WO2016205942A1 - Hpk1 inhibitors and methods of using same - Google Patents

Hpk1 inhibitors and methods of using same Download PDF

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Publication number
WO2016205942A1
WO2016205942A1 PCT/CA2016/050734 CA2016050734W WO2016205942A1 WO 2016205942 A1 WO2016205942 A1 WO 2016205942A1 CA 2016050734 W CA2016050734 W CA 2016050734W WO 2016205942 A1 WO2016205942 A1 WO 2016205942A1
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Prior art keywords
optionally substituted
compound
membered
alkyl
heterocyclyl
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PCT/CA2016/050734
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French (fr)
Inventor
Peter Brent Sampson
Narendra Kumar B. Patel
Heinz W. Pauls
Sze-Wan Li
Grace Ng
Radoslaw Laufer
Yong Liu
Yunhui Lang
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University Health Network
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University Health Network
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Priority to EA201890059A priority Critical patent/EA035421B1/en
Priority to RS20210675A priority patent/RS61919B1/en
Priority to ES16813437T priority patent/ES2872555T3/en
Priority to LTEP16813437.7T priority patent/LT3322711T/en
Priority to MX2018000048A priority patent/MX386480B/en
Priority to MA42456A priority patent/MA42456B1/en
Priority to US15/738,286 priority patent/US10501474B2/en
Priority to PL16813437T priority patent/PL3322711T3/en
Priority to DK16813437.7T priority patent/DK3322711T3/en
Priority to BR112017027241-5A priority patent/BR112017027241B1/en
Priority to SI201631189T priority patent/SI3322711T1/en
Priority to KR1020237042987A priority patent/KR102740623B1/en
Priority to BR122023006150-0A priority patent/BR122023006150B1/en
Priority to AU2016282289A priority patent/AU2016282289B2/en
Priority to CN201680048058.0A priority patent/CN107922431B/en
Priority to SM20210324T priority patent/SMT202100324T1/en
Priority to EP16813437.7A priority patent/EP3322711B1/en
Application filed by University Health Network filed Critical University Health Network
Priority to MDE20180529T priority patent/MD3322711T2/en
Priority to HK18114909.1A priority patent/HK1255833B/en
Priority to CN202110534553.5A priority patent/CN113214287B/en
Priority to IL283353A priority patent/IL283353B/en
Priority to KR1020187001327A priority patent/KR102614872B1/en
Priority to NZ738268A priority patent/NZ738268B2/en
Priority to HRP20210895TT priority patent/HRP20210895T1/en
Priority to CA2989684A priority patent/CA2989684C/en
Priority to JP2017566338A priority patent/JP6898868B2/en
Publication of WO2016205942A1 publication Critical patent/WO2016205942A1/en
Priority to IL256250A priority patent/IL256250B/en
Anticipated expiration legal-status Critical
Priority to US16/705,458 priority patent/US11059832B2/en
Priority to CY20211100498T priority patent/CY1124489T1/en
Priority to US17/372,717 priority patent/US20220002313A1/en
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4355—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having oxygen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4365—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system having sulfur as a ring hetero atom, e.g. ticlopidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5355—Non-condensed oxazines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07—Optical isomers

Definitions

  • Hematopoietic progenitor kinase 1 is a hematopoietic cell-restricted Ste20 serine/threonine kinase.
  • HPKl kinase activity can be induced by activation signals generated by various different cell surface receptors found in hematopoietic cells upon ligand engagement.
  • Ligand engagement or antibody-mediated crosslinking of T cell receptors (TCR), B cell antigen receptor (BCR) Liou et al, 2000, Immunity 12:399), transforming growth factor ⁇ receptor (TGF ⁇ R) (Wang et al., 1997. J. Biol. Chem. 272:22771; Zhou et al, 1999, J.
  • HPKl acts as a down-modulator of T and B cell functions through the AP-1, NFKB, Erk2, and Fos pathways; for example, HPKl has been implicated as a negative regulator of signal transduction in T-cells through phosphorylation and activation of the T-cell receptor adaptor protein SLP-76 (Di Bartolo et al., 2007, J. Exp. Med. 204:681), which leads to subsequent downregulation of the AP-1 and Erk2 pathways.
  • SLP-76 T-cell receptor adaptor protein
  • BCR B-cell receptor
  • HPKl is now viewed as a possible target for therapeutic intervention.
  • HPKl can be a novel target for cancer immunotherapy (Sawasdikosol et al., Immunol Res. 2012 Dec;54(l-3):262-5).
  • targeted disruption of HPKl alleles confers T cells with an elevated Thl cytokine production in response to TCR engagement.
  • HPKl (-/-) T cells proliferate more rapidly than the haplotype- matched wild-type counterpart and are resistant to prostaglandin E2 (PGE(2))-mediated suppression.
  • PGE(2) prostaglandin E2
  • mice that received adoptive transfer of HPKl (-/-) T cells became resistant to lung tumor growth.
  • the loss of HPKl from dendritic cells (DCs) endows them with superior antigen presentation ability, enabling HPKl (-/-) DCs to elicit a more potent anti-tumor immune response when used as cancer vaccine.
  • HPKl When evaluating if a small-molecule inhibitor of HPKl would capture the phenotype of mice with targeted disruption of the gene, it is important to consider the non-catalytic roles of the protein.
  • full-length HPKl can promote TCR-mediated activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF- ⁇ ) pathway
  • the catalytically inactive cleavage product HPK1-C can suppress NF- ⁇ activation upon TCR restimulation, leading to activation-induced cell death (AICD) (Brenner et al, EMBO J. 2005, 24:4279).
  • HPKl Taking together the catalytic and non-catalytic roles of HPKl, it is possible that blocking the HPKl kinase activity with a small-molecule inhibitor may promote activation of B-and T-cells, leading to superior anti-tumor immunity, while also facilitating AICD, helping to maintain peripheral immune tolerance.
  • the exact effects of an HPKl inhibitor would be borne out by testing in mouse models of cancer, such as syngeneic tumor xenografts. Given that HPKl is not expressed in any major organs, outside the hematopoietic system, it is less likely that an inhibitor of HPKl kinase activity would cause any serious side effects.
  • thienopyridinone compounds are HPKl inhibitors (see Example B). They also have inhibitory activities against FLT3 and LCK (see Example C). Additionally, it has been demonstrated that certain thienopyridinone compounds as HPKl inhibitors alone, and in combination with anti-PD-1 antibodies are effective in preclinical models with certain cancer cell types (see Example E).
  • the particular combination therapies disclosed herein demonstrate surprising biological activity with significant anticancer effects. Specifically, with the combination of HPKl inhibitors and anti-PD-1 antibodies, significant responses following PD-1/PD-L1 blockade have now been demonstrated in CT26. WT colon carcinoma. Based on these discoveries, thienopyridinone compounds, pharmaceutical compositions thereof, and methods of using the same are disclosed herein.
  • One embodiment of the invention is a compound represented by Structural Formula
  • Another embodiment of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and a compound represented by Structural Formula (I) described above or a pharmaceutically acceptable salt thereof.
  • Another embodiment of the invention is a method of treating a subject with a disease which can be regulated by HPKl comprising administering to the subject an effective amount of a compound of Structural Formula (I) or a pharmaceutically acceptable salt thereof.
  • Another embodiment of the invention is a method of inhibiting HPKl activity in a subject in need of inhibition of HPKl activity, comprising administering to the subject an effective amount of a compound represented by Structural Formula (I) or a pharmaceutically acceptable salt thereof.
  • Another embodiment of the invention is a compound represented by Structural Formula (I) or a pharmaceutically acceptable salt thereof for use in therapy.
  • the therapy is for treating a subject with cancer.
  • the therapy is for inhibiting HPKl activity in a subject in need of inhibition of HPKl activity.
  • Another embodiment of the invention is the use of a compound represented by Structural Formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a subject with cancer.
  • a compound represented by Structural Formulas (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for inhibiting HPKl activity in a subject in need of inhibition of HPKl activity.
  • the present invention is also directed to a method of treating a subject with cancer, comprising administering to the subject an effective amount of a HPKl inhibitor (e.g., a compound represented by Structural Formula (I)), or a pharmaceutically acceptable salt thereof, and an effective second anti-cancer treatment (e.g. , a chemotherapeutic agent, a targeted therapeutic agent, radiation or surgery).
  • the second anti-cancer treatment is a PD-1 inhibitor.
  • the present invention is also directed to a method of treating a subject with cancer, comprising administering to the subject an effective amount of a HPKl inhibitor (e.g., a compound represented by Structural Formula (I)), or a pharmaceutically acceptable salt thereof, and an effective amount of an immunomodulatory agent such as a checkpoint inhibitor (e.g. , anti-PD-1 antibody, anti-CTLA4 antibody or anti-PD-Ll antibody ) or an inhibitor of tryptophan oxidation (e.g. IDOl, ID02 or TD02 inhibitor).
  • a checkpoint inhibitor e.g. , anti-PD-1 antibody, anti-CTLA4 antibody or anti-PD-Ll antibody
  • an inhibitor of tryptophan oxidation e.g. IDOl, ID02 or TD02 inhibitor
  • the immunomodulatory agent is anti-PD-1 antibody.
  • the present invention further provides the use of a HPKl inhibitor (e.g., a compound represented by Structural Formula (I), or a pharmaceutically acceptable salt thereof), for the manufacture of a medicament for the treatment of a subject with cancer, in combination with a PD-1 inhibitor such as nivolumab, pembrolizumab, pidilizumab, BMS 936559, MPDL3280A, MSB0010718C or MEDI4736.
  • a PD-1 inhibitor such as nivolumab, pembrolizumab, pidilizumab, BMS 936559, MPDL3280A, MSB0010718C or MEDI4736.
  • the PD-1 inhibitor is nivolumab.
  • the PD-1 inhibitor is pembrolizumab.
  • the PD-1 inhibitor is anti-PDl antibody.
  • the HPKl inhibitor is admininstered with an effective amount of one or more other anti-cancer therapies, and preferably in combination with PD-1 inhibitor.
  • Figure 1 shows the inhibitory effect of compound example A30 against SLP-76 serine 376 phosphorylation in a-CD3 stimulated Jurkat E6.1 cells.
  • Figure 2 is a graph illustrating the tumour growth inhibition percentage following administration of compound Al alone and in combination with an anti-PDl antibody.
  • Figure 3 shows the effect of compound example A30 in the EAE disease progression model.
  • the invention is directed to a compound represented by Formula (I):
  • one of Xi, X 2 , and X3 is S, the other two are each independently CR, wherein R is -H, -F, -CI, -Br, -CN, -NH3 ⁇ 4 -OH, optionally substituted (Ci-C 6 )alkyl, optionally substituted (Ci- Ce)alkoxy, optionally substituted -(CH2) n (C3-Cio)cycloalkyl, optionally substituted -(CH 2 ) n -3- 7 membered monocyclic heterocyclyl, optionally substituted -(CH 2 ) n phenyl, optionally substituted -(CH 2 ) n -5-7 membered monocyclic heteroaryl, optionally substituted -(CH 2 ) n - bridged (C6-Ci 2 )cycloalkyl, optionally substituted -(CH 2 ) n -6-12 membered bridged heterocyclyl, optionally substituted -(
  • Ri is -NR a R b or -OR al ;
  • R a for each occurrence is independently -H, optionally substituted (Ci-Ce)alkyl, optionally substituted -(CH 2 ) n (C3-Cio)cycloalkyl, optionally substituted -(CH 2 ) n -3-10 membered heterocyclyl, optionally substituted -(CH 2 ) n (C6-Cio)aryl, optionally substituted - (CH 2 ) n -5-10 membered heteroaryl, optionally substituted -(CH 2 ) n -bridged (C6-Ci 2 )cycloalkyl, or optionally substituted -(CH 2 ) n -6-12 membered bridged heterocyclyl;
  • R b for each occurrence is independently -H or -(Ci-C6)alkyl; or,
  • R a and R b together with the nitrogen to which they are attached, form optionally substituted -(C3-Cio)heterocyclyl;
  • R al for each occurrence is independently -H, optionally substituted (Ci-C6)alkyl, optionally substituted (C3-Cio)cycloalkyl, optionally substituted 3-10 membered heterocyclyl, optionally substituted (C6-Cio)aryl, or optionally substituted 3-10 membered heteroaryl; or
  • R 2 and R3 are each independently -H or -(Ci-C6)alkyl;
  • R.4 and Rs are each independently -H, optionally substituted (Ci-C6)alkyl, optionally substituted (C3-Cio)cycloalkyl, optionally substituted 3-10 membered heterocyclyl, optionally substituted (C6-Cio)aryl, optionally substituted 5-10 membered heteroaryl, optionally substituted bridged (C6-Ci2)cycloalkyl, or optionally substituted 6-12 membered bridged heterocyclyl; or
  • R.4 and Rs together with the nitrogen to which they are attached, form optionally substituted 4-10 membered heterocyclyl, optionally substituted 5-10 membered heteroaryl, or optionally substituted 6-12 membered bridged heterocyclyl;
  • R6 for each occurrence is independently -F, -CI, -Br, -CN, -NH3 ⁇ 4 -OH, -(Ci-C6)alkyl, -(Ci-C 6 )haloalkyl, -(C 2 -C 6 )alkenyl, -(C 2 -C 6 )alkynyl, (C 3 -C 6 )cycloalkyl, -(Ci-C 6 )alkoxy, -(Ci- C 6 )haloalkoxy, -(Ci-C 6 )alkylene-OH, or -(Ci-C 6 )alkylene-NH 2 ;
  • n 0, 1, 2, or 3;
  • n 0, 1, or 2.
  • the invention provides a compound represented by structural formula (I-A)-(I-C), (II-A)-(II-C), or (III-A)-(III-C):
  • the invention provides a compound represented by structural formula (I), (I-A)-(I-C), (II-A)-(II-C), or (III-A)-(III-C), wherein R 4 and R 5 , together with the nitrogen to which they are attached, form 4-7 membered monocyclic heterocyclyl or 6-12 membered bridged heterocyclyl, wherein the 4-7 membered monocyclic heterocyclyl or 6-12 membered bridged heterocyclyl is optionally substituted with 1-3 groups selected from -F, - CI, -Br, -CN, -Nth, -OH, oxo, -(Ci-C 4 )alkyl, -(Ci-C 4 )haloalkyl, -(Ci-C 4 )alkoxy, -(Ci- C 4 )haloalkoxy, -(Ci-C 4 )alkylene-OH, or -(Ci-C 4
  • the invention provides a compound represented by structural formula (I), (I-A)-(I-C), (II-A)-(II-C), or (III-A)-(III-C), wherein R a for each occurrence is independently -H, -(Ci-Ce)alkyl, -(CH 2 ) hinder-(C3-C 7 )cycloalkyl, -(CH 2 ) admir-4-7 membered monocyclic heterocyclyl, -(CH2) n -bridged (C6-Ci2)cycloalkyl, optionally substituted -(CH2) n - 5-10 membered heteroaryl; or -(CH2) n -6-12 membered bridged heterocyclyl, wherein -(Ci- C6)alkyl, -(CH2) n -(C3-C7)cycloalkyl, -(CH2) n -4-7 membered monocyclic heterocyclyl, - (CH 2
  • the invention provides a compound represented by structural formula (I), (I-A)-(I-C), (II-A)-(II-C), or (III-A)-(III-C), wherein R is H, -F, -CI, -Br, -OH, - (Ci-C 4 )alkyl, -(Ci-C 4 )haloalkyl, -(Ci-C 4 )alkoxy, -(Ci-C )alkylene-OH or 4-7 membered monocyclic heterocyclyl optionally substituted with 1-3 groups selected from -F, -CI, -Br, - OH, -(Ci-C 4 )alkyl, -(Ci-C 4 )haloalkyl, or -(Ci-C 4 )alkoxy, and values for the remainder of the variables are as described above for Structural Formula (I) or in the third or fourth embodiment.
  • R is H, -F
  • the invention provides a compound represented by structural formula (I), (I-A)-(I-C), (II-A)-(II-C), or (III-A)-(III-C), wherein R 4 and R 5 , together with the nitrogen to which they are attached, form -N-alkyl-piperazinyl or morpholinyl, wherein the piperazinyl or morpholinyl is optionally substituted with 1-2 groups selected from -F, -CI, - Br,-OH, -(Ci-C 4 )alkyl, -(Ci-C 4 )haloalkyl, or -(Ci-C 4 )alkoxy, and values for the remainder of the variables are as described above for Structural Formula (I), or in the third, fourth, or fifth embodiment.
  • the invention provides a compound represented by structural formula (I), (I-A)-(I-C), (II-A)-(II-C), or (III-A)-(III-C), wherein R a for each occurrence is independently -H, -(CH2) n -(C3-C6)cycloalkyl, -(CH2) n -3-6 membered heterocyclyl, wherein the -(CH2) n -(C3-C6)cycloalkyl or -(CH2) n -3-6 membered heterocyclyl is optionally substituted with 1-3 groups selected from -F, -CI, -Br, -CN, -NH2, -OH, -(Ci- C 4 )alkyl, or -(Ci-C 4 )alkoxy; and n is 0 or 1, and values for the remainder of the variables are as described above for Structural Formula (I), or in the third, fourth, fifth, or sixth
  • the invention provides a compound represented by structural formula (I), (I-A)-(I-C), (II-A)-(II-C), or (III-A)-(III-C), wherein R is H, -(Ci- C4)alkyl, -(Ci-C4)alkoxy, N-piperazinyl optionally substituted with -C02-(Ci-C4)alkyl, and values for the remainder of the variables are as described above for Structural Formula (I), or in the third, fourth, fifth, sixth, or seventh embodiment.
  • R is H.
  • the invention provides a compound represented by structural formula (I), (I-A)-(I-C), (II-A)-(II-C), or (III-A)-(III-C), wherein R 4 and R 5 , together with the nitrogen to which they are attached, form -N-methyl-piperazinyl or morpholinyl, both of which are optionally substituted with one or two methyl, and values for the remainder of the variables are as described above for Structural Formula (I), or in the third, fourth, fifth, sixth, seventh, or eighth embodiment.
  • the invention provides a compound represented by structural formula (I), (I-A)-(I-C), (II-A)-(II-C), or (III-A)-(III-C), wherein R a for each occurrence is independently -H; -(C3-Ce)cycloalkyl optionally substituted with -OH; -(CH2) n -tetrahydro- 2H-pyran; morpholinyl; piperidinyl optionally substituted with -F, -OH or methyl; or tetrahydrofuran; and n is 0 or 1, and values for the remainder of the variables are as described above for Structural Formula (I), or in the third, fourth, fifth, sixth, seventh, eighth or ninth embodiment.
  • the invention also includes the compounds depicted by structure and/or described by name in the Exemplification.
  • the invention includes both the neutral form (free base) of these compounds as well as pharmaceutically acceptable salts thereof.
  • Treatments with and/or uses of these compounds includes the neutral form of these compounds as well as pharmaceutically acceptable salts thereof.
  • alkyl used alone or as part of a larger moiety, such as “alkoxy” or “haloalkyl” and the like, means saturated aliphatic straight-chain or branched monovalent hydrocarbon radical. Unless otherwise specified, an alkyl group typically has 1-6 carbon atoms, i.e. (Ci-C6)alkyl. As used herein, a "(Ci-C6)alkyl” group means a radical having from 1 to 6 carbon atoms in a linear or branched arrangement. Examples include methyl, ethyl, n- propyl, wo-propyl etc.
  • Alkoxy means an alkyl radical attached through an oxygen linking atom, represented by -O-alkyl.
  • (Ci-C4)alkoxy includes methoxy, ethoxy, propoxy, and butoxy.
  • haloalkyl and haloalkoxy means alkyl or alkoxy, as the case may be, substituted with one or more halogen atoms.
  • halogen means F, CI, Br or I.
  • the halogen in a haloalkyl or haloalkoxy is F.
  • Alkenyl means branched or straight-chain monovalent hydrocarbon radical containing at least one double bond. Alkenyl may be mono or polyunsaturated, and may exist in the E or Z configuration. Unless otherwise specified, an alkenyl group typically has 2-6 carbon atoms, i.e. (C2-Ce)alkenyl.
  • (C2-C6)alkenyl means a radical having from 2-6 carbon atoms in a linear or branched arrangement.
  • Alkynyl means branched or straight-chain monovalent hydrocarbon radical containing at least one triple bond. Unless otherwise specified, an alkynyl group typically has 2-6 carbon atoms, i.e. (C2-Ce)alkynyl.
  • (C2-C6)alkynyl means a radical having from 2-6 carbon atoms in a linear or branched arrangement.
  • Cycloalkyl means a saturated aliphatic cyclic hydrocarbon radical, typically containing from 3-8 ring carbon atoms, i.e., (C3-C8)cycloalkyl.
  • (C3-C8)cycloalkyl includes, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
  • bridged used alone or as part of a larger moiety as in “bridged cycloalkyl” or “bridged heterocyclyl” refers to a ring system which includes two rings that share at least three adjacent ring atoms.
  • Bridged cycloalkyl typically contains 6-12 ring carbon atoms.
  • Bridged heterocyclyl typically have 6-12 ring atoms selected from carbon and at least one (typically 1 to 4, more typically 1 or 2) heteroatom (e.g. , oxygen, nitrogen or sulfur).
  • aryl used alone or as part of a larger moiety as in “arylalkyl”, “arylalkoxy”, or “aryloxyalkyl”, means a carbocyclic aromatic ring. It also includes a phenyl ring fused with a cycloalkyl group.
  • aryl may be used interchangeably with the terms “aryl ring” “carbocyclic aromatic ring”, “aryl group” and “carbocyclic aromatic group”.
  • An aryl group typically has six to fourteen ring atoms.
  • Examples includes phenyl, naphthyl, anthracenyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl and the like.
  • a "substituted aryl group” is substituted at any one or more substitutable ring atom, which is a ring carbon atom bonded to a hydrogen.
  • heteroaryl when used alone or as part of a larger moiety as in “heteroarylalkyl” or “heteroarylalkoxy”, refers to aromatic ring groups having five to fourteen ring atoms selected from carbon and at least one (typically 1 to 4, more typically 1 or 2) heteroatoms (e.g. , oxygen, nitrogen or sulfur).
  • Heteroaryl includes monocyclic rings and polycyclic rings in which a monocyclic heteroaromatic ring is fused to one or more other aryl, heterocyclyl or heteroaromatic rings.
  • heteroaryl includes monocyclic, bicyclic or tricyclic ring systems.
  • Examples of monocyclic 5-6 membered heteroaryl groups include furanyl (e.g., 2- furanyl, 3-furanyl), imidazolyl (e.g., N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), isoxazolyl (e.g. , 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl), oxadiazolyl (e.g. , 2-oxadiazolyl, 5- oxadiazolyl), oxazolyl (e.g. , 2-oxazolyl, 4-oxazolyl, 5-oxazolyl), pyrazolyl (e.g.
  • pyrrolyl e.g. , 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl
  • pyridyl e.g. , 2-pyridyl, 3- pyridyl, 4-pyridyl
  • pyrimidinyl e.g., 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl
  • pyridazinyl e.g., 3-pyridazinyl
  • thiazolyl e.g. , 2-thiazolyl, 4-thiazolyl, 5-thiazolyl
  • isothiazolyl triazolyl (e.g.
  • polycyclic aromatic heteroaryl groups include carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, isobenzofuranyl, indolyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, quinolinyl, isoquinolinyl, indazolyl, isoindolyl, acridinyl, or benzisoxazolyl.
  • a "substituted heteroaryl group” is substituted at any one or more substitutable ring atom, which is a ring carbon or ring nitrogen atom bonded to a hydrogen.
  • Heterocyclyl means a saturated or unsaturated non-aromatic 3-12 membered ring radical optionally containing one or more double bonds. It can be monocyclic, bicyclic, tricyclic, or fused.
  • the heterocycloalkyl contains 1 to 4 heteroatoms, which may be the same or different, selected from N, O or S.
  • the heterocyclyl ring optionally contains one or more double bonds and/or is optionally fused with one or more aromatic rings (e.g., phenyl ring).
  • the term “heterocyclyl” is intended to include all the possible isomeric forms.
  • heterocycloalkyl examples include, but are not limited to, azetidinyl , morpholinyl, thiomorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, dihydroimidazole, dihydrofuranyl, dihydropyranyl, dihydropyridinyl, dihydropyrimidinyl, dihydrothienyl, dihydrothiophenyl, dihydrothiopyranyl, tetrahydroimidazole, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, and tetrahydrothiopyrany
  • polycyclic heterocycloalkyl groups include dihydroindolyl, dihydroisoindolyl, dihydrobenzimidazolyl, dihydrobenzothienyl, dihydrobenzofuranyl, dihydroisobenzofuranyl, dihydrobenzotriazolyl, dihydrobenzothiazolyl, dihydrobenzoxazolyl, dihydroquinolinyl, tetrahydroquinolinyl, dihydroisoquinolinyl, tetrahydroisoquinolinyl, dihydroindazolyl, dihydroacridinyl, tetrahydroacridinyl, dihydrobenzisoxazolyl, chroman, chromene, isochroman and isochromene.
  • Stereoisomers are compounds which differ only in their spatial arrangement.
  • a disclosed compound is named or depicted by structure without indicating stereochemistry, it is understood that the name or structure encompasses all possible stereoisomers, geometric isomers, including essentially pure stereo or geometric isomers, as well as combination thereof.
  • Stereoisomers are compounds that differ only in their spatial arrangement. Enantiomers are pairs of stereoisomers whose mirror images are not superimposable, most commonly because they contain an asymmetrically substituted carbon atom that acts as a chiral center. "Enantiomer” means one of a pair of molecules that are mirror images of each other and are not superimposable. Diastereomers are stereoisomers that contain two or more asymmetrically substituted carbon atoms. "Geometric isomers” are stereoisomers that differ in the orientation of substituent atoms in relationship to a carbon-carbon double bond, to a carbocyclyl ring, or to a bridged bicyclic system.
  • geometric isomeric purity of the named or depicted geometric isomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% pure by weight.
  • Geometric isomeric purity is determined by dividing the weight of the named or depicted geometric isomer in the mixture by the total weight of all of the geometric isomers in the mixture.
  • the named or depicted stereoisomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% by weight pure relative to all of the other stereoisomers. Percent by weight pure relative to all of the other stereoisomers is the ratio of the weight of one stereoisomer over the weight of the other stereoisomers.
  • the depicted or named enantiomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% by weight optically pure (also referred to as "enantiomerically pure"). Percent optical purity by weight is the ratio of the weight of the enantiomer over the weight of the enantiomer plus the weight of its optical isomer.
  • stereochemistry of a disclosed compound is named or depicted by structure, and the named or depicted structure encompasses more than one stereoisomer (e.g. , as in a diastereomeric pair), it is to be understood that one of the encompassed stereoisomers or any mixture of the encompassed stereoisomers are included. It is to be further understood that the stereoisomeric purity of the named or depicted stereoisomers at least 60%, 70%, 80%, 90%, 99% or 99.9% by weight pure relative to all of the other stereoisomers. The stereoisomeric purity in this case is determined by dividing the total weight in the mixture of the stereoisomers encompassed by the name or structure by the total weight in the mixture of all of the stereoisomers.
  • the name or structure encompasses one stereoisomer free of other stereoisomers, mixtures of stereoisomers, and mixtures of stereoisomers in which one or more stereoisomers is enriched relative to the other stereoisomer(s).
  • the name or structure may encompass one stereoisomer free of other diastereomers, mixtures of stereoisomers, and mixtures of stereoisomers in which one or more diastereomers is enriched relative to the other diastereomer(s).
  • Enantiomeric and diastereomeric mixtures can be resolved into their component enantiomers or stereoisomers by well-known methods, such as chiral-phase gas chromatography, chiral-phase high performance liquid chromatography, crystallizing the compound as a chiral salt complex, or crystallizing the compound in a chiral solvent.
  • Enantiomers and diastereomers can also be obtained from diastereomerically- or enantiomerically-pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.
  • Suitable pharmaceutically acceptable acid addition salts of the compounds described herein include salts of inorganic acids (such as hydrochloric acid, hydrobromic, phosphoric, metaphosphoric, nitric, and sulfuric acids) and of organic acids (such as acetic acid, benzenesulfonic, benzoic, ethanesulfonic, methanesulfonic, succinic, and trifluoroacetic acid acids).
  • inorganic acids such as hydrochloric acid, hydrobromic, phosphoric, metaphosphoric, nitric, and sulfuric acids
  • organic acids such as acetic acid, benzenesulfonic, benzoic, ethanesulfonic, methanesulfonic, succinic, and trifluoroacetic acid acids.
  • Compounds of the present teachings with acidic groups such as carboxylic acids can form pharmaceutically acceptable salts with pharmaceutically acceptable base(s).
  • Suitable pharmaceutically acceptable basic salts include ammonium salts, alkali metal salts (such as sodium and potassium salts) and alkaline earth metal salts (such as magnesium and calcium salts).
  • Compounds with a quaternary ammonium group also contain a counteranion such as chloride, bromide, iodide, acetate, perchlorate and the like.
  • Other examples of such salts include hydrochlorides, hydrobromides, sulfates, methanesulfonates, nitrates, acetates, succinates, benzoates and salts with amino acids such as glutamic acid.
  • Compounds described herein can inhibit HPK1.
  • compounds described herein are useful in the treatment of diseases or conditions associated with such kinases.
  • the compounds described herein are HPK1 inhibitors, and are useful for treating diseases, such as cancer, associated with such kinase(s).
  • Another aspect of the present teachings relates to a method of treating a subject with cancer comprising administering to the subject an effective amount of a compound described herein.
  • the compounds described herein inhibit the growth of a tumor.
  • Cancers that can be treated (including reduction in the likelihood of recurrence) by the methods of the present teachings include breast cancer, colorectal cancer, lung cancer, ovarian cancer, uterine cancer, prostate cancer, leukemias, lymphomas, brain cancer (including glioblastoma multiforme and neuroblastoma), head and neck cancer, pancreatic cancer, melanoma, hepatocellular carcinoma, renal cancer, and soft tissue sarcomas.
  • the cancer is breast cancer, colon cancer, and ovarian cancer.
  • the cancer is selected from leukemia, acute myeloid leukemia, chronic myelogenous leukemia, breast cancer, brain cancer, colon cancer, colorectal cancer, head and neck cancer, hepatocellular carcinoma, lung adenocarcinoma, metastatic melanoma, pancreatic cancer, prostate cancer, ovarian cancer and renal cancer.
  • the cancer is lung cancer, colon cancer, brain cancer, neuroblastoma, prostate cancer, melanoma, glioblastoma multiforme or ovarian cancer.
  • the cancer is lung cancer, breast cancer, colon cancer, brain cancer, neuroblastoma, prostate cancer, melanoma, glioblastoma multiforme or ovarian cancer.
  • the cancer is breast cancer, colon cancer and lung cancer. In another embodiment, the cancer is a breast cancer. In yet another embodiment, the cancer is a basal sub-type breast cancer or a luminal B subtype breast cancer. In yet another embodiment, the cancer is a basal sub-type breast cancer. In yet another embodiment, the basal sub-type breast cancer is ER (estrogen receptor), HER2 and PR (progesterone receptor) negative breast cancer. In yet another embodiment, the cancer is a soft tissue cancer. A "soft tissue cancer" is an art-recognized term that encompasses tumors derived from any soft tissue of the body.
  • soft tissue connects, supports, or surrounds various structures and organs of the body, including, but not limited to, smooth muscle, skeletal muscle, tendons, fibrous tissues, fatty tissue, blood and lymph vessels, perivascular tissue, nerves, mesenchymal cells and synovial tissues.
  • soft tissue cancers can be of fat tissue, muscle tissue, nerve tissue, joint tissue, blood vessels, lymph vessels, and fibrous tissues.
  • Soft tissue cancers can be benign or malignant. Generally, malignant soft tissue cancers are referred to as sarcomas, or soft tissue sarcomas.
  • soft tissue tumors including lipoma, lipoblastoma, hibernoma, liposarcoma, leiomyoma, leiomyosarcoma, rhabdomyoma, rhabdomyosarcoma, neurofibroma, schwannoma (neurilemoma), neuroma, malignant schwannoma, neurofibrosarcoma, neurogenic sarcoma, nodular tenosynovitis, synovial sarcoma, hemangioma, glomus tumor, hemangiopericytoma, hemangioendothelioma, angiosarcoma, Kaposi sarcoma, lymphangioma, fibroma, elastofibroma, superficial fibromatosis, fibrous histiocytoma, fibrosarcoma, fibromatosis, dermatofibrosarcoma protuberans (DFSP), malignant fibrous hist
  • the soft tissue cancer is a sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleomorphic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
  • the present teachings also provide methods of treating a subject with a disease comprising administering to the subject an effective amount of a compound represented by Structural Formula (I) in combination with an effective immunomodulatory therapy (also referred as immunotherapy).
  • Immunotherapy is the treatment of disease by using an immunomodulatory agent to induce, enhance, or suppress an immune response. Immunotherapies designed to elicit or amplify an immune response are classified as activation immunotherapies, while immunotherapies that reduce or suppress are classified as suppression immunotherapies.
  • the disease described herein is a cancer.
  • Immunomodulatory therapies used alone or in combination approaches, include i) immune checkpoint blockade inhibitors, including but not limited to anti-CTLA4 (cytotoxic T-lymphocyte-associated protein 4) antibodies (e.g. Ipilimumab), agents that disrupt the PD- 1/PD-Ll and PD-L2 interaction, e.g. Nivolumab (Opdivo - Bristol Myers Squibb), Pembrolizumab (Keytruda, KM-3475, Merck), Pidilizumab (CT-011, Cure Tech), BMS 936559 (BMS) and MPDL3280A (Roche); and other immune response inhibitory receptors e.g.
  • CTLA4 cytotoxic T-lymphocyte-associated protein 4
  • Ipilimumab cytotoxic T-lymphocyte-associated protein 4
  • agents that disrupt the PD- 1/PD-Ll and PD-L2 interaction e.g. Nivolumab (Opdivo - Bristol
  • the immunomodulatory agent used for the immunomodulatory therapy is a PD-1 inhibitor, for example, an anti-PDl antibody.
  • Programmed cell death protein 1 also known as PD-1 and CD279 (cluster of differentiation 279), is a protein that in humans is encoded by the PDCD1 gene.
  • PD-1 is a cell surface receptor that belongs to the immunoglobulin superfamily and is expressed on T cells and pro-B cells.
  • PD-1 binds two ligands, PD-L1 and PD-L2, both of which are members of the B7 family.
  • PD-1 and its ligands play an important role in down regulating the immune system by preventing the activation of T-cells, which in turn reduces autoimmunity and promotes self- tolerance.
  • the inhibitory effect of PD-1 is accomplished through a dual mechanism of promoting apoptosis(programmed cell death) in antigen specific T-cells in lymph nodes while simultaneously reducing apoptosis in regulatory T cells (suppressor T cells).
  • the PD-1 inhibitor used in the present invention includes, but is not limited to, nivolumab, pembrolizumab, pidilizumab, BMS 936559, MPDL3280A, MSB0010718C or MEDI4736.
  • BMS 936559, MPDL3280A, MSB0010718C, and MEDI4736 bind ligand PD-L1, all of which are antibodies.
  • Both nivolumab and pembrolizumab are approved by the Food and Drug Administration for treatment of unresectable or metastatic melanoma which no longer responds to other drugs.
  • Vaccination strategies include anti-microbial immunotherapy, which includes vaccination, involves activating the immune system to respond to an infectious agent.
  • T-cell therapy uses T cell-based cytotoxic responses to attack cancer cells.
  • T cells that have a natural or genetically engineered reactivity to a patient's cancer are generated in vitro and then transferred back into the cancer patient.
  • One study using autologous tumor- infiltrating lymphocytes was an effective treatment for patients with metastatic melanoma. This can be achieved by taking T cells that are found with the tumor of the patient, which are trained to attack the cancerous cells. These T cells are referred to as tumor-infiltrating lymphocytes (TIL) are then encouraged to multiply in vitro using high concentrations of IL-2, anti-CD3 and allo-reactive feeder cells. These T cells are then transferred back into the patient along with exogenous administration of IL-2 to further boost their anti-cancer activity.
  • TIL tumor-infiltrating lymphocytes
  • the present teachings also provide methods of treating a subject with a cancer comprising administering to the subject an effective amount of a compound represented by Structural Formula (I) in combination with an effective anti-cancer therapy.
  • the cancer is a metastatic cancer.
  • a "metastatic cancer” is a cancer that has spread from its primary site to other parts of the body.
  • the anti-cancer therapy described herein includes co-administration of an effective amount of a second anti-cancer agent together with a disclosed HPK-1 inhibitor.
  • An "anticancer agent” is a compound, which when administered in an effective amount to a subject with cancer, can achieve, partially or substantially, one or more of the following: arresting the growth, reducing the extent of a cancer (e.g., reducing size of a tumor), inhibiting the growth rate of a cancer, and ameliorating or improving a clinical symptom or indicator associated with a cancer (such as tissue or serum components) or increasing longevity of the subject.
  • the anti-cancer agents suitable for use in the methods described herein include any anti-cancer agents that have been approved for the treatment of cancer.
  • the anti-cancer agent includes, but is not limited to, a targeted antibody, an angiogenesis inhibitor, an alkylating agent, an antimetabolite, a vinca alkaloid, a taxane, a podophyllotoxin, a topoisomerase inhibitor, a hormonal antineoplastic agent and other antineoplastic agents.
  • the anti-cancer agent is a PD-1 inhibitor, for example, an anti-PDl antibody.
  • the anti-cancer agents that can be used in methods described herein include, but are not limited to, paclitaxel, docetaxel, 5-fluorouracil, trastuzumab, lapatinib, bevacizumab, letrozole, goserelin, tamoxifen, cetuximab, panitumumab, gemcitabine, capecitabine, irinotecan, oxaliplatin, carboplatin, cisplatin, doxorubicin, epirubicin, cyclophosphamide, methotrexate, vinblastine, vincristine, melphalan, cytarabine, etoposide, daunorubicin, bleomycin, mitomycin and adriamycin and a combination thereof.
  • the anti-cancer agent and the compound represented by Structural Formula (I) are administered contemporaneously.
  • the anti-cancer agent and the compound can be administered in the same formulation or in different formulations.
  • the compound and the additional anti-cancer agent are administered separately at different times.
  • treating a subject with a cancer includes achieving, partially or substantially, one or more of the following: arresting the growth, reducing the extent of the cancer (e.g., reducing size of a tumor), inhibiting the growth rate of the cancer, ameliorating or improving a clinical symptom or indicator associated with the cancer (such as tissue or serum components) or increasing longevity of the subject; and reducing the likelihood of recurrence of the cancer.
  • an "effective amount” means an amount when administered to the subject which results in beneficial or desired results, including clinical results, e.g., inhibits, suppresses or reduces the cancer (e.g., as determined by clinical symptoms or the amount of cancer cells) in a subject as compared to a control.
  • an effective amount of a compound taught herein varies depending upon various factors, such as the given drug or compound, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art.
  • An effective amount of a compound of the present teachings may be readily determined by one of ordinary skill by routine methods known in the art.
  • an effective amount of a compound taught herein ranges from about 0.1 to about 1000 mg/kg body weight, alternatively about 1 to about 500 mg/kg body weight. In another embodiment, an effective amount of a compound taught herein ranges from about 0.5 to about 5000 mg/m 2 , alternatively about from 5 to about 2500 mg/m 2 , and in another alternative from about 50 to about 1000 mg/m 2 .
  • the skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject suffering from cancer or reduce the likelihood of recurrence of a cancer. These factors include, but are not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the subject and other diseases present.
  • a "subject” is a mammal, preferably a human, but can also be an animal in need of veterinary treatment, e.g., companion animals (e.g. , dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, and the like) and laboratory animals (e.g. , rats, mice, guinea pigs, and the like).
  • companion animals e.g., dogs, cats, and the like
  • farm animals e.g., cows, sheep, pigs, horses, and the like
  • laboratory animals e.g. , rats, mice, guinea pigs, and the like.
  • the compounds taught herein can be administered to a patient in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art.
  • the compounds of the present teachings may be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump or transdermal administration and the pharmaceutical compositions formulated accordingly.
  • Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal and topical modes of administration. Parenteral administration can be by continuous infusion over a selected period of time.
  • the compounds taught herein can be suitably formulated into pharmaceutical compositions for administration to a subject.
  • the pharmaceutical compositions of the present teachings optionally include one or more pharmaceutically acceptable carriers and/or diluents therefor, such as lactose, starch, cellulose and dextrose.
  • Other excipients such as flavoring agents; sweeteners; and preservatives, such as methyl, ethyl, propyl and butyl parabens, can also be included. More complete listings of suitable excipients can be found in the Handbook of Pharmaceutical Excipients (5 th Ed., Pharmaceutical Press (2005)). A person skilled in the art would know how to prepare formulations suitable for various types of administration routes.
  • a compound of the present teachings may be incorporated with excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • solutions of a compound of the present teachings can generally be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • sterile aqueous solutions or dispersion of, and sterile powders of, a compound described herein for the extemporaneous preparation of sterile injectable solutions or dispersions are appropriate.
  • Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multi-dose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomizing device.
  • the sealed container may be a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use.
  • the dosage form comprises an aerosol dispenser, it will contain a propellant which can be a compressed gas such as compressed air or an organic propellant such as fluorochlorohydrocarbon.
  • the aerosol dosage forms can also take the form of a pump-atomizer.
  • the compounds of the present teachings can be formulated with a carrier such as sugar, acacia, tragacanth, or gelatin and glycerine, as tablets, lozenges or pastilles.
  • a carrier such as sugar, acacia, tragacanth, or gelatin and glycerine, as tablets, lozenges or pastilles.
  • the compounds described herein can be formulated in the form of suppositories containing a conventional suppository base such as cocoa butter.
  • the compounds of invention may be prepared by methods known to those skilled in the art, as illustrated by the general schemes and procedures below and by the preparative examples that follow. All starting materials are either commercially available or prepared by methods known to those skilled in the art and the procedures described below.
  • PoraPak ® Rxn CX refers to a commercial cation-exchange resin available from Waters.
  • Microwave reactions were performed with a Biotage Initiator microwave reactor. Reaction progress was generally monitored by LCMS (Bruker Exquire 4000 or Waters Acquity UPLC system). Flash column chromatographic purification of intermediates or final products was performed using a Biotage Isolera with KP-SIL or HP-SIL silica cartridges, or KP-NH basic modified silica and corresponding samplets. Reverse-phase HPLC purification was performed on a Varian PrepStar model SD-1 HPLC system with a Varian Monochrom 10 ⁇ C-18 reverse-phase column using a gradient of 10% MeOH/0.05% TFA-H 2 0 to 90% MeOH/0.05% TFA in H2O over a 40-min period at a flow rate of 40 mL/min.
  • Reverse phase purification was also performed using a Biotage Isolera equipped with a KP-C18-H column using a between 10-95% MeOH or CH3CN/ 0.1% TFA in H 2 0.
  • Proton NMRs were recorded on a Bruker 400 MHz spectrometer, and mass spectra were obtained using a Bruker Esquire 4000 spectrometer or Waters Acquity UPLC system.
  • the uncyclized addition adduct was separated by column chromatography, dissolved in THF and treated with KHMDS, LiHMDS, or LDA (3-5 equiv). The reaction was stirred at 45 °C for 1-4 h. The reaction was then cooled to rt and quenched with satd aq NH4CI. The aqueous layer was extracted with EtOAc or DCM, and the combined organic extracts were dried over MgS04, filtered and concentrated. Crude product was purified by column chromatography or prep- HPLC to give the desired product.
  • 1,4-diazepane (6.85 g, 60 mmol) and K2CO3 (8.28 g, 60 mmol) was heat at 90 °C for 20 h. After diluting with H 2 0 (500 mL) , it was extracted with EtOAc (60 mL x 3), concentrated and dried to give crude 5-(4-methyl-l,4- diazepan-l-yl)-2-nitroaniline as a dark red oil (12.50 g). NMR indicated a mixture of product and 5-chloro-2-nitroaniline (2: 1).
  • Step 1 Reagents (general method C): a mixture of 4-(4-methoxybenzyl)-6-(5 and/or 6-(4- methylpiperazin-l-yl)-l-((trifluoromethyl)sulfonyl)-lH-benzo[d]imidazol-2-yl)-5-oxo-4,5- dihydro-thieno[3,2-b]pyridin-7-yl trifluoromethanesulfonate (crude, 0.3 mmol), pyridin-4- ylmethanamine (0.09 mL, 0.89 mmol). MS ESI [M+H] + 724.2, calcd for
  • Step 2 Reagents (general method D): 4-(4-methoxybenzyl)-6-(5 and/or 6-(4-methylpiperazin- l-yl)-l-((trifluoromethyl)sulfonyl)-lH-benzo[d]imidazol-2-yl)-7-((pyridin-4- ylmethyl)amino)thieno[3,2-b]pyridin-5(4H)-one, TFA (4 mL), cone. HC1 (1 mL).
  • Step 2 The product of the previous reaction was filtered through PoraPak (2 g, using MeOH then 2 M NH 3 in MeOH) and dried.
  • the reaction was then cooled to rt, quenched with aq NH4CI, concentrated under reduced pressure and purified by prep HPLC. Filtration through PoraPak (2 g) and trituration with CH2CI2 afforded the title compound as a light yellow solid 3.6 mg (3 %).
  • Reagents (method A3): Step 1 : ethyl 2-(5-methyl-6-(4-methylpiperazin-l -yl)-lH- benzo[d]imidazol-2-yl)acetate (0. 17 g, 0.53 mmol), 2-aminothiophene-3-carbonitrile (72 mg, 0.53 mmol), LDA (1.0 M in THF/hexanes, 1.7 mL, 1.7 mmol) in anh THF (12 mL).
  • Step 2 LiHMDS (1.0 M in THF, 1.7 mL, 1.7 mmol) in anh THF (20 mL).
  • Reagents (method A3): Step ] : ethyl 2-(6-(morpholine-4-carbonyl)-lH- 3enzo[d]imidazol-2- yl)acetate (0.22 g, 0.70 mmol ) and 2-aminothiophene-3-carbonitrile (8' % mg, 0.70 mmol), LiHMDS (1.0 M in THF, 3.5 n lL, 3.5 mmol) in anh. THF (24 mL). Step 2: LiHMDS (1.0 M in THF, 1.2 mL, 1.2 mmol) in a nh THF (20 mL).
  • tert-butyl 4-(2-(2-ethoxy-2-oxoethyl)-lH-benzo[d]imidazol-6-yl)piperazine-l-carboxylate To a solution of tert-butyl 4-(3,4-diaminophenyl)piperazine-l-carboxylate (100 mg, 0.34 mmol) in EtOH (3 mL) was added ethyl 3-ethoxy-3-iminopropionate hydrochloride (190 mg, 0.68 mmol). The resulting mixture was heated at 60 °C for 3 h. After removal of solvents, it was diluted with DCM (10 mL), adjust pH ⁇ 8 with satd NaHC03 and separated.
  • A65 4-amino-5-(6-(4-(oxetan-3-yl)piperazin-l-yl)-lH-benzo[d]imidazol-2-yl)thieno[2,3- b]pyridin-6(7H)-one
  • a mixture of 4-amino-5-(6-(piperazin-l-yl)-lH- benzo[d]imidazol-2-yl)thieno[2,3-b]pyridin-6(7H)-one 45 mg, 0.123 mmol
  • oxetan-3-one 8.8 mg, 0.123
  • Step 2 Reagents (general method D): a mixture of 5-(6-((2S,6R)-2,6-dimethylmorpholino)-l-
  • MAP4K1 Active HPK1
  • GST-HPK1 (0.6 nM) was incubated in the presence of 12 ⁇ ATP (Sigma cat# A7699), 5 mM MOPS (pH 7.2), 2.5 mM ⁇ -glycerol-phosphate, 5 mM MgCb, 0.4 mM EDTA, 1 mM EGTA, 0.05 mM DTT, in a 96 well microtitre plate pre- coated with 0.5 ⁇ g/well bovine myelin basic protein (MBP) (Millipore, cat #13-110).
  • MBP bovine myelin basic protein
  • the reaction was allowed to proceed for 30 min, followed by 5 washes of the plate with Wash Buffer (phosphate buffered saline supplemented with 0.2% Tween 20), and incubation for 30 min with a 1 :3000 dilution of anti-phospho-threonine rabbit polyclonal antibody (Cell Signaling cat# 9381).
  • the plate was washed 5 times with wash buffer, incubated for 30 min in the presence of goat anti-rabbit horse radish peroxidase conjugate (BioRad cat# 1721019, 1:3000 concentration), washed an additional 5 times with wash buffer, and incubated in the presence of TMB substrate (Sigma cat# T0440).
  • the colorimetric reaction was allowed to continue for 5 min, followed by addition of stop solution (0.5 N H2SO4), and quantified by detection at 450 nm with a monochromatic plate reader (Molecular Devices M5).
  • XLfit4, IDBS non-linear 4 point logistic curve fit
  • FLT3 and LCK compound inhibition were determined using FRET based Z'-LYTE Kinase Assay Kit with Tyrosine 2 peptide as the substrate (Invitrogen cat # PV3191).
  • the FLT3 kinase assay was performed according to the manufacturer's suggested specifications with an ATP concentration of 940 ⁇ and 1 nM FLT3 (Invitrogen cat # PV3182) and 180 ⁇ ATP and 25 nM LCK (Invitrogen cat # P3043) for the LCK kinase reaction.
  • the % inhibition values were determined according to the manufacturer's directions and IC50 values were obtained using a non-linear 4 point logistic curve fit (XLfit4, IDBS).
  • IC50 value ranges for exemplary compounds are given.
  • the IC50 ranges are indicated as "A,” “B,” and “C,” for values less than or equal to 0.05 ⁇ ; those greater than 0.05 ⁇ and less than or equal to 0.5 ⁇ ; and those greater than 0.5 ⁇ , respectively.
  • Jurkat E6.1 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA), and maintained according to the supplier's instructions. Cells were washed three times and starved in RPMI 1640 medium supplemented with 0.5% fetal calf serum for 18 h at 37 °C. Serum starved cells were pretreated with the indicated concentration of inhibitor for 4 hours before stimulation with 10 ⁇ g/ml a-CD3 antibody (BioLegend, Inc., San Diego, CA) for 10 min at 37 °C.
  • ATCC American Type Culture Collection
  • VA Manassas, VA
  • Serum starved cells were pretreated with the indicated concentration of inhibitor for 4 hours before stimulation with 10 ⁇ g/ml a-CD3 antibody (BioLegend, Inc., San Diego, CA) for 10 min at 37 °C.
  • the cells were washed once in phosphate-buffered saline (pH 7.4) containing 10 mm sodium pyrophosphate, 10 mm sodium fluoride, 10 mm EDTA, and 1 mm sodium orthovanadate. Protein lysates were prepared using ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer. A total of 100 ⁇ g of cell lysate was loaded onto Bis-Tris gels (Life Technologies, Carlsbad, CA) with full-range molecular weight marker as a size reference, and resolved by SDS-PAGE electrophoresis.
  • RIPA radioimmunoprecipitation assay
  • Proteins were transferred to PVDF membrane (Millipore, Billerica, MA), blocked and probed with antibodies for phospho-SLP-76 (Ser376) (rabbit polyclonal #13177; Cell Signaling Technology Inc., Danvers, MA), SLP-76 (rabbit polyclonal #4958; Cell Signaling Technology Inc., Danvers, MA), phospho-ERK (mouse monoclonal sc-7383; Santa Cruz Biotechnology Inc., Santa Cruz, CA) and ERK1/2 (rabbit polyclonal 06-182; Millipore, Billerica, MA). Secondary antibodies were diluted 1 in 15,000 and incubated for lh at rt. Protein bands were visualized and quantified using Odyssey near infrared imager (LI-COR, Lincoln, NE).
  • LI-COR Odyssey near infrared imager
  • Table 2 below lists effects of representative compounds of the present invention against SLP- 76 serine 376 phosphorylation and ERK1/2 T202/Y204 phosphorylation in a-CD3 stimulated Jurkat E6.1 cells.
  • Example E Syngeneic CT26 cell line xenograft model.
  • the CT26 WT cell line which is an N-nitroso-N-methylurethane-(N MU) induced, mouse- derived, undifferentiated colon carcinoma cell line, was obtained from American Type Culture Collection (ATCC CRL-2638, Manassas, VA, DC, USA). Cells were grown in Roswell Park memorial Institute medium commonly referred to as RPMI 1640 Medium containing 4.5 g/L glucose, 0.11 g/L sodium pyruvate, 1.5 g/L sodium bicarbonate, L- glutamine& 2.385 g/L HEPES plus 10% fetal bovine serum.
  • mice Six to eight week old female BALB/c mice were purchased from Jackson Laboratories and received and acclimated at the MaRS-TMDT Animal Resources Centre for 1 week prior to the start of the experiment. The mice were fed ad libitum autoclaved water and Rodent Lab Diet (Harlan Teklad LM-485) consisting of 19% crude protein, 5% crude fat, and 5% crude fiber. Mice were housed in microisolator cages and maintained in an environment with a 12 h light cycle at 20-22°C and 40-60% humidity.
  • CT26 cells were harvested and re-suspended with serum free RPMI1640 to a concentration of lxl0 7 /mL and each mouse was injected subcutaneously with a volume of 0.1 mL containing lxlO 6 CT26 cells in the right rear flank.
  • animals were separated into five groups of eight animals per group such that each group contained animals bearing tumors of similar average size and treatment was initiated.
  • Example Al was dissolved in water to a concentration of 7.5 mg/mL or 15 mg/mL for dosing of the 75 mg/kg and the 150 mg/kg doses, respectively.
  • a rat IgG2b anti-PDl antibody BioXcell (NH, USA) was dosed used.
  • the five groups were treated with: i) 10 mL/kg water QD for 21 d administered by oral gavage (PO) plus 150 ⁇ g rat IgG2b isotype control antibody dosed by intraperitoneal (IP) injection on day 0, 3, 6 and 10 (the control arm); ii) 150 ⁇ g anti-PD-1 antibody dosed by intraperitoneal (IP) injection on day 0, 3, 6 and 10; iii) 75 mg/kg Example Al QD for 21 days administered PO; iv) 150 mg/kg Example Al QD for 21 days administered PO v) 150 mg/kg Example Al QD for 21 days administered PO plus 150 ⁇ g anti-PD-1 antibody dosed by intraperitoneal (IP) injection on day 0, 3, 6 and 10. Toxicity was evaluated by body weight measurements and clinical observations. Tumour measurements and body weights were taken three times per week. Percent tumor growth inhibition (TGI) was calculated by the formula:
  • %TGI 1 OO X [1 - (TVf, treated - TVi,treated)/(TVf,control - TVi, control)]
  • Tumour growth inhibition at day 21 is shown in figure 2.
  • a dose-dependent effect was observed in response to treatment with Example Al, with 75 mg/kg and 150 mg/kg QD inhibiting tumour growth by 44% and 64%, respectively.
  • Whilst the anti-PD-1 antibody alone resulted in an average TGI of 34%, when combined with 150 mg/kg QD Example Al, the TGI increased to 86%.
  • mice in efficacy experiments should be sacrificed when the tumour size is above 1500 mm 3 or if the bodyweight of the animal decreases or if the animals are displaying clinical signs that require termination for humane reasons.
  • the compound was well tolerated with all animals gaining weight over the course of the study and no animals were terminated due to clinical signs.
  • a tumour size of ⁇ 1500 mm 3 at day 21 was used as a cutoff to represent survival.
  • Example F EAE disease progression model
  • mice C57/BL6 mice were obtained from Jackson Laboratories. The Institutional Animal Care and Use Committee of the University Health Network approved all animal procedures. Mice were subcutaneously (SC) immunized with MOG35-55 peptide emulsified in Complete Freund's Adjuvant (CFA) supplemented with Mycobacterium tuberculosis. On days 0 and 2 after immunization, the mice were intraperitoneal (IP) injected with pertussis toxin. Clinical signs of EAE were monitored daily, according to the following criteria: 0, no disease; 1, decreased tail tone; 2, hind limb weakness or partial paralysis; 3, complete hind limb paralysis; 4, front and hind limb paralysis; 5, death, or sacrifice due to moribund state.
  • CFA Complete Freund's Adjuvant

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Abstract

Thienopyridinone compounds of Formula (I) and pharmaceutically acceptable salts thereof are described. In these compounds, one of X1; X2, and X3 is S and the other two are each independently CR, wherein R and all other variables are as defined herein. The compounds are shown to inhibit HPK1 kinase activity and to have in vivo antitumor activity. The compounds can be effectively combined with pharmaceutically acceptable carriers and also with other immunomodulatory approaches, such as checkpoint inhibition or inhibitors of tryptophan oxidation. Formula (I).

Description

HPKl INHIBITORS AND METHODS OF USING SAME
RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 62/184,348, filed June 25, 2015. The entire teachings of the aforementioned application are incorporated herein by reference.
BACKGROUND
Hematopoietic progenitor kinase 1 (HPKl) is a hematopoietic cell-restricted Ste20 serine/threonine kinase. HPKl kinase activity can be induced by activation signals generated by various different cell surface receptors found in hematopoietic cells upon ligand engagement. Ligand engagement or antibody-mediated crosslinking of T cell receptors (TCR), B cell antigen receptor (BCR) (Liou et al, 2000, Immunity 12:399), transforming growth factor β receptor (TGF^R) (Wang et al., 1997. J. Biol. Chem. 272:22771; Zhou et al, 1999, J. Biol. Chem. 274: 13133), erythropoietin receptor (EPOR) (Nagata et al, 1999, Blood 93:3347), and Fas (Chen et al, 1999, Oncogene 18:7370) can induce HPKl kinase activity. Each receptor utilizes unique, but sometimes overlapping, signaling mechanisms to activate HPKl . HPKl acts as a down-modulator of T and B cell functions through the AP-1, NFKB, Erk2, and Fos pathways; for example, HPKl has been implicated as a negative regulator of signal transduction in T-cells through phosphorylation and activation of the T-cell receptor adaptor protein SLP-76 (Di Bartolo et al., 2007, J. Exp. Med. 204:681), which leads to subsequent downregulation of the AP-1 and Erk2 pathways. In B-cells, HPKl downregulates B-cell receptor (BCR) signaling through phosphorylation of the SLP-76 paralog BLINK (Wang et al, 2012, J. Biol. Chem. 287: 11037).
Thus, HPKl is now viewed as a possible target for therapeutic intervention. For example, it has been reported that HPKl can be a novel target for cancer immunotherapy (Sawasdikosol et al., Immunol Res. 2012 Dec;54(l-3):262-5). Specifically, targeted disruption of HPKl alleles confers T cells with an elevated Thl cytokine production in response to TCR engagement. HPKl (-/-) T cells proliferate more rapidly than the haplotype- matched wild-type counterpart and are resistant to prostaglandin E2 (PGE(2))-mediated suppression. Most strikingly, mice that received adoptive transfer of HPKl (-/-) T cells became resistant to lung tumor growth. Also, the loss of HPKl from dendritic cells (DCs) endows them with superior antigen presentation ability, enabling HPKl (-/-) DCs to elicit a more potent anti-tumor immune response when used as cancer vaccine.
When evaluating if a small-molecule inhibitor of HPKl would capture the phenotype of mice with targeted disruption of the gene, it is important to consider the non-catalytic roles of the protein. In particular, while full-length HPKl can promote TCR-mediated activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κΒ) pathway, the catalytically inactive cleavage product HPK1-C can suppress NF-κΒ activation upon TCR restimulation, leading to activation-induced cell death (AICD) (Brenner et al, EMBO J. 2005, 24:4279). Taking together the catalytic and non-catalytic roles of HPKl, it is possible that blocking the HPKl kinase activity with a small-molecule inhibitor may promote activation of B-and T-cells, leading to superior anti-tumor immunity, while also facilitating AICD, helping to maintain peripheral immune tolerance. The exact effects of an HPKl inhibitor would be borne out by testing in mouse models of cancer, such as syngeneic tumor xenografts. Given that HPKl is not expressed in any major organs, outside the hematopoietic system, it is less likely that an inhibitor of HPKl kinase activity would cause any serious side effects.
In view of the above, there is a need in the art for novel compounds that can inhibit
HPKl .
SUMMARY OF THE INVENTION
Applicant has now discovered that certain thienopyridinone compounds are HPKl inhibitors (see Example B). They also have inhibitory activities against FLT3 and LCK (see Example C). Additionally, it has been demonstrated that certain thienopyridinone compounds as HPKl inhibitors alone, and in combination with anti-PD-1 antibodies are effective in preclinical models with certain cancer cell types (see Example E). The particular combination therapies disclosed herein demonstrate surprising biological activity with significant anticancer effects. Specifically, with the combination of HPKl inhibitors and anti-PD-1 antibodies, significant responses following PD-1/PD-L1 blockade have now been demonstrated in CT26. WT colon carcinoma. Based on these discoveries, thienopyridinone compounds, pharmaceutical compositions thereof, and methods of using the same are disclosed herein. One embodiment of the invention is a compound represented by Structural Formula
Figure imgf000004_0001
(i); or a pharmaceutically acceptable salt thereof. Values for each of the variables are provided below.
Another embodiment of the invention is a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and a compound represented by Structural Formula (I) described above or a pharmaceutically acceptable salt thereof.
Another embodiment of the invention is a method of treating a subject with a disease which can be regulated by HPKl comprising administering to the subject an effective amount of a compound of Structural Formula (I) or a pharmaceutically acceptable salt thereof.
Another embodiment of the invention is a method of inhibiting HPKl activity in a subject in need of inhibition of HPKl activity, comprising administering to the subject an effective amount of a compound represented by Structural Formula (I) or a pharmaceutically acceptable salt thereof.
Another embodiment of the invention is a compound represented by Structural Formula (I) or a pharmaceutically acceptable salt thereof for use in therapy. In some embodiments, the therapy is for treating a subject with cancer. Alternatively, the therapy is for inhibiting HPKl activity in a subject in need of inhibition of HPKl activity.
Another embodiment of the invention is the use of a compound represented by Structural Formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a subject with cancer.
Another embodiment of the invention the use of a compound represented by Structural Formulas (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for inhibiting HPKl activity in a subject in need of inhibition of HPKl activity. The present invention is also directed to a method of treating a subject with cancer, comprising administering to the subject an effective amount of a HPKl inhibitor (e.g., a compound represented by Structural Formula (I)), or a pharmaceutically acceptable salt thereof, and an effective second anti-cancer treatment (e.g. , a chemotherapeutic agent, a targeted therapeutic agent, radiation or surgery). In one example, the second anti-cancer treatment is a PD-1 inhibitor.
The present invention is also directed to a method of treating a subject with cancer, comprising administering to the subject an effective amount of a HPKl inhibitor (e.g., a compound represented by Structural Formula (I)), or a pharmaceutically acceptable salt thereof, and an effective amount of an immunomodulatory agent such as a checkpoint inhibitor (e.g. , anti-PD-1 antibody, anti-CTLA4 antibody or anti-PD-Ll antibody ) or an inhibitor of tryptophan oxidation (e.g. IDOl, ID02 or TD02 inhibitor). In one example, the immunomodulatory agent is anti-PD-1 antibody.
In an embodiment, the present invention further provides the use of a HPKl inhibitor (e.g., a compound represented by Structural Formula (I), or a pharmaceutically acceptable salt thereof), for the manufacture of a medicament for the treatment of a subject with cancer, in combination with a PD-1 inhibitor such as nivolumab, pembrolizumab, pidilizumab, BMS 936559, MPDL3280A, MSB0010718C or MEDI4736. Preferably, the PD-1 inhibitor is nivolumab. Alternatively, the PD-1 inhibitor is pembrolizumab. In one embodiment, the PD-1 inhibitor is anti-PDl antibody.
In one alternative, the HPKl inhibitor is admininstered with an effective amount of one or more other anti-cancer therapies, and preferably in combination with PD-1 inhibitor.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the inhibitory effect of compound example A30 against SLP-76 serine 376 phosphorylation in a-CD3 stimulated Jurkat E6.1 cells.
Figure 2 is a graph illustrating the tumour growth inhibition percentage following administration of compound Al alone and in combination with an anti-PDl antibody.
Figure 3 shows the effect of compound example A30 in the EAE disease progression model. DETAILED DESCRIPTION OF THE INVENTION
In a first embodiment, the invention is directed to a compound represented by Formula (I):
Figure imgf000006_0001
or a pharmaceutically acceptable salt thereof, wherein:
one of Xi, X2, and X3 is S, the other two are each independently CR, wherein R is -H, -F, -CI, -Br, -CN, -NH¾ -OH, optionally substituted (Ci-C6)alkyl, optionally substituted (Ci- Ce)alkoxy, optionally substituted -(CH2)n(C3-Cio)cycloalkyl, optionally substituted -(CH2)n-3- 7 membered monocyclic heterocyclyl, optionally substituted -(CH2)nphenyl, optionally substituted -(CH2)n-5-7 membered monocyclic heteroaryl, optionally substituted -(CH2)n- bridged (C6-Ci2)cycloalkyl, optionally substituted -(CH2)n-6-12 membered bridged heterocyclyl, optionally substituted -(CH2)n-7-12 membered bicyclic heteroaryl, or optionally substituted -(CH2)n-7-12 membered bicyclic heteroaryl;
Y is a bond, -CH2-, -C(=0)-;
Ri is -NRaRb or -ORal;
Ra for each occurrence is independently -H, optionally substituted (Ci-Ce)alkyl, optionally substituted -(CH2)n(C3-Cio)cycloalkyl, optionally substituted -(CH2)n-3-10 membered heterocyclyl, optionally substituted -(CH2)n(C6-Cio)aryl, optionally substituted - (CH2)n-5-10 membered heteroaryl, optionally substituted -(CH2)n-bridged (C6-Ci2)cycloalkyl, or optionally substituted -(CH2)n-6-12 membered bridged heterocyclyl;
Rb for each occurrence is independently -H or -(Ci-C6)alkyl; or,
Ra and Rb, together with the nitrogen to which they are attached, form optionally substituted -(C3-Cio)heterocyclyl;
Ral for each occurrence is independently -H, optionally substituted (Ci-C6)alkyl, optionally substituted (C3-Cio)cycloalkyl, optionally substituted 3-10 membered heterocyclyl, optionally substituted (C6-Cio)aryl, or optionally substituted 3-10 membered heteroaryl; or
R2 and R3 are each independently -H or -(Ci-C6)alkyl; R.4 and Rs are each independently -H, optionally substituted (Ci-C6)alkyl, optionally substituted (C3-Cio)cycloalkyl, optionally substituted 3-10 membered heterocyclyl, optionally substituted (C6-Cio)aryl, optionally substituted 5-10 membered heteroaryl, optionally substituted bridged (C6-Ci2)cycloalkyl, or optionally substituted 6-12 membered bridged heterocyclyl; or
R.4 and Rs, together with the nitrogen to which they are attached, form optionally substituted 4-10 membered heterocyclyl, optionally substituted 5-10 membered heteroaryl, or optionally substituted 6-12 membered bridged heterocyclyl;
R6 for each occurrence is independently -F, -CI, -Br, -CN, -NH¾ -OH, -(Ci-C6)alkyl, -(Ci-C6)haloalkyl, -(C2-C6)alkenyl, -(C2-C6)alkynyl, (C3-C6)cycloalkyl, -(Ci-C6)alkoxy, -(Ci- C6)haloalkoxy, -(Ci-C6)alkylene-OH, or -(Ci-C6)alkylene-NH2;
m is 0, 1, 2, or 3; and
n is 0, 1, or 2.
In a second embodiment, the invention provides a compound represented by structural formula (I-A)-(I-C), (II-A)-(II-C), or (III-A)-(III-C):
Figure imgf000007_0001
Figure imgf000008_0001
Figure imgf000009_0001
or a pharmaceutically acceptable salt thereof. Values for the variables in Structural Formulae (I-A)-(I-C), (II-A)-(II-C), and (III-A)-(III-C) are as described for Structural Formula (I).
In a third embodiment, the invention provides a compound represented by structural formula (I), (I-A)-(I-C), (II-A)-(II-C), or (III-A)-(III-C), wherein R4 and R5, together with the nitrogen to which they are attached, form 4-7 membered monocyclic heterocyclyl or 6-12 membered bridged heterocyclyl, wherein the 4-7 membered monocyclic heterocyclyl or 6-12 membered bridged heterocyclyl is optionally substituted with 1-3 groups selected from -F, - CI, -Br, -CN, -Nth, -OH, oxo, -(Ci-C4)alkyl, -(Ci-C4)haloalkyl, -(Ci-C4)alkoxy, -(Ci- C4)haloalkoxy, -(Ci-C4)alkylene-OH, or -(Ci-C4)alkylene-Nth. Values for the remainder of the variables are as described for Structural Formula (I).
In a fourth embodiment, the invention provides a compound represented by structural formula (I), (I-A)-(I-C), (II-A)-(II-C), or (III-A)-(III-C), wherein Ra for each occurrence is independently -H, -(Ci-Ce)alkyl, -(CH2)„-(C3-C7)cycloalkyl, -(CH2)„-4-7 membered monocyclic heterocyclyl, -(CH2)n-bridged (C6-Ci2)cycloalkyl, optionally substituted -(CH2)n- 5-10 membered heteroaryl; or -(CH2)n-6-12 membered bridged heterocyclyl, wherein -(Ci- C6)alkyl, -(CH2)n-(C3-C7)cycloalkyl, -(CH2)n-4-7 membered monocyclic heterocyclyl, - (CH2)n-bridged (C6-Ci2)cycloalkyl, -(CH2)n-5-10 membered heteroaryl, or -(CH2)n-6-12 membered bridged heterocyclyl, is optionally substituted with 1-3 groups selected from -F, - CI, -Br, -CN, -NH¾ -OH, oxo, -(Ci-C4)alkyl, -(Ci-C4)haloalkyl, -(Ci-C4)alkoxy, -(Ci- C4)haloalkoxy, -(Ci-C4)alkylene-OH, or -(Ci-C4)alkylene-NH2, and values for the remainder of the variables are as described above for Structural Formula (I) or in the third embodiment.
In a fifth embodiment, the invention provides a compound represented by structural formula (I), (I-A)-(I-C), (II-A)-(II-C), or (III-A)-(III-C), wherein R is H, -F, -CI, -Br, -OH, - (Ci-C4)alkyl, -(Ci-C4)haloalkyl, -(Ci-C4)alkoxy, -(Ci-C )alkylene-OH or 4-7 membered monocyclic heterocyclyl optionally substituted with 1-3 groups selected from -F, -CI, -Br, - OH, -(Ci-C4)alkyl, -(Ci-C4)haloalkyl, or -(Ci-C4)alkoxy, and values for the remainder of the variables are as described above for Structural Formula (I) or in the third or fourth embodiment.
In a sixth embodiment, the invention provides a compound represented by structural formula (I), (I-A)-(I-C), (II-A)-(II-C), or (III-A)-(III-C), wherein R4 and R5, together with the nitrogen to which they are attached, form -N-alkyl-piperazinyl or morpholinyl, wherein the piperazinyl or morpholinyl is optionally substituted with 1-2 groups selected from -F, -CI, - Br,-OH, -(Ci-C4)alkyl, -(Ci-C4)haloalkyl, or -(Ci-C4)alkoxy, and values for the remainder of the variables are as described above for Structural Formula (I), or in the third, fourth, or fifth embodiment.
In a seventh embodiment, the invention provides a compound represented by structural formula (I), (I-A)-(I-C), (II-A)-(II-C), or (III-A)-(III-C), wherein Ra for each occurrence is independently -H, -(CH2)n-(C3-C6)cycloalkyl, -(CH2)n-3-6 membered heterocyclyl, wherein the -(CH2)n-(C3-C6)cycloalkyl or -(CH2)n-3-6 membered heterocyclyl is optionally substituted with 1-3 groups selected from -F, -CI, -Br, -CN, -NH2, -OH, -(Ci- C4)alkyl, or -(Ci-C4)alkoxy; and n is 0 or 1, and values for the remainder of the variables are as described above for Structural Formula (I), or in the third, fourth, fifth, or sixth embodiment. In an eighth embodiment, the invention provides a compound represented by structural formula (I), (I-A)-(I-C), (II-A)-(II-C), or (III-A)-(III-C), wherein R is H, -(Ci- C4)alkyl, -(Ci-C4)alkoxy, N-piperazinyl optionally substituted with -C02-(Ci-C4)alkyl, and values for the remainder of the variables are as described above for Structural Formula (I), or in the third, fourth, fifth, sixth, or seventh embodiment. Alternatively, R is H.
In a ninth embodiment, the invention provides a compound represented by structural formula (I), (I-A)-(I-C), (II-A)-(II-C), or (III-A)-(III-C), wherein R4 and R5, together with the nitrogen to which they are attached, form -N-methyl-piperazinyl or morpholinyl, both of which are optionally substituted with one or two methyl, and values for the remainder of the variables are as described above for Structural Formula (I), or in the third, fourth, fifth, sixth, seventh, or eighth embodiment.
In a tenth embodiment, the invention provides a compound represented by structural formula (I), (I-A)-(I-C), (II-A)-(II-C), or (III-A)-(III-C), wherein Ra for each occurrence is independently -H; -(C3-Ce)cycloalkyl optionally substituted with -OH; -(CH2)n-tetrahydro- 2H-pyran; morpholinyl; piperidinyl optionally substituted with -F, -OH or methyl; or tetrahydrofuran; and n is 0 or 1, and values for the remainder of the variables are as described above for Structural Formula (I), or in the third, fourth, fifth, sixth, seventh, eighth or ninth embodiment.
The invention also includes the compounds depicted by structure and/or described by name in the Exemplification. The invention includes both the neutral form (free base) of these compounds as well as pharmaceutically acceptable salts thereof. Treatments with and/or uses of these compounds includes the neutral form of these compounds as well as pharmaceutically acceptable salts thereof.
The term "alkyl" used alone or as part of a larger moiety, such as "alkoxy" or "haloalkyl" and the like, means saturated aliphatic straight-chain or branched monovalent hydrocarbon radical. Unless otherwise specified, an alkyl group typically has 1-6 carbon atoms, i.e. (Ci-C6)alkyl. As used herein, a "(Ci-C6)alkyl" group means a radical having from 1 to 6 carbon atoms in a linear or branched arrangement. Examples include methyl, ethyl, n- propyl, wo-propyl etc.
"Alkoxy" means an alkyl radical attached through an oxygen linking atom, represented by -O-alkyl. For example, "(Ci-C4)alkoxy" includes methoxy, ethoxy, propoxy, and butoxy. The terms "haloalkyl" and "haloalkoxy" means alkyl or alkoxy, as the case may be, substituted with one or more halogen atoms. The term "halogen" means F, CI, Br or I. Preferably the halogen in a haloalkyl or haloalkoxy is F.
"Alkenyl" means branched or straight-chain monovalent hydrocarbon radical containing at least one double bond. Alkenyl may be mono or polyunsaturated, and may exist in the E or Z configuration. Unless otherwise specified, an alkenyl group typically has 2-6 carbon atoms, i.e. (C2-Ce)alkenyl. For example, "(C2-C6)alkenyl" means a radical having from 2-6 carbon atoms in a linear or branched arrangement.
"Alkynyl" means branched or straight-chain monovalent hydrocarbon radical containing at least one triple bond. Unless otherwise specified, an alkynyl group typically has 2-6 carbon atoms, i.e. (C2-Ce)alkynyl. For example, "(C2-C6)alkynyl" means a radical having from 2-6 carbon atoms in a linear or branched arrangement.
"Cycloalkyl" means a saturated aliphatic cyclic hydrocarbon radical, typically containing from 3-8 ring carbon atoms, i.e., (C3-C8)cycloalkyl. (C3-C8)cycloalkyl includes, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
As used herein, the term "bridged" used alone or as part of a larger moiety as in "bridged cycloalkyl" or "bridged heterocyclyl" refers to a ring system which includes two rings that share at least three adjacent ring atoms. Bridged cycloalkyl typically contains 6-12 ring carbon atoms. Bridged heterocyclyl typically have 6-12 ring atoms selected from carbon and at least one (typically 1 to 4, more typically 1 or 2) heteroatom (e.g. , oxygen, nitrogen or sulfur).
The term "aryl" used alone or as part of a larger moiety as in "arylalkyl", "arylalkoxy", or "aryloxyalkyl", means a carbocyclic aromatic ring. It also includes a phenyl ring fused with a cycloalkyl group. The term "aryl" may be used interchangeably with the terms "aryl ring" "carbocyclic aromatic ring", "aryl group" and "carbocyclic aromatic group". An aryl group typically has six to fourteen ring atoms. Examples includes phenyl, naphthyl, anthracenyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl and the like. A "substituted aryl group" is substituted at any one or more substitutable ring atom, which is a ring carbon atom bonded to a hydrogen.
The term "heteroaryl", "heteroaromatic", "heteroaryl ring", "heteroaryl group", "heteroaromatic ring", and "heteroaromatic group", are used interchangeably herein. "Heteroaryl" when used alone or as part of a larger moiety as in "heteroarylalkyl" or "heteroarylalkoxy", refers to aromatic ring groups having five to fourteen ring atoms selected from carbon and at least one (typically 1 to 4, more typically 1 or 2) heteroatoms (e.g. , oxygen, nitrogen or sulfur). "Heteroaryl" includes monocyclic rings and polycyclic rings in which a monocyclic heteroaromatic ring is fused to one or more other aryl, heterocyclyl or heteroaromatic rings. As such, "5-14 membered heteroaryl" includes monocyclic, bicyclic or tricyclic ring systems.
Examples of monocyclic 5-6 membered heteroaryl groups include furanyl (e.g., 2- furanyl, 3-furanyl), imidazolyl (e.g., N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), isoxazolyl (e.g. , 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl), oxadiazolyl (e.g. , 2-oxadiazolyl, 5- oxadiazolyl), oxazolyl (e.g. , 2-oxazolyl, 4-oxazolyl, 5-oxazolyl), pyrazolyl (e.g. , 3-pyrazolyl, 4-pyrazolyl), pyrrolyl (e.g. , 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl), pyridyl (e.g. , 2-pyridyl, 3- pyridyl, 4-pyridyl), pyrimidinyl (e.g., 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl), pyridazinyl (e.g., 3-pyridazinyl), thiazolyl (e.g. , 2-thiazolyl, 4-thiazolyl, 5-thiazolyl), isothiazolyl, triazolyl (e.g. , 2-triazolyl, 5-triazolyl), tetrazolyl (e.g. , tetrazolyl), and thienyl (e.g., 2-thienyl, 3-thienyl). Examples of polycyclic aromatic heteroaryl groups include carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, isobenzofuranyl, indolyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, quinolinyl, isoquinolinyl, indazolyl, isoindolyl, acridinyl, or benzisoxazolyl. A "substituted heteroaryl group" is substituted at any one or more substitutable ring atom, which is a ring carbon or ring nitrogen atom bonded to a hydrogen.
"Heterocyclyl" means a saturated or unsaturated non-aromatic 3-12 membered ring radical optionally containing one or more double bonds. It can be monocyclic, bicyclic, tricyclic, or fused. The heterocycloalkyl contains 1 to 4 heteroatoms, which may be the same or different, selected from N, O or S. The heterocyclyl ring optionally contains one or more double bonds and/or is optionally fused with one or more aromatic rings (e.g., phenyl ring). The term "heterocyclyl" is intended to include all the possible isomeric forms. Examples of heterocycloalkyl include, but are not limited to, azetidinyl , morpholinyl, thiomorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, dihydroimidazole, dihydrofuranyl, dihydropyranyl, dihydropyridinyl, dihydropyrimidinyl, dihydrothienyl, dihydrothiophenyl, dihydrothiopyranyl, tetrahydroimidazole, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, and tetrahydrothiopyranyl. Examples of polycyclic heterocycloalkyl groups include dihydroindolyl, dihydroisoindolyl, dihydrobenzimidazolyl, dihydrobenzothienyl, dihydrobenzofuranyl, dihydroisobenzofuranyl, dihydrobenzotriazolyl, dihydrobenzothiazolyl, dihydrobenzoxazolyl, dihydroquinolinyl, tetrahydroquinolinyl, dihydroisoquinolinyl, tetrahydroisoquinolinyl, dihydroindazolyl, dihydroacridinyl, tetrahydroacridinyl, dihydrobenzisoxazolyl, chroman, chromene, isochroman and isochromene.
Certain of the compounds described herein may exist in various stereoisomeric or tautomeric forms. Stereoisomers are compounds which differ only in their spatial arrangement. When a disclosed compound is named or depicted by structure without indicating stereochemistry, it is understood that the name or structure encompasses all possible stereoisomers, geometric isomers, including essentially pure stereo or geometric isomers, as well as combination thereof.
In certain instances tautomeric forms of the disclosed compounds exist, such as the tautomeric structures shown below:
Figure imgf000014_0001
Tautomer
It is to be understood that when a compound herein is represented by a structural formula or designated by a chemical name herein, all other tautomeric forms which may exist for the compound are encompassed by the structural formula.
Certain of the disclosed compounds may exist in various stereoisomeric forms. Stereoisomers are compounds that differ only in their spatial arrangement. Enantiomers are pairs of stereoisomers whose mirror images are not superimposable, most commonly because they contain an asymmetrically substituted carbon atom that acts as a chiral center. "Enantiomer" means one of a pair of molecules that are mirror images of each other and are not superimposable. Diastereomers are stereoisomers that contain two or more asymmetrically substituted carbon atoms. "Geometric isomers" are stereoisomers that differ in the orientation of substituent atoms in relationship to a carbon-carbon double bond, to a carbocyclyl ring, or to a bridged bicyclic system.
When a geometric isomer is depicted by name or structure, it is to be understood that the geometric isomeric purity of the named or depicted geometric isomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% pure by weight. Geometric isomeric purity is determined by dividing the weight of the named or depicted geometric isomer in the mixture by the total weight of all of the geometric isomers in the mixture.
When the stereochemistry of a disclosed compound is named or depicted by structure, the named or depicted stereoisomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% by weight pure relative to all of the other stereoisomers. Percent by weight pure relative to all of the other stereoisomers is the ratio of the weight of one stereoisomer over the weight of the other stereoisomers. When a single enantiomer is named or depicted by structure, the depicted or named enantiomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% by weight optically pure (also referred to as "enantiomerically pure"). Percent optical purity by weight is the ratio of the weight of the enantiomer over the weight of the enantiomer plus the weight of its optical isomer.
When the stereochemistry of a disclosed compound is named or depicted by structure, and the named or depicted structure encompasses more than one stereoisomer (e.g. , as in a diastereomeric pair), it is to be understood that one of the encompassed stereoisomers or any mixture of the encompassed stereoisomers are included. It is to be further understood that the stereoisomeric purity of the named or depicted stereoisomers at least 60%, 70%, 80%, 90%, 99% or 99.9% by weight pure relative to all of the other stereoisomers. The stereoisomeric purity in this case is determined by dividing the total weight in the mixture of the stereoisomers encompassed by the name or structure by the total weight in the mixture of all of the stereoisomers.
When a disclosed compound is named or depicted by structure without indicating the stereochemistry, and the compound has one chiral center, it is to be understood that the name or structure encompasses one enantiomer of compound free from the corresponding optical isomer, a racemic mixture of the compound and mixtures enriched in one enantiomer relative to its corresponding optical isomer.
When a disclosed compound is named or depicted by structure without indicating the stereochemistry and e.g. , the compound has at least two chiral centers, it is to be understood that the name or structure encompasses one stereoisomer free of other stereoisomers, mixtures of stereoisomers, and mixtures of stereoisomers in which one or more stereoisomers is enriched relative to the other stereoisomer(s). For example, the name or structure may encompass one stereoisomer free of other diastereomers, mixtures of stereoisomers, and mixtures of stereoisomers in which one or more diastereomers is enriched relative to the other diastereomer(s).
Enantiomeric and diastereomeric mixtures can be resolved into their component enantiomers or stereoisomers by well-known methods, such as chiral-phase gas chromatography, chiral-phase high performance liquid chromatography, crystallizing the compound as a chiral salt complex, or crystallizing the compound in a chiral solvent. Enantiomers and diastereomers can also be obtained from diastereomerically- or enantiomerically-pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.
Included in the present teachings are pharmaceutically acceptable salts of the compounds disclosed herein. The disclosed compounds have basic amine groups and therefore can form pharmaceutically acceptable salts with pharmaceutically acceptable acid(s). Suitable pharmaceutically acceptable acid addition salts of the compounds described herein include salts of inorganic acids (such as hydrochloric acid, hydrobromic, phosphoric, metaphosphoric, nitric, and sulfuric acids) and of organic acids (such as acetic acid, benzenesulfonic, benzoic, ethanesulfonic, methanesulfonic, succinic, and trifluoroacetic acid acids). Compounds of the present teachings with acidic groups such as carboxylic acids can form pharmaceutically acceptable salts with pharmaceutically acceptable base(s). Suitable pharmaceutically acceptable basic salts include ammonium salts, alkali metal salts (such as sodium and potassium salts) and alkaline earth metal salts (such as magnesium and calcium salts). Compounds with a quaternary ammonium group also contain a counteranion such as chloride, bromide, iodide, acetate, perchlorate and the like. Other examples of such salts include hydrochlorides, hydrobromides, sulfates, methanesulfonates, nitrates, acetates, succinates, benzoates and salts with amino acids such as glutamic acid.
Compounds described herein can inhibit HPK1. Thus, generally, compounds described herein are useful in the treatment of diseases or conditions associated with such kinases. In one embodiment, the compounds described herein are HPK1 inhibitors, and are useful for treating diseases, such as cancer, associated with such kinase(s).
Another aspect of the present teachings relates to a method of treating a subject with cancer comprising administering to the subject an effective amount of a compound described herein. In one embodiment, the compounds described herein inhibit the growth of a tumor.
Cancers that can be treated (including reduction in the likelihood of recurrence) by the methods of the present teachings include breast cancer, colorectal cancer, lung cancer, ovarian cancer, uterine cancer, prostate cancer, leukemias, lymphomas, brain cancer (including glioblastoma multiforme and neuroblastoma), head and neck cancer, pancreatic cancer, melanoma, hepatocellular carcinoma, renal cancer, and soft tissue sarcomas. In one embodiment, the cancer is breast cancer, colon cancer, and ovarian cancer. In one embodiment, the cancer is selected from leukemia, acute myeloid leukemia, chronic myelogenous leukemia, breast cancer, brain cancer, colon cancer, colorectal cancer, head and neck cancer, hepatocellular carcinoma, lung adenocarcinoma, metastatic melanoma, pancreatic cancer, prostate cancer, ovarian cancer and renal cancer. In one embodiment, the cancer is lung cancer, colon cancer, brain cancer, neuroblastoma, prostate cancer, melanoma, glioblastoma multiforme or ovarian cancer. In another embodiment, the cancer is lung cancer, breast cancer, colon cancer, brain cancer, neuroblastoma, prostate cancer, melanoma, glioblastoma multiforme or ovarian cancer. In yet another embodiment, the cancer is breast cancer, colon cancer and lung cancer. In another embodiment, the cancer is a breast cancer. In yet another embodiment, the cancer is a basal sub-type breast cancer or a luminal B subtype breast cancer. In yet another embodiment, the cancer is a basal sub-type breast cancer. In yet another embodiment, the basal sub-type breast cancer is ER (estrogen receptor), HER2 and PR (progesterone receptor) negative breast cancer. In yet another embodiment, the cancer is a soft tissue cancer. A "soft tissue cancer" is an art-recognized term that encompasses tumors derived from any soft tissue of the body. Such soft tissue connects, supports, or surrounds various structures and organs of the body, including, but not limited to, smooth muscle, skeletal muscle, tendons, fibrous tissues, fatty tissue, blood and lymph vessels, perivascular tissue, nerves, mesenchymal cells and synovial tissues. Thus, soft tissue cancers can be of fat tissue, muscle tissue, nerve tissue, joint tissue, blood vessels, lymph vessels, and fibrous tissues. Soft tissue cancers can be benign or malignant. Generally, malignant soft tissue cancers are referred to as sarcomas, or soft tissue sarcomas. There are many types of soft tissue tumors, including lipoma, lipoblastoma, hibernoma, liposarcoma, leiomyoma, leiomyosarcoma, rhabdomyoma, rhabdomyosarcoma, neurofibroma, schwannoma (neurilemoma), neuroma, malignant schwannoma, neurofibrosarcoma, neurogenic sarcoma, nodular tenosynovitis, synovial sarcoma, hemangioma, glomus tumor, hemangiopericytoma, hemangioendothelioma, angiosarcoma, Kaposi sarcoma, lymphangioma, fibroma, elastofibroma, superficial fibromatosis, fibrous histiocytoma, fibrosarcoma, fibromatosis, dermatofibrosarcoma protuberans (DFSP), malignant fibrous histiocytoma (MFH), myxoma, granular cell tumor, malignant mesenchymomas, alveolar soft-part sarcoma, epithelioid sarcoma, clear cell sarcoma, and desmoplastic small cell tumor. In a particular embodiment, the soft tissue cancer is a sarcoma selected from the group consisting of a fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a dedifferentiated liposarcoma, a pleomorphic liposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, and a synovial sarcoma.
The present teachings also provide methods of treating a subject with a disease comprising administering to the subject an effective amount of a compound represented by Structural Formula (I) in combination with an effective immunomodulatory therapy (also referred as immunotherapy). Immunotherapy is the treatment of disease by using an immunomodulatory agent to induce, enhance, or suppress an immune response. Immunotherapies designed to elicit or amplify an immune response are classified as activation immunotherapies, while immunotherapies that reduce or suppress are classified as suppression immunotherapies. The disease described herein is a cancer.
Immunomodulatory therapies, used alone or in combination approaches, include i) immune checkpoint blockade inhibitors, including but not limited to anti-CTLA4 (cytotoxic T-lymphocyte-associated protein 4) antibodies (e.g. Ipilimumab), agents that disrupt the PD- 1/PD-Ll and PD-L2 interaction, e.g. Nivolumab (Opdivo - Bristol Myers Squibb), Pembrolizumab (Keytruda, KM-3475, Merck), Pidilizumab (CT-011, Cure Tech), BMS 936559 (BMS) and MPDL3280A (Roche); and other immune response inhibitory receptors e.g. anti-CD47; ii) cell based therapies (including, but not limited to, dendritic cell therapy (e.g. Sipuleucel T (Provenge) and adoptive T-cell therapies, iii) vaccination strategies; iv) Adoptive T-cell therapy; v) agents that prevent metabolic inhibition of the immune response, including inhibitors of indoleamine 2, 3-dioxygenase (e.g. INCB024360 (Incyte), 1-methyl- D-tryptophan, indoximod (NewLink Genetics)) or arginase; and vi) cytokine-based therapy, e.g., interferons (in particular type I interferon) and interleukins (e.g. interleukin-2). In one embodiment, the immunomodulatory agent used for the immunomodulatory therapy is a PD-1 inhibitor, for example, an anti-PDl antibody.
Programmed cell death protein 1, also known as PD-1 and CD279 (cluster of differentiation 279), is a protein that in humans is encoded by the PDCD1 gene. PD-1 is a cell surface receptor that belongs to the immunoglobulin superfamily and is expressed on T cells and pro-B cells. PD-1 binds two ligands, PD-L1 and PD-L2, both of which are members of the B7 family.
PD-1 and its ligands play an important role in down regulating the immune system by preventing the activation of T-cells, which in turn reduces autoimmunity and promotes self- tolerance. The inhibitory effect of PD-1 is accomplished through a dual mechanism of promoting apoptosis(programmed cell death) in antigen specific T-cells in lymph nodes while simultaneously reducing apoptosis in regulatory T cells (suppressor T cells).
The PD-1 inhibitor used in the present invention includes, but is not limited to, nivolumab, pembrolizumab, pidilizumab, BMS 936559, MPDL3280A, MSB0010718C or MEDI4736. Among them, BMS 936559, MPDL3280A, MSB0010718C, and MEDI4736 bind ligand PD-L1, all of which are antibodies. Both nivolumab and pembrolizumab are approved by the Food and Drug Administration for treatment of unresectable or metastatic melanoma which no longer responds to other drugs.
Vaccination strategies include anti-microbial immunotherapy, which includes vaccination, involves activating the immune system to respond to an infectious agent.
Adoptive T-cell therapy uses T cell-based cytotoxic responses to attack cancer cells. T cells that have a natural or genetically engineered reactivity to a patient's cancer are generated in vitro and then transferred back into the cancer patient. One study using autologous tumor- infiltrating lymphocytes was an effective treatment for patients with metastatic melanoma. This can be achieved by taking T cells that are found with the tumor of the patient, which are trained to attack the cancerous cells. These T cells are referred to as tumor-infiltrating lymphocytes (TIL) are then encouraged to multiply in vitro using high concentrations of IL-2, anti-CD3 and allo-reactive feeder cells. These T cells are then transferred back into the patient along with exogenous administration of IL-2 to further boost their anti-cancer activity.
The present teachings also provide methods of treating a subject with a cancer comprising administering to the subject an effective amount of a compound represented by Structural Formula (I) in combination with an effective anti-cancer therapy. In one embodiment, the cancer is a metastatic cancer. A "metastatic cancer" is a cancer that has spread from its primary site to other parts of the body.
The anti-cancer therapy described herein includes co-administration of an effective amount of a second anti-cancer agent together with a disclosed HPK-1 inhibitor. An "anticancer agent" is a compound, which when administered in an effective amount to a subject with cancer, can achieve, partially or substantially, one or more of the following: arresting the growth, reducing the extent of a cancer (e.g., reducing size of a tumor), inhibiting the growth rate of a cancer, and ameliorating or improving a clinical symptom or indicator associated with a cancer (such as tissue or serum components) or increasing longevity of the subject.
The anti-cancer agents suitable for use in the methods described herein include any anti-cancer agents that have been approved for the treatment of cancer. In one embodiment, the anti-cancer agent includes, but is not limited to, a targeted antibody, an angiogenesis inhibitor, an alkylating agent, an antimetabolite, a vinca alkaloid, a taxane, a podophyllotoxin, a topoisomerase inhibitor, a hormonal antineoplastic agent and other antineoplastic agents. In one embodiment, the anti-cancer agent is a PD-1 inhibitor, for example, an anti-PDl antibody.
In one embodiment, the anti-cancer agents that can be used in methods described herein include, but are not limited to, paclitaxel, docetaxel, 5-fluorouracil, trastuzumab, lapatinib, bevacizumab, letrozole, goserelin, tamoxifen, cetuximab, panitumumab, gemcitabine, capecitabine, irinotecan, oxaliplatin, carboplatin, cisplatin, doxorubicin, epirubicin, cyclophosphamide, methotrexate, vinblastine, vincristine, melphalan, cytarabine, etoposide, daunorubicin, bleomycin, mitomycin and adriamycin and a combination thereof.
In one embodiment, the anti-cancer agent and the compound represented by Structural Formula (I) are administered contemporaneously. When administered contemporaneously, the anti-cancer agent and the compound can be administered in the same formulation or in different formulations. Alternatively, the compound and the additional anti-cancer agent are administered separately at different times.
As used herein, "treating a subject with a cancer" includes achieving, partially or substantially, one or more of the following: arresting the growth, reducing the extent of the cancer (e.g., reducing size of a tumor), inhibiting the growth rate of the cancer, ameliorating or improving a clinical symptom or indicator associated with the cancer (such as tissue or serum components) or increasing longevity of the subject; and reducing the likelihood of recurrence of the cancer.
The term an "effective amount" means an amount when administered to the subject which results in beneficial or desired results, including clinical results, e.g., inhibits, suppresses or reduces the cancer (e.g., as determined by clinical symptoms or the amount of cancer cells) in a subject as compared to a control.
Generally, an effective amount of a compound taught herein varies depending upon various factors, such as the given drug or compound, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art. An effective amount of a compound of the present teachings may be readily determined by one of ordinary skill by routine methods known in the art.
In an embodiment, an effective amount of a compound taught herein ranges from about 0.1 to about 1000 mg/kg body weight, alternatively about 1 to about 500 mg/kg body weight. In another embodiment, an effective amount of a compound taught herein ranges from about 0.5 to about 5000 mg/m2, alternatively about from 5 to about 2500 mg/m2, and in another alternative from about 50 to about 1000 mg/m2. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject suffering from cancer or reduce the likelihood of recurrence of a cancer. These factors include, but are not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the subject and other diseases present.
A "subject" is a mammal, preferably a human, but can also be an animal in need of veterinary treatment, e.g., companion animals (e.g. , dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, and the like) and laboratory animals (e.g. , rats, mice, guinea pigs, and the like).
The compounds taught herein can be administered to a patient in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. The compounds of the present teachings may be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump or transdermal administration and the pharmaceutical compositions formulated accordingly. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal and topical modes of administration. Parenteral administration can be by continuous infusion over a selected period of time.
The compounds taught herein can be suitably formulated into pharmaceutical compositions for administration to a subject. The pharmaceutical compositions of the present teachings optionally include one or more pharmaceutically acceptable carriers and/or diluents therefor, such as lactose, starch, cellulose and dextrose. Other excipients, such as flavoring agents; sweeteners; and preservatives, such as methyl, ethyl, propyl and butyl parabens, can also be included. More complete listings of suitable excipients can be found in the Handbook of Pharmaceutical Excipients (5th Ed., Pharmaceutical Press (2005)). A person skilled in the art would know how to prepare formulations suitable for various types of administration routes. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2003 - 20th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999. The carriers, diluents and/or excipients are "acceptable" in the sense of being compatible with the other ingredients of the pharmaceutical composition and not deleterious to the recipient thereof.
Typically, for oral therapeutic administration, a compound of the present teachings may be incorporated with excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
Typically for parenteral administration, solutions of a compound of the present teachings can generally be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
Typically, for injectable use, sterile aqueous solutions or dispersion of, and sterile powders of, a compound described herein for the extemporaneous preparation of sterile injectable solutions or dispersions are appropriate.
For nasal administration, the compounds of the present teachings can be formulated as aerosols, drops, gels and powders. Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multi-dose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomizing device. Alternatively, the sealed container may be a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use. Where the dosage form comprises an aerosol dispenser, it will contain a propellant which can be a compressed gas such as compressed air or an organic propellant such as fluorochlorohydrocarbon. The aerosol dosage forms can also take the form of a pump-atomizer.
For buccal or sublingual administration, the compounds of the present teachings can be formulated with a carrier such as sugar, acacia, tragacanth, or gelatin and glycerine, as tablets, lozenges or pastilles.
For rectal administration, the compounds described herein can be formulated in the form of suppositories containing a conventional suppository base such as cocoa butter.
The compounds of invention may be prepared by methods known to those skilled in the art, as illustrated by the general schemes and procedures below and by the preparative examples that follow. All starting materials are either commercially available or prepared by methods known to those skilled in the art and the procedures described below.
General synthetic approaches to the claims compounds are provided in the exemplification below, as illustrated in Schemes 1 and 2.
EXEMPLIFICATION
Example A: Synthesis
General Methods
Commercially available starting materials, reagents, and solvents were used as received. In general, anhydrous reactions were performed under an inert atmosphere such as nitrogen or Argon. PoraPak® Rxn CX refers to a commercial cation-exchange resin available from Waters.
Microwave reactions were performed with a Biotage Initiator microwave reactor. Reaction progress was generally monitored by LCMS (Bruker Exquire 4000 or Waters Acquity UPLC system). Flash column chromatographic purification of intermediates or final products was performed using a Biotage Isolera with KP-SIL or HP-SIL silica cartridges, or KP-NH basic modified silica and corresponding samplets. Reverse-phase HPLC purification was performed on a Varian PrepStar model SD-1 HPLC system with a Varian Monochrom 10μ C-18 reverse-phase column using a gradient of 10% MeOH/0.05% TFA-H20 to 90% MeOH/0.05% TFA in H2O over a 40-min period at a flow rate of 40 mL/min. Reverse phase purification was also performed using a Biotage Isolera equipped with a KP-C18-H column using a between 10-95% MeOH or CH3CN/ 0.1% TFA in H20. Proton NMRs were recorded on a Bruker 400 MHz spectrometer, and mass spectra were obtained using a Bruker Esquire 4000 spectrometer or Waters Acquity UPLC system.
Compound names were generated using the software built into CambridgeSoft- PerkinElmer's ChemBioDraw Ultra version 12.0.
Abbreviations;
aq aqueous
anh anhydrous
Ar argon
Boc ferf-butoxycarbonyl
br. broad
calcd calculated
d doublet (only when used within 1H NMR spectra)
DCM dichloromethane
de diastereomeric excess
DIPEA diisopropylethylamine
DMF N,N-dimethylformamide
DMSO dimethylsulfoxide
dppf 1,1'- bis( diphenylphosphino) ferrocene
equiv equivalent
Flt3 fms-related tyrosine kinase 3
h hour
HPK1 hematopoietic progenitor kinase 1
HPLC high performance liquid chromatography
IPA isopropanol
KHMDS potassium hexamethyldisilazide
Lck lymphocyte-specific protein tyrosine kinase
LC-MS liquid chromatography coupled to mass spectrometry LDA lithium diisopropyllamide
LiHMDS lithium hexamethyldisilazide min minute
m multiplet
MeCN acetonitrile
MS ESI mass spectra, electrospray ionization
NMR nuclear magnetic resonance
O/N overnight
PMB para-methoxybenzyl
prep preparative
rt room temperature
Rt retention time
RP reverse phase
s singlet
satd saturated
t triplet
temp. temperature
TFA trifluoroacetic acid
THF tetrahydrofuran
Figure imgf000025_0001
Scheme 1
Figure imgf000026_0001
Figure imgf000026_0002
Scheme 2
Preparation of Starting Materials
General Method Al (Base-induced cvclization using benzimidazole ester)
A solution of aryl oxazine-2,4-dione (1 equiv), or aminoaryl nitrile and substituted 1H- benzo[d]imidazol-2-yl)acetate (1-1.2 equiv) in THF was treated with KHMDS, LiHMDS, or LDA (3-5 equiv). The reaction was stirred at 45 °C for 4-24 h. The reaction was then cooled to rt and quenched with satd aq NH4CI. The aqueous layer was extracted with EtOAc or DCM, and the combined organic extracts were dried over MgS04, filtered and concentrated. Crude product was purified by column chromatography or prep-HPLC to give the desired product.
General Method A2 (Two-step, base-induced cvclization using benzimidazole ester)
A solution of aryl oxazine-2,4-dione (1 equiv), or aminoaryl nitrile and substituted 1H- benzo[d]imidazol-2-yl)acetate (1-1.2 equiv) in was treated with KHMDS, LiHMDS, KOBu1 or LDA (3-5 equiv) at 45 °C for 2-4 h. The reaction was then cooled to rt and quenched with satd aq NH4CI. The aqueous layer was extracted with EtOAc or DCM, and the combined organic extracts were dried over MgS04, filtered and concentrated. The uncyclized addition adduct was separated by column chromatography, dissolved in THF and treated with KHMDS, LiHMDS, or LDA (3-5 equiv). The reaction was stirred at 45 °C for 1-4 h. The reaction was then cooled to rt and quenched with satd aq NH4CI. The aqueous layer was extracted with EtOAc or DCM, and the combined organic extracts were dried over MgS04, filtered and concentrated. Crude product was purified by column chromatography or prep- HPLC to give the desired product.
General Method A3 (Two-step, base-induced cvclization using benzimidazole ester)
A solution of aminoaryl nitrile and substituted lH-benzo[d]imidazol-2-yl)acetate (1 equiv) in THF was treated with LiHMDS, or LDA (5 equiv) (step 1). The reaction was stirred at 35-40 °C for 1-1.5 h. The reaction was then cooled to rt and quenched with satd aq NH4CI and concentrated. Crude product was purified by prep-HPLC to give uncyclized intermediate that was neutralized, dried and subjected to the conditions described in general method Al using LiHMDS (step 2).
General Method B (Triflate formation)
A solution of benzimidazol-2-yl arylpyridinone derivate (1 equiv) and pyridine (20 equiv) in DCM was treated with Tf20 (8 equiv). The reaction was stirred at 0 °C for 2-8 h. The reaction was then quenched with satd aq NaHC03. The aqueous layer was extracted with DCM, and the combined organic extracts were dried over MgS04, filtered and concentrated. Crude product was used in the next step without further purification.
General Method C (Amine substitution)
A solution of benzoimidazol-2-yl arylpyridinone bistriflate derivate (1 equiv) in MeCN, DCM, or DMF was treated with amine (1.2-3 equiv). In the case where the amine is a salt (e.g. HCl), the amine salt was dissolved in MeOH or DMF and passed through a PoraPak Rxn CX ion exchange column to yield the free base which was added to the reaction mixture. The reaction mixture was stirred at rt or up to 45 °C for 1-48 h. Solvent was removed and the crude product was purified by column chromatography or prep-HPLC to give the desired product.
General Method D (Global deprotection)
A solution of protected benzoimidazol-2-yl arylpyridinone derivate (1 equiv) in TFA/conc. HCl (7: 1 v/v) was heated at 80-100 °C for 3-24 h. Solvent was removed and the crude product was purified by column chromatography (free base) or prep-HPLC (TFA salt) to give the desired product. To generate the desired product as a HCl salt, the free base was dissolved in MeOH and 1 M HCl-Et20 (2-4 equiv) was added at rt. The solution was stirred for 5 min and azeotroped twice with MeOH. General Method E (PMB-protection)
A solution of thiaisatoic anhydride (1 equiv), l-(chloromethyl)-4-methoxybenzene (1-1.2 equiv), K2CO3 (1-1.2 equiv) and/or KI (1-1.2 equiv) in DMF was stirred at rt for 4-24 h. The reaction mixture was then slowly added to H2O, precipitate was collected by vacuum filtration to give the desired.
Intermediates; lH-thienor3.4-din.31oxazine-2.4-dione
To a solution of 4-ieri-butoxycarbonylamino-thiophene-3-carboxylic acid (2.5 g, 10.2 mmol) in PhMe (25 mL) was added oxalyl chloride (1.29 mL, 15.3
Figure imgf000028_0001
mmol) at rt. The reaction mixture was gradually heated to 95 °C and stirred at 95 °C for 1 h. After reaction completion, the reaction was cooled to rt and filtered. The solid was washed with hexanes (2 x 5 mL), and dried under vacuum to afford the title compound as a cream solid (1.61 g, 93%). ¾ NMR (400 MHz, DMSO-d6) δ 11.57 (s, IH), 8.64 (d, J=3.2 Hz, IH), 6.89 (d, J=2.8 Hz, IH); MS ESI [M + H]+ 170.0, calcd for [C6H3NO3S+ H]+ 169.9. l-(4-methoxybenzyl)-lH-thienor3.4-diri.31oxazine-2.4-dione
According to general method E, to a solution of lH-thieno[3,4-d] [l,3]oxazine-
2,4-dione (1.6 g, 9.45 mmol) in anh DMF (20 mL), K2CO3 (1.56 g, 11.3 mmol) was added followed by KI (0.62 g, 3.78 mmol) under stirring at rt. PMBC1
Figure imgf000028_0002
(1.54 mL, 11.3 mmol) was added dropwise over 10 min and the reaction mixture was stirred for a further 2 h. After reaction completion the reaction mixture was poured into H2O (200 mL) to precipitate the product which was filtered, washed with H2O and dried to afford the title compound as an off-white solid (2.3 g, 84%). ¾ NMR (400 MHz, CDCI3) δ 8.35 (d, J=3.2 Hz, IH), 7.31 (d, J=8.8 Hz, 2H), 6.89 (d, J=8.8 Hz, 2H), 6.62 (d, J=3.2 Hz, IH), 5.08 (s, 2H), 3.80 (s, 3H); MS ESI [M + H]+ 291.2, calcd for [C14H11NO4S+ H]+ 290.0.
7-hvdroxy-4-(4-methoxybenzyl)-6-(6-(4-methylpiperazin-l-yl)-lH-benzordlimidazol-2- yl)thienoi3.2-blpyridin-5(4H)-one
According to general method Al, to a solution of l-(4- methoxybenzyl)- lH-thieno[3,2-d] [1,3] oxazine-2,4-dione
[Tetrahedron (1999) 55 6167-6174] (2.89 g, 10 mmol), ethyl 2-
Figure imgf000028_0003
(6-(4-methylpiperazin-l-yl)-lH-benzo[d]imidazol-2-yl)acetate [J.Med. Chem. (2009), 52, 278-292] (3.02 g, 10 mmol), LiHMDS (1 M in THF, 4 mL, 4 mmol) were used to generate the title compound as an orange solid (2.65 g, 51%). ¾ NMR (400 MHz, CDCh) δ 13.68 (br.s., 1H), 12.57 (s, 1H), 7.55 (dd, J=5.2, 2.0 Hz, 1H), 7.40-7.32 (m, 1H), 7.23 (d, J=8.8 Hz, 2H), 7.04-6.93 (m, 3H), 6.85 (d, J=8.8 Hz, 2H), 5.37 (s, 2H), 3.77 (s, 3H), 3.30-3.19 (m, 4H), 2.69-2.58 (m, 4H), 2.39 (s, 3H); MS ESI [M+H]+ 502.1, calcd for [C27H27N503S+H]+ 502.2.
4-hvdroxy-7-(4-methoxybenzyl)-5-(6-(4-methylpiperazin-l-yl)-lH-benzord1imidazol-2- vDthi eno [2.3 -bl pyri din-6(7H)-one
According to general method A2, a solution of l-(4- methoxybenzyl)-lH-thieno[2,3-d][l,3] oxazine-2,4-dione (0.40 g, 1.4 mmol), ethyl 2-(6-(4-methylpiperazin-l-yl)-lH-
Figure imgf000029_0001
benzo[d]imidazol-2-yl)acetate (0.46 g, 1.5 mmol), and LDA (1 M in THF, 6.2 mL, 4.5 mmol) were used to generate the title compound as a brown solid (0.220 g, 32%). ¾ NMR (400 MHz, CDCh) δ 13.87 (br.s., 1H), 12.52 (s, 1H), 7.49 (dd, J = 14.9 Hz, 1H), 7.40-7.24 (m, 3H), 7.03-6.64 (m, 5H), 5.28 (d, J = 13.8 Hz, 2H), 3.76 (s, 3H), 3.21 (d, J= 18.8 Hz, 4H), 2.65 (m, d, J= 19.1 Hz, 4H), 2.41 (s, 3H); MS ESI [M+H]+ 502.3, calcd for [C27H27N503S+H]+ 502.2.
Figure imgf000029_0002
one Reagents (General Method Al): l-(4-methoxybenzyl)-lH-thieno[2,3-d] [l,3]oxazine-2,4-dione (0.40 g, 1.4 mmol), ethyl 2-(6-morpholino-lH-benzo[d]imidazol-2-yl)acetate (0.4 g, 1.4 mmol), LDA (17 mL, 17 mmol). MS ESI [M+H]+ 489.2, calcd for [C26H24N4O4S+ H]+ 489.1. ethyl 3-(4-((4-methoxybenzyl)amino)thiophen-3-yl)-2-(6-(4-methylpiperazin-l-yl)-lH-benzo rdl imidazol -2-yl)-3-oxopropanoate
To a solution of ethyl 2-(6-(4-methylpiperazin-l-yl)-lH- benzo[d]imidazol-2-yl)acetate (2.58 g, 8.55 mmol) and l-(4- methoxy benzyl)-lH-thieno[3,4-d] [l,3]oxazine-2,4-dione (2.46 g,
Figure imgf000030_0001
8.55 mmol) in anh THF (48 mL), 1 M LDA (34 mL, 1 M in THF/hexane, 34 mmol) was added dropwise at 40 °C under Ar. The resulting brown solution was stirred at 40 °C for 1 h and then quenched with aq NH4CI (50 mL) at rt. The reaction mixture was diluted with H20 (50 mL) and extracted with DCM (2 x 200 mL). The combined organic layers were washed once with H20, dried over Na2S04, and concentrated to give crude ester. The crude product was purified by flash chromatography (gradient: EtO Ac/hex 0-40%, followed by MeOH/DCM 0-25%) to give the title compound as a light brown solid (3.05 g, 65%). MS ESI [M + H]+ 548.2, calcd for [C29H33N5O4S+ H]+ 548.2.
4-hvdroxy-l-(4-methoxybenzyl)-3-(6-(4-methylpiperazin-l-yl)-lH-benzo[dlimidazol-2- yl)thienor3.4-blpyridin-2(lH)-one
Ethyl 3-(4-((4-methoxybenzyl)amino)thiophen-3-yl)-2-(6-(4- methylpiperazin-l-yl)-lH-benzo[d]imidazol-2-yl)-3-oxopropanoate (3.05 g, 5.57 mmol), described above, was dissolved in anh THF
Figure imgf000030_0002
(30 mL) at rt under Ar. A solution of LDA (16.8 mL, 1 M in THF/hexane, 16.71 mmol) was added dropwise at 40 °C. The resulting brown solution was stirred at 40 °C for 1 h and then quenched with aq NH4CI (25 mL) at rt. The mixture was diluted with H2O (25 mL) and extracted with DCM (2 x 250 mL). The combined organic layers were washed once with H2O, dried over Na2S04, and concentrated to give crude product. The crude product was purified by flash chromatography (gradient: MeOH/DCM 0-20%) to give the title compound as a light brown solid (1.81 g, 65%). ¾ NMR (400 MHz, DMSO-de) δ 13.8-13.25 (m, 1H), 8.13 (d, J=3.6 Hz, 1H), 7.58 (d, J=8.8 Hz, 1H), 7.36-7.29 (m, 3H), 7.06-7.02 (m, 1H), 6.97 (d, J=3.6 Hz, 1H), 6.86 (d, J=8.4 Hz, 2H), 5.19 (s, 2H), 3.69 (s, 3H), 3.16 (br.s, 4H), 2.60 (br.s, 4H), 2.31 (s, 3H); a signal due to OH group cannot be readily detected. MS ESI 502.1 [M + H]+, calcd for [C27H27N5O3S+ H]+ 502.2. 7-(4-methoxybenzyl)-5-(5 and/or 6)-(4-methylpiperazin-l-yl)-l-((trifluoromethyl)sulfonyl)- lH-benzo[dlimidazol-2-yl)-6-oxo-6.7-dihvdrothieno[2.3-blpyridin-4-yl
trifluoromethanesulfonate
Synthesized according to general method B using 4-hydroxy-7-(4-methoxybenzyl)-5-(6-(4-
Figure imgf000031_0001
methy lpiper-azin- 1 -yl)-l H-benzo [d] imidazole- yl)thieno[2,3-b]py-ridin-6(7H)-one (0.22 g, 0.44 mmol), Tf2O(0.60 mL, 3.5 mmol), and pyridine (0.72 mL, 8.8 mmol). The title compounds obtained as an indeterminate mixture of regioisomers, were used in the next step without purification. MS ESI [M+H]+ 766.1, calcd for [C29H25F6N507S3+H]+ 766.1. l-(4-methoxybenzyl)-3-(5 and/or 6-(4-methylpiperazin-l-yl)-l-((trifluoromethyl)sulfonyl)- lH-benzo[dl-imidazol-2-yl)-2-oxo-1.2-dihvdrothieno[3.4-blpyridin-4-yl
trifluoromethanesulfonate According to general method B, a solution of 4-
Figure imgf000031_0002
hydroxy- 1 -(4-methoxybenzyl)-3-(6-(4- methylpiperazin-l-yl)-lH-benzo [d]imidazol-2-yl)thieno[3,4-b]pyridin-2(lH)-one (220 mg, 0.43 mmol) and pyridine (708 mL, 8.76 mmol) in DCM (12 mL) was added Tf20 (558 mL, 3.50 mmol) at -5° C. The reaction was stirred between -5 and 0 °C for lh. The reaction was quenched with satd aq NaHC03. The aqueous layer was extracted with DCM, and the combined organic extracts were dried over Na2S04, and concentrated under vacuum to give dark brown oil. The crude product, obtained as an indeterminate mixture of regioisomers, was used directly in the next step without further purification. MS ESI [M + H]+ 766.0, calcd for [C29H25F6N5O7S3+ H]+ 766.1.
7-(4-methoxybenzyl)-5-(5 and/or 6-moφholino-l- trifluoromethyl sulfonyl -lH- benzo[dlimidazol-2-yl)-6-oxo-6.7-dihvdrothieno[2.3-blpyridin-4-yl
trifluoromethanesulfonate
According to general method B, a solution of4- hydroxy-7-(4-methoxybenzyl)-5-(6-mo holino-lH-
Figure imgf000031_0003
benzo [d] imidazol-2-yl)-thieno[2,3 -b] pyridin-6(7H)- one (200 mg, 0.41 mmol) and pyridine (0.66 mL, 8.2 mmol) in DCM (20 mL) was added Tf20 (0.55 mL, 3.28 mmol) at 0°C. The reaction mixture was stirred at 0 °C for 2 h and then quenched with satd aq NaHC03. The aqueous layer was extracted with DCM. The combined organic extracts were dried over Na2S04, and concentrated to give the crude title compound (mixture of two regioisomers) as brown oil which was used directly in the subsequent step without further purification considering quantitative yield. MS ESI [M + H]+ 753.0, calcd for [C28H22F6N408S3+ H]+ 752.9.
5-methyl-lH-thieno[2.3-dl[1.31oxazine-2.4-dione
) in H20 (20 mL) was added methyl 2-
Figure imgf000032_0001
(1.0 g, 5.84 mmol) at rt. The resulting reaction was heated to 90°C for 2 h and then cooled to 0°C. A solution of triphosgene (0.866 g, 2.92 mmol) in PhMe (12 mL) was added dropwise over 10 min. The resulting solution was gradually warmed to rt and stirred for 2 h. The resulting solid was filtered, washed with H20 and dried to afford the title compound as a light pink solid (0.65 g, 61%). ¾ NMR (400 MHz, CDsOD) δ 6.65 (d, J=\2 Ηζ,ΙΗ), 2.42 (d, J=\2 Hz, 3H); MS ESI [M + H]+ 184.0, calcd for
Figure imgf000032_0002
1 -(4-methoxybenzyl)-5 -methyl- lH-thieno[2.3-dl [1.31 oxazine-2.4-dione
To a solution of 5-methyl-lH-thieno[2,3-d][l,3]oxazine-2,4-dione (0.625 g, 3.41 mmol) in anh DMF (9 mL), K2C03 (0.566 g, 4.09 mmol) was added followed
Figure imgf000032_0003
by KI (0.142 g, 0.85 mmol) under stirring at rt. PMB-C1 (0.56 mL, 4.06 mmol) was added dropwise to the reaction over 10 min and stirred for further 2 h. The reaction mixture was poured into H20 (100 mL) to precipitate the product which was filtered, washed with H20 and dried to afford the title compound as a light brown solid (0.935 g, 91%). ¾ NMR (400 MHz, CDCh) δ 7.38 (d, J=8.8 Hz, 2H), 6.90-6.88 (m, 2H), 6.46 (d, J=\2 Hz, 1H), 5.05 (s, 2H), 3.80 (s, 3H), 2.42 (d, J=1.2 Hz, 3H); MS ESI [M+H]+ 304.2, calcd for [Ci5Hi3N04S+H]+ 304.1.
4-hvdroxy-7-(4-methoxybenzyl)-3-methyl-5-(6-(4-methylpiperazin-l-yl)-lH- benzo[dlimidazol-2-yl) thieno[2.3-blpyridin-6(7H)-one
A solution of LDA (34 mL, 1 M in THF/hexane, 34 mmol) was added dropwise to a solution of ethyl 2-(6-(4-methylpiperazin-l-yl)-
Figure imgf000032_0004
lH-benzo[d]imidazol-2-yl)acetate (922 mg, 3.04 mmol) and l-(4- methoxy benzyl)-5-methyl-lH-thieno[2,3-d][l,3]oxazine-2,4-dione (925 mg, 3.04 mmol) in anh THF (28 mL) at 40°C under Ar. The resulting brown solution was stirred at 40 °C for 2 h and then quenched with aq, NH4CI (25 mL) at rt. The reaction mixture was diluted with H20 (25 mL) and extracted with DCM (2 x 100 mL). The combined organic layers were washed once with H2O, dried over Na2S04, and concentrated to give mixture of product and uncyclized ester. The crude mass was purified by flash chromatography (gradient: EtO Ac/hex 0-40%, followed by MeOH/DCM 0-25%) to give mixture of product and uncyclized ester (900 mg).
Above mixture of product and uncyclized ester (900 mg) was dissolved in anh THF (9 mL) at rt under Ar. A solution of LDA (5 mL, 1 M in THF/hexane) was added dropwise at 40°C. The resulting brown solution was stirred at 40°C for 1 h and worked up it as per above to give crude product. The crude product was purified by flash chromatography (gradient: MeOH/DCM 0-20%) to give the title compound as a cream solid (325 mg, 21%). ¾ NMR (400 MHz, CDCh) δ 12.54 (s, 1H), 7.38 (d, J=8.4 Hz, 1H), 7.33 (d, J=8.4 Hz, 2H), 7.01-6.98 (m, 2H), 6.85 (d, J=8.8 Hz, 2H), 6.40 (s, 1H), 5.26 (s, 2H), 3.80-3.61 (m, 6H), 3.60-3.51 (m, 4H), 2.89-2.88 (m, 4H), 2.63 (s, 3H); the signal due to OH group cannot be readily detected . MS ESI [M+H]+ 516.2, calcd for [C28H29N503S+H]+ 516.2.
7-(4-methoxybenzyl)-3-methyl-5-(5 and /or 6)-(4-methylpiperazin- 1 -yl)- 1 -
((trifluoromethyl)sulfonyl)-lH-benzordlimidazol-2-yl)-6-oxo-6.7-dihvdrothienor2.3- blpyridin-4-yl trifluoromethanesulfonate
The title compound was prepared according to general method B by utilizing
Figure imgf000033_0001
4-hydroxy-7-(4-methoxy-benzyl)-3- methyl-5-(6-(4-methylpiper-azin-l-yl)-lH-benzo[d]imidazol-2-yl) thieno[2,3-b]pyridin- 6(7H)-one (320 mg, 0.62 mmol), pyridine (1.0 mL, 12.4 mmol), Tf20 (0.833 mL, 4.96 mmol) in DCM (12 mL) to give a dark brown oil. The crude product, obtained as an indeterminate mixture of 2 regioisomers, was used directly in the next step without further purification. MS ESI [M + H]+ 780.0, calcd for [C30H27F6N5O7S3+ H]+ 780.1. ethyl 2-(6-((3r.5s)-rel-3.4.5-trimethylpiperazin-l-yl)-lH-benzo[dlimidazol-2-yl)acetate
A. 2-nitro-5-( (3r, 5s)-rel-3, 4, 5-trimethylpiperazin-l-yl)aniline
A mixture of 5-chloro-2-nitroaniline (1.73 g, 10 mmol), (3r,5s)-rel- 1,2,6- trimethylpiperazine (1.41 g, 11 mmol) and K2CO3 (2.72 g, 20 mmol) was
Figure imgf000033_0002
irradiated in microwave at 140 °C for 4 h. H20 (150 mL) was then added with stirring, suction filtered, rinsed with H20 and dried to give the title compound as a brown solid (2.47 g, 94%). ¾ NMR (400 MHz, DMSO-d6) δ 7.79 (d, J=10.0 Hz, 1H), 7.23 (s, 2H, NH2), 6.41 (dd, J=9.6, 1.6 Hz, 1H), 6.20 (d, J=2.4 Hz, 1H), 3.77 (d, J=12.4 Hz, 2H), 2.59 (t, J=11.8 Hz, 2H), 2.19-2.11 (m, 2H), 2.16 (s, 3H), 1.05 (d, J=6.0 Hz, 6H); MS ESI [M+H]+ 265.3, calcd for [Ci3H2oN402+H]+ 265.2.
B. 4-((3r, 5s)-rel-3, 4, 5-trimethylpiperazin-l-yl)benzene-l, 2-diamine
To a suspension of 2-nitro-5-((3r,5s)-rel-3,4,5-trimethylpiperazin-l- yl)aniline (2.47 g, 9.4 mmol) in MeOH (30 mL) was added 10% Pd/C
Figure imgf000034_0001
(247 mg, 10% wt.). The resulting mixture was hydrogenated under H2 balloon O/N. After additional 10% Pd/C (124 mg, 5% wt.) was added, it was hydrogenated under H2 balloon O/N, filtered, concentrated and dried to give the title compound as a dark brown solid (2.25 g, quantitative). ¾ NMR (400 MHz, CD3OD) δ 6.66 (d, J=8.4 Hz, 1H), 6.47 (d, J=2.4 Hz, 1H), 6.31 (dd, J=8.4, 2.8 Hz, 1H), 3.35-3.25 (m, 2H), 2.47-2.40 (m, 4H), 2.34 (s, 3H), 1.18 (d, J=5.6 Hz, 6H); MS ESI [M+H]+ 235.3, calcd for [Ci3H22N4+H]+ 235.2.
C. ethyl 2-( 6-( ( 3r, 5s)-rel-3, 4, 5-trimethylpiperazin-l-yl)-lH-benzo[d]imidazol-2-yl)acetate
To a solution of 4-((3r,5s)-rel-3,4,5-trimethylpiperazin-l- yl)benzene-l, 2-diamine (2.25g, 9.4 mmol) in EtOH (40 mL) was added ethyl 3-ethoxy-3-iminopropionate hydrochloride (2.93 g, 15
Figure imgf000034_0002
mmol). The resulting mixture was heated at 80 °C for 2 h. After removal of solvents, it was diluted with DCM/MeOH (100 mL/10 mL), basified with satd aq NaHCOs (30 mL) and separated. The aqueous layer was extracted with DCM (60 mLx2) and the combined extracts were concentrated and purified by flash chromatography (gradient: 100% EtOAc, then MeOH/DCM 0-20%) to give the title compound as a dark orange solid (2.32 g, 73%). ¾ NMR (400 MHz, CDCh) δ 10.13 (br s, 1H, NH), 7.53-6.88 (m, 3H), 4.25 (q, J=7.2 Hz, 2H), 4.03 (s, 2H), 3.43 (d, J=11.2 Hz, 2H), 2.61 (t, J=11.2 Hz, 2H), 2.50-2.41 (m, 2H), 2.35 (s, 3H), 1.32 (t, J=7.2 Hz, 3H), 1.19 (d, J=6.0 Hz, 6H); MS ESI [M+H]+ 331.3, calcd for [Ci8H26N402+H]+ 331.2.
7-hvdroxy-4-(4-methoxybenzyl)-6-(6-((3r.5s)-rel-3.4.5-trimethylpiperazin-l-yl)-lH- benzordlimidazol-2-yl)thienor3.2-blpyridin-5(4H)-one
To a mixture of l-(4-methoxybenzyl)-lH-thieno[3,2- d] [l,3]oxazine-2,4-dione (1.16g, 4 mmol) and ethyl 2-(6-((3r,5s)- rel-3 ,4,5-trimethylpiperazin- 1 -yl)-l H-benzo[d] imidazol-2-
Figure imgf000034_0003
yl)acetate (990 mg, 3 mmol) in THF (20 mL) was added LDA (1.0 M in THF/hex, 10 mL, 10 mmol) dropwise at rt. After addition, the resulting mixture was stirred at 40 °C for 1 h, diluted with DCM, quenched with satd aq NH4CI and extracted with DCM. The combined extracts were concentrated and purified by flash chromatography (gradient: 20-100% EtOAc/hex, then MeOH(0.5% NH3)/DCM 0-20%) to give a mixture of cyclized and uncyclized product as a brown foam (1.10 g). The mixture was redissolved in THF (15 mL) and LDA (1.0 M in THF/hex, 6 mL, 6 mmol) was added dropwise at rt. The process and workup were both the same as above. The title compound was obtained as an orange solid (630 mg, 40%). MS ESI [M+H]+ 530.3, calcd for
Figure imgf000035_0001
530.2.
4-(4-methoxybenzyl)-5-oxo-6-(l-((trifluoromethyl)sulfonyl)-(5 and/or 6)-((3r.5s)-rel-3.4.5- trimethyl-piperazin-l-yl)-lH-benzo[dlimidazol-2-yl)-4.5-dihydrothieno[3.2-blpyridin-7-yl trifluoromethane sulfonate
According to general method B, a solution of 7- hydroxy-4-(4-methoxybenzyl)-6-(6-((3r,5s)-rel-
Figure imgf000035_0002
3,4,5-trimethyl-piperazin-l-yl)-lH- benzo[d]imidazol-2-yl)thieno[3,2-b]pyridin-5(4H)-one (106 mg, 0.2 mmol) in DCM (15 mL) at 0 °C was added pyridine (0.32 mL, 4 mmol), followed by Tf20 (0.27 mL, 1.2 mmol). The resulting mixture was stirred at 0 °C for 1 h, diluted with DCM (10 mL), quenched with satd aq NaHC03 (15 mL), extracted with DCM (20 mLx2) and concentrated to give the crude title compound (an indeterminate mixture of two regioisomers) as a brown oil which was used directly in the subsequent steps. MS ESI [M+H]+ 794.1, calcd for
Figure imgf000035_0003
794.11.
Synthesis of 2-amino-4-ethoxythiophene-3-carbonitrile
The mixture of MeC(OMe)3 (2.26 mL, 12.3 mmol) and CH2(CN)2 (0.78 mL, 12.3 mmol) was stirred at 65 °C for 3 h before cooled down to rt. THF (10 mL)
Figure imgf000035_0004
and sulfur (395 mg) was added followed by addition of Et3N (1.72 mL, 12.3 mmol) dropwise. The resulting reaction mixture was stirred at 60 °C for 15 min and concentrated under reduced pressure. The residue was partitioned between EtOAc and H20, extracted with EtOAc, dried over Na2S04, filtered, and concentrated to dryness. The residue was triturated with DCM and filtered to give the title compound as a brown solid (1.23 g,
60%). ¾ NMR (400 MHz, CD3OD) δ 5.27 (s, 1H), 3.99 (q, J=7.0 Hz, 2H), 1.38 (t, J=7.0 Hz, 3H); MS ESI [M + H]+ 169.0, calcd for [C7H8N2OS+H]+ 169.0 . tert-Butyl 4-(5-amino-4-cvanothiophen-3-yl)piperazine- 1 -carboxylate
A mixture of MeC(OMe)3 (1.3 mL, 10 mmol) and CH2(CN)2 (0.66 g, 10 mmol) was heated in closed vial at 80 °C for 17 h. The reaction was
Figure imgf000036_0001
cooled to rt and tert-butyl piperazine-l-carboxylate (2.79 g, 15.0 mmol) was added. Heating with stirring was continued at 65 °C for 5 h. The reaction mixture was then concentrated in vacuo. Ss (0.34 g) and anh THF (10 mL) were added. The suspension was heated with stirring at 40 °C. Et3N (1.3 mL, 9.3 mmol) was added dropwise over 15 min. The oil bath temperature was increased to 60 °C and stirring was continued for 11 h. The reaction was then concentrated under reduced pressure and purified by flash chromatography (Si02, hexanes:EtOAc 5-50 %) to afford tert-butyl 4-(5-amino-4-cyanothiophen-3- yl)piperazine-l-carboxylate as a light orange solid (0.71 g, 23 %). ¾ NMR (400 MHz, DMSO-de) δ 7.12 (s, 2H), 5.46 (s, IH), 3.45-3.37 (m, 4H), 2.90-2.81 (m, 4H), 1.40 (s, 9H). MS ESI [M+H]+ 309.3, calcd for [Ci4H20N4O2S + H]+ 309.1.
tert-butyl 4-(4-amino-5-(6-(4-methylpiperazin-l-yl)-lH-benzo[dlimidazol-2-yl)-6-oxo-6.7- dihvdrothieno-r2.3-blpyridin-3-yl)piperazine-l-carboxylate
LiHMDS (1.0 M in THF, 2.8 mL, 2.8 mmol) was added dropwise at rt to a stirred suspension of ethyl 2-(6-(4- methylpiperazin-1 -yl)-lH-benzo[d]imidazol-2-yl)acetate (0.170 g, 0.56 mmol) and tert-butyl 4-(5-amino-4-
Figure imgf000036_0002
cyanothiophen-3-yl)piperazine-l-carboxylate (0.175 g, 0.56 mmol) in anh THF (10 mL) under Ar. The reaction was stirred at rt for additional 5 min and then heated in an oil bath at 40 °C for 1 h. The reaction was cooled to rt, quenched with satd aq NH4CI, concentrated under reduced pressure and purified by flash chromatography (MeOH/DCM 0-20 %) to give the title compound as a light tan solid (83 mg, 26%). ¾ NMR (400 MHz, CD3OD) δ 7.45 (d, J=8.8 Hz, IH), 7.10 (d, J=2.3 Hz, IH), 7.03 (dd, J=8.8, 2.3 Hz, IH), 6.18 (s, IH), 3.62-3.50 (m, 4H), 3.24-3.18 (m, 4H), 3.05-2.98 (m, 4H), 2.75-2.67 (m, 4H), 2.41 (s, 3H), 1.49 (s, 9H); MS ESI [M+H]+ 565.3, calcd for [C^HseNsOsS +H]+ 565.4. ethyl 2-(6-(4-methylpiperazine-l-carbonyl)-lH-benzo[dlimidazol-2-yl)acetate
A. (3, 4-Dinitrophenyl)(4-methylpiperazin-l-yl)methanone
To a suspension of 3,4-dinitrobenzoic acid (1.23 g, 5.8 mmol) in anh DCM (20 mL) at rt was added dropwise oxalyl chloride (1.0 mL, 11.7
Figure imgf000036_0003
mmol) followed by anh DMF (2 drops). The reaction was stirred overnight and then concentrated at rt. The residue was dissolved in anh THF (40 mL) at 0 °C under Ar. 1 -Methylpiperazine (1.3 mL, 11.7 mmol) was added dropwise (thick white suspension was stirred with intermittent shaking). After the addition the cooling was continued for 10 min before the cooling bath was removed. After stirring the reaction at rt for 3 h, H20 was added. THF was removed under reduced pressure and the aqueous residue was extracted (CH2CI2; 2 % MeOH in CH2CI2. 2x). The combined organic extracts were dried over Na2S04 and concentrated under reduced pressure to afford (3,4-dinitrophenyl)(4- methylpiperazin-l-yl)methanone as a light orange solid (1.77 g, quant) .¾ NMR (400 MHz, DMSO-de) δ 8.29 (d, J=8.3 Hz, IH), 8.27 (d, J=1.5 Hz, IH), 7.97 (dd, J=8.3, 1.8 Hz, IH), 3.59-3.68 (m, 2H), 3.24-2.53 ( m, 2H), 2.42-2.35 ( m, 2H), 2.21-2.32 ( m, 2H), 2.20 (s, 3H). MS ESI [M+H]+ 295.2, calcd for [C12H14N4O5 + H]+ 295.1.
B. ( 3, 4-Diaminophenyl) ( 4-methylpiperazin-l -yljmethanone
A solution of (3,4-dinitrophenyl)(4-methylpiperazin-l-yl)methanone
(0.53 g, 1.8 mmol) in THF (25 mL) and EtOH (50 mL) was degassed
Figure imgf000037_0001
with N2. Pd/C (191 mg, 0.18 mmol) was added and the reaction was stirred under H2 (1 atm) overnight at rt. The reaction mixture was then filtered through Celite and concentrated under reduced pressure to afford (3,4-diaminophenyl)(4-methylpiperazin-l- yl)methanone as a purple solid (0.44 g, quant). ¾ NMR (400 MHz, DMSO-d6) δ 6.61-6.55 (m, IH), 6.47-6.45 (m, 2H), 4.81 (br.s, 2H), 4.58 (br. s, 2H), 3.50-3.39 (m, 4H), 2.34-2.22 (m, 4H), 2.18 (s, 3H). MS ESI [M+H]+ 235.1, calcd for [C12H18N4O + H]+ 235.1.
C. ethyl 2-(6-(4-methylpiperazine-l-carbonyl)-lH-benzo[d]imidazol-2-yl)acetate
(3,4-Diaminophenyl)(4-methylpiperazin-l-yl)methanone (0.44 g, 1.8 mmol ) and 3-ethoxy-3-iminopropanoic acid hydrochloride (1.07 g, 5.5 mmol) in anh EtOH (100 mL) under Ar were heated with stirring
Figure imgf000037_0002
overnight at 65 °C. The reaction mixture was then concentrated under reduced pressure. The residue was taken in to H2O (15 mL), neutralized with 10 % aq Na2C03, extracted with CH2CI2 (2x), washed (brine) and dried over Na2S04. Purification by flash chromatography (0-50 % MeOH in CH2CI2) afforded the title compound as a yellow foam (0.31 g, 52%). ¾ NMR (400 MHz, CD3OD) δ 7.71-7.57 (m, 2H), 7.33 (d, J=8.2 Hz, IH), 4.23 (q, J=7.2 Hz, 2H), 3.91-3.40 (m, 4H), 2.62-2.38 (m, 4H), 2.34 (s, 3H), 1.28 (t, J=7.1 Hz, 3H); Signals due to CH2-ester are absent in CD3OD. MS ESI [M+H]+ 331.2, calcd for [Ci7H22N403+H]+ 331.2. ethyl 2-(6-(morpholine-4-carbonyl)-lH-benzordlimidazol-2-yl)acetate
A. (3, 4-dinitrophenyl)(morpholino)methanone
To a suspension of 3,4-dinitrobenzoic acid (1.30 g, 6.1 mmol) in anh DCM (50 mL) at rt was added dropwise (COCl)2 (1.0 mL, 11.7 mmol)
Figure imgf000038_0001
followed by anh DMF (2 drops). The reaction was stirred ovemig then concentrated at rt. The residue was dissolved in anh THF (24 mL) at 0 °C under Ar. morpholine (1.0 mL, 11.6 mmol) was added dropwise (thick white suspension was stirred with intermittent shaking). After the addition the cooling was continued for 10 min before the cooling bath was removed. After stirring the reaction at rt for 3 h, H20 was added. THF was removed under reduced pressure and the aqueous residue was extracted (CH2C12. 2x). The combined organic extracts were dried (Na2S04) and concentrated under reduced pressure to afford (3,4-dinitrophenyl)(morpholino)methanone as a light orange solid (1.8 g, quant) . ¾ NMR (400 MHz, DMSO-c e) δ 8.28 - 8.31 (m, 2 H), 8.00 (dd, J=8.28, 1.76 Hz, 1 H), 3.39 - 3.80 (m, 8 H).
B. ethyl 2-( 6-(morpholine-4-carbonyl)-lH-benzo[d]imidazol-2-yl)acetate
A solution of (3,4-dinitrophenyl)(mo holino)methanone (0.83 g, 2.9 mmol) in THF (30 mL) and EtOH (60 mL) was degassed with N2. Pd/C (0.31 mg, 0.29 mmol) was added and the reaction was stirred
Figure imgf000038_0002
under H2 (1 atm) overnight at rt. The reaction mixture was then filtered through Celite and concentrated under reduced pressure to afford (3,4- diaminophenyl)(mo holino)methanone as a purple foam. LCMS (ESI) m/z calcd for [C11H15N3O2 + H]+ 222.1 ; found 222.2. The material and ethyl 3-ethoxy-3-iminopropanoate hydrochloride (1.2 g, 6.2 mmol) in anh EtOH (100 mL) under Ar were heated with stirring overnight at 65 °C. The reaction mixture was then concentrated under reduced pressure. Purification by flash chromatography (0-20 % MeOH in CH2CI2) afforded the title compound as a pale red foam (0.43 g, 47%).. ¾ NMR (400 MHz, CD3OD) δ 7.52 - 7.76 (m, 2 H), 7.33 (dd, J=8.28, 1.51 Hz, 1 H), 4.22 (q, J=7.19 Hz, 2 H), 4.00 - 4.05 (m, 2 H), 3.70 (br. s., 8 H), 1.28 (t, J=7.15 Hz, 3 H); Signals due to CH2-ester are absent in CD3OD; MS ESI [M+H]+ 318.2, calcd for [Ci7H22N403+H]+ 318.1. ethyl 2-(5-methyl-6-(4-methylpiperazin-l-yl)-lH-benzo[dlimidazol-2-yl)acetate
A. 4-methyl-5-(4-methylpiperazin-l-yl)-2-nitroaniline
5-Chloro-4-methyl-2-nitroaniline (0.32 g, 1.7 mmol ) and 1- methylpiperazine (1.5 mL, 13.5 mmol) were heated in a sealed tube at
80 °C for 30 min followed by at 105 °C for 1 d and 120 °C for 2 d.
Figure imgf000039_0001
The reaction was later cooled, diluted with H20 and filtered. The collected solid was rinsed with H20 and dried in vacuo to afford the title compound a as a yellow solid (0.36 g, 84 %). ¾ NMR (400 MHz, DMSO-d6) δ 7.72 (s, 1 H), 7.27 (s, 2 H), 6.44 (s, 1 H), 2.97-2.86 (m 4 H), 2.49-2.39 (m, 4 H), 2.22 (s, 3 H), 2.11 (s, 3 H). LCMS (ESI) m/z calcd for [C12H18N4O2 + H]+ 251.1; found 235.3.
B. ethyl 2-(5-methyl-6-( 4-methylpiperazin-l-yl)-lH-benzo[d]imidazol-2-yl)acetate
4-methyl-5-(4-methylpiperazin-l-yl)-2-nitroaniline (0.36 g, 1.4 mmol) and Pd/C (10 %, 81 mg, 0.08 mmol) in EtOH (50 mL),
Figure imgf000039_0002
THF(25 mL) were degassed with N2 and then stirred under H2 (1 atm) for 5 d. The reaction mix was filtered through Celite, the pad was rinsed with EtOH. The filtrate was concentrated under reduced pressure to afford 4-methyl-5-(4- methylpiperazin-l-yl)benzene-l,2-diamine as a yellow tan solid (0.35 g, quant). The material (0.35 g) and ethyl 3-ethoxy-3-iminopropanoate hydrochloride (0.81 g, 4.1 mmol) in anh EtOH (70 mL) under Ar were heated with stirring overnight at 65 °C. The reaction mixture was then concentrated under reduced pressure, taken into H20 (20 mL) and basified with 2 M aq Na2C03 to pH 9. The mixture was extracted with DCM (2x); the organic extracts were dried (Na2S04) and concentrated under reduced pressure. Purification by flash chromatography (0-30 % MeOH in CH2C12) afforded the title compound as a yellow foam (0.36g, 82%). ¾ NMR (500 MHz, CD3OD) δ 7.35 (s, 1 H), 7.25 (s, 1 H), 4.22 (q, J=7.09 Hz,
2 H), 2.95 - 3.03 (m, 4 H), 2.88-2.58 (m, 4 H), 2.43 (s, 3 H), 2.41 (s, 3 H), 1.28 (t, J=7.09 Hz,
3 H); Signals due to CH2-ester are absent in CD3OD; LCMS (ESI) m/z calcd for [C17H24N4O2+ H]+ 317.2; found 317.3.
Ethyl 2-(5-fluoro-6-morpholino-lH-benzordlimidazol-2-yl)acetate
A. 4-fluoro-5-morpholino-2-nitroaniline
A mixture of 5-chloro-4-fluoro-2-nitroaniline (1.0 g, 5.24 mmol), T| T morpholine (1.37 mL, 15.7 mmol) and DMSO (5 mL) was heated in oil bath 140 °C for 3 h. Then H20 (50 niL) was added with stirring at 80°C to precipitate the product and allowed the suspension to rt, suction filtered, washed with H20 and dried to give the title compound as a yellow solid (1.25 g, 94%). ¾ NMR (400 MHz, CD3OD) δ 7.17 (d, J=14.0 Hz, IH), 6.37 (d, J=8.0 Hz, IH), 3.83 (t, J=4.4 Hz, 4H), 3.22 (t, J=4.8 Hz, 4H); MS ESI [M+H]+ 242.1, calcd for [Ci0Hi2FN3O3+H]+ 242.1.
B. 4-fluoro-5-morpholinobenzene-l , 2-diamine
To a 100 mL round-bottom flask was charged with 4-ΑυοΓθ-5^ο ηο1ίηο-2-ηίπΌ3ηί1ίη6 (1.23 g) and MeOH (37 mL) at rt under Ar blanket. Raney Nickel (0.123 g) was added under stirring with caution at rt. The reaction mass was slowly heated to 60-65° and hydrazine hydrate (0.86 mL) was added to the reaction mass dropwise in about 5 min. The reaction was stirred at 65-70°C for 2 hrs. After reaction completion, cooled it to rt and filtered the catalyst through a Celite pad under Ar and washed the Celite
Figure imgf000040_0001
pad with MeOH (5 mL * 2). The combined filtrate was concentrated and purified by flash chromatography (gradient: MeOH/DCM 0-25%) to give the title compound as a light brown solid (0.615 g, 57%). ¾ NMR (400 MHz, CD3OD) δ 6.51- 6.47 (m, 2H), 3.81 (t, J=4.8 Hz, 4H), 2.93 (t, J=4.8 Hz, 4H); MS ESI [M+H]+ 212.0, calcd for
[CioHi4FN30+H]+ 212.1.
C. ethyl 2-(5-fluoro-6-morpholino-lH-benzo[d]imidazol-2-yl)acetate
To a solution of 4-fluoro-5-mo holinobenzene-l,2-diamine (0.615 g, 2.91 mmol) in EtOH (30 mL) at 65°C was added ethyl 3-ethoxy-3-iminopropionate hydrochloride (1.14 g, 5.82 mmol) in two equal lots at an interval of 5 min each. Then stirred the reaction mass at 65°C for 2 hrs. After reaction completion concentrate the reaction mass under reduced
Figure imgf000040_0002
pressure to leaving behind thick brown oil. To the resulting oil H20 (25 mL) added and adjusted the pH ~ 10 using 2 M aq Na2C03. The resultant mixture was extracted with DCM (30 mL * 2) and the combined extracts were concentrated and purified by flash chromatography (gradient: Hex/ EtOAc 0-40%, then MeOH/DCM 0- 20%) to give the title compound as a brown solid (0.786 g, 88%). ¾ NMR (400 MHz, CD3OD) δ 7.26 (d, J=12.4 Hz, IH), 7.19 (d, J=7.6 Hz, 1H),4.25 - 4.20 (m, 2H), 3.88 (t, J=4.4 Hz, 4H), 3.08 (t, J=4.8 Hz, 4H), 1.28 (t, J=7.2 Hz, 3H); MS ESI [M+H]+ 308.1.0, calcd for [Ci5Hi8FN303+H]+ 308.1. Ethyl 2-(6-(4-methyl-1.4-diazepan-l-yl)-lH-benzordlimidazol-2-yl)acetate
A.5-(4-methyl-l, 4-diazepan-l-yl)-2-nitroaniline
A mixture of 5-chloro-2-nitroaniline (8.63 g, 50 mmol), 1-methyl-
1,4-diazepane (6.85 g, 60 mmol) and K2CO3 (8.28 g, 60 mmol) was
Figure imgf000041_0001
heat at 90 °C for 20 h. After diluting with H20 (500 mL) , it was extracted with EtOAc (60 mL x 3), concentrated and dried to give crude 5-(4-methyl-l,4- diazepan-l-yl)-2-nitroaniline as a dark red oil (12.50 g). NMR indicated a mixture of product and 5-chloro-2-nitroaniline (2: 1). ¾ NMR (400 MHz, CD3OD) δ 7.72 (d, J=10.0 Hz, 1H), 6.26 (dd, J=9.8, 2.6 Hz, 1H), 6.02 (d, J=2.4 Hz, 1H), 3.66-3.63 (m, 2H), 3.58 (t, J=6.4 Hz, 2H), 2.77-2.74 (m, 2H), 2.62-2.59 (m, 2H), 2.39 (s, 3H), 2.07-2.00 (m, 2H); MS ESI [M+H]+ 251.3, calcd for [Ci2Hi8N402+H]+ 251.15.
B. 4-( 4-methyl-l, 4-diazepan-l -yl)benzene-l, 2-diamine
To a mixture of crude 5-(4-methyl-l,4-diazepan-l-yl)-2-nitroaniline (12.50 g) and Raney-Nickel (1.25 g) in MeOH (150 mL) at 65 °C was added N2H4-H2O (12.0 mL) over 10 min. After addition, the resulting
Figure imgf000041_0002
mixture was stirred at 70 °C for 30 min. Upon cooling to rt, it was filtered through Celite and rinsed with MeOH. The filtrate was concentrated and dried to give crude 4-(4-methyl-l,4-diazepan-l-yl)benzene-l, 2-diamine as a dark red brown oil (10.57 g).
¾ NMR (400 MHz, CD3OD) δ 6.63 (d, J=8.0 Hz, 1H), 6.53 (dd, J=8.4, 2.4 Hz, 1H), 6.26 (d, J=2.4 Hz, 1H), 3.60-3.40 (m, 4H),
Figure imgf000041_0003
2.75-2.71 (m, 2H), 2.62-2.58 (m, 2H), 2.37 (s, 3H), 2.04-1.97 (m,
2H).
C. Ethyl 2-(6-(4-methyl-l, 4-diazepan-l-yl)-lH-benzo[d]imidazol-2-yl)acetate
A mixture of crude 4-(4-methyl-l,4-diazepan-l-yl)benzene-l, 2-diamine (10.57 g) and ethyl 3-ethoxy-3-iminopropionate hydrochloride (19.50 g, 100 mmol) in EtOH (200 mL) was heated at 90 °C for 2 h. After removal of solvents, it was diluted with H20 (50 mL), basified with 2 M aq Na2C03 (40 mL) and extracted with DCM (60 mL x 3). The combined extracts were concentrated and purified by flash chromatography (gradient: 0-100% EtOAc/hexane, then MeOH/DCM 0-25%) to give the title compound as a dark brown oil (7.31 g, 46% over 3 steps). ¾ NMR (400 MHz, CD3OD) δ 8.38 (d, J=8.8 Hz, 1H), 6.82-6.77 (m, 2H), 4.22 (q, J =6.8 Hz, 2H), 3.66-3.61 (m, 2H), 3.54 (t, J =6.4 Hz, 2H), 2.85-2.80 (m, 2H), 2.68-2.64 (m, 2H), 2.41 (s, 3H), 2.12-2.05 (m, 2H), 1.29 (t, J =7.0 Hz, 3H); MS ESI [M+H]+ 317.3, calcd for [Ci7H24N402+H]+ 317.20. Representative Examples:
Al : 4-amino-5 -(6-(4-methylpiperazin- 1 -yl)- 1 H-benzo [dl imidazol-2-yl)thieno [2.3-bl pyridin- 6(7H)-one
To a solution of ethyl 2-(6-(4-methylpiperazin-l-yl)-lH benzo[d]imidazol-2-yl)acetate (2.42 g, 8.05 mmol) and 2 aminothiophene-3-carbonitrile (1.0 g, 8.05 mmol) in anh
Figure imgf000042_0001
THF (40 mL) at 40 °C added LDA (40 mL, 1 M in THF/hexane, 40 mmol) dropwise over 15 min under Ar. The resulting brown solution was stirred at 40 °C for 2 h and then quenched with aq NH4CI (50 mL) at rt. The mixture was diluted with H20 (125 mL) and extracted with ethyl acetate (2 x 200 mL). The combined organic layers were washed once with H20, dried over Na2S04, and concentrated to give crude product. The crude product was triturated with DCM (20 mL) followed by MeOH (25 mL) to give the title compound as a light brown solid (1.95 g, 64%).
The free base (1.95 g) was suspended in MeOH (50 mL) and added 1 M HCl-Et20 (13 mL) at rt. The suspension was stirred for 15 min at rt and concentrated under vacuum and azeotroped with MeOH (2 x 25 mL) to give the HC1 salt as a dark brown solid (2.28 g, 62%); ¾ NMR (400 MHz, CD3OD) δ 7.69 (d, J=9.2 Hz, 1H), 7.52 (d, J=5.6 Hz, 1H), 7.36 (dd, J=8.8, 2.4 Hz, 1H), 7.30 (d, J=2.4 Hz, 1H), 7.19 (d, J=5.6 Hz, 1H), 3.97-3.93 (m, 2H), 3.70- 3.67 (m, 2H), 3.39-3.35 (m, 2H), 3.34-3.18 (m, 2H), 3.01 (s, 3H); MS ESI [M + H]+ 381.2, calcd for [C19H20 N6OS+ H]+ 381.1.
Figure imgf000042_0002
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
A22: 6-(6-(4- 16 mg (9%); methylpiperazin- 1 -yl)- yellow solid; lH-benzo[d]imidazol-2- TFA salt yl)-7-((pyridin-4- ylmethyl)amino)thieno[3,
2-b] pyridin-5 (4H)-one
Step 1 : Reagents (general method C): a mixture of 4-(4-methoxybenzyl)-6-(5 and/or 6-(4- methylpiperazin-l-yl)-l-((trifluoromethyl)sulfonyl)-lH-benzo[d]imidazol-2-yl)-5-oxo-4,5- dihydro-thieno[3,2-b]pyridin-7-yl trifluoromethanesulfonate (crude, 0.3 mmol), pyridin-4- ylmethanamine (0.09 mL, 0.89 mmol). MS ESI [M+H]+ 724.2, calcd for
[C34H32F3N704S2+H]+ 724.2.
Step 2: Reagents (general method D): 4-(4-methoxybenzyl)-6-(5 and/or 6-(4-methylpiperazin- l-yl)-l-((trifluoromethyl)sulfonyl)-lH-benzo[d]imidazol-2-yl)-7-((pyridin-4- ylmethyl)amino)thieno[3,2-b]pyridin-5(4H)-one, TFA (4 mL), cone. HC1 (1 mL). ¾ NMR (400 MHz, CDsOD) δ 8.77 (d, J=6.8 Hz, 2H), 8.05 (d, J=6.8 Hz, 2H), 7.84 (d, J=5.6 Hz, IH), 7.58 (d, J=8.8 Hz, IH), 7.25 (d, J=2.0 Hz, IH), 7.14 (dd, J=8.8, 2.0 Hz, IH), 7.06 (d, J=5.6 Hz, IH), 5.41 (s, 2H), 3.91-3.78 (m, 2H), 3.75-3.59 (m, 2H), 3.41-3.33 (m, 2H), 3.21-3.05 (m, 2H), 3.00 (m, 3H). MS ESI [M+H]+ 472.3, calcd for [C2sH25N7OS+H]+ 472.2.
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
The following compounds were prepared according to the general method A3.
A60: 4-amino-5-(5-(4-methylpiperazine-l-carbonyl)-lH-benzo[dlimidazol-2-yl)thieno[2.3- bl -pyridin-6(7H)-one
Figure imgf000065_0001
LDA (1.0 M in THF/hexanes, 2.3 niL, 2.3 mmol) was added dropwise over 15 min at rt to a stirred suspension of ethyl 2-(6-(4-methylpiperazine-l-carbonyl)-lH- benzo[d]imidazol-2-yl)acetate (0.150 g, 0.45 mmol) and 2-aminothiophene-3-carbonitrile (0.056 g, 0.45 mmol) in anh. THF (20 mL) under Ar. The addition was done initially at rt and after 5 minutes at 35 °C. The heating was continued at 35 °C for 1 h before the reaction mixture was cooled to rt, quenched with aq NH4CI and concentrated under reduced pressure. Purification by RP HPLC afforded N-(3-cyanothiophen-2-yl)-2-(5-(4-methylpiperazine-l- carbonyl)-lH-benzo[d]imidazol-2-yl)acetamide*TFA as a light brown solid (82 mg, 35 %). ¾ NMR (400 MHz, CD3OD) δ 7.96 (s, 1 H), 7.89 (d, J=8.5 Hz, 1 H), 7.68 (dd, J=8.4, 1.4 Hz, 1 H), 7.09 - 7.14 (m, 2 H), 3.25 - 3.81 (m, 8 H), 2.97 (s, 3 H).
Step 2. The product of the previous reaction was filtered through PoraPak (2 g, using MeOH then 2 M NH3 in MeOH) and dried. An anh THF (12 mL) solution of the material (0.055 g, 0.13 mmol) under Ar was treated with LiHMDS (1.0 M in THF, 0.7 mL, 0.7 mmol) over 3 min at rt„ stirred for 10 min and heated at 45 °C for 95 min. The reaction was then cooled to rt, quenched with aq NH4CI, concentrated under reduced pressure and purified by prep HPLC. Filtration through PoraPak (2 g) and trituration with CH2CI2 afforded the title compound as a light yellow solid 3.6 mg (3 %). ¾ NMR (400 MHz, CD3OD) δ 7.58 - 7.77 (m, 2 H), 7.51 (d, J=5.80 Hz, 1 H), 7.30 (dd, J=8.30, 1.30 Hz, 1 H), 7.14 (d, J=5.80 Hz, 1 H), 3.53 - 3.92 (m, 4 H), 2.48 - 2.70 (m, 4 H), 2.43 (s, 3 H). MS ESI [M + H]+ 409.2, calcd for
+ 409.2.
Figure imgf000065_0002
Reagents (method A3): Step 1 : ethyl 2-(5-methyl-6-(4-methylpiperazin-l -yl)-lH- benzo[d]imidazol-2-yl)acetate (0. 17 g, 0.53 mmol), 2-aminothiophene-3-carbonitrile (72 mg, 0.53 mmol), LDA (1.0 M in THF/hexanes, 1.7 mL, 1.7 mmol) in anh THF (12 mL). Step 2: LiHMDS (1.0 M in THF, 1.7 mL, 1.7 mmol) in anh THF (20 mL).
¾ NMR (400 MHz, CD3OD) δ 7.48 (d, J=5.77 Hz, l H), 7.44 (s, 1 H), 7.38 (s, 1 H), 7.12 (d, J=5.77 Hz, 1 H), 3.58 - 3.59 (m, 2 H), 3.43-3.23 ( m., 4 H), 3.07 - 3.22 (m, 2 H), 3.01 (s, 3 H), 2.45 (s, 3 H). MS ESI [M+H]+ 395.1, calcd for [C20H22N6OS +H]+ 395.2.
A62: 4-amino-5-(5-(morpho- 6.5 mg (2 %); line-4-carbonyl)- 1 H-benzo- white solid; [d] imidazol-2-yl)thieno [2,3 - free base b] pyridin-6(7H)-one
Figure imgf000066_0001
H
Reagents (method A3): Step ] : ethyl 2-(6-(morpholine-4-carbonyl)-lH- 3enzo[d]imidazol-2- yl)acetate (0.22 g, 0.70 mmol ) and 2-aminothiophene-3-carbonitrile (8' % mg, 0.70 mmol), LiHMDS (1.0 M in THF, 3.5 n lL, 3.5 mmol) in anh. THF (24 mL). Step 2: LiHMDS (1.0 M in THF, 1.2 mL, 1.2 mmol) in a nh THF (20 mL).
¾ NMR (400 MHz, CD3OD) δ 7.63 - 7.76 (m, 2 H), 7.48 - 7.52 (m, 1 H), 7.31 (dd, J=8.30, 1.50 Hz, 1 H), 7. 14 (d, J=5.77 Hz, 1 H), 3.58 - 3.88 (m, 8 H). MS ESI [M+H]+ 396.2, calcd for [C19H17N5O3S +H]+ 396.1
A63; 7-(cvclopropylamino)-6-(6-(4-methylpiperazin-l-yl)-lH-benzo[dlimidazol-2- yl)thienor3.2-bl-pyridin-5(4H)-one 2.2.2-trifluroaceatate
A suspension of 7-hydroxy-6-(6-(4-methylpiperazin-l - yl)-lH-benzo[d]imidazol-2-yl)thieno[3,2-b]pyridin-
5(4H)-one (58 mg, 0.152 mmol) in anhydrous DCM (1 mL) was added Tf2O(0.55 mL, 0.916 mmol) dropwise at
Figure imgf000066_0002
rt. The resulting reaction mixture was stirred at rt overnight before addition of cyclopropanamine (100 mg, 1.83 mmol) at 0 °C dropwise. The resulting reaction mixture was stirred at 40 °C overnight and diluted with DCM followed by washing with satd NaHC03. The organic layer was dried over Na2S04, filtered, and concentrated to dryness. The residue was dissolved in MeOH and run through PoraPak followed by removal of solvent under reduced pressure. The crude product was purified by prep HPLC to give the title compound as a yellow solid (5 mg, 6% yield). ¾ NMR (400 MHz, CD3OD) δ 7.98 (d, J=5.5 Hz, 1H), 7.61 (d, J=9.0 Hz, 1H), 7.26 (d, J=2.0 Hz, 1H ),
7.21 (dd, J=8.4, 2.0 Hz, 1H), 7.10 (d, J=5.5 Hz, 1H), 3.92-3.84 (m, 2H), 3.71 -3.62 (m, 2H), 3.41-3.36 (m, 2H), 3.20-3.10 (m, 2H), 3.09-3.03 (m, 1H), 3.01 (s, 3H), 1.04-0.96 (m, 2H), 0.93-0.89 (m, 2H); MS ESI [M + H]+ 421.2 , calcd for [C22H24N6OS+H]+ 421.2.
A64 ; 4-amino-5-(6-(piperazin- 1 -yl)- 1 H-benzo Tdl imidazol-2-vDthieno T2.3-bl pyridin-6(7H)- one
tert-butyl 4-(3-amino-4-nitrophenyl)piperazine-l-carboxylate
A mixture of 5-chloro-2-nitroaniline (2.5 g, 14.48 mmol), tert-butyl piperazine-l-carboxylate (3.24 g, 17.38 mmol) and K2CO3 (4.0 g, 28.96
Figure imgf000067_0001
mmol) in DMSO
(100 mL) was stirred at 100 °C for 3 days. H20 (150 mL) was then added with stirring, suction filtered, rinsed with H20 and dried to give the title compound as a brown solid (2.6 g, 57%). ¾ NMR (400 MHz, CDCh) δ 8.04 (d, J=9.79 Hz, 1 H), 6.27 (dd, J=9.66, 2.64 Hz, 1 H), 6.21 - 6.11 (m, 2 H), 5.95 (d, J=2.51 Hz, 1 H), 3.61 - 3.54 (m, 4 H), 3.40 - 3.34 (m, 4 H), 1.50 (s, 9 H); MS ESI [M+H]+ 323.2, calcd for [Ci5H22N404+H]+ 323.2. tert-butyl 4-(3, 4-diaminophenyl)piperazine-l-carboxylate
To a suspension of tert-butyl 4-(3-amino-4-nitrophenyl)piperazine-l- carboxylate
Figure imgf000067_0002
(2.6 g, 8.04 mmol) in MeOH (150 mL) was added 10% Pd/C (130 mg,
5% wt). The resulting mixture was hydrogenated under H2 balloon O/N. The resulting reaction mixture was filtered, concentrated and dried to give the title compound as a dark brown solid (2.29 g, 97%). ¾ NMR (400 MHz, CDCh) δ 6.66 (d, J=8.28 Hz, 1 H), 6.39 (d, J=2.51 Hz, 1 H), 6.34 (dd, J=8.28, 2.51 Hz, 1 H), 3.60 - 3.53 (m, 4 H), 3.46 - 3.23 (m, 4 H), 3.02- 2.95 (m, 4 H), 1.49 (s, 9 H); MS ESI [M+H]+ 293.1, calcd for [Ci5H24N402+H]+ 293.2. tert-butyl 4-(2-(2-ethoxy-2-oxoethyl)-lH-benzo[d]imidazol-6-yl)piperazine-l-carboxylate To a solution of tert-butyl 4-(3,4-diaminophenyl)piperazine-l-carboxylate (100 mg, 0.34 mmol) in EtOH (3 mL) was added ethyl 3-ethoxy-3-iminopropionate hydrochloride (190 mg, 0.68 mmol). The resulting mixture was heated at 60 °C for 3 h. After removal of solvents, it was diluted with DCM (10 mL), adjust pH ~ 8 with satd NaHC03 and separated. The aqueous was extracted with DCM (10 mLx2) and the combined extracts were dried over NaS04, then concentrated and purified by flash chromatography (gradient: 100% EtOAc, then MeOH/DCM 0-20%) to give the title compound as a dark orange solid (116 mg, 87%). ¾ NMR (400 MHz, CD3OD) δ 7.49 - 7.40 (m, 1 H), 7.15 - 7.10 (m, 2 H), 4.22 (q, J=7.11 Hz, 2 H), 3.95 (s, 1 H), 3.61 (br. s., 4 H), 3.11 (br. s., 4 H), 1.50 (s, 9 H), 1.28 (t, J=7.15 Hz, 3 H); MS ESI [M+H]+ 389.2, calcd for [C2oH28N404+H]+ 389.2. tert-butyl 4-(2-( 4-amino-6-oxo-6, 7-dihydrothieno[2, 3-b ]pyridin-5-yl)-lH-benzo[d]imidazol- 6-yl)piperazine-l-carboxylate
Figure imgf000068_0001
to general method A, to a solution of 2-amino-4- ethoxythiophene-3-carbonitrile (64 mg, 0.52 mmol), tert-butyl 4-(2-(2-ethoxy-2-oxoethyl)- lH-benzo[d]imidazol-6-yl)piperazine-l-carboxylate (200 mg, 0.52 mmol), LiHMDS (1 M in THF, 2.0 mL, 2.06 mmol).) were used to generate the title compound as a light brown solid (88 mg, 35%). ¾ NMR (500 MHz, DMSO-c e) δ 12.72 - 12.61 (m, 1 H), 12.13 - 12.02 (m, 1 H), 10.72 - 10.55 (m, 1 H), 8.01 - 7.93 (m, 1 H), 7.57 (d, J=5.62 Hz, 1 H), 7.52 - 7.43 (m, 1 H), 7.24 - 7.10 (m, 2 H), 6.93 - 6.87 (m, 1 H), 3.52 - 3.44 (m, 4 H), 3.07 - 3.00 (m, 4 H), 1.45 - 1.40 (m, 9 H); MS ESI [M+H]+ 467.2, calcd for ^s^eNeOsS+Hf 467.2.
4-amino-5-( 6-(piperazin-l-yl)-lH-benzo[d]imidazol-2-yl)thieno[2, 3-b ]pyridin-6(7H)-one
A mixture of tert-butyl 4-(2-(4-amino-6-oxo-6,7- dihydrothieno[2,3-b]pyridin-5-yl)-lH-benzo[d]imidazol-6- yl)piperazine-l-carboxylate (83 mg, 0.178 mmol) in TFA (1 mL)
Figure imgf000068_0002
was stirred at rt for 2 h before concentrated. The residue was dissolved in MeOH (20 mL) and run through PoraPak then concentrated to give the title compound as a yellow solid (45 mg, 69%). ¾ NMR (400 MHz, DMSO-c e) δ 12.65 - 12.58 (m, 1 H), 10.77 - 10.61 (m, 1 H), 8.03 - 7.94 (m, 1 H), 7.59 (d, J=5.77 Hz, 1 H), 7.54 - 7.42 (m, 1 H), 7.19 - 7.10 (m, 2 H), 6.92 - 6.86 (m, 1 H), 3.09 - 3.01 (m, 4 H), 2.94 - 2.88 (m, 4 H); the signal due to NH2 cannot be readily detected. MS ESI [M+H]+ 367.2, calcd for
[Ci8Hi8N6OS+H]+ 367.1.
A65: 4-amino-5-(6-(4-(oxetan-3-yl)piperazin-l-yl)-lH-benzo[d]imidazol-2-yl)thieno[2,3- b]pyridin-6(7H)-one A mixture of 4-amino-5-(6-(piperazin-l-yl)-lH- benzo[d]imidazol-2-yl)thieno[2,3-b]pyridin-6(7H)-one (45 mg, 0.123 mmol), oxetan-3-one (8.8 mg, 0.123
Figure imgf000069_0001
mmol), and NaBH(OAc)3 (120 mg, 0.552 mmol) in DCE (2 mL) was stirred at rt overnight then filtered. The filtrate was concentrated and purified by prep. HPLC to give the title compound as TFA salt as a yellow solid (50 mg, 76%). ¾ NMR (400 MHz, CD3OD) δ 7.66 (d, J=9.03 Hz, 1 H), 7.51 (d, J=5.77 Hz, 1 H), 7.29 (t, J=8.91 Hz, 2 H), 7.18 (d, J=6.02 Hz, 1 H), 4.98 - 4.87 (m, 4H), 4.54 - 4.45 (m, 1 H), 3.63 - 3.40 (m, 8 H); MS ESI [M+H]+ 423.2, calcd for [C2iH22N602S+H]+ 423.2.
Figure imgf000069_0002
Figure imgf000070_0001
6-(4- for
(1.5 7.45 (m, 3H), 3.03
7.69 (m, 5 H), 3.60 for
for
Figure imgf000071_0001
Step 2: Reagents (general method D): a mixture of 5-(6-((2S,6R)-2,6-dimethylmorpholino)-l-
Figure imgf000072_0001
Figure imgf000073_0001
Example B: HPK1 Inhibition Assay
Active HPK1 (MAP4K1) was purchased as an N-terminal GST fusion of human HPK1 (aa 1-346) from Invitrogen (cat # PV6355). HPK1 activity was measured using an indirect ELISA detection system. GST-HPK1 (0.6 nM) was incubated in the presence of 12 μΜ ATP (Sigma cat# A7699), 5 mM MOPS (pH 7.2), 2.5 mM β-glycerol-phosphate, 5 mM MgCb, 0.4 mM EDTA, 1 mM EGTA, 0.05 mM DTT, in a 96 well microtitre plate pre- coated with 0.5 μg/well bovine myelin basic protein (MBP) (Millipore, cat #13-110). The reaction was allowed to proceed for 30 min, followed by 5 washes of the plate with Wash Buffer (phosphate buffered saline supplemented with 0.2% Tween 20), and incubation for 30 min with a 1 :3000 dilution of anti-phospho-threonine rabbit polyclonal antibody (Cell Signaling cat# 9381). The plate was washed 5 times with wash buffer, incubated for 30 min in the presence of goat anti-rabbit horse radish peroxidase conjugate (BioRad cat# 1721019, 1:3000 concentration), washed an additional 5 times with wash buffer, and incubated in the presence of TMB substrate (Sigma cat# T0440). The colorimetric reaction was allowed to continue for 5 min, followed by addition of stop solution (0.5 N H2SO4), and quantified by detection at 450 nm with a monochromatic plate reader (Molecular Devices M5).
Compound inhibition was determined at either a fixed concentration (10 μΜ) or at a variable inhibitor concentration (typically 50 μΜ to 0.1 μΜ in a 10 point dose response titration). Compounds were pre-incubated in the presence of enzyme for 15 min prior to addition of ATP and the activity remaining quantified using the above described activity assay. The % inhibition of a compound was determined using the following formula; % inhibition = 100 x (1 - (experimental value - background value)/(high activity control - background value)). The IC50 value was determined using a non-linear 4 point logistic curve fit (XLfit4, IDBS) with the formula; (A+(B/(l+((x/C)AD)))), where A = background value, B = range, C = inflection point, D = curve fit parameter.
Example C: FLT3 Inhibition Assay
FLT3 and LCK compound inhibition were determined using FRET based Z'-LYTE Kinase Assay Kit with Tyrosine 2 peptide as the substrate (Invitrogen cat # PV3191). The FLT3 kinase assay was performed according to the manufacturer's suggested specifications with an ATP concentration of 940 μΜ and 1 nM FLT3 (Invitrogen cat # PV3182) and 180 μΜ ATP and 25 nM LCK (Invitrogen cat # P3043) for the LCK kinase reaction. The % inhibition values were determined according to the manufacturer's directions and IC50 values were obtained using a non-linear 4 point logistic curve fit (XLfit4, IDBS).
In Table 1 below, IC50 value ranges for exemplary compounds are given. The IC50 ranges are indicated as "A," "B," and "C," for values less than or equal to 0.05 μΜ; those greater than 0.05 μΜ and less than or equal to 0.5 μΜ; and those greater than 0.5 μΜ, respectively.
Table 1: Inhibition Data of HPK1, Lck and Flt3
Figure imgf000074_0001
Al A B A
A2 A B A
A3 C - -
A4 A B A
A5 C C -
A6 B - -
A7 A B A
A8 A B A
A9 A A A
A10 A B A
All A
A12 A - -
A13 A B A
A14 A A A
A15 A A A
A16 A - -
A17 A A A
A18 A B A
A19 B
A20 C
A21 A C B
A22 A B A
A23 A A A
A24 A A A
A25 A - -
A26 A - -
A27 A A A
A28 A - -
A29 A B -
A30 A A A
A31 A B A
A32 C A -
A33 A B A
A34 A A A
A35 A A A
A36 A A A
A37 A A A
A38 A A A
A39 A A A
A40 A A A
A41 A A A
A42 A A A
A43 A B A
A44 A A A
A45 A A A
A46 A B A
A47 A - -
A48 A A A A49 C - -
A50 C C -
A51 A - -
A52 A A A
A53 A A A
A54 A B A
A55 A B A
A56 A - -
A57 A B A
A58 A B A
A59 - - -
A60 B B -
A61 A B A
A62 B - -
A63 A - -
A64 A B A
A65 A B A
A66 A A A
A67 A B A
A68 A A A
A69 - B A
A70 - A A
A71 A A A
A72 A C A
A73 B - -
A74 A - -
A75 A - -
A76 A - -
A77 A - -
Example D: In vitro Phosphorylation Assays
Jurkat E6.1 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA), and maintained according to the supplier's instructions. Cells were washed three times and starved in RPMI 1640 medium supplemented with 0.5% fetal calf serum for 18 h at 37 °C. Serum starved cells were pretreated with the indicated concentration of inhibitor for 4 hours before stimulation with 10 μg/ml a-CD3 antibody (BioLegend, Inc., San Diego, CA) for 10 min at 37 °C. The cells were washed once in phosphate-buffered saline (pH 7.4) containing 10 mm sodium pyrophosphate, 10 mm sodium fluoride, 10 mm EDTA, and 1 mm sodium orthovanadate. Protein lysates were prepared using ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer. A total of 100 μg of cell lysate was loaded onto Bis-Tris gels (Life Technologies, Carlsbad, CA) with full-range molecular weight marker as a size reference, and resolved by SDS-PAGE electrophoresis. Proteins were transferred to PVDF membrane (Millipore, Billerica, MA), blocked and probed with antibodies for phospho-SLP-76 (Ser376) (rabbit polyclonal #13177; Cell Signaling Technology Inc., Danvers, MA), SLP-76 (rabbit polyclonal #4958; Cell Signaling Technology Inc., Danvers, MA), phospho-ERK (mouse monoclonal sc-7383; Santa Cruz Biotechnology Inc., Santa Cruz, CA) and ERK1/2 (rabbit polyclonal 06-182; Millipore, Billerica, MA). Secondary antibodies were diluted 1 in 15,000 and incubated for lh at rt. Protein bands were visualized and quantified using Odyssey near infrared imager (LI-COR, Lincoln, NE).
Table 2 below lists effects of representative compounds of the present invention against SLP- 76 serine 376 phosphorylation and ERK1/2 T202/Y204 phosphorylation in a-CD3 stimulated Jurkat E6.1 cells.
Table 2. Effects of HPK1 inhibitors against SLP-76 serine 376 phosphorylation and ERK1/2 T202/Y204 phosphorylation in a-CD3 stimulated Jurkat E6.1 cells.
Figure imgf000077_0001
*>75 % inhibition as estimated by immunoblot analysis
Example E: Syngeneic CT26 cell line xenograft model. The CT26 WT cell line, which is an N-nitroso-N-methylurethane-(N MU) induced, mouse- derived, undifferentiated colon carcinoma cell line, was obtained from American Type Culture Collection (ATCC CRL-2638, Manassas, VA, DC, USA). Cells were grown in Roswell Park memorial Institute medium commonly referred to as RPMI 1640 Medium containing 4.5 g/L glucose, 0.11 g/L sodium pyruvate, 1.5 g/L sodium bicarbonate, L- glutamine& 2.385 g/L HEPES plus 10% fetal bovine serum. Six to eight week old female BALB/c mice were purchased from Jackson Laboratories and received and acclimated at the MaRS-TMDT Animal Resources Centre for 1 week prior to the start of the experiment. The mice were fed ad libitum autoclaved water and Rodent Lab Diet (Harlan Teklad LM-485) consisting of 19% crude protein, 5% crude fat, and 5% crude fiber. Mice were housed in microisolator cages and maintained in an environment with a 12 h light cycle at 20-22°C and 40-60% humidity. On the day of implantation, CT26 cells were harvested and re-suspended with serum free RPMI1640 to a concentration of lxl07/mL and each mouse was injected subcutaneously with a volume of 0.1 mL containing lxlO6 CT26 cells in the right rear flank. After 6 d, palpable tumors with an average volume of -65 mm3 (calculated using the formula: tumor volume = width2 x length / 2) had formed. At this time, animals were separated into five groups of eight animals per group such that each group contained animals bearing tumors of similar average size and treatment was initiated. For dosing, Example Al was dissolved in water to a concentration of 7.5 mg/mL or 15 mg/mL for dosing of the 75 mg/kg and the 150 mg/kg doses, respectively. As a positive control and to investigate the combinatorial activity of Example Al, a rat IgG2b anti-PDl antibody (BioXcell (NH, USA)) was dosed used. The five groups were treated with: i) 10 mL/kg water QD for 21 d administered by oral gavage (PO) plus 150 μg rat IgG2b isotype control antibody dosed by intraperitoneal (IP) injection on day 0, 3, 6 and 10 (the control arm); ii) 150 μg anti-PD-1 antibody dosed by intraperitoneal (IP) injection on day 0, 3, 6 and 10; iii) 75 mg/kg Example Al QD for 21 days administered PO; iv) 150 mg/kg Example Al QD for 21 days administered PO v) 150 mg/kg Example Al QD for 21 days administered PO plus 150 μg anti-PD-1 antibody dosed by intraperitoneal (IP) injection on day 0, 3, 6 and 10. Toxicity was evaluated by body weight measurements and clinical observations. Tumour measurements and body weights were taken three times per week. Percent tumor growth inhibition (TGI) was calculated by the formula:
%TGI = 1 OO X [1 - (TVf, treated - TVi,treated)/(TVf,control - TVi, control)]
Tumour growth inhibition at day 21, is shown in figure 2. A dose-dependent effect was observed in response to treatment with Example Al, with 75 mg/kg and 150 mg/kg QD inhibiting tumour growth by 44% and 64%, respectively. Whilst the anti-PD-1 antibody alone resulted in an average TGI of 34%, when combined with 150 mg/kg QD Example Al, the TGI increased to 86%.
According to University Health Network (UHN) Animal Use Protocols (AUPs), mice in efficacy experiments should be sacrificed when the tumour size is above 1500 mm3 or if the bodyweight of the animal decreases or if the animals are displaying clinical signs that require termination for humane reasons. In this study, the compound was well tolerated with all animals gaining weight over the course of the study and no animals were terminated due to clinical signs. A tumour size of <1500 mm3 at day 21 was used as a cutoff to represent survival. Using this cutoff, at day 21 no animals survived in the control arm, 1 of 8 animals (12.5%) survived in the anti-PD-1 arm, 2 of 8 animals (25%) survived in the 75 mg/kg/day Example Al arm, 3 of 8 animals (37.5%) survived in the 150 mg/kg/day Example Al arm, and 7 of 8 animals (87.5%) survived in the 150 mg/kg/day Example Al and anti-PD-1 arm. These results demonstrate that compounds of the invention, as exemplified by compound Al, have in vivo antitumor activity and can be efficaciously combined with other immunomodulatory approaches.
Example F: EAE disease progression model
C57/BL6 mice were obtained from Jackson Laboratories. The Institutional Animal Care and Use Committee of the University Health Network approved all animal procedures. Mice were subcutaneously (SC) immunized with MOG35-55 peptide emulsified in Complete Freund's Adjuvant (CFA) supplemented with Mycobacterium tuberculosis. On days 0 and 2 after immunization, the mice were intraperitoneal (IP) injected with pertussis toxin. Clinical signs of EAE were monitored daily, according to the following criteria: 0, no disease; 1, decreased tail tone; 2, hind limb weakness or partial paralysis; 3, complete hind limb paralysis; 4, front and hind limb paralysis; 5, death, or sacrifice due to moribund state. For treatment with compound during EAE induction, mice were dosed orally (PO) with 50 mg/kg A30 (n=4) or water (vehicle control; n=5) every day (QD). Data are the mean score ± SEM. The test results is shown in Figure 3.

Claims

CLAIMS WHAT IS CLAIMED IS:
1. A compound represented by Formula (I):
Figure imgf000080_0001
(I) or a pharmaceutically acceptable salt thereof, wherein:
one of Xi, X2, and X3 is S, the other two are each independently CR, wherein R is -H, -F, -CI, -Br, -CN, -NH¾ -OH, optionally substituted (Ci-C6)alkyl, optionally substituted (Ci- Ce)alkoxy, optionally substituted -(CH2)n(C3-Cio)cycloalkyl, optionally substituted -(CH2)n-3- 7 membered monocyclic heterocyclyl, optionally substituted -(CH2)nphenyl, optionally substituted -(CH2)n-5-7 membered monocylic heteroaryl, optionally substituted -(CH2)n- bridged (C6-Ci2)cycloalkyl, optionally substituted -(CH2)n-6-12 membered bridged heterocyclyl, optionally substituted -(CH2)n-7-12 membered bicyclic heteroaryl, or optionally substituted -(CH2)n-7-12 membered bicyclic heteroaryl;
Y is a bond, -CH2-, -C(=0)-;
Ri is -NRaRb or -OR*1;
Ra for each occurrence is independently -H, optionally substituted (Ci-Ce)alkyl, optionally substituted -(CH2)n(C3-C1o)cycloalkyl, optionally substituted -(CH2)n-3-10 membered heterocyclyl, optionally substituted -(CH2)n(C6-Cio)aryl, optionally substituted - (CH2)n-5-10 membered heteroaryl, optionally substituted -(CH2)n-bridged (C6-Ci2)cycloalkyl, or optionally substituted -(CH2)n-6-12 membered bridged heterocyclyl;
Rb for each occurrence is independently -H or -(Ci-C6)alkyl; or,
Ra and Rb, together with the nitrogen to which they are attached, form optionally substituted -(C3-Cio)heterocyclyl;
Ral for each occurrence is independently -H, optionally substituted (Ci-C6)alkyl, optionally substituted (C3-Cio)cycloalkyl, optionally substituted 3-10 membered heterocyclyl, optionally substituted (C6-Cio)aryl, or optionally substituted 3-10 membered heteroaryl; or
R2 and R3 are each independently -H or -(Ci-C6)alkyl; R.4 and Rs are each independently -H, optionally substituted (Ci-C6)alkyl, optionally substituted (C3-Cio)cycloalkyl, optionally substituted 3-10 membered heterocyclyl, optionally substituted (C6-Cio)aryl, optionally substituted 5-10 membered heteroaryl, optionally substituted bridged (C6-Ci2)cycloalkyl, or optionally substituted 6-12 membered bridged heterocyclyl; or
R.4 and Rs, together with the nitrogen to which they are attached, form optionally substituted 4-10 membered heterocyclyl, optionally substituted 5-10 membered heteroaryl, or optionally substituted 6-12 membered bridged heterocyclyl;
R6 for each occurrence is independently -F, -CI, -Br, -CN, -NH¾ -OH, -(Ci-C6)alkyl, -(Ci-C6)haloalkyl, -(C2-C6)alkenyl, -(C2-C6)alkynyl, (C3-C6)cycloalkyl, -(Ci-C6)alkoxy, -(Ci- C6)haloalkoxy, -(Ci-C6)alkylene-OH, or -(Ci-C6)alkylene-NH2;
m is 0, 1, 2, or 3; and
n is 0, 1, or 2.
2. The compound of claim 1, wherein the compound is represented by formula (I -A):
Figure imgf000081_0001
3. The compound of claim 1, wherein the compound is represented by formula (I-B):
Figure imgf000081_0002
4. The compound of claim 1, wherein the compound is represented by formula (I-C):
Figure imgf000082_0001
5. The compound of claim 1, wherein the compound is represented by formula (II- A):
Figure imgf000082_0002
6. The compound of claim 1, wherein the compound is represented by formula (II-B):
Figure imgf000082_0003
7. The compound of claim 1, wherein the compound is represented by formula (II-C):
Figure imgf000083_0001
8. The compound of claim 1, wherein the compound is represented by formula (III-Al) or formula (III-A2):
Figure imgf000083_0002
9. The compound of claim 1, wherein the compound is represented by formula (III-B):
Figure imgf000083_0003
10. The compound of claim 1, wherein the compound is represented by formula (III-C):
Figure imgf000084_0001
11. The compound of any one of claims 1-10, wherein R4 and R5, together with the nitrogen to which they are attached, form 4-7 membered monocyclic heterocyclyl or 6-12 membered bridged heterocyclyl, wherein the 4-7 membered monocyclic heterocyclyl or 6-12 membered bridged heterocyclyl is optionally substituted with 1-3 groups selected from -F, - CI, -Br, -CN, -NH¾ -OH, oxo, -(Ci-C4)alkyl, -(Ci-C4)haloalkyl, -(Ci-C4)alkoxy, -(Ci- C4)haloalkoxy, -(Ci-C4)alkylene-OH, or -(Ci-C4)alkylene-NH2.
12. The compound of any one of claims 1-11, wherein Ra for each occurrence is independently -H, -(Ci-Ce)alkyl, -(CH2)„-(C3-C7)cycloalkyl, -(CH2)„-4-7 membered monocyclic heterocyclyl, -(CH2)n-bridged (C6-Ci2)cycloalkyl, optionally substituted -(CH2)n- 5-10 membered heteroaryl; or -(CH2)n-6-12 membered bridged heterocyclyl, wherein -(Ci- Ce)alkyl, -(CH2)n-(C3-C7)cycloalkyl, -(CH2)n-4-7 membered monocyclic heterocyclyl, - (CH2)n-bridged (C6-Ci2)cycloalkyl, -(CH2)n-5-10 membered heteroaryl, or -(CH2)n-6-12 membered bridged heterocyclyl, is optionally substituted with 1-3 groups selected from -F, - CI, -Br, -CN, -NH¾ -OH, oxo, -(Ci-C4)alkyl, -(Ci-C4)haloalkyl, -(Ci-C4)alkoxy, -(Ci- C )haloalkoxy, -(Ci-C )alkylene-OH, or -(Ci-C )alkylene-NH2.
13. The compound of any one of claims 1-12, wherein R is H, -F, -CI, -Br, -OH, -(Ci- C4)alkyl, -(Ci-C4)haloalkyl, -(Ci-C4)alkoxy, -(Ci-C4)alkylene-OH or 4-7 membered monocyclic heterocyclyl optionally substituted with 1-3 groups selected from -F, -CI, -Br, - OH, -(Ci-C4)alkyl, -(Ci-C4)haloalkyl, or -(Ci-C4)alkoxy.
The compound of any one of claims 1-13, wherein R4 and R5, together with the gen to which they are attached, form -N-alkyl-piperazinyl or morpholinyl, wherein the piperazinyl or morpholinyl is optionally substituted with 1-2 groups selected from -F, -CI, - Br,-OH, -(Ci-C4)alkyl, -(Ci-C4)haloalkyl, or -(Ci-C4)alkoxy.
15. The compound of any one of claims 1-14, wherein Ra for each occurrence is independently -H, -(CH2)n-(C3-C6)cycloalkyl, -(CH2)n-3-6 membered heterocyclyl, wherein the -(CH2)n-(C3-C6)cycloalkyl or -(CH2)n-3-6 membered heterocyclyl is optionally substituted with 1-3 groups selected from -F, -CI, -Br, -CN, -NH¾ -OH, -(Ci-C4)alkyl, or -(Ci-C )alkoxy; and n is 0 or 1.
16. The compound of any one of claims 1-15, wherein R is H, -(Ci-C4)alkyl, -(Ci- C4)alkoxy, N-piperazinyl optionally substituted with -C02-(Ci-C4)alkyl.
17. The compound of any one of claims 1-16, wherein R is H.
18. The compound of any one of claims 1-17, wherein R4 and Rs, together with the nitrogen to which they are attached, form -N-methyl-piperazinyl or morpholinyl, both of which are optionally substituted with one or two methyl.
19. The compound of any one of claims 1-18, wherein Ra for each occurrence is independently -H; -(C3-Ce)cycloalkyl optionally substituted with -OH; -(CH2)n-tetrahydro- 2H-pyran; morpholinyl; piperidinyl optionally substituted with -F, -OH or methyl; or tetrahydrofuran; and n is 0 or 1.
20. A pharmaceutical composition comprising a compound of any one of claims 1-19 and a pharmaceutically acceptable carrier or diluent.
21. A method for treating a subject with cancer, comprising: administering to the subject an effective amount of a compound of any one of claims 1-19, or a pharmaceutically acceptable salt thereof.
22. A method of treating a subject with cancer, comprising administering to the subject an effective amount of a compound of any one of claims 1-19, or a pharmaceutically acceptable salt thereof, and an effective second anti-cancer treatment (e.g., a chemotherapeutic agent, a targeted therapeutic agent, radiation or surgery).
23. A method of treating a subject with cancer, comprising administering to the subject an effective amount of a compound of any one of claims 1-19, or a pharmaceutically acceptable salt thereof, and an effective amount of an immunomodulatory agent such as a checkpoint inhibitor (e.g. , anti-PD-1 antibody, anti-CTLA4 antibody or anti-PD-Ll antibody ) or an inhibitor of tryptophan oxidation (e.g. IDOl, ID02 or TD02 inhibitor).
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