Indazole based compounds and associated methods of use

Bifunctional compounds, which find utility as modulators of leucine-rich repeat kinase 2 (LRRK2), are described herein. In particular, the hetero-bifunctional compounds of the present disclosure contain on one end a moiety that binds to the cereblon E3 ubiquitin ligase and on the other end a moiety which binds LRRK2, such that the target protein is placed in proximity to the ubiquitin ligase to effect degradation (and inhibition) of target protein. The hetero-bifunctional compounds of the present disclosure exhibit a broad range of pharmacological activities associated with degradation/inhibition of target protein. Diseases or disorders that result from aberrant regulation of the target protein are treated or prevented with compounds and compositions of the present disclosure.

INCORPORATION BY REFERENCE

FIELD OF THE INVENTION

The invention provides hetero-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 of leucine-rich repeat kinase 2 (LRRK2), which is then degraded and/or inhibited.

BACKGROUND

Most small molecule drugs bind enzymes or receptors in tight and well-defined pockets. On the other hand, protein-protein interactions are notoriously difficult to target using small molecules due to their large contact surfaces and the shallow grooves or flat interfaces involved. E3 ubiquitin ligases (of which hundreds are known in humans) confer substrate specificity for ubiquitination, and therefore are more attractive therapeutic targets than general proteasome inhibitors due to their specificity for certain protein substrates. The development of ligands of E3 ligases has proven challenging, in part due to the fact that they must disrupt protein-protein interactions. However, recent developments have provided specific ligands that bind to these ligases. For example, since the discovery of nutlins, the first small molecule E3 ligase inhibitors, additional compounds have been reported that target E3 ligases.

Cereblon is a protein that in humans is encoded by the CRBN gene. CRBN orthologs are highly conserved from plants to humans, which underscores its physiological importance. Cereblon forms an E3 ubiquitin ligase complex with damaged DNA binding protein 1 (DDB1), Cullin-4A (CUL4A), and regulator of cullins 1 (ROC1). This complex ubiquitinates a number of other proteins. Through a mechanism which has not been completely elucidated, cereblon ubiquitination of target proteins results in increased levels of fibroblast growth factor 8 (FGF8) and fibroblast growth factor 10 (FGF10). FGF8 in turn regulates a number of developmental processes, such as limb and auditory vesicle formation. The net result is that this ubiquitin ligase complex is important for limb outgrowth in embryos. In the absence of cereblon, DDB1 forms a complex with DDB2 that functions as a DNA damage-binding protein.

Bifunctional compounds such as those described in U.S. Patent Application Publications 2015/0291562 and 2014/0356322 (incorporated herein by reference), function to recruit endogenous proteins to an E3 ubiquitin ligase for ubiquitination and subsequent degradation in the proteasome degradation pathway. In particular, the publications cited above describe bifunctional or proteolysis-targeting chimeric (PROTAC®) protein degrader compounds, which find utility as modulators of targeted ubiquitination of a variety of polypeptides and proteins, which are then degraded and/or inhibited by the bifunctional compounds.

Leucine-rich repeat kinase 2 (LRRK2) is a member of the leucine-rich repeat kinase family and is a large multi-domain protein with an N-terminal armadillo domain, ankryin repeat region, a leucine-rich repeat (LRR) domain, a tandem Roco type GTPase domain, a kinase domain containing a DFG-like motif, and a C-terminal WD40 domain. The LRRK2 protein is 2527 amino acids and a molecular weight of 280 kDa. Catalytic activities of LRRK2 are associated with the kinase and GTPase domain, and LRRK2 is a heterodimer in its active form (Greggio E, et al.: The Parkinson disease-associated leucine-rich repeat kinase 2 (LRRK2) is a dimer that undergoes intramolecular autophosphorylation.J Biol Chem2008, 283:16906-16914). GTP binding is essential for kinase activity, and mutations that prevent GTP binding have been shown to ablate LRRK2 kinase activity (Ito G, et al.: GTP binding is essential to the protein kinase activity of LRRK2, a causative gene product for familial Parkinson's disease.Biochemistry2007, 46:1380-1388). The only validated physiological substrates (other than LRRK2 itself) are a subset of low-molecular weight G-proteins including Rab8a and Rab10, which are involved in regulation of vesicle trafficking and endosome function and trafficking on cytoskeletal networks (Steger M, et al.: Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases.Elife2016, 5. e12813). Expression levels of LRRK2 are highest in immune cells (neutrophils, monocytes and B cells), lung and kidney, with lower levels in the brain where it is expressed in dopaminergic neurons of the substantia nigra (West A B, et al.: Differential LRRK2 expression in the cortex, striatum, and substantia nigra in transgenic and nontransgenic rodents.J Comp Neurol2014, 522:2465-2480).

There are several dominant gain-of-function pathogenic and characterized mutations to LRRK2, located either in the Roco domains (N1437H, R1441G/C/H, Y1699C), effecting GTP hydrolysis, or in the kinase domain (G2019S and I2020T). The G2019S is the most common LRRK2 mutation linked to Parkinson's disease (PD), which is a progressive neurodegenerative disorder characterized by resting tremors, rigidity, decreased movement (bradykinesia), and postural instability. The histological hallmarks of PD include neurodegeneration of the dopaminergic neurons in the substantia nigra pars compacta as well as intracellular inclusions called Lewy bodies and neurites consisting of the aggregated form of the alpha-synuclein protein. G2019S is associated with 1-2% of all PD patients and causes an increase in kinase activity of 2-fold in vitro (West A B, et al.: Parkinson's disease associated mutations in leucine-rich repeat kinase 2 augment kinase activity.Proc Natl Acad Sci USA2005, 102: 16842-16847) and autophosphorylation at Ser1292 is increased 4-fold (Sheng Z, et al.: Ser1292 autophosphorylation is an indicator of LRRK2 kinase activity and contributes to the cellular effects of PD mutations.Sci Transl Med2012, 4:164ra161). The G2019S and I2020T mutations lie within the DFG motif (DYGI in the case of LRRK2), common to all kinases, which controls catalytic activity. These mutations are thought to disrupt the inactive conformation and thus increase catalytic activity (Schmidt S H, et al.: The dynamic switch mechanism that leads to activation of LRRK2 is embedded in the DFGpsi motif in the kinase domain.Proc Natl Acad Sci USA2019, 116:14979-14988). Several of the above Parkinson disease-associated mutations (R1441C/G, Y1699C and I2020T) suppress phosphorylation of LRRK2 at Ser910 and Ser935, which in turn reduces LRRK2 association with 14-3-3 proteins, thought to represent an inactive form of LRRK2 (Nichols J, et al.: 14-3-3 binding to LRRK2 is disrupted by multiple Parkinson's disease associated mutations and regulates cytoplasmic localisation.Biochem J2010, 430:393-404).

Furthermore, LRRK2 is linked to autosomal dominant inherited PD through a mutation within a region of chromosome 12, termed PARK8, which is linked to the LRRK2 gene (Funayama M, et al.: A new locus for Parkinson's disease (PARK8) maps to chromosome 12p11.2-q13.1. Ann Neurol2002, 51:296-301; Zimprich A, et al.: Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology.Neuron2004, 44:601-607; Paisan-Ruiz C, et al.: Cloning of the gene containing mutations that cause PARK8-linked Parkinson's disease.Neuron2004, 44:595-600). LRRK2 was first described as having a link to autosomal dominant inherited Parkinson's disease in 1978, where it was traced to a family in Japan (Nukada H, et al.: [A big family of paralysis agitans (author's transl)].Rinsho Shinkeigaku1978, 18:627-634). The most common pathogenic LRRK2 mutation (G2019S) occurs in 4-8% of familial and 1-3% of sporadic PD cases. In addition, the G2019S mutation is common among PD patients of select ancestry, with 30-40% of North African Berber and 14% of Jewish patients harboring the mutation.

LRRK2 kinase inhibitors have been proposed as having the potential to treat mutation-driven PD, where there is an increase in LRRK2 activity, such as G2019S, and idiopathic PD, where the activity of LRRK2 is increased (Chen J, et al.: Leucine-rich repeat kinase 2 in Parkinson's disease: updated from pathogenesis to potential therapeutic target.Eur Neurol2018, 79:256-265; Alessi D R, et al.: LRRK2 kinase in Parkinson's disease.Science2018, 360:36-37; Di Maio R, et al.: LRRK2 activation in idiopathic Parkinson's disease.Sci Transl Med2018, 10). Several therapeutics are progressing into the clinic, including LRRK2 kinase inhibitors that will directly affect phosphorylation of downstream targets, and oligonucleotides (ASO's) directly infused into the CNS to block translation of LRRK2 protein, thereby reducing LRRK2 protein levels.

Lewy bodies are the main histological hallmark of PD. Lewy bodies are composed primarily of alpha-synuclein aggregates, and mutations in alpha-synuclein that increase this aggregation also increase the risk of developing PD (Meade R M, et al.: Alpha-synuclein structure and Parkinson's disease lessons and emerging principles.Mol Neurodegener2019, 14. 29-29). Depletion of LRRK2 with ASOs (Zhao H T, et al.: LRRK2 antisense oligonucleotides ameliorate a-synuclein inclusion formation in a Parkinson's disease mouse model. Molecular therapy.Nucleic acids2017, 8:508-519) and deletion of LRRK2 at a genomic level have been shown to reduce alpha-synuclein mediated pathology in mouse models of PD (Lin X, et al.: Leucine-rich repeat kinase 2 regulates the progression of neuropathology induced by Parkinson's-disease-related mutant alpha-synuclein.Neuron2009, 64:807-827). Mutations increasing LRRK2 activity, such as G2019S, increase the aggregation of alpha-synuclein in neurons and mouse models of PD. This increase was reversed with LRRK2 kinase inhibitors (Volpicelli-Daley L A, et al. G2019S-LRRK2 Expression Augments α-Synuclein Sequestration into Inclusions in Neurons.J Neurosci.2016 Jul. 13; 36(28):7415-27. doi: 10.1523/JNEUROSCI.3642-15.2016). There is some evidence to suggest that the G2019S mutant form of LRRK2 is resistant to inhibition by kinase inhibitors in the CNS, potentially reducing their disease modifying effect (Kelly K, et al. The G2019S mutation in LRRK2 imparts resiliency to kinase inhibition.Exp Neurol.2018 November; 309:1-13). Even though most cases of PD also have Lewy bodies upon post-mortem examination, Lewy bodies are not present in a high number of LRRK2 G2019S mutation associated PD cases (Kalia L V, et al.: Clinical correlations with Lewy body pathology in LRRK2-related Parkinson disease.JAMA neurol2015, 72:100-105). In addition to Lewy bodies being a common feature of PD, Tau pathology is also a major feature of LRRK2 mutation carriers at post-mortem (Henderson M X, et al.: Alzheimer's disease tau is a prominent pathology in LRRK2 Parkinson's disease.Acta Neuropathol Commun2019, 7. 183-183). In one study, Tau pathology was observed in 100% of LRRK2 mutation carriers, thereby highlighting LRRK2 as an important target linking PD with Tau pathology in the context of PD, even though the genetic causal link was not as strong between LRRK2 and primary tauopathies, such as supranuclear palsy (PSP) or corticobasal degeneration (CBD) (Ross O A, et al. (2006) Lrrk2 R1441 substitution and progressive supranuclear palsy.Neuropathol Appl Neurobiol32(1):23-25; Sanchez-Contreras M, et al. (2017) Study of LRRK2 variation in tauopathy: progressive supranuclear palsy and corticobasal degeneration.Mov Disord32(1):115-123). A common variation at the LRRK2 locus as a genetic determinant of PSP survival was recently reported (Jabbari E, et al., Common variation at the LRRK2 locus is associated with survival in the primary tauopathy progressive supranuclear palsy. bioRxiv 2020.02.04.932335; doi: https://doi.org/10.1101/2020.02.04.932335). It has been reported that increased LRRK2 expression in PSP by expression quantitative trait loci (eQTL) analysis may result in a reactive microglia-induced proinflammatory state which drives ongoing accumulation of misfolded Tau protein and clinical disease progression. Functional variants of LRRK2 have also been linked to Crohn's Disease and leprosy type 1 inflammatory reactions (Hui K Y, et al. Functional variants in the LRRK2 gene confer shared effects on risk for Crohn's disease and Parkinson's disease.Sci Transl Med.2018 Jan. 10; 10(423). pii: eaai7795. doi: 10.1126/scitranslmed.aai7795; Fava et al. Pleiotropic effects for Parkin and LRRK2 in leprosy type-1 reactions and Parkinson's disease.Proc Natl Acad Sci USA.2019 Jul. 30; 116(31):15616-15624. doi: 10.1073/pnas.1901805116. Epub 2019 Jul. 15).

LRRK2 is highly expressed in the immune system in neutrophils, monocytes and macrophages, as well as in brain microglia, and is a modulator of the intrinsic regulation of microglial activation and of lysosomal degradation processes (Ma et al. Genetic comorbidities in Parkinson's disease.Hum Mol Genet.2014 Feb. 1; 23(3):831-41. doi: 10.1093/hmg/ddt465. Epub 2013 Sep. 20, which was reviewed in Schapansky et al. The complex relationships between microglia, alpha-synuclein, and LRRK2 in Parkinson's disease.Neuroscience.2015 Aug. 27; 302:74-88. doi: 10.1016/j.neuroscience.2014.09.049. Epub 2014 Oct. 2). Prolonged activation of these immune cells through PD disease processes or mutations in LRRK2 could increase neuroinflammation and lead to a greater risk of developing PD and/or Tau pathology. Treatment with anti-TNF agents reduces the risk of developing PD by 78% in patients with inflammatory bowel disorder (Peter I, et al.: Anti-tumor necrosis factor therapy and incidence of Parkinson disease among patients with inflammatory bowel disease.JAMA Neurol2018), thereby demonstrating the strong linkage between inflammation and PD. In addition to PD, LRRK2 has been linked to other diseases such as cancer, leprosy, and Crohn's disease (Lewis P A, Manzoni C. LRRK2 and human disease: a complicated question or a question of complexes? (2012).Sci Signal.5(207), pe2).

An ongoing need exists in the art for effective treatments for LRRK2 related disease and disorders, e.g., idiopathic PD, LRRK2 mutation associated PD (e.g., PD associated with one or more LRRK2 activating mutations), primary tauopathies (e.g., supranuclear palsy (PSP) or corticobasal degeneration (CBD)), lewy body dementia, Crohn's Disease, Leprosy (e.g., Leprosy with type 1 inflammatory reactions), and/or neuroinflammation.

SUMMARY

The present disclosure describes hetero-bifunctional compounds that function to recruit leucine-rich repeat kinase 2 (LRRK2) to an E3 ubiquitin ligase for targeted ubiquitination and subsequent proteasomal degradation, and methods of making and using the same. In addition, the description provides methods of using an effective amount of a compound of the present disclosure for the treatment or amelioration of a disease condition, such as an LRRK2-related disease or disorder, e.g., accumulation or overactivity of an LRRK2 protein or a mutated LRRK2 protein or a mis-folded LRRK2 protein, or alpha-synuclein aggregation or accumulation, or Tau aggregation or accumulation, or idiopathic PD, or a LRRK2 mutation associated PD (e.g., PD associated with one or more LRRK2 activating mutations), or a primary tauopathy (e.g., supranuclear palsy (PSP) or corticobasal degeneration (CBD)), or lewy body dementia, or Crohn's Disease, or Leprosy (e.g., Leprosy with type 1 inflammatory reactions), or neuroinflammation.

As such, in one aspect the disclosure provides hetero-bifunctional compounds, which comprise an E3 ubiquitin ligase binding moiety (i.e., a ligand for an E3 ubiquitin ligase (a “ULM” group)), and a moiety that binds LRRK2 or a mutated version thereof (i.e., a protein targeting moiety or “PTM” group, that is, a LRRK2 targeting ligand or a “LTM” group) such that the LRRK2 protein is thereby placed in proximity to the ubiquitin ligase to effect ubiquitination and subsequent degradation (and/or inhibition) of the LRRK2 protein. In a preferred embodiment, the ULM (ubiquitination ligase binding moiety) is a cereblon E3 ubiquitin ligase binding moiety (CLM). For example, the structure of the bifunctional compound can be depicted as:

The respective positions of the PTM and ULM moieties (e.g., CLM), as well as their number as illustrated herein, is provided by way of example only and is not intended to limit the compounds in any way. As would be understood by the skilled artisan, the bifunctional compounds as described herein can be synthesized such that the number and position of the respective functional moieties can be varied as desired.

In certain embodiments, the bifunctional compound further comprises a chemical linker (“L”). In this example, the structure of the bifunctional compound can be depicted as:

where PTM is a LRRK2-targeting moiety (LTM), L is a linker, e.g., a bond or a chemical linking group coupling PTM to ULM, and ULM is a cereblon E3 ubiquitin ligase binding moiety (CLM).

For example, the structure of the bifunctional compound can be depicted as:

wherein: PTM is a LRRK2-targeting moiety (LTM); “L” is a linker (e.g. a bond or a chemical linking group) coupling the PTM and CLM; and CLM is cereblon E3 ubiquitin ligase binding moiety that binds to cereblon.

In certain embodiments, the compounds as described herein comprise multiple independently selected ULMs, multiple PTMs, multiple chemical linkers or a combination thereof.

In any of the aspects or embodiments described herein, the PTM is a small molecule that binds LRRK2 or a mutant thereof. In any of the aspects or embodiments described herein, the PTM is a small molecule that binds LRRK2. In any of the aspects or embodiments described herein, the PTM is a small molecule that binds both an LRRK2 wild type protein and an LRRK2 mutant, such as a LRRK2 mutant including one or more mutation selected from G2019S, I2020T, N1437H, R1441G/C/H, and Y1699C. In any of the aspects or embodiments described herein, the PTM is a small molecule that binds both an LRRK2 wild type protein and an LRRK2 mutant such as, but not limited to, G2019S, I2020T, N1437H, R1441G/C/H, Y1699C, or a combination thereof. In any aspect or embodiment described herein, the small molecule binds the LRRK2 is as described herein.

In an embodiment, the CLM comprises a chemical group derived from an imide, a thioimide, an amide, or a thioamide. In a particular embodiment, the chemical group is a phthalimido group, or an analog or derivative thereof. In a certain embodiment, the CLM is selected from thalidomide, lenalidomide, pomalidomide, analogs thereof, isosteres thereof, and derivatives thereof. Other contemplated CLMs are described in U.S. Patent Application Publication No. 2015/0291562, which is incorporated herein by reference in its entirety.

In certain embodiments, “L” is a bond. In additional embodiments, the linker “L” is a connector with a linear non-hydrogen atom number in the range of 1 to 20. The connector “L” can contain, but is not limited to, one or more functional groups such as ether, amide, alkane, alkene, alkyne, ketone, hydroxyl, carboxylic acid, thioether, sulfoxide, and sulfone. The linker can contain aromatic, heteroaromatic, cyclic, bicyclic or tricyclic moieties. Substitution with halogen, such as Cl, F, Br and I can be included in the linker. In the case of fluorine substitution, single or multiple fluorines can be included.

In certain embodiments, CLM is a derivative of piperidine-2,6-dione, where piperidine-2,6-dione can be substituted at the 3-position, and the 3-substitution can be bicyclic hetero-aromatics with the linkage as C—N bond or C—C bond. Examples of CLM can be, but are not limited to, pomalidomide, lenalidomide and thalidomide and their analogs.

In an additional aspect, the description provides therapeutic compositions comprising an effective amount of a compound as described herein, or a salt form thereof, and a pharmaceutically acceptable carrier. The therapeutic compositions can be used to trigger targeted degradation of LRRK2 or a mutated version thereof and/or inhibition of LRRK2 or a mutated version thereof, in a patient or subject, for example, an animal such as a human, and can be used for treating or ameliorating one or more disease states, conditions, or symptoms causally related to LRRK2 or mutated version thereof, which treatment is accomplished through degradation or inhibition of the LRRK2 protein or mutated version thereof, or controlling or lowering LRRK2 protein levels or protein levels of a mutated version thereof, in a patient or subject. In certain embodiments, the therapeutic compositions as described herein may be used to effectuate the degradation of LRRK2, or a mutant form thereof, for the treatment or amelioration of a disease such as, e.g., LRRK2 accumulation or overeactivity, alpha-synuclein aggregation or accumulation, Tau aggregation or accumulation, idiopathic PD, LRRK2 mutation associated PD (e.g., PD associated with one or more LRRK2 activating mutations), primary tauopathies (e.g., supranuclear palsy (PSP) or corticobasal degeneration (CBD)), lewy body dementia, Crohn's Disease, Leprosy (e.g., Leprosy with type 1 inflammatory reactions), and/or neuroinflammation.

In yet another aspect, the present disclosure provides a method of ubiquitinating LRRK2 or a mutated form thereof in a cell. In certain embodiments, the method comprises administering a hetero-bifunctional compound as described herein comprising a PTM that binds LRRK2 or a mutant form thereof, and a CLM, preferably linked through a chemical linker moiety, as described herein, to effectuate degradation of the LRRK2 protein or mutant form thereof. Though not wanting to be limited by theory, the inventors believe that, pursuant to the invention, poly-ubiquitination of the LRRK2 wild-type or mutant protein will occur when it is placed in proximity to the E3 ubiquitin ligase via use of the hetero-bifunctional compound, thereby triggering subsequent degradation of the LRRK2 or mutant protein via the proteasomal pathway and control or reduction of LRRK2 protein levels in cells, such as cells of a subject in need of such treatment. The control or reduction in levels of the LRRK2 protein or mutated form thereof afforded by the present disclosure provides treatment of a LRRK2 causally related disease state, condition or related symptom, as modulated through a lowering of the amount of LRRK2 protein or mutated form thereof in cells of the subject.

In still another aspect, the description provides methods for treating or ameliorating a disease, condition, or symptom thereof causally related to LRRK2 or mutated form thereof in a subject or a patient, e.g., an animal such as a human, comprising administering to a subject in need thereof a composition comprising an effective amount, e.g., a therapeutically effective amount, of a hetero-bifunctional compound as described herein or salt form thereof, and a pharmaceutically acceptable carrier, wherein the composition is effective for treating or ameliorating the disease or disorder or symptom thereof in the subject.

In another aspect, the description provides methods for identifying the effects of the degradation of LRRK2 protein in a biological system using compounds according to the present disclosure.

In another aspect, the description provides processes and intermediates for making a hetero-bifunctional compound of the present disclosure capable of targeted ubiquitination and degradation of the LRRK2 protein in a cell (e.g., in vivo or in vitro).

DETAILED DESCRIPTION

Presently described are compounds, compositions and methods that relate to the surprising discovery that an E3 ubiquitin ligase (e.g., a cereblon E3 ubiquitin ligase) ubiquitinates the LRRK2 protein or mutated form thereof once the E3 ubiquitin ligase and the LRRK2 protein are placed in proximity via a bifunctional compound that binds both the E3 ubiquitin ligase and the LRRK2 protein. Accordingly the present disclosure provides compounds and compositions comprising an E3 ubiquitin ligase binding moiety (“ULM”) coupled by a bond or chemical linking group (L) to a protein targeting moiety (“PTM”) that targets the LRRK2 protein, which results in the ubiquitination of the LRRK2 protein, and which leads to degradation of the LRRK2 protein by the proteasome (seeFIG.1).

In one aspect, the description provides compounds in which the PTM binds to the LRRK2 protein and/or a mutated form thereof. The present disclosure also provides a library of compositions and the use thereof to produce targeted degradation of the LRRK2 protein in a cell.

In certain aspects, the present disclosure provides hetero-bifunctional compounds which comprise a ligand, e.g., a small molecule ligand (i.e., having a molecular weight of below 2,000, 1,000, 500, or 200 Daltons), which is capable of binding to an E3 ubiquitin ligase, such as cereblon. The compounds also comprise a small molecule moiety that is capable of binding to the LRRK2 protein or mutated form thereof in such a way that the LRRK2 protein or mutated form is placed in proximity to the ubiquitin ligase to effect ubiquitination and degradation (and/or inhibition) of the LRRK2 protein or mutated form. “Small molecule” means, in addition to the above, that the molecule is non-peptidyl, that is, it is not considered a peptide, e.g., comprises fewer than 4, 3, or 2 amino acid residues. In accordance with the present description, each of the PTM, ULM and hetero-bifunctional molecule is a small molecule.

The term “LRRK2” as used throughout the Specification, unless specifically indicated to the contrary, is intended to include both wild-type LRRK2 and mutant forms therefore, such as a LRRK2 mutant protein including one or more mutation selected from G2019S, I2020T, N1437H, R1441G/C/H, and Y1699C.

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 belongs. 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 (i.e., 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, unless otherwise indicated.

It should also be understood that, in certain methods or processes described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise.

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 two or more 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 hetero-bifunctional compounds described herein are coadministered with at least one additional bioactive agent, e.g., an anticancer agent. In particularly preferred aspects, the co-administration of such compounds results in synergistic activity and/or therapy such as, e.g., anticancer activity.

The term “compound”, as used herein, unless otherwise indicated, refers to any specific hetero-bifunctional compound disclosed herein, pharmaceutically acceptable salts and solvates thereof, and deuterated forms of any of the aforementioned molecules, where applicable. Deuterated compounds contemplated are those in which one or more of the hydrogen atoms contained in the drug molecule have been replaced by deuterium. Such deuterated compounds preferably have one or more improved pharmacokinetic or pharmacodynamic properties (e.g., longer half-life) compared to the equivalent “undeuterated” compound.

The term “ubiquitin ligase” refers to a family of proteins that facilitate the transfer of one or more ubiquitins to a specific substrate protein. Addition of a chain of several ubiquitins (poly-ubiquitination) targets the substrate protein for degradation. For example, cereblon is an E3 ubiquitin ligase that alone, or in combination with an E2 ubiquitin-conjugating enzyme, can ultimately cause the attachment of a chain of four ubiquitins to a lysine residue on the target protein, thereby targeting the protein for degradation by the proteasome. The ubiquitin ligase is involved in poly-ubiquitination such that a first ubiquitin is attached to a lysine on the target protein; a second ubiquitin is attached to the first; a third is attached to the second, and a fourth is attached to the third. Such poly-ubiquitination marks proteins for degradation by the proteasome.

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 diseases, conditions or symptoms that 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 terms “patient” and “subject” refer to a human patient unless otherwise stated or implied from the context of the use of the term.

The terms “effective” and “therapeutically effective” are used to describe an amount of a compound or composition which, when used within the context of its intended use, and either in a single dose or, more preferably after multiple doses within the context of a treatment regimen, effects an intended result such as an improvement in a disease or condition, or amelioration or reduction in one or more symptoms associated with a disease or condition. The terms “effective” and “therapeutically effective” subsume all other “effective amount” or “effective concentration” terms, which are otherwise described or used in the present application.

Compounds and Compositions

In one aspect, the description provides hetero-bifunctional compounds comprising an E3 ubiquitin ligase binding moiety (“ULM”) that is a cereblon E3 ubiquitin ligase binding moiety (a “CLM”), The CLM is covalently coupled to a protein targeting moiety (PTM) that binds to the protein, which coupling is either directly by a bond or via a chemical linking group (L) according to the structure:
PTM-L-CLM  (A)
wherein L is the bond or chemical linking group, and PTM is a protein targeting moiety that binds to the protein LRRK2 or a mutant form thereof, e.g., G2019S, where the PTM is a LRRK2 targeting moiety (LTM). The term CLM is inclusive of all cereblon binding moieties.

In any of the aspects or embodiments, the CLM demonstrates a half maximal inhibitory concentration (IC50) for the E3 ubiquitin ligase (e.g., cereblon E3 ubiquitin ligase) of less than about 200 μM. The IC50can be determined according to any suitable method known in the art, e.g., a fluorescent polarization assay.

The term “alkyl” shall mean within its context a linear, branch-chained or cyclic fully saturated hydrocarbon radical, preferably a C1-C10, preferably a C1-C6, or more preferably a C1-C3alkyl group, which may be optionally substituted with any suitable functional group or groups. Examples of alkyl groups are methyl, ethyl, n-butyl, sec-butyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, isopropyl, 2-methylpropyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclopentyl, cyclopentylethyl, cyclohexylethyl and cyclohexyl, among others. In certain embodiments, the alkyl group is end-capped with a halogen group (At, Br, Cl, F, or I).

The term “Alkenyl” refers to linear, branch-chained or cyclic C2-C10(preferably C2-C6) hydrocarbon radicals containing at least one C═C bond.

The term “Alkynyl” refers to linear, branch-chained or cyclic C2-C10(preferably C2-C6) hydrocarbon radicals containing at least one C≡C bond.

The term “alkylene” when used, refers to a —(CH2)n— group (n is an integer generally from 0-6), which may be optionally substituted. When substituted, the alkylene group preferably is substituted on one or more of the methylene groups with a C1-C6alkyl group (including a cyclopropyl group or a t-butyl group), but may also be substituted with one or more halo groups, preferably from 1 to 3 halo groups or one or two hydroxyl groups, O—(C1-C6alkyl) groups or amino acid sidechains as otherwise disclosed herein. In certain embodiments, an alkylene group may be substituted with a urethane or alkoxy group (or other suitable functional group) which may be further substituted with a polyethylene glycol chain (of from 1 to 10, preferably 1 to 6, or more preferably 1 to 4 ethylene glycol units) to which is substituted (preferably, but not exclusively on the distal end of the polyethylene glycol chain) an alkyl chain substituted with a single halogen group, preferably a chlorine group. In still other embodiments, the alkylene (e.g., methylene) group, may be substituted with an amino acid sidechain group such as a sidechain group of a natural or unnatural amino acid, for example, alanine, β-alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, glutamine, glycine, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, proline, serine, threonine, valine, tryptophan or tyrosine.

The term “unsubstituted” shall mean substituted only with hydrogen atoms. A range of carbon atoms which includes C0means that carbon is absent and is replaced with H. Thus, a range of carbon atoms which is C0-C6includes carbons atoms of 1, 2, 3, 4, 5 and 6 and for C0, H stands in place of carbon.

The term “substituted” or “optionally substituted” shall mean independently (i.e., where more than one substituent occurs, each substituent is selected independent of another substituent) one or more substituents (independently up to five substituents, preferably up to three substituents, more preferably 1 or 2 substituents on a moiety in a compound according to the present disclosure and may include substituents which themselves may be further substituted) at a carbon (or nitrogen) position anywhere on a molecule within context, and includes as possible substituents hydroxyl, thiol, carboxyl, cyano (C≡N), nitro (NO2), halogen (preferably, 1, 2 or 3 halogens, especially on an alkyl, especially a methyl group such as a trifluoromethyl), an alkyl group (preferably, C1-C10, more preferably, C1-C6), aryl (especially phenyl and substituted phenyl, for example benzyl or benzoyl), alkoxy group (preferably, C1-C6alkyl or aryl, including phenyl and substituted phenyl), thioether (preferably, C1-C6alkyl or aryl), acyl (preferably, C1-C6acyl), ester or thioester (preferably, C1-C6alkyl or aryl) including alkylene ester (such that attachment is on the alkylene group, rather than at the ester function which is preferably substituted with a C1-C6alkyl or aryl group), halogen (preferably, F or Cl), amine (including a five- or six-membered cyclic alkylene amine, further including a C1-C6alkyl amine or a C1-C6dialkyl amine which alkyl groups may be substituted with one or two hydroxyl groups) or an optionally substituted —N(C0-C6alkyl)C(O)(O—C1-C6alkyl) group (which may be optionally substituted with a polyethylene glycol chain to which is further bound an alkyl group containing a single halogen, preferably chlorine substituent), hydrazine, amido, which are preferably independently substituted with one or two C1-C6alkyl groups (including a carboxamide which is optionally substituted with one or two C1-C6alkyl groups), alkanol (preferably, C1-C6alkyl or aryl), or alkanoic acid (preferably, C1-C6alkyl or aryl). Substituents according to the present disclosure may include, for example —SiR1R2R3groups where each of R1and R2is as otherwise described herein and R3is H or a C1-C6alkyl group, preferably R1, R2, R3together is a C1-C3alkyl group (including an isopropyl or t-butyl group). Each of the above-described groups may be linked directly to the substituted moiety or alternatively, the substituent may be linked to the substituted moiety (preferably in the case of an aryl or heteroaryl moiety) through an optionally substituted —(CH2)m— or alternatively an optionally substituted —(OCH2)m—, —(OCH2CH2)m— or —(CH2CH2O)m— group, which may be substituted with any one or more of the above-described substituents. Alkylene groups —(CH2)m— or —(CH2)n— groups or other chains such as ethylene glycol chains, as identified above, may be substituted anywhere on the chain. Preferred substituents on alkylene groups include halogen or C1-C6(preferably C1-C3) alkyl groups, which may be optionally substituted with one or two hydroxyl groups, one or two ether groups (O—C1-C6groups), up to three halo groups (preferably F), or a side chain of an amino acid as otherwise described herein and optionally substituted amide (preferably carboxamide substituted as described above) or urethane groups (often with one or two C0-C6alkyl substituents, which group(s) may be further substituted). In certain embodiments, the alkylene group (often a single methylene group) is substituted with one or two optionally substituted C1-C6alkyl groups, preferably C1-C4alkyl group, most often methyl or O-methyl groups or a sidechain of an amino acid as otherwise described herein. In the present disclosure, a moiety in a molecule may be optionally substituted with up to five substituents, preferably up to three substituents. Most often, in the present disclosure moieties which are substituted are substituted with one or two substituents.

The term “substituted” (each substituent being independent of any other substituent) shall also mean within its context of use C1-C6alkyl, C1-C6alkoxy, halogen, amido, carboxamido, sulfone, including sulfonamide, keto, carboxy, C1-C6ester (oxyester or carbonylester), C1-C6keto, urethane —O—C(O)—NR1R2or —N(R1)—C(O)—O—R1, nitro, cyano and amine (especially including a C1-C6alkylene-NR1R2, a mono- or di-C1-C6alkyl substituted amines which may be optionally substituted with one or two hydroxyl groups). Each of these groups contain unless otherwise indicated, within context, between 1 and 6 carbon atoms. In certain embodiments, preferred substituents will include for example, —NH—, —NHC(O)—, —O—, ═O, —(CH2)m— (here, m and n are in context, 1, 2, 3, 4, 5 or 6), —S—, —S(O)—, SO2— or —NH—C(O)—NH—, —(CH2)nOH, —(CH2)nSH, —(CH2)nCOOH, C1-C6alkyl, —(CH2)nO—(C1-C6alkyl), —(CH2)nC(O)—(C1-C6alkyl), —(CH2)nOC(O)—(C1-C6alkyl), —(CH2)nC(O)O—(C1-C6alkyl), —(CH2)nNHC(O)—R1, —(CH2)nC(O)—NR1R2, —(OCH2)nOH, —(CH2O)nCOOH, C1-C6alkyl, —(OCH2)nO—(C1-C6alkyl), —(CH2O)nC(O)—(C1-C6alkyl), —(OCH2)nNHC(O)—R1, —(CH2O)nC(O)—NR1R2, —S(O)2—RS, —S(O)—RS(RSis C1-C6alkyl or a —(CH2)m—NR1R2group), NO2, CN or halogen (F, Cl, Br, I, preferably F or Cl), depending on the context of the use of the substituent. R1and R2are each, within context, H or a C1-C6alkyl group (which may be optionally substituted with one or two hydroxyl groups or up to three halogen groups, preferably fluorine). The term “substituted” shall also mean, within the chemical context of the compound defined and substituent used, an optionally substituted aryl or heteroaryl group or an optionally substituted heterocyclic group as otherwise described herein. Alkylene groups may also be substituted as otherwise disclosed herein, preferably with optionally substituted C1-C6alkyl groups (methyl, ethyl or hydroxymethyl or hydroxyethyl is preferred, thus providing a chiral center), a sidechain of an amino acid group as otherwise described herein, an amido group as described hereinabove, or a urethane group O—C(O)—NR1R2group where R1and R2are as otherwise described herein, although numerous other groups may also be used as substituents. Various optionally substituted moieties may be substituted with 3 or more substituents, preferably no more than 3 substituents and preferably with 1 or 2 substituents. It is noted that in instances where, in a compound at a particular position of the molecule substitution is required (principally, because of valency), but no substitution is indicated, then that substituent is construed or understood to be H, unless the context of the substitution suggests otherwise.

The term “substituted aryl” refers to an aromatic carbocyclic group comprised of at least one aromatic ring or of multiple condensed rings at least one of which being aromatic, wherein the ring(s) are substituted with one or more substituents. For example, an aryl group can comprise a substituent(s) selected from: —(CH2)nOH, —(CH2)n—O—(C1-C6)alkyl, —(CH2)n—O—(CH2)n—(C1-C6)alkyl, —(CH2)n—C(O)(C0-C6) alkyl, —(CH2)n—C(O)O(C0-C6)alkyl, —(CH2)n—OC(O)(C0-C6)alkyl, amine, mono- or di-(C1-C6alkyl) amine wherein the alkyl group on the amine is optionally substituted with 1 or 2 hydroxyl groups or up to three halo (preferably F, Cl) groups, OH, COOH, C1-C6alkyl, preferably CH3, CF3, OMe, OCF3, NO2, or CN group (each of which may be substituted in ortho-, meta- and/or para-positions of the phenyl ring, preferably para-), an optionally substituted phenyl group (the phenyl group itself is preferably connected to a PTM group, including a ULM group, via a linker group), and/or at least one of F, Cl, OH, COOH, CH3, CF3, OMe, OCF3, NO2, or CN group (in ortho-, meta- and/or para-positions of the phenyl ring, preferably para-), a naphthyl group, which may be optionally substituted, an optionally substituted heteroaryl, preferably an optionally substituted isoxazole including a methyl substituted isoxazole, an optionally substituted oxazole including a methyl substituted oxazole, an optionally substituted thiazole including a methyl substituted thiazole, an optionally substituted isothiazole including a methyl substituted isothiazole, an optionally substituted pyrrole including a methyl substituted pyrrole, an optionally substituted imidazole including a methylimidazole, an optionally substituted benzimidazole or methoxybenzylimidazole, an optionally substituted oximidazole or methyloximidazole, an optionally substituted diazole group, including a methyldiazole group, an optionally substituted triazole group, including a methylsubstituted triazole group, an optionally substituted pyridine group, including a halo-(preferably, F) or methyl substituted pyridine group or an oxapyridine group (where the pyridine group is linked to the phenyl group by an oxygen), an optionally substituted furan, an optionally substituted benzofuran, an optionally substituted dihydrobenzofuran, an optionally substituted indole, indolizine or azaindolizine (2, 3, or 4-azaindolizine), an optionally substituted quinoline, and combinations thereof.

“Carboxyl” denotes the group —C(O)OR, where R is hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, whereas these generic substituents have meanings which are identical with definitions of the corresponding groups defined herein.

The term “heteroaryl” or “hetaryl” can mean but is in no way limited to a 5-16 membered heteroaryl (e.g., 5, 6, 7 or 8 membered monocyclic ring or a 10-16 membered heteroaryl having multiple condensed rings), an optionally substituted quinoline (which may be attached to the pharmacophore or substituted on any carbon atom within the quinoline ring), an optionally substituted indole (including dihydroindole), an optionally substituted indolizine, an optionally substituted azaindolizine (2, 3 or 4-azaindolizine) an optionally substituted benzimidazole, benzodiazole, benzoxofuran, an optionally substituted imidazole, an optionally substituted isoxazole, an optionally substituted oxazole (preferably methyl substituted), an optionally substituted diazole, an optionally substituted triazole, a tetrazole, an optionally substituted benzofuran, an optionally substituted thiophene, an optionally substituted thiazole (preferably methyl and/or thiol substituted), an optionally substituted isothiazole, an optionally substituted triazole (preferably a 1,2,3-triazole substituted with a methyl group, a triisopropylsilyl group, an optionally substituted —(CH2)m—O—C1-C6alkyl group or an optionally substituted —(CH2)m—C(O)—O—C1-C6alkyl group), an optionally substituted pyridine (2-, 3, or 4-pyridine) or a group according to the chemical structure:

wherein:Scis CHRSS, NRURE, or O;RHETis H, CN, NO2, halo (preferably Cl or F), optionally substituted C1-C6alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups (e.g. CF3), optionally substituted O(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted acetylenic group —C≡C—Rawhere Rais H or a C1-C6alkyl group (preferably C1-C3alkyl);RSSis H, CN, NO2, halo (preferably F or Cl), optionally substituted C1-C6alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups), optionally substituted O—(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted —C(O)(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups);RUREis H, a C1-C6alkyl (preferably H or C1-C3alkyl) or a —C(O)(C1-C6alkyl), each of which groups is optionally substituted with one or two hydroxyl groups or up to three halogen, preferably fluorine groups, or an optionally substituted heterocycle, for example piperidine, morpholine, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, piperidine, piperazine, each of which is optionally substituted, andYCis N or C—RYC, where RYCis H, OH, CN, NO2, halo (preferably Cl or F), optionally substituted C1-C6alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups (e.g. CF3), optionally substituted O(C1-C6alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted acetylenic group —C≡C—Rawhere Rais H or a C1-C6alkyl group (preferably C1-C3alkyl).

The terms “aralkyl” and “heteroarylalkyl” refer to groups that comprise both aryl or, respectively, heteroaryl as well as alkyl and/or heteroalkyl and/or carbocyclic and/or heterocycloalkyl ring systems according to the above definitions.

The term “arylalkyl” as used herein refers to an aryl group as defined above appended to an alkyl group defined above. The arylalkyl group is attached to the parent moiety through an alkyl group wherein the alkyl group is one to six carbon atoms. The aryl group in the arylalkyl group may be substituted as defined above.

The term “Heterocycle” refers to a cyclic group which contains at least one heteroatom, e.g., N, O or S, and may be aromatic (heteroaryl) or non-aromatic. Thus, the heteroaryl moieties are subsumed under the definition of heterocycle, depending on the context of its use. Exemplary heteroaryl groups are described hereinabove.

The term “cycloalkyl” can mean but is in no way limited to univalent groups derived from monocyclic or polycyclic alkyl groups or cycloalkanes, as defined herein, e.g., saturated monocyclic hydrocarbon groups having from three to twenty carbon atoms in the ring, including, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like. The term “substituted cycloalkyl” can mean but is in no way limited to a monocyclic or polycyclic alkyl group and being substituted by one or more substituents, for example, amino, halogen, alkyl, substituted alkyl, carbyloxy, carbylmercapto, aryl, nitro, mercapto or sulfo, whereas these generic substituent groups have meanings which are identical with definitions of the corresponding groups as defined in this legend.

“Heterocycloalkyl” refers to a monocyclic or polycyclic alkyl group in which at least one ring carbon atom of its cyclic structure being replaced with a heteroatom selected from the group consisting of N, O, S or P. “Substituted heterocycloalkyl” refers to a monocyclic or polycyclic alkyl group in which at least one ring carbon atom of its cyclic structure being replaced with a heteroatom selected from the group consisting of N, O, S or P and the group is containing one or more substituents selected from the group consisting of halogen, alkyl, substituted alkyl, carbyloxy, carbylmercapto, aryl, nitro, mercapto or sulfo, whereas these generic substituent group have meanings which are identical with definitions of the corresponding groups as defined in this legend.

The term “hydrocarbyl” shall mean a compound which contains carbon and hydrogen and which may be fully saturated, partially unsaturated or aromatic and includes aryl groups, alkyl groups, alkenyl groups and alkynyl groups.

The term “independently” is used herein to indicate that the variable, which is independently applied, varies independently from application to application.

The term “lower alkyl” refers to methyl, ethyl or propyl

The term “lower alkoxy” refers to methoxy, ethoxy or propoxy.

In one aspect the description provides CLMs useful for binding and recruiting cereblon. In certain embodiments, the CLM is selected from the group consisting of chemical structures:

In any aspect or embodiment described herein, the CLM comprises a chemical structure selected from the group consisting of:

In any aspect or embodiment described herein, the CLM or ULM is selected from the structure of Formula (g):

wherein:W of Formula (g) is independently selected from the group CH2, O, C═O, NH, and N-alkyl;A of Formula (g) is selected from a H, methyl, or optionally substituted linear or branched alkyl;n is an integer from 1 to 4;R of Formula (g) is independently selected from a H, O, OH, N, NH, NH2, —Cl, —F, —Br, —I, methyl, optionally substituted linear or branched alkyl (e.g., optionally substituted linear or branched C1-C6 alkyl), optionally substitute linear or branched alkoxy (e.g., optionally substituted linear or branched C1-C6 alkoxy), -alkyl-aryl (e.g., an -alkyl-aryl comprising at least one of C1-C6 alkyl, C4-C7 aryl, or a combination thereof), aryl (e.g., C5-C7 aryl), amine, amide, or carboxy), wherein at least one R or W is modified to be covalently joined to a PTM, a chemical linking group (L), a ULM, CLM, or combination thereof; andof Formula (g) represents a bond that may be stereospecific ((R) or (S)) or non-stereospecific.

In any aspect or embodiment described herein, the CLM or ULM is selected from the group consisting of:

wherein:W is C═O or CH2;N* is a nitrogen atom that is covalently linked to the PTM or linker, or that is shared with the the PTM or linker (L) (e.g., a heteroatom shared with an optionally substituted heterocyclyl of the linker (L) or PTM); andindicates the point of attachment of the CLM or ULM to the linker (L) or PTM.

In any aspect or embodiment described herein, at least one R (e.g. an R group selected from the following H, O, OH, N, NH, NH2, C1-C6 alkyl, C1-C6 alkoxy, -alkyl-aryl (e.g., an -alkyl-aryl comprising at least one of C1-C6 alkyl, C4-C7 aryl, or a combination thereof), aryl (e.g., C5-C7 aryl), amine, amide, or carboxy) or W is modified to be covalently joined to a PTM, a chemical linker group (L), a ULM, a CLM, or a combination thereof

In any aspect or embodiment described herein, the W, X, Y, Z, G, G′, R, R′, R″, Q1-Q4, and A of Formulas (a) through (g) can independently be covalently coupled to a linker and/or a linker to which is attached one or more PTM, ULM, or CLM groups.

In any of the aspects or embodiments described herein, n is an integer from 1 to 4, and each R is independently selected functional groups or atoms, for example, O, OH, N, —Cl, —F, C1-C6 alkyl, C1-C6 alkoxy, -alkyl-aryl (e.g., an -alkyl-aryl comprising at least one of C1-C6 alkyl, C4-C7 aryl, or a combination thereof), aryl (e.g., C5-C7 aryl), amine, amide, or carboxy, on the aryl or heteroaryl of the CLM, and optionally, one of which is modified to be covalently joined to a PTM, a chemical linker group (L), a ULM, CLM or combination thereof.

More specifically, non-limiting examples of CLMs include those shown below as well as those “hybrid” molecules that arise from the combination of one or more of the different features shown in the molecules below wherein at least one R or W is modified to be covalently joined to a PTM, a chemical linking group (L), a ULM, CLM, or combination thereof.

In any aspect or embodiment described herein, the CLM comprises a chemical structure selected from the group:

In any aspect or embodiment described herein, the CLM is covalently joined to a PTM, or a chemical linker group (L) via an R group (such as, R, R1, R2, R3, R4, or R′), W, X, or a Q group (such as, Q1, Q2, Q3, Q4, or Q5).

In any aspect or embodiment described herein, the CLM is covalently joined to a PTM, or a chemical linker group (L) via W, X, R, R1, R2, R3, R4, R5, R′, Q1, Q2, Q3, Q4, and Q5.

In any aspect or embodiment described herein, the W, X, R1, R2, R3, R4, R′, Q1, Q2, Q3, Q4, and Q5can independently be covalently coupled to a linker and/or a linker to which is attached to one or more PTM, ULM, CLM groups.

More specifically, non-limiting examples of CLMs include those shown below as well as “hybrid” molecules or compounds that arise from combining one or more features of the following compounds:

wherein:W is independently selected from the group CH2, CHR, C═O, SO2, NH, and N-alkyl;R1is selected from the group absent, H, CH, CN, C1-C3 alkyl;R2is H or a C1-C3 alkyl;R3is selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy;R4is methyl or ethyl;R5is H or halo;R6is H or halo;n is an integer from 0-4;R and R′ are independently H, a functional group or an atom (e.g., H, halogen (e.g., —Cl or —F), amine, C1-C3 alkyl, C1-C3 alkyl, C1-C3 alkoxyl, NR2R3, or C(═O)OR2); or an attachment point for a PTM, or a chemical linker group (L),Q1and Q2are each independently C or N substituted with a group independently selected from H or C1-C3alkyl; andis a single or double bond.

In any aspect or embodiment described herein, the W, R1, R2, Q1, Q2, Q3, Q4, R, and R′ can independently be covalently coupled to a linker and/or a linker to which is attached one or more PTM groups.

In any aspect or embodiment described herein, the R1, R2, Q1, Q2, Q3, Q4, R, and R′ can independently be covalently coupled to a linker and/or a linker to which is attached one or more PTM groups.

In any aspect or embodiment described herein, the Q1, Q2, Q3, Q4, R, and R′ can independently be covalently coupled to a linker and/or a linker to which is attached one or more PTM groups.

As would be readily apparent, in any aspect or embodiment described herein, R, R′, R″, R1, R2, R3, R4, R5, and R6of the CLM can be a bond.

In any aspect or embodiment described herein, R is a bond or modified to be covalently joined to the linker group (L) or, a PTM or combination thereof.

In any aspect or embodiment described herein, the CLM is selected from:

wherein R′ is a halogen and R1is as described herein.

In certain cases, “CLM” can be an imide that binds to cereblon E3 ligase. These imides and linker attachment point can be, but not be limited to one of the following structures:

In any aspect or embodiment described herein, the ULM is selected from the group consisting of:

wherein:of the ULM indicates the point of attachment with a linker group or a PTM;N* is a nitrogen atom that is shared with the chemical linker group or PTM; andW, Q4, and Q5are each defined as described in any aspect or embodiment described herein.
Exemplary Linkers

In certain embodiments, the compounds as described herein include a PTM chemically linked to a ULM (e.g., CLM) via a chemical linker (L). In certain embodiments, the linker group L comprises one or more covalently connected structural units (e.g., -AL1. . . (AL)q- or -(AL)q-), wherein AL1is a group coupled to PTM, and (AL)qis a group coupled to ULM.

In any aspect or embodiment described herein, the linker (L) to a ULM (e.g., CLM) connection is a stable L-ULM connection. For example, in any aspect or embodiment described herein, when a linker (L) and a ULM are connected via a heteroatom (e.g., N, O, S), any additional heteroatom, if present, is separated by at least a carbon atom (e.g., —CH2—), such as with an acetal or aminal group. By way of further example, in any aspect or embodiment described herein, when a linker (L) and a ULM are connected via a heteroatom, the heteroatom is not part of an ester.

In any aspect or embodiment described herein, the linker group L is a bond or a chemical linker group represented by the formula -(AL)q-, wherein A is a chemical moiety and q is an integer from 6-30 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25), and wherein L is covalently bound to both the PTM and the ULM, and provides for binding of the PTM to the protein target and the ULM to an E3 ubiquitin ligase in sufficient proximity to result in target protein ubiquitination.

In certain embodiments, q is an integer greater than or equal to 1.

In any aspect or embodiment described herein, e.g., where q of the linker is greater than 2, (AL)qis a group which is AL1and (AL)qwherein the linker couples a PTM to a ULM.

In any aspect or embodiment described herein, e.g., where q of the linker is 2, AL2is a group which is connected to AL1and to a ULM.

In any aspect or embodiment described herein, e.g., where q of the linker is 1, the structure of the linker group L is -AL1-, and AL1is a group which connects a ULM moiety to a PTM moiety.

In any aspect or embodiment described herein, the unit ALof linker (L) comprises a group represented by a general structure selected from the group consisting of:—NR(CH2)n-(lower alkyl)-, —NR(CH2)n-(lower alkoxyl)-, —NR(CH2)n-(lower alkoxyl)-OCH2—, —NR(CH2)n-(lower alkoxyl)-(lower alkyl)-OCH2—, —NR(CH2)n-(cycloalkyl)-(lower alkyl)-OCH2—, —NR(CH2)n-(heterocycloalkyl)-, —NR(CH2CH2O)n-(lower alkyl)-O—CH2—, —NR(CH2CH2O)n-(heterocycloalkyl)-O—CH2—, —NR(CH2CH2O)n-Aryl-O—CH2—, —NR(CH2CH2O)n-(heteroaryl)-O—CH2—, —NR(CH2CH2O)n-(cyclo alkyl)-O-(heteroaryl)-O—CH2—, —NR(CH2CH2O)n-(cyclo alkyl)-O-Aryl-O—CH2—, —NR(CH2CH2O)n-(lower alkyl)-NH-Aryl-O—CH2—, —NR(CH2CH2O)n-(lower alkyl)-O-Aryl-CH2, —NR(CH2CH2O)n-cycloalkyl-O-Aryl-, —NR(CH2CH2O)n-cycloalkyl-O-(heteroaryl)l-, —NR(CH2CH2)n-(cycloalkyl)-O-(heterocyclyl)-CH2, —NR(CH2CH2)n-(heterocyclyl)-(heterocyclyl)-CH2, and —N(R1R2)-(heterocyclyl)-CH2; wheren of the linker can be 0 to 10;R of the linker can be H, or lower alkyl; andR1 and R2 of the linker can form a ring with the connecting N.

In any aspect or embodiment described herein, the linker (L) does not have heteroatom-heteroatom bonding (e.g., no heteroatoms are covalently linked or adjacently located).

In any aspect or embodiment described herein, the unit ALof the linker (L) comprises a structure selected from the group consisting of:

wherein N* is a nitrogen atom that is covalently linked to the ULM or the PTM, or that is shared with the ULM or the PTM.

In any aspect or embodiment described herein, the unit ALof the linker (L) comprises a structure selected from the group consisting of:

In any aspect or embodiment described herein, the unit ALof the linker (L) is selected from:

wherein N* is a nitrogen atom that is covalently linked to the ULM or PTM, or that is shared with the ULM or PTM.

In any aspect or embodiment described herein, the unit ALof linker (L) comprises a group represented by a general structure selected from the group consisting of:

where each n and m of the linker can independently be 0, 1, 2, 3, 4, 5, or 6.

In any aspect or embodiment described herein, the unit ALof linker (L) is selected from the group consisting of:

wherein each m and n is independently selected from 0, 1, 2, 3, 4, 5, or 6.

In any aspect or embodiment described herein, the unit ALof linker (L) is selected from the group consisting of:

In any aspect or embodiment described herein, the unit ALof linker (L) is selected from the group consisting of:

In any aspect or embodiment described herein, the linker (L) comprises a structure selected from the structure shown below:

wherein:WL1and WL2are each independently absent, a 4-8 membered ring with 0-4 heteroatoms, optionally substituted with RQ, each RQis independently a H, halo, OH, CN, CF3, optionally substituted linear or branched C1-C6alkyl, optionally substituted linear or branched C1-C6alkoxy, or 2 RQgroups taken together with the atom they are attached to, form a 4-8 membered ring system containing 0-4 heteroatoms;YL1is each independently a bond, optionally substituted linear or branched C1-C6alkyl and optionally one or more C atoms are replaced with O or NRYL1, optionally substituted C1-C6alkene and optionally one or more C atoms are replaced with O, optionally substituted C1-C6alkyne, and optionally one or more C atoms are replaced with O, or optionally substituted linear or branched C1-C6alkoxy;RYL1is H, or optionally substituted linear or branched C1-6alkyl;n is 0-10; andandindicates the attachment point to the PTM or the ULM.

In any aspect or embodiment described herein, the linker (L) comprises a structure selected from the structure shown below:

wherein:WL1and WL2are each independently absent, piperazine, piperidine, morpholine, optionally substituted with RQ, each RQis independently a H, —Cl—, —F—, OH, CN, CF3, optionally substituted linear or branched C1-C6alkyl (e.g. methyl, ethyl), optionally substituted linear or branched C1-C6alkoxy (e.g. methoxy, ethoxy);YL1is each independently a bond, optionally substituted linear or branched C1-C6alkyl and optionally one or more C atoms are replaced with O or NRYL1; optionally substituted C1-C6alkene and optionally one or more C atoms are replaced with O, optionally substituted C1-C6alkyne and optionally one or more C atoms are replaced with O, or optionally substituted linear or branched C1-C6alkoxy;RYL1is H, or optionally substituted linear or branched C1-6alkyl (e.g. methyl, ethyl);n is 0-10; andandindicates the attachment point to the PTM or the ULM.

In any aspect or embodiment described herein, the linker (L) comprises a structure selected from the structure shown below:

wherein:WL1and WL2are each independently absent, aryl, heteroaryl, cyclic, heterocyclic, C1-6alkyl and optionally one or more C atoms are replaced with O or NRYL1, C1-6alkene and optionally one or more C atoms are replaced with O, C1-6alkyne and optionally one or more C atoms are replaced with O, bicyclic, biaryl, biheteroaryl, or biheterocyclic, each optionally substituted with RQ, each RQis independently a H, halo, OH, CN, CF3, hydroxyl, nitro, C≡CH, C2-6alkenyl, C2-6alkynyl, optionally substituted linear or branched C1-C6alkyl, optionally substituted linear or branched C1-C6alkoxy, optionally substituted OC1-3alkyl (e.g., optionally substituted by 1 or more —F), OH, NH2, NRY1RY2, CN, or 2 RQgroups taken together with the atom they are attached to, form a 4-8 membered ring system containing 0-4 heteroatoms;YL1is each independently a bond, NRYL1, O, S, NRYL2, CRYL1RYL2, C═O, C═S, SO, SO2, optionally substituted linear or branched C1-C6alkyl and optionally one or more C atoms are replaced with O; optionally substituted linear or branched C1-C6alkoxy;QLis a 3-6 membered alicyclic, bicyclic or aromatic ring with 0-4 heteroatoms, optionally bridged, optionally substituted with 0-6 RQ, each RQis independently H, optionally substitute linear or branched C1-6alkyl (e.g., optionally substituted by 1 or more halo, C1-6alkoxyl), or 2 RQgroups taken together with the atom they are attached to, form a 3-8 membered ring system containing 0-2 heteroatoms;RYL1, RYL2are each independently H, OH, optionally substituted linear or branched C1-6alkyl (e.g., optionally substituted by 1 or more halo, C1-6alkoxyl), or R1, R2together with the atom they are attached to, form a 3-8 membered ring system containing 0-2 heteroatoms;n is 0-10; andandindicates the attachment point to the PTM or the ULM.

In any aspect or embodiment described herein, the linker (L) comprises a structure selected from the structure shown below:

wherein:WL1and WL2are each independently absent, cyclohexane, cyclopentane, piperazine, piperidine, morpholine, C1-6alkyl and optionally one or more C atoms are replaced with O or NRYL1, C1-6alkene and optionally one or more C atoms are replaced with O, C1-6alkene and optionally one or more C atoms are replaced with O, or C1-6alkyne and optionally one or more C atoms are replaced with O, each optionally substituted with RQ, each RQis independently a H, —Cl, —F, OH, CN, CF3, hydroxyl, optionally substituted linear or branched C1-C6alkyl (e.g., methyl, ethyl), or optionally substituted linear or branched C1-C6alkoxy;YL1is each independently a bond, NRYL1, O, CRYL1RYL2, C═O, optionally substituted linear or branched C1-C6alkyl and optionally one or more C atoms are replaced with O or NRYL1, C1-6alkene and optionally one or more C atoms are replaced with O, C1-6alkyne and optionally one or more C atoms are replaced with O, or optionally substituted linear or branched C1-C6alkoxy;QLis a 3-6 membered heterocyclic, heterobicyclic, or heteroaryl ring, optionally substituted with 0-6 RQ, each RQis independently H, or optionally substituted linear or branched C1-6alkyl (e.g., optionally substituted by 1 or more halo, C1-6alkoxyl);RYL1, RYL2are each independently H, optionally substituted linear or branched C1-6alkyl (e.g., methyl, ethyl, optionally substituted by 1 or more halo, C1-6alkoxyl);n is 0-10; andandindicates the attachment point to the PTM or the ULM.
Exemplary PTMs

In one aspect of the disclosure, the PTM group (also referred as the LTM group) binds to the target protein, LRRK2 or mutated form thereof.

The compositions described below exemplify members of LRRK2 binding moieties that can be used according to the present invention. These binding moieties are linked to the ubiquitin ligase binding moiety (CLM) preferably through a chemical linking group in order to present the LRRK2 protein (to which LTM is bound) in proximity to the ubiquitin ligase for ubiquitination and subsequent degradation.

In certain contexts, the term “target protein” is used to refer to the LRRK2 protein, a member of the leucine-rich repeat kinase family, which is a target protein to be ubiquitinated and degraded. In other contexts, the term “target protein” is used to refer to a mutated form of the LRRK2 protein, such as a LRRK protein having one or mutation selected from the group consisting of G2019S, I2020T, N1437H, R1441G/C/H, and Y1699C.

The term “protein target moiety” or PTM is used to describe a small molecule which binds to LRRK2 or mutated form thereof, and can be used to target the protein for ubiquitination and degradation.

The compositions described herein exemplify the use of some of these PTMs.

In any aspect or embodiment described herein, the PTM is a small molecule that binds LRRK2. For example, in any aspect or embodiment described herein, the PTM is represented by the chemical structure PTM-IA or PTM-IB:

wherein:R1is selected from a linear or branched C1-C6 alkyl (e.g., isopropyl or tert-butyl), an optionally substituted C3-C6 cycloalkyl (e.g., an optionally substituted C3-C5 cycloalkyl, a methylated C3-C5 cycloalkyl, or

is an optionally substituted 3-10 membered cycloalkyl or heterocyloalkyl containing 1-4 (e.g., 1, 2, 3, or 4) heteroatoms selected from N, O, and S (e.g., optionally substituted with one or more (e.g., 1, 2, 3, or 4) substitutions, andof the PTM indicates the point of attachment with a linker (L) or a ULM.

In any aspect or embodiment described herein, the PTM is represented by the chemical structure PTM-IIA, PTM-IIB, PTM-IIIA, and PTM-IIIB.

wherein the dashed line is the point of attachment to the M or the oxygen atom of the PTM), linear or branched C1-C6 haloalkyl (e.g., linear or branched C1-C4 haloalkyl), an optionally substituted C3-C6 halocycloalkyl (e.g., C3-C5 halocycloalkyl), an optionally substituted alkylnitrile (e.g., a C1-C4 alkyl nitrile), an optionally substituted C3-C6 cyclonitrile (e.g, a C3-C5 cyclonitrile);R2is hydrogen, halogen (e.g., F, Cl, or Br), C1-C3 alkyl, or C1-C3 fluoroalkyl;X4CH or N;M is a CH2, NH, or O;

is an optionally substituted 3-10 membered heterocyloalkyl containing 1-4 (e.g., 1, 2, 3, or 4) heteroatoms selected from N, O, and S (e.g., optionally substituted with one or more (e.g., 1, 2, 3, or 4) substitutions, andof the PTM indicates the point of attachment with a chemical linker group or a ULM.

In any aspect or embodiment described herein,

In any aspect or embodiment described herein, the PTM is covalently linked to L or ULM via an atom of the heterocycloalkyl of

or a substituent thereof.

In any aspect or embodiment described herein,

In any aspect or embodiment described herein,

In any aspect or embodiment described herein described herein,

wherein:R3and R4are each independently selected from a H, halogen, OH, NH2, N(C1-C3 alkyl)2, linear or branched C1-C4 alkyl, linear or branched C1-C4 hydroxyalkyl, linear or branched C1-C4 alkoxy, and linear or branched C1-C4 haloalkyl;indicates the point of attachment of the

(i.e., the point of attachment with the 6-membered heteroaryl of the PTM); andindicates the point of attachment of the PTM with the L or ULM, and where not present, the

may be attached to the L or ULM via an atom of the 6-membered heterocycloalkyl (e.g., a carbon or nitrogen), R3, or R4.

In any aspect or embodiment described herein, such as but not limited to that in the preceding paragraph or the following paragraph,

In any aspect or embodiment described herein, such as but not limited to that in the preceding paragraph,

In any aspect or embodiment described herein, such as but not limited to that in the preceding paragraph,

In any aspect or embodiment described herein, such as but not limited to that in the preceding paragraph,

In any aspect or embodiment described herein, such as but not limited to that in the preceding paragraph,

In any aspect or embodiment described herein described herein,

wherein:R3is H or linear or branched C1-C3 alkyl (e.g., methyl or ethyl);R4is H or linear or branched C1-C3 alkyl (e.g., methyl or ethyl);indicates the point of attachment of the

(i.e., the point of attachment with the 6-membered heteroaryl of the PTM); andindicates the point of attachment of the PTM with the L or ULM, and where not present, the

may be attached to the L or ULM via an atom of the 6-membered heterocycloalkyl (e.g., a carbon or nitrogen of the 6-membered heterocycloalkyl), R3, or R4.

In any aspect or embodiment described herein,

is selected from:

wherein R3and R4are defined as described in any aspect or embodiment described herein.

In any aspect or embodiment described herein,

is selected from:

wherein:R3and R4are defined as described in any aspect or embodiment described herein; andthe heterocycloalkyl is attached to L or PTM via an atom of the heterocycloalkyl or a substituent thereof (e.g., R3, R4, or a methyl group).

In any aspect or embodiment described herein, the PTM has the chemical structure:

wherein:X4, R1, R2, R3and R4are defined as described in any aspect or embodiment described herein; andthe PTM is attached to the L or ULM via an atom of heterocyloalkyl A (e.g, a carbon or nitrogen of the heterocycloalkyl), R3, or R4.

In any aspect or embodiment described herein, the PTM has the chemical structure:

wherein:X4, R1, R2, R3and R4are defined as described in any aspect or embodiment described herein; andof the PTM indicates the point of attachment with the L or ULM.

In any aspect or embodiment described herein, the R1is selected from an optionally substituted C3-C5 cycloalkyl and a liner or branched C1-C4 alkyl.

In any aspect or embodiment described herein, R1is

wherein: R1a, R1b, and R1Care each independently a H or a linear or branched C1-C2 alkyl, each optionally substituted with one or more halogen or nitrile group; or R1aor R1btogether with the carbon to which they are attached form a C3-C6 cycloalkyl that is optionally substituted with one or more C1-C3 alkyl, nitrile group, or halogen.

In any aspect or embodiment described herein, R1is

wherein: R1a, R1b, and R1Care each independently a H, or a linear or branched C1-C2 alkyl; or R1aor R1btogether with the carbon to which they are attached form a C3-C6 cycloalkyl.

In any aspect or embodiment described herein, the PTM is selected from:

are defined as described in any aspect or embodiment described herein; andthe L or ULM is attached via an atom of the heterocycloalkyl A (e.g., a carbon or nitrogen of the heterocycloalkyl), R3, or R4.

In any aspect or embodiment described herein, the PTM is selected from:

wherein:each of X4, R1a, R1b, R1c, R3, and R4are defined as described in any aspect or embodiment described herein; andof the PTM indicates the point of attachment with the L or ULM, and where not present.

In any aspect or embodiment described herein, the R1is selected from

wherein the dashed line is the point of attachment to the M or oxygen atom of the PTM.

In any aspect or embodiment described herein, the R2is H or F.

In any aspect or embodiment described herein, the PTM has the chemical structure:

whereinof the PTM indicates the point of attachment with a chemical linker group or a ULM.

In any aspect embodiment described herein, the PTM has the chemical structure:

wherein the PTM is covalently linked to the L or ULM via an atom of the heterocycloalkyl A or a substituent thereof.

In any aspect or embodiment described herein, the PTM has the chemical structure:

wherein theindicates the point of attachment with a L or a ULM.
Therapeutic Compositions

The present invention further provides pharmaceutical compositions comprising therapeutically effective amounts of at least one bifunctional compound as described herein, in combination with a pharmaceutically acceptable carrier, additive or excipient.

In an additional aspect, the description provides therapeutic compositions comprising an effective amount of a compound as described herein or salt form thereof, and a pharmaceutically acceptable carrier, additive or excipient, and optionally an additional bioactive agent. The therapeutic compositions effect targeted protein degradation in a patient or subject, for example, an animal such as a human, and can be used for treating or ameliorating disease states or conditions which are modulated by degrading the target protein. In certain embodiments, the therapeutic compositions as described herein may be used to effectuate the degradation of protein for the treatment or amelioration of LRRK2-mediated inflammatory diseases, autoimmune diseases or cancer. In certain additional embodiments, the disease is idiopathic PD, LRRK2 mutation-associated PD (e.g., PD associated with one or more LRRK2 activating mutations), primary tauopathies (e.g., supranuclear palsy (PSP) or corticobasal degeneration (CBD)), lewy body dementia, Crohn's Disease, Leprosy (e.g., Leprosy with type 1 inflammatory reactions), and/or neuroinflammation.

In alternative aspects, the present disclosure relates to a method for treating a disease state or ameliorating one or more symptoms of a disease or condition in a subject in need thereof by degrading the LRRK2 protein (e.g., a wildtype LRRK2 protein or an LRRK2 mutant protein (e.g., a LRRK2 mutant protein including one or more mutation selected from G2019S, I2020T, N1437H, R1441G/C/H, and Y1699C) comprising administering to said patient or subject an effective amount, e.g., a therapeutically effective amount, of at least one compound as described herein, optionally in combination with a pharmaceutically acceptable carrier, additive or excipient, and optionally coadministered with an additional bioactive agent, wherein the composition is effective for treating or ameliorating the disease or disorder or one or more symptoms thereof in the subject. The method according to the present disclosure may be used to treat certain disease states, conditions or symptoms including inflammatory disease, autoimmune disease, or cancer, by virtue of the administration of effective amounts of at least one compound described herein. For example, the method according to the present disclosure may be used to treat one or more of Parkinson's Disease (PD), idiopathic PD, LRRK2 mutation associated PD (e.g., PD associated with one or more LRRK2 activating mutations), primary tauopathies (e.g., supranuclear palsy (PSP) or corticobasal degeneration (CBD)), lewy body dementia, Crohn's Disease, Leprosy (e.g., Leprosy with type 1 inflammatory reactions), and neuroinflammation (such as is observed in Alzheimer's disease, PD, multiple sclerosis, traumatic brain injury, spinal cord injury, etc.).

The present disclosure further includes pharmaceutical compositions comprising a pharmaceutically acceptable salt, in particular, acid or base addition salts of the compounds as described herein. The acids which are used to prepare the pharmaceutically acceptable acid addition salts of the aforementioned compounds useful according to this aspect are those which form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate [i.e., 1,1′-methylene-bis-(2-hydroxy-3 naphthoate)]salts, among numerous others.

Pharmaceutically acceptable base addition salts may also be used to produce pharmaceutically acceptable salt forms of the compounds according to the present disclosure. The chemical bases that may be used as reagents to prepare pharmaceutically acceptable base salts of the present compounds are those that form non-toxic base salts with such compounds. Such non-toxic base salts include, but are not limited to those derived from such pharmacologically acceptable cations such as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium, zinc and magnesium), ammonium or water-soluble amine addition salts such as N-methylglucamine-(meglumine), and the lower alkanolammonium and other base salts of pharmaceutically acceptable organic amines, among others.

The compounds as described herein may, in accordance with the disclosure, be administered in single or divided doses by the oral, parenteral or topical routes. Administration of the active compound may range from continuous (intravenous drip) to several oral administrations per day (for example, Q.I.D.) and may include oral, topical, parenteral, intramuscular, intravenous, sub-cutaneous, transdermal (which may include a penetration enhancement agent), buccal, sublingual, intranasal, intraocular, intrathecal, vaginal, and suppository administration, among other routes of administration. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Enteric coated oral tablets may also be used to enhance bioavailability of the compounds from an oral route of administration. The most effective dosage form will depend upon the pharmacokinetics of the particular agent chosen as well as the type, location and severity of disease, condition or symptom, and the health of the patient. Administration of compounds according to the present disclosure as sprays, mists, or aerosols for intra-nasal, intra-tracheal or pulmonary administration may also be used. The present disclosure therefore also is directed to pharmaceutical compositions comprising an effective amount of compound as described herein, optionally in combination with a pharmaceutically acceptable carrier, additive or excipient. Compounds according to the present disclosure may be administered in immediate release, intermediate release or sustained or controlled release forms. Sustained or controlled release forms are preferably administered orally, but also in suppository and transdermal or other topical forms. Intramuscular injections in liposomal form or in depot formulation may also be used to control or sustain the release of compound at an injection site.

The pharmaceutical compositions as described herein may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch, among others known in the art. For oral administration in a capsule form, useful diluents include lactose and corn starch. When aqueous suspensions are required for oral use, the active ingredient may be combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added. Lubricating agents, such as magnesium stearate, are also typically added.

The pharmaceutical compositions as described herein may also be administered topically. For topical applications, the pharmaceutical composition can be formulated in a transdermal patch, which can either be a reservoir patch or a matrix patch comprising the active compound combined with one or more carriers, buffers, absorption enhancers, and providing from 1 day to two weeks of continuous administration.

Alternatively, the pharmaceutical compositions of the present disclosure may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this disclosure include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.

Alternatively, the pharmaceutical compositions of the present disclosure can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Alternatively, the pharmaceutical compositions of the present disclosure can be formulated for ophthalmic use. For example, the pharmaceutical compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutical compositions may be formulated in an ointment such as petrolatum.

The amount of active pharmaceutical ingredient in a pharmaceutical composition as described herein that may be combined with the carrier materials to produce a single dosage form will vary depending upon the condition of the subject and disease, condition or symptom treated, the particular mode of administration, and the condition of the subject. Preferably, the compositions should be formulated to contain between about 0.05 milligram and about 750 milligrams or more, more preferably about 1 milligram to about 600 milligrams, and even more preferably about 10 milligrams to about 500 milligrams of active ingredient, alone or in combination with another compound according to the present disclosure.

It should also be understood that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity and bioavailability of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease or condition being treated.

A patient or subject in need of therapy using compounds according to the methods described herein can be treated by administering to the patient (subject) an effective amount of the compound according to the present disclosure depending upon the pharmaceutically acceptable salt or solvate thereof, optionally in a pharmaceutically acceptable carrier or diluent, either alone, or in combination with another known therapeutic agent.

In certain aspects, the active compound is combined with the pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to a patient a therapeutically effective amount for the desired indication, without causing an undue degree of serious toxic effects in the patient treated. A preferred dose of the active compound for all of the herein-mentioned conditions is in the range from about 10 nanograms per kilograms (ng/kg) to 300 milligrams per kilograms (mg/kg), preferably 0.1 to 100 mg/kg per day, more generally 0.5 to about 25 mg per kilogram body weight of the recipient/patient per day. A typical topical dosage will range from 0.01-5% wt/wt in a suitable carrier.

In certain aspects, the compound is conveniently administered in any suitable unit dosage form, including but not limited to a dosage form containing less than 1 milligrams (mg), 1 mg to 3000 mg, or 5 mg to 500 mg of active ingredient per unit dosage form. An oral dosage of about 25 mg-250 mg is often convenient.

In certain aspects, the active ingredient is preferably administered to achieve peak plasma concentrations of the active compound of about 0.00001-30 millimole (mM), preferably about 0.1-30 micromole (μM). This may be achieved, for example, by the intravenous injection of a solution or formulation of the active ingredient, optionally in saline, or an aqueous medium or administered as a bolus of the active ingredient. Oral administration may also be appropriate to generate effective plasma concentrations of active agent.

Oral compositions will generally include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound or its prodrug derivative can be incorporated with excipients and used in the form of tablets, troches, or capsules. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.

The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a dispersing agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or enteric agents.

The active compound or pharmaceutically acceptable salt thereof can be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.

The active compound or pharmaceutically acceptable salts thereof can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as anti-cancer agents, as described herein among others. In certain preferred aspects of the disclosure, one or more compounds according to the present disclosure are coadministered with another bioactive agent, such as an anti-cancer agent or a wound healing agent, including an antibiotic, as otherwise described herein.

If administered intravenously, preferred carriers are physiological saline or phosphate buffered saline (PBS).

Liposomal suspensions may also be pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811 (which is incorporated herein by reference in its entirety). For example, liposome formulations may be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container. An aqueous solution of the active compound are then introduced into the container. The container is then swirled by hand to free lipid material from the sides of the container and to disperse lipid aggregates, thereby forming the liposomal suspension.

Therapeutic Methods

In an additional aspect, the description provides therapeutic methods comprising administration of an effective amount of a compound as described herein or salt form thereof, and a pharmaceutically acceptable carrier. The therapeutic methods are useful to effect protein degradation in a patient or subject in need thereof, for example, an animal such as a human, for treating or ameliorating a disease state, condition or related symptom that may be treated through targeted protein degradation.

The terms “treat”, “treating”, and “treatment”, etc., as used herein, refer to any action providing a benefit to a patient for which the present compounds may be administered, including the treatment of any disease state, condition, or symptom which is related to the protein to which the present compounds bind. Disease states or conditions, including cancer, which may be treated using compounds according to the present disclosure are set forth hereinabove.

The description provides therapeutic methods for effectuating the degradation of proteins of interest for the treatment or amelioration of a disease, e.g., Parkinson's Disease (PD), primary tauopathies, lewy body dementia, Crohn's Disease, Leprosy, and/or neuroinflammation (such as is observed in. In any aspect or embodiment, the disease is idiopathic PD, LRRK2 mutation associated PD (e.g., PD associated with one or more LRRK2 activating mutations), PSP, CBD, Leprosy with type 1 inflammatory reactions, Alzheimer's disease, PD, multiple sclerosis, traumatic brain injury, and/or spinal cord injury. As such, in another aspect, the description provides a method of ubiquitinating/degrading a target protein in a cell. In certain embodiments, the method comprises administering a bifunctional compound of the invention. The control or reduction of specific protein levels in cells of a subject as afforded by the present disclosure provides treatment of a disease state, condition, or symptom. In any aspect or embodiment, the method comprises administering an effective amount of a compound as described herein, optionally including a pharmaceutically acceptable excipient, carrier, adjuvant, another bioactive agent or combination thereof.

In additional embodiments, the description provides methods for treating or ameliorating a disease, disorder or symptom thereof in a subject or a patient, e.g., an animal such as a human, comprising administering to a subject in need thereof a composition comprising an effective amount, e.g., a therapeutically effective amount, of a compound as described herein or salt form thereof, and a pharmaceutically acceptable excipient, carrier, adjuvant, another bioactive agent or combination thereof, wherein the composition is effective for treating or ameliorating the disease or disorder or symptom thereof in the subject.

In another aspect, the description provides methods for identifying the effects of the degradation of proteins of interest in a biological system using compounds according to the present disclosure.

In another aspect, the description provides a process for making a molecule that can cause degradation of LRRK2 in a cell, comprising the steps of: (i) providing a small molecule that binds to the LRRK2 or a mutated form thereof; (ii) providing an E3 ubiquitin ligase binding moiety (ULM), preferably a CLM such as thalidomide, pomalidomide, lenalidomide or an analog thereof; and (iii) covalently coupling the small molecule of step (i) to the ULM of step (ii) via a chemical linking group (L) to form a compound which binds to both a cereblon E3 ubiquitin ligase and LRRK2 protein and/or mutated form in the cell, such that the cereblon E3 ubiquitin ligase is in proximity to, and ubiquitinates the LRRK2 protein bound thereto, such that the ubiquitinated LRRK2 is then degraded.

In another aspect, the description provides a method for detecting whether a molecule can trigger degradation of a LRRK2 protein in a cell, the method comprising the steps of: (i) providing a molecule for which the ability to trigger degradation of LRRK2 protein in a cell is to be detected, said molecule comprising the structure: CLM-L-PTM, wherein CLM is a cereblon E3 ubiquitin ligase binding moiety capable of binding a cereblon E3 ubiquitin ligase in a cell, which CLM is thalidomide, pomalidomide, lenalidomide, or an analog thereof; PTM is a protein targeting moiety, which is a small molecule that binds to LRRK2 and/or mutated LRRK form thereof, said LRRK2 having at least one lysine residue available to be ubiquitinated by a cereblon E3 ubiquitin ligase bound to the CLM of the molecule; and L is a chemical linking group that covalently links the CLM to the PTM to form the molecule; (ii) incubating a LRRK2 protein-expressing cell in the presence of the molecule of step (i); and (iii) detecting whether the LRRK2 protein in the cell has been degraded.

In any of the aspects or embodiments described herein, the small molecule capable of binding LRRK2, is a small molecule that binds of LRRK2. In certain embodiments, the small molecule that binds the LRRK2 is as described herein.

In another aspect of said treatment, the present disclosure provides a method of treating a human patient in need of said treatment of a disease state, condition, or symptom causally related to LRRK2, and/or LRRK2 mutated form, expression, over-expression, mutation, aggregation, accumulation, misfolding or dysregulation where the degradation of the LRRK2 protein will produce a therapeutic effect in the patient, the method comprising administering to the patient an effective amount of a compound according to the present disclosure, optionally in combination with another bioactive agent.

In another aspect of said treatment, the present disclosure provides a method of treating a human patient in need of said treatment of a disease state, condition, or symptom causally related to alpha-synuclein expression, over-expression, mutation, aggregation, accumulation, misfolding or dysregulation where the degradation of the LRRK2 protein and/or mutated form thereof will produce a therapeutic effect in the patient, the method comprising administering to the patient an effective amount of a compound according to the present disclosure, optionally in combination with another bioactive agent.

In another aspect of said treatment, the present disclosure provides a method of treating a human patient in need of said treatment of a disease state, condition, or symptom causally related to alpha-synuclein expression, over-expression, mutation, aggregation, misfolding or dysregulation where the degradation of the LRRK2 protein and/or mutated form thereof will produce a therapeutic effect in the patient, the method comprising administering to the patient an effective amount of a compound according to the present disclosure, optionally in combination with another bioactive agent.

In another aspect of said treatment, the present disclosure provides a method of treating a human patient in need of said treatment of a disease state, condition, or symptom causally related to Tau expression, over-expression, mutation, aggregation, misfolding or dysregulation where the degradation of the LRRK2 protein and/or mutated form thereof will produce a therapeutic effect in the patient, the method comprising administering to the patient an effective amount of a compound according to the present disclosure, optionally in combination with another bioactive agent.

The disease state, condition, or symptom may be caused by a microbial agent or other exogenous agent such as a virus, bacteria, fungus, protozoa or other microbe, or may be a disease state, which is caused by expression, overexpression, mutation, misfolding, or dysregulation of the protein, which leads to a disease state, condition, or symptom.

In another aspect, the present disclosure provides a method of treating or ameliorating at least one symptom of a disease or condition in a subject, comprising the steps of: providing a subject identified as having a symptom of a disease or condition causally related to expression, overexpression, mutation, misfolding, or dysregulation of LRRK2 protein and/or mutated form thereof in the subject, and the symptom of the disease or condition is treated or ameliorated by degrading LRRK2 protein and/or mutated form thereof in cells of the subject; and administering to the subject therapeutically effective amount of a compound comprising a small molecule of the present invention such that the LRRK2 protein and/or mutated form thereof is degraded, thereby treating or ameliorating at least one symptom of a disease or condition in the subject.

The term “disease state or condition” is used to describe any disease state or condition wherein protein expression overexpression, mutation, misfolding, or dysregulation (e.g., the amount of protein expressed in a patient is elevated) occurs and where degradation of the LRRK2 protein and/or mutated form thereof to reduce or stabilize the level of LRRK2 protein (whether mutated or not) in a patient provides beneficial therapy or relief of symptoms to a patient in need thereof. In certain instances, the disease state, condition, or symptom may be cured.

Disease state, condition, or symptom which may be treated using compounds according to the present disclosure include, for example, Parkinson's Disease (PD), idiopathic PD, LRRK2 mutation associated PD (e.g., PD associated with one or more LRRK2 activating mutations), primary tauopathies (e.g., supranuclear palsy (PSP) or corticobasal degeneration (CBD)), lewy body dementia, Crohn's Disease, Leprosy (e.g., Leprosy with type 1 inflammatory reactions), and/or neuroinflammation (such as is observed in Alzheimer's disease, PD, multiple sclerosis, traumatic brain injury, spinal cord injury, etc.).

The term “bioactive agent” is used to describe an agent, other than a compound according to the present disclosure, which is used in combination with a present compound as an agent with biological activity to assist in effecting an intended therapy, inhibition and/or prevention/prophylaxis for which the present compounds are used. Preferred bioactive agents for use herein include those agents which have pharmacological activity similar to that for which the present compounds are used or administered and include for example, anti-cancer agents, antiviral agents, especially including anti-HIV agents and anti-HCV agents, antimicrobial agents, antifungal agents, etc.

The term “additional anti-autoimmune disease agent” is used to describe an anti-autoimmune disease therapeutic agent, which may be combined with a compound according to the present disclosure to treat autoimmune disease. These agents include, for example, infliximab, tofacitinib, baricitinib, secukinumab, adalimumab, etanercept, golimumab, certolizumab pepol, anti-proliferative drugs (for example, mycophenolate mofetil) and corticosteroids.

The term “pharmaceutically acceptable derivative” is used throughout the specification to describe any pharmaceutically acceptable prodrug form (such as an ester, amide other prodrug group), which, upon administration to a patient, provides directly or indirectly the present compound or an active metabolite of the present compound.

EXAMPLES

Abbreviations

General Synthetic Approach

The synthetic realization and optimization of the bifunctional molecules as described herein may be approached in a stepwise or modular fashion. For example, identification of compounds that bind to the target protein, i.e., LRRK2 can involve high or medium throughput screening campaigns if no suitable ligands are immediately available. It is not unusual for initial ligands to require iterative design and optimization cycles to improve suboptimal aspects as identified by data from suitable in vitro and pharmacological and/or ADMET assays. Part of the optimization/SAR campaign would be to probe positions of the ligand that are tolerant of substitution and that might be suitable places on which to attach the chemical linking group previously referred to herein. Where crystallographic or NMR structural data are available, these can be used to focus such a synthetic effort.

In a very analogous way one can identify and optimize ligands for an E3 Ligase.

With PTMs and ULMs (e.g. CLMs) in hand, one skilled in the art can use known synthetic methods for their combination with or without a chemical linking group(s). Chemical linking group(s) can be synthesized with a range of compositions, lengths and flexibility and functionalized such that the PTM and ULM groups can be attached sequentially to distal ends of the linker. Thus, a library of bifunctional molecules can be realized and profiled in in vitro and in vivo pharmacological and ADMET/PK studies. As with the PTM and ULM groups, the final bifunctional molecules can be subject to iterative design and optimization cycles in order to identify molecules with desirable properties.

In some instances, protecting group strategies and/or functional group interconversions (FGIs) may be required to facilitate the preparation of the desired materials. Such chemical processes are well known to the synthetic organic chemist and many of these may be found in texts such as “Greene's Protective Groups in Organic Synthesis” Peter G. M. Wuts and Theodora W. Greene (Wiley), and “Organic Synthesis: The Disconnection Approach” Stuart Warren and Paul Wyatt (Wiley).

Synthetic Procedures

General Synthetic Scheme

X represents a suitable leaving group (e.g. OTs, OMs, Cl, Br, etc.)Y represents either a primary or secondary amine or alcoholM represents a metalated version of the TLM (Na+, Cs+, Li+, etc)PG represents a suitable protecting group

Exemplary Synthesis of Intermediate 1, 2-(2,6-dioxo-3-piperidyl)-5-hydroxy-isoindoline-1,3-dione

To a solution of 3-aminopiperidine-2,6-dione (4.1 g, 24.7 mmol, 1.50 eq, HCl salt) in acetic acid (45 mL) was added sodium acetate (4.1 g, 49.4 mmol, 3.00 eq), then the mixture was stirred at 25° C. for 1 h. Then 4-hydroxyphthalic acid (3.0 g, 16.5 mmol, 1.00 eq) was added into the mixture and heated to 120° C., stirred for additional 11 h. LCMS showed the desired MS was detected and the reaction was complete. The mixture was concentrated and then poured into water (20 mL), and then filtered. The crude product was purified by column chromatography (dichloromethane:methanol=50:1 to 10:1) to afford 2-(2,6-dioxo-3-piperidyl)-5-hydroxy-isoindoline-1,3-dione (3.9 g, 14.3 mmol, 86% yield) as a colorless solid.

To a solution of 2-bromo-1,1-dimethoxy-ethane (3.22 g, 19.04 mmol, 2 eq) in dimethyl formamide (20 mL) was added potassium carbonate (3.95 g, 28.56 mmol, 3 eq) and dimethyl 4-hydroxybenzene-1, 2-dicarboxylate (2 g, 9.52 mmol, 1 eq). The mixture was stirred at 100° C. for 3 hours. LCMS indicated 4-hydroxybenzene-1, 2-dicarboxylate was consumed completely and one new spot formed. The reaction mixture was quenched by water 200 mL at 25° C., and then extracted with ethyl acetate (50 mL×3). The combined organic layers were washed with brines (50 mL×3), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (Petroleum ether:Ethyl acetate=15:1 to 8:1). Compound dimethyl 4-(2,2-dimethoxyethoxy)benzene-1,2-dicarboxylate (2.64 g, 8.85 mmol, 92% yield) was obtained as a yellow oil.
Step 2

To a solution of dimethyl 4-(2,2-dimethoxyethoxy)benzene-1,2-dicarboxylate (2.64 g, 8.86 mmol, 1 eq) in methyl alcohol (20 mL) was added sodium hydroxide (4 M, 4.43 mL, 2 eq). The mixture was stirred at 40° C. for 12 hours. The reaction mixture was quenched by hydrochloric acid 20 mL at 20° C., and then diluted with water 100 mL and extracted with ethyl acetate (50 mL×3). The combined organic layers were washed with brines (50 mL×3), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was used into the next step without further purification. Compound 4-(2,2-dimethoxyethoxy)phthalic acid (2.2 g, 8.14 mmol, 91% yield) was obtained as a yellow oil.
Step 3

To a solution of 4-(2,2-dimethoxyethoxy)phthalic acid (2.2 g, 8.14 mmol, 1 eq) in pyridine (10 mL) was added 3-aminopiperidine-2,6-dione (2.01 g, 12.21 mmol, 1.5 eq, hydrochloride). The mixture was stirred at 100° C. for 12 hours. The reaction mixture was concentrated under reduced pressure to remove pyridine (10 mL). The residue was diluted with water 200 mL and extracted with ethyl acetate (50 mL×3). The combined organic layers were washed with brines (50 mL×3), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (Petroleum ether/Ethyl acetate=10:1 to 3:1). Compound 5-(2,2-dimethoxyethoxy)-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (1.6 g, 4.20 mmol, 51% yield, 95% purity) was obtained as a yellow oil.
Step 4

To a solution of 2-(2,6-dioxo-3-piperidyl)-5-hydroxy-isoindoline-1,3-dione (300 mg, 1.09 mmol, 1 eq) and 2-(2-hydroxyethoxy)ethyl 4-methylbenzenesulfonate (341 mg, 1.31 mmol, 1.2 eq) in N,N-dimethylformamide (4 mL) was added potassium carbonate (302 mg, 2.19 mmol, 2 eq). The mixture was stirred at 60° C. for 12 hours. LCMS showed the reaction was completed. The mixture was diluted with water (10 mL) and extracted with dichloromethane (10 mL×3). The combined organic layer was washed with brine (20 mL×2), dried with anhydrous sodium sulfate, filtered and concentrated in vacuum. The residue was purified by column chromatography (dichloromethane:methanol=1:0 to 50:1) to give 2-(2,6-dioxo-3-piperidyl)-5-[2-(2-hydroxyethoxy)ethoxy]isoindoline-1,3-dione (400 mg) as a yellow oil.
Step 2

Related intermediates 2-[2-[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]oxyethoxy]ethoxy]ethyl 4-methylbenzenesulfonate, 2-[2-[2-[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]oxyethoxy]ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate, and 2-[2-[2-[2-[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]oxyethoxy]ethoxy]ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate were prepared in a manner analogous with 2-[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]oxyethoxy]ethyl 4-methylbenzenesulfonate.

Exemplary Synthesis of Exemplary Compound 1

To a solution of 2-bromo-4-fluoro-1-nitro-benzene (16.78 g, 76.28 mmol, 1.1 eq) and 1-methylcyclopropanol (5 g, 69.34 mmol, 1 eq) in DMF (160 mL) was added NaH (4.16 g, 104.01 mmol, 60% in mineral oil, 1.5 eq) in one portion at 0° C. under N2. Then the mixture was heated to 20° C. and stirred for 4 hours. TLC showed there were new spots. The residue was poured into water (200 mL) and stirred for 10 min. The aqueous phase was extracted with ethyl acetate (3×300 mL). The combined organic phase was washed with brine (2×200 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel column chromatography (100-200 mesh silica gel, 0-2% of Ethyl acetate in Petroleum ether) to afford 2-bromo-4-(1-methylcyclopropoxy)-1-nitro-benzene (14.3 g, 52.56 mmol, 75.79% yield) as a yellow oil.
Step 2

To a mixture of 2-bromo-4-(1-methylcyclopropoxy)-1-nitro-benzene (14.3 g, 52.56 mmol, 1 eq), K2CO3(14.53 g, 105.11 mmol, 2 eq) and Cs2CO3(17.12 g, 52.56 mmol, 1 eq) in 1,4-dioxane (100 mL) was added 2,4,6-trimethyl-1,3,5,2,4,6-trioxatriborinane (32.99 g, 131.39 mmol, 36.73 mL, 50% purity in EtOAc, 2.5 eq) and Pd(PPh3)4(6.07 g, 5.26 mmol, 0.1 eq)) at 20° C., then heated to 100° C. and stirred for 16 hours to give yellow solution. TLC showed the reaction was completed. The reaction was cooled to 20° C. and concentrated under vacuum. To this residue was added PE:EtOAc (10:1, 100 mL), and the mixture was filtered through a pad of silica. The filter pad was washed with petroleum ether:EtOAc (10:1, 1000 mL) solvent. The residue was purified by silica gel chromatography (100-200 mesh silica gel, 0-1% of Ethyl acetate in Petroleum ether) to afford 2-methyl-4-(1-methylcyclopropoxy)-1-nitro-benzene (11 g, crude) as a yellow oil.
Step 3

To a mixture of 2-methyl-4-(1-methylcyclopropoxy)-1-nitro-benzene (11 g, 53.08 mmol, 1 eq) in EtOH (100 mL) was added 10% of Pd/C (4 g, 5.31 mmol, 0.1 eq) and ammonium formate (40.17 g, 636.99 mmol, 12 eq) in one portion at 20° C. under N2. The mixture was stirred at 20° C. for 2 h to give a black mixture. TLC showed the reaction was completed. The mixture was filtered through a pad of silica gel, washed with EtOAc (3×200 mL) and concentrated in vacuum. The residue was purified by silica gel chromatography (0-10% of Ethyl acetate in Petroleum ether) to afford 2-methyl-4-(1-methylcyclopropoxy) aniline (9.8 g, crude) as a red oil.
Step 4

To a mixture of 2-methyl-4-(1-methylcyclopropoxy) aniline (9.8 g, 55.29 mmol, 1 eq) and Et3N (13.99 g, 138.23 mmol, 19.24 mL, 2.5 eq) in DCM (100 mL) was added Ac2O (11.29 g, 110.58 mmol, 10.36 mL, 2 eq) in one portion at 0° C. under N2. The mixture was stirred at 0° C. for 30 min, then heated to 20° C. and stirred for 16 hours. TLC showed the reaction was completed. The reaction was quenched with a saturated solution of aqueous NaHCO3(30 mL) to adjusted pH=7-8 and extracted with DCM (3×50 mL). The combined organic phase was washed with brine (3×50 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (20-40% Ethyl acetate in Petroleum ether) to afford N-[2-methyl-4-(1-methylcyclopropoxy) phenyl] acetamide (9.3 g, 42.41 mmol, 76.71% yield) as a yellow oil.
Step 5

To a mixture of 1-[5-(1-methylcyclopropoxy)indazol-1-yl]ethanone (8 g, 34.74 mmol, 1 eq) in MeOH (80 mL) was added NH3(g/)MeOH (7 M, 24.82 mL, 5 eq) in one portion at 20° C. The mixture was stirred at 20° C. for 2 hours to give a yellow solution. TLC showed the reaction was completed. The solution was concentrated in vacuum to afford 5-(1-methylcyclopropoxy)-1H-indazole (7.8 g, crude) as a yellow solid.
Step 7

To a mixture of 5-(1-methylcyclopropoxy)-1H-indazole (7.8 g, 41.44 mmol, 1 eq) in THF (80 mL) was added N-dicyclohexylmethylamine (10.52 g, 53.87 mmol, 1.3 eq) and SEM-Cl (8.29 g, 49.73 mmol, 8.80 mL, 1.2 eq) in one portion at 20° C. The mixture was stirred at 20° C. for 16 hours to give an orange solution. TLC showed the reaction was completed. The residue was poured into water (60 mL). The aqueous phase was extracted with ethyl acetate (3×50 mL). The combined organic phase was washed with brine (2×50 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (100-200 mesh silica gel, 0-10% of ethyl acetate in Petroleum ether) to afford trimethyl-[2-[[5-(1-methylcyclopropoxy) indazol-2-yl] methoxy] ethyl] silane (5.4 g, 16.96 mmol, 40.92% yield) as a yellow oil.
Step 8

To a mixture of trimethyl-[2-[[5-(1-methylcyclopropoxy)indazol-2-yl]methoxy]ethyl]silane (4.36 g, 13.70 mmol, 5.32e-1 eq) in THF (6 mL) was dropwise added n-BuLi (2.5 M, 13.40 mL, 1.3 eq) dropwise at −70° C. under N2. The mixture was then stirred at −20° C. for 1 h, and a solution of ZnCl2(0.7 M, 55.20 mL, 1.5 eq) was dropwise added at −70° C. The mixture was stirred for 1 h at −40° C. A mixture of 4, 6-dichloropyrimidine (4.22 g, 28.34 mmol, 1.1 eq) and Pd(PPh3)4(1.49 g, 1.29 mmol, 0.05 eq) in THF (4 mL) was stirred at 20° C. for 1 h and was added to that solution. The cold bath was removed, and the mixture was stirred at 20° C. for 16 h to give a yellow solution. TLC showed there was starting material remained and at the same time some new spots were formed. The residue was poured into water (10 mL). The aqueous phase was extracted with ethyl acetate (3×20 mL). The combined organic phase was washed with brine (2×20 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (100-200 mesh silica gel, 0-10% of Ethyl acetate in Petroleum ether) to afford 2-[[3-(6-chloropyrimidin-4-yl)-5-(1-methylcyclopropoxy) indazol-2-yl] methoxy] ethyl-trimethyl-silane (2.9 g, crude) as a yellow oil.
Step 9

To a mixture of 2-[[3-(6-chloropyrimidin-4-yl)-5-(1-methylcyclopropoxy) indazol-2-yl]methoxy]ethyl-trimethyl-silane (500 mg, 1.16 mmol, 1 eq) and tert-butyl (2S)-2-methylpiperazine-1-carboxylate (697.02 mg, 3.48 mmol, 3 eq) in DMSO (5 mL) was added Et3N (704.34 mg, 6.96 mmol, 968.82 uL, 6 eq) in one portion and then the mixture was stirred at 100° C. for 1 h. TLC showed the reaction was completed. The mixture was cooled to 20° C. The residue was poured into water (5 mL). The aqueous phase was extracted with ethyl acetate (3×5 mL). The combined organic phase was washed with brine (2×5 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give tert-butyl (2S)-2-methyl-4-[6-[5-(1-methylcyclopropoxy)-2-(2-trimethylsilylethoxymethyl) indazol-3-yl] pyrimidin-4-yl] piperazine-1-carboxylate (802 mg, crude) as a yellow oil.
Step 10

To a mixture of tert-butyl (2S)-2-methyl-4-[6-[5-[(1-methylcyclopropyl)methyl]-2-(2-trimethylsilylethoxymethyl)indazol-3-yl]pyrimidin-4-yl]piperazine-1-carboxylate (802 mg, 1.35 mmol, 1 eq) in DCM (5 mL) was added TFA (771.25 mg, 6.76 mmol, 500.81 uL, 5 eq) in one portion at 25° C. The mixture was stirred at 25° C. for 16 hours. The HCl (4 M, 338.20 uL, 1 eq) in MeOH (5 mL) was added at 25° C., then heated to 60° C. and stirred for 0.5 hours. LCMS showed the reaction was completed. The mixture was cooled to 20° C. The residue was poured into NaHCO3(5 mL) to adjust pH=7-8. The aqueous phase was extracted with ethyl acetate (3×10 mL). The combined organic phase was washed with brine (2×10 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (0-40% of Ethyl acetate in MeOH) to give 5-(1-methylcyclopropoxy)-3-[6-[(3S)-3-methylpiperazin-1-yl]pyrimidin-4-yl]-1H-indazole (450 mg, 1.18 mmol, 87.41% yield, 95.77% purity) as a yellow solid.
Step 11

Exemplary Synthesis of Exemplary Compound 2

Exemplary Synthesis of Exemplary Compound 3

Exemplary Synthesis of Exemplary Compound 4

Exemplary Synthesis of Exemplary Compound 5

Exemplary Synthesis of Exemplary Compound 6

To a mixture of 2-[[3-(6-chloropyrimidin-4-yl)-5-(1-methylcyclopropoxy)indazol-2-yl]methoxy]ethyl-trimethyl-silane (500 mg, 1.16 mmol, 1 eq), tert-butyl (2S,6R)-2,6-dimethylpiperazine-1-carboxylate (248.61 mg, 1.16 mmol, 1 eq) in DMSO (5 mL) was added Et3N (352.17 mg, 3.48 mmol, 484.41 uL, 3 eq) in one portion and then the solution was stirred at 100° C. for 1 h. LCMS (EB16-35-P1A1) showed the starting material was consumed completely. The mixture was cooled to 20° C. The residue was poured into water (5 mL). The mixture was extracted with ethyl acetate (3×5 mL). The combined organic phase was washed with brine (2×5 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (Petroleum ether/Ethyl acetate=10/1 to 5/1) to give tert-butyl (2S, 6R)-2,6-dimethyl-4-[6-[5-(1-methylcyclopropoxy)-2-(2-trimethylsilylethoxymethyl)indazol-3-yl]pyrimidin-4-yl]piperazine-1-carboxylate (811 mg, crude) as a yellow oil.
Step 2

To a mixture of tert-butyl (2S,6R)-2,6-dimethyl-4-[6-[5-(1-methylcyclopropoxy)-2-(2-trimethylsilylethoxymethyl)indazol-3-yl]pyrimidin-4-yl]piperazine-1-carboxylate (811 mg, 1.33 mmol, 1 eq) in MeOH (5 mL) was added HCl(g)/dioxane (4 M, 1.67 mL, 5 eq) in one portion at 20° C. The mixture was stirred at 65° C. for 0.5 h. LCMS showed the reaction was completed. The mixture was cooled to 20° C. The residue was poured into NaHCO3(10 mL) to adjust pH=7-8. The mixture was extracted with ethyl acetate (3×10 mL). The combined organic phase was washed with brine (2×10 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (100-200 mesh silica gel, 0-25% of Ethyl acetate in MeOH) to give 3-[6-[(3S,5R)-3,5-dimethylpiperazin-1-yl]pyrimidin-4-yl]-5-(1-methylcyclopropoxy)-1H-indazole (400 mg, 978.01 umol, 73.42% yield, 92.537% purity) as a yellow solid.
Step 3

Exemplary Synthesis of Exemplary Compound 7

Exemplary Synthesis of Exemplary Compound 8

To a mixture of 5-isopropoxy-3-[6-[(3S)-3-methylpiperazin-1-yl]pyrimidin-4-yl]-1H-indazole (160 mg, 453.99 umol, 1 eq) in MeCN (5 mL) was added 2-[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]oxyethoxy]ethoxymethyl 4-methylbenzenesulfonate (347.37 mg, 635.58 umol, 1.4 eq), DIEA (293.37 mg, 2.27 mmol, 395.38 uL, 5 eq) and KI (602.90 mg, 3.63 mmol, 8 eq). The mixture was stirred at 95° C. for 12 hours to give a brown mixture. The mixture was cooled to room temperature and 20 mL water was added into reaction mixture. The resulting mixture was extracted with EtOAc (10 mL×3). The combined extracts were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue (300 mg). The residue was purified by prep-HPLC (FA) to give 2-(2,6-dioxo-3-piperidyl)-5-[2-[2-[2-[(2S)-4-[6-(5-isopropoxy-1H-indazol-3-yl)pyrimidin-4-yl]-2-methyl-piperazin-1-yl]ethoxy]ethoxy]ethoxy]isoindoline-1,3-dione (104.3 mg, 137.94 umol, 30.38% yield, 97.97% purity) as a pink solid.

Exemplary Synthesis of Exemplary Compound 9

To a mixture of 5-isopropoxy-3-[6-[(3S)-3-methylpiperazin-1-yl]pyrimidin-4-yl]-1H-indazole (160 mg, 453.99 umol, 1 eq) in MeCN (8 mL) was added 2-[2-[2-[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]oxyethoxy]ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate (384.29 mg, 635.58 umol, 1.4 eq), DIEA (293.37 mg, 2.27 mmol, 395.38 uL, 5 eq) and KI (602.90 mg, 3.63 mmol, 8 eq). The mixture was stirred at 95° C. for 12 hours to give a brown mixture. The mixture was cooled to room temperature and water (20 mL) was added into reaction mixture. The resulting mixture was extracted with EtOAc (10 mL×3). The combined extracts were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue (320 mg). The residue was purified by prep-HPLC (FA) to give 2-(2,6-dioxo-3-piperidyl)-5-[2-[2-[2-[2-[(2S)-4-[6-(5-isopropoxy-1H-indazol-3-yl)pyrimidin-4-yl]-2-methyl-piperazin-1-yl]ethoxy]ethoxy]ethoxy]ethoxy]isoindoline-1,3-dione (44.9 mg, 56.05 umol, 12.35% yield, 97.98% purity) as pink solid.

Exemplary Synthesis of Exemplary Compound 10

To a mixture of 3-[6-[(3R,5S)-3,5-dimethylpiperazin-1-yl]pyrimidin-4-yl]-5-isopropoxy-1H-indazole (0.15 g, 409.32 μmol, 1 eq.) and 2-[2-[2-[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]oxyethoxy]ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate (296.98 mg, 491.19 μmol, 1.2 eq.), DIEA (793.53 mg, 6.14 mmol, 1.07 mL, 15 eq.) in MeCN (8 mL) and DMSO (2 mL) was added KI (1.02 g, 6.14 mmol, 15 eq) in one portion at 25° C. under N2. The mixture was stirred at 100° C. and stirred for 16 hours. The reaction mixture was concentrated, cooled with an ice bath, and sat NH4Cl was added to adjust the pH to 6. Saturated brine was added thereto, followed by extraction with ethyl acetate (50 mL×2). The organic layer was dried over anhydrous magnesium sulfate and concentrated. The residue was purified by prep-TLC (silica gel, EA:MeOH=10:1) to give 2-(2,6-dioxo-3-piperidyl)-5-[2-[2-[2-[2-[(2R,6S)-4-[6-(5-isopropoxy-1H-indazol-3-yl)pyrimidin-4-yl]-2,6-dimethyl-piperazin-1-yl]ethoxy]ethoxy]ethoxy]ethoxy]isoindoline-1,3-dione (8.5 mg, 10.19 μmol, 2.49% yield, 95.81% purity) as a light yellow solid.

Exemplary Synthesis of Exemplary Compound 11

To a mixture of 1H-indazol-5-ol (5 g, 37.28 mmol, 1 eq) and Cs2CO3(18.22 g, 55.91 mmol, 1.5 eq) in DMF (50 mL) was added 2-iodopropane (8.24 g, 48.46 mmol, 4.85 mL, 1.3 eq) in one portion at 25° C. The mixture was stirred at 25° C. for 4 hours. TLC (petroleum ether:ethyl acetate=3:1, Rf=0.58) showed the reaction completed. The mixture was poured into water (50 mL) and the aqueous phase was extracted with ethyl acetate (60 mL*3). The combined organic phase was washed with brine (20 mL*2), dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (Petroleum ether/Ethyl acetate=3/1) to afford 5-isopropoxy-1H-indazole (4.1 g, 23.27 mmol, 62.42% yield) as a light yellow solid.
Step 2

To a mixture of 5-isopropoxy-1H-indazole (4 g, 22.70 mmol, 1 eq) in MeCN (80 mL) was added K2CO3(3.14 g, 22.70 mmol, 1 eq) and I2(5.76 g, 22.70 mmol, 4.57 mL, 1 eq) in one portion at 25° C. under N2. The mixture was stirred at 25° C. for 16 hours. TLC (Petroleum ether:Ethyl acetate=3:1) showed the reaction was completed. The mixture was diluted with brine (100 mL) and the aqueous phase was extracted with dichloromethane (100 mL*3). The combined organic phase was dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (Petroleum ether/Ethyl acetate=10/3) to afford 3-iodo-5-isopropoxy-1H-indazole (5.5 g, 18.21 mmol, 80.20% yield) as a light-yellow oil.
Step 3

To a mixture of 3-iodo-5-isopropoxy-1H-indazole (5.5 g, 18.21 mmol, 1 eq) in THF (100 mL) was added N-cyclohexyl-N-methyl-cyclohexanamine (4.62 g, 23.67 mmol, 5.02 mL, 1.3 eq) and SEM-Cl (3.04 g, 18.21 mmol, 3.22 mL, 1 eq) in one portion at 25° C. The mixture was stirred at 25° C. for 16 hours to give orange solution. TLC (Petroleum ether:Ethyl acetate=20/1) showed the reaction was completed. The residue was poured into water (100 mL). The aqueous phase was extracted with ethyl acetate (3×80 mL). The combined organic phase was washed with brine (2×20 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (Petroleum ether/Ethyl acetate=100/3) to afford 2-[(3-iodo-5-isopropoxy-indazol-1-yl)methoxy]ethyl-trimethyl-silane (7.4 g, 15.75 mmol, 86.47% yield, 92% purity) as a light yellow oil.
Step 4

To a mixture of 2-[(3-iodo-5-isopropoxy-indazol-1-yl)methoxy]ethyl-trimethyl-silane (7.4 g, 17.11 mmol, 1 eq) and (2-fluoro-4-pyridyl)boronic acid (3.62 g, 25.67 mmol, 1.5 eq) in dioxane (100 mL) was added K3PO4(14.53 g, 68.46 mmol, 4 eq) and Pd(dppf)Cl2(2.50 g, 3.42 mmol, 0.2 eq) in one portion at 25° C. under N2. The mixture was heated to 90° C. with stirring for 5 hours under N2. TLC (Petroleum ether:Ethyl acetate=20/1) showed the reaction was completed. The mixture was cooled to 25° C. and the residue was poured into water (80 mL). The aqueous phase was extracted with ethyl acetate (90 mL*2). The combined organic phase was washed with brine (30 mL*2), dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was purified by silica gel chromatography (Petroleum ether/Ethyl acetate=10/1) to give 2-[[3-(2-fluoro-4-pyridyl)-5-isopropoxy-indazol-1-yl]methoxy]ethyl-trimethyl-silane (5.96 g, 12.32 mmol, 71.98% yield, 83% purity) as a light yellow solid.
Step 5

A mixture of 2-[[5-isopropoxy-3-[2-[(3S)-3-methylpiperazin-1-yl]-4-pyridyl]indazol-1-yl]methoxy]ethyl-trimethyl-silane (200 mg, 415.19 umol, 1 eq) and TFA (5 mL) were stirred at 25° C. for 1 hour. The mixture was concentrated under reduced pressure at 60° C. to give a residue. The residue was purified by silica gel chromatography (Dichloromethane/Methanol=100/5) to afford 5-isopropoxy-3-[2-[(3S)-3-methylpiperazin-1-yl]-4-pyridyl]-1H-indazole (200 mg, 352.83 umol, 84.98% yield, 62% purity) as a dark oil.
Step 7

Exemplary Synthesis of Exemplary Compound 12

To a solution of 5-(1-methylcyclopropoxy)-1H-indazole (500 mg, 2.66 mmol, 1 eq) in DMF (5 mL) was added KOH (558.89 mg, 9.96 mmol, 3.75 eq) and 12 (1.35 g, 5.31 mmol, 1.07 mL, 2 eq). The mixture was stirred at 25° C. for 2 hr. The reaction mixture was diluted with sat Na2SO3(10 mL) and extracted with EtOAc (20 mL*3). The combined organic layers were washed with brine (20 mL*2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by column chromatography on silica gel (PE in EA=0 to 10%) to give 3-iodo-5-(1-methylcyclopropoxy)-1H-indazole (458 mg, 1.26 mmol, 47.53% yield, 86.592% purity) as a yellow solid.
Step 2

To a mixture of 3-iodo-5-(1-methylcyclopropoxy)-1H-indazole (458 mg, 1.30 mmol, 1 eq) in THF (20 mL) was added N-cyclohexyl-N-methyl-cyclohexanamine (760.45 mg, 3.89 mmol, 825.68 uL, 3 eq) and SEM-Cl (432.69 mg, 2.60 mmol, 459.33 uL, 2 eq) in one portion at 20° C. The mixture was stirred at 20° C. for 3 hours to give orange suspension. TLC showed the reaction was completed. The residue was poured into water (10 mL). The aqueous phase was extracted with ethyl acetate (10 mL*2). The combined organic phase was washed with brine (10 mL*2), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (100-200 mesh silica gel, 0-10% of ethyl acetate in Petroleum ether) to give [3-iodo-5-(1-methylcyclopropoxy)indazol-1-yl]methanol (300 mg, 706.09 umol, 54.41% yield, 81% purity) as a yellow solid.
Step 3

To a solution of [2-[(3S)-4-tert-butoxycarbonyl-3-methyl-piperazin-1-yl]-4-pyridyl]boronic acid (857.08 mg, 1.31 mmol, 1.5 eq) and [3-iodo-5-(1-methylcyclopropoxy)indazol-1-yl]methanol (300.00 mg, 871.72 umol, 1 eq) in 1,4-dioxane (10 mL) and H2O (2 mL) was added Pd(dppf)Cl2(95.68 mg, 130.76 umol, 0.15 eq) and Na2CO3(277.18 mg, 2.62 mmol, 3 eq). The mixture was stirred at 90° C. under N2for 1.5 hr. The mixture was cooled to 20° C. and concentrated under reduced pressure. The residue was poured into water (10 mL) and the aqueous phase was extracted with ethyl acetate (10 mL*3). The combined organic phase was washed with brine (10 mL*2), dried with anhydrous Na2SO4, filtered and concentrated under vacuum. The residue was purified by silica gel chromatography (column height: 20 g, 100-200 mesh silica gel, 0-20% of Ethyl acetate in Petroleum ether) to give tert-butyl (2S)-2-methyl-4-[4-[5-(1-methylcyclopropoxy)-1H-indazol-3-yl]-2-pyridyl] piperazine-1-carboxylate (400 mg, crude) as a yellow oil.
Step 5

To a mixture of tert-butyl (2S)-2-methyl-4-[4-[5-(1-methylcyclopropoxy)-2H-indazol-3-yl]-2-pyridyl]piperazine-1-carboxylate (400 mg, 422.80 umol, 1 eq) in MeOH (10 mL) was added HCl/dioxane (4 M, 528.51 uL, 5 eq) in one portion at 20° C. The mixture was stirred at 65° C. for 0.5 h. TLC (EtOAc, Rf=0.07) and LCMS showed the reaction was completed. The mixture was cooled to 20° C. and the residue was poured into saturated aq.NaHCO3(pH=7-8). The aqueous phase was extracted with ethyl acetate (20 mL*3). The combined organic phase was washed with brine (20 mL*2), dried with anhydrous Na2SO4, filtered and concentrated under vacuum. The residue was purified by silica gel chromatography (100-200 mesh silica gel, 0-25% of MeOH in DCM) to give 5-(1-methylcyclopropoxy)-3-[2-[(3S)-3-methylpiperazin-1-yl]-4-pyridyl]-1H-indazole (170 mg, 241.82 umol, 57.19% yield, 51.7% purity) as a yellow solid.
Step 6

Exemplary Synthesis of Exemplary Compound 13

Exemplary Synthesis of Exemplary Compound 14

To a mixture of 2-fluoro-5-methyl-phenol (7 g, 55.50 mmol, 1 eq) in AcOH (15.2 mL) and H2SO4(2 mL) was added NaNO2(3.83 g, 55.50 mmol, 1 eq) in H2O (35 mL) at 0° C. Then the mixture was stirred at 0° C. for 1 hour. The reaction mixture was poured into ice-water (100 mL). The precipitate was collected by filtration followed by washing with water (3×100 mL). The resulting solid was added portion-wise to a mixture of HNO3(12 mL) and H2O (35 mL) with stirring. The resulting suspension was stirred at 45° C. for 2 hours. After cooling to room temperature, the mixture was diluted with cold water (100 mL) and filtered. The solid was washed with water (2×100 mL) and then dissolved in ethyl acetate (100 mL). The organic layer was washed with brine (2×100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-fluoro-5-methyl-4-nitro-phenol (5.3 g, 30.35 mmol, 54.69% yield, 98% purity) as a yellow solid.
Step 2

To a stirred solution of 1-fluoro-2-isopropoxy-4-methyl-5-nitro-benzene (5 g, 23.45 mmol, 1 eq) in EtOH (120 mL) was added ammonium formate (16.27 g, 257.97 mmol, 11 eq) followed by Pd/C (2.5 g, 23.45 mmol, 10% purity, 1.00 eq). The reaction mixture was stirred at 20° C. for 4 hours. The reaction mixture was filtered and concentrated under vacuum to obtain the residue. Dichloromethane (50 mL) was added to the residue and filtered. The filtrate was concentrated under vacuum to give 5-fluoro-4-isopropoxy-2-methyl-aniline (4.2 g, 20.63 mmol, 87.97% yield, 90% purity) as a brown oil
Step 4

To a stirred solution of 5-fluoro-4-isopropoxy-2-methyl-aniline (4.2 g, 20.63 mmol, 1 eq) in AcOH (40 mL) was added NaNO2(1.57 g, 22.69 mmol, 1.1 eq) in H2O (5 mL) at 0° C. The reaction mixture was stirred at 20° C. for 16 hours. The reaction mixture color was changed from yellow to brown. The reaction mixture was concentrated under vacuum to obtain the residue. Saturated NaHCO3solution (40 mL) was added and the mixture was extracted with EA (40 mL). The combined organic layers were washed with brine 40 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography on silica gel (PE/EA=100:20, 100:30) to give 6-fluoro-5-isopropoxy-1H-indazole (3.5 g, 18.02 mmol, 87.36% yield) as a brown oil.
Step 5

To a solution of 6-fluoro-5-isopropoxy-1H-indazole (1.4 g, 7.21 mmol, 1 eq) in DMF (30 mL) was added KOH (1.52 g, 27.03 mmol, 3.75 eq) and I2(3.66 g, 14.42 mmol, 2.90 mL, 2 eq). The mixture was stirred at 25° C. for 3 hr. The reaction mixture was diluted with sat Na2S2O3(30 mL) and extracted with EtOAc (50 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by column chromatography on silica gel (0 to 10% ethyl acetate in petroleum ether) to afford 6-fluoro-3-iodo-5-isopropoxy-1H-indazole (1.62 g, 3.95 mmol, 54.76% yield, 78% purity) as a yellow solid.
Step 6

To a solution of 6-fluoro-3-iodo-5-isopropoxy-1H-indazole (1.61 g, 5.03 mmol, 1 eq) and 2-(chloromethoxy)ethyl-trimethyl-silane (838.61 mg, 5.03 mmol, 890.24 uL, 1 eq) in THF (20 mL) was added N-cyclohexyl-N-methylcyclohexanamine (1.28 g, 6.54 mmol, 1.39 mL, 1.3 eq). The mixture was stirred at 25° C. for 16 hr. The reaction mixture was diluted with water (30 mL) and extracted with EtOAc (50 mL). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by column chromatography on silica gel (0 to 5% ethyl acetate in petroleum ether) to afford 2-[(6-fluoro-3-iodo-5-isopropoxy-indazol-1-yl)methoxy]ethyl-trimethyl-silane (2.05 g, 4.23 mmol, 84.16% yield, 93% purity) as a yellow oil.
Step 7

To a solution of 4-bromo-2-fluoro-pyridine (1 g, 5.68 mmol, 1 eq) and tert-butyl (2S)-2-methylpiperazine-1-carboxylate (1.71 g, 8.52 mmol, 1.5 eq) in DMSO (8 mL) was added K2CO3(2.36 g, 17.05 mmol, 3 eq). The mixture was stirred at 100° C. for 4 hr. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (50 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by column chromatography on silica gel (0 to 10% ethyl acetate in petroleum ether) to afford tert-butyl (2S)-4-(4-bromo-2-pyridyl)-2-methyl-piperazine-1-carboxylate (1.7 g, 4.68 mmol, 82.30% yield, 98% purity) as a colorless oil.
Step 8

Exemplary Synthesis of Exemplary Compound 15

Exemplary Synthesis of Exemplary Compound 16

To a mixture of 1H-indazol-5-ol (1 g, 7.46 mmol, 1 eq) in DCM (10 mL) was added imidazole (1.52 g, 22.37 mmol, 3 eq) and TBSCl (1.69 g, 11.18 mmol, 1.37 mL, 1.5 eq) in one portion at 20° C. under N2. The mixture was stirred at 20° C. for 2 hours to give brown solution. TLC (DCM:MeOH=10:1, Rf=0.23) showed the reaction was completed. The residue was poured into water (10 mL). The aqueous phase was extracted with ethyl acetate (10 mL*3). The combined organic phase was washed with brine (10 mL*3), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (12 g, 30 mL/min, 100-200 mesh silica gel, 0-5% (10 min) of MeOH in DCM) to give tert-butyl-(1H-indazol-5-yloxy)-dimethyl-silane (1.5 g, 5.87 mmol, 78.73% yield, 97.2% purity) as a yellow oil.
Step 2

To a mixture of tert-butyl-dimethyl-[1-(2-trimethylsilylethoxymethyl)indazol-5-yl]oxy-silane (1.82 g, 4.81 mmol, 1 eq) in THF (5 mL) was dropwise added n-BuLi (2.5 M, 2.50 mL, 1.3 eq) at −70° C. under N2. The mixture was then stirred at −20° C. for 5 min, and a solution of ZnCl2(1 M, 7.21 mL, 1.5 eq) was dropwise added at −70° C. The mixture was stirred for 10 min at −40° C. A mixture of 4,6-dichloropyrimidine (787.67 mg, 5.29 mmol, 1.1 eq) and Pd(PPh3)4(277.71 mg, 240.32 umol, 0.05 eq) in THF (1 mL) was stirred at 20° C. for 30 min and added to the reaction. The cold bath was removed and the mixture was stirred at 20° C. for 2 h to give yellow solution. TLC (PE:EtOAc=10:1, Rf=0.83) showed there was a new spot. The residue was poured into water (10 mL). The aqueous phase was extracted with ethyl acetate (20 mL*3). The combined organic phase was washed with brine (20 mL*2), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (40 g, 35 mL/min, 100-200 mesh silica gel, 0-5% (30 min) of Ethyl acetate in Petroleum ether) to give tert-butyl-[3-(6-chloropyrimidin-4-yl)-1-(2-trimethylsilylethoxymethyl)indazol-5-yl] oxy-dimethyl-silane (1.18 g, 2.40 mmol, 49.98% yield) as a yellow oil.
Step 4

To a mixture of tert-butyl-[3-(6-chloropyrimidin-4-yl)-1-(2-trimethylsilylethoxymethyl)indazol-5-yl]oxy-dimethyl-silane (1.18 g, 2.40 mmol, 1 eq) in DCM (10 mL) was added TFA (3 g, 26.31 mmol, 1.95 mL, 10.95 eq) in one portion at 20° C. under N2. The mixture was stirred at 20° C. for 1 h. Then the NH3·H2O (2.55 g, 24.02 mmol, 2.80 mL, 33% purity, 10 eq) was added and the solution was stirred at 20° C. for 1 h. The solution was concentrated under vacuum. The crude was dissolved in THF (5 mL) and TBAF (1 M, 2.40 mL, 1 eq) was added and the solution was stirred for 1 h to give yellow solution. The aqueous phase was extracted with ethyl acetate (5 mL*3). The combined organic phase was washed with brine (5 mL*3), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (column height: 12 g, 30 mL/min, 0-32% (18 min) of Ethyl acetate in Petroleum ether, 32% (12 min) of Ethyl acetate in Petroleum ether) to give 3-(6-chloropyrimidin-4-yl)-1H-indazol-5-ol (140 mg, 567.60 umol, 23.63% yield) as a yellow oil.
Step 5

To a mixture of 5-tert-butoxy-3-(6-chloropyrimidin-4-yl)-1H-indazole (200 mg, 660.59 umol, 1 eq), benzyl (2S)-2-methylpiperazine-1-carboxylate (154.77 mg, 660.59 umol, 1 eq) in DMSO (10 mL) was added Et3N (200.54 mg, 1.98 mmol, 275.84 uL, 3 eq) in one portion and then was stirred at 100° C. for 1 h. TLC (PE:EtOAc=5:1, Rf=0.50) showed the starting material was consumed completely. The mixture was cooled to 20° C., then the residue was poured into water (10 mL). The aqueous phase was extracted with ethyl acetate (3×10 mL). The combined organic phase was washed with brine (2×10 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (20 g, 40 mL/min, 100-200 mesh silica gel, 0-17% (3 min) of Ethyl acetate in Petroleum ether, 17% (5 min) of Ethyl acetate in Petroleum ether) to give benzyl (2S)-4-[6-(5-tert-butoxy-1H-indazol-3-yl)pyrimidin-4-yl]-2-methyl-piperazine-1-carboxylate (230 mg, 450.27 umol, 68.16% yield, 98% purity) as a yellow gum.
Step 7

To a mixture of benzyl (2S)-4-[6-(5-tert-butoxy-1H-indazol-3-yl)pyrimidin-4-yl]-2-methyl-piperazine-1-carboxylate (230 mg, 459.46 umol, 1 eq) in EtOH (5 mL) was added Pd/C (100 mg, 459.46 umol, 10% purity, 1 eq) in one portion at 20° C. under N2. The suspension was degassed under vacuum and purged with H2several times. The mixture was stirred under H2(15 psi) at 20° C. for 1 h. The suspension was filtered through a pad of Celite and the pad was washed with EtOAc (3×50 mL) to give 5-tert-butoxy-3-[6-[(3S)-3-methylpiperazin-1-yl]pyrimidin-4-yl]-1H-indazole (140 mg, 382.03 umol, 83.15% yield) as a yellow gum. The crude product was used for next step.
Step 8

Exemplary Synthesis of Exemplary Compound 17

To a solution of ethyl 2-(4-benzylmorpholin-2-yl)acetate (5 g, 18.99 mmol, 1 eq) in THF (50 mL) was added LiAlH4(1.08 g, 28.48 mmol, 1.5 eq). After addition, the reaction mixture was stirred at 20° C. for 1 h. TLC (PE:EtOAc=5:1, Rf=0.18) showed the reaction was completed. The reaction mixture was quenched with water (5 mL), then 15% sodium hydroxide aqueous solution (5 mL) and water (15 mL) was added. The solid was removed by filtration. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (column height: 40 g, 100-200 mesh silica gel, 0-50% (20 min) of Ethyl acetate in Petroleum ether) to give 2-(4-benzylmorpholin-2-yl)ethanol (2.73 g, 12.34 mmol, 64.97% yield) as a yellow oil.
Step 3

Exemplary Synthesis of Exemplary Compound 18

To a mixture of tert-butyl (2*2)-2-[2-[2-[2-[2-(p-tolylsulfonyloxy)ethoxy]ethoxy]ethoxy]ethyl]morpholine-4-carboxylate (146 mg, 282.05 umol, 1 eq) and 2-(2,6-dioxo-3-piperidyl)-5-hydroxy-isoindoline-1,3-dione (85.08 mg, 310.26 umol, 1.1 eq) in DMF (2 mL) was added K2CO3(77.97 mg, 564.11 umol, 2 eq) in one portion at 20° C. under N2. The mixture was stirred at 70° C. for 2 hours. The mixture was cooled to 25° C. and concentrated in reduced pressure at 25° C. The residue was poured into water (5 mL). The aqueous phase was extracted with ethyl acetate (5 mL*3). The combined organic phase was washed with brine (5 mL*3), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (column height: 12 g, 100-200 mesh silica gel, 0-80% (20 min) of Ethyl acetate in Petroleum ether, 80% (10 min) of Ethyl acetate in Petroleum ether) to give tert-butyl (2*2)-2-[2-[2-[2-[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]oxyethoxy]ethoxy]ethoxy]ethyl]morpholine-4-carboxylate (118 mg, 190.43 umol, 67.51% yield) as a colourless gum.
Step 2

Exemplary Synthesis of Exemplary Compound 19

Exemplary Synthesis of Exemplary Compound 20

To a solution of 2-(2,6-dioxo-3-piperidyl)-5-fluoro-isoindoline-1,3-dione (1.15 g, 4.16 mmol, 1 eq) and tert-butyl piperazine-1-carboxylate (852.97 mg, 4.58 mmol, 1.1 eq) in NMP (10 mL) was added DIEA (1.61 g, 12.49 mmol, 2.18 mL, 3 eq). The sealed tube was heated at 140° C. for 2 hours under microwave. The mixture was combined with batch EB12-30-P1 and diluted with water (50 mL) and extracted with ethyl acetate (2×50 mL). The combined organic phase was washed with saturated brine (2×30 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0 to 50% ethyl acetate in petroleum ether) to afford tert-butyl 4-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]piperazine-1-carboxylate (1.4 g, 3.16 mmol, 76.00% yield) as a yellow solid. Based on EB12-30 (905.08 umol starting material) and EB12-32 (4.16 mmol starting material), the average yield is 62.49%.
Step 3

To a solution of tert-butyl 4-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]piperazine-1-carboxylate (1.2 g, 2.71 mmol, 1 eq) in MeOH (10 mL) was added HCl/dioxane (4 M, 2.00 mL, 2.95 eq). After addition, the reaction solution was stirred at 65° C. for 1 h. The reaction solution was combined with batch EB12-34-P1. The mixture was concentrated under reduced pressure to afford 2-(2,6-dioxopiperidin-3-yl)-5-(piperazin-1-yl)isoindoline-1,3-dione hydrochloride (1.1 g, crude) as a yellow solid. Based on EB12-34 (452.01 umol starting material) and EB12-35 (2.71 mmol starting material), the average yield is 91.04%.
Step 4

A solution of KOH (2.21 g, 39.46 mmol, 2 eq) in ethylene glycol (3.67 g, 59.17 mmol, 3.31 mL, 5 eq) was stirred at 115° C. After the potassium hydroxide dissolved, 2-bromo-1,1-dimethoxy-ethane (2 g, 11.83 mmol, 1.39 mL, 1 eq) was added dropwise over 5 minutes, and the reaction mixture was stirred for 20 hours. TLC (ethyl acetate:petroleum ether=1:1) showed a new spot. The mixture was then allowed to cool to room temperature (20° C.), and the whole was diluted with water (40 mL), then extracted with dichloromethane (3×20 mL). The organic layer was washed with brine (3×20 mL), dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0 to 50% ethyl acetate in petroleum ether) to afford 2-(2,2-dimethoxyethoxy)ethanol (200 mg, 1.33 mmol, 11.25% yield) as a light yellow oil.
Step 5

To a solution of 2-(2,2-dimethoxyethoxy)ethanol (200 mg, 1.33 mmol, 1 eq) and 4-methylbenzenesulfonyl chloride (507.81 mg, 2.66 mmol, 2 eq) in DCM (3 mL) was added TEA (269.53 mg, 2.66 mmol, 370.74 uL, 2 eq). After addition, the reaction solution was stirred at 20° C. for 16 h. TLC (Petroleum ether:Ethyl acetate=1:1) showed starting material was consumed and TLC (Petroleum ether:Ethyl acetate=5:1) showed a new spot. The reaction mixture was diluted with water (10 mL) and extracted with dichloromethane (3×10 mL). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0 to 30% ethyl acetate in petroleum ether) to afford 2-(2,2-dimethoxyethoxy)ethyl 4-methylbenzenesulfonate (350 mg, 1.15 mmol, 86.35% yield) as a light yellow oil.
Step 6

To a solution of 3-[6-[(3S)-4-[2-(2,2-dimethoxyethoxy)ethyl]-3-methyl-piperazin-1-yl]pyrimidin-4-yl]-5-(1-methylcyclopropoxy)-1H-indazole (80 mg, 161.10 umol, 1 eq) in THF (3 mL) was added H2SO4(2 M, 3.22 mL, 40 eq). After addition, the reaction solution was stirred at 70° C. for 1 h. The reaction solution was quenched with saturated NaHCO3(pH=7). The resulting mixture was extracted with ethyl acetate (3×10 mL). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure to afford 2-[2-[(2S)-2-methyl-4-[6-[5-(1-methylcyclopropoxy)-1H-indazol-3-yl]pyrimidin-4-yl]piperazin-1-yl]ethoxy]acetaldehyde (60 mg, 118.53 umol, 73.58% yield, 89% purity) as a yellow solid. The crude product was used directly.
Step 8

Exemplary Synthesis of Exemplary Compound 21

To a solution of 3-[6-[(3R,5S)-4-[2-(2,2-dimethoxyethoxy)ethyl]-3,5-dimethyl-piperazin-1-yl]pyrimidin-4-yl]-5-(1-methylcyclopropoxy)-1H-indazole (110 mg, 215.42 umol, 1 eq) in THF (5 mL) was added H2SO4(2 M, 4.31 mL, 40 eq) in one portion at 20° C. under N2. Then the solution was heated to 70° C. and stirred for 1 h to give yellow solution. TLC (DCM:MeOH=10:1, Rf=0.06) showed the reaction was completed. The solution was cooled to 20° C. The solution as poured into water (5 mL) and NaHCO3to adjusted the pH to 7-8. The aqueous phase was extracted with ethyl acetate (3×10 mL). The combined organic phase was washed with brine (2×10 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give 2-[2-[(2R,6S)-2,6-dimethyl-4-[6-[5-(1-methylcyclopropoxy)-1H-indazol-3-yl]pyrimidin-4-yl]piperazin-1-yl]ethoxy]acetaldehyde (50 mg, 75.72 umol, 35.15% yield, 70.353% purity) as a yellow solid.
Step 3

Exemplary Synthesis of Exemplary Compound 22

To a solution of 5-isopropoxy-3-[6-[(3S)-3-methylpiperazin-1-yl]pyrimidin-4-yl]-1H-indazole (320 mg, 822.85 umol, 1 eq., HCl salt) and 2-(2,2-diethoxyethoxy)ethyl 4-methylbenzenesulfonate (328.23 mg, 987.42 umol, 1.2 eq.) in CH3CN (6 mL) was added KI (1.37 g, 8.23 mmol, 10 eq.) and DIEA (1.06 g, 8.23 mmol, 1.43 mL, 10 eq.). The mixture was stirred at 90° C. for 18 hr. Several new peaks were shown on LC-MS and ˜41% of desired compound was detected. The reaction mixture was diluted with water (20 mL) and extracted with EA (30 mL). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by column chromatography on silica gel (DCM in MeOH=0 to 10%) to afford 3-[6-[(3S)-4-[2-(2,2-diethoxyethoxy)ethyl]-3-methyl-piperazin-1-yl]pyrimidin-4-yl]-5-isopropoxy-1H-indazole (250 mg, 419.40 umol, 50.97% yield, 86% purity) as a yellow oil.
Step 2

To a solution of 3-[6-[(3S)-4-[2-(2,2-diethoxyethoxy)ethyl]-3-methyl-piperazin-1-yl]pyrimidin-4-yl]-5-isopropoxy-1H-indazole (125 mg, 243.83 umol, 1 eq.) in THF (1 mL) was added H2SO4(2 M, 1.25 mL, 10.25 eq.). The mixture was stirred at 70° C. for 1 hr. TLC indicated reactant was consumed and one major new spot with larger polarity was detected. The reaction mixture was diluted with saturated NaHCO3(20 mL) and extracted with EA (30 mL). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-[2-[(2S)-4-[6-(5-isopropoxy-1H-indazol-3-yl) pyrimidin-4-yl]-2-methyl-piperazin-1-yl]ethoxy]acetaldehyde (100 mg, 207.52 umol, 85.11% yield, 91% purity) as a light yellow solid.
Step 3

Exemplary Synthesis of Exemplary Compound 23

To a solution of tert-butyl (3S)-3-methylpiperazine-1-carboxylate (3.5 g, 17.48 mmol, 1 eq) and 4-bromobutan-1-ol (3.34 g, 17.48 mmol, 1 eq) in THF (10 mL) was added K2CO3(7.25 g, 52.43 mmol, 3 eq). Then the mixture was stirred at 60° C. for 16 hours under N2. TLC (Dichloromethane:Methanol=10:1, Rf=0.2) showed the reaction new spot. The reaction mixture was filtered and the filtrate was concentrated. The crude was purified by a flash chromatography on silica (0-10% Methanol in Dichloromethane) to afford tert-butyl (3S)-4-(4-hydroxybutyl)-3-methyl-piperazine-1-carboxylate (4 g, 14.69 mmol, 84.03% yield) as a colorless liquid.
Step 2

A solution of OXALYL CHLORIDE (512.58 mg, 4.04 mmol, 353.51 uL, 1.1 eq) in DCM (10 mL) was cooled to −60° C. under an atmosphere of dry nitrogen. A solution of DMSO (717.13 mg, 9.18 mmol, 717.13 uL, 2.5 eq) in DCM (10 mL) was added dropwise, and the mixture was subsequently stirred for 15 min at −60° C. Next, a solution of tert-butyl (3R)-4-(4-hydroxybutyl)-3-methyl-piperazine-1-carboxylate (1 g, 3.67 mmol, 1 eq) in DCM (10 mL) was added dropwise and the mixture was stirred for 45 min at −60° C. Subsequently, TEA (1.11 g, 11.01 mmol, 1.53 mL, 3 eq) was added, and the mixture was warmed to −60° C. for 1 h. TLC (Dichloromethane:Methanol=10:1, Rf=0.4) showed a new spot. The reaction mixture was filtered and the filtrate was used directly in the next step. tert-butyl (3S)-3-methyl-4-(4-oxobutyl)piperazine-1-carboxylate (990 mg, crude) in DCM solution as Colorless Liquid, which was used directly in the next step.
Step 3

Exemplary Synthesis of Exemplary Compound 24

To a solution of 5-(1-methylcyclopropoxy)-3-[6-[(3S)-3-methylpiperazin-1-yl]pyrimidin-4-yl]-1H-indazole (100 mg, 274.39 umol, 1 eq) and tert-butyl 4-formylpiperidine-1-carboxylate (117.04 mg, 548.78 umol, 2 eq) in DMF (2 mL) was added CH3COOH (823.89 ug, 13.72 umol, 7.85e−1uL, 0.05 eq) and NaOAc (45.02 mg, 548.78 umol, 2 eq) in one portion at 20° C. under N2. The solution was stirred at 20° C. for 5 h. then NaBH3CN (34.49 mg, 548.78 umol, 2 eq) was added and the solution was stirred for 1 h to give pale yellow solution. The residue was poured into water (5 mL) and stirred for 5 min. The aqueous phase was extracted with ethyl acetate (3×10 mL). The combined organic phase was washed with brine (2×5 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (100-200 mesh silica gel, 50-80% of Ethyl acetate in Petroleum ether) to give tert-butyl 4-[[(2S)-2-methyl-4-[6-[5-(1-methylcyclopropoxy)-1H-indazol-3-yl]pyrimidin-4-yl]piperazin-1-yl]methyl]piperidine-1-carboxylate (110 mg, 172.72 umol, 62.95% yield, 88.199% purity) as a yellow oil.
Step 2

To a mixture of tert-butyl 4-[[(2S)-2-methyl-4-[6-[5-(1-methylcyclopropoxy)-1H-indazol-3-yl]pyrimidin-4-yl]piperazin-1-yl]methyl]piperidine-1-carboxylate (110 mg, 195.83 umol, 1 eq) in DCM (5 mL) was added TFA (66.99 mg, 587.48 umol, 43.50 uL, 3 eq) in one portion at 20° C. under N2. The mixture was stirred at 20° C. for 1 h. The residue was poured into NaHCO3(5 mL) to adjust the pH=7-8. The aqueous phase was extracted with ethyl acetate (3×5 mL). The combined organic phase was washed with brine (3×5 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give crude product (150 mg). The crude product was purified by silica gel chromatography (100-200 mesh silica gel, 0-100% of MeOH in EtOAc) to give 5-(1-methylcyclopropoxy)-3-[6-[(3S)-3-methyl-4-(4-piperidylmethyl)piperazin-1-yl]pyrimidin-4-yl]-1H-indazole (100 mg, crude) as a yellow gum.
Step 3

To a mixture of 2-(2,6-dioxo-3-piperidyl)-5-fluoro-isoindoline-1,3-dione (100 mg, 362.03 umol, 1 eq) and 4-piperidylmethanol (83.39 mg, 724.06 umol, 2 eq) in DMSO (2 mL) was added DIEA (140.37 mg, 1.09 mmol, 189.18 uL, 3 eq) in one portion at 20° C. The mixture was stirred at 100° C. for 3 h. TLC (DCM:MeOH=10:1, Rf=0.36) showed the reaction was completed. The mixture was cooled to 20° C. The residue was poured into NaHCO3(10 mL) to adjust pH=7-8. The aqueous phase was extracted with ethyl acetate (3×10 mL). The combined organic phase was washed with brine (2×10 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (100-200 mesh silica gel, 0-10% of MeOH in DCM) to give 2-(2,6-dioxo-3-piperidyl)-5-[4-(hydroxymethyl)-1-piperidyl]isoindoline-1,3-dione (120 mg, 283.18 umol, 78.22% yield, 87.640% purity) as a yellow gum.
Step 4

To a solution of 2-(2,6-dioxo-3-piperidyl)-5-[4-(hydroxymethyl)-1-piperidyl]isoindoline-1,3-dione (120 mg, 323.11 umol, 1 eq) and TEA (98.09 mg, 969.34 umol, 134.92 uL, 3 eq) in DCM (5 mL) was added TsCl (27.35 mg, 387.74 umol, 1.2 eq) in one portion at 0° C. under N2. The mixture was stirred at 20° C. for 20 hours to give yellow solution. TLC (DCM:MeOH=10:1, Rf=0.45) showed the reaction was completed. The residue was poured into water (5 mL). The aqueous phase was extracted with ethyl acetate (3×5 mL). The combined organic phase was washed with brine (2×5 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (100-200 mesh silica gel, 0-50% of Ethyl acetate in Petroleum ether for 5 min, 50-100 of Ethyl acetate in Petroleum ether for 10 min) to give [1-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]-4-piperidyl]methyl 4-methylbenzenesulfonate (160 mg, 228.91 umol, 70.85% yield, 75.194% purity) as a yellow solid.
Step 5

Exemplary Synthesis of Exemplary Compound 25

To a solution of tert-butyl 4-(2-ethoxy-2-oxo-ethoxy)piperidine-1-carboxylate (800 mg, 2.78 mmol, 1 eq) in THF (10 mL) was added LiAlH4(158.50 mg, 4.18 mmol, 1.5 eq) at 0° C. After addition, the reaction mixture was stirred at 20° C. for 2 h. TLC (petroleum ether:ethyl acetate=1:1) showed starting material consumed and a new spot formed. The reaction mixture was quenched by addition of water (0.5 mL), followed by 15% aqueous NaOH (0.5 mL) and water (1.5 mL). The solid was removed by filtration. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0 to 50% ethyl acetate in petroleum ether) to afford tert-butyl 4-(2-hydroxyethoxy)piperidine-1-carboxylate (400 mg, 1.63 mmol, 58.57% yield) as a colorless oil.
Step 3

Exemplary Synthesis of Exemplary Compound 26

To a solution of benzyl (3S)-4-[2-(4-tert-butoxycarbonylpiperazin-1-yl)ethyl]-3-methyl-piperazine-1-carboxylate (460 mg, 1.03 mmol, 1 eq) in MeOH (5 mL) was added Pd/C (200 mg, 2.06 mmol, 10% purity, 2 eq) under N2. The suspension was degassed under vacuum and purged with H2several times. The mixture was stirred under H2(15 psi) at 25° C. for 2 hours. TLC indicated no reactant was remained, and one major new spot with larger polarity was detected. The reaction mixture was filtered and the filter was concentrated to give tert-butyl 4-[2-[(2S)-2-methylpiperazin-1-yl]ethyl]piperazine-1-carboxylate (290 mg, 928.15 umol, 90.11% yield) as a light yellow oil.
Step 3

To a solution of tert-butyl 4-[2-[(2S)-2-methylpiperazin-1-yl]ethyl]piperazine-1-carboxylate (290 mg, 928.15 umol, 1 eq) and 2-[[3-(6-chloropyrimidin-4-yl)-5-isopropoxy-indazol-1-yl]methoxy]ethyl-trimethyl-silane (388.89 mg, 928.15 umol, 1 eq) in DMSO (3 mL) was added DIEA (359.87 mg, 2.78 mmol, 485.00 uL, 3 eq). The mixture was stirred at 100° C. for 2 hr. LC-MS (EB134-185-P1A) showed no reactant 1 was remained. Several new peaks were shown on LC-MS and ˜64% of desired compound was detected. The reaction mixture was diluted with water (20 mL) and extracted with EA (30 mL). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by column chromatography on silica gel (DCM in MeOH=0 to 3%) to give tert-butyl 4-[2-[(2S)-4-[6-[5-isopropoxy-1-(2-trimethylsilylethoxymethyl) indazol-3-yl]pyrimidin-4-yl]-2-methyl-piperazin-1-yl]ethyl]piperazine-1-carboxylate (590 mg, 812.44 umol, 87.53% yield, 95.7% purity) as a brown gum.
Step 4

To a solution of tert-butyl 4-[2-[(2S)-4-[6-[5-isopropoxy-1-(2-trimethylsilylethoxymethyl)indazol-3-yl]pyrimidin-4-yl]-2-methyl-piperazin-1-yl]ethyl]piperazine-1-carboxylate (350 mg, 503.61 umol, 1 eq) in DCM (3 mL) was added TFA (7.19 g, 63.03 mmol, 4.67 mL, 125.15 eq). The mixture was stirred at 25° C. for 16 h. And then NH3·H2O (211.79 mg, 1.51 mmol, 232.74 uL, 25% purity, 3 eq) was added to solution and the mixture was stirred for 2 h. LC-MS (EB134-187-P1B) showed no reactant was remained. Several new peaks were shown on LC-MS and ˜97% of desired compound was detected. The reaction mixture was diluted with water (20 mL) and extracted with EA (30 mL). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give 5-isopropoxy-3-[6-[(3S)-3-methyl-4-(2-piperazin-1-ylethyl)piperazin-1-yl]pyrimidin-4-yl]-1H-indazole (150 mg, 313.17 umol, 62.18% yield, 97% purity) as a yellow gum.
Step 5

Exemplary Synthesis of Exemplary Compound 27

To a solution of tert-butyl 4-(2-chloroethyl)piperazine-1-carboxylate (1 g, 4.02 mmol, 1 eq) and 4-(dimethoxymethyl)piperidine (960.16 mg, 6.03 mmol, 1.5 eq) in EtOH (30 mL) and water (4 mL) was added NaHCO3(1.01 g, 12.06 mmol, 469.07 uL, 3 eq) at 25° C. under N2. Then the reaction mixture was heated to 80° C. and stirred for 5 hr to give a white suspension. TLC (Dichloromethane:methanol=10:1) showed the reaction was completed. The mixture was concentrated under vacuum, the residue was putted into water (30 mL). The aqueous phase was extracted with ethyl acetate (30 mL*3). The combined organic phase was washed with brine (30 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified with silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, Dichloromethane:Methanol=100/1, 5/1) to afford tert-butyl 4-[2-[4-(dimethoxymethyl)-1-piperidyl]ethyl]piperazine-1-carboxylate (900 mg, 2.42 mmol, 60.26% yield) as a yellow oil.
Step 2

To a solution of tert-butyl 4-[2-[4-(dimethoxymethyl)-1-piperidyl]ethyl]piperazine-1-carboxylate (800 mg, 2.15 mmol, 1 eq) in THF (5 mL) was added HCl (2 M, 5 mL, 4.64 eq) at 25° C., then the reaction mixture was stirred at 50° C. for 2 h to give an off-yellow solution. TLC (Dichloromethane:methanol=10:1) showed the reaction was completed. The residue was adjusted to pH=8 with NaHCO3(s), then the mixture was poured into water (25 mL). The aqueous phase was extracted with ethyl acetate (25 mL*2). The combined organic phase was washed with brine (30 mL*2), dried with anhydrous Na2SO4, filtered and concentrated in vacuum to afford tert-butyl 4-[2-(4-formyl-1-piperidyl)ethyl]piperazine-1-carboxylate (420 mg, crude) as an off-yellow oil.
Step 3

To a solution of 2-(2,6-dioxo-3-piperidyl)-5-piperazin-1-yl-isoindoline-1,3-dione (601.45 mg, 1.55 mmol, 1.2 eq, FA) and NaOAc (423.47 mg, 5.16 mmol, 4 eq) in DCM (5 mL) and MeOH (5 mL) at 25° C., then the reaction was stirred at 25° C. for 1 h, then tert-butyl 4-[2-(4-formyl-1-piperidyl)ethyl]piperazine-1-carboxylate (420 mg, 1.29 mmol, 1 eq) was added and stirred for 1 h, then acetic acid (193.75 mg, 3.23 mmol, 184.52 uL, 2.5 eq) and sodium cyanoborohydride (162.20 mg, 2.58 mmol, 2 eq) were stirred at 25° C. for 1 h, then the reaction mixture was stirred at 25° C. for 14 h to give a yellow solution. TLC (Dichloromethane:methanol=10:1) showed there was new spot detected. The residue was poured into ice-water (w/w=1/1) (35 mL). The aqueous phase was extracted with ethyl acetate (35 mL*2). Then the aqueous phase was lyophilized to give a yellow solid. The solid was washed with MeOH/DCM (1/1, 40 mL) to give a yellow suspension. The suspension was filtered and concentrated in vacuum to give yellow oil. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, Dichloromethane:Methanol=100/1, 5/1) to afford tert-butyl 4-[2-[4-[[4-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl] piperazin-1-yl]methyl]-1-piperidyl]ethyl]piperazine-1-carboxylate (320 mg, 490.95 umol, 38.04% yield) as a yellow oil
Step 4

To a solution of tert-butyl 4-[2-[4-[[4-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]piperazin-1-yl]methyl]-1-piperidyl]ethyl]piperazine-1-carboxylate (320 mg, 490.95 umol, 1 eq) in DCM (10 mL) was added TFA (3.08 g, 27.01 mmol, 2 mL, 55.02 eq) at 25° C., then the reaction mixture was stirred at 25° C. for 0.5 h to give a yellow solution. TLC (Dichloromethane:methanol=10:1) showed starting material consumed and a new spot formed. The reaction mixture was concentrate in vacuum to give 2-(2,6-dioxo-3-piperidyl)-5-[4-[[1-(2-piperazin-1-ylethyl)-4-piperidyl]methyl]piperazin-1-yl]isoindoline-1,3-dione (480 mg, 395.33 umol, 80.52% yield, 83% purity, 4TFA) as a yellow solid.
Step 5

Exemplary Synthesis of Exemplary Compound 28

Exemplary Synthesis of Exemplary Compound 29

To a solution of benzyl (3S)-3-methylpiperazine-1-carboxylate (300 mg, 1.28 mmol, 1 eq) and 2-chloroacetaldehyde (753.84 mg, 3.84 mmol, 617.90 uL, 3 eq) in DCM (5 mL) and MeOH (5 mL) was added HOAc (7.69 mg, 128.04 umol, 7.32 uL, 0.1 eq). Then the mixture was stirred at 25° C. for 20 min. Then the NaBH3CN (241.39 mg, 3.84 mmol, 3 eq) was added to the solution and the reaction was stirred at 25° C. for 1 h. TLC (Petroleum ether:Ethyl acetate=1:1, Rf=0.5) was showed the reaction completed. The reaction mixture was poured into H2O (10 mL). The mixture was extracted with ethyl acetate (20 mL*3). The organic phase was washed with brine (20 mL), dried over anhydrous Na2SO4, concentrated in vacuum to give a residue. The residue was purified by silica gel column chromatography (0-100% Ethyl acetate in Petroleum ether) to give benzyl (3S)-4-(2-chloroethyl)-3-methyl-piperazine-1-carboxylate (180 mg, 606.49 umol, 47.37% yield) as a colorless oil.
Step 2

Exemplary Synthesis of Exemplary Compound 30

Exemplary Synthesis of Exemplary Compound 31

A mixture of benzyl (3S)-4-[2-[4-[[4-[2-(2,6-dioxo-3-piperidyl)-1-oxo-isoindolin-5-yl]piperazin-1-yl]methyl]-1-piperidyl]ethyl]-3-methyl-piperazine-1-carboxylate (70 mg, 102.06 umol, 1 eq) and TFA (3 mL) was stirred at 80° C. for 2 h. The reaction solution was concentrated under reduced pressure to give 3-[5-[4-[[1-[2-[(2S)-2-methylpiperazin-1-yl]ethyl]-4-piperidyl]methyl]piperazin-1-yl]-1-oxo-isoindolin-2-yl]piperidine-2,6-dione (110 mg, crude, TFA) as a brown gum. The crude product was used directly.
Step 3

Exemplary Synthesis of Exemplary Compound 32

A mixture of NaH (2.31 g, 57.73 mmol, 60% purity, 2 eq) in DMF (20 mL) was added tert-butyl 3-hydroxyazetidine-1-carboxylate (5 g, 28.87 mmol, 1 eq) in DMF (20 mL) at 0° C. The mixture was stirred at 25° C. for 0.5 hours and then 2-(2-bromoethoxy)tetrahydropyran (6.64 g, 31.75 mmol, 4.81 mL, 1.1 eq) in DMF (20 mL) was added into reaction mixture at 0° C. The mixture was stirred at 20° C. for 16 hours to give a brown mixture. TLC (DCM:MeOH=10:1, Rf=0.56) showed there was a new spot. The residue was poured into water (50 mL). The aqueous phase was extracted with ethyl acetate (50 mL*3). The combined organic phase was washed with brine (50 mL*2), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (45 g, 30 mL/min, 0-50% (15 min) of Ethyl acetate in Petroleum ether) to give tert-butyl 3-(2-tetrahydropyran-2-yloxyethoxy)azetidine-1-carboxylate (6.6 g, 21.90 mmol, 75.86% yield) as a yellow oil.
Step 3

To a mixture of 2-(2,6-dioxo-3-piperidyl)-5-hydroxy-isoindoline-1,3-dione (1.55 g, 5.65 mmol, 1 eq) in DMF (20 mL) was added Na2CO3(1.20 g, 11.31 mmol, 2 eq) and tert-butyl 3-[2-(p-tolylsulfonyloxy)ethoxy]azetidine-1-carboxylate (2.1 g, 5.65 mmol, 1 eq). The mixture was stirred at 70° C. for 16 hours to give a yellow mixture. The reaction mixture was cooled to room temperature and added into aq. HCl (100 mL, 2%, v/v) at 0° C., and the resulting mixture was extracted with EtOAc (30 mL*3). The combined extracts were washed with water (30 mL), brine (30 mL), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (DCM:MeOH=10:1, Rf=0.31, 80 g, 0-50% (30 min) of Ethyl acetate in Petroleum ether, 50% (60 min) of Ethyl acetate in Petroleum ether) to give tert-butyl 3-[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]oxyethoxy]azetidine-1-carboxylate (1.8 g, 3.80 mmol, 67.24% yield) as a white gum.
Step 6

To a mixture of tert-butyl 3-[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]oxyethoxy]azetidine-1-carboxylate (1.2 g, 2.53 mmol, 1 eq) in DCM (10 mL) was added TFA (866.94 mg, 7.60 mmol, 562.95 uL, 3 eq) in one portion at 20° C. under N2. The mixture was stirred at 20° C. for 30 min. TLC showed the reaction was completed. The mixture was concentrated under reduced pressure to give 5-[2-(azetidin-3-yloxy)ethoxy]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (2 g, crude) as a colourless gum.
Step 7

Exemplary Synthesis of Exemplary Compound 33

To a solution of tert-butyl 4-(2-ethoxy-2-oxo-ethoxy)piperidine-1-carboxylate (800 mg, 2.78 mmol, 1 eq) in THF (10 mL) was added LiAlH4(158.50 mg, 4.18 mmol, 1.5 eq) at 0° C. After addition, the reaction mixture was stirred at 20° C. for 2 h. TLC (petroleum ether:ethyl acetate=1:1) showed starting material consumed and a new spot formed. The reaction mixture was quenched by addition of water (0.5 mL), followed by 15% aqueous NaOH (0.5 mL) and water (1.5 mL). The solid was removed by filtration. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0 to 50% ethyl acetate in petroleum ether) to afford tert-butyl 4-(2-hydroxyethoxy)piperidine-1-carboxylate (400 mg, 1.63 mmol, 58.57% yield) as a colorless oil.
Step 3

Exemplary Synthesis of Exemplary Compound 34

but-3-yn-1-ol (1 g, 14.27 mmol, 1.08 mL, 1 eq) and 2-bromo-1,1-diethoxy-ethane (2.81 g, 14.27 mmol, 2.15 mL, 1 eq) were dissolved in dry DMF (10 mL) and then NaH (684.77 mg, 17.12 mmol, 60% purity, 1.2 eq) was added in small portions at 0° C. Then the mixture was stirred at 0° C. for 3 h. TLC (Petroleum ether:Ethyl acetate=5:1, Rf=0.2) showed the reaction new spot. The reaction was quenched by aq.NH4Cl (10 mL) solution and extracted with ethyl acetate (3*20 mL). The combined organic phases were washed with water, dried with Na2SO4, concentrated in vacuum to give a residue. The residue was purified by silica gel column chromatography (0-20% ethyl acetate in Petroleum ether) to give 4-(2,2-diethoxyethoxy)but-1-yne (600 mg, 3.22 mmol, 22.58% yield) as a colorless oil.
Step 2

To a flame-dried three-neck 100 mL round-bottomed flask equipped with an argon inlet adapter, a septum, and a stir bar was added 4-(2,2-diethoxyethoxy)but-1-yne (600 mg, 3.22 mmol, 1 eq) and THF (10 mL) via syringe. The solution was cooled at −78° C. (bath temperature) in a dry ice/acetone bath, and n-BuLi (2.5 M, 1.55 mL, 1.2 eq) was added dropwise via syringe turning the reaction brown. The reaction was stirred at −78° C. for 30 min, and DMF (470.95 mg, 6.44 mmol, 495.73 uL, 2 eq) was added dropwise via syringe turning the reaction colorless. The reaction was stirred at −78° C. for 30 min, and was then warmed to 25° C. and stirred for 2 h. TLC (Petroleum ether:Ethyl acetate=2:1, Rf=0.1) showed a new spot. The reaction was added to a cold solution of ethyl acetate (10 mL) and 10 percent KH2PO4(10 mL) and stirred for 30 min. The aqueous layer was separated and the organic layer was washed with brine (20 mL), dried over magnesium sulfate, gravity filtered, and concentrated under reduced pressure to afford 5-(2,2-diethoxyethoxy)pent-2-ynal (350 mg, crude) as a yellow oil.
Step 3

To a solution of 5-[4-[5-(2,2-diethoxyethoxy)pent-2-ynyl]piperazin-1-yl]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (95 mg, 175.73 umol, 1 eq) in THF (2 mL) was added H2SO4(2 M, 2 mL, 86.50 eq). Then the mixture was stirred at 65° C. for 1 hr. TLC (Dichloromethane:Methanol=10:1, Rf=0.37) showed starting material consumed. The reaction mixture was poured into H2O (2 mL) and basified with aqueous NaHCO3till PH=8. The mixture was extracted with ethyl acetate (15 mL*5), dried over anhydrous Na2SO4, concentrated in vacuum to give 2-[5-[4-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]piperazin-1-yl]pent-3-ynoxy]acetaldehyde (80 mg, crude) as a yellow solid.
Step 5

Exemplary Synthesis of Exemplary Compound 35

Tert-butyl 6-[2-[(2S)-4-[6-(5-isopropoxy-1H-indazol-3-yl)pyrimidin-4-yl]-2-methyl-piperazin-1-yl]ethyl]-2,6-diazaspiro[3.3]heptane-2-carboxylate (124 mg, 215.00 umol, 1 eq) was dissolved in DCM (3 mL) and TFA (3.08 g, 27.01 mmol, 2 mL, 125.63 eq). The reaction was stirred at 25° C. for 1 hr. TLC (Dichloromethane:Methanol=10:1, Rf=0.01) indicated that the reaction was complete. The reaction mixture was concentrated in vacuum to give 3-[6-[(3S)-4-[2-(2,6-diazaspiro[3.3]heptan-2-yl)ethyl]-3-methyl-piperazin-1-yl]pyrimidin-4-yl]-5-isopropoxy-1H-indazole (126 mg, crude, TFA) as a yellow gum. The crude product was used for next step directly.
Step 4

Exemplary Synthesis of Exemplary Compound 36

To a mixture of 3-[tert-butoxycarbonyl(methyl)amino]propyl 4-methylbenzenesulfonate (670 mg, 1.95 mmol, 1 eq) and 2-(2,6-dioxo-3-piperidyl)-5-piperazin-1-yl-isoindoline-1,3-dione (890.32 mg, 2.60 mmol, 1.33 eq) in MeCN (5 mL) was added KI (1.62 g, 9.75 mmol, 5 eq) and DIPEA (1.26 g, 9.75 mmol, 1.70 mL, 5 eq) in one portion at 20° C. under N2. The mixture was stirred at 80° C. for 16 hours. The mixture was cooled to 20° C. and concentrated under reduced pressure at 20° C. The residue was poured into water (10 mL). The aqueous phase was extracted with ethyl acetate (10 mL*3). The combined organic phase was washed with brine (10 mL*3), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (column height: 40 g, 100-200 mesh silica gel, 0-100% of Ethyl acetate in Petroleum ether) to give tert-butyl N-[3-[4-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]piperazin-1-yl]propyl]-N-methyl-carbamate (1 g, crude) as a yellow solid.
Step 3

Exemplary Synthesis of Exemplary Compound 37

To a mixture of tert-butyl N-(2-hydroxyethyl)-N-methyl-carbamate (200 mg, 1.14 mmol, 1 eq) in DCM (5 mL) was added DMP (968.22 mg, 2.28 mmol, 706.73 uL, 2 eq) in one portion at 20° C. under N2. The mixture was stirred at 20° C. for 16 h to give white suspension. TLC (Ethyl acetate:Petroleum ether=10:1) showed the starting material was consumed completely. The residue was poured into NaHCO3to adjust the pH=7-8, and Na2SO3(10 mL) was added. The aqueous phase was extracted with DCM (5 mL*3). The combined organic phase was washed with brine (5 mL*2), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (column height: 12 g, 100-200 mesh silica gel, 0-20% (10 min) of Ethyl acetate in Petroleum ether, 20% (5 min) of Ethyl acetate in Petroleum ether) to give tert-butyl N-methyl-N-(2-oxoethyl)carbamate (150 mg, 866.01 umol, 75.87% yield) as a colourless oil.
Step 2

To a mixture of tert-butyl N-[2-[4-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]piperazin-1-yl]ethyl]-N-methyl-carbamate (180 mg, 360.32 umol, 1 eq) in DCM (5 mL) was added TFA (123.25 mg, 1.08 mmol, 80.03 uL, 3 eq) in one portion at 25° C. under N2. The mixture was stirred at 25° C. for 30 min to give yellow solution. The solution was concentrated in vacuum to give 2-(2,6-dioxo-3-piperidyl)-5-[4-[2-(methylamino)ethyl]piperazin-1-yl]isoindoline-1,3-dione (200 mg, crude, TFA) as a yellow gum.
Step 4

To a solution of 2-(2,6-dioxo-3-piperidyl)-5-[1-[2-(methylamino)ethyl]-4-piperidyl]isoindoline-1,3-dione (144.04 mg, 361.51 umol, 1 eq) and 3-[6-[(3S)-4-(2-chloroethyl)-3-methyl-piperazin-1-yl]pyrimidin-4-yl]-5-isopropoxy-1H-indazole (165.00 mg, 397.66 umol, 1.1 eq) in CH3CN (5 mL) was added DIPEA (140.16 mg, 1.08 mmol, 188.90 uL, 3 eq). The mixture was stirred at 80° C. for 16 hr to give yellow suspension. The mixture was cooled to 20° C. and concentrated in reduced pressure at 20° C. The residue was poured into water (5 mL). The aqueous phase was extracted with ethyl acetate (5 mL*3). The combined organic phase was washed with brine (5 mL*2), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The crude product was purified by reversed-phase HPLC (column: Phenomenex Luna C18 100*30 mm*5 um; mobile phase: [water (0.225% FA)-ACN]; B %: 10%-40%, 9 min) to give crude product (16 mg). The crude was purified by prep-TLC (DCM:MeOH=10:1, Rf=0.51) to give 2-(2,6-dioxo-3-piperidyl)-5-[4-[2-[2-[(2S)-4-[6-(5-isopropoxy-1H-indazol-3-yl)pyrimidin-4-yl]-2-methyl-piperazin-1-yl]ethyl-methyl-amino]ethyl]piperazin-1-yl]isoindoline-1,3-dione (10.4 mg, 13.18 umol, 3.65% yield, 98.6% purity) as a yellow solid.

Exemplary Synthesis of Exemplary Compound 38

To a solution of 2-(2,6-dioxo-3-piperidyl)-5-[4-[3-(methylamino)propyl]piperazin-1-yl]isoindoline-1,3-dione (140.34 mg, 339.41 umol, 1 eq) and benzyl (3S)-4-(3-chloropropyl)-3-methyl-piperazine-1-carboxylate (105.49 mg, 339.41 umol, 1 eq) in MeCN (5 mL) was added DIPEA (131.60 mg, 1.02 mmol, 177.35 uL, 3 eq). The mixture was stirred at 80° C. for 16 hr to give yellow suspension. The mixture was cooled to 20° C. and concentrated in reduced pressure at 20° C. The residue was poured into water (5 mL). The aqueous phase was extracted with ethyl acetate (5 mL*3). The combined organic phase was washed with brine (5 mL*2), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The crude product was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, 0-100% of DCM in MeOH) to give benzyl (3S)-4-[3-[3-[4-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]piperazin-1-yl]propyl-methyl-amino]propyl]-3-methyl-piperazine-1-carboxylate (200 mg, crude) as a yellow solid.
Step 3

To a solution of benzyl (3S)-4-[3-[3-[4-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]piperazin-1-yl]propyl-methyl-amino]propyl]-3-methyl-piperazine-1-carboxylate (200 mg, 290.77 umol, 1 eq) in TFA (33.15 mg, 290.77 umol, 21.53 uL, 1 eq). Then the reaction mixture was stirred at 70° C. for 1 hr to give yellow solution. The reaction mixture was poured into DCM (20 mL), concentrated in rotary evaporator to give 2-(2,6-dioxo-3-piperidyl)-5-[4-[3-[methyl-[3-[(2S)-2-methylpiperazin-1-yl]propyl]amino]propyl]piperazin-1-yl]isoindoline-1,3-dione (200 mg, crude, TFA) as a yellow gum.
Step 4

Exemplary Synthesis of Exemplary Compound 39

To a solution of tert-butyl 4-(2-chloroethyl)piperazine-1-carboxylate (200 mg, 804.02 umol, 1 eq) in DCM (2 mL) was added TFA (3.08 g, 27.01 mmol, 2 mL, 33.60 eq) and the mixture was stirred at 20° C. for 1 hr. TLC (Dichloromethane:Methanol=10:1, Rf=0.02) showed no starting material and a new spot. The residue was concentrated under reduced pressure to give 1-(2-chloroethyl)piperazine (119 mg, crude, TFA) as a colorless oil.
Step 2
A solution of 1-(2-chloroethyl)piperazine (119 mg, 800.63 umol, 1 eq) and 1-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]piperidine-4-carbaldehyde (295.73 mg, 800.63 umol, 1 eq) in HOAC (1 mL) and MeOH (10 mL) was stirred at 20° C. for 20 min, then borane:2-methylpyridine (171.27 mg, 1.60 mmol, 2 eq) was added. Then the mixture was stirred at 30° C. for 16 h under N2. TLC (Dichloromethane:Methanol=10:1, Rf=0.3) showed no start material and a new spot. The residue was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (0 to 25% Dichloromethane in Methanol) to give 5-[4-[[4-(2-chloroethyl)piperazin-1-yl]methyl]-1-piperidyl]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (390 mg, 769.12 umol, 96.06% yield, 99% purity) as a yellow solid.
Step 3
To a mixture of 1H-indazol-5-ol (10 g, 74.55 mmol, 1 eq) in DMF (100 mL) was added Cs2CO3 (36.44 g, 111.83 mmol, 1.5 eq) and 2-iodopropane (19.01 g, 111.83 mmol, 11.18 mL, 1.5 eq). The mixture was stirred at 20° C. for 16 hours to give a brown mixture. LCMS showed the reaction was completed, and the desired MS value was in main peak. TLC (Petroleum ether:Ethyl acetate=2:1, UV=254 nm, Plate1) showed new spots. The mixture was filtered and the filter cake was washed with EtOAc (50 mL) and then 150 mL saturated NH4Cl (aq.) was added into the filtrate. The resulting mixture was extracted with EtOAc (50 mL*3), and the combined extracts were washed with saturated NH4Cl (50 mL*3), brine (50 mL), dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (0-20% (10 min) of Ethyl acetate in Petroleum ether, 20% (10 min) of Ethyl acetate in Petroleum ether) to give 5-isopropoxy-1H-indazole (7.6 g, 43.13 mmol, 57.85% yield) as a yellow solid.
Step 4
To a mixture of 5-isopropoxy-1H-indazole (7.6 g, 43.13 mmol, 1 eq) in THF (100 mL) was added N-cyclohexyl-N-methyl-cyclohexanamine (25.27 g, 129.39 mmol, 27.44 mL, 3 eq) and SEM-Cl (14.38 g, 86.26 mmol, 15.27 mL, 2 eq) in one portion at 20° C. The mixture was stirred at 20° C. for 2 hours to give orange suspension. TLC showed the reaction was completed. The residue was poured into water (50 mL). The aqueous phase was extracted with ethyl acetate (50 mL*3). The combined organic phase was washed with brine (50 mL*2), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (100-200 mesh silica gel, 0-3% (20 min) of ethyl acetate in Petroleum ether, 3-10% (10 min) of ethyl acetate in Petroleum ether) to give 2-[(5-isopropoxyindazol-2-yl)methoxy]ethyl-trimethyl-silane (11.5 g, 36.77 mmol, 85.26% yield, 98% purity) as a yellow oil.
Step 5
To a mixture of 2-[(5-isopropoxyindazol-2-yl)methoxy]ethyl-trimethyl-silane (11 g, 35.89 mmol, 1 eq) in THF (60 mL) was dropwise added n-BuLi (2.5 M, 15.79 mL, 1.1 eq) at −70° C. under N2. The mixture was then stirred at −20° C. for 5 minutes, and a solution of ZnCl2 (2 M, 26.92 mL, 1.5 eq) was dropwise added at −70° C. The mixture was stirred for 10 min at −40° C. A mixture of 4,6-dichloropyrimidine (5.35 g, 35.89 mmol, 1 eq) and Pd(PPh3)4 (2.07 g, 1.79 mmol, 0.05 eq) in THF (10 mL) was stirred at 20° C. for 30 minutes and was added to that solution. The cold bath was removed, and the mixture was stirred at 20° C. for 10 h to give yellow solution. TLC (Petroleum ether:Ethyl acetate=3:1, Rf=0.83) and LCMS showed the reaction was completed. The residue was poured into water (60 mL). The aqueous phase was extracted with ethyl acetate (60 mL*3). The combined organic phase was washed with brine (20 mL*2), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (100-200 mesh silica gel, 0-5% (30 minutes) of Ethyl acetate in Petroleum ether, 5% (60 min) of Ethyl acetate in Petroleum ether) to give 2-[[3-(6-chloropyrimidin-4-yl)-5-isopropoxy-indazol-2-yl]methoxy]ethyl-trimethyl-silane (8.5 g, 20.29 mmol, 56.52% yield) as a yellow oil
Step 6
To a mixture of 2-[[3-(6-chloropyrimidin-4-yl)-5-isopropoxy-indazol-2-yl]methoxy]ethyl-trimethyl-silane (2 g, 4.77 mmol, 1 eq), tert-butyl piperazine-1-carboxylate (1.07 g, 5.73 mmol, 1.2 eq) in DMSO (10 mL) was added Et3N (1.45 g, 14.32 mmol, 1.99 mL, 3 eq) in one portion and then was stirred at 100° C. for 1 h. TLC showed the starting material was consumed completely. The mixture was cooled to 20° C., the residue was poured into water (10 mL). The aqueous phase was extracted with ethyl acetate (10 mL*3). The combined organic phase was washed with brine (10 mL*2), dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give tert-butyl 4-[6-[5-isopropoxy-2-(2-trimethylsilylethoxymethyl)indazol-3-yl]pyrimidin-4-yl]piperazine-1-carboxylate (2.4 g, 4.01 mmol, 83.98% yield, 95% purity) as a yellow oil.
Step 7
To a mixture of tert-butyl 4-[6-[5-isopropoxy-2-(2-trimethylsilylethoxymethyl)indazol-3-yl]pyrimidin-4-yl]piperazine-1-carboxylate (2.4 g, 4.22 mmol, 1 eq) in MeOH (10 mL) was added HCl(g)/dioxane (4 M, 5.27 mL, 5 eq) in one portion at 20° C. The mixture was stirred at 65° C. for 0.5 h to give yellow mixture. TLC (EtOAc, Rf=0.07) showed the reaction was completed. The mixture was cooled to 20° C. The residue was poured into NaHCO3 (20 mL) to adjust pH=7-8. The aqueous phase was extracted with ethyl acetate (20 mL*3). The combined organic phase was washed with brine (20 mL*2), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (100-200 mesh silica gel, 0-25% of MeOH in DCM) to give 5-isopropoxy-3-(6-piperazin-1-ylpyrimidin-4-yl)-1H-indazole (1.6 g, crude) as a yellow gum.
Step 8
To a solution of 5-isopropoxy-3-(6-piperazin-1-ylpyrimidin-4-yl)-1H-indazole (100 mg, 295.50 umol, 1 eq) and 5-[4-[[4-(2-chloroethyl)piperazin-1-yl]methyl]-1-piperidyl]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (100 mg, 199.20 umol, 6.74e-1 eq) and DIEA (190.95 mg, 1.48 mmol, 257.35 uL, 5 eq) and KI (245.27 mg, 1.48 mmol, 5 eq) in MeCN (10 mL). Then the mixture was stirred at 100° C. for 16 hr under N2. LCMS showed desired product. The residue was diluted with H2O (20 mL) extracted with ethyl acetate (20 mL×3). The combined organic layers were washed with brine (15 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: 3_Phenomenex Luna C18 75*30 mm*3 um; mobile phase: [water (0.225% FA)-ACN]; B %: 0%-30%, 35 min) to afford 2-(2,6-dioxo-3-piperidyl)-5-[4-[[4-[2-[4-[6-(5-isopropoxy-1H-indazol-3-yl)pyrimidin-4-yl]piperazin-1-yl]ethyl]piperazin-1-yl]methyl]-1-piperidyl]isoindoline-1,3-dione (27.6 mg, 34.21 umol, 11.58% yield, 99.66% purity) as a yellow solid.

Exemplary Synthesis of Exemplary Compound 40

To a solution of tert-butyl 4-(2-bromoacetyl)piperidine-1-carboxylate (695.05 mg, 2.27 mmol, 1 eq) in MeCN (10 mL) was stirred at 20° C. for 20 min. Then the mixture was added benzyl piperazine-1-carboxylate (500 mg, 2.27 mmol, 438.60 uL, 1 eq) and stirred at 20° C. for 16 hr under N2. TLC (Dichloromethane:Methanol=10:1, Rf=0.6) showed no start material and a new spot. The residue was diluted with H2O (30 mL) extracted with ethyl acetate (50 mL×3). The combined organic layers were washed with brine (45 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (0 to 10% Dichloromethane in Methanol) to give benzyl 4-[2-(1-tert-butoxycarbonyl-4-piperidyl)-2-oxo-ethyl]piperazine-1-carboxylate (950 mg, 1.58 mmol, 69.51% yield, 74% purity) as a yellow gum.
Step 2
To a solution of benzyl 4-[2-(1-tert-butoxycarbonyl-4-piperidyl)-2-oxo-ethyl]piperazine-1-carboxylate (920 mg, 2.06 mmol, 1 eq) in DCM (30 mL) was stirred at 0° C. for 20 min. Then the mixture was added DAST (11.65 g, 72.27 mmol, 9.55 mL, 35 eq) and stirred at 20° C. for 2 hr under N2. TLC (Dichloromethane:Methanol=10:1, Rf=0.5) showed no start material and a new spot. The reaction was cooled to 0° C. and quenched with aqueous NaHCO3 (90 mL) extracted with ethyl acetate (50 mL×2). The combined organic layers were washed with brine (45 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (0 to 10% Dichloromethane in Methanol) to give benzyl 4-[2-(1-tert-butoxycarbonyl-4-piperidyl)-2,2-difluoro-ethyl]piperazine-1-carboxylate (330 mg, 515.24 umol, 24.95% yield, 73% purity) as a yellow gum.
Step 3
To a solution of benzyl 4-[2-(1-tert-butoxycarbonyl-4-piperidyl)-2,2-difluoro-ethyl]piperazine-1-carboxylate (100 mg, 213.88 umol, 1 eq) in DCM (2 mL) was added TFA (2.31 g, 20.26 mmol, 1.5 mL, 94.72 eq) and stirred at 20° C. for 1 hr. TLC (Dichloromethane:Methanol=10:1, Rf=0.01) showed no start material and a new spot. The residue was concentrated under reduced pressure to give benzyl 4-[2,2-difluoro-2-(4-piperidyl)ethyl]piperazine-1-carboxylate (78 mg, crude) as a yellow gum.
Step 4
To a solution of benzyl 4-[2,2-difluoro-2-(4-piperidyl)ethyl]piperazine-1-carboxylate (78 mg, 212.28 umol, 1 eq) and 1-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]piperidine-4-carbaldehyde (78.41 mg, 212.28 umol, 1 eq) in HOAC (1 mL) and MeOH (10 mL) was stirred at 20° C. for 20 min, then was added borane; 2-methylpyridine (45.41 mg, 424.57 umol, 2 eq). Then the mixture was stirred at 25° C. for 16 h under N2. TLC (Dichloromethane:Methanol=10:1, Rf=0.5) showed no start material and a new spot. The residue was concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (0 to 10% Dichloromethane in Methanol) to give benzyl 4-[2-[1-[[1-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]-4-piperidyl]methyl]-4-piperidyl]-2,2-difluoro-ethyl]piperazine-1-carboxylate (150 mg, 167.52 umol, 78.91% yield, 80.5% purity) as a yellow solid.
Step 5
To a solution of benzyl 4-[2-[1-[[1-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]-4-piperidyl]methyl]-4-piperidyl]-2,2-difluoro-ethyl]piperazine-1-carboxylate (150 mg, 208.10 umol, 1 eq) was added TFA (3.08 g, 27.01 mmol, 2 mL, 129.81 eq) stirred at 70° C. for 4 hr. TLC (Dichloromethane:Methanol=10:1, Rf=0.01) showed no start material and a new spot. The residue was concentrated under reduced pressure to give 5-[4-[[4-(1,1-difluoro-2-piperazin-1-yl-ethyl)-1-piperidyl]methyl]-1-piperidyl]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (140 mg, crude, TFA) as a yellow gum.
Step 6
To a solution of 5-[4-[[4-(1,1-difluoro-2-piperazin-1-yl-ethyl)-1-piperidyl]methyl]-1-piperidyl]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (140 mg, 199.80 umol, 1 eq, TFA) and 3-(6-chloropyrimidin-4-yl)-5-isopropoxy-1H-indazole (57.69 mg, 199.80 umol, 1 eq) in DMSO (5 mL) and added DIEA (258.22 mg, 2.00 mmol, 348.01 uL, 10 eq). Then the mixture was stirred at 80° C. for 16 hr under N2. LCMS showed desired product. The residue was diluted with H2O (20 mL) extracted with ethyl acetate (20 mL×3). The combined organic layers were washed with brine (15 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: 3_Phenomenex Luna C18 75*30 mm*3 um; mobile phase: [water (0.225% FA)-ACN]; B %: 10%-40%, 35 min) to afford 5-[4-[[4-[1,1-difluoro-2-[4-[6-(5-isopropoxy-1H-indazol-3-yl)pyrimidin-4-yl]piperazin-1-yl]ethyl]-1-piperidyl]methyl]-1-piperidyl]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (33.7 mg, 39.95 umol, 20.00% yield, 99.46% purity) as a yellow solid.

Exemplary Synthesis of Exemplary Compound 41

To a mixture of 1-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]piperidine-4-carbaldehyde (200 mg, 541.46 umol, 1 eq) and 4-(2,2-dimethoxyethyl)piperidine (93.81 mg, 541.46 umol, 1 eq) in MeOH (10 mL) was added borane; 2-methylpyridine (115.83 mg, 1.08 mmol, 2 eq) and CH3COOH (1 mL) in one portion at 20° C. under N2. The mixture was stirred at 20° C. for 2 hours to give yellow solution. LCMS showed there was desired MS. The residue was poured into saturated NaHCO3 to adjusted the pH=7-8. The aqueous phase was extracted with ethyl acetate (10 mL*3). The combined organic phase was washed with brine (10 mL*2), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (12 g, 0-100% (10 min) of Ethyl acetate in Petroleum ether, 1-10% (5 min) of Methanol in Dichloromethane) to give 5-[4-[[4-(2,2-dimethoxyethyl)-1-piperidyl]methyl]-1-piperidyl]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (210 mg, 338.95 umol, 62.60% yield, 85% purity) as a yellow gum.
Step 2
To a solution of 5-[4-[[4-(2,2-dimethoxyethyl)-1-piperidyl]methyl]-1-piperidyl]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (100 mg, 189.89 umol, 1 eq) in THF (5 mL) was added HCl (2 M, 10.22 mL, 107.60 eq) in one portion at 20° C. under N2. Then the solution was heated to 70° C. and stirred for 1 h to give yellow solution. TLC (DCM:MeOH=10:1, Rf=0.06) showed the reaction was completed. The solution was cooled to 20° C. The solution was poured onto water (5 mL) and NaHCO3 to adjusted the pH=7-8. The aqueous phase was extracted with ethyl acetate (10 mL*3). The combined organic phase was washed with brine (10 mL*2), dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give 2-[1-[[1-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]-4-piperidyl]methyl]-4-piperidyl]acetaldehyde (83 mg, 162.35 umol, 85.50% yield, 94% purity) as a yellow solid.
Step 3
To a mixture of 2-[1-[[1-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]-4-piperidyl]methyl]-4-piperidyl]acetaldehyde (83 mg, 172.72 umol, 1 eq) and 5-isopropoxy-3-(6-piperazin-1-ylpyrimidin-4-yl)-1H-indazole (58.45 mg, 172.72 umol, 1 eq) in MeOH (10 mL) was added CH3COOH (10.37 mg, 172.72 umol, 9.88 uL, 1 eq) and borane; 2-methylpyridine (36.95 mg, 345.43 umol, 2 eq) in one portion at 20° C. under N2. The mixture was stirred at 30° C. for 16 h. LCMS showed there was desired MS. The residue was poured into water (10 mL). The aqueous phase was extracted with ethyl acetate (10 mL*3). The combined organic phase was washed with brine (10 mL*2), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The crude product was purified by reversed-phase HPLC (Column: 3_Phenomenex Luna C18 75*30 mm*3 um; Condition: water (0.225% FA)-ACN; Begin B: 0; End B: 30; FlowRate: 25 mL/min; Gradient Time: 35 min; 100% B Hold Time: 3 min) to give 2-(2,6-dioxo-3-piperidyl)-5-[4-[[4-[2-[4-[6-(5-isopropoxy-1H-indazol-3-yl)pyrimidin-4-yl]piperazin-1-yl]ethyl]-1-piperidyl]methyl]-1-piperidyl]isoindoline-1,3-dione (25.5 mg, 30.77 umol, 17.82% yield, 96.9% purity) as a yellow gum.

Exemplary Synthesis of Exemplary Compound 42

A solution of 2-[(3R)-1-tert-butoxycarbonylpyrrolidin-3-yl]acetic acid (500 mg, 2.18 mmol, 1 eq) in THF (10 mL) was cooled to −10° C. borane; tetrahydrofuran (1 M, 2.62 mL, 1.2 eq) was added slowly to the flask while maintaining the temperature lower than 0° C. The solution was warmed to 20° C. and stirred for 1 h. TLC (PE:EtOAc=1:1) showed a new spot. The solution was cooled to 0° C., and a 15% sodium hydroxide solution (10 mL) was added drop-wise over a 5 minute period to control gas evolution. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (3×20 mL). The organic layer was washed with brine (30 mL), dried over sodium sulfate and concentrated under reduced pressure to afford tert-butyl (3R)-3-(2-hydroxyethyl)pyrrolidine-1-carboxylate (395 mg, 1.83 mmol, 84.13% yield) as a colorless oil.
Step 2
To a solution of tert-butyl (3R)-3-(2-hydroxyethyl)pyrrolidine-1-carboxylate (395 mg, 1.83 mmol, 1 eq) in DCM (5 mL) was added TosCl (699.58 mg, 3.67 mmol, 2 eq) and TEA (371.31 mg, 3.67 mmol, 510.75 uL, 2 eq). After addition, the reaction solution was stirred at 20° C. for 12 h. TLC (PE:EtOAc=1:1) showed several new spots. The reaction was diluted with water (10 mL) and extracted with dichloromethane (3×10 mL). The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0 to 30% ethyl acetate in petroleum ether) to afford tert-butyl (3R)-3-[2-(p-tolylsulfonyloxy)ethyl]pyrrolidine-1-carboxylate (600 mg, 1.58 mmol, 86.39% yield, 97.6% purity) as a light yellow oil.
Step 3
To a solution of tert-butyl (3R)-3-[2-(p-tolylsulfonyloxy)ethyl]pyrrolidine-1-carboxylate (300 mg, 811.96 umol, 1 eq) and 5-isopropoxy-3-(6-piperazin-1-ylpyrimidin-4-yl)-1H-indazole (274.77 mg, 811.96 umol, 1 eq) in CH3CN (5 mL) was added KI (269.57 mg, 1.62 mmol, 2 eq) and DIEA (209.88 mg, 1.62 mmol, 282.86 uL, 2 eq). After addition, the reaction mixture was stirred at 95° C. for 4 h. LCMS showed desired MS. After cooling, the reaction mixture was diluted with ethyl acetate (30 mL) and washed with water (20 mL). The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0 to 10% methanol in dichloromethane) to afford tert-butyl (3S)-3-[2-[4-[6-(5-isopropoxy-1H-indazol-3-yl)pyrimidin-4-yl]piperazin-1-yl]ethyl]pyrrolidine-1-carboxylate (210 mg, 376.62 umol, 46.38% yield, 96.07% purity) as a yellow solid.
Step 4
To a solution of tert-butyl (3S)-3-[2-[4-[6-(5-isopropoxy-1H-indazol-3-yl)pyrimidin-4-yl]piperazin-1-yl]ethyl]pyrrolidine-1-carboxylate (210 mg, 392.03 umol, 1 eq) in DCM (5 mL) was added TFA (1.54 g, 13.51 mmol, 1 mL, 34.45 eq). After addition, the reaction mixture was stirred at 20° C. for 30 min. LCMS showed desired MS. The reaction was concentrated under reduced pressure. Then the resulting was diluted with dichloromethane (5 mL) and treated with DIEA (1.5 mL). The mixture was concentrated in vacuo to afford 5-isopropoxy-3-[6-[4-[2-[(3R)-pyrrolidin-3-yl]ethyl]piperazin-1-yl]pyrimidin-4-yl]-1H-indazole (170 mg, crude) as a yellow solid. The crude product was used for next step directly.
Step 5
To a solution of 1-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]piperidine-4-carbaldehyde (74.63 mg, 202.04 umol, 1.1 eq) in MeOH (3 mL) and HOAc (0.3 mL) was added 5-isopropoxy-3-[6-[4-[2-[(3R)-pyrrolidin-3-yl]ethyl]piperazin-1-yl]pyrimidin-4-yl]-1H-indazole (80 mg, 183.67 umol, 1 eq) and borane; 2-methylpyridine (39.29 mg, 367.34 umol, 2 eq). After addition, the reaction solution was stirred at 25° C. for 12 h. LCMS showed desired MS. The reaction was diluted with water (5 mL) and extracted with dichloromethane (3×10 mL). The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by prep.HPLC (column: 3_Phenomenex Luna C18 75*30 mm*3 um; mobile phase: [water (0.225% FA)-ACN]; B %: 0-30%; 35 min) to afford 2-(2,6-dioxo-3-piperidyl)-5-[4-[[(3R)-3-[2-[4-[6-(5-isopropoxy-1H-indazol-3-yl)pyrimidin-4-yl]piperazin-1-yl]ethyl]pyrrolidin-1-yl]methyl]-1-piperidyl]isoindoline-1,3-dione (67.7 mg, 85.13 umol, 46.35% yield, 99.20% purity) as a yellow solid.

Exemplary Synthesis of Exemplary Compound 43

Compound 43 was prepared in a manner analogous to compound 42 starting with 2-[(3S)-1-tert-butoxycarbonylpyrrolidin-3-yl]acetic acid.

Exemplary Synthesis of Exemplary Compound 44

A 3-neck round bottom flask was charged with anhydrous DMF (3 mL) and Zn (1.28 g, 19.51 mmol, 2.5 eq). The mixture was stirred at 20° C. While a mixture of 1,2-DIBROMOETHANE (293.25 mg, 1.56 mmol, 117.77 uL, 0.2 eq) and TMSCl (169.59 mg, 1.56 mmol, 198.12 uL, 0.2 eq) was added at a rate to maintain the temperature below 65° C. over 30 min. The resulting slurry was aged for 15 min. A solution of tert-butyl 3-iodoazetidine-1-carboxylate (3 g, 10.60 mmol, 1.36 eq) in DMF (4 mL) was added dropwise over 10 min at a rate to maintain the temperature below 65° C. and the milky suspension was stirred for 30 min while slowly cooling to 20° C. Another round bottom flask was charged with Pd(dppf)Cl2·CH2Cl2 (63.74 mg, 78.05 umol, 0.01 eq) CuI (44.59 mg, 234.15 umol, 0.03 eq) and 4-iodopyridine (1.6 g, 7.81 mmol, 1 eq) in DMF (4 mL) under N2. The resulting mixture was degassed with alternate vacuum/N2 purges. The above prepared zinc iodide reagent of compound in DMF was added as a suspension. The mixture was degassed with vacuum/N2 twice and then heated to 80° C. for 2 hr. TLC (Dichloromethane:Methanol=10:1, Rf=0.5) showed the reaction a new spot. The reaction mixture was filtered and concentrated under reduced pressure. The crude product was poured into H2O (50 mL). The mixture was extracted with ethyl acetate (45 mL*3). The organic phase was washed with brine (30 mL), dried over anhydrous Na2SO4, concentrated in vacuum to give a residue. The residue was purified by silica gel column chromatography (0 to 10% Methanol in Dichloromethane) to give tert-butyl 3-(4-pyridyl)azetidine-1-carboxylate (1.2 g, 4.97 mmol, 63.65% yield, 97% purity) as a yellow oil.
Step 2
To a solution of tert-butyl 3-(4-pyridyl)azetidine-1-carboxylate (1.2 g, 5.12 mmol, 1 eq) in MeCN (10 mL) was added BnBr (892.80 mg, 5.22 mmol, 0.62 mL, 1.02 eq). The mixture was stirred at 80° C. for 1 hr to give yellow suspension. TLC (Dichloromethane:Methanol=10:1, Rf=0.2) showed the reaction a new spot. The reaction mixture was concentrated in vacuum to give tert-butyl 3-(1-benzylpyridin-1-ium-4-yl)azetidine-1-carboxylate (1.6 g, crude) as a yellow gum.
Step 3
To a solution of tert-butyl 3-(1-benzylpyridin-1-ium-4-yl)azetidine-1-carboxylate (1.6 g, 4.92 mmol, 1 eq) in EtOH (10 mL) was added NaBH4 (558.03 mg, 14.75 mmol, 3 eq). The mixture was stirred at 0° C. for 2 h. LCMS showed the formation of the product. TLC (Dichloromethane:Methanol=10:1, Rf=0.5) showed the reaction a new spot. The crude product was poured into H2O (50 mL). The mixture was extracted with ethyl acetate (45 mL*3). The organic phase was washed with brine (30 mL), dried over anhydrous Na2SO4, concentrated in vacuum to give a residue. The residue was purified by silica gel column chromatography (0 to 5% Methanol in Dichloromethane) to give tert-butyl 3-(1-benzyl-3,6-dihydro-2H-pyridin-4-yl)azetidine-1-carboxylate (1.2 g, 2.19 mmol, 44.59% yield, 60% purity) as a colorless gum.
Step 4
To a solution of tert-butyl 3-(1-benzyl-3,6-dihydro-2H-pyridin-4-yl)azetidine-1-carboxylate (1.2 g, 3.65 mmol, 1 eq) in EtOH (15 mL) and EtOAc (15 mL) was added Pd/C (120 mg, 10% purity) and Pd(OH)2 (120 mg, 85.45 umol, 10% purity, 2.34e-2 eq) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (50 psi) at 60° C. for 18 hours. TLC (Dichloromethane:Methanol=10:1, Rf=0.1) showed the reaction a new spot. The reaction mixture was filtered and concentrated under reduced pressure to afford tert-butyl 3-(4-piperidyl)azetidine-1-carboxylate (900 mg, crude) as a Light Yellow oil.
Step 5
To a solution of tert-butyl 3-(4-piperidyl)azetidine-1-carboxylate (0.4 g, 1.66 mmol, 1 eq) and 2-(2,6-dioxo-3-piperidyl)-5-fluoro-isoindoline-1,3-dione (413.74 mg, 1.50 mmol, 0.9 eq) and DIEA (1.08 g, 8.32 mmol, 1.45 mL, 5 eq) in DMSO (20. mL). Then the mixture was stirred at 100° C. for 16 hr under N2. TLC (Dichloromethane:Methanol=10:1, Rf=0.6) showed no start material and a new spot. The residue was diluted with H2O (50 mL) extracted with ethyl acetate (70 mL×3). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (0 to 10% Methanol in Dichloromethane) to give tert-butyl 3-[1-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]-4-piperidyl]azetidine-1-carboxylate (800 mg, 1.47 mmol, 88.09% yield, 91% purity) as a yellow solid.
Step 6
To a solution of tert-butyl 3-[1-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-5-yl]-4-piperidyl]azetidine-1-carboxylate (800 mg, 1.61 mmol, 1 eq) in DCM (2 mL) and added TFA (3.08 g, 27.01 mmol, 2 mL, 16.77 eq). Then the mixture was stirred at 20° C. for 1 hr under. The residue was concentrated under reduced pressure to give 5-[4-(azetidin-3-yl)-1-piperidyl]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (1.2 g, crude, 3TFA) as a yellow solid.
Step 7
To a solution of 5-[4-(azetidin-3-yl)-1-piperidyl]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (400 mg, 541.63 umol, 1 eq, 3TFA) and 2-chloroacetaldehyde (122.00 mg, 621.68 umol, 0.1 mL, 40% purity, 1.15 eq) in DCM (15 mL) and MeOH (15 mL) was added NaOAc (266.59 mg, 3.25 mmol, 6 eq) and HOAc (3.25 mg, 54.16 umol, 3.10 uL, 0.1 eq). The mixture was stirred at 25° C. for 20 min. Then the NaBH3CN (102.11 mg, 1.62 mmol, 3 eq) was added of the solution and was stirred at 25° C. for 2 hr. TLC (Dichloromethane:Methanol=10:1, Rf=0.4) showed no start material and a new spot. The reaction mixture was poured into H2O (20 mL). The mixture was extracted with ethyl acetate (30 mL*3). The organic phase was washed with brine (20 mL), dried over anhydrous Na2SO4, concentrated in vacuum to give a residue. The residue was purified by silica gel column chromatography (0-15% Methanol in Dichloromethane) to give 5-[4-[1-(2-chloroethyl)azetidin-3-yl]-1-piperidyl]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (248 mg, 502.55 umol, 92.78% yield, 93% purity) as a yellow solid.
Step 8
To a solution of 5-isopropoxy-3-(6-piperazin-1-ylpyrimidin-4-yl)-1H-indazole (79.64 mg, 235.33 umol, 0.9 eq) and 5-[4-[1-(2-chloroethyl)azetidin-3-yl]-1-piperidyl]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (120 mg, 261.47 umol, 1 eq) and DIEA (168.96 mg, 1.31 mmol, 227.71 uL, 5 eq) and KI (217.02 mg, 1.31 mmol, 5 eq) in MeCN (10 mL). Then the mixture was stirred at 100° C. for 16 hr under N2. The residue was diluted with H2O (20 mL) extracted with ethyl acetate (20 mL×3). The combined organic layers were washed with brine (15 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: 3_Phenomenex Luna C18 75*30 mm*3 um; mobile phase: [water (0.225% FA)-ACN]; B %: 0%-30%, 35 min) to give 2-(2,6-dioxo-3-piperidyl)-5-[4-[1-[2-[4-[6-(5-isopropoxy-1H-indazol-3-yl)pyrimidin-4-yl]piperazin-1-yl]ethyl]azetidin-3-yl]-1-piperidyl]isoindoline-1,3-dione (13.3 mg, 17.43 umol, 6.67% yield, 99.73% purity) as a yellow solid.

Exemplary Synthesis of Exemplary Compound 45

Exemplary Synthesis of Exemplary Compound 46

Compound 46 was prepared in a manner analogous to compound 45 starting with tert-butyl 3-(2-hydroxyethyl)azetidine-1-carboxylate.

Exemplary Synthesis of Exemplary Compound 47

Exemplary Synthesis of Exemplary Compound 48

Compound 48 was prepared in a manner analogous to compound 47 starting with tert-butyl (2S)-2-(p-tolylsulfonyloxymethyl)morpholine-4-carboxylate

Exemplary Synthesis of Exemplary Compound 49

Exemplary Synthesis of Exemplary Compound 50

Compound 50 was prepared in a manner analogous to compound 49 starting with (2R)-4-[6-(5-isopropoxy-1H-indazol-3-yl)pyrimidin-4-yl]-2-(2-piperazin-1-ylethyl)morpholine

Exemplary Synthesis of Exemplary Compound 51

Protein Level Control

This description also provides methods for the control of protein levels within a cell. The method is based on the use of compounds as described herein such that degradation of the target protein LRRK2 in vivo will result in the reducing the amount of the target protein in a biological system, preferably to provide a particular therapeutic benefit.

The following examples are used to assist in describing the present disclosure, but should not be seen as limiting the present disclosure in any way.

In certain embodiments, the description provides the following exemplary LRRK2-degrading bifunctional molecules (compounds of Table 1 or exemplary compounds 1-51), including salts, polymorphs, analogs, derivatives, and deuterated forms thereof.

Exemplary Assay for Testing LRRK2 Degradation Driven by Exemplary Hetero-Bifunctional Compounds Designed to Target LRRK2

The assay measures the degradation of wildtype and G2019S LRRK2 tagged with a HiBit tag on the C-terminus of the protein that was expressed from a mammalian expression vector, driven by the ubiquitin promoter in HEK293 cells. Each compound dose-response was repeated on two separate days, on three separate plates each day.

Plasmid Preparation. Transfection mixes were assembled as follows and incubated for 30 minutes at room temperature. In a 15 mL tube, 5.25 mL Opti-MEM (no additions) was mixed with 17 μL Firefly Luciferase plasmid at 1 μg/μL and 158 μL WT plasmid DNA at 1 μg/μL (175 μg total DNA) were mixed by flicking. In a new 15 mL tube, 5.25 mL OptiMEM was mixed with 17 μL Firefly Luciferase plasmid at 1 μg/μL and 158 μL G2019S plasmid DNA at 1 μg/μL (175 μg total DNA) were mixed by flicking. X-tremeGene HP was mixed thoroughly using a vortex. Next, 175 μL was added to each tube and flicked to mix. Both tubes were left to incubate for 30 minutes at room temperature.

While the transfection mixes were incubating, HEK293 cells (acquired from ATCC; ATCC CRL-1573) were harvested with trypsin. Once cells are detached, the cells were resuspended in 12 mL OptiMEM +5% FBS and transferred to a 50 mL tube. The cells were mixed well and counted. Using OptiMEM +5% FBS, the cells were diluted in two 250 mL conical tubes at 0.71×106cells/mL in 70 mL. One tube was labeled “WT” and the other “G2019S”. The WT and G2019S transfection mixes were added dropwise to the corresponding 250 mL tubes. The tubes were mixed first by pipetting then by swirling. The tubes were incubated at room temperature for at least 5 minutes.

Each tube was swirled before dispensing and after every three plates. Seventy microliters of cells were dispensed with WT or G2019S DNA to seven plates each. Three plates of each were tested with compound plate one (preparation described below) and three plates of each were tested with compound plate two (preparation described below). The first plate from each set served as a “prime” plate and was not used to test compounds. Each plate was incubated in the hood for 10 minutes before placing in the 37° C. incubator for 24 hours.

Preparation of Compound and Assay Plates. Two compound plates were made using 96 well polypropylene plates. Compounds were made up at 10 mM and were diluted to 1 mM in 30 μL. Each dose response curve included a well of DMSO, as a negative control and for normalization, and a well of 0.5 μM of Exemplary Compound 4 as a positive control. In addition to seven test compounds, each plate also included a dose response of Exemplary Compound 4. The compound plates were spun down along at 1200 rpm for 2 minutes.

The two compound plates were then mixed and 2 μL was diluted in intermediate plates having 248 μL of Opti-Mem in each well. Next, 10 μL diluted compounds from the intermediate plates were added to each test plate (three WT and three G2019S plates per compound plate for a total of 12 assay plates). The plates were incubated for 24 hours at 37° C.

All assay plates and all Nano-Glo Dual-Luciferase Reporter Assay System components (except for the DLR substrate) were equilibrated to room temperature. Next, the luciferase buffer was mixed with the lyophilized amber bottle until fully dissolved, and 75 μL of the luciferase mixture was added to each well of each assay plate. The assay plates were incubated for 10 minutes at room temperature with shaking for at least 5 minutes, and then read on a plate reader.

Developing Plates and Analyzing Data. One milliliter of DLR substrate and 1 mL LgBiT Protein were added to the Stop and Glo buffer, and 75 μL of the mixture was added to each well of each plate. Optically clear seals were added to each plate and each plate was incubated for 20 minutes with shaking for at least 10 minutes, and then read on a plate reader.

As mentioned above, plates were run in triplicate and assay repeated twice (total of 6 replicates per exemplary compound. Each cell was examined for firefly luciferase for cell number and viability and Nanoluc for the LRRK2-HiBit quantification.

Ratio of (HiBit/luciferase)*1000 was determined and the data was normalized to % of DMSO median value. Curve fitting was performed on each individual plate. The data for exemplary compounds of Table 1 below is shown below in Table 2 in the *G2019S DC50 (nM), **G2019S Dmax (%), *WT DC50 (nM) and **WT Dmax (%) columns.

Exemplary Assay for Testing LRRK2 Degradation Driven by Exemplary Hetero-Bifunctional Compounds Designed to Target LRRK2

The assay measures the degradation of LRRK2 in cells where the C-terminus (3′) of the endogenous gene has been tagged with a HiBit sequence in HEK293 cells. The cells also express firefly lucisferase, expressed from a Cytomegalovirus promoter and introduced into the HiBit tagged cells and stably expressed. The Nano-Glo® Dual Luciferase Reporter Assay System (Promega™, Madison, WI) was utilized.

Day 1—Preparation of Compound and Assay Plates. Two sets of plates were prepared: a triplicate set for the HiBit assay in white 384-well plates and a triplicate set of plate in black 384-well plates for the Alamar Blue cell viability assay. Briefly, the growth media (DMEM+Glutamax-10% fetal bovine serum-1% Penicillin-Streptomicin) from two T128 flasks was aspirated from the flasks. Cells were washed with Dulbecco's Phosphate Buffered Saline (dPBS) and aspirated. Trypsin (3 mL per flask) was added and the flasks were incubated for 2-3 minutes.

Ten mL of OptiMEM-10% fetal bovine-1% penicillin-streptomycin (hereinafter, “OptiMEM media”) was added to the flask and the cells and transferred to a 50 mL conical tube. A cell count (25 ul of cell into Effendorf vial +25 ul of Trypan Blue Stain) was performed and the cell density adjusted to 15,000 cell/45 μl/well (3.33×10{circumflex over ( )}5/mL) in OptiMEM media.

Forty-five microliters of the cell suspension (15,000 cells) was aliquoted to each well of the white 384-well plate. The plates incubated at room temperature for 10 minutes before being placed in the 37° C.+5% CO2incubator overnight

Day 2—Compound Treatment. Exemplary compounds were prepared at a 1 mM starting concentration and 1:3 serial dilution for 11 points CRC prepared and stored in the freezer. The Master Compound Plate was thawed overnight at room temperature. DMSO (20 μL) was added into column 24 of the Master Compound Plate for negative control and 20 μL of 300 μM of Exemplary Compound 4 in column 23 as positive control.

Intermediate Compound Plate with 4% DMSO in OptiMEM Media. DMSO was added to warm OptiMEM media to achieve a 4% DMSO solution (approximately 50 mL/plate). One-hundred microliters of the OptiMEM-4% DMSO was aliquoted to each well of 384-Well Deep Well Microplates.

The Master Compound Plate and the Intermediate Compound Plate were spun down.

One microliter of compound from the Master Compound Plate was transferred into the Intermediate plate (a 1:100 dilution). The diluted mixture was mixed and 5 μL transferred into the assay plate (a 1:10 dilution) for the final starting concentration of 1 μM. The Treated Assay plates were incubated for 24 hours at 37° C.+5% CO2. The Master Compound Plate was sealed and store at room temperature for a second run that was performed within a week.

Day 3—HiBit Assay. Five microliters of Alamar Blue was added to each well of the black 384-well plates. The plates were incubated for 2 hours in the incubator (37° C.+5% CO2) and at room temperature for one hour. Fluorescence of each plate was read on a plated reader for the Alamar Blue viability assay.

One set of white assay plates was warmed to room temperature (45 minute).

The One Glo luciferase mixture was prepared. The media from white 384-well assay plates was aspirated. Twenty-five μL of the One Glo luciferase mixture was added to each well of the assay plates. The plates were incubated on the bench (room temperature) for 45 minutes, including 10 minutes of shaking at 700 rpm. The luminescence of each plate was read on a plate reader.

1:100 DLR substrate and 1:100 LgBiT Protein dilution were added to the Promega Stop and Glo buffer and mixed just before addition to assay plates. Twenty-five microliters of Stop and Glo mixture was added to each well. Assay plates incubated for at least 45 minutes, including 10 minutes of shaking at 700 rpm. The luminescence of each plate was read on a plate reader.

Analysis of LRRK2 HiBit Screening assays. As mentioned above, plates were run in triplicate and the assay repeated twice (total of 6 replicate for exemplary compound). For each treatment, measurements were taken for firefly luciferase for cell number, cell viability (Alamar Blue), and Nanoluc for the LRRK2-HiBit quantification.

The LRRK2 HiBit and alamar blue signal was normalized to % DMSO median value for each plate. Curve fitting was performed on each compound for replicates across three plates. The date for exemplary compounds of Table 1 below is shown below in Table 2 in the Endogenous *WT DC50 (nM) and **Endogenous WT Dmax columns (%).

A novel bifunctional molecule, which contains a LRRK2 recruiting moiety and an E3 ubiquitin ligase recruiting moiety is described. The bifunctional molecules of the present disclosure actively degrades LRRK2, leading to robust cellular proliferation suppression and apoptosis induction. Protein degradation mediated by the bifunctional compounds of the present disclosure provides a promising strategy in targeting the “undruggable” pathological proteins by traditional approaches.

The preceding general areas of utility are given by way of example only and are not intended to be limiting on the scope of the present disclosure and appended claims. Additional objects and advantages associated with the compositions, methods, and processes of the present disclosure will be appreciated by one of ordinary skill in the art in light of the instant claims, description, and examples. For example, the various aspects and embodiments of the disclosure may be utilized in numerous combinations, all of which are expressly contemplated by the present description. These additional aspects and embodiments are expressly included within the scope of the present disclosure. The publications and other materials used herein to illuminate the background of the disclosure, and in particular cases, to provide additional details respecting the practice, are incorporated by reference.

Thus, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims. It is understood that the detailed examples and embodiments described herein are given by way of example for illustrative purposes only, and are in no way considered to be limiting to the disclosure. Various modifications or changes in light thereof will be suggested to persons skilled in the art and are included within the spirit and purview of this application and are considered within the scope of the appended claims. For example, the relative quantities of the ingredients may be varied to optimize the desired effects, additional ingredients may be added, and/or similar ingredients may be substituted for one or more of the ingredients described. Additional advantageous features and functionalities associated with the systems, methods, and processes of the present disclosure will be apparent from the appended claims. Moreover, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims.