EA043597B1 - LANTHIONINE C-LIKE PROTEIN 2 LIGANDS, CELLS DERIVED FROM THEM, AND THERAPIES APPLYING THEM - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to the field of treatment of diseases and disorders. More specifically, the present invention relates to classes of biologically active compounds for the treatment of 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 non-alcoholic steatohepatitis.

State of the art

Lanthionine C-like protein 2 (LANCL2) (also called lanthionine synthetase C-like protein 2 or lanthionine synthetase component C-like protein 2) is a membrane receptor expressed in immune and epithelial cells of various mucous membranes, including respiratory and GI, as well as reproductive and neural tissues, which can be controlled by direct binding of ligands. Activation of the LANCL2 pathway has been shown to be beneficial in multiple autoimmune, inflammatory and metabolic disorders, ranging from glycemic control and insulin sensitivity in diabetes, to improved survival and regulatory effects in viral and bacterial infectious diseases, to suppression of inflammation in inflammatory bowel disease.

The incidence of autoimmune diseases such as inflammatory bowel disease (IBD), systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, psoriasis and multiple sclerosis is increasing worldwide. Meanwhile, other chronic diseases of metabolic origin, including prediabetes, type 2 diabetes, and metabolic syndrome, are estimated to affect nearly half of U.S. adults. For these disorders, current treatments are moderately effective, with the potential for severe side effects such as heart attack and stroke (TZDs for type 2 diabetes) or increased incidence of cancer and infection (biologics for inflammatory bowel disease and other autoimmune diseases), and without significant improving the overall quality of life. Type 1 diabetes has no approved medications other than lifelong insulin therapy. Other diseases, such as multiple sclerosis and systemic lupus erythematosus, have very limited ability to slow the progression of the disease to debilitating physical conditions requiring organ transplantation and full-time nursing care. The LANCL2 pathway offers an innovative solution to these diseases by normalizing metabolism, restoring immune tolerance, and suppressing inflammation, which contribute to the worse prognosis of many of these diseases.

Abscisic acid (ABA) is one of the natural compounds found that binds to LANCL2. A huge number of compounds have been described in the field of synthetic organic chemistry. Various compounds are provided by the following references: WO 1997/036866 to Diana et al., WO 2006/053109 to Sun et al., WO 2006/080821 to Kim et al., WO 2007/019417 to Nunes et al., WO 2009 /067600 and WO 2009/067621 to Singh et al., WO 2008/079277 to Adams et al., JP 2008/056615 to Urasoe et al., WO 2011/066898 to Stoessel et al., US 2013/0142825 to Bassaganya- Riera et al. and US Patent No. 7741367 to Bassaganya-Riera et al. Some of the compounds described in these references are known to activate the LANCL2 pathway, while others are not.

Previous compounds targeting LANCL2, such as BT-11, are highly restricted to the gastrointestinal tract with limited systemic exposure. Although this localization may be preferable for the treatment of gastrointestinal disorders and infections, additional classes of LANCL2-based therapeutics are needed to treat systemic and non-GI diseases with varying pharmacokinetic properties and efficacy. BT-11 and its therapeutic use are described in US patent 9556146 to Bassaganya-Riera et al.; US patent 9839635 to Bassaganya-Riera et al.; US patent 10028950 to Bassaganya-Riera et al.; US patent 10201538 to Bassaganya-Riera et al.; US patent 10493072 to Bassaganya-Riera et al.; US patent 10682349 to Bassaganya-Riera et al.; US 2019/0160100 A1 to Bassaganya-Riera et al.; Bissel et al. 2016 (Bissel P, Boes K, Hinckley J, Jortner BS, Magnin-Bissel G, Werre SR, Ehrich M, Carbo A, Philipson C, Hontecillas R, Philipson N, Gandour RD, Bassaganya-Riera J. Exploratory Studies With BT- 11: A Proposed Orally Active Therapeutic for Crohn's Disease. Int J Toxicol. 2016 Sep;35(5):521-9); and Carbo et al. 2016 (Carbo A, Gandour RD, Hontecillas R, Philipson N, Uren A, Bassaganya-Riera J. An N, N-Bis(benzimidazolylpicolinoyl)piperazine (BT-11): A Novel Lanthionine Synthetase C-Like 2-Based Therapeutic for Inflammatory Bowel Disease J Med Chem 2016 Nov 23;59(22):10113-10126); Leber et al. 2018 (Leber A, Hontecillas R, Zoccoli-Rodriguez V, Bassaganya-Riera J. Activation of LANCL2 by BT-11 Ameliorates IBD by Supporting Regulatory T Cell Stability Through Immunometabolic Mechanisms. Inflamm Bowel Dis. 2018 Aug 16; 24(9): 1978-1991); Leber et al. 2019 Int J Toxicol. (Leber A, Hontecillas R, Zoccoli-Rodriguez V, Ehrich M, Davis J, Chauhan J, BassaganyaRiera J. Nonclinical Toxicology and Toxicokinetic Profile of an Oral Lanthionine Synthetase C-Like 2 (LANCL2) Agonist, BT-11. Int J Toxicol 2019 Mar/Apr;38(2):96-109); Leber et al. 2019 J Immunol. (Leber A, Hontecillas R, Zoccoli-Rodriguez V, Chauhan J, Bassaganya-Riera J. Oral Treatment with BT-11 Ameliorates Inflammatory Bowel Disease by Enhancing Regulatory T Cell Responses in the Gut. J Immunol. 2019 Apr 1; 202(7 ):2095-2104); and Leber et al. 2020 (Leber A, Hontecillas R, Zoccoli-Rodriguez V, Colombel JF, Chauhan J,

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Ehrich M, Farinola N, Bassaganya-Riera J. The Safety, Tolerability, and Pharmacokinetics Profile of BT-11, an

Oral, Gut-Restricted Lanthionine Synthetase C-Like 2 Agonist Investigational New Drug for Inflammatory

Bowel Disease: A Randomized, Double-Blind, Placebo-Controlled Phase I Clinical Trial. Inflamm Bowel Dis.

2020 Mar 4; 26(4):643-652).

The invention provides compounds that have been developed using novel medicinal chemistry approaches and tested using in silico, in vitro and in vivo methods to balance the ability to selectively bind the LANCL2 protein with systemic bioavailability and potency. These compounds may influence positive responses in a variety of disease states, including, but not limited to, autoimmune, chronic inflammatory, inflammatory, metabolic, and infectious diseases.

The essence of the invention

The invention provides compounds of the formula Z-Y-Q-Y' or pharmaceutically acceptable salts thereof, in which

Z represents:

Y represents:

Q is piperazine-1,4-diyl or piperazine-2,6-dione-1,4-diyl; Y' represents:

- absent between A ¹⁵ and A ¹⁶ ;

A ¹ , A ² , A ³ , A ⁸ , A ⁹ , A ¹¹ , A ¹² , A ¹³ , A ¹⁸ , A ²⁰ and A ²² each represent C(R ³ );

A ⁴ , A ¹⁹ and A ²¹ are each independently C(R ³ ) or N;

each of A ⁶ , A ¹⁰ and A ¹⁷ represents N;

A ⁵ represents N(R ³ ) or O;

And ⁷ represents N(R ³ );

A ¹⁴ represents O;

A ¹⁵ and A ¹⁶ each represent C(R ³ ) ₂ ;

R ¹ represents C1, C2 or C ₃ alkylene, optionally substituted with one or more C1-C6 alkyl groups;

R ³ in each case is independently hydrogen, C1-C ₆ alkyl or cyano.

In some embodiments, A ⁴ is CH. In some embodiments, A ⁵ is NH. In some embodiments, Y is:

In some embodiments, Q is piperazine-1,4-diyl.

In some embodiments, Q is piperazine-2,6-dione-1,4-diyl.

In some embodiments, Y' is:

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In some embodiments, R ¹ is -(CH ₂ )i _-3 - or -C(CH ₃ ) ₂ -. In some embodiments, A ¹⁹ and A ²¹ are each CH. In some embodiments, R ³ in A ²⁰ is cyano.

In some embodiments, each R ³ is a hydrogen atom, except that the R ³ in A ²⁰ is a hydrogen or cyano atom. In some embodiments, ZYQY' is:

A ⁴ represents CH;

And ⁵ represents NH;

each of A ¹⁹ and A ²¹ represents CH;

R ¹ represents -(CH ₂ ) _1-3 - or -C(CH ₃ ) ₂ -; and each R ³ represents a hydrogen atom, except that R ³ in A ²⁰ represents a hydrogen or cyano atom.

In some embodiments, Q is piperazine-1,4-diyl.

In some embodiments, Q is piperazine-2,6-dione-1,4-diyl.

In some embodiments, R ¹ is -(CH2)-.

In some embodiments, R ¹ is -C(CH3)2-.

In some embodiments, R ³ in A ²⁰ is cyano. In some embodiments, the connection is selected from:

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or a pharmaceutically acceptable salt of any of the above. In some embodiments, the connection is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the connection is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the connection is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the connection is:

or a pharmaceutically acceptable salt thereof.

Any option described above or elsewhere herein may be combined with any one or more other, non-mutually exclusive options described above or elsewhere herein.

The invention provides methods of treating a condition in an animal using any one or more of the compounds described in the present invention. The methods may include administering to the animal an effective amount of one or more compounds described in the present invention. An effective amount is an amount effective to treat the condition of the animal. The condition may include any one or more of the conditions described in the present invention. In some embodiments, the condition includes an autoimmune disease. In some embodiments, the condition includes an inflammatory disease. In some embodiments, the condition includes a chronic inflammatory disease. In some embodiments, the condition includes inflammatory bowel disease, such as ulcerative colitis and/or Crohn's disease. In some embodiments, the condition includes diabetes. In some embodiments, the condition includes an infectious disease. In some embodiments, the condition includes lupus, such as systemic lupus erythematosus. In some embodiments, the condition includes Sjögren's syndrome. In some embodiments, the condition includes rheumatoid arthritis. In some embodiments, the disease includes type 1 diabetes. In some embodiments, the condition includes a viral disease such as influenza, Zika virus infection, and coronavirus infection. In some embodiments, the condition includes non-alcoholic steatohepatitis.

The objects and advantages of the invention will become more apparent from the following detailed description of a preferred embodiment of the invention taken in conjunction with the accompanying drawings.

Brief description of drawings

Fig. 1A-1C. Computational prediction of binding of selected compounds to LANCL2 in kcal/mol.

Fig. 2. Pharmacokinetics of VT-63 after oral administration of 10 and 40 mg/kg.

Fig. 3. Immunological validation of BT-63 activity in CD4+ splenocytes. The percentages of IFNa+, TNFa+, and IL10+ FOXP3+ CD4+ T cells were measured by flow cytometry after in vitro treatment of BT-63 cells at concentrations of 0.1, 1, and 10 μM in wild-type and LANCL2-deficient cells. Statistical significance (p<0.05) is indicated by asterisks.

Fig. 4A and 4B. Activity of compounds in CD4+ splenocytes. The percentages of IFNu+ and FOXP3+ CD4+ T cells were measured by flow cytometry after in vitro treatment of BT63 and BT-62 cells at concentrations of 0.1, 1, and 10 microM (Fig. 4A). The percentages of IFN+ and TNF+ CD4+ T cells were measured by flow cytometry after in vitro treatment of BT-104-A, BT-104-B, and BT-104-C cells at concentrations of 0.1 microM (Fig. 4B). Statistical significance (p<0.05) is indicated by asterisks.

Fig. 5. Efficiency of VT-63 on the NOD SD1 model. Weekly monitoring of blood glucose levels and percent hyperglycemia in NOD mice treated with vehicle or 10 or 20 mg/kg BT-63 daily.

Fig. 6. Immune effects of BT-63 in vivo in the NOD model of type 1 diabetes (T1DM). Ratio of spleen cells producing IL10 and cells producing IFNu, BCL6+ IL21+ CD4+ T helpers and CD4+ PD1+ T cells of the spleen at 21 weeks of age with oral administration of vehicle or 10 or 20 mg/kg BT-63 daily.

Fig. 7. Evaluation of the plasma biomarker VT-63 in the NOD model of T1DM. Plasma concentrations of C-peptide, HbA1c, MCP-1, and TNF at 21 weeks of age with oral vehicle or 10 or 20 mg/kg BT-63 daily.

Fig. 8. Efficacy of BT-63 in the TLR7/9-induced SLE model. Plasma antibodies to double-stranded DNA, urine albumin levels, CD8+ IFNu+ and IL6+ CD45+ spleen cells after two weeks of oral administration of vehicle or 20 mg/kg BT-63.

Fig. 9. Efficiency of VT-63 on the MDR1a-/- B3K model. Colonic lamina propria Th1, Th17, CD25+ FOXP3+ CD4+ Tregs and neutrophils after four weeks of oral vehicle or 20 mg/kg BT-63.

Fig. 10. Reduction of systemic inflammation in Mdr1a-/- mice treated with BT-63. Splenic Th1 and CD25+ Tregs after four weeks of oral vehicle or 20 mg/kg BT-63.

Fig. 11. Efficacy of BT-63 in a model of influenza A viral infection in mice. Survival and disease activity index for 12 days after influenza A (H1N1) infection with daily oral administration of vehicle or 20 mg/kg BT-63.

Fig. 12. Modulation of the immune response of the lungs to influenza A infection using BT-63. IL10+ lung CD8+ T cells, alveolar macrophages, CD4+ TNF+ T cells and neutrophils on day 12 postinfection with daily oral administration of vehicle or 20 mg/kg BT-63.

Fig. 13. Efficiency of VT-63 on the NZB/W F1 SKV model. Changes in body weight from baseline during 12 weeks of vehicle or 20 mg/kg BT-63 administration. Assessment of urinary protein, plasma anti-dsDNA antibodies, and plasma IFN-α concentrations after 12 weeks of oral administration of vehicle or BT-63 (20 mg/kg).

Fig. 14. Immunological responses to BT-63 on the NZB/W F1 model. CD19+ IgD+ IgMlo spleen follicular B cells, CD138 ^hl CD19lo MHCIIlo plasma cells, CXCR3+ plasma cells, IL6+ CD11b+ CD11c+ myeloid cells, CD4+ CD25+ FOXP3+ Tregs, CD4+ IL21+ BCL6+ follicular T helpers after 12 weeks of oral vehicle administration or VT-63 (20 mg/ kg).

Fig. 15. Additional exemplary compounds of the invention (VT-104-A, VT-104-B, VT-104-C) that bind to LANCL2 and that can be used in any method described herein. Conversion of piperazine in VT-63 to piperazine-2,6-dione, as present in VT-104-A, or addition of gem-dimethyl or nitrile groups to VT-63, as present in VT-104-B or VT-104C , accordingly, serve to improve the systemic half-life of the target compound by increasing the resistance of the methylene group in BT-63 to enzymatic metabolism.

Fig. 16A and 16B. In vivo validation of the effectiveness of VT-104-B on the CCl ₄ model of non-alcoholic steatohepatitis. Liver weight (FIG. 16A) and fibrosis score (FIG. 16B) after 4 weeks of biweekly carbon tetrachloride injections in vehicle and BT-104-B (20 mg/kg)-treated mice. Statistical

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Fig. 17A-17C. In vivo validation of the effectiveness of BT-104-B in a model of collagen-induced arthritis. Percentages of IL17+ (Figure 17A), IFN+ CD4+ T cells (Figure 17B), and TNF+ CD11b+ CD11c+ myeloid cells (Figure 17C) in the spleens of mice with collagen-induced arthritis after 4 weeks of daily oral administration of vehicle or BT-104 -B (20 mg/kg). Statistical significance (P<0.05) is indicated by asterisks.

Fig. 18A-18C. In vivo validation of the effectiveness of VT-104-B in the NZB/W F1 SLE model. Percentages of IL17+ (FIG. 18A), IL21+ (FIG. 18B), and CD25+ FOXP3+ CD4+ T cells (FIG. 18C) in the spleen of NZB/W F1 mice after 12 weeks of daily oral administration of vehicle or BT-104-B (20 mg /kg). Statistical significance (P<0.05) is indicated by asterisks.

Detailed Description of the Invention

Unless otherwise specified, the following definitions are used in the present invention.

Analysis of Variance (ANOVA): An arithmetic process for allocating the total variation in data sets into specific components based on sources of variation. It was used to determine whether numerical differences between treatment groups were statistically significant.

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

Enantiomer: optical isomer; a chemical classification of molecules based on their ability to rotate the plane of polarization clockwise (+) or counterclockwise (-).

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: Inflammatory bowel disease (IBD) involves chronic inflammation of all or part of the gastrointestinal tract. IBD primarily includes ulcerative colitis and Crohn's disease. Both usually include severe diarrhea, pain, fatigue and weight loss. IBD can be severe and sometimes leads to life-threatening complications.

Ulcerative colitis (UC): UC is an IBD that causes long-term inflammation and sores (ulcerations) in the innermost lining of the colon and rectum.

Crohn's disease: Crohn's disease is an IBD that causes inflammation of the lining of the gastrointestinal tract. In Crohn's disease, inflammation often extends deep into the affected tissue. Inflammation can affect different parts of the gastrointestinal tract, the large intestine, the small intestine, or both.

IL-10: Interleukin-10 (IL-10), also known as human cytokine synthesis inhibitory factor (CSIF), is an anti-inflammatory cytokine. In humans, IL-10 is encoded by the IL10 gene.

FOXP3: FOXP3 (forkhead P3), also known as scurfin, is a protein involved in immune system responses. A member of the FOX protein family, FOXP3 appears to act as a master regulator (transcription factor) in the development and function of regulatory T cells.

TNF-alpha: Tumor necrosis factor (TNF, cachexin or cachectin and formerly known as tumor necrosis factor alpha or TNFa) is a cytokine involved in systemic inflammation and is a member of the group of cytokines that stimulate the acute phase response.

MCP1: monocyte chemoattractant protein-1. An older term for a CC cytokine that is critical for the development of atherosclerotic lesions found in endothelial cells, macrophages, and vascular smooth muscle cells of patients undergoing coronary artery bypass grafting procedures. The currently officially preferred term is chemokine (C-C motif) ligand 2.

Interferon gamma: Interferon gamma is a proinflammatory dimerized soluble cytokine that is the sole member of the type II interferon class.

Leukocyte infiltration: Leukocyte infiltration refers to the process of moving or infiltrating white blood cells into damaged tissue to begin the repair process.

Type 1 diabetes: An autoimmune disease characterized as a chronic condition in which the pancreas produces little or no insulin as a result of immunological destruction of the insulin-producing beta cells in the pancreatic islets. Insulin deficiency leads to chronic hyperglycemia, which can cause organ damage, shortened life expectancy, and decreased quality of life. The disease is also called juvenile 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, including the skin, joints, kidneys, brain, heart and cardiovascular system, and other organs.

The term halogen atom refers to a fluorine atom, a chlorine atom, a bromine atom and an iodine atom. Fluorine atom, chlorine atom and bromine atom are preferred.

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

The term alkyl includes a monovalent straight or branched hydrocarbon group containing from one to eight carbon atom(s). Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, n-heptyl, n-octyl and the like. C^^-alkyl is preferred. C1- _C4 -alkyl or C1- _C3 -alkyl is even more preferred. When the number of carbon atoms is indicated, it means alkyl having a number of carbon atoms within the range.

The term alkenyl includes a monovalent straight or branched hydrocarbon group containing from two to eight carbon atoms and one or more double bonds. Examples include vinyl, allyl, 1-propenyl, 2-butenyl, 2-pentenyl, 2-hexenyl, 2-heptenyl, 2-octenyl and the like. C2-C6 alkenyl is preferred. C ₂ -C ₄ -alkenyl is even more preferred.

The term alkynyl includes a monovalent straight or branched hydrocarbon group containing from two to eight carbon atoms and one or more triple bonds. Examples include ethynyl, 1-propynyl, 2-propynyl, 2-butynyl, 2-pentynyl, 2-hexynyl, 2-heptynyl, 2-octynyl and the like. C ₂ -C6 alkynyl is preferred. C ₂ -C ₄ -alkynyl is even more preferred.

The term cycloalkyl includes cycloalkyl containing from three to eight carbon atoms. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like. C ₃ -C _b -cycloalkyl is preferred.

The term cycloalkenyl includes cycloalkenyl containing from three to eight carbon atoms. Examples include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl and the like. C ₃ -C _b -cycloalkenyl is preferred.

The term alkyloxy includes a group in which the oxygen atom is replaced by one alkyl, as described in the present invention. Examples include methyloxy, ethyloxy, n-propyloxy, isopropyloxy, n-butyloxy, isobutyloxy, sec-butyloxy, tert-butyloxy, n-pentyloxy, isopentyloxy, 2pentyloxy, 3-pentyloxy, n-hexyloxy, isohexyloxy, 2-hexyloxy, 3- hexyloxy, n-heptyloxy, noctyloxy and the like. C1- _C6 alkyloxy is preferred. C1- _C4 alkyloxy or C1-C3 alkyloxy is even more preferred. When the number of carbon atoms is indicated, it means alkyloxy having a number of carbon atoms within the range.

The term alkenyloxy includes a group in which the oxygen atom is replaced by one alkenyl, as described in the present invention. Examples include vinyloxy, allyloxy, 1-propenyloxy, 2-butenyloxy, 2-pentenyloxy, 2-hexenyloxy, 2-heptenyloxy, 2-octenyloxy and the like. C ₂ -C ₆ alkenyloxy is preferred. Moreover, C2-C4 alkenyloxy is even more preferred. When the number of carbon atoms is indicated, it means alkenyloxy having a number of carbon atoms within the range.

The term alkynyloxy includes a group in which the oxygen atom is replaced by one alkynyl, as described in the present invention. Examples include ethynyloxy, 1-propynyloxy, 2propynyloxy, 2-butynyloxy, 2-pentynyloxy, 2-hexynyloxy, 2-heptynyloxy, 2-octynyloxy and the like. C ₂ -C ₆ -alkynyloxy is preferred. C ₂ -C ₄ -alkynyloxy is even more preferred. When the number of carbon atoms is indicated, it means alkynyloxy having a number of carbon atoms within the range.

The term cycloalkyloxy includes a group in which the oxygen atom is replaced by one cycloalkyl, as described in the present invention. Examples include cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, cycloheptyloxy and cyclooctyloxy. C ₃ -C _b -cycloalkyloxy is preferred. When the number of carbon atoms is indicated, it means cycloalkyloxy having a number of carbon atoms within the range.

The term cycloalkenyloxy includes a group in which the oxygen atom is replaced by one cycloalkenyl, as described in the present invention. Examples include cyclopropenyloxy, cyclobutenyloxy, cyclopentenyloxy, cyclohexenyloxy, cycloheptenyloxy and cyclooctenyloxy. C3-C6cycloalkenyloxy is preferred. When the number of carbon atoms is indicated, it means cycloalkenyloxy having a number of carbon atoms within the range.

The term alkylthio includes a group in which the sulfur atom is replaced by one alkyl, as described in the present invention. Examples include methylthio, ethylthio, n-propylthio, isopropylthio, nbutylthio, isobutylthio, sec-butylthio, tert-butylthio, n-pentylthio, isopentylthio, 2-pentylthio, 3pentylthio, n-hexylthio, isohexylthio, 2-hexylthio, 3-hexylthio, n-heptylthio, n-octylthio and the like. C1- _C6 -alkylthio is preferred. A C1- _C4 alkylthio is even more preferred. When the number of carbon atoms is indicated, it means an alkylthio having a number of carbon atoms within the range.

The term alkenylthio includes a group in which the sulfur atom is replaced by one alkenyl, as described in the present invention. Examples include vinylthio, allylthio, 1-propenylthio, 2-butenylthio, 2-pentenylthio, 2-hexenylthio, 2-heptenylthio, 2-octenylthio and the like. C ₂ -C ₆ -alkenylthio is preferred. C ₂ -C ₄ -alkylthio is even more preferred. When the number of carbon atoms is indicated, it means alkenylthio having a number of carbon atoms within the range.

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The term alkynylthio includes a group in which the sulfur atom is replaced by one alkynyl, as described in the present invention. Examples include ethynylthio, 1-propynylthio, 2-propynylthio, 2-butynylthio, 2-pentynylthio, 2-hexynylthio, 2-heptynylthio, 2-octynylthio and the like. C ₂ -C ₆ -alkynylthio is preferred. C ₂ -C ₄ -alkynylthio is even more preferred. When the number of carbon atoms is indicated, it means an alkynylthio having a number of carbon atoms within the range.

The term alkylsulfinyl includes a group in which sulfinyl is replaced by one alkyl, as described in the present invention. Examples include methylsulfinyl, ethylsulfinyl, npropylsulfinyl, isopropylsulfinyl, n-butylsulfinyl, isobutylsulfinyl, sec-butylsulfinyl, tert-butylsulfinyl, n-pentylsulfinyl, isopentylsulfinyl, 2-pentylsulfinyl, 3pentylsulfinyl, n-hexylsulfinyl, isohexylsulfinyl, 2-hexylsulfinyl, 3-hexylsulfinyl, nheptylsulfinyl, n-octylsulfinyl and the like. C1- _C6 -alkylsulfinyl is preferred. C1- _C4 -alkylsulfinyl is even more preferred.

The term alkylsulfonyl includes a group in which the sulfonyl is replaced by one alkyl, as described in the present invention. Examples include methylsulfonyl, ethylsulfonyl, npropylsulfonyl, isopropylsulfonyl, n-butylsulfonyl, isobutylsulfonyl, sec-butylsulfonyl, tert-butylsulfonyl, n-pentylsulfonyl, isopentylsulfonyl, 2-pentylsulfonyl, 3pentylsulfonyl, n-hexylsulfonyl, isohexylsulfonyl, 2-hexylsulfonyl, 3-hexylsulfonyl, nheptylsulfonyl, n-octylsulfonyl and the like. C1- _C6 -alkylsulfonyl is preferred. C1- _C4 -alkylsulfonyl is even more preferred.

The term alkylsulfonyloxy includes a group in which the oxygen atom is replaced by one alkylsulfonyl, as described in the present invention. Examples include methylsulfonyloxy, ethylsulfonyloxy, n-propylsulfonyloxy, isopropylsulfonyloxy, n-butylsulfonyloxy, isobutylsulfonyloxy, sec-butylsulfonyloxy, tert-butylsulfonyloxy, n-pentylsulfonyloxy, isopentylsulfonyloxy, 2-pentylsulfonyloxy, 3-pentylsulfonyloxy, n-hexylsulfonyloxy, isohexyl sulfonyloxy, 2-hexylsulfonyloxy, 3-hexylsulfonyloxy, n-heptylsulfonyloxy, noctylsulfonyloxy and the like. C ₁ -C ₆ -alkylsulfonyl is preferred. C1- _C4 alkylsulfonyl is even more preferred.

The term cycloalkylthio includes a group in which the sulfur atom is replaced by one cycloalkyl, as described in the present invention. Examples include cyclopropylthio, cyclobutylthio, cyclopentylthio, cyclohexylthio, cycloheptylthio, cyclooctylthio and the like. C ₃ -C ₆ -cycloalkylthio is preferred. When the number of carbon atoms is indicated, it means a cycloalkylthio having a number of carbon atoms within the range.

The term cycloalkylsulfinyl includes a group in which the sulfinyl is replaced by one cycloalkyl, as described in the present invention. Examples include cyclopropylsulfinyl, cyclobutylsulfinyl, cyclopentylsulfinyl, cyclohexylsulfinyl, cycloheptylsulfinyl and cyclooctylsulfinyl. C ₃ -C ₆ -cycloalkylsulfinyl is preferred.

The term cycloalkylsulfonyl includes a group in which the sulfonyl is replaced by one cycloalkyl, as described in the present invention. Examples include cyclopropylsulfonyl, cyclobutylsulfonyl, cyclopentylsulfonyl, cyclohexylsulfonyl, cycloheptylsulfonyl and cyclooctylsulfonyl. C ₃ -C ₆ -cycloalkylsulfonyl is preferred.

The term cycloalkylsulfonyloxy includes a group in which the oxygen atom is replaced by one cycloalkylsulfonyl, as described in the present invention. Examples include cyclopropylsulfonyloxy, cyclobutylsulfonyloxy, cyclopentylsulfonyloxy, cyclohexylsulfonyloxy, cycloheptylsulfonyloxy and cyclooctylsulfonyloxy. C ₃ -C ₆ -cycloalkylsulfonyloxy is preferred.

The term cycloalkenylthio includes a group in which the sulfur atom is replaced by one cycloalkenyl, as described in the present invention. Examples include cyclopropenylthio, cyclobutenylthio, cyclopentenylthio, cyclohexenylthio, cycloheptenylthio and cyclooctenylthio. C ₃ -C ₆ -cycloalkenylthio is preferred. When the number of carbon atoms is indicated, it means cycloalkenylthio having a number of carbon atoms within the range.

The term cycloalkenylsulfinyl includes a group in which the sulfinyl is replaced by one cycloalkenyl, as described in the present invention. Examples include cyclopropenylsulfinyl, cyclobutenylsulfinyl, cyclopentenylsulfinyl, cyclohexenylsulfinyl, cycloheptenylsulfinyl and cyclooctenylsulfinyl. C ₃ -C ₆ -cycloalkenylsulfinyl is preferred.

The term cycloalkenylsulfonyl includes a group in which the sulfonyl is replaced by one cycloalkenyl, as described in the present invention. Examples include cyclopropenylsulfonyl, cyclobutenylsulfonyl, cyclopentenylsulfonyl, cyclohexenylsulfonyl, cycloheptenylsulfonyl and cyclooctenylsulfonyl. Preferably C ₃ -C ₆ -cycloalkenylsulfonyl is preferred.

The term cycloalkenylsulfonyloxy includes a group in which the oxygen atom is replaced by one cycloalkenylsulfonyl, as described in the present invention. Examples include cyclopropenylsulfonyloxy, cyclobutenylsulfonyloxy, cyclopentenylsulfonyloxy, cyclohexenylsulfonyloxy, cycloheptenylsulfonyloxy and cyclooctenylsulfonyloxy. C ₃ -C _b cycloalkenylsulfonyloxy is preferred.

The term alkyloxycarbonyl includes a group in which the carbonyl is replaced by one alkyloxy, as described in the present invention. Examples include methyloxycarbonyl, ethyloxycarbonyl, npropyloxycarbonyl, isopropyloxycarbonyl, n-butyloxycarbonyl, t-butyloxycarbonyl and npentyloxycarbonyl. C1-C6 or C1- _C4 alkyloxycarbonyl is preferred. C1-C2alkyloxycarbonyl is even more preferred.

The term alkenyloxycarbonyl includes a group in which the carbonyl is replaced by one alkenyloxy, as described in the present invention. Examples include vinyloxycarbonyl, allyloxycarbonyl, 1-propenyloxycarbonyl, 2-butenyloxycarbonyl and 2-pentenyloxycarbonyl. C ₂ -C _b or C ₂ -C ₄ alkyloxycarbonyl is preferred.

The term alkynyloxycarbonyl includes a group in which the carbonyl is replaced by one alkynyloxy, as described in the present invention. Examples include ethynyloxycarbonyl, 1propynyloxycarbonyl, 2-propynyloxycarbonyl, 2-butynyloxycarbonyl and 2-pentynyloxycarbonyl. C ₂ -C _b or C ₂ -C ₄ -alkynyloxycarbonyl is preferred.

The term acyl includes alkylcarbonyl in which the alkyl portion is alkyl as described herein, alkenylcarbonyl in which the alkenyl portion is alkenyl as described in the present invention, alkynylcarbonyl in which the alkynyl portion is alkynyl as described in the present invention , cycloalkylcarbonyl, in which the cycloalkyl moiety is cycloalkyl, as described in the present invention, arylcarbonyl, in which the aryl moiety is aryl, as described in the present invention, heteroarylcarbonyl, in which the heteroaryl moiety is heteroaryl, as described in the present invention, and a non-aromatic heterocyclic carbonyl, wherein the non-aromatic heterocyclic group portion is a non-aromatic heterocyclic group as described in the present invention. An alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and non-aromatic heterocyclic group may be substituted, respectively, by substituent groups represented by optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl and optionally a substituted non-aromatic heterocyclic group as described in the present invention. Examples of the acyl group include acetyl, propionyl, butyroyl, cyclohexylcarbonyl, benzoyl, pyridinecarbonyl and the like.

The term optionally substituted amino includes an amino group that may be substituted with one or two alkyl group(s) as described herein, alkenyl as described herein, alkynyl as described herein, cycloalkyl as described herein, cycloalkynyl, as described in the present invention, aryl, as described in the present invention, heteroaryl, as described in the present invention, acyl, as described in the present invention, alkyloxycarbonyl, as described in the present invention, alkenyloxycarbonyl, as described in the present invention, alkynyloxycarbonyl, as described in the present invention, alkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, arylsulfonyl and/or heteroarylsulfonyl, as described in the present invention. Examples of the optionally substituted amino group include amino, methylamino, dimethylamino, ethylamino, diethylamino, ethylmethylamino, benzylamino, acetylamino, benzoylamino, methyloxycarbonylamino and methanesulfonylamino. Amino, methylamino, dimethylamino, ethylmethylamino, diethylamino, acetylamino and methanesulfonylamino are preferred.

The term optionally substituted carbamoyl includes an aminocarbonyl group in which the optionally substituted amino portion is an optionally substituted amino, as described in the present invention. Examples of the optionally substituted carbamoyl group include carbamoyl, N-methylcarbamoyl, N,N-dimethylcarbamoyl, N-ethyl-N-methylcarbamoyl, N,Ndiethylcarbamoyl, N-phenylcarbamoyl, N-benzylcarbamoyl, N-acetylcarbamoyl and Nmethylsulfonylcarbamoyl, etc. Carbamoyl, N-methylcarbamoyl, N,N-dimethylcarbamoyl and Nmethylsulfonylcarbamoyl, etc. are preferred.

The term optionally substituted sulfamoyl includes an aminosulfonyl group in which the optionally substituted amino portion is an optionally substituted amino, as described in the present invention. Examples of the optionally substituted sulfamoyl group include sulfamoyl, N-methylsulfamoyl, N,N-dimethylsulfamoyl, N-ethyl-N-methylsulfamoyl, N,N-diethylsulfamoyl, N-phenylsulfamoyl, N-benzylsulfamoyl, N-acetylsulfamoyl and Nmethylsulfonylsulfamoyl, etc. Sulfamoyl, N-methylsulfamoyl, N,N-dimethylsulfamoyl and Nmethylsulfonylsulfamoyl, etc. are preferred.

The term alkylene means a linear or branched alkylene group containing from one to eight carbon atom(s). Examples include methylene, ethylene, 1-methylethylene, trimethylene, 1methyltrimethylene, pentamethylene, hexamethylene and the like. C1- _C4 or _{C1_3} alkylenes are preferred. C ₁ -C ₂ -alkylene is even more preferred.

- 9 043597

The term aryl includes aromatic monocyclic or aromatic fused cyclic hydrocarbons. It may be fused with a cycloalkyl as described in the present invention, a cycloalkenyl as described in the present invention, or a non-aromatic heterocyclic group as described in the present invention, in any possible position. Both the monocyclic ring and the fused ring can be substituted in any position. Examples include phenyl, 1-naphthyl, 2-naphthyl, anthryl, tetrahydronaphthyl, 1,3-benzodioxolyl, 1,4-benzodioxanyl, etc. Phenyl, 1-naphthyl and 2-naphthyl are preferred. Phenyl is even more preferred.

The term non-aromatic heterocyclic group includes a 5-7 membered non-aromatic heterocyclic ring containing one or more heteroatom(s) independently selected from oxygen, sulfur and nitrogen atoms, or a polycyclic ring formed by the fusion of two or more rings thereof. Examples include pyrrolidinyl (eg, 1-pyrrolidinyl, 2-pyrrolidinyl), pyrrolinyl (eg, 3-pyrrolinyl), imidazolidinyl (eg, 2-imidazolidinyl), imidazolinyl (eg, imidazolinyl), pyrazolidinyl (eg, 1-pyrazolidinyl, 2- pyrazolidinyl), pyrazolinyl (e.g. pyrazolinyl), piperidyl (e.g. piperidino, 2-piperidyl), piperazinyl (e.g. 1-piperazinyl), indolinyl (e.g. 1-indolinyl), isoindolinyl (e.g. isoindolinyl), morpholinyl (e.g. morpholino, 3-morpholinyl), etc.

The term heteroaryl includes a 5-6 membered aromatic ring containing one or more heteroatom(s) independently selected from oxygen, sulfur and nitrogen atoms. It may be fused with a cycloalkyl as described in the present invention, an aryl as described in the present invention, a non-aromatic heterocyclic group as described in the present invention, or another heteroaryl in any possible position. The heteroaryl group may be substituted at any position when it is a monocyclic ring or a fused ring. Examples include pyrrolyl (eg 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl), furyl (eg 2-furyl, 3-furyl), thienyl (eg 2-thienyl, 3-thienyl), imidazolyl (eg 2 -imidazolyl, 4-imidazolyl), pyrazolyl (e.g. 1-pyrazolyl, 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 (e.g. 2-pyrimidinyl, 4-pyrimidinyl), pyridazinyl (e.g. 3-pyridazinyl), tetrazolyl (e.g. 3-tetrazolyl), oxadiazolyl (e.g. 1,3,4-oxadiazolyl), thiadiazolyl (e.g. 1,3,4thiadiazolyl), indolidinyl (e.g. 2-indolidinyl, 6-indolidinyl) , isoindolinyl (e.g. 2isoindolinyl), indolyl (e.g. 1-indolyl, 2-indolyl, 3-indolyl), indazolyl (e.g. 3-indazolyl), purinyl (e.g. 8-purinyl), quinolidinyl (e.g. 2-quinolidinyl), isoquinolyl (e.g. 3-isoquinolyl), quinolyl (e.g. 2-quinolyl, 5-quinolyl), phtarazinyl (e.g. 1ftharazinyl), naphthylidinyl (e.g. 2-naphthylidinyl), quinolanyl (e.g. 2-quinolanyl), quinazolinyl (e.g. , 2-quinazolinyl), cinnolinyl (e.g. 3-cinnolinyl), pteridinyl (e.g. 2pteridinyl), carbazolyl (e.g. 2-carbazolyl, 4-carbazolyl), phenanthridinyl (e.g. 2phenanthridinyl, 3-phenanthridinyl), acridinyl (e.g. 1-acridinyl, 2-acridinyl), dibenzofuranyl (e.g. 1-dibenzofuranyl, 2-dibenzofuranyl), benzoimidazolyl (e.g. 2-benzoimidazolyl), benzoisoxazolyl (e.g. 3-benzoisoxazolyl), benzooxazolyl (e.g. 2-benzooxazolyl), benzooxadiazolyl (e.g. 4-benzooxadiazolyl), benzoisothiazolyl (e.g. 3-benzoisothiazolyl), benzothiazolyl (e.g. 2-benzothiazolyl), benzofuryl (e.g. 3-benzofuryl), benzothienyl (e.g. 2-benzothienyl), dibenzothienyl (e.g. 2- dibenzothienyl) and benzodioxolyl (for example, 1,3benzodioxolyl), etc.

The term aryloxy includes a group in which the oxygen atom is replaced by one aryl, as described in the present invention. Examples include phenyloxy and naphthyloxy, etc.

The term arylthio includes a group in which the sulfur atom is replaced by one aryl, as described in the present invention. Examples include phenylthio and naphthylthio, etc.

The term arylsulfinyl includes a group in which sulfinyl is replaced by one aryl, as described in the present invention. Examples include phenylsulfinyl and naphthylsulfinyl, etc.

The term arylsulfonyl includes a group in which the sulfonyl is replaced by one aryl, as described in the present invention. Examples include phenylsulfonyl and naphthylsulfonyl, etc.

Examples of arylsulfonyloxy include phenylsulfonyloxy and naphthylsulfonyloxy, etc.

The term aryloxycarbonyl includes a group in which the carbonyl is replaced by one aryloxy, as described in the present invention. Examples include phenyloxycarbonyl, 1-naphthyloxycarbonyl and 2naphthyloxycarbonyl, etc.

The term heteroaryloxy includes a group in which the oxygen atom is replaced by one heteroaryl, as described in the present invention. Examples include pyrrolyloxy, furyloxy, thienyloxy, imidazolyloxy, pyrazolyloxy, isothiazolyloxy, isoxazolyloxy, oxazolyloxy, thiazolyloxy, pyridyloxy, pyrazinyloxy, pyrimidinyloxy, pyridazinyloxy, tetrazolyloxy, oxadiazolyloxy, thiadiazolyloxy, indolidinyloxy, isoindolinyloxy, indolyloxy , indazolyloxy, purinyloxy, quinolidinyloxy, isoquinolyloxy, quinolyloxy, phtharasinyloxy, naphthylidinyloxy, quinolanyloxy,

- 10 043597 quinazolinyloxy, cinnolinyloxy, pteridinyloxy, carbazolyloxy, phenanthridinyloxy, acridinyloxy, dibenzofuranyloxy, benzoimidazolyloxy, benzoisoxazolyloxy, benzooxazolyloxy, benzooxadiazolyloxy, benzoisothiazolyloxy, benzothiazolyloxy, benzofuryloxy, benzothienyloxy, dibenzothienyl oxy and benzodioxolyloxy. Preferably furyloxy, thienyloxy, imidazolyloxy, pyrazolyloxy, isothiazolyloxy, isoxazolyloxy, oxazolyloxy, thiazolyloxy, pyridyloxy, pyrazinyloxy, pyrimidinyloxy and pyridazinyloxy, etc.

The term heteroarylthio includes a group in which the sulfur atom is replaced by one heteroaryl, as described in the present invention. Examples include pyrrolylthio, furylthio, thienylthio, imidazolylthio, pyrazolylthio, isothiazolylthio, isoxazolylthio, oxazolylthio, thiazolylthio, pyridylthio, pyrazinylthio, pyrimidinylthio, pyridazinylthio, tetrazolylthio, oxadiazolylthio, thiadiazolylthio, ind. olidinylthio, isoindolinylthio, indolylthio, indazolylthio, purinylthio, quinolidinylthio, isoquinolylthio, quinolylthio, phtarazinylthio, naphthylidinylthio, quinolanylthio, quinazolinylthio, cinnolinylthio, pteridinylthio, carbazolylthio, phenanthridinylthio, acridinylthio, dibenzofuranylthio, benzoimidazolylthio, benzoisoxazolylthio, benzooxazolylthio, benzooxadiazolylthio, benzoisothiazolylthio , benzothiazolylthio, benzofurylthio, benzothienylthio, dibenzothienylthio and benzodioxolylthio, etc. Preferably, furylthio, thienylthio, imidazolylthio, pyrazolylthio, isothiazolylthio, isoxazolylthio, oxazolylthio, thiazolylthio, pyridylthio, pyrazinylthio, pyrimidinylthio and pyridazinylthio, etc.

The term heteroarylsulfinyl includes a group in which the sulfinyl is replaced by one heteroaryl, as described in the present invention. Examples include pyrrolylsulfinyl, furylsulfinyl, thienylsulfinyl, imidazolylsulfinyl, pyrazolylsulfinyl, isothiazolylsulfinyl, isoxazolylsulfinyl, oxazolylsulfinyl, thiazolylsulfinyl, pyridylsulfinyl, pyrazinylsulfinyl, pyrimidinylsulfinyl, pyridazinylsulfinyl, tetrazolylsulfinyl, oxadiazolylsulphinyl inyl, thiadiazolylsulfinyl, indolidinylsulfinyl, isoindolylsulfinyl, indolylsulfinyl, indazolylsulfinyl, purinylsulfinyl, quinolidinylsulfinyl, isoquinolylsulfinyl, quinolylsulfinyl, phtarazinylsulfinyl, naphthylidinylsulfinyl, quinolanylsulfinyl, quinazolinylsulfinyl, cinnolinylsulfinyl, pteridinylsulfinyl, carbazolylsulfinyl, phenanthridinylsulfinyl, acridinylsulfinyl, dibenzofuranylsulfinyl, benzoimidazolylsulfinyl, benzoisoxazolylsulfinyl, benzooxazolylsulfinyl, benzooxadia zolylsulfinyl, benzothiazolylsulfinyl, benzothiazolylsulfinyl, benzofurylsulfinyl, benzothienylsulfinyl, dibenzothienylsulfinyl and benzodioxolylsulfinyl, etc. Preferably, furylsulfinyl, thienylsulfinyl, imidazolylsulfinyl, pyrazolylsulfinyl, isothiazolylsulfinyl, isoxazolylsulfinyl, oxazolylsulfinyl, thiazolylsulfinyl, pyridylsulfinyl, pyrazinylsulfinyl, pyrimidinylsulfinyl and pyridazinylsulfinyl, etc.

The term heteroarylsulfonyl includes a group in which the sulfonyl is replaced by one heteroaryl, as described in the present invention. Examples include pyrrolylsulfonyl, furylsulfonyl, thienylsulfonyl, imidazolylsulfonyl, pyrazolylsulfonyl, isothiazolylsulfonyl, isoxazolylsulfonyl, oxazolylsulfonyl, thiazolylsulfonyl, pyridylsulfonyl, pyrazinylsulfonyl, pyrimidinylsulfonyl, pyridazinylsulfonyl, tetrazolylsulfonyl, oxadiazolylsulfonyl onyl, thiadiazolylsulfonyl, indolizinylsulfonyl, isoindolylsulfonyl, indolylsulfonyl, indazolylsulfonyl, purinylsulfonyl, quinolidinylsulfonyl, isoquinolylsulfonyl, quinolylsulfonyl, phtarazinylsulfonyl, naphthylidinylsulfonyl, quinolanylsulfonyl, quinazolinylsulfonyl, cinnolinylsulfonyl, pteridinylsulfonyl, carbazolylsulfonyl, phenanthridinylsulfonyl, acridinylsulfonyl, dibenzofuranylsulfonyl, benzoimidazolylsulfonyl, benzoisoxazolylsulfonyl, benzooxazolylsulfonyl, benzooxadia zolylsulfonyl, benzothiazolylsulfonyl, benzothiazolylsulfonyl, benzofurylsulfonyl, benzothienylsulfonyl, dibenzothienylsulfonyl and benzodioxolylsulfonyl, etc. Furylsulfonyl, thienylsulfonyl, imidazolylsulfonyl, pyrazolylsulfonyl, isothiazolylsulfonyl, isoxazolylsulfonyl, oxazolylsulfonyl, thiazolylsulfonyl, pyridylsulfonyl, pyrazinylsulfonyl, pyrimidinylsulfonyl and pyridazinylsulfonyl are preferred.

The term heteroarylsulfonyloxy includes a group in which the oxygen atom is replaced by one heteroarylsulfonyl, as described in the present invention. Examples include pyrrolylsulfonyloxy, furylsulfonyloxy, thienylsulfonyloxy, imidazolylsulfonyloxy, pyrazolylsulfonyloxy, isothiazolylsulfonyloxy, isoxazolylsulfonyloxy, oxazolylsulfonyloxy, thiazolylsulfonyloxy, pyridylsulfonyloxy, pyrazinylsulfonyloxy, pyrimidinylsulfonyloxy, pyridazinylsulfonyloxy, tetra zolylsulfonyloxy, oxadiazolylsulfonyloxy, thiadiazolylsulfonyloxy, indolysinylsulfonyloxy, isoindolylsulfonyloxy, indolylsulfonyloxy, indazolylsulfonyloxy, purinylsulfonyloxy, quinolidinylsulfonyloxy, isoquinolylsulfonyloxy, quinolylsulfonyloxy, phtarazinylsulfonyloxy, naphthylidinylsulfonyloxy, quinolanylsulfonyloxy, quinazolinylsulfonyloxy, cinnolinylsulfonyloxy, pteridinylsulfonyloxy, carbazolylsulfonyloxy, phenanthridinylsulfonyloxy, acridinylsulfonyloxy, dibenzofuranylsulfonyloxy, benzoimidazolylsulfonyloxy, benzoisoxazolylsulfonyloxy, benzoox azolylsulfonyloxy, benzoxadiazolylsulfonyloxy, benzoisothiazolylsulfonyloxy, benzothiazolylsulfonyloxy, benzofurylsulfonyloxy, benzothienylsulfonyloxy, dibenzothienylsulfonyloxy and benzodioxolylsulfonyloxy, etc. Preferably, furylsulfonyloxy, thienylsulfonyloxy, imidazolyl-11 043597 sulfonyloxy, pyrazolylsulfonyloxy, isothiazolylsulfonyloxy, isoxazolylsulfonyloxy, oxazolylsulfonyloxy, thiazolylsulfonyloxy, pyridylsulfonyloxy, pyrazinylsulfonyloxy, pyrimidinylsulfonyloxy and pyridazinylsulfonyl oxy, etc.

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

The term aromatic heterocyclic ring includes an aromatic monocyclic ring or an aromatic fused heterocyclic ring. Examples include pyrrole ring, furan ring, thiophene ring, pyrazole ring, imidazole ring, isothiazole ring, isoxazole ring, oxazole ring, thiazole ring, pyrazine ring, pyrimidine ring, pyridazole ring, tetrazole ring, oxadiazole ring, thiadiazole ring, indolizine ring, isoindole ring, indole ring, indazole ring, purine ring, quinolidine ring, isoquinoline ring, quinoline ring, phtarazine ring, naphthyridine ring, quinolane ring, quinazoline ring, cinnoline ring, pteridine ring, carbazole ring, phenanthridine ring, acridine ring, dibenzofuran ring , benzoimidazole ring, benzoisoxazole ring, benzooxazole ring, benzooxadiazole ring, benzoisothiazole ring, benzothiazole ring, benzofuran ring, benzothiophene ring, dibenzothiophene ring and benzodioxolane ring. A pyridine ring, a furan ring and a thiophene ring are preferred.

The term C1-Cb-alkylene includes a linear or branched alkylene group containing from one to six carbon atom(s). Examples include _-CH2- , -CH( _CH3 )-, _-C ( _CH3 ) _2- , -CH2CH2-, -CH(CH3)CH2-, -C(CH3) _2CH2- , _{-CH2CH 2} CH ₂ -, -CH ₂ CH ₂ CH ₂ CH ₂ -, -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ - and -CH2CH2CH2CH2CH2CH2-. Preferred are -CH2-, -CH2CH2-, -CH2CH2CH2- and -CH ₂ CH ₂ CH ₂ CH ₂ -.

The term alkylene, optionally containing one or two heteroatom(s) or optionally substituted alkylene, optionally containing one or two heteroatom(s) includes a linear or branched alkylene group containing from one to six carbon atoms, optionally containing one or two heteroatom(s) , which may be substituted with alkyl as described in the present invention. Examples include -CH2-, -CH( _CH3 )-, _-C ( _{CH3 ) 2- ,} -CH2CH2-, -CH2CH2CH2- _{, -CH2CH2CH2CH2CH2- , -CH2CH2CH2CH 2} CH ₂ -, -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -, -CH2O-, -OCH2-, -CH2CH2O-, -OCH2CH2-, -CH2S-, -SCH2-, -CH ₂ CH ₂ S -, -SCH2CH2-, -CH ₂ CH ₂ OCH ₂ CH ₂ -, -OCH2CH2O-, -OCH2O-, -NHCH2-, -N(CH ₃ )CH ₂ -, -N ⁺ (CH ₃ ) ₂ CH ₂ - , -NHCH2CH2CH2— and -N(CH ₃ )CH ₂ CH ₂ CH ₂ - etc. Preferred are -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -OCH2CH2O-, -OCH2O- and -M(CH3)CH2CH2CH2-.

The term alkenylene, optionally containing one or two heteroatom(s) or optionally substituted alkenylene, optionally containing one or two heteroatom(s) includes a straight or branched alkenylene group containing from two to six carbon atoms, optionally containing one or two heteroatom(s) , which may be substituted with alkyl as described in the present invention. Examples include -CH=CHCH=CH-, -CH=CHO-, -OCH=CH-, -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 alkynylene, optionally containing one or two heteroatom(s), includes a linear or branched alkynylene group containing from two to six carbon atoms, optionally containing one or two heteroatom(s), which may be substituted with alkyl, as described in the present invention. Examples include -C=CCH ₂ -, -CH ₂ C=CCH ₂ -, -CH ₂ C=CCH ₂ O-, -OCH ₂ C=CH-, -CH2C CCH2S-, -SCH2C CH-, -CH2C CCH2NH- , -NHCH2C CH-, -CH2C CCH2N(CH;)- and

-N(CH ₃ )CH ₂ C=CH-. The most preferred are -CH ₂ C=CH ₂ - and -OCH ₂ C=CH-.

The term 3-8 membered nitrogen-containing non-aromatic heterocyclic ring includes a ring of any of the formulas described as such in US Pat. No. 8,143,285, which is incorporated herein by reference in its entirety.

The term 3-8-nitrogen-containing aromatic heterocyclic ring includes a 3-8 membered aromatic heterocyclic ring containing one or more nitrogen atom(s) and additionally optionally an oxygen atom and/or a sulfur atom in the ring. Examples include pyrrolyl (eg, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl), imidazolyl (eg, 2-imidazolyl, 4-imidazolyl), pyrazolyl (eg, 1-pyrazolyl, 3-pyrazolyl), isothiazolyl (eg, 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-pyridazinyl), pyrimidinyl (e.g. 2pyrimidinyl, 4-pyrimidinyl), pyridazinyl (e.g. 3-pyridazinyl), tetrazolyl (e.g. 1Htetrazolyl), oxadiazolyl (e.g. 1,3,4-oxadiazolyl) and thiadiazolyl (e.g. 1 ,3,4-thiadiazolyl).

The term 4-8 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 Pat. No. 8,143,285, which is incorporated herein by reference in its entirety.

Optionally substituted is used interchangeably with substituted or unsubstituted herein.

In the present specification, examples of substituents in optionally substituted alkyl, optionally substituted alkyloxy, optionally substituted alkylthio, optionally substituted alkylsulfinyl, optionally substituted alkylsulfonyl, optionally substituted alkylsulfonyloxy and optionally substituted alkyloxycarbonyl include cycloalkyl, alkylene, optionally containing one or two heteroatom(s), hydroxy, oxo, alkyloxy, optionally substituted by a group of substituents A in one to three position(s), mercapto, alkylthio, halogen atom, nitro, cyano, carboxy, alkyloxycarbonyl, optionally substituted amino, optionally substituted carbamoyl, acyl, aryl (for example, phenyl), optionally substituted by a group of substituents B in one to three position(s), heteroaryl (e.g., pyridyl, furyl, thienyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl), optionally substituted by a group of substituents C in one to three position(s), optionally substituted non-aromatic heterocyclic ring group (e.g., morpholinyl, pyrrolidinyl, piperazinyl), which may be substituted by a group of substituents C in one to three position(s), aryloxy (e.g., phenyloxy), optionally substituted by a group of substituents B in one to three position(s), alkylsulfonyl and similar. The above optionally substituted moieties may be substituted with one to three of the above substituent(s) in any possible position.

In the present specification, examples of substituents in optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted alkenylthio, optionally substituted alkynylthio, optionally substituted alkenyloxycarbonyl, optionally substituted alkynyloxycarbonyl, optionally substituted cycloalkyl, optionally substituted substituted cycloalkenyl, optionally substituted cycloalkyloxy, optionally substituted cycloalkenyloxy, optionally substituted cycloalkylthio, optionally substituted cycloalkenylthio, optionally substituted cycloalkylsulfinyl, optionally substituted cycloalkenylsulfinyl, optionally substituted cycloalkylsulfonyl, optionally substituted cycloalkenylsulfonyl, optionally substituted cycloalkylsulfonyloxy, optionally substituted nom cycloalkenylsulfonyloxy, optionally substituted alkenyloxycarbonyl, optionally substituted alkylene, optionally substituted A-Sb- alkylene, optionally substituted alkylene, optionally containing one or two heteroatom(s), optionally substituted alkenylene, optionally substituted alkenylene, optionally containing one or two heteroatom(s), optionally substituted alkynylene, and optionally substituted alkynylene, optionally containing one or two heteroatom(s) ) include alkyl (such as dialkyl), optionally substituted by a group of substituents D in one to three position(s), cycloalkyl, hydroxy, oxo, alkyloxy, optionally substituted by a group of substituents A in one to three position(s), mercapto, alkylthio, atom halogen, nitro, cyano, carboxy, alkyloxycarbonyl, optionally substituted amino, optionally substituted carbamoyl, acyl, acyloxy, aryl (e.g. phenyl), optionally substituted by substituent group B in one to three position(s), heteroaryl (e.g. pyridyl, furyl , thienyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl), optionally substituted by a group of substituents C in one to three position(s), a non-aromatic heterocyclic group (for example, morpholinyl, pyrrolidinyl, piperazinyl), optionally substituted by a group of substituents C in one to three position(s) yah), aryloxy (eg phenyloxy), optionally substituted by a group of substituents C in one to three position(s), alkylsulfonyl and the like. The above optionally substituted moieties may be replaced by one or more of the above substituent(s) in any possible position.

In the present specification, examples of substituents in optionally substituted aryl, optionally substituted phenoxy, optionally substituted aryloxy, optionally substituted phenylthio, optionally substituted arylthio, optionally substituted arylsulfinyl, optionally substituted arylsulfonyl, optionally substituted arylsulfonyloxy, optionally substituted heteroaryl, optionally substituted heteroaryloxy, optionally substituted heteroarylthio, optionally substituted heteroarylsulfinyl, optionally substituted heteroarylsulfonyl, optionally substituted heteroarylsulfonyloxy, optionally substituted non-aromatic heterocyclic group, optionally substituted piperazin-1,4-diyl, substituted piperazin-1,4-diyl, optionally substituted C ₆ -arene-1,4-diamine- K ¹ ,K4-diyl and substituted C ₆ -arene-1,4-diamine-K ¹ ,K ⁴ -diyl include alkyl, optionally substituted by a group of substituents D in one to three position(s), oxo, cycloalkyl, alkenyl, alkynyl , hydroxy, alkyloxy, optionally substituted by a group of substituents A in one to three position(s), aryloxy (e.g. phenoxy), optionally substituted by a group of substituents B in one to three position(s), mercapto, alkylthio, halogen atom, nitro, cyano , carboxy, alkyloxycarbonyl, acyl, alkylsulfonyl, optionally substituted amino, optionally substituted carbamoyl, aryl (for example, phenyl), optionally substituted by a group of substituents B in one to three position(s), heteroaryl (for example, pyridyl, furyl, thienyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl), optionally substituted by a group of substituents C in one to three position(s), a non-aromatic heterocyclic group (for example, morpholinyl, pyrrolidinyl, piperazinyl), optionally substituted by a group of substituents C in one to three position(s) ), and the like. The above optionally substituted moieties may be replaced by one or more of the above substituent(s) in any possible position.

Substituent group A consists of a halogen and a phenyl atom, optionally substituted with one or three substituent(s) selected from substituent group B.

Substituent group B consists of halogen, alkyl, alkyloxy, cyano and nitro.

The group of substituents C consists of a halogen atom and an alkyl atom.

Substituent group D consists of a halogen atom and an alkyloxy.

In compounds in which R ² represents an electron pair, the oxygen is negatively charged and can form a salt with the cation.

In some compounds, R ³ in at least 1, at least 2, at least 3 of A ¹ , A ² , A ³ , A ⁴ and A ⁵ represents a hydrogen atom or C1- _C6 -alkyl.

In carrying out the methods of the present invention, a therapeutically effective amount of the compounds of the invention can be administered to an animal, including mammals and humans, by many routes. Although in a preferred embodiment, the compounds of the invention are administered orally, parenterally or topically, other forms of administration are also contemplated, such as through medicinal compounds or aerosols.

For oral administration, an effective amount of the compounds can be administered, for example, in a solid, semi-solid, liquid or gaseous state. Specific examples include tablet, capsule, powder, granule, solution, suspension, syrup and elixir. However, 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 binder, disintegrant and/or glidant. If necessary, the resulting composition can be mixed with a diluent, buffer, impregnation agent, preservative and/or flavoring agent using known methods. Examples of the binder include crystalline cellulose, cellulose derivatives, corn starch, cyclodextrins and gelatin. Examples of the disintegrant include corn starch, potato starch and sodium carboxymethylcellulose. Examples of glidant include talc and magnesium stearate. In addition, excipients that have been commonly used, such as lactose and mannitol, can also be used.

For parenteral administration, the compounds of the present invention can be administered rectally or by injection. For rectal administration, a suppository can be used. A suppository can be prepared by mixing the compounds of the present invention with a pharmaceutically acceptable excipient that melts at body temperature but remains solid at room temperature. Examples include, but are not limited to, cocoa butter, carbon wax, and polyethylene glycol. The resulting composition can be formed into any desired shape using methods known in the art.

For administration by injection, the compounds of the present invention can be administered subcutaneously, intradermally, intravenously or intramuscularly. Drug preparations for such injection can be prepared by dissolving, suspending or emulsifying the compounds of the invention in an aqueous or non-aqueous solvent such as vegetable oil, synthetic resin glyceride, higher fatty acid ester or propylene glycol by a known method. If desired, auxiliary substances such as a solubilizing agent, an osmoregulating agent, an emulsifier, a stabilizer or a preservative, which are commonly used, may also be added. Although not required, it is preferable that the composition be sterile or sterilized.

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

For topical administration, topical formulations may be in the form of gel, cream, lotion, liquid, emulsion, ointment, spray, solution, suspension and patches. Inactive ingredients in topical formulations include, for example, but are not limited to, lauryl lactate (emollient/permeation enhancer), diethylene glycol monoethyl ether (emollient/permeation enhancer), DMSO (solubility enhancer), silicone elastomer (rheology/texture modifier). ), caprylic/capric triglyceride (emollient), octisalate (emollient/UV filter), silicone fluid (emollient/diluent), squalene (emollient), sunflower oil (emollient) and silica (thickener).

The compounds of the invention can also be used together with an additional compound having other pharmaceutically useful activity to produce a drug. A medicament containing a compound of the invention, either as a separate compound or as part of a composition, can be used to treat subjects in need of such treatment.

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The compounds of the invention can also be administered in the form of an aerosol or inhaler prepared by loading the compounds in liquid or fine powder form together with a gaseous or liquid atomizing agent and, if necessary, a known adjuvant such as a blowing agent into a non-pressurized container such as an aerosol container or nebulizer. Compressed gas such as dichlorofluoromethane, propane or nitrogen can be used as the atomizing agent.

The compounds of the invention can be administered to animals, including mammals and humans, if desired, in the form of 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, their esters, their pharmaceutically acceptable salts, their metabolites, their structurally related compounds, their analogs and combinations thereof, in a single dose or multiple doses, is also provided herein. invention.

The compounds of the invention can also be administered to an animal in need of such treatment as a nutritional supplement, either in the form of a food or a nutraceutical supplement.

The terms prevention, treatment or improvement and similar terms as used herein include prevention and complete or partial treatment. The terms may also include reduction of symptoms, improvement of symptoms, reduction in severity of symptoms, reduction in the incidence of disease, or any other change in the patient's condition that improves the therapeutic effect.

The compounds described in this invention are preferably used and/or administered in the form of a composition. Suitable compositions are preferably a pharmaceutical composition, a food product or a nutritional supplement. These compositions provide a convenient form for delivering the compounds. The compositions of the invention may contain an antioxidant in an amount effective to increase the stability 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 intended to be administered in the practice of the invention is any suitable amount. Preferably it is from 1 ng/kg body weight to 20 g/kg body weight, more preferably in the range from 1 μg/kg body weight to 1 g/kg body weight, for example, from 1 mg/kg body weight to 100 mg/ kg body weight compound per day. Accordingly, suitable compositions can be formulated. Those skilled in the field of dosing biologically active agents will be able to develop specific dosing regimens for various subjects based on known and well-studied parameters.

A preferred composition according to the invention is a pharmaceutical composition, for example, in the form of tablets, pills, capsules, caplets, multiparticulates (including granules, beads, pellets and microencapsulated particles), powders, elixirs, syrups, suspensions and solutions. Pharmaceutical compositions typically contain a pharmaceutically acceptable diluent or carrier. The pharmaceutical compositions are preferably adapted for parenteral or oral administration. Compositions for oral administration may be in solid or liquid form and may take the form of tablets, powders, suspensions and syrups, among others. Optionally, the compositions contain one or more flavors and/or colors. In general, therapeutic and nutritional compositions may contain any substance that does not significantly affect the effect of the compounds on the subject.

Pharmaceutically acceptable carriers suitable for use in such compositions are well known in the pharmaceutical field. The compositions of the invention may contain 0.01-99% by weight of the compounds of the invention. The compositions of the invention are typically prepared in unit dosage form. Preferably, the unit dosage of the compounds described in the present invention is from 0.1 to 2000 mg, more preferably from 50 to 1000 mg. The excipients used in the preparation of these compositions are those known in the art.

Further examples of product forms for the composition are dietary supplements, for example in the form of a soft gel or hard capsule, containing an encapsulating material selected from the group consisting of gelatin, starch, modified starch, starch derivatives such as glucose, sucrose, lactose and fructose. The encapsulating material may optionally contain crosslinking or polymerizing agents, stabilizers, antioxidants, light absorbing agents to protect photosensitive fillers, preservatives, and the like. Preferably, the unit dosage of the compounds described in the present invention is from 0.1 to 2000 mg, more preferably from 50 to 1000 mg.

In general, the term carrier may be used in this invention to refer to a composition with which the described compounds may be admixed, whether a pharmaceutical carrier, a food product, a nutritional supplement, or a dietary supplement. The materials described above may be considered as supports for the purposes of the invention. In certain embodiments of the invention, the carrier exhibits substantially no biological activity with respect to the compounds of the invention.

Dosage: The methods of the present invention may include administering a therapeutically effective amount of the compound to an animal in need of such treatment. The effective amount of the compound depends on the form of the compound administered, the duration of administration, the route of administration (eg, oral or parenteral), the age of the animal, and the condition of the animal, including mammals and humans.

For example, the amount of a compound effective for treating or preventing type 1 diabetes, lupus, ulcerative colitis, Crohn's disease, gastrointestinal inflammation, or any other condition described herein in an animal may range from 1 ng/kg/day to 20 g/kg/day. The preferred effective amount of the compound is from 50 μg/kg/day to 5 g/kg/day, with a more preferred dose from 1 to 100 mg/kg/day. An effective amount of a compound is most effective in the treatment or prevention of type 1 diabetes, lupus, ulcerative colitis, Crohn's disease, gastrointestinal inflammation, or any other animal condition described herein when administered to the 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, with the most effective being determined to identify the induction of positive responses. An effective amount of the compound can be continued beyond these periods to maintain positive responses in chronic diseases.

The amount of compound most effective in preventing excessive activation of the immune system may range from 1 ng/kg/day to 20 g/kg/day, with a preferred dose of 1 to 100 mg/kg/day.

When an effective amount of a compound of the present invention is administered as part of a food, therapeutic, medical or veterinary composition, the preferred dose is in the range of from about 0.01 to 2.0% w/w. in relation to a food or nutraceutical product.

In certain other embodiments, the present invention provides the use of the compounds described herein for the treatment and prevention of type 1 diabetes, lupus, IBD, and gastrointestinal inflammation.

Furthermore, generally, the present invention relates to the inhibition or activation of inflammation systemically, wherein suitable components include the pancreas, spleen, lung, heart, central nervous system, joints, liver, kidney or gastrointestinal tract, wherein suitable components include the esophagus , stomach, small intestine, cecum, colon and rectum. The effect occurs as a result of the compound affecting various types of cells in the body, causing a biological effect. The cells may include gastrointestinal tissue cells, immune cells (ie, macrophages, monocytes, lymphocytes), pancreatic islet cells, endothelial cells, neurons, or epithelial cells. In certain embodiments, the invention provides treatment of subjects with a compound of the invention, for example, as a dietary supplement to reduce or prevent inflammation associated with type 1 diabetes, lupus, or inflammatory bowel disease, or Crohn's disease, or ulcerative colitis.

In practice, the methods of the invention may provide a route for administering the compounds to a subject by any suitable route of administration, using any suitable form as described above, and allowing the subject's body to distribute the compounds to the target cell through natural processes. As described above, administration can also be accomplished by direct injection into a site (eg, organ, tissue) containing a target cell (ie, the cell to be treated).

In addition, administration can be carried out in any number of treatment regimens. Thus, it may include a single dose or dosage of the experimental compound or multiple doses or dosages over a period of time. Accordingly, treatment may involve repeating the administration step one or more times until the desired result is achieved. In certain embodiments, treatment may continue for extended periods of time, such as weeks, months, or years. Dosage regimens may preferably include administration of the compound 6 times daily to once weekly, with a more preferred regimen of three times daily to once daily. One skilled in the art is fully capable of easily developing suitable dosage regimens for patients based on parameters known in the art. Dosage amounts for the compounds of the invention can be used in the methods of these embodiments of the invention. For the treatment of type 1 diabetes, lupus or IBD, it is preferable to administer the compounds in amounts of from about 10 ng/day to 10 g/day.

The amount administered will vary depending on the subject, the stage of the disease or disorder, the age of the subject, the general health of the subject, and various other parameters known and commonly appreciated by one skilled in the art of medicine. Typically, a sufficient amount of the compound is administered to cause a noticeable change in the degree of inflammation in the pancreas, gastrointestinal tract, or systemically. The reduction in inflammation may be related to the amount of pain experienced by the subject, insulin, antinuclear antigen antibodies, levels of TNF-α or CRP in the blood, the percentage of regulatory T cells in the blood, or the concentration of calprotectin in the feces. Suitable amounts are disclosed herein, and additional suitable amounts can be determined by one skilled in the art without undue or undue experimentation based on the amounts disclosed herein.

It should be obvious that the present invention provides therapy with a LANCL2 binding compound for use in contacting cells, for example, in the treatment of subject cells. The foregoing discussion focuses on the use of the compounds of the present invention as part of a composition for use in what would generally be considered a pharmaceutical or medical setting.

The compounds described herein for the treatment of an autoimmune disease, including type 1 diabetes, systemic lupus erythematosus, IBD, Sjögren's syndrome and other conditions described herein, may be formulated as a pharmaceutical, nutritional composition, functional food composition or dietary supplement, as described in more detail above.

Alternatively, or in addition to methods of treating conditions by direct administration of compounds, conditions can be treated using prepared cells derived from progenitor cells with compounds.

The term progenitor cell is used herein to generally refer to any cell that serves as a parent cell that is processed to form a prepared cell. The cell may be a cell higher in a lineage, leading to a prepared cell such as a stem cell, progenitor cell, or progenitor cell (as the term is used in the art to refer to an intermediate between a stem cell and a differentiated cell), for example with totipotent, multipotent or unipotent properties, but does not necessarily have to be so. Accordingly, in some embodiments, creating a prepared cell from a progenitor cell includes differentiating the progenitor cell into a prepared cell. In other embodiments, creating a prepared cell from a progenitor cell simply involves inducing changes, such as changes in gene expression.

Prepared cells can be obtained from progenitor cells by reacting the progenitor cells in vitro with one or more compounds of the invention to thereby obtain prepared cells. The terms in vitro and ex vivo are used interchangeably herein in contrast to in vivo and refer to the state of being outside a living organism.

Precursors and/or prepared cells of the invention may include immune cells. Examples of immune cells include granulocytes, mast cells, monocytes, macrophages, neutrophils, dendritic cells, natural killer cells, T cells and B cells, among others. Examples of granulocytes include basophils, eosinophils and neutrophils.

The precursors and/or prepared cells of the invention may include white blood cells (leukocytes). Examples of white blood cells include neutrophils, eosinophils (acidophils), basophils, lymphocytes, and monocytes.

The progenitors and/or prepared cells of the invention may include peripheral blood mononuclear cells (PBMCs) or lamina propria mononuclear cells (LPMCs). Examples of PBMCs and LPMCs include lymphocytes (T cells, B cells, NK cells) and monocytes.

Precursors and/or prepared cells of the invention may include T cells. T cells are divided into two broad categories: CD8+ T cells or CD4+ T cells, depending on which protein is present on the surface of the cell. T cells have many functions, including killing infected cells and activating or recruiting other immune cells. CD8+ T cells are also called cytotoxic T cells or cytotoxic lymphocytes (CTLs). They are critical for recognizing and eliminating virus-infected cells and cancer cells. The main subsets of CD4+ T cells are naive CD4+ T cells, TH1 cells, TH2 cells, TH17 cells and Treg cells, where TH refers to T helper cells. Naïve CD4+ T cells are T cells that do not differentiate into any of the TH1 cells, TH2 cells, TH17 cells, and Treg cells. Regulatory T cells (Tregs) control and inhibit the activity of other T cells. They prevent adverse activation of the immune system and maintain tolerance or prevent immune reactions against the body's own cells and antigens. In some embodiments, the progenitor cells include naïve CD4+ T cells and the prepared cells include Treg cells.

Producing prepared cells from progenitor cells may involve reacting an amount of one or more compounds of the invention for a time effective to induce a compound-dependent difference in the prepared cells relative to the progenitor cells. As used herein, the term compound-dependent difference refers to the difference between a prepared cell and a progenitor cell resulting from the interaction of the progenitor cell with one or more compounds of the invention. Compound-dependent differences can be determined by the interaction of cells with media in the presence or absence of one or more compounds of the invention, wherein compound-dependent differences are characteristics that appear only for cells interacting with media in the presence of one or more compounds of the invention. Compound-dependent differences may be differences not only in kind but also in degree.

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Compound-dependent differences in prepared cells may include differences in gene expression. Unless explicitly stated otherwise, gene expression is used herein broadly to refer to any or all transcription or translation. Thus, a difference in gene expression could be a difference in mRNA production, a difference in protein production, or both. Unless explicitly stated otherwise, a differentially expressed gene may be identified herein by reference to a protein derived from the gene (eg, FOXP3) or by reference to the gene itself (eg, Lag3). In various embodiments, compound-dependent differences in gene expression may include one or more of increased expression of IL-10 or an orthologue thereof, increased expression of FOXP3 or an ortholog thereof, decreased expression of TNFα or an ortholog thereof, decreased expression of IFNa or an orthologue thereof, decreased expression of Tbet or its ortholog, increased expression of Lag3 or its ortholog, increased expression of Socs2 or its ortholog, increased expression of Irf7 or its ortholog, increased expression of P2rx7 or its ortholog, increased expression of Capn3 or its ortholog, increased expression of Ikzf2 or its ortholog, increased expression of Stat5a or its ortholog, increased expression of Pten or its ortholog, increased expression of Foxo1 or its ortholog, and/or increased expression of Phlpp1 or its ortholog. Orthologs may include orthologs in animal species. Orthologs may include orthologs in mammalian species. Orthologs (eg, for the mouse genes mentioned above) may include orthologs in primates. Orthologs (eg, for the mouse genes mentioned above) may include orthologs in humans.

The compound-dependent difference in conditioned cells may include other detectable differences such as increased phosphorylation of STAT5a or its ortholog, increased phosphorylation of FOXO1 or its orthologue, and/or increased pyruvate kinase activity.

When prepared cells are prepared, progenitor cells can react with amounts of compound from about 100 nM, about 10 nM, about 1 nM or less to about 1 μM, about 10 μM, about 100 μM, about 1 mM or more. Progenitor cells can react with the compound for a period of 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 embodiments, the PBMCs or LPMCs fully react with the compound of the invention. PBMCs or LPMCs can be isolated from the animal. In some embodiments, subtypes of PBMCs or LPMCs, such as T cells, can be isolated from the PBMCs or LPMCs and then reacted with a compound of the invention. In some embodiments, PBMCs or LPMCs are reacted with a compound of the invention and then cell subtypes, such as T cells or a specific T cell type, are isolated from them. Methods for isolating PBMCs, LPMCs and their subtypes are known in the art. See, for example, Majowicz et al. 2012 (Majowicz A, van der Marel S, te Velde AA, Meijer SL, Petry H, van Deventer SJ, Ferreira V. Murine CD4+CD25 ^- cells activated in vitro with PMA/ionomycin and anti-CD3 acquire regulatory function and ameliorate experimental colitis in vivo. BMC Gastroenterol. 2012 Dec 3;12:172) and Canavan et al. 20016 (Canavan JB, Scotta C, Vossenkamper A, Goldberg R, Elder MJ, Shoval I, 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 adoptive cell therapy for Crohn's disease. Gut. 2016 Apr; 65(4):584-94). Subsets of PMBCs, for example, can be isolated using 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 CD3/CD28 T-activator DYNABEADS® from ThermoFisher Scientific (Waltham, MA).

Obtaining prepared cells may involve differentiating the prepared cells from progenitor cells. For example, prepared cells such as Treg cells can be differentiated from progenitor cells such as naïve CD4+ T cells. Such differentiation may involve the interaction of progenitor cells with differentiation factors in addition to one or more compounds of the invention. Various differentiation factors may include all-trans retinoic acid, TGF-β, phorbol myristate acetate, ionomycin, rapamycin, and/or IL2. In some embodiments, differentiation may involve increasing the proportion of Treg cells in prepared cells relative to the proportion in progenitor cells.

The progenitors and prepared cells of the invention can be isolated cells. The term isolated or purified means material that is removed from its original environment, such as the natural environment. A material is said to be purified when it is present in a particular composition at a higher or lower concentration than the concentration that exists prior to the purification step(s).

Treatment of a condition with the prepared cells of the invention may involve administering the cells to an animal in an amount sufficient 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

- 18 043597 ziyu. The prepared cells can be injected or introduced directly into the bloodstream or other parts of the body. Non-limiting forms of enteral administration include oral and rectal administration so that the prepared cells enter the gastrointestinal tract. The prepared cells may be autologous to the animal being treated (i.e., derived from a cell taken from the same animal for which the prepared cell is used to treat) or heterologous to the animal being treated (i.e., derived from from another animal for the treatment of which the prepared cell is used). The cell obtained as described above can be used in a method of treating any of the conditions described in the present invention. Examples of conditions include inflammatory bowel disease. Examples of types of bowel inflammation include inflammatory bowel disease. Exemplary types of inflammatory bowel disease include Crohn's disease and ulcerative colitis.

In one embodiment of the invention, a method of treating an immunometabolic disease includes treatment that does not cause noticeable side effects, such as significant weight gain, systemic immune suppression, Cushingoid appearance, osteopenia/osteoporosis, cellular toxicity or pancreatitis, which are common with currently available treatments (i.e. statins, antibiotics, corticosteroids, doxorubicin, methotrexate). In other words, it has been found that a method of treatment in accordance with the present invention, which provides a therapeutic effect, at least in part, by affecting the expression and/or activation of LANCL2 and/or other immunometabolic pathways in some cells, provides a beneficial effect, not causing a significant increase in weight, for example due to fluid retention, in the subject being treated compared to other similar subjects not receiving treatment.

As such, the immunometabolic methods of the present invention can provide treatment to reduce inflammation by affecting the metabolism of immune cells. The methods may reduce inflammation systemically (ie, throughout the subject's entire body) or locally (eg, at the site of administration or at the site of inflammatory cells, including, but not limited to, T cells and macrophages). When treating or preventing inflammation through immunometabolism, one effect that can be observed is a shift in glucose metabolism. In particular, the shift may occur from production of lactate from pyruvate to entry into the tricarboxylic acid cycle, which is associated with immunoinflammatory actions. More specifically, this shift in metabolism may be associated with an increased proportion of CD4+ CD25+ FOXP3+ or other regulatory CD4+ T cells compared to effector CD4+ T cells such as IL17+ Th17 cells or IFN+ Th1 cells. Another observed effect may be a decrease in cell proliferation as a result of a combination of decreased anaerobic metabolism and increased immune checkpoint pathways. Another effect of therapeutically induced metabolic shifts may be a reduction in the expression of inflammatory chemokines such as MCP-1, IL-8, or CXCL9 as a result of altered fatty acid processing and storage. Thus, the methods can also be considered as methods of affecting or altering the immune response of the target to whom the therapy is administered, thereby preventing inflammation, disease and pathology.

The invention provides methods for inhibiting inflammation in the gastrointestinal tract, wherein relevant components include the stomach, small intestine, large intestine and rectum.

The invention provides methods for treating or preventing the development of IBD in a subject suffering from IBD, or otherwise in healthy individuals, possibly with a genetic predisposition to Crohn's disease or ulcerative colitis. These methods may also include treatment of patients with remissive IBD. In accordance with the invention, the term subject suffering from IBD is used to refer to a subject (eg, animal, human) suffering from a disease or disorder exhibiting one or more clinical signs that are typical of IBD. In general, a method of treating or preventing in accordance with this aspect of the invention involves administering to a subject an amount of a compound or cell therapy that is effective to treat or prevent one or more symptoms or clinical manifestations of IBD or to prevent the development of such symptom(s) or manifestation(s). ).

Thus, in accordance with the methods of the invention, the invention may provide methods for treating IBD, inflammation associated with intestinal infection, and inflammation associated with autoimmune diseases. Treatment methods can be preventive methods. In certain embodiments, the method is a method for treating IBD, inflammation associated with intestinal infection, and inflammation associated with autoimmune diseases. In other embodiments, the method is a method for preventing IBD. In embodiments, the method is a method of preventing activation of a remissive form of IBD. In still other embodiments, the method is a method of improving the health of a subject suffering from IBD, inflammation associated with intestinal infection, and inflammation associated with autoimmune diseases. Organisms that cause gastrointestinal infections include but are not

- 19 043597 are limited to them, Escherichia coli, Shigella, Salmonella, pathogenic Vibrios, Campylobacter jejuni, Yersina enterocolitica, Toxoplasma gondii, Entamoeba histolytica and Giardia lamblia. Accordingly, in certain embodiments, the invention provides a method of protecting the health, organs and/or tissues of a subject suffering from IBD, inflammation associated with intestinal infection, and inflammation associated with autoimmune diseases, or at risk of developing IBD, inflammation associated with intestinal infection, and inflammation associated with autoimmune diseases.

In one embodiment of the invention, a method of treating IBD includes treating IBD without causing noticeable side effects, such as significant weight gain, systemic immune suppression, Cushingoid appearance, osteopenia/osteoporosis, or pancreatitis, which are common in currently available treatments for IBD (i.e. i.e. corticosteroids, tumor necrosis factor alpha inhibitors). In other words, it has been found that a treatment method according to the present invention, which provides a therapeutic effect at least in part by affecting the expression and/or activation of LANCL2 in certain cells, provides a beneficial effect without causing significant weight gain, e.g. , due to fluid retention in the subject being treated compared to other similar subjects not receiving treatment.

As such, the methods of the present invention may provide methods for reducing inflammation. The methods may reduce inflammation systemically (ie, throughout the subject's entire body) or locally (eg, at the site of administration or at the site of inflammatory cells, including, but not limited to, T cells and macrophages). When treating or preventing inflammation in accordance with the methods of the present invention, one effect that may be seen is a decrease in the number of monocytes or macrophages and blood lymphocytes infiltrating the intestine. Another may be an increase in populations of regulatory immune cells, such as CD4 ⁺ CD25 ⁺ FoxP3 ⁺ regulatory T cells, or an increase in the regulatory properties of lymphocytes or macrophages (for example, increased levels of interleukin 4 (IL-4) or IL-10 or decreased TNF-α and IL -6). Another may be a reduction in the presence of inflammatory genes and/or adhesion molecules. Thus, the methods can also be considered as methods of influencing or altering the immune response of the subject to whom the therapy is administered. The subject may have inflammatory bowel disease or another condition in which T cell immunomodulation or inhibition of cell adhesion molecules is the desired outcome.

The invention also provides methods for treating 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 caused by viruses of the Adenoviridae family, such as adenovirus; viruses of the Herpesviridae family, such as herpes simplex type 1, herpes simplex type 2, varicella zoster virus, Epstein-Barr virus, human cytomegalovirus, human herpes virus and type 8; viruses of the Papillomaviridae family, such as human papillomavirus; viruses of the Polyomaviridae family, such as BK virus and JC virus; viruses of the Poxviridae family, such as smallpox; viruses of the Hepadnaviridae family, such as hepatitis B virus; viruses of the Parvoviridae family, such as human bocavirus and parvovirus B19; viruses of the Astroviridae family, such as human astrovirus; viruses of the Caliciviridae family, such as norovirus; viruses of the Picornaviridae family, such as Coxsackie virus, hepatitis A virus, poliovirus and rhinovirus; viruses of the Coronaviridae family, such as coronavirus; viruses of the Flaviviridae family, such as hepatitis C virus, Zika virus, yellow fever virus, dengue virus and West Nile virus, viruses of the Togaviridae family, such as rubella virus; viruses of the Hepeviridae family, such as hepatitis E virus; viruses of the Retroviridae family, such as human immunodeficiency virus (HIV); viruses of the Orthomyxoviridae family, such as influenza virus; viruses of the Arenaviridae family, such as Guanarito virus, Junin virus, Lassa virus, Machupo virus and Sabia virus; viruses of the Bunyaviridae family, such as Crimean-Congo hemorrhagic fever virus; viruses of the Filoviridae family, such as Ebola virus and Marburg virus; viruses of the Paramyxoviridae family, such as measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus, Hendra virus and Nipah virus; viruses of the Rhabdoviridae family, such as rabies virus; undesignated viruses such as hepatitis D virus; and viruses of the family Reoviridae, such as rotavirus, orbivirus, colivirus and Banna virus; among other things.

Non-limiting examples of bacterial infections include infections caused by the bacteria 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 psitta ci , Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, My cobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus- 20 043597 cus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalact iae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum , 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 infections caused by fungi of the genus Aspergillus, such as Aspergillus fumigatus, which causes 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 (desert fever); fungi of the genus Cryptococcus, such as Cryptococcus neoformans and Cryptococcus gattii, which cause cryptococcosis; dermatophyte fungi that cause ringworm; fungi that cause fungal keratitis, such as Fusarium spp., Aspergillus spp. and Candida spp.; fungi of the genus Histoplasma, such as Histoplasma capsulatum, which cause histoplasmosis; fungi of the order Mucorales, which cause mucormycosis; fungi of the genus Saccharomyces, such as Saccharomyces cerevisiae; fungi of the genus Pneumocystis, such as Pneumocystis jirovecii, which cause Pneumocystis pneumonia; and fungi of the genus Sporothrix, such as Sporothrix schenckii, which cause sporotrichosis.

The invention also provides methods for treating hyperproliferative disorders with the compounds or cells described herein. Hyperproliferative disorders include conditions involving uncontrolled cell growth, such as cancer or conditions involving the growth of tumors, adenomas, or polyps. Non-limiting examples of hyperproliferative disorders include colorectal cancer, familial adenomatous polyposis (FAP), throat cancer, thyroid cancer, stomach cancer, gastrointestinal cancer, pancreatic cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia, hepatic cell carcinoma, gastrointestinal stromal tumors, acute lymphoblastic leukemia, chronic myeloproliferative disorders, hypereosinophilic syndrome, mastocytosis, among others.

The invention also provides methods for treating an inborn error of metabolism using the compounds or cells described in the present invention. Non-limiting examples of inborn errors of metabolism include Wilson's disease, Andersen's disease or other glycogen storage diseases, cystinuria, Fabry disease, adult citrullinemia type II, Zellweger syndrome, branched-chain ketonuria, Lesch-Nyhan syndrome, Niemann-Pick disease, Fanconi-Bickel disease, Gierke's disease, hereditary fructose intolerance, phenylketonuria, medium-chain acyl-CoA dehydrogenase deficiency, among others.

The invention also provides methods for treating a chronic immunometabolic disease with the compounds or cells described herein. Non-limiting examples of chronic immunometabolic diseases include cardiovascular diseases such as atherosclerosis, coronary artery disease, peripheral arterial disease, cardiopulmonary failure, endocarditis, myocarditis and hypertension.

The invention also provides methods for treating an autoimmune disease, such as an inflammatory autoimmune disease, with the compounds or cells described herein. Non-limiting examples of autoimmune diseases include inflammatory bowel disease (IBD) (eg, Crohn's disease and ulcerative colitis), lupus, systemic lupus, Sjögren's syndrome, rheumatoid arthritis, type 1 diabetes, psoriasis, multiple sclerosis, and autoimmune diseases caused by cancer immunotherapy, among others . Non-limiting examples of autoimmune diseases caused by cancer immunotherapy include rheumatic diseases caused by cancer immunotherapy.

The invention also provides methods for treating chronic inflammatory diseases with the compounds or cells described herein. Non-limiting examples of chronic inflammatory diseases include metabolic syndrome, obesity, prediabetes, cardiovascular disease and type 2 diabetes, among others.

The invention also provides methods for treating inflammatory diseases, such as acute colonic diverticulitis, radiation-induced gastrointestinal inflammation, and liver inflammation, including, but limited to, non-alcoholic steatohepatitis (NASH), with compounds or cells described herein. Non-limiting examples of radiation-induced gastrointestinal inflammation include radiation proctitis, radiation enteritis, and radiation proctosigmoiditis.

The invention also provides methods for treating diabetes with the compounds or cells described herein, including type 1 diabetes, type 2 diabetes and other types of diabetes. The term diabetes or diabetes mellitus is used to refer to metabolic disorders in which the subject has high blood sugar levels (ie, hyperglycemia). Hyperglycemic conditions have different etiologies, for example, the pancreas does not produce enough insulin or the cells do not respond to the insulin produced. There are several recognized subtypes of diabetes. Type 1 diabetes is characterized by the body's complete inability to produce insulin or the body's inability to produce enough insulin. Type 2 diabetes usually occurs as a result of insulin resistance, a condition in which cells cannot use insulin properly. Type 2 diabetes is sometimes accompanied by a deficiency of insulin. Gestational diabetes occurs when pregnant women without a prior diagnosis of diabetes develop hyperglycemia. Less common forms of diabetes include congenital diabetes (due to genetic defects related to insulin secretion), diabetes associated with cystic fibrosis, steroid-induced diabetes caused by high doses of glucocorticoids, and several forms of monogenic diabetes (including adult-onset diabetes in the young). Monogenic diabetes includes several inherited forms of diabetes caused by mutations in a single autosomal dominant gene (as opposed to the more complex polygenic etiology leading to hyperglycemia).

The invention also provides methods for treating chronic pain with the compounds or cells described herein. Non-limiting examples of chronic pain conditions include fibromyalgia, nerve damage, migraines, back pain, abdominal pain, among others.

The invention also provides methods for treating additional conditions with the compounds or cells described herein. These include chronic inflammatory diseases such as chronic granulomatous disease, graft-versus-host disease, and tumor necrosis factor receptor-associated periodic syndrome; muscle wasting such as amyotrophic lateral sclerosis, Duchenne muscular dystrophy, scoliosis, and progressive muscular atrophy; and others.

In one aspect, the invention provides methods for treating an autoimmune inflammatory disease with the compounds or cells described herein. Non-limiting examples of autoimmune inflammatory diseases include inflammatory 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 alleviating symptoms in a subject diagnosed with systemic lupus erythematosus, or preventing the development of the disease in a subject genetically predisposed to systemic lupus erythematosus. Symptoms and signs of lupus that can be treated with the invention include, but are not limited to, lupus nephritis, inflammation of the central nervous system, headaches, scleritis, optic neuritis, fever, arteriosclerosis, coronary artery disease, joint pain and macular rash . The invention also provides a method for treating additional forms of lupus, including cutaneous lupus (discoid), drug-induced lupus, and neonatal lupus.

Conditions that can be treated by the methods described herein include any condition described as being treatable with BT-11 in any of US Pat. No. 9,556,146 to Bassaganya-Riera et al.; US Pat. No. 9,839,635 to Bassaganya-Riera et al.; US patent 10028950 to Bassaganya-Riera et al.; US patent 10201538 to Bassaganya-Riera et al.; US patent 10493072 to Bassaganya-Riera et al.; US patent 10682349 to Bassaganya-Riera et al.; US 2019/0160100 A1 to BassaganyaRiera et al.; Bissel et al. 2016 (Bissel P, Boes K, Hinckley J, Jortner BS, Magnin-Bissel G, Werre SR, Ehrich M, Carbo A, Philipson C, Hontecillas R, Philipson N, Gandour RD, Bassaganya-Riera J. Exploratory Studies With BT- 11: A Proposed Orally Active Therapeutic for Crohn's Disease. Int J Toxicol. 2016 Sep;35(5):521-9); and Carbo et al. 2016 (Carbo A, Gandour RD, Hontecillas R, Philipson N, Uren A, Bassaganya-Riera J. An N, NBis(benzimidazolylpicolinoyl)piperazine (BT-11): A Novel Lanthionine Synthetase C-Like 2-Based Therapeutic for Inflammatory Bowel Disease J Med Chem 2016 Nov 23;59(22):10113-10126); Leber et al. 2018 (Leber A, Hontecillas R, Zoccoli-Rodriguez V, Bassaganya-Riera J. Activation of LANCL2 by BT-11 Ameliorates IBD by Supporting Regulatory T Cell Stability Through Immunometabolic Mechanisms. Inflamm Bowel Dis. 2018 Aug 16; 24(9): 1978-1991); Leber et al. 2019 Int J Toxicol. (Leber A, Hontecillas R, Zoccoli-Rodriguez V, Ehrich M, Davis J, Chauhan J, Bassaganya-Riera J. Nonclinical Toxicology and Toxicokinetic Profile of an Oral Lanthionine Synthetase C-Like 2 (LANCL2) Agonist, BT-11. Int J Toxicol 2019 Mar/Apr;38(2):96-109); Leber et al. 2019 J Immunol. (Leber A, Hontecillas R, Zoccoli-Rodriguez V, Chauhan J, Bassaganya-Riera J. Oral Treatment with BT-11 Ameliorates Inflammatory Bowel Disease by Enhancing Regulatory T Cell Responses in the Gut. J Immunol. 2019 Apr 1; 202(7 ):2095-2104); and Leber et al. 2020 (Leber A, Hontecillas R, ZoccoliRodriguez V, Colombel JF, Chauhan J, Ehrich M, Farinola N, Bassaganya-Riera J. The Safety, Tolerance, and Pharmacokinetics Profile of BT-11, an Oral, Gut-Restricted Lanthionine Synthetase C -Like 2 Agonist Investigational New Drug for Inflammatory Bowel Disease: A Randomized, Double-Blind, Placebo-Controlled Phase I Clinical Trial. Inflammatory Bowel Dis. 2020 Mar 4;26(4):643-652).

The elements and steps of the method described in this application can be used in any combination, regardless of whether they are explicitly described or not.

All combinations of method steps used herein may be performed in any order unless otherwise indicated or clearly implied by the context to which the combination is referred.

As used herein, the singular forms include plural references unless the content specifically states otherwise.

Numerical ranges used herein are intended to include each

- 22 043597 numbers and subsets of numbers contained in this range, whether specifically disclosed or not. Additionally, these number ranges should be viewed as supporting a statement that applies to any number or subset of numbers within the range. For example, a disclosure of 1 to 10 should be interpreted as supporting a range of 2 to 8, 3 to 7, 5 to 6, 1 to 9, 3.6 to 4.6,

3.5 to 9.9, etc.

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 specifically and individually identified as being incorporated by reference. In the event of a conflict between this disclosure and the included references, this disclosure controls.

It is obvious that the invention is not limited to the specific construction and arrangement of parts illustrated and described in the present invention, but covers such modified forms thereof as are included within the scope of the claims.

Examples

Molecular modeling.

Example 1: Molecular modeling of LANCL2 ligands.

Using previously described LANCL2 ligands, including abscisic acid and NSC61610, we identified the presence of a major small molecule binding pocket on LANCL2. These ligands were docked to a homologous model structure of LANCL2, based on the crystal structure of the closely related family homologue LANCL1, to identify important binding residues.

Formation of a compound. From the identified residues and predicted biochemical interactions, structures for high affinity ligands of LANCL2 were generated (Figure 1). The structures were generated and chemically optimized using WebMo. The structure files were created in .pdb format and converted to .pdbqt format by calculating charges using the Gasteiger method. Structures were docked using AutoDock Vina to confirm binding affinities in a specific binding pocket using a cuboid search grid of size (21 × 21 × 21 Å) to ensure predicted binding affinities and ligand conformations. Binding affinity was normalized to the molecular weight of the ligand. Top ligands were selected for further binding position investigation.

Analysis. Compounds were pre-ranked by lowest predicted binding affinity normalized to molecular weight, representing the most favorable binding position by minimizing total intermolecular energy, total internal energy, and torsional free energy. Compounds were then prioritized based on suitable distances to critical binding residues on LANCL2.

Results. Based on the virtual screening and the resulting compounds (Figure 1), BT-63, an asymmetric small molecule piperazine, provided sufficient binding affinity greater than the predicted affinity of ABA (positive control). In addition to binding, BT-63 provided favorable oral drug characteristics and had few safety concerns. Similar molecules within a small chemotype provided almost equal affinity with minor modifications in chemical properties. These include VT-64, VT-65, VT-95, VT-96 and VT-99. Meanwhile, changes on the opposite side of the molecule (BT-71, -72, -73, -74, -75) resulted in similar small changes in binding affinity. Molecular modeling of this family of molecules supports the claim that the covered chemotype has LANCL2 binding properties.

Medicinal chemistry.

Example 2A. Synthesis of VT-63.

BT-63, (2-(4-(6-(1H-benzo[d]imidazol-2-yl)picolinoyl)piperazin-1-yl)-1-phenylethanone, can be synthesized using a five-step process. Sulfur powder was added to mixtures of benzene-1,2-diamine and 2,6-dimethylpyridine, then heated to 160° C. The reaction mixture was diluted with methanol, filtered and evaporated under reduced pressure to obtain 2-(6-methylpyridin-2-yl)-1Hbenzo[d]imidazole Selenium dioxide was added to a solution of 2-(6-methylpyridin-2-yl)-1Hbenzo[d]imidazole in pyridine and heated to 110° C. The solvent from the reaction mixture was evaporated under reduced pressure and the resulting crude product was extracted with ethyl acetate. filtered and dried under vacuum to obtain 6-(1H-benzo[d]imidazol2-yl)picolinic acid.DIPEA was added to a solution of tert-butylpiperazine-1-carboxylate and 2bromo-1-phenylethanone in DCM at 0°C and stirred. the mixture was diluted with ice water and the organic layer was separated to obtain tert-butyl 4-(2-oxo-2-phenylethyl)piperazine-1-carboxylate. 4n. HCl in 1,4-dioxane was added to a solution of tert-butyl 4-(2-oxo-2-phenylethyl)piperazine-1carboxylate in DCM at 0°C and stirred at RT. The solvent was evaporated to obtain 1-phenyl-2(piperazin-1-yl)ethanoneHCl as a white to grayish or yellowish solid.

- 23 043597

HATU was added to a solution of 6-(III-benzo[d]imidazol-2-yl)picolinic acid, 1-phenyl-2-(piperazin1-yl)ethanoneHCl and DIPEA in DMF and mixed. The reaction mixture was diluted and extracted with ethyl acetate. The combined organic layer was dried over sodium sulfate and evaporated under reduced pressure. The resulting crude product was purified using a Grace purification system on a 40 μm silica column using methanol-dichloromethane as eluent to give 2-(4-(6-(N-benzo[d]imidazol-2-yl)picolinoyl) piperazin-1-yl)-1-phenylethanone. 1H NMR (400 MHz, DMSO): δ 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=8.40 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 VT-104-A.

VT -104-A, (2-(4-(6-( 1 H-benzo[d]imidazol-2-yl)picolinoyl)piperazin-1 -yl)-2-methyl-1 phenylpropan-1-one, can synthesize through a six-step process.

Dimethyl 2,6-pyridine carboxylate was stirred in methanol and cooled to 10-20°C. Potassium hydroxide was slowly added to the reaction mixture and stirred for 6 hours. After completion of the reaction, the solvent was evaporated to obtain 2,6-pyridine dicarboxylic acid monomethyl ester.

Monomethyl ester of 2,6-pyridinedicarboxylic acid was stirred in n-methylpyrrolidone and cooled to 10-20°C. DIPEA, EDC.HCl and HOBt were successively added to the reaction mixture. After adding the solvent, benzene-1,2-diamine was added and stirred for 24 hours. After completion of the reaction, the solvent was evaporated to give 6-(methoxycarbonyl)pyridine-2-(2'aminoacetanilide).

6-(Methoxycarbonyl)pyridine-2-(2'-aminoacetanilide) was stirred in acetic acid for 15 minutes. The reaction mass was heated to 60-65°C and stirred for 16 hours. After completion of the reaction, the solvent was evaporated to obtain 6-(1H-benzo[d]imidazol-2-yl)methylpicolinate.

6-(1H-benzo[d]imidazol-2-yl)methylpicolinate was added to lithium hydroxide in THF/water and stirred for 7 hours. After completion of the reaction, the solvent was evaporated to give 6-(1H-benzo[d]imidazol-2-yl )picolinic acid.

Sodium methoxide was added to a solution of 2-bromo-2-methyl-1-phenylpropan-1-one in dry methanol and heated at 40°C for 4 hours. The crude product was placed in toluene. Piperazine was added to the crude product and refluxed for 16 hours. After completion of the reaction, the solvent was evaporated under reduced pressure to obtain 2-methyl-1-phenyl-2-(piperazin-1yl)propan-1-one.

6-(III-benzo[d]imidazol-2-yl)picolinic acid in TEA was added to a solution of 2-methyl-1-phenyl2-(piperazin-1-yl)propan-1-one in THF and stirred for 10 min . T3P in ethyl acetate was added and stirred for 6 hours. After completion of the reaction, the solvent was evaporated under reduced pressure. The product was purified to give (2-(4-(6-(III-benzo[d]imidazol-2-yl)picolinoyl)piperazin-1yl)-2-methyl-1-phenylpropan-1-one (VT-104-A 1H NMR (400 MHz, DMSO- _d6 ): δ 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. Synthesis of VT-104-V.

VT-104-B, 4-(6-(1H-benzo[d]imidazol-2-yl)picolinoyl)-1-(2-oxo-2-phenylethyl)piperazine-2,6dione, can be synthesized via a six-step process.

Dimethyl 2,6-pyridine carboxylate was stirred in methanol and cooled to 10-20°C. Potassium hydroxide was slowly added to the reaction mixture and stirred for 6 hours. After completion of the reaction, the solvent was evaporated to obtain 2,6-pyridine dicarboxylic acid monomethyl ester.

Monomethyl ester of 2,6-pyridinedicarboxylic acid was stirred in n-methylpyrrolidone and cooled to 10-20°C. DIPEA, EDC.HCl and HOBt were sequentially added to the reaction mixture. After adding the solvent, benzene-1,2-diamine was added and stirred for 24 hours. After completion of the reaction, the solvent was evaporated to give 6-(methoxycarbonyl)pyridine-2-(2'aminoacetanilide).

6-(Methoxycarbonyl)pyridine-2-(2'-aminoacetanilide) was stirred in acetic acid for 15 minutes. The reaction mass was heated to 60-65°C and stirred for 16 hours. After completion of the reaction, the solvent was evaporated to obtain 6-(1H-benzo[d]imidazol-2-yl)methylpicolinate.

6-(1H-Benzo[d]imidazol-2-yl)methylpicolinate was added to lithium hydroxide in THF/water and stirred for 7 hours. After completion of the reaction, the solvent was evaporated to give 6-(1H-benzo[d]imidazol-2-yl )picolinic acid.

Piperazine-2,6-dione in TEA was added to a solution of 6-(1H-benzo[d]imidazol-2-yl)picolinic acid in THF and stirred for 10 min. T3P in ethyl acetate was then added and stirred for 2 hours. After completion of the reaction, the reaction mixture was extracted with ethyl acetate and washed with water to obtain 1-(6-(III-benzo[d]imidazol-2-yl)picolinoyl)piperazine-2,6- diona.

2-Bromo-1-phenylethan-1-one in DIPEA was added to a solution of 1-(6-(1H-benzo[d]imidazol-2

- 24 043597 yl)picolinoyl)piperazine-2,6-dione in dry DCM:DMF and stirred for 2 hours. After completion of the reaction, the reaction mixture was extracted with ethyl acetate and washed with water. The material was dried and purified to give 4-(6-(1H-benzo[d]imidazol-2-yl)picolinoyl)-1-(2-oxo-2phenylethyl)piperazine-2,6-dione (BT-104-B) . 1H NMR (400 MHz, DMSO-d ₆ ): δ 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), 7 .60-7.56 (m, 3H), 7.30-7.24 (m, 2H), 5.25 (s, 2H), 4.86 (d, J=4.40 Hz, 4H). HPLC purity: 98.82%; MS: m/z 452.26 (M-H).

Receptor binding.

Example 3 Coupling surface plasmon resonance to LANCL2.

Introduction.

Virtual screening and in silico experiments are valuable tools for identifying and prioritizing scaffolds of interest when developing new small molecule ligands for a therapeutic target. To confirm these results, a variety of in vitro methods are available to determine the affinity of a small molecule for a protein of interest. One identified method is surface plasmon resonance, which is the ability to assess binding at steady state by passing a ligand suspension over an immobilized purified protein. This method was used to evaluate potential LANCL2 ligands.

Ways.

Preparation and purification of LANCL2. LANCL2 was cloned into E. coli, amplified, and transfected into Pichia pastoris. P. pastoris was sown on adenine-selective medium. Stable transfected colonies were selected and grown in YPD broth at 30°C for 24 h with shaking at 240 rpm. The starter culture was used to inoculate a basal medium (1% yeast extract, 2% peptone, 1% sorbitol, 2% yeast nitrogen agar base) containing biotin and containing potassium phosphate buffer. The inoculated basal medium was incubated for 48 h at 30°C, 240 rpm. P. pastoris was then pelleted by centrifugation and resuspended in expression medium (1% sorbitol, 2% yeast nitrogen agar base) containing biotin and containing potassium phosphate buffer. The culture was induced daily to produce protein by adding methanol and incubated for 4 days at 28°C, 240 rpm. After incubation, cells were pelleted by centrifugation and lysed by sonication. Recombinant LANCL2 protein was purified by fast protein liquid chromatography (AktaPrime) using immobilized metal affinity chromatography. Protein fractions were eluted in 1-ml aliquots and assessed for LANCL2 content.

Surface plasmon resonance. Biacore T200 was used to assess binding to the LANCL2 protein. The protein was immobilized on the CM5 sensor chip. LANCL2 was diluted in 10 mM sodium acetate buffer at pH 4.0 and immobilized on a flow cell to ∼3700 RU using standard amine immobilization chemistry. Based on the immobilized response values, theoretical R _max values were calculated. The R _max values suggest a 1:1 interaction mechanism. Overnight kinetics were performed for all analytes binding to immobilized proteins. Kinetic experiments were carried out in the presence of working buffer + 1% DMSO. The flow rate of all solutions was maintained at 50 μL/min. Analyte concentrations were 0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM.

Results.

BT-63 bound to the LANCL2 protein with a KD of 2.74 μM. Surface plasmon resonance confirms the predicted binding of this family of molecules of the described Markush structure to LANCL2. In comparison, BT-62, a molecule identical to BT-63 except for the methylene between the piperazine and the carbonyl in BT-63, had a KD of 18.0 μM. The addition of methylene provides increased affinity for LANCL2 with a KD in a range similar to that of BT-11, a LANCL2-targeting therapeutic.

Experimental studies.

Example 4. Pharmacokinetics of VT-63.

Introduction.

In addition to inducing immune effects, the pharmaceutical compound must also reach compartments within the body in adequate concentrations to provide a therapeutic effect. In addition, early evaluation of pharmacokinetics is necessary to determine the desired route of administration.

Ways.

C57BL/6 mice were orally administered 10 and 40 mg/kg BT-63 via gavage of a methylcellulose solution containing BT-63. After gastric intubation, 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 quantified by LC-MS/MS.

Results.

Oral administration of BT-63 (10 mg/kg) resulted in a maximum plasma concentration of 585

- 25 043597 ng/ml and the area under the effect curve is 1055 chng/ml (Fig. 2). Oral administration of 40 mg/kg resulted in a dramatic increase in maximum plasma concentration (4465 ng/ml) as well as exposure (11552 ng/ml). The results indicate that BT-63 is viable as an oral small molecule therapeutic. Based on these results, BT-63 is a highly bioavailable compound, unlike 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 10 mg/kg dose).

Example 5 Detection of immune effects in CD4+ T cells.

Introduction.

Dysfunction of CD4+ T helper cells plays a central role in the pathogenesis of many autoimmune diseases. These cells are important for maintaining the health of the subject, enhancing immune responses, and maintaining homeostasis. However, in the case of autoimmune and inflammatory disease, CD4+ T helper cells may become overactive, activated in the absence of stimuli, or unable to resolve inflammation. In these scenarios, therapeutic agents that can mitigate or prevent inflammation are valuable treatments for disease control. To this end, the inventors confirmed the functional therapeutic potential of VT-63, VT-104-A (see FIG. 15), VT-104-B (see FIG. 15) and VT-104-C (see FIG. 15 ), LANCL2-binding ligands in this cell type.

Ways.

Cell culture. Spleens were dissected from C57BL/6 mice and assessed in wild-type and LANCL2-deficient states. The spleens were crushed between the frosted ends of microscope slides and filtered to obtain a cell suspension. Erythrocytes were lysed by hypotonic lysis. The remaining cells were washed and filtered. CD4+ T cells were enriched in suspension using negative selection based on magnetic sorting. Cells were harvested and seeded into anti-CD3-coated 96-well plates and cultured in the presence of BT-63 at 0, 0.1, 1, and 10 microM or BT-104A, BT-104B, or BT-104C at 0.1 microM for 48 h. During the last 6 h of culture, cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin.

Immunological analysis. Cells were collected from 96-well plates and stained with an antibody cocktail for immunophenotyping by flow cytometry. The cell culture supernatant was collected and analyzed for cytokine concentrations using a cytometric analysis system. Data were recorded on a BD FACS Celesta and analyzed using FacsDiva.

Results.

BT-63 decreased the proportions of IFNa-producing and TNFα-producing CD4+ T cells in wild-type cell culture and increased the proportions of IL10+ FOXP3+ CD4+ T cells (Fig. 3). In the absence of LANCL2, these effects were lost. Due to the suppression of inflammatory cytokines, these results suggest that BT-63 may act as an activator of LANCL2. Given the loss of activity in LANCL2-deficient cells, BT-63 exerts its dominant mechanistic effect through the LANCL2 pathway. Combined with the in silico and in vitro binding results, actions through the LANCL2 pathway are likely to result from direct binding to LANCL2.

In addition, BT-63 provided stronger immune effects compared to BT-62 (Fig. 4A). While BT-63 provided a significant increase in the number of FOXP3+ cells and a significant decrease in the number of IFN+ cells, BT-62 did not cause any changes at the concentrations tested. Therefore, the alkylene linker between the piperazine and carbonyl groups is critical for higher binding affinity to LANCL2 and greater immunological potency.

It was found that BT-104-A, BT-104-B and BT-104-C significantly reduced the proportions of IFNa+ CD4+ T cells, while BT-104-A and BT-104-B also significantly reduced the proportions of TNFa+ CD4+ T cells (Fig. 4B). Of the three compounds, BT-104-B was found to have the greatest numerical effect on IFNa+ cells and BT-104-A had the largest numerical effect on TNFa+ cells, although only statistical differences were observed with respect to vehicle.

Example 6. Use of BT-63 in a model of T1DM in NOD mice.

Introduction.

Type 1 diabetes (T1D) is an autoimmune disease in which the immune system destroys insulin-producing cells in the pancreas, requiring lifelong insulin therapy through injections or pumps. With current treatments, glycemic control is difficult, leading to prolonged periods of hyperglycemia and dysregulation of glucose metabolism, which contributes to organ damage and associated morbidities (blindness, renal failure, cardiovascular disease, limb loss). There are currently no approved treatments to prevent early progression of the disease (i.e., restoring immunological tolerance to diabetes-associated antigens so that

- 26 043597 promote regrowth of pancreatic beta cells) and very few are approved to help with glycemic control. LANCL2 is a potent receptor that promotes immune responses, cellular metabolism, and cell survival. This triple mechanism makes LANCL2 an attractive target for acute and long-term maintenance therapy in T1DM.

Ways.

NOD model. Non-obese diabetic (NOD) ShiLt mice were used in this study. NOD mice possess numerous genetic mutations that enable spontaneous onset of hyperglycemia and pancreatic pathologies associated with T1DM. Mice entered the experiment at 9 weeks of age and were observed for 12 periods. Mice were administered vehicle, 10 mg/kg BT-63, or 20 mg/kg BT-63 via gavage daily. Once a week, blood samples were collected from the tail vein to check the glucose concentration using a glucometer. After 12 weeks, mice were sacrificed to collect blood and organs for immunological testing.

Introduction of treatment. VT-63 was prepared in a 0.5% methylcellulose solution (12-15 cP). The dosage used was 10 or 20 mg/kg administered once daily. Mice were weighed weekly to update dosage composition. Dosage was calculated based on average body weight for each sex. The oral dosage was administered through the gastrointestinal tract in a volume of 0.2 ml.

Immunological analysis. Blood was collected by cardiac puncture into a heparinized tube. Plasma was separated after centrifugation and analyzed using Luminex for cytokines and hormones associated with the development of T1DM. The spleens were dissected, crushed and filtered to obtain a cell suspension. Erythrocytes were lysed. Cells were labeled with mixtures of extracellular (CD45, CD3, CD4, CD8, CD19, NK1.1, CD25, F4/80, CD11b, Gr1, CX3CR1, CD64) and intracellular (Tbet, BCL6, FOXP3, IFNu, IL21, IL10) antibodies at sequential live staining in 96-well plates in preparation for flow cytometry. Data were recorded on a BD FACS Celesta and analyzed using FacsDiva.

Results.

Oral administration of BT-63 protected against the development of hyperglycemia. Mice treated with BT-63 had significantly lower blood glucose levels from 19 weeks of age until the end of the experiment (Figure 5). The percentage of mice with hyperglycemia was reduced by 50% in the 20 mg/kg group compared to the vehicle group, while HbA1c at 21 weeks of age was reduced in both the 10 mg/kg and 20 mg/kg groups. kg. In the spleen, oral administration of BT-63 increased the ratio of CD4+ IL10+ to CD4+ IFNu+, while decreasing T follicular helper (Tfh) cells (BCL6+ IL21+) and increasing PD1+ CD4+ T cells (Fig. 6). BT-63 also increased plasma insulin and C-peptide levels and decreased MCP-1 and TNF (Fig. 7). Data support BT-63 as a preventative and restorative therapy for T1DM.

Example 7: Use of BT-63 in a TLR-induced SLE mouse model.

Introduction.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that can cause damage to the kidneys, cardiovascular system, and joints. Due to self-tolerance, standard cell apoptosis results in the production of nuclear antigens, which are processed without an immune response in non-SLE individuals. However, in SLE, the immune system reacts to these antigens by generating antibodies to double-stranded DNA (dsDNA) and other nuclear antigens, which form immune complexes that can be deposited throughout the body and cause inappropriate immune responses. Currently, the disease is treated with steroids, biologics, and other immunosuppressants with a high likelihood of harmful side effects and weakening the body's immune system. Treatment with activating ligands of LANCL2 can restore immunological tolerance and reduce the production of self-targeting antibodies.

Ways.

Resiquimod model. Resiquimod was prepared in a 1:3 ethanol:acetone mixture to provide each mouse with 85 μg of resiquimod. The resiquimod solution was mixed thoroughly and applied to the ear of C57BL6 mice three times a week for a 2-week period. Mice were monitored daily for signs of disease.

Introduction of treatment. VT-63 was prepared in a 0.5% methylcellulose solution (12-15 cP). The dosage used was 20 mg/kg once daily. Mice were weighed weekly to update dosage composition. Dosage was calculated based on average body weight for each sex. The oral dose was administered through the gastrointestinal tract in a volume of 0.2 ml.

Immunological analysis. Blood was collected by cardiac puncture into a tube with EDTA. Plasma was separated after centrifugation and analyzed by ELISA for anti-dsDNA antibodies. Urine was collected for albumin analysis to check renal function. The spleens were dissected, minced and filtered to obtain a cell suspension. Erythrocytes were lysed. Cells

- 27 043597 were labeled with mixtures of extracellular (CD45, CD3, CD4, CD8, CD19, NK1.1, CD25, F4/80, CD11b, Gr1, CX3CR1,

CD64) and intracellular (Tbet, BCL6, FOXP3, IFNu, IL6, IL10) antibodies in sequential live staining in 96-well plates in preparation for flow cytometry. Data were recorded on a BD FACS Celesta and analyzed using FacsDiva.

Results.

Oral administration of BT-63 reduces plasma anti-dsDNA antibody concentrations after 2 weeks of treatment and provides a reduction in urinary albumin, resulting in two benefits specific to the pathogenesis of SLE (Figure 8). Immunological differences are present in the spleen in the form of reduced IFN+ CD8+ T cells and IL6+ cells, indicating reduced inflammation.

Example 8. Use of VT-63 on a model of chronic IBD.

Introduction.

Crohn's disease and ulcerative colitis are chronic diseases with sporadic periods of unresolved inflammatory flare-ups leading to progressive damage to the intestinal mucosa. Loss of Mdr1a in mice impairs the ability of epithelial cells to properly process and eliminate waste products, leading to spontaneous colitis. Colitis in these mice is chronic and penetrates the layers of the intestine. Thus, the Mdr1a-/- model is an ideal model to study long-term therapeutic administration to induce and maintain reduced disease severity.

Unlike other genetic models of the disease, which can produce immunocompromised mice, Mdr1a-/- mice are immunocompetent, with the deletion instead affecting the cells' ability to repel molecules and prevent cellular stress. The accumulation of waste products and cellular byproducts leads to dysregulation of the epithelial cell life cycle and increased secretion of inflammatory cytokines and chemokines. Thus, a chronic and spontaneous onset of the disease is provided, with the primary initial events occurring within the epithelium. In addition, the MDR1 gene is a novel risk allele for IBD and influences sensitivity to glucocorticoid-based treatments. The ability of BT63 to provide therapeutic efficacy in the absence of this gene is an important indicator of resistance in the presence of genetic abnormalities and indicates the potential for human translation in a surgically salvage context.

Ways.

Model MDR1a-/-. MDR1a-deficient mice spontaneously develop colitis. MDR1a-/- began treatment with BT-63 (oral, 20 mg/kg) at 6 weeks of age and continued treatment until 10 weeks of age. Mice were weighed and scored weekly.

Introduction of treatment. VT-63 was prepared in a 0.5% methylcellulose solution (12-15 cP). The dosage used was 20 mg/kg once daily. Mice were weighed weekly to update dosage composition. Dosage was calculated based on average body weight for each sex. The oral dose was administered through the gastrointestinal tract in a volume of 0.2 ml.

Flow cytometry. The columns were collected in RPMI/FBS buffer containing collagenase (300 U/ml) and DNase (50 U/ml) for digestion. Tissues were digested for 60 min with stirring at 37°C. The resulting cell suspensions were filtered through 100 μm mesh filters, centrifuged (300xg, 8 min) and washed with fresh RPMI. After filtering the resulting single cell suspensions, immune cells were purified with a Percoll gradient containing 40% Percoll cells layered on a 70% Percoll solution. After centrifugation, the interphase was collected and washed to obtain enriched lamina propria cell fractions. Cells were labeled with mixtures of extracellular (CD45, CD3, CD4, CD8, CD19, NK1.1, CD25, F4/80, CD11b, Gr1, CX3CR1, CD64) and intracellular (Tbet, RORyT, FOXP3, IFNu, IL17, IL10) antibodies at sequential live staining in 96-well plates. Data were acquired using a FACS Celesta flow pitometer with FACSDiva software.

Results.

Oral administration of BT-63 reduces disease activity in Mdr1a-/- mice. Disease activity in a given colitis model is a summary assessment of weight loss, presence and severity of rectal bleeding, stool consistency, pain symptoms, and general behavior of the mouse. BT63 reduced disease activity throughout the infection period, with a maximum observed reduction of 90% in the fourth week of treatment. In the lamina propria of the colon, BT-63 significantly altered the proportions of immune cells (Fig. 9). Specifically, BT-63 reduces the proportions of Th1, Th17, and neutrophils, three major cell subsets responsible for inflammation of the colon mucosa. In the colon, the proportion of regulatory CD4+ CD25+ T cells increased. Meanwhile, the number of splenic Th1 cells decreased and splenic CD25+ Tregs increased after BT-63 treatment (Figure 10). The efficacy of BT-63 in a highly translated mouse model of IBD highlights the use of BT-63 as an oral therapeutic agent for the treatment of CD and UC.

Example 9. Efficiency of BT-63 on a model of viral infection.

- 28 043597

Activation of LANCL2 by specific ligands may modulate the immune response, prevent viral entry or replication processes to reduce viral load, and promote local tissue repair and homeostasis. To test the effectiveness of BT-63, the inventors will use a mouse model of influenza virus infection.

Ways.

Mouse model. Eight- to ten-week-old wild-type C57BL/6 mice were anesthetized by isoflurane inhalation. Mice were infected with influenza A (H1N1) intranasally at a permissive dose of 350 PFU/mouse. Mice were administered BT-63 daily at a dose of 20 mg/kg orally via gastric tube. Mice were weighed and assessed daily for 12 days. On day 12, mice were sacrificed to measure immune responses by flow cytometry in the lungs.

Flow cytometry. The lungs will be cut into small pieces and collected in RPMI/FBS/CaCl ₂ buffer containing collagenase (300 U/ml) and DNase (50 U/ml) for digestion. Tissues will be digested for 60 to 90 minutes while stirring at 37°C. The resulting cell suspensions will be filtered through 100 μm mesh filters, centrifuged (300xg, 8 min) and washed with fresh RPMI. Red blood cells will be lysed by hypotonic lysis and removed by filtration. Cells will be washed and plated for flow cytometry staining. Cells will be labeled with mixtures of extracellular (CD45, CD3, CD4, CD8, CD19, NK1.1, CD25, F4/80, CD11b, CD11c, Gr1, CX3CR1, CD64, SiglecF, Ly6C) and intracellular (Tbet, RORyT, FOXP3, IFNu , IL6, IL10, IFN) antibodies with sequential live staining in 96-well plates. Data will be acquired using a FACS Celesta flow pitometer with FACSDiva software.

Results.

Oral administration of BT-63 protects against mortality and reduces symptoms associated with influenza (Fig. 11). VT-63 significantly reduced the disease activity index from day 3 post-infection to day 12 post-infection compared to animals treated with vehicle. In addition, BT-63 increases survival by more than 50% compared to vehicle. Immunologically, BT-63 increased the number of IL10+ CD8+ T cells and alveolar macrophages at day 12 postinfection in the lungs (Fig. 12). These two cell types have previously been characterized as important cells in recovery from pulmonary viral infections. In addition, mice treated with BT-63 had decreased proportions of TNF-α+ CD4+ T cells and neutrophils in the lungs, cell types associated with tissue damage. Taken together, the data support the protective effect of BT-63 against viral infections.

Example 10 Ex Vivo Treatment with CD4+ T Cells for the Treatment of Inflammatory Disease.

BT-63, through immunometabolic signaling, alters the phenotypic profile of cells in vitro and immune responses in vivo. Specifically, BT-63 shapes CD4+ T cells to increase FOXP3 expression, increase suppressive capacity, and enhance the stability of these regulatory cells under inflammatory conditions. Therefore, adoptive transfer of BT-63-treated cells ex vivo is advantageous in the treatment of inflammatory diseases and disorders with inadequate CD4+ T cell responses, such as inflammatory bowel disease, graft-versus-host disease, and others described herein.

Ways.

Naïve CD4+ T cells can be isolated from mouse spleens using magnetic sorting. Isolated cells can be incubated in 96-well plates coated with anti-CD3/anti-CD28 in Treg differentiation medium. The Treg differentiation medium may be Iscove's modified Dulbecco's medium (IMDM) (ThermoFisher Scientific) supplemented with fetal bovine serum, HEPES, penicillin/streptomycin, L-glutamine and differentiating agents. Treg differentiating agents can be 10 nM all-trans retinoic acid and 5 ng/ml TGF-β. Additional experiments can be performed to compare differentiation in Treg differentiation medium with and without the addition of 10 ng/ml IL-2 or IL-12. Cells can be incubated with vehicle, 10 nM or 100 nM BT-63 in differentiation medium for 48 hours prior to analysis. Before analysis, cells can be stimulated with PMA and ionomycin for 6 hours.

In transfer experiments, donor spleens can be minced and enriched for the CD4+ fraction using magnetic sorting. CD4+CD45RB ^hl CD25- (Teff) and CD4+CD45RB ^lo CD25+ (Tregg) cells can be sorted using the 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 the specified experimental group, Rag2-/- recipient mice can receive 4x105 Teff and 1x105 Treg cells from the vehicle or BT-63 treated groups by intraperitoneal injection. Mice can be weighed and assessed weekly until sacrificed 5 weeks after transfer.

Lamina propria lymphocytes and cultured cells can be plated in 96-well plates (6x105 cells/well) and processed for immunophenotyping by flow cytometry as previously described. Briefly, cells can be incubated with antibodies,

- 29 043597 conjugated with fluorochrome, to extracellular markers: CD45, CD4, CD3, CD25, CD8. Samples requiring secondary staining can be incubated with secondary antibodies or streptavidin-conjugated fluorochrome. The samples can then be fixed and permeable. Cells can be incubated with antibodies to intracellular markers: Tbet, IFNu, IL10, FOXP3, IL17, RORyT. Data can be acquired using a BD FACS Celesta flow cytometer and analyzed using FACS Diva software (BD Pharmingen).

Results.

Given the importance of CD25+ FOXP3+ regulatory CD4+ T cells for the efficacy of BT-63, we seek to confirm the direct effect of BT-63 on their differentiation and ability to maintain phenotype under inflammatory conditions. Naïve CD4+ T cells can be differentiated into Tregs in vitro in the presence or absence of IL-2 according to the methods described above. We predict that treatment with BT-63 (100 nM) will significantly increase CD25+ FOXP3+ subtype formation in the absence of IL-2, a difference that will be further enhanced by the addition of IL-2. We predict that at concentrations as low as 10 nM, BT-63 will induce significantly more CD25+ FOXP3+ cells in the presence of IL-2. We predict that under these differentiation conditions only low levels of the mixed CD25+ Tbet+ subtype will be observed and this will not be statistically altered by BT-63. We predict that BT-63 will retain significantly higher levels of CD25+ FOXP3+ cells in IL-12-treated samples. This would contrast with the suppression of CD25+ FOXP3+ cells in IL12-treated samples in the absence of BT-63. The addition of IL-12 will also cause an increase in CD25+ Tbet+ cells in all groups, although BT-63 will provide dose-dependent protection in this mixed subset.

To identify signaling pathways modulated by BT-63 in vivo, we will isolate colonic CD4+ T cells from vehicle- and BT-63-treated Mdr1a-/- mice at the onset of colitis at 10 weeks of age. In CD4+ T cells, oral administration of BT-63 will result in significantly higher expression of Stat5a and Foxo1, two members of the IL-2 signaling pathway. Meanwhile, the expression of Pten and Phlpp1 will increase. In vitro, STAT5a will be phosphorylated at higher proportions in BT-63-treated samples, in basal Treg differentiation media, and in Treg differentiation media supplemented with either IL-2 or IL-12. FOXO1 would be similarly affected in both basal Treg differentiation media and Treg differentiation media containing IL-2, but not in Treg differentiation media containing IL-12. Cells will also differentiate in the presence of PTEN (SF1670) or STAT5 (STAT5i) inhibitors. In Treg differentiation medium containing both IL-2 and IL-12, addition of STAT5i will prevent the effects of BT-63 on CD25+ FOXP3+ and CD25+ Tbet+ cells. In contrast, SF1670 alone would prevent the effects of BT-63 on CD25+ Tbet+ cells in IL-2-containing medium.

Rag2−/− mice lack mature T and B lymphocytes. Consequently, these mice do not develop mechanisms of self-tolerance, microbial homeostasis, and general immunoregulation. Transfer of naïve CD4+ T cells into Rag2-/- mice induces intestinal inflammation, resulting from the absence of these mechanisms, by expanding the transferred cells in vivo and differentiating into inflammatory phenotypes, similar to what occurs in active inflammatory autoimmune diseases, including, but not limited to, inflammatory bowel disease. The inventors hypothesize that transfer of ex vivo BT-63-treated regulatory cells will provide homeostasis and immunoregulatory mechanisms to recipient animals.

Adoptive transfer of Tregs treated ex vivo with BT-63 (100 nM) will reduce overall disease severity and ensure maintenance of immune benefits up to the study cut-off time of 5 weeks post-transfer. In addition to overall disease improvement, ex vivo treatment of Tregs with BT-63 will result in altered colonic lamina propria cell phenotypes. In Treg groups treated with BT-63, the number of IFNu-producing and IL-17+ RORyT+ CD4+ T cells decreases. Meanwhile, the number of CD25+ Tregs will increase, indicating increased stability and increased ability to serve as a founder population of regulatory cells. In addition, interactions with the IL-2/STAT5 signaling axis will promote important changes in the cytokine and chemokine microenvironment that enhance the effects of the transferred cells.

These results will indicate that the effects of BT-63 on immune cells when administered in vivo will be reproducible when treating immune cells ex vivo. The inventors predict that administration of the prepared cells of the invention to an animal will be effective in treating any of the conditions described in the present invention, in addition to inflammatory diseases such as IBD.

Example 11. Use of BT-63 in a mouse model of genetic SLE.

Introduction.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that can cause damage to the kidneys, cardiovascular system, and joints. SLE is the result of a complex interaction of genetic factors that results in an immunological disease manifested primarily through the formation of autoantibodies. One preclinical model aimed at capturing these complex factors is the NZB/W F1 model. Crossing F1 NZB and NZW mice results in mice with progressive autoimmunity. This autoimmunity shares many features with human SLE, including the formation of antinuclear antibodies, kidney damage, and enhanced type I interferon responses.

Ways.

NZB/W F1 model. Twenty-four-week-old female NZB/W F1 mice were randomized to vehicle or BT-63 based on baseline urinary protein levels (n=10). BT63 was administered orally daily at a dose of 20 mg/kg for 12 weeks. Mice were weighed weekly to update dosage composition. The dosage was calculated based on average body weight.

Immunological analysis. Blood was collected by cardiac puncture into a tube with EDTA. Plasma was separated after centrifugation and analyzed by ELISA for anti-dsDNA antibodies and IFNα. Urine was collected for protein analysis to check renal function. The spleens were dissected, crushed and filtered to obtain a cell suspension. Erythrocytes 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, IL21, IL6, IL10) antibodies with sequential live staining in 96-well plates in preparation for flow cytometry. Data were recorded on a BD FACS Celesta and analyzed using FacsDiva.

Results.

Orally administered BT-63 protected mice from weight loss and prevented proteinuria from worsening from baseline (FIG. 13). Mice treated with BT-63 had reduced levels of anti-dsDNA and plasma IFN-α levels at 36 weeks of age (12 weeks of treatment). Treatment with BT-63 resulted in a modest reduction in follicular B cells and plasma cells in the spleen, particularly CXCR3+ plasma cells, a major pathogenic subset of lupus. However, there was a greater reduction in IL6+ myeloid cells, CD25+ FOXP3+ regulatory CD4+ T cells, and IL21+ BCL6+ follicular helper T cells compared to vehicle (Figure 14).

Example 12. Use of VT-104-B on a model of non-alcoholic steatohepatitis (NASH).

Introduction.

NASH is a progressive chronic liver disease that affects more than 140 million people worldwide, with total healthcare costs exceeding $8 billion annually in the United States alone. No current therapeutic agents are approved for the treatment of NASH. Although it is a reversible condition, ineffective treatment of NASH results in a higher risk of hepatocellular carcinoma, liver failure, and cardiac death. NASH is a complex disease associated with multiple hepatic and extrahepatic factors. However, many therapeutics in development fail to address all three major areas of dysregulation, consisting of metabolic, inflammatory, and fibrotic factors.

Ways.

WD-induced model. C57BL/6 mice were given intraperitoneal injections of 0.5 μl/g CCl ₄ every two weeks to induce steatohepatitis for 4 weeks. Mice were treated daily therapeutically after 2 weeks of injections. Treatment with BT-104-B (20 mg/kg) or vehicle control was administered via gastric tube. The dosage was calculated based on average body weight.

Analysis. The liver was excised and weighed. Liver sections were excised and stored in buffered formalin for Sirius red staining or snap-frozen for fibrosis assessment. The severity of fibrosis was assessed using the Sirius red liver staining scale during microscopic examination.

Results.

Oral administration of BT-104-B reduced liver weights (Figure 16A) and fibrosis scores (Figure 16B) after 2 weeks of treatment, indicating the ability to reduce liver inflammation and fibrosis in relation to NASH.

Example 13. Use of VT-104-B in a mouse model of rheumatoid arthritis.

Introduction.

Rheumatoid arthritis (RA) causes severe inflammation of the joints, leading to loss of mobility and severe pain. The underlying immunology of synovial inflammation is complex, involving interactions between myeloid cells, T cells, fibroblasts, and other structural cells of the synovium. High expression of TNF and IL-6 plays a central role in the pathogenesis of RA, with additional contributions of IL-1β, IL-12, IL-17, IL-21, IL-23, MCP1 and TGF-β. Together, these cytokines can lead to leukocyte recruitment, bone remodeling, pannus formation, oxidative stress, and joint mucosal hyperplasia.

Ways.

Models. Six-week-old C57B1/6 mice were immunized with 200 μg of chicken collagen, an emulsifier

Claims (22)

in Freund's complete adjuvant, by intradermal injection into the base of the tail. Mice were treated with 20 mg/kg BT-104-B or vehicle daily for four weeks. Immunological analysis. The spleens of mice were excised. The tissues were minced and filtered to obtain a cell suspension. Erythrocytes were lysed. Cells were labeled with mixtures of extracellular (CD45, CD3, CD4, CD8, CD11b, CD11c) and intracellular (IFN, IL17, TNF) antibodies in sequential live staining in 96-well plates in preparation for flow cytometry. Data were recorded on a BD FACS Celesta and analyzed using FACSDiva. Results. Oral administration of BT-104-B significantly reduced the proportion of IL-17+ CD4+ T cells (Fig. 17A), IFN+ CD4+ T cells (Fig. 17B) and TNF+ CD11b+ CD11c+ myeloid cells (Fig. 17C) in the spleen of mice with collagen-induced arthritis compared with vehicle-treated control animals. This indicates the ability of BT-104-B and related compounds to treat rheumatoid arthritis. Example 14. Use of VT-104-B in a mouse model of genetic SLE. Introduction. Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that can cause damage to the kidneys, cardiovascular system, and joints. SLE is the result of a complex interaction of genetic factors that results in an immunological disease manifested primarily through the formation of autoantibodies. One preclinical model aimed at capturing these complex factors is the NZB/W F1 model. Crossing F1 NZB and NZW mice results in mice with progressive autoimmunity. This autoimmunity shares many features with human SLE, including the formation of antinuclear antibodies, kidney damage, and enhanced type I interferon responses. Ways. NZB/W F1 model. Twenty-four-week-old female NZB/W F1 mice were randomized to vehicle or BT-104-B based on baseline urinary protein levels. VT-104B was administered daily at a dose of 20 mg/kg for 12 weeks. Mice were weighed weekly to update dosage composition. The dosage was calculated based on average body weight. Immunological analysis. The spleens were dissected, minced and filtered to obtain a cell suspension. Erythrocytes were lysed. Cells were labeled with mixtures of extracellular (CD45, CD3, CD4, CD8, CD25) and intracellular (IL21, IL17, FOXP3) antibodies in sequential live staining in 96-well plates in preparation for flow cytometry. Data were recorded on a BD FACS Celesta and analyzed using FACSDiva. Results. Oral administration of BT-104-B significantly reduced the proportion of IL-17+ CD4+ T cells (Fig. 18A) and IL21+ CD4+ T cells (Fig. 18B), while significantly increasing the proportion of CD25+ FOXP3+ CD4+ T cells (Fig. 18C) in spleens of NZB/W F1 mice compared to vehicle-treated control animals. This indicates the potential of BT-104-B and related compounds to treat SLE. CLAIM 1. A compound of the formula Z-Y-Q-Y' or a pharmaceutically acceptable salt thereof, in which: Z represents: Y represents: Q is piperazine-1,4-diyl or piperazine-2,6-dione-1,4-diyl; Y' represents: - 32 043597 - absent between A ¹⁵ and A ¹⁶ ; A ¹ , A ² , A ³ , A ⁸ , A ⁹ , A ¹¹ , A ¹² , A ¹³ , A ¹⁸ , A ²⁰ and A ²² each represent C(R ³ ); A ⁴ , A ¹⁹ and A ²¹ are each independently C(R ³ ) or N; each of A ⁶ , A ¹⁰ and A ¹⁷ represents N; A ⁵ represents N(R ³ ) or O; And ⁷ represents N(R ³ ); A ¹⁴ represents O; A ¹⁵ and A ¹⁶ each represent C(R ³ ) ₂ ; R ¹ represents C1, _C2 or _C3 alkylene, optionally substituted with one or more C1- _C6 alkyl groups; R ³ in each case represents independently a hydrogen atom, C _g C ₆ -alkyl or cyano. 2. The compound according to claim 1, in which A ⁴ represents CH. 3. The compound according to claim 1, in which A ⁵ represents NH. 4. A compound according to claim 1, wherein Y is: 5. A compound according to claim 1, wherein Q is piperazine-1,4-diyl. 6. A compound according to claim 1, wherein Q is piperazine-2,6-dione-1,4-diyl. 7. The compound according to claim 1, in which Y' represents: ABOUT 8. A compound according to claim 7, wherein R ¹ is -(CH ₂ ) ₁ . ₃ - or -C(CH ₃ ) ₂ -. 9. The compound according to claim 7, wherein each of A ¹⁹ and A ²¹ represents CH. 10. The compound according to claim 7, wherein R ³ in A ²⁰ is cyano. 11. The compound of claim 1, wherein each R ³ represents a hydrogen atom, except that the R ³ in A ²⁰ represents a hydrogen or cyano atom. 12. The compound according to claim 1, in which Z-Y-Q-Y' represents: ABOUT A ⁴ represents CH; And ⁵ represents NH; each of A ¹⁹ and A ²¹ represents CH; R ¹ represents -(CH ₂ ) ₁ . ₃ - or -C(CH ₃ ) ₂ -; and each R ³ represents a hydrogen atom, except that R ³ in A ²⁰ represents a hydrogen or cyano atom. 13. A compound according to claim 12, wherein Q is piperazine-1,4-diyl. 14. A compound according to claim 12, wherein Q is piperazine-2,6-dione-1,4-diyl. 15. The compound according to claim 12, where R ¹ represents -(CH ₂ )-. 16. The compound according to claim 12, where R ¹ represents -C(CH ₃ ) ₂ -. 17. The compound according to claim 12, wherein R ³ in A ²⁰ is cyano. 18. The compound according to claim 1, wherein the compound is selected from: - 33 043597 or a pharmaceutically acceptable salt of any of the above. 19. The connection according to claim 1, where the connection is: or a pharmaceutically acceptable salt thereof. 20. The connection according to claim 1, where the connection is: or a pharmaceutically acceptable salt thereof. 21. The connection according to claim 1, where the connection is: O or a pharmaceutically acceptable salt thereof. 22. The connection according to claim 1, where the connection is: -