BRM Targeting Compounds And Associated Methods Of Use

The disclosure is directed to compounds of Formula I   Pharmaceutical compositions comprising compounds of Formula I, as well as methods of their use and preparation, are also described.

TECHNICAL FIELD

The description provides bifunctional compounds comprising a target protein binding moiety and a E3 ubiquitin ligase binding moiety, and associated methods of use. The bifunctional compounds are useful as modulators of targeted ubiquitination, especially with respect to Switch/Sucrose Non-Fermentable (SWI/SNF)-Related, Matrix-Associated, Actin-Dependent Regulator of Chromatin, Subfamily A, Member 2 (SMARCA2) (i.e., BRAHMA or BRM), which are degraded and/or otherwise inhibited by bifunctional compounds according to the present disclosure.

BACKGROUND

The human SWItch/Sucrose Non-Fermentable (SWI/SNF) complexes are ATP-dependent chromatin remodelers. These large complexes play important roles in essential cellular processes, such as transcription, DNA repair and replication by regulating DNA accessibility.

Mutations in the genes encoding up to 20 canonical SWI/SNF subunits are observed in nearly 20% of all human cancers with the highest frequency of mutations observed in rhabdoid tumors, female cancers (including ovarian, uterine, cervical and endometrial), lung adenocarcinoma, gastric adenocarcinoma, melanoma, esophageal, and renal clear cell carcinoma.

SMARCA2 (BRM) and SMARCA4 (BRG1) are the subunits containing catalytic ATPase domains and they are essential for the function of SWI/SNF in perturbation of histone-DNA contacts, thereby providing access points to transcription factors and cognate DNA elements that facilitate gene activation and repression.

SMARCA2 and SMARCA4 shares a high degree of homology (up to 75%). SMARCA4 is frequently mutated in primary tumors (i.e., deleted or inactivated), particularly in lung cancer (12%), melanoma, liver cancer and pancreatic cancer. SMARCA2 is one of the top essential genes in SMARCA4-mutant (deleted) cancer cell line. This is because SMARCA4 deleted cancer cells exclusively rely on SMARCA2 ATPase activity for their chromatin remodeling activity for cellular functions such as cell proliferation, survival and growth. Thus, targeting SMARCA2 may be promising therapeutic approach in SMARCA4-related or deficient cancers (genetic synthetic lethality).

Previous studies have demonstrated the strong synthetic lethality using gene expression manipulation such as RNAi; downregulating SMARCA2 gene expression in SMARCA4 mutated cancer cells results in suppression of cancer cell proliferation. However, SMARCA2/4 bromodomain inhibitors (e.g., PFI-3) exhibit none to minor effects on cell proliferation inhibition [Vangamudi et al. Cancer Res 2015]. This phenotypic discrepancy between gene expression downregulation and small molecule-based approach lead us to investigating protein degradation bispecific molecules in SMARCA4 deficient cancers.

SMARCA2 is also reported to play roles in multiple myeloma expressing t(4; 14) chromosomal translocation [Chooi et al. Cancer Res abstract 2018]. SMARCA2 interacts with NSD2 and regulates gene expression such as PRL3 and CCND1. SMARCA2 gene expression downregulation with shRNA reduces cell cycle S phase and suppresses cell proliferation of t(4; 14) MM cells.

SUMMARY

The present disclosure is directed to compounds of Formula (I):

Stereoisomers of the compounds of Formula I, and the pharmaceutical salts and stereoisomers thereof, are also contemplated, described, and encompassed herein. Methods of using compounds of Formula I are described, as well as pharmaceutical compositions including the compounds of Formula I.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

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 this disclosure pertains. The terminology used in the description is for describing particular embodiments only and is not intended to be limiting of the disclosure.

The following terms are used to describe the present disclosure. In instances where a term is not specifically defined herein, that term is given an art-recognized meaning by those of ordinary skill applying that term in context to its use in describing the present disclosure.

The articles “a” and “an” as used herein and in the appended claims are used herein to refer to one or to more than one (e.g., to at least one) of the grammatical object of the article unless the context clearly indicates otherwise. By way of example, “an element” means one element or more than one element.

The terms “co-administration” and “co-administering” or “combination therapy” refer to both concurrent administration (administration of two or more therapeutic agents at the same time) and time varied administration (administration of one or more therapeutic agents at a time different from that of the administration of an additional therapeutic agent or agents), as long as the therapeutic agents are present in the patient to some extent, preferably at effective amounts, at the same time. In certain preferred aspects, one or more of the present compounds described herein, are co-administered in combination with at least one additional bioactive agent, especially including an anticancer agent. In particularly preferred aspects, the co-administration of compounds results in synergistic activity and/or therapy, including anticancer activity.

The term “compound”, as used herein, unless otherwise indicated, refers to any specific chemical compound disclosed herein and includes tautomers, regioisomers, geometric isomers, and where applicable, stereoisomers, including optical isomers (enantiomers) and other stereoisomers (diastereomers) thereof, as well as pharmaceutically acceptable salts and derivatives, including prodrug and/or deuterated forms thereof where applicable, in context. Deuterated small molecules contemplated are those in which one or more of the hydrogen atoms contained in the drug molecule have been replaced by deuterium.

Within its use in context, the term compound generally refers to a single compound, but also may include other compounds such as stereoisomers, regioisomers and/or optical isomers (including racemic mixtures) as well as specific enantiomers or enantiomerically enriched mixtures of disclosed compounds. The term also refers, in context to prodrug forms of compounds which have been modified to facilitate the administration and delivery of compounds to a site of activity. It is noted that in describing the present compounds, numerous substituents and variables associated with same, among others, are described. It is understood by those of ordinary skill that molecules which are described herein are stable compounds as generally described hereunder.

The term “ubiquitin ligase” refers to a family of proteins that facilitate the transfer of ubiquitin to a specific substrate protein, targeting the substrate protein for degradation. For example, an E3 ubiquitin ligase protein that alone or in combination with an E2 ubiquitin-conjugating enzyme causes the attachment of ubiquitin to a lysine on a target protein, and subsequently targets the specific protein substrates for degradation by the proteasome. Thus, E3 ubiquitin ligase alone or in complex with an E2 ubiquitin conjugating enzyme is responsible for the transfer of ubiquitin to targeted proteins. In general, the ubiquitin ligase is involved in polyubiquitination such that a second ubiquitin is attached to the first; a third is attached to the second, and so forth. Polyubiquitination marks proteins for degradation by the proteasome. However, there are some ubiquitination events that are limited to mono-ubiquitination, in which only a single ubiquitin is added by the ubiquitin ligase to a substrate molecule. Mono-ubiquitinated proteins are not targeted to the proteasome for degradation, but may instead be altered in their cellular location or function, for example, via binding other proteins that have domains capable of binding ubiquitin. Further complicating matters, different lysines on ubiquitin can be targeted by an E3 to make chains. The most common lysine is Lys48 on the ubiquitin chain. This is the lysine used to make polyubiquitin, which is recognized by the proteasome.

As used herein, “Cereblon (CRBN) E3 Ubiquitin Ligase” refers to the substrate recognition subunit of the Cullin RING E3 ubiquitin ligase complexes. CRBN are one of the most popular E3 ligases recruited by bifunctional Proteolysis-targeting chimeras (PROTACs) to induce ubiquitination and subsequent proteasomal degradation of a target protein (Maniaci C. et al., Bioorg Med Chem. 2019, 27(12): 2466-2479).

As used herein, the term “alkyl”, by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain hydrocarbon radical having up to twelve carbon atoms. In some embodiments, the number of carbon atoms is designated (i.e., C1-C8means one to eight carbons). Examples of alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl, iso-butyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Alkyl groups may be optionally substituted as provided herein. In some embodiments, the alkyl group is a C1-C6alkyl; in some embodiments, it is a C1-C4alkyl.

When a range of carbon atoms is used herein, for example, C1-C6, all ranges, as well as individual numbers of carbon atoms are encompassed. For example, “C1-C3” includes C1-C3, C1-C2, C2-C3, C1, C2, and C3.

The term “optionally substituted”, as used in combination with a substituent defined herein, means that the substituent may, but is not required to, have one or more hydrogens replaced with one or more suitable functional groups or other substituents as provided herein. For example, a substituent may be optionally substituted with one or more of: halo, cyano, C1-6alkyl, C3-6cycloalkyl, C2-6alkenyl, C2-6alkynyl, halo(C1-6)alkyl, C1-6alkoxy, halo(C1-6alkoxy), C1-6alkylthio, C1-6alkylamino, NH2, NH(C1-6alkyl), N(C1-6alkyl)2, NH(C1-6alkoxy), N(C1-6alkoxy)2, —C(O)NHC1-6alkyl, —C(O)N(C1-6alkyl)2, —C(O)NH2, —C(O)C1-6alkyl, —C(O)2C1-6alkyl, NHCO(C1-6alkyl), —N(C1-6alkyl)CO(C1-6alkyl), —S(O)C1-6alkyl, —S(O)2C1-6alkyl, oxo, 6-12 membered aryl, benzyl, pyridinyl, pyrazolyl, thiazolyl, isothiazolyl, or other 5 to 12 membered heteroaryl groups. In some embodiments, each of the above optional substituents are themselves optionally substituted by one or two groups.

The term “optionally substituted —CH2—,” refers to “—CH2—” or substituted —CH2—.” A substituted —CH2— may also be referred to as —CH(substituent)- or —C(substituent)(substituent)-, wherein each substituent is independently selected from the optional substituents described herein.

The term “cycloalkyl” as used herein refers to a 3-12 membered cyclic alkyl group, and includes bridged and spirocycles (e.g., adamantine). Cycloalkyl groups may be fully saturated or partially unsaturated. The term “cycloalkyl” also includes multiple condensed ring systems (e.g., ring systems comprising 2, 3 or 4 rings) wherein a single cycloalkyl ring (as defined above) can be condensed with one or more groups selected from heterocycles, carbocycles, aryls, or heteroaryls to form the multiple condensed ring system. Such multiple condensed ring systems may be optionally substituted with one or more (e.g., 1, 2, 3 or 4) oxo groups on the carbocycle or heterocycle portions of the multiple condensed ring. The rings of the multiple condensed ring system can be connected to each other via fused, spiro and bridged bonds when allowed by valency requirements. It is to be understood that the individual rings of the multiple condensed ring system may be connected in any order relative to one another. It is also to be understood that the point of attachment of a multiple condensed ring system (as defined above for a cycloalkyl) can be at any position of the cycloalkylic ring. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, cyclohexyl, cycloheptyl, cyclooctyl, indenyl, bicyclo[2.2.1]heptanyl, bicyclo[3.1.1]heptanyl, bicyclo[4.1.0]heptanyl, spiro[3.3]heptanyl, and spiro[3.4]octanyl. In some embodiments, the cycloalkyl group is a 3-7 membered cycloalkyl.

The term “akenyl” as used herein refers to C2-C12alkyl group that contains at least one carbon-carbon double bond. In some embodiments, the alkenyl group is optionally substituted. In some embodiments, the alkenyl group is a C2-C6alkenyl.

The term “akynyl” as used herein refers to C2-C12alkyl group that contains at least one carbon-carbon triple bond. In some embodiments, the alkenyl group is optionally substituted. In some embodiments, the alkynyl group is a C2-C6alkynyl.

The terms “alkoxy,” “alkylamino” and “alkylthio”, are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom (“oxy”), an amino group (“amino”) or thio group. The term “alkylamino” includes mono- di-alkylamino groups, the alkyl portions can be the same or different.

The terms “halo” or “halogen”, by itself or as part of another substituent, means a fluorine, chlorine, bromine, or iodine atom.

The term “heteroalkyl” refers to an alkyl group in which one or more carbon atom has been replaced by a heteroatom selected from S, O, P and N. Exemplary heteroalkyls include alkyl ethers, secondary and tertiary alkyl amines, alkyl amides, alkyl sulfides, and the like. The group may be a terminal group or a bridging group. As used herein reference to the normal chain when used in the context of a bridging group refers to the direct chain of atoms linking the two terminal positions of the bridging group.

The term “aryl” as used herein refers to a single, all carbon aromatic ring or a multiple condensed all carbon ring system wherein at least one of the rings is aromatic. For example, in certain embodiments, an aryl group has 6 to 12 carbon atoms. Aryl includes a phenyl radical. Aryl also includes multiple condensed ring systems (e.g., ring systems comprising 2, 3 or 4 rings) having about 9 to 12 carbon atoms in which at least one ring is aromatic and wherein the other rings may be aromatic or not aromatic. Such multiple condensed ring systems are optionally substituted with one or more (e.g., 1, 2 or 3) oxo groups on any carbocycle portion of the multiple condensed ring system. The rings of the multiple condensed ring system can be connected to each other via fused, spiro and bridged bonds when allowed by valency requirements. It is to be understood that the point of attachment of a multiple condensed ring system, as defined above, can be at any position of the aromatic ring. Non-limiting examples of aryl groups include, but are not limited to, phenyl, indenyl, naphthyl, 1, 2, 3,4-tetrahydronaphth-yl, and the like.

The term “heteroaryl” as used herein refers to a single aromatic ring that has at least one atom other than carbon in the ring, wherein the atoms are selected from the group consisting of oxygen, nitrogen and sulfur; “heteroaryl” also includes multiple condensed ring systems that have at least one such aromatic ring, which multiple condensed ring systems are further described below. Thus, “heteroaryl” includes single aromatic rings of from about 1 to 6 carbon atoms and about 1-4 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur. The sulfur and nitrogen atoms may also be present in an oxidized form provided the ring is aromatic. Exemplary heteroaryl ring systems include but are not limited to pyridyl, pyrimidinyl, oxazolyl or furyl. “Heteroaryl” also includes multiple condensed ring systems (e.g., ring systems comprising 2, 3 or 4 rings) wherein a heteroaryl group, as defined above, is condensed with one or more rings selected from heteroaryls (to form for example a naphthyridinyl such as 1,8-naphthyridinyl), heterocycles, (to form for example a 1, 2, 3, 4-tetra-hydronaphthyridinyl such as 1,2,3,4-tetrahydro-1,8-naphthyridinyl), carbocycles (to form for example 5,6,7,8-tetrahydroquinolyl) and aryls (to form for example indazolyl) to form the multiple condensed ring system. Thus, a heteroaryl (a single aromatic ring or multiple condensed ring system) has about 1-20 carbon atoms and about 1-6 heteroatoms within the heteroaryl ring. A heteroaryl (a single aromatic ring or multiple condensed ring system) can also have about 5 to 12 or about 5 to 10 members within the heteroaryl ring. Multiple condensed ring systems may be optionally substituted with one or more (e.g., 1, 2, 3 or 4) oxo groups on the carbocycle or heterocycle portions of the condensed ring. The rings of a multiple condensed ring system can be connected to each other via fused, spiro and bridged bonds when allowed by valency requirements. It is to be understood that the individual rings of the multiple condensed ring system may be connected in any order relative to one another. It is also to be understood that the point of attachment of a multiple condensed ring system (as defined above for a heteroaryl) can be at any position of the heteroaryl ring. It is also to be understood that the point of attachment for a heteroaryl or heteroaryl multiple condensed ring system can be at any suitable atom of the heteroaryl ring including a carbon atom and a heteroatom (e.g., a nitrogen). Exemplary heteroaryls include but are not limited to pyridyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrazolyl, thienyl, indolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, furyl, oxadiazolyl, thiadiazolyl, quinolyl, isoquinolyl, benzothiazolyl, benzoxazolyl, indazolyl, quinoxalyl, quinazolyl, 5,6,7,8-tetrahydroisoquinolinyl benzofuranyl, benzimidazolyl, thianaphthenyl, pyrrolo[2,3-b]pyridinyl, quinazolinyl-4(3H)-one, triazolyl, 4,5,6,7-tetrahydro-1H-indazole and 3b,4,4a,5-tetrahydro-1H-cyclopropa[3,4]cyclo-penta[1,2-c]pyrazole. In one embodiment the term “heteroaryl” refers to a single aromatic ring containing at least one heteroatom. For example, the term includes 5-membered and 6-membered monocyclic aromatic rings that include one or more heteroatoms. Non-limiting examples of heteroaryl include but are not limited to pyridyl, furyl, thiazole, pyrimidine, oxazole, and thiadiazole.

The term “heterocyclyl” or “heterocycle” as used herein refers to a single saturated or partially unsaturated ring that has at least one atom other than carbon in the ring, wherein the atom is selected from the group consisting of oxygen, nitrogen and sulfur; the term also includes multiple condensed ring systems that have at least one such saturated or partially unsaturated ring, which multiple condensed ring systems are further described below. Thus, the term includes single saturated or partially unsaturated rings (e.g., 3, 4, 5, 6 or 7-membered rings) from about 1 to 6 carbon atoms and from about 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur in the ring. The ring may be substituted with one or more (e.g., 1, 2 or 3) oxo groups and the sulfur and nitrogen atoms may also be present in their oxidized forms. Exemplary heterocycles include but are not limited to azetidinyl, tetrahydrofuranyl and piperidinyl. The term “heterocycle” also includes multiple condensed ring systems (e.g., ring systems comprising 2, 3 or 4 rings) wherein a single heterocycle ring (as defined above) can be condensed with one or more groups selected from heterocycles (to form for example a 1,8-decahydronapthyridinyl), carbocycles (to form for example a decahydroquinolyl) and aryls to form the multiple condensed ring system. Thus, a heterocycle (a single saturated or single partially unsaturated ring or multiple condensed ring system) has about 2-20 carbon atoms and 1-6 heteroatoms within the heterocycle ring. Such multiple condensed ring systems may be optionally substituted with one or more (e.g., 1, 2, 3 or 4) oxo groups on the carbocycle or heterocycle portions of the multiple condensed ring. The rings of the multiple condensed ring system can be connected to each other via fused, spiro and bridged bonds when allowed by valency requirements. It is to be understood that the individual rings of the multiple condensed ring system may be connected in any order relative to one another. Accordingly, a heterocycle (a single saturated or single partially unsaturated ring or multiple condensed ring system) has about 3-20 atoms including about 1-6 heteroatoms within the heterocycle ring system. It is also to be understood that the point of attachment of a multiple condensed ring system (as defined above for a heterocylyl) can be at any position of the heterocyclic ring. It is also to be understood that the point of attachment for a heterocycle or heterocycle multiple condensed ring system can be at any suitable atom of the heterocyclic ring including a carbon atom and a heteroatom (e.g., a nitrogen). In one embodiment the term heterocycle includes a C2-20heterocycle. In one embodiment the term heterocycle includes a C2-7heterocycle. In one embodiment the term heterocycle includes a C2-5heterocycle. In one embodiment the term heterocycle includes a C2-4heterocycle. Exemplary heterocycles include, but are not limited to aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, homopiperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, tetrahydrofuranyl, dihydrooxazolyl, tetrahydropyranyl, tetrahydrothiopyranyl, 1,2,3,4-tetrahydro-quinolyl, benzoxazinyl, dihydrooxazolyl, chromanyl, 1,2-dihydropyridinyl, 2,3-dihydrobenzo-furanyl, 1,3-benzodioxolyl, 1,4-benzodioxanyl, spiro[cyclopropane-1,11-isoindolinyl]-3′-one, isoindolinyl-1-one, 2-oxa-6-azaspiro[3.3]heptanyl, imidazolidin-2-one N-methylpiperidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, 1,4-dioxane, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S,S-oxide, pyran, 3-pyrroline, thiopyran, pyrone, tetrahydrothiophene, quinuclidine, tropane, 2-azaspiro[3.3]-heptane, (1R,5S)-3-azabicyclo[3.2.1]octane, (1s,4s)-2-azabicyclo[2.2.2]octane, (1R,4R)-2-oxa-5-azabicyclo[2.2.2]octane and pyrrolidin-2-one. In one embodiment the term “heterocycle” refers to a monocyclic, saturated or partially unsaturated, 3-8 membered ring having at least one heteroatom. For example, the term includes a monocyclic, saturated or partially unsaturated, 4, 5, 6, or 7 membered ring having at least one heteroatom. Non-limiting examples of heterocycle include aziridine, azetidine, pyrrolidine, piperidine, piperidine, piperazine, oxirane, morpholine, and thiomorpholine. The term “9- or 10-membered heterobicycle” as used herein refers to a partially unsaturated or aromatic fused bicyclic ring system having at least one heteroatom. For example, the term 9- or 10-membered heterobicycle includes a bicyclic ring system having a benzo ring fused to a 5-membered or 6-membered saturated, partially unsaturated, or aromatic ring that contains one or more heteroatoms.

As used herein, the term “heteroatom” is meant to include oxygen (O), nitrogen (N), sulfur (S) and silicon (Si). The nitrogen and sulfur can be in an oxidized form when feasible.

As used herein, the term “stereoisomers” refers to compounds which have identical chemical constitution but differ with regard to the arrangement of the atoms or groups in space, e.g., enantiomers, diastereomers, tautomers.

The term “patient” or “subject” is used throughout the specification to describe an animal, preferably a human or a domesticated animal, to whom treatment, including prophylactic treatment, with the compositions according to the present disclosure is provided. For treatment of those infections, conditions or disease states which are specific for a specific animal such as a human patient, the term patient refers to that specific animal, including a domesticated animal such as a dog or cat or a farm animal such as a horse, cow, sheep, etc. In general, in the present disclosure, the term patient refers to a human patient unless otherwise stated or implied from the context of the use of the term.

The term “effective” is used to describe an amount of a compound, composition or component which, when used within the context of its intended use, effects an intended result. The term effective subsumes all other effective amount or effective concentration terms, which are otherwise described or used in the present application.

A “pharmaceutically acceptable excipient” refers to a substance that is non-toxic, biologically tolerable, and otherwise biologically suitable for administration to a subject, such as an inert substance, added to a pharmacological composition or otherwise used as a vehicle, carrier, or diluent to facilitate administration of an agent and that is compatible therewith. Examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

A “solvate” refers to a physical association of a compound of Formula I with one or more solvent molecules.

“Treating” or “treatment” of any disease or disorder refers, in one embodiment, to ameliorating the disease or disorder (e.g., arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment “treating” or “treatment” refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, “treating” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In yet another embodiment, “treating” or “treatment” refers to delaying the onset of the disease or disorder.

In one aspect, the disclosure is directed to a compound of Formula (I):

In some embodiments, R1in Formula I is halo, C1-6alkyl, or haloalkyl. In some embodiments, R1in Formula I is halo. In some embodiments, R1in Formula I is C1-6alkyl. In some embodiments, R1in Formula I is haloalkyl.

In other embodiments, R1in Formula I is F. In other embodiments, R1in Formula I is C1. In other embodiments, R1in Formula I is methyl.

In some embodiments, each R2in Formula I is independently H, D, or F. In some embodiments, each R2in Formula I is H. In some embodiments, each R2in Formula I is D. In some embodiments, each R2in Formula I is F.

In other embodiments, at least one R2in Formula I is H. In other embodiments, at least one R2in Formula I is D. In other embodiments, at least one R2in Formula I is F.

In some embodiments, n in Formula (I) is 1, 2 or 3. In some embodiments, n in Formula (I) is 1. In other embodiments, n in Formula (I) is 2. In yet other embodiments, n in Formula (I) is 3.

In some embodiments, each R3in Formula I is independently H, D, C1-6alkyl, haloalkyl, or C3-6cycloalkyl. In some embodiments, each R3in Formula I is H. In some embodiments, each R3in Formula I is D. In some embodiments, each R3in Formula I is C1-6alkyl. In some embodiments, each R3in Formula I is haloalkyl. In some embodiments, each R3in Formula I is C3-6cycloalkyl.

In other embodiments, at least one R3in Formula I is H. In other embodiments, at least one R3in Formula I is D. In other embodiments, at least one R3in Formula I is C1-6alkyl. In other embodiments, at least one R3in Formula I is haloalkyl. In other embodiments, at least one R3in Formula I is C3-6cycloalkyl.

In some embodiments, m in Formula (I) is 1, 2, 3, 4, 5 or 6. In some embodiments, m in Formula (I) is 1. In some embodiments, m in Formula (I) is 2. In other embodiments, m in Formula (I) is 3. In other embodiments, m in Formula (I) is 4. In yet other embodiments, m in Formula (I) is 5. In yet other embodiments, m in Formula (I) is 6.

In some embodiments, R4in Formula I is H, D, C1-6alkyl, C3-6cycloalkyl, alkoxyalkyl, cyanoalkyl or haloalkyl. In some embodiments, R4in Formula I is H. In some embodiments, R4in Formula I is D. In some embodiments, R4in Formula I is C1-6alkyl. In other embodiments, R4in Formula I is haloalkyl. In other embodiments, R4in Formula I is C3-6cycloalkyl. In yet other embodiments, R4in Formula I is alkoxyalkyl. In yet other embodiments, R4in Formula I is cyanoalkyl.

In some embodiments, R5in Formula I is independently H, D, or F. In some embodiments, R5in Formula I is H. In other embodiments, R5in Formula I is D. In other embodiments, R5in Formula I is F.

In some embodiments, L1in Formula I is a bond, C(R3)2, or CO. In some embodiments, L1in Formula (I) is a bond. In some embodiments, L1in Formula (I) is C(R3)2. In other embodiments, L1in Formula (I) is CO. In other embodiments, L1in Formula (I) is methylene.

In some embodiments, L2in Formula I is a bond, C(R3)2, or CO. In some embodiments, L2in Formula (I) is a bond. In some embodiments, L2in Formula (I) is C(R3)2. In other embodiments, L2in Formula (I) is CO. In other embodiments, L2in Formula (I) is methylene.

In some embodiments, ring A1in Formula (I) is a 3-7 membered cycloalkyl group, a 4-7-membered heterocycloalkyl group, an aryl group, or a heteroaryl group.

In some embodiments, ring A1in Formula (I) is a 3-7 membered cycloalkyl group. In some embodiments, ring A1is a 4-7-membered heterocycloalkyl group. In other embodiments, ring A1is an aryl. In other embodiments, ring A1is a heteroaryl group.

In some embodiments, ring A1in Formula (I) is a cyclohexyl group. In some embodiments, ring A1in Formula (I) is a piperazine group, a morpholine group, a piperidine group, a pyrrolidine group, an azetidine group or an azabicyclo-hexane group.

In some embodiments, ring A1in Formula (I) is a piperazine group. In some embodiments, ring A1in Formula (I) is a morpholine group. In other embodiments, ring A1in Formula (I) is a piperidine group. In other embodiments, ring A1in Formula (I) is a pyrrolidine group. In yet other embodiments, ring A1in Formula (I) is an azetidine group. In yet other embodiments, ring A1in Formula (I) is an azabicyclo-hexane group.

In some embodiments, ring A2in Formula (I) is a 3-7 membered cycloalkyl group, a 4-7-membered heterocycloalkyl group, an aryl group, or a heteroaryl group.

In some embodiments, ring A2in Formula (I) is a 3-7 membered cycloalkyl group. In some embodiments, ring A2is a 4-7-membered heterocycloalkyl group. In other embodiments, ring A2is an aryl. In other embodiments, ring A2is a heteroaryl group.

In some embodiments, ring A2in Formula (I) is a cyclohexyl group. In some embodiments, ring A2in Formula (I) is a piperazine group, a morpholine group, a piperidine group, a pyrrolidine group, an azetidine group or an azabicyclo-hexane group.

In some embodiments, ring A2in Formula (I) is a piperazine group. In some embodiments, ring A2in Formula (I) is a morpholine group. In other embodiments, ring A2in Formula (I) is a piperidine group. In other embodiments, ring A2in Formula (I) is a pyrrolidine group. In yet other embodiments, ring A2in Formula (I) is an azetidine group. In yet other embodiments, ring A2in Formula (I) is an azabicyclo-hexane group.

In some embodiments, the compounds of Formula (I) are the pharmaceutically acceptable salts. In some embodiments, the compounds of Formula (I) are solvates. In some embodiments, the compounds of Formula (I) are N-oxides. In some embodiments, the compounds of Formula (I) are stereoisomers.

In some embodiments, the compounds of Formula (I) are represented by compounds of Formula II

In some embodiments, each R6in Formula II is independently H, D, C1-6alkyl, haloalkyl, or C3-6cycloalkyl. In some embodiments, each R6in Formula II is H. In some embodiments, each R6in Formula II is D. In some embodiments, each R6in Formula II is C1-6alkyl. In some embodiments, each R6in Formula II is haloalkyl. In some embodiments, each R6in Formula II is C3-6cycloalkyl.

In other embodiments, at least one R6in Formula II is H. In other embodiments, at least one R6in Formula II is D. In other embodiments, at least one R6in Formula II is C1-6alkyl. In other embodiments, at least one R6in Formula II is haloalkyl. In other embodiments, at least one R6in Formula II is C3-6cycloalkyl.

In some embodiments, p in Formula II is 1, 2, 3, 4, 5, 6, 7 or 8. In some embodiments, p in Formula II is 1. In some embodiments, p in Formula II is 2. In other embodiments, p in Formula II is 3. In other embodiments, p in Formula II is 4. In other embodiments, p in Formula II is 5. In other embodiments, p in Formula II is 6. In yet other embodiments, p in Formula II is 7. In yet other embodiments, p in Formula II is 8.

In some embodiments, Z in Formula II is N or CR6. In some embodiments, Z in Formula II is N. In some embodiments, Z in Formula II is CR6. In some embodiments, Z in Formula II is CH3.

In some embodiments, the compounds of Formula (I) are represented by compounds of Formula III

In some embodiments, the compounds of Formula (I) are represented by compounds of Formula IV

In some embodiments, the compounds of Formula (I) are represented by compounds of Formula V

or a pharmaceutically acceptable salt thereof; andwherein each R1, R2, (R3)m, R4, and (R6)pare defined with respect to Formula (I) and Formula (II).

In some embodiments, the compounds of Formula (I) are represented by compounds of Formula VI

or a pharmaceutically acceptable salt thereof; andwherein each R2, R4, and (R6)pare defined with respect to Formula (I) and Formula (II).

In yet further embodiments, the compounds of Formula (I) are:

3-(6-(4-(((3R,5S)-4-(2-(3,5-difluoro-2-hydroxyphenyl)-6a-methyl-6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carbonyl)-3,5-dimethyl-piperazin-1-yl)methyl)piperidin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione;or a pharmaceutically acceptable salt thereof.

In yet further embodiments, the compounds of Formula (I) are:

In yet further embodiments, the compounds of Formula (I) are:

(R)-3-(6-(4-(((3R,5R)-4-((R)-2-(3-fluoro-2-hydroxyphenyl)-6a-(trifluoromethyl)-6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carbonyl)-3,5-dimethylpiperazin-1-yl)methyl)piperidin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione;or a pharmaceutically acceptable salt thereof.

It will be apparent that the compounds of Formula I, including all subgenera described herein, may have multiple stereogenic centers. As a result, there exist multiple stereoisomers (enantiomers and diastereomers) of the compounds of Formula I (and subgenera described herein). The present disclosure contemplates and encompasses each stereoisomer of any compound of Formula I (and subgenera described herein), as well as mixtures of said stereoisomers.

Pharmaceutically acceptable salts and solvates of the compounds of Formula I (including all subgenera described herein) are also within the scope of the disclosure.

Isotopic variants of the compounds of Formula I (including all subgenera described herein) are also contemplated by the present disclosure.

Pharmaceutical Compositions and Methods of Administration

The subject pharmaceutical compositions are typically formulated to provide a therapeutically effective amount of a compound of the present disclosure as the active ingredient, or a pharmaceutically acceptable salt, ester, prodrug, solvate, hydrate or derivative thereof. Where desired, the pharmaceutical compositions contain pharmaceutically acceptable salt and/or coordination complex thereof, and one or more pharmaceutically acceptable excipients, carriers, including inert solid diluents and fillers, diluents, including sterile aqueous solution and various organic solvents, permeation enhancers, solubilizers and adjuvants.

The subject pharmaceutical compositions can be administered alone or in combination with one or more other agents, which are also typically administered in the form of pharmaceutical compositions. Where desired, the one or more compounds of the invention and other agent(s) may be mixed into a preparation or both components may be formulated into separate preparations to use them in combination separately or at the same time.

In some embodiments, the concentration of one or more compounds of the invention is in the range from approximately 0.0001% to approximately 50%, approximately 0.001% to approximately 40%, approximately 0.01% to approximately 30%, approximately 0.02% to approximately 29%, approximately 0.03% to approximately 28%, approximately 0.04% to approximately 27%, approximately 0.05% to approximately 26%, approximately 0.06% to approximately 25%, approximately 0.07% to approximately 24%, approximately 0.08% to approximately 23%, approximately 0.09% to approximately 22%, approximately 0.1% to approximately 21%, approximately 0.2% to approximately 20%, approximately 0.3% to approximately 19%, approximately 0.4% to approximately 18%, approximately 0.5% to approximately 17%, approximately 0.6% to approximately 16%, approximately 0.7% to approximately 15%, approximately 0.8% to approximately 14%, approximately 0.9% to approximately 12%, approximately 1% to approximately 10% w/w, w/v or v/v.

In some embodiments, the concentration of one or more compounds of the invention is in the range from approximately 0.001% to approximately 10%, approximately 0.01% to approximately 5%, approximately 0.02% to approximately 4.5%, approximately 0.03% to approximately 4%, approximately 0.04% to approximately 3.5%, approximately 0.05% to approximately 3%, approximately 0.06% to approximately 2.5%, approximately 0.07% to approximately 2%, approximately 0.08% to approximately 1.5%, approximately 0.09% to approximately 1%, approximately 0.1% to approximately 0.9% w/w, w/v or v/v.

In some embodiments, the amount of one or more compounds of the invention is in the range of 0.0001-10 g, 0.0005-9 g, 0.001-8 g, 0.005-7 g, 0.01-6 g, 0.05-5 g, 0.1-4 g, 0.5-4 g, or 1-3 g.

The compounds according to the invention are effective over a wide dosage range. For example, in the treatment of adult humans, dosages from 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg per day, and from 5 to 40 mg per day are examples of dosages that may be used. An exemplary dosage is 10 to 30 mg per day. The exact dosage will depend upon the route of administration, the form in which the compound is administered, the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician.

A pharmaceutical composition of the invention typically contains an active ingredient (e.g., a compound of the disclosure) of the present invention or a pharmaceutically acceptable salt and/or coordination complex thereof, and one or more pharmaceutically acceptable excipients, carriers, including but not limited to inert solid diluents and fillers, diluents, sterile aqueous solution and various organic solvents, permeation enhancers, solubilizers and adjuvants.

Described below are non-limiting exemplary pharmaceutical compositions and methods for preparing the same.

Pharmaceutical Compositions for Oral Administration

In some embodiments, the invention provides a pharmaceutical composition for oral administration containing a compound of the invention, and a pharmaceutical excipient suitable for oral administration.

In some embodiments, the invention provides a solid pharmaceutical composition for oral administration containing: (i) an effective amount of a compound of the invention; optionally (ii) an effective amount of a second agent; and (iii) a pharmaceutical excipient suitable for oral administration. In some embodiments, the composition further contains: (iv) an effective amount of a third agent.

In some embodiments, the pharmaceutical composition may be a liquid pharmaceutical composition suitable for oral consumption. Pharmaceutical compositions of the invention suitable for oral administration can be presented as discrete dosage forms, such as capsules, cachets, or tablets, or liquids or aerosol sprays each containing a predetermined amount of an active ingredient as a powder or in granules, a solution, or a suspension in an aqueous or non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil liquid emulsion. Such dosage forms can be prepared by any of the methods of pharmacy, but all methods include the step of bringing the active ingredient into association with the carrier, which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation. For example, a tablet can be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as powder or granules, optionally mixed with an excipient such as, but not limited to, a binder, a lubricant, an inert diluent, and/or a surface active or dispersing agent. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

This invention further encompasses anhydrous pharmaceutical compositions and dosage forms comprising an active ingredient, since water can facilitate the degradation of some compounds. For example, water may be added (e.g., 5%) in the pharmaceutical arts as a means of simulating long-term storage in order to determine characteristics such as shelf-life or the stability of formulations over time. Anhydrous pharmaceutical compositions and dosage forms of the invention can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms of the invention which contain lactose can be made anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected. An anhydrous pharmaceutical composition may be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions may be packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastic or the like, unit dose containers, blister packs, and strip packs.

An active ingredient can be combined in an intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of preparation desired for administration. In preparing the compositions for an oral dosage form, any of the usual pharmaceutical media can be employed as carriers, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like in the case of oral liquid preparations (such as suspensions, solutions, and elixirs) or aerosols; or carriers such as starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents can be used in the case of oral solid preparations, in some embodiments without employing the use of lactose. For example, suitable carriers include powders, capsules, and tablets, with the solid oral preparations. If desired, tablets can be coated by standard aqueous or nonaqueous techniques.

Examples of suitable fillers for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.

Disintegrants may be used in the compositions of the invention to provide tablets that disintegrate when exposed to an aqueous environment. Too much of a disintegrant may produce tablets which may disintegrate in the bottle. Too little may be insufficient for disintegration to occur and may thus alter the rate and extent of release of the active ingredient(s) from the dosage form. Thus, a sufficient amount of disintegrant that is neither too little nor too much to detrimentally alter the release of the active ingredient(s) may be used to form the dosage forms of the compounds disclosed herein. The amount of disintegrant used may vary based upon the type of formulation and mode of administration, and may be readily discernible to those of ordinary skill in the art. About 0.5 to about 15 weight percent of disintegrant, or about 1 to about 5 weight percent of disintegrant, may be used in the pharmaceutical composition. Disintegrants that can be used to form pharmaceutical compositions and dosage forms of the invention include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums or mixtures thereof.

Lubricants which can be used to form pharmaceutical compositions and dosage forms of the invention include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, or mixtures thereof. Additional lubricants include, for example, a syloid silica gel, a coagulated aerosol of synthetic silica, or mixtures thereof. A lubricant can optionally be added, in an amount of less than about 1 weight percent of the pharmaceutical composition.

When aqueous suspensions and/or elixirs are desired for oral administration, the active ingredient therein may be combined with various sweetening or flavoring agents, coloring matter or dyes and, if so desired, emulsifying and/or suspending agents, together with such diluents as water, ethanol, propylene glycol, glycerin and various combinations thereof.

The tablets can be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil.

Surfactant which can be used to form pharmaceutical compositions and dosage forms of the invention include, but are not limited to, hydrophilic surfactants, lipophilic surfactants, and mixtures thereof. That is, a mixture of hydrophilic surfactants may be employed, a mixture of lipophilic surfactants may be employed, or a mixture of at least one hydrophilic surfactant and at least one lipophilic surfactant may be employed.

A suitable hydrophilic surfactant may generally have an HLB value of at least 10, while suitable lipophilic surfactants may generally have an HLB value of or less than about 10. An empirical parameter used to characterize the relative hydrophilicity and hydrophobicity of non-ionic amphiphilic compounds is the hydrophilic-lipophilic balance (“HLB” value). Surfactants with lower HLB values are more lipophilic or hydrophobic, and have greater solubility in oils, while surfactants with higher HLB values are more hydrophilic, and have greater solubility in aqueous solutions.

Hydrophilic surfactants are generally considered to be those compounds having an HLB value greater than about 10, as well as anionic, cationic, or zwitterionic compounds for which the HLB scale is not generally applicable. Similarly, lipophilic (e.g., hydrophobic) surfactants are compounds having an HLB value equal to or less than about 10. However, HLB value of a surfactant is merely a rough guide generally used to enable formulation of industrial, pharmaceutical and cosmetic emulsions.

Hydrophilic surfactants may be either ionic or non-ionic. Suitable ionic surfactants include, but are not limited to, alkylammonium salts; fusidic acid salts; fatty acid derivatives of amino acids, oligopeptides, and polypeptides; glyceride derivatives of amino acids, oligopeptides, and polypeptides; lecithins and hydrogenated lecithins; lysolecithins and hydrogenated lysolecithins; phospholipids and derivatives thereof; lysophospholipids and derivatives thereof; carnitine fatty acid ester salts; salts of alkylsulfates; fatty acid salts; sodium docusate; acyl lactylates; mono- and di-acetylated tartaric acid esters of mono- and di-glycerides; succinylated mono- and di-glycerides; citric acid esters of mono- and di-glycerides; and mixtures thereof.

Within the aforementioned group, ionic surfactants include, by way of example: lecithins, lysolecithin, phospholipids, lysophospholipids and derivatives thereof; carnitine fatty acid ester salts; salts of alkylsulfates; fatty acid salts; sodium docusate; acylactylates; mono- and di-acetylated tartaric acid esters of mono- and di-glycerides; succinylated mono- and di-glycerides; citric acid esters of mono- and di-glycerides; and mixtures thereof.

Hydrophilic non-ionic surfactants may include, but are not limited to, alkylglucosides; alkylmaltosides; alkylthioglucosides; lauryl macrogolglycerides; polyoxyalkylene alkyl ethers such as polyethylene glycol alkyl ethers; polyoxyalkylene alkylphenols such as polyethylene glycol alkyl phenols; polyoxyalkylene alkyl phenol fatty acid esters such as polyethylene glycol fatty acids monoesters and polyethylene glycol fatty acids diesters; polyethylene glycol glycerol fatty acid esters; polyglycerol fatty acid esters; polyoxyalkylene sorbitan fatty acid esters such as polyethylene glycol sorbitan fatty acid esters; hydrophilic transesterification products of a polyol with at least one member of the group consisting of glycerides, vegetable oils, hydrogenated vegetable oils, fatty acids, and sterols; polyoxyethylene sterols, derivatives, and analogues thereof; polyoxyethylated vitamins and derivatives thereof; polyoxyethylene-polyoxypropylene block copolymers; and mixtures thereof; polyethylene glycol sorbitan fatty acid esters and hydrophilic transesterification products of a polyol with at least one member of the group consisting of triglycerides, vegetable oils, and hydrogenated vegetable oils. The polyol may be glycerol, ethylene glycol, polyethylene glycol, sorbitol, propylene glycol, pentaerythritol, or a saccharide.

Suitable lipophilic surfactants include, by way of example only: fatty alcohols; glycerol fatty acid esters; acetylated glycerol fatty acid esters; lower alcohol fatty acids esters; propylene glycol fatty acid esters; sorbitan fatty acid esters; polyethylene glycol sorbitan fatty acid esters; sterols and sterol derivatives; polyoxyethylated sterols and sterol derivatives; polyethylene glycol alkyl ethers; sugar esters; sugar ethers; lactic acid derivatives of mono- and di-glycerides; hydrophobic transesterification products of a polyol with at least one member of the group consisting of glycerides, vegetable oils, hydrogenated vegetable oils, fatty acids and sterols; oil-soluble vitamins/vitamin derivatives; and mixtures thereof. Within this group, preferred lipophilic surfactants include glycerol fatty acid esters, propylene glycol fatty acid esters, and mixtures thereof, or are hydrophobic transesterification products of a polyol with at least one member of the group consisting of vegetable oils, hydrogenated vegetable oils, and triglycerides.

In one embodiment, the composition may include a solubilizer to ensure good solubilization and/or dissolution of the compound of the present invention and to minimize precipitation of the compound of the present invention. This can be especially important for compositions for non-oral use, e.g., compositions for injection. A solubilizer may also be added to increase the solubility of the hydrophilic drug and/or other components, such as surfactants, or to maintain the composition as a stable or homogeneous solution or dispersion.

The amount of solubilizer that can be included is not particularly limited. The amount of a given solubilizer may be limited to a bioacceptable amount, which may be readily determined by one of skill in the art. In some circumstances, it may be advantageous to include amounts of solubilizers far in excess of bioacceptable amounts, for example to maximize the concentration of the drug, with excess solubilizer removed prior to providing the composition to a subject using conventional techniques, such as distillation or evaporation. Thus, if present, the solubilizer can be in a weight ratio of 10%, 25% o, 50%), 100% o, or up to about 200%> by weight, based on the combined weight of the drug, and other excipients. If desired, very small amounts of solubilizer may also be used, such as 5%>, 2%>, 1%) or even less. Typically, the solubilizer may be present in an amount of about 1%> to about 100%, more typically about 5%> to about 25%> by weight.

Pharmaceutical Compositions for Injection

In some embodiments, the invention provides a pharmaceutical composition for injection containing a compound of the present invention and a pharmaceutical excipient suitable for injection. Components and amounts of agents in the compositions are as described herein.

The forms in which the novel compositions of the present invention may be incorporated for administration by injection include aqueous or oil suspensions, or emulsions, with sesame oil, corn oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueous solution, and similar pharmaceutical vehicles.

Aqueous solutions in saline are also conventionally used for injection. Ethanol, glycerol, propylene glycol, liquid polyethylene glycol, and the like (and suitable mixtures thereof), cyclodextrin derivatives, and vegetable oils may also be employed. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, for the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

Pharmaceutical Compositions for Topical (e.g., Transdermal) Delivery

In some embodiments, the invention provides a pharmaceutical composition for transdermal delivery containing a compound of the present invention and a pharmaceutical excipient suitable for transdermal delivery.

Compositions of the present invention can be formulated into preparations in solid, semisolid, or liquid forms suitable for local or topical administration, such as gels, water soluble jellies, creams, lotions, suspensions, foams, powders, slurries, ointments, solutions, oils, pastes, suppositories, sprays, emulsions, saline solutions, dimethylsulfoxide (DMSO)-based solutions. In general, carriers with higher densities are capable of providing an area with a prolonged exposure to the active ingredients. In contrast, a solution formulation may provide more immediate exposure of the active ingredient to the chosen area.

The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients, which are compounds that allow increased penetration of, or assist in the delivery of, therapeutic molecules across the stratum corneum permeability barrier of the skin. There are many of these penetration-enhancing molecules known to those trained in the art of topical formulation.

Another exemplary formulation for use in the methods of the present invention employs transdermal delivery devices (“patches”). Such transdermal patches may be used to provide continuous or discontinuous infusion of a compound of the present invention in controlled amounts, either with or without another agent.

The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, e.g., U.S. Pat. Nos. 5,023,252, 4,992,445 and 5,001,139. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.

Pharmaceutical Compositions for Inhalation

Other Pharmaceutical Compositions

Pharmaceutical compositions may also be prepared from compositions described herein and one or more pharmaceutically acceptable excipients suitable for sublingual, buccal, rectal, intraosseous, intraocular, intranasal, epidural, or intraspinal administration. Preparations for such pharmaceutical compositions are well-known in the art. See, e.g., Anderson, Philip O.; Knoben, James E.; Troutman, William G, eds., Handbook of Clinical Drug Data, Tenth Edition, McGraw-Hill, 2002; Pratt and Taylor, eds., Principles of Drug Action, Third Edition, Churchill Livingston, New York, 1990; Katzung, ed., Basic and Clinical Pharmacology, Ninth Edition, McGraw Hill, 20037ybg; Goodman and Gilman, eds., The Pharmacological Basis of Therapeutics, Tenth Edition, McGraw Hill, 2001; Remingtons Pharmaceutical Sciences, 20th Ed., Lippincott Williams & Wilkins., 2000; Martindale, The Extra Pharmacopoeia, Thirty-Second Edition (The Pharmaceutical Press, London, 1999); all of which are incorporated by reference herein in their entirety.

Administration of the compounds or pharmaceutical composition of the present invention can be affected by any method that enables delivery of the compounds to the site of action. These methods include oral routes, intraduodenal routes, parenteral injection (including intravenous, intraarterial, subcutaneous, intramuscular, intravascular, intraperitoneal or infusion), topical (e.g., transdermal application), rectal administration, via local delivery by catheter or stent or through inhalation. Compounds can also be administered intraadiposally or intrathecally.

In some embodiments, the compounds or pharmaceutical composition of the present invention are administered by intravenous injection.

The amount of the compound administered will be dependent on the subject being treated, the severity of the disorder or condition, the rate of administration, the disposition of the compound and the discretion of the prescribing physician. However, an effective dosage is in the range of about 0.001 to about 100 mg per kg body weight per day, preferably about 1 to about 35 mg/kg/day, in single or divided doses. For a 70 kg human, this would amount to about 0.05 to 7 g/day, preferably about 0.05 to about 2.5 g/day. In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect, e.g., by dividing such larger doses into several small doses for administration throughout the day.

In some embodiments, a compound of the invention is administered in a single dose.

Typically, such administration will be by injection, e.g., intravenous injection, in order to introduce the agent quickly. However, other routes may be used as appropriate. A single dose of a compound of the invention may also be used for treatment of an acute condition.

In some embodiments, a compound of the invention is administered in multiple doses. Dosing may be about once, twice, three times, four times, five times, six times, or more than six times per day. Dosing may be about once a month, once every two weeks, once a week, or once every other day. In another embodiment a compound of the invention and another agent are administered together about once per day to about 6 times per day. In another embodiment the administration of a compound of the invention and an agent continues for less than about 7 days. In yet another embodiment the administration continues for more than about 6, 10, 14, 28 days, two months, six months, or one year. In some cases, continuous dosing is achieved and maintained as long as necessary.

Administration of the compounds of the invention may continue as long as necessary. In some embodiments, a compound of the invention is administered for more than 1, 2, 3, 4, 5, 6, 7, 14, or 28 days. In some embodiments, a compound of the invention is administered for less than 28, 14, 7, 6, 5, 4, 3, 2, or 1 day. In some embodiments, a compound of the invention is administered chronically on an ongoing basis, e.g., for the treatment of chronic effects.

An effective amount of a compound of the invention may be administered in either single or multiple doses by any of the accepted modes of administration of agents having similar utilities, including rectal, buccal, intranasal and transdermal routes, by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, orally, topically, or as an inhalant.

The compositions of the invention may also be delivered via an impregnated or coated device such as a stent, for example, or an artery-inserted cylindrical polymer. Such a method of administration may, for example, aid in the prevention or amelioration of restenosis following procedures such as balloon angioplasty. Without being bound by theory, compounds of the invention may slow or inhibit the migration and proliferation of smooth muscle cells in the arterial wall which contribute to restenosis. A compound of the invention may be administered, for example, by local delivery from the struts of a stent, from a stent graft, from grafts, or from the cover or sheath of a stent. In some embodiments, a compound of the invention is admixed with a matrix. Such a matrix may be a polymeric matrix and may serve to bond the compound to the stent. Polymeric matrices suitable for such use, include, for example, lactone-based polyesters or copolyesters such as polylactide, polycaprolactonglycolide, polyorthoesters, polyanhydrides, polyaminoacids, polysaccharides, polyphosphazenes, poly (ether-ester) copolymers (e.g. PEO-PLLA); polydimethylsiloxane, poly(ethylene-vinylacetate), acrylate-based polymers or copolymers (e.g. polyhydroxyethyl methylmethacrylate, polyvinyl pyrrolidinone), fluorinated polymers such as polytetrafluoroethylene and cellulose esters. Suitable matrices may be nondegrading or may degrade with time, releasing the compound or compounds. Compounds of the invention may be applied to the surface of the stent by various methods such as dip/spin coating, spray coating, dip-coating, and/or brush-coating. The compounds may be applied in a solvent and the solvent may be allowed to evaporate, thus forming a layer of compound onto the stent. Alternatively, the compound may be located in the body of the stent or graft, for example in microchannels or micropores. When implanted, the compound diffuses out of the body of the stent to contact the arterial wall. Such stents may be prepared by dipping a stent manufactured to contain such micropores or microchannels into a solution of the compound of the invention in a suitable solvent, followed by evaporation of the solvent. Excess drug on the surface of the stent may be removed via an additional brief solvent wash. In yet other embodiments, compounds of the invention may be covalently linked to a stent or graft. A covalent linker may be used which degrades in vivo, leading to the release of the compound of the invention. Any bio-labile linkage may be used for such a purpose, such as ester, amide or anhydride linkages. Compounds of the invention may additionally be administered intravascularly from a balloon used during angioplasty. Extravascular administration of the compounds via the pericard or via advential application of formulations of the invention may also be performed to decrease restenosis.

A variety of stent devices which may be used as described are disclosed, for example, in the following references, all of which are hereby incorporated by reference: U.S. Pat. Nos. 5,451,233; 5,040,548; 5,061,273; 5,496,346; 5,292,331; 5,674,278; 3,657,744; 4,739,762; 5,195,984; 5,292,331; U.S. Pat. Nos. 5,674,278; 5,879,382; 6,344,053.

The compounds of the invention may be administered in dosages. It is known in the art that due to intersubject variability in compound pharmacokinetics, individualization of dosing regimen is necessary for optimal therapy. Dosing for a compound of the invention may be found by routine experimentation in light of the instant disclosure.

When a compound of the invention is administered in a composition that comprises one or more agents, and the agent has a shorter half-life than the compound of the invention unit dose forms of the agent and the compound of the invention may be adjusted accordingly.

The subject pharmaceutical composition may, for example, be in a form suitable for oral administration as a tablet, capsule, pill, powder, sustained release formulations, solution, suspension, for parenteral injection as a sterile solution, suspension or emulsion, for topical administration as an ointment or cream or for rectal administration as a suppository. The pharmaceutical composition may be in unit dosage forms suitable for single administration of precise dosages. The pharmaceutical composition will include a conventional pharmaceutical carrier or excipient and a compound according to the invention as an active ingredient. In addition, it may include other medicinal or pharmaceutical agents, carriers, adjuvants, etc. Exemplary parenteral administration forms include solutions or suspensions of active compound in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired.

Methods of Use

The method typically comprises administering to a subject a therapeutically effective amount of a compound of the invention. The therapeutically effective amount of the subject combination of compounds may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in target cells, e.g., reduction of proliferation or downregulation of activity of a target protein. The specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.

In certain embodiment, the present invention provides a pharmaceutical composition comprising a compound of bispecific formula, or pharmaceutically acceptable salt thereof.

In certain embodiment, the present invention provides a pharmaceutical composition comprising a compound of bispecific formula for use in degrading a target protein in a cell.

In certain embodiment, a method of degrading a target protein comprising administering to a cell therapeutically effective amount of a bispecific compound, or pharmaceutically acceptable salt, wherein the compound is effective for degrading the target protein.

In certain embodiment, the present invention provides a pharmaceutical composition comprising a compound of bispecific formula, for use in treating or preventing of a disease or disorder in which SMARCA2 and/or SMARCA4 plays a role.

In certain embodiment, the present invention provides a pharmaceutical composition comprising a compound of bispecific formula, for use in treating or preventing of a disease or disorder in which SWI/SNF mutations plays a role.

In certain embodiment, target protein complex is SWI/SNF in a cell.

In certain embodiment, diseases or disorders dependent on SMARCA2 or SMARCA4 include cancers.

In certain embodiment, diseases or disorders dependent on SWI/SNF complex include cancers.

In certain embodiments, the cancers which may be treated using compounds according to the present disclosure include, for example, T-lineage Acute lymphoblastic Leukemia (T-ALL), T-lineage lymphoblastic Lymphoma (T-LL), Peripheral T-cell lymphoma, Adult T-cell Leukemia, Pre-B ALL, Pre-B Lymphomas, Large B-cell Lymphoma, Burkitts Lymphoma, B-cell ALL, Philadelphia chromosome positive ALL and Philadelphia chromosome positive CML.

In certain further embodiment, the cancer is a SMARCA2 and/or SMARAC4-dependent cancer.

In certain embodiment, the present invention provides a pharmaceutical composition comprising a compound of bispecific formula for use in the diseases or disorders dependent upon SMARCA2 and/or SMARCA4 is cancer.

Compounds of the disclosure, as well as pharmaceutical compositions comprising them, can be administered to treat any of the described diseases, alone or in combination with a medical therapy. Medical therapies include, for example, surgery and radiotherapy (e.g., gamma-radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, systemic radioactive isotopes).

In other aspects, compounds of the disclosure, as well as pharmaceutical compositions comprising them, can be administered to treat any of the described diseases, alone or in combination with one or more other agents.

In other methods, the compounds of the disclosure, as well as pharmaceutical compositions comprising them, can be administered in combination with agonists of nuclear receptors agents.

In other methods, the compounds of the disclosure, as well as pharmaceutical compositions comprising them, can be administered in combination with antagonists of nuclear receptors agents.

In other methods, the compounds of the disclosure, as well as pharmaceutical compositions comprising them, can be administered in combination with an anti-proliferative agent.

Combination Therapies

In some embodiments, the compounds of the invention can be used in combination with a therapeutic agent that targets an epigenetic regulator. Examples of epigenetic regulators include bromodomain inhibitors, the histone lysine methyltransferase inhibitors, histone arginine methyl transferase inhibitors, histone demethylase inhibitors, histone deacetylase inhibitors, histone acetylase inhibitors, and DNA methyltransferase inhibitors. Histone deacetylase inhibitors include, e.g., vorinostat. Histone arginine methyl transferase inhibitors include inhibitors of protein arginine methyltransferases (PRMTs) such as PRMT5, PRMT1 and PRMT4. DNA methyltransferase inhibitors include inhibitors of DNMT1 and DNMT3.

In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of PD-1, e.g., an anti-PD-1 monoclonal antibody. In some embodiments, the anti-PD-1 monoclonal antibody is nivolumab, pembrolizumab (also known as MK-3475), or PDR001. In some embodiments, the anti-PD-1 monoclonal antibody is nivolumab or pembrolizumab. In some embodiments, the anti-PD1 antibody is pembrolizumab. In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of PD-L1, e.g., an anti-PD-L1 monoclonal antibody. In some embodiments, the anti-PD-L1 monoclonal antibody is atezolizumab, durvalumab, or BMS-935559. In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CTLA-4, e.g., an anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody is ipilimumab.

In some embodiments, the agent is an alkylating agent, a proteasome inhibitor, a corticosteroid, or an immunomodulatory agent. Examples of an alkylating agent include cyclophosphamide (CY), melphalan (MEL), and bendamustine. In some embodiments, the proteasome inhibitor is carfilzomib. In some embodiments, the corticosteroid is dexamethasone (DEX). In some embodiments, the immunomodulatory agent is lenalidomide (LEN) or pomalidomide (POM).

Compounds of the invention can be prepared using numerous preparatory reactions known in the literature. The Schemes below provide general guidance in connection with preparing the compounds of the invention. One skilled in the art would understand that the preparations shown in the Schemes can be modified or optimized using general knowledge of organic chemistry to prepare various compounds of the invention. Example synthetic methods for preparing compounds of the invention are provided in the Schemes below.

The following Examples are provided to illustrate some of the concepts described within this disclosure. While the Examples are considered to provide an embodiment, it should not be considered to limit the more general embodiments described herein.

EXAMPLES

General Synthetic Procedures

The compounds described herein may be prepared according to the following synthetic schemes and general synthetic procedures.

The compounds of Formula (1-8) where R4=H, can be made according to the route described in Scheme 1. SNAr reaction between commercially available starting materials 1-1, where R4=H and compounds 1-2, where Q1=Cl, Br, in the presence of base (e.g., Cs2CO3, NaHCO3, DIPEA) at elevated room temperatures gives alcohol 1-3. Azidation of compounds 1-3 upon treatment with PPh3/DIAD/DPPA yields compounds 1-4. Staudinger reduction of compounds 1˜4 upon treatment with PPh3at elevated temperatures followed by SNAr in the presence of base (e.g., Cs2CO3, NaHCO3, DIPEA) yields compounds 1-5. Protection of the —NH group with an appropriate group (e.g., Boc, SEM, Bn, etc.) can give compounds 1-6, which can be converted to compounds 1-7 under standard Suzuki conditions (e.g., in the presence of a palladium catalyst, such as but not limited to tetrakis(triphenylphosphine)palladium(0) or [1,1′-bis (diphenylphosphino)ferrocene]dichloropalladium (II), complex with dichloromethane and a base (e.g., a carbonate base)) using the appropriate boronic acid or ester (e.g., 2-hydroxy-phenylboronic acid). Removal of the protecting groups can yield compounds 1-8.

The compounds of Formula (1-8) where R4≠H can be made according to the route described in Scheme 2. Protection of the —NH group of commercially available starting materials 2-1 with an appropriate group (e.g., Boc, SEM, Bn, etc.) can give compounds 2-2. Esterification of compounds 2-2 in the presence of base (e.g., Cs2CO3, NaHCO3, DIPEA) yields compounds 2-3. Alkylation of compounds 2-3 upon sequential treatment with LiHMDS and an electrophile (e.g. MeI), results in compounds 2-4, where R4≠H. One pot amide formation, selective deprotection, and SNAr between compounds 2-4 and compounds 2-5, where Q1=Cl, Br, in the presence of base (e.g., Cs2CO3, NaHCO3, DIPEA) at elevated temperature gives compounds 2-6. Compounds 2-6 can be converted to compounds 2-7 under standard Suzuki conditions (e.g., in the presence of a palladium catalyst, such as but not limited to tetrakis-(triphenylphosphine)palladium(0) or [1,1′-bis (diphenylphosphino)ferrocene]dichloropalladium (II), complex with dichloromethane and a base (e.g., a carbonate base)) using the appropriate boronic acid or ester (e.g., 2-hydroxy-phenylboronic acid). Reduction of compounds 2-7 in the presence of BH3·THF at elevated temperatures yields compounds 2-8. Removal of the protecting groups can yield compounds 1-8.

The intermediate of formula 3-6, where X1=CO and X2=CH2, can be synthesized according to the route described Scheme 3. SNAr between 3-1 and 3-2 (e.g., DIPEA) can afford compound 3-3. Reduction of the cyano in 3-3, can be achieved under appropriate conditions (e.g., Raney Nickel) to yield 3-4. Reductive amination and cyclization of 3-4 with 3-5 (e.g., DIPEA followed by AcOH and Sodium triacetoxyborohydride) affords 3-6, wherein A2and R5are defined above.

The intermediate of formula 3-6, where X1=CH2and X2=CO, can be synthesized according to the route described Scheme 4. SNAr between 4-1 and 3-2 (e.g., DIPEA) can afford compound 4-2. Reduction of the cyano in 4-2, can be achieved under appropriate conditions (e.g., Raney Nickel) to yield 4-3. Reductive amination and cyclization of 4-3 with 3-5 (e.g., DIPEA followed by AcOH and sodium triacetoxyborohydride) affords 3-6, wherein A2and R5are defined above.

The intermediate of formula 3-6, where X1=CO and X2=CO, can be synthesized according to the route described in Scheme 5. SNAr between 5-1 and 3-2 (e.g., DIPEA) can afford compound 3-6, wherein A2and R5are defined above.

The compounds of the Formula (I) can be made according to the route described in Scheme 6. The coupling between 1-8 and 6-1 either through urea formation (e.g., DIPEA), amide formation (e.g., HATU) or reductive amination (e.g., sodium triacetoxyborohydride) can afford compounds 6-2. Subsequent coupling of 6-2 with 3-6 (X1, X2=CH2or CO) either through reductive amination (e.g., sodium triacetoxyborohydride) or alkylation (e.g., DIPEA) yields compounds of the Formula (I).

To a solution of 3,4,6-trichloropyridazine (5.70 g, 31.1 mmol) in DMF (24.0 mL) was added N,N-diisopropylethylamine (5.95 mL, 34.2 mmol) and tert-butyl (R)-3-(hydroxymethyl) piperazine-1-carboxylate (7.10 g, 32.8 mmol). The reaction was stirred at 80° C. overnight. The reaction was cooled to 45° C. and water (17.0 mL) was slowly added. The resulting clear solution was stirred at 35° C. for 30 min until a precipitate formed. Another portion of water (23.0 mL) was charged slowly, and the mixture was stirred at 0° C. for an additional 1 h. The mixture was filtered, and the resulting solid was washed with water, collected and dried under vacuum to give tert-butyl (R)-4-(3,6-dichloropyridazin-4-yl)-3-(hydroxymethyl)piperazine-1-carboxylate (8.50 g, 75% yield) as an off-white solid. LCMS calcd for C14H21C12N4O3[M+H]+: m/z=363.1; Found: 363.1.

To a solution of tert-butyl (R)-4-(3,6-dichloropyridazin-4-yl)-3-(hydroxymethyl) piperazine-1-carboxylate (5.45 g, 15.0 mmol) and triphenylphosphine (4.72 g, 18.0 mmol) in THF (150 mL) was added diisopropyl azodicarboxylate (3.54 mL, 18.0 mmol) and diphenyl-phosphoryl azide (3.90 mL, 18.0 mmol) at 0° C. The reaction was then stirred at room temperature overnight. The reaction mixture was cooled to 0° C., quenched with water and extracted with EtOAc. The combined organic layers were washed with a saturated brine solution and water, dried over Na2SO4and filtered. The filtrate was concentrated under reduced pressure to give crude tert-butyl (R)-3-(azidomethyl)-4-(3,6-dichloropyridazin-4-yl)piperazine-1-carboxylate (19.4 g), which was used without further purification (30% purity by LCMS). LCMS calcd for C14H20C12N7O2[M+H]+: m/z=388.1; Found: 388.0.

Triphenylphosphine (4.94 g, 18.8 mmol) was added to a stirred solution of crude tert-butyl (R)-3-(azidomethyl)-4-(3,6-dichloropyridazin-4-yl) piperazine-1-carboxylate (20.3 g, 15.7 mmol, 30% purity) in THF (200 mL). The resulting solution was stirred at 60° C. for 3 hours. Water (20.0 mL) and N,N-diisopropylethylamine (8.20 mL, 47.1 mmol) were added sequentially. After 20 hours, the reaction mixture was diluted with EtOAc (100 mL) and water (100 mL). The aqueous layer was separated and extracted with EtOAc. The combined organic layers were washed with a saturated brine solution, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography, eluting with a gradient of 0-100% EtOAc/hexanes to give tert-butyl (S)-2-chloro-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carboxylate (3.10 g, 60% yield) as an off-white solid. LCMS calcd for C14H21ClN5O2[M+H]+: m/z=326.1; Found: 326.2.

A 4 N solution of HCl in 1,4-dioxane (4.00 mL) was added dropwise to a solution of di-tert-butyl (R)-2-(3-fluoro-2-hydroxyphenyl)-6a,7,9,10-tetrahydro-5H-pyrazino[1′,2′:4,5]pyrazino [2,3-c]pyridazine-5,8(6H)-dicarboxylate (1.10 g, 2.12 mmol) in DCM (10.0 mL). The reaction was stirred for 5 hours at room temperature. The reaction was concentrated under reduced pressure to yield (R)-2-fluoro-6-(6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazin-2-yl)phenol; dihydrochloride (602 mg, 73% yield) as an off-white solid. LCMS calcd for C15H17FN5O [M+H]+: m/z=302.1; Found: 302.0.

The title compound was prepared using the procedure analogous to that described for Intermediate 1, with (3,5-difluoro-2-hydroxyphenyl)boronic acid replacing (3-fluoro-2-hydroxyphenyl)boronic acid in step 5. LCMS calcd for C15H16F2N5O [M+H]+: m/z=320.1; Found: 320.0

To a stirred suspension of O-benzyl-N-(tert-butoxycarbonyl)-L-serine (20.0 g, 67.7 mmol) and 1-hydroxybenzotriazole hydrate (11.0 g, 81.3 mmol) in DCM (451 mL) was added N,N-diisopropylethylamine (14.2 mL, 81.3 mmol) at 0° C. 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (15.6 g, 81.3 mmol) was added to the reaction mixture and stirred at 0° C. for 15 minutes. A solution of alanine methyl ester hydrochloride (11.3 g, 81.3 mmol) and N,N-diisopropylethylamine (14.2 mL, 81.3 mmol) in DMF (30 mL) was added dropwise at 0° C. over 5 minutes to the reaction mixture. The reaction was warmed to room temperature and stirred for 3 hours. Water (500 mL) was added, and the aqueous phase was extracted with DCM (300 mL×3). The organic phases were dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography, eluting with a gradient of 0-100% EtOAC/hexanes to give methyl O-benzyl-N-(tert-butoxycarbonyl)-L-seryl-L-alaninate (26.1 g, 99.0% yield). LCMS calculated for C19H29N2O6(M+H)+: m/z=381.2; Found: 381.0.

To a solution of methyl O-benzyl-N-(tert-butoxycarbonyl)-L-seryl-L-alaninate (26.1 g, 68.6 mmol) in DCM (260 mL) was added TFA (51.4 mL, 672 mmol) at room temperature. The reaction was stirred for 3 hours. The reaction mixture was basified to between pH 7 and pH 8 via a saturated aqueous NaHCO3solution. The aqueous phase was extracted with DCM (100 mL×3) and the organics were washed with a saturated brine solution (100 mL×1). The combined organic phases were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was used directly in the next step without additional purification to give crude methyl O-benzyl-L-seryl-L-alaninate (16.9 g). LCMS calculated for C14H21N2O4(M+H)±: m/z=281.2; Found: 281.0.

A solution of crude methyl O-benzyl-L-seryl-L-alaninate (16.9 g, 60.3 mmol) in 1,4-dioxane (169 mL) was stirred at 100° C. overnight. The reaction was cooled to room temperature, upon which a white solid precipitated out. The white precipitate was filtered, collected, and washed with cold MTBE (100 mL) to give (35,65)-3-((benzyloxy)methyl)-6-methylpiperazine-2,5-dione (11.0 g, yield: 73.0% yield). LCMS calculated for C13H17N2O3(M+H)±: m/z=249.1; Found: 249.2.

To a solution of (3S,6S)-3-((benzyloxy)methyl)-6-methylpiperazine-2,5-dione (9.00 g, 36.3 mmol) in THF (201 mL) was added borane dimethyl sulfide complex (27.5 mL, 290 mmol) while on an ice-water bath. The reaction was warmed to room temperature and stirred at 60° C. overnight. The reaction was cooled on an ice-water bath and MeOH was slowly added (200 mL). The reaction mixture was warmed to room temperature and a 1 N HCl aqueous solution was added dropwise to reach a pH ˜3. The mixture was stirred at 50° C. for 3 hours. The reaction mixture was basified to pH 12 by adding a 1 N NaOH aqueous solution dropwise and the aqueous phase was extracted with CHCl3(200 mL×3). The combined organic phases were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was directly used in the next step without additional purification to give crude (2R,5S)-2-((benzyloxy)methyl)-5-methylpiperazine (9.80 g). LCMS calculated for C13H21N2O (M+H)±: m/z=221.2; Found: 221.2.

To a solution of crude (2R,5S)-2-((benzyloxy)methyl)-5-methylpiperazine (9.80, 44.5 mmol) in DCM (445 mL) was added a 1 M solution of BCl3(178 mL, 178 mmol) in DCM at −78° C. The reaction was slowly warmed to room temperature and stirred overnight. The reaction was cooled over an ice-water bath and MeOH (200 mL) was slowly added. The reaction mixture was concentrated to dryness under reduced pressure. The residue was directly used in the next step without additional purification to give crude ((2R,5S)-5-methylpiperazin-2-yl)methanol (9.0 g). LCMS calculated for C6H15N2O (M+H)+: m/z=131.1; Found: 131.0.

To a solution of crude ((2R,5S)-5-methylpiperazin-2-yl)methanol (9.00 g, 69.1 mmol) in DCM (376 mL) was added triethylamine (120 mL, 864 mmol) and di-tert-butyl dicarbonate (45.3 g, 207 mmol) at 0° C. The reaction was stirred at room temperature overnight and then concentrated to dryness under reduced pressure. The residue was directly used in the next step without additional purification to give crude di-tert-butyl (2R,5S)-2-(hydroxymethyl)-5-methylpiperazine-1,4-dicarboxylate (24.0 g). LCMS calculated for C16H31N2O5(M+H)+: m/z=331.2; Found: 331.0.

To a solution of crude di-tert-butyl (2R,5S)-2-(hydroxymethyl)-5-methylpiperazine-1,4-dicarboxylate (14.0 g, 42.4 mmol) in EtOH (78.5 mL) was added a solution of NaOH (8.50 g, 212 mmol) in water (78.5 mL). The reaction mixture was stirred at 80° C. overnight. The reaction was cooled to room temperature and pH ˜9 was reached by adding a 1 N HCl aqueous solution dropwise. The aqueous phase was extracted with CHCl3(100 mL×3) and the combined organic phases were dried over Na2SO4, filtered and concentrated under reduced pressure. The crude residue was purified by flash silica gel column chromatography eluting with a gradient of 0-10% DCM/MeOH (containing 0.1% triethylamine) to give tert-butyl (2S,5R)-5-(hydroxymethyl)-2-methylpiperazine-1-carboxylate (2.70 g, 28% yield). LCMS calculated for C11H23N2O3(M+H)+: m/z=231.2; Found: 231.1.

To a solution of 3,4,6-trichloropyridazine (406 mg, 2.21 mmol) in DMF (1.75 mL) was added N—N-diisopropylethylamine (0.59 mL, 3.39 mmol) and tert-butyl (2S,5R)-5-(hydroxy-methyl)-2-methylpiperazine-1-carboxylate (300 mg, 1.30 mmol) in DMF (1.75 mL) at room temperature. The reaction was stirred at 80° C. overnight. The reaction was cooled to room temperature, diluted with water (15.0 mL) and extracted with EtOAc (15 mL×3). The combined organic phases were dried over Na2SO4, filtered and concentrated under reduced pressure. The crude material was purified by silica gel flash column chromatography eluting with a gradient of 10-100% EtOAc/heptanes to give tert-butyl (2S,5R)-4-(3,6-dichloropyridazin-4-yl)-5-(hydroxymethyl)-2-methylpiperazine-1-carboxylate (408 mg, 83% yield), as a yellow viscous oil. LCMS calc. for C15H23C12N4O3[M+H]+: m/z=377.1; Found: 377.0.

To a solution of tert-butyl (2S,5R)-4-(3,6-dichloropyridazin-4-yl)-5-(hydroxymethyl)-2-methylpiperazine-1-carboxylate (2.70 g, 7.16 mmol) and triphenylphosphine (2.63 g, 10 mmol) in THF (35 mL) was slowly added diisopropyl azodicarboxylate (1.97 mL, 10 mmol) at 0° C. The reaction mixture was stirred at 0° C. for 10 minutes, followed by the slow addition of diphenylphosphoryl azide (2.76 g, 10 mmol). The reaction mixture was stirred at room temperature overnight. The reaction was concentrated under reduced pressure and a saturated aqueous solution of NaHCO3(15.0 mL) was added. The mixture was extracted with EtOAc (15 mL×3) and the combined organics were dried over Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified by silica gel flash column chromatography eluting with a gradient of 10-50% EtOAc/heptanes to give tert-butyl (2S,5R)-5-(azidomethyl)-4-(3,6-dichloropyridazin-4-yl)-2-methylpiperazine-1-carboxylate (2.00 g, 70% yield), as a yellow oil. LCMS calc. for C15H22C12N7O2[M+H]+: m/z=402.1; Found: 401.8.

A solution of triphenylphosphine (5.50 g, 20.8 mmol) and tert-butyl (2S,5R)-5-(azido-methyl)-4-(3,6-dichloropyridazin-4-yl)-2-methylpiperazine-1-carboxylate (7.00 g, 17.3 mmol) in THF (45.0 mL) was heated at 60° C. for 3 hours. Water (4.50 mL) and N—N-diisopropyl-ethylamine (9.00 mL, 51.9 mmol) were added and the reaction mixture was stirred at 60° C. overnight. The reaction was cooled to room temperature and concentrated under reduced pressure. The residual oil was diluted with water (40.0 mL) and extracted with EtOAc (50 mL×3). The combined organics were dried over Na2SO4, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel flash column chromatography, eluting with a gradient of 10-100% EtOAc/hexanes to give tert-butyl (6aS,9S)-2-chloro-9-methyl-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carboxylate (1.50 g, 26% yield), as a yellow solid. LCMS calc. for C15H23ClN5O2[M+H]+: m/z=340.2; Found: 340.0.

The solution of tert-butyl (6aS,9S)-2-chloro-9-methyl-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carboxylate (1.50 g, 4.41 mmol) in DCM (24.0 mL) was added triethylamine (1.23 mL, 8.83 mmol) and di-tert-butyl dicarbonate (1.35 g, 6.18 mmol) at 0° C. The reaction was stirred at room temperature overnight. The starting material was not completely consumed and 4-dimethylaminopyridine (54.0 mg, 0.440 mmol) and additional di-tert-butyl dicarbonate (675 mg, 3.09 mmol) were added. The reaction was further stirred at room temperature for 3 hours. A saturated aqueous solution of NH4Cl (30.0 mL) was added and the aqueous phase was extracted with DCM (30 mL×3). The combined organics were dried over Na2SO4, filtered and concentrated under reduced pressure. The crude material was purified by silica gel flash column chromatography, eluting with a gradient of 10-50% EtOAc/hexanes to give di-tert-butyl (6aR,9S)-2-chloro-9-methyl-6a,7,9,10-tetrahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-5,8(6H)-dicarboxylate (1.10 g, 57% yield), as white solid. LCMS calc. for C20H31ClN5O4[M+H]+: m/z=440.2; Found: 440.1.

A mixture of di-tert-butyl (6aR,9S)-2-chloro-9-methyl-6a,7,9,10-tetrahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-5,8(6H)-dicarboxylate (420 mg, 0.950 mmol), (3,5-difluoro-2-hydroxyphenyl)-boronic acid (199 mg, 1.15 mmol), cesium carbonate (622 mg, 1.91 mmol) and XPhos Pd G2 (150 mg, 0.190 mmol) was degassed. After addition of 1,4-Dioxane (4.00 mL) and water (0.500 mL), the reaction was stirred at 80° C. overnight. The mixture was diluted with DCM and washed with water. The combined organics were dried over Na2SO4, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel flash column chromatography eluting with a gradient of 0-100% EtOAc/hexanes followed by 15% MeOH/DCM to give di-tert-butyl (6aR,9S)-2-(3,5-difluoro-2-hydroxyphenyl)-9-methyl-6a,7,9,10-tetrahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-5,8(6H)-dicarboxylate (380 mg, 75% yield). LCMS calc. for C26H34F2N5O5[M+H]+: m/z=534.3; Found: 534.2.

A solution of di-tert-butyl (6aR,9S)-2-(3,5-difluoro-2-hydroxyphenyl)-9-methyl-6a,7,9,10-tetrahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-5,8(6H)-dicarboxylate (200 mg, 0.370 mmol) in DCM (1.00 mL) was treated with a 4 N HCl solution in 1,4-dioxane (2.00 mL, 64.7 mmol) at room temperature for 1 hour. The mixture was concentrated under reduced pressure to give 2,4-difluoro-6-((6aR,9S)-9-methyl-6,6a,7,8,9,10-hexahydro-5H-pyrazino [1′,2′:4,5]pyrazino[2,3-c]pyridazin-2-yl)phenol (120 mg, 79% yield) as the HCl salt. The material was used without additional purification. LCMS calc. for C16H18F2N5O [M+H]+: m/z=334.2; Found: 334.1.

To a solution of (S)-piperazine-2-carboxylic acid; dihydrochloride (2.50 g, 12.3 mmol) in a mixture of water (4.92 mL) and 1,4-dioxane (19.7 mL) was added sodium hydroxide (3.64 mL, 47.3 mmol) in water (3.00 mL), followed by di-tert butyl dicarbonate (5.64 g, 25.9 mmol) at 0° C. The reaction was stirred at room temperature overnight. Imidazole (838 mg, 12.3 mmol) was added and the reaction was stirred at room temperature for 20 minutes. The reaction mixture was diluted with DCM and the organics were washed with a 1 N aqueous solution of HCl (4×25 mL) and a saturated brine solution (1×25 mL). The combined organics were dried over MgSO4, filtered and concentrated under reduced pressure to give crude (S)-1,4-bis(tert-butoxycarbonyl) piperazine-2-carboxylic acid (4.07 g) as a white solid. The material was used in the following step without additional purification.

Iodomethane (0.283 mL, 4.55 mmol) was added to a solution of (S)-1,4-bis(tert-butoxycarbonyl)-piperazine-2-carboxylic acid (1.00 g, 3.03 mmol) and potassium carbonate (545 mg, 3.94 mmol) in DMF (10.1 mL) at 0° C. The reaction was warmed to room temperature and stirred for 2 hours. The reaction mixture was quenched with a saturated aqueous solution of NH4Cl and diluted with water. The aqueous phase was extracted with EtOAc (3×25 mL) and the combined organics were washed with a saturated brine solution (1×25 mL), dried over MgSO4, filtered and concentrated under reduced pressure to give crude 1,4-di-tert-butyl 2-methyl (S)-piperazine-1,2,4-tricarboxylate (2.14 g). The material was used in the following step without additional purification. LCMS calcd for C7H13N2O4[M+H-C9H16O2]+: m/z=189.1; Found: 189.0.

LiHMDS (7.14 mL, 7.14 mmol) was added to a solution of 1,4-di-tert-butyl 2-methyl (S)-piperazine-1,2,4-tricarboxylate (2.14 g) in THF at −78° C. The reaction was stirred for 2 hours at −78° C. Iodomethane (741 μL, 11.9 mmol) was added and the reaction was warmed to room temperature and stirred overnight. The reaction mixture was quenched with a saturated aqueous solution of NH4Cl (25 mL) and extracted with EtOAc (2×25 mL). The combined organics were washed with a saturated brine solution (1×25 mL), dried over MgSO4, filtered and concentrated under reduced pressure. The crude material was purified by silica gel flash column chromatography, eluting with a gradient of 0-100% EtOAc/hexanes to give 1,4-di-tert-butyl 2-methyl 2-methylpiperazine-1,2,4-tricarboxylate (2.10 g, 98% yield) as a clear oil. LCMS calcd for C8H15N2O4[M+H—C9H16O2]+: m/z=203.1; Found: 203.1.

To a solution of 1,4-di-tert-butyl 2-methyl 2-methylpiperazine-1,2,4-tricarboxylate (2.50 g, 7.26 mmol) in DCM (43.6 mL) was carefully added DMF (436 μL) and oxalyl chloride (1.87 mL, 21.8 mmol). The mixture was stirred at room temperature for 30 minutes. The volatiles were removed under reduced pressure and DMF (13.1 mL), N,N-Diisopropylethylamine (6.32 mL, 36.3 mmol) and 4-bromo-6-chloropyridazin-3-amine (3.03 g, 14.5 mmol) were added sequentially. The resulting mixture was stirred at 120° C. overnight. The reaction mixture was diluted with EtOAc (100 mL) and washed with a saturated brine solution (30 mL×2). The combined organics were dried over MgSO4, filtered and concentrated under reduced pressure. The crude material was purified by silica gel flash column chromatography, eluting with a gradient of 0-100% EtOAc/hexanes to give tert-butyl 2-chloro-6a-methyl-6-oxo-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carboxylate (929 mg, 36% yield) as a tan solid. LCMS calcd for C15H21C1N5O3[M+H]+: m/z=354.1; Found: 354.1.

A 1 N solution of BH3in THF (559 μL, 559 μmol) was added to a solution of tert-butyl 2-(3-fluoro-2-hydroxyphenyl)-6a-methyl-6-oxo-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carboxylate (Isomer 1) (50.0 mg, 112 μmol) in THF (1.12 mL) at room temperature. The reaction was heated to 60° C. and stirred for 24 hours. The reaction was cooled to room temperature and carefully diluted MeOH (10 mL) and then heated at 80° C. for 10 minutes. The reaction was cooled to room temperature, diluted with DCM and the organics were washed with a saturated aqueous solution of NaHCO3. The combined organics were dried over MgSO4, filtered and concentrated under reduced pressure. The crude material was purified by silica gel flash column chromatography, eluting with a gradient of 0-20% MeOH/DCM to give tert-butyl 2-(3-fluoro-2-hydroxyphenyl)-6a-methyl-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carboxylate (Isomer 1) (12.0 mg, 26% yield). LCMS calcd for C21H27FN5O3[M+H]+: m/z=416.2; Found: 416.2.

The title compound was prepared using the procedure analogous to those described for Intermediate 3, step 13, with tert-butyl 2-(3-fluoro-2-hydroxyphenyl)-6a-methyl-5,6,6a,7,9,10-hexahydro-8H-pyrazino-[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carboxylate (isomer 1) replacing di-tert-butyl (6aR,9S)-2-(3,5-difluoro-2-hydroxyphenyl)-9-methyl-6a,7,9,10-tetrahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-5,8(6H)-dicarboxylate. LCMS calcd for C16H19FN5O [M+H]+: m/z=316.2; Found: 316.1.

A solution of triphosgene (2.24 g, 7.54 mmol) in DCM (15.0 mL) was carefully added dropwise to a solution of tert-butyl (3R,5S)-3,5-dimethylpiperazine-1-carboxylate (2.15 g, 10.1 mmol) and pyridine (1.22 mL, 15.1 mmol) in DCM (25.0 mL) at 0° C. The reaction was warmed to room temperature and stirred for 2 hours. Upon completion, the reaction mixture was poured into a 1 N aqueous HCl solution (150 mL), while on ice and stirred for 15 minutes. The organic phase was separated, and the aqueous phase was extracted with DCM (3×30 mL). The combined organics were washed with a saturated brine solution (50 mL), dried over MgSO4, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel flash column chromatography, eluting with a gradient of 0-10% ethyl acetate/hexanes to give tert-butyl (3R,5S)-4-(chlorocarbonyl)-3,5-dimethylpiperazine-1-carboxylate (1.66 g, 60% yield) as a white solid. 1H NMR (300 MHz, CDCl3) 4.40 (s, 2H), 4.15-3.77 (d, J=18 Hz, 2H), 3.05 (s, 2H), 1.52 (s, 9H), 1.40-1.27 (d, J, 12 Hz, 6H).

Triethylamine (19.4 μL, 139 μmol) and tert-butyl (3R,5S)-4-(chlorocarbonyl)-3,5-dimethylpiperazine-1-carboxylate (12.8 mg, 46.4 μmol) in DCM (1.00 mL) was added to a solution of 2-fluoro-6-(6a-methyl-6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazin-2-yl)phenol; dihydrochloride (Isomer 1, 9.00 mg, 20.0 μmol) in DCM (1.15 mL). The reaction was stirred at room temperature overnight. The reaction mixture was quenched with MeOH and a saturated aqueous solution of NaHCO3was added. The aqueous phase was extracted with DCM and the combined organics were dried over MgSO4, filtered, and concentrated under reduced pressure. The crude material was dissolved in DCM (1.15 mL), 2,2,2-trifluoroacetic acid (53.2 μL, 0.695 mmol) was added and the reaction was stirred for 1 hour. The reaction mixture was concentrated under reduced pressure and the crude residue was purified by prep-HPLC (Waters CSH-C18, 5 uM, 30×100 mm, 6.1-26.1% MeCN/water (containing 0.1% TFA) over 5 min) to give the TFA salt of the title compound (3.00 mg, 19% yield) as an oil. LCMS calcd for C23H31FN7O2[M+H]+: m/z=456.2; Found: 456.1.

The title compound was prepared using the procedure analogous to those described for Intermediate 4, steps 1-10, with (2-chloro-6-hydroxyphenyl)boronic acid replacing 3-fluoro-2-hydroxyphenylboronic acid in Step 6 and tert-butyl (3R,5R)-4-(chlorocarbonyl)-3,5-dimethylpiperazine-1-carboxylate replacing tert-butyl (3R,5S)-3,5-dimethylpiperazine-1-carboxylate in Step 9. LCMS calcd for C23H31ClN7O2[M+H]+: m/z=472.2; Found: 472.0.

The intermediates shown below in Table 1 were prepared by methods analogous to that described for the preparing Intermediate 4 using the appropriate starting materials.

To a solution of methyl 2-cyano-5-fluorobenzoate (2.00 g, 11.2 mmol) and 4-piperidinemethanol (1.67 g, 14.5 mmol) in dimethyl sulfoxide (22.3 mL) was added N,N-diisopropylethylamine (5.83 mL, 33.5 mmol). The reaction mixture was heated to 110° C. and stirred for 1.5 hours. The product mixture was diluted with EtOAc (100 mL) and transferred to a separatory funnel. The diluted reaction mixture was washed with a saturated aqueous sodium chloride solution (50 mL×2). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel flash column chromatography, eluting with a gradient 0-100% EtOAc/hexanes to obtain methyl 2-cyano-5-(4-(hydroxymethyl)piperidin-1-yl)benzoate (3.02 g, 98% yield) as a yellow oil. LCMS calcd for C15H18N2O3[M+H]+: m/z=275.1; Found: 275.1.

To a solution of methyl 2-cyano-5-(4-(hydroxymethyl)piperidin-1-yl)benzoate (3.00 g, 10.9 mmol), sodium hypophosphite monohydrate (11.7 g, 111 mmol) and acetic acid (12.7 mL, 222 mmol) in pyridine (26.3 mL) was added a slurry of Raney nickel (1.97 g, 33.6 mmol) in water (28.0 mL). The reaction mixture was heated to 70° C. and stirred for 8 hours. The product mixture was filtered through celite and the celite was washed with EtOAc (50 mL×2). The filtrate was transferred to a separatory funnel and washed with water (150 mL). The aqueous layer was extracted with EtOAc (75 mL×2). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel flash column chromatography eluting with a gradient of 0-100% EtOAc/hexanes to give methyl 2-formyl-5-(4-(hydroxymethyl)piperidin-1-yl)benzoate (2.31 g, 76.0%) as a yellow oil. LCMS calcd for C15H20NO4[M+H]+: m/z=278.1; Found: 278.1.

To a stirring solution of methyl 2-formyl-5-(4-(hydroxymethyl)piperidin-1-yl)benzoate (2.40 g, 8.65 mmol) in DCM (48.8 mL) and DMF (48.8 mL) was added 3-aminopiperidine-2,6-dione hydrochloride (1.85 g, 11.3 mmol) followed by N,N-diisopropylethylamine (3.77 mL, 21.6 mmol). The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was cooled to 0° C. and acetic acid (5.94 mL, 104 mmol) followed by sodium triacetoxy-borohydride (5.50 g, 26.0 mmol) was added. The reaction mixture was allowed to slowly warm to room temperature and stirred for an additional 3 hours. The reaction mixture was diluted with water (10 mL) and the solution was basified with saturated aqueous NaHCO3solution until no further evolution of gas was observed. The basified product mixture was filtered and the solid was washed with water (10 mL×2). The solid was collected and dried under vacuum to obtain 3-(6-(4-(hydroxymethyl)piperidin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (1.95 g, 63%) as a grey-white solid. LCMS calcd for C19H23N3O4[M+H]+: m/z=358.2; found: 358.1.

The intermediates shown below in Table 2 were prepared by methods analogous to those described for the preparation of Intermediate 20 using the appropriate starting materials.

To a solution of methyl 2-formyl-5-[4-(hydroxymethyl)piperidin-1-yl]benzoate (3.0 g, 10.82 mmol) in DCM (45 mL) was charge H-Glu(OtBu)-NH2HCl (2.84 g, 11.9 mmol), acetic acid (1.24 mL, 21.64 mmol), and N,N-Diisopropylethylamine (2.07 mL, 11.9 mmol). The reaction mixture was stirred at RT for 30 min then cool to 5° C. Sodium triacetoxyborohydride (0.840 g, 3.97 mmol) was charged portion wise at 5° C. then stirred at room temp overnight. After being diluted with DCM (45 mL), the reaction was quenched with sat. NaHCO3aq solution until no more bubbling was observed, while white precipitation formed. Filtered precipitate and washed with water and DCM. Collect solid and dried under vacuum to give a white solid (4.1 g, 88% yield, 98.4% ee). LCMS calcd for C23H34N3O5[M+H]+: m/z=432.1; Found: 432.1

To a stirred solution of tert-butyl (S)-5-amino-4-(6-(4-(hydroxymethyl)piperidin-1-yl)-1-oxoisoindolin-2-yl)-5-oxopentanoate (3.00 g, 6.95 mmol) in DCM (30.0 mL) was charged N,N-Diisopropylethylamine (1.33 mL, 7.65 mmol) followed by acetyl chloride (0.74 mL, 10.4 mmol). The reaction was stirred at RT for 2 hours. The reaction was quenched with sat. NaHCO3, extracted with DCM, dried over Na2SO4, purified by Flash Chromatography with 5% MeOH in DCM to give a pale-yellow solid (3.1 g, 94%). LCMS calcd for C25H36N3O6[M+H]+: m/z=474.1; Found: 474.1.

To a stirred solution of tert-butyl (S)-4-(6-(4-(acetoxymethyl)piperidin-1-yl)-1-oxoisoindolin-2-yl)-5-amino-5-oxopentanoate (1.15 g, 2.43 mmol) in MeCN (12.0 mL) was charged benzenesulfonic acid (768.0 mg, 4.86 mmol). The reaction mixture was heated to 85° C. After 2 hours, the reaction was cooled to RT and charged with water (12 mL) then stirred at 75° C. for 4 hours until LCMS showed full conversion. The reaction mixture was evaporated to remove volatiles, the residue was cooled to 5° C. and charged with NaHCO3aq slowly until pH=6-7 during which white precipitation formed. Filtered and washed with water. Collect solid and dried under vacuum to give an off-white solid (730 mg, 84% yield, 98.2% ee). LCMS calcd for C19H24N3O4[M+H]+: m/z=358.2; Found: 358.1

The intermediate shown below in Table 3 was prepared by the method used in preparing Int-27 using the appropriate starting materials.

To a stirring solution of (R)-morpholin-2-ylmethanol (255 mg, 2.18 mmol), methyl 2-cyano-4-fluorobenzoate (300 mg, 1.67 mmol) in NMP (6.70 mL) was added N,N-diisopropyl-ethylamine (0.88 mL, 5.02 mmol). The reaction was heated to 120° C. and stirred for 1.5 hours. The reaction mixture was cooled to room temperature and diluted with DCM and a saturated aqueous brine solution. The aqueous layer was extracted with DCM twice. The organic layers were combined, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel flash column chromatography eluting with a gradient of 0-100% EtOAc/hexane to give methyl (R)-2-cyano-4-(2-(hydroxymethyl)morpholino)benzoate (401 mg, 87% yield) as a yellow oil. LCMS m/z calcd for C14H17N2O4[M+H]+: m/z=277.1; found: 277.0.

A mixture of methyl (R)-2-cyano-4-(2-(hydroxymethyl)morpholino)benzoate (401 mg, 1.45 mmol), pyridine (3.50 mL, 43.2 mmol), acetic acid (1.70 mL, 29.4 mmol) and sodium hypophosphite monohydrate (1.55 g, 14.67 mmol) was treated with a slurry of Raney nickel (262 mg, 4.46 mmol) in water (3.70 mL). The reaction was heated to 70° C. and stirred for 8 hours, while monitored by LCMS. The reaction mixture was filtered through celite and the celite was washed with EtOAc and water. The aqueous layer was extracted with EtOAc and the combined organics were dried over Na2SO4, filtered and concentrated under reduced pressure. The crude material was purified by silica gel flash column chromatography eluting with a gradient of 0-100% EtOAc/hexane. The product was obtained as a yellow oil to give methyl (R)-2-formyl-4-(2-(hydroxymethyl)morpholino)benzoate (308 mg, 76% yield). LCMS calcd for C14H18NO5[M+H]+: m/z=280.1; Found: 280.0.

N,N-diisopropylethylamine (0.58 mL, 3.31 mmol) was added to a solution of methyl (R)-2-formyl-4-(2-(hydroxymethyl)morpholino)benzoate (308 mg, 1.10 mmol) and 3-amino-piperidine-2,6-dione, HCl (272 mg, 1.65 mmol) in DCM (5.50 mL). The reaction was stirred at room temperature overnight. The reaction was cooled to 0° C. and acetic acid (0.760 mL, 13.2 mmol) and sodium triacetoxyborohydride (701 mg, 3.31 mmol) were added. The reaction was stirred at 0° C. until complete. A saturated aqueous solution of NaHCO3was added the crude reaction mixture was concentrated under reduced pressure. The crude material was dissolved in DMSO, filtered and the filtrate was purified by prep-HPLC (Waters CSH-C18 column, 7.2-27.2% MeCN/water (containing 0.1% TFA) over 5 min) to give 3-(5-((R)-2-(hydroxymethyl) morpholino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione; 2,2,2-trifluoroacetic acid (259 mg, 50% yield) as a purple oil. LCMS m/z calcd for C18H22N3O5[M+H]+: m/z=360.2; Found: 360.1.

N,N-Diisopropylethylamine (0.380 mL, 2.17 mmol) was added to a solution of 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindole-1,3-dione (200 mg, 0.720 mmol) and 4-piperidine-methanol (208 mg, 1.81 mmol) in NMP (7.24 mL). The reaction was heated to 100° C. and monitored by LCMS until complete. The reaction was cooled to room temperature and the solution was filtered and purified by prep-HPLC (Waters CSH-C18 column, 18.3-38.3% MeCN/water (containing 0.1% TFA) over 5 min) to give the TFA salt of 2-(2,6-dioxopiperidin-3-yl)-5-(4-(hydroxymethyl)piperidin-1-yl)isoindoline-1,3-dione (185 mg, 53% yield) as a yellow solid. LCMS m/z calcd for C19H22N3O5[M+H]+: m/z=372.2; Found: 372.0.

LiHMDS (1 M in THF, 31.9 mL, 31.9 mmol) was added to a solution of 1,4-di-tert-butyl 2-methyl (S)-piperazine-1,2,4-tricarboxylate (5.5 g, 16 mmol) in THF (80 mL) at −78° C. The reaction was stirred for 1 hour at −78° C. Difluoromethyl trifluoromethanesulfonate (6.07 mL, 47.9 mmol) was added to the reaction mixture and the reaction mixture was allowed to slowly warm to room temperature and stirred overnight. The reaction mixture was quenched with a saturated aqueous solution of NH4Cl (200 mL) and extracted with EtOAc (2×200 mL). The combined organics were dried over MgSO4, filtered, and concentrated under reduced pressure. The crude material was purified by silica gel flash column chromatography with a gradient of 0-30% EtOAc/hexanes to give 1,4-di-tert-butyl 2-methyl 2-(difluoromethyl)piperazine-1,2,4-tricarboxylate as an orange oil (6.0 g, 95%). LCMS calcd for C8H13F2N2O4[M+H-C9H16O2]+: m/z=239.1; Found: 239.0

Lithium hydroxide (3.3 g, 0.14 mol) was added to a solution of 1,4-di-tert-butyl 2-methyl 2-(difluoromethyl)piperazine-1,2,4-tricarboxylate (5.5 g, 13.9 mmol) in THF (100 mL), methanol (40 mL), and water (40 mL) at room temperature. The reaction mixture was heated to 60° C. and stirred overnight. The reaction mixture was quenched with a 1 M HCl solution (200 mL). The product mixture was transferred to a separatory funnel and extracted with DCM (2×200 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue was used in the next step without purification. 1,4-bis(tert-butoxycarbonyl)-2-(difluoromethyl)piperazine-2-carboxylic acid (5.3 g, 100%) was obtained as a white foam. LCMS calcd for C7H11F2N2O4[M+H-C9H16O2]+: m/z=225.1; Found: 225.0

Oxalyl chloride (1.55 mL, 18.1 mmol) was added dropwise to a stirring solution of DMF (1.4 mL, 18.1 mmol) and DCM (35 mL) at 0° C. The reaction mixture was stirred for 10 minutes at 0° C. Then a solution of 1,4-bis(tert-butoxycarbonyl)-2-(difluoromethyl)piperazine-2-carboxylic acid (5.3 g, 13.9 mmol) and pyridine (1.69 mL, 20.9 mmol) in DCM (10 mL) was added to the reaction mixture at 0° C. After the addition the reaction mixture was stirred for 30 minutes at 0° C. Then the reaction mixture was transferred to a separatory funnel and washed with water (2×30 mL) and a saturated sodium chloride aqueous solution (30 mL). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue was dissolved in DMF (28 mL), and N,N-diisopropylethylamine (7.28 mL, 41.8 mmol) and 4-bromo-6-chloropyridazin-3-amine (2.76 g, 13.2 mmol) were added sequentially. The resulting mixture was stirred at 120° C. overnight. The product mixture was diluted with EtOAc (200 mL) and washed with a saturated sodium chloride aqueous solution (2×200 mL). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel flash column chromatography with a gradient of 0-100% EtOAc/hexanes to obtain tert-butyl 2-chloro-6a-(difluoromethyl)-6-oxo-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carboxylate as a mixture with 4-bromo-6-chloropyridazin-3-amine. The mixture was dissolved in THF (40 mL), and di-tert-butyl dicarbonate (4.8 mL, 20.9 mmol) and 4-(dimethylamino)pyridine (223 mg, 1.82 mmol) were added sequentially. The reaction mixture was stirred for 5 hours. The product mixture was concentrated under reduced pressure. The resulting residue was purified by silica gel flash column chromatography with a gradient of 0-100% EtOAc/hexanes to obtain di-tert-butyl 2-chloro-6a-(difluoromethyl)-6-oxo-6a,7,9,10-tetrahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-5,8(6H)-dicarboxylate as a pale yellow foamy solid (523 mg, 8%). LCMS calcd for C20H26ClF2N5O5[M+H]+: m/z=490.2; Found: 489.9

A 20 mL scintillation vial was charged with tert-butyl (S)-2-chloro-6a-(difluoromethyl)-6-oxo-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carboxylate (326 mg, 0.66 mmol), cesium carbonate (650 mg, 2.0 mmol), XPhos Pd G2 (52.4 mg, 0.066 mmol), and 3-fluoro-2-hydroxyphenylboronic acid (239 mg, 1.5 mmol). The mixture was dissolved in 1,4-dioxane (4 mL) and water (0.5 mL). The reaction mixture was sparged with N2gas for 2 minutes, sealed, and heated to 80° C. The reaction mixture was stirred for 2 hours at 80° C. The product mixture was diluted with EtOAc (50 mL) and washed with water (100 mL). The aqueous layer was extracted with EtOAc (2×100 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel flash column chromatography with a gradient of 0-100% EtOAc/hexanes to obtain tert-butyl (S)-6a-(difluoromethyl)-2-(3-fluoro-2-hydroxyphenyl)-6-oxo-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carboxylate as a pale oil (205 mg, 66%). LCMS calcd for C21H22F3N5O4[M+H]+: m/z=466.2; Found: 466.0

Borane tetrahydrofuran complex (1 M, 7.4 mL, 7.4 mmol) was added to a solution of tert-butyl (S)-6a-(difluoromethyl)-2-(3-fluoro-2-hydroxyphenyl)-6-oxo-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carboxylate (570 mg, 1.22 mmol) in THF (8.5 mL) at room temperature. The reaction mixture was heated to 60° C. for 2 hours. The reaction mixture was cooled to 0° C. and quenched with MeOH (3 mL) slowly. The reaction mixture was diluted with EtOAc (100 mL). The diluted reaction mixture was washed with a saturated sodium bicarbonate aqueous solution (2×100 mL). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue was dissolved in THF (8.5 mL) and MeOH (3.7 mL). Acetic acid (2.1 mL, 36.7 mmol) and sodium cyanoborohydride (770 mg, 12.2 mmol) were added sequentially to the reaction mixture at room temperature. The reaction mixture was refluxed at 80° C. for 16 hours. The product mixture was cooled to room temperature and diluted with EtOAc (100 mL). The diluted reaction mixture was washed with a saturated sodium bicarbonate aqueous solution (2×100 mL). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel flash column chromatography with a gradient of 0-15% MeOH/DCM to obtain tert-butyl (R)-6a-(difluoromethyl)-2-(3-fluoro-2-hydroxyphenyl)-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carboxylate as a dark oil (455 mg, 82%). LCMS calcd for C21H25F3N5O [M+H]+: m/z=452.2; Found: 452.1

Hydrochloric acid (4 M in 1,4-dioxane, 3.8 mL, 15.2 mmol) was added to a solution of tert-butyl (R)-6a-(difluoromethyl)-2-(3-fluoro-2-hydroxyphenyl)-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carboxylate (455 mg, 1.0 mmol) in DCM (10 mL) at room temperature. The reaction was stirred overnight. The product mixture was concentrated under reduced pressure to obtain the HCl salt of (S)-2-(6a-(difluoromethyl)-6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazin-2-yl)-6-fluorophenol as a tan solid (427 mg, 100%). LCMS calcd for C16H17F3N5O [M+H]+: m/z=352.1; Found: 352.1

Triphosgene (415 mg, 1.4 mmol) was added portionwise to a stirring solution of tert-butyl 3,3-dimethylpiperazine-1-carboxylate (500 mg, 2.33 mmol) and pyridine (570 μL, 7.0 mmol) in DCM (20 mL) at 0° C. The reaction was warmed to room temperature and stirred for 2 hours. The product mixture was washed with 1 M HCl aqueous solution (50 mL). The aqueous layer was extract with DCM (2×50 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue obtained was used without further purification. tert-Butyl 4-(chlorocarbonyl)-3,3-dimethylpiperazine-1-carboxylate was obtained as a yellow oil (650 mg, 100%).

N,N-Diisopropylethylamine (701 μL, 4.0 mmol) and 4-(dimethylamino)pyridine (36.9 mg, 0.30 mmol) were added sequentially to a stirring solution of (S)-2-(6a-(difluoromethyl)-6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazin-2-yl)-6-fluorophenol (427 mg, 1.01 mmol) in dimethylacetamide (10 mL) at room temperature. The reaction mixture was stirred 15 minutes. A solution of tert-butyl 4-(chlorocarbonyl)-3,3-dimethylpiperazine-1-carboxylate (418 mg, 1.51 mmol) in dimethylacetamide (2 mL) was added to the reaction mixture. The reaction mixture was stirred 30 minutes at room temperature. The product mixture was diluted with EtOAc (100 mL) washed with saturated sodium bicarbonate aqueous solution (100 mL). The aqueous layer was extracted with EtOAc (2×100 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel flash column chromatography with a gradient of 0-15% MeOH/DCM to obtain tert-butyl (R)-4-(6a-(difluoromethyl)-2-(3-fluoro-2-hydroxyphenyl)-6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carbonyl)-3,3-dimethylpiperazine-1-carboxylate as a yellow oil (502 mg, 84.3%). LC calcd for C28F37F3N7O4[M+H]+: m/z=592.3 Found: 592.2.

Step 10. (R)-(6a-(difluoromethyl)-2-(3-fluoro-2-hydroxyphenyl)-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazin-8-yl)(2,2-dimethylpiperazin-1-yl)methanone 12841 Trifluoroacetic acid (1.54 mL, 20.1 mmol) was added to a stirring solution of tert-butyl (R)-4-(6a-(difluoromethyl)-2-(3-fluoro-2-hydroxyphenyl)-6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carbonyl)-3,3-dimethylpiperazine-1-carboxylate (502 mg, 0.85 mmol) in DCM (10 mL) at room temperature. The reaction was stirred 1 hour at room temperature. The product mixture was concentrated under reduced pressure to obtain the trifluoroacetic acid salt of (R)-(6a-(difluoromethyl)-2-(3-fluoro-2-hydroxyphenyl)-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazin-8-yl)(2,2-dimethylpiperazin-1-yl)methanone as an oil (610 mg, 100%) LCMS calcd for C23H29F3N7O2[M+H]+: m/z=492.2 Found: 492.0.

Hydrochloric acid (4 M in dioxane. 0.21 mL, 0.86 mmol) was added to a stirring solution of tert-butyl (S)-6a-(difluoromethyl)-2-(3-fluoro-2-hydroxyphenyl)-6-oxo-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carboxylate (40 mg, 0.086 mmol) in DCM (2 mL). The reaction mixture was stirred overnight. The product mixture was concentrated under reduced pressure to obtain the HCl salt of (S)-6a-(difluoromethyl)-2-(3-fluoro-2-hydroxyphenyl)-7,8,9,10-tetrahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazin-6(6aH)-one (37 mg, 99%). LCMS calcd for C16H14F3N5O2[M+H]+: m/z=366.1; Found: 366.0

N,N-Diisopropylethylamine (30 μL, 0.17 mmol) and 4-(dimethylamino)pyridine (1.6 mg, 0.013 mmol) were added to a stirring solution of (S)-2-(6a-(difluoromethyl)-6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazin-2-yl)-6-fluorophenol (19.2 mg, 0.043 mmol) in dimethylacetamide (1 mL) at room temperature. The reaction mixture was stirred 15 minutes. A solution of tert-butyl (3R,5S)-4-(chlorocarbonyl)-3,5-dimethylpiperazine-1-carboxylate (18 mg, 0.065 mmol) in dimethylacetamide (1 mL) was added to the reaction mixture. The reaction mixture was stirred 30 minutes at room temperature. The product mixture was diluted with EtOAc (25 mL) and washed with a saturated sodium bicarbonate aqueous solution (25 mL). The aqueous layer was extract with EtOAc (2×25 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue obtained was purified by silica gel flash column chromatography with a gradient of 0-10% MeOH/DCM to obtain tert-butyl (3R,5S)-4-((S)-6a-(difluoromethyl)-2-(3-fluoro-2-hydroxy-phenyl)-6-oxo-6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carbonyl)-3,5-dimethylpiperazine-1-carboxylate as a yellow oil (37 mg, 71%). LCMS ailed for C28H34F3N7O5[M+H]+: m/z=606.3; Found: 606.3

Borane tetrahydrofuran complex (1 M in THF, 0.53 mL, 0.53 mmol) was added to a stirring solution of tert-butyl (3R,5S)-4-((S)-6a-(difluoromethyl)-2-(3-fluoro-2-hydroxyphenyl)-6-oxo-6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carbonyl)-3,5-dimethylpiperazine-1-carboxylate (80 mg, 0.132 mmol) in THF (1 mL) at room temperature. The reaction mixture was heated to 60° C. and stirred overnight. The product mixture was cooled to 0° C. and quenched by slow addition of MeOH (2 mL). The quenched product mixture was heated to 80° C. and stirred for 30 minutes. The product mixture was concentrated under reduced pressure. The residue obtained was dissolved in DCM (2 mL). Trifluoroacetic acid (300 μL, 3.96 mmol) was added to the dissolved residue and stirred at room temperature for 1 hour. The product mixture was concentrated under reduced pressure. The residue obtained was dissolved in acetonitrile and purified by prep-HPLC (Waters CSH-Flouro-Phenyl, 5 uM, 30×100 mm, 5-25% MeCN/water (containing 0.1% TFA) over 5 min) to give ((R)-6a-(difluoromethyl)-2-(3-fluoro-2-hydroxyphenyl)-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazin-8-yl)((2R,6S)-2,6-dimethylpiperazin-1-yl)methanone (16 mg, 20%) as its TFA salt. LCMS calcd for C23H28F3N7O2[M+H]+: m/z=492.2 Found: 492.1

The intermediates shown below in Table 4 were prepared by methods analogous to that described for preparing Intermediate 31 using the appropriate starting materials.

To a solution of methyl 2-cyano-5-fluorobenzoate (5.0 g, 27.9 mmol) and 4-piperidine-methanol (9.64 g, 83.7 mmol) in NMP (20 mL), N, N-diisopropylethylamine (7.29 mL, 41.9 mmol) was added and the reaction was heated at 120° C. for 4 h. DIPEA was removed using rotary evaporation. The solution was further diluted by ethyl acetate and washed with H2O. The organic layer was collected and dried over Na2SO4, filtered and concentrated. The crude product was purified by FCC (0% to 40% EA/heptanes) to obtain the title compound as a yellowish solid (5.1 g, 18.6 mmol, 67% yield). LCMS: (M+H)+=275.1.

To a solution of methyl 2-cyano-5-[4-(hydroxymethyl)piperidin-1-yl]benzoate (2.5 g, 9.1 mmol) in DCM (30 mL) was added Dess-Martin Periodinane (5.8 g, 13.7 mmol) at 0° C. The reaction was stirred for 15 mins at room temperature, and TLC monitoring showed full consumption of starting material (eluent: 100% EA). The reaction mixture was diluted with DCM and quenched by saturated NaHCO3until effervescence stopped. The aqueous phase was extracted with DCM and the organic phase was dried over Na2SO4, filtered and concentrated under reduced pressure. The crude was purified by FCC (10% to 85% EA/heptanes) to obtain the title compound that was taken to next step without further purification. 1H NMR (300 MHz, CDCl3) δ 9.71 (s, 1H), 7.64-7.46 (m, 2H), 6.99 (dt, J=10.4, 5.2 Hz, 1H), 3.98 (dt, J=13.1, 4.1 Hz, 2H), 3.22-3.05 (m, 2H), 2.65-2.47 (m, 1H), 2.12-1.99 (m, 2H), 1.85-1.67 (m, 2H).

To a solution of methyl 2-cyano-5-[4-(dimethoxymethyl)piperidin-1-yl]benzoate (1.2 g, 3.77 mmol) in pyridine (2.44 mL, 30.2 mmol) and acetic acid (2.16 mL, 37.7 mmol) was added sodium phosphinate hydrate (0.8 g, 7.54 mmol) and water (5 mL). To the stirring solution, Raney nickel slurry in water (663 mg, 11.3 mmol) was added in multiple portions to avoid emulsion formation. The reaction was heated to 75° C. overnight. The heating was stopped and the reaction was cooled down, diluted with MeOH, filtered through celite, and washed with MeOH. The filtrate was concentrated to remove pyridine and methanol. The residue was dissolved in ethyl acetate and washed with water. The organic phase was dried over Na2SO4, filtered and concentrated. The product was purified by FCC (0% to 35% EA/Heptanes) to obtain the title compound (742 mg, 2.31 mmol, 61% yield).

To a solution of methyl 5-[4-(dimethoxymethyl)piperidin-1-yl]-2-formylbenzoate (450.0 mg, 1.4 mmol) in DCM (2 mL) and DMF (2 mL) was added N,N-diisopropylethylamine (0.61 mL, 3.5 mmol) and methyl (S)-4,5-diamino-5-oxopentanoate (269 mg, 1.68 mmol). The reaction was stirred at rt for 1 h and acetic acid (0.8 mL, 14. mmol) was added to this solution. The reaction was further stirred for another hour and sodium triacetoxyborohydride (890 mg, 4.2 mmol) was added, and the reaction was stirred over the weekend. The reaction was stopped, diluted with DCM (50 mL), and quenched by saturated NaHCO3solution dropwise until pH of 8-9 was maintained. The organic phase was collected, dried over Na2SO4, filtered and concentrated. The residue was purified by FCC (0% to 100% EA/Heptanes) to obtain the title compound (400 mg, 0.93 mmol, 66% yield).

2,2,2-Trifluoroacetic acid (2.38 mL, 31.1 mmol) was added to a solution of (S)-3-(6-(4-(dimethoxymethyl)piperidin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (500 mg, 1.25 mmol) in a 3 to 1 mixture of DCM (9.3 mL) and acetone (3.1 mL). The reaction was stirred overnight and concentrated under reduced pressure to obtain the title compound (440 mg, quantitative yield) as the TFA salt. LCMS calcd for C19H22N3O4(M+H)+m/z=356.2; Found: 356.2.

Trifluoroacetic acid (2.38 mL, 31.1 mmol) was added to a stirring solution of 3-(6-(4-(dimethoxymethyl)piperidin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (500 mg, 1.25 mmol) in DCM (9.3 mL) and acetone (3.1 mL) at room temperature. The reaction mixture was stirred 48 hours at room temperature. The product mixture was concentrated. The residue obtained was dissolved in DCM (100 mL) and transferred to a separatory funnel containing saturated sodium carbonate aqueous solution (50 mL) and saturated sodium bicarbonate aqueous solution (50 mL). The aqueous layer was extracted with DCM (3×100 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to obtain 1-(2-(2,6-dioxopiperidin-3-yl)-3-oxoisoindolin-5-yl)piperidine-4-carbaldehyde (440 mg, 99%) as a white solid. LCMS calcd for C19H22N3O4[M+H]+: m/z=356.2; Found: 356.1.

This compound was prepared by a procedure analogous to that described for Intermediate 37, utilizing tert-butyl piperazine-1-carboxylate instead of 4-piperidinemethanol in Step 1. LCMS calcd for C17H21N4O3(M+2H-Boc)+m/z=329.2; Found: 329.1.

LiHMDS (1 M in THF, 5.3 mL, 5.3 mmol) was added to a solution of 1,4-di-tert-butyl 2-methyl (S)-piperazine-1,2,4-tricarboxylate (1.3 g, 3.77 mmol) in THF (19 mL) at −78° C. The reaction mixture was stirred for 2 hours at −78° C. Iodoethane (0.91 mL, 11.3 mmol) was added to the reaction mixture at −78° C. The reaction mixture was stirred overnight and allowed to slowly warm to room temperature. The product mixture was quenched with saturated ammonium chloride aqueous solution (30 mL). The quenched product mixture was diluted with EtOAc (50 mL). The organic layer was washed with water (30 mL) and saturated sodium chloride solution (30 mL). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue obtained was purified by silica gel flash column chromatography with a gradient of 0-60% EtOAc/hexanes to obtain 1,4-di-tert-butyl 2-methyl 2-ethylpiperazine-1,2,4-tricarboxylate (1.2 g, 85%) as a clear oil. LCMS calcd for C9H17N2O4[M+2H—C9H16O2]+: m/z=217.1; Found: 2171

Lithium hydroxide (1.25 g, 52.3 mmol) was added to a stirring solution of 1,4-di-tert-butyl 2-methyl 2-ethylpiperazine-1,2,4-tricarboxylate (1.3 g, 3.49 mmol) in THF (30 mL), methanol (10 mL), and water (10 mL). The reaction mixture was stirred overnight. The product mixture was diluted with 1 M HCl aqueous solution (70 mL). The diluted product mixture was extracted with DCM (2×100 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain 1,4-bis(tert-butoxycarbonyl)-2-ethylpiperazine-2-carboxylic acid (1.24 g, 99%) as a clear oil. LCMS calcd for C8H15N2O4[M+2H—C9H16O2]+: m/z=203.1; Found: 20.3.1

Oxalyl chloride (0.76 mL, 8.86 mmol) was added dropwise to a stirring solution of 1,4-bis(tert-butoxycarbonyl)-2-ethylpiperazine-2-carboxylic acid (1.27 g, 3.54 mmol) and dimethylformamide (270 μL, 3.5 mmol) in DCM (27 mL) at 0° C. The reaction mixture was warmed to room temperature and stirred for 40 minutes. The reaction mixture was concentrated under reduced pressure then redissolved in DMF (8.1 mL). N,N-Diisopropylethylamine (3.1 mL, 17.7 mmol) and 4-bromo-6-chloropyridazin-3-amine (1.48 g, 7.09 mmol) were added in sequence to the stirring reaction mixture at room temperature. The reaction mixture was heated to 120° C. and stirred for 18 hours. The product mixture was diluted with EtOAc (80 mL) and washed with saturated sodium chloride aqueous solution (80 mL). The aqueous layer was extracted with EtOAc (2×80 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue obtained was purified by silica gel flash column chromatography with a gradient of 0-100% EtOAc/hexanes to obtain tert-butyl 2-chloro-6a-ethyl-6-oxo-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carboxylate (401 mg, 31%) as a white solid. LCMS calcd for C16H23ClN5O3[M+H]+: m/z=368.2; Found: 368.1.

Di-tert-butyl decarbonate (470 μL, 2.07 mmol) and 4-(dimethylamino)pyridine (38 mg, 0.31 mmol) were added in sequence to a stirring solution of tert-butyl 2-chloro-6a-ethyl-6-oxo-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carboxylate (380 mg, 1.03 mmol) in THF (10 mL) at room temperature. The reaction mixture was stirred for 5 hours. The product mixture was concentrated under reduced pressure. The residue obtained was purified by silica gel flash column chromatography with a gradient of 0-100% EtOAc/hexanes to obtain di-tert-butyl 2-chloro-6a-ethyl-6-oxo-6a,7,9,10-tetrahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-5,8(6H)-dicarboxylate (310 mg, 64%) as a tan foamy solid. LCMS calcd for C21H31ClN5O5[M+H]+: m/z=468.2; Found: 468.0.

A 20 mL scintillation vial was charged with di-tert-butyl 2-chloro-6a-ethyl-6-oxo-6a,7,9,10-tetrahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-5,8(6H)-dicarboxylate (Isomer 1) (227 mg, 0.49 mmol), cesium carbonate (474 mg, 1.46 mmol), XPhos Pd G2 (57.3 mg, 0.073 mmol), and 3-fluoro-2-hydroxyphenylboronic acid (151 mg, 0.97 mmol). The mixture was dissolved in 1,4-dioxane (4 mL) and water (0.5 mL). The reaction mixture was sparged with N2gas for 2 minutes, sealed, and heated to 80° C. The reaction mixture was stirred for 2 hours at 80° C. The product mixture was diluted with EtOAc (50 mL) and washed with water (100 mL). The aqueous layer was extracted with EtOAc (2×100 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel flash column chromatography with a gradient of 0-30% acetone/hexanes to obtain tert-butyl 6a-ethyl-2-(3-fluoro-2-hydroxyphenyl)-6-oxo-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carboxylate (Isomer 1) (130 mg, 60%).

N,N-Diisopropylethylamine (220 μL, 1.26 mmol) and 4-(dimethylamino)pyridine (11.6 mg, 0.095 mmol) were added to a stirring solution of 6a-ethyl-2-(3-fluoro-2-hydroxyphenyl)-7,8,9,10-tetrahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazin-6(6aH)-one (120 mg, 0.316 mmol) in dimethylacetamide (3 mL) at room temperature. The reaction mixture was stirred 15 minutes. A solution of tert-butyl 4-(chlorocarbonyl)-3,3-dimethylpiperazine-1-carboxylate (131 mg, 0.474 mmol) in dimethylacetamide (1 mL) was added to the reaction mixture. The reaction mixture was stirred 30 minutes at room temperature. The product mixture was diluted with EtOAc (50 mL) washed with saturated sodium bicarbonate aqueous solution (50 mL). The aqueous layer was extract with EtOAc (2×50 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue obtained was purified by silica gel flash column chromatography with a gradient of 0-100% EtOAc/hexanes to obtain tert-butyl 4-(6a-ethyl-2-(3-fluoro-2-hydroxyphenyl)-6-oxo-6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carbonyl)-3,3-dimethylpiperazine-1-carboxylate as a yellow oil (170 mg, 92%). LCMS calcd for C29H39FN7O5[M+H]+: m/z=584.3; Found: 584.2.

Borane tetrahydrofuran complex (1 M in THF, 2.33 mL, 2.33 mmol) was added to a stirring solution of tert-butyl 4-(6a-ethyl-2-(3-fluoro-2-hydroxyphenyl)-6-oxo-6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carbonyl)-3,3-dimethylpiperazine-1-carboxylate in THF (9 mL) at room temperature. The reaction mixture was heated to 60° C. and stirred for 5 hours. The reaction mixture was cooled to room temperature and borane tetrahydrofuran complex (1 M in THF, 2.33 mL, 2.33 mmol) was added to the reaction mixture. The reaction mixture was heated to 60° C. and stirred for 3 hours. The product mixture was cooled to 0° C. and quenched by slow addition of MeOH (6 mL). The quenched product mixture was heated to 70° C. and stirred for 3 hours. The product mixture was concentrated under reduced pressure. The residue obtained was purified by silica gel flash column chromatography with a gradient of 0-10% MeOH/DCM to obtain tert-butyl 4-(6a-ethyl-2-(3-fluoro-2-hydroxyphenyl)-6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carbonyl)-3,3-dimethylpiperazine-1-carboxylate (Isomer 1) (121 mg, 73%). LCMS calcd for C29H41FN7O4[M+H]+: m/z=570.3; Found: 570.3

Trifluoroacetic acid (403 μL, 5.3 mmol) was added to a stirring solution of tert-butyl 4-(6a-ethyl-2-(3-fluoro-2-hydroxyphenyl)-6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]pyrazino [2,3-c]pyridazine-8-carbonyl)-3,3-dimethylpiperazine-1-carboxylate (120 mg, 0.211 mmol) in DCM (3 mL) at room temperature. The reaction mixture was stirred 1 hour. The product mixture was concentrated under reduced pressure to obtain the TFA salt of (2,2-dimethylpiperazin-1-yl)(6a-ethyl-2-(3-fluoro-2-hydroxyphenyl)-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazin-8-yl)methanone (147 mg, 100%). LCMS calcd for C24H33FN7O2[M+H]+: m/z=470.3; Found: 470.1

The intermediates shown below in Table 5 were prepared by methods analogous to that described for the preparing Intermediate 40 using the appropriate starting materials.

LiHMDS (1 M in THF, 8.97 mL, 8.97 mmol) was added to a solution of 1,4-di-tert-butyl 2-methyl (S)-piperazine-1,2,4-tricarboxylate (2.06 g, 5.98 mmol) in THF (24 mL) at −78° C. The reaction was stirred for 1.5 h at −78° C. Fluoroiodomethane (1.2 mL, 12 mmol) was added and the reaction was allowed to slowly warm to room temperature and stirred overnight. The reaction mixture was quenched with a saturated ammonium chloride aqueous solution (60 mL) and extracted with EtOAc (2×60 mL). The combined organics were dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude material was purified by silica gel flash column chromatography with a gradient of 0-30% EtOAc/hexanes to give 1,4-di-tert-butyl 2-methyl 2-(fluoromethyl)piperazine-1,2,4-tricarboxylate as an oil (1.98 g, 88%) LCMS calcd for C8H14FN2O4[M+2H—C9H16O2]+: m/z=221.1; Found: 221.1

Step 2. Synthesis of 1,4-bis(tert-butoxycarbonyl)-2-(fluoromethyl)piperazine-2-carboxylic acid

Lithium hydroxide (2.97 g, 70.8 mmol) was added to a solution of 1,4-di-tert-butyl 2-methyl 2-(fluoromethyl)piperazine-1,2,4-tricarboxylate (2.8 g, 7.54 mmol) in THF (25 mL), methanol (25 mL), and water (2.5 mL) at room temperature. The reaction mixture was heated to 55° C. and stirred overnight. The reaction mixture was quenched with a 1 M HCl solution (75 mL). The product mixture was transferred to a separatory funnel and extracted with DCM (2×100 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue was used in the next step without purification. 1,4-bis(tert-butoxycarbonyl)-2-(fluoromethyl)piperazine-2-carboxylic acid (2.7 g, 99%) was obtained as a yellow oil. LCMS calcd for C7H12FN2O4[M+2H—C9H16O2]+: m/z=207.1; Found: 207.0

Oxalyl chloride (831 μL, 9.69 mmol) was added dropwise to a stirring solution of DMF (750 μL, 9.69 mmol) and DCM (16 mL) at 0° C. The reaction mixture was stirred for 10 minutes at 0° C. Then a solution of 1,4-bis(tert-butoxycarbonyl)-2-(fluoromethyl)piperazine-2-carboxylic acid (2.7 g, 7.45 mmol) and pyridine (904 μL, 11.2 mmol) in DCM (5 mL) was added to the reaction mixture at 0° C. After the addition the reaction mixture was stirred for 30 minutes at 0° C. Then the reaction mixture was diluted with DCM (30 mL), transferred to a separatory funnel, and washed with water (2×30 mL) and a saturated sodium chloride aqueous solution (30 mL). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue was dissolved in DMF (11 mL), and N,N-diisopropylethylamine (3.89 mL, 22.4 mmol) and 4-bromo-6-chloropyridazin-3-amine (1.4 g, 6.71 mmol) were added sequentially. The resulting mixture was stirred at 120° C. overnight. The product mixture was diluted with EtOAc (150 mL) and washed with a saturated sodium chloride aqueous solution (2×200 mL). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel flash column chromatography with a gradient of 0-100% EtOAc/hexanes to obtain tert-butyl 2-chloro-6a-(fluoromethyl)-6-oxo-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carboxylate as a mixture with 4-bromo-6-chloropyridazin-3-amine. The mixture was dissolved in THF (20 mL), and di-tert-butyl decarbonate (1.7 mL, 7.45 mmol) and 4-(dimethylamino)pyridine (120 mg, 0.97 mmol) were added sequentially. The reaction mixture was stirred for 5 hours. The product mixture was concentrated under reduced pressure. The resulting residue was purified by silica gel flash column chromatography with a gradient of 0-100% EtOAc/hexanes to obtain di-tert-butyl 2-chloro-6a-(fluoromethyl)-6-oxo-6a,7,9,10-tetrahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-5,8(6H)-dicarboxylate as a pale yellow foamy solid (465 mg, 13.2%). LCMS calcd for C20H28ClFN5O5[M+H]+: m/z=472.2; Found: 472.0

A 20 mL scintillation vial was charged with di-tert-butyl 2-chloro-6a-(fluoromethyl)-6-oxo-6a,7,9,10-tetrahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-5,8(6H)-dicarboxylate (Isomer 1) (465 mg, 0.985 mmol), cesium carbonate (963 mg, 2.96 mmol), XPhos Pd G2 (77.5 mg, 0.01 mmol), and 3-fluoro-2-methoxyphenylboronic acid (385 mg, 2.27 mmol). The mixture was dissolved in 1,4-dioxane (5.8 mL) and water (0.7 mL). The reaction mixture was sparged with N2gas for 2 minutes, sealed, and heated to 80° C. The reaction mixture was stirred for 2 hours at 80° C. The product mixture was diluted with EtOAc (50 mL) and washed with water (100 mL). The aqueous layer was extracted with EtOAc (2×100 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel flash column chromatography with a gradient of 0-100% EtOAc/hexanes to obtain tert-butyl 2-(3-fluoro-2-methoxyphenyl)-6a-(fluoromethyl)-6-oxo-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carboxylate (332 mg, 73%). LCMS calcd for C22H26F2N5O4[M+H]+: m/z=462.2; Found: 462.1

Borane tetrahydrofuran complex (1 M, 2.97 mL, 2.97 mmol) was added to a solution of tert-butyl 2-(3-fluoro-2-methoxyphenyl)-6a-(fluoromethyl)-6-oxo-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carboxylate (342 mg, 0.742 mmol) in THF (5 mL) at room temperature. The reaction mixture was heated to 60° C. The reaction mixture was stirred at 60° C. for 2 hours. The reaction mixture was cooled to 0° C. and quenched with MeOH (3 mL) slowly. The reaction mixture was diluted with EtOAc (60 mL). The diluted reaction mixture was washed with saturated sodium bicarbonate aqueous solution (2×60 mL). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue was dissolved in THF (5 mL) and MeOH (2 mL). Acetic acid (1.27 mL, 22.3 mmol) and sodium cyanoborohydride (466 mg, 7.42 mmol) were added sequentially to the reaction mixture at room temperature. The reaction mixture was refluxed at 80° C. for 16 hours. The product mixture was cooled to room temperature and diluted with EtOAc (100 mL). The diluted reaction mixture was washed with saturated sodium bicarbonate aqueous solution (2×100 mL). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue dissolved in THF (5 mL). Di-tert-butyl dicarbonate (340 μL, 1.48 mmol) and 4-(dimethylamino) pyridine (11 mg, 0.09 mmol) were added sequentially. The reaction mixture was stirred 2 hours at room temperature. The product mixture was concentrated under reduced pressure. The resulting residue was purified by silica gel flash column chromatography with a gradient of 0-100% EtOAc/hexanes to obtain di-tert-butyl 2-(3-fluoro-2-methoxyphenyl)-6a-(fluoromethyl)-6a,7,9,10-tetrahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-5.8(6H)-dicarboxylate as a dark oil (270 mg, 66%). LCMS calcd for C27H36F2N5O5[M+H]+: m/z=548.3; Found: 548.2

Hydrochloric acid (4 M in 1,4-dioxane, 0.52 mL, 2.09 mmol) was added to a solution of tert-butyl 2-(3-fluoro-2-methoxyphenyl)-6a-(fluoromethyl)-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carboxylate (Isomer 2) (62.1 mg, 0.14 mmol) in DCM (2.8 mL) at room temperature. The reaction was stirred overnight. The product mixture was concentrated under reduced pressure to obtain the HCl salt of 2-(3-fluoro-2-methoxyphenyl)-6a-(fluoromethyl)-6,6a,7,8,9,10-hexahydro-5/1-pyrazino[1′,2′:4,5]pyrazino [2,3-c]pyridazine (isomer 2) as a tan solid (58 mg, 100%). LCMS calcd for C17H20F2N5O [M+H]+: m/z=348.2; Found: 348.0

N,N-Diisopropylethylamine (98 μL, 0.56 mmol) and 4-(dimethylamino)pyridine (5.2 mg, 0.04 mmol) was added to a stirring solution of 2-(3-fluoro-2-methoxyphenyl)-6a-(fluoromethyl)-6.6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine (isomer 2) (59 mg, 0.14 mmol) in dimethylacetamide (2 mL) at room temperature. The reaction mixture was stirred 15 minutes. A solution of tert-butyl 4-(chlorocarbonyl)-3,3-dimethylpiperazine-1-carboxylate (58.7 mg, 0.21 mmol) in dimethylacetamide (1 mL) was added to the reaction mixture. The reaction mixture was stirred 30 minutes at room temperature. The product mixture was diluted with EtOAc (30 mL) and washed with saturated sodium bicarbonate aqueous solution (30 mL). The aqueous layer was extract with EtOAc (2×30 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel flash column chromatography with a gradient of 0-15% MeOH/DCM to obtain tert-butyl 4-(2-(3-fluoro-2-methoxyphenyl)-6a-(fluoromethyl)-6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carbonyl)-3,3-dimethylpiperazine-1-carboxylate (Isomer 2) (78 mg, 91%). LC MS calcd for C29H40F2N7O4[M+H]+: m/z=588.3; Found: 588.2.

Boron tribromide (119 μL, 1.25 mmol) was added to a stirring solution of tert-butyl 4-(2-(3-fluoro-2-methoxyphenyl)-6a-(fluoromethyl)-6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carbonyl)-3,3-dimethylpiperazine-1-carboxylate (Isomer 2) (74 mg, 0.125 mmol) in DCM (4 mL) at 0° C. The reaction was warmed to room temperature and stirred for 12 hours. The product mixture was cooled to 0° C. and quenched with water (1 mL). The quenched product mixture was transferred to a separatory funnel containing saturated potassium carbonate aqueous solution (30 mL) and extracted with 3:1 CHCl3:iPrOH (6×30 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure to obtain (2,2-dimethylpiperazin-1-yl)(2-(3-fluoro-2-hydroxyphenyl)-6a-(fluoromethyl)-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazin-8-yl)methanone (Isomer 2) (51 mg, 86%). LCMS calcd for C23H30F2N7O2[M+H]+: m/z=474.2; Found: 474.2.

The intermediate shown below in Table 6 was prepared by methods analogous to that described for the preparing Intermediate 55 using the appropriate starting materials.

To a vial containing tert-butyl cis-3,5-dimethylpiperazine-1-carboxylate (206 mg, 0.96 mmol) was added DCM (4.80 mL) and pyridine (0.190 mL, 2.40 mmol). The reaction was cooled to 0° C. and triphosgene (186 mg, 0.630 mmol) was added. The reaction was left to warm to room temperature and stirred for 1 hour. The reaction was diluted in DCM then washed with a 1 N aqueous solution of HCl. The layers were separated and the aqueous phase was extracted with additional DCM. The organic layers were combined and dried over Na2SO4, filtered and concentrated under reduced pressure. The crude material was dissolved in DCM (4.80 mL) and 4-fluoro-2-[(10R)-1,5,6,8,12-pentazatricyclo[8.4.0.02,7]tetradeca-2(7),3,5-trien-4-yl]phenol; dihydrochloride (180 mg, 0.480 mmol) and triethylamine (0.340 mL, 2.40 mmol) were added. The reaction was stirred overnight at room temperature. The crude residue was purified by prep-HPLC (Waters CSH-C18, 23.5-43.5% MeCN/water (containing 0.1% TFA) over 5 min) to give the Boc-intermediate as a brown oil. The residue was dissolved in DCM (4.80 mL) and 2,2,2-trifluoroacetic acid (1.10 mL, 14.4 mmol) was added and the reaction was stirred at room temperature for 1.5 hours. The reaction mixture was concentrated under reduced pressure to obtain ((2S,6R)-2,6-dimethylpiperazin-1-yl)((S)-2-(3-fluoro-2-hydroxyphenyl)-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazin-8-yl)methanone as the TFA salt (119.47 mg, 45% yield). The material was used without additional purification. LCMS calcd for C22H29FN7O2[M+H]+: m/z=442.2; found: 442.1.

Examples shown below in Table 7 were prepared as TFA salts by the method used in preparing Example 1 using the appropriate intermediates and starting materials.

A solution of acetic acid (100 μL, 1.70 mmol), (R)-2,4-difluoro-6-(6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazin-2-yl)phenol (111 mg, 0.283 mmol, Intermediate 2), tert-butyl (1R,5S,6r)-6-formyl-3-azabicyclo[3.1.0]hexane-3-carboxylate (90.0 mg, 0.424 mmol) and N,N-diisopropylethylamine (197 μL, 1.13 mmol) in DMF (1.00 mL) was stirred at room temperature for 1 hour. Sodium triacetoxyborohydride (180 mg, 0.849 mmol) was added and the reaction was stirred at room temperature for 2 hours. The reaction mixture was diluted with DCM (3.00 mL) and washed with water (2×3 mL). The organics were dried over MgSO4, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel flash column chromatography eluting with a gradient of 0-10% MeOH/DCM to obtain tert-butyl (1R,5S)-6-[[(10S)-4-(3,5-difluoro-2-hydroxyphenyl)-1,5,6,8,12-pentazatricyclo [8.4.0.02,7]tetradeca-2,4,6-trien-12-yl]methyl]-3-azabicyclo[3.1.0]hexane-3-carboxylate (140 mg, 96% yield). LCMS calc. for C26H33F2N6O3[M+H]+: m/z=515.3; Found: 515.1.

A solution of 4 N HCl in 1,4-dioxane (1.17 mL, 37.9 mmol) was added to a solution of tert-butyl (1R,5S,6s)-6-(((S)-2-(3,5-difluoro-2-hydroxyphenyl)-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazin-8-yl)methyl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (140 mg, 0.272 mmol) in DCM (1.00 mL). The reaction was stirred at room temperature for 1 hour. The mixture was concentrated to give the title compound (120 mg, 91% yield) as its HCl salt. The material was used in the following step without additional purification. LCMS calc. for C21H25F2N6O [M+H]+: m/z=415.2; Found: 415.1

To a mixture of ((3R,4R)-1-(2-(2,6-dioxopiperidin-3-yl)-3-oxoisoindolin-5-yl)-3-fluoropiperidin-4-yl)methyl 4-methylbenzenesulfonate (82.0 mg, 0.218 mmol, Intermediate 22) in pyridine (1.07 mL, 13.3 mmol) was added tosyl chloride (83.3 mg, 0.440 mmol). The mixture was stirred at room temperature for 1 hour. Additional tosyl chloride was added every 2 hours until the starting material was consumed. The mixture was diluted with DCM and the organic phase was washed with water. The combined organics were dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel flash column chromatography, eluting with a gradient of 0-100% EtOAC/DCM to give rac-((3R,4R)-1-(2-(2,6-dioxopiperidin-3-yl)-3-oxoisoindolin-5-yl)-3-fluoropiperidin-4-yl)methyl 4-methyl-benzenesulfonate (110 mg, 95% yield). LCMS calcd for C26H29FN3O6S [M+H]+: m/z=530.2; found: 530.2.

Examples shown below in Table 8 were prepared as TFA salts by the method used in preparing Examples 37 & 38 using the appropriate intermediates and starting materials.

Examples shown below in Table 9 were prepared as TFA salts by the method used in preparing Example 42 and 43 using the appropriate intermediates and starting materials.

A mixture of di-tert-butyl (10R)-4-chloro-1,5,6,8,12-pentazatricyclo[8.4.0.02,7]tetradeca-2(7),3,5-triene-8,12-dicarboxylate (300 mg, 0.700 mmol, Intermediate 1, Step 4), (3,5-difluoro-2-hydroxyphenyl)boronic acid (135 mg, 0.770 mmol), cesium carbonate (504.5 mg, 1.55 mmol) and XPhos Pd G2 (83.1 mg, 0.110 mmol) in 1,4-dioxane (4.00 mL) and water (0.500 mL) was degassed with N2, and the reaction mixture was stirred at 120° C. for 2 hours. The mixture was diluted with DCM and washed with a saturated brine solution. The aqueous phase was extracted with DCM and the combined organics were dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude residue was suspended in a mixture of 1,4-dioxane (4.00 mL) and water (0.500 mL) and 9-fluorenylmethoxycarbonyl chloride (401 mg, 1.55 mmol) was added, followed by sodium bicarbonate (355 mg, 4.23 mmol). After stirring at room temperature for 4 hours, an additional portion of 9-fluorenylmethoxycarbonyl chloride (401 mg, 1.55 mmol) and sodium bicarbonate (355 mg, 4.23 mmol) were added. The reaction was stirred at room temperature overnight. The reaction mixture was diluted with DCM and water. The layers were separated and the combined organics were dried over Na2SO4, filtered and concentrated under reduced pressure. The crude material was purified by silica gel flash column chromatography, eluting with a gradient of 0-100% EtOAc/hexanes to give 5-((9H-fluoren-9-yl)methyl) 8-(tert-butyl) (R)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)oxy)-3,5-difluorophenyl)-6a,7,9,10-tetrahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-5,8(6H)-dicarboxylate (492 mg, 81% yield). LCMS calcd for C54H44F2N5O7[M+H]+: m/z=864.3; Found: 864.3.

5-((9H-fluoren-9-yl)methyl) 8-(tert-butyl) (R)-2-(2-((((9H-fluoren-9-yl)methoxy) carbonyl)oxy)-3,5-difluorophenyl)-6a,7,9,10-tetrahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-5,8(6H)-dicarboxylate (421 mg, 0.487 mmol) was dissolved in DCM (4.87 mL) and treated with a 4 N solution of HCl in 1,4-dioxane (0.610 mL, 2.44 mmol). The reaction was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure to give crude (9H-fluoren-9-yl)methyl (R)-2-(2-((((9H-fluoren-9-yl)methoxy) carbonyl)oxy)-3,5-difluorophenyl)-6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-5-carboxylate as an HCl salt (407 mg). The material was used without additional purification. LCMS calcd for C45H36F2N5O5[M+H]+: m/z=764.3; found: 764.2.

To a vial containing tert-butyl (3R,5R)-3,5-dimethylpiperazine-1-carboxylate (82.0 mg, 0.380 mmol) was added DCM (1.90 mL) and pyridine (0.08 mL, 0.96 mmol), followed by triphosgene (68.1 mg, 0.23 mmol) at 0° C. The reaction was left to stir at room temperature for 1 hour, upon which the reaction was then was diluted in DCM and a 1 N aqueous solution of HCl. The layers were separated, and the combined organics were dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was suspended in DCM (1.90 mL) and crude (9H-fluoren-9-yl)methyl (R)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)oxy)-3,5-difluorophenyl)-6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-5-carboxylate; dihydrochloride (160 mg, 0.190 mmol) and triethylamine (0.130 mL, 0.960 mmol) were added. The reaction was stirred at room temperature for 48 hours. The crude reaction mixture was diluted in EtOAc and the organic phase was washed with a saturated brine solution. The combined organics were dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude residue was dissolved in MeOH (3.00 mL), and water (1.00 mL) and lithium hydroxide (20 eq) were added. The reaction was stirred at 85° C. for 12 hours. The reaction mixture was cooled to room temperature, filtered and the filtrate was purified by prep-HPLC (Waters CSH-C18, 5 uM, 30×100 mm, 25.6-45.6% MeCN/water (containing 0.1% TFA) over 5 min) to give boc-intermediate. 2,2,2-trifluoroacetic acid (0.439 mL) was added to a solution of Boc-intermediate in DCM (1.90 mL). The reaction was stirred at room temperature. Upon completion, the reaction mixture was concentrated under reduced pressure to give ((S)-2-(3,5-difluoro-2-hydroxyphenyl)-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazin-8-yl)((2R,6R)-2,6-dimethylpiperazin-1-yl)methanone as a TFA salt (50.0 mg, 46% yield). The material was used without additional purification. LCMS calcd for C22H28F2N7O2[M+H]+: m/z=460.2; Found: 460.1.

The title compound was prepared using the procedure analogous to those described for preparing Example 1, Step 2 with ((S)-2-(3,5-difluoro-2-hydroxyphenyl)-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazin-8-yl)((2R,6R)-2,6-dimethylpiperazin-1-yl)methanone; 2,2,2-trifluoroacetic acid replacing ((2S,6R)-2,6-dimethylpiperazin-1-yl)((S)-2-(3-fluoro-2-hydroxyphenyl)-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazin-8-yl)methanone; 2,2,2-trifluoroacetic acid. LCMS calcd for C41H49F2N10O5[M+H]+: m/z=799.4; found: 799.2.

Examples shown below in Table 10 were prepared as TFA salts by the method used in preparing Example 45 using the appropriate intermediates and starting materials.

A solution of 4 N HCl in 1,4-dioxane (20 mL, 80 mmol) was added dropwise to a solution of 1-Boc-piperidine-4-carboxaldehyde (601 mg, 2.82 mmol) in 1,4-dioxane (20 mL). The reaction was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure to afford the HCl salt of the title compound hydrochloric acid salt (398 mg, 94% yield) as a white solid.

A solution of triphosgene (82.0 mg, 0.28 mmol) in DCM (0.5 mL) was added to a solution of piperidine-4-carbaldehyde; hydrochloride (111 mg, 0.740 mmol) and pyridine (0.120 mL, 1.48 mmol) in DCM (2.50 mL) at 0° C. The reaction was stirred at 0° C. for 1 hour, then warmed to room temperature and stirred for 3 hours. The reaction was poured into a 1 N aqueous HCl solution (5.00 mL) and the aqueous phase was extracted with DCM (2×3 mL). The combined organics were dried over MgSO4, filtered and concentrated under reduced pressure. The crude residue was dissolved in DCM (3 mL) and 2,4-difluoro-6-[(10R)-1,5,6,8,12-pentazatricyclo[8.4.0.02,7]tetradeca-2(7),3,5-trien-4-yl]phenol; dihydrochloride (73.0 mg, 0.180 mmol, Intermediate 2) and triethylamine (0.10 mL, 0.74 mmol) were added. The reaction was stirred at room temperature for 1 hour. The reaction mixture was diluted in methanol (15 mL), filtered, and the filtrate was purified by prep-HPLC on a C18 column (11.5-31.5% MeCN in 0.1% TFA (aq.), pH=2) to afford the TFA salt of the title compound (27.0 mg, 26% yield) as a light yellow oil. LCMS calc. for C22H25F2N6O3[M+H]+: m/z=459.2; Found: 459.0.

A solution of (S)-1-(2-(3,5-difluoro-2-hydroxyphenyl)-6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]-pyrazino[2,3-c]pyridazine-8-carbonyl)piperidine-4-carbaldehyde; 2,2,2-trifluoroacetic acid (34.0 mg, 0.060 mmol), 3-(1-oxo-6-(piperazin-1-yl)isoindolin-2-yl)piperidine-2,6-dione; hydrochloride) (33.0 mg, 90.0 μmol), magnesium sulfate (29.0 mg, 0.240 mmol) and sodium acetate (28.0 mg, 0.340 mmol) in DMSO (1.00 mL) was stirred at 40° C. for 45 minutes. Sodium triacetoxyborohydride (55.0 mg, 0.26 mmol) was added and the reaction was stirred at room temperature for 6 hours. The reaction was quenched with water (1.0 mL) and diluted with DMSO (15.0 mL). The mixture was filtered and the filtrate was purified by prep-HPLC on a C18 column (7.6-27.6% MeCN in 0.1% TFA (aq.), pH=2) to afford the TFA salt of the title compound (32.0 mg, 30% yield) as a white solid. LCMS calc. for C39H45F2N10O5[M+H]+: m/z=771.4; Found: 771.2.

Examples shown below in Table 11 were prepared as TFA salts by the method described in the preparation of Example 49 using the appropriate intermediates and starting materials.

p-Toluenesulfonic acid (36.0 mg, 0.209 mmol) followed by 3,4-dihydro-2H-pyran (1.38 mL, 15.1 mmol) was added to a solution of ((1r,4r)-cyclohexane-1,4-diyl)dimethanol (2.00 g, 13.9 mmol) in DCM (30 mL) at 0° C. The reaction was stirred at 0° C. for 30 min, then warmed to room temperature and stirred overnight. The crude reaction mixture was poured into a saturated aqueous solution of sodium bicarbonate and the layers were separated. The organics were washed with water, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The crude material was purified by silica gel flash column chromatography, eluting with a gradient of 0-100% EtOAc/hexane to give ((1r,4r)-4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)cyclohexyl)methanol (850 mg, 27% yield) as a clear oil. Note: Product does not ionize on LCMS. Intermediate was used directly in next step.

A solution of ((1r,4r)-4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)cyclohexyl)methanol (286 mg, 1.25 mmol) and Dess-Martin periodinane (706 mg, 1.66 mmol) in DCM (12.5 mL) was stirred at room temperature for 2.5 hours. The crude reaction mixture was diluted with DCM and a saturated aqueous solution of sodium bicarbonate was added. The layers were separated and the organics were dried over magnesium sulfate, filtered and concentrated under reduced pressure to give crude (1r,4r)-4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)cyclohexane-1-carbaldehyde as a white solid. The material was used in the following step without additional purification. Note: Product does not ionize. Was used directly in next step.

The title compound was prepared using the procedure analogous to that described for preparing Example 48, Step 4 with crude (1r,4r)-4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl) cyclohexane-1-carbaldehyde replacing (5)-1-(2-(3,5-difluoro-2-hydroxyphenyl)-6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]pyra-zino[2,3-c]pyridazine-8-carbonyl)piperidine-4-carbaldehyde; 2,2,2-trifluoroacetic acid. LCMS calcd for C30H43N4O5[M+H]+: m/z=539.3; found: 539.3.

The title compound was prepared using the procedure analogous to those described for preparing Example 49 with 3-(6-(4-(((1r,4r)-4-(hydroxymethyl)cyclohexyl)methyl)piperazin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione replacing 3-[5-[2-(hydroxymethyl)morpholin-4-yl]-3-oxo-1H-isoindol-2-yl]-piperidine-2,6-dione and Intermediate 2 replacing 2-[(10S)-12-[[(1S,5R)-3-azabicyclo[3.1.0]hexan-6-yl]methyl]-1,5,6,8,12-pentazatricyclo[8.4.0.02,7]tetradeca-2(7),3,5-trien-4-yl]-4,6-difluorophenol; 2,2,2-trifluoroacetic acid. LCMS calcd for C44H48F2N9O4[M+H]+: m/z=756.4; found: 756.3.

To a 100 mL round bottom flask was added cis-4-(hydroxymethyl)cyclohexanecarboxylic acid (2.24 g, 14.2 mmol), DCM (33.0 mL) and p-TSOH·H2O (49.0 mg, 0.260 mmol). Reaction was heated to 40° C. to help partially dissolve the mixture. The reaction was cooled to −78° C. and a solution of 3,4-Dihydro-2H-pyran (1.41 mL, 15.5 mmol) in DCM (33.0 mL) was added dropwise via an addition funnel over 35 minutes. The reaction warmed to room temperature. After 4.5 hours, the reaction was quenched with 30.0 mL sat. sodium bicarbonate and stirred vigorously for 10 minutes. The solution was poured into a separatory funnel with more sat. sodium bicarbonate and DCM. The organic layer was separated, dried and concentrated under reduced pressure. The residue was purified by silica gel flash column (120 g) chromatography eluting with a gradient of 0-100% EtOAC/Hexanes to yield (1s,4s)-4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)cyclohexane-1-carboxylic acid (1.00 g, 29% yield) as a clear oil. Note: product did not ionize and was used as is directly in next step without additional purification

(1s,4s)-4-(((Tetrahydro-2H-pyran-2-yl)oxy)methyl)cyclohexane-1-carboxylic acid (135 mg, 0.560 mmol) was dissolved in THF (1.11 mL) then cooled to 0° C. Borane tetrahydrofuran (0.560 mL, 0.560 mmol) was added dropwise. The reaction was stirred at 0° C. for 10 minutes, then slowly allowed to warm to room temperature. After 5 hours, the reaction was quenched with MeOH, concentrated under reduced pressure, then taken up in EtOAc and poured into water. The aqueous phase was extracted with EtOAc (2×20 mL). The combined organic layers were washed with water followed by brine, dried over MgSO4, filtered and concentrated under reduced pressure to yield ((1s,4s)-4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)cyclohexyl)methanol (75.5 mg, 59% yield) as a clear oil. Note: product did not ionize and was used as is directly in next step without additional purification

The title compound was prepared as a TFA salt using the procedure analogous to those described for Intermediate 4, steps 1-10, with (2S,5R)-tert-butyl 2,5-dimethylpiperazine-1-carboxylate replacing tert-butyl (3R,5S)-3,5-dimethylpiperazine-1-carboxylate in Step 9 and Example 1 using the appropriate starting materials and intermediates. LCMS calcd for C42H52FN10O5[M+H]+: m/z=795.4; Found: 795.1.

The title compound was prepared as a TFA salt using the procedure analogous to those described for Example 54, steps 1-5, with Intermediate 1 replacing Intermediate 2 in Step 5. LCMS calcd for C40H49FN9O4[M+H]+: m/z=738.4; Found: 738.2.

The title compound was prepared as the TFA salt using the procedure analogous to those described for Example 1 using the appropriate starting materials and intermediates. LCMS calcd for C43H53F2N10O5[M+H]+: m/z=827.4; Found: 827.2.

The title compound was prepared as the TFA salt using the procedure analogous to those described for Example 1 using the appropriate starting materials and intermediates. LCMS calcd for C43H54FN10O5[M+H]+: m/z=809.4; Found: 809.2.

The title compound was prepared as the TFA salt using the procedure analogous to those described for Example 1 using the appropriate starting materials and intermediates. LCMS calcd for C43H53F2N10O5[M+H]+: m/z=827.4; Found: 827.1.

The title compound was prepared as the TFA salt using the procedure analogous to those described for Example 1 using the appropriate starting materials and intermediates. LCMS calcd for C43H54FN10O5[M+H]+: m/z=809.4; Found: 809.4.

The title compound was prepared as the TFA salt using the procedure analogous to those described for Example 1 using the appropriate starting materials and intermediates. LCMS calcd for C42H52ClN10O5[M+H]+: m/z=811.4; Found: 811.0.

The title compound was prepared as the TFA salt using the procedure analogous to those described for Example 1 using the appropriate starting materials and intermediates. LCMS calcd for C42H51F2N10O5[M+H]+: m/z=813.4; Found: 813.1.

Examples 66-70 shown below in Table 12 were prepared as TFA salts by the method used in preparing Example 65 using the appropriate intermediates and starting materials.

Borane tetrahydrofuran complex (1 M, 1.65 mL, 1.65 mmol) was added to (2R,4r,6S)-1-(tert-butoxycarbonyl)-2,6-dimethylpiperidine-4-carboxylic acid (170.0 mg, 0.66 mmol) in THF (3.3 mL) at 0° C. The reaction mixture was warmed to room temperature overnight. The reaction mixture was cooled to 0° C. and quenched with MeOH (2 mL). The reaction mixture was directly condensed. The residue was dissolved in water (30 mL) and EtOAc (30 mL). The aqueous layer was extracted with EtOAc (2×30 mL) and the combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to obtain tert-butyl (2R,4r,6S)-4-(hydroxymethyl)-2,6-dimethylpiperidine-1-carboxylate (158 mg, 98%) as a clear oil. LCMS calcd for C8H18NO [M+H-C5H9O2]+: m/z=144.1; Found: 144.0

Benzoyl chloride was added to a stirring solution of tert-butyl (2R,4r,6S)-4-(hydroxymethyl)-2,6-dimethylpiperidine-1-carboxylate (188 mg, 0.77 mmol), triethylamine (118 μL, 0.85 mmol), and 4-(dimethylamino)pyridine (11 mg, 0.09 mmol) in DCM (2 mL). The reaction mixture was stirred at room temperature for 6 hours. The product mixture was diluted with EtOAc (40 mL) and washed with water (30 mL) and a saturated sodium chloride solution (2×30 mL). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue obtained was dissolved in DCM (6 mL). Hydrochloric acid (4 M in 1,4-dioxane, 1.9 mL, 7.7 mmol) was added to the reaction mixture. The reaction mixture was stirred overnight at room temperature. The product mixture was concentrated under reduced pressure to obtain the HCl salt of ((2R,4r,6S)-2,6-dimethylpiperidin-4-yl)methyl benzoate (220 mg, 100%) as a white powder. LCMS calcd for C15H22NO2[M+H]+: m/z=248.2; Found: 248.1

Triphosgene (132 mg, 0.45 mmol) was added to a stirring solution of ((2R,4r,6S)-2,6-dimethylpiperidin-4-yl)methyl benzoate (330 mg, 1.16 mmol) and pyridine (0.47 μL, 5.8 mmol) in DCM (5.8 mL) at 0° C. The reaction mixture was warmed to room temperature and stirred for 2 hours. The product mixture was transferred to a separatory funnel containing 1 M aqueous HCl solution (30 mL). The diluted product mixture was extracted with DCM (2×30 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to obtain ((2R,4r,6S)-1-(chlorocarbonyl)-2,6-dimethylpiperidin-4-yl)methyl benzoate as a yellow oil. The residue was used without further purification.

((2R,4r,6S)-1-(chlorocarbonyl)-2,6-dimethylpiperidin-4-yl)methyl benzoate (252 mg, 0.81 mmol) in a solution of dimethylacetamide (1 mL) was added to a stirring solution of (R)-2-(6a-(difluoromethyl)-6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazin-2-yl)-6-fluorophenol (230.0 mg, 54 mmol), N,N-diisopropylethylamine (378 μL, 2.2 mmol), and 4-(dimethylamino)pyridine (20 mg, 0.16 mmol) in dimethylacetamide (4 mL) at room temperature. The reaction mixture was stirred at room temperature for 30 minutes. The product mixture was quenched with water (20 mL). The diluted product mixture was extracted with EtOAc (3×30 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue obtained was purified by silica gel flash column chromatography with a gradient of 0-15% MeOH/DCM to obtain ((2R,4S,6S)-1-((S)-6a-(difluoromethyl)-2-(3-fluoro-2-hydroxyphenyl)-6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carbonyl)-2,6-dimethylpiperidin-4-yl)methyl benzoate (195 mg, 58%). LCMS calcd for C32H36F3N6O4[M+H]+: m/z=625.3; Found: 625.2

Potassium carbonate (431 mg, 3.1 mmol) was added to a stirring solution of ((2R,4S,6S)-1-((S)-6a-(difluoromethyl)-2-(3-fluoro-2-hydroxyphenyl)-6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carbonyl)-2,6-dimethylpiperidin-4-yl)methyl benzoate (195 mg, 0.31 mmol) in methanol (4 mL) at room temperature. The reaction was stirred at room temperature for 3 hours. The product mixture was quenched with saturated sodium bicarbonate aqueous solution (30 mL). The diluted product mixture was extracted with EtOAc (3×30 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue obtained was purified by silica gel flash column chromatography with a gradient of 0-20% MeOH/DCM to obtain ((S)-6a-(difluoromethyl)-2-(3-fluoro-2-hydroxyphenyl)-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazin-8-yl)((2R,4S,6S)-4-(hydroxymethyl)-2,6-dimethylpiperidin-1-yl)methanone (52 mg, 32%) as a yellow oil. LCMS calcd for C25H32F3N6O3[M+H]+: m/z=521.3; Found: 521.1

Examples 72-73 shown below in Table 13 were prepared as TFA salts by the method used in preparing Example 71 using the appropriate intermediates and starting materials.

Examples 75-88 shown below in Table 14 were prepared as TFA salts by the method used in preparing Example 74 using the appropriate intermediates and starting materials

Example 90 shown below in Table 15 was prepared as the TFA salt by the method used in preparing Example 89 using the appropriate intermediates and starting materials.

Triphosgene (41.5 mg, 0.14 mmol) was added to a stirring solution of (2S,5R)-tert-butyl 2,5-dimethylpiperazine-1-carboxylate (50 mg, 0.23 mmol) and pyridine (57 μL, 0.70 mmol) at 0° C. The reaction was warmed to room temperature and stirred for 2 hours. The product mixture was washed with 1 M HCl aqueous solution (30 mL). The aqueous layer was extract with DCM (2×30 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue obtained was used without further purification. tert-butyl (2S,5R)-4-(chlorocarbonyl)-2,5-dimethylpiperazine-1-carboxylate was obtained as a yellow oil (62 mg, 96%).

tert-Butyl (2S,5R)-4-(chlorocarbonyl)-2,5-dimethylpiperazine-1-carboxylate (45 mg, 0.16 mmol) in DCM (1 mL) was added to a stirring solution of 2-(3-fluoro-2-hydroxyphenyl)-6a-methyl-7,8,9,10-tetrahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazin-6(6aH)-one (Isomer 1) (0.075 mmol) and triethylamine (62 μL, 0.45 mmol) in DCM (1 mL) at room temperature. The reaction was stirred at room temperature for 2 hours. The product mixture was purified by silica gel flash column chromatography with a gradient of 0-10% MeOH/DCM to obtain tert-butyl (2S,5R)-4-(2-(3-fluoro-2-hydroxyphenyl)-6a-methyl-6-oxo-6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carbonyl)-2,5-dimethylpiperazine-1-carboxylate (Isomer 1) (28 mg, 66%). LCMS calcd for C28H37FN7O5[M+H]+: m/z=570.3; Found: 570.1

Borane tetrahydrofuran complex (1 M in THF, 0.43 mL, 0.43 mmol) was added to a stirring solution of tert-butyl (2S,5R)-4-(2-(3-fluoro-2-hydroxyphenyl)-6a-methyl-6-oxo-6,6a,7,8,9,10-hexahydro-5H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carbonyl)-2,5-dimethylpiperazine-1-carboxylate (Isomer 1) (40 mg, 0.070 mmol) in THF (1.5 mL) at room temperature. The reaction mixture was heated to 65° C. and stirred overnight at 65° C. The product mixture was cooled to 0° C. and quenched with MeOH (3 mL). The quenched product mixture was heated to 80° C. and stirred 30 minutes and then was cooled to room temperature and concentrated under reduced pressure. The residue obtain was dissolve in DCM (2 mL). Trifluoroacetic acid (173 μL, 2.27 mmol) was added to the diluted residue at room temperature. The resulting mixture was stirred at room temperature for 1 hour. The product mixture was concentrated under reduced pressure and purified by prep-HPLC (Waters CSH-C18, 5 uM, 30×100 mm, 6.4-26.4% MeCN/water (containing 0.1% TFA) over 5 min) to give ((2R,5S)-2,5-dimethylpiperazin-1-yl)(2-(3-fluoro-2-hydroxyphenyl)-6a-methyl-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazin-8-yl)methanone (Isomer 1) (8 mg, 25%) as its TFA salt. LCMS calcd for C23H31FN7O2[M+H]+: m/z=456.3; Found: 456.0

Example 92 shown below in Table 16 was prepared as the TFA salt by the method used in preparing Example 91 using the appropriate intermediates and starting materials.

To a solution of 2-(methoxymethyl)-1,4-bis[(2-methylpropan-2-yl)oxycarbonyl]piperazine-2-carboxylic acid (0.86 g, 2.3 mmol) and pyridine (0.28 mL, 3.45 mmol) in DCM (1.5 mL) was carefully added DMF (436 μL) and oxalyl chloride (0.26 mL, 2.99 mmol). The mixture was stirred at room temperature for 30 minutes. The volatiles were removed under reduced pressure, then DMF (3 mL), N,N-Diisopropylethylamine (1.2 mL, 6.89 mmol) and 4-bromo-6-chloropyridazin-3-amine (0.48 g, 2.3 mmol) were added sequentially. The resulting mixture was stirred at 120° C. overnight. The reaction mixture was diluted with EtOAc (100 mL) and washed with a saturated brine solution (30 mL×2). The combined organics were dried over MgSO4, filtered and concentrated under reduced pressure. The crude material was purified by silica gel flash column chromatography, eluting with a gradient of 0-100% EtOAc/hexanes to give tert-butyl 2-chloro-6a-(methoxymethyl)-6-oxo-5,6,6a,7,9,10-hexahydro-8H-pyrazino[1′,2′:4,5]pyrazino[2,3-c]pyridazine-8-carboxylate (140 mg, 0.36 mmol, 72% yield). LCMS calcd for C16H23ClN5O4[M+H]+: m/z=384.1; Found: 384.1.

Example 94 was prepared as the TFA salt by the method described in preparing Example 93 using isomer 2 obtained in Step 5.

Examples 96-101 shown below in Table 17 were prepared as TFA salts by the method used in preparing Example 95 using the appropriate intermediates and starting materials.

(Methoxymethyl)triphenylphosphonium chloride (433.0 mg, 1.26 mmol) was suspended in THF (5 mL) and cooled to −78° C. A solution of LiHMDS (1.26 mL, 1.26 mmol) was added drop-wise. The reaction mixture was then stirred at −78° C. for another 2 hours followed by the addition of a solution of methyl 1-fluoro-4-oxocyclohexane-1-carboxylate (200.0 mg, 1.15 mmol) in THF (2 mL). The reaction was slowly warmed to room temperature and stirred overnight. Next, hydrochloric acid (2.0 mL, 4.0 mmol) was added and the reaction was stirred for 2 hrs. The reaction mixture was extracted with ethyl acetate. The organic layer was dried with Na2SO4, filtered, and concentrated to give crude methyl 1-fluoro-4-formylcyclohexane-1-carboxylate (216 mg, 1.15 mmol, 99% yield) which was used directly in the next step.

Example 103 was prepared as the TFA salt by the method described in preparing Example 102.

Examples 105-113 shown below in Table 18 were prepared as TFA salts by the method used in preparing Example 104 using the appropriate intermediates and starting materials.

Examples 114-118 shown below in Table 19 were prepared as TFA salts by the method described in the preparation of Example 54 using the appropriate intermediates and starting materials.

Example 119 shown in Table 20 was prepared as the TFA salt by the method described in the preparation of Example 55 using the appropriate intermediates and starting materials.

Examples 120-124 shown below in Table 21 were prepared as TFA salts by the method described in the preparation of Example 71 using the appropriate intermediates and starting materials.

Preparation of SMARCA2/4-HiBiT Knock-In Cells

HiBiT peptide knock-in of SMARCA2 in LgBiT expressing HEK293T cells was performed by CRISPR-mediated tagging system as described Promega. The homozygous HiBiT knock-in on c-terminus SMARCA2 was confirmed by sanger sequence. SMARCA2-HiBiT knock-in Hela monoclonal cell (CS302366) and SMARCA4-HiBiT knock-in Hela monoclonal cell (CS3023226) were purchased from Promega. The heterozygous HiBiT-knock-in was confirmed by sanger sequence in both SMARCA2-HiBiT and SMARCA4-HiBiT monoclonal cells.

SMARCA2 HiBiT and SMARCA4 HiBiT Degradation Assay in HeLa Cells

Compounds were dissolved in DMSO to make 10 mM stock and 3-fold series dilutions were further conducted keeping the highest concentration 10 μM. NCIH1693 and NCIH520 cells were maintained in PRMI 1640 medium (Corning Cellgro, Catalog #:10-040-CV) supplemented with 10% v/v FBS (GE Healthcare, Catalog #: SH30910.03) by splitting 1:3 twice a week.

Dispense 10 ul aliquot of prepared Hela-SMARCA2-HiBiT or Hela-SMARCA4-HiBiT cells (1:1 ratio of cells:Trypan Blue (#1450013, Bio-Rad)) onto cell counting slide (#145-0011, Bio-Rad) and obtain cell density and cell viability using cell counter (TC20, Bio-Rad). Remove appropriate volume of resuspended cells from culture flask to accommodate 2500 cells/well @ 20 μL/well. Transfer Hela-HiBiT cells to 50 mL conical (#430290, Corning). Spin down at 1000 rpm for 5 min using tabletop centrifuge (SPINCHRON 15, Beckman). Discard supernatant and resuspend cell pellet in modified EMEM (#30-2003, ATCC) cell culture media containing 10% FBS (F2422-500ML, Sigma), and 1X Penicillin/Streptomycin (200 g/L) (30-002-CI, Corning) to a cell density of 125,000 cells/mL. Dispense 20 μL of resuspended Hela-HiBit cells per well in 384-well TC treated plate (#12-565-343, Thermo Scientific) using standard cassette (#50950372, Thermo Scientific) on Multidrop Combi (#5840310, Thermo Scientific) inside laminar flow cabinet. Dispense test compounds onto plates using digital liquid dispenser (D300E, Tecan). Incubate plates in humidified tissue culture incubator @37° C. for 18 hours. Add 20 μL of prepared Nano-Glo® HiBiT Lytic detection buffer (N3050, Promega) to each well of 384-well plate using small tube cassette (#24073295, Thermo Scientific) on Multidrop Combi, incubate @ RT for 30-60 min. Read plates on microplate reader (Envision 2105, PerkinElmer) using 384 well Ultra-Sensitive luminescence mode. Raw data files and compound information reports are swept into centralized data lake and deconvoluted using automated scripts designed by TetraScience, Inc. Data analysis, curve-fitting and reporting done in Dotmatics Informatics Suite using Screening Ultra module.