WO2023055427A1 - Small molecule antagonists of pf4 - Google Patents

Small molecule antagonists of pf4 Download PDF

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Publication number
WO2023055427A1
WO2023055427A1 PCT/US2022/021370 US2022021370W WO2023055427A1 WO 2023055427 A1 WO2023055427 A1 WO 2023055427A1 US 2022021370 W US2022021370 W US 2022021370W WO 2023055427 A1 WO2023055427 A1 WO 2023055427A1
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compound
independently selected
optionally substituted
alkyl
platelet
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PCT/US2022/021370
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French (fr)
Inventor
Bruce SACHAIS
Jay E. Wrobel
Allen B. Reitz
Steven E. MCKENZIE
Mark E. Mcdonnell
Haiyan Bian
Thomas A. FORD-HUTCHINSON
Robert J. ROSANO
Yuhang ZHOU
Randall Jeffrey BINDER
Colin Tice
Sarah HYMAN
Justin Sausker
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New York Blood Center, Inc.
Fox Chase Chemical Diversity Center, Inc.
Thomas Jefferson University
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Application filed by New York Blood Center, Inc., Fox Chase Chemical Diversity Center, Inc., Thomas Jefferson University filed Critical New York Blood Center, Inc.
Publication of WO2023055427A1 publication Critical patent/WO2023055427A1/en

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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
    • C07C49/587—Unsaturated compounds containing a keto groups being part of a ring
    • C07C49/657—Unsaturated compounds containing a keto groups being part of a ring containing six-membered aromatic rings
    • C07C49/665—Unsaturated compounds containing a keto groups being part of a ring containing six-membered aromatic rings a keto group being part of a condensed ring system
    • C07C49/675—Unsaturated compounds containing a keto groups being part of a ring containing six-membered aromatic rings a keto group being part of a condensed ring system having three rings
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    • C07C211/00—Compounds containing amino groups bound to a carbon skeleton
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/70—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/82—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton with the carbon atom of at least one of the carboxamide groups bound to a carbon atom of a ring other than a six-membered aromatic ring
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/08—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon radicals, substituted by hetero atoms, attached to ring carbon atoms
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    • C07—ORGANIC CHEMISTRY
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    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/18—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member
    • C07D207/22—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/24—Oxygen or sulfur atoms
    • C07D207/26—2-Pyrrolidones
    • C07D207/263—2-Pyrrolidones with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms
    • C07D207/27—2-Pyrrolidones with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms with substituted hydrocarbon radicals directly attached to the ring nitrogen atom
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/08—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms
    • C07D211/18—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D211/30—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by doubly bound oxygen or sulfur atoms or by two oxygen or sulfur atoms singly bound to the same carbon atom
    • C07D211/32—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by doubly bound oxygen or sulfur atoms or by two oxygen or sulfur atoms singly bound to the same carbon atom by oxygen atoms
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/44—Radicals substituted by doubly-bound oxygen, sulfur, or nitrogen atoms, or by two such atoms singly-bound to the same carbon atom
    • C07D213/46—Oxygen atoms
    • C07D213/50—Ketonic radicals
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/26—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/44—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D317/46—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C07D317/48—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring
    • C07D317/50—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to atoms of the carbocyclic ring
    • C07D317/58—Radicals substituted by nitrogen atoms
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/02—Systems containing only non-condensed rings with a three-membered ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/04—Systems containing only non-condensed rings with a four-membered ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2603/00—Systems containing at least three condensed rings
    • C07C2603/02—Ortho- or ortho- and peri-condensed systems
    • C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
    • C07C2603/06—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members
    • C07C2603/10—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings

Definitions

  • TECHNICAL FIELD [0003] This invention relates to inhibitors of PF4 tetramerization useful in treating, for example, heparin induced thrombocytopenia (HIT) or heparin induced thrombocytopenia and thrombosis (HITT).
  • HIT heparin induced thrombocytopenia
  • HITT heparin induced thrombocytopenia and thrombosis
  • Heparin-induced thrombocytopenia and thrombosis is a serious complication of heparin therapy. Heparin is a naturally-occurring anticoagulant that prevents the formation of clots and extension of existing clots within the vasculature. Major medical applications of heparin include dialysis, cardiac catheterization, and cardiopulmonary bypass surgery.
  • HIT heparin induced thrombocytopenia
  • HIT is caused by an immunological reaction that targets platelets leading to a low platelet count (thrombocytopenia).
  • HIT increases the risk of blood clots forming within blood vessels and blocking the flow of blood (thrombosis), referred to as HITT when thrombosis occurs.
  • HITT develops in approximately 1-3% of patients treated with heparin for 5-10 days.
  • Affected individuals have a 20-50% risk of developing new thromboembolic events, a mortality rate of about 20%, and an additional ⁇ 10% of patients require amputations or suffer other major morbidity.
  • HITT low molecular weight heparins
  • LMWH low molecular weight heparins
  • HITT continues to be a significant medical problem. This is likely due to the fact that heparin remains the anticoagulant of choice for many patients (such as patients undergoing cardiopulmonary bypass or percutaneous coronary intervention, at high risk for bleeding, or with renal failure). HITT occurs even after treatment with LMWHs, although at a reduced rate compared to unfractionated heparin in some patients.
  • HITT is a major treatment-induced cause of morbidity and mortality in this patient population.
  • PF4 platelet factor 4
  • the present application provides compounds that inhibit platelet activation by directly inhibiting tetramerization of platelet factor 4 (PF4), and may be useful in treating diseases and conditions in which increased blood clotting is indicated. Suitable examples of such diseases include HIT and HITT.
  • the compounds of the present application have high potency at a ⁇ M level, little or no activity against other chemokines, excellent ADMET properties including microsome stability and little or no cytochrome P450 inhibition, little or no cytotoxicity, little or no hERG inhibition, good aqueous solubility and PK parameters suitable for intravenous (IV) administration, such as moderate-long half-life, and low clearance and volume of distribution. Exemplary embodiments of such compounds are described below. [0007]
  • the present disclosure provides a compound of Formula (II): or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 , R 3 and R 4 are as described herein.
  • the present disclosure provides a pharmaceutical composition comprising a compound of Formula (II), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • the present disclosure provides a method of: • preventing formation of platelet factor-4 (PF4) tetramers in a subject; and/or • disrupting platelet factor-4 (PF4) tetramers in a subject; and/or • preventing formation of an ultra-large complex (ULC) comprising a PF4 tetramer and a glycosaminoglycan (GAG) in a subject; and/or • inhibiting ULC-antibody complex binding to a Fc ⁇ RIIa receptor on a platelet in a subject; and/or • inhibiting platelet aggregation in a subject, and/or • increasing high density lipoproteins in a subject; and/or • modulating clotting or hemostasis in a subject; and/or • correcting a plate
  • the present disclosure provides a method of treating or preventing a disease or condition selected from: • heparin induced thrombocytopenia and thrombosis (HITT); • a thrombotic complication of HITT; • heparin induced thrombocytopenia (HIT); • vaccine-induced immune thrombotic thrombocytopenia (VITT); • atherosclerosis or atherosclerotic vascular disease; • decrease in platelet production; • inflammation or an inflammatory disease; • antiphospholipid syndrome; • platelet imbalance or insufficiency; and • a clotting or hemostasis disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the second general aspect.
  • HITT heparin induced thrombocytopenia and thrombosis
  • HIT heparin induced thrombocytopenia
  • VITT vaccine-induced immune thro
  • FIG. 1A is a diagram showing formation of Ultra Large Complex (ULC) of PF4 tetramers and heparin. The figure shows that formation of ULC requires tetramerization of PF4.
  • FIG.1B is a diagram showing that inhibitors of tetramerization inhibit ULC formation.
  • FIG. 2 is a diagram showing simplified mechanism of heparin-induced thrombocytopenia and thrombosis (HITT) pathophysiology.
  • FIG.3 is an image showing PF4 dimer with a hypothesized site of binding for PF4 tetramerization antagonists.
  • FIG.4A is a scheme showing equilibrium between tautomeric forms of a compound containing an indane-1,3-dione moiety substituted in the 2-position.
  • FIG.4B is a scheme showing equilibrium between tautomeric forms of a compound containing an indane-1,3-dione moiety that is unsubstituted in the 2-position.
  • FIG. 5 is a graph showing difference in P-selectin expression in human platelets treated with PF4 along and with PF4 and heparin.
  • FIG. 6 is a graph showing difference in P-selectin expression in human platelets treated with PF4 at 37 ⁇ g/mL and with PF4 at 65 ⁇ g/mL.
  • PF4 platelet factor 4
  • CXC CXC (or beta) chemokine subfamily
  • PF4 is synthesized by megakaryocytes and comprises 2-3% of the total released protein in mature platelets.
  • PF4 exists as a tetramer in the ⁇ -granules of platelets and is secreted in high concentrations when platelets are activated.
  • PF4 tetramers bind avidly to glycosaminoglycans (GAGs).
  • PF4 PF4 tetramers and heparin
  • ULCs ultra large complexes of PF4 tetramers and heparin
  • a transgenic mouse model of HITT demonstrates that heparin, PF4 (forming ULC), anti- heparin/PF4 antibody, and the platelet receptor Fc ⁇ RIIa are necessary and sufficient to recapitulate the salient features of HITT in vivo. Treatment of patients with heparin is thought to favor the formation of the ULCs, placing these patients at risk for HITT.
  • FIG. 2 A simplified mechanism of HITT pathophysiology is shown in Figure 2, and demonstrates the feed-forward nature of this disorder. Specifically, antibody recognition of ULC leads to platelet activation, releasing more PF4, which can form additional ULC to be recognized by antibody. The compounds of the present application inhibit cellular activation by limiting the ULC formation.
  • step (1) PF4 released from activated platelets as a tetramer forms a complex (ULC) with heparin which has been administered to a patient as an anticoagulant; in step (2) pathogenic antibodies bind to the complex of heparin and PF4 tetramer; in step (3) an antibody-decorated heparin-PF4 complex binds to a platelet via its Fc ⁇ RIIa receptor; and in step (4) crosslinking of Fc ⁇ RIIa leads to platelet activation and release of additional PF4, which can bind to heparin and feed the pathogenic cycle.
  • ULC complex
  • pathogenic antibodies bind to the complex of heparin and PF4 tetramer
  • step (3) an antibody-decorated heparin-PF4 complex binds to a platelet via its Fc ⁇ RIIa receptor
  • step (4) crosslinking of Fc ⁇ RIIa leads to platelet activation and release of additional PF4, which can bind to heparin and feed
  • HITT current treatment for HITT relies on removal of all heparin exposure from patients with suspected HITT and administration of a non-heparin alternative anticoagulant, typically a direct thrombin inhibitor, which may carry a significant risk of bleeding. Hence, despite discontinued heparin, the patients remain at significant risk for thrombosis and death.
  • a non-heparin alternative anticoagulant typically a direct thrombin inhibitor
  • VITT Vaccine-induced immune thrombotic thrombocytopenia
  • adenovirus-based vaccine is characterized by development of thrombocytopenia and thrombosis in atypical locations (including, but not limited to, the cerebral and/or splanchic veins) within weeks of receiving a vaccination.
  • VITT has most recently been identified in subjects receiving adenvirus-based SARS-CoV-2 vaccines, which involves, in part, antibodies directed toward PF4 and/or PF4-heparin complexes.
  • the present application provides compounds that directly target and intervene in the pathophysiology of thrombosis, and limit and/or prevent the complications of this condition and related diseases.
  • PF4 refers to platelet factor 4 which is a 70 amino acid, lysine-rich, 7.8 kDa platelet-specific protein that belongs to the CXC (or beta) chemokine subfamily, in which the first two of the four conserved cysteine residues are separated by one amino acid residue.
  • PF4 is naturally occurring, i.e., wild-type.
  • PF4 may be synthesized by recombinant or chemical methods.
  • the term PF4 also refers to mutations thereof in which one or more of the amino acids is replaced with a different amino acid.
  • PF4 mutations are described in International Patent Publication No. WO 02/006300 and the inventors’ prior publication WO2013/142328, which are incorporated herein by reference.
  • the term “compound” is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures named or depicted. Compounds herein identified by name or structure as one particular tautomeric form are intended to include other tautomeric forms unless otherwise specified.
  • tautomer refers to compounds which are capable of existing in a state of equilibrium between two isomeric forms.
  • Such compounds may differ in the bond connecting two atoms or groups and the position of these atoms or groups in the compound.
  • Suitable examples of an equilibrium between the tautomeric forms are depicted in Figures 4A and 4B.
  • the R- group may correspond to C 1-6 alkoxy, C 1-6 alkyl or an NH(R a1 ) fragment, as in any one of the substituents R 1 -R 4 in a compound of Formula (II) described herein.
  • C n-m alkyl refers to a saturated hydrocarbon group that may be straight- chain (linear) or branched, having n to m carbons.
  • alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; higher homologs such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, and the like.
  • the alkyl group contains from 1 to 6 carbon atoms, from 1 to 4 carbon atoms, from 1 to 3 carbon atoms, or 1 to 2 carbon atoms.
  • C n-m alkenyl refers to an alkyl group having one or more double carbon-carbon bonds and having n to m carbons.
  • Example alkenyl groups include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl, and the like.
  • the alkenyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.
  • the term “C n-m alkoxy”, employed alone or in combination with other terms refers to a group of formula –O-C n-m alkyl.
  • Examplary alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (for example, n-propoxy and isopropoxy), butoxy (for example, n-butoxy and tert-butoxy), and the like.
  • the alkoxy group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.
  • halo refers to a halogen atom such as F, Cl, Br, or I.
  • a halo is F, Cl, or Br.
  • halo is F, Cl, or I.
  • halo is F, I, or Br.
  • C n-m haloalkyl refers to an alkyl group having from one halogen atom to 2s+1 halogen atoms which may be the same or different, where “s” is the number of carbon atoms in the alkyl group, win the present application the alkyl group has n to m carbon atoms.
  • the haloalkyl group is fluorinated only.
  • the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.
  • C n-m haloalkoxy refers to a group of formula – O-haloalkyl having n to m carbon atoms.
  • An example haloalkoxy group is OCF 3 .
  • the haloalkoxy group is fluorinated only.
  • the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.
  • cycloalkyl refers to non-aromatic saturated or unsaturated cyclic hydrocarbons including cyclized alkyl and/or alkenyl groups.
  • Cycloalkyl groups can include mono- or polycyclic (for example, having 2, 3 or 4 fused rings) groups and spirocycles. Ring-forming carbon atoms of a cycloalkyl group can be optionally substituted by oxo or sulfido (for example, C(O) or C(S)). Also included in the definition of cycloalkyl are moieties that have one or more aromatic rings fused (that is, having a bond in common with) to the non-aromatic cyclic hydrocarbon, for example, benzo or thienyl derivatives of cyclopentane, cyclohexane, and the like.
  • a cycloalkyl group containing a fused aromatic ring can be attached through any ring-forming atom including a ring-forming atom of the fused aromatic ring.
  • Cycloalkyl groups can have 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 ring-forming atoms.
  • the cycloalkyl is a 3-12 membered monocyclic or bicyclic cycloalkyl.
  • the cycloalkyl is a C 3-7 monocyclic cycloalkyl.
  • cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcarnyl, cyclooctyl, cyclooctenyl, and the like.
  • cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, or cyclooctenyl.
  • the cycloalkyl is a cyclooctenyl ring fused with 1 or 2 benzene rings. In some embodiments, the cycloalkyl is a 3-8 membered or 3-7 membered monocyclic cycloalkyl group (for example, C 3-8 or C 3-7 cycloalkyl). In some embodiments, the cycloalkyl is a 8-12-membered bicyclic cycloalkyl. In some embodiments, the cycloalkyl is a 8-16-membered bicyclic or tricyclic cycloalkyl (for example, C 8-16 cycloalkyl).
  • the cycloalkyl is unsaturated cyclic hydrocarbon group (that is, the cycloalkyl contains at least one double bond).
  • heteroalkyl refers to branched or unbranched heteroalkyls having one or more heteroatoms selected, independently, from O, N, or S. Examples of heteroalkyls include, but are not limited to, CH 2CH2OCH2CH2OCH2CH2OCH3, CH2CH2OCH2CH2OCH3, CH 2 NHCH 2 CH 2 OCH 2 CH 2 OCH 3 , and CH 2 CH 2 CH 2 NH 2 , and the like.
  • heterocycloalkyl or “aliphatic heterocycle” refers to non-aromatic saturated or unsaturated monocyclic or polycyclic heterocycles having one or more ring- forming heteroatoms selected from O, N, or S. Included in heterocycloalkyl are monocyclic 4- , 5-, 6-, 7-, 8-, 9- or 10-membered heterocycloalkyl groups. Heterocycloalkyl groups can also include spirocycles.
  • Example heterocycloalkyl groups include pyrrolidin-2-one, 1,3- isoxazolidin-2-one, pyranyl, tetrahydropuran, oxetanyl, azetidinyl, morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, azepanyl, benzazapene, and the like.
  • Ring-forming carbon atoms and heteroatoms of a heterocycloalkyl group can be optionally substituted by oxo or sulfido groups (e.g., C(O), S(O), C(S), or S(O)2, etc.).
  • the heterocycloalkyl group can be attached through a ring-forming carbon atom or a ring-forming heteroatom.
  • the heterocycloalkyl group contains 0 to 3 double bonds.
  • the heterocycloalkyl group contains 0 to 2 double bonds.
  • the heterocycloalkyl group is unsaturated (i.e., the heterocycloalkyl contains at least one double bond).
  • heterocycloalkyl moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the non-aromatic heterocycle, for example, benzo or thienyl derivatives of piperidine, morpholine, azepine, etc.
  • a heterocycloalkyl group containing a fused aromatic ring can be attached through any ring-forming atom including a ring-forming atom of the fused aromatic ring.
  • the heterocycloalkyl is a monocyclic 4-6 membered heterocycloalkyl having 1 or 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur and having one or more oxidized ring members.
  • the heterocycloalkyl is a monocyclic or bicyclic 4-10 membered heterocycloalkyl having 1, 2, 3, or 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur and having one or more oxidized ring members.
  • the heterocycloalkyl is a 8-12-membered heterocycloalkyl (e.g., bicyclic heterocycloalkyl).
  • the heterocycloalkyl is a 8-16-membered heterocycloalkyl (e.g., bicyclic or tricyclic heterocycloalkyl).
  • the 8-12 membered bicyclic heterocycloalkyl is a 8-12 membered fused heterocycloalkylaryl group or a 8-12 membered fused heterocycloalkylheteroaryl group.
  • the heterocycloalkyl is a 9-12 membered bicyclic heterocycloalkyl.
  • the 9-10 membered bicyclic heterocycloalkyl is a 9-10 membered fused heterocycloalkylaryl group or a 9-10 membered fused heterocycloalkylheteroaryl group.
  • heterocycloalkylene refers to a divalent heterocycloalkyl linking group.
  • heteroaryl refers to a monocyclic or polycyclic aromatic heterocycle having at least one heteroatom ring member selected from sulfur, oxygen, and nitrogen.
  • the heteroaryl ring has 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen.
  • any ring-forming N in a heteroaryl moiety can be an N-oxide.
  • the heteroaryl is a 5-10 membered monocyclic or bicyclic heteroaryl having 1, 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen.
  • the heteroaryl is a 5-6 membered monocyclic heteroaryl having 1 or 2 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl is a five-membered or six-membered heteroaryl ring.
  • a five- membered heteroaryl ring is a heteroaryl with a ring having five ring atoms win the present application one or more (for example, 1, 2, or 3) ring atoms are independently selected from N, O, and S.
  • Exemplary five-membered heteroaryls are thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3- thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4- triazolyl, 1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl.
  • a six-membered heteroaryl ring is a heteroaryl with a ring having six ring atoms win the present application one or more (for example, 1, 2, or 3) ring atoms are independently selected from N, O, and S.
  • Exemplary six- membered heteroaryls are pyridyl, pyrazinyl, pyrimidinyl, triazinyl and pyridazinyl.
  • aromatic refers to a carbocycle or heterocycle having one or more polyunsaturated rings having aromatic character (that is, having (4n + 2) delocalized ⁇ (pi) electrons where n is an integer).
  • n-membered typically describes the number of ring- forming atoms in a moiety where the number of ring-forming atoms is n.
  • piperidinyl is an example of a 6-membered heterocycloalkyl ring
  • pyrazolyl is an example of a 5-membered heteroaryl ring
  • pyridyl is an example of a 6-membered heteroaryl ring
  • 1,2,3,4-tetrahydro-naphthalene is an example of a 10-membered cycloalkyl group.
  • aryl refers to an aromatic hydrocarbon group, which may be monocyclic or polycyclic (for example, having 2, 3 or 4 fused rings).
  • C n-m aryl refers to an aryl group having from n to m ring carbon atoms.
  • Aryl groups include, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl and the like.
  • aryl groups have from 6 to about 20 carbon atoms, from 6 to about 15 carbon atoms, or from 6 to about 10 carbon atoms.
  • the aryl group is phenyl.
  • C n-m alkoxycarbonyl refers to a group of formula -C(O)O-alkyl, win the present application the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.
  • alkoxycarbonyl groups include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl (for example, n-propoxycarbonyl and isopropoxycarbonyl), butoxycarbonyl (for example, n-butoxycarbonyl and tert-butoxycarbonyl), and the like.
  • C n-m alkylcarbonyl refers to a group of formula -C(O)-alkyl, win the present application the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.
  • alkylcarbonyl groups include, but are not limited to, methylcarbonyl, ethylcarbonyl, propylcarbonyl (for example, n-propylcarbonyl and isopropylcarbonyl), butylcarbonyl (for example, n-butylcarbonyl and tert-butylcarbonyl), and the like.
  • the term “carboxy” or “carboxyl” refers to a - C(O)OH group.
  • pharmaceutical and “pharmaceutically acceptable” are employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • the term “cell” is meant to refer to a cell that is in vitro, ex vivo, or in vivo.
  • an ex vivo cell can be part of a tissue sample excised from an organism such as a mammal.
  • an in vitro cell can be a cell in a cell culture.
  • an in vivo cell is a cell living in an organism such as a mammal.
  • the phrase “effective amount” or “therapeutically effective amount” refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.
  • treating refers to 1) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology), or 2) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology).
  • the term “preventing” or “prevention” of a disease, condition or disorder refers to decreasing the risk of occurrence of the disease, condition or disorder in a subject or group of subjects (e.g., a subject or group of subjects predisposed to or susceptible to the disease, condition or disorder). In some embodiments, preventing a disease, condition or disorder refers to decreasing the possibility of acquiring the disease, condition or disorder and/or its associated symptoms. In some embodiments, preventing a disease, condition or disorder refers to completely or almost completely stopping the disease, condition or disorder from occurring. [0054] As used herein, in such methods the term "biological sample” refers to a body fluid or tissue.
  • the body fluid can include, without limitation, whole blood, serum, plasma, peripheral blood, synovial fluid, cerebrospinal fluid, saliva, urine, semen, or other fluid secretion.
  • tissue can include, without limitation, bone marrow and lymph node, as well as samples of other tissues.
  • R 1 and R 3 are each independently selected from H, C 1-6 alkylcarbonyl, C(O)NH(R a1 ), and C(O)Cy 4 , wherein C 1-6 alkyl in the C 1-6 alkylcarbonyl is optionally substituted with 1, 2, or 3 substituents independently selected from Cy 4 , C 6-10 aryloxy, C 1-3 alkoxy, -NH(C 1-6 alkoxycarbonyl), and –NH(C 1-6 alkylcarbonyl), wherin C 1-6 alkoxy is optionally substituted with C 6-10 aryl; R 2 and R 4 are each independently selected from H and C 1-6 alkylcarbonyl; each R a1 is independently selected from C 1-6 alkyl, C 1-6 alkenyl, Cy 1 , C 1-6 alkoxycarbonyl, and S(O) 2 R a2
  • R 1 is H. In other embodiments, R 1 is C 1-6 alkylcarbonyl (e.g., methylcarbonyl). In yet other embodiments, R 1 is C(O)NH(R a1 ).
  • R 2 is H. In other embodiments, R 2 is C 1-6 alkylcarbonyl.
  • R 3 is H. In other embodiments, R 3 is C 1-6 alkylcarbonyl (e.g., methylcarbonyl). In yet other embodiments, R 3 is C(O)NH(R a1 ).
  • R 1 and R 3 are each independently selected from C 1-6 alkylcarbonyl and C(O)NH(R a1 ).
  • R 4 is H. In other embodiments, R 4 is C 1-6 alkylcarbonyl.
  • R 2 and R 4 are each H; and R 1 and R 3 are each independently selected from H, C 1-6 alkylcarbonyl, and C(O)NH(R a1 ).
  • R 2 and R 4 are each H; and R 1 and R 3 are each independently selected from C 1-6 alkylcarbonyl, and C(O)NH(R a1 ).
  • R 1 and R 3 are each independently selected from H and C 1-6 alkylcarbonyl; and R 2 and R 4 are each C 1-6 alkylcarbonyl. [0064] In some embodiments, R 1 and R 3 are each H; and R 2 and R 4 are each C 1-6 alkylcarbonyl. [0065] In some embodiments, R 1 , R 2 , R 3 and R 4 are each independently selected from H and C 1-6 alkylcarbonyl. [0066] In some embodiments, R 1 , R 2 , R 3 and R 4 are each H. [0067] In some embodiments, R 1 and R 3 are each C 1-6 alkylcarbonyl.
  • R 1 and R 3 are each C(O)NH(R a1 ).
  • R 2 and R 4 are each H; and R 1 and R 3 are each C(O)NH(R a1 ).
  • R a1 is C 1-6 alkyl, optionally substituted with 1 or 2 substituents independently selected from Cy 2 , carboxyl, C1-3 alkoxycarbonyl, C1-3 alkoxy, and C1-3 haloalkoxy wherein the C 1-3 alkoxy is optionally substituted with C 1-3 alkoxy.
  • R a1 is C 1-6 alkyl, optionally substituted with 1 or 2 substituents independently selected from Cy 2 , carboxyl, and C1-3 alkoxycarbonyl.
  • R a1 is methyl, ethyl, propyl, isopropyl and sec-butyl, each of which is optionally substituted with 1 or 2 substituents independently selected from Cy 2 , carboxyl, C 1-3 alkoxycarbonyl, C 1-3 alkoxy, and C 1-3 haloalkoxy wherein the C 1-3 alkoxy is optionally substituted with C 1-3 alkoxy.
  • R a1 is methyl, ethyl, propyl, isopropyl and sec-butyl, each of which is optionally substituted with 1 or 2 substituents independently selected from Cy 2 , carboxyl, and C 1-3 alkoxycarbonyl.
  • R a1 is methyl, ethyl and propyl, each of which is optionally substituted with 1 or 2 substituents independently selected from Cy 2 , carboxyl, and C 1-3 alkoxycarbonyl.
  • R a1 is C 1-6 alkenyl.
  • R a1 is Cy 1 , optionally substituted with 1 or 2 substituents independently selected from halo, C 1-3 alkyl, C 1-3 alkoxy and Cy 3 .
  • R a1 is Cy 1 , optionally substituted with 1 or 2 substituents independently selected from C 1-3 alkyl, C 1-3 alkoxy and Cy 3 .
  • R a1 is selected from phenyl, dihydrobenzodioxinyl, pyridinyl and cyclopropyl, each of which is optionally substituted with 1 or 2 substituents independently selected from C 1-3 alkyl, C 1-3 alkoxy and Cy 3 .
  • R a1 is phenyl, dihydrobenzodioxinyl and cyclopropyl, each of which is optionally substituted with 1 or 2 substituents independently selected from C 1-3 alkyl, C 1-3 alkoxy and Cy 3 .
  • R a1 is S(O) 2 R a2 .
  • R a2 is phenyl, optionally substituted with C 1-3 alkyl.
  • R a2 is phenyl, optionally substituted with methyl.
  • each R a1 is independently selected from C 1-6 alkyl, C 1-6 alkenyl, Cy 1 , C 1-6 alkoxycarbonyl, and S(O) 2 R a2 , wherein the C 1-6 alkyl is optionally substituted with 1, 2, or 3 substituents independently selected from Cy 2 , carboxyl and C1-3 alkoxycarbonyl.
  • each R a1 is independently selected from C 1-6 alkyl, C 1-6 alkenyl, Cy 1 , and S(O) 2 R a2 , wherein the C 1-6 alkyl is optionally substituted with 1, 2, or 3 substituents independently selected from Cy 2 , carboxyl, C1-3 alkoxycarbonyl.
  • each R a1 is independently selected from propyl, isopropyl, sec-butyl, allyl, phenyl, tosyl, ethoxycarbonyl, methoxyphenyl, nitrophenyl, methylphenyl, ethoxyphenyl, dihydrobenzodioxinyl, dichloropyridinyl, benzyl, fluorobenzyl, (pyrimidin-2- yl)phenyl, cyclopropyl, phenylcyclopropyl, phenylethyl, (trifluoromethoxyphenyl)ethyl, (ethoxycarbonyl)methyl, (ethoxycarbonyl)ethyl, phenylpropyl, (fluorophenyl)ethyl, (chlorophenyl)ethyl, (methylphenyl)ethyl, furanylmethyl, benzodioxolylmethyl,
  • each R a1 is independently selected from propyl, isopropyl, sec-butyl, allyl, phenyl, tosyl, ethoxycarbonyl, methoxyphenyl, methylphenyl, ethoxyphenyl, dihydrobenzodioxinyl, dichloropyridinyl, benzyl, fluorobenzyl, (pyrimidin-2-yl)phenyl, cyclopropyl, phenylcyclopropyl, phenylcyclopropyl, phenylethyl, (trifluoromethoxyphenyl)ethyl, (ethoxycarbonyl)methyl, (ethoxycarbonyl)ethyl, phenylpropyl, (fluorophenyl)ethyl, (chlorophenyl)ethyl, (methylphenyl)ethyl, furanylmethyl, benzodioxolylmethyl, benzodio
  • each R a1 is independently selected from propyl, isopropyl, sec-butyl, allyl, tosyl, methylphenyl, ethoxyphenyl, benzyl, (pyrimidin-2-yl)phenyl, cyclopropyl, phenylcyclopropyl, phenylethyl, (trifluoromethoxyphenyl)ethyl, (ethoxycarbonyl)ethyl, phenylpropyl, (fluorophenyl)ethyl, (chlorophenyl)ethyl, (methylphenyl)ethyl, benzodioxolylethyl, and (carboxyl)ethyl.
  • Cy 1 is C 3-10 cycloalkyl, optionally substituted with 1 or 2 substituents independently selected from C 1-3 alkyl, C 1-3 alkoxy and Cy 3 .
  • Cy 1 is cyclopropyl, optionally substituted with 1 or 2 substituents independently selected from C 1-3 alkyl, C 1-3 alkoxy and Cy 3 .
  • Cy 1 is C 6-10 aryl, optionally substituted with 1 or 2 substituents independently selected from C 1-3 alkyl, C 1-3 alkoxy and Cy 3 .
  • Cy 1 is selected from phenyl, dihydrobenzodioxinyl, and cyclopropyl, each of which is optionally substituted with 1 or 2 substituents independently selected from C 1-3 alkyl, C 1-3 alkoxy and Cy 3 .
  • Cy 1 is selected from phenyl and cyclopropyl, each of which is optionally substituted with 1 or 2 substituents independently selected from C 1-3 alkyl, C 1-3 alkoxy and Cy 3 .
  • Cy 1 is selected from phenyl and cyclopropyl, each of which is optionally substituted with 1 or 2 substituents independently selected from C 1-3 alkyl, C 1-3 alkoxy and Cy 3 .
  • each Cy 1 is independently selected form C 3-10 cycloalkyl and C 6-10 aryl, each of which is optionally substituted with 1 or 2 substituents independently selected from C 1-3 alkyl, C 1-3 alkoxy and Cy 3 .
  • each Cy 1 is independently selected form phenyl, methoxyphenyl, ethoxyphenyl, dihydrobenzodioxinyl, dichloropyridinyl, cyclopropyl, methylphenyl, nitrophenyl, (pyrimidin-2-yl)phenyl, and phenylcyclopropyl.
  • each Cy 1 is independently selected from ethoxyphenyl, cyclopropyl, methylphenyl, (pyrimidin-2-yl)phenyl, and phenylcyclopropyl.
  • Cy 2 is 5-10 membered heteroaryl, each of which is optionally substituted with 1 or 2 substituents independently selected from halo, C 1-3 alkyl, and C 1-3 haloalkoxy. In some aspects of these embodiments, the 5-10 membered heteroaryl is furanyl. [0098] In some embodiments, Cy 2 is C 6-10 aryl, optionally substituted with 1 or 2 substituents independently selected from halo, C 1-3 alkyl, and C 1-3 haloalkoxy.
  • Cy 2 is selected from phenyl and benzodioxolyl, each of which is optionally substituted with 1 or 2 substituents independently selected from halo, C 1-3 alkyl, and C 1-3 haloalkoxy.
  • Cy 2 is phenyl, each of which is optionally substituted with 1 or 2 substituents independently selected from halo, C 1-3 alkyl, and C 1-3 haloalkoxy.
  • each Cy 2 is independently selected from phenyl, trifluoromethoxyphenyl, fluorophenyl, chlorophenyl, methylphenyl, furanyl, and benzodioxolyl.
  • each Cy 2 is independently selected from phenyl, trifluoromethoxyphenyl, fluorophenyl, chlorophenyl, methylphenyl, and benzodioxolyl.
  • each Cy 3 is C 6-10 aryl. In some aspects of these embodiments, the C 6-10 aryl is phenyl.
  • each Cy 3 is 5-10 membered heteroaryl. In some aspects of these embodiments, the 5-10 membered heteroaryl is pyrimidinyl.
  • each Cy 3 is independently selected from phenyl and pyrimidinyl.
  • R 1 and R 3 are each independently selected from H, C 1-6 alkylcarbonyl, and C(O)NH(R a1 );
  • R 2 and R 4 are each independently selected from H and C 1-6 alkylcarbonyl;
  • each R a1 is independently selected from C 1-6 alkyl, C 1-6 alkenyl, Cy 1 , and S(O) 2 R a2 , wherein the C 1-6 alkyl is optionally substituted with 1, 2, or 3 substituents independently selected from Cy 2 , carboxyl and C 1-3 alkoxycarbonyl;
  • each Cy 1 is independently selected form C 3-10 cycloalkyl and C 6-10 aryl, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from C 1-3 alkyl, 1-3 alkoxy and Cy 3 ;
  • each Cy 2 is C 6-10 aryl, optionally substituted with 1, 2, or 3 substituents independently selected from halo, C 1-3 alkyl, and C 1-3 haloalk
  • R 1 and R 3 are each C(O)NH(R a1 ); each R a1 is independently selected from C 1-6 alkyl, C 1-6 alkenyl, Cy 1 , C 1-6 alkoxycarbonyl, and S(O) 2 R a2 , wherein the C 1-6 alkyl is optionally substituted with 1, 2, or 3 substituents independently selected from Cy 2 , carboxyl and C 1-3 alkoxycarbonyl; each Cy 1 is independently selected form C 3-10 cycloalkyl and C 6-10 aryl, each of which is optionally substituted with 1 or 2 substituents independently selected from C 1-3 alkyl, C 1-3 alkoxy and Cy 3 ; and each Cy 2 is C 6-10 aryl, optionally substituted with 1 or 2 substituents independently selected from halo, C 1-3 alkyl, and C 1-3 haloalkoxy.
  • R 1 and R 3 are each C(O)NH(R a1 ); each R a1 is independently selected from C 1-6 alkyl, C 1-6 alkenyl, Cy 1 , and S(O) 2 R a2 , wherein the C 1-6 alkyl is optionally substituted with 1, 2, or 3 substituents independently selected from Cy 2 , carboxyl and C 1-3 alkoxycarbonyl; each Cy 1 is independently selected form C 3-10 cycloalkyl and C 6-10 aryl, each of which is optionally substituted with 1 or 2 substituents independently selected from C 1-3 alkyl, C 1-3 alkoxy and Cy 3 ; and each Cy 2 is C 6-10 aryl, optionally substituted with 1 or 2 substituents independently selected from halo, C1-3 alkyl, and C1-3 haloalkoxy.
  • the compound of Formula (II) has Formula (IIa): or a pharmaceutically acceptable salt thereof.
  • Cy 4 is C 6-10 aryl, optionally substituted with C 1-6 alkoxycarbonyl.
  • Cy 4 is C 3-10 cycloalkyl, optionally substituted with C 1-6 alkoxycarbonyl.
  • Cy 4 is 4-10 membered heterocycloalkyl, optionally substituted with C 1-6 alkoxycarbonyl.
  • Cy 4 is pyrrolidinyl, optionally substituted with (t-butoxy)carbonyl.
  • Cy 4 is selected from piperidinyl and pyrrolidinyl, each of which is optionally substituted with (t-butoxy)carbonyl.
  • Cy 4 is 5-10 membered heteroaryl, optionally substituted with C 1-6 alkoxycarbonyl.
  • each Cy 4 is independently selected from cyclopentyl, pyridinyl, piperidinyl, pyrrolidinyl, and phenyl.
  • the cyclopentyl, pyridinyl, piperidinyl, pyrrolidinyl, or phenyl is optionally substituted with (t-butoxy)carbonyl.
  • each Cy 4 is independently selected from: cyclopentyl, pyridinyl, piperidinyl, and phenyl. In some aspects of these embodiments, the cyclopentyl, pyridinyl, piperidinyl, or phenyl is optionally substituted with (t-butoxy)carbonyl. [0116] In some embodiments, each Cy 4 is independently selected from C 3-10 cycloalkyl and 4-10 membered heterocycloalkyl.
  • R 2 and R 4 are each H; and R 1 and R 3 are each independently selected from C 1-6 alkylcarbonyl and C(O)Cy 4 , wherein C 1-6 alkyl in the C 1-6 alkylcarbonyl is optionally substituted with 1, 2, or 3 substituents independently selected from Cy 4 , C 6-10 aryloxy, C 1-3 alkoxy, -NH(C 1-6 alkoxycarbonyl), and –NH(C 1-6 alkylcarbonyl), wherin C 1-6 alkoxy is optionally substituted with C 6-10 aryl.
  • R 2 and R 4 are each H; and R 1 and R 3 are each independently an C 1-6 alkylcarbonyl, wherein C 1-6 alkyl in the C1-6 alkylcarbonyl is optionally substituted with 1 or 2 substituents independently selected from Cy 4 , C 6-10 aryloxy, C 1-3 alkoxy, -NH(C 1-6 alkoxycarbonyl), and –NH(C 1-6 alkylcarbonyl), wherin C 1-6 alkoxy is optionally substituted with C 6-10 aryl.
  • R 2 and R 4 are each H; and R 1 and R 3 are each independently an C 1-6 alkylcarbonyl, wherein C 1-6 alkyl in the C 1-6 alkylcarbonyl is substituted with 1 or 2 substituents independently selected from Cy 4 , C 6-10 aryloxy, C 1-3 alkoxy, -NH(C 1-6 alkoxycarbonyl), and –NH(C 1-6 alkylcarbonyl), wherin C 1-6 alkoxy is optionally substituted with C 6-10 aryl.
  • each C 1-6 alkylcarbonyl is selected from: methylcarbonyl, ethylcarbonyl, and n-propylcarbonyl.
  • the methyl, ethy, and n-propyl are each optionally substituted with 1 or 2 substituents indepenently selected from Cy 4 , phenoxy, benzoxy, -NHC(O)(t-butoxy), and -NH(acetyl).
  • R 2 and R 4 are each H; and R 1 and R 3 are each independently selected from: methylcarbonyl, ethylcarbonyl, and n-propylcarbonyl, wherein the methyl, ethy, and n-propyl are each optionally substituted with 1 or 2 substituents indepenently selected from Cy 4 , phenoxy, benzoxy, -NHC(O)(t-butoxy), and -NH(acetyl).
  • R 2 and R 4 are each H; and R 1 and R 3 are each independently selected from: methylcarbonyl, ethylcarbonyl, and n-propylcarbonyl, wherein the methyl, ethy, and n-propyl are each substituted with 1 or 2 substituents indepenently selected from Cy 4 , phenoxy, benzoxy, -NHC(O)(t-butoxy), and -NH(acetyl).
  • R 2 and R 4 are each H; and R 1 and R 3 are each independently a C 1-6 alkylcarbonyl, wherein C 1-6 alkyl in the C 1-6 alkylcarbonyl is substituted Cy 4 .
  • R 2 and R 4 are each H; and R 1 and R 3 are each independently selected from: methylcarbonyl, ethylcarbonyl, and n-propylcarbonyl, wherein the methyl, ethy, and n-propyl are each optionally substituted with Cy4 .
  • the compound of Formula (II) has Formula (IIb): or a pharmaceutically acceptable salt thereof.
  • each Cy 4 is independently selected from C 3-10 cycloalkyl and 4-10 membered heterocycloalkyl.
  • the compound of Formula (II) is not a compound selected from: [0127] In some embodiments, the compound of Formula (II) is selected from: or a pharmaceutically acceptable salt thereof. [0128] In some embodiments, the compound of Formula (II) is selected from: O . or a pharmaceutically acceptable salt thereof. [0129] In some embodiments, the compound of Formula (II) is selected from: or a pharmaceutically acceptable salt thereof. [0130] In some embodiments, a salt of a compound of Formula (II) is formed between an acid and a basic group of the compound, such as an amino functional group, or a base and an acidic group of the compound, such as a carboxyl functional group.
  • the compound is a pharmaceutically acceptable acid addition salt.
  • acids commonly employed to form pharmaceutically acceptable salts of the compounds of Formula (II) include inorganic acids such as hydrogen bisulfide, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid, as well as organic acids such as para-toluenesulfonic acid, salicylic acid, tartaric acid, bitartaric acid, ascorbic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, lactic acid, oxalic acid, para-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid and acetic acid, as well as related inorganic and organic acids.
  • Such pharmaceutically acceptable salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate, xylene sulfonate, phenylacetate, phenylpropionat
  • pharmaceutically acceptable acid addition salts include those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and especially those formed with organic acids such as maleic acid.
  • bases commonly employed to form pharmaceutically acceptable salts of the compounds of Formula (II) include hydroxides of alkali metals, including sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, organic amines such as unsubstituted or hydroxyl-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-(C 1 -C 6 )-alkylamine), such as N,N-dimethyl-N-(2- hydroxyethy
  • the compounds of Formula (II), or pharmaceutically acceptable salts thereof are substantially isolated.
  • Methods of making the compounds [0134] Compounds of Formula (II), including salts thereof, can be prepared using known organic synthesis techniques and can be synthesized according to any of numerous possible synthetic routes.
  • the compounds described herein may be prepared using methods and procedures similar to those of Examples 39-64 and 197-208 herein.
  • a person skilled in the art knows how to select and implement appropriate synthetic protocols, and appreciates that the processes described are not the exclusive means by which compounds provided herein may be synthesized, and that a broad repertoire of synthetic organic reactions is available to be potentially employed in synthesizing compounds provided herein.
  • any one of the compounds of Formula (II) may be prepared according to the synthetic routes outlined in Scheme 1, using methods and procedures similar to those of Examples 39-64 and 197-208.
  • the R- group may correspond to C 1-6 alkoxy, C 1-6 alkyl or an NH(R a1 ) fragment, as in any one of the substituents R 1 -R 4 in a compound of Formula (II) described herein.
  • Suitable synthetic methods of starting materials, intermediates and products may be identified by reference to the literature, including reference sources such as: Advances in Heterocyclic Chemistry, Vols.1-107 (Elsevier, 1963-2012); Journal of Heterocyclic Chemistry Vols.
  • the compound of Formula (II) may be prepared according to the methods and procedures similar to those described in Larsen, B.J. et al., Tetrahedron 2018, 2762-2768, which is incorporated herein by reference in its entirety.
  • the reactions for preparing the compounds provided herein can be carried out in suitable solvents which can be readily selected by one of skill in the art of organic synthesis.
  • suitable solvents can be substantially non-reactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out, e.g., temperatures which can range from the solvent's freezing temperature to the solvent's boiling temperature.
  • a given reaction can be carried out in one solvent or a mixture of more than one solvent.
  • suitable solvents for a particular reaction step can be selected by the skilled artisan.
  • the compounds described herein may be docked and scored on their ability to bind to the dimer interface pocket lined by monomer lysines and glutamine residues in the protein structure of PF4 (see, e.g., Figure 3).
  • the compound of Formula (II) prevent formation of and/or disrupt the PF4 tetramer. The prevention and/or disruption may occur in vitro, ex vivo or in vivo.
  • the prevention of disruption may occur in a subject (i.e., after administering the compound to the subject), e.g., in need thereof.
  • the compound of Formula (II) may prevent formation and/or disrupt complexes formed between glycosaminoglycan (GAG) and a PF4 tetramer.
  • GAG glycosaminoglycan
  • PF4 tetramer pathogenic complexes of GAG and PF4 are very large and are referred to as ultralarge complexes (ULC).
  • ULCs are 600 kDa or larger.
  • ULCs are 670 kDa or larger.
  • GAGs are long unbranched polysaccharides having a repeating disaccharide unit (a hexose (six-carbon sugar) or a hexuronic acid, linked to a hexosamine (six-carbon sugar containing nitrogen)).
  • the GAG is selected from among wild-type GAGs or synthetically produced GAGs.
  • the GAG is heparin, hyaluronan, hyaluronic acid, dermatan sulfate, keratan sulfate, or a chondroitin, or a salt thereof.
  • the GAG is heparin.
  • the GAG is heparan sulfate.
  • PF4:heparin ULCs are more pathogenic than heparin:PF4 SCs.
  • Heparin:PF4 ULCs are better recognized by HITT antibodies and lead to more platelet activation in the presence of these antibodies. Disruption of ULC represents a valid therapeutic target in the treatment of a disease mediated by the ULC, such as the HITT.
  • the compounds described herein bind to PF4 monomers, PF4 trimers, PF4 dimers, and/or PF4 tetramers and inhibit the formation of the PF4 tetramers and/or inhibit a ULC formed with the PF4 tetramers.
  • the present compounds also disrupt a salt bridge in a PF4 tetramer and thereby disrupt or inhibit formation of the PF4 tetramer.
  • the compound binds to a specific site (e.g., specific amino acid) at the PF4 tetramer, trimer, dimer or monomer.
  • the compounds are capable of antagonizing an electrostatic attraction between the PF4 monomers, dimers, and trimers in the PF4 tetramer, and therefore successfully disrupt the salt bridge of the tetramer.
  • a functional group of the present compound of Formula (II) binds stronger to the PF4 monomer, dimer, or trimer, than they bind to one another.
  • the salt bridge between oligomers in the PF4 tetramer is formed via electrostatic interactions of a negatively charged amino acid, such as glutamic acid, of a first PF4 monomer or PF4 dimer, and a positively charged amino acid, such as lysine, of a second PF4 monomer or PF4 dimer.
  • This salt bridge is typically formed by interaction of at least one Glu or at least one Lys on a first PF4 monomer or PF4 dimer, and at least one Lys or at least one Glu on a second PF4 monomer or PF4 dimer.
  • the salt bridge is formed via at least electrostatic interactions between Lys50 in a first PF4 monomer Glu28 in a second PF4 monomer.
  • the salt bridge is formed an electrostatic interaction between Glu128 or Lys350 of a first PF4 monomer or dimer, and Glu328 or Lys150 of a second PF4 monomer or dimer.
  • the salt bridge is formed via an electrostatic interactions between Glu228 or Lys450 of a first PF4 monomer or dimer and Glu428 or Lys250 of a second PF4 monomer or dimer.
  • the salt bridge is formed on a PF4 dimer-dimer interface. That is, the compound of the present disclosure disrupts the salt bridge on the PF4 dimer-dimer interface.
  • a PF4:heparin ULC is antigen that promotes production of an antibody that is specific to the complex. The anybody recognizes the complex composed of heparin and the PF4 tetramer, and forms a pathogenic ULC-antibody complex.
  • the compounds described herein inhibit binding of the pathogenic ULC-antibody complex to a Fc ⁇ RIIa receptor on a surface of a platelet. This leads to inhibition of platelet activation, and results in decreased production of PF4 by the platelet.
  • the inhibition of binding of ULC-antibody complex to a Fc ⁇ RIIa also leads to inhibition of platelet aggregation, increased high density lipoproteins, modulated (e.g., reduced) blood clotting or hemostasis, and corrected platelet imbalance in the subject (e.g., in need thereof).
  • the platelet imbalance results from heparin administration to the subject.
  • Such methods include measuring a first level of PF4 tetramer in a first biological sample obtained from a subject and administering a first effective amount of the compound of Formula (II) that is required to decrease the first PF4 tetramer level.
  • methods for disrupting PF4 tetramers include measuring a first level of PF4 tetramer in a first biological sample obtained from a subject, administering a first effective amount of the compound of Formula (II) required to decrease the first PF4 tetramer level, and optionally administering a medication which disrupts PF4 tetramers.
  • methods for disrupting PF4 tetramers include measuring a first level of PF4 tetramer in a first biological sample obtained from a subject, administering a first effective amount of the compound of Formula (II) required to decrease the first PF4 tetramer level, measuring a second level of PF4 tetramer in a second biological sample obtained from the subject, and administering a second effective amount of the compound of Formula (II) required to decrease the second PF4 tetramer level.
  • kits for disrupting ULCs containing PF4 tetramers and heparin are provided. These methods include measuring a first level of ULCs in a first biological sample obtained from a subject and administering a first effective amount of the compound of Formula (II) required to decrease the first ULC tetramer level.
  • methods for disrupting ULCs containing PF4 tetramers and heparin include measuring a first level of ULCs in a first biological sample obtained from a subject, administering a first effective amount of the compound of Formula (II) required to decrease the first ULC level, measuring a second level of ULCs in a second biological sample obtained from the subject, and administering a second effective amount of the compound of Formula (II) required to decrease the second ULC level.
  • methods for preventing the formation of PF4 tetramers include measuring a first level of PF4 tetramer in a first biological sample obtained from a subject, administering a first effective amount of the compound of Formula (II) required to prevent formation of the PF4 tetramer, measuring a second level of PF4 tetramer in a second biological sample obtained from the subject, and administering a second effective amount of the compound of Formula (II) required to prevent formation of PF4 tetramer.
  • these screening methods are useful in monitoring e.g., cancer patients. In another embodiment, these screening methods are useful in determining the likelihood of cancer patients being administered heparin in developing HIT or HITT. According to this method, biological samples are obtained from subjects and the level of PF4 tetramer and/or PF4 tetramer:GAG (heparin) ULC measured. The screening may be conducted using techniques commonly known and used in the art.
  • Comparison of the levels of PF4 tetramer and/or PF4 tetramer:heparin ULC to a control level and/or negative control provides evidence that the patient may be treated using one or more of the antagonistic compounds described herein.
  • antagonistic compound administration may be contemplated if the subject's PF4 tetramer and/or PF4 tetramer:heparin ULC level is higher than the PF4 tetramer and/or PF4 tetramer:heparin ULC level of a healthy subject.
  • the present disclosure provides a method of treating a disease or condition characterized by PF4 tetramerization (e.g., a disease or medical condition related directly or indirectly to the formation of PF4 tetramers).
  • the disease or medical condition may also be caused by the formation of PF4 tetramers.
  • the subject or patient has elevated levels of PF4 tetramer.
  • the subject or patient has elevated levels of PF4 tetramer:GAG ULCs.
  • Suitable examples of a disease or condition include, but are not limited to, heparin- induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HIT), thrombotic complication of HITT, atherosclerosis or atherosclerotic vascular disease, platelet imbalance or insufficiency, antiphospholipid syndrome, inflammation or inflammatory disease, vaccine-induced immune throbotic thrombocytopenia (VITT), or clotting or hemostasis disorders.
  • the disease or medical condition is heparin-induced thrombocytopenia (HIT).
  • HIT results from the development of thrombocytopenia (low platelet count), for example, due to the administration of an anticoagulant (e.g., heparin or warfarin).
  • an anticoagulant e.g., heparin or warfarin
  • the disease or medical condition is heparin-induced thrombocytopenia and/with thrombosis (HITT).
  • HITT results when HIT precedes thrombosis (abnormal antibodies and abnormal blood clots form inside a blood vessel).
  • the thrombosis is characterized by lower than normal thrombin-antithrombin complex (TAT) level.
  • TAT normal thrombin-antithrombin complex
  • the disease or medical condition is vaccine-induced immune thrombotic thrombocytopenia (VITT) also known as thrombosis with thrombocytopenia syndrome.
  • VITT is caused by an adenovirus-based vaccine.
  • the vaccine is a adenovirus-based SARS-CoV-2 vaccine.
  • Treatment of atherosclerotic vascular disease typically involves anti-platelet therapy (e.g, aspirin and Plavix® which are not well tolerated in all patients).
  • thrombopoeitin (TPO) analogs and mimetics may be used, but these drugs have significant side effects and compliance issues.
  • MPL agonists which activate MPL-the TPO receptor MPL agonists which activate MPL-the TPO receptor
  • some lipid lowering therapies are able to increase HDL, but the ability of available drugs to do so is limited.
  • the disease that may be successfully treated by the compound of Formula (II) is antiphospholipid syndrome.
  • atherosclerosis resulting from the formation of a PF4 tetramer may be treated using a compound of Formula (II) described herein.
  • the disease or medical condition is a platelet imbalance.
  • the treatment method thereby includes correcting this platelet imbalance or preventing a platelet imbalance.
  • platelet levels are increased by stimulating platelet production.
  • a decrease in platelet production is prevented.
  • the platelet imbalance e.g., low levels of platelets
  • the platelet imbalance i.e., low levels of platelets, results from heparin administration to a subject.
  • the compounds of Formula (II) discussed herein may also be an alternative therapy utilized to treat diseases related to TPO.
  • the compounds may also be contemplated for use in preventing or treating inflammation which results from the formation of PF4 tetramers.
  • the inflammation may be the caused by any number of factors. In one embodiment, the inflammation is acute or chronic. In another embodiments, the inflammation is localized or systemic. The inflammation may be the result of a variety of factors and/or conditions.
  • the compounds of Formula (II) may also be useful in therapies for subjects having atherosclerotic vascular disease in which the patient is intolerant to the conventional treatments (e.g., statins).
  • an inflammatory disease is chronic inflammatory demyelinating polyneuropathy, inflammatory myopathy, inflammatory bowel diseases (IBDs), Crohn disease (CD), ulcerative colitis (UC), chronic inflammatory condition with polygenic susceptibility, inflammation of the uvea (e.g., anterior uveitis, e.g., iridocyclitis or ulceris; intermediate uveitis (also known as pars planitis); posterior uveitis; or chorioretinitis, e.g., pan-uveitis).
  • IBDs inflammatory bowel diseases
  • CD Crohn disease
  • UC ulcerative colitis
  • chronic inflammatory condition with polygenic susceptibility inflammation of the uvea (e.g., anterior uveitis, e.g., iridocyclitis or ulceris; intermediate uveitis (also known as pars planitis); posterior uveitis; or chorioretinitis, e.g., pan-
  • the compounds of the present application may be synergistic with the conventional therapies.
  • the compounds of Formula (II) are also useful in increasing high density lipoproteins (HDL) in a subject.
  • the PF4 antagonists of the present application are useful in preventing a decrease of HDLs.
  • a PF4-associated disease or condition in a subject may occur simultaneously with another disease or medical condition.
  • a decrease in platelet production may develop in a patient diagnosed with cancer.
  • the cancer is selected from the group selected from sarcoma, angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma, myxoma, rhabdomyoma, fibroma, lipoma, teratoma, lung cancer, breast cancer, bronchogenic carcinoma squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma, alveolar bronchiolar carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma, gastrointestinal cancer, cancer of the esophagus, squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma, cancer of the stomach, carcinoma, lymphoma, leiomyosarcoma, cancer of the pancreas, ductal adenocar
  • the cancer patient having a PF4-associated disease or condition is undergoing a chemotherapy.
  • the PF4-associated disease or disorder is the result of the chemotherapy treatment.
  • chemotherapeutic agents include paclitaxel, docetaxel, daunorubicin, cis-platin, carboplatin, and others.
  • the present application provides a method of treating cancer in a subject (e.g., any one of cancers described herein), the method comprising administering to the subject a therapeutically effective amount of a compound of Formula (II), or a pharmaceutically acceptable salt thereof.
  • discosed herein are methods of inhibiting platelet factor-4 (PF4) in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound or pharmaceutically acceptable salt thereof, or pharmaceutical composition disclosed herein, wherein the inhibition of PF4 comprises preventing formation of PF4 tetramers, and/or disrupting PF4 tetramers, and/or preventing formation of an ultra-large complex (ULC) comprising a PF4 tetramer and a glycosaminoglycan (GAG), and/or inhibiting ULC-antibody complex binding to a Fc ⁇ RIIa receptor on a platelet, and/or inhibiting platelet aggregation, and/or increasing high density lipoproteins, and/or modulating clotting or hemostasis, and/or correcting a platelet imbalance in the subject.
  • ULC ultra-large complex
  • GAG glycosaminoglycan
  • a therapeutically effective amount of a compound or pharmaceutically acceptable salt thereof, or pharmaceutical composition disclosed herein, for inhibition of PF4 comprises preventing formation of PF4 tetramers, and/or disrupting PF4 tetramers, and/or preventing formation of an ULC comprising a PF4 tetramer and a GAG, and/or inhibiting ULC-antibody complex binding to a Fc ⁇ RIIa receptor on a platelet, and/or inhibiting platelet aggregation, and/or increasing high density lipoproteins, and/or modulating clotting or hemostasis, and/or correcting a platelet imbalance in the subject.
  • the compound binds to a PF4 monomer, PF4 dimer, or PF4 tetramer.
  • the compound disrupts a salt bridge between two PF4 dimers, two or more PF4 monomers, or a PF4 dimer and a PF4 monomer, in a PF4 tetramer.
  • the disruption occurs on a dimer-dimer interface.
  • the salt bridge in the PF4 tetramer is formed via an electrostatic interaction of a negatively charged amino acid of a first PF4 monomer or PF4 dimer and a positively charged amino acid of a second PF4 monomer or PF4 dimer.
  • the negatively charged amino acid of a first PF4 monomer or PF4 dimer is a glutamic acid.
  • the positively charged amino acid of a second PF4 monomer or PF4 dimer is a lysine.
  • the GAG is a heparin.
  • the platelet imbalance results from heparin administration to the subject.
  • the molecular weight of the ULC is greater than about 600 kD. In some embodiments, inhibiting the binding of a ULC-antibody complex to a Fc ⁇ RIIa receptor on a platelet in a subject inhibits an activation of the platelet.
  • an antibody in the ULC-antibody complex recognizes a complex composed of heparin and a PF4 tetramer.
  • disclosed herein are methods of treating or preventing a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound disclosed herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition disclosed herein, wherein the disease or condition is selected from heparin induced thrombocytopenia and thrombosis (HITT), a thrombotic complication of HITT, heparin induced thrombocytopenia (HIT), vaccine- induced immune thrombotic thrombocytopenia (VITT), atherosclerosis or atherosclerotic vascular disease, decrease in platelet production, inflammation or an inflammatory disease, antiphospholipid syndrome, platelet imbalance or insufficiency, and a clotting or hemostasis disorder.
  • HITT heparin induced thrombocytopenia and thrombosis
  • a therapeutically effective amount of a compound or pharmaceutically acceptable salt thereof, or pharmaceutical composition disclosed herein for treatment or prevention of a disease or condition in a subject wherein the disease or condition is selected from HITT, a thrombotic complication of HITT, HIT, VITT, atherosclerosis or atherosclerotic vascular disease, decrease in platelet production, inflammation or an inflammatory disease, antiphospholipid syndrome, platelet imbalance or insufficiency, and a clotting or hemostasis disorder.
  • the disease or condition is mediated by a PF4 tetramer.
  • the atherosclerosis results from a PF4 tetramer formation or a formation of a GAG-PF4 complex.
  • the thrombotic complication of HITT is thrombosis.
  • the thrombosis is characterized by lower than normal thrombin-antithrombin complex level.
  • PF4 platelet factor-4
  • the method comprising administering to the subject in need thereof a therapeutically effective amount of a compound selected from: or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compounds, wherein the inhibition of PF4 comprises preventing formation of PF4 tetramers, and/or disrupting PF4 tetramers, and/or preventing formation of an ultra-large complex (ULC) comprising a PF4 tetramer and a glycosaminoglycan (GAG), and/or inhibiting ULC-antibody complex binding to a Fc ⁇ RIIa receptor on a platelet, and/or inhibiting platelet aggregation, and/or increasing high density lipoproteins, and/or modulating clotting or hemostasis, and/or correcting a platelet imbalance in the subject.
  • ULC ultra-large complex
  • GAG glycosaminoglycan
  • a compound selected from: or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compounds, for inhibiting platelet factor-4 (PF4) in a subject wherein the inhibition of PF4 comprises preventing formation of PF4 tetramers, and/or disrupting PF4 tetramers, and/or preventing formation of an ultra-large complex (ULC) comprising a PF4 tetramer and a glycosaminoglycan (GAG), and/or inhibiting ULC-antibody complex binding to a Fc ⁇ RIIa receptor on a platelet, and/or inhibiting platelet aggregation, and/or increasing high density lipoproteins, and/or modulating clotting or hemostasis, and/or correcting a platelet imbalance in the subject.
  • ULC ultra-large complex
  • GAG glycosaminoglycan
  • the compound binds to a PF4 monomer, PF4 dimer or PF4 tetramer.
  • the compound disrupts a salt bridge between two PF4 dimers, two or more PF4 monomers, or a PF4 dimer and a PF4 monomer, in a PF4 tetramer.
  • the disruption occurs on a dimer-dimer interface.
  • the salt bridge in the PF4 tetramer is formed via an electrostatic interaction of a negatively charged amino acid of a first PF4 monomer or PF4 dimer and a positively charged amino acid of a second PF4 monomer or PF4 dimer.
  • the negatively charged amino acid of a first PF4 monomer or PF4 dimer is a glutamic acid.
  • the positively charged amino acid of a second PF4 monomer or PF4 dimer is a lysine.
  • the GAG is a heparin.
  • the platelet imbalance results from heparin administration to the subject.
  • the molecular weight of the ULC is greater than about 600 kD. In some embodiments, inhibiting the binding of a ULC-antibody complex to a Fc ⁇ RIIa receptor on a platelet in a subject inhibits an activation of the platelet.
  • the ULC-antibody complex recognizes a complex composed of heparin and a PF4 tetramer.
  • disclosed herein are method of treating or preventing a disease or condition in a subject, or use of the compound in prevention or treatment of a disease or condition in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound selected from: or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound(s), wherein the disease or condition is selected from heparin induced thrombocytopenia and thrombosis (HITT), a thrombotic complication of HITT, heparin induced thrombocytopenia (HIT), vaccine-induced immune thrombotic thrombocytopenia (VITT), atherosclerosis or atherosclerotic vascular disease, decrease in platelet production, inflammation or an inflammatory disease, antiphospholipid syndrome, platelet imbalance or insufficiency, and a clo
  • the disease or condition is mediated by a PF4 tetramer.
  • the atherosclerosis results from a PF4 tetramer formation or a formation of a GAG-PF4 complex.
  • the thrombotic complication of HITT is thrombosis.
  • the thrombosis is characterized by lower than normal thrombin-antithrombin complex level.
  • the compound of Formula (II) as described herein may be administered to the subject in combination with an additional therapeutic agent.
  • the additional therapeutic agent may disrupt PF4 tetramers and/or ULCs.
  • the additional agent works synergistically with the PF4 antagonist of the present application.
  • Suitable examples of such agents include cyclic peptides which inhibit the interaction of PF4 with CCL5 (CKEY2), carbohydrates such as desulfated heparin (ODSH), or a combination thereof.
  • CKEY2 CCL5
  • ODSH desulfated heparin
  • the PF4 tetramer disruption agents may be combined with the PF4 antagonist compounds of the present application either in a pharmaceutical composition as described herein, and/or kits and methods for using the same.
  • an additional therapeutic agent is an anticoagulant (e.g., rivaroxaban, dabigatran, apixaban, edoxaban, warfarin, fondaparinux, idraparinux, acenocoumarol, phenprocoumon, atromentin, or phenindione).
  • additional therapeutic agent is heparin.
  • additional therapeutic agents include an anti-HER2 agent (e.g., trastuzumab, pertuzumab, lapatinib), a pain relief agent (e.g., a nonsteroidal anti-inflammatory drug such as celecoxib or rofecoxib), an antinausea agent, a cardioprotective drug (e.g., dexrazoxane, ACE-inhibitors, diuretics, cardiac glycosides), a cholesterol lowering drug, a revascularization drug, a beta-blocker (e.g., acebutolol, atenolol, bisoprolol, metoprolol, nadolol, nebivolol, or propranolol), an angiotensin receptor blocker (also called ARBs or angiotensin II inhibitors) (e.g., azilsartan, candesartan, eprosartan, irbesartan, los
  • kits useful, for example, in the treatment of disorders, diseases and conditions referred to herein, which include one or more containers containing a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present disclosure.
  • kits can further include, if desired, one or more of various conventional pharmaceutical kit components, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers with additional therapeutic agents, diagnostic reagents, etc. Instructions, either as inserts or as labels, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, can also be included in the kit.
  • the kit may further contain instructions for monitoring blood level of the administered compound, and materials for performing such assays including, e.g., reagents, well plates, containers, markers or labels, and the like.
  • Such kits are readily packaged in a manner suitable for treatment of a desired indication.
  • the kit may also contain instructions for use of the spray pump or other delivery device.
  • a pharmaceutical kit in another embodiment, contains a medication which causes the formation of PF4 tetramers (e.g., heparin) in a first dosage unit and one or more of a PF4 antagonistic compound of the present application in a second dosage unit.
  • a pharmaceutical kit is provided and contains a therapeutic agent which disrupts PF4 tetramers in a first dosage unit, one or more of a PF4 antagonistic compound of the present disclosure in a second dosage unit, and one or more of the carriers or excipients described herein in a third dosage unit.
  • the kit may optionally contain instructions for administering the components of the kit to a subject, for example, having cancer.
  • a pharmaceutical kit contains a therapeutic agent that causes formation of a PF4 tetramer in a first dosage unit (e.g., heparin), a therapeutic agent that disrupts a PF4 tetramer in a second dosage unit, and one or more of a PF4 antagonistic compound described herein in a third dosage unit, and one or more of the carriers or excipients in a fourth dosage unit.
  • the kit may optionally contain instructions for administering the components of the kit to a subject, e.g., having cancer.
  • compositions and formulations [0191]
  • the present application also provides pharmaceutical compositions comprising an effective amount of a compound of Formula (II) disclosed herein, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.
  • the application also provides pharmaceutical compositions and dosage forms comprising any one the additional therapeutic agents described herein.
  • the carrier(s) are “acceptable” in the sense of being compatible with the other ingredients of the formulation and, in the case of a pharmaceutically acceptable carrier, not deleterious to the recipient thereof in an amount used in the medicament.
  • Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of the present application include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose- based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.
  • ion exchangers alumina, aluminum stearate, lecithin
  • serum proteins such as human serum albumin
  • buffer substances such
  • compositions or dosage forms may contain any one of the compounds and therapeutic agents described herein in the range of 0.005% to 100% with the balance made up from the suitable pharmaceutically acceptable excipients.
  • the contemplated compositions may contain 0.001%-100% of any one of the compounds and therapeutic agents provided herein, in one embodiment 0.1-95%, in another embodiment 75-85%, in a further embodiment 20-80%, wherein the balance may be made up of any pharmaceutically acceptable excipient described herein, or any combination of these excipients.
  • Routes of administration and dosage forms [0194]
  • the pharmaceutical compositions of the present application include those suitable for any acceptable route of administration.
  • Acceptable routes of administration include, but are not limited to, buccal, cutaneous, endocervical, endosinusial, endotracheal, enteral, epidural, interstitial, intra-abdominal, intra-arterial, intrabronchial, intrabursal, intracerebral, intracisternal, intracoronary, intradermal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralymphatic, intramedullary, intrameningeal, intramuscular, intranasal, intraovarian, intraperitoneal, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratesticular, intrathecal, intratubular, intratumoral, intrauterine, intravascular, intravenous, nasal, nasogastric, oral, parenteral, percutaneous, peridural, rectal, respiratory (inhalation), sub
  • compositions and formulations described herein may conveniently be presented in a unit dosage form, e.g., tablets, sustained release capsules, and in liposomes, and may be prepared by any methods well known in the art of pharmacy. See, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, Baltimore, MD (20th ed. 2000). Such preparative methods include the step of bringing into association with the molecule to be administered ingredients such as the carrier that constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers, liposomes or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • compositions of the present application suitable for oral administration may be presented as discrete units such as capsules, sachets, granules or tablets each containing a predetermined amount (e.g., effective amount) of the active ingredient; a powder or granules; a solution or a suspension in an aqueous liquid or a non- aqueous liquid; an oil-in-water liquid emulsion; a water-in-oil liquid emulsion; packed in liposomes; or as a bolus, etc.
  • Soft gelatin capsules can be useful for containing such suspensions, which may beneficially increase the rate of compound absorption.
  • carriers that are commonly used include lactose, sucrose, glucose, mannitol, and silicic acid and starches.
  • Other acceptable excipients may include: a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar- agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as
  • useful diluents include lactose and dried corn starch.
  • the active ingredient is combined with emulsifying and suspendfing agents.
  • certain sweetening and/or flavoring and/or coloring agents may be added.
  • Compositions suitable for oral administration include lozenges comprising the ingredients in a flavored basis, usually sucrose and acacia or tragacanth; and pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia.
  • compositions suitable for parenteral administration include aqueous and non- aqueous sterile injection solutions or infusion solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, saline (e.g., 0.9% saline solution) or 5% dextrose solution, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
  • the injection solutions may be in the form, for example, of a sterile injectable aqueous or oleaginous suspension.
  • This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
  • the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant.
  • the pharmaceutical compositions of the present application may be administered in the form of suppositories for rectal administration.
  • compositions can be prepared by mixing a compound of the present application with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components.
  • suitable non-irritating excipient include, but are not limited to, cocoa butter, beeswax, and polyethylene glycols.
  • suitable non-irritating excipient include, but are not limited to, cocoa butter, beeswax, and polyethylene glycols.
  • suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components.
  • Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycols.
  • the pharmaceutical compositions of the present application may be administered by nasal aerosol or inhalation.
  • Such compositions are prepared according to techniques well- known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benz
  • the topical compositions of the present disclosure can be prepared and used in the form of an aerosol spray, cream, emulsion, solid, liquid, dispersion, foam, oil, gel, hydrogel, lotion, mousse, ointment, powder, patch, pomade, solution, pump spray, stick, towelette, soap, or other forms commonly employed in the art of topical administration and/or cosmetic and skin care formulation.
  • the topical compositions can be in an emulsion form.
  • Topical administration of the pharmaceutical compositions of the present application is especially useful when the desired treatment involves areas or organs readily accessible by topical application.
  • the topical composition comprises a combination of any one of the compounds and therapeutic agents disclosed herein, and one or more additional ingredients, carriers, excipients, or diluents including, but not limited to, absorbents, anti- irritants, anti-acne agents, preservatives, antioxidants, coloring agents/pigments, emollients (moisturizers), emulsifiers, film-forming/holding agents, fragrances, leave-on exfoliants, prescription drugs, preservatives, scrub agents, silicones, skin-identical/repairing agents, slip agents, sunscreen actives, surfactants/detergent cleansing agents, penetration enhancers, and thickeners.
  • the compounds and therapeutic agents of the present application may be incorporated into compositions for coating an implantable medical device, such as prostheses, artificial valves, vascular grafts, stents, or catheters.
  • Suitable coatings and the general preparation of coated implantable devices are known in the art and are exemplified in U.S. Patent Nos. 6,099,562; 5,886,026; and 5,304,121.
  • the coatings are typically biocompatible polymeric materials such as a hydrogel polymer, polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylactic acid, ethylene vinyl acetate, and mixtures thereof.
  • the present application provides an implantable drug release device impregnated with or containing a compound or a therapeutic agent, or a composition comprising a compound of the present application or a therapeutic agent, such that the compound or therapeutic agent is released from the device and is therapeutically active.
  • a compound of Formula (II) is present in an effective amount (e.g., a therapeutically effective amount).
  • Effective doses may vary, depending on the diseases treated, the severity of the disease, the route of administration, the sex, age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents and the judgment of the treating physician.
  • an effective amount of a compound of Formula (II) can range, for example, from about 0.001 mg/kg to about 500 mg/kg (e.g., from about 0.001 mg/kg to about 200 mg/kg; from about 0.01 mg/kg to about 200 mg/kg; from about 0.01 mg/kg to about 150 mg/kg; from about 0.01 mg/kg to about 100 mg/kg; from about 0.01 mg/kg to about 50 mg/kg; from about 0.01 mg/kg to about 10 mg/kg; from about 0.01 mg/kg to about 5 mg/kg; from about 0.01 mg/kg to about 1 mg/kg; from about 0.01 mg/kg to about 0.5 mg/kg; from about 0.01 mg/kg to about 0.1 mg/kg; from about 0.1 mg/kg to about 200 mg/kg; from about 0. 0.01 mg/kg to about 500 mg/kg (e.g., from about 0.001 mg/kg to about 200 mg/kg; from about 0.01 mg/kg to about 200 mg/kg; from about 0.01 mg/kg to about
  • an effective amount of a compound of Formula (II) is about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, or about 5 mg/kg.
  • the foregoing dosages can be administered on a daily basis (e.g., as a single dose or as two or more divided doses, e.g., once daily, twice daily, thrice daily) or non-daily basis (e.g., every other day, every two days, every three days, once weekly, twice weekly, once every two weeks, once a month).
  • a daily basis e.g., as a single dose or as two or more divided doses, e.g., once daily, twice daily, thrice daily
  • non-daily basis e.g., every other day, every two days, every three days, once weekly, twice weekly, once every two weeks, once a month.
  • LC/MS data were determined with a Waters Alliance 2695 HPLC/MS (Waters Symmetry C18, 4.6 ⁇ 75 mm, 3.5 ⁇ m) or (Phenomenex C18, 4.6 ⁇ 75 mm, 3.0 ⁇ m) with a 2996 diode array detector from 210 ⁇ 400 nm; the solvent system is 5 ⁇ 95% MeCN in water (with 0.1% TFA) over nine minutes using a linear gradient, and retention times are in minutes. Mass spectrometry was performed on a Waters ZQ using electrospray in positive mode.
  • LC/MS data were determined with a Shimadzu Prominence HPLC/MS (Phenomenex Luna C18, 3.0 ⁇ 50 mm, 3 ⁇ m) with a 2996 diode array detector from 210 ⁇ 400 nm; the solvent system is 5 ⁇ 95% MeCN in water (with 0.1% formic acid) over five minutes using a linear gradient, and retention times are in minutes. Mass spectrometry was performed on a Applied Biosystems MDS Sciex API 2000 using electrospray in negative mode.
  • LC/MS data were determined with a Waters Alliance 2695 HPLC/MS (Phenomenex C18, 4.6 ⁇ 75 mm, 3.0 ⁇ m) with a 2996 diode array detector from 210 ⁇ 400 nm; the solvent system is 5 ⁇ 95% MeCN in water (with 0.1% formic acid) over nine minutes using a linear gradient, and retention times are in minutes. Mass spectrometry was performed on a Waters ZQ using electrospray in positive mode. [0211] HRMS data were determined by The University of Notre Dame Mass Spectrometry & Proteomics Facility on a Bruker micrOTOF II.
  • Preparative reversed phase HPLC was performed on a Waters Sunfire column (19 ⁇ 50 mm, C18, 5 ⁇ m) with a 10 min mobile phase gradient of 10% acetonitrile/water to 90% acetonitrile/ water with 0.1% TFA as buffer using 214 and 254 nm as detection wavelengths. Injection and fraction collection were performed with a Gilson 215 liquid handling apparatus using Trilution LC software. [0213] 1 H NMR were recorded on Varian Oxford 300 MHz, in DMSO-d 6 . Chemical shifts ( ⁇ ) are expressed in ppm downfield from tetramethylsilane (TMS) unless otherwise noted.
  • TMS tetramethylsilane
  • EXAMPLE 39 Synthesis of 2,2,6-triacetyl-s-indacene-1,3,5,7-tetraone [0214]
  • the title compound was prepared from pyromellitic dianhydride (CAS Number 89- 32-7, 218 mg, 1.0 mmol) and pentane-2,4-dione (206 ⁇ L, 2 mmol) with trimethylamine and acetic anhydride according to the conditions in Example 1 to give the title compound (154 mg, 52% yield).
  • EXAMPLE 42 Synthesis of n 2 -(2-(cyclohexa-2,4-dien-1-yl)ethyl)-1,3,5,7-tetraoxo-n 6 - phenethyl-1,2,3,5,6,7-hexahydro-s-indacene-2,6-dicarboxamide [0218] To a solution of S-indacene-1,3,5,7(2H,6H)-tetraone (prepared in Example 41, 100 mg, 0.5 mmol) in DMF (3 mL) at between -60 to -50°C was added Et 3 N (162 ⁇ L, 1.2 mmol) and phenethyl isocyanate (161 ⁇ L, 1.2 mmol).
  • EXAMPLE 45 Synthesis of 1,3,5,7-tetraoxo-n 2 ,n 6 -di-o-tolyl-1,2,3,5,6,7-hexahydro-s- indacene-2,6-dicarboxamide [0221]
  • the title compound was prepared from S-indacene-1,3,5,7(2H,6H)-tetraone as described for Example 42, except using o-tolyl isocyanate (CAS Number 614-68-6, 145 ⁇ L, 1.2 mmol) as the isocyanate (126 mg, 56%).
  • EXAMPLE 48 Synthesis of 1,3,5,7-tetraoxo-n 2 ,n 6 -bis(3-phenylpropyl)-1,2,3,5,6,7- hexahydro-s-indacene-2,6-dicarboxamide [0224] The title compound was prepared from S-indacene-1,3,5,7(2H,6H)-tetraone as described for Example 42, except using phenpropyl isocyanate (362uL, 2.3 mmol) as the isocyanate (110mg, 44%).
  • EXAMPLE 54 Synthesis of n 2 ,n 6 -bis(2-fluorophenethyl)-1,3,5,7-tetraoxo-1,2,3,5,6,7- hexahydro-s-indacene-2,6-dicarboxamide [0230] The title compound was prepared from S-indacene-1,3,5,7(2H,6H)-tetraone as described for Example 42, except using 2-fluorophenethyl isocyanate (CAS Number 480439- 39-2, 335 ⁇ L, 2.3 mmol) as the isocyanate reagent (96 mg, 38%).
  • 2-fluorophenethyl isocyanate CAS Number 480439- 39-2, 335 ⁇ L, 2.3 mmol
  • EXAMPLE 56 Synthesis of 3- ⁇ [6-(2-ethoxycarbonyl-ethylcarbamoyl)-1,3,5,7-tetraoxo- 1,2,3,5,6,7-hexahydro-s-indacene-2-carbonyl]-amino ⁇ -propionic acid ethyl ester [0232] The title compound was prepared from S-indacene-1,3,5,7(2H,6H)-tetraone as described for Example 55, except using ethyl-3-isocyanatopropionate (CAS Number 5100-34- 5, 307 ⁇ L, 1.2mmol) as the isocyanate (60mg, 26%).
  • ethyl-3-isocyanatopropionate CAS Number 5100-34- 5, 307 ⁇ L, 1.2mmol
  • EXAMPLE 60 Synthesis of 1,3,5,7-tetraoxo-n 2 ,n 6 -bis(3-(pyrimidin-2-yl)phenyl)- 1,2,3,5,6,7-hexahydro-s-indacene-2,6-dicarboxamide [0236]
  • the title compound was prepared as the bis-sodium salt from S-indacene- 1,3,5,7(2H,6H)-tetraone as described for Example 55, except using 2-(3-isocyanatophenyl) pyrimidine (CDS018140 ALDRICH, 192 ⁇ L, 1.2 mmol) as the isocyanate reagent (37 mg, 13%).
  • EXAMPLE 64 Synthesis of 1,3,5,7-tetraoxo-n 2 ,n 6 -bis(2-phenylcyclopropyl)-1,2,3,5,6,7- hexahydro-s-indacene-2,6-dicarboxamide [0240]
  • the title compound was prepared as the bis-sodium salt from S-indacene- 1,3,5,7(2H,6H)-tetraone as described for Example 55, except using (2- isocyanatocyclopropyl)benzene (CAS Registry No. 63006-15-5, 110 ⁇ L, 1.2 mmol) as the isocyanate (164.7 mg, 66%).
  • EXAMPLE 197 2,6-bis(2-phenoxyacetyl)-1,2,3,5,6,7-hexahydro-s-indacene-1,3,5,7- tetrone O O O O O O O O O [0241]
  • DMAP 183 mg, 1.50 mmol
  • 2-phenoxyacetic acid CAS number 122- 59-8, 228mg, 1.5mmol
  • 9.0 ml of anhydrous DMF was stirred for 10 min after which a solution had formed.
  • EDCI HCl (CAS number 25952-53-8, 286.5 mg, 1.50 mmol) was added and the contents were stirred for an additional 10 min.
  • EXAMPLE 202 2,6-bis[2-(piperidin-4-yl)acetyl]-1,2,3,5,6,7-hexahydro-s-indacene- 1,3,5,7-tetrone [0246]
  • the title compound was prepared by treating the title compound from Example 200 (80 mg, 0.12 mmol) with 5N HCl solution 2 (mL). The mixture was heated at 50°C for 1 hr. The resulting solid was filtered, washed by water and dried to give the title compound (46 mg, 82%).
  • EXAMPLE 205 N- ⁇ 2-[6-(2-acetamidoacetyl)-1,3,5,7-tetraoxo-1,2,3,5,6,7-hexahydro-s- indacen-2-yl]-2-oxoethyl ⁇ acetamide [0249] In a similar manner to that of Example 197, except that the sodium salt step was omitted, the title compound was prepared from 1,3,5,7(2H,6H)-tetraone (as used in Example 55, 107 mg, 0.5 mmol) and N-acetyl glycine (CAS number 543-24-8, 176 mg, 1.5 mmol) to afford the title compound (142 mg, 69%).
  • Step 1 To a 250 ml 3-neck RB flask equipped with a stirbar, temperature probe and N2 balloon was charged pyromellitic anhydride (10.01 g, 45.89 mmol, 1 eq.) and ethyl acetoacetate (18.01 g, 138.39 mmol, 3.02 eq.). To this was added acetic anhydride (81.00 g, 793.39 mmol, 17.3 eq) to produce a clear slurry.
  • the dull green/brown cake was allowed to dry via suction on the filter, prior to placement in the vacuum oven to dry at 45°C and full house vacuum. After drying, 2,6-diacetyl-1,2,3,5,6,7-hexahydro-s-indacene- 1,3,5,7-tetrone, bis triethyl amine salt was obtained as a dull green solid (13.36g, 52% yield).
  • Step 2 To a 1000 mL 3-neck flask was charged the starting material (5.07 g, 9.04 mmol) and a stirbar. The solid was dissolved by the addition of 500 mL of water to produce a deep red solution. The mixture was allowed to stir at ambient temperature for 15 min. The reaction was cooled to ⁇ 0°C in an ice water bath.
  • Example 209 s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and cyclobutyl isocyanate (CAS 5811-25-6). The compound was isolated as the bis sodium salt.
  • Example 211 Synthesis of N2,N6-bis(cyclopropylmethyl)-1,3,5,7-tetraoxo-1,2,3,5,6,7- hexahydro-s-indacene-2,6-dicarboxamide.
  • the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and (isocyanatomethyl)cyclopropane (CAS 25694-89-7). The compound was isolated as the bis sodium salt.
  • Example 197 Using the procedure in Example 197, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and pentanoic acid (CAS 109-52-4). The compound was isolated as the bis sodium salt.
  • Example 213 Synthesis of tert-butyl N- ⁇ 2-[6-(2- ⁇ [(tert-butoxy)carbonyl](methyl) amino ⁇ acetyl)-1,3,5,7-tetraoxo-1,2,3,5,6,7-hexahydro-s-indacen-2-yl]-2-oxoethyl ⁇ -N- methylcarbamate. [0258] Using the procedure in Example 197, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and 2- ⁇ [(tert- butoxy)carbonyl](methyl)amino ⁇ acetic acid (CAS 13734-36-6).
  • Example 214 Synthesis of 2,6-bis(2-methoxyacetyl)-1,2,3,5,6,7-hexahydro-s- indacene-1,3,5,7-tetrone.
  • O O O O O O O O [0259] Using the procedure in Example 197, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and methoxyacetic acid (CAS 625-45-6).
  • Example 215 Synthesis of 2,6-bis[2-(2-methoxyethoxy)acetyl]-1,2,3,5,6,7-hexahydro- s-indacene-1,3,5,7-tetrone.
  • the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and 2-methoxyethoxy acetic acid (CAS 16024-56-9). The compound was isolated as the bis sodium salt.
  • Example 216 Synthesis of 2,6-dicyclobutanecarbonyl-1,2,3,5,6,7-hexahydro-s- indacene-1,3,5,7-tetrone.
  • the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and cyclobutene carboxylic acid (CAS 3721- 95-7). The compound was isolated as the bis sodium salt.
  • Example 217 Synthesis of N2,N6-bis[2-(2-methoxyethoxy)ethyl]-1,3,5,7-tetraoxo- 1,2,3,5,6,7-hexahydro-s-indacene-2,6-dicarboxamide.
  • the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and 1-isocyanato-2-(2- methoxyethoxy)ethane (CAS 90426-82-7). The compound was isolated as the bis sodium salt.
  • Example 209 Using the procedure in Example 42, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and 1-isocyanato-2-(2- methoxyethoxy)ethane (CAS 90426-82-7). The compound was isolated as the bis sodium salt.
  • Example 219 Synthesis of 2,6-dicyclopropanecarbonyl-1,2,3,5,6,7-hexahydro-s- indacene-1,3,5,7-tetrone.
  • the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and cyclopropane carboxylic acid (CAS 1759-53-1). The compound was isolated as the bis sodium salt.
  • Example 220 Synthesis of 2,6-bis( ⁇ 2-[2-(2-methoxyethoxy)ethoxy]acetyl ⁇ )-1,2,3,5,6,7- hexahydro-s-indacene-1,3,5,7-tetrone. [0265] Using the procedure in Example 197, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and 2-[2-(2-methoxyethoxy)ethoxy]acetic acid (CAS 16024-58-1).
  • Example 221 Synthesis of 2,6-bis[2-(2-oxopyrrolidin-1-yl)acetyl]-1,2,3,5,6,7- hexahydro-s-indacene-1,3,5,7-tetrone.
  • the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and 2-oxo-1-pyrrolidineacetic acid (CAS 53934-76-2).
  • Example 209 Using the procedure in Example 197, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and N-(tert-butoxycarbonyl)-L-pipecolic acid (CAS 26250-84-0).
  • Example 223 Synthesis of tert-butyl 3- ⁇ 2-[6-(2- ⁇ 1-[(tert-butoxy)carbonyl]pyrrolidin-3- yl ⁇ acetyl)-1,3,5,7-tetraoxo-1,2,3,5,6,7-hexahydro-s-indacen-2-yl]-2- oxoethyl ⁇ pyrrolidine-1-carboxylate.
  • Example 209 Using the procedure in Example 197, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and N-(tert-butoxycarbonyl)-3- pyrrolidineaceticacid (CAS number 175526-97-3).
  • Example 224 Synthesis of tert-butyl N- ⁇ 2-[6-(2- ⁇ [(tert- butoxy)carbonyl](methyl)amino ⁇ acetyl)-1,3,5,7-tetraoxo-1,2,3,5,6,7-hexahydro-s- indacen-2-yl]-2-oxoethyl ⁇ -N-methylcarbamate. [0269] Using the procedure in Example 197, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and N-(tert-butoxycarbonyl)-N-methylglycine (CAS number 13734-36-6).
  • Example 209 Using the procedure in Example 197, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and 2-tert-butoxyacetic acid (CAS number 13211-32-0).
  • 1 H NMR ⁇ 7.63 (br s, 2H), 4.64 - 4.53 (m, 4H), 1.17 (br s, 18H).
  • Example 226 Synthesis of tert-butyl N-[(2S)-1- ⁇ 6-[(2S)-2- ⁇ [(tert- butoxy)carbonyl]amino ⁇ -4-methylpentanoyl]-1,3,5,7-tetraoxo-1,2,3,5,6,7-hexahydro-s- indacen-2-yl ⁇ -4-methyl-1-oxopentan-2-yl]carbamate. [0271] Using the procedure in Example 197, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and N-(tert-butoxycarbonyl)-leucine (CAS number 13139-15-6).
  • Example 209 Using the procedure in Example 197, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and N-acetyl-N-methylglycine (CAS number 5888-91-5).
  • Example 229 Synthesis of 2,6-bis[(2S)-piperidine-2-carbonyl]-1,2,3,5,6,7-hexahydro- s-indacene-1,3,5,7-tetrone.
  • Example 230 Synthesis of 2,6-bis[2-(pyrrolidin-3-yl)acetyl]-1,2,3,5,6,7-hexahydro-s- indacene-1,3,5,7-tetrone.
  • the title compound was prepared from tert- butyl 3- ⁇ 2-[6-(2- ⁇ 1-[(tert-butoxy)carbonyl]pyrrolidin-3-yl ⁇ acetyl)-1,3,5,7-tetraoxo-1,2,3,5,6,7- hexahydro-s-indacen-2-yl]-2-oxoethyl ⁇ pyrrolidine-1-carboxylate (Example 223) as the bis hydrogen chloride salt.
  • Example 232 Synthesis of 2,6-bis[2-(methylamino)acetyl]-1,2,3,5,6,7-hexahydro-s- indacene-1,3,5,7-tetrone.
  • O O HN NH O O O O [0277] Using the procedure in Example 229, the title compound was prepared tert-butyl N- ⁇ 2-[6-(2- ⁇ [(tert-butoxy)carbonyl](methyl)amino ⁇ acetyl)-1,3,5,7-tetraoxo-1,2,3,5,6,7-hexahydro- s-indacen-2-yl]-2-oxoethyl ⁇ -N-methylcarbamate (Example 224) as the bis hydrogen chloride salt.
  • Step 1 To a solution of triphosgene (1.36 g, 4.5 mmol) in CH2Cl2 (13.8 mL) under nitrogen at 0°C was added pyridine (2.07 mL, 25.7 mmol). After stirring 20 min, a solution of 2-(2-(2-methoxyethoxy)ethoxy) ethanamine (CAS reg. no: 74654-07-2, 734 mg, 4.5 mmol) in CH 2 Cl 2 (13.8 mL) was added dropwise to this mixture. The reaction was warmed to ambient temperature and stirred 45 min.
  • Step 2 Using the procedure in Example 42, the title compound was prepared from s-indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and 1-[2-(2-isocyanato-ethoxy)-ethoxy]-2- methoxy-ethane from step 1 above.
  • Triethylamine (0.18 mL, 1.3 mmol) was added, followed after 5 min by 1-isocyanato-2- methanesulfonylethane (CAS reg. no: 631912-32-8, 195 mg, 1.3 mmol).
  • the cooling bath was allowed to expire, and the mixture was stirred at rt overnight.5% aq HCl (15 mL) was added and the mixture was stirred for 15 min, then filtered, washing with water (5 mL).
  • the dark red solid collected was suspended in CH 2 Cl 2 (2 mL) and 1 M aq NaOH (2 mL), and stirred for 1 h. The mixture was filtered, washing with water.
  • Step 2 A stirred solution of 1,2,3,5,6,7-hexahydro-s-indacene-1,3,5,7-tetrone (CAS reg. no: 53910-13-7, 53 mg, 0.25 mmol) and 1-isocyanato-2-(2-methanesulfonylethoxy)ethane (148 mg, 0.76 mmol) in dry DMF (5 mL) was cooled to ca. -45°C in a dry ice/MeCN bath. Triethylamine (0.10 mL, 0.72 mmol) was added and stirring was continued as the cooling bath expired.
  • cDNA encoding human PF4 was cloned into the plasmid pMT/BiPN5-His A (Invitrogen Corp.) for expression in the Drosophila Expression System (Invitrogen). Cloning was performed using Bg1 II and Age I cloning sites. A hexanucleotide encoding Bg1 II site was then eliminated by site-directed mutagenesis so that the expressed protein contained full- length wild type (wt) PF4 or PF4 K50E with an identical sequence as their counterparts expressed in E. coli. See, method described in Sachais et al (Blood 119(25): 5955-5961, 2012).
  • PF4 expression was induced by adding copper sulfate (0.5 mM).
  • the induced S2 cells were incubated in serum-free Insect-XpressTM media (Lonza) for 3-5 days; supernatants were collected, sodium azide (0.02% final concentration) and EDTA (2.5 mM final concentration) were added, and the media were filtered through an Express® PLUS 0.22 ⁇ m filter (Millipore Corp.).
  • Wild-type (WT) human PF4 in the pT7-7 vector Novagen was expressed in the Escherichia coli strain BL21DE30 pLysS (Stratagene), and purified and characterized as described by Rauova et al.
  • the relative amounts of PF4 in each of the monomer, dimer, trimer and tetramer form were calculated by analyzing the samples on a 12% SDS-polyacrylamide gel under reducing conditions. Bands were quantified using the Kodak ID Image Analysis system (Kodak). The SigmaMarkerTM reagent served as the molecular weight standard. Data was compiled and bar graphed as % PF4 for the tetramer, trimer, dimer, and monomer as a function of concentration for the test and control samples. These data show that certain compounds were effective in inhibiting PF4 tetramer formation at tested concentrations.
  • the active compounds are acidic and have low log D values.
  • the clog D (pH 7.4) calculated values range from 1.7 to -13 and therefore should generally have good aqueous solubility. Since most compounds are bis-acids, with pKa values ranging from 6.5 to -1.7, most should have poor cell permeability. These characteristics are beneficial for IV administration and also for engagement of the molecular target PF4 which is a blood protein.
  • the samples were then analyzed using photon correlation spectroscopy on a DynaPro® DLS instrument and Dynamics® software (V6.7.6; Microsoft) to obtain correlation function.
  • the results of the DLS analysis indicate the percentage of ULCs and small PF4 particles.
  • Data for the small particles a population of particles with a mean hydrodynamic diameter of about 1 nm
  • data for the large particles a population of particles with a mean hydrodynamic diameter of about 300 nm
  • Data are expressed as the percent of total intensity from each measurement, the mean of 10 measurements, and representative of two such experiments. Larger amounts of heparin result in the disruption of larger particles, i.e., ULCs, demonstrated by the presence of virtually all of the PF4 as small particles (data not shown).
  • ULC were formed as described above, with the exception that the PF4 was incubated with heparin for 30 min. Certain compounds (1 mM) were then added to wells pre- coated with KKO (monoclonal hit like antibody). These solutions were then incubated in the KKO wells overnight at 37oC. Antibody binding was detected by adding HRP (horseradish peroxicase)-conjugated sheep polyclonal anti-human PF4 antibody and developed with TMB substrate.
  • HRP horseradish peroxicase
  • platelet rich plasma from healthy donors was incubated with 0.5 ⁇ L 14 C-5-hydroxytryptamine creatinine sulfate (GE Life Sciences) per milliliter of PRP for 20 min at 37°C to produce 14 C-labeled platelets. Serotonin uptake is inhibited by adding 1 mmol/mL imipramine (Sigma-Aldrich) to the PRP.
  • the radiolabeled platelets are mixed with KKO (170 ⁇ g/mL) or with known platelet-activating HIT plasma in the absence (buffer control) or presence of a selected PF4 antagonist compound of Table 1 (2.5 – 3.0 mM).
  • Negative and positive controls contain sera from patients previously known to have negative or positive serotonin release, respectively.
  • the assay was performed in the presence of heparin (1.0 U/mL), i.e., heparin added to the positive control serum, and in the absence of heparin (background). The percent release of serotonin is calculated for all conditions. Negative controls without antibody were studied in parallel. 14 C-5-hydroxytryptamine released from platelets was measured by scintillation counter. Data are expressed as % maximal release of radioactivity with release by the positive control plasma and 1.0 U/mL heparin defined as 100%. Compounds that inhibit platelet activation by KKO are measured by the serotonin release assay. (Rauova, L.
  • ADMET absorption, distribution, metabolism, excretion and toxicity
  • Fc ⁇ RIIA transgenic murine blood was drawn from inferior vena cava under anesthesia into a 1 ml syringe with 10% volume of acid citrate dextrose (ACD).
  • Platelet pellet was resuspended in Tyrode buffer with 0.02 U/ml apyrase at 2 x 108/ml concentration at 37°C.
  • PF4 antagonists are diluted in tyrode buffer, and are incubated with 10 ⁇ g/mL human PF4 purified protein at room temperature for 30 min.
  • the PF4-drug complex is then incubated with 1 ⁇ 10 8 /mL platelets with 1mM CaCl 2 , 0.1% BSA at 37°C for 10 min.40 ⁇ g/mL KKO and 10 ⁇ g/mL anti-CD62P (P-selectin) antibodies are added into the reaction.1 mL ice-cold PBS is added to each reaction after 5 min of incubation. Labelled platelets are then analyzed by a BD Accuri C6 flow cytometer. The results are represented by both the mean fluorescence intensity and by the percentage of positive platelets.
  • FIG. 5 and 6 show effect of PF4 concentration, a combination of PF4 and heparin on P-selectin levels in human platelets.
  • each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. [0312]
  • the terms “a,” “an,” “the” and similar referents used in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

Abstract

The present application provides a compound of Formula (II): or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3 and R4 are described herein. The methods of using these compounds to inhibit tetramerization of PF4 and to treat the associated diseases and conditions, such as heparin-induced thrombocytopenia and thrombosis (HITT) and vaccine-induced immune thrombotic thrombocytopenia (VITT), methods of making these compounds, and pharmaceutical compositions containing these compounds are also disclosed.

Description

SMALL MOLECULE ANTAGONISTS OF PF4 CROSS REFERENCE TO RELATED APPLICATIONS [0001] The present application claims the benefit of U.S. Provisional patent applications 63/250,020 filed September 29, 2021 and 63/284,444 filed November 30, 2021, the entire contents of both of which are incorporated by reference herein. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] This invention was made with government support under Grant Nos. R41HL123126- 01 and R42HL123126-02 awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD [0003] This invention relates to inhibitors of PF4 tetramerization useful in treating, for example, heparin induced thrombocytopenia (HIT) or heparin induced thrombocytopenia and thrombosis (HITT). BACKGROUND [0004] Heparin-induced thrombocytopenia and thrombosis (HITT) is a serious complication of heparin therapy. Heparin is a naturally-occurring anticoagulant that prevents the formation of clots and extension of existing clots within the vasculature. Major medical applications of heparin include dialysis, cardiac catheterization, and cardiopulmonary bypass surgery. However, heparin therapy may lead to a serious complication known as heparin induced thrombocytopenia (HIT). HIT is caused by an immunological reaction that targets platelets leading to a low platelet count (thrombocytopenia). HIT increases the risk of blood clots forming within blood vessels and blocking the flow of blood (thrombosis), referred to as HITT when thrombosis occurs. HITT develops in approximately 1-3% of patients treated with heparin for 5-10 days. Affected individuals have a 20-50% risk of developing new thromboembolic events, a mortality rate of about 20%, and an additional ~10% of patients require amputations or suffer other major morbidity. The rate of occurrence of HITT is about 10-20 cases/yr/hospital, and the patients with this condition are not adequately treated by existing therapies. [0005] Despite the introduction of low molecular weight heparins (LMWH) and the synthetic pentasaccharide fondaparinux, HITT continues to be a significant medical problem. This is likely due to the fact that heparin remains the anticoagulant of choice for many patients (such as patients undergoing cardiopulmonary bypass or percutaneous coronary intervention, at high risk for bleeding, or with renal failure). HITT occurs even after treatment with LMWHs, although at a reduced rate compared to unfractionated heparin in some patients. As a large number of hospitalized patients are exposed to heparin, HITT is a major treatment-induced cause of morbidity and mortality in this patient population. SUMMARY [0006] The present application provides compounds that inhibit platelet activation by directly inhibiting tetramerization of platelet factor 4 (PF4), and may be useful in treating diseases and conditions in which increased blood clotting is indicated. Suitable examples of such diseases include HIT and HITT. The compounds of the present application have high potency at a µM level, little or no activity against other chemokines, excellent ADMET properties including microsome stability and little or no cytochrome P450 inhibition, little or no cytotoxicity, little or no hERG inhibition, good aqueous solubility and PK parameters suitable for intravenous (IV) administration, such as moderate-long half-life, and low clearance and volume of distribution. Exemplary embodiments of such compounds are described below. [0007] In a first general aspect, the present disclosure provides a compound of Formula (II):
Figure imgf000003_0001
or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3 and R4 are as described herein. [0008] In a second general aspect, the present disclosure provides a pharmaceutical composition comprising a compound of Formula (II), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. [0009] In a third general aspect, the present disclosure provides a method of: • preventing formation of platelet factor-4 (PF4) tetramers in a subject; and/or • disrupting platelet factor-4 (PF4) tetramers in a subject; and/or • preventing formation of an ultra-large complex (ULC) comprising a PF4 tetramer and a glycosaminoglycan (GAG) in a subject; and/or • inhibiting ULC-antibody complex binding to a FcγRIIa receptor on a platelet in a subject; and/or • inhibiting platelet aggregation in a subject, and/or • increasing high density lipoproteins in a subject; and/or • modulating clotting or hemostasis in a subject; and/or • correcting a platelet imbalance in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the second general aspect. [0010] In a fourth general aspect, the present disclosure provides a method of treating or preventing a disease or condition selected from: • heparin induced thrombocytopenia and thrombosis (HITT); • a thrombotic complication of HITT; • heparin induced thrombocytopenia (HIT); • vaccine-induced immune thrombotic thrombocytopenia (VITT); • atherosclerosis or atherosclerotic vascular disease; • decrease in platelet production; • inflammation or an inflammatory disease; • antiphospholipid syndrome; • platelet imbalance or insufficiency; and • a clotting or hemostasis disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula (II), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the second general aspect. [0011] Certain implementations of the first, second, third, and fourth general aspects are described herein. [0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. Methods and materials are described herein for use in the present application; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. [0013] Other features and advantages of the present application will be apparent from the following detailed description and figures, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS [0014] FIG. 1A is a diagram showing formation of Ultra Large Complex (ULC) of PF4 tetramers and heparin. The figure shows that formation of ULC requires tetramerization of PF4. [0015] FIG.1B is a diagram showing that inhibitors of tetramerization inhibit ULC formation. [0016] FIG. 2 is a diagram showing simplified mechanism of heparin-induced thrombocytopenia and thrombosis (HITT) pathophysiology. [0017] FIG.3 is an image showing PF4 dimer with a hypothesized site of binding for PF4 tetramerization antagonists. [0018] FIG.4A is a scheme showing equilibrium between tautomeric forms of a compound containing an indane-1,3-dione moiety substituted in the 2-position. [0019] FIG.4B is a scheme showing equilibrium between tautomeric forms of a compound containing an indane-1,3-dione moiety that is unsubstituted in the 2-position. [0020] FIG. 5 is a graph showing difference in P-selectin expression in human platelets treated with PF4 along and with PF4 and heparin. [0021] FIG. 6 is a graph showing difference in P-selectin expression in human platelets treated with PF4 at 37 µg/mL and with PF4 at 65 µg/mL. DETAILED DESCRIPTION [0022] The clinical manifestations of HITT are caused by antibodies that recognize a complex composed of heparin and tetramers of platelet factor 4 (PF4). PF4 is a 70 amino acid, lysine-rich 7.8 kDa platelet-specific protein that belongs to the CXC (or beta) chemokine subfamily. PF4 is synthesized by megakaryocytes and comprises 2-3% of the total released protein in mature platelets. PF4 exists as a tetramer in the α-granules of platelets and is secreted in high concentrations when platelets are activated. PF4 tetramers bind avidly to glycosaminoglycans (GAGs). The interaction of PF4 with GAGs, including heparin, contributes to the pathogenesis of HITT with the formation of ultra large complexes of PF4 tetramers and heparin (ULCs) representing the major antigen recognized by pathogenic HITT antibodies. A transgenic mouse model of HITT demonstrates that heparin, PF4 (forming ULC), anti- heparin/PF4 antibody, and the platelet receptor FcγRIIa are necessary and sufficient to recapitulate the salient features of HITT in vivo. Treatment of patients with heparin is thought to favor the formation of the ULCs, placing these patients at risk for HITT. [0023] A simplified mechanism of HITT pathophysiology is shown in Figure 2, and demonstrates the feed-forward nature of this disorder. Specifically, antibody recognition of ULC leads to platelet activation, releasing more PF4, which can form additional ULC to be recognized by antibody. The compounds of the present application inhibit cellular activation by limiting the ULC formation. Referring to Figure 2, in step (1) PF4 released from activated platelets as a tetramer forms a complex (ULC) with heparin which has been administered to a patient as an anticoagulant; in step (2) pathogenic antibodies bind to the complex of heparin and PF4 tetramer; in step (3) an antibody-decorated heparin-PF4 complex binds to a platelet via its FcγRIIa receptor; and in step (4) crosslinking of FcγRIIa leads to platelet activation and release of additional PF4, which can bind to heparin and feed the pathogenic cycle. [0024] No specific treatments for heparin-induced thrombosis currently exist. For example, current treatment for HITT relies on removal of all heparin exposure from patients with suspected HITT and administration of a non-heparin alternative anticoagulant, typically a direct thrombin inhibitor, which may carry a significant risk of bleeding. Hence, despite discontinued heparin, the patients remain at significant risk for thrombosis and death. [0025] Also disclosed herein are methods of treated thrombosis induced by vaccines. Vaccine-induced immune thrombotic thrombocytopenia (VITT) is induced in otherwise healthy individuals after an adenovirus-based vaccine and is characterized by development of thrombocytopenia and thrombosis in atypical locations (including, but not limited to, the cerebral and/or splanchic veins) within weeks of receiving a vaccination. VITT has most recently been identified in subjects receiving adenvirus-based SARS-CoV-2 vaccines, which involves, in part, antibodies directed toward PF4 and/or PF4-heparin complexes. [0026] Accordingly, the present application provides compounds that directly target and intervene in the pathophysiology of thrombosis, and limit and/or prevent the complications of this condition and related diseases. Exemplary embodiments of the compounds, and methods of making and using these compositions, are described below. Definitions [0027] The term "PF4" as used herein refers to platelet factor 4 which is a 70 amino acid, lysine-rich, 7.8 kDa platelet-specific protein that belongs to the CXC (or beta) chemokine subfamily, in which the first two of the four conserved cysteine residues are separated by one amino acid residue. In one embodiment, PF4 is naturally occurring, i.e., wild-type. In other embodiments, PF4 may be synthesized by recombinant or chemical methods. The term PF4 also refers to mutations thereof in which one or more of the amino acids is replaced with a different amino acid. Examples of PF4 mutations are described in International Patent Publication No. WO 02/006300 and the inventors’ prior publication WO2013/142328, which are incorporated herein by reference. [0028] As used herein, the term “compound” is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures named or depicted. Compounds herein identified by name or structure as one particular tautomeric form are intended to include other tautomeric forms unless otherwise specified. [0029] As used herein, the term "tautomer" refers to compounds which are capable of existing in a state of equilibrium between two isomeric forms. Such compounds may differ in the bond connecting two atoms or groups and the position of these atoms or groups in the compound. Suitable examples of an equilibrium between the tautomeric forms are depicted in Figures 4A and 4B. Referring to Figure 4A, an indane-1,3-dione moiety having an R(C=O)- substituent in the 2-position may exist as an equilibrium between forms A, B, C, D and E. In this example, the R- group may correspond to C1-6 alkoxy, C1-6 alkyl or an NH(Ra1) fragment, as in any one of the substituents R1-R4 in a compound of Formula (II) described herein. Based on the analysis of literature and NMR data presented herein, a compound containing an R(C=O)- substituent as depicted in Figure 4A most likely exists in solution as an equilibrium between forms E and D. See, e.g., Liepiņš, E. et al. Magnetic Resonance in Chemistry 1989, 27, 907; Song, J. et al. Organic Letters 2007, 9, 4307; Paul, B. K.; Guchhait, N. Computational and Theoretical Chemistry 2013, 1012, 2, which are incorporated herein by reference in their entirety. Referring to Figure 4B, an indane-1,3-dione moiety that is unsubstituted in the 2- position may exist as an equilibrium between forms F, G and H. This is the case of a compound of Formula (II) described herein when either R1 and R2 are both H, or R3 and R4 are both H, or R1-R4 are all H. Based on the analysis of NMR data, form F is favored in solution of such a compound. [0030] As used herein, the term "isomer" refers to structural, geometric and stereo isomers. As the compound of the present application may have one or more chiral centers, it is capable of existing in enantiomeric forms. [0031] As used herein, the phrase “optionally substituted” means unsubstituted or substituted. As used herein, the term “substituted” means that a hydrogen atom is removed and replaced by a substituent. It is to be understood that substitution at a given atom is limited by valency. [0032] As used in the present application, the term “Cn-m alkyl”, employed alone or in combination with other terms, refers to a saturated hydrocarbon group that may be straight- chain (linear) or branched, having n to m carbons. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; higher homologs such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, and the like. In some embodiments, the alkyl group contains from 1 to 6 carbon atoms, from 1 to 4 carbon atoms, from 1 to 3 carbon atoms, or 1 to 2 carbon atoms. [0033] As used in the present application, “Cn-m alkenyl” refers to an alkyl group having one or more double carbon-carbon bonds and having n to m carbons. Example alkenyl groups include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl, and the like. In some embodiments, the alkenyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms. [0034] As used in the present application, the term “Cn-m alkoxy”, employed alone or in combination with other terms, refers to a group of formula –O-Cn-m alkyl. Examplary alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (for example, n-propoxy and isopropoxy), butoxy (for example, n-butoxy and tert-butoxy), and the like. In some embodiments, the alkoxy group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. [0035] As used in the present application, “halo” refers to a halogen atom such as F, Cl, Br, or I. In some embodiments, a halo is F, Cl, or Br. In other embodiments, halo is F, Cl, or I. In other embodiments, halo is F, I, or Br. [0036] As used in the present application, the term “Cn-m haloalkyl”, employed alone or in combination with other terms, refers to an alkyl group having from one halogen atom to 2s+1 halogen atoms which may be the same or different, where “s” is the number of carbon atoms in the alkyl group, win the present application the alkyl group has n to m carbon atoms. In some embodiments, the haloalkyl group is fluorinated only. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. [0037] As used in the present application, “Cn-m haloalkoxy” refers to a group of formula – O-haloalkyl having n to m carbon atoms. An example haloalkoxy group is OCF3. In some embodiments, the haloalkoxy group is fluorinated only. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. [0038] As used in the present application, “cycloalkyl” refers to non-aromatic saturated or unsaturated cyclic hydrocarbons including cyclized alkyl and/or alkenyl groups. Cycloalkyl groups can include mono- or polycyclic (for example, having 2, 3 or 4 fused rings) groups and spirocycles. Ring-forming carbon atoms of a cycloalkyl group can be optionally substituted by oxo or sulfido (for example, C(O) or C(S)). Also included in the definition of cycloalkyl are moieties that have one or more aromatic rings fused (that is, having a bond in common with) to the non-aromatic cyclic hydrocarbon, for example, benzo or thienyl derivatives of cyclopentane, cyclohexane, and the like. A cycloalkyl group containing a fused aromatic ring can be attached through any ring-forming atom including a ring-forming atom of the fused aromatic ring. Cycloalkyl groups can have 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 ring-forming atoms. In some embodiments, the cycloalkyl is a 3-12 membered monocyclic or bicyclic cycloalkyl. In some embodiments, the cycloalkyl is a C3-7 monocyclic cycloalkyl. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcarnyl, cyclooctyl, cyclooctenyl, and the like. In some embodiments, cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, or cyclooctenyl. In some embodiments, the cycloalkyl is a cyclooctenyl ring fused with 1 or 2 benzene rings. In some embodiments, the cycloalkyl is a 3-8 membered or 3-7 membered monocyclic cycloalkyl group (for example, C3-8 or C3-7 cycloalkyl). In some embodiments, the cycloalkyl is a 8-12-membered bicyclic cycloalkyl. In some embodiments, the cycloalkyl is a 8-16-membered bicyclic or tricyclic cycloalkyl (for example, C8-16 cycloalkyl). In some embodiments, the cycloalkyl is unsaturated cyclic hydrocarbon group (that is, the cycloalkyl contains at least one double bond). [0039] As used herein, “heteroalkyl” refers to branched or unbranched heteroalkyls having one or more heteroatoms selected, independently, from O, N, or S. Examples of heteroalkyls include, but are not limited to, CH 2CH2OCH2CH2OCH2CH2OCH3, CH2CH2OCH2CH2OCH3, CH2NHCH2CH2OCH2CH2OCH3, and CH2CH2CH2NH2, and the like. [0040] As used herein, “heterocycloalkyl” or “aliphatic heterocycle” refers to non-aromatic saturated or unsaturated monocyclic or polycyclic heterocycles having one or more ring- forming heteroatoms selected from O, N, or S. Included in heterocycloalkyl are monocyclic 4- , 5-, 6-, 7-, 8-, 9- or 10-membered heterocycloalkyl groups. Heterocycloalkyl groups can also include spirocycles. Example heterocycloalkyl groups include pyrrolidin-2-one, 1,3- isoxazolidin-2-one, pyranyl, tetrahydropuran, oxetanyl, azetidinyl, morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, azepanyl, benzazapene, and the like. Ring-forming carbon atoms and heteroatoms of a heterocycloalkyl group can be optionally substituted by oxo or sulfido groups (e.g., C(O), S(O), C(S), or S(O)2, etc.). The heterocycloalkyl group can be attached through a ring-forming carbon atom or a ring-forming heteroatom. In some embodiments, the heterocycloalkyl group contains 0 to 3 double bonds. In some embodiments, the heterocycloalkyl group contains 0 to 2 double bonds. In some embodiments, the heterocycloalkyl group is unsaturated (i.e., the heterocycloalkyl contains at least one double bond). Also included in the definition of heterocycloalkyl are moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the non-aromatic heterocycle, for example, benzo or thienyl derivatives of piperidine, morpholine, azepine, etc. A heterocycloalkyl group containing a fused aromatic ring can be attached through any ring-forming atom including a ring-forming atom of the fused aromatic ring. In some embodiments, the heterocycloalkyl is a monocyclic 4-6 membered heterocycloalkyl having 1 or 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur and having one or more oxidized ring members. In some embodiments, the heterocycloalkyl is a monocyclic or bicyclic 4-10 membered heterocycloalkyl having 1, 2, 3, or 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur and having one or more oxidized ring members. In some embodiments, the heterocycloalkyl is a 8-12-membered heterocycloalkyl (e.g., bicyclic heterocycloalkyl). In some embodiments, the heterocycloalkyl is a 8-16-membered heterocycloalkyl (e.g., bicyclic or tricyclic heterocycloalkyl). In some embodiments, the 8-12 membered bicyclic heterocycloalkyl is a 8-12 membered fused heterocycloalkylaryl group or a 8-12 membered fused heterocycloalkylheteroaryl group. In some embodiments, the heterocycloalkyl is a 9-12 membered bicyclic heterocycloalkyl. In some embodiments, the 9-10 membered bicyclic heterocycloalkyl is a 9-10 membered fused heterocycloalkylaryl group or a 9-10 membered fused heterocycloalkylheteroaryl group. The term “heterocycloalkylene” refers to a divalent heterocycloalkyl linking group. [0041] As used in the present application, “heteroaryl” refers to a monocyclic or polycyclic aromatic heterocycle having at least one heteroatom ring member selected from sulfur, oxygen, and nitrogen. In some embodiments, the heteroaryl ring has 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, any ring-forming N in a heteroaryl moiety can be an N-oxide. In some embodiments, the heteroaryl is a 5-10 membered monocyclic or bicyclic heteroaryl having 1, 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl is a 5-6 membered monocyclic heteroaryl having 1 or 2 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl is a five-membered or six-membered heteroaryl ring. A five- membered heteroaryl ring is a heteroaryl with a ring having five ring atoms win the present application one or more (for example, 1, 2, or 3) ring atoms are independently selected from N, O, and S. Exemplary five-membered heteroaryls are thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3- thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4- triazolyl, 1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl. A six-membered heteroaryl ring is a heteroaryl with a ring having six ring atoms win the present application one or more (for example, 1, 2, or 3) ring atoms are independently selected from N, O, and S. Exemplary six- membered heteroaryls are pyridyl, pyrazinyl, pyrimidinyl, triazinyl and pyridazinyl. [0042] The term “aromatic” refers to a carbocycle or heterocycle having one or more polyunsaturated rings having aromatic character (that is, having (4n + 2) delocalized π (pi) electrons where n is an integer). [0043] The term “n-membered” where n is an integer, typically describes the number of ring- forming atoms in a moiety where the number of ring-forming atoms is n. For example, piperidinyl is an example of a 6-membered heterocycloalkyl ring, pyrazolyl is an example of a 5-membered heteroaryl ring, pyridyl is an example of a 6-membered heteroaryl ring, and 1,2,3,4-tetrahydro-naphthalene is an example of a 10-membered cycloalkyl group. [0044] The term “aryl,” employed alone or in combination with other terms, refers to an aromatic hydrocarbon group, which may be monocyclic or polycyclic (for example, having 2, 3 or 4 fused rings). The term “Cn-m aryl” refers to an aryl group having from n to m ring carbon atoms. Aryl groups include, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl and the like. In some embodiments, aryl groups have from 6 to about 20 carbon atoms, from 6 to about 15 carbon atoms, or from 6 to about 10 carbon atoms. In some embodiments, the aryl group is phenyl. [0045] As used in the present application, the term “Cn-m alkoxycarbonyl” refers to a group of formula -C(O)O-alkyl, win the present application the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Examples of alkoxycarbonyl groups include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl (for example, n-propoxycarbonyl and isopropoxycarbonyl), butoxycarbonyl (for example, n-butoxycarbonyl and tert-butoxycarbonyl), and the like. [0046] As used in the present application, the term “Cn-m alkylcarbonyl” refers to a group of formula -C(O)-alkyl, win the present application the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Examples of alkylcarbonyl groups include, but are not limited to, methylcarbonyl, ethylcarbonyl, propylcarbonyl (for example, n-propylcarbonyl and isopropylcarbonyl), butylcarbonyl (for example, n-butylcarbonyl and tert-butylcarbonyl), and the like. [0047] As used in the present application, the term “carboxy” or “carboxyl” refers to a - C(O)OH group. [0048] The terms “pharmaceutical” and “pharmaceutically acceptable” are employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. [0049] As used herein, the term “cell” is meant to refer to a cell that is in vitro, ex vivo, or in vivo. In some embodiments, an ex vivo cell can be part of a tissue sample excised from an organism such as a mammal. In some embodiments, an in vitro cell can be a cell in a cell culture. In some embodiments, an in vivo cell is a cell living in an organism such as a mammal. [0050] As used herein, the term “individual”, “patient”, or “subject” used interchangeably, refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans. [0051] As used herein, the phrase “effective amount” or “therapeutically effective amount” refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician. [0052] As used herein the term “treating” or “treatment” refers to 1) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology), or 2) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology). [0053] As used herein, the term “preventing” or “prevention” of a disease, condition or disorder refers to decreasing the risk of occurrence of the disease, condition or disorder in a subject or group of subjects (e.g., a subject or group of subjects predisposed to or susceptible to the disease, condition or disorder). In some embodiments, preventing a disease, condition or disorder refers to decreasing the possibility of acquiring the disease, condition or disorder and/or its associated symptoms. In some embodiments, preventing a disease, condition or disorder refers to completely or almost completely stopping the disease, condition or disorder from occurring. [0054] As used herein, in such methods the term "biological sample" refers to a body fluid or tissue. The body fluid can include, without limitation, whole blood, serum, plasma, peripheral blood, synovial fluid, cerebrospinal fluid, saliva, urine, semen, or other fluid secretion. The term "tissue" can include, without limitation, bone marrow and lymph node, as well as samples of other tissues. Therapeutic compounds [0055] The present application provides, inter alia, a compound of Formula (II):
Figure imgf000012_0001
or a pharmaceutically acceptable salt thereof, wherein: R1 and R3 are each independently selected from H, C1-6 alkylcarbonyl, C(O)NH(Ra1), and C(O)Cy4, wherein C1-6 alkyl in the C1-6 alkylcarbonyl is optionally substituted with 1, 2, or 3 substituents independently selected from Cy4, C6-10 aryloxy, C1-3 alkoxy, -NH(C1-6 alkoxycarbonyl), and –NH(C1-6 alkylcarbonyl), wherin C1-6 alkoxy is optionally substituted with C6-10 aryl; R2 and R4 are each independently selected from H and C1-6 alkylcarbonyl; each Ra1 is independently selected from C1-6 alkyl, C1-6 alkenyl, Cy1, C1-6 alkoxycarbonyl, and S(O)2Ra2, wherein the C1-6 alkyl is optionally substituted with 1, 2, or 3 substituents independently selected from Cy2, carboxyl, C1-3 alkoxycarbonyl, C1-3 alkoxy, and C1-3 haloalkoxy wherein the C1-3 alkoxy is optionally substituted with C1-3 alkoxy; each Cy1 is independently selected form C3-10 cycloalkyl, C6-10 aryl, and 5-10 membered heteroaryl, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from halo, C1-3 alkyl, C1-3 alkoxy and Cy3; each Cy2 is independently selected from C6-10 aryl and 5-10 membered heteroaryl, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from halo, C1-3 alkyl, and C1-3 haloalkoxy; each Cy3 is independently selected form C6-10 aryl and 5-10 membered heteroaryl; each Cy4 is independently selected from C6-10 aryl, C3-10 cycloalkyl, 4-10 membered heterocycloalkyl, and 5-10 membered heteroaryl, each of which is optionally substituted with C1-6 alkoxycarbonyl; each Ra2 is C6-10 aryl, optionally substituted with C1-3 alkyl. [0056] In some embodiments, R1 is H. In other embodiments, R1 is C1-6 alkylcarbonyl (e.g., methylcarbonyl). In yet other embodiments, R1 is C(O)NH(Ra1). [0057] In some embodiments, R2 is H. In other embodiments, R2 is C1-6 alkylcarbonyl. [0058] In some embodiments, R3 is H. In other embodiments, R3 is C1-6 alkylcarbonyl (e.g., methylcarbonyl). In yet other embodiments, R3 is C(O)NH(Ra1). [0059] In some embodiments, R1 and R3 are each independently selected from C1-6 alkylcarbonyl and C(O)NH(Ra1). [0060] In some embodiments, R4 is H. In other embodiments, R4 is C1-6 alkylcarbonyl. [0061] In some embodiments, R2 and R4 are each H; and R1 and R3 are each independently selected from H, C1-6 alkylcarbonyl, and C(O)NH(Ra1). [0062] In some embodiments, R2 and R4 are each H; and R1 and R3 are each independently selected from C1-6 alkylcarbonyl, and C(O)NH(Ra1). [0063] In some embodiments, R1 and R3 are each independently selected from H and C1-6 alkylcarbonyl; and R2 and R4 are each C1-6 alkylcarbonyl. [0064] In some embodiments, R1 and R3 are each H; and R2 and R4 are each C1-6 alkylcarbonyl. [0065] In some embodiments, R1, R2, R3 and R4 are each independently selected from H and C1-6 alkylcarbonyl. [0066] In some embodiments, R1, R2, R3 and R4 are each H. [0067] In some embodiments, R1 and R3 are each C1-6 alkylcarbonyl. [0068] In some embodiments, R1 and R3 are each C(O)NH(Ra1). [0069] In some embodiments, R2 and R4 are each H; and R1 and R3 are each C(O)NH(Ra1). [0070] In some embodiments, Ra1 is C1-6 alkyl, optionally substituted with 1 or 2 substituents independently selected from Cy2, carboxyl, C1-3 alkoxycarbonyl, C1-3 alkoxy, and C1-3 haloalkoxy wherein the C1-3 alkoxy is optionally substituted with C1-3 alkoxy. [0071] In some embodiments, Ra1 is C1-6 alkyl, optionally substituted with 1 or 2 substituents independently selected from Cy2, carboxyl, and C1-3 alkoxycarbonyl. [0072] In some embodiments, Ra1 is methyl, ethyl, propyl, isopropyl and sec-butyl, each of which is optionally substituted with 1 or 2 substituents independently selected from Cy2, carboxyl, C1-3 alkoxycarbonyl, C1-3 alkoxy, and C1-3 haloalkoxy wherein the C1-3 alkoxy is optionally substituted with C1-3 alkoxy. [0073] In some embodiments, Ra1 is methyl, ethyl, propyl, isopropyl and sec-butyl, each of which is optionally substituted with 1 or 2 substituents independently selected from Cy2, carboxyl, and C1-3 alkoxycarbonyl. [0074] In some embodiments, Ra1 is methyl, ethyl and propyl, each of which is optionally substituted with 1 or 2 substituents independently selected from Cy2, carboxyl, and C1-3 alkoxycarbonyl. [0075] In some embodiments, Ra1 is C1-6 alkenyl. [0076] In some embodiments, Ra1 is Cy1, optionally substituted with 1 or 2 substituents independently selected from halo, C1-3 alkyl, C1-3 alkoxy and Cy3. [0077] In some embodiments, Ra1 is Cy1, optionally substituted with 1 or 2 substituents independently selected from C1-3 alkyl, C1-3 alkoxy and Cy3. [0078] In some embodiments, Ra1 is selected from phenyl, dihydrobenzodioxinyl, pyridinyl and cyclopropyl, each of which is optionally substituted with 1 or 2 substituents independently selected from C1-3 alkyl, C1-3 alkoxy and Cy3. [0079] In some embodiments, Ra1 is phenyl, dihydrobenzodioxinyl and cyclopropyl, each of which is optionally substituted with 1 or 2 substituents independently selected from C1-3 alkyl, C1-3 alkoxy and Cy3. [0080] In some embodiments, Ra1 is S(O)2Ra2. [0081] In some embodiments, Ra2 is phenyl, optionally substituted with C1-3 alkyl. [0082] In some embodiments, Ra2 is phenyl, optionally substituted with methyl. [0083] In some embodiments, each Ra1 is independently selected from C1-6 alkyl, C1-6 alkenyl, Cy1, C1-6 alkoxycarbonyl, and S(O)2Ra2, wherein the C1-6 alkyl is optionally substituted with 1, 2, or 3 substituents independently selected from Cy2, carboxyl and C1-3 alkoxycarbonyl. [0084] In some embodiments, each Ra1 is independently selected from C1-6 alkyl, C1-6 alkenyl, Cy1, and S(O)2Ra2, wherein the C1-6 alkyl is optionally substituted with 1, 2, or 3 substituents independently selected from Cy2, carboxyl, C1-3 alkoxycarbonyl. [0085] In some embodiments, each Ra1 is independently selected from propyl, isopropyl, sec-butyl, allyl, phenyl, tosyl, ethoxycarbonyl, methoxyphenyl, nitrophenyl, methylphenyl, ethoxyphenyl, dihydrobenzodioxinyl, dichloropyridinyl, benzyl, fluorobenzyl, (pyrimidin-2- yl)phenyl, cyclopropyl, phenylcyclopropyl, phenylethyl, (trifluoromethoxyphenyl)ethyl, (ethoxycarbonyl)methyl, (ethoxycarbonyl)ethyl, phenylpropyl, (fluorophenyl)ethyl, (chlorophenyl)ethyl, (methylphenyl)ethyl, furanylmethyl, benzodioxolylmethyl, benzodioxolylethyl, (2-methoxyethoxy)ethyl, (2,2,2-trifluoroethoxy)ethyl, (carboxyl)ethyl, and (carboxyl)methyl. [0086] In some embodiments, each Ra1 is independently selected from propyl, isopropyl, sec-butyl, allyl, phenyl, tosyl, ethoxycarbonyl, methoxyphenyl, methylphenyl, ethoxyphenyl, dihydrobenzodioxinyl, dichloropyridinyl, benzyl, fluorobenzyl, (pyrimidin-2-yl)phenyl, cyclopropyl, phenylcyclopropyl, phenylethyl, (trifluoromethoxyphenyl)ethyl, (ethoxycarbonyl)methyl, (ethoxycarbonyl)ethyl, phenylpropyl, (fluorophenyl)ethyl, (chlorophenyl)ethyl, (methylphenyl)ethyl, furanylmethyl, benzodioxolylmethyl, benzodioxolylethyl, (carboxyl)ethyl, and (carboxyl)methyl. [0087] In some embodiments, each Ra1 is independently selected from propyl, isopropyl, sec-butyl, allyl, tosyl, methylphenyl, ethoxyphenyl, benzyl, (pyrimidin-2-yl)phenyl, cyclopropyl, phenylcyclopropyl, phenylethyl, (trifluoromethoxyphenyl)ethyl, (ethoxycarbonyl)ethyl, phenylpropyl, (fluorophenyl)ethyl, (chlorophenyl)ethyl, (methylphenyl)ethyl, benzodioxolylethyl, and (carboxyl)ethyl. [0088] In some embodiments, Cy1 is C3-10 cycloalkyl, optionally substituted with 1 or 2 substituents independently selected from C1-3 alkyl, C1-3 alkoxy and Cy3. [0089] In some embodiments, Cy1 is cyclopropyl, optionally substituted with 1 or 2 substituents independently selected from C1-3 alkyl, C1-3 alkoxy and Cy3. [0090] In some embodiments, Cy1 is C6-10 aryl, optionally substituted with 1 or 2 substituents independently selected from C1-3 alkyl, C1-3 alkoxy and Cy3. [0091] In some embodiments, Cy1 is selected from phenyl, dihydrobenzodioxinyl, and cyclopropyl, each of which is optionally substituted with 1 or 2 substituents independently selected from C1-3 alkyl, C1-3 alkoxy and Cy3. [0092] In some embodiments, Cy1 is selected from phenyl and cyclopropyl, each of which is optionally substituted with 1 or 2 substituents independently selected from C1-3 alkyl, C1-3 alkoxy and Cy3. [0093] In some embodiments, Cy1 is selected from phenyl and cyclopropyl, each of which is optionally substituted with 1 or 2 substituents independently selected from C1-3 alkyl, C1-3 alkoxy and Cy3. [0094] In some embodiments, each Cy1 is independently selected form C3-10 cycloalkyl and C6-10 aryl, each of which is optionally substituted with 1 or 2 substituents independently selected from C1-3 alkyl, C1-3 alkoxy and Cy3. [0095] In some embodiments, each Cy1 is independently selected form phenyl, methoxyphenyl, ethoxyphenyl, dihydrobenzodioxinyl, dichloropyridinyl, cyclopropyl, methylphenyl, nitrophenyl, (pyrimidin-2-yl)phenyl, and phenylcyclopropyl. [0096] In some embodiments, each Cy1 is independently selected from ethoxyphenyl, cyclopropyl, methylphenyl, (pyrimidin-2-yl)phenyl, and phenylcyclopropyl. [0097] In some embodiments, Cy2 is 5-10 membered heteroaryl, each of which is optionally substituted with 1 or 2 substituents independently selected from halo, C1-3 alkyl, and C1-3 haloalkoxy. In some aspects of these embodiments, the 5-10 membered heteroaryl is furanyl. [0098] In some embodiments, Cy2 is C6-10 aryl, optionally substituted with 1 or 2 substituents independently selected from halo, C1-3 alkyl, and C1-3 haloalkoxy. [0099] In some embodiments, Cy2 is selected from phenyl and benzodioxolyl, each of which is optionally substituted with 1 or 2 substituents independently selected from halo, C1-3 alkyl, and C1-3 haloalkoxy. [0100] In some embodiments, Cy2 is phenyl, each of which is optionally substituted with 1 or 2 substituents independently selected from halo, C1-3 alkyl, and C1-3 haloalkoxy. [0101] In some embodiments, each Cy2 is independently selected from phenyl, trifluoromethoxyphenyl, fluorophenyl, chlorophenyl, methylphenyl, furanyl, and benzodioxolyl. [0102] In some embodiments, each Cy2 is independently selected from phenyl, trifluoromethoxyphenyl, fluorophenyl, chlorophenyl, methylphenyl, and benzodioxolyl. [0103] In some embodiments, each Cy3 is C6-10 aryl. In some aspects of these embodiments, the C6-10 aryl is phenyl. [0104] In some embodiments, each Cy3 is 5-10 membered heteroaryl. In some aspects of these embodiments, the 5-10 membered heteroaryl is pyrimidinyl. [0105] In some embodiments, each Cy3 is independently selected from phenyl and pyrimidinyl. [0106] In some embodiments: R1 and R3 are each independently selected from H, C1-6 alkylcarbonyl, and C(O)NH(Ra1); R2 and R4 are each independently selected from H and C1-6 alkylcarbonyl; each Ra1 is independently selected from C1-6 alkyl, C1-6 alkenyl, Cy1, and S(O)2Ra2, wherein the C1-6 alkyl is optionally substituted with 1, 2, or 3 substituents independently selected from Cy2, carboxyl and C1-3 alkoxycarbonyl; each Cy1 is independently selected form C3-10 cycloalkyl and C6-10 aryl, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from C1-3 alkyl, 1-3 alkoxy and Cy3; each Cy2 is C6-10 aryl, optionally substituted with 1, 2, or 3 substituents independently selected from halo, C1-3 alkyl, and C1-3 haloalkoxy; each Cy3 is independently selected form C6-10 aryl and 5-10 membered heteroaryl; and each Ra2 is C6-10 aryl, optionally substituted with C1-3 alkyl. [0107] In some embodiments: R1 and R3 are each C(O)NH(Ra1); each Ra1 is independently selected from C1-6 alkyl, C1-6 alkenyl, Cy1, C1-6 alkoxycarbonyl, and S(O)2Ra2, wherein the C1-6 alkyl is optionally substituted with 1, 2, or 3 substituents independently selected from Cy2, carboxyl and C1-3 alkoxycarbonyl; each Cy1 is independently selected form C3-10 cycloalkyl and C6-10 aryl, each of which is optionally substituted with 1 or 2 substituents independently selected from C1-3 alkyl, C1-3 alkoxy and Cy3; and each Cy2 is C6-10 aryl, optionally substituted with 1 or 2 substituents independently selected from halo, C1-3 alkyl, and C1-3 haloalkoxy. [0108] In some embodiments: R1 and R3 are each C(O)NH(Ra1); each Ra1 is independently selected from C1-6 alkyl, C1-6 alkenyl, Cy1, and S(O)2Ra2, wherein the C1-6 alkyl is optionally substituted with 1, 2, or 3 substituents independently selected from Cy2, carboxyl and C1-3 alkoxycarbonyl; each Cy1 is independently selected form C3-10 cycloalkyl and C6-10 aryl, each of which is optionally substituted with 1 or 2 substituents independently selected from C1-3 alkyl, C1-3 alkoxy and Cy3; and each Cy2 is C6-10 aryl, optionally substituted with 1 or 2 substituents independently selected from halo, C1-3 alkyl, and C1-3 haloalkoxy. [0109] In some embodiments, the compound of Formula (II) has Formula (IIa):
Figure imgf000018_0001
or a pharmaceutically acceptable salt thereof. [0110] In some embodiments, Cy4 is C6-10 aryl, optionally substituted with C1-6 alkoxycarbonyl. [0111] In some embodiments, Cy4 is C3-10 cycloalkyl, optionally substituted with C1-6 alkoxycarbonyl. [0112] In some embodiments, Cy4 is 4-10 membered heterocycloalkyl, optionally substituted with C1-6 alkoxycarbonyl. In some aspects of these embodiments, Cy4 is pyrrolidinyl, optionally substituted with (t-butoxy)carbonyl. In other aspects of these embodiments, Cy4 is selected from piperidinyl and pyrrolidinyl, each of which is optionally substituted with (t-butoxy)carbonyl. [0113] In some embodiments, Cy4 is 5-10 membered heteroaryl, optionally substituted with C1-6 alkoxycarbonyl. [0114] In some embodiments, each Cy4 is independently selected from cyclopentyl, pyridinyl, piperidinyl, pyrrolidinyl, and phenyl. In some aspects of these embodiments, the cyclopentyl, pyridinyl, piperidinyl, pyrrolidinyl, or phenyl is optionally substituted with (t-butoxy)carbonyl. [0115] In some embodiments, each Cy4 is independently selected from: cyclopentyl, pyridinyl, piperidinyl, and phenyl. In some aspects of these embodiments, the cyclopentyl, pyridinyl, piperidinyl, or phenyl is optionally substituted with (t-butoxy)carbonyl. [0116] In some embodiments, each Cy4 is independently selected from C3-10 cycloalkyl and 4-10 membered heterocycloalkyl. [0117] In some embodiments: R2 and R4 are each H; and R1 and R3 are each independently selected from C1-6 alkylcarbonyl and C(O)Cy4, wherein C1-6 alkyl in the C1-6 alkylcarbonyl is optionally substituted with 1, 2, or 3 substituents independently selected from Cy4, C6-10 aryloxy, C1-3 alkoxy, -NH(C1-6 alkoxycarbonyl), and –NH(C1-6 alkylcarbonyl), wherin C1-6 alkoxy is optionally substituted with C6-10 aryl. [0118] In some embodiments: R2 and R4 are each H; and R1 and R3 are each independently an C1-6 alkylcarbonyl, wherein C1-6 alkyl in the C1-6 alkylcarbonyl is optionally substituted with 1 or 2 substituents independently selected from Cy4, C6-10 aryloxy, C1-3 alkoxy, -NH(C1-6 alkoxycarbonyl), and –NH(C1-6 alkylcarbonyl), wherin C1-6 alkoxy is optionally substituted with C6-10 aryl. [0119] In some embodiments: R2 and R4 are each H; and R1 and R3 are each independently an C1-6 alkylcarbonyl, wherein C1-6 alkyl in the C1-6 alkylcarbonyl is substituted with 1 or 2 substituents independently selected from Cy4, C6-10 aryloxy, C1-3 alkoxy, -NH(C1-6 alkoxycarbonyl), and –NH(C1-6 alkylcarbonyl), wherin C1-6 alkoxy is optionally substituted with C6-10 aryl. [0120] In some embodiments, each C1-6 alkylcarbonyl is selected from: methylcarbonyl, ethylcarbonyl, and n-propylcarbonyl. In some aspects of these embodiments, the methyl, ethy, and n-propyl are each optionally substituted with 1 or 2 substituents indepenently selected from Cy4, phenoxy, benzoxy, -NHC(O)(t-butoxy), and -NH(acetyl). [0121] In some embodiments: R2 and R4 are each H; and R1 and R3 are each independently selected from: methylcarbonyl, ethylcarbonyl, and n-propylcarbonyl, wherein the methyl, ethy, and n-propyl are each optionally substituted with 1 or 2 substituents indepenently selected from Cy4, phenoxy, benzoxy, -NHC(O)(t-butoxy), and -NH(acetyl). [0122] In some embodiments: R2 and R4 are each H; and R1 and R3 are each independently selected from: methylcarbonyl, ethylcarbonyl, and n-propylcarbonyl, wherein the methyl, ethy, and n-propyl are each substituted with 1 or 2 substituents indepenently selected from Cy4, phenoxy, benzoxy, -NHC(O)(t-butoxy), and -NH(acetyl). [0123] In some embodiments: R2 and R4 are each H; and R1 and R3 are each independently a C1-6 alkylcarbonyl, wherein C1-6 alkyl in the C1-6 alkylcarbonyl is substituted Cy4. [0124] In some embodiments: R2 and R4 are each H; and R1 and R3 are each independently selected from: methylcarbonyl, ethylcarbonyl, and n-propylcarbonyl, wherein the methyl, ethy, and n-propyl are each optionally substituted with Cy4 . [0125] In some embodiments, the compound of Formula (II) has Formula (IIb):
Figure imgf000020_0001
or a pharmaceutically acceptable salt thereof. In some aspects of these embodiments, each Cy4 is independently selected from C3-10 cycloalkyl and 4-10 membered heterocycloalkyl. [0126] In some embodiments, the compound of Formula (II) is not a compound selected from:
Figure imgf000020_0002
[0127] In some embodiments, the compound of Formula (II) is selected from:
Figure imgf000021_0001
or a pharmaceutically acceptable salt thereof. [0128] In some embodiments, the compound of Formula (II) is selected from:
Figure imgf000021_0002
Figure imgf000022_0001
Figure imgf000023_0001
O
Figure imgf000024_0001
Figure imgf000025_0001
. or a pharmaceutically acceptable salt thereof. [0129] In some embodiments, the compound of Formula (II) is selected from:
Figure imgf000025_0002
or a pharmaceutically acceptable salt thereof. [0130] In some embodiments, a salt of a compound of Formula (II) is formed between an acid and a basic group of the compound, such as an amino functional group, or a base and an acidic group of the compound, such as a carboxyl functional group. According to another embodiment, the compound is a pharmaceutically acceptable acid addition salt. [0131] In some embodiments, acids commonly employed to form pharmaceutically acceptable salts of the compounds of Formula (II) include inorganic acids such as hydrogen bisulfide, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid, as well as organic acids such as para-toluenesulfonic acid, salicylic acid, tartaric acid, bitartaric acid, ascorbic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, lactic acid, oxalic acid, para-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid and acetic acid, as well as related inorganic and organic acids. Such pharmaceutically acceptable salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate, xylene sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesu1fonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2- sulfonate, mandelate and other salts. In one embodiment, pharmaceutically acceptable acid addition salts include those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and especially those formed with organic acids such as maleic acid. [0132] In some embodiments, bases commonly employed to form pharmaceutically acceptable salts of the compounds of Formula (II) include hydroxides of alkali metals, including sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, organic amines such as unsubstituted or hydroxyl-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-(C1-C6)-alkylamine), such as N,N-dimethyl-N-(2- hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; morpholine; thiomorpholine; piperidine; pyrrolidine; and amino acids such as arginine, lysine, and the like. [0133] In some embodiments, the compounds of Formula (II), or pharmaceutically acceptable salts thereof, are substantially isolated. Methods of making the compounds [0134] Compounds of Formula (II), including salts thereof, can be prepared using known organic synthesis techniques and can be synthesized according to any of numerous possible synthetic routes. For example, the compounds described herein may be prepared using methods and procedures similar to those of Examples 39-64 and 197-208 herein. A person skilled in the art knows how to select and implement appropriate synthetic protocols, and appreciates that the processes described are not the exclusive means by which compounds provided herein may be synthesized, and that a broad repertoire of synthetic organic reactions is available to be potentially employed in synthesizing compounds provided herein. [0135] In some embodiments, any one of the compounds of Formula (II) may be prepared according to the synthetic routes outlined in Scheme 1, using methods and procedures similar to those of Examples 39-64 and 197-208. [0136] Referring to Scheme 1, the R- group may correspond to C1-6 alkoxy, C1-6 alkyl or an NH(Ra1) fragment, as in any one of the substituents R1-R4 in a compound of Formula (II) described herein. [0137] Suitable synthetic methods of starting materials, intermediates and products may be identified by reference to the literature, including reference sources such as: Advances in Heterocyclic Chemistry, Vols.1-107 (Elsevier, 1963-2012); Journal of Heterocyclic Chemistry Vols. 1-49 (Journal of Heterocyclic Chemistry, 1964-2012); Carreira, et al. (Ed.) Science of Synthesis, Vols.1-48 (2001-2010) and Knowledge Updates KU2010/1-4; 2011/1-4; 2012/1-2 (Thieme, 2001-2012); Katritzky, et al. (Ed.) Comprehensive Organic Functional Group Transformations, (Pergamon Press, 1996); Katritzky et al. (Ed.); Comprehensive Organic Functional Group Transformations II (Elsevier, 2nd Edition, 2004); Katritzky et al. (Ed.), Comprehensive Heterocyclic Chemistry (Pergamon Press, 1984); Katritzky et al., Comprehensive Heterocyclic Chemistry II, (Pergamon Press, 1996); Smith et al., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 6th Ed. (Wiley, 2007); Trost et al. (Ed.), Comprehensive Organic Synthesis (Pergamon Press, 1991). [0138] In some embodiments, the compound of Formula (II) may be prepared according to the methods and procedures similar to those described in Larsen, B.J. et al., Tetrahedron 2018, 2762-2768, which is incorporated herein by reference in its entirety. [0139] The reactions for preparing the compounds provided herein can be carried out in suitable solvents which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially non-reactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out, e.g., temperatures which can range from the solvent's freezing temperature to the solvent's boiling temperature. A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected by the skilled artisan.
Scheme 1
Figure imgf000028_0001
[0140] Preparation of the compounds provided herein can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in P. G. M. Wuts and T. W. Greene, Protective Groups in Organic Synthesis, 4th Ed., Wiley & Sons, Inc., New York (2006). Methods of use [0141] Disruption of the functionally-active PF4 tetrameric state to form functionally-inactive monomers and dimers also inhibits PF4 function. To form a ULC, PF4 must be in the tetrameric state. As schematically shown in Figure 1, inhibition of formation of a PF4 tetramer (Figure 1B) consequently inhibits the formation of the ULC with heparin. [0142] Although PF4 crystallizes as a tetramer, in solution it exists in a dynamic equilibrium between monomeric, dimeric and tetrameric forms Figure 1). As such, the relative abundance of the oligomeric form of PF4 can be influenced by shifting this equilibrium. Given that the salt bridges between Glu28 and Lys50 are predicted to stabilize the tetrameric form of PF4, Lys50 was mutated to glutamic acid in an attempt to disrupt tetramer formation. PF4 with this mutation (K50E) readily forms dimers, but not tetramers. Importantly, ULCs are not formed when heparin is incubated with K50E mutated PF4. In accordance with this observation, PF4 antagonists of the present disclosure (which are thought to bind near these residues) inhibit both tetramerization and ULC formation. [0143] In some embodiments, the compounds described herein may be docked and scored on their ability to bind to the dimer interface pocket lined by monomer lysines and glutamine residues in the protein structure of PF4 (see, e.g., Figure 3). [0144] In some embodiments, the compound of Formula (II) prevent formation of and/or disrupt the PF4 tetramer. The prevention and/or disruption may occur in vitro, ex vivo or in vivo. For example, the prevention of disruption may occur in a subject (i.e., after administering the compound to the subject), e.g., in need thereof. [0145] In some embodiments, the compound of Formula (II) may prevent formation and/or disrupt complexes formed between glycosaminoglycan (GAG) and a PF4 tetramer. In some embodiments, pathogenic complexes of GAG and PF4 are very large and are referred to as ultralarge complexes (ULC). GAG (e.g., heparin):PF4 complexes smaller than 600 kDa are typically referred to as small complexes (SC). In one embodiment, ULCs are 600 kDa or larger. In another embodiment, ULCs are 670 kDa or larger. [0146] GAGs are long unbranched polysaccharides having a repeating disaccharide unit (a hexose (six-carbon sugar) or a hexuronic acid, linked to a hexosamine (six-carbon sugar containing nitrogen)). In some embodiments, the GAG is selected from among wild-type GAGs or synthetically produced GAGs. In one embodiment, the GAG is heparin, hyaluronan, hyaluronic acid, dermatan sulfate, keratan sulfate, or a chondroitin, or a salt thereof. In some embodiments, the GAG is heparin. In a further embodiment, the GAG is heparan sulfate. [0147] PF4:heparin ULCs are more pathogenic than heparin:PF4 SCs. Heparin:PF4 ULCs are better recognized by HITT antibodies and lead to more platelet activation in the presence of these antibodies. Disruption of ULC represents a valid therapeutic target in the treatment of a disease mediated by the ULC, such as the HITT. [0148] In some embodiments, the compounds described herein bind to PF4 monomers, PF4 trimers, PF4 dimers, and/or PF4 tetramers and inhibit the formation of the PF4 tetramers and/or inhibit a ULC formed with the PF4 tetramers. In some embodiments, the present compounds also disrupt a salt bridge in a PF4 tetramer and thereby disrupt or inhibit formation of the PF4 tetramer. In some embodiments, the compound binds to a specific site (e.g., specific amino acid) at the PF4 tetramer, trimer, dimer or monomer. In some embodiments, the compounds are capable of antagonizing an electrostatic attraction between the PF4 monomers, dimers, and trimers in the PF4 tetramer, and therefore successfully disrupt the salt bridge of the tetramer. For example, a functional group of the present compound of Formula (II) binds stronger to the PF4 monomer, dimer, or trimer, than they bind to one another. [0149] The salt bridge between oligomers in the PF4 tetramer is formed via electrostatic interactions of a negatively charged amino acid, such as glutamic acid, of a first PF4 monomer or PF4 dimer, and a positively charged amino acid, such as lysine, of a second PF4 monomer or PF4 dimer. This salt bridge is typically formed by interaction of at least one Glu or at least one Lys on a first PF4 monomer or PF4 dimer, and at least one Lys or at least one Glu on a second PF4 monomer or PF4 dimer. In one embodiment, the salt bridge is formed via at least electrostatic interactions between Lys50 in a first PF4 monomer Glu28 in a second PF4 monomer. In another embodiment, the salt bridge is formed an electrostatic interaction between Glu128 or Lys350 of a first PF4 monomer or dimer, and Glu328 or Lys150 of a second PF4 monomer or dimer. In another embodiment, the salt bridge is formed via an electrostatic interactions between Glu228 or Lys450 of a first PF4 monomer or dimer and Glu428 or Lys250 of a second PF4 monomer or dimer. In some embodiments, the salt bridge is formed on a PF4 dimer-dimer interface. That is, the compound of the present disclosure disrupts the salt bridge on the PF4 dimer-dimer interface. [0150] Typically, a PF4:heparin ULC is antigen that promotes production of an antibody that is specific to the complex. The anybody recognizes the complex composed of heparin and the PF4 tetramer, and forms a pathogenic ULC-antibody complex. By inhibiting formation and/or disruption PF4 tetramer, the compounds described herein inhibit binding of the pathogenic ULC-antibody complex to a FcγRIIa receptor on a surface of a platelet. This leads to inhibition of platelet activation, and results in decreased production of PF4 by the platelet. When the platelet is contacted with the compound in vitro, ex vivo, or in vivo (e.g., by administering the compound to the subject), the inhibition of binding of ULC-antibody complex to a FcγRIIa also leads to inhibition of platelet aggregation, increased high density lipoproteins, modulated (e.g., reduced) blood clotting or hemostasis, and corrected platelet imbalance in the subject (e.g., in need thereof). In some embodiments, the platelet imbalance results from heparin administration to the subject. [0151] In one general aspect, the present application provides a method for diagnostic use or for use in determination of a dosage, or for pharmaceutical evaluation of a composition containing a compound of Formula (II) as described herein. Such methods include measuring a first level of PF4 tetramer in a first biological sample obtained from a subject and administering a first effective amount of the compound of Formula (II) that is required to decrease the first PF4 tetramer level. In another embodiment, methods for disrupting PF4 tetramers are provided and include measuring a first level of PF4 tetramer in a first biological sample obtained from a subject, administering a first effective amount of the compound of Formula (II) required to decrease the first PF4 tetramer level, and optionally administering a medication which disrupts PF4 tetramers. The medication which disrupts PF4 tetramers may be administered prior to, concurrent with, or subsequent to the PF4 antagonists discussed herein. [0152] In another embodiment, methods for disrupting PF4 tetramers are provided and include measuring a first level of PF4 tetramer in a first biological sample obtained from a subject, administering a first effective amount of the compound of Formula (II) required to decrease the first PF4 tetramer level, measuring a second level of PF4 tetramer in a second biological sample obtained from the subject, and administering a second effective amount of the compound of Formula (II) required to decrease the second PF4 tetramer level. Multiple samples can be obtained from the subject at any interval required, for example, to prevent or treat the medical condition. [0153] In a further embodiment, methods for disrupting ULCs containing PF4 tetramers and heparin are provided. These methods include measuring a first level of ULCs in a first biological sample obtained from a subject and administering a first effective amount of the compound of Formula (II) required to decrease the first ULC tetramer level. [0154] In yet another embodiment, methods for disrupting ULCs containing PF4 tetramers and heparin include measuring a first level of ULCs in a first biological sample obtained from a subject, administering a first effective amount of the compound of Formula (II) required to decrease the first ULC level, measuring a second level of ULCs in a second biological sample obtained from the subject, and administering a second effective amount of the compound of Formula (II) required to decrease the second ULC level. [0155] In still a further embodiment, methods for preventing the formation of PF4 tetramers include measuring a first level of PF4 tetramer in a first biological sample obtained from a subject, administering a first effective amount of the compound of Formula (II) required to prevent formation of the PF4 tetramer, measuring a second level of PF4 tetramer in a second biological sample obtained from the subject, and administering a second effective amount of the compound of Formula (II) required to prevent formation of PF4 tetramer. [0156] Also provided are methods for using the antagonistic compounds described herein for determining a subject's sensitivity to side effects or secondary medical conditions related to heparin administration. Further provided are methods for determining the likelihood of a subject to acquire a medical condition related to the formation of PF4 tetramers or ULCs containing PF4 tetramers and heparin. In one embodiment, these screening methods are useful in monitoring e.g., cancer patients. In another embodiment, these screening methods are useful in determining the likelihood of cancer patients being administered heparin in developing HIT or HITT. According to this method, biological samples are obtained from subjects and the level of PF4 tetramer and/or PF4 tetramer:GAG (heparin) ULC measured. The screening may be conducted using techniques commonly known and used in the art. Comparison of the levels of PF4 tetramer and/or PF4 tetramer:heparin ULC to a control level and/or negative control provides evidence that the patient may be treated using one or more of the antagonistic compounds described herein. In one embodiment, if the subject's PF4 tetramer and/or PF4 tetramer:heparin ULC level is higher than the PF4 tetramer and/or PF4 tetramer:heparin ULC level of a healthy subject, then antagonistic compound administration may be contemplated. [0157] In some embodiments, the present disclosure provides a method of treating a disease or condition characterized by PF4 tetramerization (e.g., a disease or medical condition related directly or indirectly to the formation of PF4 tetramers). The disease or medical condition may also be caused by the formation of PF4 tetramers. In some embodiments, the subject or patient has elevated levels of PF4 tetramer. In yet a further embodiment, the subject or patient has elevated levels of PF4 tetramer:GAG ULCs. [0158] Suitable examples of a disease or condition include, but are not limited to, heparin- induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HIT), thrombotic complication of HITT, atherosclerosis or atherosclerotic vascular disease, platelet imbalance or insufficiency, antiphospholipid syndrome, inflammation or inflammatory disease, vaccine-induced immune throbotic thrombocytopenia (VITT), or clotting or hemostasis disorders. [0159] In some embodiments, the disease or medical condition is heparin-induced thrombocytopenia (HIT). HIT results from the development of thrombocytopenia (low platelet count), for example, due to the administration of an anticoagulant (e.g., heparin or warfarin). In another embodiment, the disease or medical condition is heparin-induced thrombocytopenia and/with thrombosis (HITT). HITT results when HIT precedes thrombosis (abnormal antibodies and abnormal blood clots form inside a blood vessel). In some embodiments, the thrombosis is characterized by lower than normal thrombin-antithrombin complex (TAT) level. [0160] In other embodiments, the disease or medical condition is vaccine-induced immune thrombotic thrombocytopenia (VITT) also known as thrombosis with thrombocytopenia syndrome. In some embodiments, the VITT is caused by an adenovirus-based vaccine. In some embodiments, the vaccine is a adenovirus-based SARS-CoV-2 vaccine. [0161] Treatment of atherosclerotic vascular disease typically involves anti-platelet therapy (e.g, aspirin and Plavix® which are not well tolerated in all patients). For the treatment of thrombocytopenia, thrombopoeitin (TPO) analogs and mimetics (MPL agonists which activate MPL-the TPO receptor) may be used, but these drugs have significant side effects and compliance issues. Finally, some lipid lowering therapies are able to increase HDL, but the ability of available drugs to do so is limited. In another specific embodiment, the disease that may be successfully treated by the compound of Formula (II) is antiphospholipid syndrome. [0162] In a further embodiment, atherosclerosis resulting from the formation of a PF4 tetramer may be treated using a compound of Formula (II) described herein. In still another embodiment, the disease or medical condition is a platelet imbalance. The treatment method thereby includes correcting this platelet imbalance or preventing a platelet imbalance. In one example, platelet levels are increased by stimulating platelet production. In another example, a decrease in platelet production is prevented. In a further example, the platelet imbalance (e.g., low levels of platelets) results from the formation of a PF4 tetramer. In yet another example, the platelet imbalance, i.e., low levels of platelets, results from heparin administration to a subject. [0163] The compounds of Formula (II) discussed herein may also be an alternative therapy utilized to treat diseases related to TPO. The compounds may also be contemplated for use in preventing or treating inflammation which results from the formation of PF4 tetramers. The inflammation may be the caused by any number of factors. In one embodiment, the inflammation is acute or chronic. In another embodiments, the inflammation is localized or systemic. The inflammation may be the result of a variety of factors and/or conditions. The compounds of Formula (II) may also be useful in therapies for subjects having atherosclerotic vascular disease in which the patient is intolerant to the conventional treatments (e.g., statins). In some embodiments, an inflammatory disease is chronic inflammatory demyelinating polyneuropathy, inflammatory myopathy, inflammatory bowel diseases (IBDs), Crohn disease (CD), ulcerative colitis (UC), chronic inflammatory condition with polygenic susceptibility, inflammation of the uvea (e.g., anterior uveitis, e.g., iridocyclitis or iritis; intermediate uveitis (also known as pars planitis); posterior uveitis; or chorioretinitis, e.g., pan-uveitis). [0164] The compounds described herein may further be useful for modulating clotting or hemostasis. In one example, the compounds may be useful in patients that are intolerant to conventional therapies. Alternatively, the compounds of the present application may be synergistic with the conventional therapies. [0165] The compounds of Formula (II) are also useful in increasing high density lipoproteins (HDL) in a subject. Alternatively, the PF4 antagonists of the present application are useful in preventing a decrease of HDLs. [0166] In some embodiments, a PF4-associated disease or condition in a subject may occur simultaneously with another disease or medical condition. In one example, a decrease in platelet production may develop in a patient diagnosed with cancer. In some embodiments, the cancer is selected from the group selected from sarcoma, angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma, myxoma, rhabdomyoma, fibroma, lipoma, teratoma, lung cancer, breast cancer, bronchogenic carcinoma squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma, alveolar bronchiolar carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma, gastrointestinal cancer, cancer of the esophagus, squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma, cancer of the stomach, carcinoma, lymphoma, leiomyosarcoma, cancer of the pancreas, ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, vipoma, cancer of the small bowel, adenocarcinoma, lymphoma, carcinoid tumors, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma, cancer of the large bowel or colon, tubular adenoma, villous adenoma, hamartoma, leiomyoma, genitourinary tract cancer, cancer of the kidney, adenocarcinoma, Wilm's tumor (nephroblastoma), lymphoma, leukemia, cancer of the bladder, cancer of the urethra, squamous cell carcinoma, transitional cell carcinoma, cancer of the prostate, cancer of the testis, seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma, liver cancer, hepatoma hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, bone cancer, osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor, chordoma, osteochrondroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma giant cell tumor, nervous system cancer, cancer of the skull, osteoma, hemangioma, granuloma, xanthoma, osteitis deformans, cancer of the meninges meningioma, meningiosarcoma, gliomatosis, cancer of the brain, astrocytoma, medulloblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors, cancer of the spinal cord, neurofibroma, meningioma, glioma, sarcoma, gynecological cancer, cancer of the uterus, endometrial carcinoma, cancer of the cervix, cervical carcinoma, pre tumor cervical dysplasia, cancer of the ovaries, ovarian carcinoma, serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma, granulosa-theca cell tumor, Sertoli Leydig cell tumor, dysgerminoma, malignant teratoma, cancer of the vulva, squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma, cancer of the vagina, clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma, embryonal rhabdomyosarcoma, cancer of the fallopian tubes, hematologic cancer, cancer of the blood, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome, Hodgkin's lymphoma, non-Hodgkin's lymphoma (malignant lymphoma), Waldenstrom's macroglobulinemia, skin cancer, malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, adrenal gland cancer, and neuroblastoma. [0167] In some embodiments, the cancer patient having a PF4-associated disease or condition is undergoing a chemotherapy. In one example, the PF4-associated disease or disorder is the result of the chemotherapy treatment. Suitable examples of chemotherapeutic agents include paclitaxel, docetaxel, daunorubicin, cis-platin, carboplatin, and others. In some embodiments, the present application provides a method of treating cancer in a subject (e.g., any one of cancers described herein), the method comprising administering to the subject a therapeutically effective amount of a compound of Formula (II), or a pharmaceutically acceptable salt thereof. [0168] In some embodiments, discosed herein are methods of inhibiting platelet factor-4 (PF4) in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound or pharmaceutically acceptable salt thereof, or pharmaceutical composition disclosed herein, wherein the inhibition of PF4 comprises preventing formation of PF4 tetramers, and/or disrupting PF4 tetramers, and/or preventing formation of an ultra-large complex (ULC) comprising a PF4 tetramer and a glycosaminoglycan (GAG), and/or inhibiting ULC-antibody complex binding to a FcγRIIa receptor on a platelet, and/or inhibiting platelet aggregation, and/or increasing high density lipoproteins, and/or modulating clotting or hemostasis, and/or correcting a platelet imbalance in the subject. [0169] In some embodiments, disclosed herein is the use of a therapeutically effective amount of a compound or pharmaceutically acceptable salt thereof, or pharmaceutical composition disclosed herein, for inhibition of PF4, wherein the inhibition of PF4 comprises preventing formation of PF4 tetramers, and/or disrupting PF4 tetramers, and/or preventing formation of an ULC comprising a PF4 tetramer and a GAG, and/or inhibiting ULC-antibody complex binding to a FcγRIIa receptor on a platelet, and/or inhibiting platelet aggregation, and/or increasing high density lipoproteins, and/or modulating clotting or hemostasis, and/or correcting a platelet imbalance in the subject. [0170] In some embodiments, the compound binds to a PF4 monomer, PF4 dimer, or PF4 tetramer. In some embodiments, the compound disrupts a salt bridge between two PF4 dimers, two or more PF4 monomers, or a PF4 dimer and a PF4 monomer, in a PF4 tetramer. In some embodiments, the disruption occurs on a dimer-dimer interface. In some embodiments, the salt bridge in the PF4 tetramer is formed via an electrostatic interaction of a negatively charged amino acid of a first PF4 monomer or PF4 dimer and a positively charged amino acid of a second PF4 monomer or PF4 dimer. In some embodiments, the negatively charged amino acid of a first PF4 monomer or PF4 dimer is a glutamic acid. In some embodiments, the positively charged amino acid of a second PF4 monomer or PF4 dimer is a lysine. In some embodiments, the GAG is a heparin. [0171] In some embodiments, the platelet imbalance results from heparin administration to the subject. [0172] In some embodiments, the molecular weight of the ULC is greater than about 600 kD. In some embodiments, inhibiting the binding of a ULC-antibody complex to a FcγRIIa receptor on a platelet in a subject inhibits an activation of the platelet. In some embodiments, an antibody in the ULC-antibody complex recognizes a complex composed of heparin and a PF4 tetramer. [0173] In some embodiments, disclosed herein are methods of treating or preventing a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound disclosed herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition disclosed herein, wherein the disease or condition is selected from heparin induced thrombocytopenia and thrombosis (HITT), a thrombotic complication of HITT, heparin induced thrombocytopenia (HIT), vaccine- induced immune thrombotic thrombocytopenia (VITT), atherosclerosis or atherosclerotic vascular disease, decrease in platelet production, inflammation or an inflammatory disease, antiphospholipid syndrome, platelet imbalance or insufficiency, and a clotting or hemostasis disorder. [0174] In some embodiments, disclosed herein is the use of a therapeutically effective amount of a compound or pharmaceutically acceptable salt thereof, or pharmaceutical composition disclosed herein for treatment or prevention of a disease or condition in a subject, wherein the disease or condition is selected from HITT, a thrombotic complication of HITT, HIT, VITT, atherosclerosis or atherosclerotic vascular disease, decrease in platelet production, inflammation or an inflammatory disease, antiphospholipid syndrome, platelet imbalance or insufficiency, and a clotting or hemostasis disorder. [0175] In some embodiments, the disease or condition is mediated by a PF4 tetramer. In some embodiments, the atherosclerosis results from a PF4 tetramer formation or a formation of a GAG-PF4 complex. In some embodiments, the thrombotic complication of HITT is thrombosis. In some embodiments, the thrombosis is characterized by lower than normal thrombin-antithrombin complex level. [0176] In some embodiments, disclosed herein are methods of inhibiting platelet factor-4 (PF4) in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a compound selected from:
Figure imgf000037_0001
or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compounds, wherein the inhibition of PF4 comprises preventing formation of PF4 tetramers, and/or disrupting PF4 tetramers, and/or preventing formation of an ultra-large complex (ULC) comprising a PF4 tetramer and a glycosaminoglycan (GAG), and/or inhibiting ULC-antibody complex binding to a FcγRIIa receptor on a platelet, and/or inhibiting platelet aggregation, and/or increasing high density lipoproteins, and/or modulating clotting or hemostasis, and/or correcting a platelet imbalance in the subject. [0177] In some embodiments, disclosed herein is the use of a compound selected from:
Figure imgf000038_0001
or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compounds, for inhibiting platelet factor-4 (PF4) in a subject, wherein the inhibition of PF4 comprises preventing formation of PF4 tetramers, and/or disrupting PF4 tetramers, and/or preventing formation of an ultra-large complex (ULC) comprising a PF4 tetramer and a glycosaminoglycan (GAG), and/or inhibiting ULC-antibody complex binding to a FcγRIIa receptor on a platelet, and/or inhibiting platelet aggregation, and/or increasing high density lipoproteins, and/or modulating clotting or hemostasis, and/or correcting a platelet imbalance in the subject. [0178] In some embodiments, the compound binds to a PF4 monomer, PF4 dimer or PF4 tetramer. In some embodiments, the compound disrupts a salt bridge between two PF4 dimers, two or more PF4 monomers, or a PF4 dimer and a PF4 monomer, in a PF4 tetramer. In some embodiments, the disruption occurs on a dimer-dimer interface. In some embodiments, the salt bridge in the PF4 tetramer is formed via an electrostatic interaction of a negatively charged amino acid of a first PF4 monomer or PF4 dimer and a positively charged amino acid of a second PF4 monomer or PF4 dimer. In some embodiments, the negatively charged amino acid of a first PF4 monomer or PF4 dimer is a glutamic acid. In some embodiments, the positively charged amino acid of a second PF4 monomer or PF4 dimer is a lysine. In some embodiments, the GAG is a heparin. [0179] In some embodiments, the platelet imbalance results from heparin administration to the subject. [0180] In some embodiments, the molecular weight of the ULC is greater than about 600 kD. In some embodiments, inhibiting the binding of a ULC-antibody complex to a FcγRIIa receptor on a platelet in a subject inhibits an activation of the platelet. In some embodiments, the ULC-antibody complex recognizes a complex composed of heparin and a PF4 tetramer. [0181] In some embodiments, disclosed herein are method of treating or preventing a disease or condition in a subject, or use of the compound in prevention or treatment of a disease or condition in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound selected from:
Figure imgf000039_0001
or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound(s), wherein the disease or condition is selected from heparin induced thrombocytopenia and thrombosis (HITT), a thrombotic complication of HITT, heparin induced thrombocytopenia (HIT), vaccine-induced immune thrombotic thrombocytopenia (VITT), atherosclerosis or atherosclerotic vascular disease, decrease in platelet production, inflammation or an inflammatory disease, antiphospholipid syndrome, platelet imbalance or insufficiency, and a clotting or hemostasis disorder. [0182] In some embodiments, the disease or condition is mediated by a PF4 tetramer. In some embodiments, the atherosclerosis results from a PF4 tetramer formation or a formation of a GAG-PF4 complex. In some embodiments, the thrombotic complication of HITT is thrombosis. In some embodiments, the thrombosis is characterized by lower than normal thrombin-antithrombin complex level. Combinations [0183] In another general aspect, the compound of Formula (II) as described herein may be administered to the subject in combination with an additional therapeutic agent. In one example, the additional therapeutic agent may disrupt PF4 tetramers and/or ULCs. In one embodiment, the additional agent works synergistically with the PF4 antagonist of the present application. Suitable examples of such agents include cyclic peptides which inhibit the interaction of PF4 with CCL5 (CKEY2), carbohydrates such as desulfated heparin (ODSH), or a combination thereof. The PF4 tetramer disruption agents may be combined with the PF4 antagonist compounds of the present application either in a pharmaceutical composition as described herein, and/or kits and methods for using the same. [0184] In some embodiments, an additional therapeutic agent is an anticoagulant (e.g., rivaroxaban, dabigatran, apixaban, edoxaban, warfarin, fondaparinux, idraparinux, acenocoumarol, phenprocoumon, atromentin, or phenindione). In some embodiments, additional therapeutic agent is heparin. Other suitable examples of additional therapeutic agents include an anti-HER2 agent (e.g., trastuzumab, pertuzumab, lapatinib), a pain relief agent (e.g., a nonsteroidal anti-inflammatory drug such as celecoxib or rofecoxib), an antinausea agent, a cardioprotective drug (e.g., dexrazoxane, ACE-inhibitors, diuretics, cardiac glycosides), a cholesterol lowering drug, a revascularization drug, a beta-blocker (e.g., acebutolol, atenolol, bisoprolol, metoprolol, nadolol, nebivolol, or propranolol), an angiotensin receptor blocker (also called ARBs or angiotensin II inhibitors) (e.g., azilsartan, candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan, or valsartan), and an anticancer agent (e.g., paclitaxel, docetaxel, daunorubicin, cis-platin, carboplatin, taxol, 5-fluorouracil, , oxaliplatin/5 FU, abiraterone, or procarbazine). [0185] In some embodiments, the compound of Formula (II) and the additional therapeutic agent may be administered to the subject simultaneously (e.g., in the same dosage form or in separate dosage forms), or consecutively (e.g., heparin may be administered before or the compound of Formula (II)). The dosages and routes of administration are well within the judgement of the treating physician. Kits [0186] The present application also includes pharmaceutical kits useful, for example, in the treatment of disorders, diseases and conditions referred to herein, which include one or more containers containing a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present disclosure. Such kits can further include, if desired, one or more of various conventional pharmaceutical kit components, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers with additional therapeutic agents, diagnostic reagents, etc. Instructions, either as inserts or as labels, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, can also be included in the kit. [0187] Optionally, the kit may further contain instructions for monitoring blood level of the administered compound, and materials for performing such assays including, e.g., reagents, well plates, containers, markers or labels, and the like. Such kits are readily packaged in a manner suitable for treatment of a desired indication. For example, the kit may also contain instructions for use of the spray pump or other delivery device. [0188] In another embodiment, a pharmaceutical kit is provided and contains a medication which causes the formation of PF4 tetramers (e.g., heparin) in a first dosage unit and one or more of a PF4 antagonistic compound of the present application in a second dosage unit. [0189] In yet another embodiment, a pharmaceutical kit is provided and contains a therapeutic agent which disrupts PF4 tetramers in a first dosage unit, one or more of a PF4 antagonistic compound of the present disclosure in a second dosage unit, and one or more of the carriers or excipients described herein in a third dosage unit. The kit may optionally contain instructions for administering the components of the kit to a subject, for example, having cancer. [0190] In yet a further embodiment, a pharmaceutical kit is provided and contains a therapeutic agent that causes formation of a PF4 tetramer in a first dosage unit (e.g., heparin), a therapeutic agent that disrupts a PF4 tetramer in a second dosage unit, and one or more of a PF4 antagonistic compound described herein in a third dosage unit, and one or more of the carriers or excipients in a fourth dosage unit. The kit may optionally contain instructions for administering the components of the kit to a subject, e.g., having cancer. Pharmaceutical compositions and formulations [0191] The present application also provides pharmaceutical compositions comprising an effective amount of a compound of Formula (II) disclosed herein, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier. In certain embodiments, the application also provides pharmaceutical compositions and dosage forms comprising any one the additional therapeutic agents described herein. The carrier(s) are “acceptable” in the sense of being compatible with the other ingredients of the formulation and, in the case of a pharmaceutically acceptable carrier, not deleterious to the recipient thereof in an amount used in the medicament. [0192] Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of the present application include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose- based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat. [0193] The compositions or dosage forms may contain any one of the compounds and therapeutic agents described herein in the range of 0.005% to 100% with the balance made up from the suitable pharmaceutically acceptable excipients. The contemplated compositions may contain 0.001%-100% of any one of the compounds and therapeutic agents provided herein, in one embodiment 0.1-95%, in another embodiment 75-85%, in a further embodiment 20-80%, wherein the balance may be made up of any pharmaceutically acceptable excipient described herein, or any combination of these excipients. Routes of administration and dosage forms [0194] The pharmaceutical compositions of the present application include those suitable for any acceptable route of administration. Acceptable routes of administration include, but are not limited to, buccal, cutaneous, endocervical, endosinusial, endotracheal, enteral, epidural, interstitial, intra-abdominal, intra-arterial, intrabronchial, intrabursal, intracerebral, intracisternal, intracoronary, intradermal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralymphatic, intramedullary, intrameningeal, intramuscular, intranasal, intraovarian, intraperitoneal, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratesticular, intrathecal, intratubular, intratumoral, intrauterine, intravascular, intravenous, nasal, nasogastric, oral, parenteral, percutaneous, peridural, rectal, respiratory (inhalation), subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transtracheal, ureteral, urethral and vaginal. [0195] Compositions and formulations described herein may conveniently be presented in a unit dosage form, e.g., tablets, sustained release capsules, and in liposomes, and may be prepared by any methods well known in the art of pharmacy. See, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, Baltimore, MD (20th ed. 2000). Such preparative methods include the step of bringing into association with the molecule to be administered ingredients such as the carrier that constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers, liposomes or finely divided solid carriers, or both, and then, if necessary, shaping the product. [0196] In some embodiments, any one of the compounds and therapeutic agents disclosed herein are administered orally. Compositions of the present application suitable for oral administration may be presented as discrete units such as capsules, sachets, granules or tablets each containing a predetermined amount (e.g., effective amount) of the active ingredient; a powder or granules; a solution or a suspension in an aqueous liquid or a non- aqueous liquid; an oil-in-water liquid emulsion; a water-in-oil liquid emulsion; packed in liposomes; or as a bolus, etc. Soft gelatin capsules can be useful for containing such suspensions, which may beneficially increase the rate of compound absorption. In the case of tablets for oral use, carriers that are commonly used include lactose, sucrose, glucose, mannitol, and silicic acid and starches. Other acceptable excipients may include: a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar- agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspendfing agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added. Compositions suitable for oral administration include lozenges comprising the ingredients in a flavored basis, usually sucrose and acacia or tragacanth; and pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia. [0197] Compositions suitable for parenteral administration include aqueous and non- aqueous sterile injection solutions or infusion solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, saline (e.g., 0.9% saline solution) or 5% dextrose solution, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets. The injection solutions may be in the form, for example, of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant. [0198] The pharmaceutical compositions of the present application may be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of the present application with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycols. [0199] The pharmaceutical compositions of the present application may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well- known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. See, for example, U.S. Patent No. 6,803,031. Additional formulations and methods for intranasal administration are found in Ilium, L., J Pharm Pharmacol, 56:3-17, 2004 and Ilium, L., Eur J Pharm Sci 11:1-18, 2000. [0200] The topical compositions of the present disclosure can be prepared and used in the form of an aerosol spray, cream, emulsion, solid, liquid, dispersion, foam, oil, gel, hydrogel, lotion, mousse, ointment, powder, patch, pomade, solution, pump spray, stick, towelette, soap, or other forms commonly employed in the art of topical administration and/or cosmetic and skin care formulation. The topical compositions can be in an emulsion form. Topical administration of the pharmaceutical compositions of the present application is especially useful when the desired treatment involves areas or organs readily accessible by topical application. In some embodiments, the topical composition comprises a combination of any one of the compounds and therapeutic agents disclosed herein, and one or more additional ingredients, carriers, excipients, or diluents including, but not limited to, absorbents, anti- irritants, anti-acne agents, preservatives, antioxidants, coloring agents/pigments, emollients (moisturizers), emulsifiers, film-forming/holding agents, fragrances, leave-on exfoliants, prescription drugs, preservatives, scrub agents, silicones, skin-identical/repairing agents, slip agents, sunscreen actives, surfactants/detergent cleansing agents, penetration enhancers, and thickeners. [0201] The compounds and therapeutic agents of the present application may be incorporated into compositions for coating an implantable medical device, such as prostheses, artificial valves, vascular grafts, stents, or catheters. Suitable coatings and the general preparation of coated implantable devices are known in the art and are exemplified in U.S. Patent Nos. 6,099,562; 5,886,026; and 5,304,121. The coatings are typically biocompatible polymeric materials such as a hydrogel polymer, polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylactic acid, ethylene vinyl acetate, and mixtures thereof. The coatings may optionally be further covered by a suitable topcoat of fluorosilicone, polysaccharides, polyethylene glycol, phospholipids or combinations thereof to impart controlled release characteristics in the composition. Coatings for invasive devices are to be included within the definition of pharmaceutically acceptable carrier, adjuvant or vehicle, as those terms are used herein. [0202] According to another embodiment, the present application provides an implantable drug release device impregnated with or containing a compound or a therapeutic agent, or a composition comprising a compound of the present application or a therapeutic agent, such that the compound or therapeutic agent is released from the device and is therapeutically active. Dosages and regimens [0203] In the pharmaceutical compositions of the present application, a compound of Formula (II) is present in an effective amount (e.g., a therapeutically effective amount). [0204] Effective doses may vary, depending on the diseases treated, the severity of the disease, the route of administration, the sex, age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents and the judgment of the treating physician. [0205] In some embodiments, an effective amount of a compound of Formula (II) can range, for example, from about 0.001 mg/kg to about 500 mg/kg (e.g., from about 0.001 mg/kg to about 200 mg/kg; from about 0.01 mg/kg to about 200 mg/kg; from about 0.01 mg/kg to about 150 mg/kg; from about 0.01 mg/kg to about 100 mg/kg; from about 0.01 mg/kg to about 50 mg/kg; from about 0.01 mg/kg to about 10 mg/kg; from about 0.01 mg/kg to about 5 mg/kg; from about 0.01 mg/kg to about 1 mg/kg; from about 0.01 mg/kg to about 0.5 mg/kg; from about 0.01 mg/kg to about 0.1 mg/kg; from about 0.1 mg/kg to about 200 mg/kg; from about 0. 1 mg/kg to about 150 mg/kg; from about 0.1 mg/kg to about 100 mg/kg; from about 0.1 mg/kg to about 50 mg/kg; from about 0.1 mg/kg to about 10 mg/kg; from about 0.1 mg/kg to about 5 mg/kg; from about 0.1 mg/kg to about 2 mg/kg; from about 0.1 mg/kg to about 1 mg/kg; or from about 0.1 mg/kg to about 0.5 mg/kg). [0206] In some embodiments, an effective amount of a compound of Formula (II) is about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, or about 5 mg/kg. [0207] The foregoing dosages can be administered on a daily basis (e.g., as a single dose or as two or more divided doses, e.g., once daily, twice daily, thrice daily) or non-daily basis (e.g., every other day, every two days, every three days, once weekly, twice weekly, once every two weeks, once a month). EXAMPLES Materials and methods [0208] Preparative and analytical methods used to generate and evaluate the compounds of the following examples included the following: [0209] LC/MS data (ESI+) were determined with a Waters Alliance 2695 HPLC/MS (Waters Symmetry C18, 4.6 × 75 mm, 3.5 μm) or (Phenomenex C18, 4.6 × 75 mm, 3.0 μm) with a 2996 diode array detector from 210−400 nm; the solvent system is 5−95% MeCN in water (with 0.1% TFA) over nine minutes using a linear gradient, and retention times are in minutes. Mass spectrometry was performed on a Waters ZQ using electrospray in positive mode. [0210] LC/MS data (ESI-) were determined with a Shimadzu Prominence HPLC/MS (Phenomenex Luna C18, 3.0 ×50 mm, 3 μm) with a 2996 diode array detector from 210−400 nm; the solvent system is 5−95% MeCN in water (with 0.1% formic acid) over five minutes using a linear gradient, and retention times are in minutes. Mass spectrometry was performed on a Applied Biosystems MDS Sciex API 2000 using electrospray in negative mode. Alternatively LC/MS data (ESI-) were determined with a Waters Alliance 2695 HPLC/MS (Phenomenex C18, 4.6 × 75 mm, 3.0 μm) with a 2996 diode array detector from 210−400 nm; the solvent system is 5−95% MeCN in water (with 0.1% formic acid) over nine minutes using a linear gradient, and retention times are in minutes. Mass spectrometry was performed on a Waters ZQ using electrospray in positive mode. [0211] HRMS data were determined by The University of Notre Dame Mass Spectrometry & Proteomics Facility on a Bruker micrOTOF II. [0212] Preparative reversed phase HPLC was performed on a Waters Sunfire column (19 × 50 mm, C18, 5 μm) with a 10 min mobile phase gradient of 10% acetonitrile/water to 90% acetonitrile/ water with 0.1% TFA as buffer using 214 and 254 nm as detection wavelengths. Injection and fraction collection were performed with a Gilson 215 liquid handling apparatus using Trilution LC software. [0213] 1H NMR were recorded on Varian Oxford 300 MHz, in DMSO-d6. Chemical shifts (δ) are expressed in ppm downfield from tetramethylsilane (TMS) unless otherwise noted. EXAMPLE 39 - Synthesis of 2,2,6-triacetyl-s-indacene-1,3,5,7-tetraone
Figure imgf000046_0001
[0214] The title compound was prepared from pyromellitic dianhydride (CAS Number 89- 32-7, 218 mg, 1.0 mmol) and pentane-2,4-dione (206 μL, 2 mmol) with trimethylamine and acetic anhydride according to the conditions in Example 1 to give the title compound (154 mg, 52% yield). LC/MS (ESI-): Rf = 3.90 min, (M-H)- = 338.8, 296.9 LC/MS (ESI+), 4.33 min, purity > 90%,(M+1) 341.1, 299.1; 1H HRMS (ESI-): calculated for C18H11O7 m/z [M-H]-: 339.051026, observed 339.053114; NMR: δ = 8.09 (s, 1H), 7.55 (s, 1H), 263 (s, 3H), 2.45 (s, 3H), 2.40 (s, 3H); 1H NMR (chloroform-d): δ = 8.40 (s, 1H), 8.22-8.30 (m, 1H), 2.75 (s, 3H), 2.65 (s, 3H), 2.55 (s, 3H). EXAMPLE 40 - Synthesis of 2,6-diacetyl-s-indacene-1,3,5,7-tetraone
Figure imgf000047_0001
[0215] Pyromellitic dianhydride (600mg) and pentane-2,4-dione (1417uL, 13.8mmol) were dissolved in Ac2O (4mL). The mixture was heated to 200°C for 3 min in the microwave to dissolve the starting materials. Et3N (957 µL, 6.9 mmol) was added and the solution and the mixture was irradiated in the microwave at 160 °C for 6 min. The solution was then added to ice and water was added. After stirring, concentrated HCl was added, and stirred for an additional 5 min. The resulting solid was filtered, and dried. The solid was then washed with 5eq NaOH and water, and the solution was filtered. The aqueous filtrate was treated with 5eq HCl and the resulting precipitant was filtered, and dried to provide the title compound (200 mg, 5%). LC/MS (ESI+): Rf = 2.67 min, purity > 95%,(M+1) 341.1, 299.1; HRMS (ESI-): calculated for C16H9O6 m/z [M-H]-: 297.040462, observed 297.039680; 1H NMR: δ = 7.86 (s, 2H), 2.52 (s, 6H); 1H NMR (methanol-d4): δ = 8.09 (s, 2H), 2.59 (s, 6H). EXAMPLE 41 - Synthesis of s-indacene-1,3,5,7(2h,6h)-tetraone
Figure imgf000047_0002
[0216] Pyromellitic anhydride (8g, 36.7mmol) and ethyl acetoacetate (9.4 mL) were added to a solution of acetic anhydride (Ac2O, 100mL). Triethylamine (40.8 mL) was added and the reaction was heated to 100°C for 2 hr. The mixture was then cooled to RT and cooled to 0°C in an ice bath to form a brownish precipitate. After sitting in a refrigerator overnight, the precipitate was washed with acetic anhydride and ether to form an orange solid. The purified mixture was dissolved in water (400mL) and cooled in an ice bath and conc. H2SO4 (8 mL) was added..The resulting orange precipitate was filtered, washed with ethanol and air dried overnight to afford an orange solid (8.3 g). [0217] This solid (4 g) was then dissolved in acetonitrile (250 mL), forming a deep red solution, and heated to 90°C for 3 hr. A small amount of concentrated HCl was added and the mixture was stirred for an additional 30 min. After cooling to RT, a greyish precipitate was filtered and recrystallized from acetonitrile. The resulting product was isolated and dried overnight to afford the title product (3.27 g, 87%). LC/MS (ESI+): Rf = 1.76 min, purity > 95%, (M+H)+ = 215.04; HRMS (ESI-): calculated for C12H5O4 m/z [M-H]-: 213.019332, observed 213.020024;1H NMR (chloroform-d): δ = 8.52 (s, 2H), 3.43 (s, 4H). EXAMPLE 42 - Synthesis of n2-(2-(cyclohexa-2,4-dien-1-yl)ethyl)-1,3,5,7-tetraoxo-n6- phenethyl-1,2,3,5,6,7-hexahydro-s-indacene-2,6-dicarboxamide
Figure imgf000048_0001
[0218] To a solution of S-indacene-1,3,5,7(2H,6H)-tetraone (prepared in Example 41, 100 mg, 0.5 mmol) in DMF (3 mL) at between -60 to -50°C was added Et3N (162 μL, 1.2 mmol) and phenethyl isocyanate (161 μL, 1.2 mmol). The mixture was stirred for 2 hr at this temperature, and warmed up to room temperature. After stirring overnight the solution was poured onto ice and then water was slowly added. Concentrated HCl was added and the resulting solid was filtered and washed with water. After drying under vacuo the solid was diluted into 1.5 mL of water and stirred in 5 eq of NaOH. After filtering the solid was washed with ethyl acetate, methanol and 2 M aqueous HCl and dried to give the title compound (206 mg, 82%). LC/MS (ESI+): Rf = 6.46 min, purity > 95%, (M+H)+ = 677.03; HRMS (ESI-): calculated for C30H24N2O6 m/z [M-H]-: 508.16344, observed 508.1674; 1H NMR δ = 8.49 (t, J=5.57 Hz, 2 H) 7.00 - 7.35 (m, 12 H) 3.40 - 3.49 (m, 4 H) 2.68-2.78 (m, 4 H). EXAMPLE 43 - Synthesis of 1,3,5,7-tetraoxo-n2,n6-dipropyl-1,2,3,5,6,7-hexahydro-s- indacene-2,6-dicarboxamide
Figure imgf000048_0002
[0219] The title compound was prepared from S-indacene-1,3,5,7(2H,6H)-tetraone as described in Example 42, except using propyl isocyanate (109 µL, 1.2mmol) as the isocyanate reagent (98 mg, 65%). LC/MS (ESI+): Rf = 5.69 min, purity > 95%, (M+H)+ = 385.01;; HRMS (ESI-): calculated for C20H19N2O6 m/z [M-H]-: 383.124860, observed 383.1343; 1H NMR δ = 8.75-8.95 (m, 2H), 7.50 (s., 2 H) 3.24-3.35 (m, 4 H), 1.48-1.61 (m, 4 H), 0.83-0.91 (m, 6 H). EXAMPLE 44 - Synthesis of n2,n6-bis(4-ethoxyphenyl)-1,3,5,7-tetraoxo-1,2,3,5,6,7- hexahydro-s-indacene-2,6-dicarboxamide
Figure imgf000048_0003
[0220] The title compound was prepared from S-indacene-1,3,5,7(2H,6H)-tetraone as described for Example 42, except using ethoxyphenyl isocyanate (CAS Number 32459-62-4, 172 µL, 1.2 mmol) as the isocyanate (104 mg, 41%). LC/MS (ESI+): Rf = 7.50 min, purity > 95%, (M+H)+ = 541.05; HRMS (ESI-): calculated for C30H23N2O8 m/z [M-H]-: 539.145989, observed 539.146119; 1H NMR δ = 8.32 (s, 2 H) 7.46 (d, J=8.79 Hz, 2 H) 7.29 (d, J=9.09 Hz, 4 H) 6.81 (d, J=8.21 Hz, 4 H) 3.77 - 4.12 (m, 4 H), 1.28 (t, J=6.45 Hz, 6 H). EXAMPLE 45 - Synthesis of 1,3,5,7-tetraoxo-n2,n6-di-o-tolyl-1,2,3,5,6,7-hexahydro-s- indacene-2,6-dicarboxamide
Figure imgf000049_0001
[0221] The title compound was prepared from S-indacene-1,3,5,7(2H,6H)-tetraone as described for Example 42, except using o-tolyl isocyanate (CAS Number 614-68-6, 145 µL, 1.2 mmol) as the isocyanate (126 mg, 56%). LC/MS (ESI+): Rf = 7.02 min, purity > 95%, (M+H)+ = 480.90; HRMS (ESI-): calculated for C28H19N2O6 m/z [M-H]-: 479.124860, observed 479.124537; 1H NMR δ = 10.51 (m, 2 H) 8.14-8.22 (m, 2 H) 7.41 (s, 2 H) 7.03 - 7.29 (m, 4 H) 6.90-6.97 (m, 2 H), 2.33 (s, 6 H). EXAMPLE 46 - Synthesis of n2,n6-diisopropyl-1,3,5,7-tetraoxo-1,2,3,5,6,7-hexahydro-s- indacene-2,6-dicarboxamide
Figure imgf000049_0002
[0222] The title compound was prepared from S-indacene-1,3,5,7(2H,6H)-tetraone as described for Example 42, except using isopropyl isocyanate (115 µL, 1.2 mmol) as the isocyanate (80 mg, 45%). LC/MS (ESI+): Rf = 5.69 min, purity > 95%, (M+H)+ = 384.98; HRMS (ESI-): calculated for C20H19N2O6 m/z [M-H]-: 383.124860, observed 383.125235; 1H NMR δ = 7.51-7.57 (m, 2 H) 3.93 - 4.05 (m, 2 H), 1.20 - 1.28 (m, 12 H). EXAMPLE 47 - Synthesis of n2,n6-dibenzyl-1,3,5,7-tetraoxo-1,2,3,5,6,7-hexahydro-s- indacene-2,6-dicarboxamide
Figure imgf000050_0001
[0223] The title compound was prepared from S-indacene-1,3,5,7(2H,6H)-tetraone as described for Example 42, except using benzyl isocyanate (288 µL, 2.3 mmol) as the isocyanate reagent (40 mg, 18%). LC/MS (ESI+): Rf = 7.12 min, purity > 95%, (M+H)+ = 481.04; HRMS (ESI-): calculated for C28H19N2O6 m/z [M-H]-: 479.124860, observed 479.124294; 1H NMR δ = 8.97-9.32 (m, 2H), 7.44 (s, 2H), 7.23-7.38 (m,10H), 4.42-4.57 (m, 4H). EXAMPLE 48 - Synthesis of 1,3,5,7-tetraoxo-n2,n6-bis(3-phenylpropyl)-1,2,3,5,6,7- hexahydro-s-indacene-2,6-dicarboxamide
Figure imgf000050_0002
[0224] The title compound was prepared from S-indacene-1,3,5,7(2H,6H)-tetraone as described for Example 42, except using phenpropyl isocyanate (362uL, 2.3 mmol) as the isocyanate (110mg, 44%). LC/MS (ESI+): Rf = 6.95 min, purity > 95%, (M+H)+ = 537.04; ; HRMS (ESI-): calculated for C32H27N2O6 m/z [M-H]-: 535.187460, observed 535.186976; 1H NMR: δ = 8.54-8.69 (m, 2H), 7.15-7.31 (m, 12H), 3.16-3.30 (m, 4H), 2.63-2.74 (m, 4H), 1.73- 1.87 (m, 4H). EXAMPLE 49 - Synthesis of n2,n6-bis(4-chlorophenethyl)-1,3,5,7-tetraoxo-1,2,3,5,6,7- hexahydro-s-indacene-2,6-dicarboxamide
Figure imgf000050_0003
[0225] The title compound was prepared from S-indacene-1,3,5,7(2H,6H)-tetraone as described for Example 42, except using 4-chlorophenethyl isocyanate (CAS Number 55121- 08-9, 375 µL, 2.3 mmol) as the isocyanate reagent (125 mg, 46%). LC/MS (ESI+): Rf = 6.97 min, purity > 95%, (M+H)+ = 577.00; HRMS (ESI-): calculated for C30H21Cl2N2O6 m/z [M- H]-: 575.078215, observed 575.077387; 1H NMR: δ = 8.81 (br. s, 2H), 7.48 (s, 2H), 7.35(d, 4H), 7.25 (d, 4H), 3.51-3.64 (m, 4H), 2.80-2.93 (m, 4H). EXAMPLE 50 - Synthesis of n2,n6-bis(3-chlorophenethyl)-1,3,5,7-tetraoxo-1,2,3,5,6,7- hexahydro-s-indacene-2,6-dicarboxamide
Figure imgf000051_0001
[0226] The title compound was prepared from S-indacene-1,3,5,7(2H,6H)-tetraone as described for Example 42, except using 3-chlorophenethyl isocyanate (CAS Number 62334- 11-6, 375 µL, 2.3 mmol) as the isocyanate reagent (182 mg, 68%). LC/MS (ESI+): Rf = 6.93 min, purity > 95%, (M+H)+ = 576.99; HRMS (ESI-): calculated for C30H21Cl2N2O6 m/z [M- H]-: 575.078215, observed 575.076950; 1H NMR: δ = 8.62-8.77 (m, 2H), 7.20-7.39 (m, 10H), 3.48-3.62 (m, 4H), 2.78-2.94 (m, 4H). EXAMPLE 51 - Synthesis of n2,n6-bis(2-chlorophenethyl)-1,3,5,7-tetraoxo-1,2,3,5,6,7- hexahydro-s-indacene-2,6-dicarboxamide
Figure imgf000051_0002
[0227] The title compound was prepared from S-indacene-1,3,5,7(2H,6H)-tetraone as described for Example 42, except using 2-chlorophenethyl isocyanate (CAS Registry No. 1943-83-5, 375 µL, 2.3 mmol) as the isocyanate reagent (85 mg, 31%). LC/MS (ESI+): Rf = 7.03 min, purity > 95%, (M+H)+ = 577.02; HRMS (ESI-): calculated for C30H21Cl2N2O6 m/z [M-H]-: 575.078215, observed 575.079822; 1H NMR: δ = 8.47-9.04 (m, 2H), 7.23-7.48 (m, 10H), 3.56-3.69 (m, 4H) 3.00 (d, J=7 Hz, 4H). EXAMPLE 52 - Synthesis of n2,n6-bis(4-methylphenethyl)-1,3,5,7-tetraoxo-1,2,3,5,6,7- hexahydro-s-indacene-2,6-dicarboxamide
Figure imgf000051_0003
[0228] The title compound was prepared from S-indacene-1,3,5,7(2H,6H)-tetraone as described for Example 42, except using 4-methylphenethyl isocyanate (CAS Number 103809- 68-3, 400 µL, 2.3 mmol) ) as the isocyanate reagent (26 mg, 10%). LC/MS (ESI+): Rf = 7.12 min, purity > 95%, (M+H)+ = 537.09; HRMS (ESI-): calculated for C32H27N2O6 m/z [M-H]-: 535.187460, observed 535.187294; 1H NMR: δ = 8.46-8.70 (m, 2H), 7.22 (d, J=4 Hz, 2H), 7.08-7.18 (m, 8H), 2.69-2.84 (m, 4H), 2.27 (br. s., 6H). EXAMPLE 53 - Synthesis of n2,n6-bis(4-fluorophenethyl)-1,3,5,7-tetraoxo-1,2,3,5,6,7- hexahydro-s-indacene-2,6-dicarboxamide
Figure imgf000052_0001
[0229] The title compound was prepared from S-indacene-1,3,5,7(2H,6H)-tetraone as described for Example 42, except using 4-fluorophenethyl isocyanate (335 µL, 2.3 mmol) as the isocyanate reagent (122 mg, 48%). LC/MS (ESI+): Rf = 6.37 min, purity > 95%, (M+H)+ = 544.99; HRMS (ESI-): calculated for C30H21F2N2O6 m/z [M-H]-: 543.137316, observed 543.134551; 1H NMR : δ = 8.73-8.87 (m, 2H), 7.46-7.57 (m, 2H), 7.22-7.35 (m, 4H), 7.08-7.19 (m, 4H), 3.49-3.64 (m, 4H), 2.80-2.94 (m, 4H). EXAMPLE 54 - Synthesis of n2,n6-bis(2-fluorophenethyl)-1,3,5,7-tetraoxo-1,2,3,5,6,7- hexahydro-s-indacene-2,6-dicarboxamide
Figure imgf000052_0002
[0230] The title compound was prepared from S-indacene-1,3,5,7(2H,6H)-tetraone as described for Example 42, except using 2-fluorophenethyl isocyanate (CAS Number 480439- 39-2, 335 µL, 2.3 mmol) as the isocyanate reagent (96 mg, 38%). LC/MS (ESI+): Rf = 6.42 min, purity > 95%, (M+H)+ = 545.06; HRMS (ESI-): calculated for C30H21F2N2O6 m/z [M-H]- : 543.137316, observed 543.136373; 1H NMR: δ = 8.69-8.82 (m, 2H), 7.12-7.41 (m, 10H), 2.84-2.98 (m, 4H). EXAMPLE 55 - Synthesis of 1,3,5,7-tetraoxo-n2,n6-bis(4-(trifluoromethoxy)phenethyl)- 1,2,3,5,6,7-hexahydro-s-indacene-2,6-dicarboxamide
Figure imgf000052_0003
[0231] S-indacene-1,3,5,7(2H,6H)-tetraone (100 mg) was dissolved in DMF (3 mL) and cooled to between -60 to -50°C. Et3N (162 µL, 1.2 mmol) was added and the solution was allowed to stir for 5 min. 1-(2-isocyanatoethyl)-4-(trifluoromethoxy)-benzene (CAS Registry Number 730981-19-8, 270 µL) was then added to the solution and the mixture was stirred for 2 hr and then allowed to warm up to RT overnight. After stirring overnight the solution was poured onto ice and then water was slowly added. Concentrated HCl was added and the resulting solid was filtered and washed with water. After drying under vacuo the solid was diluted into 1.5 mL of water and stirred in 5 eq of NaOH. The resulting sodium salt was placed in a soxhlet extractor and purged continuously with refluxing chloroform and dichloromethane. The remaining solid was dried in vacuo to afford the title compound as the bis-sodium salt (175 mg, 55%). LC/MS (ESI+): Rf = 7.12 min, purity > 95%, (M+H)+ = 677.03; HRMS (ESI-): calculated for C32H21F6N2O8 m/z [M-H]-: 675.120759, observed 675.120735; 1H NMR δ = 8.50-8.64 (t, 2H) 7.34 (d, 4H), 7.40 (d, 4H), 7.15 (s, 2H), 3.44 (q, J=7 Hz, 4H), 2.82 (t, J=7 Hz, 4H). EXAMPLE 56 - Synthesis of 3-{[6-(2-ethoxycarbonyl-ethylcarbamoyl)-1,3,5,7-tetraoxo- 1,2,3,5,6,7-hexahydro-s-indacene-2-carbonyl]-amino}-propionic acid ethyl ester
Figure imgf000053_0001
[0232] The title compound was prepared from S-indacene-1,3,5,7(2H,6H)-tetraone as described for Example 55, except using ethyl-3-isocyanatopropionate (CAS Number 5100-34- 5, 307 µL, 1.2mmol) as the isocyanate (60mg, 26%). LC/MS (ESI+): Rf = 4.88 min, purity > 95%, (M+H)+ = 501.01; HRMS (ESI-): calculated for C24H23N2O10 m/z [M-H]-: 499.135819, observed 499.137268; 1H NMR (chloroform-d): δ = 8.28-8.37 (m, 2 H) 7.87 (s, 2 H), 4.23 (q, 4 H) 3.69-3.84 (m, 4 H), 2.68 (t, 4 H) 1.31 (t, 6 H). EXAMPLE 57 - Synthesis of 1,3,5,7-tetraoxo-n2,n6-bis(2-carboxyethyl)-1,2,3,5,6,7- hexahydro-s-indacene-2,6-dicarboxamide
Figure imgf000053_0002
[0233] 3-{[6-(2-Ethoxycarbonyl-ethylcarbamoyl)-1,3,5,7-tetraoxo-1,2,3,5,6,7-hexahydro-s- indacene-2-carbonyl]-amino}-propionic acid ethyl ester (Example 56, 20 mg) was dissolved in MeOH (0.5 mL). 2M NaOH in water (80 µL) was added the reaction and the mixture was stirred overnight. The mixture was then lyophilized to yield the title compound (16 mg, 90%) as the bis sodium salt. LC/MS (ESI+): Rf = 2.92 min, purity > 95%, (M+H)+ = 444.88; HRMS (ESI-): calculated for C20H15N2O10 m/z [M-H]-: 443.073218, observed 443.073019; 1H NMR (tetrahydrofuran-d8): δ = 7.67 (s, 2H), 3.62-3.70 (m, 4H), 2.58-2.64 (m, 4H). EXAMPLE 58 - Synthesis of n2,n6-diallyl-1,3,5,7-tetraoxo-1,2,3,5,6,7-hexahydro-s- indacene-2,6-dicarboxamide
Figure imgf000054_0001
[0234] The title compound was prepared as the bis-sodium salt from S-indacene- 1,3,5,7(2H,6H)-tetraone as described for Example 55, except using allyl isocyanate (CAS Registry No.1476-23-9, 103 µL, 1.2 mmol) as the isocyanate reagent (102 mg, 52%). LC/MS (ESI+): Rf = 5.06 min, purity > 95%, (M+H)+ = 380.96; HRMS (ESI-): calculated for C20H15N2O6 m/z [M-H]-: 379.093560, observed 379.093309; 1H NMR: δ = 8.60-8.93 (m, 2H), 7.37 (s, 2H), 5.83-5.97 (m, 2H), 5.08-5.23 (m, 4H), 3.86-3.96 (m, 4H). EXAMPLE 59 - Synthesis of 1,3,5,7-tetraoxo-n2,n6-ditosyl-1,2,3,5,6,7-hexahydro-s- indacene-2,6-dicarboxamide
Figure imgf000054_0002
[0235] The title compound was prepared as the bis-sodium salt from S-indacene- 1,3,5,7(2H,6H)-tetraone as described for Example 55, except using p-toluenesulfonyl isocyanate (CAS Number 4083-64-1, 178 µL, 2.3 mmol) as the isocyanate (110 mg, 36%). LC/MS (ESI+): Rf = 4.01 min, purity > 95%, (M+H)+ = 608.97; HRMS (ESI-): calculated for C28H19N2O10S2 m/z [M-H]-: 607.048660, observed 607.047795; 1H NMRs: δ = 11.32-12.02 (m, 2H), 7.8a (d, J=8 Hz, 4H), 7.37 (d, J=8 Hz, 4H), 7.27 (s, 2H), 2.36 (s, 6H). EXAMPLE 60 - Synthesis of 1,3,5,7-tetraoxo-n2,n6-bis(3-(pyrimidin-2-yl)phenyl)- 1,2,3,5,6,7-hexahydro-s-indacene-2,6-dicarboxamide
Figure imgf000054_0003
[0236] The title compound was prepared as the bis-sodium salt from S-indacene- 1,3,5,7(2H,6H)-tetraone as described for Example 55, except using 2-(3-isocyanatophenyl) pyrimidine (CDS018140 ALDRICH, 192 µL, 1.2 mmol) as the isocyanate reagent (37 mg, 13%). LC/MS (ESI+): No peak observed; HRMS (ESI-): calculated for C34H19N6O6 m/z [M- H]-: 607.137156, observed 607.137401; 1H NMR: δ = 11.02 (s, 2H), 8.86-8.99 (m, 4H), 8.48- 8.57 (m, 2H), 7.90-8.05 (m, 4H), 7.37-7.51 (m, 6H). EXAMPLE 61 - Synthesis of n2,n6-di-sec-butyl-1,3,5,7-tetraoxo-1,2,3,5,6,7-hexahydro-s- indacene-2,6-dicarboxamide
Figure imgf000055_0001
[0237] The title compound was prepared as the bis-sodium salt from S-indacene- 1,3,5,7(2H,6H)-tetraone as described for Example 55, except using isocyanatobutane (CAS number 15585-98-5, 268 µL, 1.2 mmol) as the isocyanate reagent (4 mg, 2%). LC/MS (ESI+): Rf = 6.51 min, purity 92%, (M+H)+ = 413.05; ; HRMS (ESI-): calculated for C22H23N2O6 m/z [M-H]-: 411.156160, observed 411.155670; 1H NMR: δ = 8.34-8.46 (m, 2H), 7.11 (s, 2H), 3.77- 3.92 (m, 2H), 1.37-1.51 (m, 4H), 1.01-1.14 (m, 8H), 0.80-0.93 (m, 8H). EXAMPLE 62 - Synthesis of n2,n6-dicyclopropyl-1,3,5,7-tetraoxo-1,2,3,5,6,7-hexahydro- s-indacene-2,6-dicarboxamide
Figure imgf000055_0002
[0238] The title compound was prepared as the bis-sodium salt from S-indacene- 1,3,5,7(2H,6H)-tetraone as described for Example 55, except using cyclopropyl isocyanate (110 µL, 1.2 mmol) as the isocyanate reagent (112 mg, 58%). LC/MS (ESI+): Rf = 4.98 min, purity > 95%, (M+H)+ = 381.03; HRMS (ESI-): calculated for C20H15N2O6 m/z [M-H]-: 379.093560, observed 379.093552; 1H NMR: δ = 8.47 (d, J=4 Hz 2H), 7.13 (s, 2H), 2.68 (m, 2H), 0.59-0.67 (m, 4H), 0.38 (dd, J= 4 and 2 Hz, 4H). EXAMPLE 63 - Synthesis of n2,n6-bis(2-(benzo[d][1,3]dioxol-5-yl)ethyl)-1,3,5,7- tetraoxo-1,2,3,5,6,7-hexahydro-s-indacene-2,6-dicarboxamide
Figure imgf000055_0003
[0239] The title compound was prepared as the bis-sodium salt from S-indacene- 1,3,5,7(2H,6H)-tetraone as described for Example 55, except using 2-(benzo[d][1,3]dioxol-5- yl)ethyl isocyanate (CAS Number 62334-09-2, 110 µL, 1.2 mmol) as the isocyanate (31 mg, 11%). LC/MS (ESI+): Rf = 6.17 min, purity > 95%, (M+H)+ = 597.00; HRMS (ESI-): calculated for C32H23N2O10 m/z [M-H]-: 595.135819, observed 595.136281; 1H NMR: δ = 8.53-8.68 (2H, m), 7.29 (1H, s, J=4 Hz), 6.80-6.90 (4H, m), 6.67-6.75 (2H, m), 5.97 (4H, s), 3.44 (4H, dd, J=3 and 2 Hz), 2.67-2.81 (4H, m). EXAMPLE 64 - Synthesis of 1,3,5,7-tetraoxo-n2,n6-bis(2-phenylcyclopropyl)-1,2,3,5,6,7- hexahydro-s-indacene-2,6-dicarboxamide
Figure imgf000056_0001
[0240] The title compound was prepared as the bis-sodium salt from S-indacene- 1,3,5,7(2H,6H)-tetraone as described for Example 55, except using (2- isocyanatocyclopropyl)benzene (CAS Registry No. 63006-15-5, 110 µL, 1.2 mmol) as the isocyanate (164.7 mg, 66%). LC/MS (ESI+): Rf = 6.67 min, purity > 95%, (M+H)+ = 533.03; HRMS (ESI-): calculated for C32H23N2O6 m/z [M-H]-: 531.156160, observed 531.155198; 1H NMR: δ = 8.64-8.73 (2H, m), 7.45 (2H, s), 7.19-7.34 (4H, m), 7.05-7.17 (6H, m), 2.87-3.01 (2H, m), 1.88-2.00 (2H, m), 1.13-1.28 (4H, m). EXAMPLE 197 - 2,6-bis(2-phenoxyacetyl)-1,2,3,5,6,7-hexahydro-s-indacene-1,3,5,7- tetrone O O O O O O O O [0241] A mixture of DMAP (183 mg, 1.50 mmol), 2-phenoxyacetic acid (CAS number 122- 59-8, 228mg, 1.5mmol) and 9.0 ml of anhydrous DMF was stirred for 10 min after which a solution had formed. EDCI HCl (CAS number 25952-53-8, 286.5 mg, 1.50 mmol) was added and the contents were stirred for an additional 10 min. s-indacene-1,3,5,7(2H,6H)-tetraone (as used in Example 55, 107 mg, 0.5 mmol) was added and the reaction mixture was stirred overnight. The following day 15 ml of 2 N HCl was added and the mixture was stirred for 5 min. The resulting precipitant was filtered from solution. The product was suspended in 1N NaOH and methylene chloride and sonicated. The resulting precipitant was filtered to provide the title compound as the bis sodium salt (30 mg, 12%). LC/MS (ESI+): Rf = 6.31 min, (M+H)+ = 483.57; HRMS (ESI-): calculated for C28H18O8 m/z [M-H]-: 481.0929, observed 481.0944; 1H NMR δ 7.31-7.49 (m, 2H), 7.12-7.29 (m, 4H), 6.69-6.91 (m, 8H), 5.01-5.21 (m, 4H) EXAMPLE 198 - 2,6-bis(2-cyclopentylacetyl)-1,2,3,5,6,7-hexahydro-s-indacene-1,3,5,7- tetrone
Figure imgf000057_0001
[0242] In a similar manner to that of Example 197 the title compound was prepared from 1,3,5,7(2H,6H)-tetraone (as used in Example 55, 107 mg, 0.5 mmol) and 2-cyclopentylacetic acid (192 mg, 1.5 mmol) to provide the title compound as the bis sodium salt (64 mg, x%). This compound (32 mg) was further purified by reverse phase C18 column (acetonitrile/water/0.1 % ammonium hydroxide) to provide the title compound as the free acid (22 mg). LC/MS (ESI+): Rf = 8.67 min, (M+H)+= 435.62; HRMS (ESI-): calculated for C26H26O6 m/z [M-H]-: 433.1657, observed 433.1658; 1H NMR δ 7.12-7.38 (m, 2H), 3.92-4.14 (m, 4H), 1.32-1.80 (m, 14H), 0.99-1.31 (m, 6H) EXAMPLE 199 - 2,6-bis[2-(pyridin-4-yl)acetyl]-1,2,3,5,6,7-hexahydro-s-indacene-1,3,5,7- tetrone
Figure imgf000057_0002
[0243] In a similar manner to that of Example 197 (FC-8486),the title compound was prepared from 1,3,5,7(2H,6H)-tetraone (Example 55, 107 mg, 0.5 mmol) and 2-(pyridin-4- yl)acetic acid (CAS number, 28356-58-3, 205 mg, 1.5 mmol) to provide the title compound as the bis sodium salt (110 mg, 44%). LC/MS (ESI+): Rf = 1.96 min, (M+H)+= 453.51; HRMS (ESI- ): calculated for C26H16N2O6 m/z [M-H]-: 451.0936, observed 451.0938; 1H NMR δ 8.23-8.55 (m, 4H), 7.00-7.42 (m, 6H), 3.79-4.28 (m, 4H). EXAMPLE 200 - tert-butyl 4-{2-[6-(2-{1-[(tert-butoxy)carbonyl]piperidin-4-yl}acetyl)- 1,3,5,7-tetraoxo-1,2,3,5,6,7-hexahydro-s-indacen-2-yl]-2-oxoethyl}piperidine-1- carboxylate
Figure imgf000057_0003
[0244] In a similar manner to that of Example 197, the title compound was prepared from 1,3,5,7(2H,6H)-tetraone (as used in Example 55, 107 mg, 0.5 mmol) and 2-(1-(tert- butoxycarbonyl)piperidin-4-yl)acetic acid (CAS number,157688-46-5, 364 mg, 1.5 mmol) to provide the title compound as the bis sodium salt (180 mg, 54%). LC/MS (ESI+): Rf = 7.72 min, (M+H)+ = 665.89; HRMS (ESI-): calculated for C36H44N2O10 m/z [M-H]-: 663.2923 , observed 663.2937; 1H NMR δ 4.02-4.17 (m, 4H), 3.72-3.90 (m, 4H), 2.58-2.74 (m, 8H), 1.75- 2.00 (m, 2H), 1.39-1.71 (m, 4H), 1.26-1.44 (m, 18H), 0.88-1.12 (m, 4H) EXAMPLE 201 - 2,6-bis[4-(benzyloxy)butanoyl]-1,2,3,5,6,7-hexahydro-s-indacene- 1,3,5,7-tetrone
Figure imgf000058_0001
[0245] In a similar manner to that of Example 197, the title compound was prepared from 1,3,5,7(2H,6H)-tetraone (as used in Example 55, 107 mg, 0.5 mmol) and 4-(benzyloxy) butanoic acid (CAS number, 10385-30-5, 291 mg, 1.5 mmol) to provide the title compound as the bis sodium salt (176 mg, 62%). LC/MS (ESI+): Rf = 7.36 min, (M+H)+= 557.69; HRMS (ESI- ): calculated for C34H30O8 m/z [M-H]-: 565.1868, observed 565.1865; 1H NMR δ 7.30 (s, 10H), 7.26 (s, 2H), 4.42 (s, 4H), 3.44-3.42 (m, 4H), 2.77-2.75 (m, 4H), 1.77-1.72 (m, 4H). EXAMPLE 202 - 2,6-bis[2-(piperidin-4-yl)acetyl]-1,2,3,5,6,7-hexahydro-s-indacene- 1,3,5,7-tetrone
Figure imgf000058_0002
[0246] The title compound was prepared by treating the title compound from Example 200 (80 mg, 0.12 mmol) with 5N HCl solution 2 (mL). The mixture was heated at 50°C for 1 hr. The resulting solid was filtered, washed by water and dried to give the title compound (46 mg, 82%). LC/MS (ESI+): Rf = 2.65 min, (M+H)+= 465.55; HRMS (ESI-): calculated for C26H28N2O6 m/z [M+Na]-: 487.1840; observed 487.1832; 1H NMR δ 7.48-7.75 (m, 2H), 3.06-3.30 (m, 4H), 2.56-3.04 (m, 8H), 1.93-2.16 (m, 2H), 1.66-1.90 (m, 4H), 1.16-1.54 (m, 4H). EXAMPLE 203 - tert-butyl (2R)-2-{6-[(2R)-1-[(tert-butoxy)carbonyl]pyrrolidine-2- carbonyl]-1,3,5,7-tetraoxo-1,2,3,5,6,7-hexahydro-s-indacene-2-carbonyl}pyrrolidine-1- carboxylate
Figure imgf000058_0003
[0247] In a similar manner to that of Example 197, except that the sodium salt step was omitted, the title compound was prepared from 1,3,5,7(2H,6H)-tetraone (as used in Example 55, 107 mg, 0.5 mmol) and N-Boc-D-Proline (CAS number 37784-17-1, 323 mg, 1.5 mmol) to afford the title compound (132 mg, 44%). LC/MS (ESI+): Rf = 6.51 min, (M+H)+ = 609.65; HRMS (ESI-): calculated for C32H35N2O10 m/z [M-H]-: 607.229629, observed 607.229719; 1H NMR δ 7.61 (s, 2H), 5.24-5.47 (m, 2H), 2.13-2.28 (m, 4H), 1.78 (s, 8H), 1.37 (s, 6H), 1.19 (s, 12H). EXAMPLE 204 - tert-butyl N-{2-[6-(2-{[(tert-butoxy)carbonyl]amino}acetyl)-1,3,5,7- tetraoxo-1,2,3,5,6,7-hexahydro-s-indacen-2-yl]-2-oxoethyl}carbamate
Figure imgf000059_0001
[0248] In a similar manner to that of Example 197, except that the sodium salt step was omitted, the title compound was prepared from 1,3,5,7(2H,6H)-tetraone (as used in Example 55, 107 mg, 0.5 mmol) and 2-(tert-butoxy)-carbonylamino acetic acid (CAS number 4530-20- 5, 263 mg, 1.5 mmol) to afford the title compound (183 mg, 70%). LC/MS (ESI+): Rf = 5.36 min, (M+H)+ = 529.54; HRMS (ESI-): calculated for C26H27N2O10 m/z [M-H]-: 527.166803, observed 527.167119; 1H NMR δ 1H NMR δ 7.86-8.05 (m, 2H), 7.57 (s, J=3.62 Hz, 2H), 4.25 (s, 4H), 1.37 (s, 18H). EXAMPLE 205 - N-{2-[6-(2-acetamidoacetyl)-1,3,5,7-tetraoxo-1,2,3,5,6,7-hexahydro-s- indacen-2-yl]-2-oxoethyl}acetamide
Figure imgf000059_0002
[0249] In a similar manner to that of Example 197, except that the sodium salt step was omitted, the title compound was prepared from 1,3,5,7(2H,6H)-tetraone (as used in Example 55, 107 mg, 0.5 mmol) and N-acetyl glycine (CAS number 543-24-8, 176 mg, 1.5 mmol) to afford the title compound (142 mg, 69%). LC/MS (ESI+): Rf = 2.19 min, (M+H)+ = 413.66; HRMS (ESI-): calculated for C20H15N2O8 m/z [M-H]-: 411.082171, observed 411.083389;1H NMR δ 7.87-7.95 (m, 2H), 7.53 (s, 2H), 4.36 (d, J=3.52 Hz, 4H), 1.87 (s, 6H). EXAMPLE 206 - tert-butyl N-[(2S)-1-{6-[(2S)-2-{[(tert- butoxy)carbonyl]amino}propanoyl]-1,3,5,7-tetraoxo-1,2,3,5,6,7-hexahydro-s-indacen-2- yl}-1-oxopropan-2-yl]carbamate
Figure imgf000059_0003
[0250] In a similar manner to that of Example 197, except that the sodium salt step was omitted, the title compound was prepared from 1,3,5,7(2H,6H)-tetraone (as used in Example 55, 107 mg, 0.5 mmol) and N-Boc-L-alanine (CAS number 15761-38-3, 289 mg, 1.5 mmol) to afford the title compound (165 mg, 59%). LC/MS (ESI+): Rf = 5.74 min, (M+H)+ = 557.83; HRMS (ESI-): calculated for C28H31N2O10 m/z [M-H]-: 555.198053, observed 555.198419; 1H NMR δ 7.90-8.01 (m, 2H), 7.61 (s, J=4.24 Hz, 2H), 5.14 (d, J=7.00 Hz, 2H), 1.35 (s, 18H), 1.20 (d, J=7.03 Hz, 6H). EXAMPLE 207 - tert-butyl N-[(1R)-2-{6-[(2R)-2-{[(tert-butoxy)carbonyl]amino}-2- phenylacetyl]-1,3,5,7-tetraoxo-1,2,3,5,6,7-hexahydro-s-indacen-2-yl}-2-oxo-1- phenylethyl]carbamate
Figure imgf000060_0001
[0251] In a similar manner to that of Example 197, except that the sodium salt step was omitted, the title compound was prepared from 1,3,5,7(2H,6H)-tetraone (as used in Example 55, 107 mg, 0.5 mmol) and N-Boc-D-phenylglycine (CAS number 33125-05-2, 377 mg, 1.5 mmol). This material was further dissolved in 20% acetonitrile in water, filtered, and precipitated in 1N HCl. The material was then dissolved in methanol, filtered, and precipitated with 1N HCl. The resulting precipitant was filtered and dried as the title compound (36 mg, 11%). LC/MS (ESI+): Rf = 7.78 min, (M-Boc+H)+ = 581.83; HRMS (ESI-): calculated for C38H35N2O10 m/z [M-H]-: 679.229875, observed 679.229719; 1H NMR δ 7.46 (s, 2H), 7.41 (d, J=7.60 Hz, 4H), 7.23 (t, J=7.60 Hz, 4H), 7.15 (t, J=6.40 Hz, 2H), 6.43-6.56 (m, 2H), 1.35 (s, 18H), 1.20-1.27 (m, 2H). EXAMPLE 208 - tert-butyl (2S)-2-(6-{1-[(tert-butoxy)carbonyl]pyrrolidine-2-carbonyl}- 1,3,5,7-tetraoxo-1,2,3,5,6,7-hexahydro-s-indacene-2-carbonyl)pyrrolidine-1-carboxylate
Figure imgf000060_0002
[0252] In a similar manner to that of Example 197, except that the sodium salt step was omitted, the title compound was prepared from 1,3,5,7(2H,6H)-tetraone (as used in Example 55, 107 mg, 0.5 mmol) and N-Boc-L-proline (CAS 15761-39-4, 323 mg, 1.5 mmol). This material was further dissolved in methanol, filtered, and treated with 2N HCl. The resulting precipitant was filtered and dried as the title compound (10 mg, 3%). LC/MS (ESI+): Rf = 6.45 min, (M+H)+ = 509.82; HRMS (ESI-): calculated for C32H35N2O10 m/z [M-H]-: 607.229561, observed 607.229719; 1H NMR δ 7.61 (br. s, 2H), 5.27-5.43 (m, 2H), 3.32-3.40 (m, 4H), 2.14- 2.30 (m, 2H), 1.69-1.85 (m, 6H), 1.37 (br. s, 6H), 1.19 (br. s, 12H). Example 209. Alternative Synthesis of s-indacene-1,3,5,7(2h,6h)-tetraone.
Figure imgf000061_0001
[0253] Step 1) To a 250 ml 3-neck RB flask equipped with a stirbar, temperature probe and N2 balloon was charged pyromellitic anhydride (10.01 g, 45.89 mmol, 1 eq.) and ethyl acetoacetate (18.01 g, 138.39 mmol, 3.02 eq.). To this was added acetic anhydride (81.00 g, 793.39 mmol, 17.3 eq) to produce a clear slurry. To the slurry was charged triethylamine (40.02 g, 395.49 mmol, 8.62 eq.) dropwise via an addition funnel over ~5 min to produce a dark purple mixture. The reaction was then heated to 100°C for 2 hr. After stirring for 2 hr, the reaction was removed from the heat and allowed to cool to ambient temperature, then was placed in the freezer to age overnight. After ageing overnight, the deep purple slurry was isolated by filtration using a 70mm Buchner funnel and paper. The wet cake was then washed with once with 50 ml of acetic anhydride and then 1x50 ml if Et2O. The dull green/brown cake was allowed to dry via suction on the filter, prior to placement in the vacuum oven to dry at 45°C and full house vacuum. After drying, 2,6-diacetyl-1,2,3,5,6,7-hexahydro-s-indacene- 1,3,5,7-tetrone, bis triethyl amine salt was obtained as a dull green solid (13.36g, 52% yield). LCMS (ESI+): Rf=2.72, M+Na=381.33; 1H NMR MeOH δ: 7.58 (s, 2H), 4.88 (s, 6H), 4.20 (q, J = 7.23 Hz, 6H), 3.14-3.28 (m, 18H), 1.21-1.38 (m, 27H). [0254] Step 2) To a 1000 mL 3-neck flask was charged the starting material (5.07 g, 9.04 mmol) and a stirbar. The solid was dissolved by the addition of 500 mL of water to produce a deep red solution. The mixture was allowed to stir at ambient temperature for 15 min. The reaction was cooled to ~ 0°C in an ice water bath. To the cold reaction mixture was added 5 mL of H2SO4 causing the reaction to become a fine red slurry. The slurry was allowed to age for 15 min, then isolated by filtration. The dull red wet cake was washed with 100 mL of water. The cake was allowed to dry via suction on the filter. The red solid (3.51 g) was placed in a 500 ml flask equipped with a stirbar. To this was charged acetonitrile (200 mL) to produce a deep red slurry. The slurry was heated to reflux for 3 hr. Upon completion of the age, the reaction was removed from the heat and allowed to cool to ambient temperature. The reaction was now a fine slurry which was isolated by filtration. The fine pink solid was allowed to dry via suction on the filter, then placed in the vacuum oven to dry. The desired product was obtained as a fine red solid (7.43 g, 82% yield). LCMS (ESI+): Rf=3.08, M+H=215.411H NMR δ: 7.64 (br. s.1H), 3.24-3.67 (br. s.2H). Example 210 – Synthesis of N2,N6-dicyclobutyl-1,3,5,7-tetraoxo-1,2,3,5,6,7-hexahydro- s-indacene-2,6-dicarboxamide.
Figure imgf000062_0001
[0255] Using the procedure in Example 42, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and cyclobutyl isocyanate (CAS 5811-25-6). The compound was isolated as the bis sodium salt. LCMS (ESI+): Rf=6.84, M+H=409.48; HRMS (ESI-): calculated for C22H19N2O6 m/z [M-H]-: 407.124860, observed 407.125703; 1H NMR δ: 8.66 (d, J = 8.21 Hz, 1H), 7.15 (s, 1H), 4.35 (sxt, J = 8.32 Hz, 1H), 2.12-2.33 (m, 2H), 1.74-1.92 (m, 2H), 1.51-1.73 (m, 2H). Example 211 – Synthesis of N2,N6-bis(cyclopropylmethyl)-1,3,5,7-tetraoxo-1,2,3,5,6,7- hexahydro-s-indacene-2,6-dicarboxamide.
Figure imgf000062_0002
[0256] Using the procedure in Example 42, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and (isocyanatomethyl)cyclopropane (CAS 25694-89-7). The compound was isolated as the bis sodium salt. LCMS (ESI+): Rf=6.39, M+H=409.42; HRMS (ESI-): calculated for C22H18N2O6 m/z [M-H]-: 407.124860, observed 407.125765; 1H NMR δ: 7.16 (s, 1H), 3.03-3.14 (m, 2H), 0.84-1.01 (m, 1H), 0.35-0.49 (m, 2H), 0.11-0.24 (m, 2H). Example 212 – Synthesis of 2,6-dipentanoyl-1,2,3,5,6,7-hexahydro-s-indacene-1,3,5,7- tetrone.
Figure imgf000062_0003
[0257] Using the procedure in Example 197, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and pentanoic acid (CAS 109-52-4).. The compound was isolated as the bis sodium salt. LCMS (ESI+): RT = 7.55 min, (M+H)+ = 383.02, (M+MeCN)+ = 424.00; HRMS (ESI-): calculated for C22H21O6 m/z [M-H]-: 381.134362, observed 381.136501; 1H NMR δ: 7.30 (s, 2 H), 2.72 (t, J=7.6 Hz, 4 H), 1.39 - 1.53 (m, 4 H), 1.21-1.36 (m, 4 H), 0.86 (t, J=7.4 Hz, 6 H). Example 213 – Synthesis of tert-butyl N-{2-[6-(2-{[(tert-butoxy)carbonyl](methyl) amino}acetyl)-1,3,5,7-tetraoxo-1,2,3,5,6,7-hexahydro-s-indacen-2-yl]-2-oxoethyl}-N- methylcarbamate.
Figure imgf000063_0001
[0258] Using the procedure in Example 197, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and 2-{[(tert- butoxy)carbonyl](methyl)amino}acetic acid (CAS 13734-36-6). The compound was isolated as the bis sodium salt. LCMS (ESI+): RT = 5.35 min, (M+H)+ = 557.45; HRMS (ESI-): calculated for C28H31N2O10 m/z [M-H]-: 555.198419, observed 555.198711; 1H NMR 300 MHz DMSO δ: 7.32 (s.2H), 4.35 (s, 6 H), 2.77 (s, 2 H), 2.72 (s, 2 H), 1.39 (s, 9 H), 1.30 (s, 9 H). Example 214 – Synthesis of 2,6-bis(2-methoxyacetyl)-1,2,3,5,6,7-hexahydro-s- indacene-1,3,5,7-tetrone. O O O O O O O O [0259] Using the procedure in Example 197, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and methoxyacetic acid (CAS 625-45-6). LCMS (ESI+): RT = 2.97 min, (M+H)+ = 359.40, (M+Na)+ = 381.40; HRMS (ESI-): calculated for C18H13O8 m/z [M-H]-: 357.061591, observed 357.061752; 1H NMR δ: 7.60 (s, 2 H), 6.63- 7.11 (br. s, 2 H), 4.58 (s, 4 H), 3.31 (s, 6 H, coincident with H2O). Example 215 – Synthesis of 2,6-bis[2-(2-methoxyethoxy)acetyl]-1,2,3,5,6,7-hexahydro- s-indacene-1,3,5,7-tetrone.
Figure imgf000063_0002
[0260] Using the procedure in Example 197, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and 2-methoxyethoxy acetic acid (CAS 16024-56-9). The compound was isolated as the bis sodium salt. LCMS (ESI+): RT = 3.33 min, (M+H)+ = 447.35, (M+Na)+ = 469.36; HRMS (ESI-): calculated for C22H21O10 m/z [M- H]-: 445.114020, observed 445.115789; 1H NMR δ: 7.36 (s, 2 H), 4.50 (s, 4 H), 3.56 (t, J=5.0 Hz, 4 H), 3.45 (t, J=2.9 Hz, 4 H, coincident with solvent), 3.24 (s, 6 H). Example 216 – Synthesis of 2,6-dicyclobutanecarbonyl-1,2,3,5,6,7-hexahydro-s- indacene-1,3,5,7-tetrone.
Figure imgf000064_0001
[0261] Using the procedure in Example 197, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and cyclobutene carboxylic acid (CAS 3721- 95-7). The compound was isolated as the bis sodium salt. LCMS (ESI+): RT = 7.58 min, (M+H)+ = 379.44, (M+MeCN)+ = 420.42; HRMS (ESI-): calculated for C22H17O6 m/z [M-H]-: 377.103062, observed.377.102433; 1H NMR δ: 7.27 (s, 2 H), 4.10 - 4.25 (m, 2 H), 1.78 - 2.19 (m, 10 H), 1.60 - 1.75 (m, 2 H). Example 217 – Synthesis of N2,N6-bis[2-(2-methoxyethoxy)ethyl]-1,3,5,7-tetraoxo- 1,2,3,5,6,7-hexahydro-s-indacene-2,6-dicarboxamide.
Figure imgf000064_0002
[0262] Using the procedure in Example 42, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and 1-isocyanato-2-(2- methoxyethoxy)ethane (CAS 90426-82-7). The compound was isolated as the bis sodium salt. LCMS (ESI+): RT = 4.57 min, (M+H)+ = 505.41; HRMS (ESI-): calculated for C24H27N2O10 m/z [M-H]-: 503.167119, observed.503.165388; 1H NMR δ: 8.53 (t, J=5.6 Hz, 2 H), 7.13 (s, 2 H), 3.49 - 3.54 (m, 8 H), 3.42-3.47 (m, 8 H), 3.24 (s, 6 H). Example 218 – Synthesis of N2,N6-dicyclopentyl-1,3,5,7-tetraoxo-1,2,3,5,6,7- hexahydro-s-indacene-2,6-dicarboxamide.
Figure imgf000064_0003
[0263] Using the procedure in Example 42, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and 1-isocyanato-2-(2- methoxyethoxy)ethane (CAS 90426-82-7). The compound was isolated as the bis sodium salt. LCMS (ESI+): RT = 7.22 min, (M+H)+ = 437.43, (M+MeCN)+ = 478.40; HRMS (ESI-): calculated for C24H23N2O6 m/z [M-H]-: 435.156160, observed.435.154270; 1H NMR δ: 8.50 (d, J=7.6 Hz, 2 H), 7.13 (s, 2 H), 4.07 - 4.21 (m, 2 H), 1.77-1.89 (m, 4 H), 1.48 - 1.72 (m, 8 H), 1.30 - 1.44 (m, 4 H). Example 219 – Synthesis of 2,6-dicyclopropanecarbonyl-1,2,3,5,6,7-hexahydro-s- indacene-1,3,5,7-tetrone.
Figure imgf000065_0001
[0264] Using the procedure in Example 197, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and cyclopropane carboxylic acid (CAS 1759-53-1). The compound was isolated as the bis sodium salt. LCMS (ESI+): RT = 6.40 min, (M+H)+ = 351.36, (M+MeCN)+ = 392.40; HRMS (ESI-): calculated for C20H13O6 m/z [M-H]-: 349.071762, observed.349.070417; 1H NMR δ: 7.33 (s, 2 H), 3.36 - 3.47 (m, 2 H), 0.72 - 0.81 (m, 4 H), 0.55 - 0.66 (m, 4 H). Yield: 79%. Example 220 – Synthesis of 2,6-bis({2-[2-(2-methoxyethoxy)ethoxy]acetyl})-1,2,3,5,6,7- hexahydro-s-indacene-1,3,5,7-tetrone.
Figure imgf000065_0002
[0265] Using the procedure in Example 197, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and 2-[2-(2-methoxyethoxy)ethoxy]acetic acid (CAS 16024-58-1). LCMS (ESI+): RT = 3.43 min, (M+H)+ = 535.44, (M+Na)+ = 557.38; HRMS (ESI-): calculated for C26H29O12 m/z [M-H]-: 533.166450, observed.533.168533; 1H NMR δ: 7.54 (s, 2 H), 4.61 (s, 4 H), 3.47-3.68 (m, 10 H), 3.38 - 3.46 (m, 8 H), 3.21 (s, 6 H). Example 221 – Synthesis of 2,6-bis[2-(2-oxopyrrolidin-1-yl)acetyl]-1,2,3,5,6,7- hexahydro-s-indacene-1,3,5,7-tetrone.
Figure imgf000065_0003
[0266] Using the procedure in Example 197, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and 2-oxo-1-pyrrolidineacetic acid (CAS 53934-76-2). LCMS (ESI+): RT = 2.63 min, (M+H) = 465.70; 1H NMR δ = 7.47 (s, 2H), 4.44 (s, 4H), 3.38 - 3.29 (m, 4H), 2.29 - 2.17 (m, 4H), 1.94 (br d, J=7.0 Hz, 4H). Example 222 – Synthesis of tert-butyl (2S)-2-{6-[(2S)-1-[(tert- butoxy)carbonyl]piperidine-2-carbonyl]-1,3,5,7-tetraoxo-1,2,3,5,6,7-hexahydro-s- indacene-2-carbonyl}piperidine-1-carboxylate.
Figure imgf000066_0001
[0267] Using the procedure in Example 197, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and N-(tert-butoxycarbonyl)-L-pipecolic acid (CAS 26250-84-0). LCMS (ESI+): RT = 6.20 min, (M+H) = 637.30; HRMS (ESI-): Predicted: 635.2610, found: 635.2616. Example 223 – Synthesis of tert-butyl 3-{2-[6-(2-{1-[(tert-butoxy)carbonyl]pyrrolidin-3- yl}acetyl)-1,3,5,7-tetraoxo-1,2,3,5,6,7-hexahydro-s-indacen-2-yl]-2- oxoethyl}pyrrolidine-1-carboxylate.
Figure imgf000066_0002
[0268] Using the procedure in Example 197, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and N-(tert-butoxycarbonyl)-3- pyrrolidineaceticacid (CAS number 175526-97-3). LCMS (ESI+): RT = 6.50 min, (M+Na) = 659.89; HRMS (ESI-): Predicted: 635.2610, found: 635.2600; 1H NMR δ = 7.62 (s, 2H), 3.49 - 3.23 (m, 4H), 3.22 - 3.06 (m, 4H), 3.04 - 2.93 (m, 2H), 2.92 - 2.79 (m, 4H), 2.04 - 1.84 (m, 4H), 1.36 (br s, 18H). Example 224 – Synthesis of tert-butyl N-{2-[6-(2-{[(tert- butoxy)carbonyl](methyl)amino} acetyl)-1,3,5,7-tetraoxo-1,2,3,5,6,7-hexahydro-s- indacen-2-yl]-2-oxoethyl}-N-methylcarbamate.
Figure imgf000066_0003
[0269] Using the procedure in Example 197, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and N-(tert-butoxycarbonyl)-N-methylglycine (CAS number 13734-36-6). LCMS (ESI+): RT = 4.63 min, (M+H) = 557.32; HRMS (ESI-): Predicted: 555.1984, found: 555.1976; 1H NMR δ = 8.46 - 8.30 (m, 2H), 7.43 - 7.34 (m, 2H), 2.83 - 2.68 (m, 4H), 2.56 (s, 6H), 1.39 (s, 10H), 1.28 (s, 8H). Example 225 – Synthesis of 2,6-bis[2-(tert-butoxy)acetyl]-1,2,3,5,6,7-hexahydro-s- indacene-1,3,5,7-tetrone.
Figure imgf000067_0001
[0270] Using the procedure in Example 197, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and 2-tert-butoxyacetic acid (CAS number 13211-32-0). LCMS (ESI+): RT = 4.47 min, (M+Na) = 464.89; HRMS (ESI-): Predicted: 441.1555, found: 441.1547.1H NMRδ = 7.63 (br s, 2H), 4.64 - 4.53 (m, 4H), 1.17 (br s, 18H). Example 226 – Synthesis of tert-butyl N-[(2S)-1-{6-[(2S)-2-{[(tert- butoxy)carbonyl]amino}-4-methylpentanoyl]-1,3,5,7-tetraoxo-1,2,3,5,6,7-hexahydro-s- indacen-2-yl}-4-methyl-1-oxopentan-2-yl]carbamate.
Figure imgf000067_0002
[0271] Using the procedure in Example 197, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and N-(tert-butoxycarbonyl)-leucine (CAS number 13139-15-6). LCMS (ESI+): RT = 6.82 min, (M+H) = 641.76; HRMS (ESI-): Predicted: 639.2923, found: 639.2934; 1H NMR δ = 6.58 (s, 2H), 5.86 - 5.63 (m, 2H), 4.31 - 4.03 (m, 2H), 1.49 - 1.21 (m, 18H), 0.64 (br d, J=6.4 Hz, 2H), 0.39 - 0.04 (m, 18H), -0.05 - -0.28 (m, 4H) Example 227 – Synthesis of N-methyl-N-(2-{6-[2-(N-methylacetamido)acetyl]-1,3,5,7- tetraoxo-1,2,3,5,6,7-hexahydro-s-indacen-2-yl}-2-oxoethyl)acetamide.
Figure imgf000067_0003
[0272] Using the procedure in Example 197, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and N-acetyl-N-methylglycine (CAS number 5888-91-5). LCMS (ESI+): RT = 2.52 min, (M+Na) = 462.93; HRMS (ESI-): Predicted: 439.1147, found: 439.1141; 1H NMR δ = 7.53 (br s, 2H), 4.67 - 4.54 (m, 4H), 2.97 (d, J=5.3 Hz, 3H), 2.75 (s, 3H), 2.02 (s, 3H), 1.82 (d, J=4.1 Hz, 3H). Example 228 – Synthesis of 2,6-bis[3-(2-methoxyethoxy)propanoyl]-1,2,3,5,6,7- hexahydro-s-indacene-1,3,5,7-tetrone.
Figure imgf000068_0001
[0273] Using the procedure in Example 197, the title compound was prepared from s- indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and 3-(2-methoxyethoxy)propanoic acid (CAS number 149577-05-9). LCMS (ESI+): RT = 4.05 min, (M+H) = 475.57; HRMS (ESI-): Predicted: 473.1453, found: 473.1454. Example 229 – Synthesis of 2,6-bis[(2S)-piperidine-2-carbonyl]-1,2,3,5,6,7-hexahydro- s-indacene-1,3,5,7-tetrone.
Figure imgf000068_0002
[0274] To a solution of tert-butyl (2S)-2-{6-[(2S)-1-[(tert-butoxy)carbonyl]piperidine-2- carbonyl]-1,3,5,7-tetraoxo-1,2,3,5,6,7-hexahydro-s-indacene-2-carbonyl}piperidine-1- carboxylate (Example 222, 70 mg, 0.1 mmol) in CH2Cl2 (1 mL) was added 4 N hydrochloric acid in dioxane (0.375 mL, 1.5 mmol) and the reaction was stirred at ambient temperature. After 90 min, LCMS indicated the complete consumption of starting material. The solvent is removed under vacuum to provide 56 mg (quant.) of the title compound as a bis-hydrogen chloride salt. LCMS (ESI+): RT = 2.52 min, (M+H) = 437.03; HRMS (ESI+): Predicted: 437.1707, found: 437.1698; 1H NMR (300MHz, DMSO-d6) δ = 8.65 (br d, J=10.5 Hz, 2H), 8.44 - 8.23 (m, 2H), 7.37 (s, 2H), 4.62 - 4.48 (m, 2H), 3.26 - 3.13 (m, 2H), 2.89 (br d, J=10.0 Hz, 2H), 2.15 - 2.02 (m, 2H), 1.84 - 1.49 (m, 10H), 1.38 - 1.18 (m, 2H). Example 230 – Synthesis of 2,6-bis[2-(pyrrolidin-3-yl)acetyl]-1,2,3,5,6,7-hexahydro-s- indacene-1,3,5,7-tetrone.
Figure imgf000068_0003
[0275] Using the procedure in Example 229, the title compound was prepared from tert- butyl 3-{2-[6-(2-{1-[(tert-butoxy)carbonyl]pyrrolidin-3-yl}acetyl)-1,3,5,7-tetraoxo-1,2,3,5,6,7- hexahydro-s-indacen-2-yl]-2-oxoethyl}pyrrolidine-1-carboxylate (Example 223) as the bis hydrogen chloride salt. LCMS (ESI+): RT = 2.47 min, (M+H) = 437.63; HRMS (ESI+): Predicted: 437.1707, found 437.1704. Example 231 – Synthesis of 2,6-bis[(2S)-2-amino-4-methylpentanoyl]-1,2,3,5,6,7- hexahydro-s-indacene-1,3,5,7-tetrone.
Figure imgf000069_0001
[0276] Using the procedure in Example 229, the title compound was prepared tert-butyl N- [(2S)-1-{6-[(2S)-2-{[(tert-butoxy)carbonyl]amino}-4-methylpentanoyl]-1,3,5,7-tetraoxo- 1,2,3,5,6,7-hexahydro-s-indacen-2-yl}-4-methyl-1-oxopentan-2-yl]carbamate (Example 226) as the bis hydrogen chloride salt. LCMS (ESI+): RT = 2.78 min, (M+H) = 441.41; HRMS (ESI+): Predicted: 441.2020, found: 441.2016; 1H NMR (300MHz, METHANOL-d4) δ = 7.62 - 7.43 (m, 2H), 5.20 - 5.07 (m, 2H), 1.92 - 1.54 (m, 6H), 1.12 (br d, J=5.9 Hz, 6H), 1.00 (br d, J=5.9 Hz, 6H). Example 232 – Synthesis of 2,6-bis[2-(methylamino)acetyl]-1,2,3,5,6,7-hexahydro-s- indacene-1,3,5,7-tetrone. O O HN NH O O O O [0277] Using the procedure in Example 229, the title compound was prepared tert-butyl N- {2-[6-(2-{[(tert-butoxy)carbonyl](methyl)amino}acetyl)-1,3,5,7-tetraoxo-1,2,3,5,6,7-hexahydro- s-indacen-2-yl]-2-oxoethyl}-N-methylcarbamate (Example 224) as the bis hydrogen chloride salt. LCMS (ESI+): RT = 1.93 min, (M+H) = 356.89; 1H NMR (300MHz, DMSO-d6) δ = 7.42 - 7.33 (m, 2H), 4.12 (br t, J=5.9 Hz, 4H), 2.62 - 2.51 (m, 6H). Example 233 – Synthesis of N2,N6-bis({2-[2-(2-methoxyethoxy)ethoxy]ethyl})-1,3,5,7- tetraoxo-1,2,3,5,6,7-hexahydro-s-indacene-2,6-dicarboxamide.
Figure imgf000069_0002
[0278] Step 1) To a solution of triphosgene (1.36 g, 4.5 mmol) in CH2Cl2 (13.8 mL) under nitrogen at 0°C was added pyridine (2.07 mL, 25.7 mmol). After stirring 20 min, a solution of 2-(2-(2-methoxyethoxy)ethoxy) ethanamine (CAS reg. no: 74654-07-2, 734 mg, 4.5 mmol) in CH2Cl2 (13.8 mL) was added dropwise to this mixture. The reaction was warmed to ambient temperature and stirred 45 min. The reaction was quenched by the addition of water (35 mL) and the organic phase was extracted (2x) with chloroform (35 mL), and dried over sodium sulfate. The solvent was removed in vacuo to provide crude 1-[2-(2-isocyanato-ethoxy)- ethoxy]-2-methoxy-ethane which was used as-is in the next step. [0279] Step 2) Using the procedure in Example 42, the title compound was prepared from s-indacene-1,3,5,7(2h,6h)-tetraone (Example 209) and 1-[2-(2-isocyanato-ethoxy)-ethoxy]-2- methoxy-ethane from step 1 above. LCMS (ESI+): RT = 4.08 min, (M+H) = 593.66; HRMS (ESI-): Predicted: 591.2195, found: 591.2181; 1H NMR (300MHz, METHANOL-d4) δ = 7.66 (s, 2H), 3.67 (s, 20H), 3.56 - 3.49 (m, 4H), 3.32 (s, 6H). Example 234 – Synthesis of 2,6-bis[2-(propan-2-yloxy)acetyl]-1,2,3,5,6,7-hexahydro-s- indacene-1,3,5,7-tetrone.
Figure imgf000070_0001
[0280] To a stirred solution of 2-(propan-2-yloxy)acetic acid (CAS reg. no: 33445-07-7, 177 mg, 1.5 mmol) and DMAP (183 mg, 1.5 mmol) in dry DMF (10 mL) was added EDC.HCl (288 mg, 1.5 mmol). The mixture was stirred under N2 for 10 min and 1,2,3,5,6,7-hexahydro-s- indacene-1,3,5,7-tetrone (CAS reg. no: 53910-13-7, 107 mg, 0.5 mmol) was added. The dark mixture was stirred at rt for 20 h. 5% aq HCl (15 mL) was added, and the mixture was stirred at rt for 15 min. The mixture was filtered and the solid collected was dried under high vacuum to give a black solid (160 mg). A sample of this black solid (61 mg) was sonicated with CH2Cl2 (1 mL) and 1 M aq NaOH (1 mL) for 2 min and stirred at rt for 1 h. The mixture was filtered, washing with water (2 x 4 mL). The filter cake was dried under high vacuum to give the title compound as its disodium salt (37 mg, 16%) as a black solid. 1H NMR (300MHz, DMSO-d6) Shift = 7.33 - 7.20 (m, 2H), 4.43 (s, 4H), 3.64-3.52 (m, 2H), 1.14 – 1.02 (m, 12H). LC-MS tR 3.88 min, m/z 437 [M+Na+]. HRMS -ve ion, calcd for C22H21O8413.124191; found 413.123141. Example 235. Synthesis of N2,N6-bis[(1,1-dioxo-1λ⁶-thiolan-3-yl)methyl]-1,3,5,7- tetraoxo-1,2,3,5,6,7-hexahydro-s-indacene-2,6-dicarboxamide.
Figure imgf000070_0002
[0281] A stirred solution of 1,2,3,5,6,7-hexahydro-s-indacene-1,3,5,7-tetrone (CAS reg. no: 53910-13-7, 107 mg, 0.5 mmol) in dry DMF (10 mL) was cooled to ca. -45 °C in a dry ice/MeCN bath and triethylamine (0.18 mL, 1.3 mmol) was added, followed after 5 min by 3- (isocyanatomethyl)-1λ⁶-thiolane-1,1-dione (CAS reg. no: 28800-41-1, 230 mg, 1.30 mmol). The cooling bath was allowed to expire, and the mixture was stirred at rt for 4h. 5% aq HCl (15 mL) was added and, after stirring for 15 min, the mixture was filtered. The filter cake was washed with 5% aq HCl (5 mL) and water (5 mL), and dried under high vacuum to leave a black solid (200 mg). A portion of this black solid (168 mg) was stirred with 1 M aq NaOH (2 mL) and CH2Cl2 (2 mL) for 2 h, and filtered, washing with water. The filtrate was concentrated and the residue was purified by prep HPLC to give the title compound (35 mg, 12%) as an orange solid. 1H NMR (300MHz, DMSO-d6) Shift = 8.93 - 8.68 (m, 2H), 7.42 (s, 2H), 3.53 - 3.33 (m, 4H), 3.30 - 3.17 (m, 4H), 3.14 - 2.96 (m, 2H), 2.90 - 2.79 (m, 2H), 2.75 - 2.57 (m, 2H), 2.32 - 2.08 (m, 2H), 1.90 - 1.67 (m, 2H). LC-MS +ve ion tR 3.32 min, m/z 565. HRMS -ve ion, calcd for C24H23N2O10S2: 563.079961; found 563.079728. Example 236. Synthesis of N2,N6-bis(1,1-dioxo-1λ⁶-thiolan-3-yl)-1,3,5,7-tetraoxo- 1,2,3,5,6,7-hexahydro-s-indacene-2,6-dicarboxamide.
Figure imgf000071_0001
[0282] A stirred solution of 1,2,3,5,6,7-hexahydro-s-indacene-1,3,5,7-tetrone (CAS reg. no: 53910-13-7, 107 mg, 0.5 mmol) in dry DMF (10 mL) was cooled to ca. -45 °C in a dry ice/MeCN bath, and triethylamine (0.18 mL, 1.3 mmol) was added, followed after 5 min by 3-isocyanato- 1λ⁶-thiolane-1,1-dione (CAS reg. no: 24373-66-8, 210 mg, 1.30 mmol). The cooling bath was allowed to expire, and the stirred mixture reached rt. After 3 h, 5% aq HCl (15 mL) was added, the mixture was stirred for 15 min and filtered. The filter cake was washed with 5% aq HCl (5 mL) and water (5 mL), and dried under high vacuum to give a red solid. A portion of this red solid (152 mg) was stirred with 1 M aq NaOH (2 mL) and CH2Cl2 (2 mL) for 2 h, and filtered, washing with water (2 mL). The filter cake was dried under high vacuum to give the disodium salt of the title compound (130 mg, 44%) as a black solid. 1H NMR (300MHz, DMSO-d6) Shift = 8.88 - 8.67 (m, 2H), 7.21 (s, 2H), 4.71-4.45 (m, 2H), 3.30 - 3.15 (m, 6H), 3.10-2.90 (m, 2H), 2.46-2.30 (m, 2H), 2.21-2.00 (m, 2H). LC-MS +ve ion tR 2.83 min, m/z 537. HRMS -ve ion, calcd for C22H19N2O10S2: 535.048660; found 535.047575. Example 237 - Synthesis of ethyl 3-[6-(3-ethoxy-3-oxopropanoyl)-1,3,5,7-tetraoxo- 1,2,3,5,6,7-hexahydro-s-indacen-2-yl]-3-oxopropanoate.
Figure imgf000071_0002
[0283] To a stirred solution of 3-ethoxy-3-oxopropanoic acid (CAS reg. no: 1071-46-1, 198 mg, 1.5 mmol) and DMAP (183 mg, 1.5 mmol) in dry DMF (10 mL) was added EDC.HCl (290 mg, 1.5 mmol). The mixture was stirred under N2 for 10 min and 1,2,3,5,6,7-hexahydro-s- indacene-1,3,5,7-tetrone (CAS reg. no: 53910-13-7, 107 mg, 0.5 mmol) was added. The dark mixture was stirred at rt for 20 h. 5% aq HCl (15 mL) was added, and the mixture was stirred at rt for 15 min. The mixture was filtered, washing with water (5 mL), and the solid collected was dried under high vacuum to give a black solid (200 mg). A sample of this black solid (140 mg) was stirred with CH2Cl2 (2 mL) and satd aq NaHCO3 (2 mL) for 2 h and filtered. The filter cake was washed with CH2Cl2 and water, and dried under high vacuum to give the disodium salt of title compound (80 mg, 32%) as a black solid. 1H NMR (300MHz, DMSO-d6) Shift = 7.40 (s, 2H), 4.03 (q, J=7.0 Hz, 4H), 3.82 (s, 4H), 1.16 (t, J=7.0 Hz, 6H). LC-MS tR 3.48 min, m/z 465. HRMS -ve ion, calcd for C22H17O10: 441.082720; found 441.083125. Example 238 - Synthesis of N2,N6-bis(1-methanesulfonylpiperidin-4-yl)-1,3,5,7- tetraoxo-1,2,3,5,6,7-hexahydro-s-indacene-2,6-dicarboxamide.)
Figure imgf000072_0001
[0284] A stirred solution of 1,2,3,5,6,7-hexahydro-s-indacene-1,3,5,7-tetrone (CAS reg. no: 53910-13-7, 100 mg, 0.47 mmol) in dry DMF (9 mL) was cooled to ca. -45 °C in a dry ice/MeCN bath and triethylamine (0.16 mL, 1.16 mmol) was added, followed after 5 min by a solution 4- isocyanato-1-methanesulfonylpiperidine (CAS reg. no: 1016886-27-3, 250 mg, 1.22 mmol) in dry DMF (1 mL). The cooling bath was allowed to expire and the mixture was stirred overnight at rt. 5% aq HCl (15 mL) was added. The mixture was stirred at rt for 15 min and filtered. The solid collected was washed with water (4 mL) and dried under high vacuum to leave a black solid (320 mg). A portion of this black solid (266 mg) was stirred with CH2Cl2 (2 mL) and 1 M aq NaOH (2 mL) at rt for 2 h and filtered. The filter cake was dried under high vacuum to give the disodium salt of the title compound (100 mg, 32%) as a dark solid. 1H NMR (300MHz, DMSO-d6) Shift = 8.67 - 8.44 (m, 2H), 7.19 (s, 2H), 3.95 - 3.72 (m, 2H), 3.58-3.38 (m, 4H), 2.99-2.80 (m, 10H), 1.99-1.82 (m, 4H), 1.62 - 1.28 (m, 4H). LC-MS +ve ion tR 3.93 min, m/z 623. HRMS -ve ion, calcd for C26H29N4O10S2: 621.133059; found: 621.132367. Example 239. Synthesis of 3-[6-(2-carboxyacetyl)-1,3,5,7-tetraoxo-1,2,3,5,6,7- hexahydro-s-indacen-2-yl]-3-oxopropanoic acid.
Figure imgf000072_0002
[0285] The filtrate from Example 235 (Synthesis of ethyl 3-[6-(3-ethoxy-3-oxopropanoyl)- 1,3,5,7-tetraoxo-1,2,3,5,6,7-hexahydro-s-indacen-2-yl]-3-oxopropanoate) was concentrated to leave a grey solid which was taken up in water (4 mL) and 1 M aq NaOH (4 mL). The mixture was stirred at rt for 16 h and filtered. The black solid collected was dried under high vacuum to give the tetrasodium salt of the title compound (30 mg). 1H NMR 1H NMR (300MHz, DEUTERIUM OXIDE) Shift = 7.45 (s, 2H), 3.57 (s, 4H). LC-MS +ve ion tR 2.45 min, m/z 387. HRMS -ve ion, calcd for C18H9O10385.020120; found 385.020064. Example 240. Synthesis of N2,N6-bis(2-methanesulfonylethyl)-1,3,5,7-tetraoxo- 1,2,3,5,6,7-hexahydro-s-indacene-2,6-dicarboxamide.
Figure imgf000073_0002
[0286] A stirred solution of 1,2,3,5,6,7-hexahydro-s-indacene-1,3,5,7-tetrone (CAS reg. no: 53910-13-7, 107 mg, 0.5 mmol) in dry DMF (10 mL) was cooled to ca. -40 °C in a dry ice/MeCN bath. Triethylamine (0.18 mL, 1.3 mmol) was added, followed after 5 min by 1-isocyanato-2- methanesulfonylethane (CAS reg. no: 631912-32-8, 195 mg, 1.3 mmol). The cooling bath was allowed to expire, and the mixture was stirred at rt overnight.5% aq HCl (15 mL) was added and the mixture was stirred for 15 min, then filtered, washing with water (5 mL). The dark red solid collected was suspended in CH2Cl2 (2 mL) and 1 M aq NaOH (2 mL), and stirred for 1 h. The mixture was filtered, washing with water. The solid collected was dried under high vacuum to give the disodium salt of the title compound (110 mg, 56%) as a black solid. 1H NMR (300MHz, DMSO-d6) Shift = 8.73 - 8.48 (m, 2H), 7.19 (s, 2H), 3.60 (br d, J=6.2 Hz, 4H), 3.39 - 3.16 (m, 4H), 2.97 (s, 6H). LC-MS +ve ion tR 2.90 min, m/z 513. HRMS -ve ion, calcd for C20H19N2O10S2: 511.048660; found 511.047109. Example 242 - Synthesis of N2,N6-bis[2-(2-methanesulfonylethoxy)ethyl]-1,3,5,7- tetraoxo-1,2,3,5,6,7-hexahydro-s-indacene-2,6-dicarboxamide
Figure imgf000073_0001
[0287] Step 1 [0288] To a stirred, ice cold mixture of 2-(2-methanesulfonylethoxy)ethan-1-amine HCl salt (CAS reg. no: 947664-66-6, 341 mg, 1.7 mmol), CH2Cl2 (6 mL) and satd aq NaHCO3 (2 mL) was added a solution of triphosgene (175 mg, 0.59 mmol) in CH2Cl2 (2 mL). The mixture was stirred in the ice bath for 1.5 h and extracted with CH2Cl2 (2 x 25 mL). The combined CH2Cl2 layer was washed with brine (2 mL), dried over Na2SO4 and concentrated to leave 1- isocyanato-2-(2-methanesulfonylethoxy)ethane (148 mg, 45%) as an oil which was used directly without characterization. [0289] Step 2 [0290] A stirred solution of 1,2,3,5,6,7-hexahydro-s-indacene-1,3,5,7-tetrone (CAS reg. no: 53910-13-7, 53 mg, 0.25 mmol) and 1-isocyanato-2-(2-methanesulfonylethoxy)ethane (148 mg, 0.76 mmol) in dry DMF (5 mL) was cooled to ca. -45°C in a dry ice/MeCN bath. Triethylamine (0.10 mL, 0.72 mmol) was added and stirring was continued as the cooling bath expired. After stirring overnight at rt, 5% aq HCl (7 mL) was added and stirring was continued for 5 min. The mixture was filtered, washing with water (4 mL). The filter cake was dried in vacuo to give a dark solid (184 mg). This material was stirred with CH 2Cl2 (2 mL) and 1 M aq NaOH at rt for 1.5 h and filtered. The filter cake was dried in vacuo to give a black solid (130 mg). A sample of this material (76 mg) was stirred with 5% aq HCl (2 mL) for 1 h and filtered. The black solid collected was dried under high vacuum to give the title compound (50 mg, 33%).1H NMR (300MHz, DMSO-d6) Shift = 9.08 - 8.55 (m, 2H), 7.54 (s, 2H), 3.79 (t, J=5.6 Hz, 4H), 3.75-3.48 (m, 8H), 3.42 - 3.30 (m, 4H), 2.99 (s, 6H). LC-MS +ve ion tR 3.48 min, m/z 601. HRMS -ve ion, calcd for C24H27N2O12S2599.101090; found 599.101410. EXAMPLE A1 - Inhibition of Pf4 Tetramer Formation [0291] Compounds of Examples 39-64 and 197-208 were tested using SDS PAGE analysis of BS3-crosslinked PF4 for their ability to prevent PF4 tetramer formation. ULCs containing PF4 tetramers were distinguished from PF4 alone using dynamic light scattering (DLS). See, Suvarna et al., Throm. Res, 122:211-220 (2008), which is hereby incorporated by reference, at least for a detailed description of this technique. [0292] cDNA encoding human PF4 was cloned into the plasmid pMT/BiPN5-His A (Invitrogen Corp.) for expression in the Drosophila Expression System (Invitrogen). Cloning was performed using Bg1 II and Age I cloning sites. A hexanucleotide encoding Bg1 II site was then eliminated by site-directed mutagenesis so that the expressed protein contained full- length wild type (wt) PF4 or PF4K50E with an identical sequence as their counterparts expressed in E. coli. See, method described in Sachais et al (Blood 119(25): 5955-5961, 2012). PF4 expression was induced by adding copper sulfate (0.5 mM). The induced S2 cells were incubated in serum-free Insect-Xpress™ media (Lonza) for 3-5 days; supernatants were collected, sodium azide (0.02% final concentration) and EDTA (2.5 mM final concentration) were added, and the media were filtered through an Express® PLUS 0.22 μm filter (Millipore Corp.). [0293] Wild-type (WT) human PF4 in the pT7-7 vector (Novagen) was expressed in the Escherichia coli strain BL21DE30 pLysS (Stratagene), and purified and characterized as described by Rauova et al. (Blood 107(6): 2346 - 2353, 2006) and Park et al., (Blood, 75:1290- 1295, 1990), which are hereby incorporated by reference. Briefly, recombinant protein was isolated from the supernatant of the bacterial lysate by affinity chromatography using a HiTrap high-performance (HP) affinity column. WT PF4 was purified from the media on a heparin HiTrap® column on an ÄTKAPrime™ system (GE Healthcare) at 4°C in Tris (10 mM), EDTA (1 mM), and pH 8 buffer. Media was loaded in buffer containing NaCl (0.5 M) and PF4 was eluted at 1.8 M NaCl using a linear gradient. Fractions containing purified PF4 as detected by silver staining of 12% NuPAGE Bis-Tris gels (Invitrogen) were pooled, concentrated and buffer exchanged into 50 mM HEPES, 0.5 M NaCl, pH - 7.2 using an Amicon® Ultra centrifugal filter (3K NMWL, Millipore Corp). Protein was quantified using a BCA assay. Experiment 1 [0294] Compounds of Examples 39-64 and 197-208 were then tested for their ability to inhibit PF4 tetramer formation using a cross-linking assay as described in Rauova cited above. Specifically, hPF4 (10 µg/mL) in phosphate-buffered saline (PBS) was incubated at 37°C for 15 min in the absence of the antagonistic compounds (control sample) and presence of compounds. Cross-linking and SDS-PAGE analysis were then performed as described in “Analysis of PF4 multimerization” of Rauova. The relative amounts of PF4 in each of the monomer, dimer, trimer and tetramer form were calculated by analyzing the samples on a 12% SDS-polyacrylamide gel under reducing conditions. Bands were quantified using the Kodak ID Image Analysis system (Kodak). The SigmaMarker™ reagent served as the molecular weight standard. Data was compiled and bar graphed as % PF4 for the tetramer, trimer, dimer, and monomer as a function of concentration for the test and control samples. These data show that certain compounds were effective in inhibiting PF4 tetramer formation at tested concentrations. Experiment 2 [0295] This experiment was performed in a manner similar to experiment 1, but PF4 (10 μg/mL) in PBS was independently incubated with compounds of Examples 39-64 and 197- 208 for 60 minutes at room temperature, followed by the addition of cross-linking reagent bis(sulfosuccinimidyl)suberate (0.2 mM) for 30 minutes at room temperature. The reaction was stopped by adding NuPAGE® lithium dodecyl sulfate (LDS) sample buffer, and denatured by heating to 70°C for 10 min according to the manufacturer's instructions. The analysis and compilation of the data also was performed as described above in Experiment 1. Table 1 shows the data for the PF4 tetramerization of these compounds. [0296] The IC50 data for inhibition of PF4 tetramerization is shown in Table 1 below. Table 1
Figure imgf000076_0001
Figure imgf000076_0002
Figure imgf000077_0001
[0297] In general, the active compounds are acidic and have low log D values. The clog D (pH 7.4) calculated values range from 1.7 to -13 and therefore should generally have good aqueous solubility. Since most compounds are bis-acids, with pKa values ranging from 6.5 to -1.7, most should have poor cell permeability. These characteristics are beneficial for IV administration and also for engagement of the molecular target PF4 which is a blood protein. EXAMPLE A2 - Inhibition of formation of ultra large complexes (ULC) of PF4 tetramers and heparin [0298] Since ULCs are formed as part of a dynamic equilibrium between heparin and PF4, dynamic light scattering (DLS) was performed to measure the ability of the exemplified compounds to disrupt preformed ULCs. Samples were analyzed and included PF4 incubated in the absence or presence of unfractionated heparin (UFH) for 20 min at room temperature at PF4 to heparin ratios (PHR) of 1:0 (no heparin), 2:1, and 0.64:1. The samples were then analyzed using photon correlation spectroscopy on a DynaPro® DLS instrument and Dynamics® software (V6.7.6; Microsoft) to obtain correlation function. The results of the DLS analysis indicate the percentage of ULCs and small PF4 particles. Data for the small particles (a population of particles with a mean hydrodynamic diameter of about 1 nm) and data for the large particles (a population of particles with a mean hydrodynamic diameter of about 300 nm) is assessed. Data are expressed as the percent of total intensity from each measurement, the mean of 10 measurements, and representative of two such experiments. Larger amounts of heparin result in the disruption of larger particles, i.e., ULCs, demonstrated by the presence of virtually all of the PF4 as small particles (data not shown). [0299] Since the PF4 antagonists discussed herein are capable of disrupting preformed ULCs, the following experiment was performed to determine if the exemplified compounds inhibit formation of ULCs. [0300] ELISA measurement and gel filtration measurement of ULC inhibition are performed as described in International Patent Publication No. WO2013/142328, which is incorporated herein by reference. Similarly, assays in which heparin concentrations are varied to determine the effect on ULC inhibition are performed as described in that publication. [0301] Certain compounds were evaluated for their ability to inhibit formation of ultra large complexes (ULC) between PF4 tetramers and heparin. The results are illustrated in Table 2 below. Table 2. ULC Inhibition and Cytotoxicity f
Figure imgf000078_0001
EXAMPLE A3 – Modulation of antibody binding to ULC [0302] The effect of the exemplified compounds on antibody binding to intact ULCs was evaluated. ULC were formed as described above, with the exception that the PF4 was incubated with heparin for 30 min. Certain compounds (1 mM) were then added to wells pre- coated with KKO (monoclonal hit like antibody). These solutions were then incubated in the KKO wells overnight at 37ºC. Antibody binding was detected by adding HRP (horseradish peroxicase)-conjugated sheep polyclonal anti-human PF4 antibody and developed with TMB substrate. After stopping the enzymatic reaction with 1 M H3PO4, absorbance was measured at 450 nm in a Packard SpectraCount™ plate reader. EXAMPLE A4 - Inhibition of platelet activation and serotonin release assay [0303] Since one of the therapeutic goals of using ULC antagonists in patients with HITT is to prevent activation, compounds were evaluated for the ability to inhibit platelet activation. Data were generated using a serotonin release assay as described in Hirschman (Br. J. Haematol., 24(6):793-802, 1973), and Rauova and Cines (N. Engl. J. Med., 303:788-795, 1980), which are hereby incorporated by reference. See also WO2013/142328. [0304] In this assay, platelet rich plasma (PRP) from healthy donors was incubated with 0.5 µL 14C-5-hydroxytryptamine creatinine sulfate (GE Life Sciences) per milliliter of PRP for 20 min at 37°C to produce 14C-labeled platelets. Serotonin uptake is inhibited by adding 1 mmol/mL imipramine (Sigma-Aldrich) to the PRP. The radiolabeled platelets are mixed with KKO (170 μg/mL) or with known platelet-activating HIT plasma in the absence (buffer control) or presence of a selected PF4 antagonist compound of Table 1 (2.5 – 3.0 mM). [0305] Negative and positive controls contain sera from patients previously known to have negative or positive serotonin release, respectively. The assay was performed in the presence of heparin (1.0 U/mL), i.e., heparin added to the positive control serum, and in the absence of heparin (background). The percent release of serotonin is calculated for all conditions. Negative controls without antibody were studied in parallel.14C-5-hydroxytryptamine released from platelets was measured by scintillation counter. Data are expressed as % maximal release of radioactivity with release by the positive control plasma and 1.0 U/mL heparin defined as 100%. Compounds that inhibit platelet activation by KKO are measured by the serotonin release assay. (Rauova, L. et al. (2005). Blood 105(1): 131-138). EXAMPLE A5 - ADMET (absorption, distribution, metabolism, excretion and toxicity) properties [0306] Certain exemplified compounds were examined for ADMET (absorption, distribution, metabolism, excretion and toxicity) profiling using conventional assays with the results shown in the Table 3 below. TABLE 3 ADMET Human and Mouse Microsome Stability Data
Figure imgf000079_0001
[0307] As shown by Table 3, for representative compounds described herein, no serious ADMET issues have arisen and the compounds meet drug property criteria for intravenous administration. The tested compounds meet drug property criteria for IV administration. EXAMPLE A6 – P-Selectin Inhibition Assay Experimental protocol [0308] FcγRIIA transgenic murine blood was drawn from inferior vena cava under anesthesia into a 1 ml syringe with 10% volume of acid citrate dextrose (ACD). Whole blood was diluted with 1x Tyrode buffer (5 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid, 0.1% BSA, 134 mM NaCl, 0.3 mM NaH2PO4, 3 mM KCl, 2 mM MgCl2, 12 mM NaHCO3, 5 mM glucose) with 0.02 U/ml apyrase and 1 µM PGE1 and centrifuged at 100 g for 10 min. Platelet- rich plasma (PRP) was carefully removed and centrifuged at 1000 g for 5 min. Platelet pellet was resuspended in Tyrode buffer with 0.02 U/ml apyrase at 2 x 108/ml concentration at 37°C. PF4 antagonists are diluted in tyrode buffer, and are incubated with 10 µg/mL human PF4 purified protein at room temperature for 30 min. The PF4-drug complex is then incubated with 1×108/mL platelets with 1mM CaCl2, 0.1% BSA at 37°C for 10 min.40 µg/mL KKO and 10 µg/mL anti-CD62P (P-selectin) antibodies are added into the reaction.1 mL ice-cold PBS is added to each reaction after 5 min of incubation. Labelled platelets are then analyzed by a BD Accuri C6 flow cytometer. The results are represented by both the mean fluorescence intensity and by the percentage of positive platelets. EXAMPLE A7 - P-Selectin Levels in Human Platelets [0309] Figures 5 and 6 show effect of PF4 concentration, a combination of PF4 and heparin on P-selectin levels in human platelets. Experimental protocol: [0310] For 100 µL reaction, add 2.5 × 107/mL human platelets, add 1.5 mM CaCl2, 37.5 µg/mL or 65 µg/mL PF4, 10 µ/mL Heparin (2 µL of 500 U/mL), with or without 100 µM of the exemplified compound (5 µl of 2 mM) for 30 min. Add KKO at 50 µg/mL (2 µL 2.6 mg/mL KKO) all at RT for 20 min. FL2-A is P-selectin. [0311] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” As used herein the terms "about" and “approximately” means within 10 to 15%, preferably within 5 to 10%. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. [0312] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention. [0313] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims. [0314] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. [0315] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of” excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein. [0316] Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above-cited references and printed publications are individually incorporated herein by reference in their entirety. [0317] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

Claims

What is claimed is: 1. A compound of Formula (II):
Figure imgf000083_0001
or a pharmaceutically acceptable salt thereof, wherein: R1 and R3 are each independently selected from H, C1-6 alkylcarbonyl, C(O)NH(Ra1), and C(O)Cy4, wherein C1-6 alkyl in the C1-6 alkylcarbonyl is optionally substituted with 1, 2, or 3 substituents independently selected from Cy4, C6-10 aryloxy, C1- 3 alkoxy, -NH(C1-6 alkoxycarbonyl), and –NH(C1-6 alkylcarbonyl), wherin C1-6 alkoxy is optionally substituted with C6-10 aryl; R2 and R4 are each independently selected from H and C1-6 alkylcarbonyl; each Ra1 is independently selected from C1-6 alkyl, C1-6 alkenyl, Cy1, C1-6 alkoxycarbonyl, and S(O)2Ra2, wherein the C1-6 alkyl is optionally substituted with 1, 2, or 3 substituents independently selected from Cy2, carboxyl, C1-3 alkoxycarbonyl, C1-3 alkoxy, and C1-3 haloalkoxy wherein the C1-3 alkoxy is optionally substituted with C1-3 alkoxy; each Cy1 is independently selected form C3-10 cycloalkyl, C6-10 aryl, and 5-10 membered heteroaryl, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from halo, C1-3 alkyl, C1-3 alkoxy and Cy3; each Cy2 is independently selected from C6-10 aryl and 5-10 membered heteroaryl, each of which is optionally substituted with 1, 2, or 3 substituents independently selected from halo, C1-3 alkyl, and C1-3 haloalkoxy; each Cy3 is independently selected form C6-10 aryl and 5-10 membered heteroaryl; each Cy4 is independently selected from C6-10 aryl, C3-10 cycloalkyl, 4-10 membered heterocycloalkyl, and 5-10 membered heteroaryl, each of which is optionally substituted with C1-6 alkoxycarbonyl; each Ra2 is C6-10 aryl, optionally substituted with C1-3 alkyl, with a proviso that the compound of Formula (II) is not any one of the following compounds:
Figure imgf000084_0001
. 2. The compound of claim 1, wherein R1, R2, R3 and R4 are each independently selected from H and C1-6 alkylcarbonyl. 3. The compound of claim 1, wherein R1 and R3 are each C1-6 alkylcarbonyl. 4. The compound of claim 1, wherein R1 and R3 are each C(O)NH(Ra1). 5. The compound of claim 1 or claim 4, wherein each Ra1 is independently selected from propyl, isopropyl, sec-butyl, allyl, phenyl, tosyl, ethoxycarbonyl, methoxyphenyl, nitrophenyl, methylphenyl, ethoxyphenyl, dihydrobenzodioxinyl, dichloropyridinyl, benzyl, fluorobenzyl, (pyrimidin-2-yl)phenyl, cyclopropyl, phenylcyclopropyl, phenylethyl, (trifluoromethoxyphenyl)ethyl, (ethoxycarbonyl)methyl, (ethoxycarbonyl)ethyl, phenylpropyl, (fluorophenyl)ethyl, (chlorophenyl)ethyl, (methylphenyl)ethyl, furanylmethyl, benzodioxolylmethyl, benzodioxolylethyl, (2-methoxyethoxy)ethyl, (2,2,2- trifluoroethoxy)ethyl, (carboxyl)ethyl, and (carboxyl)methyl. 6. The compound of claim 1 or claim 4, wherein each Ra1 is independently selected from C1-6 alkyl, C1-6 alkenyl, Cy1, C1-6 alkoxycarbonyl, and S(O)2Ra2, wherein the C1-6 alkyl is optionally substituted with 1, 2, or 3 substituents independently selected from Cy2, carboxyl and C1-3 alkoxycarbonyl. 7. The compound of claim 6, wherein each Ra1 is independently selected from propyl, isopropyl, sec-butyl, allyl, phenyl, tosyl, ethoxycarbonyl, methoxyphenyl, methylphenyl, ethoxyphenyl, dihydrobenzodioxinyl, dichloropyridinyl, benzyl, fluorobenzyl, (pyrimidin-2- yl)phenyl, cyclopropyl, phenylcyclopropyl, phenylethyl, (trifluoromethoxyphenyl)ethyl, (ethoxycarbonyl)methyl, (ethoxycarbonyl)ethyl, phenylpropyl, (fluorophenyl)ethyl, (chlorophenyl)ethyl, (methylphenyl)ethyl, furanylmethyl, benzodioxolylmethyl, benzodioxolylethyl, (carboxyl)ethyl, and (carboxyl)methyl.
8. The compound of claim 1 or claim 4, wherein each Ra1 is independently selected from C1-6 alkyl, C1-6 alkenyl, Cy1, and S(O)2Ra2, wherein the C1-6 alkyl is optionally substituted with 1, 2, or 3 substituents independently selected from Cy2, carboxyl, C1-3 alkoxycarbonyl. 9. The compound of claim 8, wherein each Ra1 is independently selected from propyl, isopropyl, sec-butyl, allyl, tosyl, methylphenyl, ethoxyphenyl, benzyl, (pyrimidin-2-yl)phenyl, cyclopropyl, phenylcyclopropyl, phenylethyl, (trifluoromethoxyphenyl)ethyl, (ethoxycarbonyl)ethyl, phenylpropyl, (fluorophenyl)ethyl, (chlorophenyl)ethyl, (methylphenyl)ethyl, benzodioxolylethyl, and (carboxyl)ethyl. 10. The compound of any one of claims 1, 4, 6 or 8, wherein each Cy1 is independently selected form phenyl, methoxyphenyl, ethoxyphenyl, dihydrobenzodioxinyl, dichloropyridinyl, cyclopropyl, methylphenyl, nitrophenyl, (pyrimidin-2-yl)phenyl, and phenylcyclopropyl. 11. The compound of any one of claims 1, 4, 6 or 8, wherein each Cy1 is independently selected form C3-10 cycloalkyl and C6-10 aryl, each of which is optionally substituted with 1 or 2 substituents independently selected from C1-3 alkyl, C1-3 alkoxy and Cy3. 12. The compound of claim 11, wherein each Cy1 is independently selected from ethoxyphenyl, cyclopropyl, methylphenyl, (pyrimidin-2-yl)phenyl, and phenylcyclopropyl. 13. The compound of any one of claims 1, 4, 6, 8, or 10-12, wherein each Cy2 is independently selected from phenyl, trifluoromethoxyphenyl, fluorophenyl, chlorophenyl, methylphenyl, furanyl, and benzodioxolyl. 14. The compound of any one of claims 1, 4, 6, 8, or 10-12, wherein each Cy2 is C6-10 aryl, optionally substituted with 1 or 2 substituents independently selected from halo, C1-3 alkyl, and C1-3 haloalkoxy. 15. The compound of claim 14, wherein each Cy2 is independently selected from phenyl, trifluoromethoxyphenyl, fluorophenyl, chlorophenyl, methylphenyl, and benzodioxolyl. 16. The compound of any one of claims 1, 4, 6, 8, 11, or 13-15, wherein each Cy3 is C6-10 aryl. 17. The compound of any one of claims 1, 4, 6, 8, 11, or 13-15, wherein each Cy3 is 5-10 membered heteroaryl. 18. The compound of any one of claims 1, 4, 6, 8, 11, or 13-15, wherein each Cy3 is independently selected from phenyl and pyrimidinyl. 19. The compound of any one of claims 1, 4, 6, 8, or 10-18, wherein each Ra2 is phenyl, optionally substituted with methyl. 20. The compound of any one of claims 1 or 16-19, wherein: R1 and R3 are each C(O)NH(Ra1); each Ra1 is independently selected from C1-6 alkyl, C1-6 alkenyl, Cy1, C1-6 alkoxycarbonyl, and S(O)2Ra2, wherein the C1-6 alkyl is optionally substituted with 1, 2, or 3 substituents independently selected from Cy2, carboxyl and C1-3 alkoxycarbonyl; each Cy1 is independently selected form C3-10 cycloalkyl and C6-10 aryl, each of which is optionally substituted with 1 or 2 substituents independently selected from C1-3 alkyl, C1-3 alkoxy and Cy3; and each Cy2 is C6-10 aryl, optionally substituted with 1 or 2 substituents independently selected from halo, C1-3 alkyl, and C1-3 haloalkoxy. 21. The compound of any one of claims 1 or 16-19, wherein: R1 and R3 are each C(O)NH(Ra1); each Ra1 is independently selected from C1-6 alkyl, C1-6 alkenyl, Cy1, and S(O)2Ra2, wherein the C1-6 alkyl is optionally substituted with 1, 2, or 3 substituents independently selected from Cy2, carboxyl, C1-3 alkoxycarbonyl; each Cy1 is independently selected form C3-10 cycloalkyl and C6-10 aryl, each of which is optionally substituted with 1 or 2 substituents independently selected from C1-3 alkyl, C1-3 alkoxy and Cy3; and each Cy2 is C6-10 aryl, optionally substituted with 1 or 2 substituents independently selected from halo, C1-3 alkyl, and C1-3 haloalkoxy. 22. The compound of any one of claims 1 or 4-21, wherein the compound of Formula (II) has Formula (IIa):
Figure imgf000086_0001
or a pharmaceutically acceptable salt thereof. 23. The compound of claim 1, wherein: R2 and R 4 are each H; and R1 and R3 are each independently selected from C1-6 alkylcarbonyl and C(O)Cy4, wherein C1-6 alkyl in the C1-6 alkylcarbonyl is optionally substituted with 1, 2, or 3 substituents independently selected from Cy4, C6-10 aryloxy, C1-3 alkoxy, -NH(C1-6 alkoxycarbonyl), and –NH(C1-6 alkylcarbonyl), wherin C1-6 alkoxy is optionally substituted with C6-10 aryl; 24. The compound of claim 23, wherein: R2 and R4 are each H; and R1 and R3 are each independently an C1-6 alkylcarbonyl, wherein C1-6 alkyl in the C1-6 alkylcarbonyl is optionally substituted with 1 or 2 substituents independently selected from Cy4, C6-10 aryloxy, C1-3 alkoxy, -NH(C1-6 alkoxycarbonyl), and –NH(C1-6 alkylcarbonyl), wherin C1-6 alkoxy is optionally substituted with C6-10 aryl. 25. The compound of any one of claims 1-3, 5-19, 23, or 24, wherein C1-6 alkylcarbonyl is selected from: methylcarbonyl, ethylcarbonyl, and n-propylcarbonyl, wherein the methyl, ethy, and n-propyl are each optionally substituted with 1 or 2 substituents indepenently selected from Cy4, phenoxy, benzoxy, -NHC(O)(t-butoxy), and -NH(acetyl). 26. The compound of claim 24, wherein R1 and R3 are each independently an C1-6 alkylcarbonyl, wherein C1-6 alkyl in the C1-6 alkylcarbonyl is substituted Cy4. 27. The compound of claim 26, wherein C1-6 alkylcarbonyl is selected from: methylcarbonyl, ethylcarbonyl, and n-propylcarbonyl, wherein the methyl, ethy, and n-propyl are each substituted with Cy4. 28. The compound of claim 23, wherein the compound of Formula (II) has formula (IIb):
Figure imgf000087_0001
or a pharmaceutically acceptable salt thereof. 29. The compound of any one of claims 1 or 23-28, wherein each Cy4 is independently selected from cyclopentyl, pyridinyl, piperidinyl, pyrrolidinyl, and phenyl, each of which is optionally substituted with (t-butoxy)carbonyl. 30. The compound of any one of claims 23-27, wherein each Cy4 is independently selected from: cyclopentyl, pyridinyl, piperidinyl, and phenyl, each of which is optionally substituted with (t-butoxy)carbonyl. 31. The compound of claim 28, wherein each Cy4 is independently selected from C3-10 cycloalkyl and 4-10 membered heterocycloalkyl. 32. The compound of claim 31, wherein each Cy4 is independently selected from cyclopentyl, piperidinyl, and pyrrolidinyl, each of which is optionally substituted with (t- butoxy)carbonyl. 33. The compound of claim 28, wherein each Cy4 is independently a 4-10 membered heterocycloalkyl, which is optionally substituted with C1-6 alkoxycarbonyl.
34. The compound of claim 1, wherein the compound of Formula (II) is selected from any one of the following compounds
Figure imgf000088_0001
Figure imgf000089_0001
O O O O
Figure imgf000090_0001
Figure imgf000091_0001
or a pharmaceutically acceptable salt thereof. 35. A pharmaceutical composition comprising a compound of any one of claims 1-34, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. 36. A method of inhibiting platelet factor-4 (PF4) in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1-34, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 35, wherein the inhibition of PF4 comprises preventing formation of PF4 tetramers, and/or disrupting PF4 tetramers, and/or preventing formation of an ultra-large complex (ULC) comprising a PF4 tetramer and a glycosaminoglycan (GAG), and/or inhibiting ULC- antibody complex binding to a FcγRIIa receptor on a platelet, and/or inhibiting platelet aggregation, and/or increasing high density lipoproteins, and/or modulating clotting or hemostasis, and/or correcting a platelet imbalance in the subject.
37. The method of claim 36, wherein the compound of any one of claims 1-34 binds to a PF4 monomer, PF4 dimer, or PF4 tetramer. 38. The method of claim 36 or claim 37, wherein the compound of any one of claims 1-34 disrupts a salt bridge between two PF4 dimers, two or more PF4 monomers, or a PF4 dimer and a PF4 monomer, in a PF4 tetramer. 39. The method of claim 38, wherein the disruption occurs on a dimer-dimer interface. 40. The method of claims 38 or claim 39, wherein the salt bridge in the PF4 tetramer is formed via an electrostatic interaction of a negatively charged amino acid of a first PF4 monomer or PF4 dimer and a positively charged amino acid of a second PF4 monomer or PF4 dimer. 41. The method of claim 40, wherein the negatively charged amino acid of a first PF4 monomer or PF4 dimer is a glutamic acid. 42. The method of claim 40 or 41, wherein the positively charged amino acid of a second PF4 monomer or PF4 dimer is a lysine. 43. The method of any one of claims 36-42, wherein the GAG is a heparin. 44. The method of any one of claims 36-43, wherein the platelet imbalance results from heparin administration to the subject. 45. The method of any one of claims 36-44, wherein molecular weight of the ULC is greater than about 600 kD. 46. The method of any one of claims 36-45, wherein inhibiting the binding of a ULC- antibody complex to a FcγRIIa receptor on a platelet in a subject inhibits an activation of the platelet. 47. The method of claim 46, wherein an antibody in the ULC-antibody complex recognizes a complex composed of heparin and a PF4 tetramer. 48. A method of treating or preventing a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1-34, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 35, wherein the disease or condition is selected from heparin induced thrombocytopenia and thrombosis (HITT), a thrombotic complication of HITT, heparin induced thrombocytopenia (HIT), vaccine-induced immune thrombotic thrombocytopenia (VITT), atherosclerosis or atherosclerotic vascular disease, decrease in platelet production, inflammation or an inflammatory disease, antiphospholipid syndrome, platelet imbalance or insufficiency, and a clotting or hemostasis disorder.
49. The method of claim 48, wherein the disease or condition is mediated by a PF4 tetramer. 50. The method of claim 48 or claim 49, wherein the atherosclerosis results from a PF4 tetramer formation or a formation of a GAG-PF4 complex. 51. The method of claim 48 or claim 49, wherein the thrombotic complication of HITT is thrombosis. 52. The method of claim 51, wherein the thrombosis is characterized by lower than normal thrombin-antithrombin complex level. 53. A pharmaceutical composition comprising a compound selected from:
Figure imgf000093_0001
or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. A method of inhibiting platelet factor-4 (PF4) in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a compound selected from:
Figure imgf000093_0002
or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 53, wherein the inhibition of PF4 comprises preventing formation of PF4 tetramers, and/or disrupting PF4 tetramers, and/or preventing formation of an ultra-large complex (ULC) comprising a PF4 tetramer and a glycosaminoglycan (GAG), and/or inhibiting ULC- antibody complex binding to a FcγRIIa receptor on a platelet, and/or inhibiting platelet aggregation, and/or increasing high density lipoproteins, and/or modulating clotting or hemostasis, and/or correcting a platelet imbalance in the subject. 54. The method of claim 54, wherein the compound binds to a PF4 monomer, PF4 dimer or PF4 tetramer. 55. The method of claim 54 or claim 55, wherein the compound disrupts a salt bridge between two PF4 dimers, two or more PF4 monomers, or a PF4 dimer and a PF4 monomer, in a PF4 tetramer.
56. The method of claim 56, wherein the disruption occurs on a dimer-dimer interface. 57. The method of claims 56 or claim 57, wherein the salt bridge in the PF4 tetramer is formed via an electrostatic interaction of a negatively charged amino acid of a first PF4 monomer or PF4 dimer and a positively charged amino acid of a second PF4 monomer or PF4 dimer. 58. The method of claim 58, wherein the negatively charged amino acid of a first PF4 monomer or PF4 dimer is a glutamic acid. 59. The method of claim 58 or claim 59, wherein the positively charged amino acid of a second PF4 monomer or PF4 dimer is a lysine. 60. The method of any one of claims 54-60, wherein the GAG is a heparin. 61. The method of any one of claims 54-61, wherein the platelet imbalance results from heparin administration to the subject. 62. The method of any one of claims 54-62, wherein molecular weight of the ULC is greater than about 600 kD. 63. The method of any one of claims 54-63, wherein inhibiting the binding of a ULC- antibody complex to a FcγRIIa receptor on a platelet in a subject inhibits an activation of the platelet. 64. The method of claim 64, wherein an antibody in the ULC-antibody complex recognizes a complex composed of heparin and a PF4 tetramer. 65. A method of treating or preventing a disease or condition in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a compound selected from:
Figure imgf000094_0001
or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 53, wherein the disease or condition is selected from heparin induced thrombocytopenia and thrombosis (HITT), a thrombotic complication of HITT, heparin induced thrombocytopenia (HIT), vaccine-induced immune thrombotic thrombocytopenia (VITT), atherosclerosis or atherosclerotic vascular disease, decrease in platelet production, inflammation or an inflammatory disease, antiphospholipid syndrome, platelet imbalance or insufficiency, and a clotting or hemostasis disorder.
66. The method of claim 66, wherein the disease or condition is mediated by a PF4 tetramer. 67. The method of claim 66 or claim 67, wherein the atherosclerosis results from a PF4 tetramer formation or a formation of a GAG-PF4 complex. 68. The method of claim 66 or claim 67, wherein the thrombotic complication of HITT is thrombosis. 69. The method of claim 69, wherein the thrombosis is characterized by lower than normal thrombin-antithrombin complex level.
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