Thiazole derivatives as TAU agglutination inhibitors

Abstract

The present invention provides a compound of the following general formula (I): # imgabs0 # or a pharmaceutically acceptable salt, solvate or hydrate thereof, wherein RN is independently-H or-Me; rX is independently C1-4 alkyl, and is optionally substituted by halogen or hydroxyl; -NHRN1, wherein RN1 is a C1-4 alkyl group; or a C5-10 heteroaryl group; rY is independently: a C5-10 heteroaryl group, optionally substituted with a methyl group, a halogen group or a phenyl group; or a neopentyl group. The compounds are useful as tau agglutination inhibitors (TAIs).

Description

Technical Field

[0001] The present invention generally relates to compounds having tau aggregation inhibitor (TAI) activity and their use in methods for treating tauopathies, including but not limited to Alzheimer's disease. Background Art

[0002] Tau protein aggregates to form neurofibrillary tangles and neuritic plaques, which are associated with cognitive decline in Alzheimer's disease (AD) [1-3]. Therefore, there is interest in developing treatments that target this pathology. Hydrogen methionine methanesulfonate (HMTM; previously known as leucomethionine methanesulfonate, LMTM) has been shown to have exposure-dependent pharmacological activity against clinical decline and brain atrophy in AD and frontotemporal dementia [4,5].

[0003] A core tau fragment corresponding to one of the species isolated from proteolytically stabilized AD paired helical filaments (PHFs) containing residues 297-391 (dGAE) spontaneously assembles in vitro in the absence of multivalent anionic cofactors to form PHFs identical to native PHFs isolated from brain tissue [6-9]. Hydromethionine (HMT) inhibits the assembly of dGAE filaments in vitro at a protein:HMT stoichiometry of 1:0.1

[10] .

[0004] The PHF core isolated from AD brain tissue is an extremely stable structure that resists proteolysis and requires harsh solvents such as formic acid to release the constitutive tau protein

[11] . The assembly of tau aggregates is an autocatalytic process in which core tau oligomers act to further convert normal full-length tau into truncated toxic species, resulting in gradual degeneration of neuronal function [12,13]. The binding affinity of tau captured in vitro is approximately 20nM, which is much higher than the physiological tau-tubulin binding value (approximately 400nM;

[14] ). This drives the redistribution of the tau protein pool from the normal soluble species to the pathological assembled form

[15] .

[0005] Although the structures of the AD PHF core and the filament core present in Pick's disease are available at atomic resolution [16,17], the molecular mechanisms underlying the pathological assembly of tau and other proteins in neurodegenerative diseases remain elusive [18-20]. Many precursors and oligomeric states of some proteins have been identified [21-24]. However, the isolation and further characterization of these structurally heterogeneous and transient forms remains a challenge, as it is unclear how to isolate them and make them suitable for unbiased structural and biochemical analysis.

[0006] The present inventors have previously reported that HMT has clinical pharmacological activity at a steady-state brain concentration estimated to be approximately 0.1 μM; this is consistent with the EC of 0.6 μM seen in cell-based assays. 50 The values ​​are in the same order of magnitude [4,13].

[0007] Given the intractability of the PHF core, it is currently not possible to design small-molecule TAIs that can act on targets within brain cells at concentrations that are safely tolerated in the clinic.

[0008] The present invention has been designed in view of the above considerations. Summary of the invention

[0009] The inventors used a combination of molecular dynamics (MD) simulations, immunochemistry, biochemistry and medicinal chemistry to study the molecular mechanism of PHF core oligomer capture of monomers and how HMTs bind to the core tau unit of PHF. It has been shown that the initiation of assembly depends on a series of steps, first involving the anchoring of the monomers, followed by the unwinding of the core unit to allow the formation of the stable cross-β-sheet structure of the PHF core. The working principle of HMTs is to bind to cryptic druggable pockets within the core unit, stabilizing it in a compact configuration without participating in the unwinding required for further self-assembly.

[0010] The cryptic binding pocket identified by the present inventors provides the basis for defining a pharmacophore model, and this model is used to design alternative tau aggregation inhibitors (TAIs). A group of compounds chemically unrelated to HMTs were synthesized to test whether new inhibitors could be rationally designed to act in the druggable pocket and optimize some of the medicinal chemistry features required for inhibitory activity. New inhibitors were discovered that were active in a previously described cell-based aggregation assay

[13] .

[0011] Therefore, in one aspect, the present invention provides novel compounds useful as tau aggregation inhibitors. These compounds contain a thiazole core and have the general formula:

[0012]

[0013] Where R N are independently -H or -Me;

[0014] R X Independently for C 1-4 Alkyl, optionally substituted with halo or hydroxy; -NHR N1 , where R N1 C 1-4 Alkyl; or C 5-10 heteroaryl;

[0015] R Y Independently: C5-10 heteroaryl, optionally substituted with methyl, halo or phenyl; or neopentyl.

[0016] The present invention also encompasses pharmaceutically acceptable salts, solvates and hydrates of these compounds.

[0017] The present invention further relates to methods, uses, compositions and other materials using these compounds as tau protein aggregation inhibitors and as therapeutic agents or preventive agents for diseases associated with tau protein aggregation ("tauopathy"). The present invention further provides methods for preparing these compounds.

[0018] The present invention includes any combination of the described aspects and preferred features unless such combination is expressly impermissible or explicitly avoided. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Embodiments and experiments illustrating the principles of the present invention will now be discussed with reference to the accompanying drawings, in which:

[0020] Figure 1 Structures of compounds that have been tested for their activity in a cell-based tau aggregation assay

[13] . The core thiazole moiety is shown in the middle of the diagram, the sulfonamide substituents (labeled 1-6) are shown at the top, and the amide substituents (labeled 7-12) are shown at the bottom.

[0021] Figure 2 (A) Inhibition of tau aggregation in fibroblasts transfected with full-length tau1-441 and constitutively expressed truncated tau (residues 295-391) [13, 25] for MT and (B) compound 9. Using this data, the concentrations (EC) for 50% inhibition of the lower 12-kDa band by MT and compound 9 were derived. 50 values) were 0.6 μM and 3.65 μM, respectively (the results of repeated measurements are listed in Example 3 below; the average value of compound 9 was 4.816 μM).

[0022] Figure 3. Hierarchical assembly of a key segment (residues 306-378) of the monomeric dGAE sequence (cartoon) into a monolayer PHF stack (backbone line). (A) Frame 1, approach of hairpin loop residues 337-355 (recognized by 1D12) at the beginning of assembly. (B) Frame 44 shows the unwinding of the stable monomer and the extension of the C- and N-termini. (C) Frame 75 shows further interactions of the pre-monomer after Pro332 flipping. (D) Frame 143 shows the truncated dGAE monomer incorporated into the PHF stack. The lower part of the figure shows the sequence of the truncated dGAE monomer used in the NEB simulation, and the regions mapped to the epitopes recognized by a set of single-chain antibodies (scAbs) (in decreasing order of grayscale: CE2>1D12>1G2>CA4).

[0023] Figure 4 HMT-tau binding pose of HMT-protein complex. The lower part of the figure shows the sequence of truncated tau residues 295-391 used in the MD simulation, and the regions mapped to the epitopes recognized by the corresponding scab (in descending order of grayscale: CE2>E2E8>1D12>1G2>CA4).

[0024] Figure 5 .Immunoreactivity of core region scAbs to dGAE assembled in the presence or absence of TAI HMT and compound 9. scAb binding to dGAE samples was performed in solution phase by first capturing dGAE with mAb 423 and then detecting bound dGAE with scAb. mAb 423 specifically recognizes the Glu391-dependent C-terminus of the dGAE fragment. Binding profiles of scAbs (A) CA4, (B) 1G2, (C) CE2, and (D) 1D12 were obtained using an anti-human Cκ HRP-conjugated secondary antibody. Reactivity with preassembled soluble dGAE (dGAE 0h) is included to demonstrate maximal reactivity of scAbs to non-aggregated dGAE. Reactivity of dGAE was measured after 24h of assembly in the absence (dGAE 24h) or presence of HMT or compound 9 (C9); compound 9 was dissolved in DMSO, with solvent controls also included. Values ​​are expressed as absorbance at 450 nm (mean ± SE; n = 2; samples were run in duplicate).

[0025] Figure 6.Immunoblotting intensity analysis showed that the binding of recombinant scAb to the dGAE (tau297-391) epitope was gradually lost during the 8-hour in vitro assembly process. FIJI was used to measure the point signal intensity at each time point and subtract the background. For normalization, when a signal is observed at time 0 in the blot, it is defined as 1, and the remaining time points are quantified relative to this. In some experimental repetitions, no signal was observed. These results are incorporated into the final summary data shown, and the time 0 value of antibodies CE2, CA4 and E2E8 is <1.0, where 3 / 9, 4 / 8 and 7 / 8 repetitions give positive results respectively. If there is no signal on the blot, it is designated as zero, and the intensity of each time point is expressed as mean ± SEM. The exposure time is used to ensure that the signal is not saturated and is within the linear range.

[0026] Figure 7 . Schematic overview of the computer-aided drug design (CADD) approach. DETAILED DESCRIPTION

[0027] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated herein by reference.

[0028] Hydromethionine (HMT) is the active part in hydromethionine mesylate (HMTM) and is an effective tau aggregation inhibitor. Hydromethionine (HMT) may also be referred to as colorless methionine (LMT). For the avoidance of doubt, the two terms are synonymous and may be used interchangeably herein.

[0029] The present inventors used a combination of molecular dynamics (MD) simulations, immunochemistry, biochemistry and medicinal chemistry to investigate the molecular mechanism by which PHF core oligomers capture monomers and how HMTs bind to the core tau units of PHF.

[0030] The advantage of molecular dynamics is that it provides a means to explore a wide range of conformational space and determine the inherent heterogeneity and stability of specific states of the molecule. Although it is difficult to obtain direct structural evidence for the existence of certain conformations, especially for molecules like dGAE that have the intrinsic ability to spontaneously aggregate, structural predictions can be made and can be tested using immunochemistry, medicinal chemistry, and biochemistry. To make this approach feasible, several tools are essential: a model system that can monitor the assembly of the PHF core, a family of specific monoclonal antibodies that recognize epitopes located in the PHF core domain, and prototype inhibitors that can be used as the basis for defining the pharmacophore model for targeted medicinal chemistry. These techniques should be used to study the assembly mode of the PHF core and the pharmacological inhibition of assembly.

[0031] Initial MD analysis of the capture of monomeric patterns by the assembled PHF core template can be summarized as a series of sequential stages. First, the monomer is anchored to the template via a tight hairpin loop formed by residues 337-355, followed by a conformational switch at Pro332 that allows the monomer to unfold and compress its N- and C-terminal arms with the corresponding fragments of the existing assembly. These latter events occur in a very short time, with residues 319-331 binding in the direction from C-terminus to N-terminus and residues 355-367 binding in the direction from N-terminus to C-terminus. This folding sequence is supported by the gradual loss of immunoreactivity of a set of scAbs recognizing epitopes spanning the PHF core tau unit. The immunoreactivity of CE2 and CA4, which recognize epitopes immediately adjacent to the hairpin loop, disappears very early, while the occlusion of the epitope closer to the C-terminus recognized by 1G2 and E2E8 occurs at a later stage. Although it has been proposed that a short fragment containing residues 306-311 triggers tau assembly

[26] , the current analysis shows that the templated monomer capture process occurs over longer distances than the short peptide simulated in vitro. We found that initial capture occurs via a highly charged hairpin loop corresponding to residues 337-355. The N-terminal and C-terminal segments are then compressed onto the pre-existing template. In this sequence, the N-terminal and C-terminal arms of the core tau unit need to be flexible to allow the formation of a stable cross-β-sheet structure, which is characteristic of the PHF core.

[0032] Since HMT is known to be a potent inhibitor of dGAE assembly, the inventors used it as a molecular probe to determine whether there is a transiently stable cryptic ligand binding pocket that can be used for structure-based drug design. Among the 750,000 possible protein conformations of dGAE identified in MD simulations, a single complex was identified in which HMT remained tightly bound to the protein structure and did not change much during the 100 ns MD simulation time. Comparison with simulations in the absence of HMT showed that HMT plays a role by stabilizing one of the available conformations in the set of possible conformations of the core tau unit. The driving force for encapsulating unqualified conformations for assembly is the formation of intramolecular hydrogen bonds and hydrophobic collapse, which reduces the water accessible, polar and hydrophobic surface areas required for the alignment and accommodation of monomers on pre-existing oligomers. We do not know whether different classes of TAI can play a role by stabilizing different conformations of the core tau unit. However, it can be concluded from the current study that HMT plays a role by stabilizing conformations that compete with the disentanglement of the monomer N-terminal and C-terminal domains required for oligomer extension.

[0033] The identified cryptic binding pocket provides the basis for defining a pharmacophore model. A detailed discussion of molecular dynamics studies and pharmacophore model definition is set forth in WO2022 / 008545 (WisTa Laboratories Ltd.), the disclosure of which is hereby incorporated by reference in its entirety.

[0034] This study provides an explanation for the surprising clinical efficacy of HMTs as TAIs. In essence, HMTs act by stabilizing the endogenous assembly-incompetent conformation of tau units in the PHF core. The inventors have demonstrated that the druggable pocket within the PHF core bound by HMTs provides a basis for the rational design of chemically unrelated TAIs.

[0035] The inventors have now designed alternative TAIs based on the above pharmacophore model. They have been synthesized and tested using two bioassay systems. The HMT binding pocket is primarily hydrophobic in nature, with some sites suitable for hydrogen bonding. A group of compounds chemically unrelated to the HMT were synthesized to test whether new inhibitors could be rationally designed to act on the druggable pocket and optimize some of the medicinal chemistry features required for inhibitory activity.

[0036] Several new inhibitors were discovered that were active in a previously reported cell-based agglutination assay

[13] . The best of these were compared with HMT in a new aqueous immunoassay that examined a panel of antibodies for agglutination-dependent epitope blocking.

[0037] Therefore, in one aspect, the present invention provides a compound of the following formula:

[0038]

[0039] Where R N are independently -H or -Me;

[0040] R X Independently for C 1-4 Alkyl, optionally substituted with halo or hydroxy; -NHR N1 , where R N1 C 1-4 Alkyl; or C 5-10 heteroaryl;

[0041] R Y Independently: C 5-10 heteroaryl, optionally substituted with methyl, halo or phenyl; or neopentyl;

[0042] or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[0043] In some embodiments, R N is -H.

[0044] In some embodiments, R N For-Me.

[0045] In some embodiments, R X Independently for C 1-4 Alkyl, which is optionally substituted with halo or hydroxy.

[0046] In some embodiments, R X is -Me, -Et, -nPr, -iPr, -nBu, -iBu or -tBu, which is optionally substituted with halo or hydroxy.

[0047] In some embodiments, R X is -Me, -Et, -nPr or -iPr, which is optionally substituted with halogen or hydroxy.

[0048] In some embodiments, R X is -Me or -Et, which is optionally substituted with halogen or hydroxy.

[0049] In some embodiments, R X is -Me, which is optionally substituted with halogen.

[0050] In some embodiments, R X is -Me, which is optionally substituted with -F or -Cl.

[0051] In some embodiments, R X It is -CH2F.

[0052] In some embodiments, R X is unsubstituted -Me.

[0053] In some embodiments, R X is -Et, optionally substituted with hydroxy.

[0054] In some embodiments, R X It is -CH2CH2OH.

[0055] In some embodiments, R X Independently -NHR N1 , where R N1 C 1-4 alkyl.

[0056] In some embodiments, R N1 is -Me, -Et, -nPr, -iPr, -nBu, -iBu or -tBu.

[0057] In some embodiments, R N1 is -Me, -Et, -nPr or -iPr.

[0058] In some embodiments, R N1 It is -Me or -Et.

[0059] In some embodiments, R X independently -NHMe.

[0060] In some embodiments, R X Independently for C 5-10 Heteroaryl.

[0061] In some embodiments, R X Nitrogen-containing C 5-10 Heteroaryl.

[0062] In some embodiments, R X Selected from indolyl, benzimidazolyl, pyrrolyl, imidazolyl, pyrazolyl.

[0063] In some embodiments, R X Indolyl.

[0064] In some embodiments, R X for

[0065]

[0066] In some embodiments, R Y It is neopentyl (-CH2C(CH3)3).

[0067] In some embodiments, R Y Independently: C 5-10 Heteroaryl, which is optionally substituted with methyl, halo or phenyl.

[0068] In some embodiments, R Y Nitrogen-containing C 5-10 Heteroaryl, which is optionally substituted with methyl, halo or phenyl.

[0069] In some embodiments, R Y Nitrogen-containing C 5-10 Heteroaryl, which is optionally substituted with halo or phenyl.

[0070] In some embodiments, R Y Nitrogen-containing C 5-10 Heteroaryl, which is optionally substituted with -Me, -Cl or -Ph.

[0071] In some embodiments, R Y is a nitrogen-containing C selected from pyrrolyl, imidazolyl, pyrazolyl, indolyl and benzimidazolyl 5-10 Heteroaryl, which is optionally substituted with -Me, -Cl or -Ph.

[0072] In some embodiments, R Y is a nitrogen-containing C selected from pyrrolyl, pyrazolyl and benzimidazolyl 5-10 Heteroaryl, which is optionally substituted with -Me, -Cl or -Ph.

[0073] In some embodiments, R Y For unsubstituted C 5-10 Heteroaryl.

[0074] In some embodiments, R Y is an unsubstituted nitrogen-containing C 5-10 Heteroaryl.

[0075] In some embodiments, R Y is unsubstituted pyrrolyl, imidazolyl, pyrazolyl, indolyl or benzimidazolyl.

[0076] In some embodiments, R Y is unsubstituted pyrrolyl or indolyl.

[0077] In some embodiments, R Y Selected from:

[0078]

[0079] Preferred compounds

[0080] In general, the present invention relates to one or more compounds selected from the following compounds, and their use in medicine:

[0081]

[0082]

[0083]

[0084]

[0085] In this and all other aspects of the invention, unless the context requires otherwise, the compound may be selected from the list consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 and 26.

[0086] In one embodiment, the compound can be selected from the list consisting of: 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 18, 20, 21 and 23.

[0087] In one embodiment, the compound is selected from the list consisting of: 1, 4, 5, 6, 9, 10, 12, 15, 16 and 20.

[0088] In one embodiment, the compound is selected from the list consisting of: 5, 9, 10, 12 and 20.

[0089] In one embodiment, the compound is selected from the list consisting of: 9, 10, 14 and 25.

[0090] In one embodiment, the compound is Compound 9.

[0091] Preferred compounds of the invention are those that show activity in the assays described herein. Particularly preferred compounds have an activity of less than 0.5 in the cell-based agglutination inhibition assay described herein (ratio of truncated tau to full-length tau; tested at 2 μM).

[0092] Preferably, the compound has an EC less than 500, 250, 200, 100 or 50. 50 , as determined with reference to the Examples herein. Preferably, the compound has a B of less than 750, 500, 200 or 100. 50 , as determined with reference to the Examples herein.

[0093] Isotope variation

[0094] In one embodiment, one or more carbon atoms of the compound are 11 C or 13 C or 14 C.

[0095] In one embodiment, one or more carbon atoms of the compound are 11 C.

[0096] In one embodiment, one or more carbon atoms of the compound are 13 C.

[0097] In one embodiment, one or more carbon atoms of the compound are 14 C.

[0098] In one embodiment, the one or more nitrogen atoms of the compound are 15 N.

[0099] Use of reversing or inhibiting the aggregation of tau protein.

[0100] One aspect of the present invention is the use of thiazole compounds for reversing or inhibiting tau protein aggregation. This polymerization can be in vitro, or in vivo, and can be related to the disease state of tau protein disease discussed herein. A method for reversing or inhibiting tau protein aggregation is also provided, which comprises contacting an aggregate or protein with a compound described herein.

[0101] As described below, it has been recognized that various tauopathy disorders characterized by significant tau pathology in neurons and / or glial cells, and this term has been used in the art for several years. The similarity between these pathological inclusions and the characteristic tau inclusions in diseases such as AD shows that structural features are shared, and the topographic distribution of pathology is the reason for observing different clinical phenotypes. In addition to the specific disease discussed below, those skilled in the art can also identify tauopathy by using a suitable ligand of the visualized agglutinated tau using PET or MRI (such as those described in WO2010 / 034982) in addition to cognitive or behavioral symptoms.

[0102] Methods of treatment or prevention and first and second medical uses

[0103] One aspect of the present invention relates to a method of treating or preventing a tauopathy disorder in a patient, comprising administering to the patient a therapeutically effective amount of a thiazole-containing compound as described herein.

[0104] Aspects of the invention relate to "tauopathies." Like Alzheimer's disease (AD), the pathogenesis of neurodegenerative diseases such as Pick's disease and progressive supranuclear palsy (PSP) appears to be associated with the accumulation of pathological truncated tau aggregates in the neocortical dentate gyrus and astrocyte pyramidal cells, respectively. Other dementias include frontotemporal dementia (FTD); FTD and Parkinson's disease linked to chromosome 17 (FTDP-17); disinhibition-dementia-parkinsonism-amyotrophic complex (DDPAC); pallidum-pontine-nigral degeneration (PPND); Guam-ALS syndrome; pallidum-nigral-subthalamic degeneration (PNLD); corticobasal degeneration (CBD); dementia with argyrophilic particles (AgD); dementia pugilistica (DP), in which neurofibrillary tangles (NFTs) are similar to those observed in AD, although the topography is different

[27] ; and chronic traumatic encephalopathy (CTE), a tauopathy that includes DP as well as repetitive and sports-related concussions

[28] . Others are discussed in Wischik et al. 2000

[30] - see especially Table 5.1).

[0105] Abnormal tau in NFTs is also found in Down syndrome (DS)

[31] and dementia with Lewy bodies (DLB)

[32] . Tau-positive NFTs are also found in postencephalitic Parkinson's disease (PEP)

[33] . Glial tau tangles have been observed in subacute sclerosing panencephalitis (SSPE)

[34] .

[0106] Other tauopathies include Niemann-Pick disease type C (NPC)

[35] ; Sanfilippo syndrome type B (or mucopolysaccharidosis III B, MPS III B)

[36] ; myotonic dystrophy (DM), DM1

[37] and DM2

[38] .

[0107] In addition, there is a growing consensus in the literature that tau pathology may also contribute to cognitive deficits and decline more generally, including mild cognitive impairment (MCI) (see, e.g.,

[39] ).

[0108] All of these diseases characterized primarily or in part by abnormal tau aggregation are referred to herein as "tauopathies" or "tau aggregation diseases."

[0109] In this and all other aspects of the invention relating to tauopathy, preferably the tauopathy is selected from the list consisting of the above-mentioned indications, namely AD, Pick's disease, PSP, FTD, FTDP-17, DDPAC, PPND, Guam-ALS syndrome, PNLD, CBD, AgD, DS, SSPE, DP, PEP, SSPE, DLB, CTE and MCI.

[0110] In a preferred embodiment, the tauopathy is Alzheimer's disease (AD).

[0111] One aspect of the present invention pertains to compounds as described herein for use in a method of treatment or prevention of the human or animal body (eg, a tauopathy disorder) by therapy.

[0112] One aspect of the present invention relates to the use of a compound as described herein in the manufacture of a medicament for the treatment or prevention of a tauopathy disorder.

[0113] Another embodiment is a method of treating or preventing a tau protein aggregation disease described herein, comprising administering to a subject a compound described herein or a therapeutic composition comprising the same, so as to inhibit tau protein aggregation associated with the disease state.

[0114] Other methods and uses

[0115] In another embodiment, the compounds described herein or therapeutic compositions comprising the same are disclosed for use in a method for treating or preventing a tau protein aggregation disease as described above, the method comprising administering a thiazole-containing compound or composition to a subject so as to inhibit tau protein aggregation associated with the disease state.

[0116] In another embodiment, disclosed is the use of a compound as described herein in the preparation of a medicament for treating or preventing a tau protein aggregation disease as described above, the method comprising administering the medicament to a subject so as to inhibit tau protein aggregation associated with the disease state.

[0117] In one embodiment, a method for regulating tau protein aggregation in the brain of a mammal is disclosed, wherein the aggregation is associated with a disease state as described above, the treatment comprising the step of administering to the mammal in need of the treatment a prophylactically or therapeutically effective amount of an inhibitor of the aggregation, wherein the inhibitor is a thiazole-containing compound as described herein.

[0118] One aspect of the invention is a method of inhibiting the production of protein aggregates (eg, in the form of paired helical filaments (PHFs), optionally in the form of neurofibrillary tangles (NFTs)) in the brain of a mammal, the treatment being as described herein.

[0119] In one aspect, the present invention provides a pharmaceutical product for treating a disease state associated with tau protein aggregation in a mammal suffering from the disease, comprising a container marked or accompanied by a label indicating that the pharmaceutical product is used to treat the disease, a container containing one or more dosage units, each dosage unit comprising at least one pharmaceutically acceptable excipient and an isolated pure compound of the present invention as an active ingredient.

[0120] Composition, Formulation and Purity

[0121] In one embodiment, the compound may be provided or used in a composition that is equal to or less than 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91% or 90% pure.

[0122] One aspect of the invention pertains to a dosage unit (e.g., a pharmaceutical tablet or capsule) comprising 20 to 300 mg of a compound described herein (e.g., obtained or obtainable by the methods described herein; having a purity as described herein; etc.), and a pharmaceutically acceptable carrier, diluent, or excipient.

[0123] In one embodiment, the dosage unit is a lozenge.

[0124] In one embodiment, the dosage unit is a capsule.

[0125] Dosage units (eg, pharmaceutical tablets or capsules) containing 20 to 300 mg of a compound described herein and a pharmaceutically acceptable carrier, diluent, or excipient are discussed in more detail below.

[0126] In one embodiment, the amount is 30 to 200 mg.

[0127] In one embodiment, the amount is about 25 mg.

[0128] In one embodiment, the amount is about 35 mg.

[0129] In one embodiment, the amount is about 50 mg.

[0130] In one embodiment, the amount is about 70 mg.

[0131] In one embodiment, the amount is about 125 mg.

[0132] In one embodiment, the amount is about 175 mg.

[0133] In one embodiment, the amount is about 250 mg.

[0134] In one embodiment, the pharmaceutically acceptable carrier, diluent or excipient is or comprises a glyceride (e.g., Lauroyl Macrogol-32 Glycerides PhEur, USP) and colloidal silicon dioxide (e.g. Colloidal Silicon Dioxide PhEur, USP) one or two.

[0135] Preparation

[0136] While it is possible for a compound to be used (eg, administered) alone, it is often preferable to present it as a composition or formulation.

[0137] In one embodiment, the composition is a pharmaceutical composition (eg, formulation, preparation, medicament) comprising a thiazole-containing compound as described herein and a pharmaceutically acceptable carrier, diluent, or excipient.

[0138] In one embodiment, the composition is a pharmaceutical composition comprising at least one compound as described herein and one or more other pharmaceutically acceptable ingredients known to those skilled in the art, including but not limited to pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, colorants, flavoring agents and sweeteners.

[0139] In one embodiment, the composition further comprises other active agents, such as other therapeutic or prophylactic agents.

[0140] Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical textbooks. For example, see Handbook of Pharmaceutical Additives, 2nd edition (M. Ash and I. Ash eds.), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA), Remington's Pharmaceutical Sciences, 20th edition, pub. Lippincott, Williams & Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.

[0141] Another aspect of the present invention relates to a method for preparing a pharmaceutical composition comprising: 11 C] The radiolabeled thiazole compound as defined herein is mixed with one or more other pharmaceutically acceptable ingredients known to those skilled in the art (e.g., carriers, diluents, excipients, etc.). If formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dose) of the active compound.

[0142] As used herein, the term "pharmaceutically acceptable" refers to compounds, ingredients, materials, compositions, dosage forms, etc. that are suitable for use in contact with the tissues of relevant subjects (e.g., humans) within the scope of sound medical judgment without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0143] The formulation can be prepared by any method well known in the pharmaceutical field. Such methods include the step of associating the active compound with a carrier that constitutes one or more accessory ingredients. In general, the formulation is prepared by uniformly and intimately associating the active compound with a carrier (e.g., a liquid carrier, a finely dispersed solid carrier, etc.), and then, if necessary, shaping the product.

[0144] The formulations may be prepared to provide fast or slow release; immediate, delayed, timed or sustained release; or a combination thereof.

[0145] Formulations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). Such liquids may additionally contain other pharmaceutically acceptable ingredients such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickening agents, and solutes that render the formulation isotonic with the blood (or other relevant body fluids) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like.

[0146] Examples of suitable isotonic carriers for use in such formulations include sodium chloride injection, Ringer's solution or lactated Ringer's injection. Typically, the concentration of the active ingredient in the liquid is from about 1 ng / ml to about 10 μg / ml, for example, from about 10 ng / ml to about 1 μg / ml. The formulation can be provided in a unit dose or multiple doses in a sealed container (e.g., ampoule and vial) and can be stored under freeze drying (lyophilization) conditions that only require the addition of a sterile liquid carrier (e.g., water for injection) immediately before use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets.

[0147] dose

[0148] It will be appreciated by those skilled in the art that the appropriate dosage of the compound and the composition comprising the compound may vary between patients. Determining the optimal dosage will generally involve balancing the level of therapeutic benefit with any risk or harmful negative effects. The selected dosage level will depend on a variety of factors, including but not limited to the activity of the specific compound, the route of administration, the time of administration, the excretion rate of the compound, the duration of treatment, other drugs, compounds and / or materials used in combination, the severity of the illness, and the species, sex, age, weight, condition, general health and previous medical history of the patient. The amount of the compound and the route of administration will ultimately be at the discretion of the physician, veterinarian or clinician, but in general, the dosage will be selected to achieve local concentrations at the site of action, and these local concentrations achieve the desired effect without causing substantial harmful negative effects.

[0149] Administration can be achieved in one dose continuously or intermittently (e.g., in divided doses at appropriate intervals) during the course of treatment. Methods for determining the most effective means and dosages for administration are well known to those skilled in the art and will vary with the formulation used for therapy, the purpose of therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be performed at a dosage level and pattern selected by the attending physician, veterinarian, or clinician.

[0150] In general, suitable doses of the active compound are in the range of about 100 ng to about 25 mg (more typically, about 1 μg to about 10 mg) per kg body weight per day of the subject. In the case where the active compound is a salt, ester, amide, prodrug or its analog, the amount administered is calculated based on the parent compound, and therefore, the actual weight to be used is increased proportionally.

[0151] In one embodiment, the active compound is administered to a human patient according to the following dosage regimen: about 100 mg, 3 times a day.

[0152] In one embodiment, the active compound is administered to a human patient according to the following dosage regimen: about 150 mg, twice a day.

[0153] In one embodiment, the active compound is administered to a human patient according to the following dosage regimen: about 200 mg, twice a day.

[0154] However, in one embodiment, the compound is administered to a human patient according to the following dosage regimen: about 50 or about 75 mg, 3 or 4 times a day.

[0155] In one embodiment, the compound is administered to a human patient according to the following dosage regimen: about 100 or about 125 mg, twice a day.

[0156] Preferred combination therapy

[0157] Combination treatments and therapies are discussed in more detail below, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously. Thus, it should be understood that any medical use or method described herein can be used in combination therapy.

[0158] In one embodiment, the treatment of the invention (e.g., using a compound of the invention) is combined with a cholinesterase inhibitor such as donepezil, Aricept TM ), rivastigmine (Exelon TM ) or galantamine (Reminyl TM )combination.

[0159] In one embodiment, the treatment of the invention (e.g., using a compound of the invention) is combined with an NMDA receptor antagonist such as memantine (Ebixa TM 、Namenda TM )combination.

[0160] In one embodiment, treatments of the invention (eg, employing a compound of the invention) are combined with a muscarinic receptor agonist.

[0161] In one embodiment, treatments of the invention (eg, using a compound of the invention) are combined with an inhibitor of amyloid precursor protein to β-amyloid (eg, an inhibitor of amyloid precursor protein processing that results in increased β-amyloid production).

[0162] Ligands and Labels

[0163] The thiazole compounds discussed herein that can inhibit tau protein aggregation can also serve as ligands or labels for tau protein (or aggregated tau protein). Therefore, in one embodiment, the thiazole compound is a ligand for tau protein (or aggregated tau protein).

[0164] Such thiazole-containing compounds (ligands) may incorporate, bind, chelate or otherwise associate with other chemical groups, such as stable and unstable detectable isotopes, radioisotopes, positron emitting atoms, magnetic resonance labels, dyes, fluorescent markers, antigenic groups, therapeutic moieties or any other moiety that may aid in prognostic, diagnostic or therapeutic applications.

[0165] For example, as described above, in one embodiment, the compound is as defined above, but with the additional limitation that the compound incorporates, binds, chelates or otherwise associates with one or more (e.g., 1, 2, 3, 4, etc.) isotopes, radioisotopes, positron emitting atoms, magnetic resonance labels, dyes, fluorescent markers, antigenic groups or therapeutic moieties.

[0166] In one embodiment, the compound is a ligand and a label, such as a label for tau protein (or aggregated tau protein), and is incorporated, bound, chelated or otherwise associated with one or more (e.g., 1, 2, 3, 4, etc.) detectable labels.

[0167] For example, in one embodiment, the compound is as defined above, with the additional limitation that the compound incorporates, binds, chelates or otherwise associates with one or more (eg, 1, 2, 3, 4, etc.) detectable labels.

[0168] Labeled compounds (eg, when attached to tau protein or aggregated tau protein) can be visualized or detected by any suitable means, and those skilled in the art will appreciate that any suitable detection means known in the art may be used.

[0169] For example, thiazole-containing compounds (ligand-labeled) can be synthesized by incorporating a positron-emitting atom (e.g. 11 C) (e.g., a carbon atom that is one or more alkyl substituents, such as methyl substituents) and detecting the compound using positron emission tomography (PET) as is known in the art.

[0170] treat

[0171] As used herein in the context of treating a condition, the term "treat" generally refers to treatment and therapy, whether human or animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved (e.g., inhibition of progression of the condition), and includes a reduction in the rate of progression, a halt in the rate of progression, regression of the condition, amelioration of the condition, and cure of the condition. Treatment as a preventive measure (i.e., prophylaxis / prevention) is also included.

[0172] As used herein, the term "therapeutically effective amount" refers to that amount of an active compound, or a material, composition, or dosage form comprising an active compound, which, when administered according to a desired treatment regimen, is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit / risk ratio.

[0173] Likewise, as used herein, the term "prophylactically effective amount" refers to that amount of an active compound, or a material, composition, or dosage form comprising an active compound, which, when administered according to the desired treatment regimen, is effective for producing some desired prophylactic effect, commensurate with a reasonable benefit / risk ratio.

[0174] The term "treatment" includes combination therapy and therapy, wherein, for example, two or more treatments or therapies are combined sequentially or simultaneously. Examples of treatment and therapy include, but are not limited to, chemotherapy (administration of an active agent, including, for example, a drug, an antibody (e.g., as in immunotherapy), a prodrug (e.g., as in photodynamic therapy, gene-directed enzyme prodrug therapy (GDEPT), antibody-directed enzyme prodrug therapy (ADEPT), etc.); surgery; radiotherapy; and gene therapy.

[0175] Route of administration

[0176] The thiazole compound or pharmaceutical composition comprising the same may be administered to the subject / patient by any convenient route of administration, whether systemically / peripherally or locally (ie, at the site of desired action).

[0177] Routes of administration include, but are not limited to, oral (e.g., by ingestion); buccal; sublingual; transdermal (including, e.g., by patches, plasters, etc.); transmucosal (including, e.g., by patches, plasters, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eye drops); pulmonary (e.g., by inhalation or insufflation therapy, using, e.g., an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, e.g., by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcutaneous, intraarticular, subarachnoid, and intrasternal (including, e.g., intracatheter injection into the brain); by implantation of a reservoir or depot, e.g., subcutaneous or intramuscular.

[0178] Subjects / Patients

[0179] The subject / patient can be an animal, a mammal, a placental mammal, a marsupial (e.g., a kangaroo, a wombat), a monotreme (e.g., a platypus), a rodent (e.g., a guinea pig, a hamster, a rat, a mouse), a murine (e.g., a mouse), a lagomorph (e.g., a rabbit), an avian (e.g., a bird), a canine (e.g., a dog), a feline (e.g., a cat), an equine (e.g., a horse), a porcine (e.g., a pig), an ovine (e.g., a sheep), a bovine (e.g., a cow), a primate, a simian (e.g., a monkey or ape), a monkey (e.g., a marmoset, a baboon), an ape (e.g., a gorilla, a chimpanzee, an orangutan, a gibbon), or a human.

[0180] Furthermore, the subject / patient may be in any of its developmental forms, such as a fetus.

[0181] In a preferred embodiment, the subject / patient is a human.

[0182] Selection of subjects for this approach can be based on known factors. Thus, initial selection of patients may involve one or more of the following: rigorous evaluation by an experienced clinician; exclusion of non-AD diagnoses as much as possible through supplemental laboratory and other testing; and objective assessment of cognitive function level using a neuropathologically validated test battery.

[0183] In one embodiment, the subject / patient is not a human.

[0184] Synthesis method

[0185] Methods for chemically synthesizing the compounds of the invention are described in the Examples herein. These and / or other well-known methods may be modified and / or adapted in a known manner to facilitate the synthesis of other compounds of the invention.

[0186] Thus, in one aspect the present invention provides a method of synthesizing a compound of the present invention as described herein, as described with reference to any of the Examples hereinafter or substantially as described.

[0187] The present invention further provides a compound of the present invention obtained or obtainable by a process as described herein.

[0188] One aspect of the present invention relates to a process for preparing a thiazole-containing TAI compound as described herein. The present invention also provides intermediate compounds useful in preparing the compounds of the present invention.

[0189] General Synthesis Methods

[0190] Compounds of formula (I) can be prepared, for example, by the routes outlined in the following general schemes:

[0191]

[0192] In this scheme and all subsequent schemes in this section, substituents may be generally represented by "R". It is understood that these substituents may independently correspond to the group R N , R X and R Y , or they may represent protected forms thereof or precursor groups thereof, depending on the overall synthetic scheme.

[0193] For example, approach (i) may include reacting a sulfonamide thiazole acid with an appropriate alkylamine in an amide formation step. For example, the acid may be treated with a base such as diisopropylethylamine DIPEA, a coupling agent such as HATU, and an amine. Preferably, the reaction is carried out in a polar aprotic solvent such as DMF or MeCN.

[0194] For example, route (ii) may comprise reacting the amide thiazole amine with a suitable sulfonyl chloride, for example in the presence of a base such as triethylamine or DIPEA. Preferably, the reaction is carried out in a polar aprotic solvent such as THF.

[0195] The starting materials for synthesizing the compounds of the invention can be prepared via known routes and / or can be commercially available. Methods for synthesizing exemplary compounds are described in the Examples herein. These and / or other known methods can be modified and / or adjusted in a known manner to facilitate the synthesis of other compounds of the invention.

[0196] ***

[0197] The features disclosed in the preceding description or in the following claims and described in the drawings in a specific form or, where appropriate, in terms of a method or process for performing a disclosed function or for obtaining a disclosed result may be used separately or in any combination of such features to implement the various forms of the invention.

[0198] Although the present invention has been described in conjunction with the above exemplary embodiments, many equivalent modifications and variations will be apparent to those skilled in the art when this disclosure is given. Therefore, the above exemplary embodiments of the present invention are considered to be illustrative and non-limiting. Changes may be made to the embodiments without departing from the spirit and scope of the present invention.

[0199] To avoid any doubt, any theoretical explanations provided herein are provided to enhance the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.

[0200] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0201] Unless the context requires otherwise, throughout this specification (including the accompanying claims), the words "comprise" and "include" and variations such as "comprises", "comprising" and "including" will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0202] It must be noted that the singular forms "a", "an" and "the" used in the specification and the appended claims include plural references unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when a value is expressed as an approximation by using the antecedent "about", it will be understood that the particular value forms another embodiment. The term "about" with respect to a value is optional and means, for example, + / - 10%.

[0203] Example

[0204] Example 1

[0205] Modeling of the PHF core oligomer-captured monomer; immunochemical analysis of assembly; characterization of the HMT binding pocket

[0206] As described in detail in WO2022 / 008545, molecular dynamics simulations were performed to explore the conformational landscape of dGAE monomers. The inventors further developed a set of single-chain monoclonal antibody fragments (scAbs) that recognize linear epitopes spanning most of the core tau unit and used them to study epitope availability in dot immunoblots during dGAE in vitro assembly [6]. It was hypothesized that the inhibitory effect of HMT on dGAE assembly could be explained by stabilization of the transiently cryptic ligand binding pocket by a ligand-induced conformational constriction

[40] , and ultimately a single complex was identified in which the HMT remained tightly bound and the protein structure did not change much after 100 ns of MD simulation time. Therefore, the HMT-bound complex represents an energy well in which the conformation is maintained for a relatively long time. The conformation of the tau297-391 core unit stabilized by HMT is a compact folded state that lacks any β-pleated sheet and is extremely different from the extended conformation required for the monomer to align onto the PHF core template.

[0207] Pharmacophore modeling of cryptic druggable pockets and its use for identification of novel inhibitors

[0208] The site to which HMT binds has been determined to be 70% druggable

[41] , i.e., has a favorable ratio of hydrophobic solvent accessible surface area. As also disclosed in WO2022 / 008545, the inventors have developed a pharmacophore model based on key residues that can be used to assist in the rational design of TAIs. The HMT binding pocket is primarily hydrophobic in nature (Phe378, Phe346, Val350, Leu315, Ile354, Ile371). Many residues are likely to form hydrogen bonds with molecules bound to this pocket, including the NH of Lys347, Thr373, Leu315, and Glu372. The inventors used the shape, hydrogen bonding, and lipophilic features observed under HMT to design alternative TAIs in the pharmacophore model.

[0209] Using computer-aided drug design methods (see below), the inventors identified the thiazole core as a suitable heterocyclic replacement for the central ring of the HMT. A series of representative candidates were synthesized and tested in a cell-based tau aggregation screening assay (see below and Figure 1 ), which measures the ability of compounds to inhibit the capture of full-length tau by dGAE and its templated truncations by endogenous proteases

[13] . In this model, MT at 1 μM reduced aggregation to 9.6% of that measured in its absence. Of the compounds synthesized and tested, compound 9 reduced tau aggregation to 26.6% of that measured in the absence of compound. A single experiment achieved 50% inhibition (EC 50 ) The required concentrations were 0.6 μM for MT and 3.65 μM for compound 9 ( Figure 2 ). The data of repeated experiments are listed in Example 3 below.

[0210] Comparison of the inhibitory activity of the tested thiazole derivatives shows that the interaction with Lys343 (via hydrogen bonding and π-cation interaction pairs) is important in determining compound potency. The orientation of the sulfonamide oxygen atom is key to obtaining the required hydrogen bond with the main chain carbonyl of Lys343. When a methyl group is substituted onto amide 7 to produce amide 8, the potency changes. This is explained by the reorientation of the thiazole amide, which forms a key H-bond with the main chain NH of Lys347 in compound 2 compared to compound 1, where this interaction is disturbed by the new interaction of the pyrrole NH with Thr373. The small alkyl substituent appears to be sufficient to bind to the lipophilic pocket of Phe378. The sulfonamide affects the ability of the amide moiety to form hydrogen bonds with the NH of Thr373. Both amide substituents 9 and 11 have the ability to form additional hydrogen bonds with proteins, either the main chain carbonyl of Asp345 with 9 or Leu315 with 11. Compound 9 satisfies many of the desired binding features, including a sulfonamide oxygen that forms a hydrogen bond with the backbone NH of Glu372, an amide carbonyl that forms a hydrogen bond with the backbone NH of Lys347, and a pyrazole that forms hydrogen bonds with the NH of Thr373 and the backbone carbonyl of Leu315. In addition, the phenyl substituent on the pyrazole fits neatly into the lipophilic pocket, forms a face-edge π stack with Phe378, and is well positioned to interact with Lys343 via a π-cation interaction. Thus, a pharmacophore model developed based on HMT binding can identify compounds that are chemically unrelated to HMTs that also have the ability to inhibit tau aggregation.

[0211] Immunochemical confirmation of TAI activity

[0212] As a separate confirmation of activity, an aqueous ELISA assay was used to measure TAI activity in dGAE preparations (see Example 3 below). Figure 3 and Figure 4 (center) was measured before and after dGAE assembly in the presence or absence of HMT and compound 9. Immunoreactivity over a range of dGAE dilutions demonstrated that all epitopes in the soluble preassembled dGAE preparation were available for antibody binding ( Figure 5 In the assembled dGAE preparation, the CA4 and CE2 epitopes were almost completely unavailable, while the availability of the 1G2 and 1D12 epitopes was greatly reduced, which is consistent with Figure 6The immunoblot analysis shown in is consistent. When dGAE assembly was performed in the presence of HMT, assembly-dependent closure of these epitopes did not occur. The CE2 epitope became more accessible in the HMT-treated sample than in the soluble pre-assembled sample, suggesting that partial oligomerization in the solution before inducing assembly was reversed. The 1G2 epitope is still partially blocked in the HMT assembly preparation, consistent with Thr373 (within the 1G2 epitope), which is a key HMT binding site. The epitope exposure of compound 9 is less complete. Although comparable to HMT as measured by 1G2 immunoreactivity, the CA4 and 1D12 epitopes are only partially exposed. Although HMT binding prevents conformational changes in the CE2 epitope that lead to early loss of immunoreactivity, this is not the case with compound 9, a feature that may be the reason why the compound is less effective in cell assays.

[0213] Computer-aided drug design (CADD)

[0214] Energy minimization and 50-ns molecular dynamics simulations were performed on 30 replicas of the unliganded single-stranded filament ( Figure 7 ). Perform principal component analysis along the simulation trajectory of the identified clusters. Take representative sample protein conformations from each cluster and determine the RMSD between snapshots. Docking and identification using 380 different protein structures to obtain 15,090 ligand poses. Score the poses and find 178 binding poses for 65 binding sites. Run a short minimization followed by a 1 ns production round on each system. Recalculate ligand binding energies. Select the ligand with the highest score and extend the simulation time and repeat this process multiple times. Process the trajectory of interest for analysis.

[0215] Example 2 - Chemical Synthesis

[0216] Unless otherwise stated, all reactions were performed under a positive pressure of argon. Unless otherwise stated, glassware was oven dried at 120°C or flame dried under vacuum. Anhydrous dichloromethane (DCM), acetonitrile (MeCN), N,N-dimethylformamide (DMF) and pyridine were purchased from Sigma-Aldrich. Anhydrous tetrahydrofuran (THF) was purchased from Acros Organics or Sigma-Aldrich. Unless otherwise stated, all commercially available solvents or reagents were used without further purification. The ambient temperature stated was 20-21°C. Reactions were monitored by thin layer chromatography (TLC) using pre-coated silica gel plates from EMD Millipore (TLC Silica gel 60F254). Flash column chromatography was performed on silica gel 60 (particle size 0.04-0.063 mm) from Fluorochem or using HPLC-MS / MS from The pre-packed column is The results were performed on a Selekt instrument.1 H NMR and 13 C NMR spectra were recorded on Bruker Avance II 400 MHz and 600 MHz spectrometers at 298 K and the residual solvent peak was used as internal standard: CDCl3, 7.26 ppm ( 1 H) and 77.16ppm( 13 C); CD3OD, 3.31ppm( 1 H) and 49.00ppm( 13 C); DMSO-d6, 2.50ppm ( 1 H) and 39.52ppm( 13 C) NMR data are presented in terms of apparent multiplicity: m = multiplet, s = singlet, d = doublet, t = triplet and combinations thereof; br indicates broad, poorly resolved signals. The IR spectra were recorded on a PerkinElmer FT-IR Spectrum Two spectrometer equipped with an ATR Diamond cell. The melting points were obtained using Melting point data were obtained with a SMP50 instrument and are uncorrected.

[0217] Synthesis of tert-butyl 4-(3,3-dimethylbutylcarbamoyl)thiazol-2-ylcarbamate

[0218]

[0219] To a stirred suspension of 2-Boc-aminothiazole-4-carboxylic acid (253 mg, 1.04 mmol) in anhydrous MeCN (20 mL) was added DIPEA (269 mg, 362 μL, 2.08 mmol) under an inert atmosphere. Once the solid was completely dissolved, HATU (407 mg, 1.07 mmol) was added followed by 3,3-dimethylbutylamine (108 mg, 144 μL, 1.07 mmol). The reaction mixture was heated at 50 °C for 23 h. Water (40 mL) was added and the resulting suspension was cooled to ambient temperature. More water (20 mL) was added and the suspension was filtered under vacuum. The collected solid was washed with water (3 x 10 mL) and dried in a vacuum oven at 40 °C for 2 h; the title compound (246 mg, 73%) was obtained as an off-white solid.

[0220] 1H (400MHz, CDCl3) δ7.73 (br s, 1H), 7.67 (s, 1H), 6.98 (br s, 1H), 3.45-3.39 (m, 2H), 1.55 (s, 11H, including H2O signal), 1.53-1.49 (m, 2H), 0.96 (s, 9H);

[0221] 13 C(101MHz, CDCl3)δ161.18,158.86,151.96,145.06,117.18,83.43,43.45,36.12,30.13,29.57,28.29

[0222] Synthesis of 2-amino-N-(3,3-dimethylbutyl)thiazole-4-carboxamide

[0223]

[0224] To a stirred solution of tert-butyl 4-(3,3-dimethylbutylcarbamoyl)thiazol-2-ylcarbamate (233 mg, 0.712 mmol) in DCM (6 mL) was added TFA (2 mL) dropwise under an inert atmosphere. The reaction mixture was stirred at 20 °C for 4 h and then concentrated to leave an orange residue which was dissolved in EtOAc (20 mL). The resulting solution was washed with saturated aqueous NaHCO3 (10 mL) and brine (10 mL). The organic phase was dried (MgSO4), filtered and evaporated to leave a viscous orange oil which was purified by flash chromatography (SiO2, EtOAc: petroleum ether 40 / 60, 7:3); the title compound (171 mg) was obtained as a yellow oil. The material was used without further purification.

[0225] 1 H(400MHz, CDCI3)δ7.33(s,1H),7.01(br s,1H),4.98(br s,2H),3.42-3.37(m,2H),1.52-1.48(m,2H),0.95(s,9H);

[0226] 13 C(101MHz,CDCI3)δ168.93,159.42,141.20,112.81,42.86,36.70,30.02,29.44

[0227] Synthesis of methyl 2-(N-(4-(3,3-dimethylbutylcarbamoyl)thiazol-2-yl)sulfamoyl)acetate

[0228]

[0229] To a stirred solution of 2-amino-N-(3,3-dimethylbutyl)thiazole-4-carboxamide (123 mg, 0.541 mmol) in DCM (5 mL) under an inert atmosphere was added pyridine (65 mg, 66 μL, 0.816 mmol) and DMAP (14 mg, 0.115 mmol). Methyl 2-(chlorosulfonyl)acetate (98 mg, 66 μL, 0.568 mmol) was added dropwise and the resulting solution was stirred at 20° C. for 43 h. The reaction mixture was diluted with DCM (5 mL) and washed with 0.5 M aqueous HCl (10 mL), water (10 mL) and brine (10 mL). The brine was extracted with EtOAc (10 mL) and the organic extracts were combined, dried (MgSO ), filtered and evaporated to leave a yellow and white solid. The crude product was purified by flash chromatography (SiO2, DCM:MeOH, 19:1 then 9:1), and the isolated material was further purified by flash chromatography (SiO2, EtOAc:MeOH, 98:2 then 9:1); the title compound was obtained as a slightly impure pink-orange foam (112 mg). This material was used without further purification.

[0230] 1 H (400 MHz, CD3OD) δ7.41 (s, 1H), 4.16 (s, 2H), 3.69 (s, 3H), 3.37-3.33 (m, 3H, including MeOH), 1.51-1.47 (m, 2H), 0.94 (s, 9H)

[0231] Synthesis of N-(3,3-dimethylbutyl)-2-(2-hydroxyethylsulfonylamino)thiazole-4-carboxamide

[0232]

[0233] Under an inert atmosphere, sodium borohydride (43 mg, 1.14 mmol) was added to a crude sample of methyl 2-(N-(4-(3,3-dimethylbutylcarbamoyl)thiazol-2-yl)sulfamoyl)acetate in anhydrous THF (5 mL) under an inert atmosphere. The resulting suspension was stirred at 20 ° C for 27 h and quenched with saturated NH4Cl aqueous solution (20 mL). The mixture was extracted with EtOAc (2x 20 mL). The aqueous layer was acidified with 3 drops of 32% HCl aqueous solution, saturated with NaCl, and extracted with DCM (3x15 mL). The DCM was dried (MgSO4), filtered and evaporated to leave a white solid. The crude product was dissolved in DCM (15 mL) and extracted with a 3:1 v / v mixture (20 mL) of water and saturated NaHCO3 aqueous solution. The aqueous layer was washed with DCM (15 mL) and then acidified to pH≈1 with 1M HCl aqueous solution. The acidic solution was extracted with DCM (2 x 15 mL) and the two combined DCM extracts were washed with brine (10 mL), dried (MgSO4), filtered and evaporated to leave a white solid. After drying in a vacuum oven at 40°C for 1.5 h, the title compound was obtained as a white solid (38 mg, 40%, 23% over 2 steps).

[0234] I U max (cm -1 )3480,3313,3095,2960,1645,1544,1267,1239,1116,1041,896,659,553,535;

[0235] 1 H(400MHz,DMSO-d6)δ12.71(br s,1H),8.43(br s,1H),7.44(s,1H),4.78(br s,1H),3.73(t,J=6.7Hz,2H),3.24-3.19(m,4H),1.43-1.39(m 2H),0.91(s,9H);

[0236] 13 C(101MHz,DMSO-d6)δ166.90(br),157.34,132.98(br),111.50(br),55.83,55.41,42.51,35.59,29.65,29.22;

[0237] HRMS:m / z(ESI)C 12 H 21 Calculated value of N3NaO4S2: 358.0871 (M+Na) +; Measured value: 358.0875 Synthesis of tert-butyl (4-(((1-methyl-1H-pyrrol-2-yl)methyl)carbamoyl)thiazol-2-yl)carbamate

[0238]

[0239] To a stirred solution of 2-Boc-aminothiazole-4-carboxylic acid (526 mg, 2.05 mmol), DIPEA (530 mg, 714 μL, 4.10 mmol) and HATU (779 mg, 2.05 mmol) in a mixture of anhydrous MeCN (15 mL) and DMF (10 mL) under inert atmosphere was added 1-(1-methyl-1H-pyrrol-2-yl)methanamine (226 mg, 2.05 mmol) and the reaction mixture was stirred for 15 h at 20° C. The solvent was evaporated and the residue was dissolved in EtOAc (40 mL) and washed with water (15 mL) and brine (15 mL). The aqueous washings were extracted with EtOAc (2 x 20 mL) and the combined organic extracts were dried (MgSO4), filtered and evaporated to give a solid which was purified by flash chromatography (SiO2, hexanes / EtOAc, 1:1); the title compound was obtained as a cream solid after drying under high vacuum at 20°C for 3.5 h (561 mg, 81%).

[0240] 1 H(400MHz,CDCl3)δ7.73(br s,1H),7.72(s,1H),7.10-7.19(m,1H),6.63(s,1H),6.14(s,1H),6.09(s,1H),4.59(d,J=5.3Hz,2H),3.60(s,3H),1.55(s,9H);

[0241] 13 C(101MHz, CDCI3)δ160.55,158.76,151.84,144.41,128.48,123.10,117.50,108.90,106.95,83.29,34.92,33.80,28.11

[0242] Synthesis of 2-amino-N-((1-methyl-1H-pyrrol-2-yl)methyl)thiazole-4-carboxamide

[0243]

[0244] A solution of tert-butyl (4-(((1-methyl-1H-pyrrol-2-yl)methyl)carbamoyl)thiazol-2-yl)carbamate (265 mg, 0.788 mmol) in 2,2,2-trifluoroethanol (3.5 mL) was placed in a sealed microwave vial and heated to 150° C. with stirring (1000 rpm) for 1.5 h. The solvent was removed by evaporation and the residue was purified by flash chromatography (SiO 2 , hexanes / EtOAc, 1 :1) to give the title compound (135 mg, 73%) as a white solid after drying at 60° C. for 19 h.

[0245] 1 H(400MHz, CDCl3)δ7,39(s,1H),7,15(br s,1H),6,62(s,1H),6,13(s,1H),6,08(s,1H),4,88(br s,2H),4.57(d,J=5.6Hz,2H),3.60(s,3H);

[0246] 13 C(101MHz, CDCl3)δ166.71,160.62,145.50,128.48,123.07,113.85,108.94,106.90,34.91,33.81

[0247] Synthesis of methyl (4-(((1-methyl-1H-pyrrol-2-yl)methyl)carbamoyl)thiazol-2-yl)sulfonyl)acetate

[0248]

[0249] To a stirred solution of 2-amino-N-((1-methyl-1H-pyrrol-2-yl)methyl)thiazole-4-carboxamide (55 mg, 0.233 mmol) and pyridine (28 mg, 28 uL, 0.349 mmol) in anhydrous DCM (5 mL) was added dropwise a solution of methyl (chlorosulfonyl)acetate (42 mg, 0.245 mmol) in DCM (150 μL,) under an inert atmosphere and the resulting solution was stirred at 21 °C for 17 h. The reaction mixture was diluted with DCM (45 mL), washed with 1M aqueous HCl (10 mL), brine (15 mL) and dried (MgSO4). After filtration, the solvent was removed by evaporation to give the title compound (74 mg, 85%) as a tan solid after drying in a vacuum oven at 40 °C for 6.5 h. The material was used without any further purification.

[0250] 1H(400MHz,DMSO-d6)δ13,09(br s,1H),8,80(br s,1H),7,58(s,1H),6,68(s,1H),5,98(s,1H),5,90(s,1H),4.38(d,J=2.7Hz,2H),4.15(s,2H),3.60(s,3H),3.55(s,3H);

[0251] 13 C(101MHz,DMSO-d6)δ168.74,164,03,156.85,132.27,128.35,122.,57,112.42,108.50,106.24,57.35,52.41,34.64,33.37

[0252] Synthesis of 2-(2-hydroxyethylsulfonylamino)-N-((1-methyl-1H-pyrrol-2-yl)methyl)thiazole-4-carboxamide

[0253]

[0254] To a stirred solution of methyl (4-(((1-methyl-1H-pyrrol-2-yl)methyl)carbamoyl)thiazol-2-yl)sulfonyl)acetate (74 mg, 0.198 mmol) in anhydrous THF (5 ml) was added sodium borohydride (30 mg, 0.792 mmol) and the resulting solution was stirred at 20 °C for 18 h under an inert atmosphere. The solvent was evaporated and the residue was dissolved in water (7 mL), acidified with 1 M aqueous HCl (2 mL), saturated with NaCl, and extracted with EtOAc (5 x 10 mL). The combined organic extracts were washed with brine (15 mL), dried (MgSO4), filtered and evaporated to give a solid which was dissolved in saturated aqueous NaHCO3 (25 mL) and extracted with EtOAc (3 x 10 mL). The aqueous phase was acidified by dropwise addition of 32% aqueous HCl, saturated with NaCl, and extracted with EtOAc (4 x 25 mL). The combined organic extracts were washed with brine (15 mL), dried (MgSO 4 ), filtered and evaporated to give a solid which was purified by flash chromatography (SiO 2 , EtOAc / MeOH, 10:1) to afford the title compound (26 mg, 38%) as a white solid after drying at 60° C. for 3 h.

[0255] I U max (cm -1 ):3315,3097,2924,1627,1554,1470,1229,1102,713,504;

[0256] 1H (400 MHz, CD3OD) δ7,42 (s, 1H), 6,56-6,61 (m, 1H), 6,03 (br s, 1H), 5,91-5,96 (m, 1H), 4,49 (s, 2H), 3.94 (t, J = 6.3 Hz, 2H), 3.55 (s, 3H), 3.30 (m, 2H) (the signal partially overlaps the CD3OD signal);

[0257] 13 C(101MHz,CD3OD)δ173.93,164.50,145.35,129.62,123.93,113.63,110.09,107.80,58.13,55.83,36.25,34.14;

[0258] HRMS:m / z(ESI)C 12 H 16 N4NaO4S2,(M+Na) + Calculated value: 367.0511; Measured value: 367.0522 Synthesis of sulfonamide thiazole ester:

[0259] 2-(2-methoxy-2-oxoethylsulfonylamino)thiazole-4-carboxylic acid methyl ester

[0260]

[0261] To a stirred suspension of methyl 2-aminothiazole-4-carboxylate (525 mg, 3.15 mmol), pyridine (373 mg, 380 μL, 4.72 mmol) and DMAP (38 mg, 0.315 mmol) in anhydrous DCM (25 mL) was added methyl (chlorosulfonyl) acetate (571 mg, 3.31 mmol) dropwise and the resulting solution was stirred at 20 °C for 17 h. The reaction mixture was diluted with DCM (25 mL) and washed with 1 M aqueous HCl (40 mL). The aqueous phase was extracted with DCM (3 x 50 mL) and the combined organic extracts were washed with brine (40 mL), dried (MgSO4), filtered and evaporated to give the title compound (920 mg, 99%) as a pale yellow solid. The material was used without further purification.

[0262] 1 H(400MHz,DMSO-d6)δ13.52(br s,1H),7.76(s,1H),4.20(s,2H),3.82(s,3H),3.61(s,3H);

[0263] 13C(101MHz,DMSO-d6)δ168.89,163.95,157.88,128.49,118.79,57.40,52.69,52.44

[0264] 2-(2-Hydroxyethylsulfonylamino)thiazole-4-carboxylic acid methyl ester

[0265]

[0266] To a stirred solution of 2-(2-methoxy-2-oxoethylsulfonylamino)thiazole-4-carboxylate (750 mg, 2.55 mmol) in a mixture of THF (55 mL) and MeOH (6 mL) was added portionwise NaBH4 (578 mg, 15.29 mmol) over 1.5 h, and the reaction mixture was stirred at 20 °C for 15 h. Thereafter, NaBH4 (49 mg, 1.30 mmol) was added and stirring was continued at ambient temperature for 24 h. The reaction mixture was cooled to 0-5 °C, brine (25 mL) was added, followed by 1 M aqueous HCl solution (22 mL). The organic phase was separated, and the aqueous layer was saturated with NaCl and extracted with THF (5 x 35 mL). The combined organic extracts were dried (MgSO4), filtered and evaporated to give a residue which was purified by flash chromatography (SiO2, DCM / MeOH, 20:1) to afford the title compound (403 mg, 59%) as a white solid after drying under high vacuum at 20°C for 7 h.

[0267] 1 H(400MHz,DMSO-d6)δ13.09(br s,1H),7.74(s,1H),4.81(br s,1H),3.81(s,3H),3.74(t,J=6.6Hz,2H),3.23(m,2H);

[0268] 13 C(101MHz,DMSO-d6)δ166.80,158.34,130.04,118.82,55.72,55.45,52.53

[0269] 2-(Methylsulfonylamino)thiazole-4-carboxylic acid methyl ester

[0270]

[0271] To a stirred solution of methyl 2-aminothiazole-4-carboxylate (801 mg, 5.06 mmol) in anhydrous pyridine (8 mL) was added methanesulfonyl chloride (591 mg, 400 μL, 5.16 mmol) under an inert atmosphere. The reaction mixture was stirred at 20 °C for 20 h and then diluted with EtOAc (40 mL). The suspension was filtered and the filtrate was washed with 1 M aqueous HCl (3 x 40 mL), water (30 mL) and brine (20 mL). The organic phase was dried (MgSO44), filtered and evaporated to leave methyl 2-(bis(methylsulfonyl)amino)thiazole-4-carboxylate (190 mg, 23%) as an orange solid.

[0272] The aqueous acid was extracted with DCM (70 mL), and the DCM was washed with water (30 mL) and brine (20 mL), then dried (MgSO4), filtered and evaporated to leave an orange solid. The combined aqueous extracts were saturated with NaCl and extracted with DCM (3 x 30 mL). The combined DCM extracts were washed with brine (20 mL), dried (MgSO4), filtered and evaporated to leave a white solid. The two solids obtained from the DCM extractions were combined and purified using Biotage Selekt was purified by flash chromatography (SiO2, 5-19% MeOH in DCM); the title compound was obtained as an off-white solid (586 mg, 49%).

[0273] 2-(Bis(methylsulfonyl)amino)thiazole-4-carboxylic acid methyl ester: 1 H(400MHz,DMSO-d6)δ8.74(s,1H),3.86(s,3H),3.69(s,6H);

[0274] 13 C NMR(101MHz,DMSO-d6)δ160.45,154.18,143.28,134.21,52.43,43.11

[0275] 2-(Methylsulfonylamino)thiazole-4-carboxylic acid methyl ester: 1H (400 MHz, CDCl3) δ 7.31 (s, 1H), 3.92 (s, 3H), 3.06 (s, 3H);

[0276] 13 C(101MHz,CDCh)δ166.71,158.04,128.58,116.55,53.25,41.67

[0277] 2-(Fluoromethylsulfonylamino)thiazole-4-carboxylic acid methyl ester

[0278]

[0279] To a stirred solution of 2-Boc-aminothiazole-4-carboxylic acid methyl ester (762 mg, 2.95 mmol) in anhydrous THF (24 ml) was added sodium hydride (60% dispersion in mineral oil, 161 mg, 4.01 mmol) under an inert atmosphere. After bubbling stopped, fluoromethanesulfonyl chloride (351 mg, 2.65 mmol) was added dropwise over 5 minutes, and the reaction mixture was stirred at 20 ° C for 70 h. The solvent was evaporated and the residue was dissolved in DCM (60 mL). The reaction mixture was cooled to 0-5 ° C, TFA (8 mL) was added dropwise, and the resulting solution was stirred at ambient temperature for 22 h. The solvent was removed by evaporation and a saturated NaHCO3 aqueous solution (50 mL) was added to the residue. The mixture was ultrasonicated for 5 min, vacuum filtered, and the filtrate was extracted with DCM (10 × 50 mL). The aqueous phase was acidified by dropwise addition of 32% HCl aqueous solution and extracted with EtOAc (4 × 40 mL). The combined organic extracts were washed with brine (40 mL), dried (MgSO4), filtered and evaporated to give the title compound (365 mg, 54%) as a white solid after drying in an oven at 60°C for 2 h.

[0280] 1 H(400MHz,DMSO-d6)δ13.74(br s,1H),7.78(s,1H),5.33(d,J=46Hz,2H),3.83(s,3H);

[0281] 13 C (101MHz, DMSO-d6) δ 169.70, 157.76, 128.38, 118.96, 90.73 (d, J = 208Hz), 52.70Synthesis of 2-(methylsulfamoyl)aminothiazole-4-carboxylic acid methyl ester

[0282]

[0283] To a stirred solution of methyl 2-aminothiazole-4-carboxylate (499 mg, 3.15 mmol) in anhydrous THF (40 mL) was added DIPEA (449 mg, 605 μL, 3.47 mmol) under an inert atmosphere. N-methylsulfamoyl chloride (446 mg, 302 μL, 3.44 mmol) was added dropwise and the resulting solution was heated at 50 ° C for 116 h. After cooling to ambient temperature, THF was evaporated and the residue was distributed between EtOAc (20 mL) and saturated NaHCO3 aqueous solution (20 mL). After phase separation, the organic phase was extracted with saturated NaHCO3 aqueous solution (10 mL) and the combined aqueous phase was washed with EtOAc (20 mL). The aqueous portion was acidified to pH ≈ 1 with 2M HCl aqueous solution, extracted with DCM (3×20 mL), saturated with NaCl, and extracted with THF (3×20 mL). The combined organic extracts were dried (MgSO4), filtered and evaporated to leave a viscous orange oil. The crude material was purified by flash chromatography (SiO2, DCM:MeOH, 94:6) and the title compound (551 mg, 70%) was isolated as a viscous light yellow oil.

[0284] 1 H(400MHz,CD3OD)δ7.64(s,1H),3.89(s,3H),2.62(s,3H);

[0285] 13 C(101MHz,CD3OD)δ167.27,160.63,133.61,119.64,53.09,29.47

[0286] Synthesis of 2-(1H-indole-3-sulfonylamino)thiazole-4-carboxylic acid methyl ester

[0287]

[0288] To a stirred solution of 1H-indole-3-sulfonyl chloride (471 mg, 2.18 mmol) in anhydrous THF (20 mL) was added 2-amino-1,3-thiazole-4-carboxylic acid methyl ester (693 mg, 4.38 mmol) under an inert atmosphere. DIPEA (312 mg, 420 μL, 2.41 mmol) was added and the resulting solution was heated at 50 ° C for 116 h. After cooling to ambient temperature, THF was evaporated and the residue was distributed between EtOAc (40 mL) and saturated NaHCO3 aqueous solution (20 mL). After phase separation, the organic phase was extracted with saturated NaHCO3 aqueous solution (20 mL), and the combined aqueous extracts were washed with EtOAc (20 mL).

[0289] The aqueous phase was acidified to pH ≈ 1 with 2M aqueous HCl, and a sticky orange solid appeared, sticking to the glass container. The mixture was extracted with DCM (20 mL), which did not dissolve the sticky solid. Nevertheless, the DCM was dried (MgSO4), filtered and evaporated to leave a viscous yellow oil. This oil was dissolved in a mixture of DCM and MeOH and concentrated to leave the title compound (265 mg, 36%) as a light yellow foamy solid.

[0290] The aqueous phase was saturated with NaCl and extracted with THF (4 x 20 mL). The combined THF extracts were dried (MgSO4), filtered and evaporated to leave a pink-orange foam which was used It was purified by flash chromatography (SiO2, 4-20% MeOH in DCM). Additional title compound (138 mg, 19%) was isolated as a pale yellow solid. Total yield: 403 mg (55%).

[0291] 1 H NMR (400MHz, CD3OD) δ7.87 (s, 1H), 7.84 (d, J = 7.7Hz, 1H), 7.44-7.41 (m, 2H), 7.22-7.17 (m, 1H), 7.16-7.12 (m, 1H), 3.79 (s, 3H);

[0292] 13 C(101MHz,CD3OD)δ168.17,159.87,137.95,131.89,130.64,124.74,124.25,122.42,120.72,118.97,115.97,113.20,53.04

[0293] Synthesis of 2-(N,S-dimethylsulfonylamino)thiazole-4-carboxylic acid methyl ester

[0294]

[0295] To a stirred suspension of 2-(methylamino)thiazole-4-carboxylic acid (501 mg, 3.17 mmol) in MeOH (12 mL) was added concentrated sulfuric acid (1.0 mL) under an inert atmosphere. The resulting solution was heated at reflux for 15 h. After cooling to ambient temperature, the MeOH was evaporated and the residue was diluted with DCM (20 mL) and water (10 mL). The aqueous phase was neutralized with saturated aqueous NaHCO3 and the phases were separated. The aqueous phase was extracted with DCM (15 mL) and the combined organic phases were washed with water (15 mL) and brine (15 mL). The DCM was dried (MgSO4), filtered and evaporated to leave methyl 2-(methylamino)thiazole-4-carboxylate (430 mg, 79%) as an off-white solid.

[0296] 1 H(400MHz, CDCI3)δ8.05(s,1H),7.34(s,1H),3.85(s,3H),3.00(s,3H);

[0297] 13 C(101MHz,CDCI3)δ172.07,162.16,143.15,115.00,52.05,32.65

[0298] To a stirred suspension of methyl 2-(methylamino)thiazole-4-carboxylate (200 mg, 1.16 mmol) in anhydrous pyridine (4 mL) was added methanesulfonyl chloride (266 mg, 180 μL, 2.32 mmol) under an inert atmosphere. The reaction mixture was stirred at 20 °C for 21 h and then diluted with DCM (20 mL). The mixture was washed with 1M aqueous HCl (3 x 20 mL), water (20 mL) and brine (10 mL). The organic phase was dried (MgSO44), filtered and evaporated to leave the title compound (227 mg, 78%) as an orange solid.

[0299] 1 H(400MHz, CDCI3)δ7.84(s,1H),3.91(s,3H),3.58(s,3H),3.03(s,3H);

[0300] 13 C(101MHz,CDCI3)δ161.70,161.65,142.78,124.53,52.57,37.60,36.85

[0301] Synthesis of sulfonylaminothiazole acids: general procedure A.

[0302]

[0303] To the sulfonylaminothiazole ester (1 eq.) was added aqueous NaOH (3-6 eq.) and the resulting mixture was stirred at ambient temperature for 3-6 h. The reaction mixture was filtered, the filtrate was washed with EtOAc (15 mL), and the aqueous layer was acidified to pH≈1 with 1 M aqueous HCl. The acidic solution was saturated with NaCl and then extracted with DCM (3×10 mL) and THF (3×15 mL). The combined THF extracts were washed with brine (10 mL), dried (MgSO4), filtered and evaporated.

[0304]

[0305] Following general procedure A using methyl 2-(2-hydroxyethylsulfonylamino)thiazole-4-carboxylate (500 mg, 1.88 mmol) and 1 M NaOH (3.2 equiv) gave 2-(2-hydroxyethylsulfonylamino)thiazole-4-carboxylic acid (426 mg, 90%) as a white solid.

[0306] 1 H(400MHz,DMSO-d6)δ12.71-14.16(br s,3H),7.61(s,1H),3.73(t,J=6.8Hz,2H),3.19(t,J=6.6Hz,2H);

[0307] 13 C(101MHz,DMSO-d6)δ167.43,159.01,130.05,117.55,55.78,55.46

[0308]

[0309] Following general procedure A, using methyl 2-(methylsulfonylamino)thiazole-4-carboxylate (626 mg, 2.65 mmol) and 1 M NaOH (3.3 equiv), 2-(methylsulfonylamino)thiazole-4-carboxylic acid (473 mg, 80%) was obtained as a light yellow solid.

[0310] 1 H(400MHz,CD3OD)δ7.52(s,1H),2.99(s,3H);

[0311] 13 C(101MHz,CD3OD)δ169.48,160.24,131.36,118.13,41.15

[0312]

[0313] Following general procedure A, using methyl 2-(fluoromethylsulfonylamino)thiazole-4-carboxylate (362 mg, 1.42 mmol) and 1 M NaOH (5.6 equiv), 2-(fluoromethylsulfonylamino)thiazole-4-carboxylic acid (317 mg, 93%) was obtained as an off-white solid.

[0314] 1 H(400MHz,DMSO-d6)δ14.70-11.95(br s,2H),7.67(s,1H),5.31(d,J=46Hz,2H);

[0315] 13C(101MHz,DMSO-d6)δ169.90,158.73,129.63,118.06,90.74(d,J=208Hz)

[0316]

[0317] Following general procedure A using methyl 2-(methylsulfamoyl)aminothiazole-4-carboxylate (170 mg, 0.677 mmol) and 0.5 M NaOH (5.6 equiv) gave 2-((N-methylsulfamoyl)amino)thiazole-4-carboxylic acid (134 mg, 83%) as a white solid.

[0318] 1 H(400MHz,CD3OD)δ7.57(s,1H),2.62(s,3H);

[0319] 13 C(101MHz,CD3OD)δ167.70,161.31,134.14,118.90,29.48

[0320]

[0321] Following general procedure A, using methyl 2-(lH-indole-3-sulfonylamino)thiazole-4-carboxylate (399 mg, 1.18 mmol) and 0.5 M NaOH (3.2 equiv), 2-(lH-indole-3-sulfonylamino)thiazole-4-carboxylic acid (325 mg, 85%) was obtained as an off-white solid.

[0322] 1 H(400MHz,DMSO-d6)δ13.21(br s,2H),11.90(br s,1H),7.94(s,1H),7.74(d,J=7.8Hz,1H),7.61(s,1H),7.47(d,J=8.0Hz,1H),7.22(app t,J=7.5Hz,1H),7.16(app t,J=7.4Hz,1H);

[0323] 13 C (101MHz, DMSO-d6) δ 159.20 (br), 136.17, 129.54 (br), 123.20, 122.77, 120.99, 119.43, 117.70 (br), 115.05 (br), 112.49 (missing 2)

[0324] Synthesis of 2-(N,S-dimethylsulfonylamino)thiazole-4-carboxylic acid

[0325]

[0326] Under an inert atmosphere, 1M NaOH aqueous solution (1 mL) was added to a solution of 2-(N,S-dimethylsulfonylamino)thiazole-4-carboxylic acid methyl ester (225 mg, 0.899 mmol) in a mixture of MeOH (6 mL) and THF (3 mL). The resulting solution was stirred at 20 ° C for 2 h and then concentrated. The residue was diluted with water (10 mL) and extracted with DCM (2×10 mL). The aqueous layer was acidified with 1M HCl aqueous solution (2 mL) and stirred until a precipitate appeared. The resulting suspension was vacuum filtered, and the collected solids were washed with water (3×10 mL) and dried in a vacuum oven at 40 ° C for 13.5 h; the title compound (174 mg, 82%) was obtained as a light yellow solid.

[0327] 1 H(400MHz,CD3OD / CDCl3)δ7.95(s,1H),3.54(s,3H),3.11(s,3H);

[0328] 13 C(101MHz,CD3OD / CDCl3)δ163.85,162.81,143.97,125.36,37.41,36.95

[0329] Synthesis of test inhibitors: General procedure B.

[0330]

[0331] i; amine, DIPEA, HATU, MeCN or DMF 20 or 50℃

[0332] To a stirred suspension of sulfonylaminothiazole acid (1.0 eq) in anhydrous MeCN or DMF under inert atmosphere was added DIPEA (2.0 eq). Once the solid was completely dissolved, HATU (1.0 eq) was added followed by the amine (1.2 eq). The reaction mixture was stirred at 20°C or 50°C. Note: If the amine hydrochloride was used, add additional DIPEA equal to the moles of the hydrochloride.

[0333] The workup procedures were different for each compound and were described in detail.

[0334]

[0335] General Procedure B was followed using 2-(2-hydroxyethylsulfonylamino)thiazole-4-carboxylic acid (87 mg, 0.345 mmol) and 1-(5(6)-chloro-1H-benzo[d]imidazol-2-yl)methanamine dihydrochloride (96 mg, 0.379 mmol) in anhydrous MeCN (10 mL) and DMF (1 mL) at 21° C. for 22 h. Water (15 mL) was added and the solution was saturated with NaCl then extracted with EtOAc (4×50 mL). The combined organic extracts were dried (MgSO ), filtered and evaporated to give a pale yellow oil which was dissolved in saturated aqueous NaHCO 3 solution (25 mL) and extracted with DCM (6×40 mL). The aqueous phase was neutralized by dropwise addition of 32% aqueous HCl, saturated with NaCl and extracted with THF (3×40 mL). The combined organic extracts were dried (MgSO4), filtered and evaporated to give a solid which was purified by two additional rounds of alkaline dissolution procedures to afford N-((5(6)-chloro-1H-benzo[d]imidazol-2-yl)methyl)-2-(2-hydroxyethylsulfonylamino)thiazole-4-carboxamide (118 mg, 82%) as an off-white solid after drying in an oven at 60°C for 17 h.

[0336] IR Umax (cm -1 )3404,3105,1626,1548,1471,1418,1255,1111,1060,1016,841,727,559;

[0337] 1 H(400MHz,DMSO-d6)δ11.94-12.88(br s,2H),8.85(s,1H),7.56(s,1H),7.50(d,J=8.0Hz,1H),7.40(s,1H),7.16(d,J=8.6Hz,1H),4.45-4.79(br s,1H),4.62(d,J=5.3Hz,2H),3.71(t,J=6.8Hz,2H),3.12(t,J=6.8Hz,2H);

[0338] 13 C (101 MHz, DMSO-d6) δ 168.56, 161.01, 153.87, 140.85, 137.07 (br), 125.86, 121.75, 115.80 (br), 114.86 (br), 112.60, 56.49, 54.45, 37.31 (missing 2);

[0339] HRMS:m / z(ESI)C 14 H 15Calculated value for ClN5O4S2: 416.0250 (M+H) + ; Measured value: 416.0254

[0340]

[0341] General Procedure B was followed using 2-(2-hydroxyethylsulfonylamino)thiazole-4-carboxylic acid (86 mg, 0.341 mmol) and (5-phenyl-1H-pyrazol-3-yl)methanamine (65 mg, 0.375 mmol) in anhydrous MeCN (11 mL) and DMF (0.5 mL) at 21 °C for 18 h. Brine (25 mL) was added and the reaction mixture was extracted with THF (4 x 50 mL). The combined organic extracts were dried (MgSO4), filtered and evaporated to give a pale yellow oil which was dissolved in saturated aqueous NaHCO3 solution (25 mL) and extracted with DCM (10 x 50 mL). The aqueous phase was neutralized by dropwise addition of 32% aqueous HCl, saturated with NaCl and extracted with THF (3 x 25 mL). The combined organic extracts were dried (MgSO4), filtered and evaporated to give a solid which was suspended in DCM (15 mL) and sonicated for 10 min. The solid was collected by vacuum filtration and dried in oven at 60 °C for 19 h to give 2-(2-hydroxyethylsulfonylamino)-N-((5-phenyl-1H-pyrazol-3-yl)methyl)thiazole-4-carboxamide (92 mg, 66%) as a white solid.

[0342] IR Umax (cm -1 )3320,3209,1642,1552,1303,1115,1005,890,764,751,727,660,549;

[0343] 1 H(400MHz,DMSO-d6)δ12.61-13.20(br s,2H),8.95(br s,1H),7.74(d,J=7.6Hz,2H),7.55(s,1H),7.41(app t,J=7.6Hz,2H),7.30(t,J=7.4Hz,1H),6.59(s,1H),4.79(brs,1H),4.44(d,J=4.9Hz,2H),3.73(t,J=6.4Hz,2H),3.21(m,2H);

[0344] 13C (101 MHz, DMSO-d6) δ 166.75, 157.74, 146.04, 134.01, 131.15, 128.82, 127.71, 125.02, 112.36, 101.00, 55.84, 55.35, 35.66 (1 missing);

[0345] HRMS:m / z(ESI)C 16 H 17 Calculated value of N5NaO4S2: 430.0617 (M+Na) + ; Measured value: 430.0620

[0346]

[0347] General Procedure B was followed using 2-(Methylsulfonylamino)thiazole-4-carboxylic acid (99 mg, 0.445 mmol) and 1-(1H-pyrrol-2-yl)methanamine (47 mg, 0.490 mmol) in anhydrous MeCN (13 mL) at 20°C for 23 h. Water (30 mL) was added and the solution was saturated with NaCl then extracted with EtOAc (4 x 40 mL). The combined organic extracts were washed with brine (20 mL), dried (MgSO4), filtered and evaporated to give a pale yellow oil which was dissolved in saturated aqueous NaHCO3 (30 mL) and extracted with EtOAc (4 x 20 mL). The aqueous phase was acidified by dropwise addition of 32% aqueous HCl and extracted with EtOAc (4 x 40 mL). The combined organic extracts were washed with brine (30 mL), dried (MgSO4), filtered and evaporated to give a tan solid, which was redissolved in saturated NaHCO3 aqueous solution (35 mL) and extracted with EtOAc (4×20 mL). The aqueous phase was acidified by dropwise addition of 32% HCl aqueous solution and extracted with EtOAc (4×40 mL). The combined organics were washed with brine (30 mL), dried (MgSO44), filtered and evaporated to give 2-(methylsulfonylamino)-N-((1H-pyrrole-2-yl)methyl)thiazole-4-carboxamide (87 mg, 65%) as a tan solid after drying in a vacuum oven at 40°C for 37 h.

[0348] I U max (cm -1 )3434,3345,3142,1637,1543,1512,1265,1120,1107,738,520;

[0349] 1H(400MHz,DMSO-d6)δ12.74(br s,1H),10.65(s,1H),8.74(br s,1H),7.56(s,1H),6.62-6.69(m,1H),5.89-5.97(m,2H,),4.36(d,J=5.7Hz,2H),2.97(br s,3H);

[0350] 13 C (101MHz, DMSO-d6) δ166.28, 157.61, 128.03, 117.37, 112.62, 107.40, 106.29, 40.84, 36.02; (1 missing);

[0351] HRMS:m / z(ESI)C 10 H 12 Calculated value of N4NaO3S2: 323.0249 (M+Na) + ; Measured value: 323.0275

[0352]

[0353] General Procedure B was followed using 2-(Methylsulfonylamino)thiazole-4-carboxylic acid (80 mg, 0.360 mmol) and 1-(5(6)-chloro-1H-benzo[d]imidazol-2-yl)methanamine dihydrochloride (90 mg, 0.355 mmol) in anhydrous DMF (0.5 mL) and MeCN (10 mL) at 20° C. for 89 h. Water (25 mL) was added and the solution was saturated with NaCl then extracted with EtOAc (4×30 mL). The combined organic extracts were dried (MgSO ), filtered and evaporated to give a pale yellow solid which was dissolved in saturated aqueous NaHCO 3 (25 mL) and extracted with DCM (8×35 mL). The aqueous phase was neutralized by dropwise addition of 32% aqueous HCl and extracted with THF (3×25 mL). The combined organic extracts were dried (MgSO4), filtered and evaporated to afford N-((5(6)-chloro-1H-benzo[d]imidazol-2-yl)methyl)-2-(methylsulfonylamino)thiazole-4-carboxamide (139 mg, "100%") as an off-white solid after trituration with DCM / MeOH and drying under vacuum at 50°C for 7 h. Traces of solvent (DCM, MeOH and THF) were not reduced upon further drying.

[0354] IR Umax (cm -1 )3631,3409,3111,2988,1625,1547,1471,1252,1113,839,558;

[0355] 1 H(400MHz,DMSO-d6)δ12.42(br s,2H),8.73(br s,1H),7.55(s,1H),7.50(d,J=8.5Hz,1H),7.33(s,1H),7.16(dd,J=1.2,8.5Hz,1H),4.63(d,J=5.9Hz,2H),2.80(s,3H);

[0356] 13 C (101 MHz, DMSO-d6) δ 168.54, 160.91, 153.84, 140.72, 137.26 (br), 125.86, 121.76, 115.75 (br), 114.83 (br), 112.58, 39.99, 37.32 (1 missing);

[0357] HRMS:m / z(ESI)C 13 H 12 Calculated value for ClN5O3S2: 386.0146 (M+H) + ; Measured value: 386.0148

[0358]

[0359] General Procedure B was followed using 2-(Methylsulfonylamino)thiazole-4-carboxylic acid (129 mg, 0.580 mmol) and 3,3-dimethylbutylamine (71 mg, 94 μL, 0.699 mmol) in anhydrous MeCN (10 mL) at 20 °C for 93 h. Water (80 mL) was added and the resulting mixture was extracted with DCM (3×30 mL). The combined organic extracts were washed with 1M aqueous HCl (20 mL), water (20 mL) and brine (20 mL). The organic phase was dried (MgSO4), filtered and evaporated to leave a viscous orange oil. The isolated material was partitioned between EtOAc (40 mL) and 1M aqueous HCl (25 mL), and the organic phase was separated and extracted with NaHCO3 (2×20 mL). The combined basic extracts were acidified to pH≈1 with 32% aqueous HCl and extracted with DCM (2×15 mL). The combined organic extracts were washed with water (15 mL) and brine (10 mL), dried (MgSO4), filtered and evaporated to leave N-(3,3-dimethylbutyl)-2-(methylsulfonylamino)thiazole-4-carboxamide (109 mg, 61%) as a white solid.

[0360] I U max (cm -1)3335,3104,2955,1645,1530,1282,1125,970,886,652,529,518;

[0361] 1 H(400MHz,DMSO-d6)δ12.65(br s,1H),8.42(br s,1H),7.45(s,1H),3.24-3.19(m,2H),2.98(br s,3H),1.43-1.39(m,2H),0.91(s,9H);

[0362] 13 C(101MHz,DMSO-d6)δ166.59(br),157.43,133.74(br),111.56(br),42.52,40.81,35.55,29.64,29.21;

[0363] HRMS:m / z(ESI)C 11 H 19 Calculated value of N3NaO3S2: 328.0766 (M+Na) + ; Measured value: 328.0772

[0364]

[0365] General Procedure B was followed using 2-(Methylsulfonylamino)thiazole-4-carboxylic acid (98 mg, 0.441 mmol) and (1H-indol-2-yl)methylamine hydrochloride (96 mg, 0.526 mmol) in anhydrous MeCN (5 mL) at 50 °C for 15 h. After cooling to ambient temperature, water (25 mL) was added and the resulting suspension was cooled in an ice bath for 30 min. The liquid was decanted and the solid residue was dissolved in MeOH (10 mL). The MeOH was evaporated and the orange residue was dried in a vacuum oven at 40 °C for 80 min. The dried crude material was dissolved in EtOAc (20 mL) and the solution was extracted with 1 M aqueous HCl (20 mL) and saturated aqueous NaHCO3 (10 mL). The combined aqueous extracts contained a precipitate, which was collected by vacuum filtration. The solid was washed with water (3 x 10 mL) and dried in a vacuum oven at 40 °C for 16.5 h to afford N-((1H-indol-2-yl)methyl)-2-(methylsulfonylamino)thiazole-4-carboxamide (47 mg, 30%) as a tan solid.

[0366] I U max (cm -1)2284,2242,3099,1652,1557,1533,1251,1116,1103,789,734,645,540,513;

[0367] 1 H(600MHz,DMSO-d6)δ12.75(br s,1H),10.97(s,1H),8.97(br s,1H),7.60(br s,1H),7.45(d,J=7.8Hz,1H),7.33(d,J=8.1Hz,1H),7.05-7.02(m,1H),6.96-6.94(m,1H),6.30(s,1H),4.58(d,J=5.6Hz,2H),2.99(br s,3H);

[0368] 13 C (151 MHz, DMSO-d6) δ 167.04 (br), 157.76 (br), 136.31, 136.11, 127.87, 120.74, 119.57, 118.88, 112.11 (br), 111.09, 99.32, 40.80, 36.45 (1 missing);

[0369] HRMS:m / z(ESI)C 14 H 14 Calculated value of N4NaO3S2: 373.0405 (M+Na) + ; Measured value: 373.0408

[0370]

[0371] General Procedure B was followed using 2-(methylsulfonylamino)thiazole-4-carboxylic acid (92 mg, 0.414 mmol) and (5-phenyl-1H-pyrazol-3-yl)methanamine (85 mg, 0.491 mmol) in anhydrous MeCN (10 mL) at 50 °C for 40 h. After cooling to ambient temperature, water (40 mL) was added and the resulting suspension was cooled in an ice bath for 30 min. The solid was collected by vacuum filtration, washed with water (3 x 10 mL), and dried in a vacuum oven at 40 °C for 45 min. To remove aliphatic impurities, the dried solid was partitioned between MeOH (60 mL) and hexanes (20 mL), and the resulting suspension was vacuum filtered. The collected solid was washed with hexanes (10 mL) and MeOH (10 mL) and dried in a vacuum oven at 40 °C for 1 h; 2-(methylsulfonylamino)-N-((5-phenyl-1H-pyrazol-3-yl)methyl)thiazole-4-carboxamide (77 mg, 49%) was obtained as a light tan solid. The two distinct phases in the filtrate were separated, the MeOH phase was concentrated, and the residue was dried in a vacuum oven at 40 °C for 4 h; additional 2-(methylsulfonylamino)-N-((5-phenyl-1H-pyrazol-3-yl)methyl)thiazole-4-carboxamide (18 mg, 12%) was obtained as a light tan solid.

[0372] IR Umax (cm -1 )3305,3210,3135,3089,1642,1554,1299,1120,966,769,652,519;

[0373] 1 H(400MHz,DMSO-d6)δ12.83(br s,2H),8.95(br s,1H),7.74(d,J=7.6Hz,2H),7.57(s,1H),7.41(app t,J=7.6Hz,2H),7.30(t,J=7.4Hz,1H),6.59(s,1H),4.45(d,J=5.6Hz,2H),2.99(s,3H);

[0374] 13 C (101 MHz, DMSO-d6) δ 166.23 (br), 157.80, 128.81, 127.69, 125.01, 112.56 (br), 100.97, 40.77, 35.66 (br) (missing 4);

[0375] HRMS:m / z(ESI)C 15 H 16 Calculated value for N5O3S2: 378.0695 (M+H)+ ; Measured value: 378.0703

[0376]

[0377] General Procedure B was followed using 2-(Methylsulfonylamino)thiazole-4-carboxylic acid (100 mg, 0.450 mmol) and 1-(1-methyl-1H-pyrrol-2-yl)methanamine (54 mg, 0.495 mmol) in anhydrous DMF (7 mL) at 20° C. for 20 h. The solvent was removed under high vacuum and the residue was dissolved in 5% aqueous NaHCO solution (30 mL) and extracted with EtOAc (2×30 mL). The aqueous phase was neutralized to pH 6-7 by dropwise addition of 1 M aqueous HCl and the fine precipitate was removed by vacuum filtration. The filtrate was extracted with EtOAc (5 x 40 mL) and the combined organic extracts were washed with brine (35 mL), dried (MgSO4), filtered and evaporated to give 2-(methylsulfonylamino)-N-((1-methyl-1H-pyrrol-2-yl)methyl)thiazole-4-carboxamide (75 mg, 53%) as a light tan solid after drying in a vacuum oven at 40°C for 21 h.

[0378] IR Umax(cm -1 ):3094,2918,1645,1534,1271,1118,967,839,557,521;

[0379] 1 H(400MHz,DMSO-d6)δ12.68(br s,1H),8.69(br s,1H),7.54(s,1H),6.68(s,1H),5.97(s,1H),5.87-5.93(m,1H),4.38(d,J=4.4Hz,2H),3.55(s,3H),2.95(br s,3H);

[0380] 13 C (101MHz, DMSO-d6) δ166.46, 157.32, 128.49, 122.50, 112.32, 108.38, 106.20, 40.71, 34.54, 33.35; (1 missing);

[0381] HRMS:m / z(ESI)C 11 H 14 Calculated value of N4NaO3S2: 337.0405 (M+Na) + ; Measured value: 337.0422

[0382]

[0383] General Procedure B was followed using 2-(Fluoromethylsulfonylamino)thiazole-4-carboxylic acid (76 mg, 0.316 mmol) and 1-(1H-pyrrol-2-yl)methanamine (33 mg, 0.348 mmol) in anhydrous MeCN (14 mL) at 20°C for 20 h. Water (35 mL) was added and the solution was saturated with NaCl then extracted with EtOAc (3 x 50 mL). The combined organic extracts were washed with brine (20 mL), dried (MgSO4), filtered and evaporated to give a solid which was dissolved in saturated aqueous NaHCO3 (25 mL) and extracted with DCM (8 x 40 mL). The aqueous phase was acidified by dropwise addition of 32% aqueous HCl and extracted with EtOAc (4 x 25 mL). The combined organic extracts were washed with brine (15 mL), dried (MgSO4), filtered and evaporated to give 2-(fluoromethylsulfonylamino)-N-((1H-pyrrol-2-yl)methyl)thiazole-4-carboxamide (102 mg, "102" %) as a tan solid after drying in an oven at 60°C for 14 h. Trace amounts of EtOAc were not reduced upon further drying.

[0384] IR Umax (cm -1 )3328,3097,1644,1531,1271,1127,1116,835,728,557;

[0385] 1 H(400MHz,DMSO-d6)δ13.30(br s,1H),10.67(s,1H),8.88(s,1H),7.60(s,1H),6.66(s,1H),5.94(s,2H),5.30(d,J=46Hz,2H),4.36(d,J=5.4Hz,2H);

[0386] 13 C(101MHz,DMSO-d6)δ169.46,156.97,132.41,127.79,117.30,112.50,107.36,106.25,90.72(d,J=208Hz),36.04;

[0387] HRMS:m / z(ESI)C 10 H 11 Calculated value of FN4NaO3S2: 341.0154 (M+Na) + ; Measured value: 341.0174

[0388]

[0389] General Procedure B was followed using 2-(fluoromethylsulfonylamino)thiazole-4-carboxylic acid (78 mg, 0.323 mmol) and 1-(5(6)-chloro-1H-benzo[d]imidazol-2-yl)methanamine dihydrochloride (90 mg, 0.355 mmol) in anhydrous DMF (1.2 mL) and anhydrous MeCN (12 mL) at 20° C. for 96 h. Water (35 mL) was added and the solution was saturated with NaCl then extracted with EtOAc (3×50 mL). The combined organic extracts were washed with water (40 mL), brine (30 mL), dried (MgSO ), filtered and evaporated to give a pale yellow solid. The aqueous washes were combined and extracted with EtOAc (5×80 mL). The combined organic extracts were washed with brine (40 mL), dried (MgSO ), filtered and evaporated to give a yellow solid. The solids were combined and dissolved in saturated aqueous NaHCO3 (35 mL) and extracted with DCM (6 x 40 mL). The aqueous phase was neutralized by dropwise addition of 32% aqueous HCl and extracted with EtOAc (4 x 50 mL). The combined organic extracts were washed with brine (35 mL), dried (MgSO4), filtered and evaporated to afford N-((5(6)-chloro-1H-benzo[d]imidazol-2-yl)methyl)-2-(fluoromethylsulfonamido)thiazole-4-carboxamide (137 mg, "105" %) as a light tan solid after drying in an oven at 60 °C for 20 h. Traces of DCM and EtOAc did not decrease upon further drying.

[0390] IR Umax (cm -1 )3410,3104,2972,1648,1542,1466,1265,1224,1123,840,556;

[0391] 1 H(400MHz,DMSO-d6)δ9.05(br s,1H),7.56(s,1H),7.46-7.54(m,2H),7.50(s,1H),7.17(d,J=8.5Hz,1H),5.22(d,J=47Hz,2H),4.64(d,J=5.1Hz,2H);

[0392] 13 C (101 MHz, DMSO-d6) δ 169.41, 159.25, 153.60, 139.82, 136.93, 125.93, 121.83, 115.79, 114.73, 113.17, 90.40 (d, J = 208 Hz), 37.28 (1 missing);

[0393] HRMS:m / z(ESI)C 13 H 12 Calculated value for ClFN5O3S2: 404.0054 (M+H) + ; Measured value: 404.0068

[0394]

[0395] General Procedure B was followed using 2-(fluoromethylsulfonamido)thiazole-4-carboxylic acid (76 mg, 0.316 mmol) and 3,3-dimethylbutylamine (35 mg, 0.348 mmol) in anhydrous MeCN (12 mL) at 20 °C for 21 h. Water (30 mL) was added and the solution was cooled in an ice / water bath while NaCl was added. The solid was collected by vacuum filtration, washed with water (4×5 mL) and dried to give 38 mg of a white solid. More solid was recovered by extracting the filtrate with EtOAc (4×35 mL). The combined organic extracts were washed with brine (40 mL), dried (MgSO4), filtered and evaporated to give a white solid, which was dissolved in saturated aqueous NaHCO3 (35 mL) and extracted with DCM (6×40 mL). The aqueous phase was acidified by dropwise addition of 32% aqueous HCl and extracted with EtOAc (4×40 mL). The combined organic extracts were washed with brine (30 mL), dried (MgSO4), filtered and evaporated to give N-(3,3-dimethylbutyl)-2-(fluoromethylsulfonamido)thiazole-4-carboxamide (93 mg, 91%) as a white solid after drying in an oven at 60°C for 16.5 h.

[0396] IR Umax (cm -1 )3328,2955,1646,1524,1283,1126,1059,898,659,559,518;

[0397] 1 H(400MHz, DMSO-d6)δ13.23(br s,1H),8.53(s,1H),7.49(s,1H),5.30(d,J=46Hz,2H),3.21(m,2H),1.41(m,2H),0.91(s,9H);

[0398] 13 C (101MHz, DMSO-d6) δ169.48, 157.06, 132.97, 111.82, 90.68 (d, J = 207Hz), 42.46, 35.60, 29.63, 29.19;

[0399] HRMS:m / z(ESI)C11 H 18 Calculated value of FN3NaO3S2: 346.0689 (M+Na) + ; Measured value: 346.0671

[0400]

[0401] General Procedure B was followed using 2-(fluoromethylsulfonamido)thiazole-4-carboxylic acid (60 mg, 0.250 mmol) and 1-(1H-indol-2-yl)methanamine hydrochloride (50 mg, 0.275 mmol) in anhydrous MeCN (11 mL) and DMF (0.5 mL) at 20 °C for 19 h. Water (35 mL) was added and the solution was saturated with NaCl then extracted with EtOAc (3 x 40 mL). The combined organic extracts were washed with brine (30 mL), dried (MgSO4), filtered and evaporated to give a pale yellow oil which was dissolved in saturated aqueous NaHCO3 (35 mL) and extracted with DCM (5 x 40 mL). The aqueous phase was neutralized by dropwise addition of 32% aqueous HCl and extracted with EtOAc (4 x 40 mL). The combined organic extracts were washed with brine (35 mL), dried (MgSO4), filtered and evaporated to give a solid, which was redissolved in saturated NaHCO3 aqueous solution (20 mL) and extracted with DCM (5×40 mL). The aqueous phase was neutralized by dropwise addition of 32% HCl aqueous solution and extracted with EtOAc (5×40 mL). The combined organic extracts were washed with brine (35 mL), dried (MgSO4), filtered and evaporated to give 2-(fluoromethylsulfonamido)-N-(1H-indol-2-ylmethyl)thiazole-4-carboxamide (86 mg, 93%) as a light orange solid after drying in an oven at 60°C for 14 h.

[0402] IR Umax (cm -1 )3387,1634,1538,1471,1457,1424,1271,1233,1128,1057,962,840,741,559;

[0403] 1H(400MHz,DMSO-d6)δ13.36(br s,1H),10.93(s,1H),8.57(br s,1H),7.43(d,J=7.8Hz,1H),7.35(br s,1H),7.33(d,J=7.8Hz,1H),7.02(app t,J=7.8Hz,1H),6.93(appt,J=7.6Hz,1H),6.26(s,1H),5.11(d,J=46Hz,2H),4.54(d,J=5.8Hz,2H);

[0404] 13 C(101MHz,DMSO-d6)δ169.48,160.05,140.20(br),136.95,136.12,127.87, 120.67,119.57,118.84,112.80,111.11,99.12,90.16(d,J=207Hz),36.21;

[0405] HRMS:m / z(ESI)C 14 H 13 Calculated value of FN4NaO3S2: 391.0311 (M+Na) + ; Measured value: 391.0328

[0406]

[0407] General Procedure B was followed using 2-(fluoromethylsulfonylamino)thiazole-4-carboxylic acid (38 mg, 0.158 mmol) and (5-phenyl-1H-pyrazol-3-yl)methanamine (32 mg, 0.185 mmol) in anhydrous MeCN (5 mL) at 50 °C for 26.5 h. After cooling to ambient temperature, water (20 mL) was added and the resulting suspension was cooled in an ice bath for 15 min. The solid was collected by vacuum filtration, washed with water (3 x 5 mL), and dried in a vacuum oven at 40 °C for 16 h; 2-(fluoromethylsulfonylamino)-N-((5-phenyl-1H-pyrazol-3-yl)methyl)thiazole-4-carboxamide (48 mg, 77%) was obtained as a light tan solid.

[0408] IR Umax (cm -1 )3306,3243,3094,2955,1643,1547,1442,1423,1318,1275,1133,1007,893,766,666,590,508;

[0409] 1H(600MHz,DMSO-d6)δ13.16(br s,2H),9.10(br t,J=5.7Hz,1H),7.74(d,J=7.7Hz,2H),7.62(s,1H),7.41(app t,J=7.5Hz,2H),7.30(t,J=7.4Hz,1H),6.60(s,1H),5.31(d,J=46Hz,2H),4.45(d,J=5.7Hz,2H);

[0410] 13 C (151 MHz, DMSO-d6) δ 169.46, 157.22, 144.00 (br), 132.38, 130.86 (br), 128.83, 127.72, 125.03, 112.63, 101.02, 90.73 (d, J = 207 Hz), 35.76 (br) (1 missing);

[0411] HRMS:m / z(ESI)C 15 H 15 Calculated value for N5O3S2F: 396.0600 (M+H) + ; Measured value: 396.0596

[0412]

[0413] General Procedure B was followed using 2-(Fluoromethylsulfonylamino)thiazole-4-carboxylic acid (75 mg, 0.312 mmol) and 1-(1-methyl-1H-pyrrol-2-yl)methanamine (38 mg, 0.343 mmol) in anhydrous MeCN (12 mL) at 20°C for 23 h. Water (35 mL) was added and the solution was saturated with NaCl then extracted with EtOAc (4 x 30 mL). The combined organic extracts were washed with brine (20 mL), dried (MgSO4), filtered and evaporated to give an oil which was dissolved in saturated aqueous NaHCO3 (25 mL) and extracted with DCM (5 x 20 mL). The aqueous phase was acidified by dropwise addition of 32% aqueous HCl and extracted with EtOAc (3 x 25 mL). The combined organic extracts were washed with brine (15 mL), dried (MgSO4), filtered and evaporated to give 2-(fluoromethylsulfonylamino)-N-((1-methyl-1H-pyrrol-2-yl)methyl)thiazole-4-carboxamide (86 mg, 93%) as a gummy tan solid after drying in a vacuum oven at 40°C for 16 h.

[0414] IR Umax (cm -1)3640,3096,1652,1532,1293,1131,834,739,657,556;

[0415] 1 H(400MHz,DMSO-d6)δ13.30(br s,1H),8.84(s,1H),7.61(s,1H),6.68(s,1H),5.98(s,1H),5.90(s,1H),5.29(d,J=46Hz,2H),4.39(br s,2H),3.55(s,3H);

[0416] 13 C(101MHz, DMSO-d6)δ169.51,156.75,132.17,128.30,122.56,112.59,108.49,106.21,90.70(d,J=208Hz),34.63,33.35;

[0417] HRMS:m / z(ESI)C 11 H 13 Calculated value of FN4NaO3S2: 355.0311 (M+Na) + ; Measured value: 355.0324

[0418]

[0419] General Procedure B was followed using 2-((N-methylsulfamoyl)amino)thiazole-4-carboxylic acid (94 mg, 0.396 mmol) and 1-(5(6)-chloro-1H-benzo[d]imidazol-2-yl)methanamine dihydrochloride (122 mg, 0.479 mmol) in anhydrous MeCN (10 mL) at 50°C for 66.5 h. After cooling to ambient temperature, water (80 mL) was added and the resulting suspension was cooled in an ice bath for 30 min. The liquid was decanted and the solid residue was dissolved in MeOH (15 mL). The MeOH was evaporated and the orange residue was dried in a vacuum oven at 40°C for 13 h. The dried crude material was dissolved in saturated aqueous NaHCO3 (10 mL) and the aqueous solution was extracted with EtOAc (2×10 mL). The aqueous phase was acidified to pH≈6 with 2M aqueous HCl and then extracted with DCM (10 mL) and THF (3×10 mL). The combined THF extracts were dried (MgSO ), filtered and evaporated to leave a viscous orange oil which was dried in a vacuum oven at 40° C. for 14 h. N-((5(6)-chloro-1H-benzo[d]imidazol-2-yl)methyl)-2-((N-methylsulfamoyl)amino)thiazole-4-carboxamide (59 mg, 37%) was obtained as a tan solid.

[0420] IR Umax (cm -1 )3245,3098,2968,1649,1543,1454,1260,1116,1059,883,805,562,461;

[0421] 1 H(400MHz,CD3OD)δ7.67(s,1H),7.50(d,J=2.0Hz,1H),7.46(d,J=8.6Hz,1H),7.18(dd,J=8.6,1.9Hz,1H),4.80(s,2H),2.65(s,3H);

[0422] 13 C(101MHz,CD3OD)δ163.24,163.13,154.72,144.47,140.32,137.97,129.14,124.00,117.83,116.68,115.66,38.33,29.35;

[0423] HRMS:m / z(ESI)C 13 H 14 N6O3S2 35 Calculated value of Cl: 410.0257 (M+H) + ; Measured value: 401.0257

[0424]

[0425] General Procedure B was followed using 2-((N-methylsulfamoyl)amino)thiazole-4-carboxylic acid (95 mg, 0.400 mmol) and 3,3-dimethylbutylamine (49 mg, 65 μL, 0.483 mmol) in anhydrous MeCN (10 mL) at 50 °C for 18 h. Water (20 mL) and brine (10 mL) were added and the resulting mixture was cooled to ambient temperature. The mixture was extracted with EtOAc (2 x 15 mL) and the combined organic extracts were evaporated to leave a pink oil which was dissolved in saturated aqueous NaHCO3 solution (10 mL). The aqueous solution was extracted with DCM (3 x 15 mL) and acidified to pH ≈ 1 with 2M aqueous HCl. The aqueous solution of the acid was saturated with NaCl and extracted with THF (2 x 15 mL). The combined THF extracts were dried (MgSO4), filtered and evaporated to leave N-(3,3-dimethylbutyl)-2-((N-methylsulfamoyl)amino)thiazole-4-carboxamide (120 mg, 94%) as an off-white solid.

[0426] IR Umax (cm-1 )3345,3253,2957,1646,1565,1530,1278,1248,1175,1129,1041,840,667,556,492;

[0427] 1 H(400MHz,CD3OD)δ7.61(s,1H),3.40-3.34(m,2H),2.65(s,3H),1.54-1.48(m,2H),0.96(s,9H);

[0428] 13 C(101MHz,CD3OD)δ163.07,162.29,144.09,116.76,43.89,37.10,30.60,29.68,29.28;

[0429] HRMS:m / z(ESI)C 11 H 20 Calculated value of N4NaO3S2: 343.0875 (M+Na) + ; Measured value: 343.0876

[0430]

[0431] General Procedure B was followed using 2-((N-methylsulfamoyl)amino)thiazole-4-carboxylic acid (125 mg, 0.527 mmol) and (1H-indol-2-yl)methylamine hydrochloride (115 mg, 0.630 mmol) in anhydrous MeCN (10 mL) at 50 °C for 23 h. After cooling to ambient temperature, water (80 mL) was added and the resulting turbid mixture was cooled in an ice bath for 30 min. The liquid was decanted and the solid residue was dissolved in MeOH (25 mL). The MeOH was evaporated and the orange residue was dried in a vacuum oven at 40 °C for 16 h. The dried solid was partitioned between DCM (15 mL) and saturated aqueous NaHCO3 solution (15 mL), the phases were separated, and the aqueous phase was washed with DCM (15 mL). The aqueous portion was acidified to pH≈1 with 2M aqueous HCl and the resulting precipitate was collected by vacuum filtration. The solid was washed with water (3 x 15 mL) and then dried in a vacuum oven at 40 °C for 15 h; N-((1H-indol-2-yl)methyl)-2-((N-methylsulfamoyl)amino)thiazole-4-carboxamide (46 mg, 24%) was obtained as an off-white solid.

[0432] IR Umax (cm -1)3300,1652,1539,1340,1289,1122,884,794,750,660,568,466;

[0433] 1 H(400MHz,CD3OD)δ7.68(s,1H),7.45(d,J=7.9Hz,1H),7.31(d,J=8.1Hz,1H),7.05(app t,J=7.6Hz,1H),6.96(app t,J=7.5Hz,1H),6.35(s,1H),4.69(s,2H),2.63(s,3H);

[0434] 13 C (101MHz, CD3OD) δ162.84 (br), 138.16, 136.83, 136.74*, 129.68, 129.60*, 122.28, 120.90, 120.87*, 120.18, 117.43 (br), 111.90, 101.07, 37.84, 37.82*, 29.28 (missing 2);

[0435] *Peaks occur at approximately half the height of their neighbors; all are (indol-2-yl)methyl related and are likely rotamers

[0436] HRMS:m / z(ESI)C 14 H 15 Calculated value of N5NaO3S2: 388.0514 (M+Na) + ; Measured value: 388.0505

[0437]

[0438] General Procedure B was followed using 2-((N-methylsulfamoyl)amino)thiazole-4-carboxylic acid (110 mg, 0.464 mmol) and (5-phenyl-1H-pyrazol-3-yl)methanamine (94 mg, 0.543 mmol) in anhydrous MeCN (10 mL) at 50 °C for 40 h. After cooling to ambient temperature, water (60 mL) was added and the resulting suspension was cooled in an ice bath for 30 min. The solid was collected by vacuum filtration, washed with water (3 x 10 mL), and dried in a vacuum oven at 40 °C for 69 h. The dried solid was suspended in saturated aqueous NaHCO3 solution (20 mL) and stirred before washing with EtOAc (2 x 20 mL). The aqueous phase was acidified to pH≈1 with 2M aqueous HCl, saturated with NaCl, and extracted with THF (2 x 15 mL). The combined THF extracts were washed with brine (10 mL), dried (MgSO4), filtered and evaporated to leave 2-((N-methylsulfamoyl)amino)-N-((5-phenyl-1H-pyrazol-3-yl)methyl)thiazole-4-carboxamide (91 mg, 50%) as an off-white solid.

[0439] I U max (cm -1 )3287,3183,3134,1742,1648,1539,1306,1122,883,769,695,647,574;

[0440] 1 H(400MHz,CD3OD)δ7.71-7.69(m,3H),7.41(app t,J=7.6Hz,2H),7.32(t,J=7.4Hz,1H),6.60(s,1H),4.60(s,2H),2.64(s,3H);

[0441] 13 C (101 MHz, DMSO-d6) δ 159.17 (br), 146.86 (br), 146.18 (br), 131.21 (br), 128.83, 127.74, 125.06, 114.74 (br), 100.97, 35.58, 28.78 (missing 2);

[0442] HRMS:m / z(ESI)C 15 H 17 Calculated value for N6O3S2: 393.0804 ([M+H] + ); Measured value: 393.0807

[0443]

[0444] General Procedure B was followed using 2-(1H-indole-3-sulfonylamino)thiazole-4-carboxylic acid (100 mg, 0.309 mmol) and (5-chloro-1H-1,3-benzodiazol-2-yl)methanamine dihydrochloride (94 mg, 0.369 mmol) in anhydrous MeCN (10 mL) at 50 °C for 20 h. After cooling to ambient temperature, water (40 mL) was added and the resulting suspension was cooled in an ice bath for 30 min. The solid was collected by vacuum filtration, washed with water (3 x 15 mL), and dried in a vacuum oven at 40 °C for 13.5 h. The crude material was dissolved in a mixture of THF (5 mL) and EtOAc (10 mL) and washed with water (2 x 10 mL). The organic portion was washed with saturated aqueous NaHCO3 (3 x 5 mL) and then diluted with diethyl ether (20 mL). A dark yellow oil precipitated from the solution, and the mixture was extracted with water (10 mL). The water wash was acidified to pH≈5 with 1M aqueous HCl, and the resulting precipitate was collected by vacuum filtration. The collected solid was washed with water (3×5 mL) and dried in a vacuum oven at 40° C. for 17 h; N-((5-chloro-1H-benzo[d]imidazol-2-yl)methyl)-2-(1H-indole-3-sulfonylamino)thiazole-4-carboxamide (39 mg, 26%) was obtained as an off-white solid.

[0445] I U max (cm -1 )3275,3115,1658,1541,1420,1243,1114,1019,882,746,676,588,425;

[0446] 1 H (400MHz, CD3OD) δ7.95(s,1H),7.87(d,J=7.5Hz,1H),7.57(s,1H),7.47(d,J=1. 9Hz,1H),7.46-7.42(m,2H),7.25-7.21(m,1H),7.20-7.16(m,2H),4.73(s,2H);

[0447] 13 C(101MHz,CD3OD)δ163.83,162.48,154.55,141.91,140.00,137.98,137.68,132.06,129.3 1,124.64,124.50,124.16,122.77,120.52,117.34,116.64,115.61,114.43,113.39,38.25;

[0448] HRMS:m / z(ESI)C20 H 16 35 Calculated value for ClN6O3S2: 487.0414 ([M+H] + ); Measured value: 487.0413

[0449]

[0450] Following General Procedure B, 2-(1H-indole-3-sulfonylamino)thiazole-4-carboxylic acid (84 mg, 0.260 mmol) and (5-phenyl-1H-pyrazol-3-yl)methanamine (55 mg, 0.318 mmol) in anhydrous MeCN (9 mL) were used at 50 °C for 22 h. After cooling to ambient temperature, water (15 mL) was added and the resulting suspension was transferred to a beaker. The residual solids in the reaction flask were rinsed into the beaker with water (15 mL). The suspension was stirred and cooled in an ice bath for 30 min. The solids were collected by vacuum filtration, washed with water (3×15 mL), and dried in a vacuum oven at 40 °C for 20 h; 2-(1H-indole-3-sulfonylamino)-N-((5-phenyl-1H-pyrazol-3-yl)methyl)thiazole-4-carboxamide (105 mg, 84%) was obtained as an off-white solid.

[0451] I U max (cm -1 )3297,3105,1642,1546,1509,1424,1289,1113,1014,880,745,679,653,589;

[0452] 1 H(400MHz,DMSO-d6)δ12,80(br s,2H),11,90(s,1H),8,76(br s,1H),7,96(s,1H),7,76(d,J=8.0Hz,1H),7.72(d,J=7.7Hz,2H),7.54(s,1H),7.47(d,J=8.0Hz,1H),7.39(app t,J=7.6Hz,2H),7.29(t,J=7.4Hz,1H),7.21(app t,J=7.6Hz,1H),7.16(appt,J=7.5Hz,1H),6.55(s,1H),4.40(d,J=5.6Hz,2H);

[0453] 13C (101 MHz, DMSO-d6) δ 136.16, 129.90 (br) 128.80, 127.68, 125.01, 123.22, 122.78, 121.02, 119.43, 112.50, 100.91, 35.67 (br) (8 lost);

[0454] HRMS:m / z(ESI)C 22 H 19 Calculated value for N6O3S2: 479.0960 ([M+H] + ); Measured value: 479.0962

[0455] General proceduresC.

[0456]

[0457] i; Amine, DIPEA, HATU, MeCN, 20℃ or 50℃

[0458] To a stirred suspension of 2-(N,S-dimethylsulfonylamino)thiazole-4-carboxylic acid (1.0 equiv) in anhydrous MeCN under inert atmosphere was added DIPEA (2.0 equiv). Once the solid was completely dissolved, HATU (1.0 equiv) was added followed by the amine (1.05-1.2 equiv). The reaction mixture was stirred at 20°C or 50°C. The work-up procedure was different for each compound and is detailed.

[0459]

[0460] General Procedure C was followed using 2-(N,S-dimethylsulfonylamino)thiazole-4-carboxylic acid (81 mg, 0.344 mmol) and 1-(1H-pyrrol-2-yl)methanamine (36 mg, 0.378 mmol) in anhydrous MeCN (6.5 mL) at 20°C for 70 h. The reaction mixture was diluted with water (45 mL) and cooled to 0-5°C for 1 h. The precipitate was collected by vacuum filtration, washed with water (3 x 10 mL), and dried in an oven at 60°C for 5 h to give N-((1H-pyrrol-2-yl)methyl)-2-(N,S-dimethylsulfonylamino)thiazole-4-carboxamide (92 mg, 85%) as an off-white solid.

[0461] I U max (cm -1 )3412,3303,1665,1552,1488,1349,1150,977,744,514;

[0462] 1H(400MHz,DMSO-d6)δ10.55(s,1H),8.43(t,J=5.4Hz,1H),7.88(s,1H),6.63(s,1H),5.90(s,2H),4.39(d,J=5.9Hz,2H),3.49(s,3H),3.27(s,3H);

[0463] 13 C(101MHz,DMSO-d6)δ160.91,160.00,145.08,128.82,119.85,117.19,107.08,105.92,37.07,36.24,35.57;

[0464] HRMS:m / z(ESI)C 11 H 14 Calculated value of N4NaO3S2: 337.0405 (M+Na) + ; Measured value: 337.0421

[0465]

[0466] General Procedure C was followed using 2-(N,S-dimethylsulfonylamino)thiazole-4-carboxylic acid (84 mg, 0.356 mmol) and 3,3-dimethylbutylamine (38 mg, 50 μL, 0.372 mmol) in anhydrous MeCN (5 mL) at 50 °C for 15.5 h. Water (40 mL) was added and the resulting mixture was cooled to ambient temperature. The aqueous solution was decanted and the residual oil was dissolved in EtOAc (15 mL). The organic solution was dried (MgSO4), filtered and evaporated to leave a viscous orange-yellow oil. The decanted aqueous solution was extracted with EtOAc (2×25 mL) and the combined EtOAc extracts were washed with 1 M aqueous HCl (20 mL), saturated aqueous NaHCO3 (20 mL), water (20 mL) and brine (20 mL). The organic phase was dried (MgSO4), filtered and evaporated to leave a light yellow viscous oil. The two separated oils were combined and purified by flash chromatography (SiO2, petroleum ether 40 / 60: ethyl acetate, 1: 1). The oil thus separated was dried in a vacuum oven at 40°C for 13.5 h; N-(3,3-dimethylbutyl)-2-(N,S-dimethylsulfonylamino)thiazole-4-carboxamide (106 mg, 93%) was obtained as a white solid.

[0467] I U max (cm -1)3415,3397,3119,2996,2951,2916,2866,1657,1557,1491,1347,1156,765,514,487;

[0468] 1 H(600MHz, CDCI3)δ7.69(s,1H),7.03(br t,J=5.9Hz,1H),3.49(s,3H),3.41-3.37(m,2H),3.01(s,3H),1.50-1.47(m,2H),0.92(s,9H);

[0469] 13 C(151MHz, CDCI3)δ161.08,160.55,145.84,119.12,43.35,37.45,36.51,36.03,30.00,29.43;

[0470] HRMS:m / z(ESI)C 12 H 21 Calculated value of N3NaO3S2: 342.0922 (M+Na) + ; Measured value: 342.0925

[0471]

[0472] General Procedure C was followed using 2-(N,S-dimethylsulfonylamino)thiazole-4-carboxylic acid (88 mg, 0.372 mmol) and (5-phenyl-1H-pyrazol-3-yl)methylamine (77 mg, 0.445 mmol) in anhydrous MeCN (5 mL) at 50 °C for 19.5 h. Water (40 mL) was added and the resulting mixture was cooled to ambient temperature followed by 1 h in an ice bath. The aqueous solution was decanted and the residual oil was dissolved in EtOAc (15 mL). The organic solution was dried (MgSO4), filtered and evaporated to leave a viscous yellow-orange oil which was purified by flash chromatography (SiO2, DCM:MeOH, 92:8); 2-(N,S-dimethylsulfonylamino)-N-((5-phenyl-1H-pyrazol-3-yl)methyl)thiazole-4-carboxamide (106 mg, 93%) was obtained as a white solid.

[0473] I U max (cm -1 )3184,3139,3115,3017,1652,1553,1486,1352,1156,959,776,748,699,654,512,495;

[0474] 1 H(400MHz, CDCI3)δ7.89(br t,J=5.5Hz,1H),7.77(s,1H),7.66(d,J=7.6Hz,2H),7.37(app t,J=7.5Hz,2H),7.30(t,J=7.4Hz,1H),6.50(s,1H),4.68(d,J=5.5Hz,2H),3.41(s,3H),2.96(s,3H);

[0475] 13 C(101MHz, CDCI3)δ161.41,161.25,147.71(br),146.38(br),145.14,131.19,128.92,128.25,125.54,120.01,101.70,37.47,36.43,35.97;

[0476] HRMS:m / z(ESI)C 16 H 18 Calculated for N5O3S2: 392.0851 ([M+H] + ); Measured value: 392.0849

[0477]

[0478] General Procedure C was followed using 2-(N,S-dimethylsulfonylamino)thiazole-4-carboxylic acid (81 mg, 0.344 mmol) and 1-(1-methyl-1H-pyrrol-2-yl)methanamine (42 mg, 0.378 mmol) in anhydrous MeCN (5.7 mL) at 20° C. for 20 h. The reaction mixture was diluted with water (40 mL), saturated with NaCl, and extracted with EtOAc (3×30 mL). The combined organic extracts were washed with brine (20 mL), dried (MgSO4), filtered and evaporated to give a viscous oil which was purified by flash chromatography (SiO2, DCM / MeOH, 98:2) to afford N-((1-methyl-1H-pyrrol-2-yl)methyl)-2-(N,S-dimethylsulfonylamino)thiazole-4-carboxamide (76 mg, 68%) as a gummy white solid after drying under high vacuum at 20°C for 8.5 h.

[0479] I U max (cm -1 )3394,3076,2924,1669,1546,1483,1346,1151,965,653,513;

[0480] 1H(400MHz, CDCI3)δ7.81(s,1H),7.10-7.19(m,1H),6.65(s,1H),6.16(s,1H),6 .08-6.13(m,1H),4.63(d,J=5.6Hz,2H),3.62(s,3H),3.52(s,3H),3.03(s,3H);

[0481] 13 C(101MHz, CDCI3)δ161.09,159.97,145.19,128.37,123.14,119.85,108.86,106.84,37.26,36.50,34.84,33.81;

[0482] HRMS:m / z(ESI)C 12 H 16 Calculated value of N4NaO3S2: 351.0562 (M+Na) + ; Measured value: 351.0590 Example 3 - Bioassay

[0483] dGAE assembly and measurement of pharmacological activity of inhibitor compounds by immunodetection

[0484] (a) Sandwich ELISA

[0485] Recombinant dGAE was produced in a bacterial expression system and assembled in the presence of 10 mM DTT by incubation at 37°C with agitation for 24 h as previously described [6]. To test pharmacological activity, assembly reactions were performed in Protein Lobind tubes (Eppendorf) with 100 μM dGAE and 500 μM tau aggregation inhibitors (compound 9 or HMT). Compound 9 was dissolved in DMSO (0.5% final) and HMT was dissolved in 10 mM phosphate buffer (PB) supplemented with 10 mM DTT. Control tubes containing dGAE DTT in PB and dGAE DTT in 0.5% DMSO were also set up to compare the degree of aggregation. After 24 h of assembly reaction, the contents of each tube were mixed by aspiration and samples were removed for immunodetection of exposed epitopes using liquid-phase sandwich ELISA.

[0486] 96-well Nunc Maxisorp plates were pre-coated with mAb 423 (10 μg / ml), which specifically recognizes tau truncated at C-terminal Glu391 in the pronase-resistant PHF core

[42] , washed three times with PBS containing 0.1% Tween 20 (PBST), and then blocked with 2% milk powder (Marvel) in PBS. Two-fold dilutions of dGAE assembly samples were added from a starting concentration of 10 μg / ml in PBS and incubated at 37°C for 1 h. Bound tau was then detected using scAb (10 μg / ml) and incubated at 37°C for 1 h. Anti-human C kappa HRP-conjugated secondary antibody was diluted 1:1,000 in 2% milk powder in PBS and incubated at 37°C for 1 h. Pierce 1-Step Ultra TMB-ELISA substrate solution was used for color development, followed by the addition of 1M H2SO4 to stop the reaction. Absorbance was measured at 450 nm.

[0487] (b) Cellular tau aggregation inhibition assay

[0488] Cell-based tau aggregation assays were performed as previously described [13, 25]. The process is described in more detail in WO 02 / 055720. Essentially, fibroblasts (3T6) express full-length tau ("T40"; htau40 isoform) under the control of an inducible promoter, as well as low constitutive levels of the PHF-core tau fragment (12-kDa fragment). When T40 expression is induced, it undergoes aggregation-dependent truncation within the cell, with the N-terminus at approximately amino acid residue 295 and the C-terminus at approximately residue 390, thereby producing higher levels of the 12-kDa PHF-core domain fragment. Tau aggregation inhibitors can block the generation of the 12-kDa fragment in a dose-dependent manner. In fact, the quantification of the inhibitory activity of the compound on the proteolytic generation of the intracellular 12-kDa fragment can be described entirely based on the same parameters that describe the inhibition of tau-tau binding in vivo. In other words, the extent of proteolytic generation of the 12-kDa fragment within the cell is entirely determined by the extent of tau-tau binding via the repeat domains. The availability of relevant proteases within the cell is non-limiting.

[0489] Compounds were initially tested at a single concentration of 2 μM (at least in triplicate) for the formation of truncated tau in cells, as detected by immunoblotting of cell lysates separated by SDS-PAGE

[25] . MTC was included as a reference tau aggregation inhibitor for testing at a single concentration. The ratio of the amounts of the two protein bands was measured at different TAI concentrations to correct for the expression of full-length tau and cell density, especially at high TAI concentrations.

[0490] The inhibition ratio (IR) was calculated by the following equation:

[0491]

[0492] IR x = inhibition ratio of compound at x mM; S = immunoblot signal (mean) of truncated tau or full-length tau band of compound at 0 or x mM. A ratio of 1.0 indicates no effect, while decreasing values ​​from 1.0 indicate increasing inhibition. Curves were generated for different concentrations and the EC of TAI was 50 The value is defined as the concentration at which the ratio of truncated tau to full-length tau is 50% of the ratio measured in the absence of TAI; this was determined graphically.

[0493] Compound 9 and MTC were tested within a certain concentration range (0-20 μM for compound 9 and 0-2 μM for MTC; Figure 2 ) were further tested and the ratio of the lower truncated 12-kDa tau to full-length T40 band was calculated. The concentration at which there was 50% inhibition of the 12-kDa band relative to T40 (called the EC50 value) was calculated from a plot of the ratio of the protein band relative to the band observed in untreated cells

[25] .

[0494] (c) Used to establish B 50 In vitro cell-free assay

[0495] This is described in detail in WO 96 / 30766 and

[25] . In brief, a tau fragment corresponding to the core repeat domain has been adsorbed onto a solid substrate, which is able to capture soluble full-length tau and bind tau with high affinity. This association confers stability to proteolytic digestion on the aggregated tau molecules. The process is self-propagating and can be selectively blocked by the prototype agent

[12] .

[0496] More specifically, truncated tau (residues 297-390; dGA) diluted in carbonate buffer (pH 9.6) was bound to the assay plate, and another truncated tau species (residues 297-391; dGAE) was added to the aqueous phase. The aqueous binding buffer contained phosphate buffered saline (pH 7.4) with 0.05% Tween-20 and 1% gelatin. Bound dGAE was detected using mAb 423, which recognizes a Glu-391-dependent epitope not present in the solid phase dGA.

[0497] The concentration of compound required to inhibit tau-tau binding by 50% is referred to as B 50 value.

[0498] (d) Toxicity in cells - LD50

[0499] The toxicity of the compounds described herein was evaluated using fibroblasts used in the above cell-based assays to assess EC 50 After lysis of the remaining cells, toxicity was measured by the number of cells after 24 hours of exposure to the compound using the lactate dehydrogenase assay kit TOX-7 (Sigma Biosciences) according to the manufacturer's instructions. Alternatively, a kit from Promega UK (Cyto Tox 96) was used again according to the manufacturer's instructions. 50 The concentration of compound that killed 50% of the cells was determined.

[0500] Biological data

[0501]

[0502]

[0503] As shown, compounds were tested in a cellular tau aggregation assay at a single concentration of 2-25 μM, and the results are expressed as the ratio between truncated tau / full-length tau compared to untreated cells. Values ​​of approximately 1.0 are negative, and values ​​<0.5 are positive inhibitors. For some compounds, tau aggregation appeared to be enhanced (where the ratio increased to >2.0). NE, no effect; NT, not tested.

[0504] References

[0505] Many publications are cited above to more fully describe and disclose the present invention and the prior art in the field to which the present invention belongs. The complete citations of these references are provided below. The entire contents of each of these references are incorporated herein.

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Claims

1. A compound having the following general formula: or a pharmaceutically acceptable salt, solvate or hydrate thereof, wherein R N are independently -H or -Me; R X Independently for C 1-4 Alkyl, optionally substituted with halo or hydroxy; -NHR N1 , where R N1 C 1-4 Alkyl; or C 5-10 heteroaryl; R Y Independently: C 5-10 heteroaryl, optionally substituted with methyl, halo or phenyl; or neopentyl.

2. The compound according to claim 1, wherein R X is selected from -Me and -Et, which are optionally substituted with halo or hydroxy.

3. The compound according to claim 2, wherein R X is -Me or -Et, which is substituted by a halogen group.

4. The compound according to claim 2, wherein R X It is -Me substituted by -F or -Cl.

5. The compound according to claim 2, wherein R X It is -CH2F.

6. The compound according to claim 2, wherein R X is -Me or -Et substituted with a hydroxy group.

7. The compound according to claim 2, wherein R X It is -CH2CH2OH.

8. The compound according to claim 1, wherein R X NHR N1 , where R N1 C 1-4 alkyl.

9. The compound according to claim 8, wherein R N1 It is -Me or -Et.

10. The compound according to claim 8, wherein R X is -NHMe.

11. The compound according to claim 1, wherein R X C 5-10 Heteroaryl.

12. The compound according to claim 11, wherein R X Nitrogen-containing C 5-10 Heteroaryl.

13. The compound according to claim 12, wherein R X Indolyl.

14. The compound according to claim 13, wherein R X for 15. A compound according to any one of the preceding claims, wherein R Y It is neopentyl (-CH2C(CH3)3).

16. A compound according to any one of claims 1 to 15, wherein R Y C 5-10 Heteroaryl, which is optionally substituted with methyl, halo or phenyl.

17. The compound according to claim 16, wherein R Y For unsubstituted C 5-10 Heteroaryl.

18. The compound according to claim 17, wherein R Y Nitrogen-containing C 5-10 Heteroaryl, which is optionally substituted with -Cl or phenyl.

19. A compound according to any one of claims 16 to 18, wherein R Y is selected from pyrrolyl, imidazolyl, pyrazolyl, indolyl and benzimidazolyl.

20. The compound according to claim 19, wherein R Y Selected from:

21. A compound according to any one of the preceding claims, wherein R N is -H.

22. A compound according to any one of the preceding claims, wherein R N For-Me.

23. The compound according to claim 1, which is selected from the following compounds:

24. A compound according to any one of the preceding claims for use in a method of treating or preventing tauopathy or a tau aggregation disease.

25. The compound for use according to claim 24, wherein the method is for treating or preventing Alzheimer's disease (AD); Pick's disease; Progressive supranuclear palsy (PSP); Frontotemporal dementia (FTD); FTD and Parkinson's disease linked to chromosome 17 (FTDP-17); Disinhibition-dementia-parkinsonism-amyotrophic complex (DDPAC); Globus pallidus-ponto-nigral degeneration (PPND); Guam-ALS syndrome; Pallid-nigral-Louise degeneration (PNLD); Corticobasal degeneration (CBD); Dementia with argyrophilic particles (AgD); Chronic traumatic encephalopathy (CTE); Down syndrome (DS); Dementia with Lewy bodies (DLB); or mild cognitive impairment (MCI).

26. Use of a compound according to any one of claims 1 to 23 for preparing a medicament for treating or preventing tauopathy or tau aggregation disease.

27. The method of claim 26, wherein the medicament is used to treat or prevent Alzheimer's disease (AD); Pick's disease; progressive supranuclear palsy (PSP); frontotemporal dementia (FTD); FTD and Parkinson's disease linked to chromosome 17 (FTDP-17); disinhibition-dementia-parkinsonism-amyotrophic lateral sclerosis complex (DDPAC); globus pallidus-ponto-nigral degeneration (PPND); Guam-ALS syndrome; pallid-nigral-Louise degeneration (PNLD); corticobasal degeneration (CBD); dementia with argyrophilic particles (AgD); chronic traumatic encephalopathy (CTE); Down syndrome (DS); dementia with Lewy bodies (DLB); or Mild cognitive impairment (MCI).

28. A method for treating or preventing tauopathy or tau aggregation disease, which comprises administering a therapeutically effective amount of a compound as defined in any one of claims 1 to 23 to a patient in need thereof.

29. The method of claim 28, wherein the method is used to treat or prevent Alzheimer's disease (AD); Pick's disease; Progressive supranuclear palsy (PSP); Frontotemporal dementia (FTD); FTD and Parkinson's disease linked to chromosome 17 (FTDP-17); Disinhibition-dementia-parkinsonism-amyotrophic complex (DDPAC); Globus pallidus-ponto-nigral degeneration (PPND); Guam-ALS syndrome; Pallid-nigral-Louise degeneration (PNLD); Corticobasal degeneration (CBD); Dementia with argyrophilic particles (AgD); Chronic traumatic encephalopathy (CTE); Down syndrome (DS); Dementia with Lewy bodies (DLB); or mild cognitive impairment (MCI).

30. A pharmaceutical composition for treating tauopathy or tau aggregation disease, the composition comprising a compound as defined in any one of claims 1 to 23 and a pharmaceutically acceptable excipient, carrier or diluent.

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