a difference in the prepared cell with t to the sor cell arising from contacting the sor cell with one or more nds of the invention. Compound-dependent ences can be determined by contacting cells with media in the presence or absence the one or more compounds of the invention, wherein the compound-dependent differences are teristics that appear only with the cells contacted with media in the presence of the one or more compounds of 3O the invention. The compound-dependent differences may be ences not only in kind but also of degree.

The compound-dependent difference in the prepared cells may e a difference in gene expression. Unless explicitly stated otherwise, "gene expression" is used broadly herein to refer to any or all of transcription or translation. Thus, a difference in gene sion can be a difference in mRNA production, a difference in protein production, or both. Unless explicitly stated otherwise, the gene having differential expression may be identi?ed herein by referring to the protein produced from the gene (e.g., FOXP3) or by referring to the gene itself (e.g., Lag3). In various versions of the invention, the compound-dependent differences in gene expression may comprise one or more of an increase in expression of IL-10 or an ortholog thereof, an increase in expression of FOXP3 or an ortholog thereof, a decrease in expression of TNFOL or an ortholog thereof, a decrease in expression of IFNy or an ortholog thereof, a decrease in expression of Tbet or an ortholog f, an increase in expression of Lag3 or an ortholog f, an increase in expression of Socs2 or an ortholog thereof, an increase in expression of Irf7 or an ortholog thereof, an se in expression of P2rx7 or an ortholog thereof, an se in expression of Capn3 or an ortholog thereof, an increase in expression of Isz2 or an ortholog thereof, an increase in expression of Slal5a or an ortholog thereof, an se in sion of Pten or an ortholog thereof, an increase in expression of Foxo] or an ortholog thereof, and/or an increase in expression of Phlpp] or an ortholog thereof. The orthologs may include orthologs in animal species. The orthologs may include orthologs in mammalian species. The orthologs (such as for the mouse genes named above) may include orthologs in primates. The orthologs (such as for the mouse genes named above) may include ogs in humans.

The compound-dependent difference in the prepared cells may include other 2O able differences, such as an increase in phosphorylation of STAT5a or an ortholog thereof, an increase in FOXOl phosphorylation or an ortholog thereof, and/or an increase in pyruvate kinase activity.

In generating the prepared cells, the precursor cells may be contacted with s of the compound from about 100 nM, about 10 nM, about 1 nM or less to about 1 uM, about 10 uM, about 100 uM, about 1 mM or more. The precursor cells may be contacted with the compound for a time from about 12 hours, 6 hours, 1 hour, about 30 minutes, or less to about 24 hours, about 48 hours, about 72 hours or more.

In some ns, the PBMCs or LPMCs at large are contacted with the nd of the invention. The PBMCs or LPMCs can be isolated from an animal. In some versions, 3O subtypes of PBMCs or LPMCs, such as T cells, can be isolated from the PBMCs or LPMCs and then contacted with the compound of the ion. In some versions, the PBMCs or LPMCs are contacted with the compound of the ion, and then es of cells, such as T cells or a particular type of T cells are isolated therefrom. Methods for isolating PBMCs, LPMCs, and subtypes thereof are known in the art. See, e. g., Maj owicz et al. 2012 (Maj owicz A, van der Marel S, te Velde AA, Meij er SL, Petry H, van Deventer SJ, ra V. Murine CD4+CD25' cells activated in vitro with PMA/ionomycin and anti- CD3 acquire regulatory function and ameliorate experimental colitis in viva. BMC Gaslroenlerol. 2012 Dec 3,12:172) and Canavan et al. 20016 (Canavan JB, Scotta C, Vossenkamper A, Goldberg R, Elder MJ, Shoval 1, Marks E, Stolarczyk E, Lo JW, Powell N, Fazekasova H, Irving PM, Sanderson JD, Howard JK, Yagel S, Afzali B, MacDonald TT, Hernandez-Fuentes MP, Shpigel NY, Lombardi G, Lord GM. Developing in vitro expanded CD45RA+ regulatory T cells as an ve cell therapy for Crohn's e.

Gut. 2016 Apr,65(4):584-94). Subsets of P1V?3Cs, for example, can be isolated with anti- CD3 antibodies and anti-CD28 antibodies. Anti-CD3 antibodies and anti-CD28 antibodies can be provided in the form of anti-CD3/anti-CD28 beads, such as Human T-Activator CD3/CD28 DYNABEADS® from Fisher Scienti?c (Waltham, MA).

Generating the prepared cells can comprise entiating the prepared cells from the precursor cells. For example, prepared cells such as Treg cells can be differentiated from precursor cells such as e CD4+ T cells. Such entiating can comprise contacting the precursor cells with differentiating factors in on to one or more of the compounds of the invention. Various differentiating factors may include all-trans-retinoic acid, TGF-B, phorbol myristate e, ionomycin, rapamycin, and/or lL-2. In some versions, the differentiating can comprise expanding the proportion of Treg cells in the prepared cells with respect to the portion in the precursor cells.

The precursor and prepared cells of the invention can be isolated cells. The term "isolated" or "puri?ed" means a material that is removed from its original nment, for example, the natural environment. A material is said to be "puri?ed" when it is present in a particular composition in a higher or lower concentration than the tration that exists prior to the puri?cation step(s).

Treating the ion with the prepared cells of the invention can comprise administering the cells to the animal in an amount suf?cient to treat the condition. The prepared cells can be administered using any route or method described above for the compounds, including parenterally or enterally. Non-limiting forms of parenteral administration include injection or infusion. The prepared cells can be injected or infused directly into the bloodstream or other parts of the body. Non-limiting forms of enteral administration include oral and rectal administration, such that the prepared cells enter the gastrointestinal tract. The prepared cells may be autologous to the treated animal (1'.e., generated from a cell taken from the same animal that the prepared cell is used to treat) or heterologous to the treated animal (1'.e., ted from a different animal that the ed cell is used to treat). A cell prepared as described above can be used in a method of treating any of the conditions described herein. Exemplary conditions e intestinal in?ammation. Exemplary types of intestinal in?ammation include in?ammatory bowel disease. Exemplary types of in?ammatory bowel disease include Crohn’s disease and ulcerative colitis.

In one embodiment of the invention, the method of treating immunometabolic disease comprises treatment without causing discemable side-effects, such as signi?cant weight gain, systemic immune suppression, cushingoid appearance, osteopenia/osteoporosis, ar toxicity or pancreatitis that is common of currently available treatments (i.e. s, antibiotics, corticosteroids, doxorubicin, methotrexate).

That is, it has been found that the method of treating ing to the present ion, which es the treatment effect, at least in part, by affecting the expression and/or activation of LANCL2 and/or other immunometabolic pathways in some cells, provides the bene?cial effect t causing a signi?cant gain in weight, for example by ?uid ion, in the subject being treated, as compared to other r subjects not receiving the ent.

As such, the immunometabolic methods of the present invention can provide treatments for reducing in?ammation by affecting the metabolism of immune cells. The s can reduce in?ammation systemically (i.e., throughout the subject's body) or locally (e.g., at the site of administration or the site of in?ammatory cells, including but not limited to T cells and macrophages). In treating or preventing in?ammation through immunometabolism, one effect that may be observed is a shift in the metabolism of glucose. In particular, the shift may be from the production of lactate from pyruvate towards the entrance into the tricarboxylic acid cycle that is tied with immunoin?ammatory actions. More specifically, this shift in metabolism can be associated with an increase in the proportion of CD4+CD25+FOXP3+ or other regulatory CD4+ T-cells relative to effector CD4+ T-cells such as ILl7+ Thl7 cells or IFNy+ Th1 cells. Another observed effect may be decreased cellular proliferation resulting from the combination of decreased anaerobic metabolism and increased immune oint pathways. Another effect of shifts in metabolism triggered therapeutically may be 3O decreased expression of in?ammatory chemokines such as MCP-l, IL-8, or CXCL9 resulting from altered processing and storage of fatty acids. The methods can thus also be considered methods of ing or altering the immune response of a subject to whom the therapy is administered, thereby intercepting in?ammation, disease and pathology.

The invention provides methods of inhibiting in?ammation in the GI tract, wherein the relevant components include the stomach, small intestine, large intestine, and rectum.

The invention provides methods of treating or preventing a subject suffering from IBD, or otherwise healthy individuals, perhaps with a genetic position for Crohn’s Disease or ulcerative colitis, from developing IBD. The methods may also involve treating those with a remissive form of IBD. According to the invention, the term "a subject suffering from IBD" is used to mean a subject (e.g., animal, human) having a disease or disorder showing one or more clinical signs that are typical of IBD. In general, the method of treating or preventing according to this aspect of the invention comprises administering to the subject an amount of compound or cell therapy that is ive in treating or ting one or more symptoms or clinical manifestations of IBD, or in preventing pment of such symptom(s) or manifestation(s).

Thus, according to the s of the invention, the invention can provide methods of treatment of IBD, in?ammation associated with enteric infection and ation associated with autoimmune diseases. The s of treatment can be prophylactic methods. In certain ments, the method is a method of treating IBD, in?ammation associated with enteric infection and in?ammation associated with mune es. In other embodiments, the method is a method of preventing IBD.

In embodiments, the method is a method of ting a remissive form of IBD from becoming active. In still other embodiments, the method is a method of improving the health status of a subject suffering from IBD, in?ammation associated with enteric infection and in?ammation associated with autoimmune diseases. Organisms causing gastroenteric infections include but are not d to Escherichia coli, Shigella, Salmonella, enic Vibrios, Campylobacler jejuni, Yersina enlerocolilica, Toxoplasma gondl'l', Entamoeba histolytica and Giardia lamblia. Accordingly, in n embodiments, the invention provides a method of protecting the health, organs, and/or tissues of a subject suffering from IBD, in?ammation associated with enteric infection and in?ammation associated with autoimmune diseases or at risk from developing IBD, in?ammation associated with enteric infection and in?ammation associated with autoimmune diseases.

In one embodiment of the invention, the method of treating IBD comprises treating 3O IBD without g discemable side-effects, such as signi?cant weight gain, systemic immune suppression, cushingoid appearance, enia/osteoporosis, or pancreatitis that is common of currently available IBD ents (i.e. corticosteroids, tumor necrosis factor alpha inhibitors). That is, it has been found that the method of treating according to the present invention, which provides the treatment effect, at least in part, by affecting the expression and/or activation of LANCL2 in some cells, provides the bene?cial effect without causing a signi?cant gain in weight, for example by ?uid retention, in the subject being treated, as compared to other similar subjects not receiving the treatment.

As such, the methods of the present invention can e methods of reducing in?ammation. The methods can reduce in?ammation systemically (1'.e., throughout the subject's body) or y (e.g., at the site of administration or the site of atory cells, including but not limited to T cells and macrophages). In treating or preventing ation according to the methods of the present invention, one effect that may be seen is the decrease in the number of blood monocytes or macrophages and lymphocytes in?ltrating the intestine. Another may be the increase in regulatory immune cell populations, such as CD4+CD25+FoxP3+ regulatory T-cells, or an increase in regulatory properties of lymphocytes or macrophages (e. g. increased eukin 4 (IL-4) or lL-lO or decreased TNF-d and lL-6). Another may be the sed presence of atory genes and/or adhesion molecules. The s can thus also be considered methods of affecting or altering the immune response of a subject to whom the therapy is administered. The subject may have in?ammatory bowel disease or another condition in which the immunomodulation of T cells or gulation of cellular adhesion molecules is a desired outcome.

The invention also provides methods of ng an infectious disease with the compounds or cells described herein. Non-limiting examples of such infectious diseases include viral infections, bacterial infections, and fungal infections.

Non-limiting examples of viral infections include infections from viruses in the family adenoviridae, such as adenovirus, viruses in the family herpesviridae such as herpes simplex, type 1, herpes simplex, type 2, varicella-zoster virus, n-barr virus, human cytomegalovirus, human herpesvirus, and type 8, viruses in the family papillomaviridae such as human papillomavirus, viruses in the family polyomaviridae such as BK virus and JC virus, viruses in the family poxviridae such as smallpox, viruses in the family hepadnaviridae such as hepatitis B virus, viruses in the family parvoviridae such as human bocavirus and parvovirus B19, viruses in the family astroviridae such as human astrovirus, viruses in the family caliciviridae such as norwalk virus, viruses in the 3O family viridae such as coxsackievirus, hepatitis A virus, irus, and rhinovirus, viruses in the family coronaviridae such as coronavirus, viruses in the family ridae such as tis C virus, Zika virus, yellow fever virus, dengue virus, and West Nile virus, viruses in the family togaviridae such as rubella virus, viruses in the family hepeviridae such as hepatitis E virus, viruses in the family retroviridae such as human de?ciency virus (HIV), viruses in the family orthomyxoviridae such as in?uenza virus, viruses in the family arenaviridae such as guanarito virus, junin virus, lassa virus, machupo virus, and sabia virus, viruses in the family bunyaviridae such as n- Congo hemorrhagic fever virus, viruses in the family filoviridae such as ebola virus and marburg virus, viruses in the family xoviridae such as s virus, mumps virus, parain?uenza virus, respiratory syncytial virus, human metapneumovirus, hendra virus, and nipah virus, viruses in the family rhabdoviridae such as rabies virus, unassigned viruses such as hepatitis D virus, and viruses in the family reoviridae such as rotavirus, rus, coltivirus, and banna virus, others. , among Non-limiting examples of ial infections include infections with the ia described above, in addition to Bacillus anthracis, Bacillus cereus, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis Campylobacter jejuni Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium di?icile, Clostridium perfringens, idium tetani, Corynebacterium diphtheriae, Enterococcus is, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, bacter pylori, Legionella pneumophila, pira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, ria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, la sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, ococcus pyogenes, Treponema um, Vibrio cholerae, Yersinia pestis, Yersinia enterocolitica, Yersinia pseudotuberculosis, and other species from the genera of the above-mentioned organisms.

Non-limiting examples of fungal infections include infection with fungi of the genus Aspergillus, such as Aspergillus fumigatus, which cause aspergillosis, fungi of the genus Blastomyces, such as Blastomyces dermatitidis, which cause blastomycosis, fungi of the genus Candida, such as Candida albicans, which cause candidiasis, fungi of the genus Coccidioides, which cause coccidioidomycosis (valley fever); fungi of the 3O genus Cryptococcus, such as Cryptococcus neoformans and Cryptococcus gattii, which cause cryptococcosis, dermatophytes fungi, which cause ringworm; fungi that cause fungal keratitis, such as Fusarium species, Aspergillus species, and Candida species, fungi of the genus Histoplasma, such as Histoplasma capsulatum, which cause histoplasmosis, fungi of the order Mucorales, which cause ycosis, fungi of the genus Saccharomyces, such as Saccharomyces cerevisiae,‘ fungi of the genus Pneumocyslis, such as Pneumocyslis jirovecii, which cause pneumocystis pneumonia; and fungi of the genus Sporolhrix, such as Sporothrix schenckl'l', which cause sporotrichosis.

The invention also provides methods of treating hyperproliferative disorders with the compounds or cells bed herein. Hyperproliferative disorders include conditions involving uncontrolled growth of cells, such as cancers or conditions involving the growth of tumors, as, or polyps. Non-limiting examples of hyperproliferative ers include colorectal cancer, familial atous polyposis (PAP), throat cancer, thyroid cancer, gastric cancer, cancers of the gastrointestinal tract, atic cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia, hepatocellular cancer, gastrointestinal stromal tumors, acute lymphoblastic leukemia, chronic myeloproliferative disorders, hypereosinophilic syndrome, mastocytosis, among others.

The ion also provides methods of ng an inborn error of metabolism with the compounds or cells described herein. Non-limiting examples of inborn errors of metabolism include Wilson disease, Andersen disease or other glycogen e diseases, Cystinuria, Fabry disease, adult-onset citrullinemia type II, Zellweger syndrome, branched-chain ketoaciduria, Lesch-Nyhan syndrome, Niemann-Pick disease, Fanconi- Bickel disease, von Gierke’s disease, hereditary fructose intolerance, phenylketonuria, medium chain acyl-CoA dehydrogenase de?ciency, among others.

The invention also provides methods of treating a chronic immunometabolic disease with the nds or cells described herein. miting examples of chronic immunometabolic diseases include cardiovascular disease, such as atherosclerosis, ry artery e, peripheral artery disease, pulmonary heart disease, endocarditis, myocarditis, and hypertension.

The invention also provides methods of treating an autoimmune disease, such as an atory mune disease, with the compounds or cells described herein. Nonlimiting examples of autoimmune diseases include in?ammatory bowel disease (TBD) (e. g., Crohn’s disease and ulcerative colitis), lupus, systemic lupus, Sjogren’s syndrome, rheumatoid arthritis, type 1 diabetes, sis, multiple sclerosis, and cancer- immunotherapy-induced autoimmune diseases, among others. miting examples of cancer-immunotherapy-induced autoimmune es include cancer immunotherapy- induced rheumatic diseases.

The invention also provides methods of treating chronic in?ammatory diseases with the compounds or cells described . Non-limiting examples of chronic in?ammatory diseases includes metabolic syndrome, y, prediabetes, cardiovascular disease, and type 2 diabetes, among others.

The invention also provides methods of treating in?ammatory disorders such as acute colonic diverticulitis, radiation-induced in?ammation of the gastrointestinal tract, and in?ammation of the liver, including but limited to nonalcoholic steatohepatitis (NASH), with the compounds or cells described herein. Non-limiting es of radiation-induced in?ammation of the gastrointestinal tract include radiation proctitis, radiation enteritis, and radiation proctosigmoiditis.

The invention also provides methods of treating diabetes with the compounds or cells described herein, including type 1 diabetes, type 2 es, and other types of diabetes. The term "diabetes" or "diabetes mellitus" is used to encompass metabolic disorders in which a subject has high blood sugar (i.e., hyperglycemia). Hyperglycemic conditions have various etiologies, such as the pancreas does not produce enough insulin, or cells do not d to the insulin that is produced. There are several recognized sub- types of diabetes. Type 1 es is characterized by the complete failure of the body to produce insulin or the failure of the body to produce enough n. Type 2 diabetes generally results from insulin resistance, a condition in which cells fail to use insulin properly. Type 2 diabetes sometimes co-presents with an n de?ciency. Gestational diabetes occurs when pregnant women without a previous diagnosis of diabetes develop 2O hyperglycemia. Less common forms of diabetes include congenital diabetes (due to genetic defects relating to n secretion), cystic ?brosis-related diabetes, steroid diabetes d by high doses of glucocorticoids, and several forms of monogenic diabetes (including maturity onset diabetes of the young). Monogenic diabetes asses several tary forms of diabetes caused by mutations in a single, autosomal dominant gene (as contrasted to more compleX, polygenic etiologies resulting in hyperglycemia).

The invention also es methods of treating chronic pain with the compounds or cells described herein. miting examples of c pain diseases include algia, nerve damage, migraine headaches, back pain, abdominal pain, among others.

The invention also provides methods of treating additional conditions with the compounds or cells described herein. These include c in?ammatory diseases such as chronic granulomatous disease, graft versus host disease, and tumor necrosis factor receptor associated periodic me, muscle g, such as amyotrophic lateral sclerosis, Duchenne ar dystrophy, scoliosis, and progressive ar atrophy, and others.

In one aspect, the invention provides methods of treating an autoimmune in?ammatory disease with the compounds or cells described herein. Non-limiting examples of autoimmune atory diseases include in?ammatory bowel disease (IBD), systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, psoriasis, and multiple sclerosis, among others.

The invention also provides a method of treating or ameliorating the ms in a subject diagnosed with systemic lupus erythematosus or preventing the development of e in a subject genetically predisposed to systemic lupus erythematosus. ms and tions of lupus that may be treated with the invention include but are not limited to lupus nephritis, central nervous system in?ammation, headaches, scleritis, optic neuritis, fevers, hardening of the arteries, coronary artery disease, joint pain and malar rash. The invention also es a method of treating additional forms of lupus including cutaneous lupus (discoid), drug-induced lupus, and neonatal lupus.

The conditions that can be d with the methods described herein include any condition described as capable of being treated with BT-11 in any of US Patent 9,556,146 to Bassaganya-Riera et al., US Patent 9,839,635 to Bassaganya-Riera et al., US Patent ,028,950 to Bassaganya-Riera et al., US Patent 10,201,538 to Bassaganya-Riera et al., US Patent 10,493,072 to Bassaganya-Riera et al., US Patent 10,682,349 to Bassaganya- Riera et al., US 2019/0160100 A1 to anya-Riera et al., Bissei er al. 2016 (Bissei P, Boes K, Hinckley I, Jortner BS, MagninnBissel G, Werre SR, Ehrich M, Carbo A, son C, Hontecillas R, son N, Gandour RD, Bassagariya—Riera J, Exploratory Studies With BT—l l: A Proposed Orally Active Therapeutic for Crohn‘s Disease. [m J beicai. 2016 Sep;35(5):521~9); and Carbo et a1. 2016 (Carbo A, Gandour RD, Hontecillas R Uren A I. An 9 l’hiiipson N 7 Bassaganya—Riera NN— Bis(benzimidazolylpicolinoyl)piperazine (BT41): A Novel Lanthionine tase C— Lilie 2—Based 'l7herapeutic for ln?amma‘iory Bowel Disease, J Med Chem. 2016 Nov 23,59 22):10113—10l26); Leber et al. 2018 (Leber A, Hontecillas R, inRodriguez V, Bassaganyaelkiera 3, Activation of LAN'CLZ by BT-ll Airieliorates 181) by Supporting Regulatory T Cell Stability Through lmmunometabolic Mechanisms. In?amm Bowel Dis. 2018 Aug l6;24(9):1978~1991); Leber et al, 2019 [m J i’bxicof. (Teller A, Hontecillas R, Zoccoii—Rodriguez V, Ehrich M, Davis .1, Chauhan l, BassaganyamRiera 3'. Nonciinical logy and Toxicokinetic Pro?le of an Gral lanthioriine Synthetase C—Like 2 {1.41/XN(:.1/lu2) t, BT-ll. 1m J’ Tbxicoi. 2019 Mar/Apr;3 8121964109); Leber er. al. 2019 J Immmzoi. (Leber A, Hontecillas R, Zoccoli—Rodi‘iguez V, n l, Bassaganya—Riera J. Oral Treatment with BT~11 Ameliora’tes Inflammatory Bowel Disease by Enhancing Regulatory T Cell Responses in the Gut, J Immmmi. 20l9 Apr 1;202(7):2095—2104), and Leber et al. 2020 (Leber A, Honteeillas R, ZoceolimRodriguez V, Colombel 3F, Chanhan I, Enrich M, Farinola N Bassaganya—Riera J. The Safety, rability, and Phannacokinetics Pro?le of BT41, an Qral, Gut~Restricted Lanthionine tase CmLike 2 Agonist lnvestigational New Drug for Inflammatory Bowel Disease: A Randomized, Double Blind, Placebo—Controlled Phase I Clinical Trial. zmm Bowei Dis. 2020 Mar ):643~652).

The elements and method steps described herein can be used in any combination whether explicitly described or not.

All combinations of method steps as used herein can be performed in any order, unless otherwise speci?ed or clearly implied to the contrary by the context in which the referenced combination is made.

As used herein, the singular forms "a, 77 (L 77 an, and "the" include plural referents unless the content clearly dictates otherwise.

Numerical ranges as used herein are intended to include every number and subset of numbers ned within that range, whether speci?cally disclosed or not. Further, these numerical ranges should be construed as providing support for a claim ed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 2 to 8, from 3 to 7, from 5 to 6, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

All patents, patent publications, and peer-reviewed publications (i.e., "references") cited herein are expressly incorporated by reference to the same extent as if each individual reference were speci?cally and individually indicated as being orated by reference. In case of con?ict between the present disclosure and the orated references, the present disclosure controls.

It is understood that the invention is not con?ned to the particular construction and ement of parts herein illustrated and described, but es such modi?ed forms 3O thereof as come within the scope of the claims.

EXAMPLES MOLECULAR MODELING Example 1. Molecular Modeling of LANCL2 Ligands Using previously described ligands of LANCL2 including abscisic acid and NSC61610, we determined the existence of a main small molecule binding pocket on LANCL2. These ligands were docked onto a gy model structure of LANCL2 based on the crystal structure of close family homolog LANCLl to establish ant binding residues. nd generation. From the identi?ed residues and predicted biochemical interactions, structures were generated for high af?nity LANCL2 ligands (.

Structures were ted and chemically optimized using WebMo. Structure ?les were ted in .pdb format and converted to .pdbqt format through calculation of s by Gasteiger method. Structures were docked using AutoDock Vina to con?rm binding af?nity in the de?ned binding pocket using cuboid search grid of size (21 X 21 X 21 angstrom) to e predicted binding ies and conformations of ligands. Binding af?nity was normalized to molecular weight of the ligand. Top ligands were selected for further examination of binding pose. is. nds were preliminarily ranked by lowest predicted binding af?nity normalized to molecular weight representing the most favorable g pose 2O through a minimization of total intermolecular energy, total internal energy and torsional free energy. Compounds were then prioritized based on favorable distances to critical binding residues on LANCL2.

Results. From the virtual screening and generated nds (, BT-63 an asymmetric piperazine containing small molecule ed suf?cient binding af?nity greater than the ted af?nity of ABA (positive control). In addition to binding, BT-63 provided favorable oral drug characteristics and low safety concerns. Similar molecules within the small chemotype ed near equal af?nity with slight modi?cations of chemical properties. These included BT-64, BT-65, BT-95, BT-96 and BT-99. Meanwhile changes on the opposite side of the molecule (BT-71, -72, -73, -74, -75) resulted in 3O similarly small variations in binding af?nity. The molecular modeling of this family of molecules supports the claim that the encompassed chemotype has LANCL2-binding properties.

MEDICINAL CHEMISTRY Example 2A. Synthesis of BT-63 BT-63, (2-(4-(6-(lH-benzo[d]imidazolyl) noyl) piperazin-l-yl)—l- phenylethanone, can be synthesized through a ?ve-step process. Sulfur powder was added to a mixture of benzene-1, 2-diamine and 2, 6-dimethylpyridine, then heated to 160 oC.

Reaction mixture was diluted with methanol, ?ltered and evaporated under reduced pressure to obtain 2-(6-methylpyridinyl)—1H-benzo[d]imidazole. Selinium dioxide was added to a solution of 2-(6-methylpyridinyl)-1H-benzo[d]imidazole in pyridine and heated to 110 oC. t from the reaction mixture was evaporated under reduced pressure and the obtained crude extracted with ethyl acetate. Dried solid was ?ltered and dried under vacuum to obtain 6-(1H-benzo[d]imidazolyl) picolinic acid. DIPEA was added to a solution of tert-butyl piperazine-l-carboxylate and 2-bromophenylethanone in DCM at 0 OC and stirred. Reaction mixture was diluted with ice water and organic layer was separated to obtain tert-butyl 4-(2-oxophenylethyl) piperazine-l-carboxylate. 4N HCl in 1,4-dioxane was added to a solution of utyl 4-(2-oxophenylethyl) piperazine-l-carboxylate in DCM at 0°C and stirred at RT. t was evaporated to obtain 1-Phenyl(piperazin-l-yl) ethanoneHCl as off white solid. HATU was added to a solution of 6-(lH-benzo[d]imidazolyl) nic acid, l-Phenyl(piperazin-l-yl) ethanoneHCl and DIPEA in DMF and stirred. on mixture was diluted and extracted 2O with ethyl acetate. The combined organic layer was dried with sodium sulphate and evaporated under reduced pressure. The obtained crude was puri?ed by Grace puri?cation system in silica 40um column using methanol-dichloromethane as eluent to obtain 2-(4- -benzo[d]imidazolyl) picolinoyl) piperazin-l-yl)-l-phenylethanone. 1H NMR (400 MHz, DMSO): 5 12.98 (s, 1H), 8.37 (d, J = 8.00 Hz, 1H), 8.10 (t, J = 8.00 Hz, 1H), 7.99 (d, J = 6.80 Hz, 2H), 7.72 (d, J =8.4O Hz, 1H), 7.65-7.48 (m, 5H), 7.28-7.18 (m, 2H), 3.95 (s, 2H), 3.74 (t, J = 4.80 Hz, 2H), 3.45 (t, J = 4.80 Hz, 2H), 2.68 (t, J = 4.80 Hz, 2H), 2.55 (t, J = 4.80 Hz, 2H).

Example 2B. Synthesis of BTA 3O BT-lO4-A, (2-(4-(6-(1H-benzo[d]imidazolyl) picolinoyl) piperazin-l-yl) methyl-lphenylpropan-l-one, can be synthesized h a six-step s.

Dimethyl-2,6-pyridinecarboxylate was stirred in methanol and cooled to 10 to 20 oC. Potassium ide was slowly added to reaction mass and stirred for 6 hours. After completion of reaction, solvent was evaporated to obtain 2,6-pyridinedicarboxylic acid monomethyl ester. 2,6-pyridinedicarboxylic acid monomethyl ester was stirred in n-methyl pyrrolidone and cooled to 10 to 20 oC. DIPEA, EDCHCl and HOBt were sequentially added to reaction mixture. Following solvent addition, e-1,2-diamine was added and stirred for 24 h. After completion of reaction, solvent was evaporated to obtain 6- (methoxycarbonyl)pyridine(2'-aminoacetanilide). 6-(methoxycarbonyl)pyridine(2'-aminoacetanilide) was stirred in acetic acid for minutes. Reaction mass was heated to 60 — 65 oC and d for 16 h. After completion of reaction, solvent was evaporated to obtain 6-(lH-benzo[d]imidazol yl)methylpicolinate. 6-(lH-benzo[d]imidazolyl)methylpicolinate was added to lithium hydroxide in THF/water and stirred for 7 h. After completion of reaction, solvent was ated to obtain 6-(lH-benzo[d]imidazolyl)picolinic acid.

Sodium methoxide was added to a solution of 2-bromomethyl-l-phenylpropan- l-one in dry methanol and heated at 40 0C for 4 h. Crude product was taken in toluene. zine was added to crude product and re?uxed for 16 h. After completion of reaction, solvent was evaporated under reduced pressure to obtain 2-methyl-l-phenyl(piperazin- l-yl)propanone. 6-(lH-benzo[d]imidazolyl)picolinic acid in TEA was added to a solution of 2- methyl-l-phenyl(piperazin-l-yl)propan-l-one in THF and stirred for 10 min. T3P in ethyl acetate was added and stirred for 6 h. After completion of reaction, solvent was evaporated under reduced re. t was purified to obtain (2-(4-(6-(1H- benzo[d]imidazolyl) picolinoyl) piperazin-l-yl)methyl-lphenylpropan-l-one (BT- 104-A). 1H NMR (400 MHz, DMSO-d6): 5 10.34 (s, 1H), 8.51 (d, J = 8.0 Hz, 2H), 8.45 (d, J: 7.6 Hz, 1H), 7.95 (t, J: 8.0 Hz, 1H), 7.85 (d, J: 6.4 Hz, 1H), 7.59 (d, J: 6.4 Hz, 1H), 7.57—7.50 (m, 2H), 7.42 (t, J: 7.6 Hz, 2H), 7.35—7.30 (m, 2H), 3.88 (br s, 2H), 3.53 (t, J: 4.80 Hz, 2H), 2.77 (t, J: 4.80 Hz, 2H), 2.57 (t, J: 4.80 Hz, 2H), 1.37 (s, 6H).

Example 2C. sis of BTB BT-lO4-B, 4-(6-(lH-benzo[d]imidazolyl)picolinoyl)—l-(2-oxo phenylethyl)piperazine-2,6-dione, can be synthesized through a ep process.

Dimethyl-2,6-pyridinecarboxylate was stirred in methanol and cooled to 10 to 20 oC. Potassium hydroxide was slowly added to reaction mass and stirred for 6 hours. After completion of reaction, solvent was evaporated to obtain ridinedicarboxylic acid monomethyl ester. 2,6-pyridinedicarboxylic acid monomethyl ester was stirred in n-methyl pyrrolidone and cooled to 10 to 20 oC. DIPEA, EDCHCl and HOBt were sequentially added to reaction mixture. Following solvent addition, benzene-l,2-diamine was added and stirred for 24 h. After completion of reaction, solvent was ated to obtain 6- (methoxycarbonyl)pyridine(2'-aminoacetanilide). 6-(methoxycarbonyl)pyridine(2'-aminoacetanilide) was stirred in acetic acid for minutes. Reaction mass was heated to 60 — 65 oC and stirred for 16 h. After completion of reaction, solvent was evaporated to obtain 6-(lH-benzo[d]imidazol yl)methylpicolinate. 6-(lH-benzo[d]imidazolyl)methylpicolinate was added to lithium hydroxide in THF/water and stirred for 7 h. After tion of reaction, solvent was ated to obtain 6-(lH-benzo[d]imidazolyl)picolinic acid.

Piperazine-2,6-dione in TEA was added to a solution of 6-(lH-benzo[d]imidazol- 2-yl) picolinic acid in THF and stirred for 10 min. T3P in ethyl acetate was then added and d for 2 h. After completion of reaction, reaction mixture was extracted with ethyl e and washed with water to obtain l-(6-(lH-benzo[d]imidazolyl)picolinoyl)- piperazine-2,6-dione. o-l-phenylethan-l-one in DIPEA was added to a solution of l-(6-(lH- benzo[d]imidazolyl)picolinoyl)-piperazine-2,6-dione in dry DCM:DMF and stirred for 2O 2 h. After completion of reaction, reaction e was ted with ethyl acetate and washed with water. al was dried and puri?ed to obtain 4-(6-(lH-benzo[d]imidazol- 2-yl)picolinoyl)—l-(2-oxophenylethyl)piperazine-2,6-dione (BT-lO4-B). 1H NMR (400 MHz, DMSO-d6): 5 12.88 (s, 1H), 8.46 (d, J = 7.60 Hz, 1H), 8.20 (t, J = 8.00 Hz, 1H), 8.07 (d, J: 8.00 Hz, 2H), 7.84 (d, J: 7.60 Hz, 1H),7.75-7.71 (m, 2H), .56 (m, 3H), 7.30—7.24 (m, 2H), 5.25 (s, 2H), 4.86 (d, J = 4.40 Hz, 4H). Purity by HPLC: 98.82%, MS: m/z 452.26 (M-H).

RECEPTOR BINDING Example 3. Surface Plasmon Resonance Binding to LANCLZ 3O Introduction Virtual screening and in silico experimentation are valuable means to identify and prioritize scaffolds of interest when designing new small molecule ligands for a therapeutic . To validate these ?ndings, numerous in vitro methods exist to determine the affinity of a small molecule to the protein of interest. One particular method is surface plasmon resonance, which is ability to estimate steady state binding by ?owing a suspension of ligand over immobilized puri?ed protein. This method was used to te prospective LANCL2 ligands.

Methods LANCL2 production and puri?cation. LANCL2 was cloned into E. coli, ampli?ed and transfected in Pichia pastoris. P. pastoris was plated onto adenine selective media.

Stable transfected colonies were selected and grown within YPD broth at 30°C for 24 hours, 240 RPM shaking. Starter culture was used to ate base media (1% yeast extract, 2% peptone, l% ol, 2% yeast nitrogen base) containing biotin and buffered with potassium phosphate. Inoculated base media was incubated for 48 hours at 30°C, 240 RPM. P. pastoris was then pelleted by centrifugation and resuspended in expression media (1% sorbitol, 2% yeast nitrogen base) containing biotin and buffered with potassium phosphate. Culture was induced daily for protein production through addition of methanol and incubated for a total of 4 days at 28°C, 240 RPM. After incubation, cells were pelleted by centrifugation and lysed by sonication. Recombinant LANCL2 protein was puri?ed by fast protein liquid chromatography (AktaPrime) using immobilized metal af?nity chromatography. Fractions of protein were eluted in 1 mL aliquots and evaluated for LANCL2 content.

Surface plasmon resonance. A Biacore T200 was used to evaluate binding to the LANCL2 protein. Protein was lized onto the CM5 sensor chip. LANCL2 was diluted in 10 mM sodium acetate buffer at pH 4.0 and immobilized onto the ?ow cell to a level of ~3700 RU, using standard amine coupling chemistry. Based on the immobilized response , theoretical Rmax values were ated. The Rmax values assume l:l ction mechanism. Overnight cs were performed for all analytes binding to the immobilized proteins. The kinetics experiments were performed in the presence of running buffer + 1% DMSO. The ?ow rate of all ons was maintained at 50 uL/min. Analyte concentrations were 0 uM, 2.5 uM, 5 uM, 10 uM, 20 uM, and 40 uM.

Results BT-63 bound to the LANCL2 protein with a K13 of 2.74 uM. Surface plasmon resonance tes the predicted binding of this family of molecules of the described Markush structure to LANCL2. In comparison, BT-62, a le identical to BT-63 except for the methylene between the piperazine and yl in BT-63, had a KD of 18.0 uM. The addition of the methylene provides increased af?nity for LANCL2 with a KD in similar range to that of BT-l 1, an LANCL2 targeting therapeutic.

EXPERIMENTAL STUDIES Example 4. Pharmacokinetics of BT-63 Introduction In addition to induction of immune effects, a ceutical compound must also reach compartments within the body at adequate concentrations to provide therapeutic bene?t. r, early evaluation of the pharmacokinetics is needed to determine the desired route of administration.

Methods C57BL/6 mice were dosed orally with 10 and 40 mg/kg of BT-63 by oral gavage of methylcellulose solution containing BT-63. After gavage, blood was collected at 0.5, 1, 2, 4, 6, 8, 12 and 24 hours after treatment. Plasma was collected from blood by centrifugation. BT-63 was extracted from plasma and quanti?ed by LC-MS/MS.

Results Oral BT-63 (10 mg/kg) resulted in a maximum plasma concentration of 585 ng/mL and an area under the curve re of 1055 hr*ng/mL (. Oral dosing with 40 mg/kg resulted in a sharp increase in maximum plasma tration (4465 ng/mL) as 2O well as exposure (11552 ng/mL). The s indicate that BT-63 is viable as an oral therapeutic small molecule. Based on these results, BT-63 is a highly bioavailable compound in st to other LANCL2 targeting compounds such as BT-11. Notably, at equal oral doses BT-63 has a >100-fold increase in maximum plasma concentration in mice compared to BT-11 (4.9 ng/mL at dose of 10 mg/kg).

Example 5. Identification of immune effects in CD4+ T cells uction Central to the pathogenesis of many autoimmune diseases is the dysfunction of CD4+ T helper cells. These cells are important in maintaining the health of an individual, amplifying immune responses and promoting homeostasis. However, in the case of autoimmune and in?ammatory e, CD4+ T helper cells can become overactive, activated in the absence of stimuli or unable to resolve in?ammation. In these scenarios, therapeutics that can mitigate or prevent ation are valuable treatments for the management of disease. In this end, we validated the functional therapeutic potential of BT-63, BTA (see ), BT-lO4-B (see ), and BTC (see ), LANCL2-binding ligands, in this cell type.

Methods Cell e. Spleens were excised from 6 mice and assessed in wild-type and LANCL2 de?cient states. Spleens were crushed between the frosted ends of cope slides and ?ltered to provide a cellular suspension. Red blood cells were lysed through hypotonic lysis. Remaining cells were washed and ?ltered. CD4+ T cells were ed within the suspension using magnetic sorting based negative selection. Cells were collected and plated within 96 well plates coated with anti-CD3 and cultured in the presence of BT-63 at O, 0.1, l and 10 micromolar or BT-lO4A, BT-lO4B, or BT-lO4C at 0.1 micromolar for 48 h. During the last 6 h of culture, cells were stimulated with phorbol 12-myri state- 1 3 te (PMA) and ionomycin.

Immunological analysis. Cells were collected from 96 well plates and stained with a cocktail of antibodies for immunophenotyping by ?ow cytometry. Culture supernatant was collected and assayed for cytokine concentrations by cytometric bead array. Data was captured on a BD FACS Celesta and analyzed using FacsDiva.

Results BT-63 reduced proportions of IFNy-producing and TNFd-producing CD4+ T cells within wild-type cell culture and sed tions of lLlO+ FOXP3+ CD4+ T cells (. In the absence of LANCL2, these effects were lost. From a downregulation of in?ammatory cytokines, these results indicate that BT-63 may function as an activator of LANCL2. Given the loss of activity in LANCL2 de?cient cells, BT-63 has dominant mechanistic actions h the LANCL2 pathway. Combined with results of in silica and in vitro binding, the actions through the LANCL2 y are likely a result of direct binding to LANCL2.

Additionally, BT-63 provides greater immune effects compared to BT-62 (). While BT-63 provides a signi?cant increase in FOXP3+ cells and a signi?cant 3O decrease of IFNy+ cells, BT-62 does not induce either change at the tested concentrations.

Therefore, the alkylene linker between the piperazine and carbonyl groups is critical for higher binding af?nity to LANCL2 and greater immunological ef?cacy.

BT-lO4-A, BT-lO4-B, and BT-lO4-C were observed to cantly decrease the proportions of IFNy+ CD4+ T cells, with -A and BT-lO4-B also signi?cantly decreasing TNF01+ CD4+ T cells (). Out of the three compounds, BT-lO4-B was observed to have the greatest numerical effect on IFNv+ cells, and BT-lO4-A was observed to have the greatest numerical effect on TNFOL+ cells, though the only statistical differences observed were relative to vehicle.

Example 6. Use of BT-63 in a NOD Mouse Model of T1D Introduction Type 1 Diabetes (TlD) is an autoimmune e in which the immune system destroys insulin-producing pancreatic cells necessitating life-long insulin therapy through injections or pumps. With current treatments, glycemic control is dif?cult ing in prolonged periods of hyperglycemia and dysregulated glucose lism that contribute to organ damage and co-morbidities ness, kidney failure, cardiovascular disease, loss of extremities). Currently no treatments are approved for the prevention of disease progression at onset (i.e. restoring immunological tolerance to diabetes-associated antigens to allow regrowth of pancreatic beta cells) and very few are approved to assist in glycemic control. LANCL2 is a potent receptor that contributes to immune ses, ar metabolism and survival of cells. With this three-fold mechanism, LANCL2 is an attractive target for acute and long-term maintenance therapy in TlD.

Methods 2O NOD model. Non-obese diabetic (NOD) ShiLt mice were used in this study. NOD mice have numerous genetic mutations that enable the spontaneous onset of hyperglycemia and pancreatic pathologies associated with TlD. Mice d into the experiment at 9 weeks of age and were red for a 12 period. Mice were treated daily with vehicle, 10 mg/kg BT-63, or 20 mg/kg BT-63 by oral . Once weekly blood samples were collected from the tail vein to be tested for glucose concentration by glucometer. Mice were euthanized after 12 weeks for collection of blood and organs for immunological g.

Treatment stration. BT-63 was prepared within a 0.5% methylcellulose (12- CF) solution. Dosage used was 10 or 20 mg/kg red once daily. Mice were 3O weighed on a weekly basis to update dosage formulation. Dosage was calculated based off mean body weights for each gender. Oral dosage was red by orogastric gavage of dosage in 0.2 mL volume.

Immunological analysis. Blood was collected by cardiac puncture into heparinized tube. Plasma was separated following centrifugation and assayed by LumineX for cytokine and hormones related to the development of TlD. Spleens were excised, crushed and d to provide a cellular suspension. Red blood cells were lysed. Cells were d with mixtures of extracellular (CD45, CD3, CD4, CD8, CD19, NKl.l, CD25, F4/80, CDllb, Grl, CX3CRl, CD64) and intracellular (Tbet, BCL6, FOXP3, IFNy, IL2l, lLlO) antibodies in a sequential live staining in 96-well plates in preparation for ?ow cytometry.

Data was captured on a BD FACS Celesta and analyzed using FacsDiva.

Results Oral BT-63 protected against the development of lycemia. Mice treated with BT-63 had signi?cantly lower blood glucose levels from 19 weeks of age through the end of the experiment (. The percentage of hyperglycemic mice was reduced by 50% in the 20 mg/kg group compared to the vehicle treated group while the HbAlc at 21 weeks of age was reduced in both 10 mg/kg and 20 mg/kg dose groups. In the spleen, oral BT-63 increased the CD4--ILlO+ to CD4+IFNy+ ratio while decreasing T follicular helper (Tfh) cells (BCL6+ILZl--) and increasing PDl+ CD4+ T cells (. BT-63 also increased plasma levels of insulin and C-peptide and reduced MCP-l and TNF (. The data supports BT-63 as a tative and restorative therapy in TlD.

Example 7. Use of BT-63 in a TLR—induced mouse model of SLE Introduction 2O Systemic lupus erythematosus (SLE) is a systemic mune disease that can cause damage to kidneys, cardiovasculature, and joints. Due to self-tolerance, standard apoptosis of cells s in the generation of r antigens that are processed without immune response in non-SLE af?icted individuals. However, in SLE, the immune system ds to these antigens generating antibodies to double-stranded DNA (dsDNA) and other nuclear antigens that form immune xes that can deposit throughout the body and cause unwarranted immune responses. Disease is currently treated with steroids, biologics and other suppressants with a high likelihood of detrimental side effects and weakening of the body’s immune system. Treatment through LANCL2 activating ligands could restore immunological tolerance and reduce the production of self-targeted antibodies.

Methods Resiquimod model. Resiquimod was prepared in a 1:3 ethanol:acetone e to provide 85 micrograms of imod to each mouse. Resiquimod solution was well mixed and applied to the ear of C57BL6 mice three times weekly over a 2-week period.

Mice were monitored for signs of disease daily.

Treatment administration. BT-63 was prepared within a 0.5% methylcellulose (12- CF) on. Dosage used was 20 mg/kg delivered once daily. Mice were weighed on a weekly basis to update dosage ation. Dosage was calculated based off mean body weights for each gender. Oral dosage was delivered by orogastric gavage of dosage in 0.2 mL volume.

Immunological analysis. Blood was collected by cardiac puncture into EDTA tube.

Plasma was separated following centrifugation and assayed by ELISA for anti-dsDNA antibodies. Urine was collected for assay for albumin content to test for kidney function.

Spleens were excised, crushed and ?ltered to provide a cellular suspension. Red blood cells were lysed. Cells were labeled with mixtures of ellular (CD45, CD3, CD4, CD8, CD19, NK1.1, CD25, F4/80, CD1 1b, Grl, CX3CR1, CD64) and intracellular (Tbet, BCL6, FOXP3, IFNy, 1L6, ILlO) antibodies in a sequential live ng in 96-well plates in ation for ?ow try. Data was captured on a BD FACS Celesta and analyzed using FacsDiva.

Results Oral BT-63 reduces the concentration of anti-dsDNA dies in plasma after 2 2O weeks of treatment and es reduction of urine albumin content resulting in two bene?ts speci?c to the pathogenesis of SLE (. Immunological differences are present within the spleen in the form of reduced IFNy+ CD8+ T cells and IL6+ cells indicating decreased in?ammation.

Example 8. Use of BT-63 in a Chronic Model of IBD Introduction Crohn’s disease and ulcerative colitis are chronic diseases with ic s of unresolved in?ammatory ?ares resulting in progressive damage to the intestinal mucosa.

The loss of Mdrla in mice impairs the ability of epithelial cells to correctly process and 3O ef?ux waste products leading to spontaneous colitis. The colitis in these mice is chronic and penetrates throughout the layers of the intestine. The Mdrla-/- model is therefore an ideal model to test the chronic administration of a therapeutic for the induction and maintenance of decreased disease severity.

Unlike other genetic models of disease that can generate immunocompromised mice, Mdrla-/- mice are immunocompetent, with the on d impacting the ar ability to ef?ux molecules and prevent cellular stress. The accumulation of waste and cellular by-products leads to a dysregulation of the epithelial cell lifecycle and sed secretion of in?ammatory cytokines and chemokines. Thus, it provides a chronic and spontaneous onset of disease with primary initiating events occurring within the epithelium. onally, the MDR1 gene is an emerging risk allele for IBD and affect the siveness to glucocorticoid-based ents. The ability of BT-63 to provide therapeutic ef?cacy in the absence of this gene is an important indication of robustness in the presence of genetic abnormalities and indicate an ability for human translation in a surgery-sparing context.

Methods MDRla-/- model. Mice de?cient in MDRla spontaneously develop colitis.

MDRla-/- began receiving BT-63 treatment (oral, 20 mg/kg) at 6 weeks of age and continued treatment until 10 weeks of age. Mice were weighed and scored weekly.

Treatment administration. BT-63 was prepared within a 0.5% methylcellulose (12- CF) solution. Dosage used was 20 mg/kg delivered once daily. Mice were weighed on a weekly basis to update dosage formulation. Dosage was calculated based off mean body weights for each gender. Oral dosage was delivered by orogastric gavage of dosage in 0.2 mL volume.

Flow try. Colons were collected into RPMI/FBS buffer containing collagenase (300U/mL) and DNase (50U/mL) for digestion. Tissues were ed for 60 minutes under stirring at 37°C. Resultant cellular suspensions were ?ltered through 100 um strainers, centrifuged (300 X g, 8 min), and washed in fresh RPMI. Following ?ltration of the resulting single cell suspensions, immune cells were puri?ed by Percoll gradient of cell-containing 40% Percoll overlayed onto 70% Percoll solution. After centrifugation, hase was collected and washed to obtain enriched colonic lamina propria cell fractions. Cells were labeled with mixtures of extracellular (CD45, CD3, CD4, CD8, CD19, NK1.1, CD25, F4/80, CD1 1b, Grl, CX3CR1, CD64) and intracellular (Tbet, RORyT, FOXP3, IFNy, 1Ll7, ILlO) antibodies in a sequential live staining in 96-well plates. Data was acquired using a FACS Celesta ?ow cytometer with FAC SDiva software.

Results Oral BT-63 treatment decreases the disease ty of Mdrla-/- mice. Disease activity in this model of colitis is a summarized score of the weight loss, presence and severity of rectal ng, fecal consistency, ms of pain and l behavior of a mouse. BT-63 reduced disease ty throughout the course of the challenge with a maximal observed reduction by 90% in week four of treatment. Within the colonic lamina propria, BT-63 signi?cantly alters the tions of immune cells (. In particular, BT-63 reduces the proportions of Th1, Th17 and neutrophils, three main subsets of cells responsible for the in?ammation in the colonic mucosa. Proportions of regulatory CD4+ CD25+ T cells were increased in the colon. Meanwhile, splenic Th1 cells were decreased and splenic CD25+ Tregs were increased after BT-63 treatment (). The y of BT-63 in a highly translational mouse model of IBD ghts the use of BT-63 as an oral therapeutic for CD and UC.

Example 9. Efficacy of BT-63 in a Model Viral Infection The activation of LANCL2 by speci?c ligands may modulate the immune response, prevent the entry or replication processes of the virus to reduce viral burden and promote the repair and homeostasis of the local tissue. To validate the ef?cacy of BT-63, we will use a mouse model of in?uenza virus infection.

Methods Mouse model. Eight- to ten-week old wild type C57BL/6 mice were anesthetized by iso?urane inhalation. Mice were infected with in?uenza A (H1N1) intranasally at a 2O challenge titer of 350 pfu/mouse [37]. Mice were treated daily with BT-63 at a dose of 20 mg/kg orally via gavage. Mice were weighed and scored daily over 12 days. Mice were euthanized at days 12 to measure immune ses by ?ow cytometry in the lungs.

Flow Cytometry. Lungs will be chopped into small pieces and ted into RPMI/FBS/CaCl2 buffer containing collagenase (3OOU/mL) and DNase (50U/mL) for digestion. Tissues will be digested for 60 to 90 minutes under stirring at 37°C. Resultant cellular suspensions will be ?ltered through 100 um strainers, fuged (300 x g, 8 min), and washed in fresh RPMI. Red blood cells will be lysed by hypotonic lysis and removed by ?ltration. Cells will be washed and plated for ?ow try staining. Cells will be labeled with mixtures of extracellular (CD45, CD3, CD4, CD8, CD19, NK1.1, CD25, F4/80, CD11b, CD11c, Grl, CX3CR1, CD64, SiglecF, Ly6C) and intracellular (Tbet, RORvT, FOXP3, IFNv, 1L6, IL10, IFNb) antibodies in a sequential live ng in 96-well . Data will be acquired using a FACS Celesta ?ow cytometer with FACSDiva software.

Results Oral BT-63 protects t mortality and decreases the presentation of in?uenza- associated symptoms (). BT-63 signi?cantly reduces disease activity indeX from day 3 post-infection through day 12 post-infection when compared to vehicle treated animals. Additionally, BT-63 increases survival by over 50% ed to vehicle.

Immunologically, BT-63 increases lLlO+ CD8+ T cells and alveolar macrophages at day 12 post-infection in the lung (). These two cell types have previously been characterized as important cells in the recovery from pulmonary viral infections. Further, BT-63 treated mice have decreased proportions of TNFOH‘ CD4+ T cells and neutrophils in the lungs, cell types associated with tissue damage. Together, the data s the protective effects of BT-63 in viral infections.

Example 10. Ex Vivo Treatment CD4+ T Cells for Treatment of In?ammatory Disease BT-63 through immunometabolic signaling changes the phenotypic pro?le of cells in vitro and immune ses in vivo. In particular, BT-63 shapes CD4+ T cells to increase expression of FOXP3, increase suppressive capacity, and increase stability of these regulatory cells in in?ammatory conditions. Adoptive transfer of cells treated ex vivo with BT-63 is therefore ial in treating in?ammatory diseases and disorders with inadequate CD4+ T cell responses, such as in?ammatory bowel disease, graft versus host 2O e, and others described herein.

Methods Na'1've CD4+ T cells can be isolated from the spleens of mice by magnetic g.

The ed cells can be incubated in anti-CD3/anti-CD28 coated 96 well plates in Treg entiation media. The Treg differentiation media can be 's Modi?ed Dulbecco's Medium (HVIDM) media (ThermoFisher Scienti?c) supplemented with fetal bovine serum, HEPES, penicillin/streptomycin, L-glutamine, and differentiation agents. The Treg entiation agents can be 10 nM all-trans-retinoic acid and 5 ng/mL TGF-B. Additional experiments can be conducted comparing differentiation in the Treg differentiation media 3O with and without the addition of 10 ng/mL IL-2 or IL-l2. Cells can be incubated with vehicle, 10 nM, or 100 nM BT-63 in differentiation media for 48 hours prior to assay.

Prior to assay, cells can be stimulated with PMA and ionomycin for 6 hours.

In transfer experiments, donor spleens can be crushed and enriched for CD4-- on by magnetic sorting. CD4+CD45RBhiCD25- (Teff) and CD4+CD45RB1°CD25-- (Tregg) cells can be sorted by a FACSAria cell sorter. Isolated Tregs can be cultured for 12 h in the presence of vehicle or BT-63 (100 nM). Isolated Teff can be cultured for 12 h in vehicle. Based on indicated experimental group, Rag2-/— ent mice can receive 4x105 Teff and 1x105 Treg cells from vehicle or BT-63 treated groups by intraperitoneal injection. Mice can be weighed and scored weekly until euthanasia at 5 weeks post- transfer.

Colonic lamina propria lymphocytes and cultured cells can be plated in 96 well plates (6x105 cells/well) and processed for phenotyping by ?ow cytometry as previously described. Brie?y, cells can be incubated with ?uorochrome conjugated antibodies to extracellular s: CD45, CD4, CD3, CD25, CD8. Samples needing a secondary staining can be incubated with secondary antibodies, or streptavidin-conjugated ?uorochrome. The samples can then be ?xed and permeabilized. Cells can be incubated with antibodies to intracellular markers: Tbet, IFNy, lLlO, FOXP3, lLl7, RORyT. Data can be acquired with a BD FACS Celesta ?ow cytometer and analyzed using FACS Diva software (BD Pharmingen).

Results With the importance of CD25+ FOXP3+ regulatory CD4+ T cells to the ef?cacy of BT-63, we aim to con?rm the direct effect of BT-63 on their differentiation and ability to retain phenotype in in?ammatory conditions. Na'1've CD4+ T cells can be differentiated 2O into Tregs in vitro in the ce or absence of 1L-2 according to the methods described above. We predict BT-63 treatment (100 nM) will signi?cantly increase the establishment of a CD25+ FOXP3+ e in the absence of IL-2, a ence that will further be accentuated by the addition of lL-2. We predict that at concentrations as low as 10 nM, BT-63 will induce signi?cantly more CD25+ FOXP3+ cells in the ce of lL-2. We predict only low levels of a mixed CD25+ Tbet+ subtype will be observed under these differentiation conditions, and this will not be statistically altered by BT-63. We predict BT-63 will retain signi?cantly higher levels of CD25+ FOXP3+ cells in lL-l2-treated samples. This will contrast with the suppression of CD25+ FOXP3+ cells in treated samples in the absence of BT-63. The addition of lL-l2 will also induce an increase in 3O CD25+ Tbet+ cells in all groups, though BT-63 will provide a dose-dependent protection t this mixed subset.

To identify signaling pathways modulated by BT-63 in vivo, we will isolate colonic CD4+ T cells from vehicle- and BTtreated /— mice at presentation of s at 10 weeks of age. In CD4+ T cells, oral BT-63 treatment will result in signi?cantly higher expression of Stat5a and Foxol, two members of the 1L-2 signaling pathway. Meanwhile, expression of Pten and Phlppl will se. In vilro, STATSa will be phosphorylated in a greater ratio in BTtreated samples in the base Treg differentiation media and also in the Treg differentiation media supplemented with either 1L-2 or IL-12. FOXOl will similarly be ed in both the base Treg differentiation media and the Treg differentiation media containing 1L-2, but not in the Treg differentiation media containing 1L-12. Cells will also be differentiated in the presence of inhibitors for PTEN 0) or STATS (STATSi). In the Treg differentiation media containing both IL-2 or IL-12, the addition of STATSi will prevent the effects of BT-63 on CD25+ FOXP3+ and CD25+ Tbet+ cells. In contrast, SFl67O only will prevent effects of BT-63 on CD25+ Tbet+ cells in 1L-2 containing media.

Rag2-/— mice lack mature T and B lymphocytes. Therefore, these mice fail to develop mechanisms of self-tolerance, microbial homeostasis, and overall immunoregulation. Transfer of naive CD4+ T cells into Rag2-/— mice induces intestinal in?ammation resulting from the absence of these mechanisms through in vivo expansion of the transferred cells and differentiation into in?ammatory phenotypes in a manner similar to those experienced in active in?ammatory autoimmune diseases including but not limited to in?ammatory bowel disease. We hypothesize that the transfer of regulatory cells treated ex vivo with BT-63 will confer mechanisms of tasis and immunoregulation to recipient animals.

The adoptive transfer of Tregs treated ex vivo with BT-63 (100 nM) will decrease overall disease severity and will provide maintenance of immune bene?ts up to the tested limit duration of 5 weeks ransfer. In addition to overall improvement of disease, ex vivo treatment of Tregs with BT-63 will result in changed phenotypes of colonic lamina propria cells. In BT-63 -treated Treg groups, lFNy-producing and lL-l7+ RORyT+ CD4+ T cells will reduce. Meanwhile, CD25+ Tregs will increased, indicating an increased stability and increased ability to serve as a founder population of tory cells. Further, interaction with the 1L-2/STAT5 signaling axis will promote important changes in the cytokine and ine microenvironment that amplify the effects of transferred cells.

These results will show that the s of BT-63 on immune cells when 3O administered in vivo will be replicated when ng immune cells ex vivo. We predict that administering the prepared cells of the invention to an animal will be effective in treating any of the conditions bed herein beyond in?ammatory diseases such as IBD.

Example 11. Use of BT-63 in a c Mouse Model of SLE Introduction Systemic lupus erythematosus (SLE) is a ic autoimmune e that can cause damage to kidneys, cardiovasculature, and . SLE is a result of a complex interaction of genetic factors that results in immunological disease manifested primarily through a tion of auto-antibodies. One preclinical model aimed at captured these complex factors is the NZB/W F1 model. The F1 cross of NZB and NZW mice results in mice with autoimmunity of progressive severity. This autoimmunity shares many common features with human SLE including the generation of anti-nuclear antibodies, kidney damage and ed type I interferon responses.

Methods NZB/W F1 model. Twenty-four-week-old, female NZB/W F1 mice were randomized into vehicle or BTtreated arms based on baseline urine protein levels (11 = 10). BT-63 was orally administered daily at 20 mg/kg for 12 weeks. Mice were weighed on a weekly basis to update dosage formulation. Dosage was calculated based off mean body weights.

Immunological analysis. Blood was ted by cardiac puncture into EDTA tube.

Plasma was separated following centrifugation and assayed by ELISA for anti-dsDNA 2O antibodies and lFN—d. Urine was collected to assay for protein content to test for kidney function. Spleens were excised, d and ?ltered to provide a ar suspension. Red blood cells were lysed. Cells were labeled with mixtures of extracellular (CD45, CD3, CD4, CD8, CD19, CD138, CD25, MHCII, CD11b, CD11c, CXCR3, IgD, IgM) and intracellular (BCL6, FOXP3, 1L21, 1L6, lLlO) antibodies in a sequential live staining in 96-well plates in preparation for ?ow cytometry. Data was captured on a BD FACS Celesta and analyzed using FacsDiva.

Results Orally administered BT-63 protected mice from weight loss and prevented worsening of proteinuria score from baseline (). BTtreated mice had reduced levels of plasma anti-dsDNA and lFN—d levels at 36 weeks of age (12 weeks of treatment).

BT-63 ent resulted in moderate reduction of follicular B cells and plasma cells in the spleen, in particular CXCR3+ plasma cells, a main pathogenic subset in lupus.

However, greater reductions in 1L6+ myeloid cells, CD25+ FOXP3+ regulatory CD4+ T cells and ILZl+ BCL6+ follicular helper T cells were observed relative to vehicle (FIG.

Example 12. Use of BTB in a Model of Nonalcoholic Steatohepatitis (NASH) Introduction NASH is a progressive chronic liver disease that af?icts over 140 million people worldwide with total health care costs exceeding $8 billion annually in the US alone. No current therapeutics are ed for NASH. While a ible condition, failure to effectively treat NASH results in higher risk of hepatocellular carcinoma, liver failure, and cardiac death. With a multitude of hepatic and extrahepatic factors, NASH is a x disease. Yet, many therapeutics in development fail to address all three main areas of dysregulation, comprised of metabolic, in?ammatory, and ?brotic factors.

Methods WD-induced model. C57BL/6 mice were administered bi-weekly intraperitoneal ions of 0.5 uL/g CCl4 to induce steatohepatitis for 4 weeks. Mice were treated daily, in a therapeutic manner after 2 weeks of injections. Treatment with BT-lO4-B (20 mg/kg) or vehicle control occurred by oral gavage. Dosage was calculated based off mean body 2O Analysis. Livers were excised and weighed. Sections of livers were excised and stored in buffered formalin for Sirius red staining or snap frozen for assessment of s.

Severity of ?brosis was assessed by g of Sirius red stained liver by microscopic examination.

Results Oral BT-lO4-B d liver weights (A) and fibrotic scoring (B) after 2 weeks of treatment, indicating the ability to improve liver in?ammation and ?brosis in the context of NASH. 3O Example 13. Use of BTB in a Mouse Model of Rheumatoid Arthritis Introduction toid tis (RA) causes severe in?ammation of joints leading to loss of mobility and intense pain. The underlying immunology of synovial in?ammation is complex, involving the lay of myeloid cells, T cells, fibroblasts and other ural cells of the synovium. High expression of INF and IL-6 are central to the pathogenesis of RA, with additional contributions by lL-lB, 1L-12, lL-l7, 1L-21, 1L-23, MCPl, and TGF- B. Together these cytokines can lead to leukocytic recruitment, bone remodeling, pannus formation, oxidative stress and hyperplasia of the joint lining.

Methods . Six-week-old C57Bl/6 mice were immunized with 200 ug of chicken collagen emulsi?ed in complete Freund’s adjuvant by intradermal injections at the base of the tail. Mice were treated with 20 mg/kg of BT-lO4-B or vehicle, daily for four weeks.

Immunological analysis. Spleens were excised from mice. Tissues were d and ?ltered to provide a cellular suspension. Red blood cells were lysed. Cells were labeled with mixtures of extracellular (CD45, CD3, CD4, CD8, CDllb, CDllc) and ellular (lFNy, 1Ll7, TNF) antibodies in a sequential live staining in l plates in preparation for ?ow cytometry. Data was captured on a BD FACS Celesta and analyzed using FACSDiva.

Results Oral -B signi?cantly reduced the tion of - CD4+ T cells (A), 1FNy+ CD4+ T cells (B), and TNF+ CD11b+ - myeloid cells (C) in the spleens of mice with collagen induced arthritis in comparison to e treated controls. This tes the ability to BT-lO4-B and related compounds in the treatment of rheumatoid arthritis.

Example 14. Use of BTB in a Genetic Mouse Model of SLE Introduction Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that can cause damage to kidneys, cardiovasculature, and joints. SLE is a result of a complex interaction of genetic factors that results in immunological disease manifested primarily through a generation of auto-antibodies. One preclinical model aimed at captured these complex factors is the NZB/W Fl model. The F1 cross of NZB and NZW mice results in 3O mice with autoimmunity of progressive severity. This autoimmunity shares many common features with human SLE including the generation of anti-nuclear antibodies, kidney damage, and elevated type I interferon responses.

Methods NZB/W F1 model. -four-week-old, female NZB/W F1 mice were randomized into vehicle or BTB treated arms based on baseline urine protein levels.

BTB was administered daily at 20 mg/kg for 12 weeks. Mice were weighed on a weekly basis to update dosage formulation. Dosage was calculated based off mean body weights.

Immunological analysis. Spleens were excised, crushed and ?ltered to provide a cellular sion. Red blood cells were lysed. Cells were d with mixtures of extracellular (CD45, CD3, CD4, CD8, CD25) and intracellular (IL21, lLl7, FOXP3) antibodies in a sequential live staining in 96-well plates in preparation for ?ow try.

Data was captured on a BD FACS Celesta and analyzed using FACSDiva.

Results Oral BTB signi?cantly reduced the proportion of IL-17+ CD4+ T cells ( 18A) and lLZl+ CD4+ T cells (B) while signi?cantly increasing the proportion of CD25+ FOXP3+ CD4+ T cells (C) in the spleens of NZB/W F1 mice in ison to vehicle treated controls. This indicates the ability to BTB and related compounds in the treatment of SLE.

Claims (39)

1. A nd of formula Z-Y-Q-Y' or a pharmaceutically acceptable salt or ester thereof, wherein: Z-Y-Q-Y' is: Q is piperazine-1,4-diyl or tuted piperazine-1,4-diyl; A1, A2, A3, A11, A12, A13, A18, A20, and A22 are each independently C(R3); A4, A6, A10, A19, and A21 are each independently C(R3) or N; A5 is N(R3), C(R3)2, O, or S; R1 is ally substituted alkylene optionally containing one or two heteroatom(s); and R3 in each instance is independently a hydrogen atom, a halogen atom, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, hydroxy, y, ally substituted alkyloxy, optionally substituted loxy, optionally substituted alkynyloxy, ally substituted cycloalkyloxy, optionally substituted cycloalkenyloxy, mercapto, optionally substituted alkylthio, optionally substituted alkenylthio, optionally substituted alkynylthio, ally substituted alkylsulfinyl, optionally substituted alkylsulfonyl, optionally substituted alkylsulfonyloxy, optionally tuted cycloalkylthio, optionally substituted cycloalkylsulfinyl, optionally substituted cycloalkylsulfonyl, optionally substituted cycloalkylsulfonyloxy, optionally substituted cycloalkenylthio, optionally substituted cycloalkenylsulfinyl, optionally substituted lkenylsulfonyl, optionally substituted cycloalkenylsulfonyloxy, ally substituted amino, acyl, optionally substituted alkyloxycarbonyl, optionally substituted alkenyloxycarbonyl, optionally substituted alkynyloxycarbonyl, optionally substituted aryloxycarbonyl, optionally substituted carbamoyl, optionally substituted sulfamoyl, cyano, nitro, optionally substituted aryl, optionally substituted y, optionally substituted io, optionally substituted arylsulfinyl, optionally substituted arylsulfonyl, optionally substituted arylsulfonyloxy, optionally substituted heteroaryl, optionally substituted heteroaryloxy, optionally substituted heteroarylthio, optionally substituted heteroarylsulfinyl, ally tuted heteroarylsulfonyl, optionally substituted heteroarylsulfonyloxy, or an optionally substituted non-aromatic heterocyclic group; each optionally tuted alkyl, optionally substituted alkyloxy, optionally substituted alkylthio, optionally substituted alkylsulfinyl, optionally substituted alkylsulfonyl, optionally substituted alkylsulfonyloxy, and optionally substituted alkyloxycarbonyl, when substituted, is independently substituted with one to three substituent(s) selected from the group consisting of lkyl, alkylene optionally ning one or two heteroatom(s), hydroxy, oxo, alkyloxy optionally substituted with a substituent group A at one to three position(s), to, alkylthio, a halogen atom, nitro, cyano, carboxy, alkyloxycarbonyl, ally substituted amino, optionally substituted oyl, acyl, aryl optionally substituted with a substituent group B at one to three position(s), heteroaryl optionally substituted with a substituent group C at one to three position(s), an optionally substituted non-aromatic heterocyclic ring group optionally substituted with a substituent group C at one to three position(s), aryloxy optionally substituted with a substituent group B at one to three on(s), and alkylsulfonyl; each optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkenyloxy, optionally substituted alkynyloxy, ally substituted alkenylthio, optionally substituted alkynylthio, optionally substituted alkenyloxycarbonyl, optionally substituted alkynyloxycarbonyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkyloxy, optionally tuted cycloalkenyloxy, optionally substituted cycloalkylthio, optionally substituted cycloalkenylthio, optionally tuted cycloalkylsulfinyl, optionally substituted cycloalkenylsulfinyl, optionally substituted cycloalkylsulfonyl, optionally substituted lkenylsulfonyl, optionally substituted cycloalkylsulfonyloxy, optionally tuted cycloalkenylsulfonyloxy, and optionally substituted alkylene optionally containing one or two heteroatom(s), when substituted, is independently substituted with one or more substituent(s) selected from the group consisting of alkyl optionally substituted with a substituent group D at one to three position(s), lkyl, hydroxy, oxo, alkyloxy optionally tuted with a substituent group A at one to three on(s), mercapto, alkylthio, a halogen atom, nitro, cyano, carboxy, alkyloxycarbonyl, optionally substituted amino, optionally substituted carbamoyl, acyl, acyloxy, aryl optionally substituted with a substituent group B at one to three position(s), heteroaryl optionally substituted with a substituent group C at one to three position(s), non-aromatic heterocyclic group optionally substituted with a substituent group C at one to three position(s), aryloxy ally substituted with a substituent group C at one to three position(s), and alkylsulfonyl; the substituted piperazine-1,4-diyl and each optionally substituted aryl, optionally substituted y, optionally substituted aryloxycarbonyl, optionally substituted arylthio, ally tuted arylsulfinyl, optionally substituted arylsulfonyl, optionally substituted arylsulfonyloxy, optionally substituted heteroaryl, optionally substituted heteroaryloxy, ally substituted heteroarylthio, optionally tuted heteroarylsulfinyl, optionally substituted heteroarylsulfonyl, ally substituted heteroarylsulfonyloxy, and optionally substituted non-aromatic cyclic group, when substituted, are each independently substituted with one or more substituent(s) selected from the group consisting of alkyl optionally substituted with a substituent group D at one to three position(s), oxo, cycloalkyl, alkenyl, alkynyl, hydroxy, alkyloxy ally substituted with a substituent group A at one to three position(s), aryloxy ally substituted with a substituent group B at one to three position(s), mercapto, alkylthio, a halogen atom, nitro, cyano, carboxy, alkyloxycarbonyl, acyl, alkylsulfonyl, optionally substituted amino, ally substituted carbamoyl, aryl optionally substituted with a substituent group B at one to three on(s), heteroaryl optionally substituted with a substituent group C at one to three on(s), and non-aromatic heterocyclic group optionally substituted with a substituent group C at one to three position(s); each ally substituted amino, optionally substituted carbamoyl, and optionally substituted sulfamoyl, when substituted, is ndently substituted with one or two substituent(s) selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkynyl, aryl, heteroaryl, acyl, alkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, alkyl sulfonyl, alkenylsulfonyl, alkynylsulfonyl, arylsulfonyl, and heteroarylsulfonyl; each substituent group A is independently selected from the group consisting of a halogen atom and phenyl optionally substituted with one to three substituent(s) selected from substituent group B; each substituent group B is independently selected from the group consisting of a halogen atom, alkyl, alkyloxy, cyano, and nitro; each substituent group C is ndently selected from the group consisting of a halogen atom and alkyl; and each substituent group D is independently selected from the group consisting of a halogen atom and alkyloxy.

2. The compound of claim 1, n: A5 is N(R3); and A6 is N.

3. The compound of claim 1, whereinR1 is optionally substituted alkylene, which, when substituted, is substituted with one or more substituent(s) selected from the group consisting of alkyl optionally substituted with a substituent group D at one to three position(s), cycloalkyl, y, oxo, alkyloxy optionally substituted with a substituent group A at one to three position(s), mercapto, alkylthio, a halogen atom, nitro, cyano, carboxy, xycarbonyl, optionally substituted amino, optionally substituted oyl, acyl, acyloxy, aryl optionally substituted with a substituent group B at one to three position(s), heteroaryl optionally substituted with a substituent group C at one to three position(s), non-aromatic heterocyclic group optionally substituted with a substituent group C at one to three position(s), aryloxy optionally substituted with a substituent group C at one to three position(s), and alkylsulfonyl.

4. The compound of claim 1, n R1 is optionally substituted C1, C2, or C3 ne, which, when substituted, is substituted with one or more substituent(s) selected from the group consisting of alkyl optionally substituted with a substituent group D at one to three position(s), cycloalkyl, hydroxy, oxo, alkyloxy optionally substituted with a substituent group A at one to three position(s), mercapto, alkylthio, a halogen atom, nitro, cyano, carboxy, alkyloxycarbonyl, optionally substituted amino, optionally substituted oyl, acyl, acyloxy, aryl optionally substituted with a substituent group B at one to three position(s), heteroaryl optionally substituted with a substituent group C at one to three position(s), non-aromatic heterocyclic group optionally substituted with a substituent group C at one to three on(s), y optionally substituted with a substituent group C at one to three position(s), and alkylsulfonyl.

5. The compound of claim 1, wherein R1 is unsubstituted C1, C2, or C3 alkylene.

6. The compound of claim 1, wherein R1 is unsubstituted C1 alkylene.

7. The compound of claim 1, wherein R1 is C1, C2, or C3 alkylene substituted with one or more alkyl groups.

8. The compound of claim 1, wherein R1 is C1 alkylene substituted with one or two alkyl groups.

9. The compound of claim 1, wherein Q is substituted piperazine-1,4-diyl.

10. The compound of claim 1, wherein: Q is: ; and R5 and R5' are independently alkyl ally substituted at one to three position(s) with a substituent independently selected from a halogen atom and alkyloxy; oxo; cycloalkyl; alkenyl; alkynyl; hydroxy; alkyloxy optionally substituted at one to three on(s) with a substituent independently selected from a halogen atom and phenyl optionally substituted with one to three substituent(s) independently selected from a halogen atom, alkyl, alkyloxy, cyano, and nitro; aryloxy optionally substituted at one to three position(s) with a substituent independently selected from a halogen atom, alkyl, xy, cyano, and nitro; mercapto; alkylthio; a n atom; nitro; cyano; carboxy; alkyloxycarbonyl; acyl; ulfonyl; optionally substituted amino; optionally substituted carbamoyl; aryl optionally substituted with one to three substituent(s) independently selected from a halogen atom, alkyl, xy, cyano, and nitro; heteroaryl ally tuted at one to three position(s) with a substituent independently selected from a halogen atom and alkyl; and non-aromatic heterocyclic group optionally substituted at one to three position(s) with a substituent independently selected from a halogen atom and alkyl.

11. The compound of claim10, wherein one or both of R5 and R5' are independently oxo, hydroxy, or alkyloxy.

12. The compound of claim 10, wherein R5 and R5' are each oxo.

13. The compound of claim 1, wherein one or both of A19 and A21 is N.

14. The compound of claim 1, wherein the R3 on A20 is a halogen atom, optionally substituted alkyl, optionally substituted l, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, hydroxy, carboxy, optionally substituted alkyloxy, optionally substituted loxy, optionally substituted alkynyloxy, optionally substituted cycloalkyloxy, optionally substituted cycloalkenyloxy, mercapto, optionally substituted alkylthio, optionally substituted lthio, optionally substituted alkynylthio, optionally tuted alkylsulfinyl, optionally substituted alkylsulfonyl, optionally substituted alkylsulfonyloxy, optionally tuted cycloalkylthio, optionally substituted cycloalkylsulfinyl, optionally substituted cycloalkylsulfonyl, ally substituted cycloalkylsulfonyloxy, ally substituted cycloalkenylthio, optionally substituted cycloalkenylsulfinyl, optionally substituted cycloalkenylsulfonyl, optionally substituted cycloalkenylsulfonyloxy, optionally substituted amino, acyl, optionally substituted alkyloxycarbonyl, optionally substituted alkenyloxycarbonyl, optionally substituted alkynyloxycarbonyl, optionally substituted aryloxycarbonyl, optionally substituted carbamoyl, optionally tuted sulfamoyl, cyano, nitro, optionally substituted aryl, optionally substituted y, optionally substituted arylthio, optionally substituted arylsulfinyl, optionally tuted arylsulfonyl, optionally substituted arylsulfonyloxy, optionally tuted heteroaryl, optionally substituted heteroaryloxy, optionally tuted arylthio, optionally substituted heteroarylsulfinyl, ally substituted heteroarylsulfonyl, optionally tuted heteroarylsulfonyloxy, or an optionally substituted non-aromatic heterocyclic group.

15. The compound of claim 1, wherein the R3 on A20 is cyano.

16. The nd of claim 1, wherein: A4 is C(R3); A5 is N(R3); A6 and A10 are N; and R1 is optionally substituted alkylene, which, when substituted, is substituted with one or more tuent(s) selected from the group consisting of alkyl optionally substituted with a substituent group D at one to three position(s), cycloalkyl, y, oxo, alkyloxy optionally substituted with a substituent group A at one to three position(s), mercapto, alkylthio, a halogen atom, nitro, cyano, carboxy, alkyloxycarbonyl, optionally substituted amino, ally substituted carbamoyl, acyl, y, aryl optionally substituted with a substituent group B at one to three position(s), heteroaryl optionally substituted with a substituent group C at one to three position(s), omatic heterocyclic group optionally substituted with a substituent group C at one to three position(s), aryloxy optionally substituted with a substituent group C at one to three position(s), and alkylsulfonyl.

17. The compound of claim16, wherein R1 is optionally substituted C1, C2, or C3 alkylene, which, when substituted, is substituted with one or more substituent(s) selected from the group consisting of alkyl optionally substituted with a tuent group D at one to three position(s), cycloalkyl, hydroxy, oxo, alkyloxy ally substituted with a substituent group A at one to three position(s), mercapto, alkylthio, a halogen atom, nitro, cyano, carboxy, alkyloxycarbonyl, optionally substituted amino, optionally substituted oyl, acyl, acyloxy, aryl optionally substituted with a substituent group B at one to three position(s), heteroaryl optionally substituted with a substituent group C at one to three position(s), non-aromatic heterocyclic group optionally substituted with a substituent group C at one to three position(s), aryloxy optionally substituted with a substituent group C at one to three position(s), and alkylsulfonyl.

18. The compound of claim 16, wherein R1 is unsubstituted C1, C2, or C3 alkylene.

19. The nd of claim 16, wherein R1 is unsubstituted C1 alkylene.

20. The compound of claim 16, wherein R1 is C1, C2, or C3 alkylene substituted with one or more alkyl groups.

21. The compound of claim 16, n R1 is C1 alkylene substituted with one or two alkyl groups.

22. The nd of claim 16, wherein Q is substituted piperazine-1,4-diyl.

23. The compound of claim 16, n: Q is: ; and R5 and R5' are independently alkyl optionally substituted at one to three position(s) with a substituent ndently selected from a halogen atom and alkyloxy; oxo; cycloalkyl; alkenyl; alkynyl; hydroxy; alkyloxy optionally substituted at one to three position(s) with a substituent independently selected from a halogen atom and phenyl optionally substituted with one to three substituent(s) ndently selected from a halogen atom, alkyl, alkyloxy, cyano, and nitro; aryloxy optionally substituted at one to three position(s) with a substituent independently selected from a halogen atom, alkyl, xy, cyano, and nitro; mercapto; alkylthio; a halogen atom; nitro; cyano; carboxy; alkyloxycarbonyl; acyl; alkylsulfonyl; optionally substituted amino; optionally substituted carbamoyl; aryl optionally substituted with one to three substituent(s) independently selected from a halogen atom, alkyl, alkyloxy, cyano, and nitro; aryl optionally substituted at one to three position(s) with a substituent independently selected from a halogen atom and alkyl; and non-aromatic heterocyclic group optionally substituted at one to three position(s) with a tuent independently selected from a halogen atom and alkyl.

24. The compound of claim23, wherein one or both of R5 and R5' are independently oxo, hydroxy, or alkyloxy.

25. The compound of claim 23, wherein R5 and R5' are each oxo.

26. The compound of claim 16, wherein one or both of A19 and A21 is N.

27. The compound of claim 16, wherein the R3 on A20 is a halogen a tom, optionally substituted alkyl, ally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted lkenyl, hydroxy, carboxy, optionally substituted alkyloxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, ally substituted cycloalkyloxy, optionally substituted cycloalkenyloxy, mercapto, optionally substituted alkylthio, optionally substituted alkenylthio, ally substituted alkynylthio, optionally substituted ulfinyl, optionally substituted alkylsulfonyl, optionally substituted alkylsulfonyloxy, optionally substituted cycloalkylthio, optionally tuted cycloalkylsulfinyl, ally substituted cycloalkylsulfonyl, optionally substituted cycloalkylsulfonyloxy, optionally substituted cycloalkenylthio, optionally substituted cycloalkenylsulfinyl, optionally substituted cycloalkenylsulfonyl, ally substituted cycloalkenylsulfonyloxy, optionally substituted amino, acyl, optionally substituted xycarbonyl, optionally substituted alkenyloxycarbonyl, optionally substituted loxycarbonyl, optionally substituted aryloxycarbonyl, ally substituted carbamoyl, optionally substituted sulfamoyl, cyano, nitro, optionally substituted aryl, optionally substituted aryloxy, ally substituted arylthio, optionally substituted arylsulfinyl, optionally substituted arylsulfonyl, ally substituted arylsulfonyloxy, ally substituted heteroaryl, optionally substituted heteroaryloxy, optionally substituted heteroarylthio, optionally substituted heteroarylsulfinyl, optionally substituted heteroarylsulfonyl, ally substituted heteroarylsulfonyloxy, or an optionally substituted non-aromatic heterocyclic group.

28. The compound of claim 16, wherein the R3 on A20 is cyano.

29. The compound of claim 1, wherein: Q is piperazine-1,4-diyl or: R5 and R5' are each oxo; R1 is C1 alkylene optionally substituted with one or two alkyl groups; and R3 in each instance is independently a hydrogen atom, a halogen atom, unsubstituted alkyl, unsubstituted alkenyl, unsubstituted alkynyl, tituted cycloalkyl, unsubstituted cycloalkenyl, hydroxy, carboxy, unsubstituted alkyloxy, unsubstituted alkenyloxy, unsubstituted alkynyloxy, unsubstituted lkyloxy, unsubstituted lkenyloxy, mercapto, unsubstituted hio, unsubstituted alkenylthio, unsubstituted alkynylthio, unsubstituted alkylsulfinyl, unsubstituted alkylsulfonyl, unsubstituted ulfonyloxy, unsubstituted cycloalkylthio, unsubstituted cycloalkylsulfinyl, unsubstituted cycloalkylsulfonyl, unsubstituted lkylsulfonyloxy, tituted cycloalkenylthio, unsubstituted cycloalkenylsulfinyl, unsubstituted cycloalkenylsulfonyl, unsubstituted cycloalkenylsulfonyloxy, unsubstituted amino, acyl, unsubstituted alkyloxycarbonyl, unsubstituted alkenyloxycarbonyl, tituted alkynyloxycarbonyl, unsubstituted aryloxycarbonyl, unsubstituted carbamoyl, unsubstituted sulfamoyl, cyano, nitro, unsubstituted aryl, unsubstituted aryloxy, unsubstituted arylthio, unsubstituted arylsulfinyl, unsubstituted arylsulfonyl, unsubstituted arylsulfonyloxy, unsubstituted heteroaryl, unsubstituted heteroaryloxy, unsubstituted heteroarylthio, unsubstituted heteroarylsulfinyl, unsubstituted heteroarylsulfonyl, unsubstituted heteroarylsulfonyloxy, or an unsubstituted non-aromatic heterocyclic group.

30. The compound of claim29, n each R3 is hydrogen with the exception that the R3 on A20 is en or cyano.

31. The compound of claim 29, wherein: A4 is C(R3); A5 is N(R3); and A6 and A10 are each N.

32. The compound of claim31, wherein each R3 is hydrogen with the exception that the R3 on A20 is hydrogen or cyano.

33. The compound of claim 1, n the compound is selected from: ; ; ; ; ; ; ; ; ; ; or a pharmaceutically acceptable salt of any of the ing.

34. The compound of claim 1, wherein the compound is: or a pharmaceutically acceptable salt thereof.

35. The nd of claim 1, wherein the compound is: or a pharmaceutically acceptable salt thereof.

36. The compound of claim 1, wherein the compound is: or a pharmaceutically acceptable salt thereof.

37. The compound of claim 1, wherein the compound is: or a pharmaceutically acceptable salt thereof.

38. A method of treating a condition ina non-human animal with a compound as recited in any one of claims 1 to 37, comprising administering an effective amount of the compound to the animal, wherein the condition comprises at least one of lupus, Sjögren’s syndrome, rheumatoid arthritis, type 1 diabetes, psoriasis, inflammatory bowel disease, a viral disease, and oholic steatohepatitis.

39. Use of an ive amount of a compound as recited in any one of claims 1 to 37 in the manufacture of a medicament for treating a condition in a patient, wherein the condition comprises at least one of lupus, Sjögren’s syndrome, rheumatoid arthritis, type 1 diabetes, sis, inflammatory bowel disease, a viral e, and nonalcoholic steatohepatitis. =oE>§= .0."— SUBSTITUTE SHEET (RULE 26) =oE>§= SUBSTITUTE SHEET (RULE 26) 203333 g} or .0."— SUBSTITUTE SHEET (RULE 26) Piasma 3T~53 ..... 1i} mgikg w» 48 mgfkg .4 3653C! E“ 29:30 18:39 T {3 3 8 {3 121 2’! 24 TNFi? a»: Q S.»«Ww, LEw’l {:1} VE§¥§C§§§ S‘HQ 1 {H ga?‘é W3&833: ‘: ‘2i M: m8318*»:Ax, ii} gii‘s’i LRNQL?é - CD4+ {:23 Whigie $433 : 03 gm \\\\\