NEK7 DEGRADERS AND METHODS OF USE THEREOF

FIELD OF THE INVENTION

The present invention relates to novel compounds which can act as degraders of NEK7, and methods of use thereof.

BACKGROUND

Inflammasomes are a group of intracellular complexes located in the cytosol, which are an element of innate immunity, responsible for the detection of either pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs). Inflammasome multiprotein complexes are composed of three parts: a sensor protein, an adaptor, and pro-caspase-1, responsible for the production of pro-inflammatory cytokines - interleukin 1β (IL-1β) and IL-18 from their precursors (pro- IL-1β and pro-IL-18, respectively).

Among all the known inflammasomes, the NLRP3 inflammasome plays a central role in innate immunity. NLRP3 inflammasome is composed of NLRP3 as a sensor protein, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) as an adaptor and pro-caspase-1. The interactions among these proteins are closely associated with the formation of NLRP3 inflammasome. NLRP3 has an N-terminal pyrin domain, which interacts with the adaptor protein ASC via interactions between pyrin domains; a central adenosine triphosphatase (ATPase) domain known as NACHT, which comprises an NBD, helical domain 1 (HD1), winged helix domain (WHD) and helical domain 2 (HD2) and a C-terminal LRR domain. ASC also has a caspase recruitment domain, which recruits caspase-1 via interactions between the caspase recruitment domains, to promote caspase dimerization and activation. Caspase 1 causes maturation of pro-inflammatory cytokines - IL-1β and IL- 18 from their precursor forms (pro-IL-1β and pro-IL-18 respectively).

The formation and activation of the inflammasome requires the synergistic effect of two signals. First, as a result of the initiation signal from TOLL-like receptors (TLR), proinflammatory transcription factors are induced, especially NF-KB (nuclear factor kappa-light-chain-enhancer of activated B cells) or cytokines such as TNF or IL-1β, which upregulates the inflammasome components as well as NEK7. NEK7 has recently been identified as an important requirement in NLRP3 inflammasome activation via direct interaction with NLRP3. Human NEK7, a member of the family of mammalian NIMA-related kinases (NEK proteins), consists of a non-conserved and disordered N-terminal regulatory domain as well as a conserved C-terminal catalytic domain - serine/threonine kinase.

NEK7 binds directly to the leucine-rich repeat (LRR) domain of NLRP3. The interaction stimulates the assembly and activation of the NLRP3 inflammasome and promotes its oligomerization through the bridging of adjacent subunits of the NLRP3 protein. NLRP3 is associated with the catalytic domain of NEK7, but the catalytic activity of NEK7 was shown to be dispensable for activation of the NLRP3 inflammasome.

NEK7 is expressed in a variety of tissues and is essential for cell division and growth, as well as the survival of mammalian cells. Low activity status of NEK7 protein in resting cells is critical to the maintenance of homeostasis. However, once homeostasis is disordered, an aberrant expression of NEK7 occurs, which is closely related to neoplastic progression. Overexpression of NEK7 promotes the production of abnormal cells, including the multinucleated cells and apoptotic cells which are related to inflammation. With the inappropriate release of proinflammatory cytokines, the NLRP3 inflammasome is involved in various inflammatory diseases, such as atherosclerosis, type 2 diabetes, metabolic syndrome, multiple sclerosis, Alzheimer's disease, gout, rheumatoid arthritis, and inflammatory bowel disease. Mechanism of NLRP3 inflammasome activation by NEK7 strongly indicates promising roles for targeting NEK7 in treating inflammation-related diseases. There are several pathways that are essential for the activation of NLRP3 inflammasome, including ROS signaling, K+ efflux, Ca2+ signaling, chloride efflux and lysosomal destabilization. Thus, a great number of inhibitors have been widely used to disturb these signaling pathways. Compounds focused on NEK7 may regulate NLRP3 to abolish the inflammation response with improved specificity and potency. Apart from NLRP3 inflammasome activation, NEK7 plays significant role in mitotic entry, cell cycle progression, cell division, mitotic progression. In last years the potential role of NEK7 in the cancer development of various tissues has been demonstrated.

Although inhibitors in general can inhibit protein of interest (POI) activity, targeted degradation appears as an attractive therapeutic alternative. Protein degradation is mainly regulated by the ubiquitin-proteasome pathway, in which proteins are tagged for degradation by covalent conjugation of multiples ubiquitin molecules. Manipulation of the ubiquitin-proteasome system to achieve targeted degradation of proteins within cells is possible using chemical tools and drugs. Targeted protein degradation (TPD) rather than inhibition could provide advantages such as reduced drug exposure time required to suppress signaling, it provides more complete and lasting inactivation of downstream signaling since cell needs time to express POI in required quantity again. TPD also can overcome intrinsic feedback activation or overexpression of the target protein. Protein degraders may potentially be used as a general way to solve compensatory upregulation of proteins that contributes to illness, adverse effects, and drug resistance. Therefore, there is a great need to provide NEK7 degraders as a key to downregulate inflammasome activation in NLRP3 inflammasome-related diseases as well as in cancer treatment.

SUMMARY OF INVENTION

In accordance with a first aspect of the invention, there is provided a compound of Formula (la) or

(lb): y is O, l or 2; each of X ₁ and X ₂ is independently O or S;

L is H, -C(O)alkyl, or -CH ₂ (O)COR'; each Z is independently C=O, CH ₂ or CH(C _1-2 alkyl);

Y is S, O or NH; each R is independently halogen, alkyl, haloalkyl, hydroxy, alkoxy, -NH ₂ , -NUR' or -

NR' ₂ ; each R' is independently alkyl or aryl; each n is independently 0, 1, 2 or 3; m is 0, 1 or 2; p is 0 or 1; denotes the point of attachment to is a heterocycloalkyl group, and is a 6-membered monocyclic heteroaryl group or a 10-membered fused bicyclic heteroaryl group, wherein is either unsubstituted or is substituted with one or more R ³ , no substituents other than said one or more R ³ are present on ; and each R ³ is independently halogen, unsubstituted alkyl, haloalkyl, cycloalkyl, hydroxy, OR ¹ , aryl, benzyl, -C(O)R ¹ , or-NR ² C(O)R ¹ , wherein each R ¹ is independently unsubstituted alkyl, cycloalkyl, or aryl and each R ² is independently H, unsubstituted alkyl, or cycloalkyl; wherein in formula (lb): , Z is CH ₂ and n = 0, then is substituted with one or more R ³ ; (ii) when , Z is CH ₂ , n = 0, and s a 6-membered monocyclic heteroaryl group having two heteroatoms, the two heteroatoms are not adjacent to each other,

(iii) when , Z is CH?, n = 0 and is a 6-membered monocyclic heteroaryl group substituted with one or more R ³ , then:

(a) carbon atoms adjacent to the carbon atom through which is attached to are unsubstituted;

(b) when R ³ is alkyl or O(alkyl), then is monosubstituted;

(c) when R ³ is O(alkyl), then the substitution is at a position meta or para to a heteroatom of the heteroaryl group; and

(d) when R ³ is aryl or -NR ₂ C(O)Ri, then the substitution is at a position meta to a heteroatom of the heteroaryl group, and

(iv) when , Z is CH ₂ , n = 0 and is a 10-membered fused bicyclic heteroaryl group substituted with one or more R ³ , then: each R ³ is present on the ring which contains the point of attachment to each R ³ is positioned ortho or meta to a heteroatom of the heteroaryl group; R ³ is not Cl, methyl, iPr, cyclopropane, unsubstituted phenyl, hydroxy or OMe; when R ³ is OEt, then R ³ is positioned ortho to the heteroatom of the heteroaryl group; and when R ³ is NR ₂ COMe, then R ³ is positioned meta to the heteroatom of the heteroaryl group.

In accordance with a second aspect of the invention, there is provided a compound of Formula (I): for use in a method of treating a disease or condition in a subject in need thereof, wherein: y is 0, 1 or 2; each of X ₁ and X ₂ is independently 0 or S;

L is H, -C(O)alkyl, or -CH ₂ (O)COR'; wherein each Z is independently C=O, CH ₂ or CH(C _1-2 alkyl);

Y is S, O or NH; each R is independently halogen, alkyl, haloalkyl, hydroxy, alkoxy, -NH ₂ , -NHR' or -

NR' ₂ ; each R' is independently alkyl or aryl each n is independently 0, 1, 2 or 3; m is 0, 1 or 2; p is 0 or 1; ’denotes the point of attachment to denotes the point of attachment to and

' is a heterocyclic group; wherein the disease or condition is an inflammatory disease or condition, an auto-immune disease or condition, a respiratory disease or condition, a cardiovascular disease or condition, a gastrointestinal disease or condition, a renal disease or condition, a disease or condition of the central nervous system (CNS), a disease or condition of the endocrine system, a metabolic disease or condition, a liver disease or condition, an ocular disease or condition, a skin disease or condition, a lymphatic disease or condition, a psychological disease or condition, graft versus host disease or condition, allodynia, pain, a condition associated with diabetes, a condition associated with arthritis, or a wound or burn.

In accordance with a third aspect of the invention, there is provided a method of degrading NEK7 protein comprising contacting said protein with a compound of Formula (I): wherein: y is 0, 1 or 2; each of X ₁ and X ₂ is independently 0 or S;

L is H, -C(O)alkyl, or -CH ₂ (O)COR';

wherein each Z is independently C=O, CH2 or CH(C _1-2 alkyl);

Y is S, O or NH; each R is independently halogen, alkyl, haloalkyl, hydroxy, alkoxy, -NH ₂ , -NHR' or - NR' ₂ ; each R' is independently alkyl or aryl; each n is independently 0, 1, 2 or 3; m is 0, 1 or 2; p is 0 or 1;

As used herein the term "alkyl" is intended to include both unsubstituted alkyl groups, and alkyl groups which are substituted by one or more additional groups. The term "alkyl" is intended to include both linear alkyl groups and branched alkyl groups. In some embodiments, the alkyl group is an unsubstituted alkyl group. In some embodiments, the alkyl group is substituted by one or more groups selected from -OH, -OR ^W , -NH ₂ , -NHR ^W , -NR ^W ₂ , -SO ₂ R ^W , -C(O)R ^W , -CN, and -NO ₂ , wherein each R ^w is unsubstituted and is independently alkyl, cycloalkyl, alkenyl, cycloalkenyl, aryl, heteroaryl, or benzyl. In some embodiments, the alkyl group is a C ₁ -C ₁₂ alkyl, a C ₁ -C ₁₀ alkyl, a C ₁ -C ₈ alkyl, a C ₁ -C ₆ alkyl, or a C ₁ - C ₄ alkyl group. In some embodiments the alkyl group is a linear alkyl group. In some embodiments the alkyl group is an unsubstituted linear alkyl group. In some embodiments the alkyl group is a li near alkyl group which is substituted by one or more groups selected from -OH, -OR ^W , -NH2, -NHR^, -NR ^W ₂ , - SO ₂ R ^W , -C(O)R ^W , -CN, and -NO ₂ , wherein each R ^w is unsubstituted and is independently alkyl, cycloalkyl, alkenyl, cycloalkenyl, aryl, heteroaryl, or benzyl. In some embodiments the alkyl group is a branched alkyl group. In some embodiments the alkyl group is an unsubstituted branched alkyl group. In some embodiments the alkyl group is a branched alkyl group which is substituted by one or more groups selected from -OH, -OR ^W , -NH ₂ , -NHR ^W , -NR ^W ₂ , -SO ₂ R ^W , -C(O)R ^W , -CN, and -NO ₂ , wherein each R ^w is unsubstituted and is independently alkyl, cycloalkyl, alkenyl, cycloalkenyl, aryl, heteroaryl, or benzyl.

In some embodiments of any of the above aspects, all alkyl groups are unsubstituted alkyl groups.

As used herein the term "cycloalkyl" is intended to include both unsubstituted cycloalkyl groups, and cycloalkyl groups which are substituted by one or more additional groups. In some embodiments, the cycloalkyl group is an unsubstituted cycloalkyl group. In some embodiments, the cycloalkyl group is substituted by one or more groups selected from -OH, -OR ^W , -NH ₂ , -NHR ^W , -NR ^W 2, -SO ₂ R ^W , -C(O)R ^W , - CN, and -NO2, wherein each R ^w is unsubstituted and is independently alkyl, cycloalkyl, alkenyl, cycloalkenyl, aryl, heteroaryl, or benzyl. In some embodiments, the cycloalkyl group is a C ₃ -C12 cycloalkyl, a C ₃ -C ₈ cycloalkyl, a C ₃ -C ₆ cycloalkyl, or a C ₅ -C ₆ cycloalkyl group.

In some embodiments of any of the above aspects, all cycloalkyl groups are unsubstituted cycloalkyl groups.

As used herein the term "alkenyl" is intended to include both unsubstituted alkenyl groups, and alkenyl groups which are substituted by one or more additional groups. In some embodiments, the alkenyl group is an unsubstituted alkenyl group. In some embodiments, the alkenyl group is substituted by one or more groups selected from -OH, -OR ^W , -NH ₂ , -NHR ^W , -NR ^W 2, -SO ₂ R ^W , -C(O)R ^W , - CN, and -NO ₂ , wherein each R ^w is unsubstituted and is independently alkyl, cycloalkyl, alkenyl, cycloalkenyl, aryl, heteroaryl, or benzyl. In some embodiments, the alkenyl group is a C ₂ -C ₁₂ alkenyl, a C2-C10 alkenyl, a C ₂ -C ₈ alkenyl, a C ₂ -C ₆ alkenyl, or a C ₂ -C ₄ alkenyl group. In some embodiments the alkenyl group is a linear alkenyl group. In some embodiments the alkenyl group is an unsubstituted linear alkenyl group. In some embodiments the alkenyl group is a linear alkenyl group which is substituted by one or more groups selected from -OH, -OR ^W , -NH ₂ , -NHR ^W , -NR ^W 2, -SO ₂ R ^W , -C(O)R ^W , - CN, and -NO2, wherein each R ^w is unsubstituted and is independently alkyl, cycloalkyl, alkenyl, cycloalkenyl, aryl, heteroaryl, or benzyl. In some embodiments the alkenyl group is a branched alkenyl group. In some embodiments the alkenyl group is an unsubstituted branched alkenyl group. In some embodiments the alkenyl group is a branched alkenyl group which is substituted by one or more groups selected from -OH, -OR ^W , -NH ₂ , -NHR ^W , -NR ^W ₂ , -SO ₂ R ^W , -C(O)R ^W , -CN, and -NO ₂ , wherein each R ^w is unsubstituted and is independently alkyl, cycloalkyl, alkenyl, cycloalkenyl, aryl, heteroaryl, or benzyl.

In some embodiments of any of the above aspects, all alkenyl groups are unsubstituted alkenyl groups.

As used herein the term "alkynyl" is intended to include both unsubstituted alkynyl groups, and alkynyl groups which are substituted by one or more additional groups. In some embodiments, the alkynyl group is an unsubstituted alkynyl group. In some embodiments, the alkynyl group is substituted by one or more groups selected from -OH, -OR ^W , -NH ₂ , -NHR ^W , -NR ^W ₂ , -SO ₂ R ^W , -C(O)R ^W , -CN, and -NO ₂ , wherein each R ^w is unsubstituted and is independently alkyl, cycloalkyl, alkenyl, cycloalkenyl, aryl, heteroaryl, or benzyl. In some embodiments, the alkynyl group is a C ₂ -C ₁₂ alkynyl, a C ₂ -C ₁₀ alkynyl, a C ₂ -C ₈ alkynyl, a C ₂ -C ₆ alkynyl, or a C ₂ -C4 alkynyl group. In some embodiments the alkynyl group is a linear alkynyl group. In some embodiments the alkynyl group is an unsubstituted linear alkynyl group. In some embodiments the alkynyl group is a linear alkynyl group which is substituted by one or more groups selected from -OH, -OR ^W , -NH ₂ , -NHR ^W , -NR ^W ₂ , -SO ₂ R ^W , -C(O)R ^W , -CN, and -NO ₂ , wherein each R ^w is unsubstituted and is independently alkyl, cycloalkyl, alkenyl, cycloalkenyl, aryl, heteroaryl, or benzyl. In some embodiments the alkynyl group is a branched alkynyl group. In some embodiments the alkynyl group is an unsubstituted branched alkynyl group. In some embodiments the alkynyl group is a branched alkynyl group which is substituted by one or more groups selected from -OH, -OR ^W , - NH ₂ , -NHR ^W , -NR ^W ₂ , -SO ₂ R ^W , -C(O)R ^W , -CN, and -NO ₂ , wherein each R ^w is unsubstituted and is independently alkyl, cycloalkyl, alkenyl, cycloalkenyl, aryl, heteroaryl, or benzyl.

In some embodiments of any of the above aspects, all alkynyl groups are unsubstituted alkynyl groups.

As used herein the term "aryl" is intended to include both unsubstituted aryl groups, and aryl groups which are substituted by one or more additional groups. In some embodiments, the aryl group is an unsubstituted aryl group. In some embodiments, the aryl group is substituted by one or more groups selected from -OH, -OR ^W , -NH ₂ , -NHR ^W , -NR ^W ₂ , -SO ₂ R ^W , -C(O)R ^W , -CN, and -NO ₂ , wherein each R ^w is unsubstituted and is independently alkyl, cycloalkyl, alkenyl, cycloalkenyl, aryl, heteroaryl, or benzyl. In some embodiments, the aryl group is a C ₆ -C ₁₀ aryl, a C ₆ -C ₈ aryl, or a C ₈ aryl. In some embodiments of any of the above aspects, all aryl groups are unsubstituted aryl groups.

As used herein the term "benzyl" is intended to include both unsubstituted benzyl groups, a nd benzyl groups which are substituted by one or more additional groups. In some embodiments, the benzyl group is an unsubstituted benzyl group. In some embodiments, the benzyl group is substituted by one or more groups selected from -OH, -OR ^W , -NH ₂ , -NHR ^W , -NR ^W ₂ , -SO ₂ R ^W , -C(O)R ^W , -CN, and -NO ₂ , wherein each R ^w is unsubstituted and is independently alkyl, cycloalkyl, alkenyl, cycloalkenyl, aryl, heteroaryl, or benzyl.

In some embodiments of any of the above aspects, all benzyl groups are unsubstituted benzyl groups.

As used herein, the term "heterocyclic" is intended to include monocyclic heteroaryl, monocyclic heterocycloalkyl, fused bicyclic heteroaryl, fused bicyclic heterocycloalkyl, and fused bicyclic heterocycloalkyl-aryl groups. The term "heterocyclic" is intended to include both unsubstituted heterocyclic groups, and heterocyclic groups which are substituted by one or more additional groups. In some embodiments, the heterocyclic group is an unsubstituted heterocyclic group. In some embodiments, the heterocyclic group is substituted with one or more R ³ , wherein no substituents other than said one or more R ³ are present on the heterocyclic group; wherein each R ³ is independently halogen, unsubstituted alkyl, haloalkyl, cycloalkyl, hydroxy, OR ¹ , aryl, benzyl, -C(O)R ¹ , or -NR ² C(O)R ¹ , wherein each R ¹ is independently unsubstituted alkyl, cycloalkyl, or aryl and each R ² is independently H, unsubstituted alkyl, or cycloalkyl. In some embodiments where the heterocyclic group is a heterocycloalkyl group, two R ³ groups on adjacent atoms of the heterocycloalkyl group, together with the atoms to which they are attached, form an aryl ring. In some embodiments where the heterocyclic group is a heterocycloalkyl group, two R ³ groups on the same carbon atom of the heterocycloalkyl group, together with the carbon atom to which they are attached, form a C-O group.

As used herein the term "heterocycloalkyl" is intended to include monocyclic and fused bicyclic heterocycloalkyl groups. The term "heterocycloalkyl" is intended to include both unsubstituted heterocycloalkyl groups, and heterocycloalkyl groups which are substituted by one or more additional groups. In some embodiments, the heterocycloalkyl group is an unsubstituted heterocyclic group. In some embodiments, the heterocycloalkyl group is substituted with one or more R ³ , wherein no substituents other than said one or more R ³ are present on the heterocycloalkyl group; wherein each R ³ is independently halogen, unsubstituted alkyl, haloalkyl, cycloalkyl, hydroxy, OR ¹ , aryl, benzyl, - C(O)R ¹ , or -NR ² C(O)R ¹ , wherein each R ¹ is independently unsubstituted alkyl, cycloalkyl, or aryl and each R ² is independently H, unsubstituted alkyl, or cycloalkyl. In some embodiments, two R ³ groups on adjacent atoms of the heterocycloalkyl group, together with the atoms to which they are attached, form an aryl ring. In some embodiments, two R ³ groups on the same carbon atom of the heterocycloalkyl group, together with the carbon atom to which they are attached, form a C=O group. In some embodiments, the heterocycloalkyl group is a 5-10 membered heterocycloalkyl group (also referred to as a C5-C10 heterocycloalkyl), a 5-9 membered heterocycloalkyl group (also referred to as a C5-C9 heterocycloalkyl), a 5-8 membered heterocycloalkyl group (also referred to as a C ₅ -C ₈ heterocycloalkyl), or 5- or 6-membered heterocycloalkyl group (also referred to as a C ₅ or C ₆ heterocycloalkyl).

As used herein the term "heteroaryl" is intended to include monocyclic and fused bicyclic heteroaryl groups. The term "heteroaryl" is intended to include both unsubstituted heteroaryl groups, and heteroaryl groups which are substituted by one or more additional groups. In some embodiments, the heteroaryl group is an unsubstituted heteroaryl group. In some embodiments, the heteroaryl group is substituted with one or more R ³ , wherein no substituents otherthan said one or more R ³ are present on the heteroaryl group; wherein each R ³ is independently halogen, unsubstituted alkyl, haloalkyl, cycloalkyl, hydroxy, OR ¹ , aryl, benzyl, -C(O)R ¹ , or -NR ² C(O)R ¹ , wherein each R ¹ is independently unsubstituted alkyl, cycloalkyl, or aryl and each R ² is independently H, unsubstituted alkyl, or cycloalkyl. In some embodiments, the heteroaryl group is a 5-10 membered heteroaryl group (also referred to as a Cg-C ₁ o heteroaryl), a 5-9 membered heteroaryl group (also referred to as a C ₆ -C ₉ heteroaryl), a 6-8 membered heteroaryl group (also referred to as a C ₆ -C ₈ heteroaryl), or a 6- membered heteroaryl group (also referred to as a C ₆ heteroaryl). In some embodiments, the heteroaryl group is a monocyclic heteroaryl group. In some embodiments, the heteroaryl group is a 5- 7 membered monocyclic heteroaryl group. In some embodiments, the heteroaryl group is a 6- membered monocyclic heteroaryl group. In some embodiments, the heteroaryl group is a fused bicyclic heteroaryl group. In some embodiments, the heteroaryl group is a 9- or 10-membered fused bicyclic heteroaryl group. In some embodiments, the heteroaryl group is a 10-membered fused bicyclic heteroaryl group.

In some embodiments of any of the above aspects of the invention, all alkyl, alkenyl, alkynyl, aryl, and benzyl groups in the compounds are unsubstituted. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows representative Western blotting membrane demonstrating NEK7 protein degradation induced by Compound 2 and Compound 25 of the present invention. Loading control: 0-Actin and Vinculin.

Figure 2 shows the level of IL-1β and IL-18 release by human PBMC-derived macrophages after treatment with Compound 2 and Compound 25. The results are normalized to DMSO control sample. For Compound 2 results from two independent experiments and for Compound 25 results from one independent experiment were shown. UT - cells not treated with LPS and nigericin; LPS - cel Is treated with LPS only; LPS+NIG - cells treated with LPS and nigericin, not treated with DMSO.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect of the present invention, there is provided a compound of Formula (la) or (lb): wherein: y is 0, 1 or 2; each of X ₁ and X ₂ is independently O or S;

L is H, -C(O)alkyl, or -CH ₂ (O)COR';

wherein each Z is independently C=O, CH ₂ or CH(C _1-2 alkyl);

Y is S, O or NH; each R is independently halogen, alkyl, haloalkyl, hydroxy, alkoxy, -NH ₂ , -NH R' or -

NR' ₂ ; each R' is independently alkyl or aryl; each n is independently 0, 1, 2 or 3; m is 0, 1 or 2; p is 0 or 1; ^b/c denotes the point of attachment to in Formula (la) or in Formula is a heterocycloalkyl group, and is a 6-membered monocyclic heteroaryl group or a 10-membered fused bicyclic heteroaryl group, wherein is either unsubstituted or is substituted with one or more R ³ , no substituents other than said one or more R ³ are present on and each R ³ is independently halogen, unsubstituted alkyl, haloalkyl, cycloalkyl, hydroxy, OR ¹ , aryl, benzyl, -C(O)R ¹ , or-NR ² C(O)R ¹ , wherein each R ¹ is independently unsubstituted alkyl, cycloalkyl, or aryl and each R ² is independently H, unsubstituted alkyl, or cycloalkyl; wherein in formula (lb):

(i) when is , Z is CH ₂ and n = 0, then s substituted with one or more R ³ ; —

(ii) when , Z is CH ₂ , n = 0, and is a 6-membered monocyclic heteroaryl group having two heteroatoms, the two heteroatoms are not adjacent to each other,

(iii) when , Z is CH ₂ , n = 0 and is a 6-membered monocyclic heteroaryl group substituted with one or more R ³ , then:

(a) carbon atoms adjacent to the carbon atom through which is attached to are unsubstituted;

(b) when R ³ is alkyl or O(alkyl), then is monosubstituted;

(c) when R ³ is O(alkyl), then the substitution is at a position meta or para to a heteroatom of the heteroaryl group; and (d) when R ³ is aryl or -NR ² C(O)R ₁ , then the substitution is at a position meter to a heteroatom of the heteroaryl group, and

(iv) when -membered fused bicyclic heteroaryl group substituted with one or more R ³ , then: each R ³ is present on the ring which contains the point of attachment to each R ³ is positioned ortho or meta to a heteroatom of the heteroaryl group;

R ³ is not Cl, methyl, iPr, cyclopropane, unsubstituted phenyl, hydroxy or OMe; when R ³ is OEt, then R ³ is positioned ortho to the heteroatom of the heteroaryl group; and when R ³ is NR ₂ COMe, then R ³ is positioned meta to the heteroatom of the heteroaryl group.

In some embodiments, in formula (lb):

(i) when , then Z is CH(C _1-2 alkyl) or C=O, and

(ii) when , then n is 1, 2, or 3.

In some embodiments, Z is CH ₂ or CH(C _1-2 alkyl).

In some embodiments,

In some embodiments, In some embodiments,

In some embodiments,

In some embodiments, contains one heteroatom. In some embodiments, the heteroatom is

N, S or 0. In some embodiments, the heteroatom is N. In some embodiments, the heteroatom is O.

In some embodiments, contains two heteroatoms. The heteroatoms may be independently selected from N, S and O.

In some embodiments, is a 5-10 membered heterocycloalkyl group.

In some embodiments, is a 5- or 6-membered heterocycloalkyl group.

In some embodiments, a pyrrolidine, piperidine, or oxane group. denotes the point of attachment to In some embodiments,

In some embodiments,

In some embodiments, is unsubstituted. In other embodiments, is substituted with one or more R ³ , wherein each R ³ is independently halogen, unsubstituted alkyl, haloalkyl, cycloalkyl, hydroxy, OR ¹ , aryl, benzyl, -C(O)R ¹ , or -NR^jOjR ¹ , wherein each R ¹ is independently unsubstituted alkyl, cycloalkyl or aryl and each R ² is independently H, unsubstituted alkyl or cycloalkyl; or wherein two R ³ on adjacent atoms of the heterocycloalkyl group, together with the atoms to which they are attached, form an aromatic ring; or wherein two R ³ on the same carbon atom of the heterocycloalkyl group, together with the carbon atom to which they are attached, form a C=O group.

In some embodiments, each R ³ is independently halogen, unsubstituted alkyl, haloalkyl, aryl, benzyl or -NHC(O)R ¹ ; or wherein two R ³ on the same carbon atom of the heterocycloalkyl group, together with the carbon atom to which they are attached, form a C=O group.

In other embodiments, two R ³ on adjacent atoms of the heterocycloalkyl group, together with the atoms to which they are attached, form an aromatic ring.

In some embodiments, each R ¹ is independently unsubstituted alkyl or aryl and each R ² is independently H or unsubstituted alkyl.

In some embodiments, r is an integer from 1-7, optionally from 1-3, and s is an integer from 1-9, optionally from 1-4. wherein a denotes the point of attachment to and wherein R ₃ is unsubstituted alkyl, aryl, benzyl, or -NR ² C(O)R ¹ .

In some embodiments, wherein R ₃ is unsubstituted alkyl, benzyl, or -NR ² C(O)R ¹ . In some such embodiments R ₃ is unsubstituted alkyl or benzyl. wherein R is F or alkyl. In some such embodiment

In some embodiments. contains one heteroatom. In some embodiments, the heteroatom is

N, S or 0. In some embodiments, the heteroatom is N. In some embodiments, the heteroatom is 0.

In other embodiments, contains two heteroatoms. The heteroatoms may be independently selected from N, S and O.

In some embodiments, a 6-membered monocyclic heteroaryl group.

In some embodiments, a pyridine group.

In some embodiments, In some embodiments, is a 10-membered fused bicyclic heteroaryl group.

In some embodiments, is a quinoline or isoquinoline group.

In some embodiments, wherein a denotes the point of attachment to

In some embodiments, is unsubstituted. In other embodiments, is substituted with one or more R ³ , wherein each R ³ is independently halogen, unsubstituted alkyl, haloalkyl, cycloalkyl, hydroxy, OR ¹ , aryl, benzyl, -C(O)R ¹ , or -NR ² C(O)R ¹ , wherein each R ¹ is independently unsubstituted alkyl, cycloalkyl or aryl and each R ² is independently H, unsubstituted alkyl or cycloalkyl. In some embodiments, each R ³ is independently halogen, unsubstituted alkyl, haloalkyl, aryl, benzyl or - NHC(O)R ¹ .

In some embodiments, each R ¹ is independently unsubstituted alkyl or aryl and each R ² is independently H or unsubstituted alkyl. In some embodiments,

In some embodiments.

In some embodiments, wherein a denotes the point of attachment to , and wherein R ₃ is aryl, haloalkyl or -NR ² C(O)R ¹ . In some embodiments, R ₃ is aryl or - NR ² C(O)R ¹ . In other embodiments, R ₃ is aryl or haloalkyl.

In some embodiments, L is hydrogen.

In some embodiments, X ₁ and X ₂ are O. In other embodiments, X ₁ is O and X ₂ is S. In other embodiments, wherein X ₁ is S and X ₂ is O. In other embodiments, X ₁ and X ₂ are S.

In some embodiments, Y is S.

In some embodiments, Z is C=O, CH ₂ or CHMe. In some embodiments, Z is CH ₂ or CHMe. In some embodiments, Z is CH ₂ . In some embodiments, each R is independently unsubstituted alkyl or halogen. In some embodiments, each R is independently Me or F.

In some embodiments, n is 0 or 1. In some embodiments, n is 0.

In some embodiments, m is 0.

In some embodiments, y = 1.

In some embodiments, the compound is of Formula (la). In other embodiments, the compound is of Formula (lb).

In some embodiments, the compound is selected from:

The present invention also provides a pharmaceutical composition comprising a compound of any of the embodiments described above.

The present invention also provides a compound or pharmaceutical composition as defined above for use in medicine. The present invention also provides a compound or pharmaceutical composition as defined above for use in the treatment of an inflammatory disease or condition, an auto-immune disease or condition, a respiratory disease or condition, a cardiovascular disease or condition, a gastrointestinal disease or condition, a renal disease or condition, a disease or condition of the central nervous system (CNS), a disease or condition of the endocrine system, an infection, a metabolic disease or condition, a liver disease or condition, an ocular disease or condition, a skin disease or condition, a lymphatic disease or condition, a psychological disease or condition, graft versus host disease or condition, allodynia, pain, a condition associated with diabetes, a condition associated with arthritis, a wound or burn, or cancer.

In a second aspect of the present invention, there is provided a compound of Formula (I): for use in a method of treating a disease or condition in a subject in need thereof, wherein: y is 0, 1 or 2; each of X ₁ and X ₂ is independently O or S;

L is H, -C(O)alkyl, or -CH ₂ (O)COR'; wherein each Z is independently C=0, CH ₂ or CH(C _1-2 alkyl);

Y is S, O or NH; each R is independently halogen, alkyl, haloalkyl, hydroxy, alkoxy, -NH ₂ , -NH R' or - NR' ₂ ; each R' is independently alkyl or aryl each n is independently 0, 1, 2 or 3; m is 0, 1 or 2; p is 0 or 1; denotes the point of attachment to denotes the point of attachment to and is a heterocyclic group; wherein the disease or condition is an inflammatory disease or condition, an auto-immune disease or condition, a respiratory disease or condition, a cardiovascular disease or condition, a gastrointestinal disease or condition, a renal disease or condition, a disease or condition of the central nervous system (CNS), a disease or condition of the endocrine system, a metabolic disease or condition, a liver disease or condition, an ocular disease or condition, a skin disease or condition, a lymphatic disease or condition, a psychological disease or condition, graft versus host disease or condition, allodynia, pain, a condition associated with diabetes, a condition associated with arthritis, or a wound or burn.

In some embodiments, the disease or condition is an inflammatory disease or condition, an autoimmune disease or condition, a respiratory disease or condition, a cardiovascular disease or condition, a renal disease or condition, a disease or condition of the central nervous system (CNS), a disease or condition of the endocrine system, a metabolic disease or condition, a liver disease or condition, an ocular disease or condition, a lymphatic disease or condition, a psychological disease or condition, graft versus host disease or condition, allodynia, pain, a condition associated with diabetes, a condition associated with arthritis, or a wound or burn. In some embodiments, the disease or condition is cryopyrin-associated periodic syndromes (CAPS), Muckle-Wells syndrome (MWS), familial cold autoinflammatory syndrome (FCAS), neonatal onset multisystem inflammatory disease (NOMID), familial Mediterranean fever (FMF), pyogenic arthritis, pyoderma gangrenosum and acne syndrome (PAPA), hyperimmunoglobulinemia D and periodic fever syndrome (HIDS), Tumour Necrosis Factor (TNF) Receptor-Associated Periodic Syndrome (TRAPS), systemic juvenile idiopathic arthritis, adult-onset Still's disease (AOSD), relapsing polychondritis, Schnitzler's syndrome, Sweet's syndrome, Behcet's disease, anti-synthetase syndrome, deficiency of interleukin 1 receptor antagonist (DIRA), haploinsufficiency of A20 (HA20), lupus nephritis, pulmonary arterial hypertension, idiopathic pulmonary fibrosis, amyotrophic lateral sclerosis, or gout.

In a third aspect of the present invention, there is provided a method of degrading NEK7 protein comprising contacting said protein with a compound of Formula (I): wherein: y is 0, 1 or 2; each of X ₁ and X ₂ is independently 0 or S;

L is H, -C(O)alkyl, or -CH ₂ (O)COR'; wherein each Z is independently C=0, CHz or CH(C ₁ -z alkyl);

Y is S, O or NH; each R is independently halogen, alkyl, haloalkyl, hydroxy, alkoxy, -NH2, -NH R' or -

NR' ₂ ; each R' is independently alkyl or aryl; each n is independently 0, 1, 2 or 3; m is 0, 1 or 2; p is 0 or 1; denotes the point of attachment to a heterocyclic group.

In some embodiments of the second or third aspects of the invention, , wherein is a heterocycloalkyl group, or wherein is a 6-membered monocyclic heteroaryl group or a 10-membered fused bicyclic heteroaryl group which is either unsubstituted or is substituted with one or more R ³ , wherein no substituents other than said one or more R ³ are present on and wherein each R ³ is independently halogen, unsubstituted alkyl, haloalkyl, cycloalkyl, hydroxy, OR ¹ , aryl, benzyl, - C(O)R ¹ , or -NR ² C(O)R ¹ , wherein each R ¹ is independently unsubstituted alkyl, cycloalkyl, or aryl and each R ² is independently H, unsubstituted alkyl, or cycloalkyl.

In some embodiments of the second or third aspects of the invention:

6-membered monocyclic heteroaryl group having two heteroatoms, the two heteroatoms are not adjacent to each other;

(iii) when a 6-membered monocyclic heteroaryl group substituted with one or more R ³ , then:

(a) carbon atoms adjacent to the carbon atom through which attached to are unsubstituted;

(b) when R ³ is alkyl or O(alkyl), then monosubstituted;

(c) when R ³ is O(alkyl), then the substitution is at a position meta or para to a heteroatom of the heteroaryl group; and

(d) when R ³ is aryl or -NR ₂ C(O)R ₁ , then the substitution is at a position meta to a heteroatom of the heteroaryl group; and (iv) when Z is CH ₂ , n = 0, and is a 10-membered fused bicyclic heteroaryl group substituted with one or more R ³ , then: each R ³ is present on the ring which contains the point of attachment to each R ³ is positioned ortho or meta to a heteroatom of the heteroaryl group; R ³ is not Cl, methyl, iPr, cyclopropane, unsubstituted phenyl, hydroxy or OIVle; when R ³ is OEt, then R ³ is positioned ortho to the heteroatom of the heteroaryl group; and when R ³ is NR ₂ COMe, then R ³ is positioned meta to the heteroatom of the heteroaryl group.

In some embodiments of the second or third aspects of the invention:

(i) when , n = 0 and then Z is CH(C _1-2 alkyl) or C=O, and

(ii) when , Z is CH ₂ and , then n is 1, 2, or

In some embodiments of the second or third aspects of the invention, Z is CH ₂ or CH(C _1-2 alkyl).

In some embodiments of the second or third aspects of the invention, is

In some embodiments,

In some embodiments,

In some embodiments of the second or third aspects of the invention, is

In some embodiments of the second or third aspects of the invention, is In some embodiments of the second or third aspects of the invention

In some embodiments of the second or third aspects of the invention

In some embodiments of the second or third aspects of the invention, contains one heteroatom. In some embodiments of the second or third aspects of the invention, contains two heteroatoms. The heteroatoms may be independently selected from N, S and O.

In some embodiments of the second or third aspects of the invention, , wherein is a heterocycloalkyl group.

In some embodiments of the second or third aspects of the invention, is a 5-10 membered heterocycloalkyl group

In some embodiments of the second or third aspects of the invention, is a 5- or 6-membered heterocycloalkyl group. In some embodiments of the second or third aspects of the invention, is a pyrrolidin e, piperidine, or oxane group.

In some embodiments of the second or third aspects of the invention, wherein ^a denotes the point of attachment to

In some embodiments of the second or third aspects of the invention,

In some embodiments of the second or third aspects of the invention,

In some embodiments of the second or third aspects of the invention, other embodiments of the second or third aspects of the invention more R ³ , wherein each R ³ is independently halogen, unsubstituted alkyl, haloalkyl, cycloalkyl, hydroxy, OR ¹ , aryl, benzyl, -C(O)R ¹ , or -NR ² C(O)R ¹ , wherein each R ¹ is independently unsubstituted alkyl, cycloalkyl or aryl and each R ² is independently H, unsubstituted alkyl or cycloalkyl; or wherein two R ³ on adjacent atoms of the heterocycloalkyl group, together with the atoms to which they are attached, form an aromatic ring; or wherein two R ³ on the same carbon atom of the heterocycloalkyl group, together with the carbon atom to which they are attached, form a C=O group. In some embodiments, each R ³ is independently halogen, unsubstituted alkyl, haloalkyl, aryl, benzyl or - NHC(O)Ri; or wherein two R ³ on the same carbon atom of the heterocycloalkyl group, together with the carbon atom to which they are attached, form a C=O group. In some embodiments, two R ³ on adjacent atoms of the heterocycloalkyl group, together with the atoms to which they are attached, form an aromatic ring.

In some embodiments of the second or third aspects of the invention wherein a denotes the point of attachment to r is an integer from 1-7, optionally from 1-3, and s is an integer from 1-9, optionally from 1-4.

In some embodiments of the second or third aspects of the invention

In some embodiments of the second or third aspects of the invention

In some embodiments of the second or third aspects of the invention, wherein ^a denotes the point of attachment to and wherein R ₃ is unsubstituted alkyl, aryl, benzyl, or -NR ² C(O)R ¹ .

In some embodiments of the second or third aspects of the invention,

, wherein R ₃ is unsubstituted alkyl, benzyl, or -NR ² C(O)R ¹ . In some embodiments, R ₃ is unsubstituted alkyl or benzyl. In some embodiments of the second or third aspects of the invention,

In some embodiments of the second or third aspects of the invention, In some embodiments of the second or third aspects of the invention wherein is a 6-membered monocyclic heteroaryl group or a 10-membered fused bicyclic heteroaryl group which is either unsubstituted or is substituted with one or more R ³ , wherein no substituents other than said one or more R ³ are present on and wherein each R ³ is independently halogen, unsubstituted alkyl, haloalkyl, cycloalkyl, hydroxy, OR ¹ , aryl, benzyl, -C(O)R ¹ , or -NR ² C(O)R ¹ , wherein each R ¹ is independently unsubstituted alkyl, cycloalkyl, or aryl and each R ² is independently H, unsubstituted alkyl, or cycloalkyl

In some embodiments of the second or third aspects of the invention, is a 6-membered monocyclic heteroaryl group.

In some embodiments of the second or third aspects of the invention, is a pyridine group.

In some embodiments of the second or third aspects of the invention, wherein denotes the point of attachment to

In some embodiments of the second or third aspects of the invention, is a 10-membered fused bicyclic heteroaryl group.

In some embodiments of the second or third aspects of the invention, is a quinoline or isoquinoline group. In some embodiments, wherein denotes the point of attachment to

In some embodiments of the second or third aspects of the invention, each R ¹ is independently unsubstituted alkyl or aryl and each R ² is independently H or unsubstituted alkyl.

In some embodiments of the second or third aspects of the invention, is unsubstituted.

In other embodiments is substituted with one or more R ³ , wherein each R ³ is independently halogen, unsubstituted alkyl, haloalkyl, cycloalkyl, hydroxy, OR ¹ , aryl, benzyl, -C(O)R ¹ , or -NR ² C(O)R ¹ , wherein each R ¹ is independently unsubstituted alkyl, cycloalkyl or aryl and each R ² is independently H, unsubstituted alkyl or cycloalkyl. In some embodiments, each R ³ is independently halogen, unsubstituted alkyl, haloalkyl, aryl, benzyl or -NR ² C(O)R ¹ . In some embodiments, each R ¹ is independently unsubstituted alkyl or aryl and each R ² is independently H or unsubstituted alkyl.

In some embodiments of the second or third aspects of the invention wherein denotes the point of attachment to and q is an integer from 1-4, optionally from

In some embodiments of the second or third aspects of the invention

In some embodiments of the second or third aspects of the invention

In some embodiments of the second or third aspects of the invention, wherein R ₃ is aryl, haloalkyl or -NR ² C(O)R ¹ .

In some embodiments of the second or third aspects of the invention, wherein R ₃ is aryl or -NR ² C(O)R ¹ . In some embodiments of the second or third aspects of the invention, s wherein R ₃ is aryl or haloalkyl.

In some embodiments of the second or third aspects of the invention L is hydrogen.

In some embodiments of the second or third aspects of the invention X ₁ and X ₂ are O. In other embodiments, X ₁ is O and X ₂ is S. In other embodiments, X ₁ is S and X ₂ is O. In other embodi ments, X ₁ and X ₂ are S.

In some embodiments of the second or third aspects of the invention Y is S.

In some embodiments of the second or third aspects of the invention, Z is C=O, CH ₂ or CHMe. In some embodiments, Z is CH ₂ or CHMe. In some embodiments, Z is CH ₂ .

In some embodiments of the second or third aspects of the invention, each R is independently unsubstituted alkyl or halogen. In some embodiments, each R is independently Me or F.

In some embodiments of the second or third aspects of the invention, n is 0 or 1. In some embodiments, n is 0.

In some embodiments of the second or third aspects of the invention, m is 0.

In some embodiments of the second or third aspects of the invention, y = 1.

In some embodiments of the second or third aspects of the invention, the compound is selected from:

In some embodiments of the second or third aspects of the invention, the compound is formulated in a pharmaceutical composition.

EXAMPLES

Synthesis of compounds

The reagents and solvents were used as received from the commercial sources. Proton nuclear magnetic resonance (NMR) spectra were recorded on 500MHz or 400MHz Bruker Avance spectrometers. The spectra are reported in terms of chemical shift (δ [ppm]), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, p = quintet, m= multiplet), coupling constant (J [Hz]), and integration. Chemical shifts are reported in ppm relative to dimethyl sulfoxide-d6 (δ 2.50) or chloroform-d (δ 7.26) as indicated in NMR spectra data. The samples were prepared by dissolving a dry sample (0.2 - 2 mg) in an appropriate deuterated solvent (0.7-1 mL).

LCMS measurements were collected using either Shimadzu Nexera X2/MS-2020 or Advion Expression CMS coupled to liquid chromatograph. All masses reported are the m/z of the protonated parent ions unless otherwise stated. The sample was dissolved in an appropriate solvent (e.g. DMSO, ACN, water) and was injected directly into the column using an automated sample handler.

The chemical names were generated using ChemDraw Professional v. 18.2.0.48 from PerkinElmer Informatics, Inc.

Abbreviations used in the following examples are presented below in the alphabetical order: ACN Acetonitrile

AcOEt Ethyl acetate

AIBN Azobisisobutyronitrile

Boc Tert-butoxycarbonyl

Bpin 4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl

Bu Butyl

Cbz Benzyloxycarbonyl

CRBN Cereblon

DCE 1,2-dichloroethane

DCM Dichloromethane

DDB1 Damaged DNA binding protein 1

DIPEA N, N-Diisopropylethylamine

DMC Dimethyl carbonate

DMEM Dulbecco's modified eagle medium

DMF N, N-Dimethylformamide

DMSO Dimethyl sulfoxide

DPBS Dulbecco's phosphate buffered saline

DTT Dithiothreitol

EDTA Ethylenediaminetetraacetic acid

Et Ethyl

Et ₂ O Diethyl ether

EtOH Ethanol

FA Formic acid

FBS Fetal bovine serum

HPLC High performance liquid chromatography

HRP Horseradish peroxidase

HTRF Homogeneous time resolved fluorescence iPr ₂ NH N, N-Diisopropylamine

KOAc Potassium acetate

LCMS Liquid chromatography mass spectrometry

LPS Lipopolysaccharide

Me Methyl

MeOH Methanol

MHz Megahertz NaOAc Sodium acetate

NBS N-Bromosuccinimide

NFM Non-fat dried milk

NMR Nuclear magnetic resonance

PBMC Peripheral blood mononuclear cells

PdCI ₂ (dppf) [1,1-Bis(diphenylphosphino)ferrocene]palladium(ll) dichloride

PdCI ₂ (dtbpf) (1,1-Bis(di-tert-butylphosphino)ferrocene]palladium(ll) dichlori de

PPI Protein-protein interaction ref Relative centrifugal force

RT Room temperature (temperature of between 20°C and 25°C)

Rt Retention time

SD Standard deviation

SDS Sodium dodecyl sulfate

TEA Triethylamine

TFA Trifluoroacetic acid

THF Tetrahydrofuran

Tris Tris(hydroxymethyl)aminomethane tBu Tert-butyl

General procedures

The synthesis of the compounds can be summarized in the following general procedures as set out below:

Example method 1: Reduction of the pyridine ring

Reaction Scheme 1: Reduction of the pyridine ring

To the solution of the substituted pyridine (1 equiv.) and PtO ₂ (0.1-0.4 equiv.) was added glacial acetic acid and the resulting slurry was stirred under hydrogen atmosphere (1-5 bar) at RT for 5-24 h. The solid particles were filtered off on the pad of Celite® and washed with EtOH. The filtrate was concentrated under reduced pressure and the crude product was purified by flash column chromatography and/or preparative HPLC.

Example method 2: Stille coupling

Alkyl = Me, Bu

Reaction Scheme 2: Stille coupling

The appropriate aryl bromide (1 equiv.), aryl trialkylstannane (1-1.5 equiv.), and palladium catalyst (0.05-0.2 equiv.) were purged with argon and dissolved in appropriate solvent (DMF, toluene, or 1,4- dioxane). The reaction mixture was stirred at 90-120°C for 5-24 h in the inert atmosphere. The solution was cooled to ambient temperature, filtered through pad of Celite® and concentrated under reduced pressure. The crude product was purified by flash column chromatography and/or preparative HPLC.

Example method 3: Piperidine-2, 6-dione ring formation

Reaction Scheme 3: Piperidine-2, 6-dione ring formation

The appropriate tert-butyl 5-amino-5-oxo-4-(l-oxoisoindolin-2-yl)pentanoate (1 equiv.) was dissolved in ACN and benzenesulfonic acid (1.5-2.5 equiv.) was added. The reaction mixture was stirred at 120- 140°C for 30-60 min under microwave irradiation. The volatiles were removed under reduced pressure and the crude product was purified by flash column chromatography and/or preparative HPLC. Example method 4: Reaction of methyl o-(haloalkyl)arylester with amine

Reaction Scheme 4: Reaction of methyl o-(haloalkyl)arylester with amine

The appropriate methyl o-(haloalkyl)arylester (1 equiv.) and amine hydrochloride (1-1.5 equiv.) were suspended in dry DMF or ACN. Base (2-5 equiv.) was added and the reaction was stirred at 60-120°C for 5-48 h. The volatiles were removed under reduced pressure, water was added and the solids were agitated for 1-6 h. The product was filtered, washed with water and Et ₂ O, and dried under reduced pressure. When needed, the product was purified by flash column chromatography and/or preparative HPLC.

Example method 5: Suzuki coupling

Reaction Scheme 5: Suzuki reaction

To the appropriate arylboronic acid pinacol ester (1-1.5 equiv.) in a mixture of dioxane-H ₂ O were added base (K ₂ CO ₃ , 2-6 equiv.), aryl bromide (1-1.5 equiv.), and palladium catalyst (0.05-0.15 equiv.). The reaction mixture was heated at 70-110°C for 8-48 h. After completion, the reaction mixture was diluted with water, extracted with AcOEt, the organic fraction was dried over Na ₂ SO ₄ and concentrated under reduced pressure. The product was purified by flash column chromatography and/or preparative HPLC. Example method 6: Bromination of the benzyl position

Reaction Scheme 6: Bromination of the benzyl position

To a solution of the appropriate aryl compound (1 equiv.) in appropriate solvent, NBS (1-2 equiv.) and AIBN (0.05-0.2 equiv.) were added and the reaction mixture was then stirred at 70-100°C for 8-48 h. After completion, the reaction mixture was cooled, diluted with water and extracted with AcOEt. The combined organic fractions were washed with brine, dried over Na ₂ SO ₄ and concentrated under reduced pressure. The product was purified by flash column chromatography.

Example 1: Synthesis of 3-(l-oxo-5-(pyridin-2-yl)isoindolin-2-yl)piperidlne-2, 6-dione (Compound

2(2))

Step 1: Methyl 2-(bromomethyl)-4-(pyridin-2-yl)benzoate was synthesized using the general procedure shown in Reaction Scheme 6 and Example Method 6, above (70% yield), using methyl 2- methyl-4-(pyridin-2-yl)benzoate (10 g, 44.2 mmol, 1 equiv.) as starting material, AIBN (0.2 equiv.) as initiator and DMC as solvent.

¹ H NMR (400 MHz, CDCI ₃ ) 68.73 (dt, J = 4.8, 1.5 Hz, 1H), 8.14 (d, J = 1.8 Hz, 1H), 8.08 (d, J = 8.2 Hz, 1H), 7.98 (dd, J = 8.2, 1.9 Hz, 1H), 7.84 - 7.74 (m, 2H), 7.30 (td, J = 5.0, 3.4 Hz, 1H), 5.05 (s, 2H), 3.97 (s, 3H).

Step 2: 3-(l-Oxo-5-(pyridin-2-yl)isoindolin-2-yl)piperidine-2, 6-dione was synthesized using the general procedure shown in Reaction Scheme 4 and Example Method 4, above (70% yield), using methyl 2- (bromomethyl)-4-(pyridin-2-yl)benzoate (8.0 g, 26.1 mmol, 1 equiv.) and 3-aminopiperidine-2, 6-dione hydrochloride (1.1 equiv.) as starting materials, TEA (3 equiv.) as base and DMF as solvent.

LCMS (ESI+) m/z 322.0 [M+H] ^{+ 1} H NMR (500 MHz, DMSO-d ₆ , 300K) δ 11.00 (s, 1H), 8.76 - 8.68 (m, 1H), 8.33 (s, 1H), 8.24 (dd, J = 8.0, 1.5 Hz, 1H), 8.08 (dd, J = 8.0, 1.1 Hz, 1H), 7.94 (td, J = 7.7, 1.8 Hz, 1H), 7.83 (d, J = 7.9 Hz, 1H), 7.43 (ddd, J = 7.5, 4.8, 1.0 Hz, 1H), 5.15 (dd, J = 13.3, 5.1 Hz, 1H), 4.55 (d, J = 17.3 Hz, 1H), 4.43 (d, J = 17. 3 Hz, 1H), 2.93 (ddd, J = 17.3, 13.7, 5.4 Hz, 1H), 2.61 - 2.56 (m, 1H), 2.42 (td, J = 13.2, 4.5 Hz, 1H), 2.04 (dtt, J = 12.8, 5.4, 2.7 Hz, 1H).

Example 2: Synthesis of 3-(l-oxo-5-(piperidin-2-yl)isoindolin-2-yl)piperidine-2,6-di one (Compound

Step 1: 3-(l-Oxo-5-(piperidin-2-yl)isoindolin-2-yl)piperidine-2, 6-dione (hydrochloride salt) was synthesized using the general procedure shown in Reaction Scheme 1 and Example Method 1, above (44% yield), using 3-(l-oxo-5-(pyridin-2-yl)isoindolin-2-yl)piperidine-2, 6-dione (1.0 g, 3.1 mmol, 1 equiv.) as a starting material.

LCMS (ESI+) m/z 328.2 [M+H] ⁺

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.00 (s, 1H), 9.47 (s, 1H), 9.31 (d, J = 10.9 Hz, 1H), 7.81 (dd, J = 9.2, 6.8 Hz, 2H), 7.75 - 7.66 (m, 1H), 5.13 (dd, J = 13.4, 5.0 Hz, 1H), 4.49 (dd, J = 17.3, 9.7 Hz, 1H), 4.35 (dd, J = 17.6, 9.2 Hz, 2H), 3.35 (d, J = 12.5 Hz, 1H), 3.12 - 2.99 (m, 1H), 2.92 (ddd, J = 18.2, 13.7, 5.3 Hz, 1H), 2.60 (d, J = 17.7 Hz, 1H), 2.41 (tt, J = 13.2, 6.9 Hz, 1H), 2.06 - 1.97 (m, 1H), 1.97 - 1.75 (m, 5H), 1.65 (d, J = 12.9 Hz, 1H).

Example 3: Synthesis of 2-(2,6-dioxopiperidin-3-yl)-5-(piperidin-2-yl)isoindoline-l, 3-dione

(Compound 1) Step 1: In a vial were placed dimethyl 4-(pyridin-2-yl)phthalate (125.0 mg, 0.46 mmol, 1 equiv.), diphenylamine (311.9 mg, 1.84 mmol, 4 equiv.) and tris(pentafluorophenyl)borane (23.6 mg, 0.046 mmol, 0.1 equiv.). Dry toluene (5 mL) was added followed by diphenylsilane (0.428 mL, 2.3 mmol, 5 equiv.), and the reaction mixture was refluxed for 18 h. Water (2 mL) and NaHCOs (194 mg, 2.3 mmol, 5 equiv.) were added followed by benzyl chloroformate (0.132 mL, 0.922 mmol, 2 equiv.) and the reaction mixture was stirred at RT for 18 h. The reaction mixture was acidified by addition of 10% citric acid and extracted with DCM. The organic fractions were dried over Na ₂ SO4, concentrated under reduced pressure and the crude product was purified by flash column chromatography to give dimethyl 4-(l-((benzyloxy)carbonyl)piperidin-2-yl)phthalate (71.0 mg, 37% yield).

LCMS (ESI+) 412.1 m/z [M+H] ⁺

Step 2: Dimethyl 4-(l-((benzyloxy)carbonyl)piperidin-2-yl)phthalate (55.0 mg, 0.134 mmol, 1 equiv.) was dissolved in MeOH (5 mL) and IM LiOH (3 mL, 3 mmol, 22.4 equiv.) was added. The reaction mixture was stirred at RT for 18 h, concentrated under reduced pressure and acidified by IM HCI. The product was extracted with DCM, dried over Na ₂ SO ₄ and concentrated under reduced pressure to give

4-( l-((benzy I oxy Jcarbony I ) pi peri d i n -2-yl ) p htha lie acid (51 mg, 100% yield) which was used di rectly for the next step.

LCMS (ESI+) m/z 384.1 [M+H] ⁺

Step 3: 4-(l-((Benzyloxy)carbonyl)piperidin-2-yl)phthalic acid (72.0 mg, 0.188 mmol, 1 equiv.) was dissolved in acetic anhydride (1 mL, 10.6 equiv.), and the solution was refluxed for 1 h. The volatiles were removed under reduced pressure to obtain crude benzyl 2-(l,3-dioxo-l,3-dihydroisobenzofuran-

5-yl)piperidine-l-carboxylate (68 mg, 99% yield) which was used directly for the next step.

Step 4: Benzyl 2-(l,3-dioxo-l,3-dihydroisobenzofuran-5-yl)piperidine-l-carb oxylate (34.0 mg, 0.093 mmol, 1 equiv.), 3-aminopiperidine-2, 6-dione hydrochloride (16.8 mg, 0.1 mmol, 1.1 equiv.) and KOAc (28.3 mg, 0.29 mmol, 3.1 equiv.) were dissolved in glacial acetic acid (0.57 mL) and the reaction mixture was stirred at 90°C for 18 h. The volatiles were removed under reduced pressure and the crude product was purified by preparative HPLC to give benzyl 2-(2-(2,6-dioxopiperidin-3-yl)-l,3- dioxoisoindolin-5-yl)piperidine-l-carboxylate (16.9 mg, 38% yield).

LCMS (ESI+) m/z 476.0 [M+H] ⁺

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 11.12 (s, 1H), 7.91 (d, J = 7.9 Hz, 1H), 7.75 - 7.70 (m, 1H), 7.70 - 7.65 (m, 1H), 7.40 - 7.21 (m, 5H), 5.47 (t, J = 4.4 Hz, 1H), 5.22 - 5.05 (m, 3H), 4.06 (d, J = 12.8 Hz, 1H), 2.95 - 2.83 (m, 2H), 2.61 - 2.53 (m, 2H), 2.11 - 2.01 (m, 2H), 1.91 (td, J = 12.2, 10.6, 5.3 Hz, 1H), 1.60 (d, J = 11.6 Hz, 2H), 1.54 - 1.41 (m, 1H), 1.25 (d, J = 11.9 Hz, 1H).

Step 5: Benzyl 2-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-5-yl)pipe ridine-l-ca rboxylate (16.8 mg, 0.035 mmol, 1 equiv.) and palladium on activated carbon (5 mg, 10% wt.) were suspended in EtOH (2 mL) and bubbled with argon for 15 min. The reaction mixture was then bubbled with hydrogen for 90 min. at RT until full conversion was accomplished. The solid particles were filtered- off and the volatiles were removed under reduced pressure. The crude product was purified by preparative HPLC to give 2-(2,6-Dioxopiperidin-3-yl)-5-(piperidin-2-yl)isoindoline-l, 3-dione (as formic acid salt, 10.0 mg, 74% yield).

LCMS (ESI+) m/z 342.0 [M+H] ⁺

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 11.11 (s, 1H), 8.20 (s, 1H), 7.92 (s, 1H), 7.87 (d, J = 1.0 Hz, 2H), 5.14 (dd, J = 12.8, 5.4 Hz, 1H), 3.83 (dd, J = 11.1, 2.6 Hz, 1H), 3.11 (d, J = 11.7 Hz, 1H), 2.89 (ddd, J = 16.8, 13.7, 5.4 Hz, 1H), 2.71 (td, J = 11.8, 2.8 Hz, 1H), 2.64 - 2.54 (m, 2H), 2.53 (s, 1H), 2.11 - 2.00 (m, 1H), 1.86 - 1.73 (m, 2H), 1.61 (d, J = 10.4 Hz, 1H), 1.56 - 1.40 (m, 2H), 1.40 - 1.30 (m, 1H).

Example 4: Synthesis of 3-(l-oxo-5-((/?)-piperidin-2-yl)isoindolin-2-yl)piperidine-2 , 6-dione

(Compound 9)

Step 1: 5-(Pyridin-2-yl)isobenzofuran-1(3H)-one was synthesized using the general procedure shown in Reaction Scheme 2 and Example Method 2, above (84% yield), using 5-bromoisobenzofuran-l(3H)- one (5.0 g, 23.4 mmol, 1 equiv.) and 2-(tributylstannyl)pyridine (1.4 equiv.) as starting materials, Pd(RPh ₃ )4 (0.1 equiv.) as catalyst and 1,4-dioxane as solvent.

LCMS (ESI+) m/z 212.1 [M+H] ⁺

Step 2: To a solution of 5-(pyridin-2-yl)isobenzofuran-l(3H)-one (2.10 g, 9.94 mmol, 1 equiv.) in MeOH (5 mL) were added PtO ₂ (0.13 equiv.) and di-tert-butyl dicarbonate (4.33 g, 19.9 mmol, 2 equiv.). The reaction mixture was stirred under hydrogen atmosphere for 20 h at RT. After completion, the reaction mixture was filtered through Celite®, and the filtrate was concentrated under reduced pressure to give tert-butyl 2-(l-oxo-l,3-dihydroisobenzofuran-5-yl)piperidine-l-carboxyl ate (2.50 g, 79% yield) as off-white solid.

LCMS (ESI+) m/z 318.3 [M+H] ⁺

Step 3: To a solution of tert-butyl 2-(l-oxo-l,3-dihydroisobenzofuran-5-yl)piperidine-l-carboxyl ate (2.50 g, 7.88 mmol, 1 equiv.) in THF (10 mL) and water (40 mL) was added NaOH (788 mg, 19.7 mmol,

2.5 equiv.) at 0°C and stirred at RT for 1.5 h. After completion, the reaction mixture was acidified to pH ca. 5 by 10% HCI and the product was extracted with AcOEt. The combined organic fractions were dried over Na2SO4 and concentrated under reduced pressure to give 4-(l-(tert- butoxycarbonyl)piperidin-2-yl)-2-(hydroxymethyl)benzoic acid (2.10 g, 79% yield) as a white solid.

LCMS (ESI+) m/z 336.2 [M+H] ⁺

Step 4: To a solution of 4-(l-(tert-butoxycarbonyl)piperidin-2-yl)-2-(hydroxymethyl)b enzoic acid (700 mg, 2.08 mmol, 1 equiv.) in MeOH (8 mL) and AcOEt (8 mL) was added trimethylsilyldiazomethane (5.0 mL, 5 equiv.) at -10°C and stirred at that temperature for 30 min. After completion, the reaction was quenched with ice-water, extracted with AcOEt, dried over Na ₂ SO4 and concentrated under reduced pressure to give tert-butyl 2-(3-(hydroxymethyl)-4- (methoxycarbonyl)phenyl)piperidine-l-carboxylate (610 mg, crude), which was forwarded to next step without purification.

LCMS (ESI+) m/z 350.6 [M+H] ⁺

Step 5: To a solution of tert-butyl 2-(3-(hydroxymethyl)-4-(methoxycarbonyl)phenyl)piperidine-l- carboxylate (600 mg, 1.71 mmol, 1 equiv.) in THF (15 mL) were added CBr ₄ (850 mg, 2.57 mmol,

1.5 equiv.) and PPh ₃ (810 mg, 3.07 mmol, 1.8 equiv.) at 0°C and stirred at RT for 16 h. After completion of the reaction, solid precipitate was filtered on sintered funnel and filtrate was concentrated under reduced pressure. Crude was purified by flash column chromatography to yield tert-butyl 2-(3- (bromomethyl)-4-(methoxycarbonyl)phenyl)piperidine-l-carboxy late (500 mg, 71% yield after 2 steps) as a colourless liquid.

LCMS (ESI+) m/z 312.3 [M-BOC+H] ⁺

Step 6: The enantiomers of tert-butyl 2-(3-(bromomethyl)-4-(methoxycarbonyl)phenyl)piperidine-l- carboxylate were separated by chiral HPLC (Chiralcel OJ-H, Hexane/EtOH 85/15 + 0.1% iPr ₂ N H) to give tert-butyl (R)-2-(3-(bromomethyl)-4-(methoxycarbonyl)phenyl)piperidine- l-carboxylate R _t 5.05 min, CHCl ₃ ) and tert-butyl (S)-2-(3-(bromomethyl)-4- (methoxycarbonyl)phenyl)piperidine-l-carboxylate R _t 6.17 min,

Step 7: Tert-butyl (2R)-2-(2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindolin-5-yl)pip eridine-l-carboxylate was synthesized using the general procedure shown in Reaction Scheme 4 and Example Method 4, above (62% yield), using tert-butyl (R)-2-(3-(bromomethyl)-4-(methoxycarbonyl)phenyl)piperidine- l- carboxylate (28.0 mg, 0.068 mmol, 1 equiv.) and 3-aminopiperidine-2, 6-dione hydrochloride (1.4 equiv.) as starting materials, DIPEA (5 equiv.) as base and ACN as solvent.

LCMS (ESI+) m/z 428.3 [M+H] ⁺

Step 8: Tert-butyl (2R)-2-(2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindolin-5-yl)pip eridine-l-carboxylate (18.0 mg, 0.042 mmol, 1 equiv.) was dissolved in TFA (5 mL), stirred at RT for 30 min and concentrated under reduced pressure. The product was purified by flash column chromatography to give 3-(l-oxo- 5-((R)-piperidin-2-yl)isoindolin-2-yl)piperidine-2, 6-dione (8.0 mg, 51% yield, formic acid salt).

LCMS (ESI+) m/z 328.1 [M+H] ^{+ 1} H NMR (500 MHz, DMSO-d ₆ ) δ 11.00 (s, 1H), 7.75 (d, J = 7.9 Hz, 1H), 7.67 (s, 1H), 7.57 (dt, J = 7.8, 1.9 Hz, 1H), 5.13 (dd, J = 13.3, 5.1 Hz, 1H), 4.48 (dd, J = 17.3, 7.3 Hz, 1H), 4.35 (dd, J = 17.3, 7.6 Hz, 1H), 4.00 (d, J = 9.4 Hz, 1H), 3.00 - 2.80 (m, 2H), 2.68 - 2.58 (m, 1H), 2.42 (dd, J = 13.1, 4.5 Hz, 1H), 2.03 (dtd, J = 12.7, 5.4, 2.4 Hz, 1H), 1.86 (d, J = 10.7 Hz, 2H), 1.71 (s, 1H), 1.63 - 1.48 (m, 3H).

Example 5: Synthesis of 3-(l-oxo-5-((S)-piperidin-2-yl)isoindolin-2-yl)piperidine-2, 6-dione

(Compound 10) Step 1: Tert-butyl (2S)-2-(2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindolin-5-yl)pip eridine-l-ca rboxylate was synthesized using the general procedure shown in Reaction Scheme 4 and Example Method 4, above (67% yield), using tert-butyl (S)-2-(3-(bromomethyl)-4-(methoxycarbonyl)phenyl)piperidine- 1- carboxylate (29.0 mg, 0.07 mmol, 1 equiv.) and 3-aminopiperidine-2, 6-dione hydrochloride (1.26 equiv.) as starting materials, DIPEA (5 equiv.) as base and ACN as solvent.

LCMS (ESI+) m/z 428.2 [M+H] ⁺

Step 2: Tert-butyl (2S)-2-(2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindolin-5-yl)pip eridine-l-ca rboxylate (20.0 mg, 0.047 mmol, 1 equiv.) was dissolved in TFA (2 mL), stirred at RT for 30 min and concentrated under reduced pressure. The product was purified by flash column chromatography to give 3-(l-oxo- 5-((S)-piperidin-2-yl)isoindolin-2-yl)piperidine-2, 6-dione (12.0 mg, 69% yield, formic acid salt).

LCMS (ESI+) m/z 328.1 [M+H] ⁺

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 11.00 (s, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.68 (s, 1H), 7.58 (dt, J = 7.9, 2.0 Hz, 1H), 5.14 (dd, J = 13.3, 5.1 Hz, 1H), 4.48 (dd, J = 17.3, 7.7 Hz, 1H), 4.35 (dd, J = 17.3, 7.8 Hz, 1H), 4.07 (d, J = 10.4 Hz, 1H), 2.93 (ddt, J = 15.0, 11.9, 6.0 Hz, 3H), 2.63 (ddd, J = 17.3, 4.4, 2.2 Hz, 1H), 2.47 - 2.37 (m, 1H), 2.03 (dtd, J = 12.8, 5.4, 2.3 Hz, 1H), 1.87 (t, J = 5.1 Hz, 2H), 1.74 (d, J = 9.4 Hz, 1H), 1.60 (s, 3H).

Example 6: Synthesis of 3-(l-oxo-5-(pyrrolidin-2-yl)isoindolin-2-yl)piperidine-2, 6-dione (Compound 6)

Step 1: To a solution of tert-butyl 2-(l-oxo-l,3-dihydroisobenzofuran-5-yl)pyrrolidine-l-carboxy late (130 mg, 0.43 mmol, 1 equiv.) in a mixture of THF, MeOH and water (3 mL, 1:1:1) was added NaOH (69.0 mg, 1.71 mmol, 4 equiv.) and the reaction mixture was stirred at RT for 2 h. The volatiles were removed under reduced pressure and the residue was dissolved in water (30 mL). The solution was washed with AcOEt, and then acidified by 1M HCI. The product was extracted with AcOEt, the combined organic layers were washed with water and brine, dried over Na2SO ₄ , and concentrated under reduced pressure to give 4-(l-(tert-butoxycarbonyl)pyrrolidin-2-yl)-2-(hydroxymethyl) benzoic acid (90.0 mg, 65% yield) as white solid.

LCMS (ESI+) m/z 322.4 [M+H] ⁺

Step 2: To a solution of 4-(l-(tert-butoxycarbonyl)pyrrolidin-2-yl)-2-(hydroxymethyl) benzoic acid (250 mg, 0.78 mmol, 1 equiv.) in MeOH (3 mL) and AcOEt (3 mL) was added trimethylsilyldiazomethane (1.17 mL, 2.33 mmol, 3 equiv., 2M in Et _z O) dropwise at -10°C. The reaction mixture was then stirred for 2 h at -10°C, quenched by addition of water and extracted by AcOEt. The combined organic layer was washed with water, brine, dried over Na2SO ₄ , and concentrated under reduced pressure to give crude tert-butyl 2-(3-(hydroxymethyl)-4- (methoxycarbonyl)phenyl)pyrrolidine-l-carboxylate (250 mg, 95% yield) which was used for the next step without further purification.

Step 3: To a solution of tert-butyl 2-(3-(hydroxymethyl)-4-(methoxycarbonyl)phenyl)pyrrolidine-l - carboxylate (1.10 g, 3.284 mmol, 1 equiv.) in THF (20mL) were added PPh ₃ (2.58 g, 9.851 mmol, 3 equiv.) and CBr ₄ (3.27 g, 9.851 mmol, 3 equiv.). The reaction mixture was stirred for 1 h at RT, quenched by addition of water and the product was extracted with AcOEt. The combined organic layers were washed with water, brine, dried over Na2SO ₄ and concentrated under reduced pressure. The crude product was purified by flash column chromatography to give tert-butyl 2-(3- (bromomethyl)-4-(methoxycarbonyl)phenyl)pyrrolidine-l-carbox ylate (310 mg, 23% yield).

LCMS (ESI+) m/z 398.1 [M+H] ⁺

Step 4: Tert-butyl 2-(2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindolin-5-yl)pyrrolid ine-l-carboxylate was synthesized using the general procedure shown in Reaction Scheme 4 and Example Method 4, above (62% yield), using tert-butyl 2-(3-(bromomethyl)-4-(methoxycarbonyl)phenyl)pyrrolidine-l- carboxylate (50.0 mg, 0.126 mmol, 1 equiv.) and 3-aminopiperidine-2, 6-dione hydrochloride (1.2 equiv.) as starting materials, DIPEA (5 equiv.) as base and ACN as solvent.

LCMS (ESI+) m/z 413.8 [M+H] ⁺

¹ H NMR (500 MHz, DMSO-d ₆ , 353K) δ 10.64 (s, 1H), 7.67 (d, J = 7.9 Hz, 1H), 7.40 (s, 1H), 7.33 (d, J = 7.5 Hz, 1H), 5.12 - 5.01 (m, 1H), 4.97 - 4.86 (m, 1H), 4.45 (dd, J = 16.8, 6.8 Hz, 1H), 4.36 (dd, J = 16.9, 7.8 Hz, 1H), 3.64 - 3.52 (m, 2H), 2.95 - 2.81 (m, 1H), 2.72 - 2.61 (m, 1H), 2.44 - 2.26 (m, 2H), 2.19 - 2.02 (m, 1H), 1.93 - 1.84 (m, 2H), 1.83 - 1.74 (m, 1H), 1.27 (s, 9H). Step 5: To a solution of tert-butyl 2-(2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindolin-5-yl)pyrrolid ine-l- carboxylate (20.0 mg, 0.048 mmol, 1 equiv.) in 1,4-dioxane (2 mL) and water (0.5 mL) was added concentrated HCI (0.5 mL). The resulting mixture was stirred at RT for 3 h, concentrated unde r reduced pressure and purified by preparative HPLC to give 3-(l-oxo-5-(pyrrolidin-2-yl)isoindolin-2- yl)piperidine-2, 6-dione (12.0 mg, 67% yield, formic acid salt).

LCMS (ESI+) m/z 313.9 [M+H] ⁺

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 10.98 (s, 1H), 7.68 (d, J = 7.8 Hz, 1H), 7.64 (s, 1H), 7.53 (d, J = 7.9 Hz, 1H), 5.11 (dd, J = 13.4, 5.1 Hz, 1H), 4.44 (dd, J = 17.3, 3.2 Hz, 1H), 4.35 -4.26 (m, 2H), 3.11 (dt, J = 10.1, 6.8 Hz, 1H), 3.06 - 2.97 (m, 1H), 2.92 (ddd, J = 17.4, 13.7, 5.4 Hz, 1H), 2.61 (ddd, J = 17.2, 4.2, 2.1 Hz, 1H), 2.40 (qd, J = 13.4, 4.5 Hz, 1H), 2.24 (dtd, J = 12.3, 7.7, 4.8 Hz, 1H), 2.01 (dtd, J = 12.6, 5.2, 2.1 Hz, 1H), 1.94 - 1.77 (m, 2H), 1.61 (dq, J = 12.2, 8.4 Hz, 1H).

Example 7: Synthesis of 3-(4-methyl-l-oxo-5-(piperidin-2-yl)isoindolin-2-yl)piperidi ne-2,6-dione

(Compound 19)

Step 1: 3-(5-Bromo-4-methyl-l-oxoisoindolin-2-yl)piperidine-2, 6-dione was synthesized using the general procedure shown in Reaction Scheme 4 and Example Method 4, above (13% yield), using methyl 4-bromo-2-(bromomethyl)-3-methylbenzoate (3.00 g, 9.3 mmol, 1 equiv.) and 3- aminopiperidine-2, 6-dione hydrochloride (1.1 equiv.) as starting materials, DIPEA (5 equiv.) as base and DMF as solvent.

LCMS (ESI+) m/z 337.1 [M+H] ⁺

Step 2: 3-(4-Methyl-l-oxo-5-(pyridin-2-yl)isoindolin-2-yl)piperidine -2,6-dione was synthesized using the general procedure shown in Reaction Scheme 2 and Example Method 2, above (96% yield), using 3-(5-bromo-4-methyl-l-oxoisoindolin-2-yl)piperidine-2, 6-dione (70.0 mg, 0.21 mmol, 1 equiv.) and 2- (trimethylstannyl)pyridine (1.5 equiv.) as starting materials, Pd(PPh ₃ ) ₄ (0.05 equiv.) as catalyst and toluene as solvent.

LCMS (ESI+) m/z 336.1 [M+H] ⁺

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 11.00 (s, 1H), 8.71 (ddd, J = 4.9, 1.8, 0.9 Hz, 1H), 7.93 (td, J = 7.7, 1.8 Hz, 1H), 7.65 (d, J = 7.8 Hz, 1H), 7.61 - 7.52 (m, 2H), 7.43 (ddd, J = 7.6, 4.8, 1.1 Hz, 1H), 5.17 (dd, J =

13.3, 5.1 Hz, 1H), 4.51 (d, J = 17.2 Hz, 1H), 4.34 (d, J = 17.2 Hz, 1H), 3.00 - 2.88 (m, 1H), 2.64 - 2.59 (m, 1H), 2.48 - 2.40 (m, 1H), 2.30 (s, 3H), 2.08 - 2.00 (m, 1H).

Step 3: 3-(4-Methyl-l-oxo-5-(piperidin-2-yl)isoindolin-2-yl)piperidi ne-2,6-dione (formic acid salt) was synthesized using the general procedure shown in Reaction Scheme 1 and Example Method 1, above (32% yield), using 3-(4-methyl-l-oxo-5-(pyridin-2-yl)isoindolin-2-yl)piperidine -2, 6-dione (38.0 mg, 0.113 mmol, 1 equiv.) as starting material.

LCMS (ESI+) m/z 342.1 [M+H] ⁺

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 10.98 (s, 1H), 7.69 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 7.9 Hz, 1H), 5.12 (dd, J = 13.3, 5.1 Hz, 1H), 4.42 (d, J = 17.1 Hz, 1H), 4.25 (d, J = 17.1 Hz, 1H), 4.03 (dd, J = 11.2, 2.5 Hz, 1H), 3.20 - 3.14 (m, 1H), 2.92 (ddd, J = 17.3, 13.7, 5.4 Hz, 1H), 2.83 (td, J = 11.8, 2.9 Hz, 1H), 2.61 (ddd, J =

17.3, 4.4, 2.3 Hz, 1H), 2.42 (qd, J = 13.3, 4.5 Hz, 1H), 2.30 (s, 3H), 2.01 (dtd, J = 12.7, 5.3, 2.3 Hz, 1H), 1.83 (dt, J = 10.0, 3.2 Hz, 1H), 1.79 - 1.70 (m, 1H), 1.69 - 1.61 (m, 1H), 1.60 - 1.47 (m, 2H), 1.40 (qd, J = 12.5, 3.7 Hz, 1H).

Example 8: Synthesis of 3-(6-methyl-l-oxo-5-(piperidin-2-yl)isoindolin-2-yl)piperidi ne-2,6-dione

(Compound 20)

Step 1: 5-Bromo-6-methylisobenzofuran-l(3H)-one (1.00 g, 4.44 mmol, 1 equiv.) was dissolved in

EtOH (15 mL) and DCE (15 mL) at 0°C. Thionyl chloride (1 mL, 1.9 equiv.) was added and the reaction mixture was refluxed for 16 h. The volatiles were removed under reduced pressure and the residue was neutralized using NaHCO ₃ .The product was extracted into AcOEt, the organic layer was dried over Na ₂ SO ₄ , and concentrated under reduced pressure. The crude product purified by flash column chromatography to give ethyl 4-bromo-2-(chloromethyl)-5-methylbenzoate (800 mg, 61% yield).

¹ H NMR (400 MHz, CDCI ₃ ) 6 7.83 (s, 1H), 7.71 (s, 1H), 4.96 (s, 2H), 4.39 (q, J = 7.1 Hz, 2H), 2.43 (s, 3H), 1.41 (t, J = 7.1 Hz, 3H).

Step 2: 3-(5-Bromo-6-methyl-l-oxoisoindolin-2-yl)piperidine-2, 6-dione was synthesized using the general procedure shown in Reaction Scheme 4 and Example Method 4, above (59% yield), using ethyl 4-bromo-2-(chloromethyl)-5-methylbenzoate (800 mg, 2.77 mmol, 1 equiv.) and 3-aminopiperidine- 2, 6-dione hydrochloride (1.4 equiv.) as starting materials, DIPEA (3 equiv.) as base and ACN as solvent.

LCMS (ESI+) m/z 335.3 [M+H] ⁺

Step 3: 3-(6-Methyl-l-oxo-5-(pyridin-2-yl)isoindolin-2-yl)piperidine -2, 6-dione was synthesized using the general procedure shown in Reaction Scheme 2 and Example Method 2, above (91% yield), using 3-(5-bromo-6-methyl-l-oxoisoindolin-2-yl)piperidine-2, 6-dione (55.0 mg, 0.163 mmol, 1 equiv.) and 2-(tributylstannyl)pyridine (1.3 equiv.) as starting materials, Pd(PPh ₃ ) ₄ (0.08 equiv.) as catalyst and 1,4- dioxane as solvent.

LCMS (ESI+) m/z 336.0 [M+H] ⁺

Step 4: 3-(6-Methyl-l-oxo-5-(piperidin-2-yl)isoindolin-2-yl)piperidi ne-2, 6-dione (formic acid salt) was synthesized using the general procedure shown in Reaction Scheme 1 and Example Method 1, above (69% yield), using 3-(6-methyl-l-oxo-5-(pyridin-2-yl)isoindolin-2-yl)piperidine -2, 6-dione (45.0 mg, 0.134 mmol, 1 equiv.) as starting material.

LCMS (ESI+) m/z 342.1 [M+H] ⁺

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 10.99- 10.94 (m, 1H), 7.75 (s, 1H), 7.52 (s, 1H), 5.09 (ddd, J = 13.3, 5.1, 3.3 Hz, 1H), 4.39 (t, J = 16.8 Hz, 1H), 4.26 (dd, J = 17.0, 14.5 Hz, 1H), 3.90 (dt, J = 11.0, 2.4 Hz, 1H), 3.15 - 3.11 (m, 2H), 2.90 (ddd, J = 17.3, 13.7, 5.4 Hz, 1H), 2.81 - 2.74 (m, 1H), 2.60 (ddd, J = 17.3, 4.5, 2.4 Hz, 1H), 2.42 (s, 3H), 1.98 (dtt, J = 12.9, 5.4, 2.6 Hz, 1H), 1.82 (d, J = 7.2 Hz, 1H), 1.77 - 1.70 (m, 1H), 1.63 (d, J = 9.1 Hz, 1H), 1.54 - 1.45 (m, 2H), 1.31 (dd, J = 11.4, 5.4 Hz, 1H).

Example 9: Synthesis of 3-(7-methyl-l-oxo-5-(piperidin-2-yl)isoindolin-2-yl)piperidi ne-2,6-dione

(Compound 21)

Step 1: 3-(5-Bromo-7-methyl-l-oxoisoindolin-2-yl)piperidine-2, 6-dione was synthesized using the general procedure shown in Reaction Scheme 4 and Example Method 4, above (17% yield), using methyl 4-bromo-2-(bromomethyl)-6-methylbenzoate (1.70 g, 5.3 mmol, 1 equiv.) and 3- aminopiperidine-2, 6-dione hydrochloride (1.2 equiv.) as starting materials, DIPEA (4 equiv.) as base and ACN as solvent.

LCMS (ESI+) m/z 337.1 [M+H] ⁺

Step 2: 3-(7-Methyl-l-oxo-5-(pyridin-2-yl)isoindolin-2-yl)piperidine -2, 6-dione was synthesized using the general procedure shown in Reaction Scheme 2 and Example Method 2, above (61% yield), using 3-(5-bromo-7-methyl-l-oxoisoindolin-2-yl)piperidine-2, 6-dione (98.0 mg, 0.291 mmol, 1 equiv.) and 2-(tributylstannyl)pyridine (1.2 equiv.) as starting materials, Pd(PPh3)4 (0.05 equiv.) as catalyst and 1,4- dioxane as solvent.

LCMS (ESI+) m/z 336.0 [M+H] ⁺

Step 3: 3-(7-Methyl-l-oxo-5-(piperidin-2-yl)isoindolin-2-yl)piperidi ne-2, 6-dione (formic acid salt) was synthesized using the general procedure shown in Reaction Scheme 1 and Example Method 1, above (66% yield), using 3-(7-methyl-l-oxo-5-(pyridin-2-yl)isoindolin-2-yl)piperidine -2, 6-dione (58.0 mg, 0.173 mmol, 1 equiv.) as a starting material.

LCMS (ESI+) m/z 342.1 [M+H] ⁺

NMR (500 MHz, DMSO-d ₆ ) δ 10.96 (s, 1H), 7.40 (s, 1H), 7.27 (s, 1H), 5.06 (dd, J = 13.3, 5.2 Hz, 1H), 4.36 (dd, J = 17.2, 6.8 Hz, 1H), 4.23 (dd, J = 17.2, 7.0 Hz, 1H), 3.72 (dd, J = 10.7, 2.6 Hz, 1H), 3.11 (dt, J = 10.8, 2.5 Hz, 1H), 2.90 (ddd, J = 17.3, 13.7, 5.4 Hz, 1H), 2.72 (td, J = 11.8, 3.0 Hz, 1H), 2.63 - 2.55 (m, 4H), 2.45 - 2.31 (m, 1H), 1.98 (dtd, J = 12.7, 5.4, 2.3 Hz, 1H), 1.85 - 1.78 (m, 1H), 1.78 - 1.71 (m, 1H), 1.61 (t, J = 6.4 Hz, 1H), 1.51 - 1.42 (m, 3H).

Example 10: Synthesis of 3-(3-methyl-l-oxo-5-(piperidin-2-yl)isoindolin-2-yl)piperidi ne-2,6-dione

(Compound 25)

Step 1: 3-(5-Bromo-3-methyl-l-oxoisoindolin-2-yl)piperidine-2, 6-dione was synthesized using the general procedure shown in Reaction Scheme 4 and Example Method 4, above (80% yield), using methyl 4-bromo-2-(l-bromoethyl)benzoate (70.0 mg, 0.217 mmol, 1 equiv.) and 3-aminopiperidine- 2, 6-dione hydrochloride (1.5 equiv.) as starting materials, NaOAc (4 equiv.) as base and ACN as solvent.

LCMS (ESI+) m/z 337.2 [M+H] ⁺

Step 2: 3-(3-Methyl-l-oxo-5-(pyridin-2-yl)isoindolin-2-yl)piperidine -2, 6-dione was synthesized using the general procedure shown in Reaction Scheme 2 and Example Method 2, above (57% yield), using the 3-(5-bromo-3-methyl-l-oxoisoindolin-2-yl)piperidine-2, 6-dione (100 mg, 0.297 mmol, 1 equiv.) and 2-(tributy Ista nnyl)pyrid ine (1.2 equiv.) as starting materials, Pd(PPh ₃ )4 (0.1 equiv.) as catalyst and DMF as solvent.

LCMS (ESI+) m/z 336.1 [M+H] ⁺

Step 3: 3-(3-Methyl-l-oxo-5-(piperidin-2-yl)isoindolin-2-yl)piperidi ne-2, 6-dione (formic acid salt) was synthesized using the general procedure shown in Reaction Scheme 1 and Example Method 1, above (20% yield), using the 3-{3-methyl-l-oxo-5-(pyridin-2-yl)isoindolin-2-yl)piperidine -2, 6-dione (47.0 mg, 1 equiv.) as a starting material.

LCMS (ESI+) m/z 341.9 [M+H] ⁺

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 10.92 (s, 1H), 7.64 - 7.58 (m, 2H), 7.50 (ddd, J = 13.5, 7.8, 1.3 Hz, 1H), 4.73 (dd, J = 12.8, 5.3 Hz, 1H), 4.64 (p, J = 6.5 Hz, 1H), 3.73 (dd, J = 10.6, 2.6 Hz, 1H), 3.10 (d, J = 11.9 Hz, 1H), 2.87 - 2.77 (m, 1H), 2.74 - 2.64 (m, 1H), 2.62 - 2.56 (m, 1H), 1.98 (ddt, J = 10.4, 5.3, 2.7 Hz, 1H), 1.86 - 1.79 (m, 2H), 1.78 - 1.71 (m, 2H), 1.60 (d, J = 9.6 Hz, 1H), 1.51 - 1.35 (m, 6H). Example 11: Synthesis of 3-(6-fluoro-l-oxo-5-(piperidin-2-yl)isoindolin-2-yl)piperidi ne-2,6-dione

(Compound 23)

Step 1: 3-(6-Fluoro-l-oxo-5-(pyridin-2-yl)isoindolin-2-yl)piperidine -2, 6-dione was synthesized using the general procedure shown in Reaction Scheme 2 and Example Method 2, above (91% yield), using 3-(5-bromo-6-fluoro-l-oxoisoindolin-2-yl)piperidine-2, 6-dione (52.0 mg, 0.152 mmol, 1 equiv.) and 2- (tributylstannyl)pyridine (1.2 equiv.) as starting materials, Pd(PPh ₃ )4 (0.057 equiv.) as catalyst and 1,4- dioxane as solvent.

LCMS (ESI+) m/z 340.0 [M+H] ⁺

Step 2: 3-(6-Fluoro-l-oxo-5-(piperidin-2-yl)isoindolin-2-yl)piperidi ne-2, 6-dione (formic acid salt) was synthesized using the general procedure shown in Reaction Scheme 1 and Example Method 1, above (23% yield), using 3-(6-fluoro-l-oxo-5-(pyridin-2-yl)isoindolin-2-yl)piperidine -2, 6-dione (45.0 mg, 0.133 mmol, 1 equiv.) as starting material.

LCMS (ESI+) m/z 346.1 [M+H] ⁺

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 10.98 (s, 1H), 7.81 (d, J = 5.9 Hz, 1H), 7.47 (d, J = 9.1 Hz, 1H), 5.11 (ddd, J = 13.3, 5.1, 2.5 Hz, 1H), 4.43 (dd, J = 17.2, 13.2 Hz, 1H), 4.30 (dd, J = 17.1, 13.3 Hz, 1H), 3.98 (d, J = 10.8 Hz, 1H), 3.14 - 3.07 (m, 1H), 2.91 (ddd, J = 17.3, 13.7, 5.4 Hz, 1H), 2.73 (td, J = 11.5, 2.7 Hz, 1H), 2.65 - 2.56 (m, 1H), 2.45 - 2.32 (m, 1H), 2.00 (dtt, J = 12.9, 5.3, 2.4 Hz, 1H), 1.86 - 1.71 (m, 2H), 1.64 - 1.57 (m, 1H), 1.55 - 1.40 (m, 2H), 1.35 (qdd, J = 12.1, 9.3, 3.3 Hz, 1H).

Example 12: Synthesis of 3-(4-fluoro-l-oxo-5-(piperidin-2-yl)isoindolin-2-yl)piperidi ne-2, 6-dione

(Compound 22)

Step 1: 3-(4-Fluoro-l-oxo-5-(pyridin-2-yl)isoindolin-2-yl)piperidine -2, 6-dione was synthesized using the general procedure shown in Reaction Scheme 2 and Example Method 2, above (91% yield), using 3-(5-bromo-4-fluoro-l-oxoisoindolin-2-yl)piperidine-2, 6-dione (120 mg, 0.35 mmol, 1 equiv.) and 2- (tributylstannyl)pyridine (1.2 equiv.) as starting materials, Pd(PPh ₃ ) ₄ (0.1 equiv.) as catalyst and DMF as solvent.

LCMS (ESI+) m/z 340.2 [M+H] ⁺

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 11.02 (s, 1H), 8.77 (ddd, J = 4.8, 1.8, 1.0 Hz, 1H), 8.08 (dd, J = 7.8, 6.7 Hz, 1H), 7.97 (td, J = 7.7, 1.8 Hz, 1H), 7.87 (ddt, J = 7.9, 2.2, 1.1 Hz, 1H), 7.71 (d, J = 7.8 Hz, 1H), 7.47 (ddd, 1 = 7.5, 4.8, 1.1 Hz, 1H), 5.16 (dd, J = 13.3, 5.1 Hz, 1H), 4.65 (d, J = 17.4 Hz, 1H), 4.49 (d, J = 17.4 Hz, 1H), 2.93 (ddd, J = 17.3, 13.6, 5.4 Hz, 1H), 2.63 - 2.59 (m, 1H), 2.45 (dd, J = 13.4, 4.4 Hz, 1H), 2.04 (dtd, J = 12.6, 5.3, 2.2 Hz, 1H).

Step 2: 3-(4-Fluoro-l-oxo-5-(piperidin-2-yl)isoindolin-2-yl)piperidi ne-2, 6-dione (formic acid salt) was synthesized using the general procedure shown in Reaction Scheme 1 and Example Method 1, above (9% yield), using 3-(4-fluoro-l-oxo-5-(pyridin-2-yl)isoindolin-2-yl)piperidine -2, 6-dione (70.0 mg, 0.21 mmol, 1 equiv.) as starting material.

LCMS (ESI+) m/z 346.0 [M+H] ⁺

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 10.99 (s, 1H), 7.73 (dd, J = 7.8, 6.1 Hz, 1H), 7.56 (d, J = 7.8 Hz, 1H), 5.10 (dd, J = 13.3, 5.1 Hz, 1H), 4.53 (d, J = 17.3 Hz, 1H), 4.37 (d, J = 17.3 Hz, 1H), 3.94 (dd, J = 11.0, 2.5 Hz, 1H), 3.08 (dt, J = 11.5, 2.6 Hz, 1H), 2.91 (ddd, J = 17.3, 13.7, 5.4 Hz, 1H), 2.68 (td, J = 11.8, 2.7 Hz, 1H), 2.63 - 2.57 (m, 1H), 2.42 (td, J = 13.4, 4.5 Hz, 1H), 2.00 (dtd, J = 12.8, 5.4, 2.4 Hz, 1H), 1.81 (t, J = 6.2 Hz, 1H), 1.77 - 1.70 (m, 1H), 1.62 - 1.54 (m, 1H), 1.46 (dddt, J = 19.3, 16.0, 7.5, 3.6 Hz, 2H), 1.38 - 1.28 (m, 1H).

Example 13: Synthesis of 3-(7-fluoro-l-oxo-5-(piperidin-2-yl)isoindolin-2-yl)piperidi ne-2,6-dione

(Compound 24) Step 1: 3-(5-Bromo-7-fluoro-l-oxoisoindolin-2-yl)piperidine-2, 6-dione was synthesized using the general procedure shown in Reaction Scheme 4 and Example Method 4, above (58% yield), using methyl 4-bromo-2-(bromomethyl)-6-fluorobenzoate (600 mg, 1.84 mmol, 1 equiv.) and 3- aminopiperidine-2, 6-dione hydrochloride (1.3 equiv.) as starting materials, DIPEA (3 equiv.) as base and ACN as solvent.

LCMS (ESI+) m/z 341.0 [M+H] ⁺

Step 2: 3-(7-Fluoro-l-oxo-5-(pyridin-2-yl)isoindolin-2-yl)piperidine -2, 6-dione was synthesized using the general procedure shown in Reaction Scheme 2 and Example Method 2, above (67% yield), using 3-(5-bromo-7-fluoro-l-oxoisoindolin-2-yl)piperidine-2, 6-dione (150 mg, 0.44 mmol, 1 equiv.) and 2- (tributylstannyl)pyridine (1.2 equiv.) as starting materials, PdtPPhs)-* (0.05 equiv.) as catalyst and 1,4- dioxane as solvent.

LCMS (ESI+) m/z 340.0 [M+H] ⁺

Step 3: 3-(7-Fluoro-l-oxo-5-(piperidin-2-yl)isoindolin-2-yl)piperidi ne-2, 6-dione (formic acid salt) was synthesized using the general procedure shown in Reaction Scheme 1 and Example Method 1, above (4.4% yield), using 3-(7-fluoro-l-oxo-5-(pyridin-2-yl)isoindolin-2-yl)piperidine -2, 6-dione (99.0 mg, 0.292 mmol, 1 equiv.) as starting material.

LCMS (ESI+) m/z 346.0 [M+H] ⁺

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 10.98 (s, 1H), 7.43 (s, 1H), 7.26 (d, J = 10.8 Hz, 1H), 5.06 (dd, J = 13.3,

5.1 Hz, 1H), 4.44 (dd, J = 17.6, 5.5 Hz, 1H), 4.31 (dd, J = 17.6, 6.4 Hz, 1H), 3.69 (dd, J = 11.0, 2.6 Hz, 1H), 3.10 - 3.04 (m, 1H), 2.90 (ddd, J = 17.3, 13.6, 5.4 Hz, 1H), 2.70 - 2.64 (m, 1H), 2.59 (ddd, J = 17.4, 4.5,

2.2 Hz, 1H), 2.43 - 2.36 (m, 1H), 1.99 (dtd, J = 12.5, 5.4, 2.2 Hz, 1H), 1.80 (d, J = 10.9 Hz, 1H), 1.78 - 1.71 (m, 1H), 1.61 - 1.54 (m, 1H), 1.51 - 1.38 (m, 2H), 1.38 - 1.26 (m, 1H).

Example 14: Synthesis of 3-(l-oxo-5-(6-oxopiperidin-2-yl)isoindolin-2-yl)piperidine-2 ,6-dione (Compound 42)

Step 1: Methyl 4-(6-methoxypyridin-2-yl)-2-methylbenzoate was synthesized using the general procedure shown in Reaction Scheme 5 and Example Method 5, above (82% yield), using methyl 2- methyl-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)benzoa te (1.50 g, 5.43 mmol, 1 equiv.) and 2- bromo-6-methoxypyridine (1.2 equiv.) as starting materials, K ₂ CO ₃ (3 equiv.) as base and Pd(PPh ₃ )4 (0.06 equiv.) as catalyst.

LCMS (ESI+) m/z 257.7 [M+H] ⁺

Step 2: Methyl 2-(bromomethyl)-4-(6-methoxypyridin-2-yl)benzoate was synthesized using the general procedure shown in Reaction Scheme 6 and Example Method 6, above (76% yield), using methyl 4-(6-methoxypyridin-2-yl)-2-methylbenzoate (1.00 g, 3.89 mmol, 1 equiv.) as starting material, AIBN (0.2 equiv.) as initiator and DMC as solvent.

LCMS (ESI+) m/z 336.0 [M+H] ⁺

Step 3: 3-(5-(6-Methoxypyridin-2-yl)-l-oxoisoindolin-2-yl)piperidine -2, 6-dione was synthesized using the general procedure shown in Reaction Scheme 4 and Example Method 4, above (62% yield), using methyl 2-(bromomethyl)-4-(6-methoxypyridin-2-yl)benzoate (200 mg, 0.59 mmol, 1 equiv.) and 3- aminopiperidine-2, 6-dione hydrochloride (1.1 equiv.) as starting materials, TEA (3 equiv.) as base and DMF as solvent.

LCMS (ESI+) m/z 352.2 [M+H] ⁺

Step 4: To a solution of 3-(5-(6-methoxypyridin-2-yl)-l-oxoisoindolin-2-yl)piperidine -2, 6-dione (200 mg, 0.57 mmol, 1 equiv.) in DCE (10 mL) was added IM BBr ₃ (1.7 m L, 1.7 mmol, 3 equiv.) at 0°C dropwise and the reaction mixture was stirred at RT for 3 h. Additional BBr ₃ (0.57 mL, 0.57 mmol, 1 equiv.) was added and the reaction mixture was refluxed for 16 h. The mixture was cooled to RT, concentrated under reduced pressure and the crude product was purified by preparative HPLC to give 3-(l-oxo-5-(6-oxo-l,6-dihydropyridin-2-yl)isoindolin-2-yl)pi peridine-2, 6-dione (35.0 mg, 18% yield). LCMS (ESI+) m/z 338.2 [M+H]

Step 5: 3-(l-Oxo-5-(6-oxopiperidin-2-yl)isoindolin-2-yl)piperidine-2 , 6-dione was synthesized using the general procedure shown in Reaction Scheme 1 and Example Method 1, above (37% yield), using 3- (l-oxo-5-(6-oxo-l,6-dihydropyridin-2-yl)isoindolin-2-yl)pipe ridine-2, 6-dione (150 mg, 0. 44 mmol, 1 equiv.) as starting material.

LCMS (ESI+) m/z 342.1 [M+H] ^{+ 1} H NMR (400 MHz, DMSO-d ₆ ) δ 10.99 (s, 1H), 7.85 (d, J = 2.0 Hz, 1H), 7.71 (d, J = 7.9 Hz, 1H ), 7.52 (s, 1H), 7.43 (d, J = 7.9 Hz, 1H), 5.11 (dd, J = 13.3, 5.1 Hz, 1H), 4.65 (d, J = 7.3 Hz, 1H), 4.45 (dd, J = 17.4, 4.7 Hz, 1H), 4.32 (dd, J = 17.4, 5.6 Hz, 1H), 2.91 (ddd, J = 17.3, 13.6, 5.4 Hz, 1H), 2.65 - 2.55 (m, 1H), 2.40 (qd, J = 13.3, 4.4 Hz, 1H), 2.29 - 2.20 (m, 2H), 2.11 - 1.94 (m, 2H), 1.67 (p, J = 6.6 Hz, 2H), 1.60 (dt, 7 = 13.4, 6.8 Hz, 1H).

Example 15: Synthesis of 3-(l-oxo-5-(quinolin-2-yl)isoindolin-2-yl)piperidine-2,6-dio ne (Compound 5(2))

Step 1: Methyl 2-methyl-4-(quinolin-2-yl)benzoate was synthesized using the general procedure shown in Reaction Scheme 5 and Example Method 5, above (72% yield), using methyl 2-methyl-4- (4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)benzoate (1.00 g, 3.62 mmol, 1 equiv.) and 2- bromoquinoline (1.2 equiv.) as starting materials, K2CO3 (3 equiv.) as base and Pd(PPh ₃ )4 (0.05 equiv.) as catalyst.

LCMS (ESI+) m/z 278.8 [M+H] ⁺

Step 2: Methyl 2-(bromomethyl)-4-(quinolin-2-yl)benzoate was synthesized using the general procedure shown in Reaction Scheme 6 and Example Method 6, above (62% yield), using methyl 2- methyl-4-(quinolin-2-yl)benzoate (100 mg, 0.36 mmol, 1 equiv.) as starting material, AIBN (0.2 equiv.) as initiator and DMC as solvent. LCMS (ESI+) m/z 355.8 [M+H]

Step 3: 3-(l-Oxo-5-(quinolin-2-yl)isoindolin-2-yl)piperidine-2, 6-dione was synthesized using the general procedure shown in Reaction Scheme 4 and Example Method 4, above (24% yie ld), using methyl 2-(bromomethyl)-4-(quinolin-2-yl)benzoate (1.00 g, 2.8 mmol, 1 equiv.) and 3- aminopiperidine-2, 6-dione hydrochloride (1.1 equiv.) as starting materials, TEA (3 equiv.) as base and DMF as solvent.

LCMS (ESI+) m/z 372.3 [M+H] ⁺

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.03 (s, 1H), 8.53 (d, J = 9.2 Hz, 2H), 8.45 (d, J = 8.1 Hz, 1H), 8.26 (d, J = 8.7 Hz, 1H), 8.12 (d, J = 8.5 Hz, 1H), 8.04 (d, J = 8.1 Hz, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.82 (t, J = 7.7 Hz, 1H), 7.64 (t, J = 7.5 Hz, 1H), 5.17 (dd, J = 13.3, 5.1 Hz, 1H), 4.60 (d, J = 17.3 Hz, 1H), 4.47 (d, J = 17.3 Hz, 1H), 2.94 (ddd, J = 17.9, 13.4, 5.4 Hz, 1H), 2.66 - 2.58 (m, 1H), 2.44 (dd, J = 14.0, 9.6 Hz, 1H), 2.10 - 2.01 (m, 1H).

Example 16: Synthesis of 3-(5-(lsoquinolin-3-yl)-l-oxoisoindolin-2-yl)piperidine-2,6- dione

(Compound 6(2))

Step 1: Methyl 4-(isoquinolin-3-yl)-2-methylbenzoate was synthesized using the general procedure shown in Reaction Scheme 5 and Example Method 5, above (66% yield), using methyl 2-methyl-4- (4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)benzoate (600 mg, 2.17 mmol, 1 equiv.) and 3- bromoisoquinoline (1.2 equiv.) as starting materials, K2CO3 (3 equiv.) as base and Pd(PPh ₃ ) ₄ (0.05 equiv.) as catalyst.

LCMS (ESI+) m/z 278.0 [M+H] ⁺

Step 2: Methyl 2-(bromomethyl)-4-(isoquinolin-3-yl)benzoate was synthesized using the general procedure shown in Reaction Scheme 6 and Example Method 6, above (68% yield), using methyl 4- (isoquinolin-3-yl)-2-methylbenzoate (400 mg, 1.44 mmol, l equiv.) as starting material, AIBN (0.2 equiv.) as initiator and DMC as solvent.

LCMS (ESI+) m/z 356.2 [M+H] ⁺

Step 3: 3-(5-(lsoquinolin-3-yl)-l-oxoisoindolin-2-yl)piperidine-2, 6-dione was synthesized using the general procedure shown in Reaction Scheme 4 and Example Method 4, above (44% yield), using methyl 2-(bromomethyl)-4-(isoquinolin-3-yl)benzoate (110 mg, 0.31 mmol, 1 equiv.) and 3- aminopiperidine-2, 6-dione hydrochloride (1.1 equiv.) as starting materials, TEA (3 equiv.) as base and DMF as solvent.

LCMS (ESI+) m/z 372.2 [M+H] ⁺

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.02 (s, 1H), 9.46 (s, 1H), 8.58 (s, 1H), 8.48 (s, 1H), 8.39 (d, J = 7.9 Hz, 1H), 8.19 (d, J = 8.1 Hz, 1H), 8.07 (d, J = 8.3 Hz, 1H), 7.85 (dd, J = 17.3, 8.2 Hz, 2H), 7.72 (t, J = 7.5 Hz, 1H), 5.17 (dd, J = 13.2, 5.3 Hz, 1H), 4.58 (d, J = 17.2 Hz, 1H), 4.45 (d, J = 17.2 Hz, 1H), 2.92 (d, J = 11.1 Hz, 1H), 2.62 (d, J = 18.1 Hz, 1H), 2.45-2.32 (m, 1H), 2.05 (s, 1H).

Example 17: Synthesis of 3-(l-oxo-5-(tetrahvdro-2H-pyran-2-yl)isoindolin-2-yl)piperid ine-2,6-dione (Compound 54)

Step 1: In a vial were placed 3-(5-bromo-l-oxoisoindolin-2-yl)piperidine-2, 6-dione (240 mg, 0.743 mmol, 1 equiv.), 2-(3,4-dihydro-2H-pyran-6-yl)-4,4,5,5-tetramethyl-l,3,2-diox aborolane (187 mg, 0.891 mmol, 1.2 equiv.), PdCl ₂ (PPh ₃ ) ₂ (104 mg, 0.149 mmol, 0.2 equiv.), KOAc (146 mg, 1.48 mmol, 2 equiv.), 1,4-dioxane (4.2 mL) and water (0.16 mL). The reaction mixture was stirred at 100°C for 6 h. The mixture was diluted with ACN/AcOEt, filtered through Celite® and the filtrate was concentrated under reduced pressure. The residue was triturated in ACN/AcOEt to give 3-(5-(3,4- dihydro-2H-pyran-6-yl)-l-oxoisoindolin-2-yl)piperidine-2, 6-dione (240 mg, 99% yield) that was used directly in the next step.

LCMS (ESI+) m/z 327.2 [M+H] ⁺

Step 2: 3-(5-(3,4-Dihydro-2H-pyran-6-yl)-l-oxoisoindolin-2-yl)piperi dine-2, 6-dione (132 mg, 0.30 mmol, 1 equiv.) was dissolved in degassed 1-butanol (10 mL) and ACN (1 mL). Platinum on carbon (15 mg, 10% wt.) was added and the reaction mixture was stirred under hydrogen balloon (1 bar) for 48 h. The solids were filtered through Celite®, and the filtrate was concentrated under reduced pressure. The crude product was purified by preparative HPLC to give 3-(l-oxo-5-(tetrafiydro-2H- pyran-2-yl)isoindolin-2-yl)piperidine-2,6-dione (30.0 mg, 30% yield).

LCMS: (ESI+) m/z 329.0 [M+H] ⁺

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 10.97 (s, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.58 - 7.55 (m, 1H), 7.46 (dt, J = 8.1, 1.4 Hz, 1H), 5.10 (dd, J = 13.3, 5.1 Hz, 1H), 4.48 - 4.41 (m, 2H), 4.31 (d, J = 17.2 Hz, 1H), 4.05 (ddt, J = 11.4, 3.7, 1.8 Hz, 1H), 3.56 (ddd, J = 11.3, 9.1, 6.3 Hz, 1H), 2.91 (ddd, J = 17.3, 13.7, 5.4 Hz, 1H), 2.60 (ddd, J = 17.1, 4.5, 2.2 Hz, 1H), 2.44 - 2.37 (m, 1H), 2.00 (dtd, J = 12.6, 5.3, 2.2 Hz, 1H), 1.85 (dddd, J = 13.0, 8.8, 6.9, 3.8 Hz, 2H), 1.66 (ddtt, J = 17.4, 13.8, 7.1, 3.7 Hz, 1H), 1.57 (tq, J = 6.0, 3.6 Hz, 2H), 1.46 - 1.36 (m, 1H).

Example 18: Synthesis of 3-(l-oxo-5-(5-phenylpyridin-2-yl)isoindolin-2-yl)piperidine- 2,6-dione

(Compound 4(2))

Step 1: Methyl 2-methyl-4-(5-phenylpyridin-2-yl)benzoate was synthesized using the general procedure shown in Reaction Scheme 5 and Example Method 5, above (67% yield), using methyl 2- methyl-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)benzoa te (500 mg, 1.8 mmol, 1 equiv.) and 2- bromo-5-phenylpyridine (1.2 equiv.) as starting materials, K2CO3 (3 equiv.) as base and Pd(PPh ₃ ) ₄ (0.06 equiv.) as catalyst.

LCMS (ESI+) m/z 304.2 [M+H] ⁺

Step 2: Methyl 2-(bromomethyl)-4-(5-phenylpyridin-2-yl)benzoate was synthesized using the general procedure shown in Reaction Scheme 6 and Example Method 6, above (64% yield), using methyl 2- methyl-4-(5-phenylpyridin-2-yl)benzoate (370 mg, 1.22 mmol, 1 equiv.) as starting mate rial, AIBN (0.2 equiv.) as initiator and DMC as solvent.

LCMS (ESI+) m/z 382.0 [M+H] ⁺

Step 3: 3-(l-Oxo-5-(5-phenylpyridin-2-yl)isoindolin-2-yl)piperidine- 2, 6-dione was synthesized using the general procedure shown in Reaction Scheme 4 and Example Method 4, above (52% yield), using methyl 2-(bromomethyl)-4-(5-phenylpyridin-2-yl)benzoate (130 mg, 0.34 mmol, 1 equiv.) and 3- aminopiperidine-2, 6-dione hydrochloride (1.2 equiv.) as starting materials, TEA (3 equiv.) as base and ACN as solvent.

LCMS (ESI+) m/z 398.1 [M+H] ⁺

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.01 (s, 1H), 9.05 (d, J = 2.5 Hz, 1H), 8.40 (s, 1H), 8.31 (d, J = 8.1 Hz, 1H), 8.25 (dd, J = 8.5, 2.4 Hz, 1H), 8.18 (d, J = 8.4 Hz, 1H), 7.84 (dd, J = 10.9, 7.9 Hz, 3H), 7.54 (t, J = 7.5 Hz, 2H), 7.46 (t, J = 7.4 Hz, 1H), 5.16 (dd, J = 13.3, 5.0 Hz, 1H), 4.57 (d, J = 17.4 Hz, 1H), 4.44 (d, J = 17.4 Hz, 1H), 3.01 - 2.86 (m, 1H), 2.62 (d, J = 18.1 Hz, 1H), 2.46 - 2.36 (m, 1H), 2.06 (s, 1H).

Example 19: Synthesis of 3-(l-oxo-5-(5-phenylpiperidin-2-yl)isoindolin-2-yl)piperidin e-2,6-dione (Compound 32)

Step 1: 3-(l-Oxo-5-(5-phenylpiperidin-2-yl)isoindolin-2-yl)piperidin e-2, 6-dione (formic acid salt) was synthesized using the general procedure shown in Reaction Scheme 1 and Example Method 1, above (10.0 mg, 5% yield), using 3-(l-oxo-5-(5-phenylpyridin-2-yl)isoindolin-2-yl)piperidine- 2, 6-dione (200 mg, 0.50 mmol, 1 equiv.) as starting material.

LCMS (ESI+) m/z 404.2 [M+H] ⁺

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 10.99 (s, 1H), 7.71 - 7.63 (m, 2H), 7.55 (d, J = 7.9 Hz, 1H), 7.31 (td, J = 8.2, 6.0 Hz, 4H), 7.24 - 7.18 (m, 1H), 5.11 (dd, J = 13.3, 5.1 Hz, 1H), 4.44 (dd, J = 17.2, 4.8 Hz, 1H), 4.31 (dd, J = 17.2, 5.1 Hz, 1H), 3.77 (dd, J = 11.2, 2.6 Hz, 1H), 3.18 - 3.11 (m, 1H), 2.91 (ddd, J = 17.9, 13.5, 5.4 Hz, 1H), 2.82 - 2.69 (m, 2H), 2.65 - 2.55 (m, 1H), 2.45 - 2.34 (m, 1H), 2.04 - 1.93 (m, 1H), 1.90 (td, = 6.3, 3.0 Hz, 1H), 1.84 - 1.66 (m, 1H), 1.52 (q, J = 12.3 Hz, 1H). Example 20: Synthesis of N-(6-(2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindolin-5-yl)Dyrid in-3- vDacetamide (Compound 7(2))

Step 1: Tert-butyl 4-(5-(5-acetamidopyridin-2-yl)-l-oxoisoindolin-2-yl)-5-amino -5-oxopentanoate was synthesized using the general procedure shown in Reaction Scheme 5 and Example Method 5, above (46% yield), using tert-butyl 5-amino-5-oxo-4-(l-oxo-5-(4,4,5,5-tetramethyl-l,3,2-dioxabor olan-2- yl)isoindolin-2-yl)pentanoate (460 mg, 2.51 mmol, 1.2 equiv.) and N-(6-bromopyridin-3-yl)acetamide (1 equiv.) as starting materials, K ₃ PO ₄ (3 equiv., IM solution in water) as base and PdCI ₂ (dtbpf) (0.03 equiv.) as catalyst.

LCMS (ESI+) m/z 453.0 [M+H] ⁺

Step 2: N-(6-(2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindolin-5-yl)pyrid in-3-yl)acetamide was synthesized using the general procedure shown in Reaction Scheme 3 and Example Method 3, above (26% yield), using tert-butyl 4-(5-(5-acetamidopyridin-2-yl)-l-oxoisoindolin-2-yl)-5-amino -5-oxopentanoate (90.0 mg, 0.2 mmol, 1 equiv.) as starting material.

LCMS (ESI+) m/z 379.1 [M+H] ⁺

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.01 (s, 1H), 10.32 (s, 1H), 8.83 (d, J = 2.4 Hz, 1H), 8.27 (s, 1H), 8.21 - 8.15 (m, 2H), 8.04 (d, J = 8.7 Hz, 1H), 7.80 (d, J = 8.0 Hz, 1H), 5.14 (dd, J = 13.3, 5.1 Hz, 1H), 4.53 (d, J = 17.3 Hz, 1H), 4.41 (d, J = 17.3 Hz, 1H), 2.93 (ddd, J = 17.2, 13.6, 5.4 Hz, 1H), 2.61 (ddd, J = 17.3, 4.5, 2.3 Hz, 1H), 2.41 (td, J = 13.2, 4.5 Hz, 1H), 2.11 (s, 3H), 2.07 - 1.97 (m, 1H).

Example 21: Synthesis of N-(6-(2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindolin-5-yl)piper idin-3- yljacetamide (Compound 33)

Step 1: N-(6-(2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindolin-5-yl)piper idin-3-yl)acetamide (hydrochloride salt) was synthesized using the general procedure shown in Reaction Scheme 1 and Example Method 1, above (50 mg, 20% yield), using N-(6-(2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindolin-5-yl)pyrid in-3- yl)acetamide (250 mg, 0.66 mmol, 1 equiv.) as starting material.

LCMS (ESI+) m/z 385.2 [M+H] ^{+ 1} H NMR (400 MHz, DMSO-d ₆ ) δ 11.01 (s, 1H), 8.75 (d, J = 6.6 Hz, 1H), 7.89 (d, J = 6.4 Hz, 1H), 7.86 - 7.72 (m, 2H), 5.13 (dd, J = 13.3, 5.0 Hz, 1H), 4.55 - 4.28 (m, 3H), 4.10 (d, J = 6.6 Hz, 1H), 3. 35 (d, J = 12.3 Hz, 2H), 2.93 (ddd, J = 18.1, 13.5, 5.3 Hz, 1H), 2.65 - 2.56 (m, 1H), 2.43 (dd, J = 14.9, 4.0 Hz, 1H), 2.20 (d, J = 13.4 Hz, 1H), 2.07 - 1.99 (m, 1H), 1.88 (d, J = 22.6 Hz, 5H), 1.28 - 0.99 (m, 1H).

Example 22: Synthesis of 3-(5-(5-benzylpiperidin-2-yl)-l-oxoisoindolin-2-yl)piperidin e-2,6-dione

(Compound 35)

Step 1: Tert-butyl 5-amino-4-(5-(5-benzylpyridin-2-yl)-l-oxoisoindolin-2-yl)-5- oxopentanoate was synthesized using the general procedure shown in Reaction Scheme 5 and Example Method 5, above (34% yield), using tert-butyl 5-amino-5-oxo-4-(l-oxo-5-(4,4,5,5-tetramethyl-l,3,2-dioxabor olan-2- yl)isoindolin-2-yl)pentanoate (540 mg, 1.21 mmol, 1.2 equiv.) and 5-benzyl-2-bromopyridine (1 equiv.) as starting materials, K ₃ PO ₄ (5 equiv.) as base and PdCl ₂ (dtbpf) (0.05 equiv.) as catalyst.

LCMS (ESI+) m/z 486.2 [M+H] ⁺

Step 2: 3-(5-(5-Benzylpyridin-2-yl)-l-oxoisoindoiin-2-yl)piperidine- 2, 6-dione was synthesized using the general procedure shown in Reaction Scheme 3 and Example Method 3, above (23% yield), using tert-butyl 5-amino-4-(5-(5-benzylpyridin-2-yl)-l-oxoisoindolin-2-yl)-5- oxopentanoate (100 mg, 0.21 mmol, 1 equiv.) as starting material.

LCMS (ESI+) m/z 412.3 [M+H] ⁺

Step 3: 3-(5-(5-Benzylpiperidin-2-yl)-l-oxoisoindolin-2-yl)piperidin e-2, 6-dione (acetic acid salt) was synthesized using the general procedure shown in Reaction Scheme 1 and Example Method 1, above (37 mg, 24% yield), using 3-(5-(5-benzylpyridin-2-yl)-l-oxoisoindolin-2-yl)piperidine- 2,6-dione (140 mg, 0.32 mmol, 1 equiv.) as starting material. ).

LCMS (ESI+) m/z 418.4 [M+H] ⁺

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 10.97 (s, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.57 (s, 1H), 7.47 (d, J = 7.8 Hz, 1H), 7.29 (t, J = 7.5 Hz, 2H), 7.19 (d, J = 7.1 Hz, 3H), 5.09 (dd, J = 13.3, 5.0 Hz, 1H), 4.41 (dd, J = 17.3, 4.9 Hz, 1H), 4.27 (dd, J = 17.3, 5.5 Hz, 1H), 3.61 (d, J = 10.5 Hz, 1H), 2.97 (dd, J = 11.8, 3.7 Hz, 1 H), 2.94 - 2.83 (m, 1H), 2.64 - 2.55 (m, 1H), 2.45 - 2.29 (m, 2H), 1.98 (dt, J = 11.0, 5.1 Hz, 1H), 1.82 — 1.63 (m, 4H), 1.38 - 1.11 (m, 3H).

Example 23: Synthesis of 3-(5-(5-butylpiperidin-2-yl)-l-oxoisoindolin-2-yl)piperidine -2,6-dione

(Compound 37)

Step 1: Tert-butyl (E)-5-amino-4-(5-(5-(but-l-en-l-yl)pyridin-2-yl)-l-oxoisoind olin-2-yl)-5- oxopentanoate was synthesized using the general procedure shown in Reaction Scheme 5 and Example Method 5, above (49% yield), using tert-butyl 5-amino-5-oxo-4-(l-oxo-5-(4, 4,5,5- tetramethyl-l,3,2-dioxaborolan-2-yl)isoindolin-2-yl)pentanoa te (1.10 g, 2.54 mmol, 1.2 equiv.) and (E)-2-bromo-5-(but-l-en-l-yl)pyridine (450 mg, 2.12 mmol, 1.0 equiv.) as starting materials, K ₂ CO ₃ (2.5 equiv.) as base, PdCl ₂ (dppf) (0.1 equiv.) as catalyst.

LCMS (ESI+) m/z 449.9 [M+H] ⁺

Step 2: (E)-3-(5-(5-(but-l-en-l-yl)pyridin-2-yl)-l-oxoisoindolin-2-y l)piperidine-2, 6-dione was synthesized using the general procedure shown in Reaction Scheme 3 and Example Method 3, above (60% yield), using tert-butyl (E)-5-amino-4-(5-(5-(but-l-en-l-yl)pyridin-2-yl)-l-oxoisoind olin-2-yl)-5- oxopentanoate (70 mg, 0.16 mmol, 1 equiv.) as a starting material.

LCMS (ESI+) m/z 376.1 [M+H] ⁺ Step 3: 3-(5-(5-Butylpiperidin-2-yl)-l-oxoisoindolin-2-yl)piperidine -2, 6-dione (acetic acid salt) was synthesized using the general procedure shown in Reaction Scheme 1 and Example Method 1, above (35% yield), using (E)-3-(5-(5-(but-l-en-l-yl)pyridin-2-yl)-l-oxoisoindolin-2-y l)piperidine-2, 6-dione (160 mg, 0.37 mmol, 1 equiv.) as starting material.

LCMS (ESI+) m/z 384.4 [M+H] ⁺

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 10.97 (s, 1H), 7.68 - 7.57 (m, 2H), 7.51 (d, J = 8.6 Hz, 1H), 5.10 (dd, J = 13.4, 5.1 Hz, 1H), 4.42 (dt, J = 17.4, 4.5 Hz, 1H), 4.29 (dd, J = 17.2, 5.6 Hz, 1H), 3.74- 3.64 (m, 1H), 3.60 (dd, J = 11.1, 2.4 Hz, 0.5H), 3.08 (d, J = 11.5 Hz, 0.5H), 2.91 (ddd, J = 18.1, 13.5, 5.3 Hz, 1H), 2 .81 (d, J = 3.5 Hz, 2H), 2.65 - 2.55 (m, 1H), 2.39 (qd, J = 13.9, 5.1 Hz, 1H), 2.05 - 1.95 (m, 1H), 1.71 - 1.48 (m, 3H), 1.44 (dq, J = 12.6, 6.4 Hz, 1H), 1.29 (qq, J = 11.1, 5.8 Hz, 5H), 0.95 - 0.81 (m, 4H).

Example 24: Synthesis of 3-(5-(6-butylpiperidin-2-yl)-l-oxoisoindolin-2-yl)piperidine -2,6-dione

(Compound 40)

Step 1: Tert-butyl (E)-5-amino-4-(5-(6-(but-l-en-l-yl)pyridin-2-yl)-l-oxoisoind olin-2-yl)-5- oxopentanoate was synthesized using the general procedure shown in Reaction Scheme 5 and Example Method 5, above (45% yield), using tert-butyl 5-amino-5-oxo-4-(l-oxo-5-(4, 4,5,5- tetramethyl-l,3,2-dioxaborolan-2-yl)isoindolin-2-yl)pentanoa te (130 mg, 0.61 mmol, 1 equiv.) and (E)-2-bromo-6-(but-l-en-l-yl)pyridine (1.2 equiv.) as starting materials, K2CO3 (2.5 equiv.) as base, PdCI ₂ (dppf) (0.1 equiv.) as catalyst.

LCMS (ESI+) m/z 449.9 [M+H] ⁺

Step 2: (E)-3-(5-(6-(but-l-en-l-yl)pyridin-2-yl)-l-oxoisoindolin-2-y l)piperidine-2, 6-dione was synthesized using the general procedure shown in Reaction Scheme 3 and Example Method 3, above (79% yield), using tert-butyl (£)-5-amino-4-(5-(6-(but-l-en-l-yl)pyridin-2-yl)-l-oxoisoin dolin-2-yl)-5- oxopentanoate (120 mg, 0.27 mmol, 1 equiv.) as starting material. LCMS (ESI+) m/z 376.4 [M+H]

Step 3: 3-(5-(6-Butylpiperidin-2-yl)-l-oxoisoindolin-2-yl)piperidine -2, 6-dione (mixture of stereoisomers, formic acid salts) was synthesized using the general procedure shown in Reaction Scheme 1 and Example Method 1, above (66% yield), using (f)-3-(5-(6-(but-l-en-l-yl)pyridi n-2-yl)-l- oxoisoindolin-2-yl)piperidine-2, 6-dione (200 mg, 0.46 mmol, 1 equiv.) as a starting material.

LCMS (ESI+) m/z 384.4 [M+H] ⁺

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.07 - 10.91 (s, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.63 (s, 1H), 7.52 (d, J = 7.9 Hz, 1H), 5.11 (dd, J = 13.4, 5.0 Hz, 1H), 4.43 (dd, J = 17.2, 2.9 Hz, 1H), 4.30 (dd, J = 17.3, 3.5 Hz, 1H), 3.81 (d, J = 10.9 Hz, 1H), 2.91 (ddd, J = 18.4, 13.4, 5.2 Hz, 1H), 2.73 - 2.56 (m, 2H), 2.39 (qd, J = 13.7, 4.4 Hz, 1H), 2.05 - 1.93 (m, 1H), 1.82 (d, J = 12.1 Hz, 1H), 1.71 (t, J = 14.6 Hz, 2H), 1.56 - 1.18 (m, 8H), 1.09 (q, J = 12.5 Hz, 1H), 0.94 - 0.77 (m, 3H).

Example 25: Synthesis of 3-(l-oxo-5-(5-(trifluoromethyl)pyridin-2-yl)isoindolin-2-yl) piperidine-2,6- dione (Compound 16(2))

Step 1: 3-(l-Oxo-5-(tributylstannyl)isoindolin-2-yl)piperidine-2, 6-dione (30.0 mg, 0.056 mmol, 1 equiv.), 2-bromo-5-(trifluoromethyl)pyridine (19.1 mmol, 0.084 mmol, 1.1 equiv.), and Pd(PPh ₃ )4 (5.2 mg, 0.005 mmol, 0.08 equiv.) were dissolved in 1,4-dioxane (1.5 mL). The reaction mixture was stirred at 110°C for 18 h. Additional portions of 2-bromo-5-(trifluoromethyl)pyridine (19.1 mmol, 0.084 mmol, 1.1 equiv.) and Pd(PPh ₃ ) ₄ (5.2 mg, 0.005 mmol, 0.08 equiv.) were added and reaction was continued for another 18 h. The crude product was purified by preparative HPLC to give 3-(l-oxo-5- (5-(trifluoromethyl)pyridin-2-yl)isoindolin-2-yl)piperidine- 2, 6-dione (7.5 mg, 34% yield).

LCMS (ESI+) m/z 390.0 [M+H] ⁺

¹ H NMR (500 MHz, DMSO-d _e ) δ 11.00 (s, 1H), 9.10 (dd, J = 2.1, 1.1 Hz, 1H), 8.41 (dd, J = 1.6, 0.8 Hz, 1H), 8.38 - 8.34 (m, 1H), 8.32 (q, J = 1.3 Hz, 1H), 8.32 - 8.30 (m, 1H), 7.89 (dd, J = 8.0, 0.7 Hz, 1H), 5.16 (dd, J = 13.3, 5.1 Hz, 1H), 4.58 (d, J = 17.4 Hz, 1H), 4.45 (d, J = 17.4 Hz, 1H), 2.93 (ddd, J = 17.2, 13.6, 5.4 Hz, 1H), 2.60 (dd, J = 4.4, 2.3 Hz, 1H), 2.43 (td, J = 13.2, 4.6 Hz, 1H), 2.05 (dtd, J = 12.7, 5.4, 2.3 Hz, 1H). Example 26: Synthesis of 3-(2-oxo-5-(pyridin-2-yl)benzo[ccflindol-l(2H)-yl)piperidine -2,6-dione

(Compound 23(2))

Step 1: 5-(Pyridin-2-yl)benzo[cd]indol-2(1H)-one was synthesized using the general procedure shown in Reaction Scheme 2 and Example Method 2, above (98% yield), using 5-bromobenzo[cd]inciol-2(1H)- one (200 mg, 0.806 mmol, 1 equiv.) and 2-(tributylstannyl)pyridine (1.3 equiv.) as starting materials, Pd(PPh ₃ ) ₄ (0.08 equiv.) as catalyst and 1,4-dioxane as solvent.

LCMS (ESI+) m/z 247.1 [M+H] ⁺

Step 2: 5-(Pyridin-2-yl)benzo[cd]indol-2(lH)-one (120 mg, 0.49 mmol, 1 equiv.) was dissolved in dry DMF (4 mL) and sodium bis(trimethylsilyl)amide (2.4 mL, IM in THF, 2.4 mmol, 5 equiv.) was added in one portion. The reaction mixture stirred at RT for 1 h and 3-bromopiperidine-2, 6-dione (255 mg, 1.33 mmol, 2.5 equiv.) in DMF (2 mL) was added dropwise. The reaction mixture was stirred at 80°C for 60 h. The reaction mixture was cooled to -50°C and quenched with solid NH ₄ CI, the volatiles were removed under reduced pressure and the crude product was purified by preparative HPLC to give 3- (2-oxo-5-(pyridin-2-yl)benzo[cd]indol-l(2H)-yl)piperidine-2, 6-dione (11.5 mg, 6.6% yield) as yellow solid.

LCMS (ESI+) m/z 358.1 [M+H] ⁺

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 11.14 (s, 1H), 8.84 (dt, J = 4.9, 1.5 Hz, 1H), 8.20 (d, J = 7.3 Hz, 1H), 8.09 - 8.00 (m, 2H), 7.96 (d, J = 8.7 Hz, 1H), 7.89 (dt, J = 7.9, 1.1 Hz, 1H), 7.61 - 7.50 (m, 2H), 7.21 (d, J = 7.2 Hz, 1H), 5.49 (dd, J = 13.0, 5.4 Hz, 1H), 2.97 (ddd, J = 16.8, 13.5, 5.3 Hz, 1H), 2.80 (qd, J = 13.0, 4.4 Hz, 1H), 2.67 (ddd, J = 16.9, 4.3, 2.3 Hz, 1H), 2.14 (dtd, J = 12.8, 5.3, 2.2 Hz, 1H).

Biophysics

Ternary complex formation assay

The effect of the molecular glue compounds of the invention on the formation of a ternary complex composed of [NEK7]-[compound of formula (I)]— [CRBN/DDB1] was investigated with two methods: AlphaLISA dose response assay or HTRF ternary complex assay. AlphaLISA dose response assay:

Two types of protein solution were prepared:

- 200 nM biotinylated NEK7, 40 pg/ml AlphaScreen Streptavidin-coated Donor Beads in HBS (10 mM HEPES, 150 mM NaCL, pH 7.4) buffer with 0.1% Tween-20 and ImM DTT,

- 200 nM 6XHis-CRBN/Strep-DDBl, 40 pg/ ml AlphaLISA Anti-6xHis Acceptor beads in HBS buffer with 0.1% Tween-20 and ImM DTT.

The prepared solutions were incubated at room temperature for 30 min and then the solution containing the donor beads was mixed with the solution containing the acceptor beads.

The tested compounds were dispensed onto a white 384-well AlphaPlate 384 SW. DMSO was backfilled to all wells, resulting in a final DMSO content of 2%. Wells containing only DMSO served as background. Next, 10 pl of solution with donor and acceptor beads was added to the wells.

The plate was sealed with transparent film and shaken using a VibroTurbulator for 60 sec at room temperature, level 3. The plate was then spun down shortly (10 s, 1000 ref, room temperature) and incubated at 25°C for 30 min.

The read-out was performed with PerkinElmer Enspire Multimode Plate Reader (method for AlphaLISA 384-well low volume, Filterset: λexc = 680 nm, Aem = 615 nm).

The results were analyzed as follows:

1) an average of luminescence for background signal was calculated and used as a negative control;

2) average of the maximum measured luminescence for Compound 2 was calculated and used as an internal positive control;

3) raw luminescence values were normalized against positive and negative controls;

4) Normalized responses to Compound 2 were determined.

As illustrated in Table 1, the compounds of the present invention have the capability to induce the formation of the [NEK7]-[compound of formula (I)]-[CRBN/DDB1] complex.

HTRF ternary complex assay: The effect of the molecular glue compounds of the invention on the formation of a ternary complex composed of [NEK7]-[compound of formula (I)]-[CRBN/DDB1] was investigated.

Mix solution of proteins and reagents was prepared:

- 24 nM NEK7, 52.8 nM 6XHis-CRBN/Strep-DDBl, 3 nM of Streptavidin-Eu cryptate (acceptor) and 6.67 nM of Anti-6Xhis-d2 (donor) was prepared in PPI Europium detection buffer (Cisbio) with 1 mM DTT.

The tested compounds in dose-response were dispensed onto a white 384-well low volume plate (Greiner, 784075). DMSO was backfilled to all wells, resulting in a final DMSO content of 0.5%. Wells containing only DMSO served as background.

The plate was sealed with transparent film and shaken using a VibroTurbulator for 60 sec at level 3.

The plate was then spun down shortly (10 s, 1000 ref) and incubated at 25°C for 180 min.

The read-out was performed with plate reader (Pherastar, BMG Labtech) in time resolved fluorescence mode. Filterset: TR 337 665 620.

The results were analyzed as follows:

1) an average of fluorescence for background signal was calculated and used as a negative control;

2) raw fluorescence values for tested compounds were normalized against negative controls;

3) saturation curve was fitted to specific binding with Hill slope model;

4) EC50 and pEC50 values were determined.

As illustrated in Table 1, the compounds of the present invention have the capability to induce the formation of the [NEK7]-[compound of formula (I)]— [CRBN/DDB1] complex.

Table 1: Ternary complex assay results for the compounds of the invention

AlphaLISA ternary complex level description:

A - Normalized activity >80%

B - Normalized activity >20% and <80%

C - Normalized activity >10% and <20%

D - Normalized activity <10%

HTRF activity description:

+++++ - pEC50 > 7.0

++++ - 7.0 > pEC50 > 6.5

+++ - 6.5 > pEC50 > 6.0

++ - 6.0 > pEC50 > 5.5

+ - 5.5 > pEC50 Biological Testing Methodology

Cell culture

Human PBMC-derived macrophages

Macrophages were differentiated from human PBMCs isolated from buffy coats from healthy donors.

Buffy coats were diluted 1:1 (v/v) with DPBS (Sigma-Aldrich) in falcon tubes. After reconstitution, suspension was carefully layered on Histopaque-1077 solution (Sigma-Aldrich) and centrifuged (760 x g, RT, 20 min; Brakes Off). PBMCs were collected and washed with DPBS (3x at 350 xg, RT, 8 min and 1x at 200 xg, RT, 10 min; Brakes On). Subsequently, cells were resuspended in appropriate volume of RPMI 1640 medium (Gibco) supplemented with 10% of heat-inactivated FBS (Gibco) and 1% of Penicillin-Streptomycin Solution 100x (Biowest). Cells viability was measured using trypan blue solution (Sigma-Aldrich).

10xl0 ^A 6 of PBMCs per well were seeded on 6-well plates in complete medium supplemented with 10 ng/ml of M-CSF growth factor (R&D Systems). Differentiation was conducted for a week with medium replacement every 3-4 days. Differentiation of PBMCs into mature macrophages was confirmed by microscopic evaluation and FACS surface staining for the following markers: CD11b, CD14, CD16 (BD Pharmingen). Differentiated macrophages were subjected on NLRP3 inflammasome activation assay.

HEK293 NEK7-HIBIT cell line

HEK293 NEK7-HiBiT cells were generated using CRISPR-Cas9 system. HEK293 cells were transformed with pSpCas9-BB-2A-Puro v2.0 plasmid carrying gRNA targeting the N-terminus of NEK7 and ssODN template containing the HiBiT tag sequence with flanking homology sequences. Neon Transfection System (Thermo Fisher Scientific) was used for electroporation. HEK293 NEK7-HiBiT cells were cultured with DMEM Glutamax (Gibco) supplemented with 10% heat inactivated FBS (Gibco). After transfection the culture medium was changed to DMEM Glutamax with 10% heat-inactivated FBS and 1% Penicillin-Streptomycin (Biowest) supplemented with Puromycin (2 ug/ml; Invivogen) for clonal selection. In order to isolate single cell clones for further validation and analysis, limiting dilution cloning in 96-well plates was performed. When the single clones reached confluency, HiBiT Lytic Assay (Promega) was performed in order to identify HiBiT positive clones. The clone selected for further studies was verified and validated using genotyping and HiBiT Blotting (Promega).

Selected clone of HEK293 NEK7-HiBiT cells was maintained in the DMEM-Glutamax medium supplemented with 10% of heat-inactivated FBS (Gibco) and 1% of Penicillin-Streptomycin Solution 100x (Biowest) at 37°C, 5% CO ₂ and subcultured every 2-3 days. Nano-Gio HiBiT Lytic Assay

For Nano-Gio HiBiT Lytic Assay HEK293 NEK7-HiBiT cells were seeded at the density 2xl0 ^/v 3 cells in triplicates in the 40 pL of growth medium per single well on 384 well plate (Greiner Bio-One). Compounds or DMSO were added to treatment plates using Echo555 Liquid Handler and incubated at 37°C, 5% CO ₂ for 24 hours. After incubation 10 pL of Cell Titer-Gio Reagent (prepared accords ng to the manufacturer protocol) were added to 40 pL of the cell culture medium present in each well. The plate content was briefly mixed (460 rpm) on an orbital shaker to ensure cell lysis. The plate was left at RT protected from light for another 8 min to stabilize the luminescent signal. The luminescence signal was measured using CLARIOstar Multimode Plate Reader. Focus and gain were adjusted to DMSO treated cells. The results were calculated as the NEK7-HiBiT % relative to the DMSO control of 3 technical replicates relative to the DMSO control.

NLRP3 inflammasome activation assay

Human PBMC-derived macrophages were pre-treated for 24h with exemplary compounds in the concentration range 0.1-10 pM. Dilutions of tested compound were prepared in DMSO. Afterwards cells were primed with 1 pg/ml of LPS (Invivogen) for 3h and NLRP3 inflammasomes were activated with 5 pM of nigericin solution (Invivogen) for Ih. Supernatants were centrifuged and stored for ELISA assays and cell lysates were prepared for Western blotting analysis.

Measurement of IL-1β and IL-18

IL-lp and IL-18 level was quantified using ELISA assays (R&D Systems) according to the manufacturer's protocol. 96-well plates were coated overnight with appropriate capture antibodies. Plates were blocked and incubated at RT for a minimum of Ih. Samples or standards were added and incubated for 2h in RT. Next, biotinylated anti-human IL-ip or IL-18 detection antibodies were added for 2h in RT. Strepatividin-HRP solution was added for 20 min of incubation. Subsequently, substrate solution was added for 20 min. Between each step washing procedure was performed. The reaction was stopped, and optical density was determined using CLARIOstar Multimode Plate reader set to 450 nm with wavelength correction set to 570 nm. The analysis was performed with GraphPad Prism Software and Excel spreadsheet.

Western blotting

Cell lysates from Human PBMC-derived macrophages were prepared by direct lysis in 40 p.1 RIPA lysis buffer (50mM Tris»HCI pH 7.4, 150mM NaCI, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS and 1 mM EDTA) supplemented with protease and phosphatase inhibitors (complete EDTA-free Protease Inhibitor Cocktail, Roche; Halt™ Phosphatase Inhibitor Cocktail, Thermo Scientific). Subsequently, lysates were snap frozen in liquid nitrogen and stored in -20°C. Following thawing, lysates were centrifuged at 4°C, 19 OOOxg for 15 min for supernatants collection. The protein concentration in each sample was determined by BCA method (Pierce BCA Protein Assay Kit, Thermo Fischer Scientific). The absorbance was measured using CLARIOstar Multimode Plate Reader at 562 nm. SDS-PAGE samples were prepared by mixing the lysates with 5xSB and RIPA buffer. Denaturation of the sam ples was performed by incubation at 95° for 5 minutes.

The protein samples were resolved on 4-20% TGX Stain-Free™ protein gels (Bio-Rad) and transferred onto nitrocellulose membranes (Bio-Rad) using Trans-Blot® Turbo system (Bio-Rad). Membranes were blocked in 5% non- dried milk (NFM) in TBS-T (10 mM Tris, 150 mM NaCI, 0.1 % Tween-20) for 1 h at room temperature (RT). Membranes were incubated with primary antibodies for NEK7 (O/N, 4°C) and loading control - p-Actin (Ih, RT) diluted in 5% NFM in TBS-T, followed by incubation with the appropriate horseradish peroxidise (HRP) conjugated secondary antibody diluted in 5% NFM in TBS-T for 1 h in RT. Between each antibody incubation, the membranes were washed in TBS-T. Membranes were developed using SuperSignal West Pico PLUS chemiluminescent substrate (ThermoScientific). Membrane images were captured using Chemi Doc Imager. The analysis was performed in Image Lab software. Densitometric values for NEK7 protein were normalized to the loading control and calculated as a relative to the cells treated with DMSO control.

Results

NEK7-HiBiT degradation assay

HEK293 NEK7-HiBiT cells were treated with the compounds (cone. 0.1, 1 and 10 pM) or DMSO for 24h. After incubation with compounds NEK7-HiBiT degradation was measured as a luminescence signal using CLARIOstar Multimode Plate reader.

Most of the compounds of the invention led to degradation of the NEK7-HiBiT protein. As indicated in Table 2 below, 13 out of 16 compounds reduced NEK7-HiBiT protein level by at least 50% at 10 pM. Additionally, compounds reducing NEK7-HiBiT protein level by >= 50% at 0.1 pM, were also identified.

Table 2: Levels of NEK7-HiBiT protein presented as a % of DMSO control (mean/SD of 3 technical replicates) following treatment with the compounds of the invention at a concentration of 10 pm.

A - Levels of NEK7-HiBiT Protein < 25%

B - Levels of NEK7-HiBiT Protein > 25% and < 50%

C - Levels of NEK7-HiBiT Protein > 50% and < 75%

D - Levels of NEK7-HiBiT Protein > 75%

NEK7 protein degradation determined by Western blot

NEK7-HiBiT degradation results were further confirmed for selected compounds in human PBMC- derived macrophages. Table 3 and Figure 1 shows the results of NEK7 protein levels in the above- mentioned cells treated with exemplary compounds at 0.1, 1 and 10 pM concentration or DMSO for 24h. As presented in Table 3 and Figure 1, Compound 2 and Compound 25 of the present invention induced dose-dependent degradation of NEK7 protein in macrophages derived from the human PB MC cells, resulting in almost complete degradation of the NEK7 protein at the highest dose.

Table 3: NEK7 protein degradation results in human PBMC-derived macrophages upon treatment with Compound 2 and Compound 25 at a concentration of 10 pM. Table shows densitometric values normalized to the loading control and calculated as a % of DMSO control. For Compound 2 results from two independent experiments and for Compound 25 results from one independent experiment were shown.

A - Amount of NEK7 protein < 10%

B - Amount of NEK7 protein > 10% and < 40%

C - Amount of NEK7 protein > 40% and < 75%

D - Amount of NEK7 protein > 75%

Measurement of IL-113 and IL-18 levels

To evaluate compound effect on inflammasome activation (measured as IL-ip and IL-18 release), human PBMC-derived macrophages were treated with exemplary compounds for 24h prior to inflammasome activation. As shown on Figure 2, after 24h of pre-treatment with Compound 2 and Compound 25, a dose-dependent decrease of cytokines level was noted. For Compound 25 only measurements for IL-lp were shown.