Diaminotriazole derivatives as janus kinase 2 inhibitors and uses thereof

Abstract

There are provided compounds that may act as Janus kinase 2 (JAK2) inhibitors, compositions comprising these compounds and their use in treating cancers such as myeloproliferative neoplasm (MPN). In particular, the compounds are diaminotriazole derivatives capable of binding to the pseudokinase domain (PK or JH2 domain) and to the gain-of-function V617F mutation in the pseudokinase domain of JAK2 that is prevalent in patients with MPN.

Description

[0001] Atty. Dckt. No. DFCI 3463.W01WO

[0002] DIAMINOTRIAZOLE DERIVATIVES AS JANUS KINASE 2 INHIBITORS AND USES THEREOF

[0003] Federally Sponsored Research and Development

[0004]

[0001] This invention was made with government support under R35 CA242461 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0005] Field

[0006]

[0002] There are provided compounds that may act as Janus kinase 2 (JAK2) inhibitors, compositions comprising these compounds and their use in treating cancers such as myeloproliferative neoplasm (MPN). In particular, the compounds are diaminotriazole derivatives capable of binding to the pseudokinase domain (PK or JH2 domain) and to the gain- of-function V617F mutation in the pseudokinase domain of JAK2 that is prevalent in patients with MPN.

[0007] Background

[0008]

[0003] Janus kinase (JAK) is a family of non-receptor tyrosine kinases (JAK1, JAK2, JAK3, and TYK2) that associate with the cytoplasmic tail of cytokine receptors and are involved in signaling networks that regulate and maintain hematopoiesis and the immune responses (Nair et al., Blood Cancer Discov. 2023; 4:352-64). JAKs present four functional domains: band

[0009] 4.1 / ezrin / radixin / moesin (FERM) domain, Src Homology 2-like (SH2-like) domain, and two ATP binding domains, the kinase domain (JH1; JAK2K) and pseudokinase domain (JH2; JAK2PK). The active kinase domain phosphorylates intracellular transcription factors, and the pseudokinase domain negatively regulates the kinase domain’s activity (Baker et al., Oncogene 2007; 26:6724-6737). This tandem (pseudo-)kinase structure is particularly unique among protein kinases.

[0010]

[0004] Mutations in JAK2 are associated with a wide range of human diseases, including cancers such as hematological malignancies (Bader and Meyer, Pharmaceuticals 2022, 15: 160). The gain-of-function V617F mutation in the pseudokinase domain of JAK2 is observed in 70% of patients with myeloproliferative neoplasm (MPN). JAK2 inhibitors are being developed for the Atty. Dckt. No. DFCI 3463.W01WO treatment of MPN, however, all FDA-approved JAK2 inhibitors for MPN are non-selective ATP- competitive inhibitors, which lead to severe side effects. Thus, there is a need for JAK2 pseudokinase selective inhibitor, particularly for the V617F mutant.

[0011]

[0005] The development of V617F-selective inhibitors is uniquely challenging because the V617F mutation lies in the pseudokinase domain rather than the kinase domain. Thus, it is likely to require an allosteric approach. One promising target for allosteric inhibitors is the ATP-site of the pseudokinase domain, the TYK2 inhibitor deucravacitinib (BMS-986165) is a precedent for inhibition of a JAK-family kinase via this route and was the first pseudokinase-directed therapeutic FDA-approved for the treatment of autoimmune diseases.

[0012]

[0006] Kinase inhibitor JNJ-7706621 has been shown to bind the pseudokinase domain of JAK2 but also binds the kinase domain (Puleo et al. ACS Med. Chem. Lett. 2017, 8:618-621). More selective inhibitors of the pseudokinase domain of JAK2 and the V617F mutant have been developed (Liosi et al. J. Med. Chem. 2020, 63:5324-5340; Liosi et al. J. Med. Chem. 2022, 65:8380-8400; US 2022 / 0112166 Al and WO 2023 / 064458 Al). Covalent inhibitors of the pseudokinase domain of JAK2 have also been developed by including an “acrylamide warhead” to modify amino acid cysteine 675 (Cys675) of the JAK2 pseudokinase domain (Henry et al., ACS Med. Chem. Lett. 2022, 13: 1819-1826), but their selectivity for the pseudokinase domain was lower and their affinity for the V617F mutant was not described. Therefore, there is a need for selective inhibitors of the pseudokinase domain of JAK2 that can covalently bind the pseudokinase domain even when the V617F mutation is present.

[0013]

[0007] It is known that cancers can become resistant to covalent inhibitors by acquiring mutations at the modification site of the protein, as is seen for the Bruton’s Tyrosine Kinase inhibitor Ibrutinib (Buhimschi et al. Biochemistry 2018, 57:3564-3575) where a PROteolysis TArgeting Chimera (PROTAC) approach was employed to overcome resistance. Therefore, there is a need for inhibitors of the pseudokinase domain of JAK2 that work via different mechanisms or target different residues within the pseudokinase domain of JAK2.

[0014] Summary

[0015]

[0008] Disclosed herein are compounds of formula (I) that inhibit the pseudokinase domain of JAK2. It has been discovered that by including an acrylamide warhead at specific positions and Atty. Dckt. No. DFCI 3463.W01WO with certain linkers, it is possible to modulate the potency and selectivity of the compound within the pseudokinase domain of JAK2. One compound (17) with high affinity and selectivity for the JAK2 pseudokinase domain, was surprisingly shown to result in low levels of cysteine labelling (Table 2). Crystallographic studies confirmed that compound 17 does not form a covalent interaction with Cys675 of the JAK2 pseudokinase domain (data not shown). Mass spectrometry confirmed low cysteine labelling (Table 2). This prompted further studies to identify different warhead groups that might target other residues in the same binding cleft. Specific lysine residues that can be covalently targeted were identified (Table 2). A new class of compounds with sulfonyl fluoride, sulfurofluoridates or aldehyde moieties was produced. Covalent binding of one compound (24) was confirmed by crystallography (Figure 1).

[0016]

[0009] These compounds may provide alternative therapeutic options for cancers, such as MPNs, that become resistant to current treatments. Compounds that covalently target JAK2 pseudokinase domain may improve efficiency, lower the dosage, increase compliance and / or reduce the possibility of drug resistance (Sutanto et al. RSC Med. Chem., 2020, 11:876-84).

[0017]

[0010] In a first aspect, there is provided a compound of formula (I), or a pharmaceutically acceptable salt thereof: wherein:

[0018] Ri is -SO2NH2, -C(=O)NH(CH3) or -C(=O)N(CH3)2; one of R2 or R3 is H and the other is: m is 0 or 1;

[0019] Xi and X2 are each independently selected from the group consisting of F, Cl, and H; wherein one of X3or X4 is H and the other is selected from the group consisting of: Atty. Dckt. No. DFCI 3463.W01WO wherein n is 0, 1, 2 or 3; wherein p is 0 or 1, preferably 1; or wherein X3 and X4 join together so that R2 or R3 is: with the proviso that the following compounds are excluded:

[0020] Atty. Dckt. No. DFCI 3463.W01WO

[0021] [Oi l] The excluded compounds depicted above are disclosed in Henry et al., ACS Med. Chem. Lett. 2022, 13: 1819-1826 as 7b, 7c, 7e and 7f. These compounds can be excluded under the proviso that Ri is not -CONHCH3 (i.e., Ri is -SO2NH2, or -CON(CH3)2) and / or under the proviso that Xi and / or X2 is not F (i.e., Xi and / or X2 are H) and / or under the proviso that R2 is H and / or under the proviso that m is not 0 (i.e., m is 1).

[0022]

[0012] In a second aspect, there is provided a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein:

[0023] Ri is -SO2NH2, -CONHCH3 or -CON(CH3)2;

[0024] R2 is

[0025] R3 is H; Atty. Dckt. No. DFCI 3463.W01WO m is 1 or 0;

[0026] A is a 5- or 6-membered aryl or heteroaryl ring; n is 0, 1, 2 or 3; each Y is independently a halide;

[0027] Z is selected from the group consisting of -SO2F, -OSO2F or -CHO.

[0028]

[0013] In a third aspect, there is provided a pharmaceutical composition comprising a compound of the first or second aspect and one or more pharmaceutically acceptable carrier.

[0029]

[0014] In a fourth aspect, there is provided a compound according to any one of the first or second aspects, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to the third aspect, for use in therapy.

[0030]

[0015] In a fifth aspect, there is provided a method of treating a disease treatable by inhibiting Janus kinase 2 comprising administration to a patient in need thereof a therapeutically effective amount of a compound according to any one of the first or second aspects, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to the third aspect.

[0031]

[0016] In a sixth aspect, there is provided a compound according to any one of the first or second aspects, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to the third aspect, for use in a method of treating a disease treatable by inhibiting Janus kinase 2.

[0032]

[0017] In a seventh aspect, there is provided a use of a compound according to any one of the first or second aspects, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in a method of treating a disease treatable by inhibiting Janus kinase 2.

[0033]

[0018] The disease in the fifth, sixth and seventh aspects may be cancer, such as a myeloproliferative neoplasm (MPN).

[0034] Brief Description of The Drawings

[0035]

[0019] Figure 1: Crystal structure of JAK2 pseudokinase domain wild type reacted with sulfurofluoridate Compound 24, confirming the covalent interaction between the compound and lysine 677. Atty. Dckt. No. DFCI 3463.W01WO

[0036] Detailed Description

[0037] Definitions

[0038]

[0020] The term “myeloproliferative neoplasm”, “myeloid neoplasms” or “MPNs” are blood cancer where excess blood cells are produced in the bone marrow. The WHO has characterized seven types of MPN: “chronic myeloid leukemia” (CML), “chronic neutrophilic leukemia” (CNL), “polycythemia vera” (PV), “primary myelofibrosis” (PMF; e g., the prefibrotic stage or the overt fibrotic stage), “essential thrombocythemia” (ET), “chronic eosinophilic leukemia (not otherwise specified)”, and “unclassifiable myeloproliferative neoplasm” (MPN-U) (Arber et al. 2016 Blood 127:2391-2405).

[0039]

[0021] As used herein, the term “disease associated with a V617F mutation in the Janus kinase 2 pseudokinase domain” means a disease, preferably a cancer, in which a Valine (V) to Phenylalanine (F) somatic mutation at amino acid 617 of JAK2 is present in the affected cells, preferably blood cells. In particular, in MPNs, mutations in JAK2 are known to be driver mutations.

[0040]

[0022] As used herein, the term "treating" (or "treat" or "treatment") refers to restraining, slowing, or reversing the progression or severity of an existing symptom, condition, or disorder, in a patient in need thereof. Treating may be carried out by an attending physician or caregiver, prescribing compounds of formula (I) for administration and thereby causing the application of the compound to a subject, through ingestion, infusion, injection, or any other means, whether self-administered or administered by a clinician or other qualified provider.

[0041]

[0023] As used herein, the phrase "effective amount" means an amount of a compound of formula (I) that is sufficient to inhibit, antagonize, or modulate JAK2 to achieve the objectives of the disclosure, in particular to treat in one or more doses a cancer herein described.

[0042]

[0024] As used herein, the term "patient" refers to a mammal, preferably human to whom treatment is administered.

[0043]

[0025] As used herein, the phrase "co-administering" means the administration of a compound of formula (I) and another compound described herein, separately, simultaneously, and / or sequentially over a period of time as determined by a drug label and attending physician or healthcare provider. Additionally, “co-administering” may include the administration of one or Atty. Dckt. No. DFCI 3463.W01WO more additional therapies, for example, radiation, surgery, bone marrow transplantation, chemotherapy, immunotherapy, hormone therapy, or targeted therapy.

[0044] Compounds of Formula (I)

[0045] Diaminotriazole derivatives with acrylamide moieties or reversible analogues thereof

[0046]

[0026] In a first aspect, there is provided a compound of formula (I), or a pharmaceutically acceptable salt thereof: wherein Ri is -SO2NH2, -C(=O)NH(CH3) or -C(=O)N(CH3)2; one of R2 or R3 is H and the other is: m is 0 or 1; Xi and X2 are each independently selected from the group consisting of F, Cl, and H; wherein one of X3 or X4 is H and the other is selected from the group consisting of: Atty. Dckt. No. DFCI 3463.W01WO wherein n is 0, 1, 2 or 3; wherein p is 0 or 1, preferably 1; or wherein X3 and X4 join together so that R2 or R3 is: with the proviso that the following compounds are excluded:

[0047]

[0027] The compounds of the first aspect were designed to bind the JAK2 pseudo kinase domain, for example, covalently bind to a cysteine residue within the JAK2 pseudo kinase domain allosteric site (or to be high affinity tool compounds related thereto, e.g., when X3 or X4 is -NH2 or the acrylamide is replaced with ethylamide). Atty. Dckt. No. DFCI 3463.W01WO

[0048]

[0028] In a preferred embodiment of the first aspect, the compound is a compound of formula (1), or a pharmaceutically acceptable salt thereof, wherein one of X3 or X4 is H and the other is selected from the group consisting of or X3 and X4 join together so that one of R2 or R3 is:

[0049]

[0029] In a specific preferred embodiment of the first aspect, the compound is a compound of formula (I), or a pharmaceutically acceptable salt thereof wherein one of X3 or X4 is H and the other is selected from the group consisting of Atty. Dckt. No. DFCI 3463.W01WO preferably more preferably wherein Ri is -SO2NH2; and / or more preferably wherein each of Xi and X2 is F; and / or more preferably wherein m is 0.

[0050]

[0030] In another specific preferred embodiment of the first aspect, the compound is a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein X3 and X4 join together so that R2 or R3 is: more preferably wherein each of Xi and X2 H; and / or more preferably wherein Ri is - C(=O)NH(CH3) or -C(=O)N(CH3)2; and / or more preferably wherein m is 0.

[0051]

[0031] The compound of formula (I), or a pharmaceutically acceptable salt thereof, may form a structure wherein R2 is H and R3 is: so that the compound of formula (I) forms a compound of formula (II): Atty. Dckt. No. DFCI 3463.W01WO

[0052]

[0032] Preferably, the compound is a compound of formula (II), or a pharmaceutically acceptable salt thereof, wherein one of X3 or X4 is H and the other is selected from the group consisting of:

[0053]

[0033] More preferably the compound is a compound of formula (II), or a pharmaceutically acceptable salt thereof, wherein one of X3 or X4 is H and the other is selected from the group consisting of:

[0054]

[0034] Alternatively, the compound is a compound of formula (II), or a pharmaceutically acceptable salt thereof, wherein X3 and X4 join together so that R3 is: Atty. Dckt. No. DFCI 3463.W01WO more preferably wherein m is 0.

[0055]

[0035] The compound of formula (I), or a pharmaceutically acceptable salt thereof, may form a structure wherein R3 is H and R2 is: so that the compound of formula (I) forms a compound of formula (III):

[0056]

[0036] In a preferred embodiment of the first aspect, the compound is a compound of formula (III), or a pharmaceutically acceptable salt thereof, wherein one of X3 or X4 is H and the other is selected from the group consisting of: Atty. Dckt. No. DFCI 3463.W01WO or wherein X3 and X4 join together so that R2 is:

[0057]

[0037] In a specific preferred embodiment of the first aspect, the compound is a compound of formula (III), or a pharmaceutically acceptable salt thereof, wherein one of X3 or X4 is H and the other is selected from the group consisting of: more preferably selected from the group consisting of: Atty. Dckt. No. DFCI 3463.W01WO wherein more preferably Ri is -SO2NH2; and / or more preferably wherein each of Xi and X2 is F; and / or more preferably wherein m is 0.

[0058]

[0038] In another specific preferred embodiment of the first aspect, the compound is a compound of formula (III), or a pharmaceutically acceptable salt thereof, wherein X3 and X4 join together so that R2 is: more preferably wherein each of Xi and X2 H; and / or more preferably wherein Ri is - C(=O)NH(CH3) or -C(=O)N(CH3)2; and / or more preferably wherein m is 0.

[0059]

[0039] In certain embodiments of the first aspect, m is 0.

[0060]

[0040] In certain embodiments of the first aspect, m is 1.

[0061]

[0041] In certain embodiments of the first aspect, the compound of formula (I) forms a compound of formula (la), (lb) or (Ic): Atty. Dckt. No. DFCI 3463.W01WO

[0062]

[0042] In a preferred embodiment of the first aspect, Ri is -SO2NH2 so that the compound of formula (I) forms a compound of formula (la): wherein Ri is -SO2NH2.

[0063]

[0043] In a more preferred embodiment of the first aspect, the compound of formula (I) forms a compound of formula (III) and Ri is -SO2NH2 so that the compound of formula (III) forms a compound of formula (Illa):

[0064]

[0044] In an even more preferred embodiment of the first aspect, the compound of formula (I) forms a compound of formula (Illa), wherein X3 is selected from the group consisting of: more preferably X3 is selected from the group consisting of: even more preferably X3 is selected from the group consisting of: Atty. Dckt. No. DFCI 3463.W01WO wherein X4 is H; and preferably wherein each of Xi and X2 is F.

[0065]

[0045] In certain embodiments of the first aspect, the compound is a compound of formula (I), or a pharmaceutically acceptable salt thereof, Ri is C(=O)NH(CH3) or -C(=O)N(CH3)2.

[0066]

[0046] Preferred compounds of the first aspect are that were designed to covalently bind cysteine (or analogues thereof) are: Atty. Dckt. No. DFCI 3463.W01WO Atty. Dckt. No. DFCI 3463.W01WO

[0067] Diaminotriazole derivatives with other moieties including sulfonyl fluorides, sulfurofluoridates and aldehydes

[0068]

[0047] In a second aspect, there is provided a compound of formula (I), or a pharmaceutically acceptable salt thereof: wherein:

[0069] Ri is -SO2NH2, -CONHCH3 or -CON(CH3)2;

[0070] R2 is

[0071] R3 is H; m is 1 or 0;

[0072] A is a 5- or 6-membered aryl or heteroaryl ring; n is 0, 1, or 2; each Y is independently a halide, preferably Cl or F, more preferably F;

[0073] Z is selected from the group consisting of -SO2F, -OSO2F or -CHO. Atty. Dckt. No. DFCI 3463.W01WO

[0074]

[0048] The compounds of the second aspect were designed to bind the JAK2 pseudo kinase domain, for example, covalently bind a lysine residue within the JAK2 pseudo kinase domain allosteric site.

[0075]

[0049] Suitable examples for Ring A include phenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrrolyl, pyrazolyl or thiophenyl. Preferred examples include phenyl and pyridinyl.

[0050] In a preferred embodiment of the second aspect, the compound is a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein R2 is: wherein Yi, Y2 and Y3 are independently H or a halide (e.g., Cl or F), preferably H or F.

[0076]

[0051] In a specific preferred embodiment of the second aspect, the compound is a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein R2 is: so that the compound of formula (I) forms a compound of formula (IV): Atty. Dckt. No. DFCI 3463.W01WO

[0077]

[0052] In a preferred embodiment of the second aspect, the compound is a compound of formula (IV), or a pharmaceutically acceptable salt thereof, wherein Z is -SO2F or -OSO2F; Yi is H or F; Y2 is H or F; and / or Y3 is H or F, preferably H.

[0078]

[0053] In another preferred embodiment of the second aspect, the compound is a compound of formula (IV), or a pharmaceutically acceptable salt thereof, wherein Z is -CHO; Yi is F; Y2 is H; and / or Y3 is F.

[0079]

[0054] In a more preferred embodiment of the second aspect, the compound of formula (I) forms a compound of formula (IV) and Ri is -SO2NH2 so that the compound of formula (IV) forms a compound of formula (IVa):

[0080]

[0055] In a specific embodiment of the second aspect, the compound is a compound of formula

[0081] (I), or a pharmaceutically acceptable salt thereof, wherein R2 is: so that the compound of formula (I) forms a compound of formula (V): Atty. Dckt. No. DFCI 3463.W01WO

[0082]

[0056] In a preferred embodiment of the second aspect, the compound is a compound of formula (V), or a pharmaceutically acceptable salt thereof, wherein Z is -OSO2F; Yi is H; Y2 is H; and / or Y3is H.

[0083]

[0057] In certain embodiments of the second aspect, m is 1.

[0084]

[0058] In certain embodiments of the second aspect, m is 0.

[0085]

[0059] Preferred compounds of the second aspect that were designed to covalently bind lysine are: Atty. Dckt. No. DFCI 3463.W01WO

[0086]

[0060] Particularly preferred compounds of the second aspect are:

[0087]

[0061] It should be understood that the compounds described herein, unless otherwise specified, encompass and include all isomers and stable isotopic variants of the compounds of the present disclosure, such as deuterated compounds. All isotopic variants of the compounds provided herein, whether radioactive or not, should be included within the scope of the present disclosure.

[0088] Pharmaceutically acceptable salts of the compounds of Formula (I)

[0089]

[0062] The compounds of the present disclosure may react with any of a number of inorganic and organic acids to form pharmaceutically acceptable acid addition salts. Pharmaceutically acceptable salts and common methodology for preparing them are well known in the art. See, e.g., P. Stahl, et al. Handbook of Pharmaceutical Salts: Properties, Selection and Use, (VCHA / Wiley-VCH, 2002); S. M. Berge, et al., "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, Vol. 66, No. 1, January 1977.

[0090] Pharmaceutical formulations of compounds of Formula (I) Atty. Dckt. No. DFCI 3463.W01WO

[0091]

[0063] The compounds of the present disclosure are preferably formulated as pharmaceutical compositions administered by a variety of routes. Such pharmaceutical compositions and processes for preparing same are well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy (A. Gennaro, et. al., eds., 22nd ed., Loyd V., ed., Pharmaceutical Press, 2012). Any preference for specific compounds applies equally to their pharmaceutical formulations.

[0092] Compounds of Formula (I) for treating diseases

[0093]

[0064] The compounds of formula (I), or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, may be used in methods of treating a disease treatable by inhibiting Janus kinase 2 comprising administration to a patient in need thereof a therapeutically effective amount of a compound of formula (I). Any preference for specific compounds applies equally to their use in treatment.

[0094]

[0065] The compounds of formula (I), or a pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, may be for use in therapy, preferably for use in a method of treating a disease treatable by inhibiting Janus kinase 2.

[0095]

[0066] Provided is the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in a method of treating a disease treatable by inhibiting Janus kinase 2.

[0096]

[0067] Preferably, the disease may be associated with a V617F mutation in the Janus kinase 2 pseudokinase domain.

[0097]

[0068] Preferably, the disease may be cancer, more preferably a myeloproliferative neoplasm (MPN). Preferred MPNs include may be selected from the group consisting of chronic myeloid leukemia (CML), chronic neutrophilic leukemia (CNL), polycythemia vera (PV), primary myelofibrosis (PMF; e.g., the prefibrotic stage or the overt fibrotic stage), essential thrombocythemia (ET), chronic eosinophilic leukemia (not otherwise specified), and unclassifiable myeloproliferative neoplasm (MPN-U).

[0098] Dosages of compounds of Formula (I)

[0099]

[0069] The compounds of the present disclosure are effective over a dosage range. It will be understood that the amount of the compound administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of Atty. Dckt. No. DFCI 3463.W01WO administration, the selected compound or compounds administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms. See, for example, Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 10th Edition, A. Gilman, J. Hardman and L. Limbird, eds., McGraw-Hill Press, 155-173, 2001.

[0100]

[0070] A unit dose of 1 to 1000 mg / day may be administered in one or more, preferably one or two daily doses. In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect. The dosage appropriate for a given patient will be determined by the tending healthcare provider and informed by clinical experience.

[0101] Preparations and Examples

[0102]

[0071] The following Preparations and Examples further illustrate the disclosure and represent typical synthesis of the compound of formula (I). The reagents and starting materials are readily available or may be readily synthesized by one of ordinary skill in the art. It should be understood that the Preparations and Examples are set forth by way of illustration and not limitation, and that various modifications may be made by one of ordinary skill in the art.

[0103] Scheme 1:

[0104]

[0072] Phenyl V'-cyano- \-(4-sulfamoylphenyl)c;irb;imimidate (3): 1 (4.0 g, 23.2 mmol) and

[0105] 2 (5.5 g, 23.2 mmol) were added to anhydrous THF (25 mL). The solution was heated to reflux and stirred for 16 hours. The crude mixture was then concentrated in vacuo to remove remaining solvent and resuspended in DCM (50 mL) and filtered. The remaining solid was dried under N2 for 10 minutes, then under vacuum for 16 hours to give the desired compound as a white solid (6.6 g, 90% yield), m / z ESI expected: 316.34, observed (M+H+): 317.18;XH NMR (500 MHz, DM SO-6 / 6) 8 11.13 (s, 1H), 7.86 - 7.83 (m, 2H), 7.69 - 7.65 (m, 2H), 7.49 - 7.45 (m, 2H), 7.38 (s, 2H), 7.35 - 7.30 (m, 3H). Atty. Dckt. No. DFCI 3463.W01WO

[0106]

[0073] 4-((5-amino-LH-l,2,4-triazol-3-yl)amino)benzenesulfonamide (4): 3 (6.6 g, 21 mmol) was added to THF (50 mL) and cooled to 0 °C. Hydrazine monohydrate (1.5 mL, 31 mmol) was added dropwise. The reaction vessel was allowed to warm to room temp, heated to reflux, and stirred for 16 hours. The reaction mixture was filtered, and the remaining solid was dried under N2 for 1 hour, then under vacuum for 16 hours to give the desired compound as a while solid (3.8 g, 72% yield), m / z ESI expected: 254.27, observed (M+H+): 255.04. ’H NMR (500 MHz, DMSO-t / 6) 8 11.30 (s, 1H), 9.18 (s, 1H), 7.64-7.57 (m, 4H), 7.04 (s, 2H), 5.95 (s, 2H).

[0107]

[0074] Phenyl-7V'-cyano- / V-(4-(methylcarbamoyl)phenyl)carbamimidate (6): 5 (3.0 g, 20 mmol) and (2) (4.76 g, 20 mmol) were added to anhydrous THF (25 mL). The solution was heated to reflux and stirred for 16 hours. The crude mixture was then concentrated in vacuo to remove remaining solvent and resuspended in DCM (50 mL), filtered, and washed with DCM (2 x 100 mL ). The remaining solid was dried under Nzfor 30 minutes, then under vacuum for 72 hours to give the desired compound as a white solid (4.7 g, 80% yield), m / z ESI expected: 294.31, observed (M+H+): 294.88.JH NMR (500 MHz, DMSO-c / 6) 6 11.02 (s, 1H), 8.43 (q, J = 5 Hz, 1H), 7.85 (d, J = 9 Hz, 2H), 7.56 (d, J= 9 Hz, 2H), 7.50-7.43 (m, 2H), 7.36-7.28 (m, 3H), 2.78 (d, J = 5 Hz, 3H).

[0108]

[0075] 4-((5-amino-LH-l,2,4-triazol-3-yl)amino)- / V-methylbenzaimde (7): 6 (4.7 g, 16 mmol) was added to THF (65 mL) and cooled to 0 °C. Hydrazine monohydrate (1.2 mL, 24 mmol) was added dropwise. The reaction vessel was allowed to warm to room temp, heated to reflux, and stirred for 16 hours. The reaction mixture was filtered, and the remaining solid was dried under N2 for 1 hour, then under vacuum for 16 hours to give the desired compound as a while solid (2.8 g, 76% yield), m / z ESI expected: 232.25, observed (M+H+): 232.95. 'HNMR (500 MHz, DMSO-t / 6) 8 11.23 (s, 1H), 8.99 (s, 1H), 8.0 (q, .7= 5 Hz, 1H), 7.67 (d, .7= 9 Hz, 2H), 7.51 (d, J = 9 Hz, 2H), 5.91 (s, 2H), 2.75 (d, J= 5 Hz, 3H).

[0109]

[0076] Phenyl (Z)-JV'-cyano- / V-(4-(dimethylcarbamoyl)phenyl)carbamiinidate (9): 8 (3.284 g, 20 mmol) and (2) (4.76 g, 20 mmol) were added to anhydrous THF (25 mL). The solution was heated to reflux and stirred for 16 hours. The crude mixture was then concentrated in vacuo to remove remaining solvent and resuspended in DCM (50 mL), filtered, and washed with DCM (2 x 100 mL). The remaining solid was dried under N2 for 30 minutes, then under vacuum for 72 hours to give the desired compound as a pale yellow solid (6.0 g, 97% yield), m / z ESI expected: 308.12, observed (M+H+): 308.91. ' H NMR (500 MHz, DMSO-c / 6) 8 10.99 (s, 1H), 7.55 (d, J = Atty. Dckt. No. DFCI 3463.W01WO

[0110] 8.5 Hz, 1H), 7.47 - 7.44 (m, 2H), 7.32 (d, J= 8.6 Hz, 2H), 7.18 - 7.13 (m, 2H), 6.77 - 6.75 (m, 2H), 2.94 (s, 6H).

[0111]

[0077] 4-((5-amino-l / / -l,2,4-triazol-3-yl)amino)-AfZV-dimethylbenzamide (10): 9 (6 g, 19.5 mmol) was added to THF (65 mL) and cooled to 0 °C. Hydrazine monohydrate (1.5 mL, 30 mmol) was added dropwise. The reaction vessel was allowed to warm to room temp, heated to reflux, and stirred for 16 hours. The reaction mixture was filtered, and the remaining solid was dried under N2 for 1 hour, then under vacuum for 16 hours to give the desired compound as a while solid (4.42 g, 92% yield), m / z ESI expected: 246.12, observed (M+H+): 246.95. 'll NMR (500 MHz, DMSO-r / 6) 5 11.21 (s, 1H), 8.92 (s, 1H), 7.52 (d, J= 8.7 Hz, 2H), 7.27 (d, J= 8.7 Hz, 2H), 5.90 (s, 2H), 2.96 (s, 6H).

[0112] Scheme 2;

[0113]

[0078] 3-(l-(toT-butoxycarbonyl)-l,2,3,6-tetrahydropyridin-4-yl)-2,6-difluorobenzoic acid (13): 11 (100. mg, 0.422 mmol) and 12 (140 mg, 0.46 mmol) were added to a solution of dioxane (4 mL) and water (2 mL). 2M Na2CCh solution (1.05 mL, 2.11 mmol) was added and the solution degassed in a sonicator for 2 minutes. Pd(dppf)C12 (19 mg, 0.025 mmol) and / -BuXPhos (14 mg, 0.034 mmol) were added and sealed under N2. The reaction was stirred at 95 °C for 1 hour. The crude reaction mixture was purified by reverse phase HPLC to yield the desired compound (80 mg, 56% yield), m / z ESI expected: 339.34, observed (M+H+): 339.71.

[0114]

[0079] 3-(l-( rZ-butoxycarbonyl)piperidin-4-yl)-2,6-difluorobenzoic acid (14): 13 (80 mg, 0.235 mmol) was solubilized in methanol (5 mL). 10% Pd / C (125 mg, 0.118 mmol) was added to the solution, and the reaction vessel was purged of air and filled with H2 gas. The solution was stirred at 25 °C for 16 hours. The reaction mixture was filtered through a pad of celite and Atty. Dckt. No. DFCI 3463.W01WO concentrated in vacuo to yield the desired compound (70 mg, 87% yield), m / z ESI expected: 341.35, observed (M+H+): 342.19.

[0115]

[0080] toY-butyl 4-(3-(5-amino-3-((4-sulfamoylphenyl)amino)-177-l,2,4-triazole-l- carbonyl)-2,4-difluorophenyl)piperidine-l-carboxylate (15): 14 (70 mg, 0.21 mmol) and 4 4 (52 mg, 0.21 mmol) were solubilized in THF (5 m ). A, / V’-diisopropylcarbodiimide (0.096 m , 0.62 mmol), HOBt monohydrate (69 mg, 0.41 mmol), and DIEA (0.179 mL, 1.03 mmol) were added to the solution and stirred at 25 °C for 16 hours. The reaction was quenched with water (50 mL) and diluted with ethyl acetate (50 mL). The aqueous layer was further extracted with ethyl acetate (2 x 100 mL), and the organic layers were washed with brine, dried with MgSCh, and concentrated in vacuo. The remaining residue was purified by reverse phase HPLC to yield the desired compound (47 mg, 40% yield), m / z ESI expected: 577.60, observed (M+H+): 578.24.

[0081] 4-((l-(3-(l-acryloylpiperidin-4-yl)-2,6-difluorobenzoyl)-5-amino-l / / -l,2,4-triazol-3- yl)amino)benzenesulfonamide (16): 15 (47 mg, 0.081 mmol) was solubilized in DCM (3 mL). TFA (0.3 mL) was added to the DCM to create a 10% TFA solution by volume. The solution was stirred at 25 °C for 30 minutes. The reaction mixture was concentrated in vacuo and dried under vacuum for 1 hour. The dried mixture was then solubilized in THF (1.5 mL) and saturated NaHCCh solution (1 mL). Acryloyl chloride (7.9 / / L, 0.098 mmol) was added, and the reaction mixture was stirred at 25 °C for 1.5 hours. The reaction was quenched with water (25 mL) and diluted with ethyl acetate (50 mL). The aqueous layer was further extracted with ethyl acetate (2 x 100 mL), and the organic layers were washed with brine, dried with MgSCU, and concentrated in vacuo. The remaining residue was purified by reverse phase HPLC to yield the desired compound as a white powder (23 mg, 52% yield), m / z ESI expected: 531.54, observed (M+H+): 532.37. 'H NMR (500 MHz, DMSO-c / 6) 8 9.84 (s, 1H), 8.01 (s, 2H), 7.65 (q, ,7= 8 Hz, 1H), 7.57 (d, J= 9 Hz, 2H), 7.46 (d, J= 9 Hz, 2H), 7.28 (t, J= 8.84 Hz, 1H), 7.11 (s, 2H), 6.84 (dd, J= 10 Hz, 16 Hz, 1H), 6.12 (dd, J= 2 Hz, 17 Hz, 1H), 5.68 (dd, J= 2 Hz, 10 Hz, 1H), 4.61 (d, J= 12 Hz, 1H), 4.25-4.13 (m, 1H), 3.24-3.10 (m, 2H), 2.75 (t, J= 12 Hz, 1H), 1.88-1.77 (m, 2H), 1.72- 1.58 (m, 2H).

[0116]

[0082] An analog with the same structure except for the acrylamide being replaced by an ethylamide was produced using similar techniques (16-rev). This compound cannot covalently bind JAK2. Atty. Dckt. No. DFCI 3463.W01WO

[0117]

[0083] X-( 4-(3-( 5-mn in o-3-((4-s iilf’ainoy Ipheny 1 )ani ino )- 1 / / - 1 ,2.4-t r iazole- 1 -car bony 1 )-2,4- difluorophenyl)cyclohexyl)acrylamide (17): Prepared according to the synthesis of 16 and substituting 12 for ze / 7-butyl (4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)cyclohex-3-en-l- yl)carbamate to give the desired compound as a white powder (31 mg, 48% yield), m / z ESI expected: 545.17, observed (M+H+): 545.81. 'H NMR (500 MHz, DM SO-6 / 6) 8 9.84 (s, 1H), 8.14-7.87 (m, 3H), 7.71-7.64 (m, 1H), 7.55 (d, J= 9 Hz, 2H), 7.46 (d, J= 9 Hz, 2H), 7.31 (td, J= 9 Hz, 29 Hz, 1H), 7.10 (s, 2H), , 6.10 (dt, J= 2 Hz, 16 Hz, 1H), 5.63-5.53 (m, 1H), 3.77-3.70 (m, 2H), 2.86 (m, 1H), 1.99-1.56 (m, 6H), 1.37 (dq, J= 3 Hz, 12 Hz, 1H).

[0118]

[0084] An analog with the same structure except for the acrylamide being replaced by an ethylamide was produced using similar techniques (17-rev). This compound cannot covalently bind JAK2.

[0119] Scheme 3:

[0120]

[0085] 4'-((tert-Butoxycarbonyl)amino)-2,4-difluoro-[l,l'-biphenyl]-3-carboxyIic acid (19):

[0121] Prepared from 11 (500. mg, 2.11 mmol) according to the procedure for 13, substituting 12 by 18 to give the desired compound (357 mg, 48% yield), m / z ESI expected: 349.11, observed (M+H+): 349.76.1H NMR (500 MHz, DMSO-t / 6) 8 9.52 (s, 1H), 7.68-7.61 (m, 1H), 7.57 (d, J= 8 Hz, 2H), 7.43 (d, J = 8 Hz, 2H), 7.28 (t, J= 9 Hz, 1H), 1.50 (s, 9H).

[0122]

[0086] Butyl (3'-(5-amino-3-((4-sulfamoylphenyl)amino)-17Z-l,2,4-triazole-l-carbonyl)- 2',4'-difluoro-[l,l'-biphenyl]-4-yl)carbamate (20): Prepared from 19 (357 mg, 1.02 mmol) according to the procedure for 15 to give the desired compound as an off-white solid (343 mg, Atty. Dckt. No. DFCI 3463.W01WO

[0123] 57% yield), m / z ESI expected: 585.16, observed (M+H+): 585.82. ' H NMR (500 MHz, DMSO- d6) 8 9.86 (s, 1H), 9.53 (s, 1H), 8.02 (s, 2H), 7.84-7.76 (m, 1H), 7.60-7.54 (m, 4H), 7.50-7.45 (m, 4H), 7.41 (t, J= 9, 1H), 7.08 (s, 2H), 1.49 (s, 9H).

[0124]

[0087] 7V-(3'-(5-amino-3-((4-sulfainoylphenyl)amino)-lH-l,2,4-triazole-l-carbonyl)-2',4'- difluoro-[l,l'-biphenyl]-4-yl)acrylamide (21): Prepared from 20 (65 mg, 0.11 mmol) according to the procedure for 16 to yield the desired compound as a white solid (19 mg, 32% yield), m / z ESI expected: 539.50, observed (M+H+): 540.39. ’H NMR (500 MHz, DMSO-t / 6) 8 10.32 (s, 1H), 9.86 (s, 1H), 8.04 (s, 2H), 7.86-7.78 (m, 3H), 7.59-7.54 (m, 4H), 7.51-7.47 (m, 2H), 7.44 (t, J= 9, 1H), 7.09 (s, 2H), 6.46 (dd, J= 10 Hz, 17 Hz, 1H), 6.29 (dd, J= 2 Hz, 17 Hz, 1H), 5.79 (dd, J= 2 Hz, 10 Hz, 1H).

[0125]

[0088] 7V-(3'-(5-amino-3-((4-sulfamoylphenyl)amino)-l / / -l,2,4-triazole-l-carbonyl)-2',4'- difluoro-[l,r-biphenyl]-3-yl)acrylamide (22): Prepared according to the synthesis of 21, substituting 18 for Zc / 7-butyl (3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)phenyl)carbamate to give the desired compound as a white powder (31 mg, 48% yield), m / z ESI expected: 539.50, observed (M+H+): 540.26. 'H NMR (500 MHz, DMSO- 6) 8 10.29 (s, 1H), 9.86 (s, 1H), 8.05 (s, 2H), 7.96 (s, 1H), 7.86-7.77 (m, 1H), 7.72 (d, J= 8 Hz, 1H), 7.58 (d, J= 9 Hz, 2H), 7.52-7.43 (m, 4H), 7.28 (d, J= 8 Hz, 1H), 7.08 (s, 2H), 6.44 (dd, J= 10 Hz, 17 Hz, 1H), 6.28 (dd, J= 2 Hz, 17 Hz, 1H), 5.78 (dd, J= 2 Hz, 10 Hz, 1H).

[0126]

[0089] 3-(5-amino-3-((4-sulfamoylphenyl)amino)-lZf-l,2,4-triazole-l- carbonyl)benzenesulfonyl fluoride (23): Prepared according to the synthesis of 20 using 3- (fluorosulfonyl)benzoic acid to give the desired compound as a white powder (43 mg, 56% yield), m / z ESI expected: 440.42, observed: 441.13.

[0127]

[0090] 3-(5-amino-3-((4-sulfamoylphenyl)amino)-lH-l,2,4-triazole-l-carbonyl)phenyl sulfurofluoridate (24): Prepared according to the synthesis of 20 using 3- ((fluorosulfonyl)oxy)benzoic acid to give the desired compound as a white powder (48 mg, 59% yield), m / z ESI expected: 456.42, observed: 447.27.

[0128]

[0091] 4-((5-amino-l-(2,6-difluoro-3-formylbenzoyl)-lH-l,2,4-triazol-3- yl)ainino)benzenesulfonamide (25): Prepared according to the synthesis of 20 using 2,6- difluoro-3 -formylbenzoic acid to give the desired compound as a white powder (0.107 g, 65% yield), m / z ESI expected: 422.06, observed: 422.81. ’H NMR (500 MHz, DMSO-t / 6) 8 10.20 (s, Atty. Dckt. No. DFCI 3463.W01WO

[0129] 1H), 9.87 (s, 1H), 8.18 (dd, J= 14.9, 8.3 Hz, 1H), 8.07 (br s, 2H), 7.58 - 7.54 (m, 3H), 7.45 (d, J = 8.9 Hz, 2H), 7.09 (s, 2H).

[0130]

[0092] 4-((5-amino-l-(l-hydroxy-l,3-dihydrobenzo[c][l,2]oxaborole-6-carbonyl)-l / / -l,2,4- triazol-3-yl)amino)benzenesulfonamide (26): Prepared according to the synthesis of 20 using 1 -hydroxy-1, 3-dihydrobenzo[c][l,2]oxaborole-6-carboxylic acid to give the desired compound as a white powder (67 mg, 62% yield), m / z ESI expected: 414.09, observed: 414.85. 'H NMR (500 MHz, DMSO-t / 6) 8 9.79 (s, 1H), 9.43 (br s, 1H), 8.51 (s, 1H), 8.24 (dd, J= 8.0, 1.6 Hz, 1H), 7.88 br s, 2H), 7.67 - 7.57 (m, 5H), 7.11 (s, 2H), 5.12 (s, 2H).

[0131]

[0093] 4-((5-amino-l -(3-amino-2,6-difluorobenzoyl)-l H-l ,2,4-triazol-3- yl)amino)benzenesulfonamide (27): Prepared according to the synthesis of 20 using 3-amino-

[0132] 2.6-difluorobenzoic acid to give the desired compound as a yellow powder (0.21 g, 51% yield), m / z ESI expected: 409.08, observed: 409.81.

[0133]

[0094] / V-(3-(5-amino-3-((4-siilfainoylphenyl)amino)-17 / -l,2,4-triazole-l-carbonyl)-2,4- difluorophenyl)acrylamide (28): To a stirred solution of 27 (0.21 g, 0.51 mmol) in dichloromethane (5 mL) were added 7V, / V-diisopropylethylamine (266 / / L, 1.53 mmol) and acryloyl chloride (54.0 z / L, 0.66 mmol) at 0°C, and the reaction mixture was stirred at room temperature for 1 hour. The reaction was quenched with 1 N HC1 solution (25 mL) and extracted with ethyl acetate (50 mL). The aqueous layer was further extracted with ethyl acetate (2 x 100 mL), and the organic layers were washed with brine, dried with MgSCh, and concentrated in vacuo. The remaining residue was purified by reverse phase HPLC to yield the desired compound as a white powder (0.194 g, 82% yield), m / z ESI expected: 463.09, observed (M+H+): 463.78. 'H NMR (500 MHz, DMSO-z / 6) 8 10.17 (br s, 1H), 9.84 (s, 1H), 8.13 (dd, J= 14.9, 8.9 Hz, 1H), 8.03 (br s, 2H), 7.60 (d, J= 8.9 Hz, 2H), 7.48 (d, J= 8.9 Hz, 2H), 7.33 (dd, J= 14.3, 8.3 Hz, 1H), 7.09 (s, 2H), 6.57 (dd, J = 17.0, 10.2 Hz, 1H), 6.32 (dd, J= 17.0, 1.9 Hz, 1H), 5.82 (dd, J= 10.2, 1.8 Hz, 1H).

[0134]

[0095] 4-((5-amino-l-(2,6-difluoro-3-formylbenzoyl)-l / / -l,2,4-triazol-3-yl)amino)- / V- methylbenzamide (32): Prepared according to the synthesis of 20 substituting 4 for 7 and using

[0135] 2.6-difluoro-3 -formylbenzoic acid to give the desired compound as a pale yellow powder (71 mg, 54% yield), m / z ESI expected: 400.11, observed: 400.88. 'H NMR (500 MHz, DMSO-t / 6) 8 10.20 (s, 1H), 9.70 (s, 1H), 8.23 - 8.14 (m, 2H), 8.03 (br s, 2H), 7.62 (d, J= 8.8 Hz, 2H), 7.56 (t, J= 8.7 Hz, 1H), 7.36 (d, J= 8.8 Hz, 2H), 2.73 (d, J= 4.3 Hz, 3H). Atty. Dckt. No. DFCI 3463.W01WO

[0136]

[0096] 4-((5-amino-l-(l-hydroxy-l,3-dihydrobenzo[cJ[l,2]oxaborole-6-carbonyl)-177-l,2,4- triazol-3-yl)amino)-ALmethylbenzamide (33): Prepared according to the synthesis of 20 substituting 4 for 7 and using l-hydroxy-l,3-dihydrobenzo[c][l,2]oxaborole-6-carboxylic acid to give the desired compound as a white powder (0.12 g, 73% yield), m / z ESI expected: 392.14, observed: 392.91. 'HNMR (500 MHz, DMSO-t / 6) 5 9.62 (s, 1H), 9.43 (s, 1H), 8.52 (s, 1H), 8.26 (dd, <7= 8.0, 1.2 Hz, 1H), 8.19 (d, J= 4.4 Hz, 1H), 7.86 (br s, 2H), 7.71 (d, J= 8.7 Hz, 2H), 7.61 (d, J= 8.0 Hz, 1H), 7.56 (d, J= 8.7 Hz, 2H), 5.13 (s, 2H), 2.75 (d, J= 4.4 Hz, 3H).

[0137]

[0097] 4-((5-amino-l-(3-amino-2,6-difluorobenzoyl)-177-l,2,4-triazol-3-yl)amino)-7V- methylbenzamide (34): Prepared according to the synthesis of 20 substituting 4 for 7 and using 3-amino-2,6-difluorobenzoic acid to give the desired compound as a yellow powder (83 mg, 21% yield), m / z ESI expected: 387.13, observed: 387.88.

[0138]

[0098] 5-(5-ainiiio-3-((4-(metliylc;irb:iiii()yl)pheiiyl)ainino)-l / / -l ,2,4-triazole-l -carbonyl)-2- fluorobenzenesulfonyl fluoride (35): Prepared according to the synthesis of 20 replacing 4 for 7 and using 4-fluoro-3-(fluorosulfonyl)benzoic acid to give the desired compound as a pale yellow powder (0.11 g, 40% yield), m / z ESI expected: 436.08, observed: 436.77. ’H NMR (500 MHz, DMSO-t / 6) 8 9.71 (s, 1H), 9.14 (dd, J= 6.7, 2.1 Hz, 1H), 8.67 (ddd, J= 8.6, 4.7, 2.1 Hz, 1H), 8.20 (d, J= 4.5 Hz, 1H), 7.95 (t, J= 9.4 Hz, 3H), 7.72 (d, J= 8.7 Hz, 2H), 7.52 (d, J= 8.7 Hz, 2H), 2.76 (d, J = 4.5 Hz, 3H).

[0139]

[0099] 3-(5-amino-3-((4-(methylcarbamoyl)phenyl)amino)-177-l,2,4-triazole-l-carbonyl)-4- fluorobenzenesulfonyl fluoride (36): Prepared according to the synthesis of 20 substituting 4 for 7 and using 2-fluoro-5-(fluorosulfonyl)benzoic acid to give the desired compound as a white powder (92 mg, 58% yield), m / z ESI expected: 436.08, observed: 436.78. ’H NMR (500 MHz, DMSO-t / 6) 8 9.67 (s, 1H), 8.80 (dd, J= 5.7, 2.5 Hz, 1H), 8.48 (ddd, ,7= 8.8, 4.2, 2.6 Hz, 1H), 8.17 (d, J= 4.6 Hz, 1H), 7.94 (br s, 2H), 7.88 (t, J= 9.1 Hz, 1H), 7.64 (d, J= 8.8 Hz, 2H), 7.40 (d, J= 8.8 Hz, 2H), 2.74 (d, J= 4.5 Hz, 3H).

[0140]

[0100] 5-(5-amino-3-((4-(dimethylcarbamoyl)phenyl)amino)-lH-l,2,4-triazole-l-carbonyl)- 2-fluorobenzenesulfonyl fluoride (37): Prepared according to the synthesis of 20 substituting 4 for 10 and using 4-fluoro-3-(fluorosulfonyl)benzoic acid to give the desired compound as a white powder (0.13 g, 75% yield), m / z ESI expected: 450.09, observed: 450.78. ’H NMR (500 MHz, DMSO-t / 6) 8 9.68 (s, 1H), 9.20 (dd, J= 6.7, 2.2 Hz, 1H), 8.65 (ddd, J= 8.7, 4.7, 2.2 Hz, 1H), Atty. Dckt. No. DFCI 3463.W01WO

[0141] 8.17 (br s, 2H), 7.97 - 7.91 (m, 1H), 7.54 (d, J= 8.6 Hz, 2H), 7.31 (d, J= 8.6 Hz, 2H), 2.96 (s, 6H).

[0142]

[0101] 3-(5-amino-3-((4-(dimethylcarbamoyl)phenyl)amino)-lH-l,2,4-triazole-l-carbonyl)- 4-fluorobenzenesulfonyl fluoride (38): Prepared according to the synthesis of 20 substituting 4 for 10 and using 2-fluoro-5-(fluorosulfonyl)benzoic acid to give the desired compound as a pale yellow powder (95 mg, 58% yield), m / z ESI expected: 450.09, observed: 450.75. *H NMR (500 MHz, DMSO-t / 6) 6 9.62 (s, 1H), 8.83 (dd, J = 5.8, 2.5 Hz, 1H), 8.46 (ddd, J= 8.8, 4.3, 2.6 Hz, 1H), 7.93 (br s, 2H), 7.86 (t, J= 9.2 Hz, 1H), 7.42 (d, J= 8.7 Hz, 2H), 7.22 (d, J= 8.7 Hz, 2H), 2.93 (s, 6H).

[0143] Scheme 4:

[0144]

[0102] Ethyl l,2,3?4-tetrahydroisoquinoline-7-carboxylate (66): 1, 2,3,4- tetrahydroisoquinoline-7-carboxylic acid (65) (1.0 g, 5.5 mmol) was solubilized in ethanol (5 mb) and sulfuric acid (1.21 mL, 22.6 mmol) was added dropwise at room temperature while stirring. The solution was heated to reflux and stirred for 3 hours. The reaction mixture was allowed to cool to room temperature and concentrated in vacuo to give the desired compound as a brown paste without further purification (569 mg, 49% yield), m / z ESI expected: 205.26, observed (M+H+): 206.02. 'HNMR (500 MHz, DMSO-r / 6) 8 7.69 (d, J= 8 Hz, 1H), 7.63 (s, 1H), 7.21 (d, J= 8 Hz, 1H), 4.29 (q, J= 7 Hz, 2H), 3.92 (s, 2H), 2.97 (t, J= 6 Hz, 2H), 2.76 (t, J = 6 Hz, 2H), 1.31 (t, J= 7 Hz, 3H). Atty. Dckt. No. DFCI 3463.W01WO

[0145]

[0103] 2-(to7-butyl) 7-ethyl 3,4-dihydroisoquinoline-2,7(l / / )-dicarboxylate (67): 66 (569 mg, 2.77 mmol) and TEA (0.580 mL, 4.82 mmol) were solubilized in DCM (8 mb) and BOC2O (0.764 mL, 3.86 mmol) was added dropwise. The reaction mixture was stirred at 25 °C for 3 hours. The reaction was quenched with water (25 mL) and diluted with DCM (50 mL). The aqueous layer was further extracted with DCM (2 x 100 mL), and the organic layers were washed with brine, dried with MgSCU, and concentrated in vacuo. The remaining residue was purified by normal phase chromatography (0-60% ethyl acetate in hexanes) to give the desired compound as a clear oil (610 mg, 72% yield), m / z ESI expected: 305.37, observed: 306.11. 'H NMR (500 MHz, DMSO-t / 6) 8 7.80-7.72 (m, 2H), 7.31 (d, J= 8 Hz, 1H), 4.57 (s, 2H), 4.31 (q, J= 7 Hz, 2H), 3.57 (t, J= 6 Hz, 2H), 2.85 (t, J= 6 Hz, 2H), 1.43 (s, 9H), 1.32 (t, J= 7 Hz, 3H).

[0146]

[0104] 2-(tert-butoxycarbonyl)-l,2,3,4-tetrahydroisoquinoline-7-carboxylic acid (68): 67 (610 mg, 2.0 mmol) was solubilized in THF (8 mL). Lithium hydroxide monohydrate (420 mg, 10 mmol) was solubilized in water (10 mL). The aqueous solution was added dropwise to the THF solution at room temperature, and the reaction mixture was then heated to 55 °C and stirred for two hours. The solution was then removed from heat and allowed to cool to room temperature, and 310 mg (7.4 mmol) additional lithium hydroxide monohydrate was added. The solution was reheated to 55 °C for 3 hours. The reaction was removed from heat and allowed to cool to room temperature, quenched with water (100 mL), and extracted with ethyl acetate (100 mL). The aqueous layer was acidified with dropwise IM HC1 solution until it displayed a pH of 3 and a white precipitate had formed. The aqueous layer was further extracted with ethyl acetate (2 x 150 mL) and the organic layers were washed with brine, dried with MgSCL, and concentrated in vacuo to give the desired compound without further purification as a white solid (420 mg, 76% yield), m / z ESI expected: 277.32, observed: 277.95. ’H NMR (500 MHz, DMSO-t / 6) 8 7.80-7.70 (m, 2H), 7.29 (d, J= 8 Hz, 1H), 4.56 (s, 2H), 3.57 (t, J= 6 Hz, 2H), 2.84 (t, J= 6 Hz, 2H), 1.44 (s, 9H).

[0147]

[0105] terZ-butyl 7-((3-((4-(iiiethylcarbaiiioyl)plieiiyl)amino)-l / / -l ,2,4-triazol-5- yl)carbamoyl)-3,4-dihydroisoquinoline-2(LH)-carboxylate (69): Prepared from 68 (420 mg, 1.51 mmol) according to the procedure for 15 and purified by normal phase chromatography (30- 100% ethyl acetate in hexanes) to yield the desired compound as yellow solid (65 mg, 8.7% yield), m / z ESI expected: 491.55, observed: 491.92. ’H NMR (500 MHz, DMSO-t / 6) 8 10.29 (s, 1H), 8.35 (q, J = 4 Hz, 1H), 7.99-7.91 (m, 2H), 7.90-7.83 (m, 4H), 7.34 (d, J= 8 Hz, 1H), 6.17 (s, Atty. Dckt. No. DFCI 3463.W01WO

[0148] 2H), 4.57 (s, 2H), 3.60 (t, J= 6 Hz, 2H), 2.88 (t, J= 6 Hz, 2H), 2.79 (d, J= 5 Hz, 3H), 1.45 (s, 9H).

[0149]

[0106] / 7-butyl 7-(5-amino-3-((4-(inethylcarbamoyl)phenyl)amino)-lH-l,2,4-triazole-l- carbonyl)-3,4-dihydroisoquinoline-2(177)-carboxylate (70): Prepared from 68 (420 mg, 1.51 mmol) as the major product from the reaction that produces 69, eluding from the normal phase column after 69 to give the desired compound as a yellow-white solid (100 mg, 13% yield), m / z ESI expected: 491.55, observed: 491.92.

[0150]

[0107] 2-acryl()yl-\-(3-((4-(methylcai bamoyl)phenyl)amino)-l / / -l .2.4-triazol-5-yl)-l .2,3.4- tetrahydroisoquinoline-7-carboxamide (71): Prepared from 69 (65 mg, 0.13 mmol) according to the procedure for 16 to give the desired compound as a yellow solid (34 mg, 56% yield), m / z ESI expected: 445.48, observed: 445.91.JH NMR (500 MHz, DMSO-t / 6) 5 10.32-10.28 (m, 1H), 8.35 (q, J = 4 Hz, 1H), 8.01-7.93 (m, 2H), 7.90-7.83 (m, 4H), 7.36 (d, J = 8 Hz, 1H), 6.97-6.86 (m, 1H), 6.22-6.12 (m, 3H), 5.74 (dd, J= 2 Hz, 10 Hz, 1H), 4.87 (s, 1H), 4.76 (s, 1H), 3.89-3.75 (m, 2H), 2.99-2.88 (m, 2H), 2.79 (d, J= 4 Hz, 3H).

[0151]

[0108] 4-((l-(2-acryloyl-l, 2,3, 4-tetrahydroisoquinoline-7-carbonyl)-5-amino- 1 / 7-1, 2,4- triazoI-3-yl)amino)- / V-methylbenzamide (72): Prepared from 70 (100 mg, 0.20 mmol) according to the procedure for 16 to give the desired compound as an off-white solid (6 mg, 6.5% yield), m / z ESI expected: 445.48, observed: 445.91. 'H NMR (500 MHz, DMSO-t / 6) 8 9.63 (s, 1H), 8.20-7.93 (m, 3H), 7.84 (s, 2H), 7.79-7.69 (m, 2H), 7.60-7.52 (m, 2H), 7.40 (d, J= 7.88 Hz, 1H), 6.98-6.88 (m, 1H), 6.24-6.15 (m, 1H), 5.81-5.74 (m, 1H), 4.93-4.78 (m, 2H), 3.93-3.78 (m, 2H), 3.02-2.89 (m, 2H), 2.79-2.73 (m, 3H).

[0152]

[0109] 2-acryloyl- / V-(3-((4-(methylcarbamoyl)phenyl)amino)-lH-l,2,4-triazol-5-yl)-l,2,3,4- tetrahydroisoquinoline-6-carboxamide (73): Prepared according to the synthesis of 71, substituting 65 for l,2,3,4-tetrahydroisoquinoline-6-carboxylic acid to give the desired compound as a yellow solid (56 mg, 10% yield), m / z ESI expected: 445.48, observed: 445.95. 'H NMR (500 MHz, DMSO-t / 6) 6 10.29 (s, 1H), 8.35 (q, J = 5 Hz, 1H), 7.98 (d, J= 8 Hz, 1H), 7.94 (s, 1H), 7.89-7.83 (m, 4H), 7.38 (dd, J= 8 Hz, 22 Hz, 1H), 6.92 (dd, J= 10 Hz, 16 Hz, 1H), 6.18 (dd, J= 2 Hz, 16 Hz, 1H), 5.74 (dd, J= 2 Hz, 10 Hz, 1H), 4.92-4.73 (m, 2H), 3.87-3.76 (m, 2H), 2.97- 2.85 (m, 2H), 2.79 (d, J= 4 Hz, 3H).

[0153]

[0110] 4-((l-(2-acryloyl-l,2,3,4-tetrahydroisoquinoline-6-carbonyl)-5-amino-l / / -l,2,4- triazol-3-yl)amino)- / V-methylbenzamide (74): Prepared according to the synthesis of 72, Atty. Dckt. No. DFCI 3463.W01WO substituting 65 for l,2,3,4-tetrahydroisoquinoline-6-carboxylic acid to give the desired compound as a white solid (180 mg, 33% yield), m / z ESI expected: 445.48, observed: 445.95.!H NMR (500 MHz, DMSO-t / 6) 8 9.63 (s, 1H), 8.24-8.16 (m, 1H), 8.11-8.05 (m, 1H), 8.01 (d, J= 8.05 Hz, 1H), 7.85 (s, 2H), 7.73 (d, J= 8.84 Hz, 2H), 7.56 (d, . / = 8.72 Hz, 2H), 7.46-7.37 (m, 1H), 6.94 (dd, J = 10.37, 16.53 Hz, 1H), 6.19 (dd, J= 2.39, 16.76 Hz, 1H), 5.76 (dd, J= 2.38, 10.28 Hz, 1H), 4.95-4.75 (m, 2H), 3.95-3.80 (m, 2H), 2.04-2.89 (m, 2H), 2.76 (d, J= 4.44 Hz, 3H).

[0154] Scheme 5;

[0155]

[0111] 5-((phenoxycarbonyl)amino)pyridin-3-yl sulfurofluoridate (89): 5-aminopyridin-3-yl sulfurofluoridate (88) (200 mg, 1.04 mmol) and phenyl chloroformate (0.195 mL, 1.56 mmol) were solubilized in DCM (10 mL). The reaction mixture was cooled to 0 °C, and DIEA (0.362 mL, 2.08 mmol) was added dropwise while stirring. The mixture was stirred at 0 °C for 1 hour and was quenched with saturated NaHCCh solution (100 mL) and extracted with DCM (100 mL). The aqueous layer was further extracted with DCM (2 x 100 mL), and the organic layers were washed with brine, dried with MgSCL, and concentrated in vacuo. The remaining residue was purified by normal phase chromatography (10-90% ethyl acetate in hexanes) to give the desired compound as a yellow solid (71 mg, 22% yield), m / z ESI expected: 312.27, observed: 312.75.

[0156]

[0112] 5-(5-amino-3-((4-sulfamoylphenyl)amino)-l / / -l,2,4-triazole-l-carboxainido)pyridin- 3-yl sulfurofluoridate (90): 89 (60 mg, 0.19 mmol) was solubilized with 4 (180 mg, 0.70 mmol) were solubilized in dioxane (10 mL). TEA (0.20 mL, 1.4 mmol) was added, and the solution was degassed in a sonicator for 2 minutes, sealed under N2, and stirred at 95 °C for 3 hours. The reaction was quenched with water (25 mL) and diluted with ethyl acetate (50 mL). The aqueous Atty. Dckt. No. DFCI 3463.W01WO layer was further extracted with ethyl acetate (2 x 100 mL), and the organic layers were washed with brine, dried with MgSO4, and concentrated in vacuo. The remaining residue was purified by reverse phase HPLC to give the desired compound as white solid (28 mg, 31% yield), m / z ESI expected: 472.43, observed: 472.72. *H NMR (500 MHz, DMSO-r / 6) 8 10.16 (s, 1H), 9.74 (s, 1H), 9.08 (d, J = 2 Hz, 1H), 8.70 (d, J= 2 Hz, 1H), 8.46 (t, J= 2 Hz, 1H), 7.83 (d, J= 8 Hz, 2H), 7.72 (d, J= 9 Hz, 2H), 7.53 (s, 2H), 7.16 (s, 2H).

[0157]

[0113] 5-(5-amino-3-((4-(methylcarbauioyl)phenyl)amino)-LH-l,2,4-triazole-l- carboxamido)pyridin-3-yl sulfurofluoridate (91): Prepared from 89 (71 mg, 0.23 mmol) according to the procedure for 90, substituting 4 for 7 and purified by reverse phase HPLC to give the desired compound as a white solid (6 mg, 6% yield), m / z ESI expected: 450.41, observed: 450.95. *H NMR (500 MHz, DMSO-c / 6) 8 10.20 (s, 1H), 9.56 (s, 1H), 9.09 (d, J= 2 Hz, 1H), 8.69 (d, J = 2 Hz, 1H), 8.47 (t, J = 2 Hz, 1H), 8.22 (q, J = 4 Hz, 1H), 7.79 (d, J = 9 Hz, 2H), 7.73 (d, J= 9 Hz, 2H), 7.50 (s, 2H), 2.77 (d, J = 4 Hz, 3H).

[0158]

[0114] 3-(5-amino-3-((4-sulfamoylphenyl)amino)-lH-l,2,4-triazole-l- carboxamido)benzenesulfonyl fluoride (92): Prepared according to the synthesis of 90, using 3- aminobenzenesulfonyl fluoride to give the desired compound as a white solid (34 mg, 7.2% yield), m / z ESI expected: 455.49, observed: 455.75. ’H NMR (500 MHz, DMSO-t / 6) 8 10.13 (s, 1H), 9.73 (s, 1H), 8.61 (t, J= 2 Hz, 1H), 8.27 (dd, J= 1 Hz, 8 Hz, 1H), 7.91 (d, J= 8 Hz, 1H), 7.86-7.80 (m, 3H), 7.72 (d, J= 9 Hz, 2H), 7.52 (s, 2H), 7.16 (s, 2H).

[0159]

[0115] 3-(5-amino-3-((4-(methylcarbamoyl)phenyl)amino)-LH-l,2,4-triazole-l- carboxamido)benzenesulfonyl fluoride (93): Prepared according to the synthesis of 91, using 3- aminobenzenesulfonyl fluoride to give the desired compound as a white solid (12 mg, 8.1% yield), m / z ESI expected: 433.42, observed (M+H+): 433.88. 'H NMR (500 MHz, DMSO-t / 6) 8 10.16 (s, 1H), 9.55 (s, 1H), 8.62 (t, J= 1.89 Hz, 1H), 8.28 (d, J= 7.97 Hz, 1H), 8.22 (q, . / 4.36 Hz, 1H), 7.91 (d, J= 8.10 Hz, 1H), 7.85-7.72 (m, 5H), 7.50 (s, 2H), 2.77 (d, J= 4.51 Hz, 3H).

[0160]

[0116] 3-(5-amino-3-((4-sulfanioylphenyl)aniino)-l / / -1 ,2,4-triazole-l-carboxauiido)phenyl sulfurofluoridate (94): Prepared according to the synthesis of 90 using 3-aminophenyl sulfurofluoridate to give the desired compound as a white solid (44 mg, 29% yield), m / z ESI expected: 471.44, observed: 471.75. 'H NMR (500 MHz, DMSO-r / 6) 8 9.95 (s, 1H), 9.71 (s, 1H), 8.00 (t, J= 2 Hz, 1H), 7.87 (dd, J= 1 Hz, 8 Hz, 1H), 7.82 (d, J= 9 Hz, 2H), 7.72 (d, J= 9 Hz, 2H), 7.62 (t, J= 8 Hz, 1H), 7.48 (s, 2H), 7.40 (dd, J= 2, 8.30 Hz, 1H), 7.16 (s, 2H). Atty. Dckt. No. DFCI 3463.W01WO

[0161]

[0117] 3-(5-amino-3-((4-(methylcarbamoyl)phenyl)amino)- 177-1,2, 4-triazole-l- carboxamido)phenyl sulfurofluoridate (95): Prepared according to the synthesis of 91 using 3- aminophenyl sulfurofluoridate to give the desired compound as a white solid (31 mg, 22% yield), m / z ESI expected: 449.42, observed: 449.78. 'HNMR (500 MHz, DMSO-t / 6) 5 9.98 (s, 1H), 9.53 (s, 1H), 8.21 (q, J= 4 Hz, 1H), 8.01 (t, J= 2 Hz, 1H), 7.88 (dd, J= 1 Hz, 8 Hz, 1H), 7.78 (d, J= 8 Hz, 2H), 7.73 (d, J= 9 Hz, 2H), 7.62 (t, J= 8 Hz, 1H), 7.46 (s, 2H), 7.39 (dd, J= 2 Hz, 8 Hz, 1H), 2.77 (d, J= 4 Hz, 3H).

[0162]

[0118] 5-amino-N-(l-hydroxy-l,3-dihydrobenzo[c][l,2]oxaborol-6-yl)-3-((4- sulfamoylphenyl)amino)-17f-l,2,4-triazole-l-carboxamide (96): Prepared according to the synthesis of 90 using 6-aminobenzo[c][l,2]oxaborol-l(37 / )-ol to give the desired compound as a slightly pink solid (25 mg, 19% yield), m / z ESI expected: 429.22, observed: 429.86. 'H NMR (500 MHz, DMSO-t / 6) 5 9.68 (d, J = 6 Hz, 2H), 7.98 (s, 1H), 7.84 (d, J = 9 Hz, 2H), 7.74-7.69 (m, 3H), 7.47-7.35 (m, 3H), 7.15 (s, 2H), 5.01 (s, 2H).

[0163]

[0119] 5-amino- / V-(l-hydroxy-l,3-dihydrobenzo[c][l,2]oxaborol-6-yl)-3-((4- (methylcarbamoyl)phenyl)amino)-l H-l ,2, 4-triazole-l -carboxamide (97): Prepared according to the synthesis of 91 using 6-aminobenzo[c][l,2]oxaborol-l(3 / / )-ol to give the desired compound as a slightly pink solid (11 mg, 9% yield), m / z ESI expected: 407.20, observed (M+H+): 407.91. 'H NMR (500 MHz, DMSO-t / 6) 5 9.67 (s, 1H), 9.50 (s, 1H), 8.20 (q, J= 4 Hz, 1H), 7.99 (d, J= 2 Hz, 1H), 7.80-7.68 (m, 5H), 7.43 (d, J= 8, 1H), 7.39 (s, 2H), 5.00 (s, 2H), 2.77 (d, J = 4, 3H).

[0164]

[0120] tert-butyl (3-nitrophenethyl)carbamate (99): Prepared from 2-(3 -nitrophenyl)ethan- 1 - amine (98) (600 mg, 3.6 mmol) according to the procedure for 67, and purifying the mixture by normal phase chromatography (10-60% ethyl acetate in hexanes) to give the desired compound (680 mg, 71% yield), m / z ESI expected: 266.30, observed: 266.95. 'H NMR (500 MHz, DMSO- Atty. Dckt. No. DFCI 3463.W01WO t / 6) 8 8.11-8.03 (m, 2H), 7.68 (d, J= 8 Hz, 1H), 7.59 (t, J= 8 Hz, 1H), 6.91 (t, J= 5 Hz, 1H), 3.21 (q, J= 6 Hz, 2H), 2.84 (t, J= 7 Hz, 2H), 1.33 (s, 9H).

[0165]

[0121] tert-butyl (3-aminophenethyl)carbamate (100): Prepared from 99 (680 mg, 2.6 mmol) according to the procedure for 14, only requiring 2 hours of stirring instead of 16 to give the desired compound (567 mg, 94% yield), m / z ESI expected: 236.31, observed: 237.01.JH NMR (500 MHz, DMSO-t / 6) 8 6.91 (t, J= 8 Hz, 1H), 6.83 (t, J= 5 Hz, 1H), 6.42-6.37 (m, 2H), 6.32 (d, J= 8 Hz, 1H), 4.94 (s, 2H), 3.10-3.03 (m, 2H), 2.56-2.52 (m, 2H), 1.38 (s, 9H).

[0166]

[0122] tert-butyl (3-((phenoxycarbonyl)amino)phenethyl)carbamate (101): 100 (567 mg, 2.41 mmol) and NaHCCh (79 mg, 0.94 mmol) were solubilized in THF (6 mL) and water (4 mL). The solution was cooled to 0 °C, and phenyl chloroformate (0.112 mL, 0.862 mmol) was added dropwise while stirring. The solution was allowed to warm to room temperature and was stirred for 1 hour. The reaction was quenched with water (50 mL) and diluted with ethyl acetate (50 mL). The aqueous layer was further extracted with ethyl acetate (2 x 100 mL), and the organic layers were washed with brine, dried with MgSCM, and concentrated in vacuo without further purification to give the desired compound (870 mg, 100% yield), m / z ESI expected: 356.42, observed: 356.98.

[0167]

[0123] tert-butyl (3-(3-(3-((4-(methylcarbamoyl)phenyl)amino)-l / f-l,2,4-triazol-5- yl)ureido)phenethyl) carbamate (102): Prepared from 101 (860 mg, 2.4 mmol) according to the procedure for 90, and stirring the reaction at 95 °C for 5 hours to give the desired compound as a pink solid (60 mg, 5% yield), m / z ESI expected: 494.56, observed: 494.95.

[0168]

[0124] tert-butyl (3-(5-amino-3-((4-(methylcarbamoyl)phenyl)amino)-17 / -l,2,4-triazole-l- carboxamido)phenethyl)carbamate (103): Prepared from 101 (860 mg, 2.4 mmol) as the major product from the reaction that produces 102, and stirring the reaction at 95 °C for 5 hours. The compound eluded from the normal phase column after 102 to give the desired compound as a white solid (530 mg, 44% yield), m / z ESI expected: 494.56, observed: 494.95.JH NMR (500 MHz, DMSO-t / 6) 6 9.53 (s, 1H), 9.50 (s, 1H), 8.21 (q, J= 1 Hz, 1H), 7.78 (d, J= 9 Hz, 2H), 7.72 (d, J= 9 Hz, 2H), 7.55-7.51 (m, 2H), 7.41 (s, 2H), 7.30 (t, J= 8 Hz, 1H), 7.00 (d, J= 8 Hz, 1H), 6.93 (t, J= 5 Hz, 1H), 3.16 (q, J= 6 Hz, 2H), 2.77 (d, J= 4 Hz, 3H), 2.71 (t, J= 8 Hz, 2H), 1.38 (s, 9H).

[0169]

[0125] 4-((5-(3-(3-(2-acrylamidoethyl)phenyl)ui eido)-l / / -l .2.4-triazol-3-yl)amino)-\- methylbenzamide (104): Prepared from 102 (60 mg, 0.12 mmol) according to the procedure for Atty. Dckt. No. DFCI 3463.W01WO

[0170] 21 to give the desired compound as a white solid (30 mg, 55% yield), m / z ESI expected: 448.49, observed: 448.98.

[0171]

[0126] 7V-(3-(2-acrylamidoethyl)phenyl)-5-amino-3-((4-(methylcarbamoyl)phenyl)amino)- lH-l,2,4-triazole-l-carboxamide (105): Prepared from 103 (530 mg, 1.1 mmol) according to the procedure for 21 to give the desired compound as a white solid (101 mg, 21% yield), m / z ESI expected: 448.49, observed: 448.98. 'H NMR (500 MHz, DMSO-tZ6) 8 9.55 (s, 1H), 9.50 (s, 1H), 8.25-8.18 (m, 2H), 7.78 (d, J= 9 Hz, 2H), 7.72 (d, J= 9 Hz, 2H), 7.58-7.53 (m, 2H), 7.41 (s, 2H), 7.32 (t, J= 8 Hz, 1H), 7.03 (d, J= 7 Hz, 1H), 6.22 (dd, J= 10 Hz, 17 Hz, 1H), 6.09 (dd, J= 2 Hz, 17 Hz, 1H), 5.58 (dd, J= 2 Hz, 10 Hz, 1H), 2.80-2.75 (m, 5H).

[0172]

[0127] 4-((5-(3-(4-(2-acrylamidoethyl)phenyl)ureido)-LH-l,2,4-triazol-3-yl)amino)-JV- methylbenzamide (106): Prepared according to the synthesis of 104, substituting 98 for 2-(4- nitrophenyl)ethan-l -amine to give the desired compound as a white solid (14 mg, 39% yield), m / z ESI expected: 448.49, observed: 448.95.

[0173]

[0128] 7V-(4-(2-acrylamidoethyl)phenyl)-5-amino-3-((4-(methylcarbamoyl)phenyl)amino)- LFf-l,2,4-triazole-l-carboxamide (107): Prepared according to the synthesis of 105, substituting 98 for 2-(4-nitrophenyl)ethan-l -amine to give the desired compound as a white solid (54 mg, 13% yield), m / z ESI expected: 448.49, observed: 448.95. 'H NMR (500 MHz, DM SO-6 / 6) 8 9.54 (s, 1H), 9.49 (s, 1H), 8.24-8.14 (m, 2H), 7.77 (d, J= 9 Hz, 2H), 7.72 (d, J= 9 Hz, 2H), 7.59 (d, J = 9 Hz, 2H), 7.39 (s, 2H), 7.23 (d, J= 8 Hz, 2H), 6.21 (dd, J= 10 Hz, 17 Hz, 1H), 6.07 (dd, J= 2 Hz, 17 Hz, 1H), 5.58 (dd, J= 2 Hz, 10 Hz, 1H), 3.38 (q, J= 7 Hz, 2H), 2.80-2.72 (m, 5H).

[0174]

[0129] The structures of the synthesized inhibitors are shown in Table 1.

[0175] Table 1. Synthesized compounds of formula (I). Atty. Dckt. No. DFCI 3463.W01WO Atty. Dckt. No. DFCI 3463.W01WO

[0176] Assays

[0177]

[0130] Inhibitor binding assay: Fluorescence resonance energy transfer (FRET) was used to assess allosteric binding of the compounds to JAK2 pseudokinase domain in the presence of a Cy5 labeled competitive probe for the ATP binding site. Competitive fluorescent probes for the JAK2 JH2 allosteric site are known in the art (Newton et al. ACS Med. Chem. Lett. 2017, 8:614-617; Hammaren et al. Proc. Natl. Acad. Set. U.S.A. 2015, 112:4642-4647). Purified JAK2 pseudokinase domain wild type and V617F mutant were dispensed in black 384-well plates using a Multidrop Combi dispenser (ThermoFisher) and incubated for 30 min at room temperature with inhibitors (10 mM DMSO stocks) dispensed by an HP D300e dispenser (Hewlett-Packard). The probe was then added at a final concentration of 100 nM and incubated for 30 min at room temperature, followed by the addition of anti 6HIS-Tb cryptate antibody (Cisbio) which was incubated for 2 hours at room temperature. The FRET signal ratio was measured at 665 and 620 nm using a PHERAstar microplate reader (BMG LAB TECH). The data was graphically displayed using GraphPad Prism version 9.0 (GraphPad software). The curves were fitted using a non-linear regression analysis with a three-parameter dose-response model. Atty. Dckt. No. DFCI 3463.W01WO

[0178]

[0131] Kinase inhibition assay: Biochemical activity inhibition assays for JAK2 full-length wild type and V617F mutant, JAK2 kinase (K) domain, and pseudokinase-kinase (PK-K) domains wild type and V617F mutant, were performed by homogeneous time-resolved fluorescence (HTRF) KinEASE-TK (Cisbio) assay according to the manufacturer’s protocol. Assays were optimized for ATP concentration of 100 pM and assay buffer containing purified JAK2 at a final concentration of 10 nM for JAK2 full-length wild type and of 1 nM for JAK2 V617F mutant was dispensed in black 384-well plates using a Multidrop Combi dispenser (ThermoFisher) and incubated at room temperature for 30 min. Inhibitors (10 mM DMSO stocks) were dispensed into black 384-well plates using an HP D300e dispenser (Hewlett-Packard) and normalized to a 1% final DMSO concentration. Compound IC50 values were determined by 11 -point inhibition curves (from 30 to 0.0005 pM) in triplicate. The FRET signal ratio was measured at 665 and 620 nm using a PHERAstar microplate reader (BMG LABTECH). The data was graphically displayed using GraphPad Prism version 9.0 (GraphPad software). The curves were fitted using a nonlinear regression analysis with a three-parameter dose-response model.

[0179]

[0132] Crystallization and structure determination: Crystals of JAK2 pseudokinase domain in complex with compounds were prepared using the hanging drop vapor diffusion method over a reservoir solution containing 200 mM Sodium Acetate, 100 mM Tris-HCl, and 18%-30%(w / v) polyethylene glycol 4000 by co-crystallization. Crystals were flash frozen and X-ray diffraction data were collected at 100 K at the Advanced Light Source a part of the Northeastern Collaborative Access Team (NE-CAT) on Beamline 24-1D-C (wavelength 0.97918 A). Diffraction data were indexed, integrated, and scaled using XDS via xia2 compiled through SBGrid. Phases were obtained by molecular replacement with the program PHASER using the previously solved JAK2 pseudokinase domain structure in complex with JNJ7706621 (PDB

[0180] 5 WIN) as the initial search model. All model building and refinement were performed using WinCoot (v.0.9.6. EL) and Phenix (v.1.20.1-4487). Successive rounds were performed until acceptable statistics were achieved.

[0181]

[0133] Intact protein LC-MS analysis for labeling efficiency determination: IAK2 pseudokinase domain was incubated with compounds (1 :5 ratio) at room temperate for 24 h. Samples were flash frozen to stop the reaction. Intact proteins were acidified with formic acid and desalted over C4 resin prior to injection onto a U3000 RSLC fitted with a MAbPacTM RP column (Thermo Scientific ES907; 150 mm x 15 cm column packed with 4 mm, 1500 A resin). Atty. Dckt. No. DFCI 3463.W01WO

[0182] Proteins were eluted with a 5-50% gradient of acetonitrile in 0. 1% formic acid over 15 min and electrosprayed (1.9 kV) into an Orbitrap Eclipse mass spectrometer (Thermo Scientific). Full scan mass spectra (m / z 600-2000) were acquired in profile mode. Mass spectra were deconvoluted using UniDec (version 6.0.4). Labeling efficiency was calculated from zero charge mass spectra using peak heights according to [peak height labeled protein] / [peak height labeled protein + peak height unlabeled protein] x 100%.

[0183]

[0134] LC-MS / MS analysis for covalent labeling site identification: JAK2 pseudokinase domain was incubated with compounds (1 :2.5 ratio) at room temperate for 24 h. Samples were flash frozen to stop the reaction. Proteins were denatured in 8 M urea and reduced in 10 mM TCEP for 30 min at RT. Protein residues were alkylated with 15 mM iodoacetamide for 45 min in the dark and quenched with 10 mM DTT. Urea was diluted to 2 M prior to proteolytic digest with GluC at 37°C overnight. Digests were quenched to 1% formic acid and desalted over SOLAm HRP elution plates (Thermo Fischer Scientific). Peptides were analyzed on an Ultimate 3000 RSLCnano system coupled to an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fischer Scientific). Peptides were separated across a 70-min gradient of 6-30% acetonitrile in 0.1% formic acid over a 50-cm C18 column (ES803A, Thermo Fischer Scientific) and electrosprayed (1.9 kV, 300°C) into the mass spectrometer with an EasySpray ion source (Thermo Fischer Scientific). Precursor ion scans (375-1,325 m / z) were obtained in the orbitrap at 120,000 resolution in profile. Data dependent fragment ion scans (n = 2 in 15 s, exclusion duration = 30 s) were acquired in the orbitrap following HCD fragmentation (35% NCE, 0.7 m / z isolation, 30,000 resolution). Raw data were searched against the JAK2 engineered sequence using SEQUEST, permitting a mass tolerance of ±10 ppm, 2 missed cleavages by GluC, and the following modifications: methionine oxidation, serine / threonine / tyrosine phosphorylation, cysteine carbamidomethylation, and covalent compound modifications.

[0184]

[0135] Activity results: The results confirm that the compounds were inhibitors of JAK2:

[0185] Table 2. Activity results for compounds of formula (I) with primary labelling sites on the JAK2 PK domain. Atty. Dckt. No. DFCI 3463.W01WO Atty. Dckt. No. DFCI 3463.W01WO

[0186] ]SI: Selectivity Index.2NT: Not Tested. 'ND: Not Determined.

[0187]

[0136] As shown in Table 2, the acrylamide series provided JAK2 pseudokinase domain inhibitors with excellent selectivity of up to 200-fold for the pseudokinase domain over the kinase domain (K / PK) and up to 600-fold for the V617F mutant over the kinase domain (K / PKV617F). It also provided compounds that targeted cysteine residues such as C618, C644, C675, C723, C747 and / or C787. Surprisingly, some acrylamide compounds displayed excellent binding and selectivity for JAK2 pseudokinase domain, but showed little or no covalent binding Atty. Dckt. No. DFCI 3463.W01WO to Cys675. For example, Compound 17 displayed no detectable covalent binding. Crystallographic studies confirmed that Compound 17 does not form a covalent interaction with Cys675 of the JAK2 pseudokinase domain (data not shown). Reversible analogues of Compounds 16 and 17, 16-rev and 17-rev, which lacked the acrylamide functionality, also provided excellent selectivity (70 to 155-fold; data not shown). This confirmed that covalent binding was not essential within this series, even if it provides additional benefits (Sutanto et al. RSC Med. Chem., 2020, 11:876-84).

[0188]

[0137] Based on the structure of the JAK2 pseudokinase domain complexed to Compound 17, it was hypothesized that warheads targeting lysine residues might be particularly suited to producing covalent inhibitors targeting the same binding cleft within the JAK2 pseudokinase domain. Analogues with sulfonyl fluoride, sulfurofluoridate, aldehyde and other warheads were produced. It was surprisingly identified that certain lysine residues can be covalently targeted using a new class of compounds with sulfonyl fluoride, sulfurofluoridate or aldehyde moieties. Other potential-lysine binding moieties failed to covalently bind. As shown in Table 2, many of the lysine binding compounds displayed affinities in the nanomolar range. Covalent binding of one compound (24) to K677 was confirmed by crystallography (Figure 1).

[0189]

[0138] Therefore, this series of compounds provides selective inhibitors of the pseudokinase domain of JAK2 and / or inhibitors that covalently bind to different residues within the pseudokinase domain, in particular lysine residues. Such compounds might provide orthogonal therapeutic options to currently available JAK2 inhibitors.

Claims

Atty. Dckt. No. DFCI 3463.W01WOCLAIMS1. A compound of formula (I), or a pharmaceutically acceptable salt thereof,wherein:Ri is -SO2NH2, -CONHCH3 or -CON(CH3)2;R2 isR3 is H; m is 1 or 0;A is a 5- or 6-membered aryl or heteroaryl ring; n is 0, 1, or 2; each Y is independently a halide;Z is selected from the group consisting of -SO2F, -OSO2F or -CHO.

2. The compound of formula (I) according to claim 1, wherein R2 is:wherein Yi, Y2 and Y3are independently H or a halide, preferably Cl, F or H, more preferably H or F.

3. The compound of formula (I) according to claim 2, wherein R2 is:Atty. Dckt. No. DFCI 3463.W01WO4. The compound of formula (I) according to claim 2 or 3, wherein Z is -SO2F or -OSO2F;Yi is H or F;Y2 is H or F; and / orY3 is H or F; preferably H.

5. The compound of formula (I) according to claim 2 or 3, wherein Z is -CHO;Yi is F;Y2 is H; and / orY3is F.

6. The compound of formula (I) according to claim 2, wherein R2 is:

7. The compound of formula (I) according to claim 6, wherein:Yi is H;Y2is H;Y3is H; and / orZ is -OSO2F.Atty. Dckt. No. DFCI 3463.W01WO8. A compound, or a pharmaceutically acceptable salt thereof, according to claim 1, wherein the compound is selected from the group consisting of:

9. A compound of formula (I), or a pharmaceutically acceptable salt thereof:whereinRi is -SO2NH2, -C(=O)NH(CH3) or -C(=O)N(CH3)2; one of R2 or R3 is H and the other is:Atty. Dckt. No. DFCI 3463.W01WOm is 0 or 1;Xi and X2 are each independently selected from the group consisting of F, Cl, and H; and wherein one of X3 or X4 is H and the other is selected from the group consisting of:wherein n is 0, 1, 2 or 3; wherein p is 0 or 1; or wherein X3 and X4 join together so that R2 or R3 is:Atty. Dckt. No. DFCI 3463.W01WOwith the proviso that the following compounds are excluded:

10. The compound, or a pharmaceutically acceptable salt thereof, according to claim 9, wherein one of X3 or X4 is H and the other is selected from the group consisting ofAtty. Dckt. No. DFCI 3463.W01WO preferablyand / or preferably wherein Ri is -SO2NH2; and / or preferably wherein each of Xi and X2 isF; and / or preferably wherein m is 0; or wherein X3 and X4 join together so that one of R2 or R3 is:; preferably wherein each of Xi and X2 H; and / or preferably wherein Ri is -C(=O)NH(CH3) or -C(=O)N(CH3)2; and / or preferably wherein m is 0.

11. The compound, or a pharmaceutically acceptable salt thereof, according to any one of claim 9 or 10, wherein R2 is H and R3 is:such that the compound of formula (I) forms a compound of formula (II):Atty. Dckt. No. DFCI 3463.W01WO12. The compound, or a pharmaceutically acceptable salt thereof, according to any one of claim 9 to 11, wherein R3 is H and R2 is:so that the compound of formula (I) forms a compound of formula (III):

13. The compound, or a pharmaceutically acceptable salt thereof, according to any one of claim 9 to 12, wherein Ri is -SO2NH2 so that the compound of formula (I) forms a compound of formula (la):Atty. Dckt. No. DFCI 3463.W01WO preferably wherein R3 is H and R2 is:so that the compound of formula (I) forms a compound of formula (Illa):

14. The compound, or a pharmaceutically acceptable salt thereof, according to any preceding claim, wherein m is 0.

15. A compound, or a pharmaceutically acceptable salt thereof, according to claim 9, wherein the compound is selected from the group consisting of:Atty. Dckt. No. DFCI 3463.W01WOAtty. Dckt. No. DFCI 3463.W01WO16. A pharmaceutical composition comprising a compound according to any one of claims 1 to 15 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

17. A method of treating a disease treatable by inhibiting Janus kinase 2 comprising administration to a patient in need thereof a therapeutically effective amount of a compound according to any one of claims 1 to 15 or pharmaceutically acceptable salt thereof.

18. A compound according to any one of claims 1 to 15 or pharmaceutically acceptable salt thereof for use in treating a disease, preferably a disease treatable by inhibiting Janus kinase 2.

19. Use of a compound according to any one of claims 1 to 15 or pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a disease, preferably a disease treatable by inhibiting Janus kinase 2.

20. The method, compound for use, or use according to any one of claims 17 to 19, wherein the disease is associated with a V617F mutation in the Janus kinase 2 pseudokinase domain, preferably wherein the disease a myeloproliferative neoplasm (MPN), preferably is selected from the group consisting of chronic myeloid leukemia (CML), chronic neutrophilic leukemia (CNL), polycythemia vera (PV), primary myelofibrosis (PMF; e.g., the prefibrotic stage or the overt fibrotic stage), essential thrombocythemia (ET), chronic eosinophilic leukemia (not otherwise specified), and unclassifiable myeloproliferative neoplasm (MPN-U).

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