Compounds and methods for modulating CDK9 activity

Abstract

Heterobifunctional degrader compounds that bind to cyclin-dependent kinase 9 (CDK9) and E3 ubiquitin ligase are provided. Methods of treating a subject for a CDK9-mediated disease by administering a therapeutically effective amount of a pharmaceutical composition comprising the heterobifunctional degrader are also provided.

Description

[Background technology]

[0001] introduction The cyclin-dependent kinase (CDK) family of proteins is a key regulator of the cell cycle and gene transcription. The cell cycle is a cellular mechanism that regulates the timing of cell growth and division. The cell cycle is a multifaceted process that directs cell proliferation through a series of checkpoints that correct DNA damage, genetic abnormalities, and other errors. Nonhuman Primates in Biomedical Research (Second Edition, 2012) (Non-Patent Document 1). Each stage is controlled by a combination of cyclins and CDKs, which phosphorylate specific sets of cyclins to trigger entry into the next stage of the cell cycle. Casem, ML, Case Studies in Cell Biology, ISBN-13: 978-0128013946, 2016, Chapter: Cell Cycle (pp. 299-326) (Non-Patent Document 2). The accumulation of cyclin proteins through regulation of cyclin mRNA transcription acts as a 'biological switch' to turn CDKs on and off, transitioning cells from one stage to the next.

[0002] CDK1, 2, 3, 4, and 6 regulate the timing of the cell division cycle, while CDK7 and CDK9 regulate the transcriptional activity of RNA polymerase II through phosphorylation of its carboxy-terminal domain. Lucking, et al., ChemMedChem, 2017, 12, 1776-1793, doi:10.1002 / cmdc.201700447 (Non-Patent Document 3).

[0003] CDK9 regulates the transcriptional activity of key oncogenic proteins such as AR, MYC, MCL-1, and BCL-2, and stimulates pro-inflammatory transcription factors such as NFkB and STAT3. Gregory et al., Leukemia. 2015 June; 29(6): 1437-1441 (Non-Patent Document 4); Krystof, et al., Curr Pharm Des. 2012 July; 18(20): 2883-2890 (Non-Patent Document 5). CDK9 forms a heterodimer with one of four cyclin partners (cyclin T1, cyclin K, cyclin T2a, or cyclin T2b) called positive transcription elongation factors (P-TEFb). RNA polymerase II pauses mRNA transcription 20–40 nucleotides along the DNA template through interaction with negative elongation factors, which serve as the primary regulatory control mechanism for the transcription of rapidly induced genes. P-TEFb resolves RNA polymerase II-mediated pausing by phosphorylating the carboxy-terminal domain of RNA polymerase II and inactivating negative elongation factors. Compounds targeting CDK9 and P-TEFb are currently undergoing clinical trials. The enzymatic activity of CDK9 is important for stimulating transcription elongation of most protein-coding genes. Krystof, et al., Curr Pharm Des. 2012 July; 18(20): 2883-2890 (Non-Patent Document 5). Many CDK inhibitors with heterocyclic core structures have been developed. For example, the purine scaffold has been the source of CDK inhibitors developed for cancer treatment, including seliciclib (Cyclacel Pharmaceuticals, Inc.) and other purine derivatives. SC Wilson et al., Bioorg & Med Chem 2011 November; 19(22): 6949-6965 (Non-Patent Document 6). These purine derivatives target CDK9 as well as CDK7 and CDK2, although inhibition of CDK2 raises safety and toxicity concerns. Triazine core-based CDK9 inhibitors, such as atubeciclib, have also been developed.Lucking et al., ChemMedChem 2017, 12, 1776-1793 (Non-Patent Document 3). Unfortunately, treatment with CDK9 inhibitors remains relatively unsuccessful and is accompanied by numerous side effects. Morales et al., Cell Cycle 2016, vol. 15, no. 4, 519-527 (Non-Patent Document 7). Therefore, there is a need for novel CDK9 inhibitors for treating CDK9-mediated diseases. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Nonhuman Primates in Biomedical Research (Second Edition, 2012) [Non-patent document 2] Casem, ML, Case Studies in Cell Biology, ISBN-13: 978-0128013946, 2016, Chapter: Cell Cycle (p. 299-326) [Non-patent document 3] Lucking, et al., ChemMedChem, 2017, 12, 1776-1793, doi:10.1002 / cmdc.201700447 [Non-patent document 4] Gregory et al., Leukemia. 2015 June; 29(6): 1437-1441 [Non-Patent Document 5] Krystof, et al., Curr Pharm Des. 2012 July; 18(20): 2883-2890 [Non-patent document 6] SC Wilson et al., Bioorg & Med Chem 2011 November; 19(22): 6949-6965 [Non-Patent Document 7] Morales et al., Cell Cycle 2016, vol. 15, no. 4, 519-527 Summary of the Invention

[0005] overview Heterobifunctional degrader compounds that bind to cyclin-dependent kinase 9 (CDK9) and E3 ubiquitin ligase are provided. Methods of treating a subject for a CDK9-mediated disease by administering a therapeutically effective amount of a pharmaceutical composition comprising the heterobifunctional degrader are also provided. [The present invention 1001] A compound of formula (I) or a pharmaceutically acceptable salt thereof: XLY (I) During the ceremony, X is an E3 ubiquitin ligase binding ligand; and L is a linking group covalently bonded to X and Y; Y is a group represented by formula (II): TIFF0007814386000001.tif32128 is a cyclin-dependent kinase 9 (CDK9) binding ligand of R 1 D, halo, R at any position 7 CO 2 R 8 , CO 2 R 8 , CO 2 H, R 7 CO 2 H, NH 2 , NHR 8 , OH, OR 8 , S.H., S.R. 8 , NHCOR 8 ,NHSO 2 R 8 , SO 2 NH 2 , SO 2 NHR 8 or C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 cycloalkenyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, optionally substituted with one or more of Alternatively, R 1 and R2 together form D, halo, NH at any position. 2 , NHR 8 , N.R. 7 R 8 , OH, OR 8 , S.H., S.R. 8 , NHCOR 8 ,NHSO 2 R 8 , SO 2 NH 2 , or SO 2 NHR 8 forming a fused C5-C6 cycloaryl optionally substituted with one or more of: R 2 、R 3 , and R 5 are independently H, D, halo, or at any position D, halo, R 7 CO 2 R 8 , CO 2 R 8 , CO 2 H, R 7 CO 2 H, NH 2 , NHR 8 , OH, OR 8 , S.H., S.R. 8 , NHCOR 8 ,NHSO 2 R 8 , SO 2 NH 2 , SO 2 NHR 8 or a C1-C5 alkyl or C3-C6 cycloalkyl optionally substituted with one or more of Alternatively, R 2 and R 5 together, D, halo, R at any position 7 CO 2 R 8 , CO 2 R 8 , CO 2 H, R 7 CO 2 H, NH 2 , NHR 8 , OH, OR 8 , S.H., S.R. 8 , NHCOR 8 ,NHSO 2 R 8 , SO 2 NH 2 , SO 2 NHR 8 forming a fused C5-C6 cycloaryl optionally substituted with one or more of: R 4 D, halo, R at any position 7 CO 2 R 8 , CO 2 R 8 , CO 2 H, R 7 CO 2 H, NH 2 , NHR 8 , OH, OR 8 , S.H., S.R. 8 , NHCOR 8 ,NHSO 2 R 8 , SO 2 NH 2 , SO 2 NHR 8 H, D, halo, C1-C5 alkyl, C3-C6 cycloalkyl, cyano, hydroxyl, or —O—(C1-C5 alkyl), optionally substituted with one or more of: R 6 is H or D; R 7 is (CH 2 ) n where n is an integer from 1 to 6; and R 8 is D, halo, OH, SH, or NH at any position 2 and C1-C6 alkyl or C3-C6 cycloalkyl optionally substituted with one or more of: [The present invention 1002] Y is R 5 The compound of claim 1001, wherein the compound is covalently bonded to L via [The present invention 1003] R 5 is an optionally substituted C3 to C6 cycloalkyl group. [The present invention 1004] R 5 The compound of the present invention 1003, wherein is an optionally substituted C5 cycloalkyl group. [The present invention 1005] R 5 but, TIFF0007814386000002.tif15128 1004. A compound of the present invention having a structure selected from the group consisting of: [The present invention 1006] R 1 The compound of any one of claims 1001 to 1005, wherein is a C1 to C6 alkyl group. [The present invention 1007] R 1 but, TIFF0007814386000003.tif33128 1006. A compound of the present invention having a structure selected from the group consisting of: [The present invention 1008] R 2 、R 3 , and R 6 are each independently H or D. [The present invention 1009] R 4 The compound of any one of claims 1001 to 1008, wherein is halo, cyano, or methyl. [The present invention 1010] R 4 The compound of any one of 1001 to 1008 of the present invention, wherein is H or D. [The present invention 1011] Y is, TIFF0007814386000004.tif73128 1001. A compound of the present invention having a structure selected from the group consisting of: [The present invention 1012] 10. The compound of any one of claims 1001 to 1011, wherein X is a cereblon (CRBN) binding ligand. [The present invention 1013] 1012. The compound of claim 10, wherein X has 200 or fewer atoms. [The present invention 1014] X is, TIFF0007814386000005.tif88128 1013. A compound of the present invention having a structure derived by removing a hydrogen atom from a structure selected from the group consisting of: [The present invention 1015] X is a structure TIFF0007814386000006.tif33128 The compound of the present invention 1014, having the formula: [The present invention 1016] 1001-1011. Any of the compounds of claims 1001-1011, wherein X is a von Hippel-Lindau (VHL) binding ligand. [The present invention 1017] 1016. The compound of claim 10, wherein X has 200 or fewer atoms. [The present invention 1018] X is, TIFF0007814386000007.tif55128 1017. A compound of the present invention having a structure derived by removing a hydrogen atom from a structure selected from the group consisting of: [The present invention 1019] The compound of the present invention wherein L has the structure of formula (III): TIFF0007814386000008.tif27128 During the ceremony, a, b, c, and d are each independently an integer of 0 to 6; e is 0 or 1, R 10 does not exist or TIFF0007814386000009.tif12128 and having a structure selected from the group consisting of R 11 does not exist or TIFF0007814386000010.tif13128 It has a structure selected from the group consisting of: [The present invention 1020] 1001. A compound of the present invention, wherein said compound has a structure selected from the group consisting of compounds X.0, wherein X is an integer ranging from 1 to 59. [The present invention 1021] A method for treating a subject for a CDK9-mediated disease, comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising any one of the compounds of present inventions 1001 to 1020. [The present invention 1022] The method of claim 1021, wherein the CDK9-mediated disease is cancer. [The present invention 1023] 1023. The method of claim 1021 or 1022, wherein said cancer is caused by aberrant expression of MYC-1 or MCL-1, or is a hematological malignancy, or a solid tumor. [The present invention 1024] Any of the methods of present inventions 1021 to 1023, wherein the disease is selected from the group consisting of acute myeloid leukemia, primary peritoneal carcinoma, chronic lymphocytic leukemia, recurrent multiple myeloma, non-Hodgkin's lymphoma, acute lymphoblastic leukemia, acute mixed lineage leukemia, advanced breast cancer, non-small cell lung cancer, and liver cancer. [The present invention 1025] The method of any one of claims 1021 to 1024, wherein the compound is that of claim 1020. DETAILED DESCRIPTION OF THE INVENTION

[0006] Detailed Description Heterobifunctional degrader compounds that bind to cyclin-dependent kinase 9 (CDK9) and E3 ubiquitin ligase are provided. Methods of treating a subject for a CDK9-mediated disease by administering a therapeutically effective amount of a pharmaceutical composition comprising the heterobifunctional degrader are also provided.

[0007] Before describing the present invention in more detail, it is to be understood that the present invention may have many different embodiments and is not limited to the particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0008] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded within the range, and each range in which either, neither, or both limits are included within the smaller range is also encompassed within the invention, except where there is any specifically excluded limit within the stated range. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included within the invention.

[0009] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those usually understood by those skilled in the art to which this invention belongs.Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this invention, and some potential and exemplary methods and materials may be described below.All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials for which the publications are cited.To the extent that there is a conflict with the incorporated publications, it is understood that the present disclosure shall prevail.

[0010] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural forms of the referent unless the context clearly dictates otherwise. Thus, for example, a reference to "a droplet" includes a plurality of such droplets, and a reference to "the discrete entity" includes a reference to one or more discrete entities.

[0011] It is further noted that the claims may be drafted to exclude any element, for example, any optical element, and as such, this statement is intended to serve as a precondition for the use of exclusive terminology such as "only," "only," or the use of a "negative" limitation in connection with the recitation of claim elements.

[0012] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed. To the extent that a definition or usage of any term herein conflicts with a definition or usage of the term in an application or reference incorporated herein by reference, the present application shall control.

[0013] It will be apparent to one of ordinary skill in the art upon reading this disclosure that each of the individual embodiments described and illustrated herein has distinct components and features which are readily distinguishable from or combinable with the features of any of the other embodiments without departing from the scope or spirit of the invention. Any described method can be carried out in the order of events described or in another order which is logically possible.

[0014] definition The terms "linker" and "linking group" are used interchangeably to refer to a group that connects two groups. An exemplary type of connection includes a covalent bond. As used herein, the term "radical," such as "monoradical" or "diradical," refers to the number of groups to which a group can connect. As an example, a monoradical group can connect to only one other group; for example, methyl (-CH3) and ethyl (-CH2CH3) groups are monoradical groups. In contrast, -CH2- and -CH2CH2- groups are diradical groups because they can each connect to two different groups. Because the linker connects to two groups, the linker is a diradical group. The connection between groups can also be described by the term "valency," such as "monovalent" or "divalent," which refers to the bond order of the connection. As an example, the group -CH3 is a monovalent group because it can form a single covalent bond with another group, such as -OH, to form H3COH. The group =CH2 is a divalent group because it can form a double bond with another group, for example, an oxygen atom, to form formaldehyde (CH2O).

[0015] "Alkyl" refers to a monovalent saturated aliphatic hydrocarbyl group having 1 to 10 carbon atoms, e.g., 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3 carbon atoms. This term includes straight-chain and branched hydrocarbyl groups such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), t-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-), by way of example.

[0016] The term "substituted alkyl" refers to an alkyl group as defined herein, except that one or more carbon atoms in the alkyl chain (excluding the C1 carbon atom) are substituted with -O-, -N-, -S-, -S(O), n - (n is 0 to 2), -NR- (R is hydrogen or alkyl), and the like, optionally substituted with a heteroatom such as alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl, and -NR a R bwherein R' and R'' may be the same or different and are selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, and heterocycle. As an example, the -CH2CH2CH3 (propyl) group can be considered an alkyl group. If the second carbon atom is replaced with an oxygen atom, the resulting group is -CHOCH3. This group can be considered a substituted alkyl because it can be considered a -CH3 (methyl) group with one of the hydrogen atoms replaced with a -OCH3 (methoxy) group.

[0017] "Alkylene" is either straight chain or branched and includes -O-, -NR 10 -, -NR 10 C(O)-, -C(O)NR 10 - refers to a divalent aliphatic hydrocarbyl group, preferably having 1 to 6, more preferably 1 to 3 carbon atoms, optionally interrupted by one or more groups selected from the group consisting of methylene (-CH-), ethylene (-CHCH-), n-propylene (-CHCHCH-), isopropylene (-CHCH(CH)-), (-C(CH)CHCH-), (-C(CH)CHC(O)-), (-C(CH)CHC(O)NH-), (-CH(CH)CH-), and the like.

[0018] "Substituted alkylene" refers to an alkylene group in which one to three hydrogens have been replaced with a substituent as set forth with respect to carbon in the definition of "substituted" below.

[0019] The term "alkane" refers to alkyl and alkylene groups, as defined herein.

[0020] "Alkenyl" refers to straight or branched chain hydrocarbyl groups having from 2 to 6 carbon atoms, preferably from 2 to 4 carbon atoms, and at least 1, preferably 1 to 2, sites of double bond unsaturation. This term includes, by way of example, bivinyl, allyl, and but-3-en-1-yl. This term includes cis and trans isomers or mixtures of these isomers.

[0021] The term "substituted alkenyl" refers to an alkenyl group, as defined herein, having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0022] "Alkynyl" refers to a straight-chain or branched monovalent hydrocarbyl group having from 2 to 6 carbon atoms, preferably from 2 to 3 carbon atoms, and having at least 1, preferably 1 to 2, sites of triple bond unsaturation. Examples of such alkynyl groups include acetylenyl (-C≡CH) and propargyl (-CHC≡CH).

[0023] The term "substituted alkynyl" refers to an alkynyl group, as defined herein, having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0024] "Aryl" or "Ar" refers to a monovalent aromatic carbocyclic group of 6 to 18 carbon atoms having a single ring (e.g., present in a phenyl group) or a ring system having multiple condensed rings (examples of such aromatic ring systems include naphthyl, anthryl, and indanyl), which may or may not be aromatic, but where the point of attachment is through an atom of the aromatic ring. This term includes, by way of example, phenyl and naphthyl. Unless otherwise constrained by the definition of an aryl substituent, such an aryl group may be optionally substituted with 1 to 5 substituents, or 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and trihalomethyl.

[0025] "Cyano" or "nitrile" refers to the group --CN.

[0026] "Cycloalkyl" refers to cyclic alkyl groups of from 3 to 10 carbon atoms having mono- or polycyclic rings, including fused, bridged, and spirocyclic systems. Examples of suitable cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl.

[0027] The term "substituted cycloalkyl" refers to a cycloalkyl group having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0028] "Cycloalkenyl" refers to a non-aromatic cyclic alkyl group of 3 to 10 carbon atoms having a monocyclic or polycyclic ring and at least one double bond, preferably 1 to 2 double bonds.

[0029] The term "substituted cycloalkenyl" refers to cycloalkenyl groups having from 1 to 5, or from 1 to 3, substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0030] "Cycloalkynyl" refers to a non-aromatic cycloalkyl group having mono- or polycyclic rings and having at least one triple bond.

[0031] "Halo" or "halogen" refers to fluoro, chloro, bromo, and iodo.

[0032] "Heteroaryl" refers to an aromatic group having 1 to 15 carbon atoms, e.g., 1 to 10 carbon atoms, and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur, within the ring. Such heteroaryl groups can have a single ring (e.g., pyridinyl, imidazolyl, or furyl) or multiple condensed rings within the ring system (e.g., as in groups such as indolizinyl, quinolinyl, benzofuran, benzimidazolyl, or benzothienyl), with at least one ring within the ring system being aromatic. To satisfy valency requirements, any heteroatoms in such heteroaryl rings may or may not be bonded to H or a substituent, e.g., an alkyl group or other substituent described herein. In certain embodiments, the nitrogen and / or sulfur ring atoms of a heteroaryl group may be oxidized to provide N-oxide (N→O), sulfinyl, or sulfonyl moieties. The term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl. Unless otherwise constrained by the definition of a heteroaryl substituent, such heteroaryl groups are optionally substituted with 1 to 5 substituents, or 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and trihalomethyl.

[0033] "Heterocycle," "heterocyclic," "heterocycloalkyl," and "heterocyclyl" refer to saturated or unsaturated groups having a single ring or multiple fused rings, including fused-bridged rings and spiro ring systems, and having 3 to 20 ring atoms, including 1 to 10 heteroatoms. These ring atoms are selected from nitrogen, sulfur, or oxygen; in fused ring systems, one or more of the rings can be cycloalkyl, aryl, or heteroaryl, but the point of attachment is through a non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atoms of a heterocyclic group can be oxidized to provide an N-oxide, -S(O)-, or -SO2- moiety. To satisfy valence requirements, any heteroatom in such a heterocyclic ring may be bonded or unbonded to one or more H or one or more substituents, such as alkyl groups or other substituents described herein.

[0034] Examples of heterocycles and heteroaryls are azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazoline, These include, but are not limited to, benzo[b]thiophene, benzo[b]thiophene, benzoyl, benzo[b]thiophene ...

[0035] Unless otherwise constrained by the definition of a heterocyclic substituent, such heterocyclic groups are optionally substituted with 1 to 5 substituents, or 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and fused heterocycle.

[0036] "Oxo" refers to the atom (=O).

[0037] Further to the disclosure herein, the term "substituted," when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituents, as defined below.

[0038] In addition to the groups disclosed in connection with each individual term herein, substituents to replace one or more hydrogens on a saturated carbon atom in a specified group or radical (where any two hydrogens on a single carbon are ═O, ═NR, etc.) are also included. 70 , =N-OR 70 , =N2 or =S) is -R unless otherwise specified. 60 , halo, =O, -OR 70 , -SR 70 , -NR 80 R 80、トリハロメチル、-CN、-OCN、-SCN、-NO、-NO2、=N2、-N3、-SO2R 70 、-SO2O - M + 、-SO2OR 70 、-OSO2R 70 、-OSO2O - M + 、-OSO2OR 70 、-P(O)(O - )2(M + )2、-P(O)(OR 70 )O - M + 、-P(O)(OR 70 )2、-C(O)R 70 、-C(S)R 70 、-C(NR 70 )R 70 、-C(O)O - M + 、-C(O)OR 70 、-C(S)OR 70 、-C(O)NR 80 R 80 、-C(NR 70 )NR 80 R 80 、-OC(O)R 70 、-OC(S)R 70 、-OC(O)O - M + 、-OC(O)OR 70 、-OC(S)OR 70 、-NR 70 C(O)R 70 、-NR 70 C(S)R 70 、-NR 70 CO2 - M + 、-NR 70 CO2R 70 、-NR 70 C(S)OR 70 、-NR 70 C(O)NR 80 R 80 、-NR 70 C(NR 70 )R 70 および-NR 70 C(NR 70 )NR 80 R80 and R 60 is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl; and each R 70 are independently hydrogen or R 60 and each R 80 is independently R 70 or two R 80 together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocycloalkyl, which may contain 1 to 4 identical or different additional heteroatoms selected from the group consisting of O, N, and S, wherein N may have —H or C1-C3 alkyl substitution; each M + is a counterion with a net single positive charge. + are independent, e.g., K + , Na + , Li + Alkali ions such as +N(R 60 ) 4; or [Ca 2+ ] 0.5 , [Mg 2+ ] 0.5 , or [Ba 2+ ] 0.5 (The "0.5" subscript means that one of the counterions to such divalent alkaline earth ions is the ionized form of a compound of the invention and the other is a typical counterion such as chloride, or two ionized compounds disclosed herein can serve as counterions to such divalent alkaline earth ions, or a doubly ionized compound of the invention can serve as counterions to such divalent alkaline earth ions.) Specific examples include -NR 80 R 80 is meant to include -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, N-methyl-piperazin-1-yl, and N-morpholinyl.

[0039] Further to the disclosure herein, substituents replacing hydrogens on unsaturated carbon atoms in "substituted" alkene, alkyne, aryl, and heteroaryl groups are designated by -R unless otherwise specified. 60 , halo, -O - M + , -OR 70 , -SR 70 , -S - M + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R 70 , -SO3 - M + , -SO3R 70 , -OSO2R 70 , -OSO3 - M + , -OSO3R 70 , -PO3 -2 (M + )2, -P(O)(OR 70 )O - M + , -P(O)(OR 70 )2, -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -CO2 - M + , -CO2R 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OCO2 - M + , -OCO2R 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 CO2 - M + , -NR 70CO2R 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 and R 60 , R 70 , R 80 and M + is as defined above, but in the case of a substituted alkene or alkyne, the substituent is -O - M + , -OR 70 , -SR 70 , or -S - M + isn't it.

[0040] In addition to the groups disclosed in connection with each individual term herein, substituents replacing hydrogens on nitrogen atoms in "substituted" heteroalkyl and cycloheteroalkyl groups include, unless otherwise specified, -R 60 , -O - M + , -OR 70 , -SR 70 , -S - M + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R 70 , -S(O)2O - M + , -S(O)2OR 70 , -OS(O)2R 70 , -OS(O)2O - M + , -OS(O)2OR 70 , -P(O)(O - )2(M + )2, -P(O)(OR 70 )O - M + , -P(O)(OR 70 )(OR 70), -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -C(O)OR 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OC(O)OR 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 C(O)OR 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 and R 60 , R 70 , R 80 and M + is as defined above.

[0041] Further to the disclosure herein, in certain embodiments, a substituted group has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

[0042] For all of the substituents defined above, polymers arrived at by defining the substituents to have further substituents thereon (e.g., a substituted aryl having a substituted aryl group as a substituent, which is itself substituted with a substituted aryl group, which is further substituted with a substituted aryl group, etc.) are not intended to be encompassed herein. In such cases, the maximum number of such substitutions is 3. For example, the series of substitutions of substituted aryl groups specifically contemplated herein is limited to substituted aryl-(substituted aryl)-substituted aryl.

[0043] Unless otherwise indicated, names of substituents not explicitly defined herein are arrived at by naming the terminal portion of the functional group followed by the adjacent functional group toward the point of attachment. For example, the substituent "arylalkyloxycarbonyl" refers to the group (aryl)-(alkyl)-OC(O)-.

[0044] For any of the groups disclosed herein that contain one or more substituents, it is of course understood that such groups do not include any substitutions or patterns that are sterically impractical and / or synthetically infeasible. In addition, the subject compounds include all stereochemical isomers arising from the substitution of these compounds.

[0045] The term "pharmaceutically acceptable salt" refers to a salt that is acceptable for administration to a patient, such as a mammal (a salt whose counterion has acceptable mammalian safety for a given administration regimen). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids. "Pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of a compound, which is derived from a variety of organic and inorganic counterions known in the art, including, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc., and, if the molecule contains a basic functional group, organic or inorganic acid salts such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, etc.

[0046] The term "salt thereof" refers to a compound formed when a proton of an acid is replaced by a cation, such as a metal cation or an organic cation. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not required for salts of intermediate compounds not intended for administration to a patient. By way of example, salts of the present compounds include those in which the compound is protonated with an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid being the anionic component of the salt.

[0047] "Stereoisomer" and "stereoisomers" refer to compounds that have the same atoms connected but differ in the arrangement of the atoms in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers.

[0048] "Tautomer" refers to alternative forms of molecules that differ only in the electronic bonding and / or position of protons of the atoms, such as enol-keto and imine-enamine tautomers, or tautomeric forms of heteroaryl groups that contain the ring atom configuration -N=C(H)-NH-, such as pyrazole, imidazole, benzimidazole, triazole, tetrazole, etc. One of ordinary skill in the art will recognize that other tautomeric ring atom configurations are possible.

[0049] It will be appreciated that the term "or a salt or solvate or stereoisomer thereof" is intended to include all permutations of salts, solvates and stereoisomers, e.g., solvates of pharmaceutically acceptable salts of stereoisomers of the subject compound.

[0050] compound Heterobifunctional degrader compounds are provided that bind to cyclin-dependent kinase 9 (CDK9) and E3 ubiquitin ligase. In some cases, the compounds are of formula (I), or a pharmaceutically acceptable salt thereof: XLY (I) where: X is an E3 ubiquitin ligase binding ligand; and L is a linking group covalently bonded to X and Y; Y is a group represented by formula (II): TIFF0007814386000011.tif34128 is a cyclin-dependent kinase 9 (CDK9) binding ligand; R 1 D, halo, R at any position 7 CO2R 8 , CO2R 8 , CO2H, R 7 CO2H, NH2, NHR 8 , OH, OR 8 , S.H., S.R. 8 , NHCOR 8 , NHSO2R 8 , SO2NH2, SO2NHR 8 or C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 cycloalkenyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, optionally substituted with one or more of Alternatively, R 1 and R 2 together, D, halo, NH2, NHR at any position 8 , N.R. 7 R 8 , OH, OR 8 , S.H., S.R.8 , NHCOR 8 , NHSO2R 8 , SO2NH2, or SO2NHR 8 forming a fused C5-C6 cycloaryl optionally substituted with one or more of: R 2 , R 3 , and R 5 are independently H, D, halo, or at any position D, halo, R 7 CO2R 8 , CO2R 8 , CO2H, R 7 CO2H, NH2, NHR 8 , OH, OR 8 , S.H., S.R. 8 , NHCOR 8 , NHSO2R 8 , SO2NH2, SO2NHR 8 or a C1-C5 alkyl or C3-C6 cycloalkyl optionally substituted with one or more of Alternatively, R 2 and R 5 together, D, halo, R at any position 7 CO2R 8 , CO2R 8 , CO2H, R 7 CO2H, NH2, NHR 8 , OH, OR 8 , S.H., S.R. 8 , NHCOR 8 , NHSO2R 8 , SO2NH2, SO2NHR 8 forming a fused C5-C6 cycloaryl optionally substituted with one or more of: R 4 D, halo, R at any position 7 CO2R 8 , CO2R 8 , CO2H, R 7 CO2H, NH2, NHR 8 , OH, OR 8 , S.H., S.R. 8 , NHCOR 8 , NHSO2R 8 , SO2NH2, SO2NHR8 H, D, halo, C1-C5 alkyl, C3-C6 cycloalkyl, cyano, hydroxyl, or —O—(C1-C5 alkyl), optionally substituted with one or more of: R 6 is H or D; R 7 is (CH2) n where n is an integer from 1 to 6; and R 8 is a C1-C6 alkyl or C3-C6 cycloalkyl optionally substituted at any position with one or more of D, halo, OH, SH, or NH2.

[0051] Base Y Y is a cyclin-dependent kinase 9 (CDK9) binding ligand of formula (II). As used herein, "CDK9" refers to a protein encoded by the CDK9 gene. CDK refers to cyclin-dependent kinase, "cyclin" refers to the regulation of the cell cycle, and "kinase" refers to a protein that functions as an enzyme by phosphorylating other proteins. Several CDK genes and proteins are known, and the specific CDK discussed herein is named CDK9. Additional names for CDK9 include CDC2L4, PITALRE, C-2k, and TAK. In some cases, the CDK9 protein is encoded by the human CDK9 gene located on chromosome 9, band 9q34.11. One function of CDK9 is to regulate the transcriptional activity of key oncogenic proteins such as AR, MYC, MCL-1, and BCL-2, and stimulate proinflammatory transcription factors such as NFkB and STAT3 (Gregory et al., Leukemia. 2015 June; 29(6): 1437-1441; Krystof, et al., Curr Pharm Des. 2012 July; 18(20): 2883-2890). CDK9 can heterodimerize with one of four cyclin partners (cyclin T1, cyclin K, cyclin T2a, or cyclin T2b) called positive transcription elongation factor (P-TEFb).

[0052] In some cases, Y specifically binds to CDK9. As used herein, "specific binding" refers to the ability of a first group (or a first member of a specific binding pair) to preferentially bind to a particular analyte (or a second member of a specific binding pair) in a mixture of different analytes. For example, Y may preferentially bind to CDK9 over CDK4, i.e., Y selectively binds to CDK9 over CDK4. In some cases, the binding preference is 5-fold or more, e.g., 10-fold or more, or 100-fold or more. The term "binding ligand" means that one group can bind to another group, e.g., group Y can bind to CDK9.

[0053] In some cases, Y is R 5 Covalently bonded to L via, for example, R 5 can be an optionally substituted C3-C6 cycloalkyl group. 5 can be a substituted C3-C6 cycloalkyl group. In some cases, R 5 is an optionally substituted C5 cycloalkyl group, for example, a substituted C5 cycloalkyl group. In some cases, R 5 teeth TIFF0007814386000012.tif15128.

[0054] In some cases, Y is R 1 , R 2 , R 3 , R 4 , or R 6 In some cases, Y is covalently bonded to L through the five-membered ring of formula (II). That is, R 3 or R 4 does not exist, and R 3 or R 4 There is a covalent bond to L at the position of R. 3 or R 4 can be considered a hydrogen atom, but its removal can provide a site for covalent bonding to L. In some cases, Y is covalently bonded to L through the six-membered ring of formula (II). That is, R 1 or R 2 does not exist, and R 1 or R 2 There is a covalent bond to L at position

[0055] As discussed above, R 1 is C1-C6 alkyl, C3-C6 cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, and may be substituted. 1is heterocyclyl, e.g., heterocycloalkyl or heterocycloalkenyl. Exemplary heterocycloalkenyl groups include structures derived from dihydrofuran, dihydropyran, or dihydropyrrole by removal of a hydrogen atom. Exemplary heterocycloalkyl groups include structures derived from tetrahydrofuran, tetrahydropyran, and tetrahydropyrrole by removal of a hydrogen atom. In some cases, R 1 is a C1-C6 alkyl group. In some cases, R 1 teeth TIFF0007814386000013.tif33128.

[0056] In some cases, R 2 , R 3 , and R 6 are each independently H or D. For example, in some cases R 2 , R 3 , and R 6 are H. In other cases, R 2 , R 3 , and R 6 One or more of the groups may be D, e.g., R 2 is H and R 3 is D and R 6 is D. In some cases, R 2 and R 3 or both independently may be selected from D, halo, R at any position. 7 CO2R 8 , CO2R 8 , CO2H, R 7 CO2H, NH2, NHR 8 , OH, OR 8 , S.H., S.R. 8 , NHCOR 8 , NHSO2R 8 , SO2NH2, SO2NHR 8 and C1-C5 alkyl or C3-C6 cycloalkyl optionally substituted with one or more of the following.

[0057] In some embodiments, R4 is selected from the group consisting of halo, cyano, C1-C5 alkyl, hydroxyl, -O-(C1-C5 alkyl), and fluorinated C1-C5 alkyl. C1-C5 alkyl refers to an alkyl group having 1, 2, 3, 4, or 5 carbon atoms. Fluorinated C1-C5 alkyl refers to a C1-C5 alkyl group in which one or more hydrogen atoms have been replaced with fluorine atoms, e.g., -CH2F, CHF2, and -CF3. Exemplary -O-(C1-C6 alkyl) groups include methoxy and ethoxy. In some cases, R 4 is halo, cyano, or methyl. In some cases, R 4 is halo, e.g., chloro. In other cases, R 4 is H or D.

[0058] In some cases, Y is TIFF0007814386000014.tif76128.

[0059] Additional exemplary Y groups are described in U.S. Patent Application Publication No. 2020 / 0131189 (hereinafter, the '189 Publication), which is incorporated herein by reference. Formula (I) of the '189 Publication describes compounds having the same general structure and numbering as set forth in Formula (II) of the present application. As an example, the Y group can include the structure of any one of compounds 1-208 of the '189 Publication. In other words, a hydrogen atom can be removed from any one of compounds 1-208 of the '189 Publication, and the resulting monovalent group can be attached to an L group at the position of the removed hydrogen atom. For example, compound 1 of the '189 Publication is shown below, and a hydrogen atom (not shown) can be removed to provide a position for connecting the Y group to the L group. As an example, in the first and second options shown below, a hydrogen atom located on the cyclopentane ring can be removed, and the resulting monovalent group can be a Y group that connects to an L group at the position of the removed hydrogen atom. In a third option, a hydrogen atom on the pyrazole ring is removed to provide a connection to the L group. In some cases, the Y group is a derivative of any one of compounds 1-208 of the '189 publication, for example, where the connection to the L group is through a cycloalkyl group attached to the amino group, as shown in options 1 and 2 below. TIFF0007814386000015.tif87128

[0060] Group X As described above, X is an E3 ubiquitin ligase-binding ligand. In other words, it has the ability to bind to an E3 ubiquitin ligase. This binding, and its potential use in treating patients for disease states, can be understood by considering the role of E3 ubiquitin ligases in a biochemical process known as the ubiquitin proteasome system (UPS).

[0061] The ubiquitin proteasome system (UPS) helps regulate protein homeostasis by degrading proteins, such as damaged or misfolded proteins. The UPS system involves a series of enzymatic events called E1, E2, and E3. During the E1 step, the protein ubiquitin is activated to form a covalent intermediate with the E1 enzyme. The ubiquitin protein is then transferred from the E1 enzyme to an E2 enzyme (i.e., an E2 ubiquitin conjugating enzyme). In the final step, an E3 enzyme (i.e., an E3 ubiquitin ligase) catalyzes the transfer of ubiquitin from the E2 to the protein destined for degradation. The presence of ubiquitin or polyubiquitin chains on the target protein is then recognized by another component called the proteasome, which actually degrades the target protein. Thus, the term "E3 ubiquitin ligase" refers to an enzyme that catalyzes the transfer of a ubiquitin group from another enzyme, e.g., an E2 ubiquitin conjugating enzyme, to a target protein that is degraded, e.g., by the proteasome, upon detection of one or more ubiquitin groups.

[0062] Thus, the X group of the compounds can bind to a portion of an E3 ubiquitin ligase, and this binding allows the compounds to affect the operation of the ubiquitin ligase system in some cases, such as using the UPS to selectively degrade certain proteins, which can be used to treat patients.

[0063] In some cases, X is a cereblon (CRBN)-binding ligand, where CRBN is a protein encoded by the CRBN gene. Additional names for CRBN include MRT2 and MRT2A (mental retardation, nonsyndromic, autosomal recessive, A2). CRBN's role in UPS protein degradation occurs when CRBN binds with other proteins to form an E3 enzyme complex, i.e., an "E3 ubiquitin ligase complex." In some cases, CRBN binds with cullin 4A (CUL4A), regulator of cullin 1 (ROC1), and damaged DNA-binding protein 1 (DDB1) to form an E3 ubiquitin ligase complex. Thus, because the X group of the compound has the ability to bind to cereblon (CRBN), this interaction, in some cases, can bring the compound as a whole into close proximity with the E3 ubiquitin ligase complex. In some cases, X binds to the CRBN protein encoded by the human CRBN gene, which is located on chromosome 3 at band 3p26.2.

[0064] In some cases, X is a CRBN-binding ligand that is a small molecule group, e.g., that has 200 atoms or less. For example, X is The compound may have a structure derived by removal of a hydrogen atom from a structure selected from the group consisting of: TIFF0007814386000016.tif60128.

[0065] The X group can be attached to L at any suitable point. For example, a hydrogen atom at any one of the positions marked with an asterisk (*) in the structure below can be removed to provide a position for covalent attachment to L. Thus, in some cases, the X group is attached to the L group through an aryl carbon, an alkyl carbon, or a nitrogen atom. TIFF0007814386000017.tif20128

[0066] As another example, the X group can be derived from the molecule shown below, where, for example, the bond to L can be through the group shown as -NH. In another case, the X group has the structure shown below, except that a hydrogen atom corresponding to one of those marked with an asterisk (*) in the example above has been removed to provide a location for the bond to L. TIFF0007814386000018.tif21128

[0067] Thus, the X group can have a structure derived from the four CRBN-binding ligands shown above, with one hydrogen atom removed to provide a location for covalent attachment to L. In some cases, the X group has the formula: TIFF0007814386000019.tif33128

[0068] In addition to cereblon (CRBN), von Hippel-Lindau (VHL) is another protein involved in E3 ubiquitin ligases. The VHL protein is encoded by the VHL gene and is sometimes called the "von Hippel-Lindau tumor suppressor" because mutations in the VHL gene can cause cancerous tumors. In some cases, X binds to the VHL protein, which is encoded by the human VHL gene located on chromosome 3 at band 3p25.3. In the context of the ubiquitin proteasome system (UPS), VHL can bind to other proteins, such as elongin B, elongin C, and cullin-2, to form E3 ubiquitin ligases. Further names for VHL proteins include HRCA1, RCA1, and VHL1. Thus, because the VHL protein is part of an E3 ubiquitin ligase, if X is a VHL-binding ligand, this binding may bring the compounds described herein into proximity with the E3 ubiquitin ligase, allowing the ligase to be modified or utilized, for example, for therapeutic purposes.

[0069] In some cases, X is a von Hippel-Lindau (VHL) binding ligand. In some cases, X is a VHL binding ligand that is a small molecule group, e.g., having 200 atoms or less. As an example, X can have a structure derived by removing a hydrogen atom from the structure shown below. TIFF0007814386000020.tif55128

[0070] X can be covalently bonded to L at any suitable position. By way of example, a hydrogen atom at any of the positions marked with an asterisk (*) can be removed to provide a position for bonding to L. In some cases, X connects to L through an aryl carbon, alkyl carbon, nitrogen atom, or oxygen atom. TIFF0007814386000021.tif61128

[0071] For example, X is TIFF0007814386000022.tif55128.

[0072] In some cases, X specifically binds to an E3 ubiquitin ligase. As used herein, "specific binding" refers to the ability of a first group (or a first member of a specific binding pair) to preferentially bind to a particular analyte (or a second member of a specific binding pair) in a mixture of different analytes. For example, Y may preferentially bind to an E3 ubiquitin ligase, e.g., tumor necrosis factor alpha (TNF-α), over other proteins in a cell. In some cases, the binding preference is 5-fold or more, e.g., 10-fold or more, or 100-fold or more.

[0073] Base L As described above, L is a linking group covalently bonded to X and Y. As used herein, "linking group" is used interchangeably with "linker" to refer to a group connecting two or more other groups, e.g., X and Y. In some cases, the backbone of the linking group L is 100 atoms or less, e.g., 50 atoms or less or 25 atoms or less. The term "backbone" refers to the minimum number of atoms that connect each other through a series of covalent bonds to each of the terminal atoms that are bonded to other groups, e.g., X and Y. In some embodiments, L is a hydrocarbon group, e.g., alkyl, alkylene, alkenylene, alkynylene, aryl, heteroaryl, cycloalkyl, heterocycle, or a substituted derivative thereof, or a combination thereof. In some cases, the linking group L includes a combination of different groups, e.g., a substituted alkyl connected to a heterocycle.

[0074] In some cases, L is a group of formula (III) It has the structure TIFF0007814386000023.tif29128, During the ceremony, a, b, c, and d are each independently an integer of 0 to 6; e is 0 or 1, R 10 does not exist or TIFF0007814386000024.tif12128, and R 11 does not exist or TIFF0007814386000025.tif13128.

[0075] In some cases, a is 1 or 2. In some cases, b is 0, 1, or 2. In some cases, c is 0, 1, 2, or 3. In some cases, d is 0. In some cases, e is 0. In some cases, a is 1 or 2, b is 0, 1, or 2, c is 0, 1, 2, or 3, d is 0, and e is 0.

[0076] In some cases, R 11 does not exist, and R 10 teeth TIFF0007814386000026.tif12128.

[0077] In some cases, R 10 does not exist, and R 11 teeth TIFF0007814386000027.tif13128.

[0078] In some cases, L has a structure selected from the linking groups of the compounds discussed in the Specific Embodiments section below. For example, compound 1.0 is discussed in the Specific Embodiments section and also shown below. TIFF0007814386000028.tif48128

[0079] Compound 1.0 is seen to have the X and Y groups discussed above, and therefore the central L group is seen to have the structure shown below. TIFF0007814386000029.tif86128

[0080] This analysis can be repeated for each of the compounds in the Specific Embodiments section below, demonstrating several different identities of the L linker.

[0081] Any other suitable linking group, i.e., linker, can be used. In some cases, the linking group comprises an alkyl chain, an aryl chain, or a polyethylene glycol chain terminated at its two ends by two coupling groups, each independently selected from an amine, a carbamate, a carboxylic acid, a carboxylate, a maleimide, an activated ester, an N-hydroxysuccinimidyl, a hydrazine, a hydrazide, an azide, an alkyne, an aldehyde, or a thiol. In some cases, the two coupling groups are different from each other, allowing for selective coupling of a first end of the linker to the X group and selective coupling of a second end of the linker to the Y group. Exemplary methods for forming compounds are provided in the Examples section.

[0082] Exemplary linking methods include those used in the field of antibody-drug conjugates.For example, Tsuchikama and An, incorporated herein by reference, describe conjugation and linking chemistry (Protein Cell, 2018, 9(1):33, doi: 10.1007 / s13238-016-0323-0).Further exemplary linkers are described in U.S. Patent Nos. 9,872,924 and 10,377,837; International Publication No. WO2020 / 005754; and U.S. Patent Application Publication No. 2019 / 0388555, incorporated herein by reference.

[0083] Specific aspects In some cases, the compound is TIFF0007814386000030.tif48128TIFF0007814386000031.tif193120TIFF00 07814386000032.tif230128TIFF0007814386000033.tif202134TIFF00078143 86000034.tif219116TIFF0007814386000035.tif240113TIFF00078143860000 36.tif187128TIFF0007814386000037.tif167130TIFF0007814386000038.tif 201129TIFF0007814386000039.tif212132TIFF0007814386000040.tif176128TIFF0007814386000041.tif229133TIFF0007814386000042.tif218129TIFF0007814386000043.tif23892TIFF0007814386000044.tif202131TIFF0007814386000045.tif210129TIFF0007814386000046.tif99128

[0084] method Also provided are methods of treating a subject for a CDK9-mediated disease by administering a therapeutically effective amount of a pharmaceutical composition comprising the heterobifunctional resolving agent compounds described above.

[0085] In some cases, the CDK9-mediated disease is cancer, e.g., a cancer caused by aberrant expression of MYC-1 or MCL-1, a hematological malignancy, or a solid tumor. In some cases, the disease is selected from the group consisting of acute myeloid leukemia, primary peritoneal carcinoma, chronic lymphocytic leukemia, recurrent multiple myeloma, non-Hodgkin's lymphoma, acute lymphoblastic leukemia, acute mixed lineage leukemia, advanced breast cancer, non-small cell lung cancer, and liver cancer.

[0086] In some cases, the compound is a compound described in the Specific Embodiments section above. [Example]

[0087] The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but it should be recognized that some experimental error and deviations occur. Unless otherwise indicated, parts are parts by weight, molecular weights are weight average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric. Standard abbreviations may be used, such as bp, base pairs; kb, kilobases; pl, picoliters; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acids; nt, nucleotides; etc. The term "RT" refers to room temperature.

[0088] Compound 1.0 2-[2-[2-[[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4yl]oxyacetyl]amino]ethoxy]ethoxy]-N-[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]acetamide TIFF0007814386000047.tif46133

[0089] Synthesis of tert-butyl 2-[2-[2-[[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]oxyacetyl]amino]ethoxy]ethoxy]acetate (Compound 1.01) To a stirred solution of DIPEA (0.4 mL, 2.26 mmol) in THF (5 mL) under a nitrogen atmosphere was added HOBt (91.42 mg, 0.6800 mmol), EDC.HCl (129.34 mg, 0.6800 mmol), and 2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]oxyacetic acid (150 mg, 0.4500 mmol) at 0 °C. The reaction was stirred at the same temperature for 10 min, then tert-butyl 2-[2-(2-aminoethoxy)ethoxy]acetate hydrochloride (115.45 mg, 0.4500 mmol) was added, and the reaction mixture was stirred at ambient temperature for 16 h. The progress of the reaction was monitored by TLC (5% MeOH in DCM, product Rf = 0.7, SM Rf = 0.2) and LCMS. The reaction mixture was quenched with ice-cold water, concentrated under reduced pressure, extracted with EtOAc (50 mL × 1), and the organic layer was washed with saturated brine solution (50 mL × 1). The organic layer was dried over anhydrous Na2SO4, the solvent was removed, and purified by CombiFlash column chromatography (silica gel) eluting with 5% MeOH in DCM to give tert-butyl 2-[2-[2-[[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]oxyacetyl]amino]ethoxy]ethoxy]acetate (compound 1.01) (100 mg, 42%) as a sticky solid. LCMS-ESI (pos.) m / z: 534.1 (M+1) +

[0090] Synthesis of 2-[2-[2-[[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]oxyacetyl]amino]ethoxy]ethoxy]acetic acid (Compound 1.02) To a stirred solution of tert-butyl 2-[2-[2-[[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]oxyacetyl]amino]ethoxy]ethoxy]acetate (100 mg, 0.1900 mmol) (Compound 1.01) in DCM (1.0 mL) at 0° C. was added TFA (0.72 mL, 9.37 mmol). The reaction was allowed to warm to room temperature and stirring was continued for 3 h. After completion of the reaction, monitored by LCMS, TFA was removed under reduced pressure and the crude material was triturated with n-pentane to afford 2-[2-[2-[[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4yl]oxyacetyl]amino]ethoxy]ethoxy]acetic acid (compound 1.02) (60 mg, 67%) as an off-white solid. LCMS-ESI (pos.) m / z: 478.4 (M+1) +

[0091] Synthesis of 2-[2-[2-[[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]oxyacetyl]amino]ethoxy]ethoxy]-N-[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]acetamide (Compound 1.0) To a stirred solution of (1S,3S)-N3-[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine hydrochloride (36.04 mg, 0.1100 mmol) (CAS#2416873-83-9, prepared using the procedure described in US20200131189A1) in DMF (0.2893 mL) was added DIPEA (0.35 mL, 2.49 mmol) at 0°C. Then, 2-[2-[2-[[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]oxyacetyl]amino]ethoxy]ethoxy]acetic acid (50 mg, 0.1000 mmol) (Compound 1.02) and HATU (568.19 mg, 1.49 mmol) were added. The reaction was stirred at room temperature for 16 hours, and the reaction progress was monitored by TLC (5% MeOH in DCM, product Rf = 0.7, SM Rf = 0.4) and LCMS. The reaction mixture was quenched with ice-cold water, extracted with EtOAc (30 mL × 2), and the organic layer was washed with saturated brine solution (30 mL × 1). The organic layer was dried over anhydrous NaSO, the solvent was concentrated, and purified by preparative HPLC to give 2-[2-[2-[[2-[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]oxyacetyl]amino]ethoxy]ethoxy]-N-[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]acetamide (compound 1.0) (28 mg, 36%) as an off-white solid. LCMS-ESI (pos.) m / z: 747.8 (M+1) + TIFF0007814386000048.tif38152

[0092] Compound 2.0 2-[2-[2-[2-[[2-[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]oxyacetyl]amino]ethoxy]ethoxy]ethoxy]-N-[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]acetamide TIFF0007814386000049.tif57133

[0093] Synthesis of tert-butyl 2-[2-[2-[2-[[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]oxyacetyl]amino]ethoxy]ethoxy]ethoxy]acetate (Compound 2.01) To a stirred solution of DIPEA (0.24 mL, 1.35 mmol) in THF (5 mL) under a nitrogen atmosphere, HOBt (91.42 mg, 0.6800 mmol), EDC.HCl (129.34 mg, 0.680 mmol), and 2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]oxyacetic acid (Compound 2.03) (150 mg, 0.4500 mmol) were added at 0 °C and stirred for 10 min. 2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]tert-butyl acetate (118.88 mg, 0.4500 mmol) was added at the same temperature, and the reaction mixture was stirred at ambient temperature for 16 h. The progress of the reaction was monitored by TLC (5% MeOH in DCM, product Rf=0.7, SM Rf=0.2) and LCMS. The reaction mixture was quenched with ice-cold water, concentrated under reduced pressure, extracted with EtOAc (50 mL x 2), and the organic layer was washed with saturated brine solution (50 mL x 1). The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude residue was purified by CombiFlash column chromatography (silica gel) eluting with 5% MeOH in DCM to afford tert-butyl 2-[2-[2-[2-[[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]oxyacetyl]amino]ethoxy]ethoxy]ethoxy]acetate (compound 2.01) (110 mg, 42%) as a sticky solid. LCMS-ESI (pos.) m / z: 578.5 (M+1) +

[0094] Synthesis of 2-[2-[2-[2-[[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]oxyacetyl]amino]ethoxy]ethoxy]ethoxy]acetic acid (Compound 2.02) To a stirred solution of tert-butyl 2-[2-[2-[2-[[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]oxyacetyl]amino]ethoxy]ethoxy]ethoxy]acetate (110 mg, 0.1900 mmol) (Compound 2.01) in DCM (1 mL) at 0° C. was added TFA (0.73 mL, 9.52 mmol). The reaction was allowed to warm to ambient temperature and stirring was continued for 3 h. After completion of the reaction, monitored by LCMS, TFA was removed under reduced pressure and the crude material was triturated with n-pentane to give 2-[2-[2-[2-[[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]oxyacetyl]amino]ethoxy]ethoxy]ethoxy]acetic acid (compound 2.02) (70 mg, 70%) as an off-white solid. LCMS-ESI (pos.) m / z: 522.2 (M+1) +

[0095] Synthesis of 2-[2-[2-[2-[[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]oxyacetyl]amino]ethoxy]ethoxy]ethoxy]-N-[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]acetamide (Compound 2.0) To a stirred solution of (1S,3S)-N3-[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine hydrochloride (33 mg, 0.1000 mmol) in DMF (0.27 mL) was added DIPEA (0.05 mL, 0.3800 mmol) and 2-[2-[2-[2-[[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]oxyacetyl]amino]ethoxy]ethoxy]ethoxy]acetic acid (Compound 2.02) (50 mg, 0.1000 mmol) at 0° C. Then HATU (43.75 mg, 0.1200 mmol) was added. The reaction was stirred at ambient temperature for 16 hours, then quenched with ice-cold water, extracted with EtOAc (30 mL × 1), and the organic layer was washed with saturated brine solution (30 mL × 1). The organic layer was dried over anhydrous Na2SO4, the solvent was concentrated, and purified by preparative HPLC to give 2-[2-[2-[2-[[2-[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]oxyacetyl]amino]ethoxy]ethoxy]ethoxy]-N-[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]acetamide (compound 2.0) (19 mg, 25%) as an off-white solid. LCMS-ESI (pos.) m / z: 791.9 (M+1) + TIFF0007814386000050.tif38154

[0096] Compound 3.0 N-[(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]-2-[2-[2-[2-[[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]oxyacetyl]amino]ethoxy]ethoxy]ethoxy]acetamide TIFF0007814386000051.tif29134

[0097] Synthesis of N-[(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]-2-[2-[2-[2-[[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]oxyacetyl]amino]ethoxy]ethoxy]ethoxy]acetamide (Compound 3.0) To a stirred solution of 2-[2-[2-[2-[[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]oxyacetyl]amino]ethoxy]ethoxy]ethoxy]acetic acid (Compound 2.02) (40 mg, 0.08 mmol) in DMF (0.22 mL) was added (1S,3S)—N3-[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (25 mg, 0.07 mmol) (CAS# 2416874-47-8, prepared as described in US20200131189A1) and DIPEA (0.03 mL, 0.23 mmol) at 0° C. HATU (35 mg, 0.09 mmol) was then added and the reaction mixture was stirred at ambient temperature for 16 h. The progress of the reaction was monitored by TLC (5% MeOH in DCM, product R f =0.7, SM R f =0.4) and monitored by LCMS. The reaction mixture was quenched with ice-cold water and extracted with EtOAc (30 mL × 2). The organic layer was washed with saturated brine solution (30 mL × 1), dried over anhydrous Na2SO4, and evaporated in vacuo. The crude residue was purified by preparative HPLC using ammonium acetate buffer and MECN as the mobile phase, followed by lyophilization to give N-[(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]-2-[2-[2-[2-[[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]oxyacetyl]amino]ethoxy]ethoxy]ethoxy]acetamide (Compound 3.0) (10 mg, 15%). LCMS-ESI (pos.) m / z: 825.6 (M+1) + TIFF0007814386000052.tif46154

[0098] Compound 4.0 N-[(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]-2-[2-[2-[[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]oxyacetyl]amino]ethoxy]ethoxy]acetamide TIFF0007814386000053.tif26136

[0099] Synthesis of N-[(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]-2-[2-[2-[[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]oxyacetyl]amino]ethoxy]ethoxy]acetamide (Compound 4.0) To a stirred solution of 2-[2-[2-[[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]oxyacetyl]amino]ethoxy]ethoxy]acetic acid (36.62 mg, 0.08 mmol) (Compound 1.02) in DMF (0.22 mL) was added (1S,3S)—N3-[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (25 mg, 0.07 mmol) (CAS#2416874-47-8, prepared using the procedure described in US20200131189A1) and DIPEA (0.03 mL, 0.23 mmol) at 0° C. Then HATU (35 mg, 0.09 mmol) was added. The reaction mixture was stirred at ambient temperature for 16 hours, then quenched with ice-cold water and extracted with EtOAc (30 mL × 2). The organic layer was washed with saturated brine solution (30 mL × 1), dried over anhydrous Na2SO4, and evaporated in vacuo. The crude residue was purified by preparative HPLC using ammonium acetate buffer and MeCN as the mobile phase, followed by lyophilization to give N-[(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]-2-[2-[2-[[2-[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]oxyacetyl]amino]ethoxy]ethoxy]acetamide (compound 4.0) (17 mg, 27%). LCMS-ESI (pos.) m / z: 781.8 (M+1) + TIFF0007814386000054.tif46155

[0100] Compound 5.0 N-[2-[2-[2-[2-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]amino]ethoxy]ethoxy]ethoxy]ethyl]-2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetamide TIFF0007814386000055.tif76136

[0101] Synthesis of 2-(2,6-dioxo-3-piperidyl)-4-fluoro-isoindoline-1,3-dione (compound 5.01) To a stirred solution of 4-fluoroisobenzofuran-1,3-dione (500 mg, 3.01 mmol) and 3-aminopiperidine-2,6-dione hydrochloride (495.42 mg, 3.01 mmol) in acetic acid (10 mL) was added potassium acetate (456.02 mg, 4.52 mmol) at room temperature. The reaction mixture was then heated at 90° C. for 16 hours. After completion of the reaction, the solvent was removed under reduced pressure, and the crude residue was purified by CombiFlash column chromatography (silica gel) eluting with 5% MeOH in DCM to give 2-(2,6-dioxo-3-piperidyl)-4-fluoro-isoindoline-1,3-dione (compound 5.01) (565 mg, 68%). LCMS-ESI (pos.) m / z: 277.0 (M+1). +

[0102] Synthesis of tert-butyl N-[2-[2-[2-[2-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]amino]ethoxy]ethoxy]ethoxy]ethyl]carbamate (Compound 5.02) To a solution of 2-(2,6-dioxo-3-piperidyl)-4-fluoro-isoindoline-1,3-dione (500 mg, 1.81 mmol) (compound 5.01) and tert-butyl N-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethyl]carbamate (0.6 mL, 2.17 mmol) [CAS: 101187-40-0] in DMA (10 mL) was added DIPEA (0.97 mL, 5.43 mmol). The reaction mixture was heated at 90 °C for 16 h. The reaction mixture was taken up in EtOAc, and the organics were washed with water (2 × 20 mL), followed by saturated brine solution (1 × 20 mL). The organic layer was separated, dried over anhydrous NaSO, and concentrated to dryness in vacuo. The crude product was purified by CombiFlash column chromatography (silica gel) eluting with 60% EtOAc in hexanes. The desired fractions were concentrated to dryness in vacuo to give tert-butyl N-[2-[2-[2-[2-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]amino]ethoxy]ethoxy]ethoxy]ethyl]carbamate (compound 5.02) (600 mg, 60%). LCMS-ESI (pos.) m / z: 549.4 (M+1) +

[0103] Synthesis of 4-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethylamino]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (compound 5.03) To a stirred solution of tert-butyl N-[2-[2-[2-[2-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]amino]ethoxy]ethoxy]ethoxy]ethyl]carbamate (600 mg, 1.09 mmol) (Compound 5.02) in dioxane (3 mL) was slowly added dioxane-HCl (4 M; 30 mL, 167.44 mmol) at 0° C. and stirred at ambient temperature for 3 hours. The reaction mixture was concentrated in vacuo to give 4-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethylamino]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (Compound 5.03) (480 mg, 98%). LCMS-ESI (pos.) m / z: 448.8 (M+1) +

[0104] Synthesis of tert-butyl 2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetate (Compound 5.04) To a stirred solution of [(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-4-ium-7-yl]amino]cyclopentyl]ammonium dichloride (100 mg, 0.28 mmol) in MeCN (6.25 mL) was added KCO (76.71 mg, 0.56 mmol) and tert-butyl 2-bromoacetate (64.96 mg, 0.3300 mmol) slowly at 0 °C and stirred at ambient temperature for 3 h. The reaction mixture was evaporated and the residue was diluted with MeCN and stirred at ambient temperature for 3 h. The reaction mixture was filtered through a bed of Celite, washed with ethyl acetate, and the organic layer was concentrated to dryness in vacuo to give crude tert-butyl 2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetate (compound 5.04) (80 mg, 72%). LCMS-ESI (pos.) m / z: 401.8 (M+1) +

[0105] Synthesis of 2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetic acid (Compound 5.05) To a stirred solution of tert-butyl 2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetate (60 mg, 0.15 mmol) (Compound 5.04) in DCM (2 mL) was added TFA (0.02 mL, 0.30 mmol) at 0 °C and stirred at ambient temperature for 3 h. The reaction mixture was concentrated under reduced pressure and triturated with n-pentane to give 2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetic acid (50 mg, 97%) (Compound 5.05). LCMS-ESI (pos.) m / z: 346.3 (M+1) +

[0106] Synthesis of N-[2-[2-[2-[2-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]amino]ethoxy]ethoxy]ethoxy]ethyl]-2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetamide (Compound 5.0) To a stirred solution of 4-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethylamino]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (25 mg, 0.06 mmol) (Compound 5.03) and 2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetic acid (23.11 mg, 0.07 mmol) (Compound 5.05) in DMF (1 mL) was added DIPEA (0.04 mL, 0.22 mmol) followed by HATU (42.39 mg, 0.11 mmol) at 0° C. and stirred at ambient temperature for 16 hours. The reaction mixture was then poured into ice-cold water and extracted with ethyl acetate. The organic layer was dried over anhydrous NaSO and concentrated under vacuum to give the crude material, which was purified by preparative HPLC to give N-[2-[2-[2-[2-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]amino]ethoxy]ethoxy]ethoxy]ethyl]-2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetamide (compound 5.0) (5 mg, 11%). LCMS-ESI (pos.) m / z: 776.9 (M+1) + TIFF0007814386000056.tif29155

[0107] Compound 6.0 N-[2-[2-[2-[2-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]amino]ethoxy]ethoxy]ethoxy]ethyl]-2-[[(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetamide TIFF0007814386000057.tif55128

[0108] Synthesis of tert-butyl 2-[[(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetate (Compound 6.01) To a stirred solution of (1S,3S)-N3-[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine hydrochloride (200 mg, 0.56 mmol) in MeCN (5 mL) at 0 °C under a nitrogen atmosphere, K2CO3 (154.28 mg, 1.12 mmol) was slowly added followed by tert-butyl 2-bromoacetate (130.65 mg, 0.67 mmol) at 0 °C and stirred at ambient temperature for 3 h. The reaction mixture was diluted with water (10 mL) and the compound was extracted with EtOAc (30 mL). The solvent was removed under reduced pressure and the crude residue was purified by CombiFlash column chromatography (silica gel, eluting with 10-15% EtOAc in hexanes) to give tert-butyl 2-[[(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetate (Compound 6.01) (120 mg, 44%). LCMS-ESI (pos.) m / z: 437.2 (M+2) +

[0109] Synthesis of 2-[[(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetic acid 2,2,2-trifluoroacetic acid (Compound 6.02) To a stirred solution of tert-butyl 2-[[(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetate (120 mg, 0.28 mmol) (Compound 6.01) in DCM (2 mL) under a nitrogen atmosphere was added TFA (0.50 mL, 0.06 mmol) at 0° C., and the reaction mixture was stirred at ambient temperature for 3 hours. After completion of the reaction, the solvent and excess TFA were removed in vacuo. The residue was triturated with n-pentane (2 x 1 mL) and diethyl ether (2 x 1 mL) to give 2-[[(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetic acid 2,2,2-trifluoroacetic acid (compound 6.02) (95 mg, 85%). LCMS-ESI (pos.) m / z: 380.5 (M+1) +

[0110] Synthesis of N-[2-[2-[2-[2-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]amino]ethoxy]ethoxy]ethoxy]ethyl]-2-[[(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetamide (Compound 6.0) To a stirred solution of 2-[[(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetic acid:2,2,2-trifluoroacetic acid (Compound 6.02) (20 mg, 0.05 mmol) in DMF (1 mL) under a nitrogen atmosphere was added 4-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethylamino]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (25.97 mg, 0.06 mmol) (Compound 5.03) followed by DIPEA (0.03 mL, 0.16 mol) at 0° C., and the reaction mixture was stirred at 0° C. for 5 minutes. PyBop (32.88 mg, 0.06 mmol) was then added, and the reaction mixture was stirred at ambient temperature for 16 hours. The solvent was evaporated under reduced pressure and the crude residue was purified by preparative HPLC using ammonium bicarbonate buffer and CHCN as the mobile phase, followed by lyophilization to give N-[2-[2-[2-[2-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]amino]ethoxy]ethoxy]ethoxy]ethyl]-2-[[(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetamide (compound 6.0) (10.5 mg, 23%). LCMS-ESI (pos.) m / z: 810.3 (M+1). + TIFF0007814386000058.tif38155

[0111] Compound 7.0 2-(2,6-dioxo-3-piperidyl)-4-[2-[2-[2-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethoxy]ethylamino]isoindoline-1,3-dione: TIFF0007814386000059.tif61135

[0112] Step-1: Synthesis of tert-butyl N-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethyl]carbamate (Compound 7.01) To a stirred solution of 2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethanol (250 mg, 1.29 mmol) in DCM (5 mL) was slowly added EtN (0.36 mL, 2.59 mmol) and Boc anhydride (0.36 mL, 1.55 mmol) at 0 °C and stirred at ambient temperature for 3 h. The reaction mixture was concentrated to dryness, and the residue was taken up in DCM (30 mL) and washed with water (2 × 15 mL) followed by saturated brine solution (1 × 15 mL). The organic layer was separated, dried over anhydrous NaSO, and concentrated to dryness in vacuo. The crude material was purified by CombiFlash column chromatography (silica gel) eluting with 5% MeOH in DCM. The desired fractions were concentrated to dryness in vacuo to give tert-butyl N-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethyl]carbamate (compound 7.01) (230 mg, 61%). LCMS-ESI (pos.) m / z: 294.4 (M+1) +

[0113] Step-2: Synthesis of tert-butyl N-[2-[2-[2-(2-oxoethoxy)ethoxy]ethoxy]ethyl]carbamate (compound 7.02) To a stirred solution of tert-butyl N-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethyl]carbamate (compound 7.01) (280 mg, 0.95 mmol) in DCM (8 mL) was added Dess-Martin periodinane (485.8 mg, 1.15 mmol) at 0 °C and stirred at ambient temperature for 3 h. The reaction was concentrated to dryness in vacuo, and the residue was taken up in DCM (25 mL) and washed with saturated NaHCO solution (2 × 25 mL) followed by saturated brine solution (1 × 25 mL). The organic layer was separated, dried over anhydrous NaSO, and concentrated to dryness in vacuo. The crude material was purified by CombiFlash column chromatography (silica gel) eluting with 5% MeOH in DCM. The desired fractions were concentrated to dryness in vacuo to give tert-butyl N-[2-[2-[2-(2-oxoethoxy)ethoxy]ethoxy]ethyl]carbamate (compound 7.02) (187 mg, 67%). LCMS-ESI (pos.) m / z: 292.2 (M+1) +

[0114] Step-3: Synthesis of tert-butyl N-[2-[2-[2-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethoxy]ethyl]carbamate (Compound 7.03) To a stirred solution of [(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-4-ium-7-yl]amino]cyclopentyl]ammonium dichloride (267.15 mg, 0.74 mmol) (CAS# 2416873-83-9, prepared using the procedure described in US20200131189A1) and 4 Å MS in methanol (5 mL) at 0 °C was added sodium cyanoborohydride (116.47 mg, 1.85 mmol). The reaction mixture was allowed to warm to room temperature and continued stirring at room temperature for 0.5 h. Then tert-butyl N-[2-[2-[2-(2-oxoethoxy)ethoxy]ethoxy]ethyl]carbamate (180 mg, 0.62 mmol) was added and stirred at ambient temperature for 16 h. The reaction mixture was filtered, and the filtrate was concentrated to dryness in vacuo. The crude material was purified by CombiFlash column chromatography (silica gel) eluting with 5% MeOH in DCM. The desired fractions were concentrated to dryness in vacuo to give tert-butyl N-[2-[2-[2-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethoxy]ethyl]carbamate (compound 7.03) (67 mg, 15%). LCMS-ESI (pos.) m / z: 563.2 (M+1). +

[0115] Step-4: Synthesis of 2-[2-[2-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethoxy]ethylammonium chloride (compound 7.04): To a stirred solution of tert-butyl N-[2-[2-[2-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethoxy]ethyl]carbamate (60 mg, 0.11 mmol) (Compound 7.03) in dioxane (0.5 mL) at 0° C. was added dioxane-HCl (4 M; 0.08 mL, 0.4300 mmol). The reaction was allowed to warm to room temperature and stirring was continued for 3 h. The reaction mixture was concentrated in vacuo, washed with pentane, dried in vacuo, and subsequently lyophilized to give 2-[2-[2-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethoxy]ethylammonium chloride (compound 7.04) (20 mg, 38%). LCMS-ESI (neg.) m / z: 461.4 (M-1)+

[0116] Step-5: Synthesis of 2-(2,6-dioxo-3-piperidyl)-4-[2-[2-[2-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethoxy]ethylamino]isoindoline-1,3-dione (Compound 7.0) To a stirred solution of 2-[2-[2-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethoxy]ethylammonium chloride (20 mg, 0.04 mmol) (Compound 7.04) in DMA (2 mL) at 0 °C was added DIPEA (0.02 mL, 0.12 mmol). The reaction was allowed to warm to room temperature, and 2-(2,6-dioxo-3-piperidyl)-4-fluoro-isoindoline-1,3-dione (Compound 5.01) (22.14 mg, 0.08 mmol) was added. The reaction mixture was then stirred at 90 °C for 16 hours, at which point LC-MS indicated the reaction was incomplete. Additional 2-(2,6-dioxo-3-piperidyl)-4-fluoro-isoindoline-1,3-dione (22.14 mg, 0.08 mmol) (compound 5.01) was added and stirred at 90 °C for 16 h, at which point LC-MS indicated the reaction was complete. The reaction mixture was purified by preparative HPLC and subsequently lyophilized (MeCN:HO) to give 2-(2,6-dioxo-3-piperidyl)-4-[2-[2-[2-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethoxy]ethylamino]isoindoline-1,3-dione (compound 7.0) (1.6 mg, 5%). LCMS-ESI (pos.) m / z: 719.4 (M+1). + TIFF0007814386000060.tif38155

[0117] Compound 8.0 2-(2,6-dioxo-3-piperidyl)-4-[2-[2-[2-[2-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]isoindoline-1,3-dione: TIFF0007814386000061.tif78129

[0118] Step-1: Synthesis of 2-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate (Compound 8.01) To a stirred solution of tert-butyl N-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]carbamate (280 mg, 0.83 mmol) [CAS: 1404111-67-6] in DCM (25 mL) at 0 °C, p-toluenesulfonyl chloride (316.42 mg, 1.66 mmol) and EtN (0.47 mL, 3.32 mmol) were added, and the reaction mixture was stirred at 0 °C for an additional 30 minutes. The reaction mixture was then stirred at ambient temperature for 16 hours. After completion of the reaction, water (20 mL) was added to the reaction mixture, which was then extracted with DCM (30 mL). The organic layer was washed with brine, dried over anhydrous NaSO, and concentrated in vacuo. The crude product was purified by CombiFlash column chromatography (silica gel) eluting with 60% EtOAc in hexane to give 2-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate (compound 8.01) (250 mg, 61%). LCMS-ESI (pos.) m / z: 492.0 (M+1) +

[0119] Step-2: Synthesis of tert-butyl N-[2-[2-[2-[2-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate (Compound 8.02) To a stirred solution of (1S,3S)-N3-[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine dihydrochloride (180 mg, 0.50 mmol) (CAS#2416873-83-9, prepared using the procedure described in US20200131189A1) and 2-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate (245.57 mg, 0.50 mmol) (Compound 8.01) in MeCN (10 mL) was added potassium carbonate (689.37 mg, 5.00 mmol) and the reaction mixture was heated at 85° C. for 36 hours. The reaction mixture was then concentrated in vacuo and the crude material was purified by CombiFlash column chromatography (silica gel) eluting with 10% MeOH in DCM to give tert-butyl N-[2-[2-[2-[2-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate (compound 8.02) (165 mg, 54%). LCMS-ESI (pos.) m / z: 607.0 (M+1) +

[0120] Step-3: Synthesis of (1S,3S)-N1-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethyl]-N3-[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (Compound 8.03) To a stirred solution of tert-butyl N-[2-[2-[2-[2-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate (165 mg, 0.27 mmol) in DCM (3 mL) at 0° C. was added trifluoroacetic acid (0.55 mL, 7.19 mmol) and the reaction mixture was stirred for an additional 30 min at 0° C. The reaction was then stirred at room temperature for 3 h. After completion of the reaction, the solvent was evaporated in vacuo to give (1S,3S)-N1-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethyl]-N3-[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (compound 8.03) (125 mg, 91% crude). The compound was used in the next step without further purification. LCMS-ESI (pos.) m / z: 507.3 (M+1) +

[0121] Step-4: Synthesis of 2-(2,6-dioxo-3-piperidyl)-4-[2-[2-[2-[2-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]isoindoline-1,3-dione (Compound 8.0) To a stirred solution of 2-(2,6-dioxo-3-piperidyl)-4-fluoro-isoindoline-1,3-dione (26.17 mg, 0.09 mmol) in DMA (1 mL) in a sealed tube was added (1S,3S)-N1-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethyl]-N3-[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (40 mg, 0.08 mmol) (compound 8.03) and DIPEA (0.07 mL, 0.39 mmol) in DMA (2 mL). The reaction mixture was then heated at 95 °C for 16 hours. After completion of the reaction as indicated by LCMS, the reaction mixture was cooled, poured into ice-cold water, and stirred for several minutes. The reaction mixture was extracted with EtOAc and washed with brine. The organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo to give the crude material. The crude product was purified by preparative HPLC using an ammonium acetate buffer / MECN mobile phase, followed by lyophilization to give 2-(2,6-dioxo-3-piperidyl)-4-[2-[2-[2-[2-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]isoindoline-1,3-dione (compound 8.0) (8 mg, 13%). LCMS-ESI (pos.) m / z: 763.3 (M+1) + TIFF0007814386000062.tif46155

[0122] Compound 9.0 2-(2,6-dioxo-3-piperidyl)-4-[2-[2-[2-[4-[[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]methyl]triazol-1-yl]ethoxy]ethoxy]ethylamino]isoindoline-1,3-dione TIFF0007814386000063.tif60128

[0123] Step-1: Synthesis of 4-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethylamino]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (Compound 9.01) To a stirred solution of 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethanamine (39.51 mg, 0.1800 mmol) and 2-(2,6-dioxo-3-piperidyl)-4-fluoro-isoindoline-1,3-dione (50 mg, 0.1800 mmol) (Compound 5.01) in DMA (1 mL) was added DIPEA (0.2 mL, 1.086 mmol), and the mixture was stirred at 90° C. for 16 h. The reaction mixture was quenched with ice-cold water, extracted with EtOAc (30 mL × 2), and the organic layer was washed with saturated brine solution (50 mL × 1). The organic layer was dried over anhydrous Na2SO4, the solvent was concentrated, and purified by CombiFlash column chromatography (silica gel) eluting with 70% EtOAc in hexane to give 4-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethylamino]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (compound 9.01) (45 mg, 52%) as a bright yellow solid. LCMS-ESI (pos.) m / z: 431.2 (M+1) +

[0124] Step-2: Synthesis of (1S,3S)-N3-[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]-N1-prop-2-ynyl-cyclopentane-1,3-diamine (Compound 9.02) To a stirred solution of (1S,3S)-N3-[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine dihydrochloride (10 mg, 0.036 mmol) (CAS# 2416873-83-9, prepared as described in US20200131189A1) and EtN (0.01 mL, 0.0500 mmol) in DMF (1 mL) at 0 °C, 3-bromoprop-1-yne (2.9 μL, 0.033 mmol) was added. The reaction was heated to 70 °C and stirring was continued for 16 h, at which time LC-MS indicated the reaction was complete. The reaction mixture was concentrated to dryness, and the residue was added ice-cold water and extracted with EtOAc (1 × 20 mL). The organic layer was separated and washed with ice-cold water (2 × 10 mL) followed by saturated brine solution (1 × 10 mL). The organic layer was dried over anhydrous Na2SO4 and concentrated to dryness. The residue was purified by combiflash column chromatography (silica gel) eluting with 70% EtOAc in hexane to give (1S,3S)-N3-[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]-N1-prop-2-ynyl-cyclopentane-1,3-diamine (compound 9.02) (10 mg, 18%) as a solid compound. LCMS-ESI (pos.) m / z: 326.2 (M+1) +

[0125] Step-3: Synthesis of 2-(2,6-dioxo-3-piperidyl)-4-[2-[2-[2-[4-[[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]methyl]triazol-1-yl]ethoxy]ethoxy]ethylamino]isoindoline-1,3-dione (Compound 9.0) In an oven-dried 10 mL round-bottom flask, (1S,3S)—N3-[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]-N1-prop-2-ynyl-cyclopentane-1,3-diamine (35 mg, 0.1100 mmol) (Compound 9.02), 4-[2-[2-(2-azidoethoxy)ethoxy]ethylamino]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (46.29 mg, 0.1100 mmol) (Compound 9.01), and CuI (12.07 mg, 0.1100 mmol) were added to a mixture of MeCN (1 mL) and THF (1 mL). DIPEA (0.02 mL, 0.1100 mmol) was added, and the reaction mixture was vigorously stirred at ambient temperature under an argon atmosphere for 12 hours. The reaction mixture was concentrated to dryness, and the residue was dissolved in EtOAc (20 mL) and washed with water (2 × 10 mL) followed by saturated brine solution (1 × 10 mL). The organic layer was separated, dried over anhydrous NaSO, and concentrated to dryness. The crude residue was purified by preparative HPLC to give 2-(2,6-dioxo-3-piperidyl)-4-[2-[2-[2-[4-[[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]methyl]triazol-1-yl]ethoxy]ethoxy]ethylamino]isoindoline-1,3-dione (Compound 9.0) (7 mg, 9%). LCMS-ESI (pos.) m / z: 756.4 (M+1) + TIFF0007814386000064.tif46155

[0126] Compound 10.0 2-(2,6-dioxo-3-piperidyl)-4-[2-[2-[2-[2-[4-[[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]methyl]triazol-1-yl]ethoxy]ethoxy]ethoxy]ethylamino]isoindoline-1,3-dione TIFF0007814386000065.tif25141

[0127] Step-1: Synthesis of 4-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethylamino]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (Compound 10.01) To a stirred solution of 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethanamine (39.51 mg, 0.1800 mmol) in DMA (1 mL) was added 2-(2,6-dioxo-3-piperidyl)-4-fluoro-isoindoline-1,3-dione (50 mg, 0.1800 mmol) (compound 5.01) and stirred at 90 °C for 16 h. The reaction mixture was quenched with ice-cold water and extracted with EtOAc (30 mL × 2). The organic layer was washed with saturated brine solution (50 mL × 1), dried over anhydrous Na2SO4, and evaporated under reduced pressure. The crude product was purified by CombiFlash column chromatography (silica gel) eluting with 70% EtOAc in hexane to give 4-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethylamino]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (compound 10.01) (45 mg, 52%) as a bright yellow solid. LCMS-ESI (pos.) m / z: 475.3 (M+1) +

[0128] Step-2: Synthesis of 2-(2,6-dioxo-3-piperidyl)-4-[2-[2-[2-[2-[4-[[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]methyl]triazol-1-yl]ethoxy]ethoxy]ethoxy]ethylamino]isoindoline-1,3-dione (Compound 10.0) In an oven-dried 10 mL round-bottom flask, (1S,3S)—N3-[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]-N1-prop-2-ynyl-cyclopentane-1,3-diamine (25 mg, 0.0800 mmol) (Compound 9.02), 4-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethylamino]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (36.45 mg, 0.0800 mmol) (Compound 10.01), and CuI (8.62 mg, 0.0800 mmol) were added to a mixture of MeCN (1 mL) and THF (1 mL). DIPEA (0.01 mL, 0.0800 mmol) was then added, and the reaction mixture was vigorously stirred at ambient temperature under an argon atmosphere for 12 hours. The reaction mixture was concentrated to dryness, and the residue was dissolved in EtOAc (1 × 20 mL) and washed with water (2 × 10 mL) followed by saturated brine solution (1 × 10 mL). The organic layer was separated, dried over anhydrous NaSO, and concentrated to dryness. The crude residue was purified by preparative HPLC to give 2-(2,6-dioxo-3-piperidyl)-4-[2-[2-[2-[2-[4-[[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]methyl]triazol-1-yl]ethoxy]ethoxy]ethoxy]ethylamino]isoindoline-1,3-dione (Compound 10.0) (4 mg, 7%). LCMS-ESI (pos.) m / z: 800.6 (M+1) + TIFF0007814386000066.tif46155

[0129] Compound 11.0 2-(2,6-dioxo-3-piperidyl)-4-[2-[2-[2-[2-[[(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethoxy]ethylamino]isoindoline-1,3-dione TIFF0007814386000067.tif29128

[0130] Synthesis of 2-(2,6-dioxo-3-piperidyl)-4-[2-[2-[2-[2-[[(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethoxy]ethylamino]isoindoline-1,3-dione (Compound 11.0) A solution of (1S,3S)-N3-[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (4.94 mg, 0.02 mmol) (CAS# 2416874-47-8, prepared as described in US20200131189A1), 2-[2-[2-[2-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]amino]ethoxy]ethoxy]ethoxy]acetaldehyde (7.55 mg, 0.02 mmol) (compound 7.03), and 4 Å MS in MeCN (3 mL) was stirred at 0 °C for 2 h, followed by the addition of NaBH3CN (0.01 mL, 0.05 mmol). The reaction was allowed to warm to ambient temperature and stirred for 16 hours, at which time LCMS indicated product formation. The reaction mixture was concentrated in vacuo, purified by preparative HPLC purification, and the desired fraction was lyophilized for 16 hours to afford 2-(2,6-dioxo-3-piperidyl)-4-[2-[2-[2-[2-[[(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethoxy]ethylamino]isoindoline-1,3-dione (compound 11.0) (3.17 mg, 26%). LCMS-ESI (pos.) m / z: 752.8 (M+1) + TIFF0007814386000068.tif38155

[0131] Compound 12.0 2-(2,6-dioxo-3-piperidyl)-4-[[1-[2-[2-[2-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethoxy]ethyl]triazol-4-yl]methylamino]isoindoline-1,3-dione: TIFF0007814386000069.tif62132

[0132] Process-1:(1S,3S)-N 1 -(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-N 3 Synthesis of -(5-(pentan-3-yl)pyrazolo[1,5-a]pyrimidin-7-yl)cyclopentane-1,3-diamine (Compound 12.01) To a stirred solution of (1S,3S)-N3-[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (190 mg, 0.66 mmol) (CAS# 2416873-83-9, prepared as described in US20200131189A1) and 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate (370.3 mg, 0.99 mmol) in MeCN (15 mL) was added potassium carbonate (456.16 mg, 3.31 mmol), and the reaction mixture was heated at 95 °C for 24 h. The reaction mixture was filtered, and the filtrate was concentrated in vacuo. The crude residue was purified by CombiFlash column chromatography (silica gel) eluting with 10% MeOH in DCM to give (1S,3S)-N 1 -(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-N 3 -(5-(pentan-3-yl)pyrazolo[1,5-a]pyrimidin-7-yl)cyclopentane-1,3-diamine (compound 12.01) (130 mg, 40%) was obtained. LCMS-ESI (pos.) m / z: 489.1 (M+1) +

[0133] Step-2: Synthesis of 2-(2,6-dioxo-3-piperidyl)-4-[[1-[2-[2-[2-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethoxy]ethyl]triazol-4-yl]methylamino]isoindoline-1,3-dione (Compound 12.0) To a stirred solution of (1S,3S)-N1-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethyl]-N3-[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (Compound 12.01) (10 mg, 0.02 mmol) in THF (1 mL) and MeCN (1 mL) was added CuI (2.3 mg, 0.02 mmol) at 0 °C and stirred for several minutes. 2-(2,6-Dioxo-3-piperidyl)-4-(prop-2-ynylamino)isoindoline-1,3-dione (6.37 mg, 0.02 mmol) and DIPEA (0.01 mL, 0.02 mmol) were then added, and the reaction was stirred at ambient temperature for 16 hours. The reaction progress was monitored by crude LCMS. The reaction mixture was purified by preparative HPLC, followed by lyophilization from MeCN / water to give 2-(2,6-dioxo-3-piperidyl)-4-[[1-[2-[2-[2-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethoxy]ethyl]triazol-4-yl]methylamino]isoindoline-1,3-dione (compound 12.0) (1.1 mg, 7%). LCMS-ESI (pos.) m / z: 800.3 (M+1). + TIFF0007814386000070.tif38155

[0134] Compound 13.0 4-[2-[2-[2-[2-[2-[[(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione TIFF0007814386000071.tif47137

[0135] Step-1: Synthesis of 2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethylammonium; 2,2,2-trifluoroacetic acid (Compound 13.01) To a solution of tert-butyl N-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethyl]carbamate (320 mg, 0.95 mmol) [1404111-67-6] in DCM (6 mL) at 0 °C, TFA (3 mL, 34.65 mmol) was added and stirred at ambient temperature for 3 h. After completion of the reaction, the reaction mixture was concentrated in vacuo, coevaporated with toluene, and lyophilized from MeCN:HO to give 2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethylammonium 2,2,2-trifluoroacetate (compound 13.01) (220 mg, 98%), which was used directly in the next reaction. LCMS-ESI (pos.) m / z: 238.4 (M+1) +

[0136] Step-2: Synthesis of 2-(2,6-dioxo-3-piperidyl)-4-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethylamino]isoindoline-1,3-dione (Compound 13.02) To a stirred solution of 2-(2,6-dioxo-3-piperidyl)-4-fluoro-isoindoline-1,3-dione (Compound 5.01) (153.66 mg, 0.56 mmol) and 2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethanol (Compound 13.01) (220 mg, 0.93 mmol) in DMA (2 mL) was added DIPEA (0.81 mL, 4.64 mmol) and heated at 90° C. for 16 hours. After completion of the reaction, the reaction mixture was extracted with EtOAc and washed with water followed by saturated brine solution. The organic layer was separated and concentrated in vacuo. The crude product was purified by CombiFlash column chromatography (silica gel) eluting with 80% EtOAc in hexane to give 2-(2,6-dioxo-3-piperidyl)-4-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethylamino]isoindoline-1,3-dione (compound 13.02) (70 mg, 15%). LCMS-ESI (pos.) m / z: 493.9 (M+1). +

[0137] Step-3: Synthesis of 2-[2-[2-[2-[2-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]amino]ethoxy]ethoxy]ethoxy]ethoxy]acetaldehyde (Compound 13.03) To a solution of 2-(2,6-dioxo-3-piperidyl)-4-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethylamino]isoindoline-1,3-dione (60 mg, 0.12 mmol) (Compound 13.02) in DCM (5 mL) at 0 °C, Dess-Martin periodinane (257.83 mg, 0.61 mmol) was added, and the mixture was stirred at ambient temperature for 72 h. After completion of the reaction, aqueous NaSO was added and stirred for 15 min. The reaction was extracted with DCM and washed with saturated NaHCO solution, followed by water and saturated brine solution. The organic layer was dried over anhydrous NaSO and concentrated in vacuo to give 2-[2-[2-[2-[2-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]amino]ethoxy]ethoxy]ethoxy]ethoxy]acetaldehyde (compound 13.03) (40 mg, 22%), which was used directly in the next reaction. LCMS-ESI (pos.) m / z: 492.51 (M+1) +

[0138] Step-4: Synthesis of 4-[2-[2-[2-[2-[2-[[(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (Compound 13.0) (1S,3S)-N in MeCN (3 mL) at 0 °C 1To a solution of -(3-chloro-5-(pentan-3-yl)pyrazolo[1,5-a]pyrimidin-7-yl)cyclopentane-1,3-diamine (CAS# 2416874-47-8, prepared as described in US20200131189A1) (20.96 mg, 0.07 mmol) and 2-[2-[2-[2-[2-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]amino]ethoxy]ethoxy]ethoxy]ethoxy]acetaldehyde (Compound 13.03) (40 mg, 0.08 mmol) was added 4 Å MS (100 mg) and stirred at ambient temperature for 2 hours. NaBHCN (15.34 mg, 0.24 mmol) was then added and stirred for 16 hours. The reaction mixture was filtered, and the filtrate was concentrated in vacuo. The crude product was purified by preparative HPLC and lyophilized from MeCN / water to give 4-[2-[2-[2-[2-[2-[[(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (compound 13.0) (6 mg, 9%). LCMS-ESI (pos.) m / z: 796.4 (M+1) + TIFF0007814386000072.tif38155

[0139] Compound 14.0 (2S,4R)-4-hydroxy-N-[[4-(4-methylthiazol-5-yl)phenyl]methyl]-1-[(2S)-3,3-dimethyl-2-[[2-[2-[[1-[2-[2-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethyl]triazol-4-yl]methoxy]ethoxy]acetyl]amino]butanoyl]pyrrolidine-2-carboxamide. TIFF0007814386000073.tif69132

[0140] Synthesis of (1S,3S)-N1-[2-[2-(2-azidoethoxy)ethoxy]ethyl]-N3-[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (Compound 14.01) To a stirred solution of (1S,3S)-N3-[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (175 mg, 0.61 mmol) (CAS#2416873-83-9, prepared as described in US20200131189A1) and 2-[2-(2-azidoethoxy)ethoxy]ethyl 4-methylbenzenesulfonate (250.69 mg, 0.76 mmol) in MeCN (10 mL) was added potassium carbonate (420.15 mg, 3.04 mmol), and the reaction mixture was heated at 95° C. for 24 hours. The reaction mixture was then filtered, and the filtrate was concentrated in vacuo. The crude product was purified by Combiflash column chromatography (silica gel) eluting with 10% MeOH in DCM to give (1S,3S)-N1-[2-[2-(2-azidoethoxy)ethoxy]ethyl]-N3-[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (compound 14.01) (125 mg, 46%). LCMS-ESI (pos.) m / z: 445.4 (M+1). +

[0141] Synthesis of (2S,4R)-4-hydroxy-N-[[4-(4-methylthiazol-5-yl)phenyl]methyl]-1-[(2S)-3,3-dimethyl-2-[[2-[2-[[1-[2-[2-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethyl]triazol-4-yl]methoxy]ethoxy]acetyl]amino]butanoyl]pyrrolidine-2-carboxamide (Compound 14.0) (2S,4R)-4-hydroxy-N-[[4-(4-methylthiazol-5-yl)phenyl]methyl]-1-[(2S)-3,3-dimethyl-2-[[2-(2-prop-2-ynoxyethoxy)acetyl]amino]butanoyl]pyrrolidine-2-carboxamide (25 mg, 0.04 mmol) in DMA (3 mL) (Journal of Medicinal Chemistry (2018), 61(2), To a stirred solution of (1S,3S)-N1-[2-[2-(2-azidoethoxy)ethoxy]ethyl]-N3-[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (Compound 14.01) (described in J. Med. Chem. Soc. Soc. 453-461) was added (19.47 mg, 0.04 mmol), copper sulfate (1.8 mg, 0.01 mmol), and sodium ascorbate (2.89 mg, 0.01 mmol), and the reaction mixture was heated in a sealed tube at 90° C. for 3 hours. The progress of the reaction was monitored by LCMS. The reaction mixture was directly purified by preparative HPLC and subsequently lyophilized to give (2S,4R)-4-hydroxy-N-[[4-(4-methylthiazol-5-yl)phenyl]methyl]-1-[(2S)-3,3-dimethyl-2-[[2-[2-[[1-[2-[2-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethyl]triazol-4-yl]methoxy]ethoxy]acetyl]amino]butanoyl]pyrrolidine-2-carboxamide (compound 14.0) (7 mg, 16%). LCMS-ESI (pos.) m / z: 1015.6 (M+1) + TIFF0007814386000074.tif46155

[0142] Compound 15.0 5-[[1-[2-[2-[2-[[(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethyl]triazol-4-yl]methylamino]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione TIFF0007814386000075.tif55128

[0143] Step-1: Synthesis of (1S,3S)-N1-[2-[2-(2-azidoethoxy)ethoxy]ethyl]-N3-[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (Compound 15.01) To a stirred solution of [(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]ammonium chloride (156.65 mg, 0.44 mmol) (CAS# 2416874-47-8, prepared as described in US20200131189A1) in MeCN (10 mL) was added 2-[2-(2-azidoethoxy)ethoxy]ethyl 4-methylbenzenesulfonate (180 mg, 0.55 mmol) and KCO (603.33 mg, 4.37 mmol), and the reaction mixture was refluxed for 48 h. The reaction mixture was filtered through Celite, and the filtrate was concentrated in vacuo. The crude product was purified by CombiFlash column chromatography (silica gel) eluting with 5% MeOH in DCM to give (1S,3S)-N1-[2-[2-(2-azidoethoxy)ethoxy]ethyl]-N3-[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (compound 15.01) (120 mg, 44%). LCMS-ESI (pos.) m / z: 478.9 (M+1). +

[0144] Step-2: Synthesis of 5-[[1-[2-[2-[2-[[(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethyl]triazol-4-yl]methylamino]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (Compound 15.0) To a solution of (1S,3S)-N1-[2-[2-(2-azidoethoxy)ethoxy]ethyl]-N3-[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (Compound 15.01) (53 mg, 0.11 mmol) in THF (2 mL) was added 2-(2,6-dioxo-3-piperidyl)-4-(prop-2-ynylamino)isoindoline-1,3-dione (34.44 mg, 0.11 mmol), CuSO4 (3.53 mg, 0.02 mmol), sodium ascorbate (4.38 mg, 0.02 mmol), and water (0.2 mL), and the reaction mixture was stirred at ambient temperature for 16 hours. The reaction mixture was evaporated in vacuo and the residue was purified by preparative HPLC followed by lyophilization from MeCN:HO to give 5-[[1-[2-[2-[2-[[(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethyl]triazol-4-yl]methylamino]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (compound 15.0) (25.18 mg, 28%). LCMS-ESI (pos.) m / z: 790.4 (M+1) + TIFF0007814386000076.tif38155

[0145] Compound 16.0 5-[[1-[2-[2-[2-[2-[[(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethoxy]ethyl]triazol-4-yl]methylamino]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione TIFF0007814386000077.tif62131

[0146] Step-1: Synthesis of (1S,3S)-N1-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethyl]-N3-[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (Compound 16.01) To a stirred solution of [(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]ammonium chloride (153.53 mg, 0.43 mmol) (CAS# 2416874-47-8, prepared using the procedure described in US20200131189A1) in MeCN (10 mL) was added 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate (200 mg, 0.54 mmol) and KCO (591.29 mg, 4.28 mmol), and the reaction mixture was refluxed for 48 h. The reaction mixture was filtered through Celite, and the filtrate was concentrated in vacuo. The crude product was purified by CombiFlash column chromatography (silica gel) eluting with 5% MeOH in DCM to give (1S,3S)-N1-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethyl]-N3-[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (compound 16.01) (110 mg, 37%). LCMS-ESI (pos.) m / z: 523.3 (M+1). +

[0147] Step-2: Synthesis of 5-[[1-[2-[2-[2-[2-[[(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethoxy]ethyl]triazol-4-yl]methylamino]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (Compound 16.0) To a solution of (1S,3S)-N1-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethyl]-N3-[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (Compound 16.01) (48 mg, 0.09 mmol) in THF (2 mL) was added 2-(2,6-dioxo-3-piperidyl)-4-(prop-2-ynylamino)isoindoline-1,3-dione (28.57 mg, 0.09 mmol), CuSO4 (2.93 mg, 0.02 mmol), sodium ascorbate (3.64 mg, 0.02 mmol), and water (0.2 mL), and the reaction mixture was stirred at ambient temperature for 16 hours. The reaction mixture was evaporated in vacuo and the residue was purified by preparative HPLC followed by lyophilization from MeCN:HO to give 5-[[1-[2-[2-[2-[2-[[(1S,3S)-3-[[3-chloro-5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethoxy]ethoxy]ethoxy]ethyl]triazol-4-yl]methylamino]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (compound 16.0) (16 mg, 21%). LCMS-ESI (pos.) m / z: 834.4 (M+1) + TIFF0007814386000078.tif46155

[0148] Compound 17.0 2-(2,6-dioxo-3-piperidyl)-4-[2-[4-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]-1-methyl-ethyl]piperazin-1-yl]ethylamino]isoindoline-1,3-dione: TIFF0007814386000079.tif49136

[0149] Step-1: Synthesis of benzyl N-[2-[4-(2-hydroxy-1-methyl-ethyl)piperazin-1-yl]ethyl]carbamate (Compound 17.01) To a stirred solution of benzyl N-(2-bromoethyl)carbamate (805.42 mg, 3.12 mmol) in MeCN (15 mL) was added KCO (574.16 mg, 4.16 mmol) and 2-piperazin-1-ylpropan-1-ol (300 mg, 2.08 mmol), and the reaction mixture was heated at 80 °C for 12 h. After completion of the reaction, monitored by LCMS, the reaction mixture was filtered through a Celite bed, and the Celite bed was washed with EtOAc. The filtrate was dried over anhydrous NaSO and evaporated in vacuo. The crude product was purified by neutral alumina using 5% MeOH in DCM to give benzyl N-[2-[4-(2-hydroxy-1-methyl-ethyl)piperazin-1-yl]ethyl]carbamate (Compound 17.01) (500 mg, 75%). LCMS-ESI (pos.) m / z: 322.1 (M+1) +

[0150] Step-2: Synthesis of benzyl N-[2-[4-(2-chloro-1-methyl-ethyl)piperazin-1-yl]ethyl]carbamate (compound 17.02) To a stirred solution of benzyl N-[2-[4-(2-hydroxy-1-methyl-ethyl)piperazin-1-yl]ethyl]carbamate (Compound 17.01) (345 mg, 1.07 mmol) in DCM (10 mL) at 0 °C, triethylamine (0.46 mL, 3.22 mmol) and methanesulfonyl chloride (0.12 mL, 1.5 mmol) were added, and the reaction mixture was stirred at ambient temperature for 30 minutes. After completion of the reaction, monitored by TLC, DCM (25 mL) was added to the reaction mixture, and the organic layer was washed with saturated NaHCO solution followed by saturated brine solution. The organic layer was dried over anhydrous NaSO and evaporated in vacuo to give benzyl N-[2-[4-(2-chloro-1-methyl-ethyl)piperazin-1-yl]ethyl]carbamate (Compound 17.02) (350 mg crude material), which was used in the next reaction without purification. LCMS-ESI (pos.) m / z: 340.2 (M+1) +

[0151] Step-3: Synthesis of benzyl N-[2-[4-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]-1-methyl-ethyl]piperazin-1-yl]ethyl]carbamate (Compound 17.03): To a stirred solution of (1S,3S)—N3-[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine dihydrochloride (160 mg, 0.44 mmol) (CAS#2416873-83-9, prepared using the procedure described in US20200131189A1) and benzyl N-[2-[4-(2-chloro-1-methyl-ethyl)piperazin-1-yl]ethyl]carbamate (196.18 mg crude, approximately 0.58 mmol) in MeCN (10 mL) was added potassium carbonate (612.77 mg, 4.44 mmol), and the reaction mixture was heated at 95° C. for 16 h. The reaction mixture was filtered through Celite, and the filtrate was concentrated in vacuo. The crude product was purified by preparative HPLC to give N-[2-[4-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]-1-methyl-ethyl]piperazin-1-yl]ethyl]benzylcarbamate (compound 17.03) (95 mg, 36%). LCMS-ESI (pos.) m / z: 591.3 (M+1) +

[0152] Step-4: Synthesis of (1S,3S)-N1-[2-[4-(2-aminoethyl)piperazin-1-yl]propyl]-N3-[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (Compound 17.04) To a stirred solution of N-[2-[4-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]-1-methyl-ethyl]piperazin-1-yl]ethyl]benzylcarbamate (Compound 17.03) (95 mg, 0.16 mmol) in methanol (5 mL) was added 10% Pd—C (3.39 mg, 0.03 mmol). The reaction mixture was stirred under a hydrogen atmosphere at ambient temperature for 16 hours. After completion of the reaction, as monitored by LCMS, the reaction mixture was filtered through a celite bed, and the celite was washed with methanol. The filtrate was concentrated in vacuo to give (1S,3S)-N1-[2-[4-(2-aminoethyl)piperazin-1-yl]propyl]-N3-[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (compound 17.04) (60 mg crude, 82%). LCMS-ESI (pos.) m / z: 457.52 (M+1) +

[0153] Step-5: Synthesis of 2-(2,6-dioxo-3-piperidyl)-4-[2-[4-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]-1-methyl-ethyl]piperazin-1-yl]ethylamino]isoindoline-1,3-dione (Compound 17.0) To a stirred solution of (1S,3S)-N1-[2-[4-(2-aminoethyl)piperazin-1-yl]propyl]-N3-[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (Compound 17.04) (60 mg crude, ca. 0.13 mmol) and 2-(2,6-dioxo-3-piperidyl)-4-fluoro-isoindoline-1,3-dione (Compound 5.01) (30 mg, 0.11 mmol) in DMA (2 mL) was added DIPEA (0.06 mL, 0.33 mmol), and the reaction mixture was stirred at 90 °C for 12 hours. After completion of the reaction as monitored by LCMS, the reaction mixture was poured into ice-cold water, extracted with EtOAc (2 × 20 mL), and washed with saturated brine solution. The organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo to give the crude material. The crude product was purified by preparative HPLC using an ammonium acetate buffer / MeCN mobile phase, followed by lyophilization using MeCN / HO to give 2-(2,6-dioxo-3-piperidyl)-4-[2-[4-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]-1-methyl-ethyl]piperazin-1-yl]ethylamino]isoindoline-1,3-dione (compound 17.0) (5 mg, 6%). LCMS-ESI (pos.) m / z: 713.5 (M+1). + TIFF0007814386000080.tif38155

[0154] Compound 18.0 (2S,4R)-1-[(2S)-2-[[2-[2-[4-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethyl]piperazin-1-yl]ethoxy]acetyl]amino]-3,3-dimethyl-butanoyl]-4-hydroxy-N-[[4-(4-methylthiazol-5-yl)phenyl]methyl]pyrrolidine-2-carboxamide TIFF0007814386000081.tif67143

[0155] Step-1: Synthesis of tert-butyl 4-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethyl]piperazine-1-carboxylate (Compound 18.01) To a stirred suspension of potassium carbonate (264.07 mg, 2.61 mmol) in MeCN (10 mL) at ambient temperature was added (1S,3S)-N3-[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (300 mg, 1.04 mmol) (CAS#2416873-83-9, prepared using the procedure described in US20200131189A1) and tert-butyl 4-(2-methylsulfonyloxyethyl)piperazine-1-carboxylate (482.87 mg, 1.57 mmol). The reaction mixture was refluxed at 95°C for 16 hours. After completion of the reaction, monitored by TLC, it was filtered through a sintered funnel and evaporated to dryness in vacuo. The crude residue was purified by CombiFlash column chromatography (amine silica) eluting with 5% MeOH in DCM, and the desired fractions were evaporated in vacuo to give tert-butyl 4-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethyl]piperazine-1-carboxylate (compound 18.01) (200 mg, 38%). LCMS-ESI (pos.) m / z: 500.5 (M+1). +

[0156] Step-2: Synthesis of 2-piperazin-4-ium-1-ylethyl-[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]ammonium dichloride (Compound 18.02) tert-Butyl 4-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethyl]piperazine-1-carboxylate (Compound 18.01) (230 mg, 0.46 mmol) was placed in a round-bottom flask, and 4 M HCl in 1,4-dioxane (5 mL) was added. The reaction mixture was stirred at ambient temperature for 3 hours. The reaction mixture was concentrated to dryness in vacuo and washed with n-pentane to give 2-piperazin-4-ium-1-ylethyl-[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]ammonium dichloride (Compound 18.02) (216 mg, 99%). LCMS-ESI (pos.) m / z: 400.4 (M+1) +

[0157] Step-3: Synthesis of tert-butyl 2-[2-[4-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethyl]piperazin-1-yl]ethoxy]acetate (Compound 18.03) To a stirred suspension of potassium carbonate (122.51 mg, 1.21 mmol) in MeCN (10 mL) at ambient temperature was added tert-butyl 2-[2-(p-tolylsulfonyloxy)ethoxy]acetate (123.07 mg, 0.37 mmol) and 2-piperazin-4-ium-1-ylethyl-[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]ammonium dichloride (Compound 18.02) (160 mg, 0.34 mmol). The reaction was refluxed at 90° C. for 16 hours. After completion of the reaction, monitored by TLC, it was filtered through a sintered funnel and evaporated to dryness in vacuo. The crude residue was purified by CombiFlash column chromatography (silica gel) in 5% MeOH in DCM and the desired fractions were evaporated in vacuo to give tert-butyl 2-[2-[4-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethyl]piperazin-1-yl]ethoxy]acetate (compound 18.03) (120 mg, 64%). LCMS-ESI (pos.) m / z: 558.4 (M+1) +

[0158] Step-4: Synthesis of 2-[4-[2-(carboxymethoxy)ethyl]piperazin-1-yl]ethyl-[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]ammonium 2,2,2-trifluoroacetate (Compound 18.04) To a stirred solution of tert-butyl 2-[2-[4-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethyl]piperazin-1-yl]ethoxy]acetate (Compound 18.03) (50 mg, 0.09 mmol) in DCM (2 mL) at 0° C. was added trifluoroacetic acid (0.21 mL, 1.51 mmol). The reaction mixture was stirred at 0° C. for 3 hours. After completion of the reaction, monitored by TLC, the reaction mixture was evaporated to dryness in vacuo, washed with n-pentane, and dried to give 2,2,2-trifluoroacetate 2-[4-[2-(carboxymethoxy)ethyl]piperazin-1-yl]ethyl-[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]ammonium (compound 18.04) (44 mg, 80%). LCMS-ESI (pos.) m / z: 502.0 (M+1) +

[0159] Step-5: Synthesis of (2S,4R)-1-[(2S)-2-[[2-[2-[4-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethyl]piperazin-1-yl]ethoxy]acetyl]amino]-3,3-dimethyl-butanoyl]-4-hydroxy-N-[[4-(4-methylthiazol-5-yl)phenyl]methyl]pyrrolidine-2-carboxamide formate (Compound 18.0) To a stirred solution of 2-[4-[2-(carboxymethoxy)ethyl]piperazin-1-yl]ethyl-[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]ammonium 2,2,2-trifluoroacetate (40 mg, 0.08 mmol) (Compound 18.04) in DMF (3 mL) at 0° C. was added [(1S)-1-[(2S,4R)-4-hydroxy-2-[[4-(4-methylthiazol-5-yl)phenyl]methylcarbamoyl]pyrrolidine-1-carbonyl]-2,2-dimethyl-propyl]ammonium chloride (55.86 mg, 0.12 mmol) and HATU (60.64 mg, 0.16 mmol). N,N-Diisopropylethylamine (52.71 uL, 0.32 mmol) was then added to the reaction mixture. The reaction mixture was stirred at ambient temperature for 16 hours. After completion of the reaction, as monitored by LCMS, the reaction mixture was evaporated to dryness in vacuo. The crude residue was purified by preparative HPLC and subsequently lyophilized from MeCN / water to give (2S,4R)-1-[(2S)-2-[[2-[2-[4-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethyl]piperazin-1-yl]ethoxy]acetyl]amino]-3,3-dimethyl-butanoyl]-4-hydroxy-N-[[4-(4-methylthiazol-5-yl)phenyl]methyl]pyrrolidine-2-carboxamide formate (Compound 18.0) (8 mg, 11%). LCMS-ESI (pos.) m / z: 914.6 (M+1) + TIFF0007814386000082.tif38155

[0160] Compound 19.0 2-(2,6-dioxo-3-piperidyl)-4-[2-[4-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethyl]piperazin-1-yl]ethylamino]isoindoline-1,3-dione: TIFF0007814386000083.tif47143

[0161] Step-1: Synthesis of 2-(2,6-dioxo-3-piperidyl)-4-(2-hydroxyethylamino)isoindoline-1,3-dione (Compound 19.01) 2-(2,6-Dioxo-3-piperidyl)-4-fluoro-isoindoline-1,3-dione (1 g, 3.62 mmol) (Compound 5.01) and 2-aminoethanol (331.69 mg, 5.43 mmol) were taken up in DMA (10 mL), followed by the addition of DIPEA (1.95 mL, 10.86 mmol). The reaction mixture was heated at 90 °C for 16 h. The reaction mixture was then taken up in EtOAc (20 mL), and the organic layer was washed with water (2 × 20 mL), followed by saturated brine solution (1 × 20 mL). The organic layer was separated, dried over anhydrous NaSO, and concentrated to dryness in vacuo. The crude residue was purified by CombiFlash column chromatography (silica gel) eluting with 5% MeOH in DCM. The desired fractions were concentrated to dryness in vacuo to give 2-(2,6-dioxo-3-piperidyl)-4-(2-hydroxyethylamino)isoindoline-1,3-dione (compound 19.01) (200 mg, 17%). LCMS-ESI (pos.) m / z: 318.0 (M+1) +

[0162] Step-2: Synthesis of 2-(2,6-dioxo-3-piperidyl)-4-(2-hydroxyethylamino)isoindoline-1,3-dione (compound 19.02) 2-(2,6-Dioxo-3-piperidyl)-4-(2-hydroxyethylamino)isoindoline-1,3-dione (Compound 19.01) (80 mg, 0.25 mmol) was taken up in DCM (5 mL) and cooled to 0 °C. Methanesulfonyl chloride (97.57 uL, 0.76 mmol) and EtN (176.84 uL, 1.26 mmol) were then added. The reaction mixture was vigorously stirred at ambient temperature for 16 hours. The reaction mixture was concentrated to dryness in vacuo, and the crude material was purified by CombiFlash column chromatography (silica gel) eluting with 10% MeOH in DCM. The desired fractions were concentrated to dryness in vacuo to give 2-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]amino]ethyl methanesulfonate (Compound 19.02) (40 mg, 40%). LCMS-ESI (pos.) m / z: 396.1 (M+1) +

[0163] Step-3: Synthesis of acetic acid 2-(2,6-dioxo-3-piperidyl)-4-[2-[4-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethyl]piperazin-1-yl]ethylamino]isoindoline-1,3-dione (Compound 19.0) To a stirred solution of 2-piperazin-4-ium-1-ylethyl-[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]ammonium dichloride (Compound 18.02) (23.9 mg, 0.05 mmol) in dry MeCN (5 mL), sodium bicarbonate (42.5 mg, 0.51 mmol) and 2-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]amino]ethyl methanesulfonate (Compound 19.03) (20 mg, 0.05 mmol) were added and stirred at room temperature. KI (839.68 μg, 0.01 mmol) was then added and the reaction mixture was stirred at 95° C. for 16 hours. After completion of the reaction, the solvent was removed in vacuo. The crude reaction mixture was purified by preparative HPLC, followed by lyophilization from MeCN / water to give 2-(2,6-dioxo-3-piperidyl)-4-[2-[4-[2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]ethyl]piperazin-1-yl]ethylamino]isoindoline-1,3-dione acetate (compound 19.0) (4.22 mg, 11%). LCMS-ESI (pos.) m / z: 699.4 (M+1). + TIFF0007814386000084.tif38155

[0164] Compound 20.0 N-[3-[1-[2-[2-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]amino]ethoxy]ethyl]triazol-4-yl]propyl]-2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetamide: TIFF0007814386000085.tif43134

[0165] Step-1: Synthesis of N-(3-azidopropyl)-2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetamide (Compound 20.01) To a solution of 2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetic acid 2,2,2-trifluoroacetic acid (310 mg, 0.90 mmol) and 3-azidopropan-1-amine (0.09 mL, 0.90 mmol) in DMF (5 mL) at 0 °C, DIPEA (0.47 mL, 2.69 mmol), EDC.HCl (258.05 mg, 1.35 mmol), and HOBt (181.72 mg, 1.35 mmol) were added and stirred at RT for 16 h. The reaction mixture was extracted with EtOAc and then washed with water followed by saturated brine solution. The organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo. The crude material was purified by CombiFlash column chromatography (silica gel) eluting with 5% MeOH in DCM to give N-(3-azidopropyl)-2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetamide (compound 20.01) (210 mg, 47%). LCMS-ESI (pos.) m / z: 428.1 (M+1). +

[0166] Step-2: Synthesis of N-[3-[1-[2-[2-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]amino]ethoxy]ethyl]triazol-4-yl]propyl]-2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetamide (Compound 20.0) To a solution of N-(3-azidopropyl)-2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetamide (Compound 20.01) (34.73 mg, 0.08 mmol) in THF (2 mL) was added 4-(2-but-3-ynoxyethylamino)-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (30 mg, 0.08 mmol), CuSO4 (2.59 mg, 0.02 mmol), sodium ascorbate (3.22 mg, 0.02 mmol), and water (0.2 mL) and stirred at ambient temperature for 16 hours. The reaction mixture was evaporated in vacuo and the crude residue was purified by preparative HPLC followed by lyophilization from MeCN:HO to give N-[3-[1-[2-[2-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]amino]ethoxy]ethyl]triazol-4-yl]propyl]-2-[[(1S,3S)-3-[[5-(1-ethylpropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetamide (compound 20.0) (10 mg, 15%). LCMS-ESI (pos.) m / z: 797.5 (M+1) + TIFF0007814386000086.tif46155

[0167] Compound 21.0 2-(2,6-dioxo-3-piperidyl)-4-[3-[4-[2-[[(1S,3S)-3-[[5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetyl]piperazin-1-yl]propylamino]isoindoline-1,3-dione TIFF0007814386000087.tif47128

[0168] Step 1. Synthesis of ethyl 7-chloropyrazolo[1,5-a]pyrimidine-5-carboxylate (compound 21.01) TIFF0007814386000088.tif23128 To a solution of diethyl buta-2-inedionate (50 g, 293.84 mmol) in acetic acid (300 mL) was added 1H-pyrazol-5-amine (24.42 g, 293.84 mmol), and the mixture was stirred at 110 °C for 8 hours. The reaction was cooled to room temperature, and the mixture was filtered. The filter cake was washed with acetic acid (20 mL * 2) followed by methyl tert-butyl ether (30 mL * 5), then concentrated in vacuo to give the crude product. Compound ethyl 7-hydroxypyrazolo[1,5-a]pyrimidine-5-carboxylate (Compound 21.01) (42 g, 69%) was obtained as a light brown solid. TIFF0007814386000089.tif13138

[0169] Step 2. Synthesis of ethyl 7-chloropyrazolo[1,5-a]pyrimidine-5-carboxylate (compound 21.02) To a solution of ethyl 7-hydroxypyrazolo[1,5-a]pyrimidine-5-carboxylate (compound 21.01) (20 g, 96.53 mmol) in POCl3 (200 mL, 2139.6 mmol, 32.8 g) was slowly added dropwise N,N-dimethylaniline (8.19 g, 67.57 mmol). The mixture was stirred at 110 °C for 8 h. LCMS indicated that a small amount of starting material remained and the desired mass was detected. POCl3 was removed in vacuo. The residue was dissolved in DCM (10 mL), poured into ice-water (50 mL), and extracted with DCM (100 mL*3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated to give the crude product. The crude product was purified by Combiflash column (ISCO 120 g silica, 0-30% ethyl acetate in petroleum ether, 60 min gradient). Compound 7-chloropyrazolo[1,5-a]pyrimidine-5-carboxylate ethyl ester (compound 21.02) (18.5 g, 85%) was obtained as a yellow solid. LCMS (pos.) m / z 226.2, [M+1] + TIFF0007814386000091.tif13135

[0170] Step 3. Synthesis of ethyl 7-[[(1S,3S)-3-(tert-butoxycarbonylamino)cyclopentyl]amino]pyrazolo[1,5-a]pyrimidine-5-carboxylate (compound 21.03) To a stirred solution of ethyl 7-chloropyrazolo[1,5-a]pyrimidine-5-carboxylate (Compound 21.02) (8 g, 35.46 mmol) in MeCN (80 mL) was added TEA (4.97 mL, 35.46 mmol) and tert-butyl ((1S,3S)-3-aminocyclopentyl)carbamate (7.1 g, 35.46 mmol). The mixture was then stirred at 85 °C for 2 h. LC-MS showed complete consumption of the starting material and the desired mass was detected. TLC (petroleum ether:ethyl acetate = 1:1, Rf = 0.5, UV) showed complete consumption of the reactant and the formation of one new spot. The mixture was concentrated to give the crude product. The crude product was purified by Combiflash column (ISCO 12 g silica gel, 0 to 50% ethyl acetate in petroleum ether, 50 min gradient) to give compound 7-[[(1S,3S)-3-(tert-butoxycarbonylamino)cyclopentyl]amino]pyrazolo[1,5-a]pyrimidine-5-carboxylate (compound 21.03) (11.6 g, 82%) as a yellow solid. ee %=100 %. LCMS (pos.) m / z 390.3, [M+1] + TIFF0007814386000093.tif21155

[0171] Step 4. Synthesis of tert-butyl N-[(1S,3S)-3-[[5-(1-ethyl-1-hydroxy-propyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]carbamate (Compound 21.04) To a stirred solution of ethyl 7-[[(1S,3S)-3-(tert-butoxycarbonylamino)cyclopentyl]amino]pyrazolo[1,5-a]pyrimidine-5-carboxylate (compound 21.03) (12 g, 30.81 mmol) in THF (120 mL) was added bromo(ethyl)magnesium (41.08 mL, 123.25 mmol) (3 M in EtO) at 0 °C. The mixture was then stirred at 20 °C for 16 h. LC-MS indicated complete consumption of the starting material and the desired mass was detected. TLC (petroleum ether:ethyl acetate = 1:1, Rf = 0.5, UV) indicated complete consumption of the reactant and the formation of two major new spots. The mixture was quenched with saturated NH4Cl (30 mL) at 0 °C, stirred for an additional 10 min, and extracted with ethyl acetate (50 mL * 3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated to give the crude product. The crude product was purified by Combiflash column (ISCO 80 g silica gel, 0-30% ethyl acetate in petroleum ether, gradient over 50 min). The compound tert-butyl N-[(1S,3S)-3-[[5-(1-ethyl-1-hydroxypropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]carbamate (4.3 g, 10.341 mmol, 33.56%) was obtained as a yellow gum, and tert-butyl N-[(1S,3S)-3-[(5-propanoylpyrazolo[1,5-a]pyrimidin-7-yl)amino]cyclopentyl]carbamate (compound 21.04) (1 g, 8%) was obtained as a pale yellow solid. ee %=100 %. LCMS (pos.) m / z 404.4, [M+1] +

[0172] Step 5. Synthesis of (1S,3S)-N3-[5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (Compound 21.05) A solution of tert-butyl N-[(1S,3S)-3-[[5-(1-ethyl-1-hydroxypropyl)pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]carbamate (1 g, 2.48 mmol) in H2SO4 (6 mL, 2.48 mmol) was stirred at 110 °C for 1 h. LCMS and HPLC indicated complete consumption of the reactant and the desired MS were detected. The mixture was poured into ice water (10 mL), adjusted to pH 8-9 by adding ammonia, and extracted with CHCl3 / iPrOH = 3 / 1 (10 mL * 5). The combined organic layers were dried over anhydrous sodium sulfate and concentrated to give the crude product. The crude residue was purified by preparative HPLC (Kromasil C18 250*50 mm*10 μm column; 10 to 45% acetonitrile in 0.05% ammonia and 10 mM ammonium bicarbonate solution in water, 10 min gradient). Compound (1S,3S)-N3-[5-[(Z)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (30 mg, 4%) was obtained as a yellow oil, and (1S,3S)-N3-[5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (compound 21.05) (350 mg, 1.2 mmol, 48%) was obtained as a yellow gum. ee %=94.62 %. LCMS (pos.) m / z 286.2, [M+1] + TIFF0007814386000096.tif29155

[0173] Step 6. Synthesis of tert-butyl 2-[[(1S,3S)-3-[[5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetate (Compound 21.06) To a solution of (1S,3S)-N3-[5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (Compound 21.05) (390 mg, 1.37 mmol) in DMF (4 mL) was added K2CO3 (377.17 mg, 2.73 mmol) at 0 °C, and the mixture was stirred at 0 °C for 0.5 h. Then, tert-butyl 2-bromoacetate (266.55 mg, 1.37 mmol) was added at 0 °C, and the resulting mixture was stirred at 0 °C for 2 h. LCMS indicated a small amount of reactant remained and one main peak with the desired MS was detected. TLC (petroleum ether:ethyl acetate = 0:1, R f =0.45, UV) indicated that a small amount of reactant remained and one new spot had formed. The reaction mixture was poured into water (6 mL), and the resulting mixture was extracted with ethyl acetate (10 mL*3). The combined organic layers were washed with water (5 mL), brine (5 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo to give the crude product. The crude product was purified by CombiFlash column (ISCO 20 g silica gel, 0-35% ethyl acetate in petroleum ether, gradient over 20 min). Compound 2-[[(1S,3S)-3-[[5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetate tert-butyl ester (compound 21.06) (260 mg, 44%) was obtained as a pale yellow gum. ee=100%. LCMS (pos.) m / z 400.2, [M+1] + TIFF0007814386000098.tif29154

[0174] Step 7. Synthesis of 2-[[(1S,3S)-3-[[5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetic acid dihydrochloride (Compound 21.07) A solution of tert-butyl 2-[[(1S,3S)-3-[[5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetate (Compound 21.06) (250 mg, 0.6300 mmol) in HCl (5 mL, 20 mmol) (4 M in EtOAc) was stirred at 25° C. for 2 hours. LCMS indicated a small amount of reactant remained and one main peak with the desired MS was detected. The compound 2-[[(1S,3S)-3-[[5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetic acid; dihydrochloride (Compound 21.07) (210 mg, 75%) was obtained as a pale yellow solid. ee=100%. LCMS (pos.) m / z 344.1, [M+1] + TIFF0007814386000100.tif21148

[0175] Step 8. Synthesis of tert-butyl 4-[3-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]amino]propyl]piperazine-1-carboxylate (compound 21.08) To a stirred solution of 2-(2,6-dioxo-3-piperidyl)-4-fluoro-isoindoline-1,3-dione (50 mg, 0.1800 mmol) and tert-butyl 4-(3-aminopropyl)piperazine-1-carboxylate (44.05 mg, 0.1800 mmol) in DMSO (0.5000 mL) was added DIPEA (0.06 mL, 0.3600 mmol) and stirred at 80 °C for 4 h. LC-MS showed complete consumption of the reactants and the main desired mass was detected. The mixture was poured into water (4 mL) and extracted with ethyl acetate (3 × 5 mL). The organic layer was washed with brine (4 mL), dried over anhydrous NaSO, then filtered and concentrated in vacuo. The crude product was purified by preparative TLC (dichloromethane:methanol = 10 / 1, R f=0.3) to give tert-butyl 4-[3-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]amino]propyl]piperazine-1-carboxylate (compound 21.08) (50 mg, 55%). LCMS (pos.) m / z 486.2 [M+1] +

[0176] Step 9. Synthesis of 2-(2,6-dioxo-3-piperidyl)-4-(3-piperazin-1-ylpropylamino)isoindoline-1,3-dione (Compound 21.09) TIFF0007814386000102.tif30128 To a round-bottom flask containing tert-butyl 4-[3-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]amino]propyl]piperazine-1-carboxylate (Compound 21.08) (30 mg, 0.0600 mmol) was added HCl / EtOAc (2 mL, 8 mmol). The mixture was stirred at 20 °C for 2 h. TLC (DCM:MeOH = 10:1, R f =0.1) indicated complete consumption of the starting material and the formation of one new spot. The reaction mixture was concentrated under reduced pressure to give 2-(2,6-dioxo-3-piperidyl)-4-(3-piperazin-1-ylpropylamino)isoindoline-1,3-dione (compound 21.09) (25 mg, 89%) as a yellow solid.

[0177] Step 10. Synthesis of 2-(2,6-dioxo-3-piperidyl)-4-[3-[4-[2-[[(1S,3S)-3-[[5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetyl]piperazin-1-yl]propylamino]isoindoline-1,3-dione; 2,2,2-trifluoroacetic acid (Compound 21.0) TIFF0007814386000103.tif23128212821282128212821282128212821282128212 To a solution of oxo-3-piperidyl)-4-(3-piperazin-1-ylpropylamino)isoindoline-1,3-dione dihydrochloride (compound 21.09) (20.42 mg, 0.0400 mmol) and triethanolamine (TEA) (0.03 mL, 0.1800 mmol), T3P (45.85 mg, 0.0700 mmol) was added, and the mixture was stirred at 25 °C for 0.5 h. LCMS showed complete consumption of the reactant and one main peak with the desired MS peak. The reaction mixture was filtered, and the filtrate was purified by preparative HPLC (Phenomenex Luna C18 100*40 mm*5 um column; 5-30% acetonitrile in 0.1% aqueous trifluoroacetic acid, 8-minute gradient). The compound 2-(2,6-dioxo-3-piperidyl)-4-[3-[4-[2-[[(1S,3S)-3-[[5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetyl]piperazin-1-yl]propylamino]isoindoline-1,3-dione; 2,2,2-trifluoroacetic acid (Compound 21.0) (10 mg, 28%) was obtained as a yellow solid. LCMS (pos.) m / z 363.2 [1 / 2M+1] + . TIFF0007814386000104.tif29162

[0178] The compounds in Table 1 were prepared using the synthetic procedures and corresponding reagents described above for Compounds 1.0 to 21.0.

[0179] (Table 1) TIFF0007814386000105.tif40144TIFF0007814386000106.tif195144

[0180] Compound 57.0 4-[3-[4-[2-[[(1S,3S)-3-[[3-chloro-5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetyl]piperazin-1-yl]propylamino]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione TIFF0007814386000107.tif44128

[0181] Step-1. Synthesis of tert-butyl N-[(1S,3S)-3-[[5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]carbamate (Compound 57.01) To a solution of (1S,3S)-N3-[5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (Compound 21.05) (900 mg, 3.15 mmol) in DCM (10 mL) was added TEA (0.88 mL, 6.3 mmol) and di-tert-butyl dicarbonate (826 mg, 3.8 mmol), and the mixture was stirred at 25 °C for 2 h. LCMS indicated complete consumption of the starting material (Compound 21.05) and one main peak with the desired MS was detected. TLC (petroleum ether:ethyl acetate = 2:1, R f= 0.56, UV) indicated complete consumption of the starting material and the formation of one new spot. The reaction mixture was poured into water (8 mL), and the resulting mixture was extracted with dichloromethane (10 mL * 3). The combined organic layers were washed with brine (5 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo to obtain the crude product. The crude product was purified by flash column chromatography (ISCO 20 g silica, 0 to 30% ethyl acetate in petroleum ether, gradient over 20 min). Compound N-[(1S,3S)-3-[[5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]tert-butyl albamate (Compound 57.01) (1.17 g, 3.035 mmol, 96% yield) was obtained as a pale yellow gum. ee %= 99.32 %. LCMS-ESI (pos.) m / z: 386.4 [M+H] + TIFF0007814386000109.tif29155

[0182] Step-2. Synthesis of tert-butyl N-[(1S,3S)-3-[[3-chloro-5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]carbamate (Compound 57.02) To a solution of tert-butyl N-[(1S,3S)-3-[[5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]carbamate (Compound 57.01) (900 mg, 2.33 mmol) in DCM (45 mL) was slowly added NCS (327 mg, 2.45 mmol) at 0 °C, and the reaction was stirred for 1 h. TLC (petroleum ether:ethyl acetate = 3:1, R f=0.5, UV) indicated that traces of starting material (compound 57.01) remained and several new spots had formed. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by flash column (ISCO 20 g silica, 0-20% ethyl acetate in petroleum ether, gradient over 20 min). Compound N-[(1S,3S)-3-[[3-chloro-5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]carbamate tert-butyl ester (compound 57.02) (750 mg, 1.79 mmol, 76% yield) was obtained as a pale yellow oil. LCMS-ESI (pos.) m / z: 420.2 [M+H] + TIFF0007814386000111.tif29155

[0183] Step-3. Synthesis of (1S,3S)-N3-[3-chloro-5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (Compound 57.03) A solution of tert-butyl N-[(1S,3S)-3-[[3-chloro-5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]carbamate (Compound 57.02) (750 mg, 1.79 mmol) in HCl / EtOAc (4 M, 3.57 mL, 14.29 mmol) was stirred at 25 °C for 2 h. LCMS indicated complete consumption of the starting material and the main desired MS was detected. The reaction mixture was concentrated under reduced pressure to give the crude product. The crude product (1S,3S)-N3-[3-chloro-5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine (Compound 57.03) (700 mg, 1.96 mmol, crude material) was obtained as the hydrochloride salt as a pale yellow solid. LCMS-ESI (pos.) m / z: 320.1 [M+H] +

[0184] Step-4. Synthesis of tert-butyl 2-[[(1S,3S)-3-[[3-chloro-5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetate (Compound 57.04) To a solution of (1S,3S)-N3-[3-chloro-5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]cyclopentane-1,3-diamine; hydrochloride (Compound 57.03) (600 mg, 1.31 mmol) in DMF (6 mL) was added K2CO3 (543.8 mg, 3.94 mmol) and tert-butyl 2-bromoacetate (256.2 mg, 1.31 mmol) at 0 °C, and the reaction was stirred at 25 °C for 3 h. LCMS indicated traces of starting material remained and the desired MS was detected. The mixture was poured into water (10 mL) and extracted with ethyl acetate (3 x 20 mL). The organic layer was washed with brine (20 mL), dried over anhydrous Na2SO4, then filtered and concentrated under reduced pressure. The crude product was purified by flash column (ISCO 20 g silica, 0-40% ethyl acetate in petroleum ether, gradient over 20 min). Compound 2-[[(1S,3S)-3-[[3-chloro-5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]tert-butyl acetate (compound 57.04) (360 mg, 0.83 mmol, 63% yield) was obtained as a yellow oil. LCMS-ESI (pos.) m / z: 434.2 [M+H] +

[0185] Step-5. Synthesis of 2-[[(1S,3S)-3-[[3-chloro-5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetic acid (Compound 57.05) A solution of tert-butyl 2-[[(1S,3S)-3-[[3-chloro-5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetate (compound 57.04) (200 mg, 0.46 mmol) in 4 M HCl in EtOAc (3 mL, 12 mmol) was stirred at 20 °C for 2 h. LC-MS showed complete consumption of the starting material (compound 57.04) and the desired mass was detected. The mixture was concentrated in vacuo. The residue was purified by preparative HPLC (Phenomenex Luna C18 80*40 mm*3 um column; 22-42% acetonitrile in 0.04% HCl solution, 7 min gradient). 2-[[(1S,3S)-3-[[3-chloro-5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetic acid (compound 57.05) (100 mg, 0.2646 mmol, 57% yield) was obtained as a yellow solid. ee %= 96.86 %. LCMS-ESI (pos.) m / z: 378.1 [M+H] + TIFF0007814386000115.tif21155

[0186] Step-6. Synthesis of 4-[3-[4-[2-[[(1S,3S)-3-[[3-chloro-5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetyl]piperazin-1-yl]propylamino]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (Compound 57.0) To a solution of 2-(2,6-dioxo-3-piperidyl)-4-(3-piperazin-1-ylpropylamino)isoindoline-1,3-dione; hydrochloride salt (Compound 21.09) (27.69 mg, 0.06 mmol) in DCM (0.5 mL) was added 2-[[(1S,3S)-3-[[3-chloro-5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetic acid (Compound 57.05) (20 mg, 0.05 mmol), T3P (101.04 mg, 0.16 mmol), and TEA (0.52 mL, 3.7 mmol), and the reaction was then stirred at 25 °C for 1 hour. LCMS showed complete consumption of the starting material and the main desired product was detected. The reaction was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (Phenomenex Luna C18 80*30mm*3um column; 15-40% acetonitrile in 0.1% TFA solution, 8-minute gradient). Compound 4-[3-[4-[2-[[(1S,3S)-3-[[3-chloro-5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetyl]piperazin-1-yl]propylamino]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione (Compound 57.0) (7 mg, 0.009 mmol, 17% yield) was obtained as a yellow solid. LCMS-ESI (pos.) m / z: 380.2 [M+2H] 2+ TIFF0007814386000117.tif29155

[0187] Compound 58.0 4-[2-[4-[2-[[(1S,3S)-3-[[3-chloro-5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetyl]piperazin-1-yl]ethylamino]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione TIFF0007814386000118.tif49128

[0188] Compound 58.0 was prepared using the synthetic procedure and corresponding reagents described above for Compound 57.0. LCMS-ESI (pos.) m / z: 373.2 [M+2H] 2+ TIFF0007814386000119.tif29155

[0189] Compound 59.0 4-[4-[4-[2-[[(1S,3S)-3-[[3-chloro-5-[(E)-1-ethylprop-1-enyl]pyrazolo[1,5-a]pyrimidin-7-yl]amino]cyclopentyl]amino]acetyl]piperazin-1-yl]butylamino]-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione TIFF0007814386000120.tif46128

[0190] Compound 59.0 was prepared using the synthetic procedure and corresponding reagents described above for Compound 57.0. LCMS-ESI (pos.) m / z: 387.3 [M+2H] 2+ TIFF0007814386000121.tif38155

[0191] Experimental method: Degradation of CDK9 by HiBit assay Exemplary compounds of the invention were tested for CDK9 degradation. Compounds were tested in a 10-dose DC50 duplicate mode using a 3-fold serial dilution starting at 10 μM in either NCIH211 cells with endogenously tagged N-terminal CDK9-HiBit or NCIH1048 cells with endogenously tagged N-terminal CDK9-HiBit at a 24-hour culture time using the HiBit assay (Promega, Inc.) as a direct measure of CDK9 presence. A control compound (THAL-SNS-032, CAS2139287-33-3) was tested in a 10-dose DC50 mode using a 3-fold serial dilution starting at 10 μM. Results for the tested compounds are shown in Table 2. Data were normalized to positive (media only) and negative (untreated cells) controls, and curve fits were analyzed for CDK9 as % response versus log [compound concentration] using a 4-parameter logistic model in GraphPad software. For Table 2, A is <100, B is <1000, C is <10,000, D is >10,000, and ND is undetermined.

[0192] Table 2: HiBit DC for representative compounds of the present invention 50 data TIFF0007814386000122.tif98128TIFF0007814386000123.tif240105TIFF0007814386000124.tif65128

[0193] In the table above, A is <100, B is <1000, C is <10,000, D is >10,000, and ND is undecided.

[0194] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art that certain changes and modifications may be made in light of the teachings of the invention without departing from the spirit or scope of the appended claims.

[0195] Thus, the foregoing merely illustrates the principles of the invention. Those skilled in the art will recognize that, although not explicitly described or shown herein, they can devise various configurations that embody the principles of the invention and are within its spirit and scope. Furthermore, all examples and language expressing conditions described herein are intended primarily to aid the reader in understanding the principles of the invention and concepts provided by the inventors to advance the art, and should not be construed as being limited to such specifically described examples and conditions. Furthermore, all descriptions herein of principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents. Additionally, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Furthermore, nothing disclosed herein is intended to be a public benefit, regardless of whether such disclosure is expressly recited in the claims.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof: XLY (I) During the ceremony, X is an E3 ubiquitin ligase binding ligand; and L is a linking group covalently bonded to X and Y; Y is a group represented by formula (II): is a cyclin-dependent kinase 9 (CDK9) binding ligand of R 1 D, halo, R at any position 7 CO 2 R 8 , CO 2 R 8 , CO 2 H, R 7 CO 2 H, NH 2 , NHR 8 , OH, OR 8 , S.H., S.R. 8 , NHCOR 8 ,NHSO 2 R 8 , SO 2 NH 2 , SO 2 NHR 8 or C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 cycloalkenyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl, optionally substituted with one or more of Alternatively, R 1 and R 2 together form D, halo, NH at any position. 2 , NHR 8 , N.R. 7 R 8 , OH, OR 8 , S.H., S.R. 8 , NHCOR 8 ,NHSO 2 R 8 , SO 2 NH 2 , or SO 2 NHR 8 forming a fused C5-C6 cycloaryl optionally substituted with one or more of: R 2 , R 3 , and R 5 are independently H, D, halo, or at any position D, halo, R 7 CO 2 R 8 , CO 2 R 8 , CO 2 H, R 7 CO 2 H, NH 2 , NHR 8 , OH, OR 8 , S.H., S.R. 8 , NHCOR 8 ,NHSO 2 R 8 , SO 2 NH 2 , SO 2 NHR 8 or C1-C5 alkyl or C3-C6 cycloalkyl, optionally substituted with one or more of Alternatively, R 2 and R 5 together, D, halo, R at any position 7 CO 2 R 8 , CO 2 R 8 , CO 2 H, R 7 CO 2 H, NH 2 , NHR 8 , OH, OR 8 , S.H., S.R. 8 , NHCOR 8 ,NHSO 2 R 8 , SO 2 NH 2 , SO 2 NHR 8 forming a fused C5-C6 cycloaryl optionally substituted with one or more of: R 4 D, halo, R at any position 7 CO 2 R 8 , CO 2 R 8 , CO 2 H, R 7 CO 2 H, NH 2 , NHR 8 , OH, OR 8 , S.H., S.R. 8 , NHCOR 8 ,NHSO 2 R 8 , SO 2 NH 2 , SO 2 NHR 8 H, D, halo, C1-C5 alkyl, C3-C6 cycloalkyl, cyano, hydroxyl, or —O—(C1-C5 alkyl), optionally substituted with one or more of: R 6 is H or D; R 7 is (CH 2 ) n where n is an integer from 1 to 6; and R 8 is D, halo, OH, SH, or NH at any position 2 C1-C6 alkyl or C3-C6 cycloalkyl, optionally substituted with one or more of: Y is covalently bonded to L via R 5 ; X is or having a structure derived by removal of a hydrogen atom from a structure selected from the group consisting of: X is having a structure derived by removal of a hydrogen atom from a structure selected from the group consisting of: L has the structure of formula (III): During the ceremony, a, b, c, and d are each independently an integer from 0 to 6; e is 0 or 1, R 10 is absent or and having a structure selected from the group consisting of R11 is It has a structure selected from the group consisting of:

2. R 5 The compound of claim 1, wherein is an optionally substituted C3-C6 cycloalkyl group.

3. R 5 The compound of claim 2, wherein is an optionally substituted C5 cycloalkyl group.

4. R 5 but, 4. The compound of claim 3, having a structure selected from the group consisting of:

5. R 1 The compound of any one of claims 1 to 4, wherein is a C1 to C6 alkyl group.

6. R 1 but, 6. The compound of claim 5, having a structure selected from the group consisting of:

7. R 2 , R 3 , and R 6 and each independently represent H or D.

8. R 4 8. The compound of any one of claims 1-7, wherein is halo, cyano, or methyl.

9. R 4 is H or D.

10. Y is, 2. The compound of claim 1, having a structure selected from the group consisting of:

11. X is a structure 2. The compound of claim 1, having the formula:

12. The compound is selected from the group consisting of:

10. The compound of claim 1, having a structure selected from the group consisting of:

13. 13. A pharmaceutical composition for treating a subject for a CDK9-mediated disease, comprising a therapeutically effective amount of a compound according to any one of claims 1 to 12.

14. 14. The pharmaceutical composition of claim 13, wherein the CDK9-mediated disease is cancer.

15. 15. The pharmaceutical composition of claim 13 or 14, wherein the cancer is caused by aberrant expression of MYC-1 or MCL-1, is a hematological malignancy, or is a solid tumor.

16. 16. The pharmaceutical composition of any one of claims 13 to 15, wherein the disease is selected from the group consisting of acute myeloid leukemia, primary peritoneal carcinoma, chronic lymphocytic leukemia, relapsed multiple myeloma, non-Hodgkin's lymphoma, acute lymphoblastic leukemia, acute mixed lineage leukemia, advanced breast cancer, non-small cell lung cancer, and liver cancer.

17. 17. The pharmaceutical composition of any one of claims 13 to 16, wherein the compound is as defined in claim 12.

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