A series of substituted-3H-imidazo[4,5-c]pyridines and 1H-pyrrolo[2,3-c]pyridines as modulators of ectonucleotide pyrophosphatase / phosphodiesterase-1 and stimulator of interferon genes for cancer immunotherapy

Abstract

A novel series of substituted ‑3H‑imidazo[4,5‑c]pyridines and 1H‑pyrrolo[2,3‑c]pyridines of ectonucleotide pyrophosphatase / phosphodiesterase‑1 (ENPP1) and related compounds that are useful as inhibitors of ENPP1; synthetic methods for preparing the compounds; pharmaceutical compositions comprising the compounds; and methods of using the compounds and compositions to treat conditions associated with ENPP1 dysfunction.

Description

Background Art

[0001] The ectonucleotide pyrophosphatase / phosphodiesterase (ENPP) family members include seven isoforms, ENPP1-7, which are type II transmembrane glycoproteins or exoenzymes. Mass spectrometry and proteomic analysis from more than 370 protein targets led to the identification of the extracellular protein ENPP1 as one of the hot proteins showing high hydrolytic activity. ATP is the substrate of the identified ENPP1, which is hydrolyzed to AMP and PPi. CD73 converts AMP into adenosine and inorganic phosphate (Pi). Kinetic experimental data show that ENPP1 can hydrolyze ATP. These ectonucleotide enzymes participate in the hydrolysis of pyrophosphate (PPi) and phosphodiester bonds in extracellular nucleotides (such as triphosphates, oligonucleotides), and they produce nucleoside 5'-monophosphates. One of the key isoforms, ENPP1 (plasmacyte membrane glycoprotein-1, PC-1) is involved in many physiological processes, such as development, formation and transportation, as well as pathophysiological conditions. Aberrant ENPP1 expression has been detected in breast cancer relative to normal breast epithelium, supporting its potential for development in bone metastases (occurring in approximately 80% of cases), Hodgkin's lymphoma, hepatocellular carcinoma, follicular lymphoma, glioblastoma, and other malignant tumor tissues.

[0002] Recent reports show that cyclic dinucleotides (CDNs), substrates of ENPP1, stimulate innate immunity through STING-dependent activation of interferon genes. ENPP1 inhibition of STING pathway activation is critical for tumor control, similar to checkpoint inhibitors, such as inhibition of anti-PD-1 or PD-L1, which are promising immunotherapies for various cancers. In addition, mutations in ENPP1 are associated with several conditions, including infantile arterial calcification (generalized arterial calcification in infancy or GACI), ossification of the posterior longitudinal ligament of the spine, and insulin signaling and resistance. ENPP1 expression is high in bone and cartilage, and is involved in lung and kidney fibrosis. It is also found that the expression of ENPP1 is correlated with the grade of astrocytic tumors. Another study reports that ENPP1 is needed to maintain the undifferentiated and proliferative state of glioblastoma stem-like cells. Therefore, ENPP1 is an attractive druggable target for the development of new anticancer, cardiovascular, diabetes, obesity and anti-fibrosis therapies.

[0003] The importance of ENPP1 activity was further studied based on both direct binding assays and in vitro cell efficacy on MDA-MB231 cells. ENPP1 knockdown based on siRNA significantly reduced its catalytic activity in both cell-specific and in vivo experiments. These experiments demonstrated that ENPP1 activity was eliminated after treatment with siRNA. This further supports the effectiveness of this target in certain diseases. It has recently been shown that dithiophosphate analogs of endogenous cGAMP are resistant to hydrolysis by ENPP1 phosphodiesterases, and in particular cyclic dinucleotides (CDNs) are more effective in inducing IFN-β secretion in human THP1 cells by inhibiting ENPP1 activity and simultaneously STING activation responses.

[0004] There is ample evidence that ENPP1 expression is significant in human primary breast tumors relative to normal mammary epithelium, with the highest levels observed in breast-bone metastases. These data support not only a potential role for ENPP1 in breast-bone metastases, but also as a potential prognostic marker for breast cancer. These results from target validation experiments clearly support that the pharmacological role of ENPP1 for the development of novel immunotherapies for cancer is a proposed opportunity to obtain small molecule compound libraries and HTS support.

[0005] Furthermore, ENPP1 activity has also been implicated in diseases caused by bacteria and / or viruses, and thus modulators of ENPP1 may be useful in treating bacterial and / or viral diseases and conditions. Summary of the invention

[0006] In one aspect, the present invention relates to compounds of formula I:

[0007]

[0008] in

[0009] Z is -H or -N;

[0010] Y is

[0011] n is 0 or 1;

[0012] X1 is -C=O or -NH,

[0013] X2 is -C=O or -NH, provided that if X1 is -C=O, then X2 is -NH; and if X1 is -NH, then X2 is -C=O;

[0014] R1 is selected from the group consisting of: -H, halogen, -OCF3, -CF3, -CN, OCH3 and -CH3;

[0015] R2 is selected from the group consisting of: -H, -NH2, -OH and -CH3;

[0016] R3 is selected from the group consisting of: -H, -NH2, -OH and -CH3;

[0017] R4 is selected from the group consisting of: -H, -CH2-CH3 and -CH3;

[0018] R5 is selected from the group consisting of:

[0019] Wherein R5 can be further substituted by one or more R6, and wherein each R6 is independently selected from the group consisting of: -OCH3, OH, F, Cl, -CH3, O-CH2-CH3, OCH2-CF3, OCH2-CF3OH, OCHF2, ONa, S(O)2, S(O)2NH2,

[0020] or an isomer or a pharmaceutically acceptable salt thereof.

[0021] In a preferred embodiment,

[0022] Z is -N;

[0023] n is 0;

[0024] X1 is -C=O;

[0025] X2 is -NH;

[0026] R1 is selected from the group consisting of halogen and -CN;

[0027] R2 is selected from the group consisting of -H and -CH3;

[0028] R3 is -H;

[0029] R4 is -H;

[0030] R5 is and

[0031] R6 is selected from the group consisting of -OCH3 and OH.

[0032] In another preferred embodiment, R1 is selected from the group consisting of -Cl and -CN.

[0033] Examples of provided compounds include:

[0034]

[0035]

[0036]

[0037]

[0038] as well as

[0039]

[0040] The present invention also includes hydrates, solvates, polymorphs, isomers, tautomers, pharmaceutically acceptable salts of the compounds, and pharmaceutically acceptable salts of the tautomers.

[0041] The present invention also provides pharmaceutical formulations, medicaments comprising the compounds, methods of preparing the pharmaceutical formulations, medicaments, compounds, and methods of treating patients with the provided pharmaceutical formulations and compounds.

[0042] Compounds of the invention were identified by structure-based, computational docking and binding free energy.

[0043] Also disclosed are pharmaceutical compositions comprising a therapeutically effective amount of a disclosed compound and a pharmaceutically acceptable carrier.

[0044] Also disclosed are synthetic methods for preparing the disclosed compounds. In additional aspects, disclosed are products of the disclosed synthetic methods.

[0045] Also disclosed are methods for treating a disorder associated with ENPP1 activity dysfunction in a mammal, comprising the step of administering to the mammal a therapeutically effective amount of a disclosed compound, or a pharmaceutically acceptable salt, tautomer, isomer, hydrate, solvate or polymorph thereof.

[0046] Also disclosed is a method for inhibiting ENPP1 activity in a mammal, comprising the step of administering to the mammal a therapeutically effective amount of at least one disclosed compound, or a pharmaceutically acceptable salt, tautomer, isomer, hydrate, solvate or polymorph thereof.

[0047] Also disclosed are methods for inhibiting ENPP1 activity of at least one cell, comprising the step of contacting the at least one cell with an effective amount of at least one disclosed compound, or a pharmaceutically acceptable salt, tautomer, isomer, hydrate, solvate, or polymorph thereof.

[0048] Also disclosed is a method for treating a condition associated with ENPP1 activity dysfunction in a mammal by eliciting an immunotherapy response in the mammal, the method comprising administering to the mammal a therapeutically effective amount of a disclosed compound, or a pharmaceutically acceptable salt, tautomer, isomer, hydrate, solvate or polymorph thereof, wherein the compound induces an immunotherapy response that is beneficial in treating the condition associated with ENPP1 activity. Such a condition may be, but is not limited to, any type of cancer or any disease caused by bacteria and / or viruses involving ENPP1 activity.

[0049] Also disclosed are pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an effective amount of a disclosed compound, or a pharmaceutically acceptable salt, tautomer, isomer, hydrate, solvate, or polymorph thereof.

[0050] Also disclosed are kits comprising at least one disclosed compound, or a pharmaceutically acceptable salt, tautomer, isomer, hydrate, solvate, or polymorph thereof.

[0051] Also disclosed are methods for making a medicament comprising combining at least one disclosed compound or at least one disclosed product with a pharmaceutically acceptable carrier or diluent. In a further aspect, the invention relates to the use of the disclosed compounds in the manufacture of a medicament for treating a condition associated with ENPP1 activity dysfunction. In a further aspect, the invention relates to the use of the disclosed compounds in the manufacture of a medicament for treating a condition of uncontrolled cell proliferation.

[0052] Also disclosed is the use of a disclosed compound or a disclosed product in the manufacture of a medicament for treating a disorder associated with ENPP1 dysfunction in a mammal.

[0053] Although aspects of the invention may be described and claimed in specific statutory categories, such as the system statutory category, this is for convenience only, and those skilled in the art will understand that each aspect of the invention may be described and claimed in any statutory category. Unless expressly stated otherwise, it is not intended that any method or aspect described herein be interpreted as requiring that its steps be performed in a particular order. Therefore, in the event that a method claim does not specifically state in the claim or specification that the steps are limited to a particular order, in no way is it intended to infer an order in any respect. This applies to any possible non-express basis for interpretation, including matters regarding the logic of the arrangement of steps or operational flow, ordinary meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1is a graph of ENPP1 inhibition assay of TMP for some of the compounds of the present invention.

[0055] Figure 2A This is a diagram of a positive control in an ENPP2 inhibition assay.

[0056] Figure 2B is a graph of ENPP2 inhibition assay of some of the compounds of the present invention.

[0057] Figure 3 It is a graph of ENPP1 thermal shift measurement of the compounds of the present invention.

[0058] Figure 4 is a bar graph of ENPP1 mineralization assays for some of the compounds of the present invention.

[0059] Figure 5A is a graph of ENPP1 inhibition assay of TMP for some of the compounds of the present invention.

[0060] Figure 5B is a graph of ENPP1 inhibition assay with ATP for some of the compounds of the present invention.

[0061] Fig. 6A is a graph of B16F10 tumor volume versus time following in vivo administration for some of the compounds of the invention.

[0062] Figure 6B is a graph of B16F10 tumor volume versus time following in vivo administration for some of the compounds of the invention.

[0063] Figure 7 It is a graph of ENPP1 thermal shift measurement of the compounds of the present invention.

[0064] Figure 8 is a graph of ENPP1 inhibition assay of TMP for some of the compounds of the present invention.

[0065] Fig. 9 are photographs of ENPP1 mineralization assays of some of the compounds of the present invention.

[0066] Fig.10 is a bar graph of a 24 hour THP1 IRF-3 reporter gene assay for compounds of the invention.

[0067] Fig.11 is a bar graph of a 72 hour THP1 IRF-3 reporter gene assay for compounds of the invention.

[0068] Fig. 12Aare immunofluorescence images showing the localization of phospho-IRF3 in MDA-MB-231 cells.

[0069] Fig. 12B Is a bar graph of interferon-stimulated reporter genes in cells treated with compounds of the invention.

[0070] Fig. 12C is a bar graph of IFNβ expression in cells treated with compounds of the present invention.

[0071] Fig.12D is a bar graph of ISG15 expression in cells treated with compounds of the invention.

[0072] Fig.13A is a tumor growth inhibition curve of one of the compounds of the present invention after in vivo administration.

[0073] Fig. 13B is a tumor growth inhibition curve of one of the compounds of the present invention after in vivo administration.

[0074] Fig.14 is a tumor growth inhibition curve of one of the compounds of the present invention after in vivo administration.

[0075] Fig.15A is a pharmacokinetic (PK) plasma concentration profile of one of the compounds of the present invention.

[0076] Fig. 15B is a tumor growth inhibition curve of one of the compounds of the present invention after in vivo administration.

[0077] Fig. 15C is a flow cytometric graph of a tumor excised after administration of one of the compounds of the present invention.

[0078] Fig.15D is a flow cytometric graph of a tumor excised after administration of one of the compounds of the present invention. DETAILED DESCRIPTION

[0079] A. Definition

[0080] As used herein, the nomenclature of compounds, including organic compounds, may be given using common names, IUPAC, IUBMB, or CAS nomenclature suggestions. When one or more stereochemical features are present, the Cahn-Ingold-Prelog rules for stereochemistry may be employed to assign stereochemical priority, E / Z specifications, etc. Given a name, one skilled in the art can readily determine the structure of the compound by systematic reduction of the compound structure using the naming convention or by commercially available software such as ChemDraw. TM (Cambridge Software, USA).

[0081] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a functional group," "an alkyl," or "a residue" includes a mixture of two or more such functional groups, alkyl groups, or residues, etc.

[0082] In this article, ranges can be expressed as from "about" a specific value and / or to "about" another specific value. When expressing such a range, additional aspects include from a specific value and / or to another specific value. Similarly, when a value is expressed as an approximation by using the antecedent "about", it should be understood that the specific value forms additional aspects. It should be further understood that the endpoints of each range in the range are important relative to the other endpoint and independently of the other endpoint. It should also be understood that multiple values ​​are disclosed herein, and each value is also disclosed herein as "about" for the specific value in addition to the value itself. For example, if the value "10" is disclosed, "about 10" is also disclosed. It should also be understood that each unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13 and 14 are also disclosed.

[0083] References in the specification and the subsequent claims to parts by weight of a particular element or component in a composition denote the weight relationship between that element or component and any other elements or components in the composition or article, expressed as parts by weight. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight of component Y, X and Y are present in a weight ratio of 2:5, and are present in this ratio regardless of whether the compound contains additional components.

[0084] Unless specifically stated to the contrary, the weight percent (wt. %) of a component is based on the total weight of the formulation or composition in which the component is included.

[0085] As used herein, the term "ENPP1" refers to ectonucleotide pyrophosphatase / phosphodiesterase.

[0086] As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0087] As used herein, the term "subject" can be a vertebrate, such as a mammal, fish, bird, reptile or amphibian. Therefore, the subject of the method disclosed herein can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The term does not indicate a specific age or gender. Therefore, it is intended to cover adult and newborn subjects and fetuses, whether male or female. In one aspect, the subject is a mammal. A patient refers to a subject suffering from a disease or condition. The term "patient" includes human and livestock subjects. In some aspects of the disclosed method, prior to the administering step, the subject has been diagnosed with a condition of uncontrolled cell proliferation associated with ENPP1 dysfunction. In some aspects of the disclosed method, prior to the administering step, the subject has been diagnosed with a need to inhibit ENPP1.

[0088] As used herein, the term "treatment" refers to the medical management of a patient, intended to cure, improve, stabilize or prevent a disease, pathological condition or illness. This term includes active treatment, i.e., treatment specifically for the improvement of a disease, pathological condition or illness, and also includes etiological treatment, i.e., treatment for the removal of the cause of the disease, pathological condition or illness. In addition, this term includes palliative treatment, i.e., treatment designed to relieve symptoms rather than cure a disease, pathological condition or illness; preventive treatment, i.e., treatment for minimizing or partially or completely inhibiting the development of a disease, pathological condition or illness; and supportive treatment, i.e., treatment for another specific therapy for supplementing the improvement of a disease, pathological condition or illness. In various aspects, the term encompasses any treatment of a subject, including mammals (e.g., humans), and includes: (i) preventing the disease from occurring in a subject who may be prone to the disease but has not yet been diagnosed as having the disease; (ii) inhibiting the disease, i.e., curbing its development; or (iii) alleviating the disease, even if the disease subsides. On the one hand, the subject is a mammal, such as a primate, and in another aspect, the subject is a human. The term "subject" also includes domestic animals (eg, cats, dogs, etc.), livestock (eg, cows, horses, pigs, sheep, goats, etc.), and laboratory animals (eg, mice, rabbits, rats, guinea pigs, fruit flies, zebrafish, etc.).

[0089] As used herein, the term "prevent" or "preventing" means to exclude, avoid, eliminate, stop, stop or hinder something from happening, especially by preemptive action. It should be understood that unless otherwise expressly indicated, in the case of reducing, inhibiting or preventing as used herein, the use of the other two words is also expressly disclosed.

[0090] As used herein, the term "diagnosed" means having been subjected to a physical examination by a technician (e.g., a physician) and found to have a condition that can be diagnosed or treated by a compound, composition, or method disclosed herein. For example, "diagnosed with a disorder of uncontrolled cell proliferation" means having been subjected to a physical examination by a technician (e.g., a physician) and found to have a condition that can be diagnosed or treated by a compound or composition that can inhibit ENPP1. As another example, "diagnosed as requiring inhibition of ENPP1" means having been subjected to a physical examination by a technician (e.g., a physician) and found to have a condition characterized by ENPP1 dysfunction. As discussed herein, such a diagnosis can refer to a condition such as uncontrolled cell proliferation, cancer, and the like. For example, "diagnosed as requiring treatment of one or more conditions of uncontrolled cell proliferation associated with ENPP1 dysfunction" means having been subjected to a physical examination by a technician (e.g., a physician) and found to have one or more conditions of uncontrolled cell proliferation associated with ENPP1 dysfunction.

[0091] As used herein, the phrase "identified as requiring treatment of a condition" and the like refers to selecting a subject based on the need for treatment of the condition. For example, based on an early diagnosis by a technician, a subject may be identified as requiring treatment of a condition (e.g., a condition associated with dysfunction of ENPP1) and thereafter undergo treatment for the condition. It is contemplated that, in one aspect, the identification may be performed by a person different from the person making the diagnosis. It is also contemplated that, in other aspects, administration may be performed by a person who subsequently performs the administration.

[0092] As used herein, the terms "administering" and "administration" refer to any method of providing a pharmaceutical formulation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, intraocular administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, oral administration, intraurethral administration, and parenteral administration, including injection, such as intravenous administration, intraarterial administration, intramuscular administration, and subcutaneous administration. Administration may be continuous or intermittent. In various aspects, the formulation may be administered therapeutically; that is, the formulation is administered to treat an existing disease or condition. In various other aspects, the formulation may be administered prophylactically; that is, it is administered to prevent a disease or condition.

[0093] The term "contacting" as used herein means bringing a disclosed compound and a cell, target receptor or other biological entity together in such a way that the compound can directly or indirectly affect the activity of the target (e.g., receptor, cell, etc.); i.e., by interacting with the target itself; i.e., by interacting with another molecule, cofactor, factor or protein on which the activity of the target depends.

[0094] As used herein, the terms "effective amount" and "amount effective" refer to an amount sufficient to achieve a desired result or have an effect on an undesirable condition. For example, a "therapeutically effective amount" refers to an amount sufficient to achieve a desired therapeutic result or have an effect on an undesirable symptom but generally insufficient to cause adverse side effects. The specific therapeutically effective dosage level for any particular patient will depend on a variety of factors, including the condition being treated and the severity of the condition; the specific composition employed; the patient's age, weight, general health, sex and diet; the time of administration; the route of administration; the excretion rate of the specific compound employed; the duration of treatment; the drugs used in combination or concomitantly with the specific compound employed, and similar factors well known in the medical field. For example, it is entirely within the technical scope of the art to start using a compound at a dosage level lower than the dosage level required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. If necessary, for the purpose of administration, the effective daily dose can be divided into multiple doses. Therefore, a single dosage composition can contain such an amount or a submultiple thereof to constitute a daily dose. In the case of any contraindications, the dosage can be adjusted by the individual physician. The dosage can vary and can be administered once or multiple times a day for one or several days. Guidance on appropriate dosages for a given class of pharmaceutical products can be found in the literature.In further various aspects, the formulations may be administered in a "prophylactically effective amount"; that is, an amount effective for preventing a disease or condition.

[0095] As used herein, “EC 50 " is intended to refer to the concentration of a substance (e.g., a compound or drug) required for 50% agonism or activation of a biological process or component of a process, including a protein, subunit, organelle, ribonucleoprotein, etc. In one aspect, EC 50 Can refer to the concentration of a substance required for 50% agonism or activation in vivo, as further defined elsewhere herein. In a further aspect, EC 50 Refers to the concentration of agonist or activator that elicits a response halfway between the baseline and maximal response.

[0096] As used herein, “IC 50" is intended to refer to the concentration of a substance (e.g., a compound or drug) required for 50% inhibition of a biological process or component of a process, including a protein, subunit, organelle, ribonucleoprotein, etc. For example, IC 50 It may refer to the concentration of a substance required for 50% inhibition in vivo or inhibition measured in vitro, as further defined elsewhere herein. Alternatively, IC 50 Refers to the half-maximal (50%) inhibitory concentration (IC) of a substance. Inhibition can be measured in cell lines such as AN3 CA, BT-20, BT-549, HCT 116, HER218, MCF7, MDA-MB-231, MDA-MB-235, MDA-MB-435S, MDA-MB-468, PANC-1, PC-3, SK-N-MC, T-47D, and U-87MG.

[0097] The term "pharmaceutically acceptable" describes a material that is not biologically or otherwise undesirable, ie, does not cause unacceptable levels of undesired biological effects or interact in a deleterious manner.

[0098] As used herein, the term "stable" refers to compounds that are not substantially altered when subjected to conditions that allow them to be produced, detected, and in some aspect recovered, purified, and used for one or more of the purposes disclosed herein.

[0099] As used herein, the term "derivative" refers to a compound whose structure is derived from the structure of a parent compound (e.g., a compound disclosed herein), and whose structure is sufficiently similar to that disclosed herein, and based on this similarity, a person skilled in the art would expect the compound to exhibit the same or similar activity and utility as the claimed compound, or to induce the same or similar activity and utility as a precursor to the claimed compound. Exemplary derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of the parent compound.

[0100] As used herein, the term "pharmaceutically acceptable carrier" refers to a sterile aqueous or non-aqueous solution, dispersion, suspension or emulsion, and a sterile powder for reconstitution into a sterile injectable solution or dispersion before use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethyl cellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters, such as ethyl oleate. Appropriate fluidity can be maintained by, for example, using a coating material (such as lecithin) in the case of a dispersion by maintaining the required particle size and by using a surfactant. These compositions can also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. Various antibacterial and antifungal agents can be included, such as parabens, chlorobutanol, phenol, sorbic acid, etc. to ensure the prevention of microbial effects. It can also be desirable to include isotonic agents, such as sugar, sodium chloride, etc. Prolonged absorption of injectable drug forms can be achieved by containing agents that delay absorption (such as aluminum monostearate and gelatin). Injectable reservoir-type (depot) forms are prepared by forming a microcapsule matrix of the drug in a biodegradable polymer, such as polylactide-polyglycolide, poly(orthoester) and poly(anhydride). According to the ratio of the drug to the polymer and the properties of the specific polymer used, the rate of drug release can be controlled. Depot-type injectable formulations are also prepared by trapping the drug in liposomes or microemulsions compatible with body tissues. Injectable formulations can be sterilized, for example, by filtering through a bacterial retention filter, or by incorporating a sterilizing agent in the form of a sterile solid composition, which can be dissolved or dispersed in sterile water or other sterile injectable media before use. Suitable inert carriers can include sugars, such as lactose. Desirably, the effective particle size of at least 95% by weight of the active ingredient particles is within the range of 0.01 to 10 microns.

[0101] As used in the specification and in the claims which follow, a residue of a chemical species refers to a moiety that is the resultant product of the chemical species in a particular reaction scheme or subsequent formulation or chemical product, regardless of whether the moiety is actually obtained from the chemical species. Thus, an ethylene glycol residue in a polyester refers to one or more -OCH2CH2O- units in the polyester, regardless of whether ethylene glycol was used to prepare the polyester. Similarly, a sebacic acid residue in a polyester refers to one or more -CO(CH2)8CO- moieties in the polyester, regardless of whether the residue was obtained by reacting sebacic acid or an ester thereof to obtain the polyester.

[0102] As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad sense, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. For appropriate organic compounds, permissible substituents may be one or more and may be the same or different. For the purposes of this disclosure, heteroatoms (such as nitrogen) may have hydrogen substituents and / or any permissible substituents of organic compounds described herein, which will satisfy the valence of heteroatoms. This disclosure is not intended to be limited in any way to permissible substituents of organic compounds. Moreover, the term "substituted" or "substituted by ... " includes implicit conditions: this substitution is consistent with the permissible valence of the substituted atom and substituent, and the substitution results in a stable compound, such as a compound that does not spontaneously undergo transformation (such as by rearrangement, cyclization, elimination, etc.). It is also contemplated that, in certain aspects, individual substituents may be further optionally substituted (ie, further substituted or unsubstituted) unless expressly indicated to the contrary.

[0103] In defining various terms, “A 1 ”, “A 2 ”, “A 3 ” and “A 4 " are used herein as general symbols to represent various specific substituents. These symbols can be any substituent, not limited to those disclosed herein, and when these symbols are defined as certain substituents in one instance, they can be defined as some other substituents in another instance.

[0104] As used herein, the term "alkyl" is a branched or unbranched saturated hydrocarbon group having 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, etc. The alkyl group can be cyclic or acyclic. The alkyl group can be branched or unbranched. The alkyl group can also be substituted or unsubstituted. For example, the alkyl group can be substituted by one or more groups, including but not limited to alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxyl, nitro, silyl, sulfo-oxo or mercaptan, as described herein. "Low alkyl" is an alkyl group containing one to six (e.g., one to four) carbon atoms.

[0105] For example, "C1-C3 alkyl" can be selected from methyl, ethyl, n-propyl, isopropyl and cyclopropyl, or a subset thereof. In some aspects, "C1-C3 alkyl" can optionally be further substituted. As another example, "C1-C4 alkyl" can be selected from methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl and cyclobutyl, or a subset thereof. In some aspects, "C1-C4 alkyl" can optionally be further substituted. As another example, "C1-C6 alkyl" can be selected from methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, n-pentyl, isopentyl, sec-pentyl, tert-pentyl, neopentyl, cyclopentyl, n-hexyl, isohexyl, 3-methylpentane, 2,3-dimethylbutane, neohexane and cyclohexane, or a subset thereof. In some aspects, "C1-C6 alkyl" can be optionally further substituted. As another example, "C1-C8 alkyl" can be selected from methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, isobutyl, cyclobutyl, n-pentyl, isopentyl, sec-pentyl, tert-pentyl, neopentyl, cyclopentyl, n-hexyl, isohexyl, 3-methylpentane, 2,3-dimethylbutane, neohexane, cyclohexane, heptane, cycloheptane, octane and cyclooctane, or selected from a subset thereof. In some aspects, "C1-C8 alkyl" can be optionally further substituted. As another example, "C1-C8 alkyl" can be selected from methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, isobutyl, cyclobutyl, n-pentyl, isopentyl, sec-pentyl, tert-pentyl, neopentyl, cyclopentyl, n-hexyl, isohexyl, 3-methylpentane, 2,3-dimethylbutane, neohexane, cyclohexane, heptane, cycloheptane, octane and cyclooctane, or selected from a subset thereof. As another example, "C1-C12 alkyl" can be selected from methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, isobutyl, cyclobutyl, n-pentyl, isopentyl, sec-pentyl, tert-pentyl, neopentyl, cyclopentyl, n-hexyl, isohexyl, 3-methylpentane, 2,3-dimethylbutane, neohexane, cyclohexane, heptane, cycloheptane, octane, cyclooctane, nonane, cyclononane, decane, cyclodecane, undecane, cycloundecane, dodecane and cyclododecane, or a subset thereof. In certain aspects, "C1-C12 alkyl" can optionally be further substituted.

[0106] Throughout this specification, "alkyl" is generally used to refer to both unsubstituted alkyl and substituted alkyl; however, substituted alkyl is also specifically referred to herein by identifying one or more specific substituents on the alkyl. For example, the term "halogenated alkyl" or "haloalkyl" specifically refers to an alkyl substituted with one or more halides (e.g., fluoride, chloride, bromide, or iodide). The term "alkoxyalkyl" specifically refers to an alkyl substituted with one or more alkoxy groups, as described below. The term "alkylamino" specifically refers to an alkyl substituted with one or more amino groups, etc., as described below. When "alkyl" is used in one case and a specific term such as "alkyl alcohol" is used in another case, this is not meant to imply that the term "alkyl" also does not refer to a specific term such as "alkyl alcohol", etc.

[0107] This practice also applies to other groups described herein. That is, although terms such as "cycloalkyl" refer to both unsubstituted and substituted cycloalkyl moieties, the substituted moieties may be specifically identified in addition herein; for example, a specific substituted cycloalkyl may be referred to as, for example, "alkylcycloalkyl". Similarly, a substituted alkoxy may be specifically referred to as, for example, "haloalkoxy", a specific substituted alkenyl may be, for example, "alkenyl alcohol", etc. Again, the practice of using a general term such as "cycloalkyl" and a specific term such as "alkylcycloalkyl" is not meant to imply that the general term also does not include the specific term.

[0108] As used herein, term "cycloalkyl" is a non-aromatic carbon-based ring consisting of at least three carbon atoms. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, etc. Term "heterocycloalkyl" is a type of cycloalkyl as defined above, and is included in the meaning of term "cycloalkyl", wherein at least one carbon atom in the carbon atom of the ring is replaced by a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur or phosphorus. Cycloalkyl and heterocycloalkyl can be substituted or unsubstituted. Cycloalkyl and heterocycloalkyl can be substituted by one or more groups, such as, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxyl, nitro, silyl, sulfo-oxo, nitrile, sulfonamide or thiol, as described herein.

[0109] As used herein, the term "aryl" is a group containing any carbon-based aromatic group, including but not limited to benzene, naphthalene, phenyl, biphenyl, phenoxybenzene, etc. The term "aryl" also includes "heteroaryl", which is defined as a group containing an aromatic group, and the aromatic group has at least one heteroatom incorporated into the ring of the aromatic group. Examples of heteroatoms include but are not limited to nitrogen, oxygen, sulfur and phosphorus. Similarly, the term "non-heteroaryl", which is also included in the term "aryl", defines a group containing an aromatic group that does not contain heteroatoms. The aryl group can be substituted or unsubstituted. The aryl group can be substituted by one or more groups, and the one or more groups include but are not limited to alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxyl, ketone, azide, nitro, silyl, sulfo-oxo, nitrile, sulfonamide or thiol, as described herein. The term “biaryl” is a specific type of aryl group and is included in the definition of “aryl.” A biaryl group refers to two aryl groups bound together by a fused ring structure as in naphthalene or linked by one or more carbon-carbon bonds as in biphenyl.

[0110] As used herein, the terms "halogen", "halide" and "halo" refer to the halogens fluorine, chlorine, bromine and iodine. It is also contemplated that, in various aspects, the halogen may be selected from fluorine, chlorine, bromine and iodine. For example, the halogen may be selected from fluorine, chlorine and bromine. As a further example, the halogen may be selected from fluorine and chlorine. As a further example, the halogen may be selected from chlorine and bromine. As a further example, the halogen may be selected from bromine and iodine. As a further example, the halogen may be selected from chlorine, bromine and iodine. In one aspect, the halogen may be fluorine. In another aspect, the halogen may be chlorine. In still another aspect, the halogen is bromine. In yet another aspect, the halogen is iodine.

[0111] It is also contemplated that, in some aspects, pseudo-halogens (e.g., trifluoromethanesulfonate, mesylate, toluenesulfonate, brosylate, etc.) may be used in place of halogens. For example, in some aspects, halogens may be replaced by pseudo-halogens. As another example, pseudo-halogens may be selected from trifluoromethanesulfonate, mesylate, toluenesulfonate, and brosylate. On the one hand, pseudo-halogens are trifluoromethanesulfonates. In another aspect, pseudo-halogens are mesylates. In another aspect, pseudo-halogens are toluenesulfonates. In another aspect, pseudo-halogens are brosylates.

[0112] As used herein, the term "heterocycle" refers to monocyclic and polycyclic aromatic or non-aromatic ring systems in which at least one of the ring members is not carbon. The heterocyclic ring includes azetidine, dioxane, furan, imidazole, isothiazole, isoxazole, morpholine, oxazole, oxazole (including 1,2,3-oxadiazole, 1,2,5-oxadiazole and 1,3,4-oxadiazole), piperazine, piperidine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolidine, tetrahydrofuran, tetrahydropyran, tetrazine (including 1,2,4,5-tetrazine), tetrazole (including 1,2,3,4-tetrazole and 1,2,4,5-tetrazole), thiadiazole (including 1,2,3-thiadiazole, 1,2,5-thiadiazole and 1,3,4-thiadiazole), thiazole, thiophene, triazine (including 1,3,5-triazine and 1,2,4-triazine), triazole (including 1,2,3-triazole, 1,3,4-triazole) and the like.

[0113] The term "hydroxyl" as used herein is represented by the formula -OH.

[0114] As used herein, “R 1 ”, “R 2 ”, “R 3 ”, “R n " ”(where n is an integer) may independently have one or more of the groups listed above. For example, if R 1If the first group is a straight chain alkyl group, one of the hydrogen atoms of the alkyl group may be optionally substituted by a hydroxyl group, an alkoxy group, an alkyl group, a halide, etc. Depending on the selected group, the first group may be incorporated into the second group, or alternatively, the first group may be a side group (i.e., connected) of the second group. For example, for the phrase "an alkyl group including an amino group", the amino group may be incorporated into the main chain of the alkyl group. Alternatively, the amino group may be connected to the main chain of the alkyl group. The nature of the selected one or more groups will determine whether the first group is embedded in the second group or connected to the second group.

[0115] As described herein, the compounds of the present invention may contain "optionally substituted" moieties. In general, the term "substituted", whether or not preceded by the term "optionally", means that one or more hydrogens of the specified moiety are replaced by suitable substituents. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted by more than one substituent selected from a specified group, the substituent may be the same or different at each position. The substituent combinations contemplated by the present invention are preferably substituent combinations that form stable or chemically feasible compounds. It is also contemplated that, in certain aspects, unless expressly indicated to the contrary, individual substituents may be further optionally substituted (i.e., further substituted or unsubstituted).

[0116] The compounds described herein may contain one or more double bonds and may therefore give rise to cis / trans (E / Z) isomers, as well as other conformational isomers. Unless otherwise specified, the present invention encompasses all such possible isomers, as well as mixtures of such isomers.

[0117] Unless otherwise stated, formulas with chemical bonds shown only as solid lines, rather than wedges or dashed lines, encompass every possible isomer, such as every enantiomer and diastereomer, as well as mixtures of isomers, such as racemic mixtures or scalemic mixtures. The compounds described herein may contain one or more asymmetric centers, and thus may produce diastereomers and optical isomers. Unless otherwise stated, the present invention includes all of these possible diastereomers and racemic mixtures thereof, their substantially pure resolved enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts thereof. Mixtures of stereoisomers are also included, as well as isolated specific stereoisomers. During the course of synthetic processes for preparing such compounds, or using racemization or epimerization processes known to those skilled in the art, the products of these processes may be mixtures of stereoisomers.

[0118] Many organic compounds exist in optically active forms with the ability to rotate the plane of plane polarized light. When describing optically active compounds, prefixes D and L or R and S are used to represent the absolute configuration of the molecule around one or more chiral centers. The prefixes d and / or (+) and (-) are used to represent the rotation sign of the compound to plane polarized light, where (-) or / represent that the compound is left-handed. The compound prefixed with (+) or d is right-handed. For a given chemical structure, these compounds referred to as stereoisomers are identical, except that they are mirror images that cannot be superimposed on each other. Specific stereoisomers may also be referred to as enantiomers, and mixtures of such isomers are commonly referred to as enantiomeric mixtures. A 50:50 mixture of enantiomers is referred to as a racemic mixture.

[0119] Many compounds described herein can have one or more chiral centers, and therefore can exist in different enantiomeric forms. If desired, a chiral carbon can be represented by an asterisk (*). When the bond to the chiral carbon is depicted as a straight line in the disclosed formula, it should be understood that both (R) and (S) configurations of the chiral carbon, and therefore both enantiomers and mixtures thereof are included in the formula. As used in the art, when it is desired to specify the absolute configuration about the chiral carbon, one of the bonds to the chiral carbon can be depicted as a wedge (bond to the atom above the plane), and the other can be depicted as a series or wedge (bond to the atom below the plane) of short parallel lines. The Cahn-Inglod-Prelog system can be used to assign (R) or (S) configurations to chiral carbons.

[0120] The compounds described herein include atoms in both their natural isotopic abundance and in unnatural abundance. The disclosed compounds may be isotopically labeled or isotopically substituted compounds, which are identical to those described, but for the fact that one or more atoms are replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as 2 H. 3 H. 13 C. 14 C. 15 N. 18 O. 17 O. 35 S. 18 F and 36 The compounds further include prodrugs thereof, and pharmaceutically acceptable salts of the compounds or prodrugs containing the above-mentioned isotopes and / or other isotopes of other atoms are within the scope of the present invention. Certain isotopically labeled compounds of the present invention (e.g., in which a radioactive isotope is incorporated, such as 3 H and14 C) are useful in drug and / or substrate tissue distribution assays. Tritiated isotopes (i.e., 3 H) and carbon-14 isotopes (i.e., 14 C) is particularly preferred due to its ease of preparation and detectability. Further, the use of heavier isotopes, such as deuterium (i.e., 2 H) can offer certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and thus may be preferred in some circumstances. Isotopically labeled compounds of the invention and prodrugs thereof can generally be prepared by carrying out the following procedure: substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.

[0121] The compounds described in the present invention can exist as solvates. In some cases, the solvent for preparing solvates is an aqueous solution, and therefore solvates are generally referred to as hydrates. Compounds can exist as hydrates, and the hydrates can be obtained, for example, by crystallization from a solvent or an aqueous solution. In this regard, one, two, three or any arbitrary number of solvates or water molecules can be combined with compounds according to the present invention to form solvates and hydrates. Unless otherwise indicated, the present invention includes all these possible solvates.

[0122] It is also understood that certain compounds described herein may exist as an equilibrium of tautomers. For example, a ketone with an α-hydrogen may exist in an equilibrium of a keto form and an enol form.

[0123]

[0124] Likewise, amides having an N-hydrogen may exist in equilibrium with the amide form and the imidic acid form. Unless stated to the contrary, the present invention encompasses all such possible tautomers.

[0125] Chemical substances are known to form solids that exist in different sequential states, which are referred to as polymorphic forms or modifications. Different modifications of polymorphic materials may differ greatly in their physical properties. Compounds according to the present invention may exist in different polymorphic forms, wherein a particular modification may be metastable. Unless otherwise indicated, the present invention encompasses all these possible polymorphic forms.

[0126] In some aspects, the structure of the compound can be represented by the following formula:

[0127]

[0128] It is understood to be equivalent to the following formula:

[0129]

[0130] where n is usually an integer. That is, R n It is understood to represent five independent substituents, R n(a) , R n(b) , R n(c) , R n(d) , R n(e) "Independent substituents" means that each R substituent can be independently defined. For example, if in one example R n(a) is halogen, then in the example described, R n(b) Not necessarily halogen.

[0131] Certain materials, compounds, compositions and components disclosed herein are either commercially available or readily synthesized using techniques generally known to those skilled in the art. For example, starting materials and reagents used in preparing the disclosed compounds and compositions are available from commercial suppliers such as Sigma-Aldrich Chemical Co. (Milwaukee, Wisconsin), Acros Organics (Morris Plains, New Jersey), Fisher Scientific (Pittsburgh, Pennsylvania), or Sigma (St. Louis, Missouri) or are prepared by methods known to those skilled in the art following the procedures set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis, Vols. 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Vols. 1-5 and Supplements (Elsevier Science Press, 1991); Publishers, 1989); Organic Reactions, Vols. 1-40 (John Wiley & Sons, 1991); March's Advanced Organic Chemistry, (John Wiley & Sons, 4th ed.); and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989).

[0132] Unless otherwise expressly stated, it is not intended that any method described herein be construed as requiring that its steps be performed in a particular order. Therefore, in the event that a method claim does not actually recite the order in which its steps are to be followed or the steps are not otherwise specifically stated in the claims or specification to be limited to a particular order, no order is intended to be inferred in any respect. This applies to any possible non-express basis for interpretation, including: matters of logic regarding arrangement of steps or operational flow; ordinary meaning derived from grammatical organization or punctuation; and the number or type of embodiments described in the specification.

[0133] Disclosed are components for preparing the composition of the present invention and the composition itself used in the method disclosed herein.These and other materials are disclosed herein, and it should be understood that when the combination, subset, interaction, group, etc. of these materials are disclosed, although it is impossible to explicitly disclose specific references to each of the various individual and collective combinations and arrangements of these compounds, each combination and arrangement is specifically envisioned and described herein.For example, if a specific compound is disclosed and discussed, and many modifications that may be made to a plurality of molecules comprising the compound are discussed, then unless specifically indicated to the contrary, each combination and arrangement and possible modifications in the compound are specifically covered.Therefore, if a class of molecules A, B and C and a class of molecules D, E and F are disclosed, and the example of the combined molecule AD is disclosed, then even if each is not narrated individually, it is still individually and collectively envisioned, thereby meaning that it is considered to disclose combinations AE, AF, BD, BE, B-F, CD, CE and CF.Similarly, any subset or combination of these is also disclosed.Therefore, for example, it is considered to disclose a subgroup of AE, B-F and CE. This concept applies to all aspects of the present application, including but not limited to steps in the method of making and using the composition of the present invention. Therefore, if there are various additional steps that can be performed, it should be understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the method of the present invention.

[0134] It is understood that the compositions disclosed herein have certain functions. Certain structural requirements for performing the disclosed functions are disclosed herein, and it is understood that there are multiple structures that can perform the same function related to the disclosed structures, and these structures will generally achieve the same result.

[0135] B. Compounds

[0136] In one aspect, the present invention relates to compounds useful as ENPP1 inhibitors. In addition, in one aspect, the compounds of the present invention are useful for treating a disorder of uncontrolled cell proliferation. In another aspect, the disorder of uncontrolled cell proliferation is cancer or a tumor. In still another aspect, the disorder of uncontrolled cell proliferation is associated with ENPP1 dysfunction, as further described herein.

[0137] In another aspect, the compounds of the invention can be used to treat diseases of bacterial or viral origin. Thus, in one aspect, the invention provides a method of treating a disease caused by bacteria or viruses, the method comprising administering to a subject a therapeutically effective amount of a compound of the invention.

[0138] The inventors have found that viral or bacterial infection can cause an increase or overexpression of ENPP1, and can be blocked by ENPP1 inhibitors of Formula 1. Further, there is some evidence that the presence of such infections leads to a decrease in IFN-β and NF-κB, particularly in cells expressing high ENPP1. Examination of the molecular mechanism of ENPP1 during viral or bacterial infection revealed that ENPP1 is involved in the hydrolysis of cGAMP in infected or transfected cells and leads to inhibition of IRF3 phosphorylation, thereby reducing IFN-β secretion. These results, combined with human cGAS, further validate the hypothesis that ENPP1 acts through cGAS to maintain cGAMP levels and contribute to viral or bacterial infection.

[0139] It is contemplated that each disclosed derivative may optionally be further substituted. It is also contemplated that the present invention may optionally omit any one or more derivatives. It is understood that the disclosed compounds may be provided by the disclosed methods. It is also understood that the disclosed compounds may be used in the disclosed methods of use.

[0140] 1. Structure

[0141] In one aspect, the present invention relates to a compound of formula I:

[0142]

[0143] in

[0144] Z is -H or -N;

[0145] Y is

[0146] n is 0 or 1;

[0147] X1 is -C=O or -NH,

[0148] X2 is -C=O or -NH, provided that if X1 is -C=O, then X2 is -NH; and if X1 is -NH, then X2 is -C=O;

[0149] R1 is selected from the group consisting of: -H, halogen, -OCF3, -CF3, -CN, OCH3 and -CH3;

[0150] R2 is selected from the group consisting of: -H, -NH2, -OH and -CH3;

[0151] R3 is selected from the group consisting of: -H, -NH2, -OH and -CH3;

[0152] R4 is selected from the group consisting of: -H, -CH2-CH3 and -CH3;

[0153] R5 is selected from the group consisting of:

[0154] Wherein R5 can be further substituted by one or more R6, and wherein each R6 is independently selected from the group consisting of: -OCH3, OH, F, Cl, -CH3, O-CH2-CH3, OCH2-CF3, OCH2-CF3OH, OCHF2, ONa, S(O)2, S(O)2NH2,

[0155] or an isomer or a pharmaceutically acceptable salt thereof.

[0156] In a preferred embodiment,

[0157] Z is -N;

[0158] n is 0;

[0159] X1 is -C=0;

[0160] X2 is -NH;

[0161] R1 is selected from the group consisting of halogen and -CN;

[0162] R2 is selected from the group consisting of -H and -CH3;

[0163] R3 is -H;

[0164] R4 is -H;

[0165] R5 is and

[0166] R6 is selected from the group consisting of -OCH3 and OH.

[0167] In another preferred embodiment, R1 is selected from the group consisting of -Cl and -CN.

[0168] Examples of provided compounds include:

[0169]

[0170]

[0171]

[0172]

[0173] as well as

[0174] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of any compound of the present invention and a pharmaceutically acceptable carrier.

[0175] The present invention also provides a method for treating a disorder of uncontrolled cell proliferation in a mammal, said method comprising the step of administering to said mammal an effective amount of any of the compounds of the present invention.

[0176] The present invention also provides a method for reducing ENPP1 activity in a mammal, comprising the step of administering an effective amount of any compound of the present invention to the mammal.

[0177] The present invention also provides a method for inhibiting ENPP1 activity in a mammal, comprising the step of administering to the mammal an effective amount of any compound of the present invention.

[0178] C. Methods of preparing compounds

[0179] In one aspect, the present invention relates to methods of preparing compounds useful as ENPP1 inhibitors. In other aspects, the products of the disclosed methods of preparation are modulators of ENPP1 activity.

[0180] In addition to other standard operations known in the literature, exemplified in the experimental section or apparent to those skilled in the art, the compounds of the present invention can be prepared by using reactions as shown in the following schemes. For clarity, examples with a single substituent are shown, where multiple substituents are allowed under the definitions disclosed herein.

[0181] In addition to other standard operations known in the literature or to those skilled in the art, the reactions for producing compounds of the present invention are also prepared by using the reactions shown in the following reaction schemes. The following examples are provided so that the present invention can be more fully understood, and the examples are illustrative only and should not be construed as limiting.

[0182] In one aspect, the disclosed compounds include products of the synthetic methods described herein. In another aspect, the disclosed compounds include compounds produced by the synthetic methods described herein. In still another aspect, the present invention includes a pharmaceutical composition comprising a therapeutically effective amount of a product of the disclosed method and a pharmaceutically acceptable carrier. In still another aspect, the present invention includes a method for manufacturing a medicament comprising combining at least one compound of any disclosed compound or at least one product of the disclosed method with a pharmaceutically acceptable carrier or diluent.

[0183] In the absence of description of the reaction conditions and the amounts of the ingredients, it is believed to be determined to be within the technical scope of the art. It is contemplated that each disclosed method may further include additional steps, operations and / or components. It is also contemplated that the present invention may optionally omit any one or more steps, operations and / or components. It should be understood that the disclosed methods may be used to provide the disclosed compounds. It should also be understood that the products of the disclosed methods may be used in the disclosed methods of use.

[0184] D. Pharmaceutical Composition

[0185] In one aspect, the present invention relates to pharmaceutical compositions comprising the disclosed compounds. That is, a pharmaceutical composition comprising a therapeutically effective amount of at least one disclosed compound or at least one product of the disclosed method and a pharmaceutically acceptable carrier can be provided.

[0186] In another aspect, the present invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a product of the disclosed synthesis method. In another aspect, the effective amount is a therapeutically effective amount. In another aspect, the effective amount is a preventive effective amount. In another aspect, the compound is a disclosed compound.

[0187] In some aspects, the disclosed pharmaceutical composition includes the disclosed compound (including one or more pharmaceutically acceptable salts thereof) as an active ingredient, a pharmaceutically acceptable carrier, and optionally other therapeutic ingredients or adjuvants. The present composition includes those compositions suitable for oral administration, rectal administration, topical administration, and parenteral administration (including subcutaneous, intramuscular, and intravenous administration), but the most suitable approach in any given case will depend on the nature and severity of the condition of the specific host and the application of the active ingredient. The pharmaceutical composition can be conveniently presented in unit dosage form and can be prepared by any method known in the pharmaceutical field.

[0188] As used herein, the term "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable non-toxic base or acid. When the compound of the present invention is acidic, its corresponding salt can be conveniently prepared from a pharmaceutically acceptable non-toxic base (including inorganic bases and organic bases). Salts derived from such inorganic bases include salts such as aluminum, ammonium, calcium, copper (-ic and -ous), iron, ferrous, lithium, magnesium, manganese (-ic and -ous), potassium, sodium, zinc, etc. Particularly preferred are ammonium salts, calcium salts, magnesium salts, potassium salts and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include primary amines, secondary amines and tertiary amines, as well as cyclic amines and substituted amines, such as naturally occurring and synthetic substituted amines. Other pharmaceutically acceptable organic non-toxic bases from which salts can be formed include ion exchange resins such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

[0189] As used herein, the term "pharmaceutically acceptable non-toxic acid" includes inorganic acids, organic acids and salts prepared therefrom, such as acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid, etc. Preferred are citric acid, hydrobromic acid, hydrochloric acid, maleic acid, phosphoric acid, sulfuric acid and tartaric acid.

[0190] In practice, according to conventional pharmaceutical compounding techniques, the compounds of the present invention or pharmaceutically acceptable salts thereof can be closely mixed with pharmaceutical carriers as active ingredients. The carrier can take a variety of forms, depending on the form of the desired preparation for administration, such as oral or parenteral (including intravenous). Therefore, the pharmaceutical composition of the present invention can be presented as discrete units suitable for oral administration, such as capsules, cachets or tablets each containing a predetermined amount of active ingredients. Further, the composition can exist as a powder, granules, solution, suspension in an aqueous liquid, non-aqueous liquid, oil-in-water emulsion or water-in-oil liquid emulsion. In addition to the common dosage forms described above, the compounds of the present invention and / or one or more pharmaceutically acceptable salts thereof can also be administered by a controlled release device and / or a delivery device. The composition can be prepared by any pharmaceutical method. Generally, such methods include the step of combining the active ingredient with a carrier constituting one or more essential ingredients. Generally, the composition is prepared by uniformly and closely mixing the active ingredient with a liquid carrier or a finely powdered solid carrier or both. The product can then be conveniently formed into the desired appearance.

[0191] Therefore, the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier and a compound of the present invention or a pharmaceutically acceptable salt thereof. The compound of the present invention or a pharmaceutically acceptable salt thereof may also be included in a pharmaceutical composition in combination with one or more other therapeutically active compounds.

[0192] The pharmaceutical carrier used can be, for example, solid, liquid or gas. Examples of solid carriers include lactose, terra alba, sucrose, talcum powder, gelatin, agar, pectin, gum arabic, magnesium stearate and stearic acid. Examples of liquid carriers are syrup, peanut oil, olive oil and water. Examples of gaseous carriers include carbon dioxide and nitrogen.

[0193] In preparing compositions for oral dosage forms, any convenient pharmaceutical medium may be used. For example, water, ethylene glycol, oils, alcohols, flavoring agents, preservatives, colorants, and the like may be used to form oral liquid preparations, such as suspensions, elixirs, and solutions; and carriers such as starch, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrants, and the like may be used to form oral solid preparations, such as powders, capsules, and tablets. Tablets and capsules are preferred oral dosage units because of ease of administration, and solid pharmaceutical carriers are thus employed. Optionally, tablets may be coated by standard aqueous or non-aqueous techniques.

[0194] Tablets containing the compositions of the present invention can be prepared by compression or molding, optionally with one or more auxiliary ingredients or adjuvants. Can be prepared by compressing the active ingredient (such as powder or granules) in a free-flowing form optionally mixed with a binder, lubricant, inert diluent, surfactant or dispersant in a suitable machine. Molded tablets can be manufactured by molding a mixture of powdered compounds moistened with an inert liquid diluent in a suitable machine.

[0195] The pharmaceutical composition of the present invention includes a compound of the present invention (or a pharmaceutically acceptable salt thereof) as an active ingredient, a pharmaceutically acceptable carrier and optionally one or more other therapeutic agents or adjuvants. The present composition includes compositions suitable for oral administration, rectal administration, topical administration and parenteral (including subcutaneous, intramuscular and intravenous) administration, but the most suitable approach in any given case will depend on the nature and severity of the condition of the specific host and the administration of the active ingredient. The pharmaceutical composition can be conveniently presented in unit dosage form and can be prepared by any method known to the pharmaceutical field.

[0196] The pharmaceutical composition of the present invention suitable for parenteral administration can be prepared as a solution or suspension of the active compound in water. Suitable surfactants, such as hydroxypropylcellulose, can be included. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils. Further, preservatives can be included to prevent harmful growth of microorganisms.

[0197] The pharmaceutical composition of the present invention suitable for injectable use comprises a sterile aqueous solution or dispersion. In addition, the composition can be in the form of a sterile powder for the temporary preparation of such sterile injectable solutions or dispersions. In all cases, the final injectable form must be sterile and must be an effective fluid for easy injection. The pharmaceutical composition must be stable under manufacturing and storage conditions; therefore, it is preferably preserved against the contamination of antimicrobial agents (such as bacteria and fungi). The carrier can be a solvent or a dispersion medium containing, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol), a vegetable oil and a suitable mixture thereof.

[0198] The pharmaceutical compositions of the present invention may be in a form suitable for topical use, such as an aerosol, cream, ointment, lotion, dusting powder, mouthwash, gargle, etc. Further, the composition may be in a form suitable for use in a transdermal device. These formulations may be prepared by conventional processing methods using the compounds of the present invention or pharmaceutically acceptable salts thereof. As an example, a cream or ointment is prepared by mixing a hydrophilic material and water with about 5 wt % to about 10 wt % of the compound to produce a cream or ointment with a desired consistency.

[0199] Pharmaceutical compositions of the present invention can be in a form suitable for rectal administration, wherein the carrier is a solid. Preferably, the mixture forms a unit dose suppository. Suitable carriers include cocoa butter and other materials commonly used in the art. Suppositories can be conveniently formed by first mixing the composition with one or more softened or molten carriers, then cooling and molding in a mold.

[0200] In addition to the above-mentioned carrier components, the above-mentioned pharmaceutical formulations may optionally include one or more additional carrier components, such as diluents, buffers, flavoring agents, adhesives, surfactants, thickeners, lubricants, preservatives (including antioxidants), etc. In addition, other adjuvants may be included to make the formulation isotonic with the blood of the intended recipient. Compositions containing the compounds of the present invention and / or pharmaceutically acceptable salts thereof may also be prepared in the form of powders or liquid concentrates.

[0201] In the treatment of conditions requiring inhibition or negative regulation of ENPP1 protein activity, appropriate dosage levels will generally be about 0.01 to 500 mg per kg of patient body weight per day, and may be administered in single or multiple doses. Preferably, the dosage level will be about 0.1 to about 250 mg / kg per day; more preferably 0.5 to 100 mg / kg per day. Suitable dosage levels may be about 0.01 to 250 mg / kg per day, about 0.05 to 100 mg / kg per day, or about 0.1 to 50 mg / kg per day. Within this range, the dosage may be 0.05 to 0.5, 0.5 to 5.0, or 5.0 to 50 mg / kg per day. For oral administration, the composition is preferably provided in the form of tablets containing 1.0 to 1000 mg of active ingredient, specifically 1.0, 5.0, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900 and 1000 mg of active ingredient, for symptomatic adjustment of the dosage of the patient to be treated. The compound can be administered according to a regimen of 1 to 4 times per day, preferably once or twice per day. This dosing regimen can be adjusted to provide the best therapeutic response.

[0202] However, it should be understood that the specific dosage level for any particular patient will depend on a variety of factors. These factors include the patient's age, weight, general health, sex and diet. Other factors include the time and route of administration, excretion rate, drug combination, and the type and severity of the specific disease being treated.

[0203] The present invention further relates to a method for manufacturing a medicament for inhibiting or negatively regulating ENPP1 protein activity in a mammal (e.g., a human) (e.g., treating a disorder of uncontrolled cell proliferation or one or more neurodegenerative disorders associated with ENPP1 dysfunction), the method comprising combining one or more disclosed compounds, products or compositions with a pharmaceutically acceptable carrier or diluent. Thus, in one aspect, the present invention relates to a method for manufacturing a medicament, the method comprising combining at least one disclosed compound or at least one disclosed product with a pharmaceutically acceptable carrier or diluent.

[0204] The disclosed pharmaceutical compositions may further include other therapeutically active compounds, which are generally applied in the treatment of the above-mentioned pathological conditions.

[0205] It is understood that the disclosed compositions can be prepared from the disclosed compounds. It is also understood that the disclosed compositions can be used in the disclosed methods of use.

[0206] E. Methods of Using Compounds and Compositions

[0207] The disclosed compounds can be used as a single agent or in combination with one or more other drugs for treating, preventing, controlling, improving the above-mentioned diseases, diseases and symptoms to which the compounds of formula I or other drugs are effective or reducing their risks, wherein the drug combination is safer or more effective than any single drug. Therefore, one or more other drugs can be used simultaneously or sequentially with the disclosed compounds by way and in a commonly used amount. When the disclosed compounds are used simultaneously with one or more other drugs, a pharmaceutical composition containing a unit dosage form of such drugs and the disclosed compounds is preferred. However, combination therapy can also be applied according to an overlapping schedule. It is also envisioned that the combination of one or more active ingredients with the disclosed compounds will be more effective than as a single agent.

[0208] The pharmaceutical compositions and methods of the present invention may further comprise other therapeutically active compounds as described herein, which are generally applied in the treatment of the above-mentioned pathological conditions.

[0209] 1. Treatment Methods

[0210] The compounds disclosed herein can be used to treat, prevent, improve, control or reduce the risk of a variety of conditions in which patients or subjects would benefit from the inhibition or negative regulation of ENPP1. In one aspect, a method of treating or preventing a condition in a subject is provided, the method comprising administering to the subject at least one disclosed compound in an effective dose and in an effective amount; at least one disclosed pharmaceutical composition; and / or at least one disclosed product to treat the condition in the subject.

[0211] Also provided is a method for treating one or more conditions in a subject for which ENPP1 inhibition is predicted to be beneficial, the method comprising the step of administering to the subject at least one disclosed compound; at least one disclosed pharmaceutical composition; and / or at least one disclosed product in an effective dose and in an effective amount to treat the condition in the subject.

[0212] In one aspect, a method for treating a disorder of uncontrolled cell proliferation is provided, the method comprising: administering to a subject at least one disclosed compound in an effective dose and an effective amount; at least one disclosed pharmaceutical composition; and / or at least one disclosed product to treat the disorder in the subject. In another aspect, a method for treating or preventing a neurodegenerative disorder is provided, the method comprising: administering to a subject at least one disclosed compound in an effective dose and an effective amount; at least one disclosed pharmaceutical composition; and / or at least one disclosed product to treat the disorder in the subject. A method for treating a disorder in a mammal is also provided, the method comprising the step of administering to the mammal at least one disclosed compound, composition, or drug.

[0213] The present invention relates to the use of chemical compositions for treating diseases or disorders in a patient, preferably a human, by administering one or more disclosed compounds or products, wherein ENPP1 inhibition is predicted to have a therapeutic effect, such as disorders of uncontrolled cell proliferation (e.g., cancer), neurodegenerative disorders, such as Alzheimer's disease, Huntington's disease and Parkinson's disease, diseases caused by bacteria and / or viruses.

[0214] The compounds of the present invention may also be used in immunotherapy.

[0215] In one embodiment, the compounds of the invention treat disorders of uncontrolled cell proliferation and / or diseases caused by bacteria and / or viruses by immunotherapy, meaning that the compounds elicit an immunotherapy response which results in the treatment of these diseases.

[0216] The compounds disclosed herein can be used to treat, prevent, ameliorate, control or reduce the risk of a variety of disorders involving uncontrolled cell proliferation.

[0217] Also provided is a method for using the disclosed compound, composition or medicine. On the one hand, the method for using relates to the treatment of illness. In other aspects, the disclosed compound can be used as a single agent or in combination with one or more other drugs for treating, preventing, controlling, improving the above-mentioned diseases, illnesses and symptoms for which the compound or other drugs have effectiveness or reducing its risk, wherein the drug combination is safer or more effective than any single drug. Therefore, one or more other drugs can be administered simultaneously or sequentially with the disclosed compound by a route and in a commonly used amount. When the disclosed compound is used simultaneously with one or more other drugs, a pharmaceutical composition in a unit dosage form containing such drugs and the disclosed compound is preferred. However, combination therapy can also be administered according to an overlapping schedule. It is also envisioned that the combination of one or more active ingredients with the disclosed compound can be more effective than as a single agent.

[0218] Examples of conditions that can be treated with provided compounds include conditions of uncontrolled cell proliferation. In yet another aspect, the condition of uncontrolled cell proliferation is cancer. In yet another aspect, the cancer is a leukemia. In even another aspect, the cancer is a sarcoma. In still another aspect, the cancer is a solid tumor. In yet another aspect, the cancer is a lymphoma.

[0219] It should be understood that cancer refers to or describes a physiological condition characterized by unregulated cell growth in mammals. Cancer can have multidrug resistance (MDR) or drug sensitivity. Examples of cancer include but are not limited to carcinoma, lymphoma, blastoma, sarcoma and leukemia. More specific examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, cervical cancer, ovarian cancer, peritoneal cancer, liver cancer, such as liver cancer, bladder cancer, colorectal cancer, endometrial cancer, kidney cancer and thyroid cancer.

[0220] In various aspects, additional examples of cancer are basal cell carcinoma, biliary tract cancer; bone cancer; brain and CNS cancer; choriocarcinoma; connective tissue cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer; intraepithelial neoplasia; laryngeal cancer; lymphoma, including Hodgkin's lymphoma and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cancer (e.g., lip, tongue, mouth and pharynx); retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory cancer; sarcoma; skin cancer; stomach cancer; testicular cancer; uterine cancer; urinary system cancer, and other cancers and sarcomas.

[0221] In other aspects, cancer is a hematological cancer. In still other aspects, hematological cancer is selected from acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myelomonocytic leukemia (CMML), childhood myelomonocytic leukemia (JMML), Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, solitary myeloma, limited myeloma and extramedullary myeloma. In still other aspects, cancer is selected from chronic lymphocytic leukemia, small lymphocytic lymphoma, B cell non-Hodgkin's lymphoma and large B cell lymphoma.

[0222] In other aspects, cancer is brain cancer. In still other aspects, brain cancer is selected from glioma, medulloblastoma, primitive neuroectodermal tumor (PNET), acoustic neuroma, glioma, meningioma, pituitary adenoma, schwannoma, CNS lymphoma, primitive neuroectodermal tumor, craniopharyngioma, chordoma, medulloblastoma, brain neuroblastoma, central neurocytoma, pineal cell tumor, pinealoblastoma, atypical teratoid rhabdoid tumor (atypicalteratoid rhabdoid tumor), chondrosarcoma, chondroma, choroid plexus carcinoma, choroid plexus papilloma, craniopharyngioma, dysembryoplastic neuroepithelioma, gangliocytoma, germ cell tumor, hemangioblastoma, hemangiopercytoma (hemangiopercytoma) and metastatic brain tumor. In yet other aspects, glioma is selected from ependymoma, astrocytoma, oligodendroglioma and oligoastrocytoma. In an even further aspect, the glioma is selected from the group consisting of infantile pilocytic astrocytoma, subependymal giant cell astrocytoma, ganglioglioma, subependymoma, pleomorphic xanthoastrocytoma, anaplastic astrocytoma, glioblastoma multiforme, brain stem glioma, oligodendroglioma, ependymoma, oligoastrocytoma, cerebellar astrocytoma, infantile desmoplastic astrocytoma, subependymal giant cell astrocytoma, diffuse astrocytoma, mixed glioma, optic nerve glioma, brain glioma, multifocal gliomatous tumor, multicentric glioblastoma multiforme tumor, paraganglioma and ganglioglioma.

[0223] In one aspect, the cancer can be a cancer selected from the following: blood cancer, brain cancer, urogenital tract cancer, gastrointestinal cancer, colon cancer, rectal cancer, breast cancer, kidney cancer, lymphatic system cancer, gastric cancer, lung cancer, pancreatic cancer and skin cancer. In another aspect, the cancer is selected from prostate cancer, glioblastoma multiforme, endometrial cancer, breast cancer and colon cancer. In another aspect, the cancer is selected from breast cancer, ovarian cancer, prostate cancer, head cancer, neck cancer and kidney cancer. In still another aspect, the cancer is selected from blood cancer, brain cancer, urogenital tract cancer, gastrointestinal cancer, colon cancer, rectal cancer, breast cancer, liver cancer, kidney cancer, lymphatic system cancer, gastric cancer, lung cancer, pancreatic cancer and skin cancer. In yet another aspect, the cancer is selected from lung cancer and liver cancer. In even another aspect, the cancer is selected from breast cancer, ovarian cancer, testicular cancer and prostate cancer. In still another aspect, the cancer is breast cancer. In yet another aspect, the cancer is ovarian cancer. In even another aspect, the cancer is prostate cancer. In still another aspect, the cancer is testicular cancer.

[0224] In various aspects, the disorder associated with ENPP1 dysfunction comprises a neurodegenerative disorder. In further aspects, the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease and Huntington's disease.

[0225] The compounds can be further used in methods for preventing, treating, controlling, improving or reducing the risk of the diseases, disorders and conditions described herein. The combination of the compounds with other agents can be further used in methods for preventing, treating, controlling, improving or reducing the risk of the above-mentioned diseases, disorders and conditions.

[0226] The invention further relates to the administration of ENPP1 inhibitors to improve treatment outcomes in the context of disorders of uncontrolled cell proliferation, including cancer. That is, in one aspect, the invention relates to a co-therapeutic method comprising the step of administering to a mammal an effective amount and dosage of at least one compound of the invention relevant to cancer therapy.

[0227] In other aspects, in the context of cancer therapy, administration improves treatment outcomes. Administration related to cancer therapy can be continuous or intermittent. Administration does not need to be performed simultaneously with therapy and can be performed before, during and / or after therapy. For example, cancer therapy can be provided within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days before or after the administration of the compound. As another example, cancer therapy can be provided within 1 week, 2 weeks, 3 weeks or 4 weeks before or after the administration of the compound. As still another example, cognitive or behavioral therapy can be provided before or after administration within a time period of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 half-lives of the administered compound.

[0228] On the one hand, the disclosed compounds can be used in combination with one or more other drugs for treating, preventing, controlling, improving the diseases or conditions for which the disclosed compounds or other drugs can be effective or reducing their risks, wherein the drugs are combined together safer or more effective than any single drug. Therefore, such one or more other drugs can be administered simultaneously or sequentially with the compound of the present invention by way and in a commonly used amount. When the compound of the present invention is used simultaneously with one or more other drugs, a pharmaceutical composition containing a unit dosage form of such other drugs and the disclosed compound is preferred. However, combination therapy can also include a therapy in which the disclosed compound and one or more other drugs are administered according to different overlapping schedules. It is also envisioned that when used in combination with one or more other active ingredients, the disclosed compounds and other active ingredients can be used in a lower dosage than when each is used alone.

[0229] Accordingly, pharmaceutical compositions include those that contain one or more other active ingredients, in addition to a compound of the present invention.

[0230] The above combination not only comprises the combination of the disclosed compound and a kind of other active compound, but also comprises the combination of the disclosed compound and two or more other active compounds. Similarly, the disclosed compound can be used in combination with other drugs, and the other drugs are used to prevent, treat, control, improve the diseases or conditions to which the disclosed compound is useful or reduce its risk. Therefore, such other drugs can be used simultaneously or sequentially with the compound of the present invention by way and in a commonly used amount. When the compound of the present invention is used simultaneously with one or more other drugs, it is preferred that the pharmaceutical composition also contains such other drugs in addition to the disclosed compound. Therefore, the pharmaceutical composition includes those that also contain one or more other active ingredients in addition to the compound of the present invention.

[0231] The weight ratio of the disclosed compound to the second active ingredient can vary and will depend on the effective dose of each ingredient. Typically, each effective dose will be used. Thus, for example, when the compound of the present invention is combined with another agent, the weight ratio of the disclosed compound to the other agent will typically range from about 1000:1 to about 1:1000, preferably about 200:1 to about 1:200. The combination of the compound of the present invention with other active ingredients will also typically be within the above range, but in each case, an effective dose of each active ingredient should be used.

[0232] In such combinations, the disclosed compounds and other active agents may be administered separately or in combination. In addition, the administration of one element may be prior to, simultaneously with, or after the administration of one or more other agents.

[0233] Therefore, the subject compound can be used alone or in combination with other medicaments known to be beneficial to the subject's indication or other drugs that affect receptors or enzymes, which improve the efficacy, safety, convenience or reduce its unwanted side effects or toxicity of the disclosed compounds. The subject compound and other medicaments can be co-administered in concomitant therapy or in a fixed combination.

[0234] In one aspect, the compounds can be used in combination with anti-cancer therapeutics or other known therapeutic agents.

[0235] In the treatment of conditions requiring inhibition or negative regulation of ENPP1, appropriate dosage levels will generally be about 0.01 to 1000 mg per kg of patient body weight per day, which can be administered in single or multiple doses. Preferably, the dosage level will be about 0.1 to about 250 mg / kg per day; more preferably about 0.5 to about 100 mg / kg per day. Suitable dosage levels may be about 0.01 to 250 mg / kg per day, about 0.05 to 100 mg / kg per day, or about 0.1 to 50 mg / kg per day. Within this range, the dosage may be 0.05 to 0.5, 0.5 to 5, or 5 to 50 mg / kg per day. For oral administration, the composition is preferably provided in the form of tablets containing 1.0 to 1000 mg of active ingredient, specifically 1.0, 5.0, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900 and 1000 mg of active ingredient, for symptomatic adjustment of the dosage of the patient to be treated. The compound can be administered according to a regimen of 1 to 4 times a day, preferably once or twice a day. This dosage regimen can be adjusted to provide an optimal therapeutic response. However, it should be understood that the specific dosage level and dosage frequency for any particular patient can vary and will depend on a variety of factors, including the activity of the specific compound used, the metabolic stability of the compound and the length of action time, age, body weight, overall health, sex, diet, mode and time of administration, excretion rate, drug combination, severity of a particular condition and the host undergoing therapy.

[0236] Thus, in one aspect, the invention relates to a method for inhibiting or negatively regulating ENPP1 in at least one cell in an effective amount to modulate or activate an ENPP1 activity response, for example in the at least one cell, the method comprising the step of contacting the at least one cell with at least one compound of the invention. In a further aspect, the cell is a mammal, such as a human. In a further aspect, the cell has been isolated from a subject prior to the contacting step. In a further aspect, the contacting is performed by administering to a subject.

[0237] a. Treatment of disorders involving uncontrolled cell proliferation

[0238] In one aspect, the invention relates to a method for treating a disorder of uncontrolled cell proliferation in a mammal, comprising the step of administering to the mammal an effective amount of at least one disclosed compound or a product of a disclosed method of preparing a compound, or a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof, thereby treating the disorder of uncontrolled cell proliferation.

[0239] In still other aspects, the effective amount is a therapeutically effective amount. In still other aspects, the effective amount is a prophylactically effective amount.

[0240] In other aspects, the mammal is a human. In yet other aspects, the method further comprises the step of identifying a mammal in need of treatment for a disorder of uncontrolled cell proliferation. In still other aspects, prior to the administering step, the mammal has been diagnosed as needing treatment for a disorder of uncontrolled cell proliferation.

[0241] In another aspect, the disorder of uncontrolled cell proliferation is cancer. In another aspect, cancer is leukemia. In an even further aspect, cancer is a sarcoma. In an even further aspect, cancer is a solid tumor. In an even further aspect, cancer is a lymphoma. In an even further aspect, cancer is selected from chronic lymphocytic leukemia, small lymphocytic lymphoma, B-cell non-Hodgkin lymphoma and large B-cell lymphoma. In an even further aspect, cancer is selected from blood cancer, brain cancer, genitourinary tract cancer, gastrointestinal cancer, colon cancer, rectal cancer, breast cancer, liver cancer, kidney cancer, lymphatic system cancer, gastric cancer, lung cancer, pancreatic cancer and skin cancer. In an even further aspect, cancer is selected from lung cancer and liver cancer. In an even further aspect, cancer is selected from breast cancer, ovarian cancer, testicular cancer and prostate cancer. In an even further aspect, cancer is breast cancer. In an even further aspect, cancer is ovarian cancer. In an even further aspect, cancer is prostate cancer. In an even further aspect, cancer is testicular cancer.

[0242] Examples

[0243] F. Experiment

[0244] The following examples are presented to provide a person of ordinary skill in the art with a complete disclosure and description of how to prepare and evaluate the compounds, compositions, articles, devices and / or methods claimed herein, and are intended to be purely illustrative of the present invention and are not intended to limit the scope of what the inventors believe to be their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, temperatures are in ° C or at ambient temperature, and pressures are at or near atmospheric pressure.

[0245] Several methods for preparing compounds of the invention are illustrated in the following examples. Starting materials and necessary intermediates are in some cases commercially available or can be prepared according to literature procedures or as demonstrated herein.

[0246] The following exemplary compounds of the present invention are synthesized. Examples are provided herein to illustrate the present invention and should not be construed as limiting the present invention in any way. According to the IUPAC naming convention, examples are generally depicted in free base form. However, some of the examples are obtained or isolated in the form of salts.

[0247] Some of the examples described are obtained as racemic mixtures of one or more enantiomers or diastereomers. One skilled in the art can separate compounds to separate individual enantiomers. Separation can be performed by coupling the racemic mixture of the compound to an enantiomerically pure compound to form a diastereomeric mixture, followed by separation of the individual diastereomers by standard methods (e.g., fractional crystallization or chromatography). The racemic or diastereomeric mixture of the compound can also be directly separated by a chromatographic method using a chiral stationary phase.

[0248] Experimental Chemistry

[0249] Synthetic Schemes, Methods and Procedures:

[0250] 6-Chloro-3H-imidazo[4,5-c]pyridine-2-thiol (Compound 2)

[0251] The reaction mixture of 6-chloropyridine-3,4-diamine (100 mg, 0.697 mmol), di(1H-imidazol-1-yl)methanthione (124 mg, 0.697 mmol) in tetrahydrofuran (4 mL, 0.697 mmol) was stirred at room temperature overnight. The precipitate was filtered and 6-chloro-3H-imidazo[4,5-c]pyridine-2-thiol (100 mg, 0.539 mmol, 77% yield) was obtained.

[0252] 1 HNMR(400MHz,CD3OD)δ7.76(s,1H),7.07(s,1H),ESI-MS:m / z 185.82(M+H) +

[0253]

[0254] 2-Chloro-N-(3,4-dimethoxyphenyl)acetamide (Compound 1)

[0255] To a solution of 3,4-dimethoxyaniline (200 mg, 1.306 mmol), TEA (0.546 mL, 3.92 mmol) in DCM (5 mL) was added 2-chloroacetyl chloride (0.125 mL, 1.567 mmol) at room temperature. After 1 hour, concentration and purification on combiflash (4 g, EtOAc / hexanes) gave 2-chloro-N-(3,4-dimethoxyphenyl)acetamide (232 mg, 1.010 mmol, 77% yield). 1 HNMR (400MHz, CDCl3) δ7.28 (J=2Hz, 1H), 6.95 (d, J=6Hz, 1H), 6.85 (d, J=8.8Hz, 1H), 4.19 (s, 2H) 3.90 (s, 3H), 3.87 (s, 3H).

[0256] 2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3,4-dimethoxyphenyl)acetamide (Compound 3)

[0257] The reaction mixture of 6-chloro-3H-imidazo[4,5-c]pyridine-2-thiol (13 mg, 0.070 mmol), 2-chloro-N-(3,4-dimethoxyphenyl)acetamide (16.08 mg, 0.070 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.029 mL, 0.210 mmol) in acetonitrile (1 mL) was heated at 65° C. for 1.5 h. Concentration and purification on combiflash (4 g, MeOH / DCM) gave 2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3,4-dimethoxyphenyl)acetamide (13 mg, 0.034 mmol, 49.0% yield). 1 HNMR(400MHz, CDCl3)δ9.81(s,1H),8.70(m,1H),7.31(s,1H),6.86(m,1H),6.77(m,2H),3.99(s,2H),3.84(m,6H).ESI-MS:m / z 379.02(M+H) +

[0258]

[0259] N-(3,4-dimethoxyphenyl)-2-((5-methoxythiazolo[4,5-b]pyridin-2-yl)thio)acetamide (Compound 4)

[0260] The reaction mixture of 5-methoxythiazolo[4,5-b]pyridine-2-thiol (17.27 mg, 0.087 mmol), 2-chloro-N-(3,4-dimethoxyphenyl)acetamide (20 mg, 0.087 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.036 mL, 0.261 mmol) in acetonitrile (1 mL) was heated at 65 °C for 2 hours. Concentration and purification on combiflash (4 g, MeOH / DCM) gave N-(3,4-dimethoxyphenyl)-2-((5-methoxythiazolo[4,5-b]pyridin-2-yl)thio)acetamide (13.5 mg, 0.034 mmol, 39.6% yield). 1 HNMR(400MHz, CD3OD), δ8.01(d,J=8.8Hz,1H),7.82(s,1H),7.31(d,J=2Hz,1H),7.0 2(d,J=8.8Hz,1H),6.86(m,2H),3.95(s,2H),3.80(s,3H),3.30(m,6H).ESI-MS:m / z 392.04(M+H) +

[0261]

[0262] N-(Benzo[d][1,3]dioxol-5-yl)-2-chloroacetamide (Compound 5)

[0263] To a solution of benzo[d][1,3]dioxol-5-amine (200 mg, 1.458 mmol), TEA (0.610 mL, 4.38 mmol) in DCM (5 mL) at room temperature was added 2-chloroacetyl chloride (0.139 mL, 1.75 mmol). After 1 hour, concentration and purification on combiflash (4 g, EtOAc / hexanes) gave N-(benzo[d][1,3]dioxol-5-yl)-2-chloroacetamide (239 mg, 1.119 mmol, 77% yield).

[0264] 1 HNMR (400MHz, CDCl3) δ8.12 (br-s, 1H), 7.23 (s, 1H), 6.85 (d, J = 8Hz, 1H), 6.78 (d, J = 8Hz, 1H), 5.97 (s, 2H), 4.18 (s, 2H) ESI-MS: m / z 213.96 (M+H) +

[0265] N-(Benzo[d][1,3]dioxol-5-yl)-2-((5-methoxythiazolo[4,5-b]pyridin-2-yl)thio)acetamide (Compound 6)

[0266] A reaction mixture of 5-methoxythiazolo[4,5-b]pyridine-2-thiol (18.56 mg, 0.094 mmol), N-(benzo[d][1,3]dioxol-5-yl)-2-chloroacetamide (20 mg, 0.094 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.039 mL, 0.281 mmol) in acetonitrile (1 mL) was heated at 65 °C for 1.5 h. Concentration and purification on combiflash (4 g, MeOH / DCM) gave N-(benzo[d][1,3]dioxol-5-yl)-2-((5-methoxythiazolo[4,5-b]pyridin-2-yl)thio)acetamide (17.8 mg, 0.047 mmol, 50.6% yield). 1 HNMR(400MHz, CDCl3)δ9.63(s,1H),8.01(d,J-8.8Hz,2H),7.22(s,1H),6.86(d ,J=8.8Hz,1H),6.72(m,3H),5.93(s,2H),4.02(s,2H),3.99(s,3H),ESI-MS:m / z 376.0(M+H) +

[0267]

[0268] N-(Benzo[d][1,3]dioxol-5-yl)-2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide (Compound 7)

[0269] A reaction mixture of 6-chloro-1H-imidazole [4,5-c] pyridine-2-thiol (17.38 mg, 0.094 mmol), N-(benzo [d] [1,3] dioxol-5-yl) -2-chloroacetamide (20 mg, 0.094 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.039 mL, 0.281 mmol) in acetonitrile (1 mL) was heated at 65 ° C for 1.5 hours. Concentration and purification on combiflash (4 g, MeOH / DCM) gave N- (benzo [d] [1,3] dioxol-5-yl) -2- ((6-chloro-1H-imidazo [4,5-c] pyridin-2-yl) thio) acetamide (11.8 mg, 0.033 mmol, 34.7% yield). 1HNMR (400MHz, CDCl3) δ8.44(s,1H),7.34(s,1H),7.13(s,1H),6.75(d,J=8.4Hz,1H),6.64(d,J=8.0Hz 1H),5.84(s,2H),3.94(s,2H), ESI-MS: m / z 362.98(M+H) +

[0270]

[0271] N-(3,4-dimethoxyphenyl)-2-((7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide (Compound 9)

[0272] The reaction mixture of 7-methyl-1H-imidazo[4,5-c]pyridine-2-thiol (14.39 mg, 0.087 mmol), 2-chloro-N-(3,4-dimethoxyphenyl)acetamide (20 mg, 0.087 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.036 mL, 0.261 mmol) in acetonitrile (1 mL) was heated at 65° C. for 2 hours. Concentration and purification on combiflash (4 g, DCM / MeOH) gave N-(3,4-dimethoxyphenyl)-2-((7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide (33 mg, 0.092 mmol, 100% yield). 1 HNMR(400MHz, CDCl3), δ10.8(s,1H),8.66(s,1H),7.99(s,1H),7.35(s,1H),7.18(s,2H),4.11(s,2H),3.80(s,6H),2.59(s,3H).ESI-MS: m / z 359.05(M+H) +

[0273]

[0274] N-(Benzo[d][1,3]dioxol-5-yl)-2-((7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide (Compound 10)

[0275] A reaction mixture of 7-methyl-1H-imidazo[4,5-c]pyridine-2-thiol (15.47 mg, 0.094 mmol), N-(benzo[d][1,3]dioxol-5-yl)-2-chloroacetamide (20 mg, 0.094 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.039 mL, 0.281 mmol) in acetonitrile (1 mL) was heated to 65 °C for 2 hours. Concentration and purification on combiflash (4 g, DCM / MeOH) gave N-(benzo[d][1,3]dioxol-5-yl)-2-((7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide (33.7 mg, 0.098 mmol, 100% yield). 1 HNMR(400MHz, CDCl3)δ8.01(m,2H),7.09(m,3H),6.69(m,1H),5.88(s,2H),3.94(s,2H).2.52(s,3H).ESI-MS:m / z 342.98(M+H) +

[0276]

[0277] 6-Chlorothiazolo[4,5-c]pyridine-2-thiol (Compound 11)

[0278] The reaction mixture of 4,6-dichloropyridin-3-amine (50 mg, 0.307 mmol), potassium O-ethyldithiocarbonate (73.8 mg, 0.460 mmol) in NMP (2 mL) was heated to 150 ° C overnight. Acetic acid (60 uL) and water were added. The precipitate was filtered and dried to give 6-chlorothiazolo[4,5-c]pyridine-2-thiol (52 mg, 0.257 mmol, 84% yield). 1 HNMR(400MHz,CD3OD)δ8.35(s,1H),7.44(s,1H).ESI-MS:m / z 202.78(M+H) +

[0279] 2-((6-chlorothiazolo[4,5-c]pyridin-2-yl)thio)-N-(3,4-dimethoxyphenyl)acetamide (Compound 12)

[0280] The reaction mixture of 6-chlorothiazolo[4,5-c]pyridine-2-thiol (17.65 mg, 0.087 mmol), 2-chloro-N-(3,4-dimethoxyphenyl)acetamide (20 mg, 0.087 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.036 mL, 0.261 mmol) in acetonitrile (1 mL) was heated at 65° C. for 1.5 hours. Concentration and purification by preparative TLC (MeOH / DCM) gave 2-((6-chlorothiazolo[4,5-c]pyridin-2-yl)thio)-N-(3,4-dimethoxyphenyl)acetamide (5.7 mg, 0.014 mmol, 16.53% yield). 1 HNMR(400MHz, CDCl3)δ8.97(s,1H),7.78(s,1H),7.34(s,1H),6.79(m,2H),4.11(s,2H),3.86(s,3H),3.84(s,3H).ESI-MS:m / z 395.97(M+H) +

[0281]

[0282] 2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3,4-dimethoxyphenyl)propionamide (Compound 14)

[0283] The reaction mixture of 6-chloro-1H-imidazo[4,5-c]pyridine-2-thiol (15.24 mg, 0.082 mmol), 2-chloro-N-(3,4-dimethoxyphenyl)propanamide (20 mg, 0.082 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.034 mL, 0.246 mmol) in acetonitrile (1 mL) was heated at 65 °C overnight. The solid was filtered and dried to give 2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3,4-dimethoxyphenyl)propanamide (17 mg, 0.043 mmol, 52.7% yield). ESI-MS: m / z 393.06 (M+H) +

[0284]

[0285] 7-Methyl-1,3-dihydro-2H-imidazo[4,5-c]pyridine-2-thione (Compound 8): To a solution of 5-methylpyridine-3,4-diamine (100 mg, 0.812 mmol) and N,N-dimethylpyridin-4-amine (109 mg, 0.893 mmol) in MeCN was added di(1H-imidazol-1-yl)methylthione (217 mg, 1.218 mmol) in portions. The mixture was stirred at room temperature for 4 hours. The formed precipitate was filtered, washed with water, Et2O, DCM and dried to give 7-methyl-1H-imidazo[4,5-c]pyridine-2(3H)-thione (100 mg, 75% yield) as a light red solid. 1 H-NMR (400MHz, DMSO-d6) δppm 12.73 (brs, 2H), 8.20 (s, 1H), 8.06 (s, 1H), 2.33 (s, 3H); ESI-MS: m / z 165.84 (M+H) + ·

[0286] 2-Chloro-N-(3-hydroxy-4-methoxyphenyl)acetamide (Compound 15): A solution of 5-amino-2-methoxyphenol (100 mg, 0.719 mmol) and triethylamine (0.150 ml, 1.078 mmol) in DCM was cooled to 0°C and 2-chloroacetyl chloride (0.060 ml, 0.755 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 2 hours. The solvent was removed and the crude product was purified by combiflash SiO2 chromatography (0-5% MeOH-DCM) to give 2-chloro-N-(3-hydroxy-4-methoxyphenyl)acetamide (58 mg, 37%) as a brown solid. 1 H-NMR (400MHz, CDCl3) δppm 8.11 (brs, 1H), 7.14 (d, 1H, J = 4Hz), 7.04 (dd, 1H, J = 4Hz and J = 8Hz), 6.81 (d, 1H, J = 12Hz), 4.18 (s, 2H), 3.88 (s, 3H); ESI-MS: m / z 215.90(M+H) + ·

[0287] N-(3-Hydroxy-4-methoxyphenyl)-2-((7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide (Compound 16): A mixture of 2-chloro-N-(3-hydroxy-4-methoxyphenyl)acetamide (15 mg, 0.070 mmol), 7-methyl-1H-imidazo[4,5-c]pyridine-2(3H)-thione (12.64 mg, 0.077 mmol), sodium iodide (10.43 mg, 0.070 mmol) and triethylamine (0.015 ml, 0.104 mmol) in MeCN was heated to 65 °C for 2 hours. The solvent was removed and the crude product was purified by combiflash SiO2 chromatography (0-10% MeOH-DCM) to afford N-(3-hydroxy-4-methoxyphenyl)-2-((7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide (11 mg, 46%) as an off-white solid. 1 H-NMR(400MHz,CD3OD / CDCl3)δppm 8.52(s,1H),7.93(s,1H),7.08(d,1H,J=4Hz),6.92(dd,1H,J=4Hz,J=8Hz),6.77(d,1H,J=8Hz),4.07(s,2H),3.80(s,3H),2.54(s,3H); ESI-MS: m / z 345.03(M+H) + .

[0288]

[0289] The reaction mixture of 7-methyl-1H-imidazo[4,5-c]pyridine-2-thiol (14.39 mg, 0.087 mmol), 2-chloro-N-(3,4-dimethoxyphenyl)acetamide (20 mg, 0.087 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.036 mL, 0.261 mmol) in acetonitrile (1 mL) was heated at 65° C. for 2 hours. Concentration and purification on combiflash (4 g, DCM / MeOH) gave N-(3,4-dimethoxyphenyl)-2-((7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide (33 mg, 0.092 mmol, 100% yield). 1 HNMR(400MHz, CDCl3),10.8(s,1H),8.66(s,1H),7.99(s,1H),7.35(s,1H),7.18(s,2H),4.11(s,2H),3.80(s,6H),2.59(s,3H).ESI-MS:m / z 359.05(M+H)+

[0290]

[0291] 2-Chloro-N-(4-hydroxy-3-methoxyphenyl)acetamide (Compound 17): A solution of 4-amino-2-methoxyphenol (100 mg, 0.719 mmol) and triethylamine (0.150 ml, 1.078 mmol) in DCM was cooled to 0°C and 2-chloroacetyl chloride (0.060 ml, 0.755 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 2 hours. The solvent was removed and the crude product was purified by combiflash SiO2 chromatography (0-5% MeOH-DCM) to give 2-chloro-N-(4-hydroxy-3-methoxyphenyl)acetamide (55 mg, 36% yield) as a brown solid. 1 H-NMR (400MHz, CDCl3) δppm 8.14 (brs, 1H), 7.38 (d, 1H, J = 2.4Hz), 6.87 (d, 1H, J = 8Hz), 6.80 (dd, 1H, J = 4Hz and J = 8Hz), 4.18 (s, 2H), 3.90 (s, 3H); ESI-MS: m / z 215.90(M+H) + .

[0292] N-(4-Hydroxy-3-methoxyphenyl)-2-((7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide (Compound 18): To a mixture of 7-methyl-1H-imidazo[4,5-c]pyridine-2(3H)-thione (13.79 mg, 0.083 mmol), 2-chloro-N-(4-hydroxy-3-methoxyphenyl)acetamide (15 mg, 0.070 mmol), sodium iodide (10.43 mg, 0.070 mmol) in MeCN was added triethylamine (0.012 ml, 0.083 mmol) and heated to 65 °C for 1 hour. The solvent was removed and the crude product was purified by combiflash (0-10% DCM-MeOH) to afford N-(4-hydroxy-3-methoxyphenyl)-2-((7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide (14 mg, 58%) as a white solid. 1H-NMR(400MHz,CD3OD / CDCl3)δppm 8.51(s,1H),7.91(s,1H),7.27(d,1H,J=4Hz),6.77(dd,1H,J=4Hz,J=8Hz),6.71(d,1H,J=8Hz),4.06(s,2H),3.80(s,3H),2.53(s,3H); ESI-MS: m / z 345.04(M+H) + .

[0293]

[0294] N-(3-Hydroxy-4-methoxyphenyl)-2-((5-methoxythiazolo[4,5-b]pyridin-2-yl)thio)acetamide (Compound 19): To a mixture of 5-methoxythiazolo[4,5-b]pyridine-2-thiol (20 mg, 0.101 mmol), 2-chloro-N-(3-hydroxy-4-methoxyphenyl)acetamide (23.93 mg, 0.111 mmol) and sodium iodide (15.02 mg, 0.101 mmol) in MeCN (1 ml) was added triethylamine (0.021 ml, 0.151 mmol) and stirred at 65 °C for 2 hours. The solvent was removed and the crude product was subjected to combiflash chromatography using 0-10% MeOH-DCM to afford N-(3-hydroxy-4-methoxyphenyl)-2-((5-methoxythiazolo[4,5-b]pyridin-2-yl)thio)acetamide (23 mg, 60%) as a light orange solid. 1 H-NMR(400MHz,CD3OD / CDCl3)δppm 9.56(brs,1H),7.98(d,1H,J=12Hz),7.05(dd,1H,J=1.2Hz,J=4Hz),6.92(dd,1H,J=4Hz,J=8Hz ), 6.83 (d, 1H, J = 8Hz), 6.74 (d, 1H, J = 12Hz), 4.00 (s, 2H), 3.96 (s, 3H), 3.81 (s, 3H); ESI-MS: m / z 378.02(M+H) + .

[0295]

[0296] N-(4-Hydroxy-3-methoxyphenyl)-2-((5-methoxythiazolo[4,5-b]pyridin-2-yl)thio)acetamide (Compound 20): To a mixture of 2-chloro-N-(4-hydroxy-3-methoxyphenyl)acetamide (20 mg, 0.093 mmol), 5-methoxythiazolo[4,5-b]pyridine-2-thiol (20.23 mg, 0.102 mmol) and sodium iodide (13.81 mg, 0.093 mmol) in MeCN was added triethylamine (0.019 ml, 0.139 mmol) and stirred at 65 °C for 2 hours. The solvent was removed and the crude product was chromatographed using 0-10% MeOH-DCM to give N-(4-hydroxy-3-methoxyphenyl)-2-((5-methoxythiazolo[4,5-b]pyridin-2-yl)thio)acetamide (22 mg, 63%) as a light orange solid. 1 H-NMR(400MHz,CD3OD / CDCl3)δppm 7.88(d,1H,J=12Hz),7.22(d,1H,J=2Hz),6.73(d,1H,J=12Hz),6.65(d,1H,J=8Hz ), 6.61 (dd, 1H, J = 4Hz, J = 8Hz), 3.97 (s, 2H), 3.85 (s, 3H), 3.72 (s, 3H); ESI-MS: m / z 378.04(M+H) + .

[0297]

[0298] 2-((6-Chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-hydroxy-4-methoxyphenyl)acetamide (Compound 21): A mixture of 2-chloro-N-(3-hydroxy-4-methoxyphenyl)acetamide (15 mg, 0.070 mmol), 6-chloro-3H-imidazo[4,5-c]pyridine-2-thiol (15.50 mg, 0.083 mmol), sodium iodide (10.43 mg, 0.070 mmol) and triethylamine (0.015 ml, 0.104 mmol) in MeCN was heated to 65 °C for 2 hours. The crude product was subjected to combiflash silica gel chromatography (0-10% MeOH-DCM) to afford 2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-hydroxy-4-methoxyphenyl)acetamide (15 mg, 59%) as a white solid. 1H-NMR(400MHz,CD3OD / CDCl3)δppm 8.49 (s, 1H), 7.48 (s, 1H), 7.11 (d, 1H, J = 2.4Hz), 6.94 (dd, 1H, J = 4Hz, J = 8Hz), 6.83 (d, 1H, J = 8Hz), 4.18 (s, 2H), 3.81 (s, 3H); ESI-MS: m / z 365.02(M+H) + .

[0299]

[0300] 2-((6-Chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-hydroxy-3-methoxyphenyl)acetamide (Compound 22): A mixture of 2-chloro-N-(4-hydroxy-3-methoxyphenyl)acetamide (17.42 mg, 0.081 mmol), 6-chloro-3H-imidazo[4,5-c]pyridine-2-thiol (15 mg, 0.081 mmol), sodium iodide (12.11 mg, 0.081 mmol) and triethylamine (0.011 ml, 0.081 mmol) in MeCN was heated to 65 °C for 2 hours. The crude product was purified by combiflash silica gel chromatography (0-10% MeOH-DCM) to give 2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-hydroxy-3-methoxyphenyl)acetamide (17 mg, 58%) as a white solid. 1 H-NMR(400MHz,CD3OD / CDCl3)δppm 8.49(s,1H),7.45(s,1H),7.28(d,1H,J=2.4Hz),6.84(dd,1H,J=4Hz,J=8Hz),6.73(d,1H,J=8Hz),4.15(s,2H),3.81(s,3H); ESI-MS: m / z 365.01(M+H) + .

[0301]

[0302] 2-Chloro-N-(3,4-dimethoxyphenyl)propionamide (Compound 23): To a cooled solution of 3,4-dimethoxyaniline (100 mg, 0.653 mmol) and triethylamine (0.136 ml, 0.979 mmol) in DCM was added 2-chloropropionyl chloride (0.076 ml, 0.783 mmol) and stirred at room temperature for 12 hours. The solvent was removed and the crude product was purified by combiflash chromatography (0-50% EtOAc-hexanes) to give 2-chloro-N-(3,4-dimethoxyphenyl)propionamide (140 mg, 88%) as a fluffy white solid. 1 H-NMR(400MHz,CDCl3)δppm 8.19(brs,1H),7.33(d,1H,J=4Hz),6.93(dd,1H,J=4Hz,J=8Hz),6.83(d,1H,J=8H z), 4.55 (q, 1H, J = 8Hz), 3.90 (s, 3H), 3.87 (s, 3H), 1.83 (d, 3H, J = 8Hz); ESI-MS: m / z 243.90(M+H) + .

[0303]

[0304] 2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-hydroxyphenyl)acetamide (Compound 25)

[0305] The reaction mixture of 6-chloro-1H-imidazo[4,5-c]pyridine-2-thiol (15.00 mg, 0.081 mmol), 2-chloro-N-(3-hydroxyphenyl)acetamide (15 mg, 0.081 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.034 mL, 0.242 mmol) in acetonitrile (1 mL) was heated at 65° C. for 2 h. Concentration and purification on combiflash (4 g, MeOH / DCM) gave 2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-hydroxyphenyl)acetamide (5 mg, 0.015 mmol, 18.48% yield). 1 HNMR (400MHz, CD3OD) δ8.49 (s, 1H), 7.47 (s, 1H), 7.13 (m, 2H), 6.96 (s, J = 8Hz, 1H), 6.53 (d, J = 8Hz, 1H), 4.19 (s, 1H), ESI-MS: m / z 334.96 (M+H) +

[0306]

[0307] 2-Chloro-N-(3-fluoro-4-methoxyphenyl)acetamide (Compound 26)

[0308] To a solution of 3-fluoro-4-methoxyaniline (200 mg, 1.417 mmol) and TEA (0.593 mL, 4.25 mmol) in DCM (5 mL) was added 2-chloroacetyl chloride (0.135 mL, 1.700 mmol). After 1 hour, the reaction mixture was concentrated and purified on combiflash (12 g, DCM / MeOH) to give 2-chloro-N-(3-fluoro-4-methoxyphenyl)acetamide (208 mg, 0.956 mmol, 67.5% yield).

[0309] 1 HNMR (400MHZ, CDCl3) δ8.14 (s, 1H), 7.46 (d, J = 12.4Hz, 1H), 7.16 (d, J = 8.4Hz, 1H), 6.93 (m, 1H), 4.18 (s, 2H), 3.88 (s, 3H), ESI-MS: m / z 217.88 (M+H) +

[0310] 2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-fluoro-4-methoxyphenyl)acetamide (Compound 27)

[0311] The reaction mixture of 6-chloro-1H-imidazo[4,5-c]pyridine-2-thiol (17.06 mg, 0.092 mmol), 2-chloro-N-(3-fluoro-4-methoxyphenyl)acetamide (20 mg, 0.092 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.038 mL, 0.276 mmol) in CH3CN was heated at 65°C for 2 hours. Concentrated and purified on combiflash (4 g, MeOH / DCM) to give 2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-fluoro-4-methoxyphenyl)acetamide (16 mg, 0.044 mmol, 47.5% yield). 1 HNMR (400MHz, CD3OD) δ8.50 (s, 1H), 7.48 (m, 2H), 7.18 (d, J = 8Hz, 1H), 7.03 (t, J = 8.8Hz, 1H), 4.22 (s, 2H), 3.84 (s, 3H), ESI-MS: m / z 366.97 (M+H) +

[0312]

[0313] 2-Chloro-N-(4-(difluoromethoxy)-3-methoxyphenyl)acetamide (Compound 28)

[0314] To the reaction mixture of 4-(difluoromethoxy)-3-methoxyaniline (150 mg, 0.793 mmol), TEA (0.334 mL, 2.379 mmol) in DCM (4 mL) was added 2-chloroacetyl chloride (0.076 mL, 0.952 mmol) dropwise. After 1 hour at room temperature, the mixture was concentrated and purified on combiflash (12 g, DCM / MeOH) to give 2-chloro-N-(4-(difluoromethoxy)-3-methoxyphenyl)acetamide (134 mg, 0.504 mmol, 63.6% yield). 1 HNMR (400MHz, CDCl3) δ8.23 (s, 1H), 7.51 (s, 1H), 7.13 (d, J = 8.4Hz, 1H), 6.92 (m, 1 H),6.7,6.51(d,J=75Hz,1H),4.2(s,2H),3.89(s,3H).ESI-MS:m / z,265.92(M+H) +

[0315] 2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-(difluoromethoxy)-3-methoxyphenyl)acetamide (Compound 29)

[0316] The reaction mixture of 6-chloro-1H-imidazo[4,5-c]pyridine-2-thiol (13.98 mg, 0.075 mmol), 2-chloro-N-(4-(difluoromethoxy)-3-methoxyphenyl)acetamide (20 mg, 0.075 mmol), sodium iodide (11.29 mg, 0.075 mmol), triethylamine (0.031 mL, 0.226 mmol) in acetonitrile (1 mL) was heated to 65 °C for 2 hours. Concentration and purification by combiflash (4 g, DCM / MeOH) gave 2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-(difluoromethoxy)-3-methoxyphenyl)acetamide (24 mg, 0.058 mmol, 77% yield). 1HNMR (400MHz, CDCl3) δ8.50 (s, 1H), 7.56 (s, 1H), 7.38 (s, 1H), 7.02 (d, J = 8.4Hz, 1H ),6.81(d,J=8.4Hz,1H),6.43(t,J=76Hz,1H),3.99(s,1H),3.80(s,1H)ESI-MS:m / z 414.97(M+H) +

[0317]

[0318] 6-(Trifluoromethyl)-3H-imidazo[4,5-c]pyridine-2-thiol (Compound 30)

[0319] A reaction mixture of 6-(trifluoromethyl)pyridine-3,4-diamine (100 mg, 0.565 mmol), di(1H-imidazol-1-yl)methanthione (101 mg, 0.565 mmol) in tetrahydrofuran (4 mL, 0.565 mmol) was stirred overnight, the precipitate was filtered, and 6-(trifluoromethyl)-3H-imidazo[4,5-c]pyridine-2-thiol (70 mg, 0.319 mmol, 56.6% yield) was obtained. 1 HNMR (400MHz, CDCl3-CD3OD) δ8.36 (s, 1H), 7.42 (s, 1H), ESI: m / z 219.8 (M+H) +

[0320] N-(3-Hydroxy-4-methoxyphenyl)-2-((6-(trifluoromethyl)-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide (Compound 31)

[0321] The reaction mixture of 6-(trifluoromethyl)-1H-imidazo[4,5-c]pyridine-2-thiol (15.25 mg, 0.070 mmol), 2-chloro-N-(3-hydroxy-4-methoxyphenyl)acetamide (15 mg, 0.070 mmol), sodium iodide (10.43 mg, 0.070 mmol), triethylamine (0.029 mL, 0.209 mmol) in acetonitrile (1 mL) was heated to 65 °C for 2 hours. Concentration and purification by combiflash (4 g, DCM / MeOH) gave N-(3-hydroxy-4-methoxyphenyl)-2-((6-(trifluoromethyl)-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide (24 mg, 0.060 mmol, 87% yield). 1HNMR (400MHz, CDCl3) δ9.03 (s, 1H), 8.00 (s, 1H), 7.50 (d, J = 8.4Hz, 1H), 7.29 (s, 1H), 7.14 (s, 1H), 6.96 (s, 1H), 4.22 (s, 2H), 4.03 (m, 3H). ESI-MS: m / z 399.06(M+H) +

[0322]

[0323] 2-Chloro-N-(4-hydroxyphenyl)acetamide (Compound 32)

[0324] To the reaction mixture of 4-aminophenol (200 mg, 1.833 mmol), TEA (0.772 mL, 5.50 mmol) in DCM (4 mL) was added 2-chloroacetyl chloride (0.146 mL, 1.833 mmol) dropwise. After 1 hour at room temperature, the mixture was concentrated and purified on combiflash (12 g, DCM / MeOH) to give 2-chloro-N-(4-hydroxyphenyl)acetamide (134 mg, 0.722 mmol, 39.4% yield). 1 HNMR (400MHz, CDCl3) δ7.32 (d, J=8.8Hz, 2H), 7.79 (d, J=8.4Hz, 2H), 4.15 (s, 2H) ESI-MS: m / z 185.84 (M+H) +

[0325] 2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-hydroxyphenyl)acetamide (Compound 33)

[0326] The reaction mixture of 6-chloro-1H-imidazo[4,5-c]pyridine-2-thiol (15.00 mg, 0.081 mmol), 2-chloro-N-(4-hydroxyphenyl)acetamide (15 mg, 0.081 mmol), sodium iodide (12.11 mg, 0.081 mmol), triethylamine (0.034 mL, 0.242 mmol) in acetonitrile (1 mL) was heated to 65° C. for 2 hours. Concentration and purification by combiflash (4 g, DCM / MeOH) gave 2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-hydroxyphenyl)acetamide (24 mg, 0.072 mmol, 89% yield). 1HNMR (400MHz, CD3OD) δ8.48(s,1H),7.38(s,1H),7.30(d,J=8.8Hz,2H)7.19(s,1H),6.71(d,J=8.8Hz,2H),3.98(s,2H), ESI-MS: m / z 334.99(M+H) +

[0327]

[0328] 2-Chloro-N-(4-methoxy-3-(trifluoromethoxy)phenyl)acetamide (Compound 34)

[0329] To the reaction mixture of 4-methoxy-3-(trifluoromethoxy)aniline (200 mg, 0.965 mmol), TEA (0.407 mL, 2.90 mmol) in DCM (4 mL) was added 2-chloroacetyl chloride (0.092 mL, 1.159 mmol) dropwise. After 1 hour at room temperature, the mixture was concentrated and purified on combiflash (12 g, DCM / MeOH) to give 2-chloro-N-(4-methoxy-3-(trifluoromethoxy)phenyl)acetamide (212 mg, 0.747 mmol, 77% yield). 1 HNMR (400MHz, CDCl3) δ8.16 (s, 1H), 7.51 (s, 1H), 7.43 (d, J = 6.4Hz, 1H), 6.97 (d, J = 8.8Hz, 1H) 4.19 (s, 2H), 3.88 (s, 3H). ESI-MS: m / z 283.87 (M+H) +

[0330] 2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-methoxy-3-(trifluoromethoxy)phenyl)acetamide (Compound 35)

[0331] The reaction mixture of 6-chloro-1H-imidazo[4,5-c]pyridine-2-thiol (9.82 mg, 0.053 mmol), 2-chloro-N-(4-methoxy-3-(trifluoromethoxy)phenyl)acetamide (15 mg, 0.053 mmol), sodium iodide (7.93 mg, 0.053 mmol), triethylamine (0.022 mL, 0.159 mmol) in acetonitrile (1 mL) was heated to 65 °C for 2 hours. Concentration and purification by combiflash (4 g, DCM / MeOH) gave 2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-methoxy-3-(trifluoromethoxy)phenyl)acetamide (25 mg, 0.058 mmol, 100% yield). 1 HNMR (400MHz, CD3OD) δ8.50(s,1H),7.65(s,1H),7.49(s,1H),7.43(d,J=6.8Hz,1H),7.10(d,J=8.8Hz,1H),4.23(s,2H),3.85(s,3H).ESI-MS: m / z 432.97(M+H) +

[0332]

[0333] 2-Chloro-N-(4-fluoro-3-hydroxyphenyl)acetamide (Compound 36)

[0334] To the reaction mixture of 5-amino-2-fluorophenol (200 mg, 1.573 mmol), TEA (0.663 mL, 4.72 mmol) in DCM (4 mL) was added 2-chloroacetyl chloride (0.125 mL, 1.573 mmol) dropwise. After 1 hour at room temperature, the mixture was concentrated and purified on combiflash (12 g, DCM / MeOH) to give 2-chloro-N-(4-fluoro-3-hydroxyphenyl)acetamide (169 mg, 0.830 mmol, 52.8% yield). 1 HNMR(400MHz,CD3OD)δ7.22(m,1H),6.93(m,1H),6.83(m,1H),4.07(s,2H)ESI-MS:m / z 203.79(M+H) +

[0335] 2-((6-Chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-fluoro-3-hydroxyphenyl)acetamide (Compound 37)

[0336] The reaction mixture of 6-chloro-1H-imidazo[4,5-c]pyridine-2-thiol (13.68 mg, 0.074 mmol), 2-chloro-N-(4-fluoro-3-hydroxyphenyl)acetamide (15 mg, 0.074 mmol), sodium iodide (11.04 mg, 0.074 mmol), triethylamine (0.031 mL, 0.221 mmol) in acetonitrile (1 mL) was heated to 65 °C for 2 hours. Concentration and purification by combiflash (4 g, DCM / MeOH) gave 2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-fluoro-3-hydroxyphenyl)acetamide (24 mg, 0.068 mmol, 92% yield). 1 HNMR(400MHz,CD3OD)δ8.50(s,1H),7.5(s,1H),7.28(m,1H)6.95(m,2H),4.21(s,2H).ESI-MS:m / z 352.93(M+H) +

[0337]

[0338] N-(3-Fluoro-4-methoxyphenyl)-2-((6-(trifluoromethyl)-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide (Compound 38)

[0339] The reaction mixture of 6-(trifluoromethyl)-1H-imidazo[4,5-c]pyridine-2-thiol (15.11 mg, 0.069 mmol), 2-chloro-N-(3-fluoro-4-methoxyphenyl)acetamide (15 mg, 0.069 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.029 mL, 0.207 mmol) in acetonitrile (1 mL) was heated to 65 °C for 2 hours. Concentration and purification by combiflash (4 g, DCM / MeOH) gave N-(3-fluoro-4-methoxyphenyl)-2-((6-(trifluoromethyl)-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide (23 mg, 0.057 mmol, 83% yield). 1 HNMR (400MHz, CD3OD) δ8.81(s,1H),7.89(s,1H),7.51(m,1H),7.20(m,1H),7.02(t,J=8.8Hz,1H),4.26(s,2H),3.33(s,3H).ESI-MS:m / z 400.99(M+H) +

[0340]

[0341] 2-Chloro-N-(4-(difluoromethoxy)phenyl)acetamide (Compound 39): To a solution of 4-(difluoromethoxy)aniline (0.078 ml, 0.628 mmol) and triethylamine (0.131 ml, 0.943 mmol) in DCM was added 2-chloroacetyl chloride (0.060 ml, 0.754 mmol) at 0°C. The mixture was stirred at room temperature for 2 hours. The solvent was removed and the crude product was purified by combiflash chromatography (0-30% EtOAc-hexanes) to give 2-chloro-N-(4-(difluoromethoxy)phenyl)acetamide (110 mg, 0.467 mmol, 74.3% yield) as a light orange solid. 1 H-NMR (400MHz, CDCl3) δppm 8.24 (brs, 1H), 7.55 (dd, 2H, J = 2Hz, J = 4Hz), 7.13 (d, 2H, J = 8Hz), 6.48 (t, 1H, J = 71.5Hz), 4.20 (s, 2H); ESI-MS: m / z 235.86 (M+H) + .

[0342] 2-((6-Chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-(difluoromethoxy)phenyl)acetamide (Compound 40): To a mixture of 2-chloro-N-(4-(difluoromethoxy)phenyl)acetamide (15 mg, 0.064 mmol), 6-chloro-3H-imidazo[4,5-c]pyridine-2-thiol (13.00 mg, 0.070 mmol) and sodium iodide (9.55 mg, 0.064 mmol) in MeCN was added triethylamine (0.013 ml, 0.095 mmol), and the mixture was stirred at 65 °C for 2 h. The solvent was removed and the crude product was purified by combiflash SiO2 chromatography (0-100% EtOAc-hexanes) to give 2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-(difluoromethoxy)phenyl)acetamide (21 mg, 0.055 mmol, 86% yield) as a white solid. 1 H-NMR(400MHz,CD3OD-CDCl3)δppm 8.48 (s, 1H), 7.52 (d, 2H, J = 8Hz), 7.42 (s, 1H), 7.05 (d, 1H, J = 8Hz), 6.53 (t, 1H, J = 76Hz), 4.12 (s, 2H); ESI-MS: m / z384.97 (M+H) + .

[0343]

[0344] 2-Chloro-N-(3-hydroxy-4-methoxyphenyl)propionamide (Compound 41): A solution of 5-amino-2-methoxyphenol (100 mg, 0.719 mmol) and potassium carbonate (109 mg, 0.791 mmol) in acetone / water was cooled and 2-chloropropionyl chloride (0.084 ml, 0.862 mmol) was added. The mixture was stirred at -10 °C for 1 hour. The reaction was diluted with EtOAc, washed with 0.5 M HCl, water, brine and dried over anhydrous Na2SO4. The solvent was removed and the crude brown solid, 2-chloro-N-(3-hydroxy-4-methoxyphenyl)propionamide (130 mg, 0.566 mmol, 79% yield) was used in the next step without purification. 1 H-NMR (400MHz, CDCl3) δppm 7.09 (d, 1H, J = 2.4Hz), 6.96 (dd, 1H, J = 2.4Hz and J=8.8Hz), 6.77(d,1H,J=8.8Hz), 4.47(q,1H,J=6.8Hz), 3.81(s,3H),1.68(d,3H,J=6.8Hz); ESI-MS: m / z 229.89(M+H) + .

[0345] N-(3-Hydroxy-4-methoxyphenyl)-2-((6-(trifluoromethyl)-3H-imidazo[4,5-c]pyridin-2-yl)thio)propanamide (Compound 42): A mixture of 2-chloro-N-(3-hydroxy-4-methoxyphenyl)propanamide (12 mg, 0.052 mmol), 6-(trifluoromethyl)-3H-imidazo[4,5-c]pyridine-2-thiol (13.74 mg, 0.063 mmol), sodium iodide (3.92 mg, 0.026 mmol) and triethylamine (10.92 μl, 0.078 mmol) in acetonitrile (1 ml) was stirred at 65°C for 4 hours. The solvent was removed and the crude product was purified by combiflash chromatography (0-5% MeOH-DCM) to afford N-(3-hydroxy-4-methoxyphenyl)-2-((6-(trifluoromethyl)-3H-imidazo[4,5-c]pyridin-2-yl)thio)propanamide (10 mg, 0.024 mmol, 46.4% yield) as an orange solid. 1H-NMR(400MHz,CD3OD)δppm 8.84(s,1H),7.91(s,1H),7.12(d,1H,J=2.4Hz),6.94(dd,1H,J=2.4Hz,J=8.8Hz),6.8 4(d,1H,J=8.4Hz), 4.78(q,1H,J=7.2), 3.81(s,3H),1.72(d,3H,J=7.2Hz); ESI-MS: m / z 413.02(M+H) + .

[0346]

[0347] 2-Chloro-N-(3,5-difluoro-4-methoxyphenyl)acetamide (Compound 43): A solution of 3,5-difluoro-4-methoxyaniline (100 mg, 0.628 mmol) and triethylamine (131 μl, 0.943 mmol) in DCM was cooled and 2-chloroacetyl chloride (60.0 μl, 0.754 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. The reaction was diluted with DCM, washed with 0.5 M HCl, water, brine, dried over Na2SO4 and evaporated. The crude product, a brown solid, 2-chloro-N-(3,5-difluoro-4-methoxyphenyl)acetamide (120 mg, 0.509 mmol, 81% yield) was used in the next step without purification. 1 H-NMR (400MHz, CDCl3) δppm 8.15 (s, 1H), 7.21 (d, 1H, J = 8Hz), 4.18 (s, 1H), 3.97 (s, 3H); ESI-MS: m / z 235.82 (M+H) + .

[0348] N-(3,5-difluoro-4-methoxyphenyl)-2-((6-(trifluoromethyl)-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide (Compound 44): A mixture of 2-chloro-N-(3,5-difluoro-4-methoxyphenyl)acetamide (14.19 mg, 0.060 mmol), 6-(trifluoromethyl)-3H-imidazo[4,5-c]pyridine-2-thiol (12 mg, 0.055 mmol), sodium iodide (4.10 mg, 0.027 mmol) and triethylamine (0.011 ml, 0.082 mmol) in MeCN was stirred at 65 °C for 1 hour. The crude product was purified by combiflash chromatography (0-100% EtOAc-hexanes) to give N-(3,5-difluoro-4-methoxyphenyl)-2-((6-(trifluoromethyl)-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide (20 mg, 0.048 mmol, 87% yield) as a white solid. 1 H-NMR (400MHz, CD3OD) δppm 8.81 (s, 1H), 7.89 (s, 1H), 7.27 (d, 2H, J = 8Hz), 4.27 (s, 2H), 3.89 (s, 3H); ESI-MS: m / z 419.00 (M+H) + .

[0349]

[0350] N-(4-Fluoro-3-hydroxyphenyl)-2-((6-(trifluoromethyl)-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide (Compound 45)

[0351] The reaction mixture of 6-(trifluoromethyl)-1H-imidazo[4,5-c]pyridine-2-thiol (12 mg, 0.055 mmol), 2-chloro-N-(4-fluoro-3-hydroxyphenyl)acetamide (12.26 mg, 0.060 mmol), sodium iodide (8.21 mg, 0.055 mmol), triethylamine (0.023 mL, 0.164 mmol) in acetonitrile (1 mL) was heated to 65 °C for 2 hours. Concentration and purification by combiflash (4 g, DCM / MeOH) gave N-(4-fluoro-3-hydroxyphenyl)-2-((6-(trifluoromethyl)-1H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide (15 mg, 0.039 mmol, 70.9% yield). 1HNMR (400MHz, CD3OD) δ8.82 (s, 1H), 7.90 (s, 1H), 7.28 (dd, J1=2.4Hz, J2=8Hz, 1H), 6.94 (m, 2H), 4.25 (s, 2H), ESI-MS: m / z 387.03 (M+H) +

[0352]

[0353] 2-Chloro-3-methyl-5-nitropyridin-4-amine (Compound 46)

[0354] To a solution of 2-chloro-3-methylpyridin-4-amine (250 mg, 1.753 mmol) in sulfuric acid (2.63 mL, 49.3 mmol) at 0°C was added potassium nitroperoxide (355 mg, 3.51 mmol). The mixture was warmed to room temperature and kept stirring overnight, poured into ice water and extracted with EtOAc. The organic layer was dried over MgSO4 and concentrated. Purification on combiflash (12 g, hexane / EtOAc) gave 2-chloro-3-methyl-5-nitropyridine-4-amine (157 mg, 0.837 mmol, 47.7% yield). 1 HNMR (400MHz, CDCl3), δ8.96 (s, 1H), 2.32 (s, 3H) ESI-MS: m / z187.84 (M+H) +

[0355] 6-Chloro-5-methylpyridine-3,4-diamine (Compound 47)

[0356] The reaction mixture of 2-chloro-3-methyl-5-nitropyridin-4-amine (10 mg, 0.053 mmol), iron (14.89 mg, 0.267 mmol), NH4Cl (14.26 mg, 0.267 mmol) in water (0.5 mL) and ethanol (0.5 mL) was heated to 70°C for 1.5 hours. Purification by preparative TLC (hexanes / EtOAc) gave 6-chloro-5-methylpyridine-3,4-diamine (8 mg, 0.051 mmol, 95% yield). 1 HNMR(400MHz, CDCl3)δ7.644(s,1H),2.22(s,3H)ESI-MS:m / z157.83(M+H) +

[0357] 6-Chloro-7-methyl-3H-imidazo[4,5-c]pyridine-2-thiol (Compound 48)

[0358] The reaction mixture of 6-chloro-5-methylpyridine-3,4-diamine (8 mg, 0.051 mmol), di(1H-imidazol-1-yl)methanone (9.05 mg, 0.051 mmol) in tetrahydrofuran (0.5 mL, 0.051 mmol) was stirred overnight. Preparative TLC gave 6-chloro-7-methyl-3H-imidazo[4,5-c]pyridine-2-thiol (6 mg, 0.030 mmol, 59.2% yield). 1 HNMR(400MHz,CD3OD)δ7.996(s,1H).2.44(s,3H)ESI-MS:m / z 199.79(M+H) +

[0359]

[0360] 2-((6-chloro-7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-hydroxy-4-methoxyphenyl)acetamide (Compound 49)

[0361] The reaction mixture of 6-chloro-7-methyl-1H-imidazo[4,5-c]pyridine-2-thiol (6 mg, 0.030 mmol), 2-chloro-N-(3-hydroxy-4-methoxyphenyl)acetamide (6.48 mg, 0.030 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.013 mL, 0.090 mmol) in acetonitrile (1 mL) was heated to 65 °C for 2 hours. Concentration and purification by preparative TLC (10%, DCM / MeOH) gave 2-((6-chloro-7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-hydroxy-4-methoxyphenyl)acetamide (7 mg, 0.018 mmol, 61.5% yield). 1 HNMR (400MHz, CD3OD) δ8.36 (s, 1H), 7.11 (d, J = 2Hz, 1H), 6.95 (dd, J1 = 2Hz, J2 = 8. 8Hz,1H),6.84(8.8Hz,1H),4.19(s,2H),3.011(s,3H),2.54(s,3H).ESI-MS:m / z 379.00(M+H) +

[0362]

[0363] 2-((6-chloro-7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-fluoro-4-methoxyphenyl)acetamide (Compound 50)

[0364] The reaction mixture of 6-chloro-7-methyl-1H-imidazo[4,5-c]pyridine-2-thiol (10 mg, 0.050 mmol), 2-chloro-N-(3-fluoro-4-methoxyphenyl)acetamide (10.90 mg, 0.050 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.021 mL, 0.150 mmol) in acetonitrile (1 mL) was heated at 65° C. for 2 h. Concentration and purification on preparative TLC (15%, MeOH / DCM) gave 2-((6-chloro-7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-fluoro-4-methoxyphenyl)acetamide (3.1 mg, 8.14 μmol, 16.25% yield). 1 HNMR (400MHz, CD3OD) δ8.36 (s, 1H), 7.50 (d, J = 13.4Hz, 1H), 7.18 (d, J = 8.4Hz, 1H),7.03(t,J=9.2Hz,1H),4.21(s,2H),3.84(s,3H)2.53(s,3H),ESI-MS:m / z 381.05(M+H) +

[0365]

[0366] 2-((6-chloro-7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-fluoro-3-hydroxyphenyl)acetamide (Compound 51)

[0367] The reaction mixture of 6-chloro-7-methyl-1H-imidazo[4,5-c]pyridine-2-thiol (10 mg, 0.050 mmol), 2-chloro-N-(4-fluoro-3-hydroxyphenyl)acetamide (10.20 mg, 0.050 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.021 mL, 0.150 mmol) in acetonitrile (1 mL) was heated at 65° C. for 2 hours. Concentration and purification by preparative TLC (15%, MeOH / DCM) gave 2-((6-chloro-7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-fluoro-3-hydroxyphenyl)acetamide (14.5 mg, 0.040 mmol, 79% yield). 1 HNMR(400MHz,CD3OD),8.36(s,1H),7.28(m,1H),6.97(m,2H),4.20(s,2H),2.54(s,3H),ESI-MS:m / z 367.00(M+H) +

[0368]

[0369] 2-((6-chloro-7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3,4-dimethoxyphenyl)acetamide (Compound 52)

[0370] The reaction mixture of 6-chloro-7-methyl-1H-imidazo[4,5-c]pyridine-2-thiol (10 mg, 0.050 mmol), 2-chloro-N-(3,4-dimethoxyphenyl)acetamide (11.50 mg, 0.050 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.021 mL, 0.150 mmol) in acetonitrile (1 mL) was heated at 65° C. for 2 hours. 2-((6-chloro-7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3,4-dimethoxyphenyl)acetamide (7.4 mg, 0.019 mmol, 37.6% yield) was obtained by filtration. 1 HNMR(400MHz,CD3OD)δ8.37(s,1H),7.30(d,J=2.4Hz,1H),7.01(m,1H),6.88(d,J=8.8Hz,1H),4.22(s,2H),3.79(s,6H),2.54(s,3H)ESI-MS:m / z 393.05(M+H) +

[0371]

[0372] 2-Chloro-N-(5,6-dimethoxybenzo[d]thiazol-2-yl)acetamide (Compound 53)

[0373] To the reaction mixture of 5,6-dimethoxybenzo[d]thiazol-2-amine (50 mg, 0.238 mmol), TEA (0.100 mL, 0.713 mmol) in DCM (1 mL) was added 2-chloroacetyl chloride (0.023 mL, 0.285 mmol) at room temperature overnight, concentrated and purified on combiflash (4 g, MeOH / DCM) to give 2-chloro-N-(5,6-dimethoxybenzo[d]thiazol-2-yl)acetamide (15 mg, 0.052 mmol, 22.00% yield). 1 HNMR(400MHz, CDCl3),9.73(s,1H),7.26(s,1H),7.25(s,1H),4.30(s,2H),3.969s,6H)ESI-MS:m / z 286.94(M+H) +

[0374] 2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(5,6-dimethoxybenzo[d]thiazol-2-yl)acetamide (Compound 54)

[0375] The reaction mixture of 6-chloro-3H-imidazo[4,5-c]pyridine-2-thiol (6.47 mg, 0.035 mmol), 2-chloro-N-(5,6-dimethoxybenzo[d]thiazol-2-yl)acetamide (10 mg, 0.035 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.015 mL, 0.105 mmol) in CH3CN (1 mL) was heated at 65°C for 2 h. Concentration and combiflash (4 g, DCM / MeOH) gave 2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(5,6-dimethoxybenzo[d]thioxo-2-yl)acetamide (8 mg, 0.018 mmol, 52.6% yield). 1 HNMR(400MHz,CD3OD)δ8.12(s,LI),7.41(s,1H),7.33(s,2H),4.38(s,2H),3.89(s,3H),3.88(s,3H),ESI-MS:m / z436.00(M+H) +

[0376]

[0377] (2-(Trifluoromethoxy)phenyl)methyl carbonate (Compound 55)

[0378] To a cooled solution of 2-(trifluoromethoxy)phenyl (0.51 ml, 3.93 mmol) and triethylamine (1.1 ml, 7.86 mmol) in DCM was slowly added methyl chloroformate (0.36 ml, 4.72 mmol) and the reaction was stirred at 25°C for 2 hours. The reaction was diluted with DCM and washed with water, brine, dried over anhydrous Na2SO4 and evaporated. The crude turbid oil (750 mg, 81%) was used in the next step without further purification. 1 H-NMR (400MHz, CDCl3) δppm 7.30 (m, 4H), 3.93 (s, 3H); 19 F-NMR (376.5MHz, CDCl3) δppm-58.10 (s, 3F).

[0379] (5-Nitro-2-(trifluoromethoxy)phenyl)methyl carbonate (Compound 57)

[0380] The suspension of sulfuric acid (2.0ml, 37.5mmol) containing (2-(trifluoromethoxy) phenyl) methyl carbonate (750mg, 3.18mmol) is cooled and solid potassium nitrate (250mg, 2.473mmol) is added partly. The mixture is stirred at room temperature for 2 hours. The reaction mixture is poured into ice water and extracted with DCM. The extracts merged are washed with water, salt water and through Na2SO4 dry. Remove solvent, and the crude product is passed through combiflash SiO2 chromatography (0-5% EtOAc-hexane) is purified to produce (4-nitro-2-(trifluoromethoxy) phenyl) methyl carbonate (250mg, 28%) and (5-nitro-2-(trifluoromethoxy) phenyl) methyl carbonate (553mg, 62%) in tan solid.

[0381] Methyl (5-nitro-2-(trifluoromethoxy)phenyl) carbonate (less polar): 1 H-NMR (400MHz, CDCl3) δppm8.23 (m, 1H), 8.21 (dd, 1H, J = 2.4, 8.8Hz), 7.52 (dd, 1H, J = 1.2, 9.2Hz), 3.98 (s, 3H); 19 F-NMR (376.5MHz, CDCl3) δppm-57.77 (s, 3F)

[0382] (4-nitro-2-(trifluoromethoxy)phenyl)methyl carbonate (Compound 56) (polar): 1 H-NMR (400MHz, CDCl3) δppm 8.25 (m, 2H), 7.52 (dd, 1H, J = 4.4, 5.2Hz), 3.97 (s, 3H); 19 F-NMR (376.5MHz, CDCl3) δppm-58.23 (s, 3F).

[0383] 5-Amino-2-(trifluoromethoxy)phenyl methyl carbonate (Compound 58)

[0384] To a solution of methyl (5-nitro-2-(trifluoromethoxy)phenyl)carbonate (190 mg, 0.676 mmol) and ammonium chloride (108 mg, 2.027 mmol) in EtOH-water (1:1) was added iron (113 mg, 2.027 mmol), and the mixture was stirred in a microwave reactor at 100° C. for 30 minutes. The solvent was removed, and the crude product was purified by combiflash SiO chromatography (0-30% EtOAc-hexanes) to give methyl 5-amino-2-(trifluoromethoxy)phenylcarbonate (66 mg, 39%) as a brown solid. 1H-NMR (400MHz, CDCl3) δppm 7.09 (dd, 1H, J = 0.8, 8.4Hz), 6.54 (d, 1H, J = 2.8Hz), 6.51 (dd, 1H, J = 2.8, 8.8Hz), 3.91 (s, 3H); 19 F-NMR (376.5MHz, CDCl3) δppm-58.82 (s, 3F).

[0385] 5-Amino-2-(trifluoromethoxy)phenol (Compound 59)

[0386] The solution of MeOH-water (2:1) containing 5-amino-2-(trifluoromethoxy)phenyl methyl carbonate (66mg, 0.263mmol) and potassium carbonate (73mg, 0.526mmol) was stirred at room temperature for 1 hour.Remove MeOH, and react with 0.5M HCl neutralization and extract with EtOAc.The organic layer merged is washed with salt water, through Na2SO4 dry and evaporated.By the crude product brown solid, 5-amino-2-(trifluoromethoxy)phenol (40mg, 79%) is used for next step without purification. 1 H-NMR (400MHz, CD3OD) δppm 6.88 (dd, 1H, J = 0.8, 8.8Hz), 6.29 (d, 1H, J = 2.8Hz), 6.17 (dd, 1H, J = 2.4, 8.8Hz); 19 F-NMR (376.5MHz, CDCl3) δppm-59.92 (s, 3F); ESI-MS: m / z 193.85 (M+H) + .

[0387] 2-Chloro-N-(3-hydroxy-4-(trifluoromethoxy)phenyl)acetamide (Compound 60)

[0388] To a cooled solution of 5-amino-2-(trifluoromethoxy)phenol (90 mg, 0.466 mmol) and potassium carbonate (77 mg, 0.559 mmol) in acetone-water (3:1) was slowly added 2-chloroacetyl chloride (0.044 ml, 0.559 mmol) and stirred for 2 hours at 0° C. The solvent was removed and the crude product was purified by combiflash SiO chromatography (0-50% EtOAc-hexanes) to give 2-chloro-N-(3-hydroxy-4-(trifluoro-methoxy)phenyl)acetamide (98 mg, 78%) as a brown solid. 1H-NMR (400MHz, CD3OD) δppm 7.40 (d, 1H, J = 2.4Hz), 7.10 (d, 1H, J = 8.8Hz), 6.95 (dd, 1H, J = 2.0, 8.8Hz, 4.12 (s, 2H); 19 F-NMR (376.5MHz, CDCl3) δppm-59.92 (s, 3F); ESI-MS: m / z269.92 (M+H) + .

[0389] N-(3-Hydroxy-4-(trifluoromethoxy)phenyl)-2-((6-(trifluoromethyl)-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide (Compound 61)

[0390] A mixture of 2-chloro-N-(3-hydroxy-4-(trifluoromethoxy)phenyl)acetamide (12 mg, 0.045 mmol), 6-(trifluoromethyl)-3H-imidazo[4,5-c]pyridine-2-thiol (11.71 mg, 0.053 mmol), triethylamine (9.31 μl, 0.067 mmol) and sodium iodide (3.34 mg, 0.022 mmol) in MeCN was stirred at 65° C. for 1 h. The crude product was purified by combiflash SiO chromatography (0-5% MeOH-DCM) to give N-(3-hydroxy-4-(trifluoromethoxy)phenyl)-2-((6-(trifluoromethyl)-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide (11 mg, 55%) as an off-white solid. 1 H-NMR (400MHz, CD3OD) δppm 8.79 (s, 1H), 7.87 (s, 1H), 7.39 (d, 1H, J = 2.4Hz), 7.12 (dd, 1H, J = 1.2, 8.8Hz), 6.97 (dd, 1H, J = 2.8, 8.8Hz), 4.23 (s, 2H); 19 F-NMR (376.5MHz, CDCl3) δppm-60.12 (s, 3F), -67.41 (s, 3F); ESI-MS: m / z 453.01 (M+H) + .

[0391]

[0392] N-(Benzo[d]thiazol-2-yl)-2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide (Compound 62)

[0393] To a mixture of 6-chloro-3H-imidazo[4,5-c]pyridine-2-thiol (10 mg, 0.054 mmol), N-(benzo[d]thiazol-2-yl)-2-chloroacetamide (14.7 mg, 0.065 mmol) and sodium iodide (4.0 mg, 0.027 mmol) in MeCN was added triethylamine (11 μL, 0.081 mmol) and the reaction was stirred at 65° C. for 1 hour. The solvent was removed and the crude product was purified by combiflash SiO chromatography (0-5% MeOH-DCM) to give N-(benzo[d]thiazol-2-yl)-2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide (16 mg, 79%) as a white solid. 1 H-NMR(400MHz,DMSO-d6)δppm 8.50 (s, 1H), 7.95 (d, 1H, J = 8Hz), 7.75 (d, 1H, J = 8Hz), 7.51 (s, 1H), 7.43 (t, 1H, J = 7.2Hz), 7.29 (t, 1H, J = 7.2Hz), 4.46 (s, 2H); ESI-MS: m / z 375.98(M) + ,377.99(M+2H) + .

[0394]

[0395] 2-Bromo-5-nitropyridin-4-amine (Compound 63)

[0396] To a solution of 2,4-dibromo-5-nitropyridine (200 mg, 0.709 mmol) and triethylamine (0.2 ml, 1.419 mmol) in THF 7M ammonia (0.2 ml, 1.419 mmol) was added and stirred at room temperature for 24 hours. The solvent was removed and the crude product was purified by combiflash SiO2 chromatography (0-50% EtOAc-hexanes) to give 2-bromo-5-nitropyridine-4-amine (135 mg, 87%) as a tan solid. 1 H-NMR (400MHz, CD3OD) δppm 8.82 (s, 1H), 7.1 1 (s, 1H); ESI-MS: m / z 219.69 (M+H) + .

[0397] 4-Amino-5-nitropyridinecarbonitrile (Compound 64)

[0398] A microwave reactor was used to heat the solution in DMA containing 2-bromo-5-nitropyridine-4-amine (135 mg, 0.619 mmol) and cupric cyanide (67 mg, 0.743 mmol) to 200 ° C for 1 hour. The reaction mixture was distributed between water and EtOAc and tilted through diatomaceous earth. The aqueous layer was extracted with EtOAc. The combined organic layers were washed with water, brine, dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by combiflash SiO2 chromatography using (0-50% EtOAc-hexane) to obtain 4-amino-5-nitropyridine carbonitrile (70 mg, 69%) as a light brown solid. 1 H-NMR (400MHz, CD3OD) δppm 9.07 (s, 1H), 7.37 (s, 1H); ESI-MS: m / z 164.77 (M+H) + .

[0399] 4,5-Diaminopyridinecarbonitrile (Compound 65)

[0400] To the slurry of EtOH containing 4-amino-5-nitropyridine carbonitrile (70mg, 0.427mmol) and iron (72mg, 1.280mmol), water containing ammonium chloride (68mg, 1.280mmol) was added, and the mixture was heated to 65°C for 1 hour. The reaction mixture was filtered through diatomaceous earth and the solvent was removed. The crude product was purified by combiflash SiO2 chromatography (0-5% MeOH-DCM) to obtain 4,5-diaminopyridine carbonitrile (32mg, 56%) as a light brown solid. 1 H-NMR (400MHz, CD3OD) δppm 7.72 (s, 1H), 6.92 (s, 1H); ESI-MS: m / z 134.85 (M+H) + .

[0401] 2-Mercapto-3H-imidazo[4,5-c]pyridine-6-carbonitrile (Compound 66)

[0402] A solution of 4,5-diaminopyridinecarbonitrile (32 mg, 0.239 mmol), di(1H-imidazol-1-yl)methanthione (51 mg, 0.286 mmol) and N,N-dimethylpyridin-4-amine (29 mg, 0.239 mmol) in MeCN was stirred at room temperature for 16 hours. MeCN was removed and the crude product was purified by combiflash SiO2 chromatography (0-5% MeOH-DCM) to give 2-mercapto-3H-imidazo[4,5-c]pyridine-6-carbonitrile (30 mg, 71%) as a tan solid. 1H-NMR (400MHz, CD3OD) δppm 8.43 (s, 1H), 7.64 (s, 1H); ESI-MS: m / z 176.91 (M+H) + .

[0403]

[0404] 2-((6-cyano-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-hydroxy-4-methoxyphenyl)acetamide (Compound 67)

[0405] To a mixture of 2-chloro-N-(3-hydroxy-4-methoxyphenyl)acetamide (3.3 mg, 0.015 mmol), 2-mercapto-3H-imidazo[4,5-c]pyridine-6-carbonitrile (3 mg, 0.017 mmol) and sodium iodide (1.2 mg, 7.74 μmol) in MeCN was added triethylamine (2.6 μL, 0.019 mmol) and the solution was stirred at 65° C. for 1 hour. The solvent was removed and the crude product was purified by combiflash SiO chromatography (0-5% MeOH-DCM) to give 2-((6-cyano-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-hydroxy-4-methoxyphenyl)acetamide (5 mg, 91%) as an off-white solid. 1 H-NMR(400MHz,CD3OD-CDCl3)δppm 8.77(s,1H),7.93(s,1H),7.10(d,1H,J=2.8Hz),6.94(dd,1H,J=2.8,8.8H z), 6.79 (d, 1H, J = 8.8Hz), 4.15 (s, 2H), 3.80 (s, 3H); ESI-MS: m / z375.98 (M) + ,356.04(M+2H) + .

[0406]

[0407] 2-((6-cyano-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3,4-dimethoxyphenyl)acetamide (Compound 68)

[0408] To a mixture of 2-chloro-N-(3,4-dimethoxyphenyl)acetamide (14 mg, 0.062 mmol), 2-mercapto-3H-imidazo[4,5-c]pyridine-6-carbonitrile (10 mg, 0.057 mmol) and sodium iodide (4.3 mg, 0.028 mmol) in MeCN was added triethylamine (12 μL, 0.085 mmol) and stirred at 65° C. for 1 hour. MeCN was removed and the crude product was purified by combiflash SiO chromatography (0-5% MeOH-DCM) to give 2-((6-cyano-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3,4-dimethoxy-phenyl)acetamide (15 mg, 72%) as a white solid. 1 H-NMR(400MHz,CD3OD-CDCl3)δppm 8.80(s,1H),7.98(s,1H),7.33(d,1H,J=2.4Hz),7.01(dd,1H,J=2.4,8.8Hz),6.87 (d,1H,J=8.8Hz),4.24(s,2H),3.81(s,3H),3.30(s,3H); ESI-MS:m / z370.05(M+H) + .

[0409]

[0410] 2-((6-cyano-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-fluoro-3-hydroxyphenyl)acetamide (Compound 69)

[0411] To a mixture of 2-chloro-N-(4-fluoro-3-hydroxyphenyl)acetamide (13 mg, 0.062 mmol), 2-mercapto-3H-imidazo[4,5-c]pyridine-6-carbonitrile (10 mg, 0.057 mmol) and sodium iodide (4.3 mg, 0.028 mmol) in MeCN was added triethylamine (12 μL, 0.085 mmol) and stirred at 65° C. for 1 hour. MeCN was removed and the crude product was purified by combiflashSiO chromatography (0-5% MeOH-DCM) to give 2-((6-cyano-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-fluoro-3-hydroxyphenyl)acetamide (11 mg, 57%) as a white solid. 1H-NMR (400MHz, CD3OD-CDCl3) δppm 8.79 (s, 1H), 7.95 (s, 1H), 7.25 (dd, 1H, J = 2.4, 7.6Hz), 6.95 (d, 1H, J = 8.8Hz), 6.91 (m, 1H), 4.19 (s, 2H); ESI-MS: m / z 344.01(M+H) + .

[0412]

[0413] 2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)sulfonyl)-N-(3-fluoro-4-methoxyphenyl)acetamide (Compound 70)

[0414] To a solution of 2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-fluoro-4-methoxyphenyl)acetamide (5 mg, 0.014 mmol) in methanol (1 mL, 0.014 mmol) was added Oxone (20.95 mg, 0.068 mmol). The product was purified on preparative TLC (MeOH / DCM) overnight to give 2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)sulfonyl)-N-(3-fluoro-4-methoxyphenyl)acetamide (2.1 mg, 5.27 μmol, 38.6% yield). 1 HNMR (400MHz, CD3OD) δ8.72 (s, 1H), 7.62 (s, 1H), 7.40 (M, 1H), 7.13 (d, J = 7.6Hz, 1H), 7.00 (m, 1H), 3.83 (s, 3H) ESI-MS: m / z 399.0 (M+H) +

[0415]

[0416] 2-Chloro-N-(6-methoxypyridin-3-yl)acetamide (Compound 71)

[0417] To a solution of 6-methoxypyridin-3-amine (53 mg, 0.427 mmol), TEA (0.179 mL, 1.281 mmol) in DCM (2 mL) was added 2-chloroacetyl chloride (0.037 mL, 0.470 mmol) at room temperature. After 1 hour, concentration and purification on combiflash (4 g, EtOAc / hexanes) gave 2-chloro-N-(6-methoxypyridin-3-yl)acetamide (51 mg, 0.254 mmol, 59.5% yield). 1HNMR (400MHz, CDCl3) δ8.30 (d, J=2.4Hz, 1H), 7.88 (m, 1H), 6.79 (d, J=8.8Hz, 1H), 4.18 (s, 2H). ESI-MS: m / z 200.98 (M+H) +

[0418] 2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(6-methoxypyridin-3-yl)acetamide (Compound 72)

[0419] The reaction mixture of 6-chloro-3H-imidazo[4,5-c]pyridine-2-thiol (9.25 mg, 0.050 mmol), 2-chloro-N-(6-methoxypyridin-3-yl)acetamide (10 mg, 0.050 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.021 mL, 0.150 mmol) in CH3CN (1 mL) was heated at 65° C. for 2 hours. 2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(6-methoxypyridin-3-yl)acetamide (12 mg, 0.034 mmol, 68.8% yield) was obtained by filtration. 1 HNMR (400MHz, CD3OD) δ8.63(s,1H),8.43(d,J=2.4Hz,1H),8.01(m 1H),7.62(s,1H),6.90(d,J=8.8Hz,1H),4.38(s,2H),4.00(s,3H)

[0420] ESI-MS: m / z 349.95 (M+H) +

[0421]

[0422] 2-Bromo-3-methyl-5-nitropyridine-4-amine (Compound 73): A mixture of 2-bromo-3-methylpyridine-4-amine (100 mg, 0.535 mmol) in sulfuric acid (849 μl, 16.04 mmol) was cooled to 0 ° C, and potassium nitrate (162 mg, 1.604 mmol) was added. The mixture was stirred at 0 ° C for another 1 hour and at room temperature for 16 hours. The reaction mixture was poured into ice water, basified with saturated NaHCO3 and extracted with EtOAc. The combined EtOAc layer was washed with water, brine, dried over Na2SO4 and evaporated. The crude product was purified by combiflash SiO2 chromatography (0-20% EtOAc-hexanes) to give 2-bromo-3-methyl-5-nitropyridine-4-amine (54 mg, 44%) as a yellow solid.1 H-NMR (400MHz, CDCl3-CD3OD) δppm 8.69 (s, 1H), 2.22 (s, 3H); ESI-MS: m / z 231.86 (M + )233.85(M+2H) + .

[0423] 4-Amino-3-methyl-5-nitropyridinecarbonitrile (Compound 74): A solution of 2-bromo-3-methyl-5-nitropyridine-4-amine (52 mg, 0.224 mmol) and cupric cyanide (24.09 mg, 0.269 mmol) in DMA was heated to 200 ° C in a microwave reactor for 1 hour. The reaction mixture was poured into water and extracted with EtOAc. The combined organic layers were washed with water, brine, dried over Na2SO4 and evaporated. The crude product was purified by combiflash SiO2 chromatography (0-30% EtOAc-hexanes) to give 4-amino-3-methyl-5-nitropyridinecarbonitrile (30 mg, 75%) as a light yellow solid. 1 H-NMR (400MHz, CD3OD) δppm 8.99 (s, 1H), 2.44 (s, 3H); ESI-MS: m / z 178.93 (M+H) + .

[0424] 4,5-Diamino-3-methylpicolinonitrile (Compound 75): To a solution of 4-amino-3-methyl-5-nitropyridinecarbonitrile (30 mg, 0.168 mmol) in EtOH was added iron (9.40 mg, 0.168 mmol), followed by a solution of water containing ammonium chloride (9.01 mg, 0.168 mmol) and stirred at 65 ° C for 1 hour. The reaction mixture was filtered through celite and evaporated. The crude product was purified by combiflash SiO2 chromatography (0-5% MeOH-DCM) to give 4,5-diamino-3-methylpicolinonitrile (15 mg, 60%) as a brown solid. 1 H-NMR (400MHz, CD3OD) δppm 7.77 (s, 1H), 2.29 (s, 3H). ESI-MS: m / z 148.97 (M+H) +

[0425] 2-Mercapto-7-methyl-3H-imidazo[4,5-c]pyridine-6-carbonitrile (Compound 76): A mixture of 4,5-diamino-3-methylpyridinecarbonitrile (15 mg, 0.101 mmol), di(1H-imidazol-1-yl)methanone (27.1 mg, 0.152 mmol) and N,N-dimethylpyridin-4-amine (14.84 mg, 0.121 mmol) in MeCN was stirred at room temperature for 16 hours. The solvent was removed and the crude product was purified by combiflash SiO2 chromatography (0-5% MeOH-DCM) to give 2-mercapto-7-methyl-3H-imidazo[4,5-c]pyridine-6-carbonitrile (17 mg, 88%) as a light brown solid. 1 H-NMR (400MHz, CD3OD) δppm 8.31 (s, 1H), 2.59 (s, 3H); ESI-MS: m / z 190.93 (M+H) + .

[0426]

[0427] 2-((6-Cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3,4-dimethoxyphenyl)acetamide (Compound 77): To a mixture of 2-mercapto-7-methyl-3H-imidazo[4,5-c]pyridine-6-carbonitrile (3 mg, 0.016 mmol), 2-chloro-N-(3,4-dimethoxyphenyl)acetamide (4.35 mg, 0.019 mmol) and sodium iodide (1.182 mg, 7.89 μmol) in MeCN was added triethylamine (3.30 μl, 0.024 mmol) and heated to 65 °C for 1 hour. The solvent was removed and the crude product was purified by combiflash SiO2 chromatography (0-10% MeOH-DCM) to give 2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3,4-dimethoxyphenyl)acetamide (4 mg, 66%) as a white solid. 1 H-NMR(400MHz,CDCl3-CD3OD)δppm 8.64(s,1H),7.32(s,1H),6.98(d,1H,J=7.6Hz),6.84(dd,1H,J=3.2,8.8Hz),4.20(s,2H),3.82(s,3H),3.81(s,3H),2.71(s,3H); ESI-MS: m / z 384.06(M+H) + .

[0428]

[0429] 2-((6-Cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-hydroxy-4-methoxyphenyl)acetamide (Compound 78): To a mixture of 2-mercapto-7-methyl-3H-imidazo[4,5-c]pyridine-6-carbonitrile (7 mg, 0.037 mmol), 2-chloro-N-(3-hydroxy-4-methoxyphenyl)acetamide (8.73 mg, 0.040 mmol), sodium iodide (2.76 mg, 0.018 mmol) in MeCN was added triethylamine (7.69 μl, 0.055 mmol) and heated to 65 °C for 1 hour. The solvent was removed and the crude product was purified by combiflash SiO2 chromatography (0-10% MeOH-DCM) to give 2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-hydroxy-4-methoxyphenyl)acetamide (7 mg, 52%) as a white solid. 1 H-NMR(400MHz,CDCl3-CD3OD)δppm 8.64(s,1H),7.10(d,1H,J=2.4Hz),6.94(dd,1H,J=2.4,8.8Hz),6.84(d,1H ,J=8.8Hz),4.18(s,2H),3.82(s,3H),3.81(s,3H),2.71(s,3H); ESI-MS:m / z 370.05(M+H) + .

[0430]

[0431] 2-((6-Cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-fluoro-3-hydroxyphenyl)acetamide (Compound 79): To a mixture of 2-mercapto-7-methyl-3H-imidazo[4,5-c]pyridine-6-carbonitrile (11.21 mg, 0.059 mmol), 2-chloro-N-(4-fluoro-3-hydroxyphenyl)acetamide (10 mg, 0.049 mmol) and sodium iodide (3.68 mg, 0.025 mmol) in MeCN was added triethylamine (10.27 μl, 0.074 mmol) and heated at 65 °C for 1 hour. MeCN was removed and the crude product was purified by combiflash SiO2 chromatography (0-5% MeOH-DCM) to give 2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-fluoro-3-hydroxyphenyl)acetamide (10 mg, 57%) as a white solid. 1H-NMR (400MHz, CDCl3-CD3OD) δppm 8.22 (s, 1H), 6.82 (dd, 1H, J = 2.4, 8.0Hz), 6.49 (m, 2H), 3.76 (s, 2H), 2.29 (s, 3H); ESI-MS: m / z 358.03 (M+H) + .

[0432]

[0433] 2-Chloro-N-(4-methoxy-3-nitrophenyl)acetamide (Compound 80): A solution of 4-methoxy-3-nitroaniline (100 mg, 0.595 mmol) in acetone was mixed with a solution of potassium carbonate (123 mg, 0.892 mmol) in water and cooled before adding 2-chloroacetyl chloride (0.061 ml, 0.773 mmol). The reaction mixture was stirred at 0°C for 1 hour. The reaction mixture was diluted with EtOAc, washed with 1M HCl, saturated NaHCO3, water, brine and dried over Na2SO4. The solvent was removed and the crude product 2-chloro-N-(4-methoxy-3-nitrophenyl)acetamide (120 mg, 82%) as a yellow solid was used in the next step without further purification. 1 H-NMR(400MHz,CDCl3)δppm 8.25(brs,1H),8.06(d,1H,J=2.4Hz),7.80(dd,1H,J=2.4,8.8Hz),7.09(d,1H,J=8.8Hz),4.21(s,2H),3.96(s,3H),3.81(s,3H); ESI-MS: m / z 244.98(M+H) + ,266.94(M+Na) + .

[0434] N-(3-amino-4-methoxyphenyl)-2-chloroacetamide (Compound 81): A mixture of 2-chloro-N-(4-methoxy-3-nitrophenyl)acetamide (50 mg, 0.204 mmol) and iron (34.2 mg, 0.613 mmol) in EtOH was used as a solution of ammonium chloride (32.8 mg, 0.613 mmol), and the mixture was stirred at 65 ° C for 1 hour. The reaction was filtered through celite and the solvent was removed. The crude product, a brown solid, N-(3-amino-4-methoxyphenyl)-2-chloroacetamide (30 mg, 68%) was used in the next step without further purification. 1H-NMR(400MHz,CDCl3-CD3OD)δppm 7.08(s,1H),6.89(d,1H,J=8Hz),6.79(d,1H,J=8.8Hz),4.15(s,2H),3.82(s,3H),3.81(s,3H); ESI-MS:m / z214.99(M+H) + .

[0435] N-(3-Amino-4-methoxyphenyl)-2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide (Compound 82): To a mixture of N-(3-amino-4-methoxyphenyl)-2-chloroacetamide (15 mg, 0.070 mmol), 6-chloro-3H-imidazo[4,5-c]pyridine-2-thiol (10.81 mg, 0.058 mmol) and sodium iodide (4.36 mg, 0.029 mmol) in MeCN was added triethylamine (12.18 μl, 0.087 mmol) and stirred at 65 °C for 1 hour. MeCN was removed and the crude product was purified by combiflash SiO2 chromatography (0-5% MeOH-DCM) to give N-(3-amino-4-methoxyphenyl)-2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide (5.5 mg, 26%) as a white solid. 1 H-NMR(400MHz,CDCl3-CD3OD)δppm 8.51(s,1H),7.50(s,1H),7.01(d,1H,J=2.4Hz),6.83(dd,1H,J=2.4,8.8Hz ), 6.76 (d, 1H, J = 8.8Hz), 4.19 (s, 2H), 3.82 (s, 3H), 3.81 (s, 3H); ESI-MS: m / z 363.97(M+H) + .

[0436]

[0437] 2-((6-Chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-methoxy-3-nitrophenyl)acetamide (Compound 83): To a mixture of 2-chloro-N-(4-methoxy-3-nitrophenyl)acetamide (15.8 mg, 0.065 mmol), 6-chloro-3H-imidazo[4,5-c]pyridine-2-thiol (10 mg, 0.054 mmol) and sodium iodide (8.07 mg, 0.054 mmol) in MeCN was added triethylamine (11 μl, 0.081 mmol) and stirred at 65 °C for 1 hour. MeCN was removed and the crude product was purified by combiflash SiO2 chromatography (0-5% MeOH-DCM) to give 2-((6-chloro-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-methoxy-3-nitro-phenyl)acetamide (17 mg, 80%) as a white solid. 1 H-NMR(400MHz,DMSO-d6)δppm 10.72(s,1H),8.51(s,1H),8.22(d,1H,J=2.4Hz),7.72(dd,1H,J=2.4,8.8Hz ),7.52(s,1H),7.35(d,1H,J=8.8Hz),4.32(s,2H),3.87(s,3H); ESI-MS:m / z 393.98(M + ).

[0438]

[0439] 2-Chloro-N-(4,5-dimethoxypyridin-2-yl)acetamide (Compound 84) To a solution of 4,5-dimethoxypyridin-2-amine (50 mg, 0.324 mmol), TEA (0.113 mL, 0.811 mmol) in DCM (2 mL) at room temperature was added 2-chloroacetyl chloride (0.028 mL, 0.357 mmol). After 1 hour, the mixture was concentrated and purified on combiflash (4 g, EtOAc / hexanes) to give 2-chloro-N-(4,5-dimethoxypyridin-2-yl)acetamide (19 mg, 0.082 mmol, 25.4% yield). 1 HNMR(400MHz, CDCl3)δppm 8.72(s,1H),7.87(s,1H),7.8(s,1H),4.18(s,2H),3.97(s,3H),3.92(s,3H)ESI-MS: m / z 230.91(M+H) +

[0440] 2-((6-Chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4,5-dimethoxypyridin-2-yl)acetamide (Compound 85): A reaction mixture of 6-chloro-1H-imidazo[4,5-c]pyridine-2-thiol (7.24 mg, 0.039 mmol), 2-chloro-N-(4,5-dimethoxypyridin-2-yl)acetamide (9 mg, 0.039 mmol), sodium iodide (1 mg, 6.67 μmol), triethylamine (0.016 mL, 0.117 mmol) in CH3CN (1 mL) was heated to 65°C for 2 hours. Concentration and purification on combiflash (4 g, MeOH / DCM) gave 2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4,5-dimethoxypyridin-2-yl)acetamide (9 mg, 0.024 mmol, 60.7% yield). 1 HNMR (400MHz, CD3OD) δppm 8.52(s,1H),7.82(s,1H),7.78(s,1H),7.51(s,1H),4.26(s,2H),3.88(s,3H),3.85(s,3H).ESI-MS: m / z 379.96(M+H) +

[0441]

[0442] 2-Chloro-N-(5,6-dimethoxypyridin-2-yl)acetamide (Compound 86) To a solution of 5,6-dimethoxypyridin-2-amine (100 mg, 0.649 mmol), TEA (0.226 mL, 1.622 mmol) in DCM (3 mL) at room temperature was added 2-chloroacetyl chloride (0.057 mL, 0.714 mmol). After 1 hour, the mixture was concentrated and purified on combiflash (4 g, EtOAc / hexanes) to give 2-chloro-N-(5,6-dimethoxypyridin-2-yl)acetamide (85 mg, 0.369 mmol, 56.8% yield). 1 HNMR (400MHz, CDCl3) δppm 8.75 (s, 1H), 7.95 (d, J = 8Hz, 1H), 7.54 (s, 1H), 7.37 (d, J = 8Hz, 1H), 4.46 (s, 2H), 4.25 (s, 3H), 4.20 (s, 3H). ESI-MS: m / z 231.12(M+H) +

[0443] 2-((6-Chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(5,6-dimethoxypyridin-2-yl)acetamide (Compound 87) A reaction mixture of 6-chloro-1H-imidazo[4,5-c]pyridine-2-thiol (8.05 mg, 0.043 mmol), 2-chloro-N-(5,6-dimethoxypyridin-2-yl)acetamide (10 mg, 0.043 mmol), sodium iodide (1 mg, 6.67 μmol) and triethylamine (0.018 mL, 0.130 mmol) in CH3CN (1 mL) was heated to 65°C overnight. Both new species were purified by pre-TLC (MeOH / DCM) to give 2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(5,6-dimethoxypyridin-2-yl)acetamide (9 mg, 0.024 mmol, 54.7% yield). 1 HNMR (400MHz, CD3OD) δppm 8.51(s,1H),7.58(d,J=8.4Hz,1H),7.51(s,1H),7.22(d,J=8.4Hz,1H),4.23(s,2H),3.89(s,3H),3.80(s,3H).ESI-MS m / z,380.09(M+H) +

[0444]

[0445] 2-Chloro-N-(4-methoxy-3-(2,2,2-trifluoroethoxy)phenyl)acetamide (Compound 88) To a solution of 4-methoxy-3-(2,2,2-trifluoroethoxy)aniline (50 mg, 0.226 mmol), TEA (0.079 mL, 0.565 mmol) in DCM (2 mL) at room temperature was added 2-chloroacetyl chloride (0.020 mL, 0.249 mmol). After 1 hour, concentration and purification on combiflash (4 g, EtOAc / hexanes) gave 2-chloro-N-(4-methoxy-3-(2,2,2-trifluoroethoxy)phenyl)acetamide (49 mg, 0.165 mmol, 72.8% yield). 1 HNMR(400MHz,CDCl3)δppm 8.13(s,1H),7.36(s,1H),7.26(s,1H),7.13(d,J=8.8Hz,1H),6.89(d,J=8.8Hz,1H),4.39(m,2H),4.18(s,2H),3.87(s,3H)ESI-MS:m / z 297.92(M+H) +

[0446] 2-((6-Chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-methoxy-3-(2,2,2-trifluoroethoxy)phenyl)acetamide (Compound 89) A reaction mixture of 6-chloro-1H-imidazo[4,5-c]pyridine-2-thiol (10 mg, 0.054 mmol), 2-chloro-N-(4-methoxy-3-(2,2,2-trifluoroethoxy)phenyl)acetamide (16.04 mg, 0.054 mmol), sodium iodide (1 mg, 6.67 μmol) and triethylamine (0.023 mL, 0.162 mmol) in CH3CN (1 mL) was heated to 65°C. Both new species were purified by pre-TLC (MeOH / DCM) to give 2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-methoxy-3-(2,2,2-trifluoroethoxy)phenyl)acetamide (9 mg, 0.020 mmol, 37.4% yield). 1 HNMR(400MHz,CD3OD)δppm 8.50(s,1H),7.50(s,1H),7.37(s,1H),7.16(d,J=8.8Hz,1H),6.97(d,J=8.8Hz,1H),4.47(m,2H),4.22(s,2H),3.83(s,3H).ESI-MS m / z,447.01(M+H) +

[0447]

[0448] 2-Chloro-N-(5,6-dimethoxypyridin-3-yl)acetamide (Compound 90) To a solution of 5,6-dimethoxypyridin-3-amine (50 mg, 0.324 mmol), TEA (0.113 mL, 0.811 mmol) in DCM (3 mL) at room temperature was added 2-chloroacetyl chloride (0.028 mL, 0.357 mmol). After 1 hour, the mixture was concentrated and purified on combiflash (4 g, EtOAc / hexanes) to give 2-chloro-N-(5,6-dimethoxypyridin-3-yl)acetamide (56 mg, 0.243 mmol, 74.9% yield). 1 HNMR (400MHz, CDCl3) δppm 8.16 (s, 1H), 7.71 (s, J = 2Hz, 1H), 7.62 (d, J = 2Hz, 1H), 4.21 (s, 2H), 4.01 (s, 3H), 3.90 (s, 3H) ESI-MS: m / z 230.99 (M+H) +

[0449] 2-((6-Chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(5,6-dimethoxypyridin-3-yl)acetamide (Compound 91) A reaction mixture of 6-chloro-1H-imidazo[4,5-c]pyridine-2-thiol (8.05 mg, 0.043 mmol), 2-chloro-N-(5,6-dimethoxypyridin-3-yl)acetamide (10 mg, 0.043 mmol), sodium iodide (1 mg, 6.67 μmol) and triethylamine (0.018 mL, 0.130 mmol) in CH3CN (1 mL) was heated to 65°C overnight. Both new species were purified by pre-TLC (MeOH / DCM) to give 2-((6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(5,6-dimethoxypyridin-3-yl)acetamide (13 mg, 0.034 mmol, 79% yield). 1 HNMR(400MHz,CD3OD)δppm8.50(s,1H),7.82(d,J=2Hz,1H),7.58(d,J=2Hz,1H),7.50(s,1H),4.25(s,2H),3.91(s,3H),3.82(s,3H),ESI-MS m / z,380.04(M+H) +

[0450]

[0451] 2-((6-Cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-fluoro-3-hydroxyphenyl)acetamide (Compound 92): To a mixture of 2-mercapto-7-methyl-3H-imidazo[4,5-c]pyridine-6-carbonitrile (11.21 mg, 0.059 mmol), 2-chloro-N-(4-fluoro-3-hydroxyphenyl)acetamide (10 mg, 0.049 mmol) and sodium iodide (3.68 mg, 0.025 mmol) in MeCN was added triethylamine (10.27 μl, 0.074 mmol) and stirred at 65 °C for 1 hour. MeCN was removed and the crude product was purified by combiflash SiO2 chromatography (0-5% MeOH-DCM) to give 2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-fluoro-3-hydroxyphenyl)acetamide (10 mg, 57%) as a white solid. 1H-NMR (400MHz, CDCl3-CD3OD) δppm 8.22 (s, 1H), 6.82 (dd, 1H, J = 2.4, 8.0Hz), 6.49 (m, 2H), 3.76 (s, 2H), 2.29 (s, 3H); ESI-MS: m / z 358.03 (M+H) + .

[0452]

[0453] 2-((6-Cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-fluoro-4-methoxyphenyl)acetamide (Compound 93): To a mixture of 2-chloro-N-(3-fluoro-4-methoxyphenyl)acetamide (13.73 mg, 0.063 mmol), 2-mercapto-7-methyl-3H-imidazo[4,5-c]pyridine-6-carbonitrile (10 mg, 0.053 mmol), sodium iodide (3.94 mg, 0.026 mmol) in MeCN was added triethylamine (10.99 μl, 0.079 mmol) and heated to 65 °C for 1 hour. MeCN was removed and the crude product was purified by combiflash SiO2 chromatography (0-5% MeOH-DCM) to give 2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-fluoro-4-methoxyphenyl)acetamide (11 mg, 56%) as a white solid. 1 H-NMR(400MHz,CDCl3-CD3OD)δppm 8.65(s,1H),7.49(dd,1H,J=2.8,13.2Hz),7.18(d,1H,J=8.4Hz),7.01(t,1H ,J=9.2Hz),4.24(s,2H),3.83(s,3H),2.70(s,3H); ESI-MS:m / z372.02(M+H) + .

[0454]

[0455] 6-Amino-2-methoxypyridine-3-ol (Compound 94): To a cooled solution of DCM containing 5,6-dimethoxypyridine-2-amine (70 mg, 0.454 mmol) was slowly added tribromoborane (1.362 ml, 1.362 mmol) (1 M in DCM), and the mixture was stirred at room temperature for 3 hours. The reaction mixture was quenched with saturated Na2CO3, followed by addition of water. The aqueous layer was extracted with DCM, and the combined organic layers were washed with water, brine, dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by combiflash SiO2 chromatography to give 6-amino-2-methoxypyridine-3-ol (24 mg, 38%) as a brown solid. 1 H-NMR (400MHz, CD3OD) δppm 6.91 (d, 1H, J = 8Hz), 6.03 (d, 1H, J = 8.4Hz), 3.87 (s, 3H); ESI-MS m / z 141.17 (M+H) + .

[0456] 2-Chloro-N-(5-hydroxy-6-methoxypyridin-2-yl)acetamide (Compound 95): To a cooled mixture of acetone-water containing 6-amino-2-methoxypyridin-3-ol (24 mg, 0.171 mmol) and potassium carbonate (28.4 mg, 0.206 mmol) was slowly added 2-chloroacetyl chloride (0.015 ml, 0.188 mmol) at 0°C and stirred for 1 hour. The reaction was diluted with EtOAc and washed with water, brine, dried over Na2SO4 and evaporated. The crude product, 2-chloro-N-(5-hydroxy-6-methoxypyridin-2-yl)acetamide (33 mg, 89%) as a brown solid was used in the next step without further purification. 1 H-NMR (400MHz, CD3OD) δppm 7.50 (d, 1H, J = 8.4Hz), 7.07 (d, 1H, J = 8.4Hz), 4.19 (s, 2H), 3.93 (s, 3H); ESI-MS: m / z 217.00 (M+H) + .

[0457] 2-((6-Cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(5-hydroxy-6-methoxypyridin-2-yl)acetamide (Compound 96): To a mixture of 2-chloro-N-(5-hydroxy-6-methoxypyridin-2-yl)acetamide (10 mg, 0.046 mmol), 2-mercapto-7-methyl-3H-imidazo[4,5-c]pyridine-6-carbonitrile (10.54 mg, 0.055 mmol) and sodium iodide (3.46 mg, 0.023 mmol) in MeCN was added triethylamine (9.65 μl, 0.069 mmol) and the reaction was stirred at 65 °C for 1 hour. The solvent was removed and the crude product was purified by combiflash SiO2 chromatography (0-5% MeOH-DCM) to give 2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(5-hydroxy-6-methoxypyridin-2-yl)acetamide (13 mg, 76%) as a white solid. 1 H-NMR(400MHz,DMSO-d6)δppm13.69(s,1H),10.43(s,1H),9.22(s,1H),8.70(s,1H),7.40( d,1H,J=8.4Hz),7.05(d,1H,J=8.0Hz),4.34(s,2H),3.84(s,3H),2.62(s,3H); ESI-MS:m / z 371.07(M+H) + .

[0458] General Synthesis of 2-Mercapto-7-methyl-3H-imidazo[4,5-c]pyridine-6-carbonitrile (Compound 76)

[0459]

[0460] Synthesis of 2-bromo-3-methylpyridine 1-oxide (Compound 98)

[0461]

[0462] At 0 ℃, in 30 minutes time period, to the stirring solution of DCM (1000mL) containing 2-bromo-3-methylpyridine (100g, 0.581mol, 1.00 equivalent), water (200g, 1.16mol, 2.0 equivalent) containing 60% meta-chloroperbenzoic acid was added batch by batch. The reaction mixture was stirred at room temperature for 16 hours. After the reaction was completed by TLC, the volatiles in the reaction mixture were evaporated to the minimum volume, and the precipitated solid was filtered and washed with dichloromethane. The filtrate was diluted with saturated NaHCO solution and extracted with (2 × 1000mL) DCM. The combined organic layer was washed with water (1000mL), saline solution (1000mL), dried over sodium sulfate and concentrated to produce a solid, which was ground with diethyl ether to produce 2-bromo-3-methylpyridine 1-oxide (compound 98) (91g, yield: 83%) as an off-white solid. TLC system: EtOAc (100), R f Value: ~0.15; LCMS (m / z): 188.0 (M+H) + ; 1 HNMR(400MHz,DMSO-d6)δ8.29-8.27(m,1H),7.16-7.10(m,2H).2.47(s,3H).

[0463] Synthesis of 2-bromo-3-methyl-4-nitropyridine 1-oxide (Compound 99)

[0464]

[0465] To a stirred solution of 2-bromo-3-methylpyridine 1-oxide (2) (4 x 22 g, 0.465 mol, 1 eq.) in H2SO4 (610 mL) was added potassium nitrate (70.6 g, 0.698 mol, 1.5 eq.) in a nitrogen atmosphere at 0 °C. The reaction mixture was stirred at 80 °C for 20 hours. The reaction mixture was quenched with ice-cold water and extracted with (2 x 800 mL) ethyl acetate. The combined organic layers were washed with water (300 mL), brine (300 mL), dried over sodium sulfate and concentrated to give 2-bromo-3-methyl-4-nitropyridine 1-oxide (compound 98) (63 g, yield: 62%) as a yellow solid. TLC system: EtoAc: Hexane (50:50), R f Value: ~0.4; LCMS (m / z): 232.9 (M+H) + ; 1 HNMR(400MHz,DMSO-d6)δ8.32(d,1H),7.83(d,1H),2.76(s,3H).

[0466] Synthesis of 2-bromo-3-methylpyridin-4-amine (Compound 100)

[0467]

[0468] Iron powder (163 g, 2.33 mol, 10 equiv) and ammonium chloride (125 g, 2.33 mol, 10 equiv) were added in portions to a stirred solution of ethanol: water (2: 1, 945 mL) containing 2-bromo-3-methyl-4-nitropyridine 1-oxide (compound 99) (63 g, 0.233 mol, 1 equiv) at room temperature. The reaction mixture was then stirred at 90 ° C for 6 hours. The reaction mixture was filtered through a celite bed and washed with ethyl acetate (500 mL), and the filtrate was washed with water (300 mL), dried over sodium sulfate and concentrated to give 2-bromo-3-methylpyridin-4-amine (compound 100) (32 g, yield: 63%) as a black solid. TLC system: EtoAc: hexane (30: 70), R f Value: ~0.2 (twice eluted); LCMS (m / z): 187.0 (M+H) + ; 1 H NMR (400MHz, DMSO-d6) δ7.85 (d, J = 5.2Hz, 1H), 6.48 (d, J = 5.2Hz, 1H), 4.34 (brs, 2H), 2.23 (s, 3H).

[0469] Synthesis of 2-bromo-3-methyl-5-nitropyridine-4-amine (Compound 73)

[0470]

[0471] Under nitrogen atmosphere at 0 ° C, to the stirring solution of H2SO4 containing 2-bromo-3-methylpyridine-4-amine (compound 100) (32g, 0.172mol, 1 equivalent) was added KNO3 (19.1g, 0.189mmol, 1.1 equivalent) in portions for 15 minutes, and stirred at room temperature for 20 hours. The reaction mixture was quenched with ice-cold aqueous solution (600mL) and extracted with (3×500mL) ethyl acetate. The combined organic layer was washed with saturated NaHCO3 solution (500mL), dried over sodium sulfate and concentrated. The crude compound was purified by grace column chromatography [eluted with 20% ethyl acetate / hexane gradient] to produce 2-bromo-3-methyl-5-nitropyridine-4-amine (compound 73) (11g, yield: 28%) as a yellow solid. TLC system: EtoAc: hexane (50:50), R f Value: ~0.6; LCMS (m / z): 232.0 (M+H) + ;1 HNMR(400MHz,DMSO-d6)δ8.91(s,1H),6.426.72(br,2H),2.37(s,3H).

[0472] Synthesis of 4-amino-3-methyl-5-nitropyridinecarbonitrile (Compound 74)

[0473]

[0474] In a sealed tube, a stirred and degassed solution of DMF (53 mL) containing 2-bromo-3-methyl-5-nitropyridine-4-amine (compound 73) (11 g, 47.6 mmol, 1.0 equiv) was treated with Zn(CN)2 (11.1 g, 95.2 mmol, 2.0 equiv), Pd(PPh3)4 (10.99 g, 9.52 mmol, 0.2 equiv). The reaction mixture was then stirred at 110 ° C under N2 for 16 hours. The reaction mixture was filtered through a celite bed and washed with ethyl acetate (250 mL), and the filtrate was washed with cold water (70 mL), dried over sodium sulfate and concentrated to give a crude product. The crude compound was purified by grace column chromatography [eluted with a 20% ethyl acetate / hexane gradient] to produce 4-amino-3-methyl-5-nitropyridinecarbonitrile (compound 74) (8.5 g, yield: 47%) as a yellow solid. TLC system: EtoAc: Hexane (60:40), R f Value: ~0.2; LCMS (m / z): 179.1 (M+H) + ; 1 HNMR(400MHz,DMSO-d6)δ8.93(s,1H),8.09-8.15(br,2H),2.37(s,3H)along with DMFtraces.

[0475] Synthesis of 4,5-diamino-3-methylpyridinecarbonitrile (Compound 75)

[0476]

[0477] 10% wet Pd / C (1.6 g 20%) was added to a stirred solution of methanol (30 ml) containing 4-amino-3-methyl-5-nitropyridinecarbonitrile (compound 74) (8.0 g, 44.9 mmol, 1.0 equiv), and the reaction mixture was stirred at room temperature for 16 hours under H2 balloon pressure. The reaction mixture was filtered through a diatomaceous earth bed and washed with methanol (100 mL), and the filtrate was concentrated to provide a crude product. The crude product was ground with hexane to produce 4,5-diamino-3-methylpyridinecarbonitrile (compound 75) (5.1 g, yield: 77%). TLC system: EtoAc: hexane (80: 30), R f Value: ~0.2; LCMS (m / z): 149.1 (M+H) + ; 1 H NMR (400MHz, DMSO-d6) δ7.63(s,1H),5.52(s,2H),5.41(s,2H),2.18(s,3H).

[0478] Synthesis of 2-mercapto-7-methyl-3H-imidazo[4,5-c]pyridine-6-carbonitrile (Compound 76)

[0479]

[0480] To a stirred solution of 4,5-diamino-3-methylpicolinonitrile (compound 75) (5.0 g, 33.7 mmol, 1 eq) in acetonitrile (50 ml) was added thioCDI (12.0 g, 67.6 mmol, 2 eq) at room temperature and stirred in a sealed tube at 85 °C for 16 hours. The reaction mixture was concentrated and purified by neutral alumina column chromatography [eluted with a 50-80% ethyl acetate / hexane gradient] to give 2-mercapto-7-methyl-3H-imidazo[4,5-c]pyridine-6-carbonitrile (compound 76) (2.5 g, yield: 40%) as a brown solid. TLC system: DCM:MeOH, R f Value: ~0.2; LCMS (m / z): 191.0 (M+H) + ; 1 HNMR (400MHz, DMSO-d6), δ8.29 (s, 1H), 2.52 (s, 3H), exchangeable protons 1 It is not obvious in HNMR.

[0481] Synthesis of sodium 5-(2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)propionamido)-2-methoxyphenol (Compound 102)

[0482]

[0483] Synthesis of 5-amino-2-methoxyphenol (Compound 102-01)

[0484]

[0485] At 0 ° C, over a 30-minute period, ammonium chloride (19.9 g, 372.9 mmol, 6.3 equivalents) was added batch by batch to a stirred solution of EtOH / H2O (2: 1) (100 mL / 50 mL) containing 2-methoxy-5-nitrophenol (compound 101) (10.0 g, 59.2 mmol, 1.0 equivalents), followed by iron powder (19.9 g, 358.2 mmol, 6.05 equivalents). The reaction mixture was heated at 90 ° C for 16 hours. After the reaction was completed as determined by TLC, the reaction mixture was filtered through a diatomaceous earth bed and washed with EtOH. The filtrate was concentrated to produce 5-amino-2-methoxyphenol (compound 102-01) (6.2 g, yield: 75%) as a brown solid. TLC system: EtOAc / hexane (50: 50), R f Value: ~0.2; LCMS (m / z): 140.3 (M+H) + ; 1 H NMR (400MHz, DMSO-d6) δ8.50(s,1H),6.58(d,J=8.4Hz,1H),6.08(d,J=2.8Hz,1H),5.93(dd,J=2.4Hz,8.4Hz,1H),4.50(s,2H),3.62(s,3H).

[0486] Synthesis of 2-chloro-N-(3-hydroxy-4-methoxyphenyl)propionamide (Compound 102-02)

[0487]

[0488] At 0 ° C, K2CO3 (750 mg, 7.20 mmol, 1.5 equivalents) was added to a stirred solution of acetone (10 mL) containing 5-amino-2-methoxyphenol (compound 102-01) (500 mg, 3.60 mmol, 1 equivalent). Then 2-chloropropionyl chloride (0.4 mL, 4.32 mmol, 1.2 equivalents) was added dropwise, and the reaction mixture was stirred at room temperature for 16 hours. After the reaction was completed as determined by TLC, the reaction mixture was quenched with ice-cold water and extracted with EtOAc (2×50 mL). The combined organic layer was washed with brine (30 mL), dried over sodium sulfate, and concentrated to produce 2-chloro-N-(3-hydroxy-4-methoxyphenyl) propionamide (compound 102-02) (380 mg, yield: 46%) as a brown solid. TLC system: EtOAc / hexane (50:50), R f Value: ~0.4; LCMS (m / z): 230.0 (M+H) + . 1 H NMR (400MHz, CDCl3) δ8.13 (s, 1H), 7.13 (s, 1H), 7.06 (dd, J = 2.8Hz, 8.8Hz, 1H) ,6.81(dd,J=8.8Hz,1H),5.67(s,1H),4.53(q,1H),3.89(s,3H),1.82(d,3H).

[0489] Synthesis of 2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-hydroxy-4-methoxyphenyl)propionamide (Compound 102-03)

[0490]

[0491] To a stirred solution of acetone (10 mL) containing 2-mercapto-7-methyl-3H-imidazo[4,5-c]pyridine-6-carbonitrile (compound 76) (200 mg, 1.05 mmol, 1 eq) and 2-chloro-N-(3-hydroxy-4-methoxyphenyl)propionamide (compound 102-02) (260 mg, 1.14 mmol, 1.1 eq) was added KCO (290 mg, 2.10 mmol, 2.0 eq) and the reaction mixture was heated at 60 ° C for 2 hours. After the reaction was completed as determined by TLC, the reaction mixture was filtered and concentrated to produce a crude product. The crude product was purified by preparative HPLC. The collected pure fractions were lyophilized to give 2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-hydroxy-4-methoxyphenyl)propanamide (compound 102-03) (75 mg, yield: 19%) as an off-white solid. TLC system: MeOH / DCM (5:95), R f Value: ~0.2; LCMS (m / z): 384.1 (M+H) + ; 1 H NMR (400MHz, DMSO-d6) δ13.69(brs,1H),10.25(s,1H),9.07(s,1H),8.76(s,1H),7.14(d,J=2.4Hz,1H),6. 95(dd,J=2.4Hz,J=8.4Hz,1H),6.83(d,J=8.4Hz,HI),4.88(s,1H),3.71(s,3H),2.67(s,3H),1.67(d,3H).

[0492] Synthesis of sodium 5-(2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)propionamido)-2-methoxyphenol (Compound 102)

[0493]

[0494] To a solution of 2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-hydroxy-4-methoxyphenyl)propanamide (compound 102-03) (45 mg, 0.117 mmol, 1 eq) in water (1 mL) was added an aqueous solution of NaOH (4.7 mg, 0.117 mmol, 1 eq) and stirred at room temperature for 1 hour. The reaction mixture was lyophilized to produce sodium 5-(2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)propanamido)-2-methoxyphenol (compound 102) (47 mg, yield: 99%). LCMS (m / z): 384.1 [(M-Na)+H] + ; 1 HNMR (400 MHz, DMSO-d6) δ 11.51 (br, 1H), 8.46 (s, 2H), 6.61 (br, 3H), 4.54 (q, 1H), 3.62 (s, 3H), 2.61 (s, 3H), 1.45 (d, 3H), the aromatic protons are broad due to the sodium salt and indicate the required number of protons.

[0495] Synthesis of 2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-methoxy-3-(methylsulfonyl)phenyl)acetamide (Compound 103)

[0496]

[0497] Synthesis of (2-methoxy-5-nitrophenyl)(methyl)sulfane (Compound 103-01)

[0498]

[0499] To a stirred solution of 2(2-methoxyphenyl)(methyl)sulfane (compound 105) (5 g, 32.4 mmol, 1.00 equiv) in H2SO4 (125 mL) was added potassium nitrate (6.52 g, 64.9 mmol, 2.0 equiv) batchwise at 0°C over a period of 30 min. The reaction mixture was stirred at room temperature for 16 h. After completion of the reaction as determined by TLC, it was quenched with aqueous ammonia and extracted with (2×200 mL) ethyl acetate. The combined organic layers were washed with water (100 mL), brine solution (100 mL), dried over sodium sulfate and concentrated to yield (2-methoxy-5-nitrophenyl)(methyl)sulfane (compound 103-01) (crude product 2.6 g, yield: 40%). TLC system: EtOAc: Hexane (50:50), R f Value: ~0.6; 1H NMR (400MHz, DMSO-d6) δ8.73(d,J=2.8Hz,1H),8.37(dd,J=2.8Hz,9.2Hz,1H),7.04(d,J=9.2Hz,1H),4.03(s,3H),2.82(s,3H).

[0500] Synthesis of 1-methoxy-2-(methylsulfonyl)-4-nitrobenzene (Compound 103-02)

[0501]

[0502] To a stirred solution of (2-methoxy-5-nitrophenyl)(methyl)sulfane (compound 103-01) (2.6 g, 13.1 mmol, 1 eq) in DCM (26 mL) was slowly added mCPBA (5.62 g, 32.6 mmol, 2.5 eq) at 0 °C. The reaction mixture was then stirred at room temperature under N2 for 16 h. The reaction mixture was quenched with saturated aqueous NaHCO3 and extracted with (2×100 mL) DCM. The combined organic layers were washed with water (50 mL), brine (50 mL), dried over sodium sulfate and concentrated to give 1-methoxy-2-(methylsulfonyl)-4-nitrobenzene (compound 103-02) (crude product 1.5 g, yield: 50%). TLC system: EtoAc: Hexane (70:30), R f Value: ~0.5; 1 H NMR (400MHz, DMSO-d6) δ8.89(d,J=2.8Hz,1H),8.50(dd,J=2.8Hz,9.2Hz,1H),7.18(d,J=9.2Hz,1H),4.13(s,3H),3.26(s,3H).

[0503] Synthesis of 4-methoxy-3-(methylsulfonyl)aniline (Compound 103-03)

[0504]

[0505] 10% Pd / C (0.7 g) was slowly added to a stirred solution of methanol (30 mL) containing 1-methoxy-2-(methylsulfonyl)-4-nitrobenzene (compound 103-02) (1.5 g, 6.49 mmol, 1 equivalent) at room temperature under H2 balloon pressure. The reaction mixture was then stirred at room temperature for 16 hours. The reaction mixture was filtered through a diatomaceous earth bed and washed with methanol (50 mL), and the filtrate was concentrated to produce a crude product. The crude product was purified by silica gel column chromatography [eluted with a 10-30% ethyl acetate / hexane gradient] to produce 4-methoxy-3-(methylsulfonyl)aniline (compound 103-03) (240 mg, yield: 18%). TLC system: EtoAc, R f Value: ~0.4; LCMS (m / z): 202.0 (M+H) + ; 1 H NMR (400MHz, DMSO-d6) δ7.07(d,J=2.8Hz,1H),7.00(d,J=8.8Hz,1H),6.83(dd,J=2.8Hz,8.8Hz,1H),5.14(s,2H),3.80(s,3H),2.91(s,3H).

[0506] Synthesis of 2-chloro-N-(4-methoxy-3-(methylsulfonyl)phenyl)acetamide (Compound 103-04)

[0507]

[0508] To a stirred solution of 4-methoxy-3-(methylsulfonyl)aniline (compound 103-03) (240 mg, 1.19 mmol, 1 eq) in acetone was added K2CO3 (246 mg, 1.78 mmol, 1.5 eq) followed by chloroacetyl chloride (148 mg, 1.31 mmol, 1.1 eq) at 0°C under nitrogen atmosphere. The reaction mixture was then stirred at room temperature for 4 hours. The reaction mixture was quenched with water and extracted with (2×60 mL) ethyl acetate. The combined organic layers were washed with water (50 mL), brine (50 mL), dried over sodium sulfate and concentrated to give a crude product. The crude product was purified by silica gel column chromatography [eluted with a 10-30% ethyl acetate / hexane gradient] to give 2-chloro-N-(4-methoxy-3-(methylsulfonyl)phenyl)acetamide (compound 103-04) (200 mg, yield: 60%). TLC system: EtoAc: Hexane (50:50), R f Value: ~0.4; LCMS (m / z): 278.0 (M+H) + ; 1H NMR (400MHz, DMSO-d6) δ10.47(s,1H),8.09(d,J=2.8Hz,1H),7.88(dd,J=2.8 Hz,9.2Hz,1H),7.29(d,J=9.2Hz,1H),4.24(s,2H),3.92(s,3H),3.24(s,3H).

[0509] Synthesis of 2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-methoxy-3-(methylsulfonyl)phenyl)acetamide (Compound 103)

[0510]

[0511] To a stirred solution of 2-chloro-N-(4-methoxy-3-(methylsulfonyl)phenyl)acetamide (compound 103-04) (200 mg, 0.72 mmol, 1 eq) in acetone under nitrogen atmosphere at room temperature was added 2-mercapto-7-methyl-3H-imidazo[4,5-c]pyridine-6-carbonitrile (compound 76) (205 mg, 1.08 mmol, 1.5 eq) and K2CO3 (149 mg, 1.08 mmol, 1.5 eq). The reaction mixture was stirred at 60°C for 8 hours. The solid material was filtered and washed with dichloromethane. The filtrate was evaporated and the crude compound was purified by preparative HPLC to give 2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-methoxy-3-(methylsulfonyl)phenyl)acetamide (Compound 76) (30 mg, yield: 10%). TLC system: MeOH:DCM (5:95), R f Value: ~0.3; LCMS (m / z): 430.0 (MH) + ; 1 HNMR(400MHz,DMSO-d6)δ13.70(brs,1H),10.66(s,1H),8.71(s,1H),8.11(d,J=2.8Hz,1H),7.84(d d,J=2.8Hz,9.2Hz,1H),7.28(s,J=4.8Hz,1H),4.36(s,2H).3.92(s,3H),3.23(s,3H),2.63(s,3H).

[0512] Synthesis of 2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-fluoro-3-sulfamoylphenyl)acetamide (Compound 104)

[0513]

[0514] Synthesis of 2-Fluoro-5-nitrobenzenesulfonamide (Compound 104-01)

[0515]

[0516] A solution of chlorosulfonic acid (4 mL, 4 volumes) containing 1-fluoro-4-nitrobenzene (Compound 106) (1 g, 7.09 mmol, 1.0 equiv) was heated at 110 ° C for 16 hours. The reaction mixture was cooled to room temperature and quenched with saturated NaHCO3 solution and extracted with diethyl ether (2×100 mL). The combined organic layers were dried over sodium sulfate and concentrated to produce a crude product. The crude product was dissolved in EtOAc (40 mL), and ammonia water (40 mL) was added dropwise at 0 ° C and stirred at the same temperature for 2 hours. After the reaction was completed as determined by TLC, the reaction mixture was diluted with water and extracted with EtOAc (2×100 mL). The combined organic layers were dried over sodium sulfate and concentrated to produce 2-fluoro-5-nitrobenzenesulfonamide (Compound 104-01) (800 mg, yield: 51%) as a brown solid. TLC system: EtOAc: Hexane (50:50), R f Value: ~0.4; LCMS (m / z): 219.0 (Μ-H) + ; 1 H NMR (400MHz, DMSO-d6) δ8.56-8.52(m,2H),8.06(s,2H),7.78-7.74(m,1H).

[0517] Synthesis of 5-amino-2-fluorobenzenesulfonamide (Compound 104-02)

[0518]

[0519] 10% Pd / C (800 mg) was added to a stirred solution of MeOH (8 mL) containing 2-fluoro-5-nitrobenzenesulfonamide (compound 104-01) (800 mg, 3.63 mmol, 1 eq). The reaction mixture was hydrogenated under H2 balloon pressure and stirred at room temperature for 2 hours. After completion of the reaction as determined by TLC, the reaction mixture was filtered through a celite bed and washed with MeOH. The filtrate was concentrated to produce 5-amino-2-fluorobenzenesulfonamide (compound 104-02) (500 mg, yield: 72%). TLC system: EtOAc: Hexane (50:50), R f Value: ~0.1; LCMS (m / z): 191.0 (M+H) + ; 1H NMR (400MHz, DMSO-d6) δ7.41(s,2H),7.04-6.97(m,2H),6.73-6.69(m,1H),5.38(brs,2H).

[0520] Synthesis of 2-chloro-N-(4-fluoro-3-sulfamoylphenyl)acetamide (Compound 104-03)

[0521]

[0522] At 0 ° C, K2CO3 (723 mg, 5.23 mmol, 2.0 equiv) was added to a stirred solution of 5-amino-2-fluorobenzenesulfonamide (compound 104-02) (500 mg, 2.61 mmol, 1.0 equiv) in acetone (5 mL), followed by 2-chloroacetyl chloride (354 mg, 3.13 mmol, 1.2 equiv). The reaction mixture was stirred at room temperature for 2 hours. After the reaction was completed as determined by TLC, the reaction mixture was diluted with water and extracted with EtOAc (2×10 mL). The combined organic layers were dried over sodium sulfate and concentrated to produce 2-chloro-N-(4-fluoro-3-aminosulfonylphenyl)acetamide (compound 104-03) (500 mg, yield: 71%) as a brown solid. TLC system: EtOAc / hexane (100), R f Value: ~0.6; LCMS (m / z): 267.0 (M+H) + ; 1 HNMR (400MHz, DMSO-d6) δ10.60(s,1H),8.12-8.09(m,1H),7.83-7.79(m,1H),7.69(s,2H),7.40(t,J=9.4Hz,1H),4.27(s,2H).

[0523] Synthesis of 2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-fluoro-3-sulfamoylphenyl)acetamide (Compound 104)

[0524]

[0525] To a stirred solution of 2-mercapto-7-methyl-3H-imidazo[4,5-c]pyridine-6-carbonitrile (compound 76) (150 mg, 0.789 mmol, 1 eq) and 2-chloro-N-(4-fluoro-3-sulfamoylphenyl)acetamide (compound 104-03) (252 mg, 0.95 mmol, 1.2 eq) in acetone (3.0 mL) was added KCO (219 mg, 1.578 mmol, 2.0 eq) and the reaction mixture was stirred at room temperature for 16 hours. After the reaction was completed as determined by TLC, the reaction mixture was filtered and concentrated to obtain a crude product. The crude product was purified by preparative HPLC, and the collected pure fractions were lyophilized to give 2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-fluoro-3-sulfamoylphenyl)acetamide (Compound 104) (50 mg, yield: 15%) as an off-white solid. TLC system: EtOAc / hexane (50:50), R f Value: ~0.15; LCMS (m / z): 419.0 (MH) + ; 1 HNMR(400MHz,DMSO-d6)δ13.75-13.61(2s,1H),10.74(s,1H),8.73-8.70(m,1H),8.12(dd,J=6.4 Hz,2.4Hz,1H),7.79-7.75(m,1H),7.67(s,2H),7.38(t,J=9.2Hz,1H),4.39(s,2H).2.63(s,3H).

[0526] Synthesis of Sodium 5-(2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetylamino)-2-methoxyphenyl phosphate (Compound 107)

[0527]

[0528] Synthesis of 2-chloro-N-(3-hydroxy-4-methoxyphenyl)acetamide (Compound 107-01)

[0529]

[0530] At 0 ° C, K2CO3 (8.93 g, 64.74 mmol, 1.5 equivalents) was added to a stirred solution of acetone (60 mL) containing 5-amino-2-methoxyphenol (compound 102-01) (6 g, 43.16 mmol, 1 equivalent). Then 2-chloroacetyl chloride (1) (5 mL, 51.79 mmol, 1.2 equivalents) was added dropwise, and the reaction mixture was stirred at room temperature for 16 hours. After the reaction was completed by TLC, the reaction mixture was quenched with ice-cold water and extracted with EtOAc (2×100 mL). The combined organic layer was washed with brine (50 mL), dried over sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography [eluted with a 20% ethyl acetate / hexane gradient] to produce 2-chloro-N-(3-hydroxy-4-methoxyphenyl)acetamide (compound 107-01) (4.2 g, yield: 45%) as a light brown solid. TLC system: EtOAc / hexane (50:50), R f Value: ~0.6; LCMS (m / z): 216.0 (M+H) + . 1 H NMR (400MHz, DMSO-d6) δ10.02 (s, 1H), 9.09 (s, 1H), 7.14 (d, J = 2.4Hz, 1H), 6.9 3(dd,J=2.4Hz,J=8.8Hz,1H),6.84(d,J=8.8Hz,1H),4.18(s,2H),3.72(s,3H).

[0531] Synthesis of Diallyl Chlorophosphate (Compound 107-03)

[0532]

[0533] To a stirred solution of diethyl ether (100 mL) containing POCl3 (4.45 mL, 65.35 mmol, 2 eq) at 0°C was added triethylamine (9.1 mL, 65.35 mmol, 2 eq) followed by allyl alcohol (2) (5 g, 32.67 mmol, 1 eq). The reaction mixture was stirred at room temperature for 16 hours and the solid was filtered. The filtrate was evaporated under reduced pressure to produce diallyl chlorophosphate (compound 107-03) (3.1 g, yield: 18%). TLC system: EtoAc: Hexane (30:70; KMnO4 staining), R f Value: ~0.6; 1 H NMR (400MHz, CDCl3) δ5.99-5.90(m,2H),5.40-5.35(m,2H),5.28-5.24(m,2H),4.58-4.52(m,4H).

[0534] Synthesis of (5-(2-chloroacetylamino)-2-methoxyphenyl)diallyl phosphate (Compound 107-04)

[0535]

[0536] To a stirred solution of 2-chloro-N-(3-hydroxy-4-methoxyphenyl)acetamide (compound 107-01) (2 g, 9.3 mmol, 1 eq) in acetone (20 mL) was added KCO (1.9 g, 13.95 mmol, 1.5 eq) and diallyl chlorophosphate (compound 107-03) (2.18 g, 11.1 mmol, 1.2 eq) at 0°C. The reaction mixture was stirred at room temperature for 16 hours and quenched with ice-cold water, extracted with ethyl acetate (2×100 mL). The combined organic layers were washed with water (50 mL), brine (50 mL), dried over sodium sulfate and concentrated to afford the crude product. The crude compound was purified over 100-200 mesh silica gel by eluting with 1% MeOH / DCM to yield (5-(2-chloroacetylamino)-2-methoxyphenyl)diallyl phosphate (compound 107-04) (800 mg, yield: 23%). TLC system: MeOH:DCM (5:95), R f Value: ~0.3; LCMS (m / z): 376.0 (M+H) + ; 1 HNMR(400MHz,DMSO-d6)δ8.39(s,1H),7.45-7.41(m,2H),6.91(d,J=8.4Hz,1H),5.98-5.94 (m,2H),5.42-5.36(m,2H),5.28-5.25(m,2H),4.71-4.67(m,4H),4.17(s,2H),3.86(s,3H).

[0537] Synthesis of 5-(2-chloroacetylamino)-2-methoxyphenyl dihydrogen phosphate (Compound 107-05)

[0538]

[0539] To a stirred solution of (5-(2-chloroacetylamino)-2-methoxyphenyl)diallyl phosphate (compound 107-04) (500 mg, 1.33 mmol, 1 eq) in DCM (10 mL) was degassed for 10 minutes and phenylsilane (0.35 mL, 2.66 mmol, 2 eq) was added, followed by Pd(PPh3)4 (153 mg, 0.133 mmol, 0.1 eq). The reaction mixture was stirred at room temperature for 30 minutes and diluted with pentane. The precipitated solid was filtered and dried to yield 5-(2-chloroacetylamino)-2-methoxyphenyl dihydrogen phosphate (compound 107-05) [380 mg (crude product)]. LCMS (m / z) 296.0 (M+H) + .

[0540] Synthesis of 5-(2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetylamino)-2-methoxyphenyl dihydrogen phosphate (Compound 107-02)

[0541]

[0542] At 0 ° C, K2CO3 (217 mg, 1.575 mmol, 1.5 eq) and 5-(2-chloroacetylamino)-2-methoxyphenyl dihydrogen phosphate (compound 107-05) (372 mg, 1.26 mmol, 1.2 eq) were added to a stirred solution of acetone:DMF (8:2) (5 mL) containing 2-mercapto-7-methyl-3H-imidazo[4,5-c]pyridine-6-carbonitrile (compound 76) (200 mg, 1.05 mmol, 1 eq). The reaction mixture was stirred at room temperature for 16 hours and diluted with DCM. The precipitated solid was filtered under vacuum to give the crude compound. The crude compound was purified by preparative HPLC to produce 5-(2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamido)-2-methoxyphenyl dihydrogen phosphate (Compound 107-02) (35 mg, yield: 7%). LCMS (m / z): 448.1 (MH) + ; 1 HNMR(400MHz,DMSO-d6)δ13.74(brs,1H),10.38(s,1H),8.73(s,1H),7.59(s,1H),7.34 (dd,J=8.8Hz,2.4Hz,1H),6.98(d,J=8.8Hz,1H),4.34(s,2H).3.73(s,3H),2.63(s,3H); 31 PNMR (162MHz)δ-6.38.

[0543] Synthesis of Sodium 5-(2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetylamino)-2-methoxyphenyl phosphate (Compound 107)

[0544]

[0545] To a stirred suspension of 5-(2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamido)-2-methoxyphenyl dihydrogen phosphate (Compound 107-02) (35 mg, 0.077 mmol, 1 eq) in water (0.5 mL) at 0°C was added NaOH (6.2 mg, 0.155 mmol, 2 eq) and stirred at room temperature for 15 minutes. The solution was evaporated under vacuum to produce sodium 5-(2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamido)-2-methoxyphenyl phosphate (Compound 107) (38 mg, yield: 99%). The product was hygroscopic and stored under nitrogen atmosphere. LCMS (m / z): 450.0 (M+H) + HPLC: 95.4%; 1 HNMR (400 MHz, DMSO-d6) δ 11.5 (brs, 1H), 8.46 (s, 1H), 7.85-7.71 (m, 2H), 7.06-6.76 (m, 2H), 3.92 (s, 2H). 3.47 (s, 3H), 2.73 (s, 3H). The protons in the spectrum are broad. 31 PNMR (162MHz)δ1.42.

[0546] Synthesis of sodium 4-(2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetylamino)-2-methoxyphenyl phosphate (Compound 108)

[0547]

[0548] Synthesis of 4-amino-2-methoxyphenol (Compound 108-01)

[0549]

[0550] To a solution of 2-methoxy-4-nitrophenol (1) (5 g, 29.5 mmol, 1.0 equiv) in methanol (50 mL) was added 10% Pd / C (3 g) and stirred at room temperature for 16 hours under H2 balloon pressure. The reaction mixture was filtered through a celite pad and concentrated under reduced pressure to give 4-amino-2-methoxyphenol (compound 108-01) (3.5 g, yield: 85%) as a brown solid. TLC system: EtOAc: Hexane (50:50), R f Value: ~0.2; LCMS (m / z): 140.1 (M+H) + ; 1 HNMR (400MHz, DMSO-d6) δ7.78(s,1H),6.45(d,J=8.8Hz,1H),6.22(d,J=2.4Hz,1H),5.98(dd,J=8.8Hz,J=2.4Hz,1H),4.42(s,2H),3.66(s,3H).

[0551] Synthesis of 2-chloro-N-(4-hydroxy-3-methoxyphenyl)acetamide (Compound 108-02)

[0552]

[0553] To a stirred solution of 4-amino-2-methoxyphenol (compound 108-01) (3.5 g, 25.1 mmol, 1.0 equiv) in acetone (35 mL) at 0°C was added KCO (10.3 g, 75.3 mmol, 3.0 equiv) followed by chloroacetyl chloride (2) (3 mL, 37.7 mmol, 1.5 equiv). The reaction mixture was stirred at room temperature for 16 hours and quenched with ice-cold water, extracted with ethyl acetate (3×80 mL). The combined organic layers were washed with water (50 mL), brine (50 mL), dried over sodium sulfate and concentrated to afford the crude product. The crude product was purified by silica gel column chromatography (eluent: 30% EtOAc / hexanes) to yield 2-chloro-N-(4-hydroxy-3-methoxyphenyl)acetamide (compound 108-02) (2.7 g, yield: 50%). TLC system: EtOAc: hexane (50:50), R f Value: ~0.4; LCMS (m / z): 216.0 (M+H) + ; 1H NMR (400 MHz, DMSO-d6) δ 10.06 (s, 1H), 8.82 (s, 1H), 7.24 (d, J = 2.4 Hz, 1H), 6.95 (dd, J = 2.4 Hz, 8.4 Hz, 1H), 6.71 (d, J = 8.4 Hz, 1H), 4.19 (s, 2H), 3.72 (s, 3H) and aliphatic impurities were observed.

[0554] Synthesis of (4-(2-chloroacetylamino)-2-methoxyphenyl)diallyl phosphate (Compound 108-03)

[0555]

[0556] K2CO3 (1.9 g, 13.95 mmol, 1.5 equiv) and diallyl chlorophosphate (compound 107-03) (2.18 g, 11.1 mmol, 1.2 equiv) were added to a stirred solution of 2-chloro-N-(4-hydroxy-3-methoxyphenyl)acetamide (compound 108-02) (2 g, 9.3 mmol, 1 equiv) in acetone (20 mL) at 0 ° C. The reaction mixture was stirred at room temperature for 16 hours. After the starting material was completed, the reaction was quenched with ice-cold water and extracted with ethyl acetate (3×50 mL). The combined organic layers were washed with water (50 mL), brine (50 mL), dried over sodium sulfate and concentrated. The crude compound was purified by 100-200 mesh silica gel by eluting with 1% MeOH / DCM to give (4-(2-chloroacetylamino)-2-methoxyphenyl)diallyl phosphate (Compound 108-03) (750 mg, yield: 22%). TLC system: MeOH:DCM (5:95), R f Value: ~0.4; LCMS (m / z): 376.0 (M+H) + ; 1 HNMR(400MHz,DMSO-d6)δ10.34(s,1H),7.43(d,J=2.0Hz,1H),7.17-7.12(m,2H),5.99-5.92 (m,2H),5.38-5.33(m,2H),5.27-5.23(m,2H),4.64-4.60(m,4H),4.25(s,2H),3.79(s,3H); 31 PNMR (162MHz) δ-5.7

[0557] Synthesis of 4-(2-chloroacetylamino)-2-methoxyphenyl dihydrogen phosphate (Compound 108-04)

[0558]

[0559] To a solution of (4-(2-chloroacetylamino)-2-methoxyphenyl)diallyl phosphate (compound 108-03) (500 mg, 1.33 mmol, 1 eq) in DCM (10 mL) was degassed for 10 minutes and then phenylsilane (0.35 mL, 2.66 mmol, 2 eq), Pd(PPh3)4 (153 mg, 0.133 mmol, 0.1 eq) were added. The reaction mixture was stirred at room temperature for 30 minutes and diluted with pentane, the precipitated solid was filtered and dried to give 4-(2-chloroacetylamino)-2-methoxyphenyl dihydrogen phosphate (compound 108-04) [400 mg (crude product)]. TLC system: MeOH:DCM (5:95), R f Value: ~0.2; LCMS (m / z): 296.0 (M+H) + .

[0560] Synthesis of 4-(2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetylamino)-2-methoxyphenyl dihydrogen phosphate (Compound 108-05)

[0561]

[0562] 2-Mercapto-7-methyl-3H-imidazo[4,5-c]pyridine-6-carbonitrile (compound 76) (200 mg, 1.05 mmol, 1.0 eq.), K2CO3 (217 mg, 1.57 mmol, 1.5 eq.) were added to a stirred solution of 4-(2-chloroacetylamino)-2-methoxyphenyl dihydrogen phosphate (compound 108-04) (372 mg, 1.26 mmol, 1.2 eq.) in acetone:DMF (8:2) (10 mL) at 0° C. The reaction mixture was stirred at room temperature for 16 h and diluted with DCM, the precipitated solid was filtered and dried to give a crude product. The crude compound was purified by preparative HPLC to produce 4-(2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamido)-2-methoxyphenyl dihydrogen phosphate (Compound 108-05) (25 mg, yield: 5%). LCMS (m / z): 448.1 (MH) + ; 1 HNMR(400MHz,DMSO-d6)δ13.78(br,1H),10.44(s,1H),8.72(s,1H),7.35(s,1H),7 .22(d,J=8.4Hz,1H),7.01(d,J=7.2Hz,1H),4.37(s,2H).3.70(s,3H),2.63(s,3H);31 PNMR (162MHz) δ-6.06

[0563] Synthesis of sodium 4-(2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetylamino)-2-methoxyphenyl phosphate (Compound 108)

[0564]

[0565] To a stirred suspension of 4-(2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamido)-2-methoxyphenyl dihydrogen phosphate (compound 108-05) (25 mg, 0.055 mmol, 1 eq) in water (0.5 mL) at 0°C was added NaOH (4.45 mg, 0.11 mmol, 2 eq) and stirred for 15 minutes. The solution was concentrated under reduced pressure to give sodium 4-(2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamido)-2-methoxyphenyl phosphate (compound 108) (25 mg, yield: 91%). LCMS (m / z): 450.0 (M-Na+H) + ; observed 1 HNMR (400MHz, DMSO-d6) δ broad peak, but D2O exchange NMR is cleaner and the observed protons are as follows: 8.46 (s, 1H), 7.39 (d, J = 8.8 Hz, 1H), 7.09 (d, J = 2.0 Hz, 1H), 6.79 (d, J = 8.8 Hz, 2.4 Hz, 1H), 3.68 (s, 3H), 2.61 (s, 3H), CH2 protons merged with solvent peak; 31 PNMR (162MHz)δ1.62.

[0566] Synthesis of 2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-(2-hydroxyethoxy)-4-methoxyphenyl)acetamide (Compound 109)

[0567]

[0568] Synthesis of 2-(2-methoxy-5-nitrophenoxy)ethan-1-ol (Compound 109-01)

[0569]

[0570] To a stirred solution of 2-methoxy-5-nitrophenol (1) (1.0 g, 5.90 mmol, 1.0 equiv) in acetone (10 mL) was added potassium carbonate (1.22 g, 8.86 mmol, 1.5 equiv) followed by 2-bromoethane-1-ol (1.10 g, 8.86 mmol, 1.5 equiv). The reaction mixture was heated at 50 °C for 16 hours. After completion of the reaction as determined by TLC, the reaction mixture was filtered and washed with EtOAc. The filtrate was concentrated to produce 2-(2-methoxy-5-nitrophenoxy)ethane-1-ol (compound 109-01) (810 mg, yield: 64%). TLC system: EtOAc / hexane (50:50), R f Value: ~0.4; 1 H NMR (400 MHz, CDCl3) δ 7.94 (dd, J = 2.8 Hz, 9.2 Hz, 1H), 7.79 (d, J = 2.4 Hz, 1H), 6.93 (d, J = 9.2 Hz, 1H), 4.20 (t, J = 4.4 Hz, 2H), 4.04-4.02 (m, 2H), 3.97 (s, 3H), 2.27 (t, J = 6.4 Hz, 1H) and bromoethanol impurity was observed.

[0571] Synthesis of 2-(5-amino-2-methoxyphenoxy)ethane-1-ol (Compound 109-02)

[0572]

[0573] 10% Pd / C (160 mg) was added to a stirred solution of MeOH (5 mL) containing 2-(2-methoxy-5-nitrophenoxy)ethane-1-ol (compound 109-01) (800 mg, 3.75 mmol, 1 equivalent). The reaction mixture was hydrogenated under H2 balloon pressure and stirred at room temperature for 3 hours. After the reaction was completed as determined by TLC, the reaction mixture was filtered through a diatomaceous earth bed and washed with MeOH. The filtrate was concentrated to produce 2-(5-amino-2-methoxyphenoxy)ethane-1-ol (compound 109-02) (640 mg, yield: 93%). TLC system: EtOAc (100), R f Value: ~0.1; 1 H NMR (400 MHz, DMSO-d6) δ 6.64 (d, J = 8.4 Hz, 1H), 6.27 (d, J = 2.4 Hz, 1H), 6.08 (dd, J = 2.4 Hz, 8.4 Hz, 1H), 4.81-4.78 (m, 3H), 3.86 (t, J = 5.2 Hz, 2H), 3.70-3.61 (m, 2H), 3.32 (s, 3H) and bromoethanol impurity was observed.

[0574] Synthesis of 2-chloro-N-(3-(2-hydroxyethoxy)-4-methoxyphenyl)acetamide (Compound 109-03)

[0575]

[0576] At 0 ° C, K2CO3 (712 mg, 5.16 mmol, 1.5 equivalents) was added to a stirred solution of acetone (5 mL) containing 2-(5-amino-2-methoxyphenoxy)ethane-1-ol (compound 109-02) (630 mg, 3.44 mmol, 1.0 equivalents), followed by 2-chloroacetyl chloride (466 mg, 4.13 mmol, 1.2 equivalents). The reaction mixture was stirred at room temperature for 1 hour. After the reaction was completed by TLC, the reaction mixture was filtered through a diatomaceous earth bed and washed with acetone. The filtrate was concentrated to produce a crude compound. The crude product was dissolved in MeOH (5 mL) and K2CO3 (1 equivalent) was added and stirred at room temperature for 1 hour. After the reaction was completed by TLC, the reaction mixture was filtered and concentrated to produce 2-chloro-N-(3-(2-hydroxyethoxy)-4-methoxyphenyl)acetamide (compound 109-03) (610 mg, yield: 68%) as a brown solid. TLC system: EtOAc / hexane (70:30), R f Value: ~0.6; 1 H NMR (400MHz, DMSO-d6) δ10.12(s,1H),7.26(d,J=9.4Hz,1H),7.10(dd,J=2.4Hz,8.4Hz,1H) ,6.91(d,J=8.8Hz,1H),4.85(t,1H),4.21(s,2H),3.92(t,J=5.2Hz,2H),3.73-3.71(m,5H).

[0577] Synthesis of 2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-(2-hydroxyethoxy)-4-methoxyphenyl)acetamide (Compound 109)

[0578]

[0579] To a stirred solution of acetone (3 mL) containing 2-mercapto-7-methyl-3H-imidazo[4,5-c]pyridine-6-carbonitrile (Compound 76) (200 mg, 1.05 mmol, 1 eq.) and 2-chloro-N-(3-(2-hydroxyethoxy)-4-methoxyphenyl)acetamide (Compound 109-03) (325 mg, 1.25 mmol, 1.2 eq.) was added KCO (217 mg, 1.57 mmol, 1.5 eq.), and the reaction mixture was stirred at room temperature for 18 hours. After the reaction was completed as determined by TLC, the reaction mixture was filtered and concentrated, dissolved in water (10 mL) and extracted with EtOAc (2×10 mL). The aqueous layer was acidified to a pH of about 4 with a 1N HCl aqueous solution and extracted twice with 10% MeOH / DCM (2×10 mL). The combined organic layers were dried over sodium sulfate, concentrated to give a crude product, which was purified by preparative HPLC to give 2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-(2-hydroxyethoxy)-4-methoxyphenyl)acetamide (Compound 109) (70 mg, yield: 16%) as an off-white solid. TLC system: EtOAc (100), R f Value: ~0.2; LCMS (m / z): 414.1 (M+H) + ; 1 H NMR (400MHz, DMSO-d6) δ13.62(brs,1H),10.36(s,1H),8.71(s,1H),7.28(d,J=2.4Hz,1H),7.07(dd,J=2.4Hz,8. 8Hz,1H),6.89(d,J=8.8Hz,1H),4.84(s,1H),4.32(s,2H),3.90(t,J=5.2Hz,2H),3.72-3.70(d,5H),2.63(s,3H).

[0580] Synthesis of 2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-fluoro-3-(2-hydroxyethoxy)phenyl)acetamide (Compound 110)

[0581]

[0582] Synthesis of 2-(2-fluoro-5-nitrophenoxy)ethanol (Compound 110-01)

[0583]

[0584] Potassium carbonate (1.32 g, 9.54 mmol, 1.5 equiv) and 2-bromoethane-1-ol (0.95 g, 7.63 mmol, 1.2 equiv) were added to a stirred solution of acetone (20 mL) containing 2-fluoro-5-nitrophenol (1) (1.0 g, 6.36 mmol, 1.0 equiv) at room temperature. The reaction mixture was stirred at room temperature for 16 hours. After completion of the reaction as determined by TLC, the reaction mixture was filtered and washed with EtOAc. The filtrate was concentrated and triturated with pentane to produce 2-(2-fluoro-5-nitrophenoxy)ethanol (Compound 110-01) (700 mg, yield: 58%). TLC system: EtOAc / hexane (50:50), R f Value: ~0.5; 1 H NMR (400MHz, DMSO-d6) δ8.00 (dd, J=2.8Hz, 7.2Hz, 1H), 7.91-7.87 (m, 1H), 7.52 (dd, 8.8Hz,10.8Hz,1H),4.99(t,J=5.2Hz,1H),4.23(t,J=4.8Hz,2H),3.78-3.75(m,2H).

[0585] Synthesis of 2-(5-amino-2-fluorophenoxy)ethanol (Compound 110-02)

[0586]

[0587] At room temperature, 10% Pd / C (700 mg) was added to a stirred solution of MeOH (10 mL) containing 2-(2-fluoro-5-nitrophenoxy)ethanol (compound 110-01) (700 mg, 3.48 mmol, 1 equivalent). The reaction mixture was hydrogenated under H2 balloon pressure and stirred at room temperature for 16 hours. After the reaction was completed as determined by TLC, the reaction mixture was filtered through a diatomaceous earth bed and washed with MeOH. The filtrate was concentrated and ground with diethyl ether to produce 2-(5-amino-2-fluorophenoxy)ethanol (compound 110-02) (480 mg, yield: 80%). TLC system: EtOAc / hexane (50:50), R f Value: ~0.2; 1 H NMR(400MHz,DMSO-d6)δ6.81(dd,J=8.8Hz,11.6Hz,1H),6.33(dd,J=2.4Hz,7.6Hz,1H), 6.07-6.04(m,1H),4.98(brs,2H),4.86(brs,1H),3.92(t,5.2Hz,2H),3.71-3.69(m,2H)

[0588] Synthesis of 2-chloro-N-(4-fluoro-3-(2-hydroxyethoxy)phenyl)acetamide (Compound 110-03)

[0589]

[0590] At 0 ° C, K2CO3 (460 mg, 3.33 mmol, 1.5 equiv) was added to a stirred solution of acetone (5 mL) containing 2-(5-amino-2-fluorophenoxy)ethanol (compound 110-02) (380 mg, 2.22 mmol, 1.0 equiv) followed by 2-chloroacetyl chloride (338 mg, 2.66 mmol, 1.2 equiv). The reaction mixture was stirred at room temperature for 1 hour. After the reaction was completed as determined by TLC, the reaction mixture was filtered through a diatomaceous earth bed and washed with acetone. The filtrate was concentrated to produce a crude compound having both a mono-N-alkylated product and a di-N / O-alkylated product. Without purification, the crude product was dissolved in MeOH (5 mL) and K2CO3 (1 equiv) was added, which was stirred at room temperature for 1 hour to produce the desired product. After being completely converted into the product as determined by TLC, the reaction mixture was filtered and concentrated to produce a crude mixture. The crude product was purified by normal phase grace instrument (eluent: 43% EtOAc / hexane), and the pure fractions were collected and concentrated to give 2-chloro-N-(4-fluoro-3-(2-hydroxyethoxy)phenyl)acetamide (compound 110-03) (400 mg, yield: 58%) as an off-white solid. TLC system: EtOAc / hexane (50:50), R f Value: ~0.6; 1 H NMR (400MHz, DMSO-d6) δ10.30 (s, 1H), 7.46 (dd, J = 8.0Hz, 2.4Hz, 1H), 7.19-7.09 (m, 2 H), 4.91 (t, J = 5.6Hz, 1H), 4.23 (s, 2H), 4.01 (t, J = 5.2Hz, 2H), 3.74 (q, J = 5.2Hz, 2H).

[0591] Synthesis of 2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-fluoro-3-(2-hydroxyethoxy)phenyl)acetamide (Compound 110)

[0592]

[0593] To a stirred solution of 2-mercapto-7-methyl-3H-imidazo[4,5-c]pyridine-6-carbonitrile (compound 76) (250 mg, 1.32 mmol, 1.0 eq) and 2-chloro-N-(4-fluoro-3-(2-hydroxyethoxy)phenyl)acetamide (compound 110-03) (400 mg, 1.65 mmol, 1.2 eq) in acetone (2.5 mL) was added KCO (363 mg, 2.63 mmol, 2.0 eq) and the reaction mixture was heated to 50° C. for 8 hours. After completion of the reaction as determined by TLC, the reaction mixture was filtered and concentrated to obtain the crude compound. The crude product was purified by Grace instrument (C18 column, reverse phase; eluent: 40% ACN / water containing 1% FA), and the pure fractions were collected and lyophilized to yield 2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-fluoro-3-(2-hydroxyethoxy)phenyl)acetamide (Compound 110) (52 mg, yield: 10%) as an off-white solid. TLC system: EtOAc (100%), R f Value: ~0.2; LCMS (m / z): 401.9 (M+H) + ; 1 HNMR(400MHz,DMSO-d6)δ11.72(s,1H),8.53(s,1H),8.23(s,1H),7.43(dd,J=2.4Hz,8.0Hz,1H),7.13-7.10(m,1H),7.02 6.99(m,1H),4.89(brs,1H),3.99-3.96(m,4H),3.71(t,J=4.8Hz,2H),2.61(s,3H).

[0594] Synthesis of 2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-methoxy-3-((1-methylpiperidin-4-yl)methoxy)phenyl)acetamide hydrochloride (Compound 111)

[0595]

[0596] Synthesis of tert-butyl 4-(hydroxymethyl)piperidine-1-carboxylate (Compound 111-01)

[0597]

[0598] At 0 ° C, 1-(tert-butyloxycarbonyl)piperidine-4-carboxylic acid (1) (3g, 13.1mmol, 1.0 equivalent) in THF (30mL) was added dropwise to a stirred solution of THF (39mL, 39.3mmol, 3 equivalents) containing 1M BH3 over a period of 15 minutes. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was completed as determined by TLC, the reaction mixture was slowly quenched with saturated NH4Cl solution at 0 ° C and stirred for 10 minutes. The compound was extracted with ethyl acetate (2×100mL), and the combined organic layers were washed with water (50mL), dried over sodium sulfate and concentrated to produce tert-butyl 4-(hydroxymethyl)piperidine-1-carboxylate (compound 111-01) (2.5g, yield: 88%). TLC system: EtOAc: Hexane (50:50; KMnO4 staining), R f Value: ~0.3; 1 H NMR (400MHz, CDCl3) δ4.11 (brs, 2H), 3.50 (d, J = 6.0Hz, 2H), 2.73-2.67 (m, 2H). 1.69-1.60 (m, 4H), 1.46 (s, 9H), 1.19-1.12 (m, 2H).

[0599] Synthesis of tert-butyl 4-((tosyloxy)methyl)piperidine-1-carboxylate (Compound 111-02)

[0600]

[0601] At 0 ° C, triethylamine (2.94g, 29.1mmol, 2.5 equivalents), DMAP (0.28g, 2.32mmol, 0.2 equivalents) and p-toluenesulfonyl chloride (2.39g, 13.9mmol, 1.2 equivalents) were added to a stirred solution of DCM (20mL) containing tert-butyl 4-(hydroxymethyl)piperidine-1-carboxylate (compound 111-01) (2.5g, 11.6mmol, 1 equivalent). The reaction mixture was stirred at room temperature under N2 for 2 hours. After the reaction was completed as determined by TLC, the reaction mixture was quenched with ice-cold water and extracted with (2×100mL) DCM. The combined organic layer was washed with water (50mL), brine (50mL), dried over sodium sulfate and concentrated to produce tert-butyl 4-((tosyloxy)methyl)piperidine-1-carboxylate (compound 111-02) (3.2g, yield: 75%). TLC system: EtOAc: hexane (50:50), R f Value: ~0.7; 1H NMR(400MHz, CDCl3)δ7.78(d,J=8.0Hz,2H),7.35(d,8.0Hz,2H),4.09(brs,2H),3.85(d,J=6.4Hz,2H), 2.68-2.65(m,2H),2.46(s,3H),1.84-1.82(m,1H),1.66-1.63(m,2H),1.43(s,9H),1.12-1.06(m,2H).

[0602] Synthesis of tert-butyl 4-((2-methoxy-5-nitrophenoxy)methyl)piperidine-1-carboxylate (Compound 111-03)

[0603]

[0604] Under N2 atmosphere at room temperature, 2-methoxy-5-nitrophenol (1.22g, 7.23mmol, 1.0 equivalent) and K2CO3 (1.99g, 14.4mmol, 2.0 equivalent) were added to a stirred solution of acetonitrile (32mL) containing tert-butyl 4-((tosyloxy)methyl)piperidine-1-carboxylate (compound 111-02) (3.2g, 8.67mmol, 1.2 equivalents). The reaction mixture was stirred at 90°C for 16 hours. After the reaction was completed as determined by TLC, the reaction mixture was diluted with water and extracted with (2×100mL) DCM. The combined organic layer was washed with water (50mL), brine (50mL), dried over sodium sulfate and concentrated to provide a crude product. The crude product was purified by trituration with diethyl ether to give tert-butyl 4-((2-methoxy-5-nitrophenoxy)methyl)piperidine-1-carboxylate (Compound 111-03) (2.1 g, yield: 66%). TLC system: EtOAc: Hexane (50:50), R f Value: ~0.5; 1 H NMR (400MHz, CDCl3) δ7.91(dd,J=8.8Hz,2.8Hz,1H),7.72(d,J=2.8Hz,1H),6.90(d,J=8.8Hz,1H),4.16(brs,2H),3.96( s,3H),3.91(d,J=7.2Hz,2H),2.80-2.74(m,2H),2.09-2.05(m,1H),1.89-1.85(m,2H),1.46(s,9H),1.34-1.30(m,2H).

[0605] Synthesis of 4-((2-methoxy-5-nitrophenoxy)methyl)-1-methylpiperidine (Compound 111-04)

[0606]

[0607] At 0 ° C, under nitrogen flushing, to a stirred solution of THF containing 4-((2-methoxy-5-nitrophenoxy) methyl) piperidine-1-carboxylic acid tert-butyl ester (compound 111-03) (2.1g, 5.74mmol, 1.0 equivalent), 1M LiAlH4 THF (11.5mL, 11.5mmol, 2 equivalents) was slowly added and continued to stir at room temperature for 8 hours. After the reaction was determined to be complete by TLC, the reaction mixture was quenched with saturated Na2SO4 solution at 0 ° C and stirred for 30 minutes. The resulting mixture was filtered through a diatomaceous earth bed and washed with EtOAc. The filtrate was concentrated and the obtained crude product was ground with diethyl ether dried under vacuum to produce 1.2g of crude product 4-((2-methoxy-5-nitrophenoxy) methyl)-1-methylpiperidine (compound 111-04), which was used directly in the next step. TLC system: EtOAc: hexane (50: 50), R f Value: ~0.1;

[0608] Synthesis of 4-methoxy-3-((1-methylpiperidin-4-yl)methoxy)aniline (Compound 111-05)

[0609]

[0610] Under nitrogen atmosphere, 10% Pd / C (1.2 g) was added to a stirred and degassed solution of MeOH (12 mL) containing 4-((2-methoxy-5-nitrophenoxy)methyl)-1-methylpiperidine (compound 111-04) (1.2 g, crude product). The reaction was stirred at room temperature for 5 hours under hydrogen balloon pressure. After completion of the reaction by LCMS, it was filtered through a celite bed and washed with methanol. The organic layer was evaporated and the residue was triturated with diethyl ether to produce 4-methoxy-3-((1-methylpiperidin-4-yl)methoxy)aniline (compound 111-05) (0.71 g, yield: 50% over two steps). TLC system: MeOH:DCM (10:90), R f Value: ~0.2; LCMS (m / z): 251.1 (M+H) + .

[0611] Synthesis of 2-chloro-N-(4-methoxy-3-((1-methylpiperidin-4-yl)methoxy)phenyl)acetamide (Compound 111-06)

[0612]

[0613] At 0 ° C, K2CO3 (772 mg, 5.6 mmol, 2.0 equiv) and chloroacetyl chloride (3) (376 mg, 3.36 mmol, 1.2 equiv) were added to a stirred solution of 4-methoxy-3-((1-methylpiperidin-4-yl)methoxy)aniline (compound 111-05) (700 mg, 2.8 mmol, 1.0 equiv) in acetone (7 mL). The reaction mixture was stirred at room temperature for 2 hours. After the reaction was completed as determined by TLC, the reaction mixture was diluted with water and extracted with ethyl acetate (3×50 mL). The combined organic layer was washed with brine (20 mL), dried over sodium sulfate, and concentrated. The crude compound was purified by grace column chromatography [reverse phase, 5-10% acetonitrile: water containing 1% FA] to give 2-chloro-N-(4-methoxy-3-((1-methylpiperidin-4-yl)methoxy)phenyl)acetamide (Compound 111-06) (362 mg, purity: 64%). TLC system: EtOAc (100), R f Value: ~0.2; LCMS (m / z): 327.3 (M+H) + ;

[0614] Synthesis of 2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-methoxy-3-((1-methylpiperidin-4-yl)methoxy)phenyl)acetamide hydrochloride (Compound 111)

[0615]

[0616] To a solution of 2-chloro-N-(4-methoxy-3-((1-methylpiperidin-4-yl)methoxy)phenyl)acetamide (compound 111-06) (350 mg, 1.07 mmol, 1.2 eq) in acetone (4 ml) at 0° C. was added KCO (247 mg, 1.78 mmol, 2.0 eq) and compound 76 (170 mg, 0.89 mmol, 1.0 eq). The reaction mixture was stirred at room temperature for 2 hours, and the solid was filtered and the filtrate was evaporated to afford the crude product. The crude product was purified by preparative HPLC to yield SR-8319 as a TFA salt. The solid was treated with aqueous HCl and evaporated to give 2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(4-methoxy-3-((1-methylpiperidin-4-yl)methoxy)phenyl)acetamide hydrochloride (Compound 111) (25 mg, yield: 2% over two steps) as an off-white solid. TLC system: MeOH:DCM (10:90), R fValue: ~0.1; LCMS (m / z): 481.2 (M-HC1+H) + ; 1 HNMR (400 MHz, DMSO-d6), δ10.36 (s, 1H), 9.70 (brs, 1H), 8.72 (s, 1H), 7.38 (d, J = 2.4 Hz, 1H), 7.04 (dd, J = 8.4 Hz, 2.4 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 4.36 (s, 2H), 3.78 (d, J = 5.6 Hz, 2H), 3.72 (s, 3H), 3.44-3.42 (m, 2H), 3.00-2.92 (m, 2H), 2.76-2.73 (m, 3H), 2.64 (s, 3H), 1.98-1.95 (m, 3H), 1.58-1.49 (m, 2H). No imidazole NH proton was observed in the spectrum.

[0617] Synthesis of 2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-(4-hydroxypiperidin-1-yl)-4-methoxyphenyl)acetamide hydrochloride (Compound 112)

[0618]

[0619] Synthesis of 4-((tert-butyldimethylsilyl)oxy)piperidine (Compound 112-01)

[0620]

[0621] At 0 ° C, pyridine (3.5 mL, 45.0 mmol, 1.5 equiv) was added to a stirred solution of DCM (20 mL) containing piperidine-4-ol (1) (3.0 g, 29.7 mmol, 1.0 equiv) followed by dropwise addition of tert-butyldimethylsilyl chloride (5.37 g, 35.0 mmol, 1.2 equiv). The reaction mixture was stirred at room temperature for 4 hours. After completion of the reaction as determined by TLC, the reaction mixture was evaporated and the resulting solid was ground with 20% EtOAc: hexane (20 mL) and filtered, dried under vacuum to produce 4-((tert-butyldimethylsilyl)oxy)piperidine (compound 112-01) (4.8 g, yield: 75%). TLC system: EtOAc: hexane (100: 0), R f Value: ∼0.5 (ninhydrin activity); 1H NMR (400MHz, CDCl3) δ4.08(s,1H),3.35-3.28(m,2H).3.22-3.17(m,2H),2.16-2.08(m,2H),1.78-1.73(m,3H),0.88(s,9H),0.05(s,6H).

[0622] Synthesis of 1-(2-methoxy-5-nitrophenyl)piperidin-4-ol (Compound 112-02)

[0623]

[0624] 4-((tert-butyldimethylsilyl)oxy)piperidine (compound 112-01) (1.50 g, 6.97 mmol, 1.2 equiv), NaOtBu (1.12 g, 11.6 mmol, 2.0 equiv) and BINAP (0.72 g, 1.16 mmol, 0.2 equiv) were added to a degassed, stirred solution of 2-bromo-1-methoxy-4-nitrobenzene (2) (1.34 g, 5.81 mmol, 1.0 equiv) in toluene (15 mL) at room temperature. The mixture was degassed for 30 minutes, followed by the addition of Pd2(dba)3 (0.53 g, 0.58 mmol, 0.1 equiv) and the reaction was stirred at 110°C for 16 hours. After the reaction was complete as determined by TLC, the reaction mixture was filtered through a bed of celite and the filtrate was evaporated to provide the crude product. The crude product was purified twice by silica gel column chromatography [eluted with 40% ethyl acetate / hexane gradient] to give 1-(2-methoxy-5-nitrophenyl)piperidin-4-ol (Compound 112-02) (430 mg, yield: 27%). TLC system: EtOAc: Hexane (60:40), R f Value: ~0.3; 1 H NMR (400 MHz, CDCl3) δ 7.93 (dd, J = 8.8 Hz, 2.8 Hz, 1H), 7.81 (d, J = 2.8 Hz, 1H), 6.89 (d, J = 8.8 Hz, 1H), 3.98 (s, 3H), 3.91-3.87 (m, 1H), 3.42-3.37 (m, 2H), 2.87-2.81 (m, 2H), 2.08-2.04 (m, 2H), 1.83-1.74 (m, 2H). No OH protons were observed.

[0625] Synthesis of 1-(5-amino-2-methoxyphenyl)piperidin-4-ol (Compound 112-03)

[0626]

[0627] At room temperature, 10% Pd / C (80 mg) was slowly added to a stirred solution of methanol (5 mL) containing 1-(2-methoxy-5-nitrophenyl)piperidin-4-ol (compound 112-02) (430 mg, 1.70 mmol, 1 equivalent). The reaction mixture was stirred at room temperature under H2 balloon pressure for 16 hours. After the reaction was completed as determined by TLC, the reaction mixture was filtered through a diatomaceous earth bed and washed with methanol (50 mL), and the filtrate was concentrated to produce 1-(5-amino-2-methoxyphenyl)piperidin-4-ol (compound 112-03) (300 mg, yield: 79%). TLC system: EtOAc: hexane (100: 0), R f Value: ~0.2; LCMS (m / z): 223.1 (M+H) + ; 1 H NMR (400MHz, DMSO-d6) δ6.59(d,J=8.4Hz,1H),6.20(d,J=2.8Hz,1H),6.10(dd,J=2.8Hz,8.4Hz,1H),4.59(d,J=4.4Hz,2.4Hz, 1H),4.51(s,2H),3.64(s,3H),3.55-3.52(m,1H),3.20-3.16(m,2H),2.59-2.53(m,2H),1.81-1.74(m,2H),1.52-1.48(m,2H).

[0628] Synthesis of 2-chloro-N-(3-(4-hydroxypiperidin-1-yl)-4-methoxyphenyl)acetamide (Compound 112-04)

[0629]

[0630] To a stirred solution of 1-(5-amino-2-methoxyphenyl)piperidin-4-ol (compound 112-03) (300 mg, 1.35 mmol, 1.0 equiv) in acetone (3 mL) was added potassium carbonate (279 mg, 2.02 mmol, 1.5 equiv) and chloroacetyl chloride (181 mg, 1.62 mmol, 1.2 equiv) at 0°C under nitrogen flushing and stirring was continued for 2 hours at room temperature. The reaction mixture was diluted with water and extracted with (2×50 mL) ethyl acetate. The combined organic layers were washed with water (20 mL), brine (20 mL), dried over sodium sulfate and concentrated to provide a crude product. The crude compound was purified by trituration with pentane and diethyl ether to produce 2-chloro-N-(3-(4-hydroxypiperidin-1-yl)-4-methoxyphenyl)acetamide (compound 112-04) (310 mg, yield: 77%). TLC system: EtOAc: Hexane (100:0), R fValue: ~0.4; LCMS (m / z): 299.3 (M+H) + ; 1 HNMR(400MHz,DMSO-d6)δ10.06(s,1H),7.18-7.16(m,2H),6.85(d,J=9.2Hz,1H),4.64-4.63(m,1H),4.41(s,2H ),3.80(s,3H),3.58-3.56(m,1H),3.23-3.14(m,2H),2.66-2.60(m,2H),1.83-1.80(m,2H),1.55-1.50(m,2H).

[0631] Synthesis of 2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-(4-hydroxypiperidin-1-yl)-4-methoxyphenyl)acetamide hydrochloride (Compound 112)

[0632]

[0633] To a stirred solution of compound 76 (200 mg, 1.05 mmol, 1.0 equiv) in acetone was added potassium carbonate (219 mg, 1.58 mmol, 1.5 equiv) and 2-chloro-N-(3-(4-hydroxypiperidin-1-yl)-4-methoxyphenyl)acetamide (compound 112-04) (344 mg, 1.16 mmol, 1.1 equiv) at 0° C. under nitrogen flushing and stirring was continued for 16 hours at room temperature. The reaction mixture was filtered and evaporated to give a crude product. The crude product was purified by preparative HPLC to give the TFA salt of SR-8321, which was treated with aqueous HCl and evaporated under reduced pressure to yield 2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)-N-(3-(4-hydroxypiperidin-1-yl)-4-methoxyphenyl)acetamide hydrochloride (Compound 112) (41 mg, yield: 9%). TLC system: MeOH:DCM (10:90), R f Value: ~0.1; LCMS (m / z): 453.0 (M+H) + ; 1HNMR (400 MHz, DMSO-d6) δ was not a clean spectrum, 10.60 (brs, 1H), 8.72 (s, 1H), 8.15-7.87 (brs, 1H), 7.45 (brs, 1H), 7.19-7.16 (m, 1H), 4.38 (s, 2H), 3.89 (s, 3H), 2.64 (s, 3H), 2.07-1.99 (m, 2H), 1.78-1.76 (m, 2H), not all protons were observed. D2O exchange NMR was clean, 8.72 (s, 1H), 7.94 (s, 1H), 7.47 (dd, J = 9.2 Hz, 2.4 Hz, 1H), 7.24 (d, J = 9.2 Hz, 1H), 4.35 (s, 2H), 3.91 (s, 3H), 3.86-3.84 (m, 2H), 3.39-3.37 (m, 2H), 2.64 (s, 3H), 2.09-2.04 (m, 2H), 1.83-1.81 (m, 2H).

[0634] Synthesis of N-(3-(((1r,4r)-4-aminocyclohexyl)oxy)-4-methoxyphenyl)-2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide hydrochloride (Compound 113)

[0635]

[0636] Synthesis of (1s,4s)-4-((tert-butoxycarbonyl)amino)cyclohexyl methanesulfonate (Compound 113-01)

[0637]

[0638] To a stirred solution of tert-butyl ((1s, 4s)-4-hydroxycyclohexyl)carbamate (1) (2.50 g, 11.6 mmol, 1.0 equiv) in DCM (25 mL) at 0°C was added triethylamine (1.92 g, 17.4 mmol, 1.5 equiv), DMAP (0.28 g, 2.29 mmol, 0.2 equiv), followed by dropwise addition of methanesulfonyl chloride (1.60 g, 13.9 mmol, 1.5 equiv). The reaction mixture was stirred at room temperature for 1 hour. After completion of the reaction as determined by TLC, it was diluted with water and extracted with DCM (2×70 mL). The combined organic layers were washed with water (50 mL), brine solution (50 mL), dried over sodium sulfate and concentrated to yield (1s, 4s)-4-((tert-butoxycarbonyl)amino)cyclohexyl methanesulfonate (compound 113-01) (3.3 g, yield: 95%). TLC system: EtOAc: hexane (30:70), R f Value: ∼0.6 (KMnO4 staining);1 H NMR (400MHz, CDCl3) δ4.90-4.87(m,1H),4.47(brs,1H),3.49(brs,1H).3.01(s,3H),2. 07-2.03(m,2H),1.86-1.82(m,2H),1.77-1.73(m,2H),1.70-1.61(m,2H),1.48(s,9H).

[0639] Synthesis of tert-butyl ((1r,4r)-4-(2-methoxy-5-nitrophenoxy)cyclohexyl)carbamate (Compound 113-02)

[0640]

[0641] To a stirred solution of 2-methoxy-5-nitrophenol (2) (3 x 0.50 g, 8.87 mmol, 1 eq) in DMF (15 mL) was added (1s, 4s)-4-((tert-butoxycarbonyl)amino)cyclohexyl methanesulfonate (compound 113-01) (3.12 g, 10.6 mmol, 1.2 eq) and CsCO (5.78 g, 17.7 mmol, 2.0 eq) at room temperature. The reaction mixture was irradiated under microwave at 120 °C for 3 h. The reaction mixture was quenched with ice-cold water and extracted with (3 x 100 mL) ethyl acetate. The combined organic layers were washed with water (100 mL), brine (50 mL), dried over sodium sulfate and concentrated to provide the crude product. The crude product was purified by silica gel column chromatography [eluted with 20% ethyl acetate / hexane gradient] to produce tert-butyl ((1r,4r)-4-(2-methoxy-5-nitrophenoxy)cyclohexyl)carbamate (compound 113-02) (0.65 g, yield: 21%). TLC system: EtOAc:hexane (50:50), R f Value: ~0.4; 1 H NMR (400MHz, CDCl3) δ7.90(dd,J=9.2Hz,2.4Hz,1H),7.75(d,J=2.4Hz,1H),6.91(d,J=9.2Hz,1H),4.41(brs,1H),4. 28-4.24(m,1H),3.95(s,3H),3.54(brs,1H),2.19-2.11(m,4H),1.72-1.63(m,2H),1.45(s,9H),1.31-1.25(m,2H).

[0642] Synthesis of tert-butyl ((1r,4r)-4-(5-amino-2-methoxyphenoxy)cyclohexyl)carbamate (Compound 113-03)

[0643]

[0644] At room temperature, 10% Pd / C (650 mg) was added to a stirred solution of methanol (7 mL) containing tert-butyl ((1r, 4r)-4-(2-methoxy-5-nitrophenoxy)cyclohexyl)carbamate (compound 113-02) (0.65 g, 1.77 mmol, 1 equivalent). The reaction mixture was stirred at room temperature under H2 balloon pressure for 4 hours. After the reaction was complete, the mixture was filtered through a diatomaceous earth bed and washed with methanol (50 mL). The filtrate was concentrated to produce tert-butyl ((1r, 4r)-4-(5-amino-2-methoxyphenoxy)cyclohexyl)carbamate (compound 113-03) (0.46 g, yield: 78%). TLC system: EtOAc (100), R f Value: ~0.2; LCMS (m / z): 337.2 (M+H) + ; 1 H NMR (400 MHz, DMSO-d6) δ 6.76 (d, J = 7.6 Hz, 1H), 6.63 (d, J = 8.8 Hz, 1H), 6.27 (d, J = 2.4 Hz, 1H), 6.07 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 4.60 (s, 2H), 4.01-3.96 (m, 1H), 3.59 (s, 3H), 2.01-1.98 (m, 2H), 1.82-1.79 (m, 2H), 1.38 (s, 9H), 1.32-1.22 (m, 4H), 1H proton merged with the solvent peak.

[0645] Synthesis of tert-butyl ((1r,4r)-4-(5-(2-chloroacetylamino)-2-methoxyphenoxy)cyclohexyl)carbamate (Compound 113-04)

[0646]

[0647] To a stirred solution of tert-butyl ((1r,4r)-4-(5-amino-2-methoxyphenoxy)cyclohexyl)carbamate (compound 113-03) (460 mg, 1.37 mmol, 1.0 equiv) in acetone at 0°C under nitrogen flushing was added potassium carbonate (378 mg, 2.74 mmol, 2.0 equiv) and chloroacetyl chloride (3) (184 mg, 1.64 mmol, 1.2 equiv) and stirring was continued at room temperature for 16 hours. The reaction mixture was diluted with water and extracted with (2×50 mL) ethyl acetate. The combined organic layers were washed with water (40 mL), brine (40 mL), dried over sodium sulfate and concentrated to provide the crude product. The crude compound was purified by trituration with pentane and diethyl ether to give tert-butyl ((1r,4r)-4-(5-(2-chloroacetylamino)-2-methoxyphenoxy)cyclohexyl)carbamate (Compound 113-04) (460 mg, yield: 84%). TLC system: EtOAc, R f Value: ~0.6; LCMS (m / z): 413.4 (M+H) + ; 1 HNMR (400MHz, CDCl3) δ8.11(s,1H),7.30(d,J=2.4Hz,1H),6.96(dd,J=8.8Hz,2.4Hz,1H),6.84(d,J=8.8Hz,1H),4.38(brs,1H),4 .18(s,2H),4.16-4.14(m,1H),3.52(brs,1H),3.84(s,3H),2.16-2.07(m,4H),1.65-1.59(m,2H),1.45(s,9H),1.26-1.22(m,2H).

[0648] Synthesis of tert-butyl ((1r,4r)-4-(5-(2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetylamino)-2-methoxyphenoxy)cyclohexyl)carbamate (Compound 113-05)

[0649]

[0650] Potassium carbonate (301 mg, 2.18 mmol, 2.0 equiv) and tert-butyl ((1r, 4r)-4-(5-(2-chloroacetylamino)-2-methoxyphenoxy)cyclohexyl)carbamate (compound 113-04) (450 mg, 1.09 mmol, 1.0 equiv) were added to a stirred solution of acetone containing compound 76 (269 mg, 1.41 mmol, 1.3 equiv) at 0°C under nitrogen flushing and stirring was continued for 16 hours at room temperature. The reaction mixture was diluted with water and extracted with (2×50 mL) ethyl acetate. The combined organic layers were washed with water (20 mL), brine (20 mL), dried over sodium sulfate and concentrated to provide the crude product. The crude product was purified by silica gel column chromatography [eluted with a 50% ethyl acetate / hexane gradient] to produce tert-butyl ((1r,4r)-4-(5-(2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetylamino)-2-methoxyphenoxy)cyclohexyl)carbamate (Compound 113-05) (155 mg, yield: 24%). TLC system: EtOAc, R f Value: ~0.4; LCMS (m / z): 567.2 (M+H) + ; 1 HNMR (400 MHz, DMSO-d6) δ13.68 (brs, 1H), 10.26 (s, 1H), 8.72 (s, 1H), 7.26 (d, J = 2.4 Hz, 1H), 7.10 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 6.89 (d, J = 8.8 Hz, 1H), 6.77 (d, J = 7.6 Hz, 1H), 4.35 (s, 2H), 4.06-4.00 (m, 1H), 3.71 (s, 3H), 2.59 (s, 3H), 2.02-1.98 (m, 2H), 1.83-1.80 (m, 2H), 1.44-1.41 (m, 2H), 1.38 (s, 9H), 1.29-1.20 (m, 2H), 1H may be merged with the solvent peak.

[0651] Synthesis of N-(3-(((1r,4r)-4-aminocyclohexyl)oxy)-4-methoxyphenyl)-2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide hydrochloride (Compound 113)

[0652]

[0653] To a stirred solution of tert-butyl ((1r,4r)-4-(5-(2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamido)-2-methoxyphenoxy)cyclohexyl)carbamate (compound 113-05) (100 mg, 0.176 mmol, 1.0 equiv) in dioxane (1 mL) was added 4 M HCl in dioxane (1.0 mL; 10 volumes) at room temperature under N2 and stirred for 2 h. The volatiles were evaporated and triturated with diethyl ether to give N-(3-(((1r,4r)-4-aminocyclohexyl)oxy)-4-methoxyphenyl)-2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide hydrochloride (Compound 113) (70 mg, yield: 81%) as an off-white solid. LCMS (m / z): 467.2 (M-HCl+H) + ; 1 H NMR (400 MHz, DMSO-d6) δ10.34 (s, 1H), 8.72 (s, 1H), 7.95 (s, 3H), 7.34 (d, J = 2.4 Hz, 1H), 7.10 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 6.92 (d, J = 8.8 Hz, 1H), 4.36 (s, 2H), 4.06-4.04 (m, 1H), 3.71 (s, 3H), 3.07 (brs, 1H), 2.64 (s, 3H), 2.08-1.97 (m, 4H), 1.48-1.40 (m, 4H). No exchangeable protons of imidazole were observed.

[0654] Synthesis of N-(3-(((1r,4r)-4-aminocyclohexyl)amino)-4-methoxyphenyl)-2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide hydrochloride (Compound 114)

[0655]

[0656] Synthesis of tert-butyl ((1r,4r)-4-aminocyclohexyl)carbamate (Compound 114-01)

[0657]

[0658] To a stirred solution of MeOH (50 mL) containing (1r, 4r)-cyclohexane-1,4-diamine (1) (2 g, 17.54 mmol, 3.6 equiv) was added (Boc)2O (1.1 mL, 4.91 mmol, 1.0 equiv) at 0°C. The reaction mixture was stirred at room temperature for 16 hours. After completion of the reaction as determined by TLC, the volatiles were evaporated, and the residue was diluted with water and extracted with ethyl acetate (2×100 mL). The combined organic layers were washed with brine solution (50 mL), dried over sodium sulfate and concentrated to produce tert-butyl ((1r, 4r)-4-aminocyclohexyl)carbamate (compound 114-01) (900 mg, yield: 86%). TLC system: MeOH / DCM (5:95), R f Value: ∼0.3 (ninhydrin staining); 1 H NMR(400MHz, CDCl3)δ4.35(brs,1H),3.39(brs,1H),2.67-2.62(m,1H).2.00-1. 98(m,2H),1.87-1.84(m,2H),1.49-1.46(m,2H),1.44(s,9H),1.18-1.12(m,4H).

[0659] Synthesis of tert-butyl ((1r,4r)-4-((2-methoxy-5-nitrophenyl)amino)cyclohexyl)carbamate (Compound 114-02)

[0660]

[0661] To a stirred solution of toluene (18 mL) containing 2-bromo-1-methoxy-4-nitrobenzene (2) (1.20 g, 5.60 mmol, 1.2 equivalents) in a sealed tube, tert-butyl ((1r, 4r)-4-aminocyclohexyl)carbamate (compound 114-01) (1.0 g, 4.67 mmol, 1.0 equivalents) and sodium tert-butoxide (672 mg, 7.0 mmol, 1.5 equivalents) were added. The reaction mixture was degassed with argon for 20 minutes and then Pd2(dba)3 (428 mg, 0.46 mmol, 0.1 equivalents) was added, followed by BINAP (582 mg, 0.93 mmol, 0.2 equivalents) and heated at 120 ° C for 16 hours. After the reaction was completed by TLC, the reaction mixture was filtered through a diatomaceous earth bed, and the filtrate was concentrated to obtain a crude product. The crude product was purified by Grace flash chromatography [eluted with 20% ethyl acetate / hexanes gradient] to produce tert-butyl ((1r,4r)-4-((2-methoxy-5-nitrophenyl)amino)cyclohexyl)carbamate (Compound 114-02) (900 mg, yield: 53%). TLC system: EtOAc:hexanes (30:70), R f Value: ~0.5; LCMS (m / z): 366.2 (M+H) + ; 1 H NMR (400MHz, CDCl3) δ7.59(dd,J=8.8Hz,2.8Hz,1H),7.35(d,J=2.4Hz,1H),6.74(d,J=8.8Hz,1H),4.44(brs,1H),4.28(d,J=8. 0Hz,1H),3.94(s,3H),3.50(brs,1H),3.31-3.28(m,1H),2.18-2.16(m,2H),2.10-2.09(m,2H),1.46(s,9H),1.32-1.28(m,4H).

[0662] Synthesis of tert-butyl ((1r,4r)-4-((5-amino-2-methoxyphenyl)amino)cyclohexyl)carbamate (Compound 114-03)

[0663]

[0664] At room temperature, 10% Pd / C (900 mg) was slowly added to a stirred solution of methanol (9 mL) containing tert-butyl ((1r, 4r)-4-((2-methoxy-5-nitrophenyl) amino) cyclohexyl) carbamate (compound 114-02) (900 mg, 2.45 mmol, 1.0 equivalent). The reaction mixture was stirred at room temperature under H2 balloon pressure for 16 hours. After the reaction was completed as determined by TLC, the reaction mixture was filtered through a diatomaceous earth bed and washed with methanol (20 mL), and the filtrate was concentrated and triturated with diethyl ether to produce tert-butyl ((1r, 4r)-4-((5-amino-2-methoxyphenyl) amino) cyclohexyl) carbamate (compound 114-03) (750 mg, yield: 91%). TLC system: EtOAc: hexane (60: 40), R f Value: ~0.4; LCMS (m / z): 336.2 (M+H) + ; 1 H NMR (400MHz, DMSO-d6) δ6.75(d,J=7.6Hz,1H),6.48(d,J=8.4Hz,1H),5.91(d,J=2.4Hz,1H),5.73(dd,J=8.4Hz,2.4Hz,1H),4.38( s,2H),3.63(s,3H),3.23-3.22(m,2H),3.03-2.99(m,1H),1.96-1.93(m,2H),1.80-1.77(m,2H),1.38(s,9H),1.26-1.16(m,4H).

[0665] Synthesis of tert-butyl ((1r,4r)-4-((5-(2-chloroacetylamino)-2-methoxyphenyl)amino)cyclohexyl)carbamate (Compound 114-04)

[0666]

[0667] At 0 ° C, potassium carbonate (462 mg, 3.34 mmol, 1.5 equivalents) and chloroacetyl chloride (0.21 mL, 2.67 mmol, 1.2 equivalents) were added to a stirred solution of acetone containing tert-butyl ((1r, 4r)-4-((5-amino-2-methoxyphenyl) amino) cyclohexyl) carbamate (compound 114-03) (750 mg, 2.23 mmol, 1.0 equivalents) and stirred at room temperature for 16 hours. After the reaction was completed as determined by TLC, the reaction mixture was diluted with water and extracted with diethyl ether (2×50 mL). The combined organic layer was washed with brine (20 mL), dried over sodium sulfate and concentrated to obtain a crude product. The crude compound was purified by Grace column chromatography [eluted with 30% ethyl acetate / hexane gradient] to produce tert-butyl ((1r,4r)-4-((5-(2-chloroacetylamino)-2-methoxyphenyl)amino)cyclohexyl)carbamate (Compound 114-04) (630 mg, yield: 68%). TLC system: EtOAc: Hexane (50:50), R f Value: ~0.6; LCMS (m / z): 412.1 (M+H) + ; 1 HNMR(400MHz, CDCl3)δ8.06(s,1H),6.86(s,1H),6.70-6.67(m,2H),4.40(brs,1H),4.17(s,2H),4.15-4.10(m,1H) ,3.82(s,3H),3.47(brs,1H),3.22(brs,1H),2.17-2.15(m,2H),2.09-2.05(m,2H),1.45(s,9H),1.32-1.24(m,4H).

[0668] Synthesis of tert-butyl ((1r,4r)-4-((5-(2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetylamino)-2-methoxyphenyl)amino)cyclohexyl)carbamate (Compound 114-05)

[0669]

[0670] Potassium carbonate (108 mg, 0.78 mmol, 1.5 eq) and tert-butyl ((1r,4r)-4-((5-(2-chloroacetylamino)-2-methoxyphenyl)amino)cyclohexyl)carbamate (compound 114-04) (217 mg, 0.52 mmol, 1.0 eq) were added to a stirred solution of acetone (1 mL) containing compound 76 (100 mg, 0.52 mmol, 1.0 eq) at 0°C and stirred at room temperature for 16 hours. The reaction mixture was diluted with water and extracted with dichloromethane (3×30 mL). The combined organic layers were washed with brine (20 mL), dried over sodium sulfate and concentrated to obtain the crude product. The crude product was purified by Grace flash column chromatography [eluted with 5% MeOH / DCM gradient] to give tert-butyl ((1r,4r)-4-((5-(2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetylamino)-2-methoxyphenyl)amino)cyclohexyl)carbamate (SR-8323-05) (102 mg, yield: 35%). TLC system: MeOH / DCM (5:95), R f Value: ~0.4; LCMS (m / z): 566.2 (M+H) + ; 1 HNMR(400MHz,DMSO-d6)δ13.70(brs,1H),10.09(s,1H),8.72(s,1H),6.81-6.69(m,4H),4.43(d,J=8.4Hz,1H),4.33(s,2H),3.7 2(s,3H),3.27-3.25(m,1H),3.01(brs,1H),2.64(s,3H),1.97-1.95(m,2H),1.80-1.78(m,2H),1.38(s,9H),1.26-1.22(m,4H).

[0671] Synthesis of N-(3-(((1r,4r)-4-aminocyclohexyl)amino)-4-methoxyphenyl)-2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide hydrochloride (Compound 114)

[0672]

[0673] To a stirred solution of tert-butyl ((1r,4r)-4-((5-(2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamido)-2-methoxyphenyl)amino)cyclohexyl)carbamate (compound 114-05) (90 mg, 0.15 mmol, 1.0 equiv) in dioxane (1 mL) was added 4 M HCl in dioxane (1.0 mL; 10 volumes) at room temperature under N2 and stirred for 2 h. The volatiles were evaporated and triturated with diethyl ether to give N-(3-(((1r,4r)-4-aminocyclohexyl)amino)-4-methoxyphenyl)-2-((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamide hydrochloride (Compound 114) (60 mg, yield: 81%) as an off-white solid. LCMS (m / z): 464.2 (M-HCl+H) + ; 1 H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.72 (s, 1H), 7.99 (s, 3H), 7.39-6.96 (m, 3H), 4.37 (s, 2H), 3.80 (s, 3H), 3.17 (brs, 1H), 2.98 (brs, 1H), 2.64 (s, 3H), 2.01-1.98 (m, 4H), 1.42-1.38 (m, 4H), no imidazole NH protons were observed and one aliphatic proton merged with the solvent peak.

[0674] Synthesis of sodium 5-(2-(((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)methyl)thiazole-5-carboxamido)-2-methoxybenzoate (Compound 115)

[0675]

[0676] Synthesis of tert-butyl 2-methylthiazole-5-carboxylate (Compound 115-01)

[0677]

[0678] To a stirred solution of tBuOH:DMF (1:1; 40 mL) containing 2-methylthiazole-5-carboxylic acid (1) (5.0 g, 34.9 mmol, 1.0 equiv) was added di-tert-butyl dicarbonate (20 mL, 87.3 mmol, 2.5 equiv) and DMAP (1.06 g, 8.73 mmol, 0.25 equiv) at room temperature. The reaction mixture was stirred at 70 °C for 16 hours. After the reaction was completed as determined by TLC, the volatiles in the reaction mixture were evaporated and diluted with water and extracted with (2×100 mL) ethyl acetate. The combined organic layers were washed with water (50 mL), brine solution (50 mL), dried over sodium sulfate and concentrated to give a crude product, which was purified by using a Grace instrument (normal phase, eluent: 15% EtOAc / hexane) to give tert-butyl 2-methylthiazole-5-carboxylate (Compound 115-01) (6.1 g, yield: 88%). TLC system: EtOAc: hexane (30:70), R f Value: ~0.7; LCMS (m / z): 200.1 (M+H) + ; 1 H NMR (400MHz, CDCl3) δ8.14(s,1H),2.73(s,3H),1.57(s,9H).

[0679] Synthesis of tert-butyl 2-(bromomethyl)thiazole-5-carboxylate (Compound 115-02)

[0680]

[0681] At room temperature, NBS (13.6 g, 76.6 mmol, 2.5 equiv) and AIBN (2.02 g, 12.3 mmol, 0.4 equiv) were added to a stirred solution of CCl4 (60 mL) containing tert-butyl 2-methylthiazole-5-carboxylate (compound 115-01) (6.1 g, 30.6 mmol, 1 equiv) . The reaction mixture was stirred at 50 ° C for 24 hours. After 24 hours, TLC indicated both the starting material and the product. The volatiles were evaporated under reduced pressure and diluted with ice-cold water and extracted with (3×100 mL) ethyl acetate. The combined organic layers were washed with water (100 mL), brine (50 mL), dried over sodium sulfate and concentrated to provide a crude product. The crude product was dissolved in THF and diisopropyl phosphite (7.6 mL, 45.9 mmol, 1.5 equiv), DIPEA (8.2 mL, 45.9 mmol, 1.5 equiv) were added at 0 ° C and stirred for 2 hours. The mixture was neutralized with 2N HCl and extracted with diethyl ether. The organic layer was concentrated and the crude product obtained was purified by silica gel column chromatography [eluted with 0%-10% ethyl acetate / hexane gradient] to produce tert-butyl 2-(bromomethyl)thiazole-5-carboxylate (Compound 115-02) (3.0 g, yield: 35%). TLC system: EtoAc: hexane (20:80), R f Value: ~0.6; 1 H NMR (400MHz, CDCl3) δ8.22(s,1H),4.69(s,2H),1.57(s,9H).

[0682] Synthesis of tert-butyl 2-(((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)methyl)thiazole-5-carboxylate (Compound 115-03)

[0683]

[0684] To a stirred solution of 2-mercapto-7-methyl-3H-imidazo[4,5-c]pyridine-6-carbonitrile (compound 76) (239 mg, 1.26 mmol, 1.0 eq) in acetone (8 mL) was added KCO (258 mg, 1.89 mmol, 1.5 eq) followed by dropwise addition of a solution of tert-butyl 2-(bromomethyl)thiazole-5-carboxylate (compound 115-02) (350 mg, 1.26 mmol, 1 eq) in acetone (2 mL) for 30 min at 0° C. Upon completion of the addition, TLC indicated product formation and consumption of starting material. The solid was filtered, and the filtrate was evaporated and purified by silica gel column chromatography [eluted with a 20%-40% ethyl acetate / hexane gradient] to produce tert-butyl 2-(((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)methyl)thiazole-5-carboxylate (compound 115-03) (170 mg, yield: 34%). TLC system: EtoAc: Hexane (50:50), R f Value: ~0.2; LCMS (m / z): 388.0 (M+H) + ; 1 H NMR(400MHz,DMSO-d6)δ13.78(brs,1H),8.76(s,1H),8.24(s,1H),5.00(s,2H),2.66(s,3H),1.49(s,9H)

[0685] Synthesis of 2-(((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)methyl)thiazole-5-carboxylic acid (Compound 115-04)

[0686]

[0687] A stirred solution of TFA (10 mL) containing tert-butyl 2-(((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)methyl)thiazole-5-carboxylate (compound 115-03) (160 mg, 0.41 mmol, 1.0 equiv) was stirred at room temperature for 1 hour. After the reaction was complete, all volatiles were evaporated and triturated with diethyl ether to produce 2-(((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)methyl)thiazole-5-carboxylic acid (compound 115-04) (90 mg, yield: 66%). TLC system: MeOH:DCM (5:95), R f Value: ~0.05; LCMS (m / z): 332.0 (M+H) + ; 1HNMR (400 MHz, DMSO-d6) δ13.85 (s, 1H), 8.77 (s, 1H), 8.25 (s, 1H), 5.02 (s, 2H), 2.65 (s, 3H), no COOH proton was observed.

[0688] Synthesis of 2-(((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)methyl)-N-(3-hydroxy-4-methoxyphenyl)thiazole-5-carboxamide (Compound 115-05)

[0689]

[0690] To a stirred solution of 2-(((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)methyl)thiazole-5-carboxylic acid (compound 115-04) (90 mg, 0.27 mmol, 1.3 equiv) in DMF (0.3 mL) at room temperature under nitrogen flushing was added DIPEA (0.12 mL, 0.68 mmol, 2.5 equiv), 5-amino-2-methoxyphenol (compound 102-01) (45.3 mg, 0.33 mmol, 1.2 equiv), HATU (155 mg, 0.41 mmol, 1.5 equiv) and DMAP (1.6 mg, 0.014 mmol, 0.05 equiv) and stirring was continued at room temperature for 1 hour. The reaction mixture was diluted with ice-cold water and extracted with (2×50 mL) ethyl acetate. The combined organic layers were washed with water (20 mL), brine (20 mL), dried over sodium sulfate and concentrated to provide a crude product. The crude product was purified by preparative HPLC to produce 2-(((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)methyl)-N-(3-hydroxy-4-methoxyphenyl)thiazole-5-carboxamide (compound 115-05) (32 mg, yield: 26%). TLC system: EtoAc (100), R f Value: ~0.3; LCMS (m / z): 453.0 (M+H) + ; 1 HNMR(400MHz,DMSO-d6)δ13.78(brs,1H),10.12(s,1H),9.08(s,1H),8.76(s,1H),8.43(s,1H),7.18(d,J =2.0Hz,1H),7.02(dd,J=8.8Hz,2.0Hz,1H),6.86(d,J=8.8Hz,1H),5.01(s,2H),3.73(s,3H),2.65(s,3H).

[0691] Synthesis of sodium 5-(2-(((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)methyl)thiazole-5-carboxamido)-2-methoxybenzoate (Compound 115)

[0692]

[0693] To a stirred suspension of 2-(((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)methyl)-N-(3-hydroxy-4-methoxyphenyl)thiazole-5-carboxamide (compound 115-05) (32 mg, 0.071 mmol, 1 eq) in water (1.4 mL) at 0°C was added NaOH (2.8 mg, 0.071 mmol, 1 eq) and stirred for 15 minutes. The solution was concentrated under reduced pressure to give sodium 5-(2-(((6-cyano-7-methyl-3H-imidazo[4,5-c]pyridin-2-yl)thio)methyl)thiazole-5-carboxamido)-2-methoxybenzoate (compound 115) (29 mg, yield: 88%). LCMS (m / z): 453.0 (M-Na+H) + ; 1 HNMR (400MHz, DMSO-d6) δ9.52(brs,2H),8.38(s,1H),8.31(s,1H),6.42-6.33(m,3H),4.78(s,2H),3.59(s,3H),2.56(s,3H).

[0694] The following compounds (Group 1, Group 2 and Group 3) can also be prepared similarly to the above compounds.

[0695]

[0696] Specifically, the syntheses of Group 1, Group 2, and Group 3 compounds can be prepared by the following general schemes outlined herein:

[0697] General Scheme for the Synthesis of Group 1, Group 2 and Group 3 Compounds

[0698]

[0699] Example 1

[0700] 1. ENPP1 Inhibition Assay

[0701] Materials: Assay buffer: 1 mM CaCl2, 0.2 mM ZnCl2, 50 mM Tris, pH 9.0. Substrate: 8 mM thymidine 5'-monophosphate p-nitrophenolate sodium salt (Sigma catalog number T4510). Enzyme: 5 ng / μL recombinant human ENPP-1 protein (R&D catalog number 6136-EN-010) in DMSO in a 96-well clear assay plate.

[0702] Methods: Eight-point serial dilutions of drug were prepared in 10x assay buffer, with final assay concentrations starting at 10μM, 3μM, 1μM, 0.3μM...0μM. Dilutions containing DMSO were used as controls. The assay plate was set up as follows, with duplicates for each well: 81μL assay buffer + 10μL ENPP1 inhibitor or DMSO + 5μL substrate + 4μL enzyme. Both enzyme and substrate were added to opposite sides of the well to ensure that there was no interaction until all wells had both components. The plate was then gently centrifuged for 10 seconds and then incubated at 37°C for 45 minutes. The reaction was quantified by measuring absorbance at 405nm using Envision.

[0703] IC 50 Calculation: IC 50 The values ​​were determined using GraphPad Prism 5 software. The data were entered into the software as an XY curve as the percentage of inhibition for each drug concentration. The drug concentration values ​​were logarithmically transformed and nonlinear regression was performed using the "Sigmoidal dose-response (variable slope)" option in GraphPad software to model the data and calculate IC 50 The reported IC 50 Values ​​are drug concentrations that achieve 50% inhibition.

[0704] The results of this experiment are shown in Figure 1 middle. Figure 1 It was demonstrated that compounds 21, 68 and 78 were effective in inhibiting the activity of ENPP1. The following Table 1 lists the IC 50 value.

[0705] Table 1

[0706] DMSO 21 68 78 <![CDATA[IC 50 ]]> N / A 0.08684 0.1142 0.07967

[0707] Figure 8 Depicted are the results of a similar experiment. 5.0 ng of ENPP1 protein was exposed to increasing doses of inhibitor (compound 78 or compound 107-02) in solution for 10 minutes, followed by the addition of 150 μM of TMP substrate. The assay was then incubated for 20 minutes and then read at 405 nm on the envision plate reader. Both compounds 78 and 107-02 showed a significant reduction in enzyme activity.

[0708] 2. ENPP2 Inhibition Assay

[0709] Materials: Assay buffer: 10 mM CaCl2, 5 mM MgCl2, 50 mM Tris, 0.02% Brij-35, pH 8.5. Substrate: 2 mM di(p-nitrophenyl)phosphate sodium salt (Sigma catalog number N3002). Enzyme: 0.2 ng / μL recombinant human ENPP-2 / Autotaxin Protein (R&D catalog number 5255-EN), PF-3080 in DMSO in a 96-well clear assay plate.

[0710] Methods: Eight-point serial dilutions of drug were prepared in 10x assay buffer, with final assay concentrations starting at 10μM, 3μM, 1μM, 0.3μM...0μM. Dilutions containing DMSO were used as controls. The assay plate was set up as follows, with duplicates for each well: 15μL assay buffer + 10μL ENPP1 inhibitor or DMSO + 50μL substrate + 25μL enzyme. Both enzyme and substrate were added to opposite sides of the well to ensure that there was no interaction until all wells had both components. The plate was then gently centrifuged for 10 seconds and then incubated at 37°C for 45 minutes. The reaction was quantified by measuring absorbance at 405nm using Envision.

[0711] IC 50 Calculation: IC 50 The values ​​were determined using GraphPad Prism 5 software. The data were entered into the software as an XY curve as the percentage of inhibition for each drug concentration. The drug concentration values ​​were logarithmically transformed and nonlinear regression was performed using the "Sigmoidal dose-response (variable slope)" option in GraphPad software to model the data and calculate IC 50 The reported IC 50 Values ​​are drug concentrations that achieve 50% inhibition.

[0712] The results of this experiment are shown in Figure 2A and 2B . Figure 2A and 2B Compounds 21 and 78 and their corresponding sodium salts were ineffective in inhibiting the activity of ENPP2. These results suggest that compounds 21 and 78 are selective in inhibiting ENPP1 but not ENPP2. Figure 2A ,IC 50 The value is 0.0007626 (compound 21), and for Figure 2B IC not calculated 50 value.

[0713] 3. ENPP1 thermal excursion assay

[0714] Materials: Recombinant human ENPP-1 protein (R&D catalog number 6136-EN-010). Assay buffer (1 mM CaCl2, 0.2 mM ZnCl2, 50 mM Tris, pH 9.0) 5000x SYPRO Orange (ThermoFisher catalog number S6651) 384-well PCR plate

[0715] Methods: Each drug was prepared as a 10x solution in assay buffer and SYPRO Orange was diluted to 10x concentration in water. Wells were set up in duplicate in a 384-well PCR plate as follows: 14 μL assay buffer, 2 μL ENPP1 inhibitor or DMSO, 2 μL (0.5 μg) ENPP1 protein. Each well was mixed and incubated on ice for 5 minutes. After incubation, 2 μL of SYPRO Orange was mixed into each well and then gently centrifuged. The protein melting reaction was run using ViiA7 software at a temperature starting from 25°C and increasing to a maximum temperature of 99°C at 0.05°C / second.

[0716] The results of this experiment are shown in Figure 3 middle. Figure 3 Compound 21 was shown to be effective in increasing the stability of recombinant ENPP1 protein. The Tm (midpoint of protein unfolding transition) of ENPP1 was 64.2°C and increased to 67.84°C in the presence of compound 21. These results suggest that compound 21 directly binds to ENPP1 and modulates its activity.

[0717] Figure 7 Depicted are the results of a similar thermal shift binding assay. In a 384-well plate, 1.0 μg of ENPP1 protein was incubated with 100 μM of the corresponding drug for 5 minutes. Each well was exposed to increasing temperatures to determine the unfolding temperature of the protein. As indicated, wells exposed to ENPP1 inhibitors showed increased unfolding temperatures, and therefore increased protein stability. 2'3' cGAMP was used as a positive control because it is a substrate for ENPP1.

[0718] ENPP1 protein melting temperatures are depicted in Table 2 below.

[0719] Table 2

[0720] Compound Tm(℃) ΔTm(℃) NT 60.05 0.00 2'3'cGAMP 61.90 1.84 Compound 78 61.16 1.11 Compound 22 61.37 1.32 Compound 107-02 63.89 3.84 Compound 108 63.60 3.54

[0721] 4. ENPP1 Mineralization Assay

[0722] Materials: Saos-2 cells (ATCC catalog number HTB-85). Culture medium. 24-well tissue culture plates. Ascorbic acid, 5 mg / mL (100X). β-Glycerol phosphate, 1 M (100X). Phosphate buffered saline. Alizarin red, 40 mM pH 4.2. 70% ethanol. Dye extraction reagent, 10% methanol + 10% acetic acid

[0723] Methods: Saos-2 cells were plated at a density of 1.5×106 cells / well and allowed to grow to 100% confluence over the next 1-2 days. Once fully confluent, the culture medium was replaced with fresh culture medium supplemented with 1× ascorbic acid, 1×β-glycerophosphate, and different concentrations of ENPP1 inhibitors (if applicable). Addition of mineralization components was considered on day 1. On day 3, the culture medium was gently aspirated without washing, followed by addition of fresh culture medium, mineralization components, and ENPP1 inhibitors (if applicable). On day 5, the culture medium was gently aspirated and the cells were washed twice with PBS. The cells were then fixed with ice-cold 70% ethanol at 4°C for 1 hour. The cells were washed once with PBS and stained with Alizarin Red for 30 minutes at room temperature with gentle rotation. The stained cells were washed three times with water at room temperature for 10 minutes with gentle rotation. After imaging the cells, dye extraction reagent was added to each well and incubated at room temperature for 30 minutes. Alizarin Red was quantified by measuring absorbance at a wavelength of 405nm using Envision.

[0724] The results of this experiment are shown in Figure 4 Data presented were normalized to DMSO control. Figure 4 Compounds 21 and 68 were shown to be effective in inhibiting the ability of ENPP1 to generate mineralized nodules. These results suggest that inhibition of ENPP1 may alter the pyrophosphate / phosphate ratio, which has been previously reported to regulate matrix mineralization in cells.

[0725] Fig. 9 A picture depicting the results of a similar SAOS-2 mineralization assay. SAOS-2 cells were cultured in the presence and absence of ENPP1 inhibitors (compounds 78, 107-02 or 108) and exposed to mineralization components for 3 days. Wells treated with ENPP1 inhibitors showed a significant reduction in total mineralization.

[0726] 5.THP1 IRF-3 reporter gene assay

[0727] Fig.10 and 11Depicted are the results of a THP1 IRF-3 reporter assay. THP1 WT and STING knockout cells were primed with 2'3' cGAMP and treated with 10 μM ENPP1 inhibitor for 24 h ( Fig.10 ) or 72 hours ( Fig.11 ). The culture medium was collected to determine the induction of the IRF-3 reporter gene using the luminescent reagent QuantiLuc. Compound 107-02 showed a significant increase in the IRF-3 response.

[0728] Fig. 12A is an immunofluorescence image of MDA-MB-231 cells treated with compound 107-02. Immunofluorescence was used to determine the localization of phospho-IRF3 (IFN regulatory factor 3) in MDA-MB-231 cells treated with 10 μM compound 107-02 for 48 hours. Fig. 12B Shown is the activity of an interferon-stimulated reporter gene measured in THP1 bicellular cells (Invivogen, USA) treated with various doses of compound 107-02 for 48 hours.

[0729] Fig. 12C Shown is the expression of IFNβ measured in THP1 bi-cells treated with various doses of compound 107-02 for 48 hours. Fig.12D Shown is the expression of ISG15 measured in THP1 bi-cells treated with different doses of compound 107-02 for 48 hours. *p<0.05.

[0730] The ability of representative disclosed compounds to modulate various biochemical and cellular activities was determined using the ENPP1 and ENPP2 inhibition assays, ENPP1 protein stability assay, and ENPP1 mineral assay described above.

[0731] IC for inhibiting any ENPP1 activity 50 (μM) are shown in Tables 3 and 4.

[0732] If IC 50 or another assay result is indicated as "ND," then not determined in the indicated assay.

[0733] Table 3: 3H-imidazo[4,5-c]pyridine series 1 of ENPP1 inhibitors

[0734]

[0735]

[0736]

[0737]

[0738]

[0739]

[0740] Table 4: 3H-imidazo[4,5-c]pyridine series 1 of ENPP1 inhibitors

[0741]

[0742] NA: no activity; NT: not tested; +: active at 50 μM concentration

[0743] Example 2. Additional experimental data - in vitro biology

[0744] 1. ENPP1 assay (TMP) Materials: Assay buffer: 1 mM CaCl2, 0.2 mM ZnCl2, 50 mM Tris, pH 9.0 Substrate: 1.5 mM thymidine 5'-monophosphate disodium salt hydrate (Sigma: T4510) - assay concentration: 150 μM. Enzyme: 1 ng / μL recombinant human ENPP-1 protein (purified in-house) - assay concentration: 5 ng / well of DMSO in a 96-well clear assay plate.

[0745] Methods: Ten-point serial dilutions of the drug were prepared in 10X assay buffer, with final assay concentrations starting at 10 μM, 3 μM, 1 μM, 0.3 μM ... 0 μM. Dilutions containing DMSO were used as controls. The assay plate was set up as follows, with duplicates for each well: 75 μL assay buffer + 10 μL ENPP1 inhibitor or DMSO dilution + 10 μL substrate + 5 μL enzyme (5 ng). Both enzyme and substrate were added to the opposite side of the well to ensure that there was no interaction until all wells had both components. The plate was then gently centrifuged for 10 seconds and then incubated at 37 ° C for 45 minutes. The reaction was quantified by measuring the absorbance at 405 nm using an Envision plate reader.

[0746] 2. ENPP1 assay (ATP) Materials: Assay buffer: 1 mM CaCl2, 0.2 mM ZnCl2, 50 mM Tris, pH 9.0. Substrate: 100 μM adenosine 5'-triphosphate disodium salt hydrate (Promega: V703A) - Assay concentration: 10 μM. Enzyme: 1 ng / μL recombinant human ENPP-1 protein (purified in-house) - Assay concentration: 5 ng / well DMSO in a 96-well white assay plate. Cell Titer Glo (Promega, catalog number G7572).

[0747] Methods: Ten-point serial dilutions of the drug were prepared in 10X assay buffer, with final assay concentrations starting at 10 μM, 3 μM, 1 μM, 0.3 μM ... 0 μM. Dilutions containing DMSO were used as controls. The assay plate was set up as follows, with duplicates for each well: 75 μL assay buffer + 10 μL ENPP1 inhibitor or DMSO dilution + 10 μL substrate + 5 μL enzyme (5 ng). Both enzyme and substrate were added to opposite sides of the well to ensure that there was no interaction until all wells had both components. The plate was then gently centrifuged for 10 seconds and then incubated at room temperature for 20 minutes. To detect the level of ATP, Cell Titer Glo was added to each well at 1: 1 and incubated for 10 minutes. The reaction was quantified by measuring luminescence using an Envision plate reader.

[0748] The results are shown in Table 5 and Figure 5A and 5B middle.

[0749] Table 5

[0750]

[0751] Figure 5A and 5B To demonstrate, ENPP1 enzyme assays using TMP or ATP as substrates were performed with or without test compounds at various concentrations, and the IC 50 Values ​​were determined using GraphPad Prism.

[0752] IC 50 The values ​​are as follows. Figure 5A :

[0753] 78μM 107μM 108μM 115μM <![CDATA[IC 50 ]]> 0.2714 0.2318 2.045 1.608

[0754] Figure 5B :

[0755] 78μM 107μM 108μM 115μM <![CDATA[IC 50 ]]> 0.07312 0.116 0.3393 0.1906

[0756] 3. ENPP1 thermal excursion assay

[0757] Materials: Recombinant human ENPP-1 protein (purified in-house). Assay buffer (1 mM CaCl2, 0.2 mM ZnCl2, 50 mM Tris, pH 9.0). 5000x SYPRO Orange (Thermo Fisher Scientific Catalog No. S6651). 384-well PCR plate

[0758] Methods: Each drug was prepared as a 10x solution in assay buffer and SYPRO Orange was diluted to 10x concentration in water. Wells were set up in duplicate in a 384-well PCR plate as follows: 14 μL assay buffer, 2 μL ENPP1 inhibitor or DMSO, 2 μL (0.5 μg) ENPP1 protein. Each well was mixed and incubated on ice for 5 minutes. After incubation, 2 μL of SYPRO Orange was mixed into each well and then gently centrifuged. The protein melting reaction was run using ViiA7 software at a temperature starting from 25°C and increasing to a maximum temperature of 99°C at 0.05°C / second.

[0759] The results are shown in Table 6 below.

[0760] Table 6: ENPP1 protein melting temperature

[0761] drug Concentration (μM) Tm(℃) ΔTm(℃) NT 60.05 2'3'cGAMP 100 61.90 1.84 Compound 58 100 63.89 3.84 Compound 59 100 63.60 3.54

[0762] Table 6 shows that the binding affinities of compounds 58 and 59 exhibited thermal shifts of 3.84 and 3.54, respectively, indicative of target binding to ENPP1. This thermal shift assay is confirmatory and supports greater binding of these compounds to the natural 2'3' cGAMP substrate.

[0763] 4. Chou-Talalay synergy study using compound 78 and olaparib

[0764] Materials: Cell lines: MDA-MB-468 and UWB289.1 (ATCC). Cell Titer Glo (Promega catalog number: G7572). 96-well TC-treated white assay plates

[0765] Methods: Cells were cultured according to ATCC tissue culture recommendations. Cells were plated at a density of 1500 cells / well in 96-well tissue culture plates and allowed to settle overnight. The next day, two drugs were prepared at 10X concentrations in the following ratios (compound 22: olaparib): 1:1, 10:1, and 1:10. The drugs were serially diluted to produce an 8-point dose curve, and 10 μL of each drug was added to the cells in a total volume of 100 μL / well. The plates were placed in a tissue culture incubator for 72 hours. After incubation, each plate was treated with Cell Titer Glo according to the supplier's recommendations, and luminescence was measured with an Envision plate reader. Data were analyzed using CalcuSyn software according to the methods described in Chou, Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res. 2010 Jan 15;70(2):440-6.

[0766] The results are shown in Table 7 below.

[0767] Table 7: Chou-Talalay synergy value

[0768] Table 7 shows the synergy study of compound 78 and olaparib in three cancer cell lines representing breast cancer, ovarian cancer and colon cancer. Combination index (CI) values ​​greater than 1 indicate competitiveness, equal to 1 indicate additivity, and less than 1 indicate synergy.

[0769]

[0770] 5. In vivo biology

[0771] Anti-tumor efficacy of compound 22 in the B16F10 (melanoma treatment model) synaptic model of C57BL / 6 mice Experimental design:

[0772] Animals: Female C57BL / 6 (4-6 weeks old from Taconic).

[0773] B16F10 cells were propagated in DMEM cell culture medium containing 10% fetal bovine serum. Cells were harvested during the logarithmic growth phase and implanted subcutaneously (1×106) into the lower abdomen of mice. Tumor growth was monitored daily and after tumors had reached 80 to 100 mm3, mice were randomly divided into groups consisting of 8 animals per group. When tumors reached approximately 2000 mm 3Animals were euthanized upon completion of the treatment regimen or 20% body weight loss or development of ulceration and necrosis. After termination, tumor samples and blood were collected and analyzed for biomarkers.

[0774] Table 8 further illustrates the study design.

[0775] Table 8

[0776]

[0777] ADU-S100 is a cyclic dinucleotide used as a reference compound, which was purchased from Invitrogen, USA, and used in animal model experiments.

[0778] Groups 1 to 7 were female C57BL / 6 mice, wherein each group contained 8 mice, and compound ADU-S100 was used as a reference agent in this study.

[0779] The results of this experiment are shown in Fig. 6A and 6B These figures show in vivo antitumor efficacy studies in the B16F10 syngeneic model in C57BL / 6 mice. Compound 78 was administered by oral (PO), intraperitoneal (IP) or intratumoral (IT) routes. ADU-S100 was administered by the intratumoral route. Fig. 6A Shown is the 28% B16F10 tumor growth inhibition observed with Compound 78 at a 30 mg / kg dose. Figure 6B 27% B16F10 tumor growth inhibition was shown. Synergy was observed between compound 78 and ADU-S100.

[0780] Table 9 illustrates the design of a similar study.

[0781] Table 9

[0782]

[0783] Fig.13A and 13B The results of this study are depicted. Fig.13A It is shown that 28% B16F10 tumor growth inhibition was observed with Compound 22 at a dose of 30 mg / kg. Fig. 13B It is stated that 27% B16F10 tumor growth inhibition was observed with Compound 22. Further, synergy was observed between Compound 22 and ADU-S100.

[0784] Table 10 below illustrates the design of a similar study.

[0785] Table 10

[0786] Group Number of animals Dosage (mg / kg) way Dosage regimen Medium G1 8 - PO Compound 22G22 8 30mg PO QD

[0787] Fig.14 The results of this study are depicted. Using compound 22, 39% CT26 tumor growth inhibition was observed.

[0788] Table 11 below illustrates the pharmacokinetic parameters of Compound 107-02.

[0789] Table 11

[0790]

[0791] Fig.15A It is the pharmacokinetic (PK) plasma concentration graph of compound 107-02. Fig. 15B is the tumor growth inhibition curve of compound 107-02 after in vivo administration. Compound 107-02 inhibited tumor growth in the KPC mouse model of PDAC, and compound 107-02 was administered once daily via IP at a dose of 20 mg / kg. Fig. 15C and 15D is a flow cytometric image of a tumor resected after administration of compound 107-02. At the end of the study, tumors were resected and analyzed for T cells and tumor-associated macrophages using flow cytometry. *p<0.05.

[0792] Prophetic Pharmaceutical Composition Examples

[0793] As used throughout these examples, "active ingredient" refers to a compound of the invention, or one or more of its pharmaceutically acceptable salts, solvates, polymorphs, hydrates, and stereochemically isomeric forms. The following examples of formulations of the compounds of the invention in tablets, suspensions, injectables, and ointments are prophetic.

[0794] Typical examples of formulations of the present invention are as follows. Various other dosage forms can be used herein, such as filled gelatin capsules, liquid emulsions / suspensions, ointments, suppositories or chewable tablets, and the disclosed compounds are used in the desired dosage according to the present invention. Predicted pharmaceutical compositions can be prepared using various conventional techniques for preparing suitable dosage forms, such as those disclosed herein and in standard reference texts, such as British and US Pharmacopoeias, Remington's Pharmaceutical Sciences (Mack Publishing Co.) and Martindale The Extra Pharmacopoeia (Martindale The Extra Pharmacopoeia) (London The Pharmaceutical Press). The disclosure of this reference is hereby incorporated by reference into this document.

[0795] a. Pharmaceutical compositions for oral administration

[0796] Tablets may be prepared as follows:

[0797]

[0798] Alternatively, about 100 mg of the disclosed compound, 50 mg of lactose (monohydrate), 50 mg of corn starch (native), 10 mg of polyvinyl pyrrolidone (PVP 25) (e.g., from BASF, Ludwigshafen, Germany) and 2 mg of magnesium stearate are used per tablet. The mixture of active ingredients, lactose and starch is granulated with a 5% solution (m / m) of PVP in water. After drying, the granules are mixed with magnesium stearate for 5 minutes. This mixture is molded using a conventional tablet press (e.g., tablet form: 8 mm diameter, 12 mm radius of curvature). The applied molding force is typically about 15 kN.

[0799] Alternatively, the disclosed compounds can be administered in the form of a suspension formulated for oral use. For example, about 100-5000 mg of the desired disclosed compound, 1000 mg of ethanol (96%), 400 mg of xanthan gum and 99 g of water are combined under stirring. A single dose of about 10-500 mg of the desired disclosed compound can be provided by 10 ml of an oral suspension.

[0800] In these examples, the active ingredient can be replaced by the same amount of any compound according to the invention, specifically the same amount of any exemplified compound. In some cases, it may be desirable to use capsules, such as filled gelatin capsules instead of tablet form. The choice of tablet or capsule will depend in part on the physicochemical properties of the specific disclosed compound used.

[0801] Examples of alternative useful carriers for preparing oral formulations are lactose, sucrose, starch, talc, magnesium stearate, crystalline cellulose, methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, glycerol, sodium alginate, gum arabic, etc. These alternative carriers may be substituted for those given above as required for the desired solubility, absorption and manufacturing characteristics.

[0802] The amount of the disclosed compound in the pharmaceutical composition for human use per tablet is determined based on both toxicology and pharmacokinetic data obtained in appropriate animal models, such as rats and at least one non-rodent species, and adjusted based on human clinical trial data. For example, it may be appropriate for the disclosed compound to be present at a level of about 10 to 1000 mg per tablet dosage unit.

[0803] b. Pharmaceutical compositions for injectable use

[0804] The parenteral composition may be prepared as follows:

[0805]

[0806] * In the context of the amount of active ingredient and the form of the active ingredient, e.g., a specific salt form of the active ingredient, the amount is adjusted as needed to maintain physiological pH.

[0807] Alternatively, a pharmaceutical composition for intravenous injection may be used, wherein the composition comprises about 100-5000 mg of the disclosed compound, 15 g of polyethylene glycol 400 and 250 g of saline solution, wherein optionally up to about 15% Cremophor EL, and optionally up to 15% ethanol and optionally up to 2 equivalents of a pharmaceutically suitable acid (such as citric acid or hydrochloric acid) are used. The preparation of such an injectable composition may be accomplished as follows: the disclosed compound and polyethylene glycol 400 are dissolved in water under stirring. The solution is sterile filtered (pore size 0.22 μm) and filled into a heat-sterilized infusion bottle under aseptic conditions. The infusion bottle is sealed with a rubber seal.

[0808] In another example, a pharmaceutical composition for intravenous injection can be used, wherein the composition includes about 10-500 mg of the disclosed compound, a standard saline solution, optionally with up to 15% by weight of Cremophor EL, and optionally up to 15% by weight of ethanol and optionally up to 2 equivalents of a pharmaceutically suitable acid, such as citric acid or hydrochloric acid. The preparation can be completed as follows: the desired disclosed compound is dissolved in a saline solution under stirring. Cremophor EL, ethanol or acid is optionally added. The solution is sterile filtered (pore size 0.22 μm) and filled into a heat-sterilized infusion bottle under aseptic conditions. The infusion bottle is sealed with a rubber seal.

[0809] In this example, the active ingredient may be replaced by the same amount of any compound according to the present invention, specifically the same amount of any exemplified compound.

[0810] The amount of the disclosed compound in each ampoule of the pharmaceutical composition for human use is determined based on both toxicological and pharmacokinetic data obtained in appropriate animal models, such as rats and at least one non-rodent species, and adjusted based on human clinical trial data. For example, it may be appropriate for the disclosed compound to be present at a level of about 10 to 1000 mg per tablet dosage unit.

[0811] Suitable carriers for parenteral preparations are, for example, water, physiological saline solution, etc., which may be used together with tris(hydroxymethyl)aminomethane, sodium carbonate, sodium hydroxide, etc. as solubilizers or pH adjusters. Parenteral preparations preferably contain 50 to 1000 mg of the disclosed compound per dosage unit.

[0812] It will be apparent to those skilled in the art that various modifications and variations may be made in the present invention without departing from the scope or spirit of the present invention. Other embodiments of the present invention will be apparent to those skilled in the art by considering the specification and practice of the present invention disclosed herein. The specification and examples are intended to be considered exemplary only, and the true scope and spirit of the present invention are indicated by the appended claims.

Claims

1. A compound selected from the group consisting of: and pharmaceutically acceptable salts thereof.

2. A pharmaceutical composition comprising a therapeutically effective amount of the compound according to claim 1 and a pharmaceutically acceptable carrier.

3. Use of a compound according to claim 1 for the preparation of a medicament for treating a disorder of uncontrolled cell proliferation in a mammal.

4. Use of the compound according to claim 1 for preparing a medicament for treating cancer in a mammal.

5. Use of the compound according to claim 1 for preparing a medicament for treating bacterial infection or viral infection in mammals.

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