Patent Application
20240101542
The present invention relates to novel compounds which are useful as inhibitors of endoplasmic reticulum aminopeptidases (ERAP), in particular as inhibitors of ERAP2. This invention also relates to the therapeutic use of these compounds.
The elemental task of the adaptive immune system is to fight against diseases by the recognition of infected or abnormal cells. For that the immune system detects antigenic peptides (epitopes) on cell surface using blood cells that implement specialized cytotoxic responses. Antigenic peptides are produced by proteolytic hydrolysis of either intracellular proteins or previously endocytosed extracellular proteins. The composition of the pool of presented peptides (repertoire) is key to a well-functioning immune system and changes in the processing of peptides promote diseases. For instance, tumour cells typically halt the generation of antigenic peptides, while overactive processing produces epitopes that lead the immune system to cause auto-immune, auto-inflammatory diseases.
A significant proportion of the precursor peptides are processed by the endoplasmic reticulum (ER) aminopeptidases ERAP enzymes (ERAP1, ERAP2, and ERAP1,2 heterodimer; referred to as ERAP). ERAP trim the N-terminally extending precursors to mature epitopes.1,2 These epitopes are loaded onto HLA class I molecules (MHC-I) and presented on the surface of antigen-presenting and tumour cells to instruct the immune system and engage immune response. Importantly some antigenic epitopes can be also destroyed by ERAP into peptides that are no longer able to bind the MHC-class I molecules. The overall composition of the antigen repertoire can have profound effects on cytotoxic response of the immune system and ERAP enzymes have emerged as key proteins for influencing its formation (and adjust T-cell and NK-cell cytotoxic responses.3,4
Polymorphisms in ERAP1 and ERAP2 have been associated with predisposition to various human diseases.5 In particular these diseases include viral infections, cancer and proliferative diseases, autoimmune diseases.6,7
Examples of relevant autoimmune diseases are, but not limited to, MHC-Class I inflammatory diseases spondyloarthritis, Behcet's disease, Birdshot uveitis, psoriasis, type-1 diabetes. These correlations have been associated to changes in the activity and specificity of ERAP.
It has been shown that ERAP are important targets to boost T-cell and NK-cell cytotoxic responses in cancer.8, 9, 10 The ERAP genotype associates with the immune infiltration of tumours, and strongly predicts the overall survival in cancer. Recent studies highlight that low levels of ERAP2 can be associated with improved response to anti-PD-L1 (immunotherapy) in patients with the luminal subtype of bladder cancer.11 In the same line, an in vivo loss-of-function CRISP genetic screen showed that deletion of ERAP1 in a mouse transplantable tumor model increased the efficacy of anti-PD-1 immunotherapy.12 Finally, ERAP modulate response to infections (HCV, HCMV, influenza virus, HPV, HIV, SARS-CoV-2, tuberculosis).13,14
ERAP inhibitors have been shown to modulate antigen presentation and immune responses.15, 16, 17 An inhibitor was shown to suppress a Th17 response in a cellular model of spondyloarthritis.18 The same compound modulates the immunopeptidome of patient cancer cells.19
These clinical and preclinical results enlighten that ERAP enzymes have important functions in vivo, and support that they are targets for therapeutic targeting of these diseases.
Accordingly, there is thus a need for efficient ERAP inhibitors, in particular for selective ERAP inhibitors. More specifically, it would be advantageous to provide selective and efficient ERAP inhibitors for use in the treatment or prophylaxis of disorders in which recognition by T-cells and/or NK-cells of antigenic peptides loaded on MHC-1 is implicated, such as proliferative, autoimmune and autoinflammatory disorders.
The present invention relates to compounds of formula (I):
The present invention also relates to the compounds of formula (I) for use in medicine, in particular for use in treatment or prophylaxis of proliferative disorders, autoinflammatory disorders and autoimmune disorders.
Further aspects of the invention are as disclosed herein and in the claims.
The present invention relates to compounds of formula (I):
Preferred salts in the context of the present invention are physiologically acceptable salts of the compounds of formula (I). However, the invention also encompasses salts which themselves are unsuitable for pharmaceutical applications but which can be used, for example, for the isolation or purification of the compounds according to the invention.
The term “physiologically acceptable salt” refers to a relatively non-toxic, inorganic or organic acid addition salt of the compound of formula (I). A suitable pharmaceutically acceptable salt of the compound of formula (I) may be, for example, an acid-addition salt of a compound of formula (I), such as an acid-addition salt with an inorganic acid, such as hydrochloric, hydrobromic, hydroiodic, sulfuric, bisulfuric, phosphoric, or nitric acid, for example, or with an organic acid, such as formic, acetic, acetoacetic, pyruvic, trifluoroacetic, propionic, butyric, hexanoic, heptanoic, undecanoic, lauric, benzoic, salicylic, 2-(4-hydroxybenzoyl)-benzoic, camphoric, cinnamic, cyclopentanepropionic, digluconic, 3-hydroxy-2-naphthoic, nicotinic, pamoic, pectinic, persulfuric, 3-phenylpropionic, picric, pivalic, 2-hydroxyethanesulfonate, itaconic, sulfamic, trifluoromethane sulfonic, dodecylsulfuric, ethansulfonic, benzenesulfonic, para-toluene sulfonic, methansulfonic, 2-naphthalenesulfonic, naphthalinedisulfonic, camphorsulfonic acid, citric, tartaric, stearic, lactic, oxalic, malonic, succinic, malic, adipic, alginic, maleic, fumaric, D-gluconic, mandelic, ascorbic, glucoheptanoic, glycerophosphoric, aspartic, sulfosalicylic, hemisulfuric, or thiocyanic acid, for example.
Solvates in the context of the invention are described as those forms of the compounds which form a complex in the solid or liquid state by coordination with solvent molecules. Hydrates are a specific form of the solvates in which the coordination is with water.
The present invention includes all possible stereoisomers of the compounds of formula (I) as single stereoisomer, or as any mixture of said stereoisomers, in any ratio. Isolation of a single stereoisomer, e.g. a single enantiomer or a single diastereomer, of a compound of formula (I) can be achieved by any suitable state of the art method, such as chromatography, especially chiral chromatography, for example.
The term “substituted by one or more” means that one or more hydrogen atoms on the designated atom or group are replaced with a selection from the indicated group, provided that the designated atom's normal valency under the existing circumstances is not exceeded. Combinations of substituents are permissible.
The term “C1-C6-alkyl” as used herein refers to straight or branched, saturated aliphatic chains of 1 to 6 carbon atoms and includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, and hexyl.
The term “C1-C6-hydroxyalkyl” as used herein designates a C1-C6-alkyl as defined herein which is substituted by one or more hydroxyl groups. Examples of C1-C6-hydroxyalkyl groups include 2-hydroxy-ethyl.
The term “C1-C6-halogenoalkyl” as used herein designates a C1-C6-alkyl as defined herein, which is substituted by one or more halogens. Examples of C1-C6-halogenolalkyl include trifluoromethyl.
The term “halogen” as used herein designates chlorine, bromine, iodine and fluorine.
The term “C3-C6-cycloalkyl” as used herein designates a monocyclic ring system containing from 3 to 6 carbon atoms, where such groups can be saturated or unsaturated, but not aromatic. In some embodiments, cycloalkyl groups are fully saturated. Examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.
The term “C1-C6-alkoxy” as used herein designates a group of formula —O—C1-C6-alkyl wherein C1-C6-alkyl is as defined herein.
The term “C1-C6-halogenoalkoxy” as used herein designates a group of formula —O—C1-C6-halogenoalkyl wherein C1-C6-halogenoalkyl is as defined herein.
The term “C1-C6-hydroxyoalkoxy” as used herein designates a group of formula —O—C1-C6-hydroxyalkyl wherein C1-C6-hydroxyalkyl is as defined herein.
The term “C1-C6-alkoxy-C1-C6-alkoxy” designates a group of formula —O—C1-C6-alkyl-O—C1-C6-alkyl wherein C1-C6-alkyl is as defined herein.
The term “carbamoyl” as used herein designates a group of formula —C(═O)—NH2.
The term “C1-C6-alkylcarbamoyl” as used herein designates a group with formula —C(═O)NH—C1-C6-alkyl.
The term “C1-C6-alkylcarbonylamino” as used herein designates a group with formula —NH—C(═O)—C1-C6-alkyl wherein C1-C6-alkyl is as defined herein.
The term “C1-C6-aminoalkyl” as used herein designates a group with formula —(CH2)n—NH2 with n ranging from 1 to 6.
The term “amidoxime” or “hydroxycarbamimidoyl” means a radical —C(═NH)NHOH or —C(═NOH)NH2.
“indolyl” as used herein includes indol-1-yl, indol-2-yl, indol-3-yl, indol-4-yl, indol-5-yl, indol-6-yl and indol-7-yl.
“pyridyl” as used herein includes pyrid-2-yl, pyrid-3-yl and pyrid-4-yl.
“benzodioxolyl” as used herein includes 1,3-benzodioxol-5-yl and 1,4-benzodioxan-6-yl.
In the above formula (I), when R1 is a phenyl, R1 may be mono-, di- or tri-substituted. Substituents may be at any position on the phenyl ring (i.e. ortho, meta and/or para). The substituents may be as disclosed herein above. Typically, R1 may be substituted by one or more substituents selected from the group consisting of hydroxyl, halogen, cyano, C1-C6-alkyl, C1-C6-halogenoalkyl, C1-C6-alkoxy, C1-C6-halogenoalkoxy C1-C6-hydroxyalkoxy, ethynyl, carbamoyl, C1-C6-alkylcarbamoyl, polyoxyethylenyl and phenoxy. In some embodiments, R1 is a phenyl which is monosubstituted in position ortho, meta or para.
In some embodiments, R1 is a phenyl which is ortho- and para-substituted. In some embodiments, R1 is a phenyl which is para-substituted and di-ortho substituted.
In some embodiments, R1 is:
wherein Ra, Rb and Rc are independently selected from the group consisting of hydrogen, hydroxyl, halogen, cyano, C1-C6-alkyl, C1-C6-halogenoalkyl, C1-C6-alkoxy, C1-C6-halogenoalkoxy, C1-C6-hydroxyalkoxy, C1-C6-alkoxy-C1-C6-alkoxy, ethynyl, carbamoyl, C1-C6-alkylcarbamoyl, polyoxyethylenyl, amidoxime and phenoxy.
In some embodiments, R1 is:
wherein Ra and Rb are independently selected from the group consisting of hydrogen, hydroxyl, halogen, cyano, C1-C6-alkyl, C1-C6-halogenoalkyl, C1-C6-alkoxy, C1-C6-halogenoalkoxy, C1-C6-hydroxyalkoxy, ethynyl, carbamoyl, C1-C6-alkylcarbamoyl, polyoxyethylenyl, and phenoxy. In some embodiments, Ra and Rb are independently selected from the group consisting of hydrogen, hydroxyl, halogen, C1-C6-alkyl, C1-C6-alkoxy, C1-C6-halogenoalkoxy and phenoxy. In some embodiments, Ra is hydroxyl, cyano, C1-C6-alkoxy (e.g. methoxy, t-butoxy), C1-C6-halogenoalkoxy (e.g. trifluoromethoxy), C1-C6-alkoxy-C1-C6-alkoxy (e.g. methoxyethoxy), amidoxime or phenoxy, typically hydroxyl, C1-C6-alkoxy (e.g. methoxy, t-butoxy), C1-C6-halogenoalkoxy (e.g. trifluoromethoxy) or phenoxy, and Rb is hydrogen. In some embodiments, Ra is hydroxyl or C1-C6-alkoxy (e.g. methoxy) and Rb is halogen (e.g. chlorine or fluorine) or C1-C6-alkoxy (e.g. methoxy).
In the above formula (I), wherein R1 is indolyl, R1 is preferably indol-3-yl that may be substituted by one or more substituents as disclosed herein above. In some embodiments, R1 is unsubstituted indol-3-yl.
In the above formula (I), wherein R1 is naphthalenyl, R1 is preferably naphthalen-2-yl that may be substituted by one or more substituents as disclosed herein above. In some embodiments, R1 is unsubstituted naphthalen-2-yl.
In the above formula (I), wherein R1 benzodioxolyl, R1 is preferably 1,3-benzodioxol-5-yl that may be substituted by one or more substituents as disclosed herein above. In some embodiments, R1 is unsubstituted 1,3-benzodioxol-5-yl.
In the above formula (I), R2 may be hydrogen, C1-C3-alkyl (e.g. methyl), cyclopropyl, typically hydrogen or C1-C3-alkyl (e.g. methyl).
In the above formula (I), R3 may be a 5- or 6-membered heteroaryl selected from the group consisting of thiophenyl, thiazolyl, isothiazolyl, thiopyranyl, dithiinyl and thiazinyl, wherein said 5- or 6-membered heteroaryl may be substituted as disclosed herein.
In some embodiments, in the above formula (I), R3 may be a 5-membered heteroaryl selected from the group consisting of thiophenyl, thiazolyl and isothiazolyl, wherein said thiophenyl, thiazolyl and isothiazolyl may be substituted as disclosed herein.
In some embodiments, in the above formula (I), R3 is a thiophenyl that may be substituted by one or more substituents selected from the group consisting of halogen, C1-C6-alkyl, phenyl and pyridyl, wherein said phenyl and pyridyl may be substituted by one or more substituents selected from the group consisting of halogen, C1-C6-alkyl, phenyl, pyridyl, C1-C6-aminoalkyl and C1-C6-alkylcarbamoyl.
In some embodiments, R3 is
wherein R5 is selected from the group consisting of hydrogen, halogen, C1-C6-alkyl, phenyl and pyridyl, wherein said phenyl and pyridyl may be substituted by one or more substituents selected from the group consisting of halogen, C1-C6-alkyl, phenyl, pyridyl, C1-C6-aminoalkyl and C1-C6-alkylcarbamoyl.
In some embodiments, R3 is
wherein R5 is selected from the group consisting of hydrogen, halogen, C1-C6-alkyl, phenyl and pyridyl (e.g. pyrid-2-yl, pyrid-3-yl and pyrid-4-yl), wherein said phenyl and pyridyl may be substituted by one or more substituents selected from the group consisting of halogen, C1-C6-alkyl, phenyl, pyridyl, C1-C6-aminoalkyl and C1-C6-alkylcarbamoyl.
In some embodiments, R3 is
wherein R6 is selected from the group consisting of hydrogen, halogen, C1-C6-alkyl, phenyl, pyridyl, C1-C6-aminoalkyl and C1-C6-alkylcarbamoyl.
In some embodiments, R3 is thiazolyl wherein said thiazolyl may be substituted as disclosed herein.
In the above formula (I), R4 may be hydrogen, C1-C3-alkyl (e.g. methyl) or C3-C6-alkyl (e.g. cyclopropyl), typically hydrogen or C1-C3-alkyl (e.g. methyl).
In some particular embodiments, the compounds of the invention are compounds of formula (I):
wherein Ra and Rb are independently selected from the group consisting of hydrogen, cyano, hydroxyl, halogen, C1-C6-alkyl, C1-C6-alkoxy, C1-C6-halogenoalkoxy and phenoxy. In some embodiments, Ra is hydroxyl, cyano, C1-C6-alkoxy (e.g. methoxy, t-butoxy), C1-C6-halogenoalkoxy (e.g. trifluoromethoxy), C1-C6-alkoxy-C1-C6-alkoxy (e.g. methoxyethoxy), amidoxime or phenoxy, typically hydroxyl, C1-C6-alkoxy (e.g. methoxy, t-butoxy), C1-C6-halogenoalkoxy (e.g. trifluoromethoxy) or phenoxy, and Rb is hydrogen. In some embodiments, Ra is hydroxyl or C1-C6-alkoxy (e.g. methoxy) and Rb is halogen (e.g. chlorine or fluorine) or C1-C6-alkoxy (e.g. methoxy).
In some of these particular embodiments, R3 is
wherein R5 is selected from the group consisting of hydrogen, halogen, C1-C6-alkyl, phenyl and pyridyl (e.g. pyrid-2-yl, pyrid-3-yl and pyrid-4-yl), wherein said phenyl and pyridyl may be substituted by one or more substituents selected from the group consisting of halogen, C1-C6-alkyl, phenyl, pyridyl, C1-C6-aminoalkyl and C1-C6-alkylcarbamoyl.
In some of these particular embodiments, R3 is
wherein R6 is selected from the group consisting of hydrogen, halogen, C1-C6-alkyl, phenyl, pyridyl, C1-C6-aminoalkyl and C1-C6-alkylcarbamoyl.
In some embodiments, the compounds of the present invention are compounds of formula (I):
The present invention includes any of the compounds of formula (I) disclosed in the “Examples” section (i.e. any of the compounds 1 to 48 disclosed herein as well as their mixtures).
The compounds of formula (I) can be prepared according to the following schemes (schemes 1, 2, 3 and 4). The schemes and procedures described below illustrate synthetic routes to the compounds of formula (I) of the invention and are not intended to be limiting. In the following, n, R1, R2, R3 and R4, unless provided differently, have the meaning as disclosed herein above.
Scheme 1: Route for the preparation of compounds of formula (I) from compounds of formula (VIII), in which n, R1, R2, R3 and R4 have the meaning as defined supra. Compounds of formulae (VIII) can be readily prepared from commercially available precursors by known methods such as disclosed in the Examples section.
Reagent and conditions: (a) KCN, NH2OH/H2O (1/1: w/w), methanol, room temperature, overnight; (b) Sulfonamides-Alkynes (compound of formula (V)), CuSO4·5H2O, Sodium ascorbate, dimethylformamide/H2O or dioxane/H2O, room temperature, overnight.
Should the reaction sequence require an amino group to be protected, the amino protecting group can be cleaved at the final stage by any conventional methods. For instance, tert-butyloxycarbonyl group (Boc) can cleaved by adding hydrochloric acid to the compound of formula (I) (HCl 4N dioxane, 1 day, room temperature). The compound may be then isolated as potassium salt by adding potassium carbonate (pH 10) in water.
“Sulfonamides-alkynes” as used herein designates a compound of formula (V):
wherein R2, R3 and R4 are as disclosed herein. Compounds of formulae (V) can be readily prepared from commercially available precursors by known methods such as disclosed in the Examples section. In some aspects, the present invention relates to a process for preparing a compound of formula (I) which comprises the steps of:
Scheme 2: Route for the preparation of compounds of formula (I) from compounds of formula (VIII), in which n, R1, R2, R3 and R4 have the meaning as defined supra. Compounds of formulae (VIII) can be readily prepared from commercially available precursors by known methods such as disclosed in the Examples section.
Reagent and conditions: (b) Sulfonamides-Alkynes (compound of formula (V), see supra), CuSO4·5H2O, Sodium ascorbate, dimethylformamide/H2O or dioxane/H2O, room temperature, overnight; (a) KCN, NH2OH/H2O (1/1: w/w), methanol, room temperature, overnight.
As indicated above, should the reaction sequence require an amino group to be protected, the amino protecting group can be cleaved at the final stage by any conventional methods. For instance, tert-butyloxycarbonyl group (Boc) can cleaved by adding hydrochloric acid to the compound of formula (I) (HCl 4N dioxane, 1 day, room temperature). Trimethylsilyl groups can be cleaved by TBAF in THF. The compound may be then converted to potassium salt by adding potassium carbonate (pH 10) in water.
In some aspects, the present invention relates to a process for preparing a compound of formula (I) which comprises the steps of:
Scheme 3: Route for the preparation of compounds of formula (I) from compounds of formula (VIII), in which n, R1, R2, R3 and R4 have the meaning as defined supra. Compounds of formulae (VIII) can be readily prepared from commercially available precursors by known methods such as disclosed in the Examples section.
Reagent and conditions: (b) Sulfonamides-Alkynes (compound of formula (V)), CuSO4·5H2O, Sodium ascorbate, dimethylformamide/H2O or dioxane/H2O, room temperature, overnight; (c) Boronic acid (R5—B(OH)2 wherein R5 is a phenyl or pyridyl optionally substituted as disclosed herein), Pd(PPh3)4, Cs2CO3, Dioxane/H2O (4/1: v/v), 70° C., 1 h45 to 4 h; (a) KCN, NH2OH/H2O (1/1: w/w), methanol, room temperature, overnight. (x) if necessary for Boc deprotection: HCl 4N dioxane, 1 day, room temperature; then potassium carbonate (pH 10) in water,
As indicated above, should the reaction sequence require an amino group to be protected, the amino protecting group can be cleaved at the final stage by any conventional methods. For instance, tert-butyloxycarbonyl group (Boc) can cleaved by adding hydrochloric acid to the compound of formula (I) (HCl 4N dioxane, 1 day, room temperature). The compound may be then converted to potassium salt by adding potassium carbonate (pH 10) in water.
In some aspects, the present invention relates to a process for preparing a compound of formula (I) which comprises the steps of:
Scheme 4: Route for the preparation of compounds of formula (I) from compounds of formula (VII), in which n, R1, R2 and R3 have the meaning as defined supra. Compounds of formulae (VII) can be readily prepared from commercially available precursors by known methods such as disclosed in the Examples section.
Reagent and conditions: (i) R2—N-propargylamine, 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), triethylamine, dimethylformamide, room temperature and overnight; (j) dimethylformamide, reflux, overnight; (k) HCl, H2O, 85° C., MW, 3 h; (1) SOCl2, methanol, 0° C. to room temperature, overnight; (m) R3-SulfonylChloride (R3—SO2Cl), N,N-Diisopropylethylamine (DIPEA), dimethylformamide, 0° C. to room temperature, overnight; (p) KCN, NH2OH/H2O (1/1: w/w), methanol, room temperature, overnight.
In some aspects, the present invention relates to a process for preparing a compound of formula (I) which comprises the steps of:
The compounds of formula (I) can be converted to any salt, particularly pharmaceutically acceptable salts, as described herein, by any method which is known to the person skilled in the art. Similarly, any salt of a compound of formula (I) can be converted into the free compound, by any method which is known to the person skilled in the art.
The present invention includes all possible salts of the compounds of the present invention as single salts, or as any mixture of said salts, in any ratio.
The present invention also relates to any intermediates as disclosed supra and in the Examples section.
In the following, the expression “a compound of formula (I)” refers to a compound of formula (I) as described herein, including any compounds of formula (I) disclosed in the “Examples” section, as well as any salts, hydrates, solvates, isomers, mixtures of isomers in any ratio and any combinations thereof. Therefore, the term “a compound of formula (I)” may refer to a single compound of formula (I) or a combination of two or more compounds of formula (I) or salts, hydrates, solvates, isomers or mixtures of isomers thereof.
The compounds of formula (I) have been found to be efficient inhibitors of ERAP, in particular to be efficient ERAP2 inhibitors. “Inhibitors” refer to molecules that are able to reduce or suppress the activity of the enzyme. The compounds of formula (I) affect the antigenic peptide repertoire presented to cytotoxic T-cells and/or NK-cells. Resultantly, the compounds of formula (I) may be useful in medicine. In particular, the compounds of formula (I) may be useful for modulating the adaptive immune response in humans and animals. More specifically, the compounds of formula (I) may be useful for the treatment or prophylaxis of disorders in which recognition by T-cells and/or NK-cells of antigenic peptides loaded on MHC-1 is implicated, such as proliferative disorders, autoinflammatory disorders and autoimmune disorders. In other terms, the compounds of formula (I) may be useful for the treatment or prophylaxis of disorders in which ERAP2 activity is implicated. The compounds of formula (I) may also be useful for the treatment or prophylaxis of infectious disorders.
The term “disorder” as used herein refers to a disease, condition or illness.
The term “treatment” as used herein refers to combating, alleviating, reducing, relieving, suppressing, repelling, healing or improving the condition of a disorder.
The terms “prophylaxis” as used herein refers to the avoidance or reduction of the risk of contracting, experiencing, suffering from or having a disorder.
The treatment or prophylaxis of a disorder may be partial or complete.
In one aspect, the present invention relates to a compound of formula (I) for use in medicine.
In accordance with a further aspect, the present invention relates to a compound of formula (I) for use in the treatment or prophylaxis of a disorder in which recognition by T-cells and/or NK-cells of antigenic peptides loaded on MHC-1 is implicated/plays a role. In other terms, the present invention relates to a compound of formula (I) for use in the treatment or prophylaxis of a disorder in which ERAP2 activity is implicated.
In another aspect, the present invention relates to a compound of formula (I) for use in the treatment or prophylaxis of a disorder wherein abnormal activity of ERAPs is observed.
In another aspect, the present invention relates to a compound of formula (I) for modulating the antigenic peptide repertoire presented by MHC-1.
In another aspect, the present invention relates to a compound of formula (I) for use as ERAP2 inhibitors.
In another aspect, the present invention relates to a compound of formula (I) for use in the treatment or prophylaxis of proliferative disorders, autoinflammatory disorders and autoimmune disorders. In other words, the present invention relates to the use of a compound of formula (I) for the treatment or prophylaxis of proliferative disorders, autoinflammatory disorders and auto immune disorders.
In one embodiment, the present invention relates to a compound of formula (I) for use in the treatment or prophylaxis of a proliferative disorder, such as cancers. Cancers include, but are not limited to, colon cancer, breast cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, bladder cancer, glioblastoma, lung cancer (e.g. non-small cell lung cancer), neuroblastoma, inflammatory myofibroblastic tumor, leukemia (e.g. acute myeloid leukemia, myelodysplastic syndrome or chronic myelomonocytic leukemia), melanoma, and lymphoma (e.g. diffuse B-cell lymphoma or anaplastic large-cell lymphoma). In particular, cancers include melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, squamous cell lung cancer, renal cell carcinoma, Hodgkin's lymphoma, cutaneous squamous cell carcinoma (CSCC), urothelial carcinoma, merkel-cell carcinoma, urothelial carcinoma, renal cell carcinoma, colorectal cancer, hepatocellular carcinoma and malignant pleural mesothelioma.
In particular, the compound of formula (I) may be useful in cancer immunotherapy and/or radiotherapy.
In one embodiment, the present invention relates to a compound of formula (I) for use in the treatment or prophylaxis of an autoimmune disorder, such as spondyloarthritis (e.g. ankylosing spondylitis, psoriatic arthritis, reactive arthritis, enteropathic arthritis), psoriasis, Birdshot retinochoroidopathy or type-1 diabetes.
In one embodiment, the present invention relates to a compound of formula (I) for use in the treatment or prophylaxis of an autoinflammatory disorder, such as Behcet's disease or psoriasis.
In accordance with a further aspect, the present invention relates a method of treatment or prophylaxis of disorders, in particular proliferative disorders, inflammatory disorders and immune disorders, particularly cancers, spondyloarthritis (e.g. ankylosing spondylitis, psoriatic arthritis, reactive arthritis, enteropathic arthritis), Birdshot retinochoroidopathy, type-1 diabetes, Behcet's disease or psoriasis, using a therapeutically effective amount of a compound of formula (I). In other words, the present invention relates to a method of treating disorders, in particular proliferative disorders, autoimmune and autoinflammatory disorders, particularly cancers, spondyloarthritis (e.g. ankylosing spondylitis, psoriatic arthritis, reactive arthritis, enteropathic arthritis), Birdshot retinochoroidopathy, type-1 diabetes, Behcet's disease or psoriasis, in a subject comprising administering to the subject, that may be human or animal, a therapeutically effective amount of at least one compound of formula (I).
A “therapeutically effective amount” as used herein refers to an amount that (i) treats or prevents the particular disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disorder described herein. The amount of a compound which constitutes a therapeutically effective amount will vary depending on many factors, such as for instance the compound and its biological activity, the composition used for administration, the route of administration, the type of disorder being treated and its severity, drugs used in combination with or coincidentally with the compounds, and the age, body weight, general health, sex, and diet of the patient. Such an effective amount can be determined routinely by one of ordinary skill in the art having regard to their own knowledge.
In another aspect, the present invention relates to a compound of formula (I) for use in a method of treating disorders, particularly proliferative disorders, autoinflammatory disorders and autoimmune disorders, particularly cancers, spondyloarthritis (e.g. ankylosing spondylitis, psoriatic arthritis, reactive arthritis, enteropathic arthritis), Birdshot retinochoroidopathy, type-1 diabetes, Behcet's disease or psoriasis.
In accordance with a further aspect, the present invention relates to the use of a compound of formula (I) for the preparation of a pharmaceutical composition, preferably a medicament, for the prophylaxis or treatment of disorders, in particular proliferative disorders, autoinflammatory disorders and autoimmune disorders, more particularly cancers, spondyloarthritis (e.g. ankylosing spondylitis, psoriatic arthritis, reactive arthritis, enteropathic arthritis), Birdshot retinochoroidopathy, type-1 diabetes, Behcet's disease or psoriasis.
The present invention also relates to pharmaceutical compositions, in particular a medicament, comprising a compound of formula (I) and one or more excipients, in particular one or more pharmaceutically acceptable excipient(s) and to their uses for the above mentioned purpose.
In one aspect, the present invention relates to a pharmaceutical composition, in particular a medicament, comprising a therapeutically effective amount of a compound of formula (I) and a pharmaceutically acceptable excipient. The pharmaceutical composition is particularly useful in the treatment or prophylaxis of disorders, in particular proliferative disorders, autoinflammatory disorders and autoimmune disorders, particularly cancers, spondyloarthritis (e.g. ankylosing spondylitis, psoriatic arthritis, reactive arthritis, enteropathic arthritis), Birdshot retinochoroidopathy, type-1 diabetes, Behcet's disease or psoriasis.
Pharmaceutically acceptable excipients include fillers and carriers, ointment bases, bases for suppositories, solvents, surfactants, emulsifiers, dispersants or wetting agents, buffers, acids and bases, isotonicity agents, adsorbent, viscosity-increasing agents, gel formers, thickeners and/or binders, disintegrants, coating materials and film formers for films or diffusion membranes, capsule materials, natural or synthetic polymers, plasticizers, penetration enhancers, stabilizers, preservatives, colourants, flavourings, sweeteners, flavour- and/or odour-masking agents.
The pharmaceutical composition may further comprise one or more additional pharmaceutically active agents, such as anticancer agents, in particular anticancer agents useful in immunotherapy or radiotherapy, disease-modifying anti-rheumatic drugs (DMARD) and current immunotherapy for immune disorders.
The pharmaceutical composition may further comprise one or more antiviral or antibacterial agents.
The pharmaceutical composition may also further comprise other ERAP1 or ERAP2 or IRAP inhibitors.
The one or more compounds of formula (I) can be administered in therapeutically effective amounts in a combinational therapy with one or more pharmaceutically active agents (pharmaceutical combinations).
Therefore, the present invention also relates to such pharmaceutical combinations. For example, the compounds of the present invention can be combined with anticancer agents, in particular anticancer agents useful in immunotherapy or radiotherapy, disease-modifying anti-rheumatic drugs (DMARD), current immunotherapy for immune disorders or other ERAP1 or ERAP2 or IRAP inhibitors.
The compounds can be administered simultaneously (as a single preparation or separate preparation), sequentially or separately.
In one aspect of the invention, a compound of formula (I) is administered to prior to administration of one or more other pharmaceutically active agents.
In another aspect of the invention, a compound of formula (I) is administered concomitantly with the administration of one or more other pharmaceutically active agents
In yet another aspect of the invention, a compound of formula (I) of the invention is administered immediately after administration of one or more other pharmaceutically active agents.
The single pharmaceutically active agents (compounds of formula (I) and other pharmaceutically active agents) may be packaged in a kit or separately.
The compounds of formula (I) can have systemic and/or local activity. For this purpose, they can be administered in a suitable manner, such as, for example, via the oral, dermal, transdermal or parenteral route.
Suitable administration forms for oral administration include for example, tablets (uncoated or coated tablets, for example with enteric or controlled release coatings that dissolve with a delay or are insoluble), orally-disintegrating tablets, films/wafers, films/lyophilisates, capsules (for example hard or soft gelatine capsules), sugar-coated tablets, granules, pellets, powders, emulsions, suspensions, aerosols or solutions.
Suitable administration forms for parenteral administration are preparations for injection and infusion in the form of solutions, suspensions, emulsions, lyophilisates or sterile powders.
Suitable administration forms for the dermal or transdermal administration routes are, for example, pharmaceutical forms for aqueous suspensions (lotions, shaking mixtures), lipophilic suspensions, emulsions, ointments, creams, transdermal therapeutic systems (for example patches), milk, pastes, foams or dusting powders.
Embodiments of the present invention will now be described by way of the following examples which are provided for illustrative purposes only, and not intended to limit the scope of the disclosure.
A flask was charged with methyl (2S)-2-(tert-butoxycarbonylamino)-3-(4-hydroxyphenyl)propanoate (50 mg, 0.169 mmol, 1 eq) phenylboronic acid (41 mg, 0.339 mmol, 2 eq), copper diacetate (31 mg, 0.169 mmol, 1 eq) and with molecular sieve. Dichloromethane and pyridine were added and the mixture was stirred at room temperature for 2 days. The mixture was diluted in water (5 mL) and extracted three times with ethyl acetate. Combined organic layers were washed three times with brine, dried over MgSO4, filtered and concentrated under reduced pressure. Crude was purified by chromatography eluting with cyclohexane/ethyl acetate (100/0 to 90/10) to give methyl (2S)-2-(tert-butoxycarbonylamino)-3-(4-phenoxyphenyl)propanoate.
Aspect: Colorless oil. Yield: 40%. Purity: 100%. LC tR=3.33 min. MS (ESI−): m/z=372 [M−H]−.
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.36-7.28 (m, 2H), 7.22-7.17 (m, 1H), 7.08 (td, J=7.4 and 0.9 Hz, 2H), 6.96-6.88 (m, 4H), 4.35 (dd, J=5.5 and 9.0 Hz, 1H), 3.70 (s, 3H), 3.09 (dd, J=5.5 and 13.8 Hz, 1H), 2.87 (dd, J=9.0 and 13.8 Hz, 1H), 1.39 (s, 9H).
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 174.1, 158.9, 157.8, 157.5, 133.4, 131.7, 130.8, 124.2, 119.9, 119.7, 80.6, 56.6, 52.6, 38.1 and 28.7.
To a solution of methyl (2S)-2-(tert-butoxycarbonylamino)-3-(4-phenoxyphenyl)propanoate (300 mg, 0.808 mmol, 1 eq) in Methanol (4 mL) and Water (3 mL) was added sodium hydroxide (129 mg, 3.23 mmol, 4 eq) and the mixture was stirred overnight at room temperature. The mixture was acidified to pH=2 using HCl 1N, the product was extracted three times with ethyl acetate, the organic phases were dried over MgSO4, filtered and concentrated under reduced pressure to furnish (2S)-2-(tert-butoxycarbonylamino)-3-(4-phenoxyphenyl)propanoic acid.
Aspect: Blue foam. Yield: 91%. Purity: 100%. LC tR=2.78 min. MS (ESI−): m/z=356 [M−H]−.
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.34-7.28 (m, 2H), 7.21 (m, 2H), 7.08 (tt, J=1.0 and 7.4 Hz, 1H), 6.96-6.87 (m, 4H), 4.34 (br, 1H), 3.16 (br, 1H), 2.89 (br, 2H), 1.38 (s, 9H).
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 158.9, 157.9 (br, 2C), 157.4, 134.0, 131.8, 130.8, 124.2, 119.8, 119.6, 80.5, 38.3, 28.7.
To an ice-cold solution of (2S)-2-(tert-butoxycarbonylamino)-3-(4-phenoxyphenyl)propanoic acid (254 mg, 0.711 mmol) in Dichloromethane (2.8 mL) was added a solution of HCl in dioxane (1.8 mL, 10 eq). The mixture was stirred at room temperature for overnight. Solvents were evaporated under vacuum to furnish (S)-2-amino-3-(4-phenoxyphenyl)propanoic acid, hydrochloric acid salt.
Aspect: Yellow solid. Yield: 93%. Purity: 97%. LC tR=1.92 min. MS (ESI+): m/z=258 [M+H]+.
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 13.82 (br, 1H), 8.50 (br, 3H), 7.42-7.35 (m, 2H), 7.33-7.28 (m, 2H), 7.15-7.10 (m, 1H), 7.01-6.94 (m, 4H), 4.13 (t, J=6.1 Hz, 1H), 3.14 (d, J=6.1 Hz, 1H).
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 170.3, 156.8, 155.7, 131.2, 130.1, 130.0, 123.3, 118.9, 118.4, 53.2, 34.9.
Propargylamine or R2—N-propargylamine (1.1-1.5 eq) and N,N-diisopropylethylamine or triethylamine (1.2-3 eq) were solubilized in dichloromethane or in dimethylformamide (0.08-0.34M). The reaction media was cooled at 0° C. and R3-sulfonyl chloride (1-1.2 eq.) was added. The mixture was allowed to reach room temperature and was stirred 1 h to one night. Then, products were washed twice with 0.5 or 1M HCl (aq), dried over MgSO4, filtered and concentrated under reduced pressure. Then, the residues were filtered on silica gel (4 g cartridge) or purified by flash chromatography to afford the desired products of formula (V):
wherein R2 and Ra are as disclosed herein and R4 is hydrogen.
Compounds of formula (V) wherein R4 is different from hydrogen may be obtained by reacting a compound of formula NH2—CHR4—CCH or NHR2—CHR4—CCH with R3-sulfonyl chloride.
Exemplary procedures are disclosed herein below.
To a stirring solution of tert-butyl N-[(1S)-1-methylprop-2-ynyl]carbamate (176 mg, 0.96 mmol, 1.0 eq.) in Methanol (4 mL, 0.25 M) was added HCl (1.2 mL of 4M solution in 1,4-dioxane, 4.81 mmol, 5.0 eq.). The resulting mixture was stirred for 3 h at room temperature before being evaporated to dryness. The obtained residue was dissolved in CH2Cl2 (5.2 mL), NEt3 (0.26 mL, 2.12 mmol, 2.1 eq.) and DMAP (1 crystal) were added and the resulting mixture was cooled to 0° C. 5-(2-pyridyl)thiophene-2-sulfonyl chloride (250 mg, 0.96 mmol, 1.0 eq.) was added. The reaction mixture was allowed to warm to room temperature and stirred for 4 h before being quenched with water. The two layers were separated and the aqueous one was extracted three times with CH2Cl2. Combined organic layers were dried with MgSO4, filtered and concentrated under reduced pressure to afford N-[(1S)-1-methylprop-2-ynyl]-5-(2-pyridyl)thiophene-2-sulfonamide (224 mg, 80%) as a beige powder which was directly used as such.
To a stirring solution of tert-butyl N-[(1S)-1-methylprop-2-ynyl]carbamate (141 mg, 0.77 mmol, 1.0 eq.) in Methanol (3.2 mL, 0.25 M) was added HCl (0.96 mL of a 4M solution in 1,4-dioxane, 3.88 mmol, 5.0 eq.). The resulting mixture was stirred for 3 h at room temperature before being evaporated to dryness. The obtained residue was dissolved in CH2Cl2 (4.10 mL), NEt3 (0.23 mL, 1.69 mmol, 2.1 eq.) and 4-dimethylaminopyridine (DMAP) (1 crystal) were added and the resulting mixture was cooled to 0° C. 5-(2-pyridyl)thiophene-2-sulfonyl chloride (200 mg, 0.77 mmol, 1.0 eq.) was added. The reaction mixture was allowed to warm to room temperature and stirred for 4 h before being quenched with water. The two layers were separated and the aqueous one was extracted three times with CH2Cl2. Combined organic layers were dried with MgSO4, filtered and concentrated under reduced pressure. The obtained residue was purified by flash chromatography on silica gel (cyclohexane/ethyl acetate 1:0 to 1:1 (v/v)) to afford N-[(1R)-1-methylprop-2-ynyl]-5-(2-pyridyl)thiophene-2-sulfonamide (150 mg, 67%) as a beige powder.
Compounds of formula (VIII) were prepared according to the following schemes:
A mixture of R1-α or β amino acids of formula (VI) (1 eq), ZnCl2 (0.06 eq.) and K2CO3 (4 eq.) in anhydrous methanol (0.6 M) under inert atmosphere was cooled to 0° C. with an ice-bath. Besides, anhydrous N,N-Diisopropylethylamine (1.1 eq.) was slowly added to a solution of 1H-Imidazole-1-sulfonyl azide hydrogenosulfate (1.2 eq.) solubilized in anhydrous methanol (0.3 M) under inert atmosphere (solution A). The azide-containing solution was immediately added dropwise to the first mixture at 0° C. Then, the cooling bath was removed and the white mixture was stirred at room temperature for one night. The mixture was then cooled down to 0° C., diluted with water (10 mL) and carefully acidified to pH=2 with diluted aq. HCl (1N). It was extracted with ethyl acetate and the aqueous layer was once again extracted with ethyl acetate. Combined organic layers were concentrated under reduced pressure to give a compound of formula (VII).
Compound of formula (VII) (1 eq.) was dissolved in methanol (0.07-0.48M) and cooled down to 0° C. and thionyl chloride (2.0 eq) was added dropwise. The resulting solution was stirred overnight in the melting ice bath. Then, solvents were evaporated under reduced pressure to give the desired products or residues were dissolved in a mixture of ethyl acetate and saturated aqueous NaHCO3 and extracted twice. Organic layers were mixed, dried over MgSO4, filtered and concentrated under reduced pressure to afford desired products of formula (VIII).
To a solution of amino-acid (1.0 eq.) of formula (VI) and TEA (1.5 eq.) in a 1:1 (v/v) mixture of 1,4-dioxane/water (15 mL), cooled to 0° C. was added Boc2O (1.2 eq.). After stirring at 0° C. for 30 min, the solution was allowed to warm to room temperature and stirred overnight. 1,4-dioxane was then removed under reduced pressure and the remaining aqueous mixture was cooled with an ice bath. If necessary, pH was adjusted to 2 by dropwise addition of 1 M HCl. The product was then extracted with EtOAc. The combined organic layers were washed with brine and dried over MgSO4, filtered and concentrated under reduced pressure to N-Boc protected amino-acid as a light oil.
To a stirring solution of the N-Boc protected amino-acid (1.0 eq.) and K2CO3 (1.3 eq.) in DMF was added MeI (2.2 eq or 1 eq.). The resulting mixture was stirred overnight before being concentrated under reduced pressure. The obtained residue was portioned between water and EtOAc. The aqueous layer was further extracted with EtOAc and combined organic layers were washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure. The crude mixture was purified by flash column chromatography on silica gel (cyclohexane/EtOAc 1:0 to 0:1 (v/v)) to afford compounds of formula (XX)
To a solution of (2S)-2-amino-3-(4-iodophenyl)propanoic acid (VI) (1.00 g, 3.44 mmol, 1 eq.) in dry methanol (9.0 mL) and cooled to 0° C. was slowly added thionyl chloride (1.0 mL, 13.7 mmol, 4.0 eq.).
The reaction mixture was allowed to reach room temperature before being concentrated under reduced pressure to afford [(1S)-1-[(4-iodophenyl)methyl]-2-methoxy-2-oxo-ethyl]ammonium; chloride (1.20 g, quant.) as a yellowish salt.
The amine function is then protected. To a solution of the amine (1.0 eq.) and TEA (1.5 eq.) in a 1:1 (v/v) mixture of 1,4-dioxane/water (15 mL), cooled to 0° C. was added Boc2O (1.2 eq.). After stirring at 0° C. for 30 min, the solution was allowed to warm to room temperature and stirred overnight. 1,4-dioxane was then removed under reduced pressure and the remaining aqueous mixture was cooled with an ice bath. The product was then extracted with EtOAc. The combined organic layers were washed with brine and dried over MgSO4, filtered and concentrated under reduced pressure to give the Boc-protected compound (XX).
If needed, a phenol group can be alkylated: to a solution of compound XX (1 eq.), brominated compound (4 eq.) and NaI (0.1 eq.) in dry DMF was added K2CO3 (2.7 eq.). The reaction vessel was equipped with a condenser and the reaction mixture stirred overnight at 85° C. It was then allowed to cool down to rt and evaporated to dryness. The resulting oil was suspended into a mixture of CH2Cl2 and sat. aq. NH4Cl. The aqueous layer was extracted twice with CH2Cl2, combined organic layers were washed with brine several times, dried over MgSO4, filtered and concentrated under reduced pressure to afford the alkylated compound.
Then Boc-group is deprotected in dry DCM using dropwise added TFA (6 eq.) or in dry MeoH using HCl (4M solution in 1,4-dioxane, 1 eq.). The resulting solution was stirred overnight and evaporated to dryness to afford the amine as TFA or HCl salt. Then the amino group is transformed to the corresponding azide (VIII) using conditions described in VIII-a protocol.
Then a solution of the amine (1 eq), ZnCl2 (0.06 eq.) and K2CO3 (4 eq.) in anhydrous methanol (0.6 M) under inert atmosphere was cooled to 0° C. with an ice-bath. Besides, anhydrous N,N-Diisopropylethylamine (1.1 eq.) was slowly added to a solution of 1H-Imidazole-1-sulfonyl azide hydrogenosulfate (1.2 eq.) solubilized in anhydrous methanol (0.3 M) under inert atmosphere. The azide-containing solution was immediately added dropwise to the amine solution at 0° C. Then, the cooling bath was removed and the white mixture was stirred at room temperature for one night. The mixture was then cooled down to 0° C., diluted with water (10 mL) and carefully acidified to pH=2 with diluted aq. HCl (1N). It was extracted with ethyl acetate and the aqueous layer was once again extracted with ethyl acetate. Combined organic layers were concentrated under reduced pressure to give a compound of formula (VIII).
If needed, a iodo-aromatic group can undergo a sonogashira coupling: To a stirring & degassed solution of methyl (2S)-2-(tert-butoxycarbonylamino)-3-(4-iodophenyl)propanoate (250 mg, 0.62 mmol, 1.0 eq.), CuI (24 mg, 0.12 mmol, 0.2 eq) and PdCl2(PPh3)2 (45 mg, 0.06 mmol, 0.1 eq.) in TEA (7 mL) was added ethynyl(trimethyl)silane (0.10 mL, 0.74 mmol, 1.2 eq.). The resulting solution was stirred at rt for 6 h before being quenched with water and diluted with Et2O. It was filtered through a celite pad and the two layers were separated. The aqueous layer was extracted with Et2O and combined organic layers washed with water, dried over MgSO4, filtered and concentrated under reduced pressure. The obtained residue was purified by flash column chromatography (cyclohexane/EtOAc 1:0 to 1:1 (v/v)) to afford methyl (2S)-2-(tert-butoxycarbonylamino)-3-[4-(2-trimethylsilylethynyl)phenyl]propanoate (166 mg, 72%) as a light oil.
Then Boc-group is deprotected in dry DCM using dropwise added TFA (6 eq.) or in dry MeoH using HCl (4M solution in 1,4-dioxane, 1 eq.). The resulting solution was stirred overnight and evaporated to dryness to afford the amine as TFA or HCl salt. Then the amino group is transformed to the corresponding azide (VIII) using conditions described in VIII-a protocol.
To a solution of Compound of formula (VIII) (1 eq.) in methanol (0.04-0.35M) was added aqueous hydroxylamine (50% w/w in water, 0.04-0.35M) and KCN (0.1-0.5 eq.). The mixture was stirred overnight. Then, the solvents were removed under reduced pressure and residue was purified through flash silica gel column or through C18 gel column.
Compound of formula (IX) (0.9-1 eq.) and compound of formula (V) (1.0-1.1 eq) were mixed in dioxane/H2O (2/1, 0.07M) or in dimethylformamide/water (2/1 or 1/0.8, 0.02-0.11M) before the addition of copper sulfate pentahydrate (0.1 or 0.2 eq) followed by sodium ascorbate (0.5 eq). The resulting mixture was stirred at room temperature overnight. Then, the mixture was diluted in water and extracted with ethyl acetate, organic layers were mixed and concentrated to dryness in vacuo. Residues were purified by flash chromatography to furnish desired 1,4 triazoles.
Compound of formula (VIII) (0.9-1 eq.) and compound of formula (V) (1.0-1.1 eq) were mixed in dioxane/H2O (2/1, 0.07M) or in dimethylformamide/water (2/1 or 1/0.8, 0.02-0.11M) before the addition of copper sulfate pentahydrate (0.1 or 0.2 eq) followed by sodium ascorbate (0.5 eq). The resulting mixture was stirred at room temperature overnight. Then, mixture was diluted in water and extracted with ethyl acetate, organic layers were mixed and concentrated to dryness in vacuo. Residues were purified by flash chromatography to furnish desired 1,4 triazoles.
To a solution of Compound of formula (X) (1 eq.) in methanol (1 eq) was added aqueous hydroxylamine (50% w/w in water, 18-300 eq) and KCN (0.1-0.5 eq.) at room temperature or 0° C. The mixture was stirred overnight. Then, the solvents were removed under reduced pressure and residue was purified through flash silica gel column or through C18 gel column.
Compound of formula (VIII) (0.9-1 eq.) and compound of formula (V) wherein R3 is a bromine-substituted thiophenyl (1.0-1.1 eq) were mixed in dioxane/H2O (2/1, 0.07M) or in dimethylformamide/water (2/1 or 1/0.8, 0.02-0.11M) before the addition of copper sulfate pentahydrate (0.1 or 0.2 eq) followed by sodium ascorbate (0.5 eq). The resulting mixture was stirred at room temperature overnight. Then, mixture was diluted in water and extracted with ethyl acetate, organic layers were mixed and concentrated to dryness in vacuo. Residues were purified by flash chromatography to furnish desired 1,4 triazoles.
A flask was charged with the compound of formula (X) (1 eq), boronic acid (R5—B(OH)2 wherein R5 is a phenyl or pyridyl optionally substituted as disclosed herein) (1.5 to 2.1 eq), palladium tetrakis (0.15 eq) and with cesium carbonate (1.05 eq). Flask was degassed and flushed with argon three times. Degassed dioxane and water (3.5/1, 0.15-022M) were added, the mixture was warmed up to 70° C. and stirred for 1 h45-4 h. The mixture was diluted in water, treated with a solution of HCl 1N (pH=1-2) and extracted three times with ethyl acetate. Combined organic layers were washed three times with brine, dried over MgSO4, filtered and concentrated under reduced pressure. Crude was purified by chromatography eluting with cyclohexane/ethyl acetate/methanol to furnish desired intermediates of formula (XI).
To a solution of compound of formula (XI) (1 eq.) in methanol (0.04-0.35M) was added aqueous hydroxylamine (50% w/w in water, 0.04-0.35M) and KCN (0.1-0.5 eq.). The mixture was stirred overnight. Then, the solvents were removed under reduced pressure and residue was purified through flash silica gel column or through C18 gel column.
If deprotection of an amino-protected group is required to arrive at a compound of formula (I), deprotection by can be performed according to well-known methods. For instance, Boc groups can be cleaved by addition of a solution of HCl 4N in dioxane. Trimethylsilyl groups can be cleaved by TBAF in THF. If appropriate, compounds of formula (I) may be isolated in salt, for instance a potassic salt.
2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (1.1 eq.) and triethylamine (3 eq.) were added to a mixture of azido amino acids (compound of formula VII) and R2—N-propargylamine (1.1-1.3 eq.) in dimethylformamide (0.24-0.30M). The mixture was stirred overnight at room temperature, ethyl acetate was added and the organic phase was washed with aq. HCl (0.1 or 1M), NaHCO3, saturated NaCl, and H2O. The organic phase was dried over MgSO4, filtered and concentrated under reduced pressure to give azido-alkynes (compound of formula (XII)).
The compound of formula (XII) was dissolved in dimethylformamide (0.009-0.04M), was heated to reflux and left stirring overnight to allow cyclisation. After cooling down, the mixture was diluted with ethyl acetate and was washed with water (three times). The organic phase was dried with MgSO4, filtered and concentrated under reduced pressure. Then, the crude product was purified by flash chromatography on silica gel column to provide compound of formula (XIII).
Compound of formula (XIII) (1 eq.) was diluted in a 6M aqueous HCl solution (0.35-0.8M) and heated with microwaves at 85° C. for 1-3 hours. Then, solvents were evaporated under reduced pressure to give compound of formula (XIV) as hydrochloric acid salts which was used in the next step without further purification.
Crude intermediate of formula (XIV) was dissolved in methanol (0.09-0.25M) and the mixture was cool down to 0° C. before the addition of thionyl chloride (2 eq). The solution was stirred at room temperature overnight and solvents were evaporated to give the desired compound as hydrochloric acid salt.
Compound of formula (XV) (1.1-1.5 eq) and N,N-diisopropylethylamine or triethylamine (1.2-3 eq) were solubilized in dichloromethane or in dimethylformamide (0.08-0.34M). The reaction media was cooled at 0° C. and R3-sulfonyl chloride (1-1.2 eq.) was added. The mixture was allowed to reach room temperature and was stirred 1 h to one night. Then, products were washed twice with 0.5 or 1M HCl (aq), dried over MgSO4, filtered and concentrated under reduced pressure. Then, the residues were filtered on silica gel (4 g cartridge) or purified by flash chromatography to afford the desired products.
To a solution of compound of formula (XVI) (1 eq.) in methanol (0.04-0.35M) was added aqueous hydroxylamine (50% w/w in water, 0.04-0.35M) and KCN (0.1-0.5 eq.). The mixture was stirred overnight. Then, the solvents were removed under reduced pressure and residue was purified through flash silica gel column or through C18 gel column.
1H NMR, 300 MHz, CDCl3-d1, δ (ppm): 7.63-7.68 (m,
13C NMR, 75 MHz, CDCl3-d1, δ (ppm): 152.1, 138.3,
1H NMR, 300 MHz, CDCl3-d1, δ (ppm): 7.62-7.58 (m,
13C NMR, 75 MHz, CDCl3-d1, δ (ppm): 151.6, 135.5,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 8.49 (s, 1H),
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 142.1, 132.7,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.77 (dd, J =
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 142.4, 133.4,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.42 (d, J =
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 149.0, 139.2,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 8.08 (dd, J =
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 141.6, 131.8,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.74 (d, J =
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 133.8, 133.7,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 8.53 (ddd,
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 152.4, 152.0,
1H NMR (300 MHz, CDCl3) δ (ppm) 8.60 (ddd,
13C NMR (75 MHz, CDCl3) δ (ppm) 151.7, 151.0,
1H NMR (300 MHz, CDCl3) δ (ppm) 8.66 (ddd,
13C NMR (75 MHz, CDCl3) δ (ppm) 150.9, 149.8,
1H NMR (300 MHz, acetone-d6) δ (ppm): 8.56 (ddd,
13C NMR (75 MHz, acetone-d6) δ (ppm) 152.0, 151.7,
1H NMR (CDCl3, 300 MHz) δ: 7.62-7.57 (m, 2H), 7.54
13C NMR (CDCl3, 75 MHz) δ: 151.1, 136.9,
1H NMR (CDCl3, 300 MHz) δ: 7.60 (m, 2H), 7.60
13C NMR (CDCl3, 75 MHz) δ: 151.1, 136.1,
1H NMR (CDCl3, 300 MHz) δ: 8.27 (s, 1H), 7.96
13C NMR (CDCl3, 75 MHz) δ: 174.2, 148.4, 135.8,
1H NMR (CDCl3, 300 MHz) δ: 8.60 (ddd,
13C NMR (CDCl3, 300 MHz) δ: 151.0, 149.8
1H NMR, 300 MHz, MeOD-d4, δ (ppm):
13C NMR, 75 MHz, MeOD-d4, δ (ppm):
1H NMR, 300 MHz, MeOD-d4, δ (ppm):
13C NMR, 75 MHz, MeOD-d4, δ (ppm):
1H NMR, 300 MHz, MeOD-d4, δ (ppm):
13C NMR, 75 MHz, MeOD-d4, δ (ppm):
1H NMR, 300 MHz, CDCl3-d1 + TMS, δ (ppm):
13C NMR, 75 MHz, CDCl3-d1 + TMS, δ (ppm):
1H NMR, 300 MHz, MeOD-d4, δ (ppm):
13C NMR, 75 MHz, MeOD-d4, δ (ppm):
1H NMR, 300 MHz, MeOD-d4, δ (ppm):
13C NMR, 75 MHz, MeOD-d4, δ (ppm):
1H NMR, 300 MHz, MeOD-d4, δ (ppm):
13C NMR, 75 MHz, MeOD-d4, δ (ppm):
1H NMR, 300 MHz, MeOD-d4, δ (ppm):
13C NMR, 75 MHz, MeOD-d4, δ (ppm):
19F NMR (282 MHz, MeOD-d4) δ: −58.5
1H NMR, 300 MHz, MeOD-d4, δ (ppm):
13C NMR, 75 MHz, MeOD-d4, δ (ppm):
1H NMR, 300 MHz, MeOD-d4, δ (ppm):
13C NMR, 75 MHz, MeOD-d4, δ (ppm):
1H NMR, 300 MHz, MeOD-d4, δ (ppm):
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 173.2,
1H NMR (MeOD, 300 MHz) δ (ppm):
13C NMR (MeOD, 75 MHz) δ (ppm):
1H NMR (CD3OD, 300 MHz) δ (ppm):
13C NMR (MeOD, 75 MHz) δ (ppm): 173.1,
1H NMR (CD3OD, 300 MHz) δ (ppm):
13C NMR (MeOD, 75 MHz) δ (ppm):
1H NMR (MeOD, 300 MHz) δ (ppm):
19F NMR (MeOD, 282 MHz) δ: −140.1.
13C NMR (MeOD, 75 MHz) δ (ppm):
1H NMR, 300 MHz, CDCl3-d1 + TMS, δ (ppm):
13C NMR, 75 MHz, CDCl3-d1 + TMS, δ (ppm):
1H NMR, 300 MHz, CDCl3-d1 + TMS, δ (ppm):
13C NMR, 75 MHz, CDCl3-d1 + TMS, δ (ppm):
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.08-
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 173.0,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.54
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 172.5,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 7.52
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 170.5,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.07-
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 172.2,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.07-
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 172.2,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.37-
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 171.8,
19F NMR (282 MHz, MeOD-d4) δ: −60.5.
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.37-
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 172, 158.7,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.16-
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 172.1,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.18-
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 172.0,
1H NMR (MeOD, 300 MHz) δ (ppm): 6.74
13C NMR (MeOD, 75 MHz) δ (ppm): 172.0, 149.2,
1H NMR (MeOD-d4, 300 MHz) δ (ppm): 7.18
13C NMR (MeOD-d4, 300 MHz) δ (ppm): 171.9,
1H NMR (MeOD-d4, 300 MHz) δ (ppm): 6.88
13C NMR (MeOD-d4, 300 MHz) δ (ppm): 172.1,
1H NMR (MeOD, 300 MHz) δ (ppm): 6.97-
19F NMR (MeOD, 282 MHz) δ: −139.9.
13C NMR (MeOD, 75 MHz) δ (ppm): 172.0, 152.7
1H NMR (acetone-d6, 300 MHz) δ: 7.23 (m, 1H),
13C NMR (acetone-d6, 75 MHz) δ: 170.1, 159.7,
1H NMR (acetone-d6, 300 MHz) δ: 7.25 (td, J =
13C NMR (acetone-d6, 75 MHz) δ: 171.5, 158.5,
1H NMR (acetone-d6 300 MHz) δ: 7.19 (m, 2H),
13C NMR (acetone-d6 75 MHz) δ: 171.1, 159.0,
1H NMR (300 MHz, CDCl3) δ: 7.24 (d, J = 2.0
13C NMR (75 MHz, CDCl3) δ: 170.3, 154.4,
1H NMR (Acetone-d6, 300 MHz) δ: 7.11-7.01
19F NMR (Acetone-d6, 282 MHz) δ: −137.14.
13C NMR (Acetone-d6, 75 MHz) δ: 170.9,
1H NMR (CDCl3, 300 MHz) δ: 7.43 (m, 2H),
1H NMR (CDCl3, 300 MHz) δ: 7.62 (m, 2H),
13C NMR (CDCl3, 75 MHz) δ: 169.9, 141.6,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 8.57
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 170.8,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 7.83-7.77
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 168.7,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 8.56
13C NMR, 75 MHz, MeOD-d4, δ (ppm):
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 9.27 (br, 1H), 8.56 (d, J = 1.6 Hz,1H), 8.08 (s, 1H), 8.02 (d,
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 163.8, 156.2, 150.4, 150.0, 149.7, 143.2, 142.0, 137.5, 132.6,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.72 (s, 1H), 7.66-7.63 (m, 2H), 7.46 (d, J = 3.7 Hz, 1H), 7.44-
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 170.0, 143.2, 134.6, 130.3, 130.3, 127.2, 125.7, 124.8, 122.6,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.71 (s, 1H), 7.59-7.56 (m, 2H), 7.41 (d, J = 3.9 Hz, 1H), 7.37-
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 170.3, 151.9, 145.0, 140.8, 137.8, 134.1, 134.0, 130.3, 130.1,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 10.29 (br, 1H), 7.76 (s, 1H), 7.86 (d, J = 7.9 Hz, 1H), 7.32-7.28
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 170.3, 144.8, 143.8, 138.0, 137.8, 133.4, 131.9, 128.1, 124.9,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 9.25 (s, 1H), 8.46 (s, 1H), 8.03 (s, 1H), 7.74-7.71 (m, 2H), 7.58
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 168.7, 156.2, 149.1, 143.2, 139.6, 132.8, 132.2, 129.9, 129.4,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.68-7.63 (m, 2H), 7.58 (s, 1H), 7.55 (d, J = 4.0 Hz, 1H), 7.45-
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 172.0, 157.5, 152.5, 142.8, 136.9, 134.8, 133.9, 131.1,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 9.24 (s, 1H), 8.45 (s, 1H), 8.02 (s, 1H), 7.74-7.69 (m, 2H), 7.58-
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 168.7, 156.2, 149.1, 143.2, 139.6, 132.7, 132.2, 129.9, 129.4,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.84 (s, 1H), 7.66-7.63 (m, 2H), 7.53 (d, J = 3.9 Hz, 1H), 7.44-
13C NMR, 75 MHz, MeOD-d4, δ (ppm): δ: 170.0, 157.7, 152.5, 143.4, 136.8, 134.8, 133.9, 131.1, 130.4,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 9.25 (s, 1H), 8.52 (t, J = 5.8 Hz, 1H), 8.03 (s, 1H), 7.40 (d, J =
13C NMR, 75 MHz, DMSO-d6, δ (ppm): δ: 168.7, 156.2, 142.9, 142.3, 132.3, 131.3, 129.9, 125.6, 123.5,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 9.25 (s, 1H), 8.36 (t, J = 6.0 Hz, 1H), 8.00 (s, 1H), 7.91 (dd, J =
13C NMR, 75 MHz, DMSO-d6, δ (ppm): δ: 168.8, 156.2, 143.2, 141.1, 132.5, 131.7, 129.9, 127.7, 125.7,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.86-7.82 (m, 2H), 7.75 (s, 1H), 7.63-7.50 (m, 3H), 6.90-6.85
13C NMR, 75 MHz, MeOD-d4, δ (ppm): δ: 170.1, 157.7, 145.4, 141.7, 133.7, 131.1, 130.2, 128.0, 127.2,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.80 (s, 1H), 7.37 (d, J = 3.7 Hz, 1H), 6.90-6.86 (m, 2H), 6.77
13C NMR, 75 MHz, MeOD-d4, δ (ppm): δ: 170.0, 157.6, 149.0, 145.2, 139.1, 133.6, 131.1, 127.2, 127.0,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 8.01 (dd, J = 1.3 and 3.1 Hz, 1H), 7.77 (s, 1H), 7.51 (dd, J = 3.1
13C NMR, 75 MHz, MeOD-d4, δ (ppm): δ: 170.1, 157.6, 145.3, 141.5, 131.6, 131.1, 129.5, 127.2,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.79 (br s, 1H), 7.65 (d, J = 5.2 Hz, 1H), 7.06 (d, J = 5.2 Hz, 1H),
13C NMR, 75 MHz, MeOD-d4, δ (ppm): δ: 170.0, 157.5, 145.1 (br), 137.6, 133.8, 132.6, 131.1, 127.2,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 9.25 (s, 1H), 8.57 (ddd, J = 1.0, 1.7 and 4.8 Hz, 1H), 8.46 (br s,
13C NMR, 75 MHz, DMSO-d6, δ (ppm): δ: 168.7, 156.2, 150.4, 150.0, 149.7, 143.2, 142.0, 137.5, 132.6,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 9.30 (br, 1H), 8.57 (ddd, J = 0.9, 1.6 and 4.8 Hz, 1H), 8.03 (dt,
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 168.7, 156.2, 150.5, 149.9, 149.7, 143.3, 142.2, 137.5, 132.5,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.74-7.71 (m, 3H), 7.37-7.33 (m, 2H), 6.89-6.84 (m, 2H), 6.65-
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 170.1, 157.7, 149.4, 145.5, 139.0, 131.1, 130.0, 128.1, 127.2,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 8.57 (ddd, J = 0.9, 1.6 and 4.9 Hz, 1H), 8.45 (br, 1H), 8.05 (s,
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 168.4, 150.4, 150.0, 149.6, 147.2 (m), 143.4; 142.0, 137.5,
19F NMR (282 MHz, DMSO-d6) δ: −57.3.
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 8.43 (br, 1H), 8.07 (s, 1H), 7.73-7.70 (m, 2H), 7.58 (d, J = 3.9
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 168.4, 149.1, 147.2 (m), 143.4, 139.5, 135.3, 132.7, 132.2,
19F NMR (282 MHz, DMSO-d6) δ: −57.3.
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 8.56 (ddd, J = 0.9, 1.6 and 4.8 Hz, 1H), 8.46 (t, J = 6 Hz, 1H),
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 169.1, 157.1, 155.9, 150.9, 150.5, 150.1, 143.7, 142.4, 138.0,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 8.45 (br, 1H), 8.04 (s, 1H), 7.74-7.70 (m, 2H), 7.58 (d, J = 3.9
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 168.7, 158.1, 149.1, 143.2, 139.6, 132.7, 132.2, 129.9, 129.4,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 8.57 (ddd, J = 1.0, 1.7 and 4.9 Hz, 1H), 8.46 (br, 1H), 8.04 (s,
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 168.7, 158.1, 150.4, 150.0, 149.7, 143.2, 142.0, 137.5, 132.6,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 8.45 (t, J = 5.9 Hz, 1H), 8.02 (s, 1H), 7.74-7.70 (m, 2H), 7.57 (d,
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 168.7, 153.9, 149.1, 143.2, 139.6, 132.8, 132.2, 130.2, 129.5,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 8.57 (ddd, J = 1.0, 1.7 and 4.8 Hz, 1H), 8.44 (br s, 1H), 8.04 (dt,
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 168.6, 153.8, 150.4, 150.0, 149.6, 143.2, 142.0, 137.5, 132.6,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 8.52 (t, J = 5.6 Hz, 1H), 8.03 (s, 1H), 7.41 (d, J = 4.0 Hz, 1H),
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 168.7, 158.0, 142.9, 142.2, 132.3, 131.2, 129.9, 127.4, 123.6,
1H NMR ((CD3)2CO-d6, 300 MHz) δ: 8.56 (ddd, J = 1.0, 1.7 and 4.9 Hz, 1H), 7.96 (dt, J = 1.1 and 8 Hz,
13C NMR ((CD3)2CO-d6, 75 MHz) δ: 169.4, 154.4, 152.0, 150.7, 148.7, 147.6,144.6, 142.9, 138.3,
1H NMR (DMSO-d6, 300 MHz) δ: 10.01 (s, 1H), 8.57 (ddd, J = 1, 1.7 and 4.9 Hz, 1H), 8.46 (t, J = 6 Hz,
13C NMR (DMSO-d6, 75 MHz) δ: 168.6, 151.9, 150.4, 150.1, 149.7, 143.3, 142.0, 137.5, 132.6, 130.2,
1H NMR (DMSO-d6, 300 MHz) δ: 10.02 (br, 1H), 8.45 (br, 1H), 8.05 (s, 1H), 7.71-7.74 (m, 2H), 7.55
13C NMR (DMSO-d6, 75 MHz) δ: 168.6, 151.9, 149.0, 143.2, 139.6, 132.7, 132.2, 130.2, 129.3, 129.1,
1H NMR (DMSO, 300 MHz) δ: 8.57 (ddd, J = 0.96, 1.7 and 4.8 Hz, 1H), 8.46 (br, 1H), 8.06 (s, 1H), 8.04
13C NMR (DMSO, 300 MHz) δ: 168.7, 150.4, 150.0, 149.6, 148.4, 147.6, 143.2, 141.9, 137.5, 132.6,
1H NMR (DMSO, 300 MHz) δ: 8.46 (t, J = 6.0 Hz, 1H), 8.06 (s, 1H), 7.74-7.71 (m, 2H), 7.58 (d, J = 3.9
13C NMR (DMSO, 300 MHz) δ: 169.2, 149.5, 148.9, 146.1, 143.7, 140.0, 133.2, 132.7, 129.8, 129.6,
1H NMR (DMSO, 300 MHz) δ: 9.68 (br, 1H), 8.57 (ddd, J = 0.9, 1.7 and 4.8Hz, 1H), 8.45 (br, 1H), 8.05
19F NMR (DMSO, 282 MHz) δ: −136.90.
13C NMR (DMSO, 75 MHz) δ: 168.6, 150.6 (d, J = 240.4 Hz), 150.4, 150.0, 149.7, 143.6 (d, J = 12.1 Hz),
1H NMR (DMSO, 300 MHz) δ: 8.04 (s, 1H), 7.74-7.70 (m, 2H), 7.55 (d, J = 3.9 Hz, 1H), 7.54 (d, J =
19F NMR (DMSO, 282 MHz) δ: −136.89.
13C NMR (DMSO, 75 MHz) δ: 168.6, 150.6 (d, J = 240.4 Hz), 149.0, 143.6 (d, J = 12.0 Hz), 143.7,
1H NMR (acetone-d6, 300 MHz) δ (ppm) 8.57 (ddd, J = 4.8, 1.7 and 1.0 Hz, 1H), 8.57 (ddd, J = 8.0, 1.0
13C NMR (acetone-d6, 75 MHz) δ (ppm) 169.5, 159.7, 152.4, 151.8, 150.6, 142.8, 139.4, 138.2, 134.0,
1H NMR (acetone-d6, 300 MHz) δ (ppm) 8.55 (ddd, J = 4.9, 1.7 and 1.0 Hz, 1H), 7.95 (ddd, J = 8.0, 1.1
13C NMR (acetone-d6, 75 MHz) δ (ppm) 169.4, 159.7, 151.9, 151.5, 150.5, 149.8, 144.6 (0.75C, maj),
1H NMR (acetone-d6, 300 MHz) δ (ppm): 8.55 (ddd, J = 4.9, 1.7 and 1.0 Hz, 1H), 7.95 (m, 1H), 7.86 (m,
13C NMR (acetone-d6, 75 MHz) δ (ppm): 169.5, 159.7, 152.0, 151.6, 150.6, 150.0, 144.6, 138.1, 133.4
1H NMR (acetone-d6, 300 MHz) δ: 8.56 (ddd, J = 4.9, 1.8 and 1.0 Hz, 1H), 7.97 (ddd, J = 8.0, 1.0 and
13C NMR (acetone-d6, 300 MHz) δ: 169.5, 160.7, 151.9, 150.6 (2C), 144.5,143.4, 138.2, 138.1, 133.5,
1H NMR (acetone-d6, 300 MHz) δ: 8.55 (ddd, J = 4.9, 1.8 and 1.0 Hz, 1H), 7.96 (ddd, J = 8.0, 1.0 and
13C NMR (acetone-d6, 75 MHz) δ: 169.7, 158.5, 151.9, 151.8, 150.5, 144.3, 143.4, 138.1, 133.5,
1H NMR (acetone-d6, 300 MHz) δ: 8.56 (ddd, J = 4.8, 1.7 and 1.0 Hz, 1H), 7.96 (ddd, J = 8.0, 1.1 and
13C NMR (acetone-d6, 75 MHz) δ: 169.5, 159.0, 151.9, 151.8, 150.6, 144.4, 143.4, 138.1, 133.5,
1H NMR (acetone-d6, 300 MHz) δ: 8.56 (ddd, J = 4.9, 1.8 and 1.0 Hz, 1H), 7.96 (ddd, J = 8.0, 1.0 and
19F NMR (acetone-d6, 282 MHz) δ: −136.8 (dd, J = 12.3 and 9.1 Hz).
13C NMR (acetone-d6, 75 MHz) δ: 169.3, 152.7 (d, J = 244 Hz), 151.9, 151.8, 150.5, 147.6 (d, J = 10.5
1H NMR (300 MHz, CDCl3) δ: 8.56 (d, J = 4.5 Hz, 1H), 7.76 (dt, J = 7.5 Hz, 2.0 Hz, 1H), 7.67 (d, J = 8.0
13C NMR (CDCl3, 75 MHz) δ: 168.4, 154.5, 151.0, 150.7, 149.6, 143.8, 141.6, 137.5, 133.6, 130.6,
1H NMR (MeOD-d4, 300 MHz) δ: 7.83 (s, 1H), 7.65 (m, 2H), 7.53 (d, J = 3.9 Hz, 1H), 7.41 (m, 3H),
13C NMR (MeOD-d4, 75 MHz) δ: 169.8, 152.0, 145.5, 141.0, 137.6, 134.2, 134.1, 133.2 (2C), 130.3
1H NMR (acetone-d6, 300 MHz) δ: 7.94 (s, 1H), 7.72 (m, 2H), 7.60 (m, 3H), 7.45 (m, 4H), 7.34 (m,
13C NMR (acetone-d6, 75 MHz) δ: 169.0, 150.9, 144.7, 142.5, 140.8, 133.8, 133.6, 133.0 (2C), 131
1H NMR (CDCl3, 300 MHz) δ: 8.17 (s, 1H), 7.90 (m, 2H), 7.60 (s, 1H), 7.45 (m, 3H), 6.88 (m, 2H), 6.74
13C NMR (CDCl3, 75 MHz) δ: 173.9, 168.6, 159.1, 147.8, 143.1, 136.1, 132.4, 131.7, 130.0 (2C),
1H NMR (acetone-d6, 300 MHz) δ: 8.57 (ddd, J = 4.9, 1.8 and 1.1 Hz, 1H), 7.99 (ddd, J = 8.0, 1.2 and
13C NMR (acetone-d6, 75 MHz) δ: 169.5, 159.6, 152.0, 151.6, 150.3, 143.4, 140.5, 138.4, 134.3,
1H NMR (acetone-d6, 300 MHz) δ: 7.78 (d, J = 0.8 Hz, 1H), 7.73-7.69 (m, 4H), 7.71 (d, J = 0.7 Hz, 1H),
13C NMR (acetone-d6, 75 MHz) δ: 169.5, 169.4, 159.8, 159.7, 150.9, 150.9, 146.8, 146.6, 141.1,
1H NMR (CDCl3, 300 MHz) δ: 7.76 (s, 1H), 7.60 (m, 2H), 7.51 (d, J = 3.9 Hz, 1H), 7.40 (m, 3H), 7.28 (d,
13C NMR (CDCl3, 75 MHz) δ: 168.7, 159.0, 151.3, 149.3, 135.6, 133.4, 132.7, 130.2 (2C), 129.4
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.88-7.84 (m, 2H), 7.77 (s, 1H), 7.76-7.72 (m, 2H), 7.53
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 170.0, 169.7, 157.7, 150.4, 145.2, 142.2, 137.0,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.77 (s, 1H), 7.63 (d, J = 8.1 Hz, 2H), 7.51 (d, J = 3.9 Hz,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 8.54-8.50 (m, 2H), 8.04 (s, 1H), 7.91 (d, J = 8.4 Hz, 2H),
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 168.7, 165.8, 158.1, 147.9, 148.2, 140.5, 134.6, 134.55,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 9.26 (br, 1H), 9.98 (br, 1H), 8.61 (d, J = 4.8 Hz, 1H), 8.53
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 168.7, 156.2, 149.7, 146.5, 145.2, 143.1, 140.9, 133.5,
1H NMR (MeOD, 300 MHz) δ: 8.57 (d, J = 5.0 Hz, 2H), 7.82 (s, 1H), 7.96 (m, 2H), 7.64 (d, J = 4.0
13C NMR (MeOD, 75 MHz) δ: 170.0, 160.3, 151.1, 147.5, 145.1, 144.4, 142.3, 134.0, 131.0, 128.4,
1H NMR (DMSO-d6, 300 MHz) δ: 8.96 (d, J = 1.9 Hz, 1H), 8.59 (d, J = 1.4 and 4.7 Hz, 1H), 8.52 (t,
13C NMR (DMSO-d6, 75 MHz) δ: 168.7, 158.1, 149.8, 146.6, 145.3, 143.1, 140.9, 133.4, 132.7,
1H NMR (DMSO, 300 MHz) δ: 8.60 (q, J = 4.5 Hz, 1H), 8.49 (s, 1H), 8.14 (t, J = 1.9 Hz, 1H), 8.04
13C NMR (DMSO, 300 MHz) δ: 168.7, 165.8, 158.1, 148.3, 143.2, 140.0, 135.4, 132.7, 132.3,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.59-7.56 (m, 1H), 7.36-7.33 (m, 1H), 7.13 (s, 1H), 7.10-7.00 (m,
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 172.6, 138.1, 128.8, 124.7, 122.4, 119.9, 119.3, 112.3, 111.4, 80.4,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.09-7.04 (m, 2H), 6.76-6.71 (m, 2H), 4.03-3.97 (m, 1H),
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 172.4, 157.4, 131.5, 129.4, 116.3, 80.4, 72.2, 62.6, 41.3, 40.9,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.10-7.07 (m, 2H), 6.81-6.76 (m, 2H), 4.20-4.18 (m, 1H),
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 171.1, 170.8, 156.0, 130.2, 128.3, 115.0, 78.1, 77.6, 73.1, 71.9,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.09-7.03 (m, 2H), 6.74-6.69 (m, 2H), 3.98-3.92 (m, 3H), 3.08
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 171.6, 157.6, 131.4, 128.2, 116.3, 80.1, 72.3, 65.7, 38.1, 29.4.
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.09-7.04 (m, 2H), 6.76-6.70 (m, 2H), 3.96 (dd, J = 6.1 and 7.9
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 171.6, 157.5, 131.4, 128.2, 116.3, 80.1, 72.3, 65.7, 38.1, 29.4.
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.56 (s, 1H), 7.54-7.51 (m, 1H), 7.36-7.32 (m, 1H), 7.12-6.99
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 175.4, 138.0, 135.5, 131.8, 128.7, 125.6, 122.6, 120.1, 119.1,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.57 (s, 1H), 7.03-6.99 (m, 2H), 6.74-6.70 (m, 2H), 5.05-4.97
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 175.1, 157.7, 135.5, 131.9, 131.8, 127.6, 116.4, 59.8, 42.1, 36.1,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 9.31 (s, 1H), 7.62 (s, 1H), 7.01-7.00 (m, 2H), 6.71-6.68 (m, 2H),
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 169.9, 156.2, 132.9, 131.2, 130.7, 126.0, 115.2, 57.6, 42.0, 40.6,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 9.31 (s, 1H), 8.42 (br d, J = 2.0 Hz, 1H), 7.53 (s, 1H), 6.52 (d,
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 165.9, 156.6, 130.2, 129.2, 128.5, 124.2, 115.0, 60.0, 37.8, 35.4.
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 9.30 (br s, 1H), 8.42 (br d, J = 2.0 Hz, 1H), 7.53 (s, 1H),
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 165.9, 156.6, 130.2, 129.2, 128.5, 124.2, 115.0, 60.0, 37.8, 35.4.
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.90 (s, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H),
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 173.3, 137.8, 134.4, 132.7, 127.9, 125.0, 122.9, 120.4, 118.6,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.87 (s, 1H), 6.78 (d, J = 8.5 Hz, 2H), 6.63 (d, J = 8.5 Hz, 2H),
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 173.3, 158.0, 134.1, 133.3, 131.1, 128.0, 116.6, 60.3, 52.8, 41.8,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 7.83 (s, 1H), 6.88-6.79 (m, 2H), 6.67-6.59 (m, 2H), 5.08-4.99
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 170.8, 156.3, 133.7, 130.5, 130.2 (2C), 126.4, 115.1, 56.4, 51.7,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.81 (s, 1H), 6.90-6.85 (m, 2H), 6.66-6.62 (m, 2H), 5.70 (dd,
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 169.6, 157.8, 134.2, 133.6, 131.2, 127.4, 116.5, 64.1, 53.8, 37.6,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.78 (s, 1H), 6.90-6.85 (m, 2H), 6.65-6.61 (m, 2H), 5.70-5.65
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 169.6, 157.9, 131.6, 131.2, 131.0, 127.4, 116.5, 64.2, 53.8, 37.6,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 10.90-10.89 (m, 1H), 8.45 (t, J = 5.9 Hz, 1H), 7.75-7.71 (m,
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 170.7, 162.3, 149.5, 138.6, 136.0, 134.0, 133.0, 132.2, 132.1,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.71-7.66 (m, 4H), 7.63-7.61 (m, 2H), 7.44-7.41 (m, 1H),
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 172.7, 162.6 (d, J = 248.1 Hz, 1C), 145.7, 139.6, 137.8, 136.8
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.50-7.47 (m, 2H), 7.43-7.40 (m, 1H), 7.35-7.29 (m, 2H), 7.18
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 172.7, 149.5, 138.4, 137.8, 136.7, 133.0, 130.3, 128.2, 128.1,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.71-7.67 (m, 2H), 7.52-7.40 (m, 5H), 7.37 (s, 1H), 6.77-6.74
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 172.5, 161.0, 157.7, 152.5, 136.4, 134.5, 134.0, 133.2, 131.3,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.70-7.68 (m, 2H), 7.40-7.38 (m, 2H), 7.27 (br s, 1H),
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 172.5, 164.99, 157.7, 149.7, 138.4, 136.7, 131.2, 130.4, 128.5,
1H NMR, 300 MHz, CDCl+hd 3+l -d+hd 1+l , δ (ppm): 7.62-7.59 (m, 2H), 7.51 (d, J = 3.9 Hz, 1H),
13C NMR, 75 MHz, CDCl+hd 3+l -d+hd 1+l , δ (ppm): 170.9, 155.7, 152.1, 134.0, 133.8, 133.6, 132.7,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.81 (dd, J = 1.3 and 5.0 Hz, 1H), 7.56 (dd, J = 1.3 and 3.8 Hz,
13C NMR, 75 MHz MeOD-d4, δ (ppm): 172.5, 157.7, 141.8, 136.4, 133.7, 133.6, 133.1, 131.2, 128.7,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 9.43-9.04 (m, 1H), 8.60-8.56 (m, 2H), 8.09-7.92 (m, 2H), 7.88
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 170.5, 156.3, 150.4, 150.3, 149.7, 141.1, 137.6, 134.0, 133.0,
1H NMR, 300 MHz, DMF-d+hd 7+l , δ (ppm): 9.51 (br s, 1H), 8.43 (br s, 1H), 7.82-7.78 (m, 2H), 7.64
13C NMR, 75 MHz, DMF-d+hd 7+l , δ (ppm): 168.9, 157.2, 150.5, 139.6, 135.3, 133.6, 133.0, 130.4,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 9.28 (br s, 1H), 8.51 (br s, 1H), 7.74-7.71 (m, 2H), 7.56 (d,
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 168.3, 156.2, 149.5, 138.8, 134.8, 133.1, 132.4, 132.2, 129.9,
1H NMR (300 MHz, CDCl+hd 3+l ) δ: 7.02 (m, 2H), 6.84 (m, 2H), 4.95 (d, J = 7.9 Hz, 1H), 4.52 (m, 1H),
13C NMR (75 MHz, CDCl+hd 3+l ) δ: 172.6, 158.0, 155.2, 130.4 (2C), 128.3, 114.8 (2C), 80.0, 71.2, 67.3,
1H NMR (300 MHz, CDCl+hd 3+l ) δ: 6.94-6.77 (m, 3H), 5.14 (d, J = 8.8 Hz, 1H), 4.48 (m, 1H), 3.79 (s,
13C NMR (75 MHz, CDCl+hd 3+l ) δ: 172.1, 155.0, 152.0 (d, J = 245 Hz), 146.5 (d, J = 10.7 Hz), 129.0 (d,
1H NMR (300 MHz, CDCl+hd 3+l ) δ: 7.13 (d, J = 2.0 Hz, 1H), 6.99 (dd, J = 8.5, 2.0 Hz, 1H), 6.85 (d,
1H NMR (CDCl+hd 3+l , 300 MHz) δ: 7.58 (m, 2H), 7.25 (m, 2H), 5.03 (d, J = 7.4 Hz, 1H), 4.60 (m, 1H),
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 10.82 (s, 1H), 10.49 (s, 1H), 8.79 (s, 1H), 8.39 (s, 1H),
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 166.2, 149.5, 143.0, 140.0, 136.5, 133.2, 132.7, 129.8,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 11.17 (br, 1H), 10.85 (br, 1H), 9.21 (br, 1H), 8.45 (br,
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 164.2, 149.0, 143.1, 139.6, 136.0, 132.9, 132.3, 129.4,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 11.24 (br, 1H), 10.85 (br, 1H), 9.24 (br, 1H), 8.35 (s, 1H),
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 164.3, 149.9, 141.2, 136.0, 134.8, 133.8, 132.0, 129.4,
1H NMR, 500 MHz, DMSO-d6, δ (ppm): 11.20 (br s, 1H), 10.86 (d, J = 1.8 Hz, 1H), 9.22 (s, 1H),
13C NMR, 125 MHz, DMSO-d6, δ (ppm): 164.2, 149.0, 143.1, 139.6, 136.0, 132.8, 132.3, 129.4,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 10.87 (br, 1H), 9.22 (br, 1H), 9.24 (br, 1H), 8.18 (s, 1H),
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 164.2, 142.9, 142.3, 136.0, 132.3, 131.2, 126.8, 124.0,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 10.5 (s, 1H), 8.81 (d, J = 1.3 Hz, 1H), 8.41 (t, J = 5.4 Hz,
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 165.5, 149.0, 142.7, 139.6, 134.5, 132.9,132.7,132.2,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 10.47 (s, 1H), 9.22 (s, 1H), 8.78 (s, 1H), 8.41 (s, NH),
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 166.0, 156.5, 149.5, 143.0, 140.0, 133.2, 132.7, 130.3,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 9.32 (br s, 3H), 8.09 (s, 1H), 7.72 (br d, J = 6.9 Hz, 2H),
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 163.8, 156.2, 149.0, 143.2, 139.7, 132.8, 132.2, 129.9,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 10.49 (s, 1H), 9.22 (s, 1H), 8.80 (s, 1H), 7.85 (s, 1H),
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 165.5, 156.0, 150.0, 140.4, 134.7, 133.8, 132.0, 129.8,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 8.09 (s, 1H), 7.73-7.71 (m, 2H), 7.57-7.53 (m, 2H),
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 163.8, 156.2, 149.1, 143.2, 139.7, 132.8, 132.3, 130.0,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.95 (s, 1H), 7.85 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 8.4 Hz,
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 165.8, 163.8, 156.2, 147.9, 143.1, 140.5, 134.53, 134.51,
1H NMR, 500 MHz, DMSO-d6, δ (ppm): 9.30 (br, 1H), 8.09 (s, 1H), 7.66 (d, J = 8.2 Hz, 2H), 7.55
13C NMR, 125 MHz, DMSO-d6, δ (ppm): 163.8, 156.2, 149.3, 143.2, 139.0, 132.9, 130.3, 130.0,
1H NMR, 500 MHz, DMSO-d6, δ (ppm): 11.12 (br s, 1H), 9.26 (s, 1H), 9.23 (br s, 1H), 8.26 (s,
13C NMR, 125 MHz, DMSO-d6, δ (ppm): 164.0, 156.2, 150.0, 141.3, 134.9, 133.8, 132.1, 130.0,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 10.98 (br s, 1H), 9.28-9.17 (m, 2H), 8.10 (s, 1H), 7.39 (d,
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 163.8, 156.2, 143.0, 142.3, 132.4, 131.3, 130.0 (2C),
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 11.03 (br s, 1H), 9.27 (s, 1H), 9.19 (br s, 1H), 8.33 (br s,
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 163.8, 156.2,143.2, 141.2, 132.5, 131.7, 130.0, 127.7,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 9.27 (br s, 2H), 9.13 (br s, 1H), 8.04 (s, 1H), 7.39 (d,
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 163.8, 156.2, 146.7, 143.3, 138.0, 132.0, 130.0, 126.2,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 9.27 (br s, 2H), 9.14 (br s, 1H), 8.14 (dd, J = 1.3 and 3.0
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 163.8, 156.2, 143.4, 140.3, 130.5, 130.0, 129.0, 125.8,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 11.07 (br s, 1H), 9.27-9.19 (m, 2H), 8.71 (br s, 1H), 8.00
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 163.7, 156.2, 143.4, 136.8, 132.6, 132.3, 130.0 (2C),
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 9.27 (s, 2H), 8.09 (s, 1H), 7.74-7.71 (m, 2H), 7.57 (d,
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 163.8, 156.2, 149.1, 143.2, 139.6, 132.8, 132.2, 130.0,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 11.07 (br, 1H), 9.25 (br, 1H), 9.17 (br, 1H), 8.56 (d,
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 163.8, 156.2, 150.4, 150.0, 149.7, 143.2, 142.0, 137.5,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 11.11 (br, 1H), 9.23 (s, 1H), 8.56 (ddd, J = 0.9, 1.7 and
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 163.0, 150.4, 150.0, 149.6, 147.3 (m), 143.4, 142.0, 137.5,
19F NMR (282 MHz, DMSO-d6) δ: - 57.6.
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 11.12 (br, 1H), 9.23 (br, 1H), 8.46 (br, 1H), 8.13 (s, 1H),
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 163.5, 149.1, 147.3 (m), 143.4, 139.6, 135.3, 132.8, 132.2,
19F NMR (282 MHz, DMSO-d6) δ: - 57.3.
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 11.10 (br, 1H), 9.22 (s, 1H), 8.56-8.55 (m, 1H), 8.47 (t,
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 163.7, 156.6, 155.5, 150.4, 150.1, 149.7, 143.3, 142.1,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 9.31 (br, 1H), 8.11 (s, 1H), 7.74-7.71 (m, 2H), 7.58 (d,
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 163.7, 156.6, 155.5, 150.4, 150.1, 149.7, 143.3, 142.1,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 11.60 (br, 1H), 9.19 (br, 1H), 8.56 (ddd, J = 0.9, 1.7 and
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 163.7, 158.1, 150.4, 150.0, 149.7, 143.2, 142.1, 137.5,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 8.11 (s, 1H), 7.75-7.72 (m, 2H), 7.58 (d, J = 3.9 Hz, 1H),
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 163.7, 153.9, 149.1, 143.3, 139.7, 132.8, 132.2, 130.2,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 11.09 (s, 1H), 9.21 (s, 1H), 8.56 (ddd, J = 0.9, 1.7 and 4.9
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 163.7, 153.9, 150.4, 150.0, 149.7, 143.3, 142.1, 137.5,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 9.20 (s, 1H), 8.53 (q, J = 4.7 Hz, 1H), 8.10 (s, 1H), 7.90
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 165.7, 163.7, 158.1, 147.9, 143.2, 140.5, 134.5, 134.5,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 9.27 (br, 1H), 8.97 (dd, J = 0.6 and 1.8 Hz, 1H), 8.60 (dd,
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 163.8, 156.2, 149.8, 146.6, 145.2, 143.1, 141.0, 133.4,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 10.87 (br s, 1H), 8.79 (br s, 1H), 7.75-7.71 (m, 2H),
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 166.0, 149.5, 138.6, 136.0, 133.8, 133.1, 132.2, 131.9,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): . 71-7.67 (m, 2H), 7.53 (d, J = 3.9 Hz, 1H), 7.48-7.40 (m,
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 168.7, 157.7, 152.4, 140.0, 136.3, 134.7, 134.0, 133.2,
1H NMR, 300 MHz, MeOD-d4, δ (ppm): 7.74-7.71 (m, 2H), 7.60 (t, J = 3.9 Hz, 1H), 7.52-7.41 (m,
13C NMR, 75 MHz, MeOD-d4, δ (ppm): 168.7, 157.6, 153.0, 135.5, 135.2, 135.0, 134.2, 133.9,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 10.54 (br s, 1H), 9.26 (s, 1H), 8.79 (br s, 1H), 8.60 (ddd,
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 165.8, 156.1, 150.4, 150.3, 149.7, 141.0, 137.6, 133.7,
1H NMR, 300 MHz, DMSO-d6, δ (ppm): 9.31 (br, 1H), 8.11 (s, 1H), 7.74-7.71 (m, 2H), 7.58 (d,
13C NMR, 75 MHz, DMSO-d6, δ (ppm): 163.7, 156.6, 155.5, 150.4, 150.1, 149.7, 143.3, 142.1,
1H NMR, 500 MHz, DMSO-d4, δ (ppm): 11.13 (br s, 1H), 9.35 (s, 1H), 9.17 (s, 1H), 7.73 (d,
13C NMR, 125 MHz, MeOD-d4, δ (ppm): 163.5, 156.2, 149.4, 139.1, 134.5, 133.1, 132.8, 132.2,
1H NMR (DMSO-d6, 300 MHz) δ: 11.08 (br, 1H), 9.21 (br, 1H), 8.64-8.62 (m, 2H), 8.56 (br, 1H),
13C NMR (DMSO-d6, 75 MHz) δ: 163.8, 158.1, 150.6, 145.6, 143.1, 142.2, 139.2, 132.7, 130.0,
1H NMR (DMSO-d6, 500 MHz) δ: 11.08 (s, 1H), 9.22 (s, 1H), 8.56 (ddd, J = 0.9, 1.8 and 4.8 Hz,
13C NMR (DMSO-d6, 75 MHz) δ: 163.6, 150.4, 150.0, 149.6, 147.1, 146.1, 143.2, 142.0, 137.5,
1H NMR (DMSO-d6, 300 MHz) δ: 11.08 (br, 1H), 9.21 (br, 1H), 8.96 (dd, J = 0.8 and 2.4 Hz, 1H),
13C NMR (DMSO-d6, 75 MHz) δ: 163.7, 158.1, 149.8, 146.6, 145.3, 143.2, 140.9, 133.4, 132.7,
1H NMR (DMSO-d6, 300 MHz) δ: 11.03 (br, 1H), 10.03 (br, 1H), 9.21 (br, 1H), 8.56 (ddd, J = 4.8,
13C NMR (DMSO-d6, 75 MHz) δ: 163.6, 151.9, 150.4, 150.0, 149.7, 143.3, 142.1, 137.5, 132.6,
1H NMR (DMSO-d6, 300 MHz) δ: 11.01 (br, 1H), 10.04 (br, 1H), 8.46 (br, 1H), 8.09 (s, 1H),
13C NMR (DMSO-d6, 75 MHz) δ: 163.6, 161.9, 149.0, 143.3, 139.6, 132.6, 132.2, 130.0, 129.4,
1H NMR (DMSO-d6, 300 MHz) δ: 11.07 (br, 1H), 9.21 (br, 1H), 8.57 (ddd, J = 4.8, 1.7 and 1.0 Hz,
13C NMR (DMSO-d6, 75 MHz) δ: 163.8, 150.4, 150.0, 149.7, 148.4, 147.7, 143.3, 142.0, 137.5,
1H NMR (DMSO-d6, 300 MHz) δ: 11.09 (br, 1H), 9.20 (br, 1H), 8.47 (br, 1H), 8.14 (s, 1H),
13C NMR (DMSO-d6, 75 MHz) δ: 163.8, 149.1, 148.4, 147.7, 143.3, 139.6, 132.8, 132.2, 129.4,
1H NMR (DMSO, 300 MHz) δ: 11.09 (br, 1H),9.20 (br, 1H), 8.49 (br, 1H), 8.6 (q, J = 4.5 Hz, 1H),
13C NMR (DMSO, 75 MHz) δ: 166.3, 164.2, 158.6, 148.8, 143.7, 140.6, 135.9, 133.2, 132.8,
1H NMR (300 MHz, DMSO-d6) δ: 8.57 (ddd, J = 0.9, 1.7 and 4.8 Hz, 1H), 8.08 (s, 1H), 8.02 (dt,
19F NMR (282, DMSO-d6) δ: - 136.96.
13C NMR (75, DMSO-d6) δ: 163.6, 150.6 (d, J = 240.4 Hz), 150.4, 150.0, 149.7, 143.6 (d, J = 12.0
1H NMR (300 MHz, DMSO-d6) δ: 8.09 (s, 1H), 7.74-7.70 (m, 2H), 7.56 (d, J = 3.9 Hz, 1H), 7.54
19F NMR (75 MHz, DMSO-d6) δ: - 136.96.
13C NMR (75 MHz, DMSO-d6) 6: 163.6, 150.6 (d, J = 240.4 Hz), 149.1, 143.6 (d, J = 12.1 Hz),
1H NMR (DMSO-d6, 300 MHz) 8 (ppm) 11.10 (br s, 1H), 9.22 (br s, 1H), 8.58 (ddd, J = 4.9, 1.7
13C NMR (DMSO-d6, 75 MHz) 8 (ppm) 163.8, 158.1, 150.9, 150.2, 149.7, 141.2, 137.6, 137.3,
1H NMR (DMSO-d6, 300 MHz) 8 (ppm) 11.05 (br s, 1H), 9.17 (br s, 1H), 8.55 (ddd, J = 4.8, 1.7
13C NMR (DMSO-d6, 75 MHz) δ (ppm) 163.7, 158.1, 150.5 (0.75C, maj), 150.4 (0.25C, min),
1H NMR (DMSO-d6, 300 MHz) 8 (ppm) 11.09 (br s, 1H), 9.25 (br s, 1H), 8.53 (ddd, J = 4.8, 1.6
13C NMR (DMSO-d6, 75 MHz) 8 (ppm) 163.7, 158.1, 150.5 (0.7C, maj), 150.4 (0.3C, min), 149.8
1H NMR (DMSO-d6, 300 MHz) δ: 8.55 (ddd, J = 4.8, 1.8 and 1.0 Hz, 1H), 8.06 (s, 1H), 8.01 (ddd,
13C NMR (DMSO-d6, 75 MHz) δ: 163.6, 159.0, 150.6, 149.6, 149.5, 143.1, 142.9, 138.7, 137.5,
1H NMR (DMSO-d6, 300 MHz) δ: 11.14 (brs, 1H), 9.17 (brs, 1H), 8.55 (ddd, J = 4.9, 1.7 and 0.9
13C NMR (DMSO-d6, 75 MHz) δ: 163.8, 157.3, 150.4, 150.1, 149.6, 143.3, 142.0, 137.5, 132.6,
1H NMR (DMSO-d6, 300 MHz) δ: 11.08 (br s, 1H), 9.19 (brs), 8.56 (ddd, J = 4.8, 1.6, and 0.9 Hz,
13C NMR (DMSO-d6, 75 MHz) δ: 163.7, 157.3, 150.4, 150.0, 149.7, 143.2, 142.1, 137.5, 132.6,
1H NMR (500 MHz, DMSO-d6) δ: 11.12 (br s, 1H), 9.26 (br s, 1H), 8.55 (ddd, J = 4.8, 1.6 and 0.9
19F NMR (282 MHz, DMSO-d6) δ: - 136.05 (dd, J = 12.2 and 9.2 Hz).
13C NMR (125 MHz, DMSO-d6) δ: 163.7, 151.1 (d, J = 241.0 Hz), 150.5, 150.1, 149.8, 146.1 (d,
1H NMR (DMSO-d6, 300 MHz) δ: 11.04 (br, 1H), 9.23 (br, 1H), 8.56 (ddd, J = 5.0, J = 1.5, J = 1.0
13C NMR (DMSO-d6, 75 MHz) δ: 163.5, 153.4, 150.4, 150.0, 149.7, 143.3, 142.0, 137.5, 132.6,
1H NMR (DMSO-d6, 300 MHz) δ: 11.09 (br s), 1H), 8.46 (br s, 1H), 8.12 (s, 1H), 7.72 (m, 2H),
13C NMR (DMSO-d6, 75 MHz) δ: 163.5, 149.1, 143.3, 142.5, 139.6, 136.8, 132.8, 132.2, 131.7,
1H NMR (DMSO-d6, 300 MHz) δ: 9.59 (s, 1H), 9.28 (br s, 1H), 8.13 (s, 1H), 7.72 (m, 2H), 7.47
13C NMR (DMSO-d6, 75 MHz) δ: 163.6, 150.5, 149.1, 143.3, 139.6, 136.5, 132.8, 132.3, 132.0,
1H NMR (DMSO-d6, 300 MHz) δ: 9.19 (br s, 1H), 8.90 (br s, 1H), 8.29 (s, 1H), 8.15 (s, 1H), 7.99
13C NMR (DMSO-d6, 75 MHz) δ: 172.0, 163.7, 158.1, 146.9, 143.0, 137.0, 131.9, 131.7, 130.0
1H NMR (DMSO-d6, 300 MHz) δ: 11.08 (br s, 1H), 9.23 (br s, 1H), 8.56 (ddd, J = 4.8, 1.7 and 0.9
13C NMR (DMSO-d6, 75 MHz) δ: 163.9, 158.1, 151.0, 150.2, 149.6, 141.9, 138.4, 137.6, 133.8,
1H NMR (DMSO-d6, 300 MHz) 8: 11.10 (s, 2H), 9.24 (d, J = 11.5 Hz, 2H), 8.16 (d, J = 7.4 Hz,
13C NMR (DMSO-d6, 75 MHz) 8: 163.9, 163.7, 158.1, 158.1, 149.4 (2C), 145.5, 145.1, 139.0,
1H NMR (DMSO-d6, 300 MHz) δ: 11.14 (br s, 1H), 9.25 (br s, 1H), 8.26 (br s, 1H), 7.78 (m, 2H),
13C NMR (DMSO-d6, 75 MHz) δ: 163.6, 158.1, 149.9, 148.8, 134.8, 133.9, 132.0, 130.1 (2C),
1H NMR (DMSO-d6, 500 MHz) δ: 11.13 (d, J = 1.1 Hz, 1H), 9.26 (d, J = 1.1 Hz, 1H), 8.47 (t,
13C NMR (DMSO-d6, 125 MHz) δ: 163.8, 149.5, 143.9, 142.2, 140.0, 133.3, 132.7 (2C), 132.6,
The enzymatic activity of ERAP1 or 2 was assayed using L-AMC (L-Leucine-7-amido-4-methylcoumarin hydrochloride) or R-AMC (L-Arginine-7-amido-4-methylcoumarin hydrochloride) respectively. Hepes at 50 mM with 100 mM NaCl at pH 7 was used as buffer. Briefly, 60 nL of test compounds were added in 384-wells plates (dark, non-binding surface) by acoustic dispensing with nanoacoustic dispenser Echo (Labcyte) and pre-incubated 30 minutes at ambient temperature with 10 μL of ERAP 0.8 μg/mL or 1 μg/mL or vehicle. The reaction was then started with the addition of 10 μL of substrate at 10 μM. The final concentration of ERAP, substrate and DMSO was 0.5 μg/mL, 5 μM and 0.4% respectively. For the kinetic readout a Victor 3V (Perkin-Elmer) was used with excitation at 380 nm and emission at 450 nm. The fluorescence was measured each 3 minutes during one hour.
The Z and Z′ factors were calculated according to J.-H. Zhang, T. D. Y. Chung, K. R. Oldenburg, A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays, J. Biomol. Screen., 4 (1999) 67-73. Data analysis was performed using Xlfit® v 5.0 or GraphPad Prism® v 4.0.
Percentages of inhibition at different concentrations were obtained as described above and IC50s were carried out as 8-point dose response curves and reported as the average of at least three independent measurements. Bestatin was used as a reference inhibitor (100% inhibition at 2 mM). Data analysis was performed using Xlfit® v 5.0 or GraphPad Prism® v 4.0. Nonlinear curve fitting and statistical analysis was done using built-in functions.
Results are presented in tables 1 and 2.
Enzymatic reactions were performed using KSIINFEKL peptide (from Proteogenix, Schiltigheim, FR). The enzymatic reactions were stopped at the desired time-point by dilution using iced acetonitrile (×100 dilution), before injection in LC-MS/MS to measure AUC. LC-MS/MS analysis were performed on an UPLC system Acquity I Class (Waters®), combined with a triple quadrupole mass spectrometer Xevo TQD (Waters®). The column was an Acquity BEH C18 50*2.1 mm, 1.7 μm column (Waters®) and the following mobile phases were used: 5 mM ammonium formate pH 3.75 buffer for solvent (A) and 5 mM ammonium formate pH 3.75 in acetonitrile for solvent (B). At a flow rate of 600 μL/min, the analytical method starts at 98% (A) for 10 s, then the percentage of B gradually increases at 98% till 2 minutes, hold at 98% (B) for 30 s before returning to the initial conditions, hold 1.5 minutes. The injection volume was 1 μL. MS analyses were performed under MRM detection using the parameters optimized for each peptide (capillary voltage, product ions, collision energy, desolvation temperature). The control of the equipment as well as the reprocessing of the analyses were carried out using MassLynx software (Waters®). For XSIINFEKL, 100% corresponds to the AUC of the peptide at t=0 without enzyme. Dose-response curves with compounds were performed at t=60 min.
Results are presented in table 3.
Using Jetprime according to the manufacturer's recommendations, HEK293 cells are transiently transfected with a single plasmid encoding i) the 14 amino-acids HiBiT tag (part of the nanoluciferase) fused to the N-terminal end of mouse H2kB, ii) an ER-targeted N terminally extended antigenic precursor peptide for L-SIINFEKL, and iii) a TAP inhibitor (UL49.5 protein). The cells are harvested 4 h post-transfection and plated in 96 wells-plate format in which the studied compounds are previously dispensed. The amount of a HiBiT-tagged H2kB protein present on the cell surface is determined 24 h later using the Nano-Glo® HiBiT Extracellular Detection System (Promega). The results are presented on
| Number | Date | Country | Kind |
|---|---|---|---|
| 20306621.2 | Dec 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/086617 | 12/17/2021 | WO |
